INSTANT DOWNLOAD WITH ANSWERS
Immunobiology 9th Edition By by Kenneth Murphy -Test Bank
Janeway’s Immunobiology, 9th Edition
Chapter 6: Antigen Presentation to T Lymphocytes
The generation of a:b T-cell receptor ligands
6-1 Antigen presentation functions both in arming effector T cells and in triggering their effector functions to attack pathogen-infected cells
6.1 Matching: The diagram in Figure Q6.1 shows a pathogen (in red) that is present in different cellular compartments of each of the cell types shown. In each case, a specific T cell subset will recognize peptides of that pathogen presented on MHC molecules on the surface of the cell, and will execute its effector function. From the list below, match the appropriate T cell effector response to the cell type and location of the pathogen.
- CD4 T cell killing of target cell
- CD8 T cell killing of target cell
iii. CD4 T cell activation of target cell’s antibody production
- CD8 T cell activation of target cell’s antibody production
- CD4 T cell activation of target cell’s ability to kill intracellular pathogen
- CD8 T cell activation of target cell’s ability to kill intracellular pathogen
6.2 Multiple choice: The mechanism of cross-presentation by dendritic cells is an essential pathway for generating CD8 T cell responses to some intracellular pathogens. If this pathway did not exist, we would be highly susceptible to:
- Intracellular pathogens that can survive inside macrophage endocytic vesicles
- Intracellular pathogens that are able to evade antibody responses
- Intracellular pathogens that do not infect and replicate in dendritic cells
- Intracellular pathogens that can spread from cell to cell by inducing cell fusion
- Intracellular pathogens that infect and replicate in red blood cells
6-2 Peptides are generated from ubiquitinated proteins in the cytosol by the proteasome
6.3 Multiple choice: The adaptive immune system developed a strategy for monitoring the proteins synthesized in virtually any cell in the body, thereby preventing pathogens from ‘hiding out’ by adopting an intracellular lifestyle. To accomplish this, the immune system:
- Co-opted the ubiquitin-proteasome system used by cell for protein turnover
- Created a novel pathway using the immunoproteasome for generating peptides
- Created a novel pathway to express foreign proteins on the cell surface
- Took advantage of proteolytic enzymes present in endocytic vesicles
- Engineered an immune-specific ubiquitin molecule for tagging foreign proteins
6.4 Multiple choice: Virus infections induce production of interferons that act on infected cells to enhance their recognition by CD8 cytotoxic T cells. To counter these mechanisms, viruses often encode proteins that interfere with antigen processing and presentation. In an experiment, cells infected with Virus X are treated with interferon and compared with uninfected cells treated with interferon. Proteasomes are isolated from the two cell populations and their enzymatic activities are compared. The data in Figure Q6.4 show the amino acid preferences for cleavage of peptides by the two samples of proteasomes.
Based on these data, Virus X most likely encodes a protein that interferes with:
- The expression of MHC class I on the surface of the infected cell
- The rate at which peptides are produced from intact proteins in the infected cell
- The transport of peptides from the cytosol to the endoplasmic reticulum in the infected cell
- The replacement of constitutive proteasome subunits with immunoproteasome subunits in the infected cell
- The development of CD8 T cells in the thymus by inhibiting the thymoproteasome
6-3 Peptides from the cytosol are transported by TAP into the endoplasmic reticulum and further processed before binding to MHC class I molecules
6.5 Short answer: A cell line carrying a mutation in a single gene is found to express very low levels of MHC class I on its surface. When infected with influenza virus, these cells are not recognized nor are they killed by a CD8 T cell line specific for an influenza peptide bound to the MHC class I protein expressed by these cells. Incubation of the mutant cell line with a large excess of this peptide in the cell culture medium overnight leads to the results shown in Figure Q6.5.
What is the most likely candidate for the gene that is defective in the mutant cell line?
6-4 Newly synthesized MHC class I molecules are retained in the endoplasmic reticulum until they bind a peptide
6.6 Multiple choice: During MHC class I synthesis and folding in the endoplasmic reticulum (ER), a process of peptide editing takes place as the newly synthesized MHC class I protein is held in a ‘peptide receptive’ state by binding to the calreticulin:ERp57:tapasin complex. Peptide editing ensures that the MHC class I molecules that reach the cell surface have stable, high affinity binding for their peptide cargo. Peptide editing is important to the immune response because it:
- Maintains high levels of surface MHC class I expression
- Ensures that MHC class I molecules are not degraded in the ER
- Retains the nascent MHC class I molecule in a peptide receptive state
- Allows surface MHC class I molecules to bind new peptides from the extracellular milieu
- Prevents surface MHC class I molecules from undergoing peptide exchange at the cell surface
6.7 True/False: MHC class I surface expression is dependent on an abundant source of pathogen-derived peptides. Thus, in uninfected cells, nearly all of the MHC class I proteins are degraded and never reach the cell surface.
6-5 Dendritic cells use cross-presentation to present exogenous proteins on MHC class molecules to prime CD8 T cells
6.8 Multiple choice: The virus shown in the diagram below is only able to infect and replicate in epithelial cells. In order for the cross-presenting dendritic cell to display viral peptides, rather than self peptides on its surface MHC class I proteins, which of the following procedures could be utilized, starting with the components shown in Figure Q6.8?
- Mix epithelial cells with heat-killed virus, wait 24 hrs, wash away any virus particles outside the epithelial cells, then add epithelial cells to dendritic cells.
- Mix epithelial cells with viral peptides, wait 24 hrs, wash away any viral peptides not bound to the epithelial cells, then add epithelial cells to dendritic cells.
- Mix epithelial cells with live virus particles, wait 24 hrs, wash away any virus particles outside the epithelial cells, then add epithelial cells to dendritic cells.
- Mix dendritic cells with viral nucleic acids and epithelial cells for 24 hrs.
- MIx epithelial cells will viral nucleic acids, wait 24 hrs, wash away any viral nucleic acid remaining outside the epithelial cells, then add epithelial cells to dendritic cells.
6.9 Multiple choice: Some viruses have mechanisms to down-regulate MHC class I protein expression on the surface of cells in which the virus is replicating. This immune evasion strategy might prevent effector CD8 cytotoxic T cells from recognizing and killing the virus-infected cells. Would this immune evasion strategy also prevent the initial activation of virus-specific CD8 T cells?
- Yes, because no viral peptide:MHC class I complexes would form to activate CD8 T cells.
- No, because dendritic cells would take up infected cells and cross-present viral peptides to activate CD8 T cells.
- No, because some presentation of MHC class I complexes with viral peptides would occur before the virus could down-regulate all the surface MHC class I protein.
- Yes, because this immune evasion strategy would also function in dendritic cells, even if the virus doesn’t replicate in dendritic cells.
- No, because the type I interferon response induced by the virus infection will up-regulate MHC class I expression and override the immune evasion mechanism.
6-6 Peptide:MHC class II complexes are generated in acidified endocytic vesicles from proteins obtained through endocytosis, phagocytosis, and autophagy
6.10 Multiple choice: Three major cell types, dendritic cells, macrophages, and B cells, present peptides bound to MHC class II molecules for recognition by CD4 T cells. In general, these peptides are derived from proteins or pathogens taken up by the cell by endocytosis, phagocytosis, or macropinocytosis. Based on these pathways of antigen uptake, some of the enzymes that degrade proteins to generate peptides for MHC class II presentation are:
- Ubiquitin ligases that tag proteins for degradation by the proteasome
- ATP transporter proteins that deliver endocytic proteins into the cytosol for degradation
- Cysteine proteases like cathepsins that function at acidic pH
- The lysosomal thiol reductase found in the endosomes
- The lysosome-associated membrane trafficking protein, LAMP-2
6-7 The invariant chain directs newly synthesized MHC class II molecules to acidified intracellular vesicles
6.11 True/False: The invariant chain protein, Ii, has only one function in MHC class II antigen presentation. This function entails Ii protein occupying the peptide-binding site of each newly synthesized class II protein, thereby preventing nascent MHC class II proteins from binding peptides or misfolded proteins in the endoplasmic reticulum.
6-8 The MHC class II-like molecules HLA-DM and HLA-DO regulate exchange of CLIP for other peptides
6.12 Multiple choice: Peptide editing is an important component of antigen presentation for both MHC class I and MHC class II pathways, as it drives the preferential presentation of high-affinity binding peptides. For MHC class II peptide editing, HLA-DM plays a key role. In the absence of HLA-DM:
- MHC class II molecules traffic to the cell surface with CLIP in their binding sites.
- No MHC class II molecules are released to traffic to the cell surface.
- MHC class II molecules bind to HLA-DO and are inhibited from binding peptides.
- Pathogens can evade the immune system by blocking peptide exchange on MHC class II.
- HLA-DO competes for high-affinity binding peptides with MHC class II molecules and blocks antigen presentation.
6.13 Multiple choice: Empty MHC class I and MHC class II molecules are rapidly removed from the cell surface. This process prevents:
- The accumulation of empty MHC molecules on the cell surface which would interfere with T cells recognizing pathogen-derived peptide:MHC complexes
- Pathogens from evading the immune response by inducing peptide release from cell surface MHC molecules
- MHC class I molecules from being internalized into endosomes and binding endosome-derived peptides
- HLA-DM from trafficking to the cell surface with MHC class II
- Inappropriate T cell recognition of healthy cells that are not infected, nor have ingested a pathogen
6-9 Cessation of antigen processing occurs in dendritic cells after their activation through reduced expression of the MARCH-1 E3 ligase
6.14 Multiple choice: The MARCH-1 E3-ubiquitin ligase is expressed in B cells, dendritic cells, and macrophages. The pathway regulated by MARCH-1 is targeted by some pathogens in an immune evasion strategy. In this strategy, the pathogens encode:
- A protein that induces degradation of MARCH-1
- A protein that mimics MARCH-1 and functions similarly
- A protein that binds to MARCH-1 and inhibits its function
- A protein that is induced by IL-10 in macrophages and dendritic cells
- A protein that induces degradation of CD86
The major histocompatibility complex and its function
6-10 Many proteins involved in antigen processing and presentation are encoded by genes within the MHC
6.15 True/False: The genes encoding MHC proteins are closely linked with genes encoding proteins involved in antigen processing and presentation. This genetic linkage facilitates the coordinate regulation of these genes by interferons.
6-11 The protein products of MHC class I and class II genes are highly polymorphic
6.16 Multiple choice: The adaptive immune system uses multiple strategies to generate diversity in our ability to mount responses to a wide array of infectious microorganisms. These strategies include the generation of diverse repertoires of B-cell and T-cell antigen receptors, as well as polymorphism of MHC genes. The polymorphism of MHC genes differs from the diversity of lymphocyte antigen receptors in that:
- It involves DNA rearrangements at multiple gene segments in the MHC locus.
- It requires different enzymes than the RAG1/RAG2 recombinase required for antigen receptor rearrangements.
- It results in a diverse repertoire of clonally distributed receptors on dendritic cells, rather than on lymphocytes.
- It creates diversity between individuals in the population rather than within a single individual.
- It does not contribute to the transplant rejection responses that occur after organ transplantation between unrelated individuals.
6.17 Short answer: Multiple mechanisms contribute to create a wide diversity in MHC protein expression between different individuals in the population. In addition to the genetic polymorphism of MHC genes, what additional mechanism(s) contribute to this diversity?
6-12 MHC polymorphism affects antigen recognition by T cells by influencing both peptide binding and the contacts between T-cell receptor and MHC molecule
6.18 Multiple choice: MHC polymorphism at individual MHC genes appears to have been strongly selected by evolutionary pressures. In other words, there appears to be selection for maintaining hundreds to thousands of different alleles of each MHC gene in the population. This notion is based on the observation that nucleotide differences between alleles that lead to amino acid substitutions are more frequent than those that are silent substitutions (i.e., not changing the amino acid sequence of the protein). In addition, the positions within the MHC protein where most of the allelic sequence variation occurs are not randomly distributed, but are concentrated in certain regions of the MHC protein. This latter point indicates:
- That some nucleotide sequences within the MHC genes are hot-spots for mutation
- That MHC genes are more susceptible to point mutations than to larger nucleotide deletions
- That MHC allelic polymorphism has been driven by selection for diversity in peptide binding specificity
- That MHC genes are more susceptible to all types of mutations than are other genes in the genome
- That MHC polymorphism has evolved to prevent pathogens that infect non-human primates from infecting humans
6.19 Multiple choice: The experiment shown in Figure Q6.19 uses two strains of mice that differ in their MHC genes. Strain A is H-2a and Strain B is H-2b. Mice of each strain are infected with the virus LCMV, and T cells are isolated at day 8 post-infection. These T cells are mixed with target cells that express either H-2a or H-2b; in each case, the target cells are either uninfected or infected with LCMV. After a four-hour incubation of T cells with target cells, the percentage of target cells lysed by the T cells is shown in the graph.
The explanation for the results of this experiment is:
- Mice of strain B do not make a T cell response to LCMV.
- Mice of strain A make a more robust T cell response to LCMV than mice of strain B.
- Target cells that express H-2b cannot be infected with LCMV.
- T cells from mice of strain A only recognize viral peptides on target cells expressing H-2a.
- LCMV peptides do not bind to MHC class I molecules from H-2b mice.
6-13 Alloreactive T cells recognizing nonself MHC molecules are very abundant
6.20 Multiple choice: In a mixed lymphocyte reaction, T cells from individual A make a robust response to antigen-presenting-cells from individual B, as long as the two individuals express different alleles of MHC molecules. Estimates indicate that up to 10% of the T cells from individual A may contribute to this response. If one performed this assay using responder T cells from a child and antigen-presenting cells from one parent, the result would be:
- A massive proliferative response made by the antigen-presenting cells of the parent
- A very weak response by the child’s T cells, involving only 0.1% of their T cells
- The complete absence of any proliferative response by the child’s T cells
- A robust cytolytic response that kills all of the parent’s antigen-presenting cells
- A robust response by the child’s T cells
6.21 True/False: Alloreactivity refers to the ability of T cells to respond to allelic polymorphisms in MHC molecules when mixed with antigen-presenting cells from a genetically different individual. The T-cell receptors involved in alloreactive responses are recognizing amino acid sequences on foreign MHC molecules and do not interact at all with the peptides bound to these MHC molecules.
6-14 Many T cells respond to superantigens
6.22 Multiple choice: Several types of pathogens encode proteins that function as superantigens, which activate massive numbers of T cells in an individual. One example is the staphylococcal enterotoxins that cause food poisoning. These superantigens are the exception to the general rule that T cells only recognize specific peptide:MHC complexes, because they:
- Induce activation of any T cell whose T-cell receptor uses a particular Vβ region bound by that superantigen
- Simultaneously stimulate all of the T-cell receptors on a given T cell
- Cover up the peptide-binding site, preventing MHC molecules from binding peptides
- Activate a large number of T cells that are specifically recognizing peptides derived from the superantigen protein
- Induce anti-microbial cytokine production that aids the immune system in clearing the pathogen
6-15 MHC polymorphism extends the range of antigens to which the immune system can respond.
6.23 Multiple choice: The extensive polymorphism of MHC genes in the population is thought to represent an evolutionary response to outflank the evasive strategies of pathogens. This polymorphism makes it difficult for pathogens to eliminate all potential MHC binding epitopes from their proteins. Based on this reasoning, it would seem advantageous for each individual to encode more than three different MHC class I and three different MHC class II genes per chromosome copy. If some individuals in the population had MHC loci that encoded 10 different MHC class I and 10 different MHC class II genes, the T cell repertoire in those individuals would likely be:
- Much more diverse than in the rest of the individuals of that population
- Much better at recognizing rare pathogens not encountered by most individuals in that population
- Much less diverse than the rest of the individuals in that population
- Much more alloreactive than the T cells found in the other individuals of that population
- Very reactive to bacterial and viral superantigens
Generation of ligands for unconventional T-cell subsets
6-16 A variety of genes with specialized functions in immunity are also encoded in the MHC
6.24 True/False: The MHC locus encodes a large number of genes spanning over four million bp of DNA. These include many genes involved in antigen processing and presentation, as well as receptors recognized by non-conventional T cells and natural killer (NK) cells. In addition, the MHC locus encodes genes with no function in immunity at all.
6-17 Specialized MHC class I molecules act as ligands for the activation and inhibition of NK cells and unconventional T-cell subsets
6.25 Multiple choice: NKG2D is an activating receptor expressed on NK cells, g:d T cells, and some cytotoxic a:b T cells. When stressed or infected cells up-regulate receptors that bind to and activate NKG2D molecules, the stressed or infected cells will be killed. This pathway relies on the fact that stressed or infected cells up-regulate:
- All classical MHC class I molecules
- HLA-C molecules that bind KIRs
- MHC class Ib genes such as MICA, MICB, and RAET1
- Qa-1 and HLA-E molecules that bind leader peptides of other HLA class I molecules
- HLA-G molecules just like those expressed on the fetal-derived cells in the placenta
6-18 Members of the CD1 family of MHC class I-like molecules present microbial lipids to invariant NKT cells
6.26 Multiple choice: Some CD1 molecules bind to glycosphingolipids, and are recognized by a subset of T cells known as invariant NKT (iNKT) cells. The ability of these T cells to recognize different glycolipid constituents from microorganisms when they are bound to CD1d places these cells in the ‘innate immune’ category. While iNKT cells do express a fully rearranged a:b T-cell receptor, one key feature of the T-cell receptors expressed on iNKT cells also places them in the ‘innate immune’ category. This feature is:
- iNKT cells have a highly restricted T-cell receptor repertoire, with the majority of cells utilizing the same Va and Ja rearrangement.
- iNKT cells express receptors that are also expressed on NK cells.
- iNKT cells express T-cell receptors that induce inhibitory, rather than activating signals.
- iNKT cells do not generally express CD4 or CD8.
- The T-cell receptors expressed on iNKT cells recognize both MHC class I and MHC class II molecules.
6-19 The nonclassical MHC class I molecule MR1 presents microbial folate metabolites to MAIT cells
6.27 Short answer: NKT cells that recognize microbial glycolipids bound to CD1 molecules comprise a class of T cells that shares features of both innate and adaptive immune cells. A second class of such cells are MAIT cells, that recognize antigens bound to the MHC class Ib molecule, MR1. What is the class of PAMP recognized by MAIT cells?
6-20 g:d T cells can recognize a variety of diverse ligands
6.28 Multiple choice: T cells expressing g:d T-cell receptors have been found to recognize a diversity of ligands, including pathogen-derived proteins, self-peptides, and stress-induced molecules. This pattern of antigen recognition shows similarity to that of iNKT cells and MAIT cells, suggesting that g:d T cells:
- Do not play an important role in immunity, but likely have a non-immune function
- Share features of both innate and adaptive immune cells
- Are only able to respond when the host is infected with a virus such as herpes simplex virus
- Are involved in maintaining the integrity of endothelial cells in the host
- Are most important in responses to tumor cells that show stress responses
6.29 Synthesis question: A family of six (mother, father, and four children) had two children with a history of chronic illness. Both children had repetitive infections of the sinuses, middle ears, and lungs due to a variety of respiratory viruses. Their other siblings were generally healthy and showed no signs of persistent or recurrent virus infections.
The two affected children had normal numbers of B cells, T cells, and NK cells in their blood. They also showed no defects in neutrophil function or in complement protein levels. The two children also had normal antibody levels to vaccine protein antigens, such as tetanus toxoid, and had normal T cell responses to antigens from the vaccine strain of Mycobacterium tuberculosis after being vaccinated.
Blood cells from one parent, one healthy child and the two affected children were examined for surface MHC protein expression by flow cytometry using two antibodies, one that recognizes all HLA class I proteins, and one that recognizes all HLA class II proteins. The results are shown in Figure Q6.29A.
- a) Analysis of HLA genotypes from the two affected children showed that they shared one haplotype of this locus. This haplotype encodes a common HLA-A allele, HLA-A2. Based on these data, is it likely that the two affected children have a point mutation (or mutations) in the coding sequence for HLA-A2? Why or why not?
- b) Name two proteins that could be candidates for the defective gene in the two affected children.
To address which gene defect might be present in the affected children, peripheral blood cells were isolated from one healthy child and one affected child, and mRNA was isolated from the cells. Quantitative RT-PCR was performed to assess the levels of mRNA for the three HLA class I heavy chain genes (HLA-A, -B, and –C) and for the b2-microglobulin gene. The results are shown in Figure Q6.29B.
In a second experiment, Western blots were performed, confirming that cell lysates from both affected children contained normal levels of all three HLA class I heavy chain proteins and the b2-microglobulin protein.
- c) Do these data eliminate any of your answers to part (b)?
In a final experiment, peripheral blood cells from the two affected children and one HLA-A2+ parent were transfected with a construct encoding the Hepatitis B virus surface antigen (HepBsAg), a protein that is currently used in the vaccine against Hepatitis B. This protein is normally synthesized and transported to the cell surface. In addition to the full length HepBsAg construct, cells were also transfected with a mini-gene, encoding just a single HLA-A2-binding peptide derived from Hepatitis B, amino acids 338–347. Using a cytolytic CD8 T cell clone specific for HepBsAg(338–347) peptide bound to HLA-A2, the transfected cells were tested for recognition by the CD8 T cell clone using an assay that measures target cell killing. Figure Q6.29C shows the results of this experiment.
- d) What is the most likely gene that is defective in the affected children?
6.30 Synthesis question: In the 1980s, a mutant strain of mice was identified, carrying amino acid changes in the MHC class II gene. This mutant strain was derived from C57Bl/6 mice, which carry the H-2b haplotype. Inbred H-2b mice express only one MHC class II protein, called Ab. The mutant strain, called ‘bm12’ was found to have 3 amino acid changes in the Ab protein, at positions 67, 70, and 71 of the Aβ chain. The positions of these amino acid changes on the MHC class II structure are shown below by the red circles in Figure Q6.30A. On the right, the side view diagram of MHC class II shows the direction of these three amino acid side chains.
Initial experiments with wild-type C57Bl/6 mice and bm12 mice showed that the wild-type mice made a robust CD4 T cell response after immunization with the insulin protein isolated from a cow; in contrast, the bm12 mice failed to make any detectable response to this foreign protein. Epitope mapping studies identified amino acid residues 1–14 of the bovine insulin A chain as the peptide recognized by CD4 T cells from wild-type mice.
- a) What is the most likely explanation for the failure of bm12 mice to make a CD4 T cell response to bovine insulin?
In a second set of experiments, T cells from wild-type (WT) or bm12 mice were mixed in vitro with antigen-presenting cells (APCs), in the presence or absence of the superantigen staphylococcal enterotoxin B (SEB), and T cell proliferation was measured. The data from these experiments are shown in Figure Q6.30B.
- b) What is the explanation for the results in Rows 1–4 of the table?
- c) Why does the T cell response to SEB (Rows 5–8) show a different pattern than the response to bovine insulin?
- d) In the table above, T cell proliferation was measured after 4 days of incubation of T cells, APCs, +/- SEB. If one isolated the T cells at the end of the incubation for the six conditions in which robust proliferation was seen (Rows 2, 3, 5–8), and stained the T cells with each antibody (separately) from a panel of antibodies that recognize each of the mouse Vb domains (i.e., an antibody to Vb1, an antibody to Vb2, etc), what result would be expected?
6.1: A = ii
B = v
C = iii
Viruses and some bacteria replicate in the cytosolic compartment, as shown in panel A. Their antigens are presented by MHC class I molecules to activate killing by cytotoxic CD8 T cells. Other bacteria and some parasites are taken up into endosomes, usually by specialized phagocytic cells such as macrophages, as shown in panel B. Here they are killed and degraded, or in some cases are able to survive and proliferate within the vesicle. Their antigens are presented by MHC class II molecules to activate cytokine production by CD4 T cells. In response to these CD4 T cell-derived cytokines, macrophages up-regulate their mechanisms for killing intracellular pathogens. Extracellular pathogens (or their protein productions) may bind to cell-surface receptors and enter the vesicular system by endocytosis, illustrated in panel C for antigens bound by the surface immunoglobulin of B cells. These antigens are presented by MHC class II molecules to CD4 helper T cells, which can then stimulate the B cells to produce antibody.
Some pathogens may infect somatic cells but not directly infect phagocytes such as dendritic cells. In this case, dendritic cells must acquire antigens from exogenous sources in order to process and present antigens to T cells. For example, to eliminate a virus that infects only epithelial cells, activation of CD8 T cells will require that dendritic cells load MHC class I molecules with peptides derived from viral proteins taken up from virally infected cells. This exogenous pathway of loading MHC class I molecules is called cross-presentation, and is carried out very efficiently by some specialized types of dendritic cells.
Proteins in cells are continually being degraded and replaced with newly synthesized
proteins. Much cytosolic protein degradation is carried out by a large, multicatalytic protease complex called the proteasome. Proteins in the cytosol are tagged for degradation via the ubiquitin–proteasome system (UPS). This begins with the attachment of a chain of several ubiquitin molecules to the target protein, a process called ubiquitination. The general degradative functions of the ubiquitin–proteasome system have been co-opted for antigen presentation, so that MHC molecules have evolved to work with the peptides that the proteasome can produce.
The constitutive b1, b2, and b5 subunits of the catalytic chamber of the proteasome are sometimes replaced by three alternative catalytic subunits that are induced by interferons. These induced subunits are called b1i (or LMP2), b2i (or MECL-1), and b5i (or LMP7). Thus, the proteasome can exist both as both a constitutive proteasome present in all cells and as the immunoproteasome, which is present in cells stimulated with interferons. The replacement of the b subunits by their interferon-inducible counterparts alters the enzymatic specificity of the proteasome such that there is increased cleavage of polypeptides after hydrophobic residues, and decreased cleavage after acidic residues. This produces peptides with carboxy-
terminal residues that are preferred anchor residues for binding to most MHC class I molecules and are also the preferred structures for transport by TAP.
6.5: Tap-1 or Tap-2.
A defect in either of these proteins would substantially reduce the availability of MHC-binding peptides in the endoplasmic reticulum (ER), leading to poor expression of MHC class I on the cell surface. This defect can be rectified by adding an excess of peptide to the outside of the cell. Another possible, though less likely, candidate is ERAAP. A defect in ERAAP also affects the peptides available in the ER for binding to MHC class I, but overall, cells do not show a reduction in total MHC class I expression on the cell surface when ERAAP is missing. Instead, the ERAAP defect alters the repertoire of MHC class I-bound peptides. A third, even less likely, possibility is a defect in the proteasome. While this defect would reduce peptide availability, cells lacking this important proteolytic machinery would not be viable.
In order for MHC class I molecules to faithfully display information about the internal state of the cell to T cells, the surface MHC class I molecules must make stable long-lasting interactions with their peptide cargo. In other words, it is critical that very little peptide exchange occurs at the cell surface. Should peptide exchange at the cell surface happen, infected cells would fail to be recognized by pathogen-specific T cells; in addition, healthy uninfected cells might bind a pathogen peptide from the extracellular milieu, and find itself a target of CD8 cytotoxic T cell killing.
In uninfected cells, peptides derived from self proteins fill the peptide-binding groove of the mature MHC class I molecules and are carried to the cell surface. Some of the newly synthesized MHC proteins fail to find a peptide cargo, and do undergo degradation, but this is not the majority of MHC class I molecules synthesized.
Cross-presentation is a specialized pathway found in a subset of dendritic cells. This pathway provides a mechanism for these dendritic cells to present peptides derived from extracellular sources on their MHC class I molecules. In other words, for these dendritic cells, the peptide source for MHC class I presentation does not have to derive from proteins synthesized in that dendritic cell’s cytosol. Cross-presenting dendritic cells can present peptides derived from viruses or bacteria taken up from the extracellular milieu. This example represents an additional mechanism for cross-presentation, where dendritic cells present peptides derived from phagocytosed dying cells infected with a cytosolic pathogen. If the virus is inactivated, it will not replicate in the epithelial cell, and therefore, will not generate proteins to be cross-presented by the dendritic cell.
The initial activation of CD8 T cells requires that the T cell recognizes viral peptide MHC class I complexes on dendritic cells, not on the infected cell itself. The mechanism that the dendritic cell uses to present viral peptides on its MHC class I proteins does not require that the virus replicate in the dendritic cell. Instead, the virus particles can be taken up from the extracellular milieu, or the dendritic cell can phagocytose dying infected cells. In either case, the viral proteins will be degraded into peptides and cross-presented on MHC class I molecules. In these scenarios, the virus is not replicating in the dendritic cell, and therefore cannot synthesize the proteins it might use to down-regulate MHC class I in the dendritic cell.
Drugs that raise the pH of endosomes inhibit the presentation of intravesicular antigens, suggesting that acid proteases are responsible for processing internalized antigen. These proteases include the cysteine proteases, known as cathepsins B, D, S, and L, of which L is the most active. Antigen processing can be mimicked to some extent by the digestion of proteins with these enzymes in vitro at acid pH. Asparagine endopeptidase (AEP), another cysteine protease, is important for processing some antigens, such as the tetanus toxin antigen for MHC class II presentation. It is likely that the overall repertoire of peptides produced within the vesicular pathway reflects the activities of the many proteases present in endosomes and lysosomes.
Trafficking of membrane proteins is controlled by cytosolic sorting tags. In this regard, Ii has a second function, which is to target delivery of the MHC class II molecules to a low-pH endosomal compartment where peptide loading can occur. The complex of MHC class II a:b heterodimers with Ii trimers is retained for 2–4 hours in this compartment. During this time, the Ii molecules are cleaved, ultimately leaving a single peptide (CLIP) bound to MHC class II molecules. Peptide exchange can then occur, leading to release of CLIP and binding of peptides derived from internalized pathogen proteins or self proteins.
HLA-DM binds to and stabilizes empty MHC class II molecules and catalyzes the release of CLIP, thus allowing the binding of other peptides to the empty MHC class II molecule. The HLA-DM molecule does not contain the open groove found in other MHC class II molecules, and it does not bind peptides. Instead, HLA-DM binds to the α chain of the MHC class II molecule near the region of the floor of the peptide-binding site. This binding induces changes in the structure of the MHC class II molecule, and holds this part of the peptide binding groove in a partially ‘open’ configuration. In this way, HLA-DM catalyzes the release of CLIP and of other unstably bound peptides from MHC class II molecules. In the absence of HLA-DM, MHC class II molecules assemble correctly with Ii and seem to follow the normal vesicular route, but fail to bind peptides derived from internalized proteins and often arrive at the cell surface with the CLIP peptide still bound. HLA-DO is as a negative regulator of HLA-DM. HLA-DO binds to HLA-DM in the same manner as MHC class II molecules, and thereby inhibits HLA-DM from mediating peptide editing of MHC class II.
MHC class I molecules display peptides derived largely from cytosolic proteins, so it is important that these MHC molecules do not bind to extracellular peptides. MHC class II molecules present peptides derived from pathogens living in the vesicles, or from pathogens or antigens they have ingested. Again, it is important that these MHC molecules do not bind to extracellular peptides. In either case, surface MHC molecules binding to extracellular peptides could lead to healthy cells mistakenly being targeted for destruction or activation. Fortunately, when an MHC class I molecule at the surface of a living cell loses its peptide, its conformation changes, the b2-microglobulin dissociates, and the a chain is internalized and rapidly degraded. Thus, most empty MHC peptide class I molecules are quickly lost from the cell surface, largely preventing them from acquiring peptides directly from the surrounding extracellular fluid. This helps ensure that primed T cells target infected cells while sparing surrounding healthy cells. Empty MHC class II molecules are also removed from the cell surface. Although at neutral pH, empty MHC class II molecules are more stable than empty MHC class I molecules, they aggregate readily, and internalization of such aggregates is thought to account for their removal.
MARCH-1 is expressed constitutively in B cells and induced by the cytokine IL-10 in macrophages and dendritic cells. It resides in the membrane of a recycling endosomal compartment, where it ubiquitinates the cytoplasmic tail of MHC class II molecules,
leading to their eventual degradation in lysosomes, thereby regulating their steady-state level of expression. The MARCH-1 pathway is shut down during infection to increase the stability of peptide:MHC complexes. In addition to regulating MHC class II expression in dendritic cells, MARCH-1 also regulates expression in dendritic cells of the co-stimulatory molecule CD86 (or B7-2), which like MHC class II molecules, is also regulated by ubiquitination. This means that by the time dendritic cells arrive at lymph nodes, they express peptides derived from the pathogens that activated them and have higher CD86 levels that provide signals for greater CD4 T cell activation. Some pathogens have taken advantage of this pathway by producing MARCH-1-like proteins to down-regulate MHC class II molecules as a means of evading adaptive immunity.
When cells are treated with the interferons IFN-a, -b, or -g, there is a marked increase in the transcription of MHC class I a-chain and b2-microglobulin genes and of the proteasome, tapasin, and TAP genes. The increases in MHC expression these cytokines produce helps all cells to process viral proteins and present the resulting virus-derived peptides on their surface. On dendritic cells, this helps to activate the appropriate T cells and initiate the adaptive immune response to the virus. In addition, gene expression of the classical MHC class II proteins, along with the invariant-chain, DMa, DMb, and DOa is coordinately increased by IFN-g, which is produced by activated
TH1 cells, CD8 T cells, and NK cells. This form of regulation allows dendritic cells and macrophages to up-regulate molecules involved in processing of intravesicular antigens when presenting antigens to T cells and NK cells. The coordinated regulation of the genes encoding these components is likely facilitated by the linkage of many of them in the MHC.
Polymorphism refers to within-species variation at a gene locus, and thus in the gene’s protein product; the variant genes that can occupy the locus are termed alleles. For several MHC class I and class II genes, there are more than 1000 alleles in the human population, far more than the number of alleles for other genes found within the MHC region. This polymorphism results in broad diversity of MHC protein expression between individuals in the population. In contrast, a single individual may express a dozen or so different MHC proteins (MHC class I and MHC class II), but all of the cells within the individual will express the same set of these MHC proteins.
6.17: Polygeny and co-dominant expression of MHC proteins.
The high polymorphism of the classical MHC genes ensures diversity in MHC gene expression in the population as a whole. However, no matter how polymorphic a gene is, no individual can express more than two alleles at a single gene locus. Polygeny, the presence of several different related genes with similar functions, along with the co-dominant expression of both alleles of each MHC gene, ensures that each individual produces a number of different MHC molecules. The combination of polymorphism, polygeny, and co-dominant expression produces the diversity of MHC molecules seen both within an individual and in the population at large.
Pathogen-driven selection can account for the large number of MHC alleles. The products of individual MHC alleles, often known as protein isoforms, can differ from one another by up to 20 amino acids, making each variant protein quite distinct. Most of the differences are localized to exposed surfaces of the extracellular domain furthest from the membrane, and to the peptide-binding groove in particular. Peptides bind to MHC class I and class II molecules through the interaction of specific anchor residues with peptide-binding pockets in the peptide-binding groove. Many of the polymorphisms in MHC molecules alter the amino acids that line these pockets and thus change the pockets’ binding specificities. This in turn changes the anchor residues of peptides that can bind to each MHC isoform.
This experiment illustrates the concept of MHC restriction. When T cells are primed in a mouse expressing H-2a, they will only recognize and kill target cells expressing H-2a MHC molecules, if those target cells are infected with the same virus used to prime the mice. The same holds true for mice of strain B. The fact that T cells from each mouse strain recognize their syngeneic MHC molecules on LCMV-infected target cells rules out any lack of response of one strain to the virus, and rules out any lack of infectivity of target cells by the virus.
A child will generally share only 50% identity of MHC alleles with either parent. In this case, the child’s T cells will respond vigorously to the non-shared 50% of peptide:MHC complexes expressed on the parent’s antigen-presenting cells. This will lead to a robust proliferative response by the child’s T cells.
In principle, alloreactive T cells might depend on recognizing either a foreign peptide antigen or the nonself MHC molecule to which it is bound for their reactivity against nonself MHC; these options have been called peptide-dependent and peptide-independent allorecognition. But as the number of individual alloreactive T-cell clones studied has increased, it seems that most alloreactive T cells actually recognize both; that is, most individual alloreactive T-cell clones respond to a foreign MHC molecule only when a particular peptide is bound to it. In this sense, the structural basis of allorecognition may be quite similar to normal MHC-restricted peptide recognition and be dependent on contacts with both peptide and MHC molecule, but in this case a foreign MHC molecule.
Superantigens are unlike other protein antigens, in that they are recognized by T cells without being processed into peptides that are captured by MHC molecules. Indeed, fragmentation of a superantigen destroys its biological activity, which depends on binding as an intact protein to the outside surface of an MHC class II molecule that has already bound peptide. In addition to binding MHC class II molecules, superantigens are able to bind the Vb region of many T-cell receptors. Bacterial superantigens bind mainly to the Vb CDR2 loop, and, to a smaller extent, to the Vb CDR1 loop and an additional loop called the hypervariable 4 or HV4 loop. Thus, the a-chain V region and the CDR3 of the b chain of the T-cell receptor have little effect on superantigen recognition, which is determined largely by the germline-encoded V gene segments that encode the expressed Vb chain. Each superantigen is specific for one or a few of the different Vb gene products, of which there are 20–50 in mice and humans; a superantigen can thus stimulate 2–20% of all T cells. This mode of stimulation does not prime an adaptive immune response specific for the pathogen. Instead, it causes a massive production of cytokines by CD4 T cells, the predominant responding population of T cells. These cytokines have two effects on the host: systemic toxicity and suppression of the adaptive
immune response. Both these effects contribute to microbial pathogenicity.
These arguments raise a question: if having three MHC class I loci is better than having one, why are there not far more? The probable explanation is that each time a distinct MHC molecule is added to the repertoire, all T cells that can respond to self peptides bound to that MHC molecule must be removed to maintain self tolerance. It seems that the number of MHC genes present in humans and mice is about optimal to balance the advantages of presenting an increased range of foreign peptides with the disadvantages of losing T cells from the repertoire.
In addition to the highly polymorphic ‘classical’ MHC class I and class II genes, there are many ‘nonclassical’ MHC genes encoded in the MHC (as well as others encoded outside this region). The MHC class I-type molecules show comparatively little polymorphism; many of these have yet to be assigned a function. These genes have been termed MHC class Ib genes. Their expression on cells is variable, both in the amount present at the cell surface and in tissue distribution. The functions of some MHC class Ib proteins are unrelated to the adaptive immune response, but many have a role in innate immunity by interacting with receptors on NK cells. The other genes that map within the MHC include some that encode complement components (for example, C2, C4, and factor B) and some that encode cytokines—for example, tumor necrosis factor-a (TNF-a) and lymphotoxin—all of which have important functions in immunity. Finally, some genes residing within the MHC have no known or suspected immunological function.
Members of the MIC gene family are MHC class Ib genes that are expressed under a different regulatory control than the classical MHC class I genes and are induced in response to cellular stress (such as heat shock). The MICA and MICB proteins are recognized by the NKG2D receptor expressed by NK cells. In addition, NKG2D is also expressed by g:d T cells and some CD8 T cells, and it can activate these cells to kill MIC-expressing targets. NKG2D is an ‘activating’ member of the NKG2 family of NK-cell receptors; its cytoplasmic domain lacks the inhibitory sequence motif found in other members of this family, which act as inhibitory receptors. Even more distantly related to MHC class I genes is a small family of proteins known in humans as the UL16-binding proteins (ULBPs) or the RAET1 proteins. These proteins also bind NKG2D. They also seem to be expressed under conditions of cellular stress, such as when cells are infected with pathogens or have undergone transformation to tumor cells. By expressing ULBPs, stressed or infected cells can bind and activate NKG2D molecules expressed on NK cells, g:d T cells, and CD8 cytotoxic a:b T cells, and so be recognized and eliminated.
The T cells that recognize lipids presented by CD1 molecules are largely negative for CD4 and CD8 expression, although some express CD4. CD1d-restricted T cells have a T-cell receptor repertoire that is substantially less diverse than conventional a:b T cells. In fact, the majority of these cells use the same TCRa chain (Va 24–Ja 18 in humans). In addition, these cells also express NK-cell receptors. These CD1-restricted T cells are called invariant NKT (iNKT) cells. The ability of iNKT cells to recognize different glycolipid constituents from microorganisms presented by CD1d molecules places them in an ‘innate’ category, while their possession of a fully rearranged T-cell receptor, despite its relatively limited repertoire, makes them ‘adaptive’.
6.27: Microbial products specific to folate metabolism.
Analysis of MR1 proteins that were refolded in the presence of supernatants from cultures of Salmonella typhimurium eventually led to the identification of several riboflavin metabolites that are formed by biosynthetic pathways in most bacteria and yeast. These metabolites not only bind to MR1, but also activate MAIT cells. Thus, MAIT cells are activated in response to infection by these organisms by detecting products specific to their folate metabolism. As such, MAIT cells appear to hold an intermediate place in the spectrum of innate and adaptive immunity, similar to iNKT cells, in that they use an antigen receptor assembled by somatic gene rearrangement, but recognize a molecular structure that falls within the definition of a PAMP.
The function of g:d T cells has remained somewhat obscure, due primarily to difficulty in identifying the ligands they recognize. Yet the abundance of g:d T cells across vertebrate species, their rapid expansion to form more than 50% of the blood lymphocytes during infections, and their abundant cytokine production all argue for an important role in immunity. Over time, many different ligands recognized by g:d T-cell clones have been identified, and their diversity suggests that, like iNKT and MAIT cells, they hold an intermediate, or transitional, position in the spectrum of innate versus adaptive immunity.
- a) The antibody used to detect MHC class I protein expression on the surface of cells will recognize all MHC class I proteins, including HLA-A, HLA-B, and HLA-C proteins. Since the affected children show no MHC class I expression at all, their cells are unable to express any of the genes. Therefore, the defect cannot be in just the gene encoding HLA-A. This is likely a genetically inherited immunodeficiency disease, with each parent being heterozygous for gene defect. It is unlikely that a single HLA locus would have inactivating mutations in all three HLA class I genes. Instead, it is more likely that the affected children inherited a deficiency in a single gene that is encoded outside of the HLA locus; furthermore, this gene must encode a protein that is required for all surface HLA class I protein expression.
- b) β2-microglobulin or TAP-1 (or TAP-2). Two proteins required for MHC class I surface expression are b2-microglobulin and a component of the TAP complex (TAP-1 or TAP-2). In the absence of b2-microglobulin, HLA class I heavy chain proteins will not fold properly, will not bind peptide, and will not traffic to the cell surface. In the absence of the TAP complex, there will be an insufficient supply of peptides in the endoplasmic reticulum. In this case, again, HLA class I proteins will not fold properly and will not traffic to the cell surface.
- c) These data rule out a defect in the b2-microglobulin gene, as both the mRNA and protein for b2-microglobulin are expressed normally in the cells from the affected children.
- d) TAP-1 or TAP-2. The data from the cytolysis experiment show that the parent’s cells will present the HepBsAg 338–347 peptide on HLA-A2 when they are transfected with either the full-length HepBsAg gene or the mini-gene encoding just the peptide epitope. In contrast, the cells from the affected children will only present this peptide from the full-length construct, not from the mini-gene. This indicates that the cells are able to present the HepBsAg epitope when the protein containing that epitope is able to get into the endoplasmic reticulum (ER). Since all surface proteins have signal sequences that target them for translocation into the ER, the full-length HepBsAg protein will access this mechanism. Once in the ER, the normal proteolytic enzymes will degrade the protein, and peptides will bind normally to HLA-A2 during synthesis and assembly. The mini-gene encodes only the 338–347 amino acids of the HepBsAg, and therefore lacks any signal sequence for translocation into the ER. This peptide will be synthesized and remain in the cytosol. These data localize the defect in the affected children’s cells to the mechanism that normally mediates transfer of cytosolic peptides into the ER, i.e., the TAP complex.
- a) The bovine insulin A (1–14) peptide most likely fails to bind to bm12. The amino acid changes in bm12 compared to wild-type Ab will alter the peptide binding specificity of this MHC class II molecule. The lack of CD4 T cell response to other regions of the bovine insulin protein in bm12 mice is due to the fact that the majority of sequences comprising the bovine insulin protein are conserved between mouse and cow insulin. The amino-terminal region of the insulin A chain is the major area of divergence between the insulin proteins from these two species.
- b) The T cells from WT mice recognize peptide:MHC complexes on bm12 antigen presenting cells as ‘foreign,’ and vice versa for the T cells from bm12 mice. This is due to the fact that the WT Ab molecules bind a distinct set of peptides compared to the bm12 Ab Even though the T-cell receptors on the T cells do not directly contact the altered amino acid residues, they ‘see’ this amino acid variation due to the broad effect of these substitutions on the global repertoire of peptides bound to WT Ab versus bm12 Ab.
- c) The T cell response to SEB occurs due to SEB binding to the side of the MHC class II protein. Since the amino acid differences between WT and bm12 Ab lie inside the peptide binding groove, they are not exposed to the surfaces that directly contact the SEB or the T-cell receptor. Therefore, T cells from each strain of mice will recognize SEB when bound to APCs from either strain of mice.
- d) The proliferating T cells in Rows 2 and 3 would be heterogeneous. All (or nearly all) of the different Vbs would be detected in the population, but at a variety of different frequencies. Some would be more abundant, and others very rare in the overall population. In contrast, the T cells in Rows 5–8 would all show predominant expansion of cells with one single Vb In the case of C57Bl/6 mice and SEB, this is Vb8. In some cases T cells expressing one of two different Vbs may be expanded. The key point is that all four populations should look the same, and show nearly homogeneous expansion of T cells whose T-cell receptors use a single type of Vb domain.
Janeway’s Immunobiology, 9th Edition
Chapter 7: Lymphocyte Receptor Signaling
7-1 Transmembrane receptors convert extracellular signals into intracellular biochemical events
7.1 Short answer: Many receptors of the immune system activate protein kinases as a mechanism of initiating signaling. For antigen receptors on lymphocytes, ligand binding induces receptor clustering, and the enzymes activated are protein tyrosine kinases. Based on this mechanism, predict the outcome of expressing a mutant form of the receptor-associated tyrosine kinase in cells that still express the wild-type version of this enzyme, and explain your reasoning. This mutant is unable to bind ATP and therefore is catalytically inactive; assume the mutant and wild-type forms of the kinase are expressed in equimolar amounts.
7-2 Intracellular signal propagation is mediated by large multiprotein signaling complexes
7.2 True/False: All of the modular protein domains used for signaling protein interactions bind to ligands that are transiently generated following receptor stimulation.
7-3 Small G proteins act as molecular switches in many different signaling pathways
7.3 Multiple choice: Small GTPases, such as Ras, Rho, and cdc42, are activated when they exchange their bound GDP for GTP. In the GTP-bound state, these proteins contribute to signaling by:
- Hydrolyzing the bound GTP back to GDP
- Interacting with GTPase-activating proteins (GAPs)
- Interacting with target proteins and altering their activity
- Diffusing from the membrane and entering the nucleus
- Inducing calcium release from the endoplasmic reticulum
7-4 Signaling proteins are recruited to the membrane by a variety of mechanisms
7.4 Short answer: Antigen receptors use multiple mechanisms to recruit signaling proteins to the plasma membrane, where they can propagate downstream signals. In some cases, recruitment of proteins to the membrane is induced following antigen receptor stimulation, whereas other proteins are constitutively associated with the membrane.
Name one mechanism that is induced by antigen receptor stimulation, and one that is constitutive, and give an example a protein recruited by each mechanism.
7-5 Post-translational modifications of proteins can both activate and inhibit signaling responses
7.5 Multiple choice: Scaffold proteins are often phosphorylated at multiple sites, allowing the recruitment of several different signaling proteins. In antigen receptor signaling pathways, this mechanism is used to bring enzymes in close proximity to their substrates. Termination of this signaling mechanism would be most efficiently accomplished by:
- Ubiquitination of the scaffold protein, leading to its degradation
- Binding of the enzyme to a GTPase activating protein (GAP)
- Depletion of the substrate due to enzyme catalysis
- Dephosphorylation of the scaffold by a phosphatase
- Ubiquitination of the enzyme by K63-linkage of polyubiquitin
7-6 The activation of some receptors generates small-molecule second messengers
7.6 Multiple choice: Second messengers, such as calcium ions (Ca2+), are chemical mediators commonly used in intracellular signaling pathways. Despite its common usage in many different cell types in the body, Ca2+ has specific effects in lymphocytes following antigen receptor stimulation. The specific responses of lymphocytes to increased concentrations of intracellular Ca2+ are determined by:
- The expression of a specific subset of Ca2+-responsive enzymes in lymphocytes compared to other cell types
- The increased expression of calmodulin in lymphocytes compared to other cell types
- The presence of enzymes that bind calmodulin in lymphocytes but not other cell types
- The high levels of Ca2+ in the endoplasmic reticulum of lymphocytes compared to other cell types
- The ability of Ca2+ to amplify signaling pathways in lymphocytes but not other cell types
7-7 Antigen receptors consist of variable antigen-binding chains associated with invariant chains that carry out the signaling function of the receptor
7.7 Multiple choice: The TCR and BCR are multi-subunit receptor complexes. Experiments examining the synthesis and transport of these receptors to the lymphocyte cell surface have shown that the signaling subunits of each receptor complex are required for transport of the ligand-binding receptor subunits to the cell surface. One possible reason for this stringent control on cell surface expression is:
- To ensure that very few complete TCRs or BCRs are expressed on the lymphocyte surface
- To ensure that each lymphocyte expresses only a single specificity of antigen receptor
- To prevent surface expression of receptors that will bind ligand but fail to induce signals
- To prevent lymphocytes from triggering antigen receptor signaling pathways from intracellular forms of the receptors
- To ensure that equimolar amounts of all antigen receptor signaling subunits are produced
7-8 Antigen recognition by the T-cell receptor and its co-receptors transduces a signal across the plasma membrane to initiate signaling
7-9 Antigen recognition by the T-cell receptor and its co-receptors leads to phosphorylation of ITAMs by Src-family kinases, generating the first intracellular signal in a signaling cascade
7.8 Short answer: Antigen receptor signaling pathways are regulated by a balanced equilibrium between tyrosine kinases and tyrosine phosphatases. In general, activation of signaling proceeds when the kinase activities leading to auto-phosphorylation of Lck, to phosphorylation of ZAP-70, and to phosphorylation of downstream adapters and scaffolds exceeds the activity of phosphatases acting on these substrates. Therefore, it came as a surprise when T cells lacking the membrane tyrosine phosphatase, CD45, were first generated, and were found to be unable to be activated by TCR stimulation. Name one important function of CD45 in T cells that explains the requirement for this phosphatase in TCR signaling.
7.9 Multiple choice: Antigen receptor signaling pathways are initiated by the action of a Src-family kinase. In T cells, the predominant Src-kinase is Lck. In resting T cells, Lck is maintained in an inactive state by allosteric interactions involving multiple domains of the enzyme. When T cells are treated with a small molecule inhibitor of the tyrosine kinase Csk, TCR signaling is initiated even in the absence of a ligand to stimulate the TCR. This occurs because:
- Csk phosphorylates Lck in its kinase domain, leading to Lck activation.
- Csk phosphorylates ZAP-70, maintaining ZAP-70 in an auto-inhibited state.
- Csk phosphorylates the ITAM motifs in the TCR z chain, leading to ZAP-70 recruitment.
- Csk phosphorylates and activates the membrane tyrosine phosphatase CD45.
- Csk phosphorylates the C-terminal negative regulatory tyrosine in Lck.
7-10 Phosphorylated ITAMs recruit and activate the tyrosine kinase ZAP-70
7.10 Multiple choice: Immunoreceptor signaling proteins, such as the TCR z chain and CD3 subunits, have conserved ITAM motifs in their cytoplasmic tails. When fully phosphorylated, the ITAM recruits a tyrosine kinase with a tandem SH2 domain structure at the amino-terminal end of the protein. Tandem SH2 domain-containing kinases do not bind to sequences in other proteins, even if they contain a phosphorylated tyrosine because:
- The amino acid sequence adjacent to the phosphorylated tyrosines in the ITAM motif is unique, and not found in any other proteins.
- The affinity of a single SH2 domain within these kinases for a tyrosine phosphorylated sequence is too low for efficient binding.
- The amino-terminal SH2 domain of the kinase has very high affinity for both of the phosphorylated tyrosines in the ITAM motif, so will not bind to other proteins.
- The amino-terminal SH2 domain of the kinase is in an autoinhibited conformation and can only bind to a phosphorylated ITAM.
- The tandem SH2 domain-containing kinase phosphorylates the tyrosines in the ITAM itself, so can only bind to these sequences.
7-11 ITAMs are also found in other receptors on leukocytes that signal for cell activation
7.11 Short answer: The TCR and BCR are each composed of two modules, an antigen-binding module and a signaling module; furthermore, in each case, the two functional modules are encoded by distinct polypeptides. In addition, the tyrosine kinases that initiate antigen receptor signaling are also separate proteins from those of each receptor. This is a different strategy for receptor signaling than the case of receptor tyrosine kinases, where the enzyme is an intrinsic component of the ligand-binding receptor protein. Name one advantage of this organization of the TCR and BCR that accounts for the expression of ZAP-70 and Syk, as well as ITAM-containing immunoreceptors, in many different subsets of immune cells.
7-12 Activated ZAP-70 phosphorylates scaffold proteins and promotes PI 3-kinase activation
7.12 True/False: Following TCR or BCR signaling, the most important events downstream of the activation of ZAP-70 or SYK, respectively, are the activation of transcription factors leading to new gene expression.
7.13 True/False: The LAT:Gads:SLP-76 complex that assembles following TCR stimulation provides the scaffold for initiating multiple downstream signaling modules, leading to actin polymerization, integrin activation, and gene expression.
7-13 Activated PLC-g generates the second messengers diacylglycerol and inositol trisphosphate that lead to transcription factor activation
7.14 Multiple choice: The TCR signaling module leading to transcription factor activation is dependent on the enzyme phospholipase-C-g (PLC-g). The mechanism by which PLC-g activates multiple transcription factors is by:
- Generating two small second messengers that act on multiple target proteins in the T cell
- Directly cleaving inhibitory subunits of multiple transcription factors, thereby releasing the active transcription factors
- Generating two small second messengers that diffuse to the nucleus and activate transcription factors present there
- Generating two small second messengers that act as chaperones to promote nuclear localization of transcription factors
- Directly cleaving the lipid binding domain from membrane-tethered transcription factors, allowing them to migrate to the nucleus
7-14 Ca2+ entry activates the transcription factor NFAT
7.15 Multiple choice: Using an antibody that recognizes the phosphorylated, but not the non-phosphorylated form of the transcription factor, NFAT, T cells are permeabilized, stained with this antibody, and analyzed by flow cytometry. Which of the data in Figure Q7.15 represent the expected pattern of staining from wild-type T cells before and after TCR stimulation.
7.16 Multiple choice: Human patients with genetic defects that result in a failure to produce the calcium channel protein ORAI1, or the ER calcium sensor protein STIM1, have severe immunodeficiency diseases. An immunosuppressive drug that would most closely mimic these primary immunodeficiencies is:
- Rituximab, a drug that depletes B cells
- Cyclosporin A, a calcineurin inhibitor
- Rapamycin, an mTOR inhibitor
- Tysabri, an inhibitor of integrin binding
- Enbrel or Humira that inhibit TNF
7.17 Multiple choice: A new strain of immunodeficient mice has been discovered, and found to have T cells that are unresponsive to TCR stimulation. The T cells from these mice have normal levels of the TCR complex on their surface, but when this TCR is stimulated, the cells fail to secrete IL-2. As a first step in determining the signaling defect responsible for this immunodeficiency, the T cells are stimulated with a phorbol ester (PMA) and Ionomycin. It is found that this treatment elicits IL-2 production by the immunodeficient T cells. Based on this information, candidate genes that could be mutated in these T cells include all of the following EXCEPT:
7-15 Ras activation stimulates the mitogen-activated protein kinase (MAPK) relay and induces expression of the transcription factor AP-1
7.18 Multiple choice: Following TCR stimulation, the small GTPase Ras is activated. Ras activation is induced by the Ras GTP-exchange factor (GEF), RasGRP. Both Ras and RasGRP are constitutively expressed in resting T cells. The reason Ras activation is only induced following TCR stimulation is:
- RasGRP undergoes a Ca2+-dependent conformational change required for its activity.
- RasGRP requires tyrosine phosphorylation for its activity.
- RasGRP is ubiquitinated and degraded in the absence of TCR stimulation.
- RasGRP recruitment to the plasma membrane requires TCR stimulation.
- Ras is only recruited to the activated TCR following assembly of the LAT:Gads:SLP-76 complex.
7.19 True/False: Diacyl-glycerol (DAG) is one of the two products generated when PLC-g cleaves the membrane phospholipid, PIP2. This small lipid mediator remains associated with the plasma membrane and functions to inhibit tyrosine phosphatases that remove activating phosphate groups from ZAP-70 and the Tec-family kinase, ITK.
7.20 True/False: Several small GTPases play critical roles in antigen receptor signaling pathways. When activated by binding to GTP, these mediators induce changes in cytoskeletal organization, adhesion, and metabolism, but have no role in transcription factor activation.
7.21 Multiple choice: T cells with defective TCR signaling are discovered, and found to have an inactivating mutation in a key TCR signaling protein. Using an antibody that recognizes the phosphorylated (activated) form of the Erk Map-kinase, stimulated T cells are permeabilized, stained with this antibody, and analyzed on the flow cytometer. These data are shown in Figure Q7.21.
Additional experiments examining Ca2+ influx into T cells following TCR stimulation show a normal response in the mutant T cells. One likely candidate gene that could be mutated in the defective cells is:
7-16 Protein kinase C activates the transcription factors NFkB and AP-1
7.22 Multiple choice: An important transcription factor activated by antigen receptor signaling in lymphocytes is an NFkB heterodimer of the two subunits, p50 and p65Rel. Defects in the IkB-kinase complex (NEMO) or mutations in IkB that prevent its phosphorylation interfere with NFkB activation and result in severe immunodeficiency diseases. This is due to the important function of:
- NEMO in targeting p50:p65Rel for ubiquitination and degradation
- NEMO in ubiquitinating IkB causing its release from NFkB
- IkB in blocking the DNA binding activity of NFkB
- IkB as a chaperone to promote NFkB nuclear localization
- NEMO in phosphorylating IkB inducing its degradation, thereby releasing NFkB
7-17 PI 3-kinase activation up-regulates cellular metabolic pathways via the serine/threonine kinase Akt
7.23 Multiple choice: Lymphocyte activation leads to robust proliferation and effector cell differentiation. The metabolic demands of these processes are met, in part, by up-regulation of glycolytic enzymes and nutrient transporters on the activated cell membrane. A key intermediate in the signaling pathway leading to enhanced glucose metabolism following antigen receptor stimulation is:
- The lipid mediator diacyl-glycerol (DAG)
- The phosphoinositide, PIP3
- Increases in cytoplasmic Ca2+
- Cleavage of the membrane phospholipid, PIP2
- The mitochondrial protein, Bcl-2
7.24 Multiple choice: The immunosuppressive drug rapamycin acts by inhibiting mTOR. When activated T cells are treated with rapamycin in a cell culture assay, they show greatly diminished proliferation, and accumulate to much lower numbers than control-treated cells. This is because:
- Rapamycin inhibits cells from increasing their synthesis of lipids and proteins.
- Rapamycin inhibits cells from activating the pro-survival protein, Bcl-2.
- Rapamycin inhibits DNA synthesis in activated T cells.
- Rapamycin inhibits cell cycle progression in activated T cells.
- Rapamycin inhibits the T cell’s production of the growth factor, IL-2.
7.25 True/False: Phosphorylation of signaling proteins can have activating or inhibitory effects on protein function. In many cases, such as the activation of mTOR, the phosphorylation of an inhibitory protein leads to inactivation of the inhibitor, resulting in downstream signaling.
7-18 T-cell receptor signaling leads to enhanced integrin-mediated cell adhesion
7.26 Multiple choice: The integrin LFA-1 is constitutively expressed on the surface of resting T cells. Yet, integrin-dependent T cell adhesion to antigen-presenting cells increases substantially following TCR stimulation. This increased integrin-dependent adhesion is mediated in part by:
- Increased synthesis of the LFA-1 protein
- Increased transport of intracellular pools of LFA-1 to the cell surface
- LFA-1 conversion to a high affinity binding state
- Increased phosphorylation of the LFA-1 cytoplasmic tail
- Activation of cdc42 and WASp
7.27 Multiple choice: Humans with defective expression of the integrin LFA-1 have an immunodeficiency disease characterized by the failure of lymphocytes and granulocytes to migrate to tissues at sites of infection or inflammation. A similar immunodeficiency would be expected if individuals had mutations disrupting the gene for:
- The complement receptor, CD21
7.28 Short answer: TCR stimulation was shown to affect ICAM-1 (integrin ligand) binding to LFA-1 (integrin) on T cells. To demonstrate this, varying concentrations of purified ICAM-1 were added to unstimulated or TCR-stimulated T cells, and the amount of ICAM-1 binding was measured. The data from such an experiment are displayed on Figure Q7.28. Assign the red or blue lines correctly to ‘unstimulated’ or ‘TCR-stimulated’ T cells, and explain the reasoning for your answer.
7-19 T-cell receptor signaling induces cytoskeletal reorganization by activating the small GTPase Cdc42
7.29 Multiple choice: Wiskott–Aldrich syndrome is an immunodeficiency disease due to mutations in the gene encoding WASp. Individuals with this disease make poor antibody responses to protein antigens, due to impaired T cell help for B cells. WASp-deficient T cells are likely impaired in providing adequate help to B cells due to:
- Defects in up-regulating expression of genes encoding cytokines required by B cells
- Defects in up-regulating metabolic pathways for T cell macromolecular synthesis
- Defects in up-regulating expression of genes needed for T cell survival
- Defects in cytoskeletal reorganization needed for directed T cell cytokine secretion
- Defects in up-regulating T cell integrin adhesion for stable interactions with B cells
7-20 The logic of B-cell receptor signaling is similar to that of T-cell receptor signaling, but some of the signaling components are specific to B cells
7.30 True/False: Unlike TCR signaling, B cell receptor (BCR) signaling is not initiated by a Src-family kinase phosphorylating tyrosine resides in ITAM motifs of BCR signaling subunits.
7.31 Matching: BCR stimulation and TCR stimulation generally activate similar downstream signaling modules, but do so using related, but not identical, signaling proteins. From the list below, match each B cell protein to its T cell counterpart.
2. Lyn, Blk or Fyn
6. Ig-a or Ig-b
7.32 Multiple choice: A mutant B cell line is examined by confocal microscopy after incubation with a microbial pathogen recognized by the BCR on these B cells. The B cells have been stained with antibodies to visualize the localization of polymerized actin and microtubules. As a control, wild-type B cells are examined. The results are shown in Figure Q7.32, with the numbers indicating the proportion of cells examined that show each pattern of staining.
To identify the specific signaling defect in these mutant B cells, a reasonable biochemical assay would be to:
- Determine if BCR stimulation of mutant B cells produces enhanced binding of the B cell to the microbe
- Determine whether the mutant B cells have reduced levels of the enzyme Protein kinase C-q
- Determine whether the mutant B cells are overexpressing the enzyme Vav
- Determine whether BCR stimulation of mutant B cells promotes exchange of GDP for GTP on cdc42
- Determine whether BCR stimulation of mutant B cells produces increased levels of DAG
7.33 Multiple choice: The B cell co-receptor, composed of CD19/CD21/CD81, is a receptor that binds to complement fragments such as C3dg. When an antigen bound by the BCR on a B cell has also been tagged with C3dg, the B cell co-receptor is stimulated together with the BCR. Signaling through the co-receptor:
- Inhibits BCR signaling by leading to ITAM dephosphorylation
- Inhibits BCR signaling by leading to PIP3 dephosphorylation
- Enhances BCR signaling by recruiting and activating PI 3-kinase
- Enhances BCR signaling by bringing the Src-kinase together with Ig-a and Ig-b.
- Inhibits BCR signaling by sequestering the antigen away from the BCR.
7-21 The cell-surface protein CD28 is a required co-stimulatory signaling receptor for naive T cell activation
7-22 Maximal activation of PLC-g, which is important for transcription factor activation, requires a co-stimulatory signal induced by CD28
7.34 True/False: The only mechanism by which CD28 co-stimulation enhances T cell activation is by recruiting and activating PI 3-kinase, leading to Akt activation.
7.35 Multiple choice: TCR and CD28 signaling together lead to maximal production of IL-2 by the activated T cell. Experiments investigating the mechanism underlying the CD28 co-stimulation-mediated increase in IL-2 production show that T cells stimulated through the TCR plus CD28 have increased levels of IL-2 mRNA compared to cells stimulated through the TCR alone. One important component contributing to increased IL-2 mRNA levels is:
- Increased protein synthesis due to increased production of ribosomes
- Increased glucose metabolism due to increased production of glycolytic enzymes
- Increased mRNA stability after transcription and splicing
- Enhanced mRNA transport from the nucleus to the cytoplasm
- Increased levels of splicing enzymes that increase IL-2 mRNA splicing efficiency
7-23 TNF receptor superfamily members augment T-cell and B-cell activation
7.36 Multiple choice: In patients with ‘CD40 ligand deficiency’, T cell-dependent B cell activation is impaired, leading to poor antibody responses to protein antigens. The signaling pathway missing in these patients’ B cells is important for:
- Inducing integrin activation to promote adhesion
- Inducing NFkB activation by the noncanonical pathway
- Inducing WASp activation and actin polymerization
- Inducing Ca2+ influx leading to NFAT activation
- Inducing Ras activation and Erk Map-kinase signaling
7.37 Multiple choice: TNF-receptor signaling commonly includes several steps that are regulated by ubiquitination. One important step following TNF-receptor stimulation is the:
- K48-linked ubiquitination and degradation of a TRAF protein, itself a ubiquitin-ligase
- K48-linked ubiquitination of the TNF-receptor cytoplasmic tail, leading to its degradation
- K63-linked ubiquitination of the TNF-receptor, providing a docking site for TRAF protein binding
- K48-linked ubiquitination of NIK, the NFkB-inducing kinase
- K63-linked ubiquitination of cIAP, leading to its binding to NIK, the NFkB-inducing kinase
7-24 Inhibitory receptors on lymphocytes down-regulate immune responses by interfering with co-stimulatory signaling pathways
7.38 True/False: The mechanism by which CTLA-4 inhibits T cell activation is by recruiting inhibitory phosphatases.
7.39 Multiple choice: ‘Checkpoint blockade’ is a therapeutic strategy based on enhancing T cell responses by inhibiting the function of inhibitory receptors, such as CTLA-4, and PD-1. Patients being treated with these protein-based therapeutics would likely be suffering from:
- An autoimmune disease
- An immunodeficiency disease
- Inflammatory bowel disease
- A neurodegenerative disease
7-25 Inhibitory receptors on lymphocytes down-regulate immune responses by recruiting protein or lipid phosphatases
7.40 Multiple choice: BCR signaling on B cells is initiated by antigen binding, leading to mTOR activation. This occurs, for instance, when the antigen is a live microbe that binds to the BCR on the B cells. Which one of the forms of antigen shown below the graph would correctly account for the data shown in Figure Q7.40.
7.41 Synthesis question: Antigen receptor signaling and lymphocyte activation.
A commonly used assay to measure Ca2+ influx in response to TCR stimulation involves loading T cells with a Ca2+–sensitive dye, and then stimulating the TCR using anti-CD3 antibody coupled to biotin, followed by cross-linking with Streptavidin (S-Av). As the antibody and then S-Av are added, the cells are run on the flow cytometer to examine the fluorescence of the Ca2+-sensitive dye. After several minutes of analysis, the cells are stimulated with ionomycin (Iono), to induce Ca2+ influx; this is used as a positive control to ensure that the cells are loaded with the dye. In Figure Q7.41A, the characteristic pattern of Ca2+ influx is shown in the red line (wild-type; WT), where TCR stimulation causes a sharp rise in cytoplasmic Ca2+, followed by a slow decline over hours. As shown below, cytoplasmic Ca2+ concentrations do not normally return to baseline for the timecourse of this experiment. A mutant mouse is identified with a defect in T cell activation in response to TCR stimulation. The calcium response of T cells from the mutant mouse is shown in the blue line.
- a) Given these data, name three T cell signaling proteins that could be defective in the mutant T cells. Then name three T cell signaling proteins that could not be responsible for this defect, even if mutated.
Additional experiments are performed to analyze protein tyrosine phosphorylation in response to TCR stimulation. For these experiments, T cells are stimulated with anti-CD3 antibody, and then lysates are prepared and run on a protein (SDS-PAGE) gel to separate the proteins by molecular weight. The proteins are transferred from the gel to a membrane for immunoblotting using an antibody that binds to all phosphorylated tyrosine residues in any protein; this antibody is called ‘anti-phospho-tyrosine antibody,’ and is abbreviated as anti-P-Y. The results are shown in Figure Q7.41B.
You confirm that the mutant T cells express normal levels of all the proteins detected in the WT cells, including PLC-g, SLP-76, ITK, ZAP-70, LCK, LAT, and the CD3 and TCRz proteins.
- b) Based on these additional data, which of the candidate proteins in your answer to part (a) are ruled out? Briefly explain your answer.
- c) What protein is most likely defective in the mutant cells and why?
- d) For the protein you named in your answer to part (c), which amino acids or domain of the protein could be mutated to account for all the data.
7.42 Synthesis question: Co-stimulatory and inhibitory receptors modulate antigen receptor signaling in T and B lymphocytes. A new receptor is discovered, expressed on the surface of T cells, and called ‘X’. An antibody to X is generated, and used in T cell stimulation experiments. In these experiments, antibodies to the TCR complex (anti-CD3) and to CD28 (anti-CD28) are known to stimulate signaling through those receptors, as does the antibody to X. The data from an experiment measuring IL-2 secretion by the T cells stimulated with different combinations of antibodies are shown in Figure Q7.42.
- a) Does stimulation of receptor X alone induce IL-2 production by T cells? Does it enhance or inhibit TCR signaling? Indicate the evidence supporting your answers.
- b) If you examined the amino acid sequence of the receptor X cytoplasmic tail, what motif would you expect to find?
Biochemical studies show that when receptor X is stimulated, a tyrosine residue in the cytoplasmic tail becomes phosphorylated.
- c) From these data, what are the two most likely signaling proteins that might be recruited to receptor X when it is stimulated? Does the T cell stimulation data shown in the graph rule in or out either of your candidate proteins? Why or why not?
- d) Describe a biochemical experiment (analysis of proteins) that would indicate which enzyme is recruited to receptor X when it is stimulated.
7.1: Following stimulation of the antigen receptor, downstream signaling would be greatly diminished. This would be visible as reduced auto-phosphorylation of the kinase and as reduced phosphorylation of downstream substrates of the pathway. In cells expressing equimolar amounts of wild-type and inactive forms of the kinase, the majority of the receptors would be associated with two inactive proteins, or one active plus one inactive; few receptors would have two active kinases associated with them. Therefore, after receptor clustering, kinase activation that normally occurs by the two associated kinases phosphorylating each other would fail to occur in the majority of receptor complexes. In this situation, the inactive form of the kinase is known as a ‘dominant-negative’ mutant, referring to its ability to poison signaling even in the presence of the wild-type kinase.
SH3 domains, which bind proline (PXXP) motifs, and PDZ domains, which bind C termini of proteins, are modular domains that bind constitutive ligands present even in the absence of receptor stimulation.
Small GTPases undergo a conformational change after releasing GDP and binding GTP. In the GTP-bound state, they bind to target proteins, and change the activity or function of these target proteins.
Recruitment induced by antigen receptor stimulation:
- binding to a membrane associated scaffold protein that is phosphorylated in response to antigen receptor stimulation (examples: Grb2, Gads, SLP-76)
- binding to the membrane phospholipid PIP3 that is generated by phosphorylation of PIP2 in response to antigen receptor stimulation (examples: Akt, Itk, PLC-g)
Recruitment that is constitutive:
- lipid modified proteins that associate constitutively with the plasma membrane (examples: small GTPases such as Ras, Rap1)
Multiple mechanisms contribute to the termination of signaling. Ubiquitination of a target protein can lead to that protein’s degradation. However, for a phosphorylated scaffold protein, the most immediate mechanism to terminate its signaling function is by dephosphorylation, thereby eliminating the binding sites for recruited proteins.
The Ca2+-calmodulin complex binds to many different enzymes in many cell types. Its specific signaling effects will depend on the exact identify of which Ca2+-responsive enzymes are expressed in each cell type.
The TCR and BCR are complex signaling receptors that require all of their subunits for proper ligand binding and optimal signaling. The surface expression of antigen receptors lacking one or more of their domains would interfere with T or B cell activation. This would be particularly detrimental in the case of cells expressing the ligand-binding domains of an antigen receptor without the signaling subunits; such a receptor would bind the ligand, but fail to signal. If antigen levels are low, this might result in a failure to mount a T or B cell response altogether.
7.8: CD45 is the phosphatase that de-phosphorylates the C-terminal negative regulatory tyrosine of Lck. When this negative regulatory tyrosine is phosphorylated and binds to the Lck SH2 domain, Lck is held in an inactive conformation. TCR signaling initiated by Lck cannot occur without CD45 to dephosphorylate this site.
Csk is a tyrosine kinase that phosphorylates the C-terminal negative regulatory tyrosine in Lck, maintaining Lck in an inactive state. In the absence of Csk, tyrosine phosphatases in the T cell will dephosphorylate this reside, leading to Lck being in a primed state. Full Lck activation will occur when Lck autophosphorylates on its activation loop tyrosine in the kinase domain, a process that takes place at a low level, even in the absence of TCR stimulation. This low basal autophosphorylation activity of Lck is normally prevented in non-stimulated T cells by the phosphorylation of the C-terminal negative regulatory tyrosine, which locks Lck in an inactive state.
Tandem SH2-containing tyrosine kinases, such as ZAP-70 and Syk, have SH2 domains that individually have very low affinity for their tyrosine-phosphorylated binding sites. Therefore, these kinases will not bind to a tyrosine-phosphorylated sequence that contains a binding site for only one of their SH2 domains. It is only when both tyrosines in an ITAM are phosphorylated, recruiting both SH2 domains together, that these kinases can stably bind to ITAM-containing signaling subunits.
7.11: Since the ITAM-containing signaling subunits and the enzymes are each present as separate polypeptides, these proteins can be re-used by other immune cells to mediate signaling via distinct ligand-binding receptors (i.e., mix and match the receptors, signaling subunits and tyrosine kinases). For instance, Fc receptors on NK cells can use the TCR z chain as a signaling subunit, and can use ZAP-70 as an enzyme. As another example, neutrophils use ITAM-containing subunits DAP-12 or Fcr-g to activate Syk downstream of the PSGL-1 (P-selectin binding) receptor.
Explanation: Four important signaling modules are activated downstream of the TCR or BCR. In addition to transcription factor activation, the three additional modules lead to increased cellular metabolic activity, actin polymerization and cytoskeletal reorganization, and increased integrin adhesiveness and clustering.
PLC-g cleaves the membrane phospholipid PIP2 into two second messengers, DAG and IP3. DAG remains tethered to the plasma membrane, and leads to activation of Ras and Protein kinase C. IP3 diffuses to the endoplasmic reticulum (ER), and binds to and activates the IP3 receptor, ultimately leading to calcium influx into the cell. Collectively, signaling pathways arising from these two second messengers activate multiple transcription factors, including NFAT, NFkB, and AP-1.
In resting T cells, NFAT is heavily phosphorylated on serine/threonine residues. Following TCR stimulation, calcineurin is activated and dephosphorylates NFAT.
Patients with defects in ORAI1 or STIM1 have impaired TCR-induced Ca2+ influx, leading to defective activation of calcineurin and its downstream target NFAT. The immunosuppressive drug cyclosporin A inhibits calcineurin and causes a similar defect in NFAT activation.
Since PMA plus Ionomycin restore normal IL-2 production in response to TCR stimulation, the defect must lie in PLC-g activation or a step upstream of this. Possible candidates would be ZAP-70, PLC-g, SLP-76 or ITK. Calcineurin would not be a possible candidate since cells with defective calcineurin would not show normal responses to PMA plus Ionomycin. This is because the Ca2+ influx induced by Ionomycin would fail to activate NFAT in the absence of calcineurin.
Although Ras is constitutively present at the plasma membrane, it requires interaction with a GEF such as RasGRP for activation. While RasGRP is also constitutively expressed in non-stimulated T cells, it is not present at the membrane, and therefore cannot interact with Ras. TCR stimulation generates DAG, a second messenger that remains associated with the plasma membrane. DAG recruits Ras GRP, which can activate Ras.
DAG remains associated with the plasma membrane and recruits RasGRP and the serine/threonine kinase, Protein kinase C (PKC)-q. RasGRP activates Ras, leading to the formation of the active transcription factor, AP-1, and PKC-q leads to the activation of a second transcription factor, NKkB.
The defect in these cells is failure to activate the Ras/Map-kinase pathway. Since Ca2+ influx in response to TCR stimulation is normal, this indicates that activation of PLC-g is normal. This information rules out PLC-g and ITK as candidates. Calcineurin is not required for Erk Map-kinase activation, so it is not a correct answer. WASp is also not required for Erk Map-kinase activation. The correct answer is RasGRP. When DAG is produced following PLC-g activation, RasGRP is required to bind DAG and activate Ras, leading to Erk Map-kinase activation.
In non-stimulated T cells, the p50/p65Rel NFkB heterodimer is inactive due to retention in the cytoplasm by binding to the inhibitory subunit, IkB. Following TCR stimulation, NEMO is activated and phosphorylates IkB. This leads to IkB ubiquitination and degradation, thereby releasing NFkB. The NFkB heterodimer then enters the nucleus where it activates transcription of target genes.
Increased glucose metabolism is induced by activation of the serine/threonine kinase Akt following TCR stimulation. Akt is activated by PDK1. Both Akt and PDK1 are recruited to the plasma membrane by binding to PIP3, as both kinases have PH domains. Binding to PIP3 activates PDK1 to phosphorylate and activate Akt.
mTOR activation leads to increases in several macromolecular synthesis pathways in T cells. They include increases in lipid metabolism, protein synthesis, and RNA transcription. Without these increases, activated T cells are unable to produce the components needed for cells to undergo the robust proliferation seen normally. mTOR is not required to activate Bcl2, nor is it required for DNA synthesis, cell cycle progression or IL-2 synthesis.
In response to antigen receptor stimulation, mTOR is activated by the small GTPase Rheb. In non-stimulated cells, Rheb is held in an inactive conformation by the protein complex TSC1/2. Activation of the serine/threonine kinase Akt leads to phosphorylation of TSC1/2, causing release of Rheb and activation of mTOR.
TCR stimulation induces increased binding of the integrin LFA-1 to its ligand, ICAM-1, expressed on antigen-presenting-cells. In large part, increased LFA-1 binding is mediated by a conformational change that converts LFA-1 from a low-affinity to a high-affinity binding state. TCR stimulation also induces LFA-1 clustering on the plasma membrane, which also contributes to increased integrin-dependent adhesion.
Integrin activation is induced by activation of the small GTPase Rap1 following antigen receptor stimulation. Therefore a defect in Rap1 would mimic a defect in LFA-1 itself. Neither CD21 nor WASp are required for integrin activation. Defects in CD3z or SLP-76 would lead to T cell defects that are much more severe than failure of cells to migrate to tissues.
7.28: Solid line = TCR-stimulated; dashed line = unstimulated.
When the TCR is stimulated, T cells bind the integrin ligand with higher affinity; hence, lower concentrations of ligand are required for maximal binding to activated T cells.
WASp is required for actin polymerization and formation of the Immune Synapse. These cytoskeletal changes are required for directed secretion of cytokines from the T cell to the B cell during the T–B interactions that are essential for T cell-dependent antibody responses. WASp is not required for cytokine or survival gene expression, up-regulation of metabolic pathways, or for integrin-mediated adhesion.
BCR signaling, like TCR signaling, is initiated by a Src-family kinase phosphorylating ITAM tyrosines on BCR signaling subunit proteins, Ig-a and Ig-b.
These mutant B cells are defective in cytoskeletal reorganization following BCR stimulation. In this case, BCR stimulation is induced by binding the antigen, which is a microbe. In wild-type cells, BCR stimulation will induce polarization of the cytoskeletal elements, the microtubule organizing center and the polymerized actin, to the immune synapse where the activated BCR is localized. BCR-induced actin polymerization and cytoskeletal reorganization is activated by the Vav GTP-exchange factor (GEF) activating the small GTPase, cdc42, a reasonable experiment would be to determine whether BCR stimulation is functioning to activate cdc42. Cdc42 activation is dependent on release of GDP and binding of GTP, so the experiment would require assessing whether this nucleotide exchange process is occurring following BCR stimulation.
CD28 co-stimulation does lead to activation of PI 3-kinase, and to production of PIP3 at the plasma membrane. In addition to Akt activation, PIP3 recruits PLC-g, ITK, and Vav. These enzymes contribute to T cell activation by enhancing transcription factor activation and actin polymerization and cytoskeletal reorganization, pathways that are not dependent on Akt.
TCR plus CD28 stimulation together lead to enhanced activation of transcription factors compared to TCR stimulation alone. This leads to increased mRNA synthesis of the IL-2 gene. In addition, CD28 co-stimulation leads to activation of Akt. Akt phosphorylates the RNA binding protein, NF-90. When phosphorylated, NF-90 translocates from the nucleus to the cytoplasm, where it binds to and stabilizes the IL-2 mRNA.
CD40 ligand deficiency results in a failure to stimulate CD40 on B cells during T–B interactions for antibody responses to protein antigens. CD40 is a TNF-receptor family member. Stimulation of CD40 leads to NFkB activation by the non-canonical pathway.
TNF-receptors stimulate NFkB activation by the noncanonical pathway. This pathways includes several steps regulated by ubiquitination. These include K63-linked ubiquitination of cIAP, inducing cIAP to ubiquitinate TRAF3 with K-48-linked poly-ubiquitin. This causes degradation of TRAF3 and release of NIK. NIK then activates IkB-kinase-a, which phosphorylates the NFkB precursor protein, p100. Phosphorylation of p100 leads to its K48-linked ubiquitination, inducing cleavage of p100 to generate p52. P52 binds to relB to form the active NFkB heterodimer.
Unlike the ITIM- and ITSM-containing receptors, CTLA-4 is no longer believed to recruit inhibitor phosphatases as a mechanism of inhibitory signaling. Instead, a major mechanism of CLTA-4 action is by competing with CD28 for binding to the shared B7 ligands.
Checkpoint blockade aims to enhance T cell activation, by inhibiting the activation of inhibitory receptors. This would be beneficial in patients where the goal is to enhance or increase their T cell activation and function. This strategy is currently being investigated for use in cancer patients, to enhance the ability of their immune system to destroy their tumor cells.
When the microbe is already coated with antibody proteins prior to incubation with the B cells, the inhibitory Fc receptor (FcgRIIb) on the B cell is stimulated along with the BCR. The co-engagement of FcgRIIb recruits the lipid phosphatase, SHIP, leading to dephosphorylation of PIP3. Loss of PIP3 reduces Akt activation, leading to reduced activation of mTOR. As a result, much higher concentrations of antigen are required to generate the same level of mTOR activation as would be seen with the live microbe alone. When the microbe is coated with C3dg, BCR signaling would be enhanced, and this form of antigen would stimulate mTOR activation at lower doses than unmodified antigen. The heat-killed microbe and membrane fragments would stimulate at similar antigen doses to live microbes.
- a) Any TCR signaling protein required for PLC-g activation could be responsible. This would include Lck, ZAP-70, SLP-76, LAT, ITK, or PLC-g Additional candidates could be PI 3-kinase, ORAI1, or STIM1. Proteins that could not be responsible for the calcium signaling defect include: WASp, cdc42, Vav, Akt, mTOR, Rheb, Protein kinase C-q, Rap1, Nck, Ras, RasGRP, Erk-MAP-kinase or calcineurin. This is not a complete list, but the most common answers.
- b) LCK is normal, as CD3 and TCRz phosphorylation are normal. Also, increased LCK phosphorylation in response to TCR stimulation (autophosphorylation) is also normal. In addition, ZAP-70 phosphorylation is normal, indicating normal LCK activity. Since there is no phosphorylation of LAT or SLP-76, there is likely a defect in ZAP-70 kinase activity. These data cannot directly rule out a defect in ITK or PLC-g, but given the lack of phosphorylation of SLP-76 and LAT, a more likely explanation is a defect in ZAP-70.
- c) ZAP-70 for reasons explained in (b).
- d) ZAP-70 could have a mutation in its kinase domain that prevents kinase activity. This could be a mutation that prevents ATP binding, or one that prevents phosphorylation on the activation loop tyrosine. Alternatively, ZAP-70 could have a mutation in the linker region between the SH2 domains and the kinase domain that prevents phosphorylation of this linker region. In the absence of phosphorylation of this linker region, ZAP-70 remains in an auto-inhibited conformation, and would not phosphorylate its downstream substrates, ZAP-70 and LAT.
- a) Stimulation of X does not induce IL-2 production by itself. Stimulation of X inhibits TCR signaling because anti-CD3 + anti-X stimulation leads to reduced IL-2 secretion compared to stimulation with anti-CD3 alone.
- b) An ITIM or ITSM motif.
- c) Inhibitory receptors that contain ITIM or ITSM motifs often recruit a phosphatase. This could be a protein tyrosine phosphatase, such as SHP or a lipid phosphatase, SHIP. The T cell stimulation data are not sufficient to identify which type of phosphatase is recruited. It could be SHP2, which would dephosphorylate signaling proteins such as ZAP-70, SLP-76 or ITK; dephosphorylation of any of these (or other) TCR signaling proteins would lead to reduced IL-2 production. Alternatively, it could be SHIP, which would dephosphorylate PIP3; this would prevent recruitment of ITK and PLC-g, either of which would lead to reduced IL-2 production.
- d) Possible answers:
- Stimulate T cells with anti-X antibody and then perform an immunoprecipitation of receptor X. Analyze the proteins in the immunoprecipitation for SHP or SHIP by Western blotting.
- Stimulate T cells with anti-CD3 + anti-CD28 or with anti-CD3 + anti-CD28 + anti-X. Lyse the T cells and examine candidate proteins for reduced tyrosine phosphorylation when stimulation of receptor X is included compared to stimulation of CD3 + CD28 alone. If X recruits SHP, cells stimulated with anti-X plus the other antibodies should show reduced tyrosine phosphorylation compared to cells stimulated with anti-CD3 + anti-CD28 alone.
In parallel, prepare lipids from T cells stimulated with anti-CD3 + anti-CD28 versus anti-CD3 + anti-CD28 + anti-X, and analyze for amounts of PIP3. If X recruits SHIP, then cells stimulated with all three antibodies should have reduced levels of PIP3 compared to cells stimulated with anti-CD3 + anti-CD28 alone.