Chapter 5: Antigen Recognition by T Lymphocytes

A. (i) Describe the structure of an MHC class I molecule, identifying the different polypeptide chains and domains. (ii) What are the names of the MHC class I molecules produced by humans? Which part of the molecule is encoded within the MHC region of the genome? (iii) Which domains or parts of domains participate in the following: antigen binding; binding the T-cell receptor; and binding the T-cell co-receptor? (iv) Which domains are the most polymorphic? B. Repeat this for an MHC class II molecule.

A. (i) The complete MHC class I molecule is a heterodimer made up of one a chain and a smaller chain called 2 - microglobulin. The a chain consists of three extracellular domains-a1 1 a2, and a3-a transmembrane region and a cytoplasmic tail. 2 -Microglobulin is a single - domain protein noncovalently associated with the extracellular portion of the a chain, providing support and stability. (ii) The polymorphic class I molecules in humans are called HLA-A, HLA-B, and HLA- C . The a chain is encoded in the MHC region by an Answers to Questions A : 7 MHC class I gene. The gene for 2 - microglobulin is elsewhere in the genome. (iii) The antigenbinding site is formed by the a1 and a2 domains, the ones farthest from the membrane, which cre ate a peptide -binding groove. The region of the MHC molecule that binds to the T- cell receptor encompasses the a helices of the a1 and a2 domains that make up the outer surfaces of the peptide -binding groove. The a3 domain binds to the T- cell co -receptor CD8 . (iv) The most p olymorphic parts of the a chain are the regions of the a1 and a2 domains that bind antigen and the T- cell receptor. 2 - Microglobulin is invariant; that is, it is the same in all individuals. B. (i) MHC class II molecules are heterodimers made up of an a chain and a chain. The a chain consists of a1 and a2 extracellular domains, a trans membrane region, and a cytoplasmic tail. The chain contains 1 and 2 extracellular domains, a transmembrane region, and a cytoplasmic tail. (ii) In humans there are three polymorphic MHC class II molecules called HLA-DP, HLA- DQ, and HLA-DR. Both chains of an MHC class II mole cule are encoded by genes in the MHC region. (iii) Antigen binds in the peptide -binding groove formed by the a1 and 1 domains. The a helices of the a1 and 1 domains interact with the T- cell receptor. The 2 domain binds to the T- cell co receptor CD4. (iv) With the exception of HLADRa, which is dimorphic, both the a and chains of MHC class II molecules are highly polymorphic. Polymorphism is concentrated around the regions that bind antigen and the T- cell receptor in the a1 and 1 domains.

What is meant by the terms (a) antigen processing and (b) antigen presentation? (c) Why are these proc esses required before T cells can be activated?

A. Antigen pro cessing is the intracellular breakdown of pathogen - derived proteins into peptide fragments that are of the appropriate size and specificity required to bind to MHC molecules. B. Antigen presentation is the assembly of pep tides with MHC molecules and the display of these complexes on the surface of antigen-presenting cells. C . Antigen processing and presentation must occur for T cells to be activated because ( 1 ) T- cell recep tors cannot bind to intact protein, only to pep tides, and (2) T- cell receptors do not bind antigen directly, but rather must recognize antigen bound to MHC molecules on the surface of antigen-pre senting cells.

A. Describe in chronological order the steps of the antigen-processing pathway for extracellular pathogens. B. What would be the outcome (i) if invariant chain were defective or missing, or (ii) if HLA-DM were not expressed?

A. Extracellular pathogens are taken up by endocytosis or phagocyto sis and degraded by enzymes into smaller peptide fragments inside acidified intracellular vesicles called phagolysosomes. MHC class II molecules delivered into the ER and being transp orted to the cell surface intersect with the phagolysosomes, where these peptides are encountered and loaded into the antigenbinding groove. To prevent MHC class II mole cules from binding to peptides prematurely, invariant chain (Ii) binds to the MHC class II antigen- binding site in the ER. Ii is also involved in transporting MHC class II molecules to the phagolysosomes via the Golgi as part of the interconnected vesicle system. Ii chain is removed from MHC class II molecules once the phagolyso some is reached. Removal is achieved in two steps : ( I ) proteolysis cleaves Ii into smaller fragments, leaving a small peptide called CLIP (class II - asso ciated invariant chain peptide) in the antigen- binding groove of the MHC class II molecule; and (2) CLIP is then released by HLA-DM catalysis. Once CLIP is removed, peptides derived from endocytosed material will bind (if the correct peptide -binding motif is possessed) and the peptide : MHC class II molecule complex will progress to the cell surface. B. (i) Defects in the invariant chain would impair normal MHC class II function because invariant chain not only protects the peptide -binding groove from binding prematurely to peptides present in the ER but is also required for trans port of MHC class II molecules to the phagolyso some. (ii) If HLA- DM were not expressed, most MHC class II molecules on the cell surface would be occupied by CLIP rather than endocytosed material. This would compromise the presentation of extracellular antigens at the threshold levels required for T- cell activation.

A. What is the difference between MHC variation due to multigene family and allelic polymorphism influence the antigens that a person's T cells can recognize? B. How do MHC variation due to multigene family and allelic polymorphism influence the antigens that a person's T cells can recognize?

A. Multigene family refers to the presence of multiple genes for MHC class I and MHC class II molecules in the genome, encoding a set of structurally similar proteins with similar functions. MHC polymorphism is the presence of multiple alleles (in some cases several hundreds) of most of the MHC class I and class II genes in the human p opulation. B . T cells recognize peptide antigens in the form of peptide : MHC complexes, which they bind using their T- cell receptors. To bind specifically, the T- cell receptor must fit b oth the peptide and the part of the MHC molecule surrounding it in the peptide -binding gro ove. (i) Because each individual produces a number of different MHC molecules from their MHC class I and class II multigene family, the T- cell receptor repertoire is not restricted to recognizing pep tides that bind to just one MHC molecule (and thus all must have the same peptide -binding motif) . Instead, the T- cell receptor rep ertoire can recognize peptides with different peptide -binding motifs during an immune response, increasing the likelihood of antigen recognition and, hence, T- cell activation. (ii) The polymorphism in MHC molecules is localized in the regions affecting T- cell receptor and peptide binding. Thus, a T- cell receptor that recognizes a given peptide bound to variant 'a' of a particular MHC molecule is likely not to recognize the same peptide b ound to variant 'b' of the same MHC molecule. Polymorphism also means that the MHC molecules of one person will bind a different set of peptides from those in another person. Taken together, these outcomes mean that because of MHC polymorphism, each individual recognizes a somewhat different range of peptide antigens using a different repertoire of T- cell receptors.

A. Describe in chronological order the steps of the endogenous antigen-processing pathway for intracellular, cytosolic pathogens. B. (i) What would be the outcome if a mutant MHC class I a chain could not associate with P2-mi croglo bulin, and (ii) what would happen if the TAP transporter were lacking as a result of mutation? Explain your answers.

A. Proteins from pathogens growing in the cytosol are broken down into small peptide fragments in proteasomes. The peptides are transported into the lumen of the endoplasmic reticulum (ER) using TAP (transporter associated with antigen processing) , which is a heterodimer of TAP- 1 and TAP-2 proteins located in the ER membrane. Pep tides bearing the appropriate peptide -binding motif bind to MHC class I molecules already delivered into the ER. MHC class I a chains are bound to the chaperone calnexin until 2 - microglobulin binds, and then are bound by the chaperones calreticulin and tapasin until peptide binds. Tapasin binds to TAP- I , positioning the MHC class I molecule near the peptide source. MHC class I molecules bound to peptide dissociate from the chaperone molecules and progress to the Golgi apparatus for completion of glycosylation and transport to the cell surface in membrane- bound vesicles. B. (i) If an MHC class I a chain is unable to bind 2 - microglobulin, it will be retained in the ER and will not be transported to the cell surface. It will remain bound to calnexin and will not fold into the conformation needed to bind to peptide. Thus, antigens will not be presented using that particular MHC class I molecule. (ii) If TAP- I or TAP-2 proteins are mutated and not expressed, then peptides will not be transported into the lumen of the ER. Without peptide, an MHC class I molecule cannot complete its assembly and will not leave the ER. A rare immunodeficiency dis ease called bare lymphocyte syndrome (MHC class I immunodeficiency) is characterized by a defective TAP protein, causing less than I % of MHC class I molecules to be expressed on the cell surface in comparison with normal. Thus, T- cell responses to all pathogen antigens that would normally be recognized on MHC class I molecules will be impaired.

What evidence supports the proposal that MHC diversity evolved by natural selection caused by infectious pathogens rather than exclusively by random DNA mutations?

MHC polymorphisms are non-randomly localized predominantly in the region of the molecule that makes contact with peptide and T- cell receptors. Random DNA mutations, in contrast, would be scattered through the gene, giving rise to amino acid changes throughout MHC molecules and not just in those areas important for pep tide binding and presentation.

Brittany Hudson, 16 years of age, was seen by her physician after the development of a small pustule around her nostrils that had expanded and was now showing signs of ulceration typical of chronic granulo matous inflammation. In the past year, Brittany had experienced a similar lesion on her left thigh that healed slowly, leaving a hyperpigmented scar. She also had a history of chronic bacterial infections of the upper and lower respiratory tract. Flow cytometric analysis of peripheral blood revealed abnormally low numbers of MHC class I molecules on cell surfaces and abnormally low numbers of CD8 T cells. A diagnosis of type I bare lymphocyte syndrome was made. A deficiency in which of the following would explain this etiology? a. HLA-DM b. Invariant chain c. Class II -associated invariant chain peptide (CLIP) d. TAP1 or TAP2 e. MHC class II transactivator (CIITA).

Rationale: The correct answer is d. A deficiency in either TAP- 1 or TAP-2, whose function is to transport peptide fragments to the lumen of the endoplasmic reticulum, would inhibit the expression of MHC class I on the cell surface because peptide must be bound to an MHC class I molecule to permit its transport from the endoplasmic reticulum to the cell membrane. Low levels of MHC class I account for the low levels of CD8 T cells; MHC class ! - restricted CD8 T cells would fail to undergo positive selection in the thymus if MHC class I levels were abnormally low. Deficiencies of HLA- DM, invariant chain (which gives rise to CLIP) , or CIITA would affect the MHC class II pathway of antigen presentation, but would have no effect on the MHC class I pathway.

Describe (A) five ways in which T-cell receptors are similar to immunoglobulins, and (B) five ways in which they are different (other than the way in which they recognize antigen).

Similarities. ( 1 ) The T- cell receptor has a similar overall structure to the membrane -bound Fab fragment of immunoglobulin, containing an antigen-binding site, two variable domains and two constant domains. (2) T- cell receptors and immunoglobulins are both generated through somatic recombination of sets of gene segments. (3) The variable region of the T- cell receptor contains three complementarity- determining regions (CDRs) encoded by the Va domain and three CDRs encoded by the V domain, analogous to the CDRs encoded by the VH and VL domains. (4) There is huge diversity in the T- cell receptor repertoire and it is generated in the same way as that in the B - cell repertoire (by combination of different gene segments, junctional diversity due to Pand N-nucleotides, and combination of two different chains) . (5) T- cell receptors are not expressed at the cell surface by themselves but require association with the CD3 y, 8, £, and s chains for stabilization and signal transduction, analogous to the Iga and Igp chains required for immunoglobulin cell - surface expression and signal transduction. B. Differences. ( 1 ) A T- cell receptor has one antigenbinding site; an immunoglobulin has at least two. (2) T- cell receptors are never secreted. (3) T- cell receptors are generated in the thymus, not the b one marrow. (4) The constant region of the T- cell receptor has no effector function and it do es not switch isotype. (5) T- cell receptors do not undergo somatic hypermutation.

T-cell receptors do not undergo isotype switching. Suggest a possible reason for this.

T- cell receptors are not made in a secreted form and their constant regions do not contribute to T- cell effector function. Other molecules secreted by T cells are used for effector functions. There is therefore no need for isotype switching in T cells, and the T- cell receptor loci do not contain numerous alternative C genes.

Compare the organization of T-cell receptor a and p genes (the TCRa and TCRP loci) with the organization of immunoglobulin heavy-chain and light-chain genes.

The organization of the TCRa locus resembles that of an immunoglobulin light- chain locus, in that both contain V and J gene segments and no D gene segments. The TCRa locus on chromosome 1 4 contains about 80 V gene segments, 6 1 J gene segments, and one C gene. The immunoglobulin light- chain loci, A and K, are encoded on chromosomes 22 and 2, respectively. The A locus contains about 30 V gene segments and four J gene segments, each paired with a C gene. The K locus contains ab out 35 V gene segments, five J segments, and one C gene segment. The arrangement of the K locus more closely resembles that of the TCRa locus except that there are more J segments in the T- cell receptor locus. The organization of the TCR locus resembles that of the immunoglobulin heavy- chain locus; both contain V, D, and J gene segments. The TCR locus contains about 52 V gene segments, two D gene segments, 1 3 J gene segments, and two C genes, encoded on chromosome 7 . Each C gene is associated with a set o f D and J gene segments. The immunoglobulin heavy- chain locus on chromosome 1 4 contains about 40V segments, 23 D segments, and six J segments, followed by nine C genes, each specifying a different immunoglobulin isotype. The heavychain C genes determine the effector function of the antibody.

Which of the following accurately completes this statement: ((The function of T cells is to make contact with and "? (Select all that apply.) a. CD8; virus-infected cells; kill virus-infected cells b. CD8; B cells; stimulate B cells to differentiate into plasma cells c. CD4; macrophages; enhance microbicidal powers of macro phages d. CD4; B cells; stimulate B cells to differentiate into plasma cells e. All of the above are accurate.

a. CD8; virus-infected cells; kill virus-infected cells c. CD4; macrophages; enhance microbicidal powers of macro phages d. CD4; B cells; stimulate B cells to differentiate into plasma cells

Which of the following removes CLIP from MHC class II molecules? a. HLA-DM b. HLA-DO c. HLA-DP d. HLA-DQ e. HLA-DR.

a. HLA-DM

The immunological consequence of severe combined immunodeficiency disease (SCID) caused by a genetic defect in either RAG-1 or RAG-2 genes is a. lack of somatic recombination in T-cell receptor and immunoglobulin gene loci b. lack of somatic recombination in T-cell receptor loci c. lack of somatic recombination in immunoglobulin loci d. lack of somatic hypermutation in T-cell receptor and immunoglobulin loci e. lack of somatic hypermutation in T-cell receptor loci

a. lack of somatic recombination in T-cell receptor and immunoglobulin gene loci

The role of the CD3 proteins and C chain on the surface of the cell is to a. transduce signals to the interior of the T cell b. bind to antigen associated with MHC molecules c. bind to MHC molecules d. bind to CD4 or CD8 molecules e. facilitate antigen processing of antigens that bind to the surface ofT cells

a. transduce signals to the interior of the T cell

Amino acid variation among MHC class II aHa types that present antigens to CD4 T cells is concentrated a. where MHC contacts the co-receptors CD4 or CD8 b. in the p chain, because the a chain is mono morphic c. where the MHC molecule contacts peptide and the T-cell receptor d. in the a chain, because the p chain is mono morphic e. throughout both the a and p chains in all domains.

c. where the MHC molecule contacts peptide and the T-cell receptor