INFECTIOUS DISEASE

IMMUNOLOGY - CHAPTER TEN 

MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) AND T-CELL RECEPTORS - ROLE IN IMMUNE RESPONSES

Gene Mayer, Ph.D
Emertius Professor of Pathology, Microbiology and Immunology
University of South Carolina

Jennifer Nyland, Ph.D
Assistant Professor of Pathology, Microbiology and Immunology
University of South Carolina

 

TEACHING OBJECTIVES
To give an overview of the role of the major histocompatibility complex in immune responses.
To describe the structure and function of class I and class II MHC molecules.
To discuss the nature of polymorphisims in class I and class II MHC molecules.
To describe the structure of the T cell receptor for antigen
To discuss the genetic basis for the generation of diversity in the TCR.
To discuss the role of the CD3 complex and co-stimulatory molecules.
To describe the nature of the immunological synapse.
To discuss the requirements for T cell activation.
 

Historical Overview 

Cell-cell interactions of the adaptive immune response are critically important in protection from pathogens. These interactions are orchestrated by the immunological synapse whose primary components are the T cell antigen receptor (TCR) and the Major histocompatibility complex (MHC) molecule. The major function of the TCR is to recognize antigen in the correct context of MHC and to transmit an excitatory signal to the interior of the cell. Since binding of peptide within the MHC is not covalent, there are many factors while help stabilize the immunological synapse.

Gene products encoded in the MHC were first identified as being important in rejection of transplanted tissues.  Furthermore, genes in the MHC were found to be highly polymorphic (i.e. in the population there were many different allelic forms of the genes).  Studies with inbred strains of mice showed that genes in the MHC were also involved in controlling both humoral and cell-mediated immune responses.  For example, some strains of mice could respond to a particular antigen but other strains could not and these strains differed only in one or more of the genes in the MHC.  Subsequent studies showed that there were two kinds of molecules encoded by the MHC – Class I molecules and class II molecules which are recognized by different classes of T cells.  Class I molecules were found on all nucleated cells (not red blood cells) whereas class II molecules were found only on antigen presenting cells, (APCs) which included dendritic cells, macrophages, B cells and a few other types (Figure 1).

It was not until the discovery of how the TCR recognizes antigen that the role of MHC genes in immune responses was understood.  The TCR was shown to recognize antigenic peptides in association with MHC molecules.  T cells recognize portions of protein antigens that are bound non-covalently to MHC gene products.  Cytotoxic T cells (Tc) recognize peptides bound to class I MHC molecules and helper T cells (Th) recognize peptides bound to class II MHC molecules.  The three dimensional structures of MHC molecules and the TCR have been determined by X-ray crystallography so that a clear picture of how the TCR, MHC gene products and antigen interact has emerged.

Figure 2
The MHC class 1 molecule has three globular domains alpha 1 (yellow), alpha 2 (green) and alpha 3 (blue). The alpha 3 domain is closely associated with the non-MHC -encoded beta 2 microglobulin (pink). The latter is stabilized by a disulfide bridge (red) and is similar to an immunoglobulin domain in three-dimensional structure. The alloantigenic sites which carry determinants specific to each individual are found in the alpha 1 and 2 domains. The latter also has a carbohydrate chain (blue, CHO). There is a phosphate in the cytoplasmic domain. Papain cleaves near the outer surface of the plasma membrane

Structure of Class I MHC Molecules  

The molecule

         Class I MHC molecules are composed of two polypeptide chains, a long α chain and a short β chain called β2-microglobulin (figure 2).  The α chain has four regions. 

• A cytoplasmic region, containing sites for phosphoylation and binding to cytoskeletal elements. 

• A transmembrane region containing hydrophic amino acids by which the molecule is anchored in the cell membrane.

• A highly conserved α3 immunoglubilin-like domain to which CD8 binds. 

• A highly polymorphic peptide binding region formed from the α1 and α2 domains.  The β2- microglobulin associates with the α chain and helps maintain the proper conformation of the molecule. 

The antigen-binding groove

An analysis of which part of the class I MHC molecules is most variable demonstrates that variability is most pronounced in the α1 and α2 domains, which comprise the peptide binding region (Figure 3).  The structure of the peptide binding groove, revealed by X-ray crystallography, shows that the groove is composed of two α helices forming a wall on each side and eight β-pleated sheets forming a floor.  The peptide is bound in the groove and the residues that line the groove make contact with the peptide (Figure 4).  These are the residues that are the most polymorphic.  The groove will accommodate peptides of approximately 8-10 amino acids long.  Whether a particular peptide will bind to the groove will depend on the amino acids that line the groove.  Because class I molecules are polymorphic, different class I molecules will bind different peptides.  Each class I molecule will bind only certain peptides and will have a set of criteria that a peptide must have in order to bind to the groove.  For example, Figure 5 shows that one class I molecule will bind peptides that have a leucine (L) as the carboxy-terminal amino acid and either tyrosine (Y) or phenylalanine (F) as the 4^th amino acid from the carboxy-terminal end. As long as these two conditions are met a peptide will bind, regardless of what the other amino acids are.  Similarly a different  class I molecule will bind any peptide that has a tyrosine (Y) as the second amino acid from the amino-terminal end and either a valine (V), isoleucine (I) or leucine (L) at the carboxy-terminal end (Figure 5).  Thus, for every class I molecule, there are certain amino acids that must be a particular location in the peptide before it will bind to the MHC molecule.  These sites in the peptide are referred to as the “anchor sites”. The ends of the peptide are buried within the closed ends of the class I binding groove while the center bulges out for presentation to the TCR.

         Within the MHC there are 6 genes that encode class I molecules HLA-A, HLA –B, HLA-C, HLA-E, HLA-F and HLA-G.  Among these HLA-A, HLA –B, and HLA-C are the most important and are most polymorphic.  Table 1 shows the degree of polymorphism at each of these loci.

Figure 5
Anchor sites in peptides that bind to class I MHC molecules (adapted from Janeway et al. Immunobiology 6th Edition
 

CHIME
 
Chime presentation showing the regions of variability of MHC I molecules and the interaction of the alpha chain with other subunits of the MHC I complex and the bound peptide  (requires Chime plug-in. Get Chime here)

The molecule

Class II MHC molecules are composed of two polypeptide chains an α and a β chain of approximately equal length (Figure 6). Both chains have four regions:

• A cytoplasmic region containing sites for phosphoylation and binding to cytoskeletal elements
• A transmembrane region containing hydrophic amino acids by which the molecule is anchored in the cell membrane
• A highly conserved α2 domain and a highly conserved β2 domain to which CD4 binds
• A highly polymorphic peptide binding region formed from the α1 and β1 domains

The antigen-binding groove

As with Class I MHC molecules, an analysis of which part of the class II MHC molecule is most variable demonstrates that variability is most pronounced in the α1 and β1 domains, which comprise the peptide binding region (Figure 7). The structure of the peptide binding groove, revealed by X-ray crystallography, shows that, like class I MHC molecules, the groove is composed of two α helices forming a wall on each side and eight β-pleated sheets forming a floor. Both the α1 and β1 chain contribute to the peptide binding groove. The peptide is bound in the groove and the residues that line the groove make contact with the peptide. These are the residues that are the most polymorphic. The groove of Class II molecules is open at one end so that the groove can accommodate longer peptides of approximately 13-25 amino acids long with some of the amino acids located outside of the groove. Whether a particular peptide will bind to the groove will depend on the amino acids that line the groove. Because class II molecules are polymorphic, different class II molecules will bind different peptides. Like class I molecules, each class II molecule will bind only certain peptides and will have a set of criteria that a peptide must have in order to bind to the groove (i.e. “anchor sites”).

Within the MHC there are 5 loci that encode class II molecules, each of which contains a gene for an α chain and at least one gene for a β chain. The loci are designated as HLA-DP, HLA –DQ, HLA-DR, HLA-DM, and HLA-DO. Among these, HLA-DP, HLA –DQ, and HLA-DR are the most important and are most polymorphic. Table 2 shows the degree of polymorphism at each of these loci.
 

Important Aspects of MHC 

• Although there is a high degree of polymorphism for a species, an individual has maximum of six different class I MHC products and only slightly more class II MHC products (considering only the major loci).  

• Each MHC molecule has only one binding site.  The different peptides a given MHC molecule can bind all bind to the same site, but only one at a time. 

• Because each MHC molecule can bind many different peptides, binding is termed degenerate. 

• MHC polymorphism is determined only in the germline.  There are no recombinational mechanisms for generating diversity. 

• MHC molecules are membrane-bound; recognition by T cells requires cell-cell contact. 

• Alleles for MHC genes are co-dominant.  Each MHC gene product is expressed on the cell surface of an individual nucleated cell. 

• A peptide must associate with a given MHC of that individual, otherwise no immune response can occur.  That is one level of control.

• Mature T cells must have a T cell receptor that recognizes the peptide associated with MHC.  This is the second level of control. 

• Cytokines (especially interferon-γ) increase level of expression of MHC. 

• Peptides from the cytosol associate with class I MHC and are recognized by Tc cells.  Peptides from within vesicles associate with class II MHC and are recognized by Th cells. 

• Polymorphism in MHC is important for survival of the species.

How do peptides get into the MHC groove?

Peptides from the cytosol associate with class I MHC and are recognized by CTL cells. The peptides enter the endoplasmic reticulum and bind in the MHC class I groove. This complex is then exported to the cell surface through the Golgi. MHC class II molecules are formed with an invariant (Ii) chain as a place holder while in the ER and Golgi. The Ii chain is cleaved and removed once the complex is in a vesicle. Peptides from within the vesicle associate with class II MHC and are then exported to the cell surface where they are recognized by Th cells.

The role of TCR in the immune response

The TCR is a surface molecule found on T cells that recognizes antigen presented in the correct MHC context. The TCR is similar to immunoglobulin and is part of the immunoglobulin superfamily. There are two types of TCRs, the predominant αβ which is commonly found in lymphoid tissues, and the γδ which is found at mucosal surfaces.

Structure of A6-T cell receptor bound to MHC class I molecule complexed with an altered Htlv-1 Tax Peptide Y8a.   The HIV peptide is shown in gray. MHC class I molecule is in dark blue, the associated beta 2 microglobulin in light blue. T cell receptor is in green and yellow.  Y. H.Ding, B. M.Baker, D. N.Garboczi, W. E.Biddison & D. C.Wiley MMDB Id: 11766 PDB Id: 1QSF Image prepared using RasMol

Figure 9
Rearrangement of the TCR beta  chain genes

The TCR is a heterodimer composed of one α and one β chain of approximately equal length (Figure 8).  Each chain has a short cytoplasmic tail but it is to small to be able to transduce an activation signal to the cell.  Both chains have a transmembrane region comprised of hydrophobic amino acids by which the molecule is anchored in the cell membrane.  Both chains have a constant region and a variable region similar to the immunoglobulin chains.  The variable region of both chains contains hypervariable regions that determine the specificity for antigen.  Each T cell bears a TCR of only one specificity (i.e. there is allelic exclusion).

THE GENETIC BASIS FOR RECEPTOR GENERATION

The genetic basis for the generation of the vast array of antigen receptors on B cells has been discussed previously (see lecture on Ig genetics).   The generation of a vast array of TCRs is accomplished by similar mechanism.  The germline genes for the TCR β genes are composed of V, D and J gene segments that rearrange during T cell development to produce many different TCR β chains (Figure 9).  The germline genes for the TCR α genes are composed of V and J gene segments which rearrange to produce α chains.  The specificity of the TCR is determined by the combination of α and β chains.

There is a small population of T cells that express TCRs that have γ and δ chains instead of α and β chains. These gamma/delta T cells predominate in the mucosal epithelium and have a repertoire biased toward certain bacterial and viral antigens. The genes for the δ chains have V, D and J gene segments whereas the genes for the γ chains have only V and J gene segments but the repertoire is considerably smaller that than that of the alpha/beta T cells. The gamma/delta T cells recognize antigen in an MHC-independent manner unlike the alpha/beta T cells.

Important aspects of the TCR

• Each T cell bears a TCR of only one specificity (i.e. there is allelic exclusion).

• The αβ TCR recognizes antigen only in the context of cell-cell interaction and in the correct MHC.

• The γδ TCR recognizes antigen in an MHC-independent manner in response to certain viral and bacterial antigen.

CHIME
  
Click on the image above to view rotatable structure and identify protein chains of MHC I and TCR interacting with HTLV tax peptide  (requires Chime plug-in. Get Chime here)

CHIME  
 
Click on the image above to view a rotatable structure and identify protein chains of MHC II and TCR interacting with an influenza HA peptide (requires Chime plug-in. Get Chime here) 

TCR and CD3 Complex  

The TCR is closely associated with a group of 5 proteins collectively called the CD3 complex (Figure 10).  The CD3 complex is composed of one γ, one δ, two ε and 2 ξ chains.  All of the proteins of the CD3 complex are invariant and they do not contribute to the specificity in any way.  The CD 3 complex is necessary for cell surface expression of the TCR during T cell development.  In addition, the CD3 complex transduces activation signals to the cell following antigen interaction with the TCR.

B. Ligands involved in the interaction of cytotoxic T cells and their target cells

Figure 12a
Activation of T cells only occurs when both TCR and co-stimulatory molecules are engaged with their respective ligands

Figure 12b
Down regulation occurs if CTLA-4 interacts with B7:
CTLA-4 sends an inhibitory signal

Figure 12c
Engagement of TCR and antigen/MHC in the absence of co-stimulation may lead to anergy

Figure 12d
Engagement of co-stimulatory molcules in the absence of TCR engagement results in no response

The “Immunological synapse”

The interaction between the TCR and MHC molecules are not very strong. Accessory molecules are necessary to help stabilize the interaction (Figure 11a,b). These include:

• CD4 binding to Class II MCH, which ensures that Th cells only interact with APCs

• CD8 binding to class I MHC, which ensures that Tc cells can interact with target cells

• CD2binding to LFA-3

• LFA-1 binding to ICAM-1

The accessory molecules are invariant and do not contribute to the specificity of the interaction, which is solely determined by the TCR. The expression of accessory molecules can be increased in response to cytokine, which is one way that cytokines can modulate immune responses.

In addition to accessory molecules which help stabilize the interaction between the TCR and antigen in association with MHC molecules, other molecules are also needed for T cell activation. Two signals are required for T cell activation – one is the engagement of the TCR with Ag/MHC and the other signal comes from the engagement of co-stimulatory molecules with their ligands. One of the most important (but not the only) co-stimulatory molecule is CD28 on T cells which must interact with B7-1 (CD80) or B7-2 (CD81) on APCs . Like accessory molecules the co-stimulatory molecules are invariant and do not contribute to the specificity of the interaction. The multiple interactions of TCR with Ag/MHC and the accessory and co-stimulatory molecules with their ligands have been termed the “immunological synapse.”

Not only is co-stimulation necessary for T cell activation, a lack of co-stimulation may result in anergy (i.e., inability to respond to antigen) or down-regulation of the response. Figure 12 shows the possible outcomes of a T cell receiving one or both of the signals necessary for activation. Engagement of the TCR with Ag/MHC but no co-simulation results in anergy. Engagement of only the co-stimulatory molecule has no effect. Engagement of TCR with Ag/MHC and co-stimulatory molecules with their ligand results in activation. Engagement of the TCR with Ag/MHC and engagement of B7 ligand with CTLA-4, molecules similar to CD28, results in down-regulation of the response. CTLA-4/B7 interaction sends an inhibitory signal to the T cell rather than an activating signal. This is one of the ways that immune responses are regulated. CTLA-4 is expressed on T cells later in an immune response and this helps to turn off the response.


Key Steps in T cell Activation

• APC must process and present peptides to T cells

• T cells must receive a co-stimulatory signal - usually from CD28/B7

• Accessory adhesion molecules must help to stabilize the binding of T cells and APC. (CD4/class II MHC, CD8/classs I MHC, LFA-1/ICAM-1 and CD2/LFA-3)

• Signals from cell surface must be transmitted to the nucleus via second messengers

• Cytokines, including IL-2, must help drive cell division
   

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