22 T-cell receptors

Dr. M. N. Gupta

1. Objectives

•  To understand the challenges and learn about early finding about TcR
•  To learn further about differentiation of a progenitor into CD4 and CD8 T-cells in the context of TcR
•  To understand the differences between BcR and TcR
•  To learn about Tregs, γ:δ T cells and NKT cells.

2. Concept Map

3. Description

The actual role and development of T-cells is best understood in the context of T-cell receptor. Discussion on TCR also is a good place to introduce some other minor T-cell classes and variants.

In early days of immunology, T-cells were classified into three classes: Th, TC and Tregulatory. Interestingly, as our understanding of T-cell functions increased, (and we learnt more about cell-cell cooperation in immunology!), Th and Tc turned out to be two main classes. Neverthless, Tregs (as these are called now) are not unimportant, may be it is just that we know less about them.

Just like our knowledge about T-cells was gathered much after B-cells, so was the case with TcR.

In order to appreciate it, we should realise that separation and characterization of soluble proteins was possible much earlier before we had method to even isolate and characterize membrane bound proteins. The corresponding tools for cells and probing their cell surfaces came up much later.

Understandably, highly specific binding probes, monoclonal antibodies facilitated looking at cell surfaces in a dramatic way.

Appreciation of the challenges in understanding nature of TcR is best done by looking at the early findings. This also serves the purpose to illustrate that even before best tools are available, perseverance and brilliance can help making breakthroughs!

T cell receptors were difficult to isolate and there was a long period during which immunologists had gathered lot of information about antibodies and Ig molecules as B-cell receptors. However, they knew little about nature of T-cell receptors.

One clear information which was available early is that there are more than one kind of T-cells and Th cells are necessary for an effective immune response by B-cells in many cases. Furthermore, if B-cells and Th cells are both specific to the same antigens, then receptors may be similar.

The Ig molecules were easy to identify as B-cell receptors as these are ultimately secreted as antibodies in modified form. So, these could be isolated and characterized as by that time basic techniques of proteins isolation and characterization were in place. T-cell receptors do not have secretory forms!

Collaborative efforts by James P. Allison (Univ. of Texas, Austin), John W. Kappler (Natl. Jewish hospital, Denver) and Ellis L. Reinherz (Harvard Medical school) provided initial clues to nature of T-cell receptors.

Then approach was to prepare antibodies which bind to T-cell surface. A protein was found whose structure varied with each T-cell clone. This looked like a receptor. This protein had two subunits which were named α and β. The total mol. wt. of this protein was about 2/3rd of IgG.

Around the same period, in 1984 Tak. W. Mak (univ.of Toronto) and Mark M. Davis (Stanford univ. school of medicine) cloned a gene (and determined its sequence) which is expressed and rearranged in T-cells. It did not do so in B-cells.

Based upon similar behaviour of B-cell receptors rearrangement, this looked like a promising molecule. Mak was using leukemia T-cells and Davis was using a hybridoma of mouse Th cell with malignant T-cell. Both different system were found to encode same protein.

The sequence of this gene was found to be homologous to Ig genes. Furthermore, the gene had upstream variable segments and downstream constant segment. Reinherz‟s group determined the amino acid sequence of the gene product and found that this gene was β subunit of their presumed T-cell receptor.

Susumo Tonegawa by this time had shifted to MIT. His group along with Herman N. Eisen‟s group in the same institute were working with mouse Tc cells. These groups isolated two more T-cell DNA clones. Even, in their clones, the downstream part of one gene was similar to β-subunit.

The second molecule had sequence similar to β-subunit, had segments which rearranged in T-cells and even had a hydrophobic anchor component. Based upon this strong evidence and putting together all information available, it was assumed that this was α-subunit of Allison, Kappler and Reinherz.

This looked like end of the puzzle. However, Haruo saito, who had been doing this work in Tonegawa‟s lab isolated a third gene from same clone of Tc cells. It also had many properties expected of a Tc cell receptor. In addition it had Glycosylated asparagine residues. this facilitated its identification as true α-subunit of Allison, Kappler and Reinherz.

This left no role what was supposed to be α-subunit by saito and kranz (in Eisen‟s lab). So, they labelled it as γ-chain. Why it had so many properties which are expected of a T-cell receptor? Did it have some role in T-cell recognition of antigens?

Let us divert to MHC molecules again for a while. Ig, T-cell receptors and MHC are most diverse classes of proteins. Ig and TcR vary from one clone of cell to another. MHC proteins vary from one individual to another.

Transfer of tissues in vertebrates is not a natural phenomenon. So, while MHC proteins were discovered in the context of graft rejection, why were these actually created by nature? In other words what role these have in vivo?

MHC restriction:

As we have learnt earlier, T-cell receptor recognizes an antigen along with a MHC protein as a self marker. Thus T-cells are said to be MHC restricted.

Just to recollect, Tc cells require antigen along with a class I MHC molecule. This evolution of TcR may have taken place since Tc cells evolved probably for protection against viral infection. So, the pattern Tc cells need to see is viral coat +MHC class I protein. This makes sure that TcR does not get saturated with many soluble antigens. So, MHC restriction evolved to ensure division of labour.

Th cells recognise B-cells and macrophages which are carrying antigen+class II MHC molecules. In this case MHC restriction may have two reasons. Firstly, the overall structure of class I and II MHC molecules are not vastly different. So, TcR of Th cells did not need search for an ab initio design. It could be tweaked just to be different from TcR of Tc cells and yet serve the distinct function required of it in view of Th cell functions. Secondly, this strengthens binding and facilitate intercellular communications.

Thus, MHC molecules, which till work on cell mediated immunity started were only known to have physiological formation of involvement of graft rejection! When a redundant chain/subunit was identified, an early model which was suggested was based upon γ-β to α-β switch.

David Raulet and Susumn Tonegawa proposed one possible model for TcR development during thymic education. According to the γ-β to α-β switch model, immature T-cells have TcR containing. γ+β chains. AS a result of MHC restriction learnt by T-cells during thymic education, the affinity of TcR to MHC protein must be decreased so that self cells are not attached, this is ensured by switch over to α-β combination.

The availability of monoclonal antibodies provided a powerful tool to probe cell surfaces. TcR structure was also determined by use of mAb. These mAbs recognized only one cloned T-cell lines. These monoclonals also inhibited binding of the antigen to these T-cells. Such mab are called clonotypic antibodies as these did not bind to other clones of T-cells.

It was found that each T-cell bears about 30,000 antigen binding receptor molecules. Furthermore, each such receptor contains two polypeptides TcRα and TcRβ linked by a S-S bond. These heterodimers were in fact analogous to Fab fragment of IgG.

A minority population of T-cells instead had a different receptor composition. These γ:δ TcRs have different properties from α:β TcRs. We will talk more about these γ:δ T-cells later. Unless otherwise mentioned, TcR will be discussed in the context of α:β TcR.

It is to be noted that unlike IgG which has two binding sites, TcR has only one binding site for the antigen. Also, unlike IgG, it has no secretory isoform. The function of T-cells is different.

Both α and β chains have extracellular domains analogous to Fab, a transmembrane domain and a short cytoplasmic tail.

Both α and β chains are glycoprotein. It is interesting to note that transmembrane domain of α has two basic amino acid and β-chain has single basic amino acid residue in the hydrophobic environments.

The crystal structure of TcR has been available. Not surprisingly, TcR chains have folds as Fab. Overall structure is somewhat wider in dimension. A distinctive feature is that in Cα domain where half the domain not juxtraposed with Cβ domain has strands which are loosely packed and have a short α-helical segment.

The inter domain contacts are also different than in the case of Fab. Both extracellular domains in the two chains have higher interfacial contact. Interesting enough, Cα-Cβ domain interaction is assisted by carbohydrate on Cα H-bonded to Cβ domain.

Unlike the aptopes which are often formed because of discontinuous part of the polypeptide chain brought together in protein conformation, TcR recognizes short continuous sequence of amino acids.

Whereas epitopes recognized by cells are mostly on the antigen surface, the antigenic peptides recognized by TcR can often be the ones which are buried. This is become possible as T-cell recognizes these peptides on APC which have “processed” the antigen.

For example, T-cell is able to recognize a peptide from the „core‟ portion of HEW lysozyme molecule.

So, the epitopes recognized by T-cells do not necessarly lie on the protein surface. This implies that a T-cell is able to direct itself in large number of „epitopes‟ arising out of the antigen.

We have frequently referred to CD4 and CD8 T-cells. Let us now look at these coreceptors in little more detail. Originally, CD4 and CD8 were identified as surface markers for functionally different Tc and Th cells. It was much later that it was discovered that these markers play an important role in recognition of two different classes of MHC molecules (class I and II).

As we have discussed, CD4 recognizes MHC class II molecules and CD8 recognizes MHC class I molecules. The CD4/CD8 interaction with MHC molecule is required for T-cell response. This is part of how MHC restriction operates, Hence, CD4 and CD8 are called co-receptors.

CD4 is a single chain polypeptide with 4 domains similar to IG. However, there are some differences. D2 is not similar to either V or C domains of Ig and is sometime called C2 domain. MHC class II molecules bind via D1+D2 domains.

CD8 on the other hand has two chains α and β which are linked by an interchain –S-S-bonds. CD8 isoform with two α-chains also exist. even CD8α and CD8β chains are not very different. Both have a domain similar to V domain and extended chain which anchors these to the T-cell membrane.

CD4 and CD8 as coreceptors are estimated to enhance T-cell response to antigen carrying APC by about 100-fold.

On their own, the binding of CD4 and CD8 with MHC molecules is not strong. Both, through their cytoplasmic portion interact with a protein tyrosine kinase and bring it closer to TcR cytoplasmic tail mediated signal transduction molecules.

The similarity between TcR and immunoglobulin receptor on B-cells has been pointed out. T-cells are also clonally selected in response to the presence of antigen. In case of T-cells, what they see is the complex of antigenic peptide: MHC complex on the surface of APC.

TcR is also thus needed to be in variety of forms. It turns out the inherent mechanism by which TcR diversity is created are quite similar to the corresponding mechanism which generate antibody or receptor from the antibody.

The α and β chains have already been described as consisting of a variable region (V) and a constant region (C). the TcR α gene locus is similar to L chains of IgG, with Vα and Jα gene segments. This is present on chromosome 14.

The TcRβ gene locus is similar to H-chains of Ig and consist of Vβ, Jβ and D gene segments. Present on chromosome 7, it is has a cluster of 52 functional gene segments and followed by two separate clusters where each cluster has one D gene segment, 6-7 J gene segments and a single C gene.

The TcRα gene locus has about 70-80 Vα, 61 J gene segments followed by a single C gene segment. The TcRα gene locus has a TcRδ locus in between J and V gene segments.

Somatic recombination during development of T-cells creates diversity of TcR and involves formation of functional α and β chains.

Α-chain formation involves a Vα gene segment combining with one of the J gene segment to creates complete V exon. Transcription and splicing of this exon to Cα produces the mRNA for α chain Similarly, during β-chain formation, recombination of Vβ, Dβ and Jβ produces V exon which with transcription and splicing lead to mRNA for β-chain. The formation of α:β heterodimer follows.

There are, thus, similarities in the way Ig and TcR produced in diverse forms. Both have P and N- nucleotides at the junction of V, D, and J gene segments. While in TcR α, P and N nucleotides are there between V and J gene segments, in Ig only half of L chains have addition of N and no P is present.

P nucleotides have palindromic segments present at the end of the gene segments. N nucleotides are nontemplate coded and are added by terminal deoxynucleotidyl transferase to a single strand of the coding DNA.

In TcR, C gene segment codes for the anchoring transmembrane part of the chains.

Hence, there is not much diversity and only one gene segment is there. Thus is unlikely Ig where C of H-chain has many segments as it is involved in effecter functions.

There is a difference between the variability expected of a B-cell receptor and a TcR. B-cell recognises the epitopes which often vary throughout their sequence/structure. TcR recognises variability in the processed peptide predominantly.

In TcR, equivalent of the CDR3 regions (of Ab) is contributed by D and J gene segments. This is centre of the binding site for the antigenic peptide. The peripheralof TcR (equivalent to CDR1 and CDR2 loops of Ab) are encoded by the V gene segments of the α and β chains.

Unlike B-cells, T-cells do not undergo hypermutation of V region. So, TcR region corresponding to CDR1 and CDR2 are less variable. That is functionally relevant as these regions recognise less variable MHC component of the MHC peptide complex.

The structural information about TcR revels that structural diversity in these molecule is maximum in the junctions of V, D and J gene segments. As we have discussed, P- and N- nucleotides present at these junctions contribute to these diversity.

The number of J gene segments are far more in TcRα (as compared to BcR) and it codes for CDR3. Hence, much of the TcR variability is concentrated in its centre.

In the absence of hypermutation in T-cells, diversity in CDR1 and CDR2 is limited by germline V gene segments. Not involving hypermutation in diversity generation perhaps is a safeguard that a random mutation later on does not generate T-cells against self antigens.

In fact, thymic education eliminates T-cells which show high affinity towards self antigens. Hence, any random mutation to enhance thus affinity will undo the crucial preventive measure enforced by thymic education.

In case of B-cells, even if hypermutation does lead to limited recognition of self antigen absence of Th cells with same specificity will result in poor B-cell response.

T-cells already, by being MHC restricted, have the capacity of recognising.the self antigen. Also, B-cells show affinity maturation which is necessary for capturing toxins in extracellular fluids with high affinity.

So, hypermutation for B-cells is a case of adaptive specialization. we will shortly discuss Tregs involved in tolerance.

Alloreactivity of T-cells

We have repeatedly mentioned that T-cells need to see a self marker in the form of MHC molecules. If that is so how come T-cells respond to foreign MHC molecules.

It seems that 1-10% of T-cells show this “alloreactivity”. So, T-cells during development may use self MHC to develop MHC- restriction but they are evolved to recognise MHC molecules in general.

There are few modes by which this “crossreactivity” may arise. The foreign peptide (+ nonself MHC molecules) is different on APC and then interaction dominates the T-cell response. In another mode, nonself MHC are so different. As MHC have high density on cell surface, thus MHC dominate binding of TcR triggers swift Graft rejection.

The common lymphoid progenitor differentiates into B-cell and Pre-T-cells in bone marrow itself. The precursor T-cells migrate to the thymus. It is here that TcR genes are rearranged in a process which includes thymic education.

T-cell precursors are enmeshed in the thymic stroma- the network of epithelial cells. The large number of “thymocytes” are formed. This is also the place where intrethymic dendritic cells are produced from haematoposite cells.

The interaction of thymocytes in turn induces the formation of reticular epithelial structure to further surround the thymocytes. In the thymus, cortex contains immature thymocytes and the medulla contains mature thymocytes, dendritic cells and macrophages.

In diGeorge‟s syndrome (in humans) and nude mutation (in mice which is accompanied by absence of hair), absence of proper thymus results in absence of any significant population of the T-lymphocytes.

Thymus is fully developed at birth.

The T-cell development in thymus is accompanies by presence of different surfaces markers. The differentiation into α:β and γ:δ T-cells happens early. Interactions with stroma (about a weak) results in CD2 expression but these are “double negative” thymocytes as these lack either CD4 or CD8. Some cells express NK 1.1+ receptor in addition to α:β TcR and are called NKT cells. NKT recognise CD1 rather than MHC molecules.

The β-chain on CD44 low CD25+ thymocytes pairs with α-chain to become pre T cell α. Proliferation and formation of double positive CD4 CD8 cells takes place. Most of these form α:β T-cells.

γ:δ cells

We had referred to switch of γ:β to γ:β T-cells as visualized by Tonegawa. The actual picture turned out to somewhat different. In addition to α, β, γ, a δ surface marker of T-cells was also identified.

Multiple gene code for α, β, γ, δ chains of TcR. In some cases of T-cells, the rearrangement to γ and δ genes occurs during the development. The number of Vγ and Vδ gene segments are fewer than Vα and Vβ.

Morphologically γ:δ T cells are granular like NK cells. The V gene segment in these cells has broader specificity and γ:δ cells recognise heat shock proteins, phospholipids and phosphoprotiens. These T-cells are not MHC restricted and can act as regulatory or cytotoxic depending upon where the subset is located.

In some ways γ:δ T cells resemble NKT cells and can be viewed as parts of both innate and adaptive immunity.

The last common precursor of α:βand γ:δ cells is a cell where in some TcR gene segment rearrangements have already taken place. This is indicated by evidence that α:β T-cells contain rearranged or more often out of frame γ-chain genes. Similarity, γ:δ T-cells are sometime found to have correct β-chain rearrangement.

It appears that there is a race to rearrange β, γ and δ loci rearrangement and if γ or δ wins, it become γ:δ T cell. most precursors successfully rearrange β-chain before γ or δ genes are formed. The β gene pairs up with PTα to produce β:PTα pre TcR. This commits the cell to becomes α:β T-cell. hence in away, Tonegawa‟s model was a brilliant hypothesis.

γ:δ T-cells differ in the pattern of expression of co-receptor molecules. Mature γ:δ T-cells goes into peripheral circulation after emerging out of thymus. Some γ:δ T-cells are found in nude mice.

In mice γ:δ T-cells reach epidermis and are wedged among the keratinocytes, their morphology here become like dendrites and hence these are called dendritic epidermal T-cells (dETCs)

The second organ to be populated is the reproductive tract. The subsequent production of γ:δ T-cells is not in burst but continuously at a rate much slower than α:β T cells. These use different V gene segmasnts then earlier γ:δ T-cells and also have extensive N-nuclotides. These populate peripheral lymphoid organs.

In humans, these are dETCs but γ:δ T-cells are present in reproductive and GI tracts.

NK T cells

These cells share some properties of both NK cells and T-cells. NKT cells express CD3 but respond to lipid and glycolipid antigen. CD1d, a non polymorphic molecule, instead of MHC constitute presentation motif. These are cytotoxic and produce wider spectrum of cytokines than NK cells.

NKT cells (alongwith γδT cells and Tregs) are present in the epithelial layer of endometrium. These cells increase in secretory phase of the menstrual cycle and are also a part of immune cell population after the second trimester of pregnancy.

Just like NK cells NKT cells play a role during microbial infection and against tumors. NKT cells, increase in number in old age but produce less IFNγ. With Both NK and NKT, the cytotoxicity decrease in old ages.

Tregs

Apart from Th and Tc cells, these is a third category of T-cells which are regulatory T-cells called Tregs. These are subset of CD4+ T-cells which express high level of CD25.

CD-25 is the α-chain of IL-2 receptor on T-cells. While both activated B and T cells also express CD25 but Tregs do not express several markers of these lymphocytes.

There are two types of Tregs: natural (Thymus derived) and induced Tregs. Natural Tregs are able to respond to self antigens and become effector suppressor T-cells. The latter type also evolve from naïve CD4+ T-cells upon exposure to the antigen. While effector T-cells are formed due to the presence of IL-2, IFN-γ and IL-4, presence of TGFβ converts CD4+ T-cells into induced Tregs which are effective T-suppressor cells.

Tregs also are MHC restricted but have a high affinity for MHC. Normally such T-cells undergo apoptosis but Tregs escape than. Tregs are anergic and contributed to peripheral tolerance. In experimental models, absence of Tregs is shown to cause wide range of autoimmune diseases.

Tregs are present in blood but constitute only 1-4% of total CD4+ T-cells. With age, this % goes down to 0.5-1.5% how Tregs suppress immune response is not completely understood.

One possible mechanism is that Tregs compete with Th and Tc cells for APC, another possible explanation is that Tregs induce apoptosis of Th and Tc cells. Tregs also express CTLA-4 which down regulates expression of CD80 and CD86 on APCs. Tregs also release IL-10 and TGFβ which affects APC role.

Tregs along with γδ T-cells and NKT cells regulate immune response. Tregs have invited attention in the context of allergy, autoimmune diseases and graft rejection.

In the T-cell story, the main proponents are its own receptors co-receptors and other surface ligands, presented antigen and MHC which are involved in the presentation of the antigen on APC or viral infected cells.

The next thing to look at in detail is how antigen is processed and presented by APC or infected cells.

Only after this, we will get a full picture about role of co-receptors and other surface ligands present on various kinds of T-cells.

Summary

• Discovery of nature of TcR
• Differences between TcR and BcR
• TcR of CD4 and CD8 T cells
• Tregs, γ:δ T cells and NKT cells