Structural and functional characterization of T and NK cell receptors

National Institute Of Allergy And Infectious Diseases

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Abstract

We have established an animal model system to investigate allogenic T cells. We use mix-lymphocyte reactions (MLR) to evaluate allogenic activities between B6 and kbm1 (a B6-related strain that contains three amino acid mutations in the alpha 2 helix region of H2-Kb) mice. In this model system, T cells do not proliferate on spleen cells (as antigen presenting cells) from syngeneic animals, but proliferate on allogenic spleen cells. Indeed, CD8 T cells from B6 mice did not proliferate on B6 spleen cells, but proliferated on kbm1 spleen cells and vice versa. We propose to define allogenic T cell repertoires in both B6 and kbm1, and by doing so, we hope to define the structural and molecular mechanism of allogenic T cells, and provide insight to what triggers T cell allogenic proliferation. MHC-restriction is a feature of antigen recognition unique to T cell receptors (TCR). It is a basis for immune surveillance and self-tolerance, namely activation against foreign and tumor antigens but avoid autoimmunity. Because of its critical importance, it is important to investigate how thymus identifies MHC-restricted TCRs from a diverse pool of randomly generated TCRs. Through comparative sequence analyses of pre- and post-selection TCRs, we found that MHC-restricted and MHC-independent TCRs are primarily distinguished by their hyper-variable non-germline CDR3 segments rather than germline-encoded segments. We showed that thymic selection of MHC-independent TCRs from pre-selection sequences is largely unconstrained, whereas thymic selection of MHC-restricted TCRs is strictly constrained by both CDR3 length and CDR3 specific amino acid usage, with thymic selection of MHC-restricted TCR disfavoring cysteine-containing CDR3 or longer CDR3 with more than 13 amino acids. Our findings provided for the first time a molecular basis between MHC-restricted and MHC-independent TCRs and identified hallmarks of MHC-restricted TCR repertoires. T cells mature through positive and negative selections to gain ability to recognize foreign antigens and to tolerate self. There is, however, no physical parameter to measure the outcome of a thymic selection other than by default that all circulating T cells are positively selected. Unlike antibodies that undergo affinity maturation through somatic hypermutation resulting in amino acid changes in antigen binding sites, T cell receptors maintain their antigen binding sequences throughout maturation. Our research revealed a new measure for individual T cell maturation using their repertoire frequencies. We showed that T cell receptor frequencies correlated with MHC-genotypes and depended on MHC-I expressions. The absence of MHC-I expression in b2m-/- animals resulted in the loss of CD8 TCR repertoire frequency correlations among b2m-/- animals. However, their frequencies can be rescued by maturation of b2m-/- precursors in MHC-I sufficient host. We showed that CD8 T cells developed their frequencies during thymic maturation. Analyses of common TCR sequences in all maturation stages allowed the tracking of frequencies on individual sequences throughout their development, thus, provided an unprecedented view of TCR development at the sequence level. Assuming both CD4 and CD8 ligands (MHC-II and MHC-I) are engaged in DP thymocytes and only MHC-I is selected in CD8 SP thymocytes, some sequences showed characteristic of favorable binding to MHC-I ligands as evidenced from their frequency increases from DP to SP when CD4 expression is down regulated. Majority of TCR sequences appear to exhibit non-optimal binding to either MHC-I or II ligands, thus remained low frequencies throughout their thymic development. We conclude that TCR frequencies are developed during thymic maturation and they bear imprints of thymic selection on individual receptor sequences.