Germline-encoded amino acids in the alphabeta T-cell receptor control thymic selection

Abstract

An alphabeta T-cell response depends on the recognition of antigen plus major histocompatibility complex (MHC) proteins by its antigen receptor (TCR). The ability of peripheral alphabeta T cells to recognize MHC is at least partly determined by MHC-dependent thymic selection, by which an immature T cell survives only if its TCR can recognize self MHC. This process may allow MHC-reactive TCRs to be selected from a repertoire with completely random and unbiased specificities. However, analysis of thymocytes before positive selection indicated that TCR proteins might have a predetermined ability to bind MHC. Here we show that specific germline-encoded amino acids in the TCR promote 'generic' MHC recognition and control thymic selection. In mice expressing single, rearranged TCR beta-chains, individual mutation of amino acids in the complementarity-determining region (CDR) 2beta to Ala reduced development of the entire TCR repertoire. Altogether, these results show that thymic selection is controlled by germline-encoded MHC contact points in the alphabeta TCR and indicate that the diversity of the peripheral T-cell repertoire is enhanced by this 'built-in' specificity.

Figure 1

Vβ8.2 amino acids Y46, Y48 and E54 are required for TCR recognition of specific MHC/peptide and allo-MHC complexes.a. IL-2 response of 5KCα-β-hybridomas transduced with retroviruses encoding WT DO-11.10 TCRα plus WT DOβ or Y46A, Y48A, and E54A mutant DOβ after stimulation with 0.4 µg/ml OVA323-339 peptide plus IA^b-expressing Chb-2.4.4 cells or IA^d-expressing A20.2J cells, no peptide with IA^b-expressing Chb-2.4.4 cells, or 1.5 µg/ml plate bound anti-TCRβ (H57-597).b,c. IL-2 response of 5KCα-β- hybridomas transduced with retroviruses encoding WT 2W1S-20.4 TCRα chain (b) or WT 75-55 TCRα chain (c), plus appropriate partner WT, Y46A, Y48A, and E54A TCRβ chain after stimulation with fibroblasts expressing IA^b with linked 3K peptide, or splenocytes expressing H2-b (C57BL/6), bm12 (B6.C-H2^bm12), d (BALB/c), f (B10.M), q (B10.D1), r (B10.RIII), s (B10.S), u (B10.PL), left unstimulated (No APC), or activated with 10 µg/ml plate-bound anti-TCRβ (H57-597). Data are mean of three independent experiments (a) or two independent experiments (b, c).

Figure 2

Vβ8.2 amino acids Y46, Y48, and E54 promote efficient thymic selection.a. Schematic illustrating the generation of retroviral bone marrow chimeras.b. Thymic cellularity of bone marrow chimeras expressing WT, Y46A, Y48A, or E54A DOβ analyzed between d29 and d34 post reconstitution. Data are mean plus s.e.m. and are cumulative from 3 independent experiments for WT (n=10) and 2 independent experiments for Y46A (n=5), Y48A (n=4), and E54A (n=6) with TCRβ−/−TCRδ−/− donor bone marrow.c,d,e. FACS analysis of CD4 and CD8 staining (c), CD5 and CD69 staining (d), and HSA (CD24) and TCRβ staining (e) of GFP+ thymocytes from chimeras expressing WT, Y46A, Y48A, or E54A DOβ analyzed between d29 and d34 post reconstitution. Representative FACS plots are shown from 3 independent experiments for WT and 2 independent experiments for Y46A, Y48A, and E54A.f. Absolute number of total mature (HSA^loTCRβ^hi) thymocytes (left panel), CD4SP mature thymocytes (middle panel), and CD8SP mature thymocytes (right panel) for chimeras expressing WT, Y46A, Y48A, or E54A DOβ analyzed between d29 and d34 post reconstitution. Data are mean plus s.e.m. and are cumulative from 3 independent experiments for WT (n=10) and 2 independent experiments for Y46A (n=5), Y48A (n=4), and E54A (n=6); (*, p<0.05;**, p<0.01; ***,P<0.001; NS, not significant).

Figure 3

Vβ6 Y46 promotes MHC recognition and thymic selection.a. IL-2 response of 5KCα-β- hybridomas transduced with retroviruses encoding TEa TCRα plus WT TEaβ (filled symbols) or Y46A TEaβ (open symbols) after stimulation with the indicated concentration of Eα52–68 peptide plus IA^b-expressing Chb-2.4.4 cells (left panel) or 10 µg/ml plate bound anti-TCRβ (right panel). Data are representative of two independent experiments.b. Thymic cellularity of bone marrow chimeras expressing WT or Y46A TCliβ analyzed between d25 and d33 post reconstitution.c. FACS analysis of CD4 and CD8 staining, CD5 and CD69 staining and HSA (CD24) and TCRβ staining of GFP+ thymocytes from chimeras expressing WT or Y46A TCliβ analyzed between d25 and d33 post reconstitution. Representative FACS plots are shown from 3 independent experiments.d. Absolute number of total mature (HSA^loTCRβ^hi) thymocytes (left panel), CD4SP mature thymocytes (middle panel), and CD8SP mature thymocytes (right panel) for chimeras expressing WT or Y46A Tcliβ analyzed between d25 and d33 post reconstitution. For (b) and (d), data points are shown for each mouse, with mean plus s.e.m., from 2 independent experiments for WT (n=3) and Y46A (n=2) with TCRβ−/− donor bone marrow (squares) and 1 experiment for WT (n=3) and Y46A (n=3) with TCRβ−/−TCRδ−/− donor bone marrow (circles); (**, p<0.01; NS, not significant).

Figure 4

Altered TCRα repertoire in chimeras expressing mutant TCRβ chains. a,b. Frequency of Vα2 and Vα8.3 expression on LN CD4+ T cells (a) or LN CD8+ T cells (b) from chimeras expressing WT, Y46A, Y48A, or E54A DOβ (white bars) and WT or Y46A TCliβ (grey bars) analyzed between d25 and d34 post reconstitution. For Vβ8.2chimeras, data are mean plus s.e.m. from 3 independent experiments for WT (n=10) and 2 independent experiments for Y46A (n=5), Y48A (n=4), and E54A (n=6). For Vβ6 chimeras, data are mean plus s.e.m. from a representative experiment for WT (n=3) and Y46A (n=3) from TCRβ−/− donors.c. IL-2 response of 5KCα-β-hybridomas transduced with retroviruses encoding Y48A DOβ (left panel) or WT DOβ (right panel) plus DO-11.10 TCRα (filled symbols) or S93A TCRα (open symbols) after stimulation with the indicated concentration of OVA323-339 peptide plus IA^b-expressing Chb-2.4.4 cells. Data are mean of 2 independent experiments.