Highly restricted T cell repertoire shaped by a single major histocompatibility complex-peptide ligand in the presence of a single rearranged T cell receptor beta chain

Abstract

The T cell repertoire is shaped by positive and negative selection of thymocytes through the interaction of alpha/beta-T cell receptors (TCR) with self-peptides bound to self-major histocompatibility complex (MHC) molecules. However, the involvement of specific TCR-peptide contacts in positive selection remains unclear. By fixing TCR-beta chains with a single rearranged TCR-beta irrelevant to the selecting ligand, we show here that T cells selected to mature on a single MHC-peptide complex express highly restricted TCR-alpha chains in terms of Valpha usage and amino acid residue of their CDR3 loops, whereas such restriction was not observed with those selected by the same MHC with diverse sets of self-peptides including this peptide. Thus, we visualized the TCR structure required to survive positive selection directed by this single ligand. Our findings provide definitive evidence that specific recognition of self-peptides by TCR could be involved in positive selection of thymocytes.

Figure 1

CD4⁺ T cell differentiation in B2L TKO and B2L DKO mice. (A) Thymocytes were prepared from six different lines (DKO, B2L DKO, B6, TKO, B2L TKO, and β2^0/0) at 6–7 wk old and analyzed for CD4 and CD8 expression. The percentages of CD4⁺CD8⁻, CD4⁺CD8^int, CD4⁺CD8⁺, and CD4⁻CD8⁺ thymocytes are indicated. The data represent at least three independent experiments. The mean ± one SD for the percentage of CD4⁺CD8⁻ and CD4⁺CD8^int thymocytes in each line are as follows: DKO, 0.7 ± 0.1% and 1.9 ± 0.1%; B2L DKO, 2.0 ± 0.4% and 2.5 ± 0.4%; B6, 7.2 ± 0.5% and 2.8 ± 0.3%; TKO, 0.4 ± 0.1% and 0.3 ± 0.0%; B2L TKO, 2.0 ± 0.3% and 0.4 ± 0.1%; β2^0/0, 8.3 ± 0.5% and 1.7 ± 0.0%. (B) The expression of NK1.1 on CD4⁺CD8⁻ thymocytes is compared between B2L DKO (left, dotted line) and B2L TKO (left, solid line) or between B6 (right, dotted line) or β2^0/0 (right, solid line). The percentage of CD4⁺CD8⁻NK1.1⁺ thymocytes in mice with or without MHC class I expression are indicated above or below the line, respectively. For each line, at least two mice were analyzed. The each value for the percentage of CD4⁺CD8⁻NK1.1⁺ thymocytes to total CD4⁺CD8⁻ thymocytes was as follows: B2L DKO, 11.3, 7.9, 8.9; B2L TKO, 0.4, 0.8, 0.7; B6, 1.6, 2.1; β2^0/0, 0.1, 0.1.

Figure 2

Vα usage (A) and CDR3 length distribution (B) of TCR-α repertoire shaped by the I-A^b–Eα52-68 complex. CD4⁺CD8⁻ thymocytes were sorted from B2L TKO or β2^0/0 mice, and TCR-α transcripts were analyzed using anchored PCR followed by sequencing of the cloned PCR products. The results are shown as the percentages of clones originating from different templates. The clones encoding undefined TCR Vα are indicated as U.D. in A. CDR3 length in (B) is indicated as the number of amino acid residues between two amino acids downstream from the conserved cysteine at position 90 in the V region and two amino acids upstream from the conserved GXG motif (where G is glycine and X is any amino acid) in the J region (64).

Figure 3

Phenotypic and functional analysis for CD4⁺ T cell differentiation directed by the I-A^b–Eα52-68 complex in the presence of the 2B4 TCR-β chain. (A) Thymocytes were prepared from TKO/2B4β, B2L TKO/2B4β, and β2^0/0/2B4β mice at 6–7 wk old and analyzed for CD4 and CD8 expression. The percentages of CD4⁺CD8⁻, CD4⁺CD8^int, CD4⁺CD8⁺, CD4⁻CD8⁺ thymocytes are indicated. For each line, at least three mice were analyzed. The each value for the percentage of CD4⁺CD8⁻ thymocytes was as follows: TKO/2B4β, 0.1, 0.1, 0.1; B2L TKO/2B4β, 1.3, 0.9, 0.7, 0.7; β2^0/0/2B4β, 2.6, 2.3, 2.7. (B) Lymph node CD4⁺ T cells from B2L TKO/2B4β or B2L TKO mice were cultured with irradiated spleen cells from B2H TKO, B6, and β2^0/0 mice, and [³H]thymidine incorporation was measured. The data indicate the mean plus one SD of triplicate cultures. (C) Thymocytes were prepared from B2L TKO/2B4β or β2^0/0/2B4β mice at 6–7 wk old, and the expression of TCR Vβ3 was analyzed for gated CD4⁺CD8⁻ thymocytes (top). The percentages of CD4⁺CD8⁻Vβ3⁻, CD4⁺CD8⁻Vβ3^int, CD4⁺CD8⁻Vβ3^hi thymocytes are indicated. For each line, four or three mice were analyzed. The each value for the percentage of CD4⁺ CD8⁻Vβ3^hi thymocytes was as follows: B2L TKO/2B4β, 64.7, 71.0, 65.2, 75.0; β2^0/0/2B4β, 69.3, 75.3, 70.8. The expression of TCR Vβ8 on CD4⁺CD8⁻Vβ3^hi, CD4⁺CD8⁻Vβ3^int, CD4⁺CD8⁻Vβ3⁻ thymocytes and the percentages of positive population are shown below.

Figure 4

Vα usage (A) and CDR3 length distribution (B) of TCR-α repertoire shaped by the I-A^b–Eα52-68 complex in the presence of 2B4 TCR-β chain. CD4⁺CD8⁻Vβ3^hi thymocytes were sorted from B2L TKO/ 2B4β, β2^0/0/2B4β, or Eα-B6/ 2B4β mice, and TCR-α transcripts were analyzed, as described for Fig. 2. The results are shown as percentages of clones originating from different templates. The closed and hatched boxes represent clones obtained from independent experiments using different mice. The clones encoding undefined Vα are indicated as U.D. in A.

Figure 5

Predicted amino acid sequence of Vα17.A2-encoding TCR-α chains. (A) The NH₂-terminal structure of Vα17.A2 from cysteine at position 90 is compared with that of AV18S2. CDR1 and CDR2 are boxed. (B) The clones originating from different templates were analyzed for CDR3 loops and Jα gene segments.