Altered positive selection due to corecognition of floppy peptide/MHC II conformers supports an integrative model of thymic selection

Abstract

Thymocytes bearing the E alpha 52-68/I-A(b) complex-specific 1H3.1 alpha beta T cell antigen receptor are positively selected in Ab-Ep [Ab-Ep transgenic, invariant chain (Ii)(-/-), I-A beta(b-/-)] mice, where I-A(b) molecules present only E alpha 52-68. Although Ii reintroduction led to deletion, I-A beta(b) reintroduction disrupted positive selection. T cell antigen receptor transgenic Ab-Ep I-A beta(b+) mice had a large thymus with an increased absolute number of CD4(+)CD8(+) cells and no overt signs of deletion. Unlike Ab-Ep Ii(+) antigen-presenting cells, Ab-Ep I-A beta(b+) antigen-presenting cells did not activate 1H3.1 T cells. However, their capacity to present E alpha 52-68 was intact. Thus, positive selection of 1H3.1 thymocytes on the tight compact E alpha 52-68/I-A(b) complex is neutralized by the corecognition of loose compact self-peptide/I-A(b) conformers that do not interfere with the cognate activation of mature 1H3.1 T cells. The data support the notion that the integration of distinct signals generated by the simultaneous recognition of multiple self-peptide/MHC complexes directs intrathymic selection of T cells.

Figure 1

Ii reintroduction in TCR Ab-Ep mice converts positive into negative intrathymic selection. Analysis of thymocytes (A) and splenocytes (B) from TCR Ab-Ep and TCR Ab-Ep Ii⁺ mice. (Center) The Vβ6 histograms. (Left and Right) The CD4/CD8 distribution with and without gating on Vβ6^high cells. Quadrant statistics are indicated. In this analysis, the cellularity was: TCR Ab-Ep, 75 × 10⁶; TCR Ab-Ep Ii⁺, 3.8 × 10⁶. The deletion pattern is representative of 12 TCR Tg Ab-Ep Tg Ii⁺ mice analyzed.

Figure 2

I-Aβ^b reintroduction in TCR Ab-Ep mice disrupts intrathymic positive selection. Shown is analysis of TCR Ab-Ep (positive selection), TCR Ab-Ep I-Aβ^b+ (altered positive selection), TCR I-Aβ^b−/− (neglect), TCR I-Aβ^b+ Ii^−/− (deficient positive selection), and TCR Ab-Ep I-Aβ^b+ Ii⁺ (deletion) thymocytes. Data are representative of 6, 10, 5, 7, and 3 mice, respectively. The cell counts were: TCR Ab-Ep, 65 × 10⁶; TCR Ab-Ep I-Aβ^b+, 62.5 × 10⁶; TCR I-Aβ^b−/− Ii^−/−, 180 × 10⁶; TCR I-Aβ^b+ Ii^−/−, 145 × 10⁶; TCR Ab-Ep I-Aβ^b+ Ii⁺, 5.2 × 10⁶. Note that those numbers translate into an increased absolute number of CD4⁺CD8⁺ cells (≈50%) in TCR Ab-Ep I-Aβ^b+ mice relative to TCR Ab-Ep mice.

Figure 3

Arrest of thymocyte maturation at the CD4⁺CD8⁺ stage in TCR Ab-Ep I-Aβ^b+ mice. (A) Analysis of TCR Ab-Ep and TCR Ab-Ep I-Aβ^b+ splenocytes. (B) CD69 expression by and FSC of CD4⁺CD8⁺ thymocytes from TCR Ab-Ep (positive selection), TCR Ab-Ep I-Aβ^b+ (altered positive selection), TCR I-Aβ^b−/− (neglect), and TCR I-Aβ^b+ Ii^−/− (deficient positive selection) mice. Cell counts were: 74.5 × 10⁶, 158 × 10⁶, 76 × 10⁶, and 112 × 10⁶, respectively. In this analysis, the increase in the absolute number of CD4⁺CD8⁺ in Ab-Ep I-Aβ^b+ mice relative to TCR Ab-Ep mice was ≈40%. Note the lack of CD4^highCD8^high cells in the TCR Ab-Ep I-Aβ^b+ thymus. Mean fluorescence values for CD69 expression and percent of enlarged cells are indicated. Data are representative of three experiments. (C) Apoptosis among CD4⁺CD8⁺ thymocytes from TCR Ab-Ep (positive selection), TCR I-Aβ^b−/− (neglect), TCR Ab-Ep I-Aβ^b+ (altered positive selection), and TCR Ab-Ep Ii⁺ (deletion) mice. Annexin V log fluorescence was plotted as histograms after gating on CD4⁺CD8⁺ cells.

Figure 4

Ab-Ep Ii⁺, but not Ab-Ep I-Aβ^b+, APCs specifically stimulate 1H3.1 T cells. (A) Proliferative response of 1H3.1 TCR Tg Rag-1^−/− T cells to Ab-Ep, Ab-Ep I-Aβ^b+ (Ab-Ep I-Ab+), and Ab-Ep Ii^± (Ab-Ep Ii+) irradiated splenocytes. 5R cells were used as positive control. (B) 1H3.1 T cell reactivity to Ab-Ep Ii⁺ APCs is inhibited by Y3JP and Y-Ae but not by Y17 or 25.9.17 mAbs. (C) The original 1H3.1 hybridoma recapitulates the response of 1H3.1 TCR Tg T cells to Ab-Ep, Ab-Ep I-Aβ^b+, and Ab-Ep Ii^± APCs. The values represent the mean ± SD of duplicate microcultures. The data are representative of four (A), two (B), and three (C) experiments.

Figure 5

Surface expression of loose compact (25.9.17⁺) self-peptide/I-A^b complexes by MHC class II⁺ cells and their capacity to interfere with positive selection of 1H3.1 thymocytes under the Ab-EP Tg I-Aβ^b+ conditions in vivo. (A) Analysis of B220⁺ splenocytes from B6 (I-A^bu/I-Eα⁻), Ab-EP, Ab-EP Ii, and Ab-EP I-A^bβ⁺ mice for surface expression of the Y-Ae, Y3JP, and 25.9.17 epitopes. Control histograms (open) were obtained by using anti-I-E (Y17 or 14.4.4S) mAb. (B) Y-Ae epitope expression by 2′-deoxyguanosine-resistant Ab-Ep and Ab-Ep I-A^b+ thymic cells. (C) Thymocytes from 12- to 15-day-old TCR Ab-Ep I-Aβ^b+ mice treated with 25.9.17 versus the isotype-matched anti-I-E 14.4.4S mAb. The data are representative of three experiments. (D) CD4⁺CD8⁻ thymocytes emerging on 25.9.17 infusion display a mature phenotype. Vβ6 and CD24/HSA expression levels (mean fluorescence indicated) as well as the expression of Vα (2, 3.2, 8.3, 11.1/.2) (percent of positive cells indicated) were plotted after gating on CD4⁺CD8⁻ cells. For comparison, the CD24/HSA fluorescence intensity on CD4⁺CD8⁺ cells was 2,014 in TCR Ab-Ep mice and 2,102 in 25.9.17-treated TCR Ab-Ep I-Aβ^b+ mice.