Differential thymic selection outcomes stimulated by focal structural alteration in peptide/major histocompatibility complex ligands

Abstract

The T lineage repertoire is shaped by T cell receptor (TCR)-dependent positive and negative thymic selection processes. Using TCR-transgenic (N15tg) beta2-microglobulin-deficient (beta2m-/-) RAG-2(-/-) H-2(b) mice specific for the VSV8 (RGYVYQGL) octapeptide bound to Kb, we identified a single weak agonist peptide variant V4L (L4) inducing phenotypic and functional T cell maturation. The cognate VSV8 peptide, in contrast, triggers negative selection. The crystal structure of L4/Kb was determined and refined to 2.1 A for comparison with the VSV8/Kb structure at similar resolution. Aside from changes on the p4 side chain of L4 and the resulting alteration of the exposed Kb Lys-66 side chain, these two structures are essentially identical. Hence, a given TCR recognizes subtle distinctions between highly related ligands, resulting in dramatically different selection outcomes. Based on these finding and the recent structural elucidation of the N15-VSV8/Kb complex, moreover, it appears that the germ-line Valpha repertoire contributes in a significant way to positive selection.

Figure 1

The L4 peptide induces positive selection of N15tg thymocytes. FTOC was performed by using N15tg/RAG-2^−/−/β₂m^−/− (H-2^b) thymic lobes in media containing 5 μg/ml human β₂m with or without 10 μM of the indicated peptides. After 7 days, thymocytes were released from the lobes by pressing through a steel mesh, counted, and triple-stained with PE-conjugated anti-CD4, Red613-conjugated anti-CD8, and mAb R53 (anti-N15 β chain clonotype) plus FITC-conjugated anti-rat IgG (19). (A) The CD8 versus CD4 staining profile of total thymocytes is shown. In this representative experiment the yield of CD8⁺ SP cells after L4 incubation was 13.5 × 10⁴ cells compared with 0.3–2.2 × 10⁴ cells in FTOC incubated in the absence of exogenous peptides or in the presence of the other indicated peptides. The total thymocyte number recovered from VSV8-exposed FTOC was significantly lower (10⁵ cells per lobe) than from FTOC incubated with any of the other peptides (4–6 × 10⁵ cells per lobe). (B) The histograms of the N15 TCRβ chain expression on DP and CD8⁺ SP thymocytes derived from FTOC incubated with K1 or L4 peptides are shown. Note that the K1 histogram represents data similar to that obtained with the other peptides (except VSV8). The CD8⁺ SP thymocytes that mature on L4 express a higher level of the TCR than the DP thymocytes harvested from the same lobe. (C) Thymocytes selected on L4 are functionally responsive to VSV8. Thymocytes from the organ cultures described above [cultured with (+) or without (−) L4] or fresh splenocytes from N15tg/RAG-2^−/− mouse were assayed for their proliferative response to irradiated EL-4 cells, in the present of rIL-2 and 10 nM VSV8 or 10 μM L4 or no peptide. After 48 h, each well was pulsed for 18 h with [³H]thymidine, harvested on filter discs, and counted. The proliferative responses for the peptides are shown. Results are mean values of triplicate samples with SD noted.

Figure 2

L4/K^b electron density map in the region of the peptide. A portion of the final 2F_o − F_c electron density map calculated with the residue at the p4 position of the peptide omitted. The refined models of both the L4/K^b (yellow) and the VSV8/K^b (red) are superimposed on the map. The map shows how similar the L4/K^b and VSV8/K^b structures are, except for the labeled p4 and Lys-66 side chains of K^b. The figure was prepared by using the program setor (55).

Figure 3

Comparison of L4/K^b and VSV8/K^b structures reveals the p4 methylene extension and conformational change in the K^b Lys-66 side chain in L4/K^b. Shown here is the schematic representation of the α1+α2 domain of the K^b molecule with side chains of Lys-66 and Glu-63 as well as the two peptides. The color codes are: green for the two K^b helical regions and the side chain of Glu-63; red for the remaining Cα trace of the α1+α2 domains; white for VSV8 peptide and the Lys-66 side chain with which it interacts; and blue for the L4 peptide and the corresponding Lys-66 residue. The figure was prepared with setor (55). Note that the view is such that the TCR would dock roughly from the top of the figure and the K^b α3 and β₂m domain would be located at the bottom of the figure, i.e., below this MHC peptide-presenting platform.