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TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8+ T cells

Abstract

In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8+ T cells is fine tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on major histocompatibility complex class I–associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8+ T cells results in aberrant TCR responsiveness, homeostatic maintenance and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8+ T cells in the periphery.

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Figure 1: TCR responsiveness is impaired in monoclonal TCR-expressing T cells generated in β5t-deficient thymus.
Figure 2: Impaired proximal TCR signaling events in T cells generated in the β5t-deficient (Psmb11−/−) thymus.
Figure 3: β5t-dependent positive selection optimizes the peripheral maintenance of T cells.
Figure 4: β5t-dependent positive selection affects the immune response to infection.
Figure 5: In β5t-deficient mice, thymocytes are positively selected through reduced TCR signals.
Figure 6: Positively selecting peptides precondition antigen responsiveness in monoclonal T cells.

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Acknowledgements

We thank H. Kosako, I. Ohigashi, M. Kozai and B. Kim for helpful discussion and reading the manuscript. This work was supported by grants from MEXT-JSPS (24111004 and 23249025 to Y.T. and 24790475 and 26460576 to K. Takada), Naito Foundation (to Y.T. and K. Takada), Takeda Science Foundation (to K. Takada), and Uehara Memorial Foundation (to K. Takada).

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Contributions

K. Takada and Y.T. conceived the study. K. Takada, S.C.J., A.S. and Y.T. designed the experiments. K. Takada, F.V.L., Y.X., K.A., H.S., S.M. and K. Tanaka performed the experiments. K. Takada, S.C.J., A.S. and Y.T. wrote the manuscript.

Corresponding author

Correspondence to Yousuke Takahama.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 TCR responsiveness of monoclonal T cells generated in the β5t-deficient thymus.

CD44loCD8+ OT-I-TCR-transgenic T cells generated in control or β5t-deficient mice were stimulated with OVAp–H-2Kb tetramers. Numbers in histograms indicate mean fluorescence intensity (MFI) of CD69 in CD69+ cells. Representative data from four independent experiments are shown. Bars indicate average ± standard errors of the mean. *P < 0.05.

Supplementary Figure 2 Impaired Erk phosphorylation in T cells generated in the β5t-deficient thymus.

ERK1 and 2 phosphorylation in response to OVAp–H-2Kb tetramer stimulation was examined in CD44loCD8+ OT-I-TCR-transgenic T cells at indicated time. Representative data from two independent experiments are shown.

Supplementary Figure 3 Peripheral T cells generated in a β5t-deficient thymus are abnormal.

(a) Frequencies of CD44loCD122lo and CD44hiCD122hi cells in CD8+ T cells from the spleens and lymph nodes of β5t-deficient Rag2-deficient OT-I-TCR-transgenic mice. (b) CD44 and CD122 expression in CD4CD8+Vα2hi mature OT-I-TCR-transgenic thymocytes. (c) Splenic CD8+ T cells in β5t-deficient mice at different ages were analyzed for the ratios of CD44lo to CD44hi cells. (d) Absolute numbers of naïve (CD44loCD122lo) and memory-like (CD44hiCD122hi) CD8+ T cells in polyclonal TCR-expressing β5t-deficient mice at different ages. (e) Thymocytes from β5t-deficient and control mice were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and adoptively transferred to sublethally irradiated (6.0 Gy) B6-Ly5.1 mice (1×106 SP thymocytes/recipient). CD45.2+ donor cells in recipient spleens were analyzed for CFSE dilution as well as CD4, CD8, and CD44 expression 14 days after transfer. (f) Representative histograms of CD8+ T cells bound to gp33–H-2Db tetramers in the experiments shown in Fig. 3c. Accumulated results from more than six (a and b) or three (c and d) mice/group and representative data from two (e) or three (f) independent experiments are shown. Graphs show data of individual mice (circles) and means (bars) (a and b). Circles and bars indicate average ± standard errors of the mean (c-e). ***P < 0.001, **P < 0.01 *P < 0.05.

Supplementary Figure 4 Increased frequency of memory-like cells in CD8+ T cell compartment.

CD44 and CD122 expression in T cells from the spleens and lymph nodes of β5t-deficient mice were analyzed. Graphs show data of individual mice (circles) and means (bars). Accumulated results from 5-6 mice per group. *P < 0.001.

Supplementary Figure 5 β5t-dependent positive selection affects monoclonal T cell responses to infection.

(a) Graphical scheme of LM-OVA infection experiments. β5t-dependent or -independent OT-I-TCR-transgenic T cells were obtained from bone marrow chimeras (Rag1−/− OT-I > Psmb11+/+ or Rag1−/− OT-I > Psmb11−/−). Bone marrow chimera-derived OT-I-TCR-transgenic T cells (CD45.2+CD90.2+) were co-transferred with normal OT-I T cells (CD45.2+CD90.1+) into B6-Ly5.1 mice (CD45.1+CD45.2). Blood lymphocytes were analyzed 5 and 8 days following LM-OVA infection. (b) Expression of CD44 and KLRG1 in donor OT-I T cells was examined 8 days after infection in the experiments shown in Fig. 4. Graphs show data of individual mice (circles) and means (bars). Cumulative data from two independent experiments. *P < 0.001.

Supplementary Figure 6 Analysis of thymocyte development according to CD69 and CCR7 expression.

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Takada, K., Van Laethem, F., Xing, Y. et al. TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8+ T cells. Nat Immunol 16, 1069–1076 (2015). https://doi.org/10.1038/ni.3237

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