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Timing and duration of MHC I positive selection signals are adjusted in the thymus to prevent lineage errors

Nature Immunology volume 17, pages 1415–1423 (2016)Cite this article

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Abstract

Major histocompatibility complex class I (MHC I) positive selection of CD8+ T cells in the thymus requires that T cell antigen receptor (TCR) signaling end in time for cytokines to induce Runx3d, the CD8-lineage transcription factor. We examined the time required for these events and found that the overall duration of positive selection was similar for all CD8+ thymocytes in mice, despite markedly different TCR signaling times. Notably, prolonged TCR signaling times were counter-balanced by accelerated Runx3d induction by cytokines and accelerated differentiation into CD8+ T cells. Consequently, lineage errors did not occur except when MHC I–TCR signaling was so prolonged that the CD4-lineage-specifying transcription factor ThPOK was expressed, preventing Runx3d induction. Thus, our results identify a compensatory signaling mechanism that prevents lineage-fate errors by dynamically modulating Runx3d induction rates during MHC I positive selection.

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Figure 1: Developmental progression during MHC I positive selection.
Figure 2: Runx3d induction during MHC class I positive selection.
Figure 3: Effect of TCR-CD8 signaling on phase 1 duration.
Figure 4: Duration time of phase 1, phase 2 and overall positive selection.
Figure 5: Phase 1 duration affects cytokine-response potential and Runx3d induction rate.
Figure 6: Prolongation of phase 1 results in lineage errors.
Figure 7: Time limit for error-free CD8 lineage specification.

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Acknowledgements

We thank H. Park, D. Singer and N. Taylor for critical reading of the manuscript; M. Nussenzweig (Rockefeller University) for Rag2GFPTg mice; D. Littman (New York University) for Runx3dYFP knock-in reporter mice; R. Bosselut (National Cancer Institute) for ThpokGFP mice; M. Kubo (Tokyo University of Science, Japan) for SOCS1Tg mice; and S. Sharrow and L. Granger for flow cytometry. Supported by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Center for Cancer Research, and the Ministry of Education, Culture, Science and Technology (Grant-in-Aid for Research Activity Start-up 26893033; and Scientific Research (C) 15K08524).

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  1. Motoko Y Kimura

    Present address: Present address: Department of Immunology, Chiba University, Chiba, Japan.,

Authors and Affiliations

  1. Experimental Immunology Branch, National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA

    Motoko Y Kimura, Julien Thomas, Xuguang Tai, Terry I Guinter, Miho Shinzawa, Ruth Etzensperger & Alfred Singer

  2. Laboratory of Mammalian Genes and Development, Eunice Kennedy Schriver National Institute of Child Health and Human Development, US National Institutes of Health, Bethesda, Maryland, USA

    Zhenhu Li & Paul Love

  3. Department of Immunology, Chiba University, Chiba, Japan

    Toshinori Nakayama

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Contributions

M.Y.K. designed the study, performed experiments, analyzed data and contributed to the writing the manuscript. J.T., X.T., T.I.G., M.S., R.E., Z.L., P.L. and T.N. performed experiments, analyzed data and provided helpful discussions. A.S. designed the study, analyzed data and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Thymocyte differentiation defined by expression of CD69 and CCR7.

Frequency of MHCII−/−Rag2GFP thymocytes from Fig. 1a at stages 1-6 defined by CD69 and CCR7 expression (top left). TCRβ expression (top right) and CD4–CD8 expression (bottom) of the same cells are also shown. Numbers in two color contour plots depict the frequency of cells within each gate.

Supplementary Figure 2 Runx3d expression during MHC I positive selection.

Runx3d-YFP expression in CD69+DP, IM1, IM2, IM3, and SP8 thymocytes from OT-I Rag2−/− Runx3dYFP mice is displayed (left panels). CCR7 expression on gated Runx3d-YFP− and Runx3d-YFP+ cells is also displayed (right panels).

Supplementary Figure 3 TCR-ligand affinity affects phase 1 duration.

(a) Profiles of CD5 expression on just-signaled CD69+CCR7− DP thymocytes from monoclonal TCR transgenic mice (dark line) and polyclonal B6 mice (shaded curve). (b) Schematic models of MHC class I positive selection of thymocytes bearing relatively low (left) or high (right) affinity TCR to intra-thymic self-ligands.

Supplementary Figure 4 Effect of TCR signaling duration on cytokine-response potential.

Schematic model illustrating decreasing SOCS1 and increasing IL-7Rα expression with time of MHC class I TCR signaling. Thymocytes with greater cytokine response potential are more strongly signaled by cytokines which induce greater amounts of Runx3d.

Supplementary Figure 5 Lineage errors during MHC I positive selection.

(a) Stronger signaling CD8.4 coreceptors induce ThPOK expression during positive selection of OT-I thymocyes. Thpok-GFP expression was quantified in pre-selection DP and IM (CD4+CD8loCD69+) thymocytes (left), and in CD4 LNT cells (right). (b) Comparison of the number (top) and frequency (bottom) of CD4 LNT cells in OT-I mice expressing CD8wt or CD8.4 coreceptors. (c) CD69 expression on CD4 and CD8 LNT cells stimulated overnight with either medium or immobilized anti-TCR+anti-CD28 antibodies. (d) TCR-coreceptor mismatching decreases signaling and survival of peripheral OT-I CD4 T cells. CD5 expression on OT-I CD4 and CD8 LNT cells (left). Recent thymic emigrants (Rag2-GFP+ cells) are 4-times more frequent among OT-I CD4 mismatched LNT cells than OT-I CD8 matched LNT cells (right). (e) CD4 lineage errors upon CD5 deletion. Peripheral OT-I CD4 T cell frequencies in MHCII−/− chimeric host mice whose OTI T cells are CD5-sufficient or CD5-deficient. * P<0.05 and **P<0.001.

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Kimura, M., Thomas, J., Tai, X. et al. Timing and duration of MHC I positive selection signals are adjusted in the thymus to prevent lineage errors. Nat Immunol 17, 1415–1423 (2016). https://doi.org/10.1038/ni.3560

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