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T cell receptor and cytokine signal integration in CD8+ T cells is mediated by the protein Themis

Nature Immunology volume 21, pages 186–198 (2020)Cite this article

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Abstract

T cell homeostasis and functional responsiveness require signals from self-peptide–major histocompatibility complex (self-pMHC) and cytokines, but the mechanisms controlling this signal integration are unknown. Using a conditional deletion of the T cell lineage-specific protein Themis, we show that Themis is required for the maintenance of peripheral CD8+ T cells and for proliferative CD8+ T cell responses to low-affinity pMHC aided by cytokines. Themis-deficient peripheral T cells show a phenotype indicative of reduced tonic signaling from self-pMHC, strongly suggesting that Themis is a positive regulator of T cell receptor signal strength in response to low-affinity self-pMHC in peripheral T cells. Signals from low-affinity pMHC and cytokines synergistically induce phosphorylation of the kinase Akt, metabolic changes and c-Myc transcription factor induction in CD8+ T cells only in the presence of Themis. This function of Themis is mediated through Shp1 phosphatase, as peripheral Themis and Shp1 double deletion rescues the peripheral CD8+ T cell maintenance.

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Fig. 1: Post-selection deletion of Themis reduces the peripheral CD8+ T cell compartment.
Fig. 2: Post-selection deletion of Themis reduces tonic signaling in OT-I CD8+ T cells, but does not alter responses to agonistic pMHC in vitro.
Fig. 3: Themis is required for LIP in vivo.
Fig. 4: Themis is required for proliferative synergy of low-affinity pMHC and cytokines.
Fig. 5: Themis-deficient OT-I CD8+ T cells show reduced Akt signaling and c-Myc upregulation in response to stimulation with low-affinity pMHC and cytokines.
Fig. 6: Themis enhances OT-I CD8+ T cell expansion during LM-OVA infection in vivo.
Fig. 7: Themis–Shp1 axis in the regulation of CD8+ T cell homeostasis.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data for Fig. 1 and Extended Data Fig. 2 are provided with the paper.

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Acknowledgements

This research was supported by Singapore Ministry of Education grant 2014-T2-1-136 and the Singapore Ministry of Health’s National Medical Research Council under grant CBRG/0097/2015 (to N.R.J.G.). The authors thank S. Kupriyanov from the Scripps Research Institute Mouse Genetics Core for the initial derivation of the Themis cKO mice, P. Hutchinson and G.H. Teo from the NUS Immunology Programme Flow Cytometry Core Facility for cell sorting, and the NIH tetramer core facility for the H-2Kb monomers.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Joanna Brzostek, Namrata Gautam, Xiang Zhao, Elijah W. Chen, Monika Mehta, Desmond W. H. Tung, Yen Leong Chua, Jiawei Yap, Su H. Cho, Shvetha Sankaran, Vasily Rybakin & Nicholas R. J. Gascoigne

  2. Laboratory of Immunobiology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium

    Vasily Rybakin

  3. State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, China

    Guo Fu

  4. Immunology Programme, Life Sciences Institute, National University of Singapore, Singapore, Singapore

    Nicholas R. J. Gascoigne

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Contributions

J.B., N.G., X.Z. and E.W.C. designed and conducted the experiments with contributions from V.R., G.F., S.S. and M.M. J.B., X.Z. and N.R.J.G. analyzed the data. Y.L.C., D.W.H.T., S.H.C. and J.Y. provided technical help. J.B., X.Z. and N.R.J.G. wrote the paper, with input and final approval from all authors.

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Correspondence to Nicholas R. J. Gascoigne.

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Extended data

Extended Data Fig. 1 Phenotype of Themis cKO mice.

(A) Themis cKO mice have normal thymic development. Percentages of DN, PD, CD4 and CD8 SP, TCRintCD69int DP, CD69low CD4 SP, CD69low CD8 SP and TCRhiCD24low CD8 SP thymocytes. Data from 13-15 mice/genotype, from 3-4 independent experiments. (B) Peripheral phenotype of Themis cKO mice. Percentages of CD8+ T lymphocytes, CD25+ CD4+ regulatory T cells, and percentages of CD44lowCD62Lhi, CD44hiCD62low, Cd44hiCD62Lhi and CD44lowCD62low populations in CD25−CD4+ T cells and CD8+ T cells. Data from 13-15 mice/genotype, from 3-4 independent experiments. (C) TCR MFI on the indicated thymocyte and lymphocyte populations. Data from 13 mice/genotype from 3 independent experiments. (D) dlLck-Cre expression does not alter percentages of CD4+ and CD8+ T cells in Themis WT mice. Percentages of CD4+ and CD8+ in the lymphocytes gate in dlLc-Cre+ and dLck-Cre- Themis WT mice. The numbers show mean percentage +/- SEM from 4-6 mice/genotype from 2 independent experiments. WT and cKO samples were compared using unpaired t-test, with Welch’s correction if required, with *** indicating p-values from 0.0001 to 0.001, ** indicating p-values from 0.001 to 0.01. Mean +/- SEM values are shown.

Extended Data Fig. 2 Phenotype of OTI TCR transgenic Themis cKO mice.

(A) Anti-Themis Western blotting of cell lysates from FACS-sorted DP and CD8 SP thymocytes,with anti-tubulin as aloading control. Data representative of 2 independent experiments. (B) Percentages of CD8 SP thymocytes, CD69low CD8 SP thymocytes and TCRhiCD24low CD8 SP thymocytes from OTI WT and cKO mice. Mean +/- SEM from 13 mice/genotype, from 4 independent experiments. (C) (left) CD5 MFI on CD8+ T cells from Themis cKO ERT2-Cre+ and Cre-mice control mice after tamoxifen treatment. Mean +/- SEM from 12 mice/genotype from 4 independent experiments. (right) CD5 MFI as percentage of ERT2-Cre- mean CD5 MFI. Mean +/- SEM from 21 mice from 7 independent experiments. (D) CD8 and TCRβ MFI on OTI cKO CD8 SP thymocytes and CD8+ lymphocytes as percentage of WT mean MFI. Mean +/- SEM from 13-17 mice/genotype from 4-5 independent experiments. (E) Ex vivo Eomes nuclear staining. SEM +/- SEM from data from 7-8 mice from 3 independent experiments. (F) Percentages of CD44lowCD62Lhi, CD44hiCD62low, Cd44hiCD62Lhi and CD44lowCD62low populations. Mean +/- SEM from 13 mice/experiment, from 4 independent experiments. (G) Increase in Va2low CD8+ T lymphocytes and CD8 SP thymocytes in Themis cKO OTI mice. The flow cytometry plots representative of 13 mice/genotype from 4 independent experiments, and the numbers shown indicate mean +/- SEM of 13 mice/genotype from 4 independent experiments. Graphs show percentages of the indicated populations from 13 mice/genotype from 4 independent experiments (Rag1 WT) or 10 mice/genotype from 2 independent experiments (Rag1 KO). Graph showing CD5 MFI on Vα2low cells as percentage of CD5 MFI on Vα2hi cells shows data from 11 mice/genotype from 3 independent experiments. WT and cKO samples were compared using unpaired t-test, with *** indicating p-values from 0.0001 to 0.001, ** indicating p-values from 0.001 to 0.01. Mean +/- SEM values are shown.

Source data

Extended Data Fig. 3 Activation of OTI TCR transgenic Themis cKO mice.

(A) H-2Kb staining on CHO cells expressing scH-2Kb. Data representative of 4 independent experiments. Lymphocytes from Themis WT and cKO OT-I mice were stimulated for the indicated times.(B) Representative Indo-1 kinetics plots, area under curve (AUC) and peak values normalized to cKO sample after tetramer stimulation. Each data point represents one WT sample normalized to its same-tube cKO sample. Data from 9 mice/genotype from 5 independent experiments. (C) OTI CD8+ T cell survival after 24h incubation with the indicated peptides. Mean +/- SEM from 3 mice/genotype from 1 experiment, representative of 2 independent experiments. (D) OTI Rag1KO Themis WT or cKO lymphocytes were labelled with CTV (cKO) or CTR (WT), mixed at 1:1 ratio and injected into β2m KO recipients. Control sample was incubated in vitro in 5ng/ml IL7. 2 days later, cell survival was analyzed. The flow cytometry plots show are representative of 1 experiment, out of two independent experiments. The percentages on the plots indicate mean +/- SEM from 3 technical replicates (IL7 in vitro culture) or 5 recipient mice/donor genotype. (E) CD5 surface expression on OTI WT and cKO CD8+ T cells after β2m KO “parking”. Data from 9-10 donor mice/genotype; 10 recipient mice/donor genotype and 4 technical replicates (IL-7) from 2 independent experiments. (F) β2m “parking” does not alter OTI cKO T cell responses to antigenic pMHC. 2 days post-transfer, lymphocytes from β2m KO recipients were co-incubated with β2m-sufficient splenocytes (Ly5a+) and the indicated peptides for 3h (CD69 upregulation) or 5h (TNF production). Data from 8 mice/recipient genotype, from 2 independent experiments. The indicated samples were compared using two-tailed unpaired t-test, with **** indicating p-value <0.0001. Mean +/- SEM values are shown.

Extended Data Fig. 4 Unimpaired IL-7 signaling and pro-survival function in Themis cKO OTI CD8+ T cells.

A) CD127 staining on ex vivo OTI CD8+ T cells, or after 6h incubation in the presence or absence of 10ng/ml IL-7. Mean +/- SEM from 7-8 mice/genotype from 2 experiments. (B) CD127 surface expression on ex vivo CD4+ and CD8+ T cells from TCR polyclonal Themis WT and cKO mice. Mean +/- SEM from 7 mice/genotype from 2 experiments. (C,D) STAT5 phosphorylation in CD8+ T cells after 15 min incubation with 10ng/ml IL-7 analyzed by imaging flow cytometry (C), and flow cytometry (D). Data from 6 mice/genotype, pooled from 2 independent experiments. (E,F) IL-7 mediated OTI CD8+ T cell survival. (E) OTI lymphocytes were incubated for 24h with or without 50ng/ml IL-7 in the presence or absence of 10μg/ml CD8β blocking antibody or 50ng/ml IL-12. Cell death was assessed by flow cytometry. Mean +/- SEM from 3 mice/genotype from 1 experiment, data representative of 2 independent experiments. (F) OTI lymphocytes were incubated for 24h with the indicated concentrations of IL-7. Cell death was assessed by flow cytometry. Data from 14 mice/genotype from 3 independent experiments. The indicated samples were compared using unpaired t-test, with Welch’s correction if required, with *** indicating p-values from 0.0001 to 0.001, ** indicating p-values from 0.001 to 0.01, and * indicating p-values from 0.01 to 0.05. Mean +/- SEM values are shown.

Extended Data Fig. 5 OTI CD8+ T cell proliferation induced by IL-7 and IL-12 requires recognition of self pMHC.

(A) WT OTI lymphocytes were incubated with 50ng/ml each IL-7 and IL-12, with or without 50ng/ml E1 peptide in the presence or absence of 10μg/ml CD8β blocking antibody for 7 days. The numbers shown denote mean +/- SEM from 3 mice from 1 experiment, representative of 2 independent experiments. (B) Acute Themis deletion reduces responses to IL-7 and IL-12. FACS-sorted naïve CD44low CD8+ T cells from tamoxifen-treated ERT2-Cre+ (Themis deletion) or ERT2-Cre- mice were Cell Trace Violet labelled and incubated in 50ng/ml IL-7 and IL-12 with or without 100 ng/ml E1 for 7 days. Cell proliferation was assessed by flow cytometry. Representative flow cytometry plots are shown, with numbers indicating mean +/- SEM of triplicates, obtained from 3-4 pooled lymphocytes/genotype. Data representative of two independent experiments. (C) WT or cKO OTI lymphocytes were incubated with 10ng/ml IL-7 and the indicated concentrations of IL-7 for 3 days. The percentage of divided cells were determined based on CTV dilution. Mean +/- SEM from 9 mice/genotype, pooled from 3 independent experiments.

Extended Data Fig. 6 Themis-deficient OTI CD8+ T cells show reduced nutrient transporter expression in response to stimulation with subthreshold affinity pMHC and cytokines.

(A) WT OTI lymphocytes were incubated with 50ng/ml each IL-7 and IL-12, in the presence or absence of 10μM Akt inhibitor Triciribine for 7 days. Mean +/- SEM from 3 mice/genotype from 1 experiment, data representative of 2 independent experiments. (B) Themis WT and cKO OTI lymphocytes were incubated with 50ng/ml of peptide and/or 50ng/ml of the indicated cytokine for 24h or 3 days, followed cell surface staining to detect large neutral amino acid transporter CD98 and the transferrin receptor CD71. Data from 7-9 mice/genotype from 3 independent experiments (CD98), and 6-14 mice from 4 independent experiments (CD71). WT and cKO samples were compared using unpaired t-test, with Welch’s correction if required, with ** indicating p-values from 0.001 to 0.01. Mean +/- SEM values are shown.

Extended Data Fig. 7 Phenotype and activation of OTI cKO CD8+ T cells during LM-OVA infection.

(A) Normal effector and memory precursor development in OTI cKO CD8+ T cells during primary LM- OVA infection. CD127 and KLRG1 staining on donor-derived (CD45.2+) CD8+ T cells at day 7 post-infection. Mean +/- SEM of effector phenotype (KLRG1+CD127low) and memory precursors (KLRG1− CD127high) from 5 mice/donor genotype, data representative of two independent experiments. (B) Eomes and T-bet expression in donor-derived (CD45.2+) CD8+ T cells at day 7 post-infection. Data from 7 mice/genotype from 2 independent experiments (Eomes), or 3-4 mice/genotype from 1 experiment (T-bet). (C) Splenocytes from day 7 LM-OVA infected mice were re-stimulated in vitro with the indicated concentrations of OVA, Q4R7 or G4 for 6h, followed by intracellular staining to detect TNF. The plots show percentage of and TNF+ donor-derived CD45.2+ CD8+ T cells. Data from 14 mice/donor genotype from 3 independent experiments (OVA), 9-14 mice/donor genotype from 2-3 experiments (Q4R7) and 5-10 mice from 1-2 experiments (G4). WT and cKO samples were compared using unpaired t-test, with Welch’s correction if required, with *** indicating p-values from 0.0001 to 0.001, ** indicating p-values from 0.001 to 0.01, and * indicating p-values from 0.01 to 0.05. Mean +/- SEM values are shown.

Extended Data Fig. 8 Themis Shp1 double cKO data.

A) Shp1 deficiency rescues CD5 expression on SP thymocytes. CD5 cell surface expression on CD4 SP and TCRhi CD8 SP thymocytes, as percentage of WT expression. Data from 7-12 mice/genotype, from 3 independent experiments. (B) Shp1-deficiency rescues Akt phosphorylation defect in Themis cKO CD8+ T cells after cytokine stimulation. Lymphocytes from the indicated mice were cultured in 50 ng/ml IL7 and IL12 for 3 days, followed by fixation, permeabilization and flow cytometry analysis. pAkt MFI are shown as % of WT values. Data from 9-11 mice/genotype from 3 independent experiments. Shp1 cKO with CD4-Cre mediated deletion were used in as controls. (C) Normal thymocyte development in Themis Shp1 double cKO mice. Percentages of TCRintCD69int DP, CD8 SP and CD4 SP thymocytes. Data from 7-12 mice/genotype, from 3 independent experiments. (D) Themis Shp1 double cKO T cells show increase in CD44hi memory phenotype, comparable to that in Shp1 cKO T cells. Percentages of CD25+CD4+ T cells, CD44hiCD25-CD4+ T cells and CD44hi CD8+ cells from 8-12 mice/genotype, from 3 independent experiments. (E) TCRβ MFI on CD44lowCD8+ T cells and CD44low CD25-CD4+ T cells from 6-8 mice/genotypes, from 2 independent experiments. (F) CD212 and CD15 cell surface expression onCD44lo CD8+ T cells from 8-12 mice/genotype, from 3 independent experiments. (G) Functional redundancy between Shp1 and Shp2 in peripheral T cells. Percentages of CD4+ and CD8+ T lymphocytes in Shp1 cKO (CD4-Cre), Shp2 cKO (CD4-Cre), Shp1 Shp2 double cKO (CD4-Cre) mice. Cre negative mice from either genotype were used as WT controls. Data from 9-11 mice/genotype, from 3 independent experiments. Data analyzed using one-way ANOVA with Tukey’s multiple comparisons test.

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Supplementary Fig. 1 (flow cytometry gating strategy).

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Brzostek, J., Gautam, N., Zhao, X. et al. T cell receptor and cytokine signal integration in CD8+ T cells is mediated by the protein Themis. Nat Immunol 21, 186–198 (2020). https://doi.org/10.1038/s41590-019-0570-3

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