PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity (original) (raw)

Data availability

The data and materials that support the findings of this study are available from the corresponding author upon reasonable request. All sequencing data from this study has been deposited in the National Center for Biotechnology Information Gene Expression Omnibus and are accessible through the Gene Expression Omnibus Series accession code GSE134413.

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Acknowledgements

We thank N. Collins (Dana-Farber Cancer Institute), C. Kadoch (Dana-Farber Cancer Institute), D. Vignali (University of Pittsburgh), E.J. Wherry (University of Pennsylvania) and G. Dranoff (Novartis Institutes for BioMedical Research) for sharing cancer cell lines and F. Zhang (Broad Institute of MIT and Harvard) for sharing loxP-STOP-loxP-Cas9 mice. This work was supported by funding from grant no. T32CA207021 from the National Cancer Institute to M.W.L., the 2016 AACR-Bristol-Myers Squibb Fellowship in Translational Immuno-oncology grant no. 16-40-15-MILL, National Center for Advancing Translational Sciences/National Institutes of Health Award no. KL2 TR002542, the Jane C. Wright, MD, Endowed Young Investigator Award from ASCO to B.C.M. and grant nos. P50CA101942 from the National Cancer Institute to G.J.F., U19AI133524 from the National Institute of Allergy and Infectious Diseases to A.H.S and W.N.H. and U54 CA225088 from the National Cancer Institute, R01 CA229851 from the National Cancer Institute and P01 AI108545 from the National Institute of Allergy and Infectious Diseases to A.H.S.

Author information

Author notes

  1. These authors contributed equally: W. Nicholas Haining, Arlene H. Sharpe.

Authors and Affiliations

  1. Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA
    Martin W. LaFleur, Thao H. Nguyen, Matthew A. Coxe, Brian C. Miller, Jacob E. Gillis, Emily F. Gaudiano & Arlene H. Sharpe
  2. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
    Martin W. LaFleur, Brian C. Miller, Kathleen B. Yates, Debattama R. Sen, Rose Al Abosy & W. Nicholas Haining
  3. Evergrande Center for Immunological Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, USA
    Martin W. LaFleur, Thao H. Nguyen, Matthew A. Coxe, Brian C. Miller, Jacob E. Gillis, Emily F. Gaudiano & Arlene H. Sharpe
  4. Division of Medical Sciences, Harvard Medical School, Boston, MA, USA
    Martin W. LaFleur & Debattama R. Sen
  5. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA
    Brian C. Miller, Kathleen B. Yates, Debattama R. Sen, W. Nicholas Haining & Arlene H. Sharpe
  6. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
    Brian C. Miller & Gordon J. Freeman

Authors

  1. Martin W. LaFleur
  2. Thao H. Nguyen
  3. Matthew A. Coxe
  4. Brian C. Miller
  5. Kathleen B. Yates
  6. Jacob E. Gillis
  7. Debattama R. Sen
  8. Emily F. Gaudiano
  9. Rose Al Abosy
  10. Gordon J. Freeman
  11. W. Nicholas Haining
  12. Arlene H. Sharpe

Contributions

M.W.L., W.N.H. and A.H.S. conceived the project and wrote the manuscript with assistance from T.H.N., M.A.C., B.C.M., E.F.G., J.E.G. and G.J.F.. M.W.L., T.H.N., W.N.H. and A.H.S. designed experiments. M.W.L. and T.H.N. performed and analyzed all experiments with assistance from M.A.C., J.E.G. and E.F.G.. K.B.Y. performed bulk RNA-seq sample processing and analyzed the RNA-seq data. B.C.M. and R.A. performed 10× single-cell RNA-seq sample processing and analyzed the single-cell RNA-seq data. D.R.S. performed ATAC-seq sample processing and analyzed the ATAC-seq data. G.J.F. contributed to anti-PD-1 experiments.

Corresponding authors

Correspondence toW. Nicholas Haining or Arlene H. Sharpe.

Ethics declarations

Competing interests

A.H.S. has patents on the PD-1 pathway licensed by Roche/Genentech and Novartis, consults for Novartis, is on the scientific advisory boards for Surface Oncology, Sqz Biotech, Elstar Therapeutics, Elpiscience, Selecta and Monopteros and has research funding from Merck, Novartis, Roche, Ipsen, UCB and Quark Ventures. W.N.H. has a patent application on T cell exhaustion-specific enhancers held by the Dana-Farber Cancer Institute and now is employed by Merck. W.N.H. is also a founder of Arsenal Biosciences. A.H.S. and W.N.H. have a patent application on PTPN2 as a therapeutic target held/submitted by the Dana-Farber Cancer Institute. G.J.F. has a consulting or advisory role for Novartis, Lilly, Roche/Genentech, Bristol-Myers Squibb, Bethyl Laboratories, Xios Therapeutics, Quiet Therapeutics and Seattle Genetics; patents, royalties or other intellectual property from Novartis, Roche/Genentech, Bristol-Myers Squibb/Medarex, Amplimmune/Astrazeneca, Merck, EMD Serono and Boehringer Ingelheim and research funding from Bristol-Myers Squibb. The remaining authors declare no competing interests.

Additional information

Peer review information Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 _Ptpn2-_deleted naive CD8+ T cells do not express effector molecules at baseline or polyfunctional cytokines following infection.

(a) Representative flow cytometry plots of CD44 and CD62L expression on naive control or _Ptpn2-_deleted P14 CD8+ T cells used in co-transfer experiments. Representative of two independent experiments, n = 1 mouse. (b-d) Representative flow cytometry plots of (b) Granzyme B expression, (c) Ki-67 expression, and (d) BrdU incorporation on naive control or _Ptpn2-_deleted P14 CD8+ T cells used in co-transfer experiments. Representative of two independent experiments, n = 5 mice. (e) Gating strategy for analyzing co-transferred CD8+ T cells from the spleen following LCMV Clone 13 infection as in Fig. 1c. (f) Quantification of frequency of IFN-γ+ TNF+ CD8+ T cells of co-transferred control and _Ptpn2-_deleted P14 T cells on days 8, 15, 22, and 30 post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (f) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001).

Supplementary Figure 2 Deletion of Ptpn2 decreases the frequency of cells expressing progenitor-associated markers.

(a) Representative flow cytometry plots of Tim-3 and Slamf6 expression on naive control or _Ptpn2-_deleted P14 CD8+ T cells used in co-transfer experiments. Representative of two independent experiments, n = 1 mouse. (b) Representative flow cytometry plots of Tim-3 and CXCR5 expression on control or _Ptpn2_-deleted P14 T cells in the spleen 8 days post LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (c) Frequency of Tim-3+ CXCR5– and Tim-3– CXCR5+ control or _Ptpn2_-deleted P14 T cells in the spleen 8 days post LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (d) Frequency of TCF1 and CD127 expressing control or _Ptpn2_-deleted P14 T cells in the spleen 8 days post LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (e) Number of Tim-3+ CXCR5– and Tim-3– CXCR5+ control or _Ptpn2_-deleted P14 T cells in the spleen 8 days post LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (f) Quantification of Tim-3+ Slamf6– and Tim-3– Slamf6+ subsets 8 days post LCMV Clone 13 infection of non-TCR transgenic control or _Ptpn2-_deleted bone marrow chimeras. Subsets in (f) are pre-gated on PD-1 to identify responding cells and Vex. Representative of two independent experiments, n = 3 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (c-e) or two-way ANOVA (f) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001).

Supplementary Figure 3 Ptpn2 deletion does not change the overall transcriptional or epigenetic states of the exhausted subpopulations.

(a) Heat map of the tSNE clusters in Fig. 3a with representative genes highlighted. Representative of one experiment, n = 4 pooled mice. (b) Expression of indicated genes in individual cells overlaid on the defined clusters. Representative of one experiment, n = 4 pooled mice. (c) Quantification of Slamf6 and Tim-3 expression assessed by flow cytometry on co-transferred control and _Ptpn2_-deleted cells analyzed by single-cell RNA-seq. Representative of one experiment, n = 4 pooled mice. (d) Plots depicting the intra-cluster density for control or _Ptpn2_-deleted cells. Representative of one experiment, n = 4 pooled mice. (e) Principal components analysis of transcriptional profiles of co-transferred control and _Ptpn2_-deleted cells day 8 post LCMV Clone 13 infection. Representative of one experiment, n = 2 mice with n = 2 technical replicates per mouse. (f) Representative GSEA curves for bulk RNA-seq of control and _Ptpn2-_deleted co-transferred cells day 8 post LCMV Clone 13 infection. LCMV Slamf6 vs. Tim-3 Up top 50 and LCMV Slamf6 vs. Tim-3 Down top 50 signatures are depicted. Representative of one experiment, n = 2 mice with n = 2 technical replicates per mouse. (g) Venn diagrams of ATAC-seq peak overlaps of Tim-3+ and Slamf6+ co-transferred control and _Ptpn2_-deleted cells day 8 post LCMV Clone 13 infection. Representative of one experiment, n = 2 mice. (h-i) Representative ATAC-seq tracks for (h) Tcf7 and (i) Tox for Tim-3+ and Slamf6+ populations in co-transferred control and _Ptpn2_-deleted cells day 8 post LCMV Clone 13 infection. For comparison green depicts ATAC-seq tracks from CD8+ T cells day 30 post LCMV Armstrong infection10. Representative of one experiment, n = 2 mice. Statistical significance was assessed by two-sided Student’s paired t-test (c), two-sided Kolmogorov–Smirnov test (f), or hypergeometric test (g) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001).

Supplementary Figure 4 _Ptpn2-_deleted CD8+ T cells have increased proliferative capacity and in vivo persistence.

(a) Representative flow cytometry histogram of CTV dilution following restimulation of Slamf6+ control or Slamf6+ _Ptpn2-_deleted cells with αCD3/CD28 and IL-2. Vertical black lines separate peak numbers. Representative of two independent experiments, n = 3 mice. (b) Quantification of number of CD8+ T cells per division following in vitro restimulation (αCD3/CD28 with IL-2) of CTV-labeled Slamf6+ control or Slamf6+ _Ptpn2_-deleted CD8+ T cells isolated on day 8 post LCMV Clone 13 infection. Representative of two independent experiments, n = 3 mice. (c) Representative flow cytometry histogram of CTV dilution following restimulation of Tim-3+ control or Tim-3+ _Ptpn2-_deleted cells with αCD3/CD28 and IL-2. Vertical black lines separate peak numbers. Representative of two independent experiments, n = 5 mice. (d) Quantification of number of CD8+ T cells per division following in vitro restimulation (αCD3/CD28 with IL-2) of CTV-labeled Tim-3+ control or Tim-3+ _Ptpn2_-deleted CD8+ T cells isolated on day 8 post LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (e) Quantification of number of recovered Tim-3+ CD8+ T cells in the liver day 6 post LCMV Clone 13 infection following transfer of control or _Ptpn2-_deleted Tim-3+ CD8+ T cells isolated on day 8 post LCMV Clone 13 infection. Representative of two pooled experiments, n = 8 mice. (f) Frequency of dead cells of transferred Tim-3+ control or Tim-3+ _Ptpn2-_deleted cells day 6 post LCMV Clone 13 infection as in (e). Representative of two pooled experiments, n = 8 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (b, d), two-sided Student’s unpaired t-test (e), or two-way ANOVA (f) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001).

Supplementary Figure 5 Costimulation and IFN-γ contribute to the increased differentiation of _Ptpn2-_deleted cells into Tim-3+ cells.

(a) Quantification of CD25 MFI on CD8+ CD25+ control or _Ptpn2_-deleted CD8+ T cells following in vitro stimulation (αCD3/CD28) in the presence of indicated cytokines or blocking antibodies. Representative of two pooled experiments, n ≥ 4 technical replicates. (b) Representative flow cytometry plots of Slamf6 and Tim-3 expression on control or _Ptpn2_-deleted CD8+ T cells following in vitro stimulation (αCD3/CD28) in the presence of IL-2 and IFN-α. Representative of two independent experiments, n ≥ 2 technical replicates. (c) Quantification of Slamf6+ Tim-3–, Slamf6+ Tim-3+, Slamf6– Tim-3+ subsets in control or _Ptpn2_-deleted CD8+ T cells following in vitro stimulation (αCD3 without αCD28) in the presence of IL-2 and IFN-α. Representative of one experiment, n ≥ 2 technical replicates. (d) Quantification of Slamf6+ Tim-3+ (left graph) and Slamf6– Tim-3+ (right graph) subsets in control cells following in vitro stimulation (αCD3/CD28) in the presence of indicated cytokines or blocking antibodies and addition of pre-conditioned supernatant from stimulated control or _Ptpn2_-deleted CD8+ T cells. Representative of one experiment, n ≥ 2 technical replicates. (e) Representative histograms of IFNAR1 expression on naive control or _Ptpn2_-deleted CD8+ T cells. Representative of one experiment, n = 1 mouse. (f) Quantification of frequencies of co-transferred control or _Ptpn2_-deleted CD8+ T cells day 4 post LCMV Clone 13 infection following treatment with isotype (left graph) or IFN-γ blocking antibody (right graph). Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 5 mice. (g) Quantification of Slamf6+ Tim-3–, Slamf6+ Tim-3+, and Slamf6– Tim-3+ subsets in _Ptpn2-_deleted P14 CD8+ T cells day 4 post LCMV Clone 13 infection in mice that received isotype or IFN-γ blocking antibody as in (f). Representative of two independent experiments, n = 5 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-way ANOVA (a, g) or two-sided Student’s paired t-test (f) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001).

Supplementary Figure 6 _Ptpn2-_deleted CD8+ T cells are more activated in response to MC38-OVA tumors.

(a) Gating strategy for analyzing transferred OT-1 CD8+ T cells from the tumor, tumor-draining lymph node, and spleen following tumor challenge. (b) Quantification of CD25 expression in OT-1 CD8+ T cells day 7 post MC38-OVA implantation in the tumor-draining lymph node for control and _Ptpn2_-deleted co-transferred cells as in Fig. 6a. Representative of two independent experiments, n = 7 mice. (c) Quantification of CD127 expression in OT-1 CD8+ T cells day 7 post MC38-OVA implantation in the tumor-draining lymph node for control and _Ptpn2_-deleted co-transferred cells as in Fig. 6a. Representative of two independent experiments, n = 7 mice. (d) Quantification of IFN-γ and TNF cytokine expression in co-transferred OT-1 CD8+ T cells in tumors on day 7 post MC38-OVA implantation for co-transferred control and _Ptpn2_-deleted cells as in Fig. 6a. Representative of two independent experiments, n = 6 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (b-d) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001).

Supplementary Figure 7 Deletion of Ptpn2 induces a systemic cytotoxic CD8+ T cell response following tumor challenge.

(a) Quantification of Vex expression from control or _Ptpn2-_deleted chimeras. Representative of two independent experiments, n = 9 mice. (b) Quantification of Ly6chi, MHC-II+, and Foxp3+ subsets in control or _Ptpn2-_deleted chimeras at steady state. Representative of two independent experiments, n ≥ 8 mice. (c) Quantification of inflammatory cytokines in the serum of control or _Ptpn2-_deleted chimeras at steady state. Nd: not detectable. Representative of two independent experiments, n = 10 mice. (d) Survival curves from Fig. 7b experiment. Representative of two independent experiments, n ≥ 8 mice. (e) Quantification of immune infiltrate in MC38 tumors 9 days post implantation in control or _Ptpn2_-deleted chimeras. Representative of two independent experiments, n ≥ 7 mice. (f) Quantification of CD127 expression in control or _Ptpn2_-deleted chimeras day 14 post MC38 tumor implantation. Representative of two independent experiments, n ≥ 7 mice. (g) Quantification of Slamf6+ and Tim-3+ subsets in control or _Ptpn2_-deleted chimeras day 12 post MC38 tumor implantation. Representative of two independent experiments, n ≥ 8 mice. (h-i) (h) Representative flow cytometry plots and (i) quantification of CD44 and CD62L subsets in control or _Ptpn2_-deleted chimeras day 14 post MC38 tumor implantation. Representative of two independent experiments, n = 5 mice. (j) Quantification of CD8+ T cells in control or _Ptpn2_-deleted chimeras treated with isotype or CD8-depleting antibody day 10 post MC38 tumor challenge. Representative of two independent experiments, n ≥ 4 mice. (k-l) Tumor growth curves for control or _Ptpn2-_deleted chimeras (k) untreated or (l) treated with a CD8-depleting antibody or isotype control rechallenged with 5 x 106 MC38-WT tumor cells, 60-days post primary MC38 tumor clearance. Representative of two independent experiments, (k) n ≥ 7 mice, (l) n ≥ 4 mice. (m) Survival curves from Fig. 7i experiment. Representative of two independent experiments, n ≥ 10 mice. Bar graphs represent mean and error bars represent standard deviation (except k-l which represent standard error). Statistical significance was assessed by two-way ANOVA (b-c, e, g, i, k-l), two-sided log-rank Mantel–Cox test (d, m), or by two-sided Student’s unpaired t-test (f) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001).

Supplementary information

Reporting Summary

Supplementary Table 1

GSEA full report for RNA-seq profiling of _Ptpn2_-deleted and control Slamf6+ and Tim-3+ cells 8 d after LCMV clone 13 infection

Supplementary Table 2

Chromatin-accessible regions for ATAC-seq profiling of _Ptpn2_-deleted and control Slamf6+ and Tim-3+ cells 8 d after LCMV clone 13 infection

Supplementary Table 3

GSEA full report for RNA-seq profiling of _Ptpn2_-deleted and control cells 7 d after MC38-OVA tumor injection

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LaFleur, M.W., Nguyen, T.H., Coxe, M.A. et al. PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity.Nat Immunol 20, 1335–1347 (2019). https://doi.org/10.1038/s41590-019-0480-4

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