Abstract
CD8+ T cells are central mediators of immune responses against infections and cancer. Here we identified Dapl1 as a crucial regulator of CD8+ T cell responses to cancer and infections. Dapl1 deficiency promotes the expansion of tumour-infiltrating effector memory-like CD8+ T cells and prevents their functional exhaustion, coupled with increased antitumour immunity and improved efficacy of adoptive T cell therapy. Dapl1 controls activation of NFATc2, a transcription factor required for the effector function of CD8+ T cells. Although NFATc2 mediates induction of the immune checkpoint receptor Tim3, competent NFATc2 activation prevents functional exhaustion of CD8+ T cells. Interestingly, exhausted CD8+ T cells display attenuated NFATc2 activation due to Tim3-mediated feedback inhibition; Dapl1 deletion rescues NFATc2 activation and thereby prevents dysfunction of exhausted CD8+ T cells in chronic infection and cancer. These findings establish Dapl1 as a crucial regulator of CD8+ T cell immunity and a potential target for cancer immunotherapy.
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Data availability
The scRNAseq data that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession code GSE175689. Previously published scRNAseq data that were re-analysed here are available under the accession code GSE139829. Previously published RNAseq data that were re-analysed here are available under the accession code GSE126777. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.
Code availability
All the code will be available from the corresponding authors on reasonable request, including but not limited to the following: scRNAseq analysis and bulk RNAseq analysis.
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Acknowledgements
We thank J. P. B. Viola for NFAT expression vectors, E. J. Wherry for LCMV clone 13 and the Mutant Mouse Resource & Research Centers for Nfatc2-KO mice. We thank the Genetically Engineered Mouse Facility as well as the flow cytometry, sequencing and microarray, and animal facilities of the shared resources at The MD Anderson Cancer Center, supported by the NIH/NCI Cancer Center Support Grant (CCSG) P30CA016672. The Advanced Technology Genomics Core (ATGC) is supported by grant no. CA016672 (ATGC) and NIH grant no. 1S10OD024977-01. This study was supported by a grant from the National Institutes of Health (grant no. AI64639 to S.-C.S.). T.G. was a visiting student supported by a scholarship from the China Scholarship Council (grant no. 201906380080).
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Contributions
L.Z. designed and performed the research, prepared the figures and wrote the manuscript. X.Z. performed scRNAseq data analysis, made significant contributions to data interpretation and organization, and provided critical reagents, scientific advice and expertise. M.G. provided essential mouse models, scientific advice and contributed to the experiments. J.K. designed and performed the research and made significant contributions to the initial characterization of Dapl1 function. Y.L., C.-J.K., X.X., T.G. and X.C. contributed to the performance of the experiments. S.-C.S. supervised the work and wrote the manuscript.
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Competing interests
Although they participated in this research while employed in the University of Texas MD Anderson Cancer Center, X.Z. is presently an employee of Flagship Labs 91, Inc, J.K. is presently an employee of Bristol Myers Squibb and X.X. is presently an employee of AbbVie. All competing interests are unrelated to the current study. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Characterization of Dapl1 expression and Dapl1-KO mice.
a, b, qRT-PCR (a) and immunoblot (b) analysis of Dapl1 expression in CD4 and CD8 T cells, B cells, bone marrow DCs (BMDC), and bone marrow derived macrophages (BMDM). c, Dapl1 knockout strategy using CRISPR–Cas9, indicating introduction of a stop codon (taa) and an EcoRI restriction site (gaattc) to the 3’ boundary of exon 3. d, Genotyping PCR using tail DNA from wild-type (WT,+/+), Dapl1 KO (–/–), and heterozygous (+/–) mice. The reverse PCR primer binds to the mutated region to distinguish WT and KO alleles. e,f, RT-PCR (e) and immunoblot (f) analysis of Dapl1 expression using WT or Dapl1 KO (KO) CD8 T cells. g-i, Flow cytometry analysis of the frequency and absolute cell numbers of CD4 and CD8 T cells (g), naive (CD44loCD62Lhi) and memory (CD44hiCD62Llo) CD4 T cells (h), and naive (CD44lo) and memory (CD44hi) CD8 T cells (i) in the spleen of wild-type (WT) or Dapl1 KO mice. Data are presented as a representative plot (left) and summary graphs (right). n = 5 per genotype. j,k, ELISA analysis of secreted IL-2 and IFN-γ (j) and qRT-PCR analysis of the indicated mRNAs (k) in wild-type and Dapl1 KO naive CD8 or CD4 T cells, stimulated with anti-CD3 and anti-CD28 for 48 h (j) or for the indicated time points (k). n = 6 per genotype. l, Immunoblot analysis of Dapl1 in human CD8 T cells transduced with a control shRNA (Ctrl) or three different Dapl1 shRNAs. m, n, qRT-PCR analysis of IFNG and IL-2 mRNAs (m) and flow cytometry analysis of intracellular IFN-γ and IL-2 proteins (n) in human CD8 T cells transduced with control or Dapl1 shRNA (#3) and stimulated with anti-CD3 plus anti-CD28 for the indicated time points (m) or 48 h (n). Data are pooled from two independent experiments. Summary data are shown as the mean±s.d. with P values determined using a two-tailed unpaired Student’s t-test (a, g-k, m, n). ns, not significant.
Extended Data Fig. 2 Dapl1 deficiency promotes antitumour responses and Tim3 expression in CD8, but not CD4, T cells.
a, Flow cytometry analysis of the frequency of IFN-γ-producing CD4 T cells in the tumour (upper) and draining lymph nodes (dLN, lower) of wild-type (WT) or Dapl1 KO (KO) mice injected subcutaneously with B16-OVA for 20 days. n = 5 per genotype. b,c, Flow cytometry analysis of the frequency and absolute cell number of CD4 and CD8 T cells in draining lymph nodes and tumours (TILs) of MC38 colon cancer cell-implanted wild-type or Dapl1 KO mice. n = 5 per genotype. d-g, Schematic of experimental design (d), flow cytometry analysis of the frequency of donor Pmel1 CD8 T cells (CD45.2+) and host CD8 T cells (CD45.1+) (e) and donor (CD45.2+) IFN-γ-, IL-2- or TNF-a-producing Pmel1 CD8 T cells (f,g) in the tumour of B16F10-implanted B6.SJL mice (CD45.1+) adoptively transferred with in vitro activated wild-type or Dapl1 KO Pmel1 (CD45.2+) CD8 T cells (collected on day 5, 10 and 15 after Pmel1 cells transfer). n = 5 per genotype. h, Flow cytometry analysis of Tim3, PD1, CTLA4 or Lag3 expression levels in CD8 TILs of wild-type (WT) or Dapl1 KO mice implanted with B16-OVA for 20 days. n = 5 per genotype. i,j, Flow cytometry analysis of the frequency of PD1+, Tim3+, CTLA4+ or Lag3+ CD8 (i) or CD4 TILs (j) in wild-type or Dapl1 KO mice implanted with MC38 colon cancer cells for 24 days. n = 5 per genotype. k-m, Immunoblot analysis of Dapl1 expression in Dapl1 KO Pmel1 CD8 T cells transduced with either an empty vector or HA-Dapl1 expression vector (k), schematic of experimental design for adoptive transfer of the transduced Dapl1 KO Pmel1 cells (CD45.2+) into B16F10-implanted B6.SJL mice (CD45.1+) (l), and flow cytometry analysis of the frequency of IFN-γ- and TNF-a-producing CD8 T cells in the tumour (m). n = 5 per genotype. n,o, Flow cytometry analysis of the frequency and absolute cell numbers of CD8 TILs in day-24 B16-OVA-implanted wild-type and Dapl1 KO mice injected i.p. with anti-PD1 (100 mg/mouse) or an IgG isotype control (n) or anti-Tim3 (200 mg/mouse) or an IgG isotype control (o) on days 7, 9, and 11, n = 5 (n) or 4 (o) per genotype. Summary data are shown as the mean ± s.d. with P values determined using a two-tailed unpaired Student’s t-test (a-c, e, g-j, m-o). ns, not significant.
Extended Data Fig. 3 Kinetic analysis of Dapl1 function in regulating anti-LCMV CD8 T cell responses.
a, Flow cytometry analysis of the frequency of H2Db/GP33−41 Tetramer+ CD8 T cells in the spleen of day 7, 14, 21, and 30 LCMV clone 13-infected wild-type (WT) and Dapl1 KO mice. Data are presented as a representative FACS plot (left) and summary graph (right). n = 5 per genotype. b,c, Flow cytometry analysis of the frequency of Tim3+, PD1+, Lag3+, or TIGIT+ CD8 T cells in the spleen of day 7, 14, 21, and 30 LCMV clone 13-infected wild-type and Dapl1 KO mice. Data are presented as a representative FACS plot (b) and summary graphs (c). n = 5 per genotype. d,e, Flow cytometry analysis of the frequency of TCF1+Tim3–, TCF1+Tim3+ and TCF1–Tim3+ subsets in gated CD8+ GP33+ PD1+ T cells (d) and CX3CR1+ cytotoxic effector CD8 cells in gated CD8+GP33+ T cells (e) in the spleen of day 7, 14, 21, and 30 LCMV clone 13-infected wild-type and Dapl1 KO mice. Data are presented as a representative FACS plot (left) and summary graphs (right). n = 5 per genotype. Data are pooled from two independent experiments. Summary data are shown as the mean±s.d. with P values determined using a two-tailed unpaired Student’s t-test (a, c-e). ns, not significant.
Extended Data Fig. 4 Characterization of in vitro generated exhausted CD8 T cells.
a, b, Flow cytometry analysis of the indicated ICRs, TOX, T-bet, and TCF1 (a) or effector-related molecules (b) in wild-type OT1 CD8 T cells cultured in vitro under mock or OVA257-264-stimulated effector or exhaustion conditions. c-g, Immunoblot analysis of Dapl1 knockdown (c) and flow cytometry analysis of the expression of PD1+ (d), Tim3+ (e), TOX (f), and TCF1 (g) in human CD8 T cells transduced with a control (Ctrl) shRNA or a Dapl1−specific shRNA and cultured under unexhaustion (cultured with 50 U/ml hIL-2) and exhaustion (three round of stimulation with anti-CD3/anti-CD28 Dynabeads) conditions. Data are representative of three independent experiments.
Extended Data Fig. 5 Dapl1 has a cell-intrinsic function in regulating CD8 T cell functions.
a, Schematic of experimental design. b-g Flow cytometry analysis of the frequency of splenic (Spl) or TIL CD45.1+CD8+ or CD45.2+CD8+ T cells (b), Granzyme B-, IFN-γ-, TNF-a-producing or Ki67+ CD8 TILs (c), PD1+,Tim3+, Lag3+ or TIGIT+ CD8 TILs (d), TCF1+Tim3–, TCF1+Tim3+ and TCF1–Tim3+ subsets in gated CD8+PD1+ TILs (e), Granzyme B-, IFN-γ-, TNF-a-producing cells in gated PD1+Tim3+ CD8 TILs (f), or Ki67+ CD8 + cells in gated PD1+Tim3+ CD8+ TILs (g) of B16-OVA tumour-bearing Rag1 KO recipient mice adoptively transferred with a mixture of BM cells derived from B6.SJL mice (CD45.1+) and Dapl1 KO (CD45.2+) or wild-type mice (CD45.2+). Data are presented as representative FACS plots and summary graphs. n = 5 chimeric mice. h-l, Flow cytometry analysis of the frequency of CD45.1+CD8+ or CD45.2+CD8+ T cells (h), H2Db/GP33-41 Tetramer+ CD8+ T cells (i), IL-2-, IFN-γ-, Granzyme B-, TNF-a-, or Ki67+-producing CD8 T cells (j), PD1+, Tim3+, Lag3+, TIGIT+, PD1+Tim3+ CD8 T cells or TCF1+Tim3-, TCF1+Tim3+, TCF1−Tim3+ subsets (k), and IFN-γ-, IL-2-, TNF-a- or Granzyme B-producing CD8 T cells (l), gated on PD1+Tim3+ CD8+T cells in the spleen of Rag1 KO recipient mice adoptively transferred with a mixture of BM cells derived from B6.SJL mice (CD45.1+) and Dapl1 KO (CD45.2+) or wild-type (CD45.2+) mice, infected i.v. with LCMV clone 13 for 30 days. Data are presented as representative FACS plots and summary graphs. n = 5 chimeric mice. Summary data are shown as the mean±s.d. with P values determined using a two-tailed unpaired Student’s t-test (b-l). ns, not significant.
Extended Data Fig. 6 Dapl1 has a cell-intrinsic role in regulating CD8 T cell responses to LM-OVA infection and cancer.
a, Schematic of experimental design for generating mixed bone marrow chimeric mice and LM-OVA infection. b,c, Flow cytometry analysis of the frequency of IFN-γ- or Granzyme B-producing CD8 effector T cells (b) and TCF1+ CD8+, TCF1- CD8+ cells (c) in the spleen of Rag1 KO recipient mice adoptively transferred (for 6 week) with a mixture of BM cells derived from B6.SJL mice (CD45.1+) and Dapl1 KO (CD45.2+) or wild-type (WT, CD45.2+) mice, infected i.v. with LM-OVA for 7 days. Data are presented as representative FACS plots and summary graphs. n = 5 chimeric mice. d-k, Schematic of experimental design (d) and flow cytometry analysis of the frequency of wild-type (CD45.1+CD45.2+) and Dapl1 KO (CD45.2+) CD8 T cells (e), Ki67+ CD8 T cells (f), effector CD8 T cells (g), Tim3+ or PD1+ CD8 T cells (h), TCF1+Tim3–, TCF1+Tim3+ and TCF1–Tim3+ subsets in gated PD1+ CD8 TILs (i), Ki67+ cells in gated PD1+Tim3+ CD8 T cells (j), and Granzyme B-, IFN-γ-, or TNF-a-producing cells in gated PD1+Tim3+ CD8 T cells (k) in the spleen (Spl) or the tumour (TIL) of B16F10 tumour-bearing B6.SJL mice (CD45.1+) adoptively transferred with a mixture (1:1) of wild-type (CD45.1+CD45.2+) and Dapl1 KO (CD45.2+) Pmel1 CD8 T cells. Data are presented as representative FACS plots and summary graphs. n = 5 recipient mice. Summary data are shown as the mean ± s.d. with P values determined using a two-tailed unpaired Student’s t-test (b, c, e-k). ns, not significant.
Extended Data Fig. 7 Dapl1 regulates progenitor-like CD8 T cell proliferation and conversion to effector-like CD8 T cells.
a, tSNE plot based on unsupervised clustering analysis of scRNAseq data from human uveal melanoma dataset GSE139829 showing 6 clusters of CD8 TILs. b,c, Expression profile of the indicated genes in each cluster of CD8 TILs (b) and Violin plots showing predominant expression of Tcf7 and Dapl1 in the cluster 1 progenitor population (c). d, qRT-PCR analysis of Dapl1 expression in PD1– Tim3– progenitor (TPro) or PD1+Tim3+ CD8 TEX cells isolated from LCMV clone 13−infected wild-type or Dapl1 KO mice. n = 6 per genotype. e-j, Schematic of experimental design (e) and flow cytometry analysis of the frequency of TCF1hi and TCF1+Tim3−progenitor exhausted CD8 T cells (f,g), CXCR3+, Ly6c+ or CCR2+ effector CD8 T cells in gated PD1lowTCF1low CD8 T cells (h), apoptotic cells in gated PD1–Tim3−CD8 T cells (i), and Ki67+ proliferative cells in gated TCF1+Tim3- CD8 T cells (j) derived from the tumour of B16F10 LCMVmini tumour-bearing B6.SJL mice on day 3 and day 10 following adoptive transfer with antigen-specific progenitor CD8 T cells sorted from the spleen of LCMV clone 13-infected wild-type and Dapl1 KO mice. Data are shown as representative plots and summary graphs. n = 5 per genotype. Summary data are shown as the mean±s.d. with P values determined using a two-tailed unpaired Student’s t-test (d, f-j). ns, not significant.
Extended Data Fig. 8 NFATc2 is regulated by Dapl1 and mediates the hyper-responses of Dapl1-KO CD8 T cells.
a-c, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates (a) or in cytoplasmic (CE) and nuclear (NE) extracts (b, c) of wild-type (WT) or Dapl1 KO (KO) CD8 (a, b) or CD4 (c) naïve T cells stimulated with anti-CD3 plus anti-CD28 for the indicated time points. d, Flow cytometry analysis of nuclear NFATc2 in human CD8 cells transduced with a non-silencing control shRNA (Ctrl shRNA) or Dapl1−specific shRNA, either not treated (NT) or stimulated for 30 min with ionomycin (Iono). Data are presented as representative FACS plots (upper) and a summary graph (lower), n = 5 biologically independent repeats. e,f, CoIP analysis of Dapl1 interaction with NFATc2 (e) or with NFATc1 (f) in 293 T cells transfected with (+) HA-Dapl1 along with (+) wild-type NFATc2 or a constitutive active NFATc2 (CA-NFATc2) (e), wild-type NFATc1 or a constitutive active NFATc1 (CA-NFATc1) (f). g, Flow cytometry analysis of intracellular IFN-γ in naive CD8 T cells from the indicated genotypes, stimulated for 48 h with anti-CD3 and anti-CD28. Data are presented as representative FACS plots (upper) and summary graphs (lower). n = 5 per genotype. h, Flow cytometry analysis of IFN-γ- or granzyme B-producing CD8 T cells in the tumour of the indicated mouse strains injected subcutaneously with 2 × 105 B16-OVA cells for 16 days. i, ICS and flow cytometry analysis of GP33−41 antigen-specific CD8 T cells producing the indicated cytokines in the spleen of LCMV clone 13−infected (for 30 days) wild-type (WT), Dapl1 KO, NFATc2 KO and Dapl1-NFATc2 double KO (dKO) mice. The splenocytes were restimulated in vitro for 6 h with GP33-41 peptide in the presence of monensin before ICS. Summary data are shown as the mean±s.d. with P values determined using a two-tailed unpaired Student’s t-test (d,g). Western blots are representative of three independent experiments (a-c, e,f).
Extended Data Fig. 9 Attenuated activation of NFATc2 and NFATc1 in exhausted CD8 T cells.
a, b Flow cytometry analysis of sorted PD1+Tim3+ exhausted (TEX) and PD1–Tim3– progenitor (TPro) CD8 TILs (a) and the expression of TCF1 (b) in PD1+Tim3+ exhausted (TEX) and PD1–Tim3– progenitor (TPro) CD8 cells derived from day 20 tumour of B16-OVA tumour-bearing wild-type (WT) or Dapl1 KO (KO) mice. c-e, Flow cytometry analysis of nuclear NFATc2 (c) and NFATc1 (d,e) in wild-type and Dapl1 KO TPro CD8 TILs (c), wild-type TEX and TPro CD8 TILs (d), or wild-type and Dapl1 KO TEX CD8 TILs (e), either untreated (NT) or ionomycin-stimulated (Iono, 30 min). n = 5 per genotype. f, g, Flow cytometry analysis of sorted PD1+Tim3+ exhausted (TEX) and PD1–Tim3– progenitor (TPro) CD8 T cells (f) and the expression of TCF1 (g) in PD1+Tim3+ exhausted (TEX) and PD1–Tim3– progenitor (TPro) CD8 cells from LCMV clone 13-infected wild-type or Dapl1 KO mice. h-j, Flow cytometry analysis of nuclear NFATc2 (h) and NFATc1(i,j) in wild-type and Dapl1 KO progenitor (TPro) CD8 T cells (h), wild-type TEX and TPro cells (i), and wild-type and Dapl1 KO TEX cells (j) from the spleen of LCMV clone 13-infected mice, either not treated (NT) or stimulated with ionomycin (Iono) for 30 min. n = 5 per genotype. k, l, Gating strategies for sorting PD1+Tim3+ exhausted (TEX) and PD1–Tim3– progenitor (TPro) Pmel1 CD8 TILs from the tumour (k) of B16F10-implanted B6.SJL mice (CD45.1+) adoptively transferred with Dapl1 KO Pmel1 CD8 T cells (CD45.2+) transduced with either an empty vector or HA-Dapl1 expression vector, and flow cytometry analysis of nuclear NFATc1 in untreated (NT) or ionomycin-stimulated (Iono, 30 min) PD1+Tim3+ exhausted (TEX) and PD1–Tim3– progenitor (TPro) Pmel1 CD8 TILs (l). n = 5 per genotype. m, Flow cytometry analysis of nuclear NFATc2 in in vitro generated exhausted human CD8 T cells transduced with a non-silencing control shRNA (Ctrl shRNA) or a Dapl1-specific shRNA, either not treated (NT) or stimulated for 30 min with ionomycin. n = 5 biologically independent repeats. n-p, Flow cytometry analysis of nuclear NFATc2 (n) and nuclear NFATc1 (o,p) in PD1+Tim3+ CD8 T cells in the tumour of B16-OVA-bearing wild-type or Dapl1 KO mice injected with anti-Tim3 or IgG isotype control on days 7, 9, and 11. n = 5 per genotype. Summary data are shown as the mean±s.d. with P values determined using a two-tailed unpaired Student’s t-test (c-e, h-j, l, m, o). ns, not significant.
Extended Data Fig. 10 The gating strategy.
Live immune cell populations were gated on the FSC-A and SSC-A. Single cells were gated basing on FSC-A and FAS-H. The subpopulations of the indicated immune cell were gated basing on specific markers as indicated in the individual panels.
Supplementary information
Supplementary Table
Supplementary Table 1. Primers for qPCR, genotyping and ChIP assays.
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Zhu, L., Zhou, X., Gu, M. et al. Dapl1 controls NFATc2 activation to regulate CD8+ T cell exhaustion and responses in chronic infection and cancer. Nat Cell Biol 24, 1165–1176 (2022). https://doi.org/10.1038/s41556-022-00942-8
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DOI: https://doi.org/10.1038/s41556-022-00942-8
