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Mechanisms of CD40-dependent cDC1 licensing beyond costimulation

Nature Immunology volume 23, pages 1536–1550 (2022)Cite this article

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Abstract

CD40 signaling in classical type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1s, including Cd70, Tnfsf9, Ptgs2 and Bcl2l1, and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T cell expansion. Loss of 4-1BB, alone or in Cd27−/− mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased caspase activation in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation by Cd40−/− cDC1s to CD8+ T cells, which were reversed by re-expression of Bcl2l1. Thus, CD40 signaling in cDC1s not only induces costimulatory ligands for CD8+ T cells but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses.

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Fig. 1: Transcriptional targets of CD40 signaling in cDC1s.
Fig. 2: CD70 expression in cDC1s partially contributes to CD40-dependent responses to tumors.
Fig. 3: Costimulatory CD27 and 4-1BB signaling do not fully mediate CD40 help.
Fig. 4: Ptgs2 signaling in cDC1s contributes to CD8+ T cell expansion, but not tumor rejection.
Fig. 5: CD40 signaling in cDC1s mediates cDC1 survival during tumor challenge.
Fig. 6: CD40 signaling mediates cDC1 survival during antigen presentation.
Fig. 7: CD40 help induces Bcl-xL for cDC1 survival for antigen presentation.
Fig. 8: Loss of Bcl-xL in cDC1s reduces cDC1 and CD8+ T cell expansion during tumor challenge.

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Data availability

Microarray data generated in the current study are available in the Gene Expression Omnibus database with the accession number GSE211426. Source data are provided with this paper.

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Acknowledgements

This publication is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health (NIH). This work was supported by the NIH (R01AI150297, R01CA248919, and R21AI164142 to K.M.M., R01CA190700 and T32CA009547 to R.D.S, and F30CA247262 to R.W.) S.T.F. is a Cancer Research Institute Irvington Fellow supported by the Cancer Research Institute. We thank J. M. White at the Department of Pathology & Immunology Transgenic Mouse Core at Washington University in St. Louis and the Genetic Editing and iPS Cell Center at Washington University in St. Louis for the generation of mouse models, the Genome Technology Access Center at the Department of Genetics at Washington University School of Medicine in St. Louis for help with genomic analysis, the Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs and Alvin J. Siteman Comprehensive Cancer Center for help with tetramer production, and the Diabetes Research Core (NIH P30 DK020579) for help with extracellular flux assays.

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  1. Timothy Gershon

    Present address: Department of Pediatrics, ECC Room 254, Emory University School of Medicine, Atlanta, GA, USA

Authors and Affiliations

  1. Department of Pathology and Immunology, Washington University in St. Louis, School of Medicine, St. Louis, MO, USA

    Renee Wu, Ray A. Ohara, Suin Jo, Tian-Tian Liu, Stephen T. Ferris, Feiya Ou, Sunkyung Kim, Derek J. Theisen, David A. Anderson III, Robert D. Schreiber, Theresa L. Murphy & Kenneth M. Murphy

  2. Department of Surgery, Washington University in St. Louis, School of Medicine, St. Louis, MO, USA

    Brian W. Wong

  3. Department of Neurology, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Timothy Gershon

  4. The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St Louis, MO, USA

    Robert D. Schreiber

  5. Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA

    Robert D. Schreiber

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Contributions

R.W., T.L.M. and K.M.M. designed the study. R.W., R.A.O. and S.J performed experiments, with advice from T.T. L., S.K., D.J.T and D.A. F.O., S.T.F., B.W.W. and R.D.S. provided assistance with experimental design. T.G. kindly provided BclxLfl/fl mice. R.W. and K.M.M. wrote the manuscript.

Corresponding author

Correspondence to Kenneth M. Murphy.

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Competing interests

R.D.S. is a co-founder, paid consultant and stockholder of Jounce Therapeutics and Asher Biotherapeutics and paid consultant and stockholder of A2 Biotherapeutics, Arch Oncology, Asher Biotherapeutics, Codiak Biosciences, NGM Biotherapeutics, Meryx and Sensei Biotherapeutics. K.M.M. is a paid member of the scientific advisory board of Harbour BioMed. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 CD40 stimulation induces CD70 and 4-1BBL expression in cDCs.

a, Hierarchical clustering of 89 genes induced at least two-fold in SDLN cDC1s treated in vitro in the absence or presence of agonistic αCD40 antibody (results averaged from three independent experiments). b, Day 8 Flt3L-treated WT (B6) bone marrow cultures were treated with no stimulation, agonistic αCD40 antibody, poly(I:C), or both. cDC1 surface expression of CD40 (top), CD70 (middle) and 4-1BBL (bottom) was analyzed by flow cytometry after 24 h. Pre-gate: B220− Siglec H− MHC class II+ CD11c+ XCR1+ Sirpα-. Numbers represent the percentage of cells in the indicated gates. Data represent two independent experiments. c, d, f, WT mice were injected i.p. with PBS, agonistic αCD40 antibody, poly(I:C), or both. c, Spleens were harvested 24 h after injection and cDC1 were analyzed for surface expression of CD40 and CD70 by flow cytometry. Pre-gate: B220− F4/80− MHC class II+ CD11c+ XCR1+ Sirpα-. Numbers represent the percentage of cells in the indicated gates. Data represent two independent experiments. d, SDLN were harvested 24 h after injection and cDC1 were analyzed for surface expression of CD40 and CD70 by flow cytometry. Pre-gate: B220− CD326− MHC class II+ CD11cint XCR1+ Sirpα-. Numbers represent the percentage of cells in the indicated gates. Data represent two independent experiments. e, Day 8 Flt3L-treated bone marrow cultures were treated with no stimulation, agonistic αCD40 antibody, poly(I:C), or both. cDC2 surface expression of CD40 and CD70 were analyzed by flow cytometry after 24 h. Pre-gate: B220− Siglec H− MHC class II+ CD11c+ XCR1−Sirpα +. Numbers represent the percentage of cells in the indicated gates. Data represent two independent experiments. f, SDLN were harvested 24 h after injection and cDC2 were analyzed for surface expression of CD40 and CD70 by flow cytometry. Pre-gate: B220− CD326− MHC class IIhi CD11cint XCR1− Sirpα +. Numbers represent the percentage of cells in the indicated gates. Data represent two independent experiments.

Source data

Extended Data Fig. 2 Analysis of migratory and resident cDCs for tumor-antigen presentation.

a, Experimental setup for Fig. 1c, d and Extended Data Fig. 2c. Migratory and resident cDCs from TDLN of B6 WT day 4 tumor-bearing mice were isolated and cultured in vitro with naïve CTV-labeled OT-I CD8 T cells in the presence or absence of agonistic CD40 antibody. Proliferation of OT-Is was analyzed 72 hours later. b, Gating strategy for migratory and resident cDCs. c, Representative flow plots depicting CD44 surface expression and CTV dilution of OT-Is as described in a. d, Experimental setup for Fig. 4d and Extended Data Fig. 2e to assess whether resident cDCs access tumor antigens at late stages of tumor challenge and prime naïve CD8 T cells. Migratory and resident cDCs were isolated from TDLNs of WT (black), Cd40cKO (Xcr1Cre/+ Cd40fl/fl, green), Cd70cKO (Xcr1Cre/+ Cd70fl/fl, orange), or Ptgs2cKO (red) mice that were injected with 106 1956-mOVA 14 days previously, although only Cd40cKO mice still had tumor, and cultured in vitro with naïve CTV-labeled OT-I CD8 T cells. Proliferation of OT-Is was analyzed 72 hours later. e, Representative flow plots depicting CD44 surface expression and CTV dilution of OT-Is as described in d.

Extended Data Fig. 3 CD70 deficiency does not impair cDC1 development.

a, Percentages of splenic cDCs (left) and T and B cells (right) between WT (Xcr1+/+ Cd70fl/fl, black circles) and Cd70cKO (Xcr1Cre/+ Cd70fl/fl, orange circles) mice at homeostasis. b, Representative flow plots depicting CD70 expression in cDC1 (left) and cDC2 (right) from Flt3L-treated BM cultures of WT (top) and Cd70cKO (bottom) stimulated with αCD40 + poly(I:C). c, Representative flow plots showing CD70 and CD40 expression in migratory cDC1s of naive SDLN (left) and day 6 TDLNs (1969 fibrosarcoma, top; 1956-mOVA, bottom) of WT (Xcr1+/+ Cd40fl/fl) and Cd40cKO (Xcr1+/+ Cd40fl/fl) mice. d, Proliferation of CTV-labeled OT-I CD8 T cells adoptively transferred into WT (Xcr1+/+ Cd70fl/fl, black circles) or Cd70cKO (orange circles) mice was analyzed 72 h after immunization with OVA-loaded splenocytes. Data represent pooled biologically independent samples from two independent experiments (n = 4 for WT and Cd70cKO –OVA splenocytes, n = 5-6 for WT and Cd70cKO + OVA splenocytes). Data represented as mean +/− s.d. e, Cd40WT (Xcr1+/+ Cd40fl/fl), Cd40cKO, Cd70WT (Xcr1+/+ Cd70fl/fl), and Cd70cKO mice were injected with 106 1969 cells, and spleens were stained for the presence of mGpd2 tetramer+ CD8 T cells on day 10. Data represent pooled biologically independent samples from three independent experiments (n = 3 for naive, n = 5 for Cd40WT, n = 5 for Cd40cKO, n = 6 for Cd70WT, and n = 5 for Cd70cKO mice). f, Individual tumor curves of WT (Xcr1+/+ Cd70fl/fl), Cd40cKO, and Cd70cKO mice during primary and secondary implantation with 1956 progressor tumor. d: Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Extended Data Fig. 4 Early and late CD8 T cell responses support anti-tumor immunity.

a, Schematic diagrams of the depletion of CD8 T cells at early (red) or late (blue) time point during tumor response. b, T cell populations in peripheral blood after early or late CD8 T cell depletion. c, Tumor growth curves of mice depleted of CD8 T cells early (red) or later (blue) in tumor response as described in a. Data represented pooled biologically independent samples from two independent experiments (n = 5 for no depletion, n = 3 for early depletion, and n = 5 for late depletion) Data represented as mean +/− s.d.

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Extended Data Fig. 5 CD8 T cell responses in Cd27-/-, Tnfrsf9-/-, and Cd27-/- Tnfrsf9-/-mice.

a, Schematic diagram showing generation of Tnfrsf9-/-mice. CRISPR/Cas9 and sgRNAs were used to target the first coding exon, exon II, resulting in a Tnfrsf9 null gene via indel. b, WT, Cd27-/-, and Tnfrsf9-/- mice were injected with 106 1969 cells, and spleens were stained for the presence of mGpd2 tetramer+ CD8 T cells on day 10. Data represent pooled biologically independent samples from five independent experiments (n = 6 for naive, n = 9 for WT, n = 8 for Cd27-/-, n = 5 for Tnfrsf9-/- mice). Data are represented as mean values +/− s.d. **P = 0.0023; ns = not significant. c, CD127, CD44, and CD62L geometric mean MFI of SPLENIC SIINFEKL-Kb-tetramer+ CD8 T cells on d10 of 1956-mOVA in WT, Cd27-/-, Tnfrsf9-/-, and Cd27-/-Tnfrsf9-/- mice. Data represent pooled biologically independent samples from four independent experiments (n = 12 for WT, n = 10 for Cd27-/-, n = 6 for Tnfrsf9-/- mice, and n = 8 for Cd27-/-Tnfrsf9-/- mice). Data are represented as mean values +/− s.d. **P = 0.0094, ***P = 0.0004, ****P = < 0.0001, ns = not significant. d, Individual tumor curves of WT (Cd27-/-) and Cd27-/- mice during primary and secondary implantation with 1956 progressor tumor. b,c: Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Extended Data Fig. 6 cDC1s during homeostasis and tumor challenge in Cd40cKO and Cd70cKO mice.

a, Quantification of migratory cDC1 number in TDLNs of tumor-bearing Cd40WT (Xcr1+/+ Cd40fl/fl), Cd40cKO (Xcr1Cre/+ Cd40fl/fl), Cd70WT (Xcr1+/+ Cd70fl/fl), and Cd70cKO (Xcr1Cre/+ Cd70fl/fl) mice as depicted in Fig. 5c. Data represent pooled biologically independent samples from five independent experiments (n = 7-8 for all groups). Data are represented as mean values +/− s.d. **P = 0.0022; ns, not significant. b, Left, Representative flow plots of resident cDC1s (red boxes) and cDC2s (black boxes) in TDLNs of day 6 1956-mOVA-bearing Cd40WT (Xcr1+/+ Cd40fl/fl), Cd40cKO (Xcr1Cre/+ Cd40fl/fl), Cd70WT (Xcr1+/+ Cd70fl/fl), and Cd70cKO (Xcr1Cre/+ Cd70fl/fl) mice. Cells are pregated as B220− CD326−MHC-II+ CD11chi. Numbers are percentages of cells in the indicated gates. Right, quantification of cDC1s as a percentage of resident cDCs. Data represent pooled biologically independent samples from five independent experiments (n = 8 for all groups). Data are represented as mean values +/− s.d. c, Quantification of cDC1s as a percentage of splenic (left), SDLN migratory (middle), and SDLN resident (right) cDCs in WT (Xcr1+/+ Cd40fl/fl) and Cd40cKO mice at homeostasis. Data represent pooled biologically independent samples from five independent experiments (n = 7 for all groups). Data are represented as mean values +/− s.d. ns, not significant. d, Day 6 and Day 14 tumor areas of WT (Xcr1+/+ Cd70fl/fl) and Cd70cKO mice injected with 106 1956-mOVA cells. Data represent pooled biologically independent samples from three independent experiments (n = 6-8 for WT and n = 10 Cd70cKO mice). ns, not significant. e, Mitotracker Deep Red FM (right) and Mitotracker Green FM (right) geometric mean MFI in TDLN migratory cDC2s of d6 1956-mOVA-bearing Cd40WT,Cd40cKO, Cd70WT, and Cd70cKO mice. Data represent pooled biologically independent samples from two independent experiments (n = 4 for all groups). Data are represented as mean values +/− s.d. ns, not significant. f, WT (Xcr1+/+ Cd40fl/fl) and Cd40cKO mice were injected with 106 1956-mOVA cells. On day 6, mice were injected with the fluorescent activated poly-caspase probe FAM-FLIVO, and then TDLNs were harvested after 1 h. Quantification of FLIVO + cells in migratory and resident cDC1s of WT and Cd40cKO mice. Data represent pooled biologically independent samples from four independent experiments (n = 7 for WT and n = 8 for Cd40cKO mice). Data are represented as mean values +/− s.d. *P = 0.0340, ns = not significant. g-h, Basal (g) and maximal (h) OCR from extracellular flux analysis of cDC1s from WT (black), Cd40-/- (green), and Ptgs2cKO (red) Flt3L-treated bone marrow cultures. Data represent mean values of four biologically independent experiments. Data are represented as mean values +/− s.d. *P < 0.05. i, Enrichment analysis of mitochondrial complex I biogenesis genes in cDC1 in the absence (red) or presence (blue) of CD40 stimulation. a-b, d-g: Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. c: Mann–Whitney test.

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Extended Data Fig. 7 Bcl-xL rescue of CD40 deficiency in cDC1.

a, Flt3L-treated BM cultures from Cd40-/- mice were transduced with Bcl-xL, and cDC1s were sorted on day 10 of culture. Representative FACS plots depicting the full gating strategy (top) and the post-sort analysis for Bcl-xL-GFP+ expressing cDC1s (middle)and untransduced Bcl-xL-GFP− cDC1s (bottom) and. b, Flt3L-treated BM cultures from Cd40-/- mice were transduced with EV, and cDC1s were sorted on day 10 of culture. Representative FACS plots depicting the full gating strategy (top) and the post-sort analysis for EV-GFP+ expressing cDC1s (middle) and untransduced EV-GFP− cDC1s (bottom).

Extended Data Fig. 8 Conditional loss of Bcl-xL impairs survival of migratory, but not resident, cDC1.

a, Representative histogram of Bcl-xL staining in cDC2 from mesenteric lymph nodes of WT and BclxLcKO mice injected i.p. with PBS (black) or agonistic CD40 antibody (red, blue). b, Representative histograms of CD40 and MHC-II surface expression in migratory cDC1 of TDLN from tumor-bearing WT (Xcr1+/+ Bclxfl/fl), Cd40cKO (Xcr1Cre/+ Cd40fl/fl), and BclxLcKO (Xcr1Cre/+ Bclxfl/fl) mice. c-d, Quantification of migratory cDC1 as a percentage (c) and number (d) from TDLNs of day 6 tumor-bearing BclxLWT, BclxLcKO, Cd40WT, Cd40cKO, and Ptgs2cKO (Xcr1Cre/+ Ptgs2fl/fl) mice. Data represent pooled biologically independent samples from two independent experiments (n = 4-5 for BclxLWT, n = 8 for BclxLcKO, n = 5 for Cd40WT, n = 4 for Cd40cKO, and n = 2 for Ptgs2cKO). Data are represented as mean values +/− s.d. *P = 0.0249, 0.0240; ***P = 0.0001; *P = 0.0257; ns, not significant. e, Representative flow plots of resident cDC1s (red boxes) and cDC2s (black boxes) in TDLNs of day 6 1956-mOVA-bearing BclxLWT, BclxLcKO, Cd40WT, Cd40cKO, and Ptgs2cKO mice. Cells are pregated as B220− CD326−MHC-II+ CD11chi. Numbers are percentages of cells in the indicated gates. Data represent pooled biologically independent samples from two independent experiments. f-g, Quantification of resident cDC1s in e as a percentage (f) and number (g) from TDLNs of day 6 tumor-bearing BclxLWT, BclxLcKO, Cd40WT, Cd40cKO, and Ptgs2cKO mice. Data represent pooled biologically independent samples from two independent experiments (n = 4-5 for BclxLWT, n = 8 for BclxLcKO, n = 5 for Cd40WT, n = 4 for Cd40cKO, and n = 2 for Ptgs2cKO). Data are represented as mean values +/− s.d. ns, not significant. c,d,f,g: Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

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Wu, R., Ohara, R.A., Jo, S. et al. Mechanisms of CD40-dependent cDC1 licensing beyond costimulation. Nat Immunol 23, 1536–1550 (2022). https://doi.org/10.1038/s41590-022-01324-w

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