7 months ago
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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.
References
Cantor, H. & Boyse, E. A. Functional subclasses of T lymphocytes bearing different Ly antigens. II. Cooperation between subclasses of Ly+ cells in the generation of killer activity. J. Exp. Med 141, 1390–1399 (1975).
Clark, E. A. & Ledbetter, J. A. Activation of human B cells mediated through two distinct cell surface differentiation antigens, Bp35 and Bp50. Proc. Natl Acad. Sci. USA 83, 4494–4498 (1986).
Armitage, R. J. et al. Molecular and biological characterization of a murine ligand for CD40. Nature 357, 80–82 (1992).
Spriggs, M. K. et al. Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion. J. Exp. Med 176, 1543–1550 (1992).
Hollenbaugh, D. et al. The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EMBO J. 11, 4313–4321 (1992).
Bennett, S. R. et al. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393, 478–480 (1998).
Schoenberger, S. P. et al. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393, 480–483 (1998).
Ridge, J. P., Di Rosa, F. & Matzinger, P. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393, 474–478 (1998).
Bourgeois, C., Rocha, B. & Tanchot, C. A role for CD40 expression on CD8+ T cells in the generation of CD8+ T cell memory. Science 297, 2060–2063 (2002).
Lee, B. O., Hartson, L. & Randall, T. D. CD40-deficient, influenza-specific CD8 memory T cells develop and function normally in a CD40-sufficient environment. J. Exp. Med 198, 1759–1764 (2003).
Sun, J. C. & Bevan, M. J. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300, 339–342 (2003).
Smith, C. M. et al. Cognate CD4+ T cell licensing of dendritic cells in CD8+ T cell immunity. Nat. Immunol. 5, 1143–1148 (2004).
Eickhoff, S. et al. Robust anti-viral immunity requires multiple distinct T cell-dendritic cell interactions. Cell 162, 1322–1337 (2015).
Hor, J. L. et al. Spatiotemporally distinct interactions with dendritic cell subsets facilitates CD4+ and CD8+ T cell activation to localized viral infection. Immunity 43, 554–565 (2015).
Hildner, K. et al. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science 322, 1097–1100 (2008).
Theisen, D. J. et al. WDFY4 is required for cross-presentation in response to viral and tumor antigens. Science 362, 694–699 (2018).
Ferris, S. T. et al. cDC1 prime and are licensed by CD4+ T cells to induce anti-tumour immunity. Nature 584, 624–629 (2020).
Kawabe, T. et al. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1, 167–178 (1994).
Renshaw, B. R. et al. Humoral immune responses in CD40 ligand-deficient mice. J. Exp. Med 180, 1889–1900 (1994).
Xu, J. et al. Mice deficient for the CD40 ligand. Immunity 1, 423–431 (1994).
Ahonen, C. et al. The CD40-TRAF6 axis controls affinity maturation and the generation of long-lived plasma cells. Nat. Immunol. 3, 451–456 (2002).
Gallagher, E. et al. Kinase MEKK1 is required for CD40-dependent activation of the kinases Jnk and p38, germinal center formation, B cell proliferation and antibody production. Nat. Immunol. 8, 57–63 (2007).
Luo, W., Weisel, F. & Shlomchik, M. J. B cell receptor and CD40 signaling are rewired for synergistic induction of the c-Myc transcription factor in germinal center B cells. Immunity 48, 313–326 (2018).
Laidlaw, B. J. & Cyster, J. G. Transcriptional regulation of memory B cell differentiation. Nat. Rev. Immunol. 21, 209–220 (2021).
Feau, S. et al. The CD4+ T-cell help signal is transmitted from APC to CD8+ T-cells via CD27-CD70 interactions. Nat. Commun. 3, 948 (2012).
Borst, J. et al. CD4+ T cell help in cancer immunology and immunotherapy. Nat. Rev. Immunol. 18, 635–647 (2018).
Bullock, T. N. & Yagita, H. Induction of CD70 on dendritic cells through CD40 or TLR stimulation contributes to the development of CD8+ T cell responses in the absence of CD4+ T cells. J. Immunol. 174, 710–717 (2005).
Ardouin, L. et al. Broad and largely concordant molecular changes characterize tolerogenic and immunogenic dendritic cell maturation in thymus and periphery. Immunity 45, 305–318 (2016).
Munitic, I. et al. CD70 deficiency impairs effector CD8 T cell generation and viral clearance but is dispensable for the recall response to lymphocytic choriomeningitis virus. J. Immunol. 190, 1169–1179 (2013).
Hendriks, J. et al. CD27 is required for generation and long-term maintenance of T cell immunity. Nat. Immunol. 1, 433–440 (2000).
Ahrends, T. et al. CD4+ T cell help confers a cytotoxic T cell effector program including coinhibitory receptor downregulation and increased tissue invasiveness. Immunity 47, 848–861 (2017).
Oba, T. et al. A critical role of CD40 and CD70 signaling in conventional type 1 dendritic cells in expansion and antitumor efficacy of adoptively transferred tumor-specific T cells. J. Immunol. 205, 1867–1877 (2020).
Theisen, D. J. et al. Batf3-dependent genes control tumor rejection induced by dendritic cells independently of cross-presentation. Cancer Immunol. Res. 7, 29–39 (2019).
Zelenay, S. et al. Cyclooxygenase-dependent tumor growth through evasion of immunity. Cell 162, 1257–1270 (2015).
Ishikawa, T. O. & Herschman, H. R. Conditional knockout mouse for tissue-specific disruption of the cyclooxygenase-2 (Cox-2) gene. Genesis 44, 143–149 (2006).
Ruhland, M. K. et al. Visualizing synaptic transfer of tumor antigens among dendritic cells. Cancer Cell 37, 786–799 (2020).
Scharping, N. E. et al. The tumor microenvironment represses T cell mitochondrial biogenesis to drive intratumoral T cell metabolic insufficiency and dysfunction. Immunity 45, 374–388 (2016).
Daniels, N. J. et al. Antigen-specific cytotoxic T lymphocytes target airway CD103+ and CD11b+ dendritic cells to suppress allergic inflammation. Mucosal Immunol. 9, 229–239 (2016).
Darzynkiewicz, Z. et al. Fluorochrome-labeled inhibitors of caspases: expedient in vitro and in vivo markers of apoptotic cells for rapid cytometric analysis. Methods Mol. Biol. 1644, 61–73 (2017).
Kim, S. et al. High amount of transcription factor IRF8 engages AP1-IRF composite elements in enhancers to direct type 1 conventional dendritic cell identity. Immunity 53, 1–16 (2020).
Zhang, N. & He, Y. W. The antiapoptotic protein Bcl-xL is dispensable for the development of effector and memory T lymphocytes. J. Immunol. 174, 6967–6973 (2005).
Ohl, L. et al. CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity 21, 279–288 (2004).
Ryan, E. P. et al. Activated human B lymphocytes express cyclooxygenase-2 and cyclooxygenase inhibitors attenuate antibody production. J. Immunol. 174, 2619–2626 (2005).
Remes Lenicov, F. et al. Prostaglandin E2 antagonizes TGF-beta actions during the differentiation of monocytes into dendritic cells. Front Immunol. 9, 1441 (2018).
Krause, P. et al. Prostaglandin E(2) enhances T-cell proliferation by inducing the costimulatory molecules OX40L, CD70, and 4-1BBL on dendritic cells. Blood 113, 2451–2460 (2009).
Sreeramkumar, V., Fresno, M. & Cuesta, N. Prostaglandin E2 and T cells: friends or foes? Immunol. Cell Biol. 90, 579–586 (2012).
Yu, Y. et al. Targeted cyclooxygenase gene (ptgs) exchange reveals discriminant isoform functionality. J. Biol. Chem. 282, 1498–1506 (2007).
Matsue, H. et al. Dendritic cells undergo rapid apoptosis in vitro during antigen-specific interaction with CD4+ T cells. J. Immunol. 162, 5287–5298 (1999).
Bjorck, P., Banchereau, J. & Flores-Romo, L. CD40 ligation counteracts Fas-induced apoptosis of human dendritic cells. Int Immunol. 9, 365–372 (1997).
Lundqvist, A. et al. Mature dendritic cells are protected from Fas/CD95-mediated apoptosis by upregulation of Bcl-X(L). Cancer Immunol. Immunother. 51, 139–144 (2002).
Miga, A. J. et al. Dendritic cell longevity and T cell persistence is controlled by CD154-CD40 interactions. Eur. J. Immunol. 31, 959–965 (2001).
Riol-Blanco, L. et al. Immunological synapse formation inhibits, via NF-kappaB and FOXO1, the apoptosis of dendritic cells. Nat. Immunol. 10, 753–760 (2009).
Chen, M. et al. Dendritic cell apoptosis in the maintenance of immune tolerance. Science 311, 1160–1164 (2006).
Chen, M. et al. Regulation of the lifespan in dendritic cell subsets. Mol. Immunol. 44, 2558–2565 (2007).
Chen, M., Huang, L. & Wang, J. Deficiency of Bim in dendritic cells contributes to overactivation of lymphocytes and autoimmunity. Blood 109, 4360–4367 (2007).
Zhang, X. et al. Up-regulation of Bcl-xL expression protects CD40-activated human B cells from Fas-mediated apoptosis. Cell Immunol. 173, 149–154 (1996).
Grillot, D. A. M. et al. bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice. J. Exp. Med. 183, 381–391 (1996).
Chao, D. T. & Korsmeyer, S. J. BCL-2 family: regulators of cell death. Annu. Rev. Immunol. 16, 395–419 (1998).
Nopora, K. et al. MHC class I cross-presentation by dendritic cells counteracts viral immune evasion. Front Immunol. 3, 348 (2012).
Pirtskhalaishvili, G. et al. Cytokine-mediated protection of human dendritic cells from prostate cancer-induced apoptosis is regulated by the Bcl-2 family of proteins. Br. J. Cancer 83, 506–513 (2000).
Pirtskhalaishvili, G. et al. Transduction of dendritic cells with Bcl-xL increases their resistance to prostate cancer-induced apoptosis and antitumor effect in mice. J. Immunol. 165, 1956–1964 (2000).
Borrow, P. et al. CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8+ CTL response. J. Exp. Med 183, 2129–2142 (1996).
Borrow, P. et al. CD40 ligand-mediated interactions are involved in the generation of memory CD8+ cytotoxic T lymphocytes (CTL) but are not required for the maintenance of CTL memory following virus infection. J. Virol. 72, 7440–7449 (1998).
Hernandez, M. G., Shen, L. & Rock, K. L. CD40 on APCs is needed for optimal programming, maintenance, and recall of CD8+ T cell memory even in the absence of CD4+ T cell help. J. Immunol. 180, 4382–4390 (2008).
Taraban, V. Y. et al. Requirement for CD70 in CD4+ Th cell-dependent and innate receptor-mediated CD8+ T cell priming. J. Immunol. 177, 2969–2975 (2006).
Ahrends, T. et al. CD27 agonism plus PD-1 blockade recapitulates CD4+ T-cell help in therapeutic anticancer vaccination. Cancer Res. 76, 2921–2931 (2016).
Lybarger, L. et al. Virus subversion of the MHC class I peptide-loading complex. Immunity 18, 121–130 (2003).
Diamond, M. S. et al. Type I interferon is selectively required by dendritic cells for immune rejection of tumors. J. Exp. Med. 208, 1989–2003 (2011).
Koebel, C. M. et al. Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450, 903–907 (2007).
Toebes, M. et al. Design and use of conditional MHC class I ligands. Nat. Med. 12, 246–251 (2006).
Andersen, R. S. et al. Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers. Nat. Protoc. 7, 891–902 (2012).
Kretzer, N. M. et al. RAB43 facilitates cross-presentation of cell-associated antigens by CD8alpha + dendritic cells. J. Exp. Med 213, 2871–2883 (2016).
Ouyang, W. et al. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism. Immunity 9, 745–755 (1998).
Cheng, E. H. et al. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell 8, 705–711 (2001).
Choi, K. et al. A common precursor for hematopoietic and endothelial cells. Development 125, 725–732 (1998).
Anderson, D. A. III et al. The MYCL and MXD1 transcription factors regulate the fitness of murine dendritic cells. Proc. Natl Acad. Sci. USA 117, 4885–4893 (2020).
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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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.
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.
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.
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.
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.
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 (2022). https://doi.org/10.1038/s41590-022-01324-w
Received: 21 December 2021
Accepted: 07 September 2022
Published: 21 October 2022
DOI: https://doi.org/10.1038/s41590-022-01324-w
