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As a cell-based cancer vaccine, dendritic cells (DC), derived from peripheral blood monocytes or bone marrow (BM) treated with GM-CSF (GMDC), were initially thought to induce antitumor immunity by presenting tumor antigens directly to host T cells. Subsequent work revealed that GMDCs do not directly prime tumor-specific T cells, but must transfer their antigens to host DCs. This reduces their advantage over strictly antigen-based strategies proposed as cancer vaccines. Type 1 conventional DCs (cDC1) have been reported to be superior to GMDCs as a cancer vaccine, but whether they act by transferring antigens to host DCs is unknown. To test this, we compared antitumor responses induced by GMDCs and cDC1 in Irf8 +32–/– mice, which lack endogenous cDC1 and cannot reject immunogenic fibrosarcomas. Both GMDCs and cDC1 could cross-present cell-associated antigens to CD8+ T cells in vitro. However, injection of GMDCs into tumors in Irf8 +32–/– mice did not induce antitumor immunity, consistent with their reported dependence on host cDC1. In contrast, injection of cDC1s into tumors in Irf8 +32–/– mice resulted in their migration to tumor-draining lymph nodes, activation of tumor-specific CD8+ T cells, and rejection of the tumors. Tumor rejection did not require the in vitro loading of cDC1 with antigens, indicating that acquisition of antigens in vivo is sufficient to induce antitumor responses. Finally, cDC1 vaccination showed abscopal effects, with rejection of untreated tumors growing concurrently on the opposite flank. These results suggest that cDC1 may be a useful future avenue to explore for antitumor therapy.See related Spotlight by Hubert et al., p. 918

Supplementary Figure from cDC1 Vaccines Drive Tumor Rejection by Direct Presentation Independently of Host cDC1

The divergence of the common dendritic cell progenitor

The events that initiate autoimmune diabetes in nonobese diabetic (NOD) mice remain poorly understood. CD4 + and CD8 + T cells are both required to develop disease, but their relative roles in initiating disease are unclear. To test whether CD4 + T cell infiltration into islets requires damage to β cells induced by autoreactive CD8 + T cells, we inactivated Wdfy4 in nonobese diabetic (NOD) mice (NOD. Wdfy4 −/−- ) using CRISPR/Cas9 targeting to eliminate cross-presentation by type 1 conventional dendritic cells (cDC1s). Similar to C57BL/6 Wdfy4 −/− mice, cDC1 in NOD. Wdfy4 −/− mice are unable to cross-present cell-associated antigens to prime CD8 + T cells, while cDC1 from heterozygous NOD. Wdfy4 +/− mice cross-present normally. Further, NOD. Wdfy4 −/− mice fail to develop diabetes while heterozygous NOD. Wdfy4 +/− mice develop diabetes similarly to wild-type NOD mice. NOD. Wdfy4 −/− mice remain capable of processing and presenting major histocompatibility complex class II (MHC-II)-restricted autoantigens and can activate β cell-specific CD4 + T cells in lymph nodes. However, disease in these mice does not progress beyond peri-islet inflammation. These results indicate that the priming of autoreactive CD8 + T cells in NOD mice requires cross-presentation by cDC1. Further, autoreactive CD8 + T cells appear to be required not only to develop diabetes, but to recruit autoreactive CD4 + T cells into islets of NOD mice, perhaps in response to progressive β cell damage.

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.

The transcriptional repressor Bcl6 has been reported as required for development of a subset of classical dendritic cell (cDCs) called cDC1, which is responsible for cross-presentation. However, mechanisms and in vivo functional analysis have been lacking. We generated a system for conditional deletion of Bcl6 in mouse cDCs. We confirmed the reported in vitro requirement for Bcl6 in cDC1 development and the general role for Bcl6 in cDC development in competitive settings. However, deletion of Bcl6 did not abrogate the in vivo development of cDC1. Instead, Bcl6 deficiency caused only a selective reduction in CD8α expression by cDC1 without affecting XCR1 or CD24 expression. Normal cDC1 development was confirmed in Bcl6cKO mice by development of XCR1+ Zbtb46-GFP+ cDC1 by rejection of syngeneic tumors and by priming of tumor-specific CD8 T cells. In summary, Bcl6 regulates a subset of cDC1-specific markers and is required in vitro but not in vivo for cDC1 development.

The events that initiate autoimmune diabetes in NOD mice remain poorly understood. CD4 and CD8 T cells are both required but whether either cell initiates disease is unclear. To test whether CD4 T cell infiltration into islet required damage to β cells induced by autoreactive CD8 T cells, we selectively inactivated Wdfy4 in NOD mice (NOD.Wdfy4-/-) using CRISPR/Cas9 targeting. Similar to C57BL/6 Wdfy4-/- mice NOD.Wdfy4-/- mice develop type 1 conventional dendritic cells (cDC1) that are unable to cross-present cell-associated antigens required to activate CD8 T cells. By contrast, cDC1 from heterozygous Wdfy4+/- mice can cross-present normally. Heterozygous NOD.Wdfy4+/- mice develop diabetes similar to NOD mice, but NOD.Wdfy4-/- mice neither develop diabetes nor prime autoreactive CD8 T cells in vivo. By contrast, NOD.Wdfy4-/- mice can process and present MHC-II-restricted autoantigens and can activate β cell specific CD4 T cells in lymph nodes, and yet do not develop CD4 T cell infiltration in islets. These results indicate that the priming of autoreactive CD8 T cells in NOD mice requires cross-presentation by cDC1. Further, autoreactive CD8 T cells are required not only to develop diabetes, but to recruit autoreactive CD4 T cells into islets of NOD mice, perhaps in response to progressive β cell damage.

Conventional type 1 dendritic cells (cDC1)1 are thought to perform antigen cross-presentation, which is required to prime CD8+ T cells2,3, whereas cDC2 are specialized for priming CD4+ T cells4,5. CD4+ T cells are also considered to help CD8+ T cell responses through a variety of mechanisms6–11, including a process whereby CD4+ T cells ‘license’ cDC1 for CD8+ T cell priming12. However, this model has not been directly tested in vivo or in the setting of help-dependent tumour rejection. Here we generated an Xcr1Cre mouse strain to evaluate the cellular interactions that mediate tumour rejection in a model requiring CD4+ and CD8+ T cells. As expected, tumour rejection required cDC1 and CD8+ T cell priming required the expression of major histocompatibility class I molecules by cDC1. Unexpectedly, early priming of CD4+ T cells against tumour-derived antigens also required cDC1, and this was not simply because they transport antigens to lymph nodes for processing by cDC2, as selective deletion of major histocompatibility class II molecules in cDC1 also prevented early CD4+ T cell priming. Furthermore, deletion of either major histocompatibility class II or CD40 in cDC1 impaired tumour rejection, consistent with a role for cognate CD4+ T cell interactions and CD40 signalling in cDC1 licensing. Finally, CD40 signalling in cDC1 was critical not only for CD8+ T cell priming, but also for initial CD4+ T cell activation. Thus, in the setting of tumour-derived antigens, cDC1 function as an autonomous platform capable of antigen processing and priming for both CD4+ and CD8+ T cells and of the direct orchestration of their cross-talk that is required for optimal anti-tumour immunity. Conventional type 1 dendritic cells perform antigen processing and priming of CD4+ and CD8+ T cells against tumour antigens, orchestrating their cross-talk to effect anti-tumour immunity.

Last year was the 10th anniversary of Ralph Steinman’s Nobel Prize awarded for his discovery of dendritic cells (DCs), while next year brings the 50th anniversary of that discovery. Current models of anti-viral and anti-tumor immunity rest solidly on Steinman’s discovery of DCs, but also rely on two seemingly unrelated phenomena, also reported in the mid-1970s: the discoveries of “help” for cytolytic T cell responses by Cantor and Boyse in 1974 and “cross-priming” by Bevan in 1976. Decades of subsequent work, controversy, and conceptual changes have gradually merged these three discoveries into current models of cell-mediated immunity against viruses and tumors.

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

As a cell-based cancer vaccine, dendritic cells (DC), derived from peripheral blood monocytes or bone marrow (BM) treated with GM-CSF (GMDC), were initially thought to induce antitumor immunity by presenting tumor antigens directly to host T cells. Subsequent work revealed that GMDCs do not directly prime tumor-specific T cells, but must transfer their antigens to host DCs. This reduces their advantage over strictly antigen-based strategies proposed as cancer vaccines. Type 1 conventional DCs (cDC1) have been reported to be superior to GMDCs as a cancer vaccine, but whether they act by transferring antigens to host DCs is unknown. To test this, we compared antitumor responses induced by GMDCs and cDC1 in Irf8 +32–/– mice, which lack endogenous cDC1 and cannot reject immunogenic fibrosarcomas. Both GMDCs and cDC1 could cross-present cell-associated antigens to CD8+ T cells in vitro. However, injection of GMDCs into tumors in Irf8 +32–/– mice did not induce antitumor immunity, consistent with their reported dependence on host cDC1. In contrast, injection of cDC1s into tumors in Irf8 +32–/– mice resulted in their migration to tumor-draining lymph nodes, activation of tumor-specific CD8+ T cells, and rejection of the tumors. Tumor rejection did not require the in vitro loading of cDC1 with antigens, indicating that acquisition of antigens in vivo is sufficient to induce antitumor responses. Finally, cDC1 vaccination showed abscopal effects, with rejection of untreated tumors growing concurrently on the opposite flank. These results suggest that cDC1 may be a useful future avenue to explore for antitumor therapy. See related Spotlight by Hubert et al., p. 918

SummaryShortly after priming, the fate of activated CD4 T cells is segregated into BCL6+ follicular helper T (Tfh) and BCL6− effector (Teff) cells. However, it remains unknown how these subsets are sustained in the presence of chronic antigen stimulation. Using a combination of single cell- and population-based approaches, we show that in chronic viral infection, activated CD4 T cells differentiate into BCL6-dependent TCF-1+ progenitor cells with superior capacity to expand and give rise to both Teff and Tfh. They share properties with progenitor-exhausted CD8 T cells and are required for the continued generation of Teff cells as antigen persists. In response to tumors, an analogous CD4 T cell population develops in draining lymph nodes. Our study reveals the heterogeneity and plasticity of CD4 T cells upon encountering persistent antigen and highlights their population dynamics through a stable bipotent intermediate state.

Soon after activation, CD4

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification. The transcription factor IRF8 is essential for classical type 1 dendritic cell (cDC1) development. Murphy and colleagues investigate in detail the molecular control of cDC1 fate specification by systematically unpicking the IRF8 enhancer regions.

CD40 signaling in conventional 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 cDC1 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 apoptosis in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation, which were reversed by re-expression of Bcl-xL. Thus, CD40 signaling in cDC1 not only induces co-stimulatory ligands for CD8 T cells, but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses. This work was supported by grants from the NIH (R01AI150297, R01CA248919, and R21AI164142 to K.M.M., and F30CA247262 to R.W.).

Efforts to determine the unique functions of the two types of classical dendritic cells, cDC1 and cDC2, are hindered by limited understanding of the divergence of the common dendritic cell progenitor (CDP). Some transcription factors act in commitment of already specified progenitors, such as Batf3 which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer, but how other factors control CDP divergence remains unknown. Here, we report the transcriptional mechanism of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis suggested that Nfil3 acts upstream of Id2, Batf3, and Zeb2 in cDC1 development but has not revealed its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and ChIP-seq analysis identified NFIL3 binding in the −165 kb Zeb2 enhancer at sites that also bind CCAAT-enhancer-binding proteins (C/EBPs). Mutation of these NFIL3/C/EBP sites by in vivo CRISPR/Cas9 targeting revealed functional redundancy, with C/EBPs and NFIL3 competing in binding these sites to support or repress Zeb2 expression, respectively. Mutation of these three NFIL3/C/EBP sites eliminated Zeb2 expression in myeloid, but not lymphoid progenitors, producing mice that completely lacked pre-cDC2 specification and mature cDC2 in vivo. These mice failed to make an appropriate TH2 response against H. polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths. Notably, Zeb2 expression is not required to maintain cDC2 identity, but acts only to block cDC1 specification. Thus, the competition between C/EBPs and NFIL3 binding at the −165 kb Zeb2 enhancer determines CDP divergence.

Background Treatment options are limited for patients with advanced melanoma who have progressed on checkpoint inhibitors and targeted therapies such as BRAF/MEK inhibitors (if BRAF-V600E mutated). Adoptive cell therapy utilizing tumor-infiltrating lymphocytes (TIL) has shown antitumor efficacy with durable responses in heavily pretreated melanoma patients. Safety and efficacy of lifileucel, a centrally manufactured cryopreserved autologous TIL therapy assessed by both investigator and an independent review committee (IRC), are presented. Methods C-144-01 is a global Phase 2 open-label, multicenter study of the safety and efficacy of lifileucel in patients with unresectable metastatic melanoma. We report on Cohort 2 (N = 66) patients with Stage IIIC/IV unresectable melanoma who received lifileucel. Tumors resected at local institutions were processed in central GMP facilities for TIL production in a 22-day process. Final TIL infusion product was cryopreserved and shipped to sites. Patients received one week of cyclophosphamide/fludarabine preconditioning lymphodepletion, a single lifileucel infusion, followed by up to 6 doses of IL-2. All responses were assessed by RECIST 1.1. Results 66 patients had the following baseline characteristics: 3.3 mean prior therapies (anti-PD1 100%; anti-CTLA-4 80%; BRAF/MEK inhibitor 23%), relatively high tumor burden (106 mm mean target lesion sum of diameters), 44% with liver and/or brain lesions, median LDH 244 U/L. Objective Response Rate (ORR) by investigator was 36.4% (2 CR, 22 PR, 1 previously confirmed PR is now changed to SD) and Disease Control Rate (DCR) of 80.3%. At a median follow up of 9.7 months, median Duration of Response (DOR) has not been reached. The adverse event profile was generally consistent with the underlying advanced disease and the profile of the lymphodepletion and IL-2 regimens. The ORR per IRC was 34.8% (2 CR, 21 PR) and DCR was 72.7%. At a median follow up of 6.9 months, the median IRC DOR has not been reached. Overall concordance rate of investigator and IRC read of response was 89.4%. The concordance compares favorably with literature reports in a metastatic disease.1 Conclusions Lifileucel treatment resulted in a 36.4% ORR in heavily pretreated metastatic melanoma patients with high baseline disease burden who had received prior anti-PD1 and BRAF/MEK inhibitors, if tumor BRAF mutated. The high concordance of 89.4% between investigator and IRC confirms the original assessment of lifileucel efficacy in metastatic melanoma.2 Acknowledgements The authors would like to thank the patients and their families for participation in the study. The authors would also like to acknowledge the support and dedication of all investigators and site team members from all participating clinical trial institutions. Trial Registration ClinicalTrials. gov Identifier: NCT02360579 Ethics Approval Ethics Approval This trial was approved by Western Institutional Review Board - IRB Tracking Number: 20160198. References Ghiorghiu DC, et al. Comparison of central and site review of RECIST data in an open randomised phase II trial in advanced melanoma. 10.1594.ecr 2009/C-075. Sarnaik A, et al. Safety and efficacy of cryopreserved autologous tumor infiltrating lymphocyte therapy (LN-144, lifileucel) in advanced metastatic melanoma patients who progressed on multiple prior therapies including anti-PD-1. J Clin Oncol 2019;37:2518–2518.

Conventional type 1 dendritic cells (cDC1)

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx+ colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx+ CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx+ CARSCs. This system can reenact the “homeostasis-injury-regeneration” cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.

10006 Background: Treatment options are limited for patients with advanced melanoma who have progressed on checkpoint inhibitors and targeted therapies. Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) leverages and enhances the body’s natural defense against cancer and has shown durable responses in heavily pretreated melanoma patients. Methods: C-144-01 is a global Phase 2 open-label, multicenter study of efficacy & safety of lifileucel in patients with unresectable metastatic melanoma who have progressed on checkpoint inhibitors and BRAF/MEK inhibitors, if BRAFv600 mutant. We report on Cohort 2 (N = 66) patients who have received TIL. Tumors were resected at local institutions, processed in central GMP facilities for TIL production, manufactured, cryopreserved & shipped back to sites in a 22-day process. Therapy consisted of one week of lymphodepletion, a single lifileucel infusion, and up to 6 IL-2 doses. ORR was based on RECIST v1.1 by investigator assessment. Data cutoff was Feb 2, 2020. Results: Baseline characteristics: 3.3 mean prior therapies (anti-PD1 100%; anti-CTLA-4 80%; BRAF/MEK inhibitor 23%), high baseline tumor burden (106 mm mean target lesion sum of diameters), 44% liver/brain lesions, 40.9% LDH > ULN. ORR by investigator was 36.4% (2 CR, 22 PR) and DCR was 80.3%. Mean time to response was 1.9 months (range: 1.3-5.6). After a median study follow-up of 17.0 months, median DOR (mDOR) was still not reached. Six responders have progressed, 2 have died and 2 started other anti-cancer therapy without progression. The adverse event profile was consistent with the underlying advanced disease and the lymphodepletion and IL-2 regimens. Additional follow-up data will be available for presentation. Conclusions: Lifileucel treatment results in a 36.4% ORR and mDOR was not reached at 17.0 months of median study follow up in a heavily pretreated metastatic melanoma patients with high baseline disease burden who progressed on multiple prior therapies, including anti-PD1 and BRAF/MEK inhibitors, if BRAFv600 mutant. Clinical trial information: NCT02360579.

CD4 T cells are thought to help promote anti-tumour responses by ‘licensing’ antigen presenting cells (APCs) that activate CD8 T cells. Conventional type 1 dendritic cells (cDC1s) are responsible for cross-presentation of tumour-derived antigens to CD8 T cells. Prevailing models presume that the cDC1 is licensed by CD4 T cells that are themselves activated by a distinct cDC subset, the cDC2. The recent finding that neoantigens presented by major histocompatibility complex (MHC) class II molecules can promote rejection of tumours that lack MHC class II (MHC-II) surface expression is consistent with an indirect action of CD4 T cells, such as cDC1 licensing. However, no study has directly identified the APC that primes the CD4 T cells responsible for licensing or clearly identified the target of CD4 licensing in vivo. Here, we generated cDC1-specific Cre expressing mouse strain to inactivate or induce expression of MHC-I, MHC-II, or CD40 specifically within the cDC1 lineage. Using a tumour model that relies on CD8 T cells and CD4 T cells for rejection, we discovered that priming of CD4 T cells against tumour-derived antigens, in contrast to soluble antigens, relied overwhelmingly on the cDC1 and not the cDC2. cDC1 do not simply transport antigen to lymph nodes for processing by cDC2, since lack of MHC-II expression on cDC1 prevented CD4 T cell priming. We also found that CD40 signaling not only affects licensing of cDC1 for CD8 T cell priming, but is also critical for the activation of CD4 T cells. Thus, in the setting of tumour-derived antigens, cDC1 function as an autonomous platform capable of priming both CD4 and CD8 T cells and orchestrating their cross-talk required for optimal anti-tumour immunity.

T follicular helper (Tfh) cells are a subset of CD4+ T cells that promote antibody production during vaccination. Conventional dendritic cells (cDCs) efficiently prime Tfh cells; however, conclusions regarding which cDC instructs Tfh cell differentiation have differed between recent studies. We found that these discrepancies might exist because of the unusual sites used for immunization in murine models, which differentially bias which DC subsets access antigen. We used intranasal immunization as a physiologically relevant route of exposure that delivers antigen to all tissue DC subsets. Using a combination of mice in which the function of individual DC subsets is impaired and different antigen formulations, we determined that CD11b+ migratory type 2 cDCs (cDC2s) are necessary and sufficient for Tfh induction. DC-specific deletion of the guanine nucleotide exchange factor DOCK8 resulted in an isolated loss of CD11b+ cDC2, but not CD103+ cDC1, migration to lung-draining lymph nodes. Impaired cDC2 migration or development in DC-specific Dock8 or Irf4 knockout mice, respectively, led to reduced Tfh cell priming, whereas loss of CD103+ cDC1s in Batf3−/− mice did not. Loss of cDC2-dependent Tfh cell priming impaired antibody-mediated protection from live influenza virus challenge. We show that migratory cDC2s uniquely carry antigen into the subanatomic regions of the lymph node where Tfh cell priming occurs—the T-B border. This work identifies the DC subset responsible for Tfh cell–dependent antibody responses, particularly when antigen dose is limiting or is encountered at a mucosal site, which could ultimately inform the formulation and delivery of vaccines.

T follicular helper (Tfh) cells are a subset of CD4(+) T cells that promote antibody production during vaccination. Conventional dendritic cells (cDCs) efficiently prime Tfh cells; however, conclusions regarding which cDC instructs Tfh cell differentiation have differed between recent studies. We found that these discrepancies might exist because of the unusual sites used for immunization in murine models, which differentially bias which DC subsets access antigen. We used intranasal immunization as a physiologically relevant route of exposure that delivers antigen to all tissue DC subsets. Using a combination of mice in which the function of individual DC subsets is impaired and different antigen formulations, we determined that CD11b(+) migratory type 2 cDCs (cDC2s) are necessary and sufficient for Tfh induction. DC-specific deletion of the guanine nucleotide exchange factor DOCK8 resulted in an isolated loss of CD11b(+) cDC2, but not CD103(+) cDC1, migration to lung-draining lymph nodes. Impaired cDC2 migration or development in DC-specific Dock8 or Irf4 knockout mice, respectively, led to reduced Tfh cell priming, whereas loss of CD103(+) cDC1s in Batf3(−/−) mice did not. Loss of cDC2-dependent Tfh cell priming impaired antibody-mediated protection from live influenza virus challenge. We show that migratory cDC2s uniquely carry antigen into the subanatomic regions of the lymph node where Tfh cell priming occurs—the T-B border. This work identifies the DC subset responsible for Tfh cell–dependent antibody responses, particularly when antigen dose is limiting or is encountered at a mucosal site, which could ultimately inform the formulation and delivery of vaccines.

Dendritic cells (DCs) are required to prime T cells during an immune response. The dogma in the field is that Batf3-dependent DCs (DC1s) prime CD8 T cells and IRF4-dependent DCs (DC2s) prime CD4 T cells. However, recent studies have shown that DC1s are required for induction of Th1 immune responses and to prime autroreactive CD4 T cells in the NOD mouse model of diabetes. Therefore, this segregation of priming theory is flawed. To further study the contribution of DC1s to CD4 T cell priming, we generated an XCR1-cre mouse and crossed it to the MHC class II floxed mouse to generate MHC class II deficient DC1s. We found that the form of antigen directs the priming of T cells. DC2s are superior at presenting soluble antigen as evidenced by normal CD4 and CD8 OVA specific T cell responses in mice lacking DC1s. However, DC1s are superior at presenting cell-associated antigen as evidenced by a lack of OVA specific CD4 and CD8 T cell priming in mice lacking DC1s. Furthermore, CD4 priming during tumor immune responses is absent when DC1s lack MHC class II. Our findings show that the DC subsets differ in the form of antigen that they present. DC1s present cell-associated material; whereas DC2s present soluble antigen.

Activation-induced cytidine deaminase (AID), the enzyme- mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID(+) immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells. United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Allergy & Infectious Diseases (NIAID) (AI061093) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Allergy & Infectious Diseases (NIAID) (AI071087) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Allergy & Infectious Diseases (NIAID) (AI082713) INSERM, CEE EUROPAD-contract 7th Framework Program (201549) Assiociation Contre le Cancer Rubicon program, Netherlands Organization for Scientific Research United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Allergy & Infectious Diseases (NIAID) (R01AI071087) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Allergy & Infectious Diseases (NIAID) (P01AI061093) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of Allergy & Infectious Diseases (NIAID) (U19AI082713)

Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naive T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.