T-cell expression of CXCL13 is associated with immunotherapy response in a sex-dependent manner in patients with lung cancer.

Emerging evidence in preclinical models demonstrates that antitumor immunity is not equivalent between males and females. However, more investigation in patients and across a wider range of cancer types is needed to fully understand sex as a variable in tumor immune responses. We investigated differences in T-cell responses between male and female patients with lung cancer by performing sex-based analysis of single cell transcriptomic datasets. We found that the transcript encoding CXC motif chemokine ligand 13 (CXCL13), which has recently been shown to correlate with T-cell tumor specificity, is expressed at greater levels in T cells isolated from female compared to male patients. Furthermore, increased expression of CXCL13 was associated with response to PD-1-targeting immunotherapy in female but not male patients. These findings suggest that there are sex-based differences in T-cell function required for response to anti-PD-1 therapy in lung cancer that may need to be considered during patient treatment decisions.

Obesity-related T cell dysfunction impairs immunosurveillance and increases cancer risk.

Obesity is a well-established risk factor for human cancer, yet the underlying mechanisms remain elusive. Immune dysfunction is commonly associated with obesity but whether compromised immune surveillance contributes to cancer susceptibility in individuals with obesity is unclear. Here we use a mouse model of diet-induced obesity to investigate tumor-infiltrating CD8 + T cell responses in lean, obese, and previously obese hosts that lost weight through either dietary restriction or treatment with semaglutide. While both strategies reduce body mass, only dietary intervention restores T cell function and improves responses to immunotherapy. In mice exposed to a chemical carcinogen, obesity-related immune dysfunction leads to higher incidence of sarcoma development. However, impaired immunoediting in the obese environment enhances tumor immunogenicity, making the malignancies highly sensitive to immunotherapy. These findings offer insight into the complex interplay between obesity, immunity and cancer, and provide explanation for the obesity paradox observed in clinical immunotherapy settings.

Enhanced in vitro type 1 conventional dendritic cell generation via the recruitment of hematopoietic stem cells and early progenitors by Kit ligand.

In vitro culture of bone marrow (BM) with Fms-like tyrosine kinase 3 ligand (Flt3L) is widely used to study development and function of type 1 conventional dendritic cells (cDC1). Hematopoietic stem cells (HSCs) and many progenitor populations that possess cDC1 potential in vivo do not express Flt3 and thus may not contribute to Flt3L-mediated cDC1 production in vitro. Here, we present a KitL/Flt3L protocol that recruits such HSCs and progenitors into the production of cDC1. Kit ligand (KitL) is used to expand HSCs and early progenitors lacking Flt3 expression into later stage where Flt3 is expressed. Following this initial KitL phase, a second Flt3L phase is used to support the final production of DCs. With this two-stage culture, we achieved approximately tenfold increased production of both cDC1 and cDC2 compared to Flt3L culture. cDC1 derived from this culture are similar to in vivo cDC1 in their dependence on IRF8, ability to produce IL-12, and induction of tumor regression in cDC1-deficient tumor-bearing mice. This KitL/Flt3L system for cDC1 production will be useful in further analysis of cDC1 that rely on in vitro generation from BM.

IL-6 selectively suppresses cDC1 specification via C/EBPß.

Cytokines produced in association with tumors can impair antitumor immune responses by reducing the abundance of type 1 conventional dendritic cells (cDC1), but the mechanism remains unclear. Here, we show that tumor-derived IL-6 generally reduces cDC development but selectively impairs cDC1 development in both murine and human systems through the induction of C/EBPß in the common dendritic cell progenitor (CDP). C/EBPß and NFIL3 compete for binding to sites in the Zeb2 -165 kb enhancer and support or repress Zeb2 expression, respectively. At homeostasis, pre-cDC1 specification occurs upon Nfil3 induction and consequent Zeb2 suppression. However, IL-6 strongly induces C/EBPß expression in CDPs. Importantly, the ability of IL-6 to impair cDC development is dependent on the presence of C/EBPß binding sites in the Zeb2 -165 kb enhancer, as this effect is lost in Δ1+2+3 mutant mice in which these binding sites are mutated. These results explain how tumor-associated IL-6 suppresses cDC1 development and suggest therapeutic approaches preventing abnormal C/EBPß induction in CDPs may help reestablish cDC1 development to enhance antitumor immunity.

WDFY4 deficiency in NOD mice ameliorates autoimmune diabetes and insulitis.

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 ß 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 ß 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 ß cell damage.

Mechanisms of CD40-dependent cDC1 licensing beyond costimulation.

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.

cDC1 Vaccines Drive Tumor Rejection by Direct Presentation Independently of Host cDC1.

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.

Ablation of cDC2 development by triple mutations within the Zeb2 enhancer.

The divergence of the common dendritic cell progenitor1-3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors-such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer4,6-but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the -165 kb Zeb2 enhancer8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPß. In vivo mutational analysis using CRISPR-Cas9 targeting showed that these NFIL3-C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3-C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (TH2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths9-11. Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the -165 kb Zeb2 enhancer.

Bcl6-Independent In Vivo Development of Functional Type 1 Classical Dendritic Cells Supporting Tumor Rejection.

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.

Differential usage of transcriptional repressor Zeb2 enhancers distinguishes adult and embryonic hematopoiesis.

The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2Δ-165mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2Δ-165 mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2Δ-165 mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at +164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.

cDC1 prime and are licensed by CD4+ T cells to induce anti-tumour immunity.

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.

Batf3-Dependent Genes Control Tumor Rejection Induced by Dendritic Cells Independently of Cross-Presentation.

The BATF3-dependent cDC1 lineage of conventional dendritic cells (cDC) is required for rejection of immunogenic sarcomas and for rejection of progressive sarcomas during checkpoint blockade therapy. One unique function of the cDC1 lineage is the efficient cross-presentation of tumor-derived neoantigens to CD8+ T cells, but it is not clear that this is the only unique function of cDC1 required for tumor rejection. We previously showed that BATF3 functions during cDC1 lineage commitment to maintain IRF8 expression in the specified cDC1 progenitor. However, since cDC1 progenitors do not develop into mature cDC1s in Batf3 -/- mice, it is still unclear whether BATF3 has additional functions in mature cDC1 cells. A transgenic Irf8-Venus reporter allele increases IRF8 protein concentration sufficiently to allow autonomous cDC1 development in spleens of Batf3 -/- mice. These restored Batf3 -/- cDC1s are transcriptionally similar to control wild-type cDC1s but have reduced expression of a restricted set of cDC1-specific genes. Restored Batf3 -/- cDC1s are able to cross-present cell-associated antigens both in vitro and in vivo However, Batf3 -/- cDC1 exhibit altered characteristics in vivo and are unable to mediate tumor rejection. These results show that BATF3, in addition to regulating Irf8 expression to stabilize cDC1 lineage commitment, also controls expression of a small set of genes required for cDC1-mediated tumor rejection. These BATF3-regulated genes may be useful targets in immunotherapies aimed at promoting tumor rejection.

Notch2-dependent DC2s mediate splenic germinal center responses.

CD4+ T follicular helper (TFH) cells support germinal center (GC) reactions promoting humoral immunity. Dendritic cell (DC) diversification into genetically distinct subsets allows for specialization in promoting responses against several types of pathogens. Whether any classical DC (cDC) subset is required for humoral immunity is unknown, however. We tested several genetic models that selectively ablate distinct DC subsets in mice for their impact on splenic GC reactions. We identified a requirement for Notch2-dependent cDC2s, but not Batf3-dependent cDC1s or Klf4-dependent cDC2s, in promoting TFH and GC B cell formation in response to sheep red blood cells and inactivated Listeria monocytogenes This effect was mediated independent of Il2ra and several Notch2-dependent genes expressed in cDC2s, including Stat4 and Havcr2 Notch2 signaling during cDC2 development also substantially reduced the efficiency of cDC2s for presentation of MHC class II-restricted antigens, limiting the strength of CD4 T cell activation. Together, these results demonstrate a nonredundant role for the Notch2-dependent cDC2 subset in supporting humoral immune responses.

Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice.

Treatment of C57BL/6 or NOD mice with a monoclonal antibody to the CSF-1 receptor resulted in depletion of the resident macrophages of pancreatic islets of Langerhans that lasted for several weeks. Depletion of macrophages in C57BL/6 mice did not affect multiple parameters of islet function, including glucose response, insulin content, and transcriptional profile. In NOD mice depleted of islet-resident macrophages starting at 3 wk of age, several changes occurred: (i) the early entrance of CD4 T cells and dendritic cells into pancreatic islets was reduced, (ii) presentation of insulin epitopes by dispersed islet cells to T cells was impaired, and (iii) the development of autoimmune diabetes was significantly reduced. Treatment of NOD mice starting at 10 wk of age, when the autoimmune process has progressed, also significantly reduced the incidence of diabetes. Despite the absence of diabetes, NOD mice treated with anti-CSF-1 receptor starting at 3 or 10 wk of age still contained variably elevated leukocytic infiltrates in their islets when examined at 20-40 wk of age. Diabetes occurred in the anti-CSF-1 receptor protected mice after treatment with a blocking antibody directed against PD-1. We conclude that treatment of NOD mice with an antibody against CSF-1 receptor reduced diabetes incidence and led to the development of a regulatory pathway that controlled autoimmune progression.

The islet-resident macrophage is in an inflammatory state and senses microbial products in blood.

We examined the transcriptional profiles of macrophages that reside in the islets of Langerhans of 3-wk-old non-obese diabetic (NOD), NOD.Rag1-/-, and B6.g7 mice. Islet macrophages expressed an activation signature with high expression of Tnf, Il1b, and MHC-II at both the transcript and protein levels. These features are common with barrier macrophages of the lung and gastrointestinal tract. Moreover, injection of lipopolysaccharide induced rapid inflammatory gene expression, indicating that blood stimulants are accessible to the macrophages and that these macrophages can sense them. In NOD mice, the autoimmune process imparted an increased inflammatory signature, including elevated expression of chemokines and chemokine receptors and an oxidative response. The elevated inflammatory signature indicates that the autoimmune program was active at the time of weaning. Thus, the macrophages of the islets of Langerhans are poised to mount an immune response even at steady state, while the presence of the adaptive immune system elevates their activation state.

Macrophages and dendritic cells in islets of Langerhans in diabetic autoimmunity: a lesson on cell interactions in a mini-organ.

Islets of Langerhans of all species harbor a small number of resident macrophages. These macrophages are found since birth, do not exchange with blood monocytes, and are maintained by a low level of replication. Under steady state conditions, the islet macrophages are in an activated state. Islet macrophages have an important homeostatic role in islet physiology. At the start of the autoimmune process in the NOD mouse, a small number of CD103+ dendritic cells (DC) are found at about the same time that CD4+ T cells also appear in islets. In the absence of the CD103+ DC in the Batf3 deficient mice, autoimmunity never develops. We discuss the interactions among the two phagocytes and beta cells that result in autoimmune diabetes in NOD mice.

The role of islet antigen presenting cells and the presentation of insulin in the initiation of autoimmune diabetes in the NOD mouse.

We have been examining antigen presentation and the antigen presenting cells (APCs) in the islets of Langerhans of the non-obese diabetic (NOD) mouse. The purpose is to identify the earliest events that initiate autoimmunity in this confined tissue. Islets normally have a population of macrophages that is distinct from those that inhabit the exocrine pancreas. Also found in NOD islets is a minor population of dendritic cells (DCs) that bear the CD103 integrin. We find close interactions between beta cells and the two APCs that result in the initiation of the autoimmunity. Even under non-inflammatory conditions, beta cells transfer insulin-containing vesicles to the APCs of the islet. This reaction requires live cells and intimate contact. The autoimmune process starts in islets with the entrance of CD4(+) T cells and an increase in the CD103(+) DCs. Mice deficient in the Batf3 transcription factor never develop diabetes due to the absence of the CD103/CD8α lineage of DCs. We hypothesize that the 12-20 peptide of the beta chain of insulin is responsible for activation of the initial CD4(+) T-cell response during diabetogenesis.

Antigen presentation events during the initiation of autoimmune diabetes in the NOD mouse.

This is a brief summary of our studies of NOD autoimmune diabetes examining the events during the initial stage of the process. Our focus has been on antigen presentation events and the antigen presenting cells (APC) inside islets. Islets of non-diabetic mice contain resident macrophages that are developmentally distinct from those in the inter-acinar stroma. The autoimmune process starts with the entrance of CD4+ T cells together with a burst of a subset of dendritic cells (DC) bearing CD103. The CD103+ DC develop under the influence of the Batf3 transcription factor. Batf3 deficient mice do not develop diabetes and their islets are uninfiltrated throughout life. Thus, the CD103+ DC are necessary for the progression of autoimmune diabetes. The major CD4+ T cell response in NOD are the T cells directed to insulin. In particular, the non-conventional 12-20 segment of the insulin B chain is presented by the class II MHC molecule I-A(g7) and elicits pathogenic CD4+ T cells. We discuss that the diabetic process requires the CD103+ DC, the CD4+ T cells to insulin peptides, and NOD specific I-Ag(7) MHC-II allele. Finally, our initial studies indicate that beta cells transfer insulin containing vesicles to the local APC in a contact-dependent reaction. Live images of beta cells interactions with the APC and electron micrographs of islet APCs also show the transfer of granules.

The pancreas anatomy conditions the origin and properties of resident macrophages.

We examine the features, origin, turnover, and gene expression of pancreatic macrophages under steady state. The data distinguish macrophages within distinct intrapancreatic microenvironments and suggest how macrophage phenotype is imprinted by the local milieu. Macrophages in islets of Langerhans and in the interacinar stroma are distinct in origin and phenotypic properties. In islets, macrophages are the only myeloid cells: they derive from definitive hematopoiesis, exchange to a minimum with blood cells, have a low level of self-replication, and depend on CSF-1. They express Il1b and Tnfa transcripts, indicating classical activation, M1, under steady state. The interacinar stroma contains two macrophage subsets. One is derived from primitive hematopoiesis, with no interchange by blood cells and alternative, M2, activation profile, whereas the second is derived from definitive hematopoiesis and exchanges with circulating myeloid cells but also shows an alternative activation profile. Complete replacement of islet and stromal macrophages by donor stem cells occurred after lethal irradiation with identical profiles as observed under steady state. The extraordinary plasticity of macrophages within the pancreatic organ and the distinct features imprinted by their anatomical localization sets the base for examining these cells in pathological conditions.

Beta cells transfer vesicles containing insulin to phagocytes for presentation to T cells.

Beta cells from nondiabetic mice transfer secretory vesicles to phagocytic cells. The passage was shown in culture studies where the transfer was probed with CD4 T cells reactive to insulin peptides. Two sets of vesicles were transferred, one containing insulin and another containing catabolites of insulin. The passage required live beta cells in a close cell contact interaction with the phagocytes. It was increased by high glucose concentration and required mobilization of intracellular Ca2+. Live images of beta cell-phagocyte interactions documented the intimacy of the membrane contact and the passage of the granules. The passage was found in beta cells isolated from islets of young nonobese diabetic (NOD) mice and nondiabetic mice as well as from nondiabetic humans. Ultrastructural analysis showed intraislet phagocytes containing vesicles having the distinct morphology of dense-core granules. These findings document a process whereby the contents of secretory granules become available to the immune system.