cDC1 Cells Research Articles

Abstract 5694: Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells

Abstract Introduction: IL-15 cytokine stimulates the proliferation and cytotoxic function of CD8+ T and NK cells. Several preclinical models have supported the anti-tumor activity of IL-15. We have produced the native heterodimeric IL-15 (hetIL-15) of human, macaque or mouse and also fusions to the Fc antibody fragments (hetIL-15FC). Experimental procedures: We studied the therapeutic efficacy of hetIL-15 or hetIL-15FC immunotherapy in the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice. The effects of hetIL-15FC on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC) and gene expression profiling (Nanostring). We also assessed the metabolic profile of tumor infiltrating T cells. Results: hetIL-15 therapy delayed the primary tumor growth and increased overall survival. hetIL-15 or hetIL-15FC peritumoral administration resulted in tumor regression in 40% of the treated animals and resistance to tumor re-challenge. Therefore, monotherapy induced long term immunological memory able to block tumor growth. Both CD8+ T and NK cells were increased in hetIL-15FC treated tumors; phenotypic analysis showed they were activated and in an active proliferation state. Transcriptomic analysis confirmed the activated state of the T and NK cells, whereas metabolic flux analysis of the infiltrated CD8+T cells from treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity (as a measure of mitochondrial reserve), which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. In addition, peritumoral hetIL-15FC administration resulted in an increase of infiltrating, conventional type 1 dendritic cells (cDC1s), suggesting mechanisms of DC-lymphocyte interactions in the tumor. We also observed a new population of intra-tumoral DCs induced by hetIL-15, CD24+ F4/80high cDC2s (F4/80high DCs). The new DC population was negatively correlated with the tumor size of EO771 tumor bearing mice. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103 and IRF8 and t-SNE analysis clustered F4/80high DCs close to cDC1s and macrophages. Conclusion: Our results show complete eradication of EO771 tumors by hetIL-15FC treatment, correlating with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1s and a novel subset of antigen presenting cells with a distinct phenotypic profile. hetIL-15 supported a favorable metabolic profile of intra-tumoral effector lymphocytes, important for their function. Effective hetIL-15 treatment leads to development of long-term anti-tumor memory. Citation Format: Sevasti Karaliota, Dimitris Stellas, Vasiliki Stravokefalou, Bethany Nagy, Cristina Bergamaschi, Barbara K. Felber, George N. Pavlakis. Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5694.

CDC1 dysregulation in cancer: An opportunity for intervention.

Conventional dendritic cells driven by the transcription factor Batf3 (cDC1 cells) are critical for the activation and maintenance of tumor-specific CD8+ T cells. In this issue of JEM, Lin et al. (https://doi.org/10.1084/jem.20190673) demonstrate systemic dysfunction of cDC1 cells in pancreatic cancer, which offers potential treatment strategies to expand the benefit of checkpoint blockade immunotherapy.

NCoR1 fine-tunes type-I IFN response in cDC1 dendritic cells by directly regulating Myd88-IRF7 axis under TLR9.

Plasmacytoid dendritic cells (DCs) are reported to induce robust type-I interferon (IFN) response, whereas cDC1 DCs develop moderate type-I IFN response upon TLR9 stimulation. It is very interesting to understand how this signaling under TLR9 is tightly regulated for the induction of type-I IFNs. Here, we report co-repressor protein NCoR1 as the major factor fine-tuning the signaling pathways regulating IFN-β expression under TLR9 in cDC1 DCs. We found that NCoR1 knockdown induced a robust IFN-β-mediated antiviral response upon TLR9 activation in cDC1 DCs. At the molecular level, we showed that NCoR1 directly repressed MyD88-IRF7 signaling axis in cDC1 cells. Therefore, NCoR1 depletion enhanced pIRF7 levels, IFN-β secretion, and downstream pSTAT1-pSTAT2 signaling, leading to sustained induction of IFN stimulatory genes. Integrative genomic analysis depicted strong enrichment of an antiviral gene-module in CpG-activated NCoR1 knockdown DCs upon TLR9 activation. Moreover, we confirmed our findings in primary DCs derived from splenocytes of WT and NCoR1 DC-/- animals, which showed protection from Sendai and Vesicular Stomatitis viruses upon CpG activation. Ultimately, we identified that NCoR1-HDAC3 complex is involved in repressing the type-I IFN response in cDC1 DCs.

Airway dendritic cell maturation in children exposed to air pollution.

Urban particulate matter (PM) enhances airway dendritic cell (DC) maturation in vitro. However, to date, there are no data on the association between exposure to urban PM and DC maturation in vivo. We sought to determine whether exposure of school-age children (8 to 14 y) to PM was associated with expression of CD86, a marker of maturation of airway conventional DCs (cDC). Healthy London school children underwent spirometry and sputum induction. Flow cytometry was used to identify CD86 and CCR7 expression on cDC subsets (CD1c+ cDC2 and CD141+ cDC1). Tertiles of mean annual exposure to PM ≤ 10 microns (PM10) at the school address were determined using the London Air Quality Toolkit model. Tertiles of exposure from the 409 children from 19 schools recruited were; lower (23.1 to 25.6 μg/m3, n = 138), middle (25.6 to 26.8 μg/m3, n = 126), and upper (26.8 to 31.0 μg/m3, n = 145). DC expression was assessed in 164/370 (44%) children who completed sputum induction. The proportion (%) of cDC expressing CD86 in the lower exposure tertile (n = 47) was lower compared with the upper exposure tertile (n = 49); (52% (44 to 70%) vs 66% (51 to 82%), p<0.05). There was a higher percentage of cDC1 cells in the lower tertile of exposure (6.63% (2.48 to 11.64) vs. 2.63% (0.72 to 7.18), p<0.05). Additionally; children in the lower exposure tertile had increased FEV1 compared with children in the upper tertile; (median z-score 0.15 (-0.59 to 0.75) vs. -0.21 (-0.86 to 0.48), p<0.05. Our data reveal that children attending schools in the highest areas of PM exposure in London exhibit increased numbers of “mature” airway cDCs, as evidenced by their expression of the surface marker CD86. This data is supportive of previous in vitro data demonstrating an alteration in the maturation of airway cDCs in response to exposure to pollutants.

Conventional type 1 dendritic cells and natural killer cells demonstrate strong correlation to cytotoxic T lymphocyte infiltration in cervical cancer tumors

Abstract Professional antigen presenting cells such as dendritic cells (DCs) are vital for priming naive CD8+ T cells and developing a sustainable anti-tumor immune response. Natural killer (NK) cells within the tumor microenvironment (TME) recruit a specific population of DCs called conventional type 1 DCs (cDC1s). However, these cells are low in abundance making their detection in the tissue context challenging. To interrogate the presence of cDC1 and NK cells in the TME and reveal their spatial relationship to each other we utilized the highly sensitive and specific RNAscope Multiplex Fluorescence in situ hybridization (ISH) assay. NK cells and cDC1 cells were identified by using cell specific marker probes in 4 cervical cancer samples. Similarly, CTLs were visualized to determine if there is a correlation between the presence of cDC1 and NK cells and infiltration of CTLs within the cervical cancer tumors. Our results revealed a strong correlation between the presence of NK cells, cDC1 cells, and CTLs within 3 out of 4 cervical cancer samples. The NK cells showed expression of the chemokines XCL1 and CCL5, suggesting that the XCR1+/CCR5+ cDC1 cells may have been potentially recruited by these NK cells in the TME. Regions high in cDC1 and NK cells also showed significantly higher levels of CTL recruitment, indicated by the presence of CD8+/IFNG+ T cells. Conversely, 1 of the 4 cervical cancer samples demonstrated relatively lower levels of NK cells which correlated with lower cDC1 cells and in turn lower CTL infiltration. In conclusion, by utilizing the RNAscope Multiplex ISH assay we identified and visualized the spatial relationship between NK cells, CTLs, and cDC1 cells, highlighting the strength of this technology to spatially interrogate the TME.

Virus-based vaccines with unique replication mechanisms differentially infect and activate dendritic cells leading to distinctive CD8+ T cell responses

Abstract The precise mechanism by which many virus-based vectors activate immune responses remains unknown. Dendritic cells (DCs) play key roles in priming T cell responses and controlling virus replication, but their functions in generating protective immunity following vaccination with viral vectors are not well understood. We hypothesized that highly immunogenic viral vectors with unique replication mechanisms but identical cell entry pathways differentially infect and activate DCs and promote antigen presentation that leads to efficient induction of distinctive antigen-specific T cell responses. To evaluate differences in replication mechanisms, we utilized a rhabdovirus vector (VSV) and an alphavirus-rhabdovirus hybrid vector (VLV), which replicates like an alphavirus but enters the cell via the VSV glycoprotein. We found that while virus replication promotes CD8+ T cell activation by VLV, replication is absolutely required for VSV-induced responses. DC subtypes were differentially infected in vitro with VSV and VLV, and displayed differences in activation following infection that were dependent on vector replication. Although bone marrow-derived DCs produced type I IFN when infected with the vectors, the mechanism of DC infection and activation was independent of type I IFN receptor signaling. Additionally, the ability of the alphavirus-based vector to generate CD8+ T cells in the absence of replication relied on cDC1 cells that promote antigen cross-presentation. These results highlight the differential activation of DCs following infection with unique viral vectors, and indicate potentially distinct roles of DC subtypes in activating the immune response following immunization.

Liposomes represent a perfect tool to enhance the cross-presentation effectivity of viral HIV-1 p24 protein fused with heat shock protein 70

Abstract Current approaches focusing on anti-viral and anti-tumor vaccines developement tend to induce strong CD4 T cell response leading in antibodies production and simultaneously, efficient CD8 T cell activation responsible for cytotoxic elimination of infected cells. The p24 protein from HIV-1 virus is a relatively conserved but poorly immunogenic protein. Its fusion with host heat shock protein 70 (hsp70) enhanced the immune response to p24 including cross-presentation on MHC I molecules, despite the recognition by antigen presenting cells as an exogenous antigen. Cross-presentation efficacy and immune response was supported by binding this construct to liposomes containing aminooxy lipids allowing further modification by click chemistry. Hsp70-p24 was bound to liposome surface and used in cDC1 cell line (DCs from murine spleen) and mice vaccination model. Activation markers, MHCI expression, apoptosis assay and proliferation efficacy was analyzed. Expression of DC cell-surface activation markers CD40, CD86 and also MHC I was increased in hsp70-p24 pulsed cells as much as LPS, but when antigens coupled to liposome expression was significantly higher. None of the constructs activated CD86 expression as much as the LPS. Studies on hepatocytes transfected by hsp70-p24 encoding plasmid DNA showed increased death of hepatocytes co-cultivated with splenocytes from mice vaccinated by hsp70-p24-liposome in comparison to hsp70-p24 alone. Activation markers on CD8 T cells from vaccinated mice were enhanced after co-cultivation with hsp70-p24-liposome pulsed cDC1 cells or bone marrow derived dendritic cells. All these results clearly show that coupling of antigen with liposomes leads to effective CD4 and also CD8 T cell activation.

INKT cell modulation of tumor microenvironment (TME) and antitumor immunity: the role of antitumorigenic and protumorigenic iNKT cells

Abstract Preclinical and clinical data indicate that iNKT cells play important roles in antitumor immunity. High number of iNKT cells in the PBL of cancer patients usually correlates with a favorable prognosis. We found that compared to wild-type (WT) mice, depletion of iNKT cells did not affect tumor progression or metastasis in mice bearing melanoma or Lewis lung cancer; restoring iNKT cells by transferring thymocytes into iNKT KO mice dramatically enhanced antitumor immunity. The underlying mechanism is no known. Herein, we report progress on the mechanistic study. Thymocyte transfer only reconstituted iNKT cells in liver, spleen and blood, but not in LN of iNKT KO mice. This suggests that two functionally opposite iNKT subsets exist in the immune system, and thymocyte transfer only restored antitumorigenic iNKT, but not protumorigenic iNKT cells. Transferring IFNγ KO thymocytes diminished iNKT reconstitution-induced antitumor immunity; transferring IL-17A KO thymocytes enhanced iNKT reconstitution-enhanced antitumor immunity. Compared to WT mice or iNKT KO mice, iNKT reconstitution in iNKT KO mice up regulated the expression of XCL1, FLT3L and CCL5, down regulated IL-6, TGFβ1, TGFβ2, COX2 expression, increased IRF8+ cDC1 cells and Siglec F− DC, and decreased αSMA+ FAP+ cancer-associated fibroblasts (CAF) and MDSC in the tumor. Taken together, these results support that antitumorigenic iNKT cells produce IFNγ dominant cytokine and facilitate NK/cDC1 infiltration and activation in the tumor to induce antitumor immunity; protumorigenic iNKT cells induce CAF to produce TGFβ, which induces a myeloid cell-mediated inhibitory TME. The balance between antitumorigenic and protumorigenic iNKT cells shapes the TME and antitumor immunity.

Regression of murine breast tumors after hetIL-15 monotherapy correlates with a novel population of intratumoral dendritic cells, in addition to increased infiltration of T, NK and cDC1 cells

Abstract Background We studied mechanisms of therapeutic efficacy of the heterodimeric IL-15 (hetIL-15) of human, macaque or mouse in several mouse tumor models, including the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice. Methods The effects of hetIL-15 on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC), gene expression profiling (Nanostring) and metabolic profiling of tumor infiltrating T cells. Results hetIL-15 administration resulted in tumor regression in 40% of the treated animals and resistance to tumor rechallenge, inducing long term immunological memory. Activated NK and CD8+ T cells were increased in hetIL-15 treated tumors. hetIL-15 administration resulted in increased intratumoral infiltration of conventional Dendritic cells (cDC1) and also induced a new population of intratumoral DC, (F4/80high DCs), which were correlated negatively with the tumor size. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103, IRF8 and XCR1. tSNE analysis clustered F4/80high DCs close to cDC1s and macrophages. Conclusion Complete eradication of EO771 tumors by hetIL-15 treatment correlates with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1 and a novel subset of antigen presenting cells with a distinct phenotypic profile. Effective hetIL-15 treatment leads to development of long term anti-tumor memory. hetIL-15 supported a favorable metabolic profile of intratumoral effector lymphocytes, important for their function.

CD73 Blockade Promotes Dendritic Cell Infiltration of Irradiated Tumors and Tumor Rejection

The ability of focal radiotherapy to promote priming of tumor-specific CD8+ T cells and increase responses to immunotherapy is dependent on infiltration of the tumor by Batf3-dependent conventional dendritic cell type 1 (cDC1) cells. Such infiltration is driven by radiotherapy-induced IFN type I (IFN-I). Other signals may also modulate cDC1 infiltration of irradiated tumors. Here we found increased expression of adenosine-generating enzymes CD38 and CD73 in irradiated mouse and human breast cancer cells and increased adenosine in mouse tumors following radiotherapy. CD73 blockade alone had no effect. CD73 blockade with radiotherapy restored radiotherapy-induced cDC1 infiltration of tumors in settings where radiotherapy induction of IFN-I was suboptimal. In the absence of radiotherapy-induced IFN-I, blockade of CD73 was required for rejection of the irradiated tumor and for systemic tumor control (abscopal effect) in the context of cytotoxic T-lymphocyte-associated protein 4 blockade. These results suggest that CD73 may be a radiation-induced checkpoint, and that CD73 blockade in combination with radiotherapy and immune checkpoint blockade might improve patient response to therapy.

Abstract 1474: Repurposing the yellow fever vaccine for intratumoral immunotherapy

Abstract Introduction: Yellow Fever attenuated virus vaccine (17D) induces very potent cellular and humoral immunity. Its delivery in situ in the tumor bed may induce an immune response leading to the killing of infected cells. Experimental procedures: 17D virus was propagated in Vero cells and infectious supernatants were harvested. To evaluate in vitro sensitivity of tumors to 17D infection, a panel of human and mouse tumor cell lines were infected with increasing MOIs and viability was quantified. To evaluate the antitumor efficacy of intratumor (i.t.) injection of 17D, 4x106pfu/50μl were injected (4 doses) in MC38- and in B16-OVA-tumor bearing C57BL/6 mice. Systemic antitumor effects of i.t 17D were examined in bilateral MC38 models in which one of the tumors was left uninjected. Depletion experiments were performed to assess the contribution of CD4, CD8, Gr1 and NK cells. To determine the role of conventional type-I dendritic cells (cDC1), identical experiments were performed in BATF3-/- mice. Tumor and lymph node immune infiltrates were examined by multiparametric flow cytometry. Combination with intraperitoneal anti-PD1 or anti-CD137 was performed to improve antitumor efficacy. To evaluate the effects of 17D-preexisting immunity, mice were preimmunized with 17D prior to MC38 bilateral inoculation and were subsequently treated. To assess the individual cell type contribution to the antitumor effects induced upon 17D pre-immunization, CD8, CD4, CD8+CD4 or sera from preimmunized mice were passively transferred 24h prior to first 17D i.t injection to MC38-tumor bearing mice. Results: 17D live attenuated strain is able to induce cell death of mouse and human tumor cell lines. In MC38 or B16-OVA mice models, repeated i.t. injections of 17D clearly delayed tumor progression. Regarding the systemic effect of i.t. 17D, certain contralateral therapeutic efficacy was induced upon i.t injection in bilateral MC38 models. These local and distant effects were dependent on CD8+ and cDC1 cells. 17D i.t injection increased in CD8+ T cell infiltrates and decreased in Tregs in injected tumors, while in contralateral tumors induced an increase of CD8+ and NK cells expressing key immunomodulatory receptors. I.t. 17D with anti-CD137 monoclonal antibody (mAb) induced a strong synergistic effect, while combination with anti-PD-1 mAb showed mild local additive efficacy compared to 17D monotherapy. Both combinatorial treatments delayed contralateral tumor growth. Notably, 17D preimmunization induced an important improvement of local and distant antitumor immunity. Such beneficial effects of preimmunization were transferred along with CD8+ T cells. Conclusions: 17D can be safely injected i.t. in preclinical models, giving rise to immune-mediate antitumor effects that can be combined with other immunotherapy agents. The repurposed use of this GMP-grade vaccine in preimmunized patients represents a promising and feasible immunotherapy approach. Citation Format: M. Angela Aznar, Carmen Molina, Alvaro Teijeira, Arantza Azpilikueta, Saray Garasa, Alfonso R. Sanchez-Paulete, Iñaki Etxeberria, Maite Alvarez, Pedro Berraondo, Ignacio Melero. Repurposing the yellow fever vaccine for intratumoral immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1474.

Splenic CD169+ marginal zone macrophages directly cross-prime CD8+ T cells in vivo using a vacuolar processing pathway

Abstract Cross-priming of CD8+ T cells is of critical importance in triggering CD8+ T cell responses against pathogens and tumors. It is commonly thought that conventional type 1 dendritic cells (cDC1) cross-prime with unequalled efficacy, conferred to them by a proteasome and TAP-dependent pathway. Here we present data challenging the unique cross-priming efficacy of both the cDC1 subset and the proteasome/TAP-dependent pathway. Splenic CD169 macrophages are strategically placed in the marginal zone for capture of blood-borne pathogens. However, they are considered unable to directly cross-present and to depend on antigen transfer to cDC1 for cross-priming. We have developed a protocol for purification of CD169 macrophages. Extensive characterization shows that these cells display morphology, phenotype and gene expression distinguishing them clearly from cDC1, cDC2, plasmacytoid DC and red pulp macrophages. In vitro, CD169 cells cross-present soluble, receptor-targeted and phagocytosed antigen with equal or better efficiency than cDC1. Remarkably, this is achieved through a vacuolar, BFA and epoxomicin-independent pathway. Using intravital biphoton microscopy, 3D light sheet imaging and antigen targeting to CD169, we show that CD169 macrophages establish long-lasting antigen-dependent contacts with cognate CD8+ T cells that result in T cell activation in situ and generation of triple-positive effector as well as memory cells. Therefore, splenic CD169 macrophages represent a second set of myeloid cells equipped for highly efficient cross-priming. The biological role of cross-priming by these cells as well as their cooperation with cDC1 cells are under investigation.

Monocyte-lineage dendritic cell (DC) precursors are pre-positioned in normal lymph nodes and rapidly differentiate into Batf3+ cross-presenting APCs during inflammation

Abstract We have shown (Immunity 48:91–106, 2018) that a subset of murine monocytic precursor cells are plastic, and can up-regulate a Batf3-driven differentiation program resembling classical cDC1 cells. The resulting inflammatory DCs are important in tumor immunity, but it has been unclear whether they played a role in normal immune responses. We now identify the precursor cells for these inflammatory DCs as a population of Ly6c+ ckit+ c− fms+ CD11b-neg cells. These are constitutively present in all resting LNs (comprising up to half of the Ly6c+ cells). In response to a standard vaccination model of cross-presented antigen (whole OVA protein plus CpG adjuvant in IFA), the precursor cells rapidly up-regulated Batf3 and differentiated into CD103+ IRF8+ DCs. Just as in tumors, maturation was dependent on intracellular p53 signaling in the precursor cells. Targeted disruption of p53 (LysMcre/p53-KO) had no effect on the number of precursors, but abrogated their ability to mature into Batf3+ DCs. In KO mice, the loss of this single monocytic DC population entirely abrogated the ability to create an inflammatory milieu in the vaccine-draining LN, and abolished cross-presentation of protein antigen to T cells. In contrast, direct presentation (soluble siinfekl peptide) remained intact. We hypothesize that the ability of monocyte-lineage cells to differentiate into Batf3+ APCs during inflammation is an important component of the normal cross-presentation process.

Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro.

The multistep differentiation process from hematopoietic stem cells through common myeloid progenitors into committed dendritic cell (DC) subsets remains to be fully addressed. These studies now show that Allograft Inflammatory Factor-1 (AIF1) is required for differentiation of classical DC type 1 (cDC1) subsets and monocyte-derived DC (Mo-DC). Phenotypic studies found that AIF1 expression increased in committed subsets differentiating from common myeloid progenitors (CMP). However, silencing AIF1 expression in hematopoietic stem progenitors restrained the capacity to differentiate into Mo-DC and cDC1 cell subsets under GM-CSF or Flt3-L stimuli conditions, respectively. This was further marked by restrained expression of IRF8, which is critical for development of Mo-DC and cDC1 subsets. As a result, absence of AIF1 restrained the cells at the Lin−CD117+FcγR−CD34+ CMP stage. Further biochemical studies revealed that abrogating AIF1 resulted in inhibition of the NFκB family member RelB expression and p38 MAPK phosphorylation during differentiation of Mo-DC. Lastly, protein binding studies identified that AIF1 interacts with protein kinase C (PKC) to influence downstream signaling pathways. Taken together, this is the first report showing a novel role of AIF1 as a calcium-responsive scaffold protein that supports IRF8 expression and interacts with PKC to drive NFκB-related RelB for successfully differentiating hematopoietic progenitor cells into cDC and Mo-DC subsets under Flt3-L and GM-CSF stimuli, respectively.

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.

Novel mechanism for estrogen receptor alpha modulation of murine lupus

Female sex is a risk factor for lupus. Sex hormones, sex chromosomes and hormone receptors are implicated in the pathogenic pathways in lupus. Estrogen receptor alpha (ERα) knockout (KO) mice are used for defining hormone receptor effects in lupus. Prior studies of ERα KO in lupus have conflicting results, likely due to sex hormone levels, different lupus strains and different ERα KO constructs. Our objective was to compare a complete KO of ERα vs. the original functional KO of ERα (expressing a short ERα) on disease expression and immune phenotype, while controlling sex hormone levels. We studied female lupus prone NZM2410 WT and ERα mutant mice. All mice (n = 44) were ovariectomized (OVX) for hormonal control. Groups of each genotype were estrogen (E2)-repleted after OVX. We found that OVXed NZM mice expressing the truncated ERα (ERα short) had significantly reduced nephritis and prolonged survival compared to both wildtype and the complete ERαKO (ERα null) mice, but surprisingly only if E2-repleted. ERα null mice were not protected regardless of E2 status. We observed significant differences in splenic B cells and dendritic cells and a decrease in cDC2 (CD11b+CD8−) dendritic cells, without a concomitant decrease in cDC1 (CD11b-CD8a+) cells comparing ERα short to ERα null or WT mice. Our data support a protective role for the ERα short protein. ERα short is similar to an endogenously expressed ERα variant (ERα46). Modulating its expression/activity represents a potential approach for treating female-predominant autoimmune diseases.

Metabolic control of cell fate bifurcations in a hematopoietic progenitor population.

Growth signals drive hematopoietic progenitor cells to proliferate and branch into divergent cell fates, but how unequal outcomes arise from a common progenitor is not fully understood. We used steady-state analysis of in vivo hematopoiesis and Fms-related tyrosine kinase 3 ligand (Flt3L)-induced in vitro differentiation of dendritic cells (DCs) to determine how growth signals regulate lineage bias. We found that Flt3L signaling induced anabolic activation and proliferation of DC progenitors, which was associated with DC differentiation. Perturbation of processes associated with quiescence and catabolism, including AMP-activated protein kinase signaling, fatty acid oxidation, or mitochondrial clearance increased development of cDC2 cells at the expense of cDC1 cells. Conversely, scavenging anabolism-associated reactive oxygen species skewed differentiation toward cDC1 cells. Sibling daughter cells of dividing DC progenitors exhibited unequal expression of the transcription factor interferon regulatory factor 8, which correlated with clonal divergence in FoxO3a signaling and population-level bifurcation of cell fate. We propose that unequal transmission of growth signals during cell division might support fate branches during proliferative expansion of progenitors.

Sterile-activated cDC1 cells in neonatal lung induce T cell IL-4 production and lymph node maturation

Abstract Neonates are susceptible to respiratory tract infections, possibly due to immature pulmonary dendritic cells (cDCs). Here we assessed the development and function of conventional DCs in the lung and the lung-draining mediastinal lymph node (MedLN). We found that neonatal lungs, from days 3 to 10 of life, contained a higher proportion of CD103+ cDC1 cells than adult lungs and that these cells could be divided into a small population of CD103hi cDC1 and a larger population of CD103int cDC1, which were not found in adult lungs. A similar expansion of cDC1 cells was observed in the MedLN migratory cDCs during the first 2 weeks after birth. Transcriptional and functional assays showed that neonatal CD103int cDC1 cells had an activated phenotype and efficiently migrated to the MedLN, their formation was dependent on the transcription factor Batf3 but CD103int cDC1 cells developed independently of the microbiota or MyD88 signaling. Neonatal cDC1 cells express high levels of OX-40L and produce IL-12p40, but not IL-12p70, and stimulated the differentiation of IL-4-producing OT-II cells. Importantly, the MedLNs of neonatal Batf3−/− had poorly developed HEVs and FRCs, which were restored by transfer of neonatal lung CD103int into Batf3−/− mice. These changes were not observed following transfer of CD103hi or CCR7−/− CD103int DCs. Overall our results indicate that neonatal lung CD103int cDCs are activated as part of a developmental program that promotes MedLN maturation and biases CD4 T cell activation towards IL-4 production.

The role of cDC1s in vivo: CD8 T cell priming through cross-presentation.

The cDC1 subset of classical dendritic cells is specialized for priming CD8 T cell responses through the process of cross-presentation. The molecular mechanisms of cross-presentation remain incompletely understood because of limited biochemical analysis of rare cDC1 cells, difficulty in their genetic manipulation, and reliance on in vitro systems based on monocyte- and bone-marrow-derived dendritic cells. This review will discuss cross-presentation from the perspective of studies with monocyte- or bone-marrow-derived dendritic cells while highlighting the need for future work examining cDC1 cells. We then discuss the role of cDC1s as a cellular platform to combine antigen processing for class I and class II MHC presentation to allow the integration of “help” from CD4 T cells during priming of CD8 T cell responses.

The Phenotypic Characterization of the Human Renal Mononuclear Phagocytes Reveal a Co-Ordinated Response to Injury.

Mammalian tissues contain networks of mononuclear phagocytes (MPh) that sense injury and orchestrate the response to it. In mice, this is affected by distinct populations of dendritic cells (DC), monocytes and macrophages and recent studies suggest the same is true for human skin and intestine but little is known about the kidney. Here we describe the analysis of MPh populations in five human kidneys and show they are highly heterogeneous and contain discrete populations of DC, monocytes and macrophages. These include: plasmacytoid DC (CD303+) and both types of conventional DC—cDC1 (CD141+ cells) and CD2 (CD1c+ cells); classical, non-classical and intermediate monocytes; and macrophages including a novel population of CD141+ macrophages clearly distinguishable from cDC1 cells. The relative size of the MPh populations differed between kidneys: the pDC population was bi-modally distributed being less than 2% of DC in two kidneys without severe injury and over 35% in the remaining three with low grade injury in the absence of morphological evidence of inflammation. There were profound differences in the other MPh populations in kidneys with high and low numbers of pDC. Thus, cDC1 cells were abundant (55 and 52.3%) when pDC were sparse and sparse (12.8–12.5%) when pDC were abundant, whereas the proportions of cDC2 cells and classical monocytes increased slightly in pDC high kidneys. We conclude that MPh are highly heterogeneous in human kidneys and that pDC infiltration indicative of low-grade injury does not occur in isolation but is part of a co-ordinated response affecting all renal DC, monocyte and macrophage populations.