Transcription Factor Batf3 Research Articles

Activation of regulatory dendritic cells by Mertk coincides with a temporal wave of apoptosis in neonatal lungs.

Multiple mechanisms restrain inflammation in neonates, most likely to prevent tissue damage caused by overly robust immune responses against newly encountered pathogens. Here, we identify a population of pulmonary dendritic cells (DCs) that express intermediate levels of CD103 (CD103int) and appear in the lungs and lung-draining lymph nodes of mice between birth and 2 weeks of age. CD103int DCs express XCR1 and CD205 and require expression of the transcription factor BATF3 for development, suggesting that they belong to the cDC1 lineage. In addition, CD103int DCs express CCR7 constitutively and spontaneously migrate to the lung-draining lymph node, where they promote stromal cell maturation and lymph node expansion. CD103int DCs mature independently of microbial exposure and TRIF- or MyD88-dependent signaling and are transcriptionally related to efferocytic and tolerogenic DCs as well as mature, regulatory DCs. Correlating with this, CD103int DCs show limited ability to stimulate proliferation and IFN-γ production by CD8+ T cells. Moreover, CD103int DCs acquire apoptotic cells efficiently, in a process that is dependent on the expression of the TAM receptor, Mertk, which drives their homeostatic maturation. The appearance of CD103int DCs coincides with a temporal wave of apoptosis in developing lungs and explains, in part, dampened pulmonary immunity in neonatal mice. Together, these data suggest a mechanism by which DCs sense apoptotic cells at sites of noninflammatory tissue remodeling, such as tumors or the developing lungs, and limit local T cell responses.

Establishment of isotype-switched, antigen-specific B cells in multiple mucosal tissues using non-mucosal immunization

Although most pathogens infect the human body via mucosal surfaces, very few injectable vaccines can specifically target immune cells to these tissues where their effector functions would be most desirable. We have previously shown that certain adjuvants can program vaccine-specific helper T cells to migrate to the gut, even when the vaccine is delivered non-mucosally. It is not known whether this is true for antigen-specific B cell responses. Here we show that a single intradermal vaccination with the adjuvant double mutant heat-labile toxin (dmLT) induces a robust endogenous, vaccine-specific, isotype-switched B cell response. When the vaccine was intradermally boosted, we detected non-circulating vaccine-specific B cell responses in the lamina propria of the large intestines, Peyer’s patches, and lungs. When compared to the TLR9 ligand adjuvant CpG, only dmLT was able to drive the establishment of isotype-switched resident B cells in these mucosal tissues, even when the dmLT-adjuvanted vaccine was administered non-mucosally. Further, we found that the transcription factor Batf3 was important for the full germinal center reaction, isotype switching, and Peyer’s patch migration of these B cells. Collectively, these data indicate that specific adjuvants can promote mucosal homing and the establishment of activated, antigen-specific B cells in mucosal tissues, even when these adjuvants are delivered by a non-mucosal route. These findings could fundamentally change the way future vaccines are formulated and delivered.

Suppression of FOXP3 expression by the AP-1 family transcription factor BATF3 requires partnering with IRF4.

FOXP3 is the lineage-defining transcription factor for Tregs, a cell type critical to immune tolerance, but the mechanisms that control FOXP3 expression in Tregs remain incompletely defined, particularly as it relates to signals downstream of TCR and CD28 signaling. Herein, we studied the role of IRF4 and BATF3, two transcription factors upregulated upon T cell activation, to the conversion of conventional CD4+ T cells to FOXP3+ T cells (iTregs) in vitro. We found that IRF4 must partner with BATF3 to bind to a regulatory region in the Foxp3 locus where they cooperatively repress FOXP3 expression and iTreg induction. In addition, we found that interactions of these transcription factors are necessary for glycolytic reprogramming of activated T cells that is antagonistic to FOXP3 expression and stability. As a result, Irf4 KO iTregs show increased demethylation of the critical CNS2 region in the Foxp3 locus. Together, our findings provide important insights how BATF3 and IRF4 interactions integrate activating signals to control CD4+ cell fate decisions and govern Foxp3 expression.

Abstract 1293: AZD0171 (anti-LIF) combines productively with chemotherapy and anti-PD-L1 in mouse models of cancer

Abstract Leukemic inhibitory factor (LIF) is an IL-6 family member involved in embryonic stem cell maintenance and protection from maternal immune-attack. Emerging evidence indicates that LIF biology is hijacked by some tumors to promote immunosuppressive tumor associated macrophages, cancer initiating cells and epithelial-mesenchymal transition (EMT). In patients treated with anti-PD-1/anti-PD-L1 antibodies (IO), baseline high LIF expression correlated with poor outcome (Loriot et al. 2021). In preclinical models, LIF blockade has separately been reported to sensitize tumors to IO and to chemotherapy (chemo), but the impact of LIF blockade on chemo/IO combination activity is unknown. We therefore evaluated the hypothesis that LIF blockade sensitizes tumors to chemo/IO combinations. AZD0171 is a monoclonal antibody that binds to LIF and prevents signaling through LIF receptor. A murine surrogate of AZD0171 (mAZD0171) was generated and tested in vivo using three syngeneic murine cancer models, and AZD0171 was tested in patient derived xenograft models. Monotherapy mAZD0171 induced tumor gene expression changes consistent with its proposed mechanism of action in the syngeneic CT26 colon tumor model; including elevated CD8 attracting chemokines (CXCL9/10), cDC1 transcription factor (Batf3) and reduced stem cell (CD44), EMT (CY61) and hypoxic/angiogenic signatures, including CXCL12. Flow cytometry analysis highlighted changes consistent with suppressive macrophage re-education (reduced CD206/elevated MHCII). mAZD0171 sensitized mouse tumors to both IO and chemo. mAZD0171 + anti-PD-L1 treatment reversed CD8 T cell exclusion in the D2A1-m2 model; a LIF expressing cancer associated fibroblast rich, anti-PD-L1 resistant mouse mammary tumor syngeneic model (Jungwirth et al. 2018). mAZD0171 + anti-PD-L1 treatment further inhibited D2A1-m2 growth versus (vs) anti-PD-L1 treatment alone. In a non-small cell lung carcinoma patient derived xenograft model, AZD0171 improved the tumor growth inhibitory effect of Carboplatin + Paclitaxel. The triplet combination of mAZD0171 plus chemo and IO had the most marked impact on both tumor microenvironment and tumor growth. mAZD0171 addition to oxaliplatin (OHP) or Docetaxel (DTX) chemotherapy plus anti-PD-L1 significantly elevated CD8 abundance and granzyme B expression in MC38 colon tumors. mAZD0171 + anti-PD-L1 + OHP or DTX improved inhibition of MC38 tumor growth (63% for OHP triplet vs 34% for anti-PD-L1 + OHP; 49% for DTX triplet vs 36% for anti-PD-L1 + DTX) and extended median survival times (P<0.05 vs anti-PD-L1+OHP/DTX). Together these data support the hypothesis that AZD0171 has the potential to sensitize solid tumors to chemotherapy/IO combinations. The safety and activity of AZD0171 is currently being assessed in a Ph2 clinical trial in combination with durvalumab (anti-PD-L1) and chemotherapy in metastatic pancreatic cancer (NCT04999969). Citation Format: Juliana Candido, Gozde Kar, Bairu Zhang, Grace Opoku-Ansah, Amit Grover, Elizabeth A. Kuczynski, Tim Slidel, Doug Palmer, Robert W. Wilkinsom, Nadia Luheshi, Jim Eyles. AZD0171 (anti-LIF) combines productively with chemotherapy and anti-PD-L1 in mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1293.

Fate mapping of single NK cells identifies a type 1 innate lymphoid-like lineage that bridges innate and adaptive recognition of viral infection

SummaryUpon viral infection, natural killer (NK) cells expressing certain germline-encoded receptors are selected, expanded, and maintained in an adaptive-like manner. Currently, these are thought to differentiate along a common pathway. However, by fate mapping of single NK cells upon murine cytomegalovirus (MCMV) infection, we identified two distinct NK cell lineages that contributed to adaptive-like responses. One was equivalent to conventional NK (cNK) cells while the other was transcriptionally similar to type 1 innate lymphoid cells (ILC1s). ILC1-like NK cells showed splenic residency and strong cytokine production but also recognized and killed MCMV-infected cells, guided by activating receptor Ly49H. Moreover, they induced clustering of conventional type 1 dendritic cells and facilitated antigen-specific T cell priming early during MCMV infection, which depended on Ly49H and the NK cell-intrinsic expression of transcription factor Batf3. Thereby, ILC1-like NK cells bridge innate and adaptive viral recognition and unite critical features of cNK cells and ILC1s.

Usefulness of BATF3 Immunohistochemistry in Diagnosing Classical Hodgkin Lymphoma.

It is well recognized that the AP-1 transcription factor BATF3 is constitutively expressed in Hodgkin/Reed-Sternberg (HRS) cells, but its potential as a diagnostic marker for classical Hodgkin lymphoma (cHL) has not yet been addressed. In this study, we performed immunohistochemistry and analyzed the BATF3 expression in lymphoma cells on 218 lymphoma samples belonging to 14 different lymphoma entities. We observed varying degrees of BATF3 expression in nearly half of the cases (n = 100) with BATF3 expression being a constitutive feature of cHL (n = 53) and anaplastic large cell lymphoma (ALCL). By scoring BATF3 expression (BATF3-score) we observed constitutively high BATF3-scores in cHL and ALCL and low to moderate BATF3-scores in all other entities examined. Western blot analysis confirmed BATF3 protein expression in cell lysates from cHL cell lines (n = 7). Thus, BATF3 can be considered a useful IHC marker for the diagnosis of cHL as it is highly sensitive and sufficiently specific when analyzed by BATF3-scoring.

BATF3 programs CD8+ T cell memory.

Antiviral CD8+ T cell responses are characterized by an initial activation/priming of T lymphocytes followed by a massive proliferation, subset differentiation, population contraction and the development of a stable memory pool. The transcription factor BATF3 has been shown to play a central role in the development of conventional dendritic cells, which in turn are critical for optimal priming of CD8+ T cells. Here we show that BATF3 was expressed transiently within the first days after T cell priming and had long-lasting T cell-intrinsic effects. T cells that lacked Batf3 showed normal expansion and differentiation, yet succumbed to an aggravated contraction and had a diminished memory response. Vice versa, BATF3 overexpression in CD8+ T cells promoted their survival and transition to memory. Mechanistically, BATF3 regulated T cell apoptosis and longevity via the proapoptotic factor BIM. By programing CD8+ T cell survival and memory, BATF3 is a promising molecule to optimize adoptive T cell therapy in patients.

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.

An Important Role for CD4+ T Cells in Adaptive Immunity to Toxoplasma gondii in Mice Lacking the Transcription Factor Batf3.

Immunity to Toxoplasma gondii at early stages of infection in C57BL/6 mice depends on gamma interferon (IFN-γ) production by NK cells, while at later stages it is primarily mediated by CD8 T cells. We decided to explore the requirement for CD4 T cells during T. gondii infection in Batf3-/- mice, which lack CD8α+ dendritic cells (DCs) that are necessary for cross-presentation of cell-associated antigens to CD8 T cells. We show that in this immunodeficient background on a BALB/c background, CD4 T cells become important effector cells and are able to protect Batf3-/- mice from infection with the avirulent strain RHΔku80Δrop5 Independently of the initial NK cell activation, CD4 T cells in wild-type and Batf3-/- mice were the major source of IFN-γ. Importantly, memory CD4 T cells were sufficient to provide protective immunity following transfer into Batf3-/- mice and secondary challenge with the virulent RHΔku80 strain. Collectively, these results show that under situations where CD8 cell responses are impaired, CD4 T cells provide an important alternative immune response to T. gondiiIMPORTANCEToxoplasma gondii is a widespread parasite of animals that causes zoonotic infections in humans. Although healthy individuals generally control the infection with only moderate symptoms, it causes serious illness in newborns and those with compromised immune systems such as HIV-infected AIDS patients. Because rodents are natural hosts for T. gondii, laboratory mice provide an excellent model for studying immune responses. Here, we used a combination of an attenuated mutant strain of the parasite that effectively vaccinates mice, with a defect in a transcriptional factor that impairs a critical subset of dendritic cells, to studying the immune response to infection. The findings reveal that in BALB/c mice, CD4 memory T cells play a dominant role in producing IFN-γ needed to control chronic infection. Hence, BALB/c mice may provide a more appropriate model for declining immunity seen in HIV-AIDS patients where loss of CD4 cells is associated with emergence of opportunistic infections.

Splenic Red Pulp Macrophages Cross-Prime Early Effector CTL That Provide Rapid Defense against Viral Infections.

Cross-presentation allows dendritic cells (DCs) to present peptides derived from endocytosed Ags on MHC class I molecules, which is important for activating CTL against viral infections and tumors. Type 1 classical DCs (cDC1), which depend on the transcription factor Batf3, are considered the main cross-presenting cells. In this study, we report that soluble Ags are efficiently cross-presented also by transcription factor SpiC-dependent red pulp macrophages (RPM) of the spleen. In contrast to cDC1, RPM used the mannose receptor for Ag uptake and employed the proteasome- and TAP-dependent cytosolic cross-presentation pathway, previously shown to be used in vitro by bone marrow-derived DCs. In an in vivo vaccination model, both cDC1 and RPM cross-primed CTL efficiently but with distinct kinetics. Within a few days, RPM induced very early effector CTL of a distinct phenotype (Ly6A/E+ Ly6C(+) KLRG1- CD127- CX3CR1- Grz-B+). In an adenoviral infection model, such CTL contained the early viral spread, whereas cDC1 induced short-lived effector CTL that eventually cleared the virus. RPM-induced early effector CTL also contributed to the endogenous antiviral response but not to CTL memory generation. In conclusion, RPM can contribute to antiviral immunity by generating a rapid CTL defense force that contains the virus until cDC1-induced CTL are available to eliminate it. This function can be harnessed for improving vaccination strategies aimed at inducing CTL.

Absence of conventional dendritic cells type 1 (cDC1) impairs intestinal homeostasis and permeability and leads to the development of obesity

Abstract The maintenance of intestinal homeostasis depends on a complex interaction between microbiota, intestinal epithelial barrier and immune system. Alteration in of one of these components could lead to the development of chronic inflammatory diseases such as inflammatory bowel diseases (IBD). Recent studies have associated obesity with more severe IBD. In obesity, pro-inflammatory macrophages accumulate in adipose tissue and their numbers correlate with inflammation and insulin resistance. BATF3 is a transcription factor implicated in the development of conventional dendritic cells type 1 (cDC1). Here, we show that aged Batf3−/− mice developed obesity as characterized by increased body weight, fasting glucose levels (Batf3−/−: 213, WT: 115 mg/dl, p < 0.005), size of abdominal white adipocyte, and development of hepatosteatosis. We also observed increased intestinal permeability in Batf3−/− mice (FITC-dextran gavage; p < 0.01) suggesting impaired epithelial barrier function. Feeding high-fat diet for 2 months further enhanced the metabolic phenotype in Batf3−/− mice, leading to increased fasting glucose levels (300 vs. 246 mg/dl, p < 0.05), and increased adipocyte size. Moreover, during high-fat diet Batf3−/− mice were more susceptible to dextran sulfate sodium (DSS) colitis with more severe cecal inflammation, increased gut and mesenteric fat M1 macrophages (p < 0.05). Our data suggest that deficiency of the transcription factor BATF3 and lack of cDC1 alters the intestinal epithelial barrier, promotes the development of obesity, and increases susceptibility to DSS. In conclusion, BATF3-dependent cDC1 play an important role in maintaining the intestinal epithelial barrier and are protective for the development of obesity.

Batf3-Dependent Dendritic Cells Promote Optimal CD8 T Cell Responses against Respiratory Poxvirus Infection

Respiratory infection with vaccinia virus (VacV) elicits robust CD8+ T cell responses that play an important role in host resistance. In the lung, VacV encounters multiple tissue-resident antigen-presenting cell (APC) populations, but which cell plays a dominant role in priming of virus-specific CD8+ effector T cell responses remains poorly defined. We used Batf3-/- mice to investigate the impact of CD103+ and CD8α+ dendritic cell (DC) deficiency on anti-VacV CD8+ T cell responses. We found that Batf3-/- mice were more susceptible to VacV infection, exhibiting profound weight loss, which correlated with impaired accumulation of gamma interferon (IFN-γ)-producing CD8+ T cells in the lungs. This was largely due to defective priming since early in the response, antigen-specific CD8+ T cells in the draining lymph nodes of Batf3-/- mice expressed significantly reduced levels of Ki67, CD25, and T-bet. These results underscore a specific role for Batf3-dependent DCs in regulating priming and expansion of effector CD8+ T cells necessary for host resistance against acute respiratory VacV infection.IMPORTANCE During respiratory infection with vaccinia virus (VacV), a member of Poxviridae family, CD8+ T cells play important role in resolving the primary infection. Effector CD8+ T cells clear the virus by accumulating in the infected lungs in large numbers and secreting molecules such as IFN-γ that kill virally infected cells. However, precise cell types that regulate the generation of effector CD8+ T cells in the lungs are not well defined. Dendritic cells (DCs) are a heterogeneous population of immune cells that are recognized as key initiators and regulators of T-cell-mediated immunity. In this study, we reveal that a specific subset of DCs that are dependent on the transcription factor Batf3 for their development regulate the magnitude of CD8+ T cell effector responses in the lungs, thereby providing protection during pulmonary VacV infection.

Abstract SY18-02: The relationship between Batf3-DC and antitumor T-cell responses

Abstract Cancer immunotherapy has attracted enormous attention and investment, stimulated by successes of immune checkpoint blockade drugs, such as anti-CTLA4 and anti-PD-1. Despite the potential of immunotherapy to eradicate even metastatic cancer, only a minority of cancer patients respond to checkpoint blockade agents. Clinical and preclinical data provide evidence that the presence of tumor-specific CD8 T cells within the tumor microenvironment (TME) is critical for the response to checkpoint blockade. Another major component of tumor-infiltrating immune systems is the mononuclear phagocytic cell compartment, which provides both pro- and antitumoral properties, depending on its composition. A rare subset of conventional dendritic cells, marked by CD103 and the transcription factor Batf3, is required for the uptake, transport, and presentation of tumor-derived antigens to CD8 T cells in the tumor-draining lymph node. Tumors lacking CD103+ dendritic cells do not mount spontaneous antitumor immune responses and fail to respond to checkpoint blockade therapy. Moreover, we observed that absence of this subset of DC is mediating resistance to adoptive T-cell transfer. Analysis of tumor-residing CD103+ dendritic cells revealed that this small subset of cells provides the predominant source of effector T-cell recruiting chemokines (CXCL9 and 10) and is a critical component of the TME. This observation imposes a more detailed and refined analysis of the contribution of tumor-residing dendritic cells to the local antitumor immune response. Citation Format: Stefani Spranger. The relationship between Batf3-DC and antitumor T-cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY18-02.

Abstract 3781: A focused dendritic cell CRISPR/Cas9 screen to identify novel regulators of cross-presentation and antitumor immunity

Abstract Introduction: Dendritic cells (DCs) are professional antigen-presenting cells with two major subsets, the classical DC type 1 and 2 (cDC1 and cDC2). The cDC1, also known as the CD8α+ DC, is especially equipped for the process of cross-presentation in which dead cell antigen is presented on MHC-I to prime cytotoxic T cells. This process is relevant to anti-cancer immunity, as mice deficient in the transcription factor BATF3, which specifically lack the cDC1, succumb to highly immunogenic tumors that are normally rejected and cleared in wild-type mice. The mechanism of cross-presentation by the cDC1 is poorly understood. In this study, we developed a focused CRISPR/Cas9 screen to identify novel genes that endow the cDC1 with the ability to cross-prime cytotoxic T cells. Methods: A curated list of target cDC1 genes was constructed using the Immunological Genome Project Database as the primary resource (www.immgen.org). 120 genes specifically expressed by the cDC1 in comparison to other murine immune cells were selected. Guide RNAs (gRNAs) were designed to early exon sequences using the MIT CRISPR Design Tool to minimize off-target effects (www.crispr.mit.edu), and 2-3 different gRNAs were evaluated in tandem for each candidate. These gRNAs were transduced via retroviral vector into lineage depleted c-Kithi bone marrow cells isolated from C57BL/6J Cas9 knock-in mice. These cells were differentiated in vitro for 8 days in the presence of the cytokine Flt3L. Transduced DCs were sorted by FACS and co-cultured in the presence of a source of dead cell antigen (heat-killed L. monocytogenes expressing ovalbumin, or HKLM-OVA) as well as CFSE-labelled transgenic CD8+ T cells with an ovalbumin peptide-specific T cell receptor (OT-1). The proliferation of OT-1 cells was measured by CFSE dilution. Results: To validate our CRISPR/Cas9-based screening approach, we targeted the first exon of the beta-2 microglobulin gene (B2M), which is required for cell surface expression of MHC-I. B2M knockout DCs were co-cultured in the presence of HKLM-OVA and CSFE-labelled OT-1 cells for 3 days. Controls included DCs transduced with a scramble gRNA with or without antigen. As expected, targeting B2M in DCs led to near-complete reduction in OT-1 proliferation compared to scramble control (3.7% vs. 20.6%, p=0.003), similar to the absence of antigen (3.7% vs. 0.96%, p=0.99). We then expanded our assay to our curated list. Promising candidate genes have been discovered and are currently being validated in murine knockout models. Conclusions: The CRISPR/Cas9 system can be adapted to screen the cDC1 for novel genes involved in cross-priming cytotoxic T cells. Validation of candidates in vivo, including tumor assays, will be presented at the meeting. These findings may have important implications for adaptive immunity against cancer. Citation Format: Jesse T. Davidson, Derek J. Theisen, Carlos G. Briseño, Vivek Durai, William E. Gillanders, Theresa L. Murphy, Kenneth M. Murphy. A focused dendritic cell CRISPR/Cas9 screen to identify novel regulators of cross-presentation and antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3781.

The transcription factor Batf3 inhibits the differentiation of regulatory T cells in the periphery

Naive CD4 T cells activated by antigen-presenting cells (APCs) undergo terminal differentiation in the periphery. Multiple mechanisms determine their fates, that is, whether they differentiate into conventional T (Tconv) cells or regulatory T (Treg) cells. The key event during Treg generation is expression of the transcription factor Foxp3, which is the lineage-determining regulator for Treg differentiation and function. Here we show that the transcription factor Batf3 acts as a fate-decision factor with respect to Tconv versus Tregs by restraining Treg differentiation. Batf3 was preferentially expressed in effector CD4 T cells but not in Treg cells, and ectopic expression of Batf3 inhibited Foxp3 induction. Batf3-deficient CD4 T cells favorably differentiated into Treg cells in vitro and in colonic lamina propria. Batf3 KO mice also showed enhanced Treg function in gut-associated immune disease models (for example, ovalbumin tolerance and inflammatory bowel disease models). Batf3 bound to the CNS1 region of the Foxp3 locus and reduced expression of the gene. Thus, Batf3 is a transcriptional suppressor of Treg differentiation.

A CD103+ Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis

Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ Tcell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets, defined their origins, and revealed an essential role for CD103+ cDCs in antigen-specific Tcell responses during subclinical viral myocarditis.

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.

CD103+ Kidney Dendritic Cells Protect against Crescentic GN by Maintaining IL-10-Producing Regulatory T Cells.

Kidney dendritic cells (DCs) regulate nephritogenic T cell responses. Most kidney DCs belong to the CD11b+ subset and promote crescentic GN (cGN). The function of the CD103+ subset, which represents <5% of kidney DCs, is poorly understood. We studied the role of CD103+ DCs in cGN using several lines of genetically modified mice that allowed us to reduce the number of these cells. In all lines, we detected a reduction of FoxP3+ intrarenal regulatory T cells (Tregs), which protect against cGN. Mice lacking the transcription factor Batf3 had a more profound reduction of CD103+ DCs and Tregs than did the other lines used, and showed the most profound aggravation of cGN. The conditional reduction of CD103+ DC numbers by 50% in Langerin-DTR mice halved Treg numbers, which did not suffice to significantly aggravate cGN. Mice lacking the cytokine Flt3L had fewer CD103+ DCs and Tregs than Langerin-DTR mice but exhibited milder cGN than did Batf3-/- mice presumably because proinflammatory CD11b+ DCs were somewhat depleted as well. Conversely, Flt3L supplementation increased the number of CD103+ DCs and Tregs, but also of proinflammatory CD11b+ DCs. On antibody-mediated removal of CD11b+ DCs, Flt3L supplementation ameliorated cGN. Mechanistically, CD103+ DCs caused cocultured T cells to differentiate into Tregs and produced the chemokine CCL20, which is known to attract Tregs into the kidney. Our findings show that CD103+ DCs foster intrarenal FoxP3+ Treg accumulation, thereby antagonizing proinflammatory CD11b+ DCs. Thus, increasing CD103+ DC numbers or functionality might be advantageous in cGN.

Transcription factor Batf3 is important for development of CD8+ T-cell response against a phagosomal bacterium regardless of the location of antigen.

Salmonella enterica serovar Typhimurium (ST) is a virulent intracellular bacterium that conceals itself in the phagosomes of infected cells. Although CD8(+) T cells promote protection against various intracellular pathogens, the role of CD8(+) T cells against virulent ST has been unclear due to early fatality of susceptible (B6) mice. Herein, we generated MHC I-deficient mice on the resistant (129SvJ) and susceptible (Nramp1 transgenic B6) background to evaluate the role of CD8(+) T cells against virulent ST. Our results indicate that CD8(+) T cells have a critical protective role in host survival during infection with virulent ST. As antigen presentation and CD8(+) T-cell activation against phagosomal antigens are considered to operate through the cross-presentation pathway, we have evaluated CD8(+) T-cell response against ST in Batf3-deficient mice that lack CD8α dendritic cells (DCs). Using a recombinant of ST that expresses antigen (ST-OVA) mainly in the phagosomes of infected cells, we show that CD8(+) T-cell response is compromised throughout the duration of infection in Batf3-deficient mice. In contrast, when ST delivers antigen to the cytosol of infected cells (ST-OVA-C), CD8(+) T-cell response against the cytosolic antigen was compromised only in the short term in the absence of CD8α DCs, with wild-type and Batf3-deficient mice generating similar CD8(+) T-cell response in the long term. Thus, Batf3 has an important role in CD8(+) T-cell priming regardless of antigenic location; however, its role is redundant at later time intervals against cytosolic antigen.

Abstract LB-236: Imprime PGG conjugated directly to protein enables cross-presentation of antigen that generates multifunctional cytotoxic T cells

Abstract Imprime PGG is a soluble yeast-derived β-1,3/1,6 glucan innate immune cell modulator that is in phase 3 and multiple phase 2 clinical trials in combination with complement activating monoclonal antibodies (e.g. bevacizumab, cetuximab). We have previously shown that Imprime binds to complement receptors and modulates the function of a variety of immune cells, including monocytes, neutrophils, and B cells. We have also demonstrated that Imprime PGG binds to various subsets of dendritic cells (DCs) and can cause intermediate upregulation of MHC class II and the co-stimulatory molecules CD80/86 critical for antigen presentation and T cell activation. Based on these findings, we hypothesized that Imprime PGG conjugated to a protein would efficiently deliver antigen to DCs and prime a cytotoxic CD8+ T cell response. To test this hypothesis, we employed the model antigen chicken ovalbumin (OVA) in a C57BL/6 mouse model to examine the generation of OVA-specific CD8+ T cell responses. We covalently linked OVA to Imprime PGG to generate a β-glucan/protein conjugate (Imprime-OVA). Using T cell receptor transgenic OT-I CD8+ T cells to track responses to OVA, we treated mice with Imprime-OVA intravenously and examined the expansion and functional quality of the T cell response 7 days later at the peak of expansion. Following Imprime-OVA treatment, OVA-specific CD8 T cells underwent vigorous expansion, upregulated the transcription factor Tbet, which is central to developing effector functions, and gained the ability to produce the cytokines IFN-γ, TNF-α and IL-2. By comparison, OVA alone did not generate a functional CD8+ T cell response and instead induced anergy. To determine if cross-presenting DCs are required for CD8+ T cell activation, we used mice deficient in the transcription factor Batf3, which selectively eliminates CD8α+ cross-presenting DCs. Following treatment with the Imprime-OVA conjugate in these Batf3-/- mice, the expansion of OVA-specific CD8+ T cells was more than 10-fold reduced compared to that in wild-type mice. Further, these cells failed to develop effector functions, indicating that CD8α+ cross-presenting DCs are crucial for this response. Together, these data show that an Imprime PGG-protein conjugate can effectively elicit the expansion and functional activation of cytotoxic T cells and may have utility as a potential cancer vaccine platform. Citation Format: Ross B. Fulton, Steven Leonardo, Kyle Michel, Lindsay Wurst, Trinda Phelon, Mike Danielson, Keith Gorden. Imprime PGG conjugated directly to protein enables cross-presentation of antigen that generates multifunctional cytotoxic T cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-236. doi:10.1158/1538-7445.AM2015-LB-236