Abstract Classical dendritic cells (cDCs) are professional antigen-presenting cells that play a key role in shaping appropriate immune responses. DCs are a potent T cell activators but they are also involved in maintaining immune homeostasis and self-tolerance. DCs can be classified into three major types: pDC, DC1 and DC2. One mechanism by which DCs regulate tolerance involves indoleamine 2,3-dioxygenase 1 (IDO1) a tryptophan (Trp) metabolizing enzyme. In this study, we analyzed the ability of L-Kyn to induce tolerogenic IDO1 pathway in different DCs subsets in vitro and in vivo model of experimental autoimmune encephalomyelitis (EAE). We show that inflammatory stimuli, like LPS, was able to induce IDO1 only in DC1, but not in DC2 or pDC, when DCs were treated as isolated cultures. In contrast, when LPS was added to cultures containing all three DC subsets, LPS could also induce IDO1 expression in DC2, which acquired tolerogenic function. Induction of IDO1 in DC2 involved a novel DC1-DC2 communication pathway mediated by a Kyn-AhR-RelB axis. Kynurenine produced by DC1 activates AhR in DC2 inducing IDO1 in a RelB-dependent manner. In vitro L-Kyn treatment impaired DC2 T cells priming ability causing suppression of MOG-specific reactivity with an increment of Foxp3+ CD4+ T cells. In vivo, oral administration of L-Kyn induces functional Treg cells that suppress EAE and this effect is completely abrogated in Ahrflox/floxCD11C Cre+ mice. These data suggest that in specific microenvironments, small numbers of IDO1-expressing DC1 may spread tolerogenic activity to DC2 cells through a kynurenine-AhR axis and L-Kyn could constituting a unique endogenous molecule for therapeutic immunomodulation of inflammatory and autoimmune diseases.
Individual elements within a superenhancer can act in a cooperative or temporal manner, but the underlying mechanisms remain obscure. We recently identified an Irf8 superenhancer, within which different elements act at distinct stages of type 1 classical dendritic cell (cDC1) development. The +41-kb Irf8 enhancer is required for pre-cDC1 specification, while the +32-kb Irf8 enhancer acts to support subsequent cDC1 maturation. Here, we found that compound heterozygous Δ32/Δ41 mice, lacking the +32- and +41-kb enhancers on different chromosomes, show normal pre-cDC1 specification but, surprisingly, completely lack mature cDC1 development, suggesting cis dependence of the +32-kb enhancer on the +41-kb enhancer. Transcription of the +32-kb Irf8 enhancer-associated long noncoding RNA (lncRNA) Gm39266 is also dependent on the +41-kb enhancer. However, cDC1 development in mice remained intact when Gm39266 transcripts were eliminated by CRISPR/Cas9-mediated deletion of lncRNA promoters and when transcription across the +32-kb enhancer was blocked by premature polyadenylation. We showed that chromatin accessibility and BATF3 binding at the +32-kb enhancer were dependent on a functional +41-kb enhancer located in cis . Thus, the +41-kb Irf8 enhancer controls the subsequent activation of the +32-kb Irf8 enhancer in a manner that is independent of associated lncRNA transcription.
Abstract Dendritic cells (DCs) are required to prime T cells during an immune response. The dogma in the field is that Batf3-dependent DCs (DC1s) prime CD8 T cells and IRF4-dependent DCs (DC2s) prime CD4 T cells. However, recent studies have shown that DC1s are required for induction of Th1 immune responses and to prime autroreactive CD4 T cells in the NOD mouse model of diabetes. Therefore, this segregation of priming theory is flawed. To further study the contribution of DC1s to CD4 T cell priming, we generated an XCR1-cre mouse and crossed it to the MHC class II floxed mouse to generate MHC class II deficient DC1s. We found that the form of antigen directs the priming of T cells. DC2s are superior at presenting soluble antigen as evidenced by normal CD4 and CD8 OVA specific T cell responses in mice lacking DC1s. However, DC1s are superior at presenting cell-associated antigen as evidenced by a lack of OVA specific CD4 and CD8 T cell priming in mice lacking DC1s. Furthermore, CD4 priming during tumor immune responses is absent when DC1s lack MHC class II. Our findings show that the DC subsets differ in the form of antigen that they present. DC1s present cell-associated material; whereas DC2s present soluble antigen.
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.
Abstract Relatively little is known with regard to CD4 T cell memory formation and metabolism, in part because CD4 T cell numbers remain low during a recall infection. Further, the contribution of self-peptide-MHC signaling (during both thymic T cell development and peripheral maintenance) to metabolic programming, if any, remains unknown. To address these questions, we have employed LLO-Hi5 and LLO-Lo5, two TCRtg CD4 T cells that recognize the same Listeria epitope with the same affinity. LLO-Hi5 CD4 T cells are highly self-reactive and respond poorly in a primary infection but robustly in a secondary infection. Less self-reactive LLO-Lo5 cells respond well during primary infection but poorly during secondary infection. We performed metabolic profiling to determine whether differences in response during infection were linked to metabolic differences between LLO-Lo5 and LLO-Hi5. We found that LLO-Lo5 had dramatically higher respiration and glycolytic rates relative to LLO-Hi5, and hypothesized that the decreased metabolism resulting from a strong interaction with self was mediated through TCR signaling. To test this hypothesis, we generated a knockin mouse expressing the Scn5a voltage gated sodium channel. This channel, when normally expressed in DP thymocytes, enhances TCR-mediated signaling. We found that overexpression of Scn5a in peripheral T cells increased TCR-proximal signaling. Further, Scn5a-expressing LLO-Lo5 cells displayed an impaired response during a primary infection. In this way, we demonstrate that tuning of TCR sensitivity to self can be used to alter in vivo immune responses. These studies highlight the critical relationship between TCR:self-pMHC interaction, metabolism, and the immune response to infection.
Abstract Batf3-dependent classical dendritic cells (cDCs) are essential for cross-presentation and clearance of intracellular pathogens through priming of CD8 T cells. By analyzing differences in gene expression between cDC subsets we identified vesicular transport molecules unique to cross-presenting dendritic cell subsets. RAB43, a GTPase that is localized to the golgi, is selectively expressed in Batf3-dependent CD8a+ and CD103+ cDCs and was highly conserved between mouse and human. Mice with germline and conditional Rab43 deletions are viable and fertile with normal hematopoiesis. However, mice lacking Rab43 showed reduced cross-presentation of cell-associated, but not soluble, antigens both in vitro and in vivo. Bone-marrow derived DCs (BMDCs) from Rab43 deficient mice show normal cross-presentation efficiency, suggesting that Rab43 is involved in optimizing cross-presentation in vivo through a pathway unique to CD8a+ DCs.
Abstract The antigen presenting cell that primes T cells in the central nervous system (CNS) remains unknown. Outside the CNS, the conventional dendritic cell 1 (cDC1) subset presents antigen to and primes CD8 T cells. However, the steady-state CNS parenchyma is relatively devoid of all dendritic cell subsets, including cDC1. cDC1 are required for anti-tumor immunity a variety of other tumor types, but their role CNS tumors remains undefined. Using the orthotopic preclinical glioblastoma models, GL261 and CT2A, we characterized the role of cDC1 in the CNS anti-tumor immune response. While cDC1 are absent in the steady state brain, tumor presence drove recruitment of cDC1 into extravascular spaces within the tumor and adjacent brain parenchyma. We further found that while GL261-bearing wildtype mice experienced survival benefit following anti-PDL1 checkpoint blockade treatment, mice with cDC1 genetically deleted experienced no survival benefit. cDC1-deficient mice completely lacked neoantigen-specific CD8 T cells against the endogenously-primed GL261-neoantigen mutant Imp3, and possessed broad CD8 effector T cell defects compared to wild type mice. Furthermore, using a fluorescent tumor-associated reporter, we detected tumor-derived material within dendritic cells from the tumor, the lymphatic vessel-containing dura, and the cervical lymph nodes. We observed the human cDC1-equivalent CD141+ cDC within human brain tumors (not limited to GBM) and dura as well. We used the GBM-specific reporter, 5-aminoilevulinic acid/protoporphyrin IX (PPIX) fluorescent metabolite to resect the tumor, and observed PPIX specifically in conventional dendritic cell subsets that had infiltrated the resected tumor, but not within those same cell subsets in the periphery, nor in T cells within the tumor. These findings comport with the canonical understanding that cDC1 uptake antigen at the effector site and migrate to draining lymph nodes to prime effector CD8 T cells, and highlight the significant role that cDC1 play in CNS anti-tumor immunity in mice and humans.
