TIM-3 blockade enhances IL-12-dependent antitumor immunity by promoting CD8+ T cell and XCR1+ dendritic cell spatial co-localization

BackgroundT cell immunoglobulin and mucin domain containing−3 (TIM-3) blocking antibodies are currently being evaluated in clinical trials for solid and hematological malignancies. Despite its identification on T cells, TIM-3 is predominantly expressed by myeloid cells, including XCR1+ type I conventional dendritic cells (cDC1s). We have recently shown that TIM-3 blockade promotes expression of CXCR3 chemokine ligands by tumor cDCs, but how this drives a CD8+ T cell-dependent response to therapy is unclear.MethodsT cell infiltration, effector function, and spatial localization in relation to XCR1+ cDC1s were evaluated in a murine orthotopic mammary carcinoma model during response to TIM-3 blockade and paclitaxel chemotherapy. Mixed bone marrow chimeras and diphtheria toxin depletion were used to determine the role of specific genes in cDC1s during therapeutic responses.ResultsTIM-3 blockade increased interferon-γ expression by CD8+ T cells without altering immune infiltration. cDC1 expression of CXCL9, but not CXCL10, was required for response to TIM-3 blockade. CXCL9 was also necessary for the increased proximity observed between CD8+ T cells and XCR1+ cDC1s during therapy. Tumor responses were dependent on cDC1 expression of interleukin-12, but not MHCI.ConclusionsTIM-3 blockade increases exposure of intratumoral CD8+ T cells to cDC1-derived cytokines, with implications for the design of therapeutic strategies using antibodies against TIM-3.

Biological Aging of CNS-Resident Cells Alters the Clinical Course and Immunopathology of Autoimmune Demyelinating Disease

Biological aging is the strongest factor associated with the clinical phenotype of multiple sclerosis (MS). Relapsing remitting MS (RRMS) typically presents in the third or fourth decade, while the mean age of presentation of progressive MS (pMS) is 45 years old. Here we show that experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer of encephalitogenic CD4+ Th17 cells, is more severe, and less like to remit, in middle-aged compared with young adult mice. Donor T cells and neutrophils are more abundant, while B cells are relatively sparse, in central nervous system (CNS) infiltrates of the older mice. Experiments with reciprocal bone marrow chimeras demonstrate that radio-resistant, non-hematopoietic cells play a dominant role in shaping age-related features of the neuroinflammatory response, as well as the clinical course, during EAE. Reminiscent of pMS, EAE in middle-aged adoptive transfer recipients is characterized by widespread microglial activation. Microglia from older mice express a distinctive transcriptomic profile, suggestive of enhanced chemokine synthesis and antigen presentation. Collectively, our findings suggest that drugs that suppress microglial activation, and acquisition or expression of aging-associated properties, may be beneficial in the treatment of progressive forms of inflammatory demyelinating disease.

Effects of PARP Inhibitor Therapy on p53-Deficient Hematopoietic Stem and Progenitor Cell Fitness

Poly (ADP-ribose) polymerase (PARP) inhibitors are an important new class of anti-cancer therapies. Therapy-related myeloid neoplasia (tMN) has been reported following PARPi therapy and is associated with adverse outcomes. We have previously shown, in retrospective data, that prior chemotherapy increases the incidence of clonal hematopoiesis (CH), especially in DNA damage response (DDR) pathway genes including TP53, PPM1D, and CHEK2 and is associated with progression to tMN. In particular, patients who receive PARPi therapy are more likely to have CH compared to other therapies or untreated patients. In the IMPACT study of CH in 10,156 cancer patients, exposure to PARPi were more likely to have CH (33%) compared to untreated patients (16%). This was particularly pronounced for DDR gene mutations, with 25% of PARPi treated patients with DDR CH compared to 2% of untreated patients. In multivariate analysis accounting for demographics and exposure to other chemotherapy or radiation therapy, exposure to PARPi conferred an increased risk of DDR CH (OR = 3.6, 95% CI 1.5-8.5, p = 0.004). From these data, we hypothesize that mutations in DDR pathway genes provide a fitness advantage to hematopoietic stem/progenitor cells (HSPCs) following PARPi treatment, leading to clonal hematopoiesis. A major limitation, however of our previous work in retrospective clinical samples, is the inability to completely adjust for the confounding effect of prior exposure to cytotoxic therapy (in particular platinum therapies) and germline BRCA1/2 mutations; both which have been shown or hypothesized to increase the risk of tMN. To test whether PARPi exposure might provide a fitness advantage to HSPCs independent of prior exposure to other therapies, we first examined the response of CRISPR-gene edited TP53-/- MOLM13 cells to the PARPi Olaparib and, as a control, Cisplatin. As expected, TP53-/- cells had increased resistance to both agents, though the response was much more pronounced in Cisplatin-treated cells (Figure 1A,B). Next, we implemented a mouse model of TP53-mutant clonal hematopoiesis, by generating mixed bone marrow chimeras transplanted with a 1:9 ratio of wildtype (CD45.1) to TP53 R172H+/- (CD45.2) cells. The "baseline" contribution of TP53 R172H+/- (CD45.2) cells to peripheral blood leukocytes 8 weeks after transplantation was determined by flow cytometry. Mice were then randomized into the following three cohorts: 1) Cisplatin (6mg/kg on days 1, 8, and 15); 2) Olaparib (50mg/kg daily for 3 weeks); and 3) vehicle alone. Peripheral blood chimerism was assessed 3, 9, and 12 weeks after initiating treatment. In addition, the contribution of TP53 R172H+/- to lineage -Sca1 +Kit + (LSK) cells in the bone marrow was determined. Cisplatin treatment resulted in a significant increase in the contribution of TP53 R172H+ to peripheral blood total leukocytes, granulocytes, and bone marrow LSK cells (Figure 1C-E). In contrast, Olaparib treated mice showed no change in CD45 chimerism. From these results we conclude that p53-deficiency does not confer a strong fitness advantage to mouse HSPCs in response to PARPi treatment. This suggests that the strong association observed between prior PARPi therapy, CH and tMN in clinical cohorts may in part be due to the confounding effects of prior (often heavy) exposure to platinum-based therapy. However, the majority of patients receiving PARPi have germline heterozygous BRCA1/2 mutations that could be contributing to their hematopoietic response to PARPi therapy. Experiments are underway to test this possibility by analyzing mixed bone marrow chimeras carrying heterozygous mutations of both Brca1 and Trp53. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

328 Batf3 dendritic cells and 4–1BB/4–1BB ligand axis are required at the effector phase within the tumor microenvironment for anti-PD-L1 efficacy

BackgroundPD-1/PD-L1 blockade has shown clinical benefit across many cancer types. However, a large fraction of patients are resistant to immune checkpoint blockade therapy and others eventually relapse. Understanding the mechanisms involved in αPD1/PD-L1 immunotherapy efficacy may enable new strategies for improving clinical outcomes. Given that Batf3-lineage dendritic cells (DCs) are needed for spontaneous T cell priming in the tumor-draining lymph node and for recruitment of effector CD8+ T cells to the tumor, in the current work we examined whether Batf3+ DCs are also required during the effector phase of the anti-tumor immune response at the time of anti-PD-L1 administration for therapeutic efficacy.MethodsWe utilized the B16-SIY melanoma model, CD11c-DTR-GFP, and CD11c-DTR-GFP/Batf3 KO bone marrow chimeras to study the role Batf3+ DCs play during anti-PD-L1 immunotherapy. To focus on the effector phase of the immune response, we depleted CD11c+ cells with diphtheria toxin from day seven of tumor injection while simultaneously blocking new T cell entry with FTY720. As flow cytometry revealed high 4-1BBL expression on intratumoral Batf3-DCs, 4-1BB KO mice and anti-4-1BBL blocking antibodies were used. Tumor growth and phenotypic analysis of the tumor infiltrate were evaluated.ResultsStrikingly, we observed that CD11c+ cells, and specifically Batf3+ DCs, were required in the tumor prior to αPD-L1 treatment for immunotherapy efficacy. The normal intratumoral expansion of antigen (Ag)-specific CD8+ tumor-infiltrating T cells (TILs) and increased ratio between Ag-specific CD8+ TILs and regulatory T cells following anti-PD-L1 therapy was eliminated with Batf3+ DC depletion. Batf3+ DCs expressed high levels of 4-1BBL, and increased expression of 4-1BB on antigen-specific CD8+ TILs upon αPD-L1 treatment required Batf3+ DCs. Mechanistic experiments confirmed a requirement for 4-1BB expression on immune cells for αPD-L1 efficacy, and blocking antibodies against 4-1BBL eliminated anti-PD-L1 efficacy as well. Using appropriate bone marrow chimeras, agonistic 4-1BB antibodies were sufficient to bypass the need for CD11c+ DCs at the effector phase for tumor control. In human melanoma samples, co-localization of Batf3+ DCs and CD8+ T cells was observed in T cell-inflamed tumors, which correlated with anti-PD-1 efficacy in metastatic melanoma.ConclusionsOur results indicate that Batf3+ DCs are necessary during the effector phase of the anti-tumor immune response for anti-PD-L1 efficacy to occur, at least in part through 4-1BB/4-1BBL-mediated reinvigoration of Ag-specific CD8+ TILs.Ethics ApprovalThe study obtained ethics approval, IRB protocol 15-0837.

Heightened turnover and failed maturation of monocyte-derived macrophages in murine Chronic Granulomatous Disease

Loss of NADPH oxidase activity leads to altered phagocyte responses and exaggerated inflammation in Chronic Granulomatous Disease (CGD). We sought to assess the effects of Nox2 absence on monocyte-derived macrophages (MoMacs) in gp91phox-/y mice during zymosan-induced peritonitis. MoMacs from CGD and wild type (WT) peritonea were characterized over time after zymosan injection. Though numbers lavaged from both genotypes were virtually identical, there were marked differences in maturation: newly recruited WT MoMacs rapidly enlarged and matured, losing Ly6C and gaining MHCII, CD206 and CD36, while CGD MoMacs remained small and were mostly Ly6C+MHCII-. RNAseq analyses showed few intrinsic differences between genotypes in newly recruited MoMacs but significant differences with time. WT MoMacs demonstrated changes in metabolism, adhesion and reparative functions, while CGD MoMacs remained inflammatory. PKH dye labeling demonstrated that while WT MoMacs were mostly recruited within the first 24h and remained in the peritoneum while maturing and enlarging, CGD monocytes streamed into the peritoneum for days with many migrating to the diaphragm where they were found in fibrin(ogen) clots surrounding clusters of neutrophils in nascent pyogranulomata. Importantly, these observations appeared to be driven by milieu: adoptive transfer of CGD MoMacs into inflamed peritonea of WT mice resulted in immunophenotypic maturation and normal behavior, whereas altered maturation/behavior of WT MoMacs resulted from transfer into inflamed peritonea of CGD mice. Additionally, Nox2-deficient MoMacs behaved similarly to their Nox2-sufficient counterparts within the largely WT milieu of mixed bone marrow chimeras. These data demonstrate persistent recruitment with fundamental failure of MoMac maturation in CGD.

Cytotoxic CD4+ T cells driven by T-cell intrinsic IL-18R/MyD88 signaling predominantly infiltrate Trypanosoma cruzi-infected hearts

Increasing attention has been directed to cytotoxic CD4+ T cells (CD4CTLs) in different pathologies, both in humans and mice. The impact of CD4CTLs in immunity and the mechanisms controlling their generation, however, remain poorly understood. Here, for the first time, we showed that CD4CTLs abundantly differentiate during mouse infection with an intracellular parasite. CD4CTLs appear in the spleen in parallel to Th1 cells, display pathogen-derived peptide-specific cytotoxicity against antigen-presenting cells and express immunoregulatory and/or exhaustion markers. We demonstrated that CD4CTL absolute numbers and activity are severely reduced in both Myd88-/- and Il18ra-/- mice. Of note, the infection of mixed-bone marrow chimeras revealed that WT, but not Myd88-/-, cells transcribe the CD4CTL gene signature and that Il18ra-/-CD4+ phenocopy Myd88-/-CD4+ T cells. Moreover, the adoptive transfer of WT CD4+GzB+ T cells to susceptible Il18ra-/- mice increased their survival. Importantly, cells expressing the CD4CTL phenotype predominate among CD4+ T cells infiltrating the infected cardiac tissue, are increased in the circulation of Chagas patients and their frequency correlates with severe cardiomyopathy. Our findings describe CD4CTLs as a major player in immune response to a relevant human pathogen and disclose T-cell intrinsic IL-18R/MyD88 signaling as a key pathway controlling the magnitude of the CD4CTL response.

Functional role of brain-engrafted macrophages against brain injuries

Background Brain-resident microglia have a distinct origin compared to macrophages in other organs. Under physiological conditions, microglia are maintained by self-renewal from the local pool, independent of hematopoietic progenitors. Pharmacological depletion of microglia during whole-brain radiotherapy prevents synaptic loss and long-term recognition memory deficits. However, the origin or repopulated cells and the mechanisms behind these protective effects are unknown. Methods CD45low/int/CD11b+ cells from naïve brains, irradiated brains, PLX5622-treated brains and PLX5622 + whole-brain radiotherapy-treated brains were FACS sorted and sequenced for transcriptomic comparisons. Bone marrow chimeras were used to trace the origin and long-term morphology of repopulated cells after PLX5622 and whole-brain radiotherapy. FACS analyses of intrinsic and exotic synaptic compartments were used to measure phagocytic activities of microglia and repopulated cells. In addition, concussive brain injuries were given to PLX5622 and brain-irradiated mice to study the potential protective functions of repopulated cells after PLX5622 + whole-brain radiotherapy. Results After a combination of whole-brain radiotherapy and microglia depletion, repopulated cells are brain-engrafted macrophages that originate from circulating monocytes. Comparisons of transcriptomes reveal that brain-engrafted macrophages have an intermediate phenotype that resembles both monocytes and embryonic microglia. In addition, brain-engrafted macrophages display reduced phagocytic activity for synaptic compartments compared to microglia from normal brains in response to a secondary concussive brain injury. Importantly, replacement of microglia by brain-engrafted macrophages spare mice from whole-brain radiotherapy-induced long-term cognitive deficits, and prevent concussive injury-induced memory loss. Conclusions Brain-engrafted macrophages prevent radiation- and concussion-induced brain injuries and cognitive deficits.

Interferon-γ-Producing CD4+ T Cells Drive Monocyte Activation in the Bone Marrow During Experimental Leishmania donovani Infection

Ly6Chi inflammatory monocytes develop in the bone marrow and migrate to the site of infection during inflammation. Upon recruitment, Ly6Chi monocytes can differentiate into dendritic cells or macrophages. According to the tissue environment they can also acquire different functions. Several studies have described pre-activation of Ly6Chi monocytes in the bone marrow during parasitic infection, but whether this process occurs during experimental visceral leishmaniasis and, if so, the mechanisms contributing to their activation are yet to be established. In wild type C57BL/6 (B6) mice infected with Leishmania donovani, the number of bone marrow Ly6Chi monocytes increased over time. Ly6Chi monocytes displayed a highly activated phenotype from 28 days to 5 months post infection (p.i), with >90% expressing MHCII and >20% expressing iNOS. In comparison, in B6.Rag2-/- mice <10% of bone marrow monocytes were MHCII+ at day 28 p.i., an activation deficiency that was reversed by adoptive transfer of CD4+ T cells. Depletion of CD4+ T cells in B6 mice and the use of mixed bone marrow chimeras further indicated that monocyte activation was driven by IFNγ produced by CD4+ T cells. In B6.Il10-/- mice, L. donovani infection induced a faster but transient activation of bone marrow monocytes, which correlated with the magnitude of CD4+ T cell production of IFNγ and resolution of the infection. Under all of the above conditions, monocyte activation was associated with greater control of parasite load in the bone marrow. Through reinfection studies in B6.Il10-/- mice and drug (AmBisome®) treatment of B6 mice, we also show the dependence of monocyte activation on parasite load. In summary, these data demonstrate that during L. donovani infection, Ly6Chi monocytes are primed in the bone marrow in a process driven by CD4+ T cells and whereby IFNγ promotes and IL-10 limits monocyte activation and that the presence of parasites/parasite antigen plays a crucial role in maintaining bone marrow monocyte activation.

Role of Tim4 in the regulation of ABCA1+ adipose tissue macrophages and post-prandial cholesterol levels

Dyslipidemia is a main driver of cardiovascular diseases. The ability of macrophages to scavenge excess lipids implicate them as mediators in this process and understanding the mechanisms underlying macrophage lipid metabolism is key to the development of new treatments. Here, we investigated how adipose tissue macrophages regulate post-prandial cholesterol transport. Single-cell RNA sequencing and protected bone marrow chimeras demonstrated that ingestion of lipids led to specific transcriptional activation of a population of resident macrophages expressing Lyve1, Tim4, and ABCA1. Blocking the phosphatidylserine receptor Tim4 inhibited lysosomal activation and the release of post-prandial high density lipoprotein cholesterol following a high fat meal. Both effects were recapitulated by chloroquine, an inhibitor of lysosomal function. Moreover, clodronate-mediated cell-depletion implicated Tim4+ resident adipose tissue macrophages in this process. Thus, these data indicate that Tim4 is a key regulator of post-prandial cholesterol transport and adipose tissue macrophage function and may represent a novel pathway to treat dyslipidemia.

Heterogeneity of meningeal B cells reveals a lymphopoietic niche at the CNS borders

The meninges contain adaptive immune cells that provide immunosurveillance of the CNS. These cells are thought to derive from the systemic circulation. Through single-cell analyses, confocal imaging, bone marrow chimeras, and parabiosis experiments, we show that meningeal B cells derive locally from the calvaria, which harbors a bone marrow niche for hematopoiesis. B cells reach the meninges from the calvaria through specialized vascular connections. This calvarial–meningeal path of B cell development may provide the CNS with a constant supply of B cells educated by CNS antigens. Conversely, we show that a subset of antigen-experienced B cells that populate the meninges in aging mice are blood-borne. These results identify a private source for meningeal B cells. which may help maintain immune privilege within the CNS.