Low-Salt Diet Attenuates B-Cell- and Myeloid-Cell-Driven Experimental Arthritides by Affecting Innate as Well as Adaptive Immune Mechanisms

A link between high sodium chloride (salt) intake and the development of autoimmune diseases was previously reported. These earlier studies demonstrated exacerbation of experimental autoimmune encephalomyelitis and colitis by excess salt intake associated with Th17- and macrophage-mediated mechanisms. Little is known about the impact of dietary salt intake on experimental arthritides. Here, we investigated if salt restriction can exert beneficial effects on collagen-induced arthritis (CIA) and K/BxN serum transfer-induced arthritis (STIA). CIA depends on both adaptive and innate immunity, while STIA predominantly mimics the innate immune cell-driven effector phase of arthritis. In both models, low salt (LS) diet significantly decreased arthritis severity compared to regular salt (RS) and high salt (HS) diet. We did not observe an aggravation of arthritis with HS diet compared to RS diet. Remarkably, in STIA, LS diet was as effective as IL-1 receptor blocking treatment. Complement-fixing anti-CII IgG2a antibodies are associated with inflammatory cell infiltration and cartilage destruction. LS diet reduced anti-CII IgG2a levels in CIA and decreased the anti-CII IgG2a/IgG1 ratios pointing toward a more Th2-like response. Significantly less inflammatory joint infiltrates and cartilage breakdown associated with reduced protein concentrations of IL-1 beta (CIA and STIA), IL-17 (CIA), and the monocyte chemoattractant protein-1 (MCP-1) (CIA) were detected in mice receiving LS diet compared to HS diet. However, we did not find a reduced IL-17A expression in CD4+ T cells upon salt restriction in CIA. Analysis of mRNA transcripts and immunoblots revealed a link between LS diet and inhibition of the p38 MAPK (mitogen-activated protein kinase)/NFAT5 (nuclear factor of activated T-cells 5) signaling axis in STIA. Further experiments indicated a decreased leukodiapedesis under LS conditions. In conclusion, dietary salt restriction ameliorates CIA and STIA, indicating a beneficial role of LS diet during both the immunization and effector phase of immune-mediated arthritides by predominantly modulating the humoral immunity and the activation status of myeloid lineage cells. Hence, salt restriction might represent a supportive dietary intervention not only to reduce cardiovascular risk, but also to improve human inflammatory joint diseases like rheumatoid arthritis.

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.

Programming isotype specific plasma cell differentiation

Antibodies are produced across multiple isotypes with distinct properties that coordinate initial antigen clearance and confer long-term antigen-specific immune protection. Here, we interrogate the molecular programs of isotype-specific murine plasma cells (PC) following helper T cell dependent immunization and within established steady-state immunity. Using integrated single cell strategies, we reveal conserved and divergent components of the rapid effector phase of antigen-specific IgM+ versus inflammation modulating programs dictated by IgG2a/b+ PC differentiation. During antibody affinity maturation, the germinal center (GC) cycle imparts separable programs for post-GC inhibitory IgG1+ and inflammatory IgG2a/b+ PC to direct long-term cellular function. In the steady-state, two subsets of IgM+ and separate IgG2b+ PC programs clearly segregate from splenic IgA+ PC programs that emphasize mucosal barrier protection. These diverse isotype-specific molecular pathways of PC differentiation control complementary modules of antigen clearance and immune protection that could be selectively targeted for immunotherapeutic applications and vaccine design.

The extracellular ATP receptor P2RX7 imprints a pro-memory transcriptional signature in effector CD8+ T cells

Development of central memory (Tcm) and resident memory (Trm) CD8+ T cells, which respectively promote immunity in the circulation and in barrier tissues, is not completely understood. Tcm and Trm cells may arise from common precursors, however their fate-inducing signals are elusive. We found that virus-specific effector CD8+ T cells display heterogeneous expression of the extracellular ATP sensor P2RX7. P2RX7-high expression is confined, at peak effector phase, to CD62L+ memory precursors which preferentially form Tcm cells. Among early effector CD8+ T cells, asymmetrical P2RX7 distribution correlated with distinct transcriptional signatures, with P2RX7-high cells enriched for memory and tissue-residency sets. P2RX7-high early effectors preferentially form both Tcm and Trm cells. Defective Tcm and Trm formation in P2RX7 deficiency is significantly reverted when the transcriptional repressor Zeb2 is ablated. Our study indicates that unequal P2RX7 upregulation in effector CD8+ T cells is a foundational element of the early Tcm/Trm fate.

CD4 Effectors Need to Recognize Antigen Locally to Become Cytotoxic CD4 and Follicular Helper T Cells

SummaryT follicular helper (TFH) and Cytotoxic CD4 (ThCTL) are tissue-restricted CD4 effector subsets, functionally specialized to mediate optimal Ab production and cytotoxicity of infected cells. Influenza infection generates robust CD4 responses, including lung ThCTL and SLO TFH, that protect against reinfection by variant strains. Antigen (Ag) presentation after infection, lasts through the effector phase of the response. Here, we show that this effector phase Ag presentation, well after priming, is required to drive CD4 effectors to ThCTL and TFH. Using in vivo influenza models, we varied Ag presentation to effectors acutely, just at the effector phase. Ag presentation was required in the tissue of effector residence. We suggest these requirements contain unnecessary or potentially pathogenic CD4 responses, only allowing them if infection is uncleared. The results imply that providing effector phase Ag, would lead to stronger humoral and CD4 tissue immunity and thus can be applied to improve vaccine design.

AB0098 THE EFFECT OF MALT1-DEFICIENCY ON THE EFFECTOR PHASE OF EXPERIMENTAL AUTOIMMUNE ARTHRITIS

Background:The paracaspase Malt1 is a cysteine protease, which forms a complex leading to the activation of the in proinflammatory transcription factor NF-κB in lymphocytes with CARMA1 and Bcl10. Previously, we showed that the myeloid equivalent of CARMA1, Card9 is important in neutrophils in Fcγ receptor-mediated cytokine release together with Bcl10 and Malt1. In line with these findings, we observed a significant decrease in the severity of autoantibody-triggered arthritis in the absence of Card9 and Bcl10.Objectives:Our aim was to directly investigate whether the genetic deficiency of Malt1, the third component of the complex altered the process of the K/BxN serum transfer arthritis (that resembles to the effector phase of rheumatoid arthritis).Methods:We used wild type and Malt1–/–mice for our experiments. Autoantibody-mediated arthritis was induced by a single intraperitoneal injection of K/BxN serum. Clinical signs of joint inflammation were scored on a scale based on the cardinal inflammatory clues for two weeks. Ankle thickness was measured by a spring-loaded caliper.Results:Similar to the deficiency of the other two components of the complex, Malt1–/–mice showed a partial, but significant decrease in the macroscopic joint inflammation compared to arthritic serum-treated wild type animals during the entire experimental process. In line with this phenomenon, Malt1–/–animals had reduced autoantibody-triggered ankle thickening.Conclusion:Our results show that Malt1 seems to be an important molecule in the development and progression of experimental autoantibody-induced arthritis in mice, highlighting the role of the molecule as a potential therapeutic target in the future.Disclosure of Interests:None declared