GSK-3b in dendritic cells exerts opposite functions in regulating cross-priming and memory CD8 T cell responses independent of b-catenin
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ABSTRACT: GSK-3b plays a critical role in regulating the Wnt/b-catenin signaling pathway, and manipulating GSK-3b in dendritic cells (DCs) has been shown to improve anti-tumor efficacy of DC vaccines. Since inhibition of GSK-3b leads to activation of b-catenin, we hypothesize that blocking GSK-3b in DCs negatively regulates DC-mediated CD8 T cell immunity and anti-tumor immunity. Using CD11c-GSK-3b-/- conditional knockout mice in which GSK-3b is genetically deleted in CD11c-expressing DCs, we surprisingly found that deletion of GSK-3b in DCs resulted in increased anti-tumor immunity, which contradicted our initial expectation of reduced anti-tumor immunity due to presumed upregulation of b-catenin in DCs. Indeed, we found by both western blot and flow cytometry that deletion of GSK-3b in DCs did not lead to augmented expression of b-catenin protein, suggesting that GSK-3b exerts its function independent of b-catenin. Supporting this notion, our single cell RNA sequencing (scRNA-seq) analysis revealed that GSK-3b-deficient DCs exhibited distinct gene expression patterns with minimally overlapping differentially expressed genes (DEGs) compared to DCs with activated b-catenin. This suggests that deletion of GSK-3b in DCs is unlikely to lead to upregulation of b-catenin at the transcriptional level. Consistent with enhanced anti-tumor immunity, we also found that CD11c-GSK-3b-/- mice exhibited significantly augmented cross-priming of antigen-specific CD8 T cells following DC-targeted vaccines. We further found that deletion of GSK-3b in DCs completely abrogated memory CD8 T cell responses, suggesting that GSK-3b in DCs also plays a negative role in regulating the differentiation and/or maintenance of memory CD8 T cells. scRNA-seq analysis further revealed that although deletion of GSK-3b in DCs positively regulated transcriptional programs for effector differentiation and function of primed antigen-specific CD8 T cells in CD11c-GSK-3b-/- mice during the priming phase, it resulted in significantly reduced antigen-specific memory CD8 T cells, consistent with diminished memory responses. Taken together, our data demonstrate that GSK-3b in DCs has opposite functions in regulating cross-priming and memory CD8 T cell responses, and GSK-3b exerts its functions independent of its regulation of b-catenin.
Project description:Recent studies have demonstrated that b-catenin in dendritic cells (DCs) serves as a key mediator in promoting both CD4 and CD8 T cell tolerance, although the mechanisms underlying how b-catenin exerts its functions remains incompletely understood. Here we report that activation of b-catenin leads to the up-regulation of inhibitory molecule T-cell immunoglobulin and mucin domain 3 (Tim-3) in type 1 conventional DCs (cDC1s). Using a cDC1-targeted vaccine model with anti-DEC-205 engineered to express the melanoma antigen--human gp100 (anti-DEC-205-hgp100), we demonstrated that CD11c-b-cateninactive mice exhibited impaired cross-priming and memory re-sponses of gp100-specific CD8 T (Pmel-1) cells upon immunization with anti-DEC-205-hgp100. Sin-gle cell RNA sequencing (scRNA-seq) analysis revealed that b-catenin in DCs negatively regulated transcription programs for effector function and proliferation of primed Pmel-1 cells, correlating with suppressed CD8 T cell immunity in CD11c-b-cateninactive mice. Further experiments showed that treating CD11c-b-cateninactive mice with anti-Tim-3 antibody upon anti-DEC-205-hgp100 vac-cination led to restored cross-priming and memory responses of gp100-specific CD8 T cells, sug-gesting that anti-Tim-3 treatment likely synergizes with DC vaccines to improve their efficacy. In-deed, treating B16F10-bearing mice with DC vaccines using anti-DEC-205-hgp100 in combination with anti-Tim-3 treatment resulted in significantly reduced tumor growth, compared to treatment with DC vaccine alone. Taken together, we have identified the b-catenin/Tim-3 axis as a novel mech-anism to inhibit anti-tumor CD8 T cell immunity, and that combination immunotherapy of a DC-targeted vaccine with anti-Tim-3 treatment leads to improved anti-tumor efficacy.
Project description:The two vertebrate Gsk-3 isoforms, Gsk-3a and Gsk-3b, are encoded by distinct genetic loci and exhibit mostly redundant function in murine embryonic stem cells (ESCs). Here we report that deletion of both Gsk-3a and Gsk-3b in mouse ESCs results in significant changes in gene expression. In contrast, deletion of either Gsk-3a or Gsk-3b individually had little effect on gene expression. These data support the notion that Gsk-3 isoforms are functionally redundant in embryonic stem cells. In addition, we did not find the expected upregulation of known Wnt target genes. Our data suggests that Gsk-3-meidated regulation of gene expression in embryonic stem cells is complex, and likely involves affects on numerous signaling pathways. The study was designed to examine the changes in gene expression between wild-type, Gsk-3a-/-, Gsk-3b-/-, and Gsk-3a-/-;Gsk-3b-/- mouse embryonic stem cells.
Project description:The two vertebrate Gsk-3 isoforms, Gsk-3a and Gsk-3b, are encoded by distinct genetic loci and exhibit mostly redundant function in murine embryonic stem cells (ESCs). Here we report that deletion of both Gsk-3a and Gsk-3b in mouse ESCs results in significant changes in gene expression. In contrast, deletion of either Gsk-3a or Gsk-3b individually had little effect on gene expression. These data support the notion that Gsk-3 isoforms are functionally redundant in embryonic stem cells. In addition, we did not find the expected upregulation of known Wnt target genes. Our data suggests that Gsk-3-meidated regulation of gene expression in embryonic stem cells is complex, and likely involves affects on numerous signaling pathways.
Project description:The unfolded protein response (UPR) sensor IRE1 and its target, the transcription factor XBP1s critically regulate the function of dendritic cell (DC) subtypes. However, the contribution of the IRE1/XBP1s axis in DCs to the antitumor immunity is not entirely understood. Here, using reporter mice we found that DCs, in particular type 1 conventional DCs (cDC1s), are major targets of IRE1 RNase activity in melanoma tumors. Deletion of XBP1s in CD11c+ cells resulted in augmented tumor growth, impaired effector T cell responses and decreased accumulation of TCF-1+CD8+ T cells with a precursor exhausted profile. Transcriptomic studies revealed that XBP1 deletion in tumor cDC1s induced regulated IRE1 dependent decay (RIDD) of mRNAs, which accounted for the dysregulated T cell immunity in melanoma. Thus, a strict regulatory circuit involving IRE1 RNase and XBP1s in DCs ensures optimal antitumor T cell immunity in melanoma models.
Project description:The two vertebrate Gsk-3 isoforms, Gsk-3a and Gsk-3b, are encoded by distinct genetic loci and exhibit mostly redundant function in murine embryonic stem cells (ESCs). Here we report that deletion of both Gsk-3a and Gsk-3b in mouse ESCs results in misregulated expression of imprinted genes and hypomethylation of corresponding imprinted loci. Treatment of wild-type ESCs with small molecule inhibitors of Gsk-3 phenocopies the DNA hypomethylation of imprinted loci observed in Gsk-3 null ESCs. We provide evidence that DNA hypomethylation in Gsk-3 null ESCs is due to a reduction in the levels of the de novo DNA methyltransferase, Dnmt3a2. Gsk-3 activity serves as a node for several signal transduction pathways, and its regulation of Dnmt3a2 expression raises the possibility that DNA methylation could be transiently affected by different types of environmental stimuli. Our data suggest that modulating Gsk-3 activity could have further reaching effects in the regulation of the epigenome. Keywords: Gene expression array-based The study was designed to examine the changes in gene expression between wild-type and Gsk-3a-/-;Gsk-3b-/- mouse embryonic stem cells.
Project description:Dendritic cells play a vital role in initiating robust immunity against pathogens as well as maintaining immunological tolerance to self antigens, food antigens and intestinal commensals. However, the intracellular signaling networks that program DCs to become tolerogenic are largely unknown. To address this, we analyzed gene expression profiles using microarray analysis of purified intestinal lamina propria DCs (CD11c+ CD11b+ DCs and CD11c+ CD11b- DCs) from mice. Keywords: Lamina propria, DCs, cell type comparison We sought to determine the expression profile of small intestine lamina propria CD11c+ cells. RNA was extracted from DCs sorted from mouse small intestine (CD11c+CD11b- and CD11c+CD11b+ cells) and hybridized on Affymetrix microarrays.
Project description:Dendritic cells (DCs) play a vital role in innate immunity. Transcriptome of DCs isolated from mouse spleen was obtained and deposited here. Keywords: Spleen, DCs We sought to determine the expression profile of splenic CD11c+ cells. RNA was extracted from DCs sorted from mouse spleen (CD11c+ cells) and hybridized on Affymetrix microarrays.
Project description:DC progenitors adapt their transcriptional program during development generating different subsets. How chromatin modifications modulate these processes is unclear. Here we investigate the impact of histone deacetylation on DCs by genetically deleting HDAC1 or HDAC2 in hematopoietic progenitors and CD11c-expressing cells. While HDAC2 isn’t critical for DC development, HDAC1 deletion impairs pro- and mature pDC generation and affects ESAM+cDC2 differentiation from tDC and pre-cDC2, whereas cDC1s are unchanged. HDAC1 knock-down in human hematopoietic cells also impairs cDC2 development highlighting its crucial role across species. Multi-omics analyses reveal that HDAC1 controls expression, chromatin accessibility and histone-acetylation of the transcription factors IRF4, IRF8 and SPIB required for efficient development of cDC2 subsets. Without HDAC1, DCs switch immunologically enhancing tumor surveillance through increased cDC1 maturation and IL-12 production driving Th1-mediated immunity and CD8+T-cell recruitment. Our study reveals the importance of histone-acetylation in DC development and anti-tumor immunity suggesting DC-targeted therapeutic strategies for immuno-oncology.
Project description:DC progenitors adapt their transcriptional program during development generating different subsets. How chromatin modifications modulate these processes is unclear. Here we investigate the impact of histone deacetylation on DCs by genetically deleting HDAC1 or HDAC2 in hematopoietic progenitors and CD11c-expressing cells. While HDAC2 isn’t critical for DC development, HDAC1 deletion impairs pro- and mature pDC generation and affects ESAM+cDC2 differentiation from tDC and pre-cDC2, whereas cDC1s are unchanged. HDAC1 knock-down in human hematopoietic cells also impairs cDC2 development highlighting its crucial role across species. Multi-omics analyses reveal that HDAC1 controls expression, chromatin accessibility and histone-acetylation of the transcription factors IRF4, IRF8 and SPIB required for efficient development of cDC2 subsets. Without HDAC1, DCs switch immunologically enhancing tumor surveillance through increased cDC1 maturation and IL-12 production driving Th1-mediated immunity and CD8+T-cell recruitment. Our study reveals the importance of histone-acetylation in DC development and anti-tumor immunity suggesting DC-targeted therapeutic strategies for immuno-oncology.
Project description:DC progenitors adapt their transcriptional program during development generating different subsets. How chromatin modifications modulate these processes is unclear. Here we investigate the impact of histone deacetylation on DCs by genetically deleting HDAC1 or HDAC2 in hematopoietic progenitors and CD11c-expressing cells. While HDAC2 isn’t critical for DC development, HDAC1 deletion impairs pro- and mature pDC generation and affects ESAM+cDC2 differentiation from tDC and pre-cDC2, whereas cDC1s are unchanged. HDAC1 knock-down in human hematopoietic cells also impairs cDC2 development highlighting its crucial role across species. Multi-omics analyses reveal that HDAC1 controls expression, chromatin accessibility and histone-acetylation of the transcription factors IRF4, IRF8 and SPIB required for efficient development of cDC2 subsets. Without HDAC1, DCs switch immunologically enhancing tumor surveillance through increased cDC1 maturation and IL-12 production driving Th1-mediated immunity and CD8+T-cell recruitment. Our study reveals the importance of histone-acetylation in DC development and anti-tumor immunity suggesting DC-targeted therapeutic strategies for immuno-oncology.