Cell-intrinsic Role Research Articles

APOE‐4 dependent dysfunctional neutrophil‐microglia crosstalk accelerates Alzheimer’s disease

AbstractBackgroundAPOE4 is the strongest genetic risk factor for late‐onset Alzheimer’s disease (AD). The role of human APOE variants in AD has been studied extensively in the regulation of brain cells but not in peripheral immunity. APOE is also expressed in neutrophils and controls their activation. Moreover, neutrophils have been shown to play a negative role in AD mice via the induction of neutrophil extracellular traps (NETs)MethodHere we aimed to dissect the impact of APOE4‐neutrophils on AD pathology, using Ly6g‐CRE mice crossed to APOE‐KI(E3 and E4)fl/fl:APP/PS1.ResultWe identified a cell intrinsic role of APOE3 in controlling neutrophil maturation and degranulation, which is exacerbated in APOE4‐KI neutrophils and reduced in APOE4‐KO neutrophils. Moreover, APOE4 neutrophils accumulated in the brains of AD mice and in AD patients, associated with microglia at sites of plaque pathology. Here we show that genetic deletion of APOE4 in neutrophils suppressed their activation and reduced their recruitment to the brains of APP/PS1 mice. RNAseq analysis showed increased proportion of disease associated microglia (DAM/MGnD) in APP/PS1: Ly6g‐CRE:APOE4fl/fl mice, associated with reduced plaque pathology. Furthermore, we show impaired induction of MGnD signature in AD brains of APOE4 carriers. Our findings show that APOE4‐induced inflammatory neutrophils are recruited to the diseased brain and impair MGnD response leading to accelerated AD pathology.ConclusionTaken together, these findings identify a cell‐intrinsic role of APOE4 in the induction of hyperactivated neutrophils recruited to the brain, which may provide new molecular targets to modulate and restore functional microglia in AD.

24. Hepatic stellate cell-intrinsic role for IRF3 in TGFβ-induced fibrogenesis

24. Hepatic stellate cell-intrinsic role for IRF3 in TGFβ-induced fibrogenesis

Defining a Targetable Leukemia Intrinsic Dependency on Noncanonical PI3Kgamma Signaling

There are overlapping clinical and pathologic characteristics between poor prognosis subsets of acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN), but common dependencies are ill-defined. We hypothesized that identifying novel shared targets and biomarkers could elucidate biology and nominate new therapies for both malignancies. We performed genome-wide CRISPR interference in BPDCN cells and identified 327 dependency genes, which were then analyzed informatically and functionally in AML, ALL, and BPDCN cells and patient-derived xenografts (PDXs). We defined a leukemia subset characterized by PIK3R5 activation and sensitivity to phosphoinositide 3-kinase gamma (PI3Kγ) inhibition via a noncanonical downstream pathway. The PI3Kγ complex includes an enzymatic subunit p110γ encoded by PIK3CG and a regulatory subunit p101 encoded by PIK3R5. Intriguingly, despite elevation of both p110γ and p101 proteins in this leukemia subset, only the PIK3R5 mRNA is overexpressed. To investigate this disconnection, we measured protein half-life and found that p110γ stability is positively regulated by PIK3R5/p101, associated with reduced p110γ ubiquitination. This suggested that increased expression of PIK3R5/p101 leads to posttranslational p110γ elevation by protecting it from ubiquitin proteasome-mediated degradation. To identify the upstream activator of PIK3R5, we analyzed RNA-seq data in >200 leukemia PDXs and found that AMLs with activated PIK3R5 are enriched for an innate immune response signature (IIRS). Supporting this model and indicating potential clinical relevance, analysis of TCGA AML patients revealed that the IIRS, which includes PIK3R5, was associated with M4/M5 monocytic AML and poor prognosis, independent of age, cytogenetics, or somatic mutations. Furthermore, in cells with low baseline expression, PIK3R5 was induced by the Toll-like receptor agonist resiquimod, showing that PIK3R5 is activated by inflammatory signaling and consistent with a monocytic/dendritic lineage association. By integrated analysis of ATAC-seq and ChIP-seq in PIK3R5 high vs low leukemias, as well as gene dependency scores, we identified the transcription factor PU.1 as responsible for PIK3R5 activation. Treatment with resiquimod enhanced the interaction between PU.1 and the PIK3R5 promoter, while depletion of PU.1 nullified the ability of resiquimod to activate PIK3R5. To elucidate molecular events downstream of p101/PI3Kγ, we studied consequences of pathway inactivation using PIK3R5 interference or the PI3Kγ-selective inhibitor eganelisib. Surprisingly, there was no effect of PI3Kγ inactivation on AKT phosphorylation or canonical PI3K-AKT-mTOR mRNA targets. Therefore, we performed phosphoproteomics and found that PAK1 S144, a phosphorylation site not previously associated with PI3K signaling, was suppressed by PIK3R5/PI3Kγ inhibition ( panel a). Selective inhibitors of PAK1, but not AKT, replicated the effect of eganelisib. Furthermore, introduction of constitutively active PAK1, but not inactive PAK1, eliminated sensitivity of leukemias to eganelisib, supporting the functional relationship between PAK1 phosphorylation and PI3Kγ dependency. PI3Kγ inhibitors are being tested in solid tumor trials to repolarize macrophages and potentiate immune checkpoint blockade, but their cancer cell-intrinsic role has not been explored. To assess PI3Kγ inhibition in vivo, we first performed intradermal xenografting assays using BPDCN derived from a patient's skin tumor. Eganelisib achieved comparable efficacy to cytarabine chemotherapy and the combination of eganelisib and cytarabine exerted significant synergy (combination index=0.63, p<0.0001). Next, we measured the effect of PI3Kγ inhibition on survival in disseminated PDX models. As predicted, single agent eganelisib significantly prolonged survival in AML, ALL, and BPDCN with high PIK3R5, while having no benefit in cases with low PIK3R5. To our surprise, the combination of eganelisib/cytarabine synergistically increased survival in all cases, regardless of baseline PIK3R5 ( panel b). We conclude that inflammatory signaling-activated PIK3R5 is a biomarker for sensitivity to PI3Kγ inhibition via an unappreciated PI3Kγ-PAK1 axis. Synergy with cytarabine unmasked eganelisib sensitivity in additional leukemias, expanding the therapeutic potential of targeting PI3Kγ.

Cutting Edge: TLR2 Signaling in B Cells Promotes Autoreactivity to DNA via IL-6 Secretion.

Autoantibodies to chromatin and dsDNA are a hallmark of systemic lupus erythematosus (SLE). In a mouse model of monogenic human SLE caused by DNASE1L3 deficiency, the anti-DNA response is dependent on endosomal nucleic acid-sensing TLRs TLR7 and TLR9. In this study, we report that this response also required TLR2, a surface receptor for microbial products that is primarily expressed on myeloid cells. Cell transfers into lymphopenic DNASE1L3-deficient mice showed that TLR2 was required for anti-DNA Ab production by lymphocytes. TLR2 was detectably expressed on B cells and facilitated the production of IL-6 by B cells activated in the presence of microbial products. Accordingly, treatment with broad-spectrum antibiotics or Ab-mediated blockade of IL-6 delayed the anti-DNA response in DNASE1L3-deficient mice. These studies reveal an unexpected B cell-intrinsic role of TLR2 in systemic autoreactivity to DNA, and they suggest that microbial products may synergize with self-DNA in the activation of autoreactive B cells in SLE.

Hepatic stellate cell-intrinsic role of SOCS1 in controlling hepatic fibrogenic response and the pro-inflammatory macrophage compartment during liver fibrosis.

Hepatic stellate cells (HSC) become activated, differentiate to myofibroblasts and produce extracellular fibrillar matrix during liver fibrosis. The hepatic fibrogenic response is orchestrated by reciprocal interactions between HSCs and macrophages and their secreted products. SOCS1 can regulate several cytokines and growth factors implicated in liver fibrosis. Here we investigated the role of SOCS1 in regulating HSC activation. Mice lacking SOCS1 in HSCs (Socs1ΔHSC) were generated by crossing Socs1fl/fl and LratCre mice. Liver fibrosis was induced by carbon tetrachloride and evaluated by Sirius red staining, hydroxyproline content and immunostaining of myofibroblasts. Gene expression of pro-fibrogenic factors, cytokines, growth factors and chemokines were quantified by RT-qPCR. The phenotype and the numbers of intrahepatic leukocyte subsets were studied by flow cytometry. The impact of fibrosis on the development of diethyl nitrosamine-induced hepatocellular carcinoma was evaluated. Socs1ΔHSC mice developed more severe liver fibrosis than control Socs1fl/fl mice that was characterized by increased collagen deposition and myofibroblast differentiation. Socs1ΔHSC mice showed a significant increase in the expression of smooth muscle actin, collagens, matrix metalloproteases, cytokines, growth factors and chemokines in the liver following fibrosis induction. The fibrotic livers of Socs1ΔHSC mice displayed heightened inflammatory cell infiltration with increased proportion and numbers of Ly6ChiCCR2+ pro-inflammatory macrophages. This macrophage population contained elevated numbers of CCR2+CX3CR1+ cells, suggesting impaired transition towards restorative macrophages. Fibrosis induction following exposure to diethyl nitrosamine resulted in more numerous and larger liver tumor nodules in Socs1ΔHSC mice than in Socs1fl/fl mice. Our findings indicate that (i) SOCS1 expression in HSCs is a critical to control liver fibrosis and development of hepatocaellular carcinoma, and (ii) attenuation of HSC activation by SOCS1 regulates pro-inflammatory macrophage recruitment and differentiation during liver fibrosis.

Renal tubular epithelial cells are constitutive non-cognate stimulators of resident T cells

Understanding the roles of different cell types in regulating Tcell homeostasis in various tissues is critical for understanding adaptive immunity. Here, we show that RTECs (renal tubular epithelial cells) are intrinsically programmed to polyclonally stimulate proliferation of kidney αβ Tcells by a cell-cell contact mechanism that is major histocompatibility complex (MHC) independent and regulated by CD155, αVβ3-integrin, and vitronectin. Peripheral CD4 and CD8 are resistant to RTEC-mediated stimulation, while the minor subset of double-negative (DN) Tcells are responsive. This functional property of RTEC is discovered by using a coculture system that recapitulates spontaneous invivo polyclonal proliferation of kidney Tcells, which are mainly comprised of central memory T (TCM) and effector memory T (TEM) cells. This robust cell-intrinsic stimulatory role of RTECs could be underlying the steady-state spontaneous proliferation of kidney Tcells. The results have conceptual implications for understanding roles of different cell types in regulating systemic and organ-specific Tcell homeostasis.

Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification

AbstractHematopoiesis produces diverse blood cell lineages to meet the basal needs and sudden demands of injury or infection. A rapid response to such challenges requires the expansion of specific lineages and a prompt return to balanced steady-state levels, necessitating tightly coordinated regulation. Previously we identified a requirement for the zinc finger and broad complex, tramtrak, bric-a-brac domain–containing 11 (ZBTB11) transcription factor in definitive hematopoiesis using a forward genetic screen for zebrafish myeloid mutants. To understand its relevance to mammalian systems, we extended these studies to mice. When Zbtb11 was deleted in the hematopoietic compartment, embryos died at embryonic day (E) 18.5 with hematopoietic failure. Zbtb11 hematopoietic knockout (Zbtb11hKO) hematopoietic stem cells (HSCs) were overabundantly specified from E14.5 to E17.5 compared with those in controls. Overspecification was accompanied by loss of stemness, inability to differentiate into committed progenitors and mature lineages in the fetal liver, failure to seed fetal bone marrow, and total hematopoietic failure. The Zbtb11hKO HSCs did not proliferate in vitro and were constrained in cell cycle progression, demonstrating the cell-intrinsic role of Zbtb11 in proliferation and cell cycle regulation in mammalian HSCs. Single-cell RNA sequencing analysis identified that Zbtb11-deficient HSCs were underrepresented in an erythroid-primed subpopulation and showed downregulation of oxidative phosphorylation pathways and dysregulation of genes associated with the hematopoietic niche. We identified a cell-intrinsic requirement for Zbtb11-mediated gene regulatory networks in sustaining a pool of maturation-capable HSCs and progenitor cells.

Theprogrammed death ligand 1 interactome demonstrates bidirectional signaling coordinating immune suppression and cancer progression in head and neck squamous cell carcinoma.

The programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) are validated cancer targets; however, emerging mechanisms and impact of PD-L1 intracellular signaling on cancer behavior are poorly understood. We investigated the cancer cell intrinsic role of PD-L1 in multiple patient-derived models in vitro and in vivo. PD-L1 overexpression, knockdown, and PD-L1 intracellular domain (PD-L1-ICD) deletion (Δ260-290PD-L1) models were assessed for key cancer properties: clonogenicity, motility, invasion, and immune evasion. To determine how PD-L1 transduces signals intracellularly, we used the BioID2 platform to identify the PD-L1 intracellular interactome. Both human papillomavirus-positive and negative patient-derived xenografts were implanted in NOD-scid-gamma and humanized mouse models to investigate the effects of recombinant PD-1, anti-PD-L1, and anti-signal transducer and activator of transcription 3 (STAT3) in vivo. PD-L1 intracellular signaling increased clonogenicity, motility, and invasiveness in multiple head and neck squamous cell carcinoma (HNSCC) models, and PD-1 binding enhanced these effects. Protein proximity labeling revealed the PD-L1 interactome, distinct for unbound and bound PD-1, which initiated cancer cell-intrinsic signaling. PD-L1 binding partners interleukin enhancer binding factors 2 and 3 (ILF2-ILF3) transduced their effect through STAT3. Δ260-290PD-L1 disrupted signaling and reversed pro-growth properties. In humanized HNSCC in vivo models bearing T-cells, PD-1 binding triggered PD-L1 signaling, and dual PD-L1 and STAT3 inhibition were required to achieve tumor control. Upon PD-1 binding, the PD-L1 extracellular and intracellular domains exert a synchronized effect to promote immune evasion by inhibiting T-cell function while simultaneously enhancing cancer cell-invasive properties.

USP51/PD-L1/ITGB1-deployed juxtacrine interaction plays a cell-intrinsic role in promoting chemoresistant phenotypes in non-small cell lung cancer.

Programmed death ligand 1 (PD-L1) has been demonstrated to facilitate tumor progression and therapeutic resistance in an immune-independent manner. Nevertheless, the function and underlying signaling network(s) of cancer cell-intrinsic PD-L1 action remain largely unknown. Herein, we sought to better understand how ubiquitin-specific peptidase 51 (USP51)/PD-L1/integrin beta-1 (ITGB1) signaling performs a cell-intrinsic role in mediating chemotherapeutic resistance in non-small cell lung cancer (NSCLC). Western blotting and flow cytometry were employed for PD-L1 detection in NSCLC cell lines. Coimmunoprecipitation and pulldown analyses, protein deubiquitination assay, tissue microarray, bioinformatic analysis and molecular biology methods were then used to determine the significance of PD-L1 in NSCLC chemoresistance and associated signaling pathways in several different cell lines, mouse models and patient tissue samples. Ubiquitin-7-amido-4-methylcoumarin (Ub-AMC)-based deubiquitinase activity, cellular thermal shift and surface plasmon resonance (SPR) analyses were performed to investigate the activity of USP51 inhibitors. We provided evidence that cancer cell-intrinsic PD-L1 conferred the development of chemoresistance by directly binding to its membrane-bound receptor ITGB1 in NSCLC. At the molecular level, PD-L1/ITGB1 interaction subsequently activated the nuclear factor-kappa B (NF-κB) axis to elicit poor response to chemotherapy. We further determined USP51 as a bona fide deubiquitinase that targeted the deubiquitination and stabilization of the PD-L1 protein in chemoresistant NSCLC cells. Clinically, we found a significant direct relationship between the USP51, PD-L1 and ITGB1 contents in NSCLC patients with chemoresistant potency. The elevated USP51, PD-L1 and ITGB1 levels were strongly associated with worse patient prognosis. Of note, we identified that a flavonoid compound dihydromyricetin (DHM) acted as a potential USP51 inhibitor and rendered NSCLC cells more sensitive to chemotherapy by targeting USP51-dependent PD-L1 ubiquitination and degradation in vitro and in vivo. Together, our results demonstrated that the USP51/PD-L1/ITGB1 network potentially contributes to the malignant progression and therapeutic resistance in NSCLC. This knowledge is beneficial to the future design of advanced cancer therapy.

The inhibitor of MyoD Family A (I-MFA) regulates megakaryocyte lineage commitment and terminal differentiation

Hematopoiesis and lineage commitment are regulated by several conserved cell-intrinsic signaling pathways, including MAPKs and β-catenin/TCF/LEF. The Inhibitor of MyoD Family A (I-MFA), a transcriptional repressor and tumor suppressor gene, interacts with these pathways and is dysregulated in chronic and acute myeloid leukemias, suggesting it may play a role in development and differentiation during hematopoiesis. To study this, immune cell populations in the bone marrow (BM) and periphery were analyzed in mice lacking Mdfi, encoding I-MFA (I-MFA−/−), and wild type (WT) controls. I-MFA−/− mice had reduced spleen and BM cellularity, with significant hyposplenism, compared to WT mice. In blood, total red blood cells and platelet counts were significantly reduced in I-MFA−/− mice, accompanied by a reduction in megakaryocyte (MK)/erythrocyte progenitor cells and an increase in myeloid progenitors in BM compared to WT mice. The K562 cell line exhibits PMA-induced MK differentiation, and shRNA knockdown of I-MFA resulted in reduced differentiation compared to control, with an increase and prolongation in phospho-JNK and phospho-ERK signaling. Overexpression of I-MFA promoted MK differentiation. These results suggest I-MFA plays a cell-intrinsic role in the response to differentiation signals, an effect that can be explored in the context of hematological cancers or other blood proliferative disorders.

The calcium channel Orai1 is required for osteoblast development: Studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1.

The calcium-selective ion channel Orai1 has a complex role in bone homeostasis, with defects in both bone production and resorption detected in Orai1 germline knock-out mice. To determine whether Orai1 has a direct, cell-intrinsic role in osteoblast differentiation and function, we bred Orai1 flox/flox (Orai1fl/fl) mice with Runx2-cre mice to eliminate its expression in osteoprogenitor cells. Interestingly, Orai1 was expressed in a mosaic pattern in Orai1fl/fl-Runx2-cre bone. Specifically, antibody labeling for Orai1 in vertebral sections was uniform in wild type animals, but patchy regions in Orai1fl/fl-Runx2-cre bone revealed Orai1 loss while in other areas expression persisted. Nevertheless, by micro-CT, bones from Orai1fl/fl-Runx2-cre mice showed reduced bone mass overall, with impaired bone formation identified by dynamic histomorphometry. Cortical surfaces of Orai1fl/fl-Runx2-cre vertebrae however exhibited patchy defects. In cell culture, Orai1-negative osteoblasts showed profound reductions in store-operated Ca2+ entry, exhibited greatly decreased alkaline phosphatase activity, and had markedly impaired substrate mineralization. We conclude that defective bone formation observed in the absence of Orai1 reflects an intrinsic role for Orai1 in differentiating osteoblasts.

CD4 T cell intrinsic arginase 1 controls the kinetics of Th1 induction and contraction

Abstract Arginase 1 (ARG1), the enzyme catalyzing the conversion of arginine to ornithine and urea, is a hallmark of IL-10 producing immunosuppressive M2 macrophages, however ARG1 activity in T cells is disputed. Here we demonstrate that Arg1, but not Arg2, expression induction is a key feature of lung CD4 T cells during mouse in vivo influenza infection. Ablation of CD4 T cell-intrinsic Arg1 unexpectedly accelerated both the virus-specific Th1 effector response and its IL-10-associated contraction. Biologically, this led to efficient viral clearance, yet significantly reduced lung pathology. Surprisingly, loss of Arg1 in CD4 T cells did not result in disturbed intracellular ornithine or polyamine levels. Instead, by employing unbiased transcriptomic and metabolomic approaches, we found that Arg1 deficiency triggered altered glutamine metabolism, and rebalancing the glutamine flux normalized the Arg1 deficient T cell response. Further, the role of Arg1 in CD4 T cells was distinct from that of its’ isoenzyme Arg2, as ablation of CD4 T cell intrinsic Arg2 resulted in normal Th1 responses, and instead altered Th2 and Th17 responses. Finally, CD4 T cells from rare patients with a deficiency in ARG1, or from healthy donors with CRISPR-Cas9-mediated ARG1 deletion, recapitulated the mouse data, demonstrating that ARG1 also plays a CD4 T cell intrinsic role in human Th1 responses. Collectively, CD4 T cell-intrinsic ARG1, functions as an unexpected pace-keeper of human and mouse T helper 1 (Th1) responses with implications for Th1-associated tissue pathologies. Supported by grants from NIH (5K22HL125593 to M.K.) the Intramural Research Program of the NIH (NIDDK ZIA/DK075149 to B.A. and NHLBI ZIA/HL006223 to C.K.)

B cell Tet2 promotes maturation of the antibody response through active DNA demethylation and OGT-mediated histone glycosylation of Aicdaand Prdm1loci

Abstract Epigenetic DNA methylation regulates gene expression and, therefore, cellular functions. It typically represses gene transcription, which can be re-activated by demethylation in the absence of cell division by the recently characterized Tet1, Tet2 and Tet3 dioxygenases. Tet2, the most abundant Tet protein in B cells, not only actively demethylates DNA but also promotes Ogt recruitment to chromatin to effect Ogt-dependent histone O-GlcNAcylation. This potentiates catalytic DNA demethylation to activate gene expression. To define the role of Tet2 in B cell functions and antibody responses, we generated Aicda creTet2 fl/flmice, which specifically delete Tet2 in activated B cells. Aicda creTet2 fl/flmice showed significantly reduced antibody responses to T-dependent and T-independent antigens, due to downregulation of AID and Blimp-1 expression, CSR/SHM and plasma cell differentiation. Accordingly, activated Aicda creTet2 fl/flB cells reduced DNA hydroxymethylation (resulting in increased methylation) and histone O-GlcNAcylation of Aicda and Prdm1 promoters and enhancers, thereby dampening CSR/SHM and plasma cell differentiation. These were partially rescued by enforced expression of Tet2 mutants defective in catalytic activity or Ogt-binding, but not by a double mutant lacking both activities. Accordingly, Tet2 and Ogt inhibitors synergistically inhibited Aicda and Prdm1 expression in both human and mouse B cells. Conversely, Tet2 activation by vitamin C enhanced CSR and plasma cell differentiation. Thus, Tet2 plays a B cell-intrinsic role in modulation of CSR/SHM and plasma cell differentiation through catalytic active DNA demethylation and (non-catalytic) Ogt-mediated histone O-GlcNAcylation. Supported by NIH grants AI 079705, AI 105813/105813S1, AI138944, AI 167416 and Lupus Research Alliance grant LRA 641363 to PC.

PD-1 immune checkpoint blockade can synergize with PSGL-1 inhibition to reinvigorate exhausted T cells

Abstract P-selectin glycoprotein ligand-1 (PSGL-1) is an immune checkpoint regulator that is highly expressed on T cells. PSGL-1- deficient mice infected with chronic lymphocytic choriomeningitis virus (LCMV) were shown to control chronic viral infection due to increased effector function by anti-viral T cells. However, the cell-intrinsic role of PSGL-1 expression on CD4 +and CD8 +T cell exhaustion is not fully known. Using adoptive transfers of WT or PSGL-1-deficient TCR transgenic CD4 +and CD8 +T cells in mice infected with chronic LCMV, we characterized the differentiation and effector response. We found increased expansion and effector function by PSGL-1-deficient T cells early during infection, however, at later stages of infection, PSGL-1-deficient T cells were functionally exhausted. We found that exhausted CD4 +and CD8 +PSGL-1-deficient T cells were reinvigorated more effectively than WT cells after PD-L1 blockade due to their increased proliferation, cytokine production, and accumulation. We also observed increased responses of CD8 +PSGL-1-deficient T cells in melanoma tumors. Our findings showed that PSGL-1 expression was required for the maintenance of both CD8 +and CD4 +exhausted T cells and that PSGL-1 deletion synergized with PD-1 blockade to reinvigorate these cells. These findings highlight an important cell-intrinsic role for PSGL-1 expression in the maintenance and reinvigoration of exhausted T cells. T32 AI 141346 R01 AI13723

TIGIT agonism alleviates costimulation blockade-resistant CD8 memory T cell responses in transplant

Abstract Tcell Immunoreceptor with Igand ITIM domains (TIGIT) is an inhibitory molecule that has been well characterized in cancer and autoimmunity. We recently published that TIGIT agonism prolongs graft survival in a mouse model of transplantation and showed that this was in part dependent on TIGIT-expressing Tregs. However, we have also observed that TIGIT expression is upregulated on memory T cell subsets that mediate costimulation blockade-resistant rejection in transplantation. In the present study, we aimed to determine the cell intrinsic role of TIGIT agonism on memory CD8 T cells in a murine model of transplant. To do this, we induced immunological memory by performing an adoptive transfer of OT-I T cells prior to infecting animals with Listeria monocytogenesthat expresses the model antigen OVA. 30-days after infection, we performed OVA-expressing skin grafts and treated with the costimulation blockade therapeutic CTLA-4Ig or CTLA-4Ig + TIGIT agonist. TIGIT agonist + CTLA-4Ig prolonged graft survival (MST= 14d) compared to CTLA-4Ig treatment alone (MST= 11d, p=0.077) and reduced antigen-specific CD8 T cell infiltration into the graft (4-fold reduction, p=0.017). TIGIT agonist also led to the sequestration of CXCR3 expressing OT-I cells in the draining lymph nodes and impaired trafficking of CXCR3 +OT-I cells to the graft compared to CTLA-4Ig alone. Additionally, TIGIT agonism in vivoled to a decrease in the frequency of TIGIT +IFNg +OT-I cells during ex vivo restimulation (2-fold decrease in CTLA-4Ig + TIGIT agonist vs. CTLA-4Ig alone, p=0.028). TIGIT agonism suppresses memory CD8 T cells in transplant by inhibiting their trafficking and decreasing cytokine production to alleviate costimulation blockade-resistant rejection. Supported by grants from NIH (5R01AI073707-10, 1F31AI161911 - 01A1)

Dendritic cell intrinsic role of Ptpn22and its pro-autoimmune allele enhance anti-viral immunity

Abstract The tyrosine phosphatase PTPN22 encodes the enzyme Lyp (PEP in mice) in all immune cells. 5–10% of the North American population expresses the PTPN22 autoimmunity risk allele 1858C>T (rs2476601) causing amino acid substitution R620W (R619W in mice). This mutation alters lymphocyte activation, toll-like receptor signaling, and cytokine production. Ptpn22 knockout (PEP-null) and alternative allele-expressing (PEP-R619W) mice clear persistent virus LCMV-cl13, have enhanced anti-viral T cell function, and a more immunostimulatory dendritic cell (DC) phenotype compared to wildtype (PEP-WT). Adoptive transfer studies suggest a T cell extrinsic mechanism driving enhanced T cell function in PEP-null and -R619W animals. However, the relative contribution of PEP-differing non-T cells during virus infection is not known. We hypothesize that the DC, as the sole PEP-differing cell, is the critical non-T cell driving increased T cell function and enhanced anti-viral immunity.To interrogate this, we employ CD11c Ptpn22 conditional knock out (cKO) mice and bone marrow derived DC (BMDC) cultures from PEP-WT, -null, and -R619W mice. During LCMV-cl13 infection, CD11c Ptpn22 cKO mice have improved disease outcome compared to WT mice. In a T cell: DC co-culture assay, both PEP-null and PEP-R619W DCs lead to more CD4 T cell function over WT DCs. Also, PEP-null and PEP-R619W BMDCs have changed response to virus infection. Taken together, these data suggest Ptpn22 and its allele have DC intrinsic mechanisms that alter anti-viral immunity. Results of this study lead to a more complete understanding of the cellular and molecular mechanisms, particularly in the DC, this common allele alters the immune response during infection. Supported by grants from the University of Kansas (KU), KU CBID CoBRE Research Award, and KU Center for Genomics.

STINGing CD4+ T cells to induce refractoriness to HIV infection

Abstract HIV rapidly infects CD4+ T cells where it can establish latent infection. Identifying immune mechanisms which can render CD4+ T cells refractory to infection represents a critical step in limiting infection and viral spread. Using purified memory CD4+ T cells from healthy donors, we found that activation of the STING signaling pathway with 0.5 mM of the STING agonist diABZI was sufficient to induce a state of refractoriness to in vitro HIV infection in memory CD4+ T cells, a major source of the HIV reservoir. We observed that STING activation in CD4+ T cells induces Type I IFN production and activates p-IRF3. CRISPR KD of STING in memory CD4+ T cells led to a significant increase in HIV infection, demonstrating a cell intrinsic role for STING signaling in limiting HIV infection/replication. Using proteomics, we identified that diABZI significantly and potently induces restriction factors and proteins involved in antiviral immunity and Type 1 IFN signaling. Importantly, proteins that were significantly reduced mapped to pathways including RNA processing and protein translation which are aspects of the host machinery which HIV needs to co-opt to replicate and produce infectious virions. Our data provide mechanistic insight into the STING pathway as a means to render CD4+ T cells refractory to HIV infection and highlights that this pathway lands a “1–2” punch by increasing expression of antiviral proteins that directly target the virus while suppressing the cellular machinery needed for the viral life cycle. The potential to use a STING agonist as a post-exposure therapeutic which directly limits viral infection while promoting global antiviral immunity represents a novel strategy that may stop HIV infection in its tracks.

Novel mechanisms of androgen receptor-centered transcriptional regulatory network in regulating CD8 +T cell exhaustion and sex bias in cancer

Abstract Sex bias in cancers arising from nonreproductive organs is known but poorly understood. We recently reported on a T cell-intrinsic role of androgen receptor (AR) in driving CD8 +T cell exhaustion which underlies the poorer tumor control in males (Kwon et al., Sci Immunol, 2022). This study aims to understand how AR regulates CD8 +tumor-infiltrating lymphocytes (TILs) by identifying its genome-wide targets. We created a CD8 +T cell-specific AR knockout (KO) mouse model by crossing E8I-cre mice with Ar-floxed mice, which we challenged with syngeneic bladder tumor MB49. We then monitored tumor growth and performed spectral flow cytometry using a T cell exhaustion panel. Further, we used Cleavage Under Targets and Tagmentation – sequencing (CUT&Tag-seq) to map the entire AR targets in CD8 +TILs. Loss of AR in CD8 +T cells significantly slowed the growth of MB49 in male but not female mice. CD8 +TILs from male CD8 AR KO mice showed reduced TOX +TCF1 −terminally exhausted subset (69%; P≤ 0.05) and TOX expression (72%; P = 0.0583) compared to controls. Similarly, TOX expression decreased by 43% in AR-deleted CD8 +T cells following chronic TCR stimulation in vitro (P ≤ 0.0001). Finally, using CUT&Tag-seq, we found that AR binds directly to promoters of multiple key transcriptional regulators of T cell exhaustion, including Tcf7 and Tox. Through identifying the genome-wide targets of AR in CD8 +TILs, we characterized the cell-intrinsic mechanism by which AR regulates CD8 +T cell exhaustion. Our work suggests AR is a master transcriptional regulator that negatively impacts CD8 +T cell immunity, which may contribute to poorer survival in males and supports combining androgen deprivation therapy with immune checkpoint blockage for cancer treatment. Special Institutional Funding

MiR-21 promotes B cell autoreactivity, inflammatory signaling, and metabolism in TLR7-driven systemic autoimmunity

Abstract MicroRNA-21 (miR-21) is upregulated in SLE patients and promotes disease in multiple autoimmune mouse models; however, mechanisms by which miR-21 promote autoimmunity are unknown. Here we show that TLR7 overexpressing SLE-prone B6.Sle1b Yaamice deficient in miR-21 (Sle1b YaamiR-21 −/−) resisted autoimmunity development through reduced germinal center (GC) B cell, T follicular helper cell, and plasma cell responses. Sle1b YaamiR-21 −/−mice had reduced serum autoantibody titers and numbers of autoantibody-producing antibody forming cells (AFCs) in the spleen and bone marrow. Downstream of TLR7 signaling, PELI1, a negative regulator of the NF-κB family member c-Rel and a direct target of miR-21, was increased in Sle1b YaamiR-21 −/−B cells. Consequently, Sle1b YaamiR-21 −/−B cells had reduced c-Rel expression and proinflammatory cytokines IL-6 and IL-1β. Additionally, Sle1b YaamiR-21 −/−B cells had reduced oxidative phosphorylation, glycolysis, and mitochondrial membrane potential compared to Sle1b YaaB cells. To define the cell-intrinsic mechanisms, we generated B cell-, T cell- and DC-specific miR-21 conditional knockout (cKO) mice on the Sle1b Yaabackground. Reduced B cell autoreactivity, inflammatory signaling, and metabolism in Sle1b YaamiR-21 −/−mice were recapitulated in B cell-specific miR-21 cKO mice. T cell-specific miR-21 cKO mice had a moderate reduction in cellular responses due to reduced co-stimulatory molecules and glycolysis; however, autoimmune responses remained intact. miR-21 deficiency in DCs had no deficit in cellular or autoimmune responses. Our data substantiate a B cell-intrinsic role for miR-21 in promoting inflammatory signaling and metabolic activity in B cells, leading to autoimmunity.

Investigating the Role of HDAC8 in CD4 +T Cells

Abstract T cell exclusion restricts immune checkpoint blockade (ICB) efficacy for some hepatocellular carcinoma (HCC) patients. Epigenetic therapy provides new opportunities to rewire transcriptional programs. We have recently elucidated that pharmacological inhibition of histone deacetylase 8 (HDAC8) resulted in increased CD8 +T cell infiltration and enhanced ICB therapy efficacy in HCC. Moreover, this combined immunotherapy induced durable survival benefits even after tumor rechallenge accompanied by elevated memory T cells (Sci Transl Med, 2021). In this study, we found that HDAC8 inhibition increased memory T cells in HCC tumor-bearing mice. Interestingly, HDAC8 knockdown in tumor cells did not influence the effect of pharmacological HDAC8 inhibition in T cell activation and cytotoxic function, suggesting an immune cell-intrinsic role of HDAC8. HDAC8 was highly expressed in T cells instead of other immune cells. Notably, HDAC8 inhibition upregulated memory-related gene expression in CD4 +T cells, but not in CD8 +T cells. Besides, HDAC8 inhibition increased acetylation of structural maintenance of chromosome 3 (SMC3ac), a ring-shaped cohesin component in topologically entrapping enhancers and promoters which may modulate gene expression. Moreover, inhibiting HDAC8 increased SMC3ac binding onto the targeting gene promoter region and induced mRNA expression in CD4-expressing Jurkat cells. These data suggest that HDAC8 inhibition exerts memory T cell formation through SMC3ac-mediated epigenome reprogramming in CD4 +T cells, thus providing insights into developing effective cancer immunotherapy. Acknowledgment: This work is supported by the RGC Collaborative Research Fund (C4045-18W), Li Ka Shing Foundation, TRS T11-706/18-N and EpiHK.