Expression Of BATF Research Articles

BATF-Activated AIM2 Mediates Immune Escape in Lung Adenocarcinoma by Regulating PD-L1

Introduction: Immunotherapy has demonstrated encouraging outcomes in tackling lung adenocarcinoma (LUAD), but immune escape may bring negative impacts. Only a single study has demonstrated the function of AIM2 in LUAD and reported that NF-κB and STAT1 are the chief transcription factors, this study is designed to analyze the role of AIM2 and examine the transcription factor, BATF in LUAD immunotherapy. Methods: Bioinformatics methods to analyze the expression and binding sites of AIM2 and BATF in LUAD, as well as the correlation between AIM2 and PD-L1. Dual-luciferase and chromatin immunoprecipitation assays were used to verify the binding of AIM2 and BATF. qRT-PCR and Western blot assayed expression of AIM2, BATF, and PD-L1 in LUAD. MTT measured cell viability, flow cytometry detected cell apoptosis, cytotoxicity assays measured the toxicity of CD8+ T cells to cancer cells, and enzyme-linked immunosorbent assay measured the expression of related cytokines. Immunohistochemistry detected the protein expression levels of AIM2, BATF, PD-L1, and CD8 in tumor tissue. Results: AIM2 and BATF were both highly expressed in LUAD, and there was a targeted binding relationship. BATF promoted LUAD cell proliferation and inhibited apoptosis by affecting AIM2 expression. The downregulation of AIM2 and PD-L1 expression inhibited PD-L1 and activated CD8+ T cells. The rescue experiment manifested that increased BATF weakened repression of AIM2 silencing on LUAD tumor immune escape in vitro and in vivo. Conclusion: BATF promoted AIM2 expression, upregulated PD-L1, inhibited CD8+ T cell activity, and ultimately led to immune escape in LUAD. Our research uncovered an innovative outlook on the intricate regulation of immune checkpoint molecules and proposed a new approach to target the BATF/AIM2 axis in tumor immunotherapy.

BRD4 inhibitor reduces exhaustion and blocks terminal differentiation in CAR-T cells by modulating BATF and EGR1

BackgroundExhaustion is a key factor that influences the efficacy of chimeric antigen receptor T (CAR-T) cells. Our previous study demonstrated that a bromodomain protein 4 (BRD4) inhibitor can revise the phenotype and function of exhausted T cells from leukemia patients. This study aims to elucidate the mechanism by which a BRD4 inhibitor reduces CAR-T cell exhaustion using single-cell RNA sequencing (scRNA-Seq).MethodsExhausted CD123-specific CAR-T cells were prepared by co-culture with CD123 antigen-positive MV411 cells. After elimination of MV411 cells and upregulation of inhibitory receptors on the surface, exhausted CAR-T cells were treated with a BRD4 inhibitor (JQ1) for 72 h. The CAR-T cells were subsequently isolated, and scRNA-Seq was conducted to characterize phenotypic and functional changes in JQ1-treated cells.ResultsBoth the proportion of exhausted CD8+ CAR-T cells and the exhausted score of CAR-T cells decreased in JQ1-treated compared with control-treated cells. Moreover, JQ1 treatment led to a higher proportion of naïve, memory, and progenitor exhausted CD8+ CAR-T cells as opposed to terminal exhausted CD8+ CAR-T cells accompanied by enhanced proliferation, differentiation, and activation capacities. Additionally, with JQ1 treatment, BATF activity and expression in naïve, memory, and progenitor exhausted CD8+ CAR-T cells decreased, whereas EGR1 activity and expression increased. Interestingly, AML patients with higher EGR1 and EGR1 target gene ssGSEA scores, coupled with lower BATF and BATF target gene ssGSEA scores, had the best prognosis.ConclusionsOur study reveals that a BRD4 inhibitor can reduce CAR-T cell exhaustion and block exhausted T cell terminal differentiation by downregulating BATF activity and expression together with upregulating EGR1 activity and expression, presenting an approach for improving the effectiveness of CAR-T cell therapy.

BATF-dependent Th17 cells act through the IL-23R pathway to promote prostate adenocarcinoma initiation and progression.

The role of Th17 cells in prostate cancer is not fully understood. The transcription factor BATF controls the differentiation of Th17 cells. Mice deficient in Batf do not produce Th17 cells. In this study, we aimed to characterize the role of Batf-dependent Th17 cells in prostate cancer by crossbreeding Batf knockout mice with mice conditionally mutant for Pten. We found that Batf knockout mice had changes in the morphology of prostate epithelial cells compared with normal mice, and Batf knockout mice deficient in Pten (called Batf-) had smaller prostate size and developed fewer invasive prostate adenocarcinomas than Pten-deficient mice with Batf expression (called Batf+). The prostate tumors in Batf- mice showed reduced proliferation, increased apoptosis, decreased angiogenesis and inflammatory cell infiltration, and activation of nuclear factor-κB signaling. Moreover, Batf- mice showed significantly reduced interleukin 23 (IL-23)-IL-23R signaling. In the prostate stroma of Batf- mice, IL-23R-positive cells were decreased considerably compared with Batf+ mice. Splenocytes and prostate tissues from Batf- mice cultured under Th17 differentiation conditions expressed reduced IL-23/IL-23R than cultured cells from Batf+ mice. Anti-IL-23p19 antibody treatment of Pten-deficient mice reduced prostate tumors and angiogenesis compared with control immunoglobulin G-treated mice. In human prostate tumors, BATF messenger RNA level was positively correlated with IL-23A and IL-23R but not RORC. Our novel findings underscore the crucial role of IL-23-IL-23R signaling in mediating the function of Batf-dependent Th17 cells, thereby promoting prostate cancer initiation and progression. This finding highlights the BATF-IL-23R axis as a promising target for the development of innovative strategies for prostate cancer prevention and treatment.

C-Jun targets miR-451a to regulate HQ-induced inhibition of erythroid differentiation via the BATF/SETD5/ARHGEF3 axis

Benzene, a widely used industrial chemical, has been clarified to cause hematotoxicity. Our previous study suggested that miR-451a may play a role in benzene-induced impairment of erythroid differentiation. However, the mechanism underlying remains unclear. In this study, we explored the role of miR-451a and its underlying mechanisms in hydroquinone (HQ)-induced suppression of erythroid differentiation in K562 cells. 0, 1.0, 2.5, 5.0, 10.0, and 50 μM HQ treatment of K562 cells resulted in a dose-dependent inhibition of erythroid differentiation, as well as the expression of miR-451a. Bioinformatics analysis was conducted to predict potential target genes of miR-451a and dual-luciferase reporter assays confirmed that miR-451a can directly bind to the 3'-UTR regions of BATF, SETD5, and ARHGEF3 mRNAs. We further demonstrated that over-expression or down-regulation of miR-451a altered the expression of BATF, SETD5, and ARHGEF3, and also modified erythroid differentiation. In addition, BATF, SETD5, and ARHGEF3 were verified to play a role in HQ-induced inhibition of erythroid differentiation in this study. Knockdown of SETD5 and ARHGEF3 reversed HQ-induced suppression of erythroid differentiation while knockdown of BATF had the opposite effect. On the other hand, we also identified c-Jun as a potential transcriptional regulator of miR-451a. Forced expression of c-Jun increased miR-451a expression and reversed the inhibition of erythroid differentiation induced by HQ, whereas knockdown of c-Jun had the opposite effect. And the binding site of c-Jun and miR-451a was verified by dual-luciferase reporter assay. Collectively, our findings indicate that miR-451a and its downstream targets BATF, SETD5, and ARHGEF3 are involved in HQ-induced erythroid differentiation disorder, and c-Jun regulates miR-451a as a transcriptional regulator in this process.

CD8 T cells with an exhaustion-like phenotype remain highly functional in hematologic tumors

Abstract Loss of immunosurveillance in solid tumors is associated with CD8 T cell dysfunction/exhaustion (TEX). Immunotherapies target TEX differentiation, which may be modified by differences in microenvironments across cancer types and tissue sites. We characterized TEX differentiation in bone marrow (BM), where liquid tumors reside, using mouse models of myeloma and B cell leukemia progressing after autologous stem cell transplantation and CD19-directed CAR T cells respectively. We used flow cytometry and concurrent analysis of RNA expression and DNA accessibility in single cells. We identified a novel CD8 T cell subset with a terminally exhausted phenotype that retained functional capacity, including secretion of cytotoxic molecules and IFNγ. These cells are referred to as interferon-Gamma-secreting, Phenotypically Exhausted (TG-PhEX). Expression of functional genes in TG-PhEX correlated with Batf expression and accessibility within the BATF motif. Chromatin remodeling in stemness associated genes indicated a loss of self-renewal capacity. Indeed, disease progression was associated with a lack of TG-PhEX expansion and not dysfunction as TG-PhEX effectively killed myeloma with comparable activity to CX3CR1+ TEX. Importantly, we observed analogous cells in myeloma and lymphoma patients. TG-PhEX are a clinically relevant target for immunotherapy of hematologic cancers and demonstrate differences in TEX differentiation across tumor sites and cancer subtypes.

IL-17-producing cells in ankylosing spondylitis patients show gender-based differences in gene expression.

Gender has been shown to impact disease expression in ankylosing spondylitis (AS) and Th17 cells play a key role in AS pathogenesis. To better understand what Th17-associated immune pathways are different between men and women, we compared the transcriptome of IL-17-enriched peripheral blood mononuclear cells (PBMCs) in male and female AS patients, with a particular focus on inflammatory cytokine genes. PBMCs were collected from 10 female and 11 male AS patients at the Clinical Research Unit of MetroHealth Medical Center. IL-17-enriched PBMCs were isolated and stimulated with CytoStim. RNA-sequencing (RNA-seq) was performed on the samples, and the data were analysed using iPathwayGuide. Inflammatory markers and genes related to Th17 differentiation and function were identified based on previous studies. RNA-seq identified 12,893 genes with 2,851 genes with p-values <0.05 with distinct patterns of gene expression between male and female AS patients. TGF-β, PGE2, and S100 proteins were significantly upregulated in males. Levels of IL-12B, a Th17 inducer, were lower in males compared to females. Additionally, receptors of IL-6, 12, 23, TGF-β, and PGE2 were downregulated in males, except for IL-17RC, which was upregulated. Genes involved in Th17 differentiation showed differential expression between genders, with elevated expression of BATF, SOCS1, NKD2, and ARID5A in men and decreased expression of FOXO1. Transcriptomic analysis revealed that male AS patients exhibit distinct expression patterns of IL-17 pro-inflammatory genes, which may contribute to the phenotypic differences observed between genders in AS.

Batf3 is a critical transcription factor in IL-2 mediated programming of CD8+ T cell memory.

Abstract CD8+ T cell activation triggers chromatin remodeling and transcription factor (TF) binding, leading to differentiation into effector (TEFF) and a spectrum of memory (TMEM) phenotypes. Previous studies have found modified chromatin accessibility at loci encoding Basic Leucine Zipper (bZIP) family motifs; however, specific mechanisms of transcriptional regulation by these TFs to program TMEM are not fully understood. During TCR stimulation, transient upregulation of bZIP TF Batf3 is accompanied by binding of genomic loci encoding its motif. Nascent RNA sequencing of IL-2Rα-/- CD8+T cells shows that Batf3 upregulation upon TCR stimulation is highly dependent on IL-2R signaling. Constitutive loss of either IL-2Rα or Batf3 results in decreased survival of CD8+ T cells during the transition from effector to memory phase of acute LCMVArm infection, and reduced immunological memory towards infection re-challenge. We find that enforced overexpression of Batf3 cDNA using a retroviral vector in virus-specific IL-2Rα-/- CD8+ T cells greatly enhanced their capacity for survival into the contraction phase of primary infection. Upon secondary infection, ectopic Batf3 expression rescued the proliferation defect seen in IL-2Rα-/- CD8+ T cells. These results suggest that Batf3 plays a crucial role within the IL-2R signal transduction cascade during CD8+ T cell activation to promote enhanced cell survival into the memory phase of infection response.

Integrative Analyses of Bulk and Single-Cell RNA Seq Identified the Shared Genes in Acute Respiratory Distress Syndrome and Rheumatoid Arthritis.

Acute respiratory distress syndrome (ARDS), a progressive status of acute lung injury (ALI), is primarily caused by an immune-mediated inflammatory disorder, which can be an acute pulmonary complication of rheumatoid arthritis (RA). As a chronic inflammatory disease regulated by the immune system, RA is closely associated with the occurrence and progression of respiratory diseases. However, it remains elusive whether there are shared genes between the molecular mechanisms underlying RA and ARDS. The objective of this study is to identify potential shared genes for further clinical drug discovery through integrated analysis of bulk RNA sequencing datasets obtained from the Gene Expression Omnibus database, employing differentially expressed genes (DEGs) analysis and weighted gene co-expression network analysis (WGCNA). The hub genes were identified through the intersection of common DEGs and WGCNA-derived genes. The Random Forest (RF) and least absolute shrinkage and selection operator (LASSO) algorithms were subsequently employed to identify key shared target genes associated with two diseases. Additionally, RA immune infiltration analysis and COVID-19 single-cell transcriptome analysis revealed the correlation between these key genes and immune cells. A total of 59 shared genes were identified from the intersection of DEGs and gene clusters obtained through WGCNA, which analyzed the integrated gene matrix of ALI/ARDS and RA. The RF and LASSO algorithms were employed to screen for target genes specific to ALI/ARDS and RA, respectively. The final set of overlapping genes (FCMR, ADAM28, HK3, GRB10, UBE2J1, HPSE, DDX24, BATF, and CST7) all exhibited a strong predictive effect with an area under the curve (AUC) value greater than 0.8. Then, the immune infiltration analysis revealed a strong correlation between UBE2J1 and plasma cells in RA. Furthermore, scRNA-seq analysis demonstrated differential expression of these nine target genes primarily in T cells and NK cells, with CST7 showing a significant positive correlation specifically with NK cells. Beyond that, transcriptome sequencing was conducted on lung tissue collected from ALI mice, confirming the substantial differential expression of FCMR, HK3, UBE2J1, and BATF. This study provides unprecedented evidence linking the pathophysiological mechanisms of ALI/ARDS and RA to immune regulation, which offers novel understanding for future clinical treatment and experimental research.

TIM-3+ CD8 T cells with a terminally exhausted phenotype retain functional capacity in hematological malignancies.

Chronic antigen stimulation is thought to generate dysfunctional CD8 T cells. Here, we identify a CD8 T cell subset in the bone marrow tumor microenvironment that, despite an apparent terminally exhausted phenotype (TPHEX), expressed granzymes, perforin, and IFN-γ. Concurrent gene expression and DNA accessibility revealed that genes encoding these functional proteins correlated with BATF expression and motif accessibility. IFN-γ+ TPHEX effectively killed myeloma with comparable efficacy to transitory effectors, and disease progression correlated with numerical deficits in IFN-γ+ TPHEX. We also observed IFN-γ+ TPHEX within CD19-targeted chimeric antigen receptor T cells, which killed CD19+ leukemia cells. An IFN-γ+ TPHEX gene signature was recapitulated in TEX cells from human cancers, including myeloma and lymphoma. Here, we characterize a TEX subset in hematological malignancies that paradoxically retains function and is distinct from dysfunctional TEX found in chronic viral infections. Thus, IFN-γ+ TPHEX represent a potential target for immunotherapy of blood cancers.

Abstract 6325: Lineage tracing defines responding CAR T cells in patients with B cell malignancies

Abstract Autologous T cells engineered to express a chimeric antigen receptor (CAR) have transformed the standard of care for patients with B cell malignancies, but &amp;gt;50% of patients progress following therapy. Here, we sought to understand key T cell intrinsic factors impacting efficacy: CAR T cell expansion, persistence, and homing to the tumor. Using an endogenous T cell receptor (TCR) sequence as a ‘barcode’, we followed individual T cell clonotypes at the single-cell level from pre-manufacture apheresis and infusion products to tumor-involved lymph nodes and blood at peak and late expansion in 22 adult patients with relapsed or refractory large B cell lymphoma (LBCL) or acute lymphoblastic leukemia (ALL) treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T cell therapy, or bispecific CD19/CD22-CAR T cells on an investigator-initiated trial (NCT03233854). The resulting CAR T cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 846,344 cells from 97 samples, with 251,175 unique TCR clonotypes identified, including 17,479 clonotypes that could be traced across ≥2 timepoints in CAR+ cells, enabling lineage tracing at scale for the first time. We found that T cells present in apheresis that preferentially expand during manufacturing primarily comprise T central memory (Tcm) cells and are enriched for predicted specificity to common viruses, including influenza, but also include a subset of T regulatory (Treg) cells. In infusion products, CAR T conventional (non-Treg) cells with superior abilities to home to the tumor, expand, and persist post-infusion share features of activated T effector memory (Tem) phenotype enriched for expression of BATF3 and high TCF4 transcription factor activity. Baseline apheresis and infusion products were enriched for cells classified as Tregs in patients with poor response. This observation aligned with our recent work linking post-infusion CAR Tregs to progression following CD19-CAR T cell therapy. We therefore performed lineage tracing and methylation analysis of the Treg-specific demethylated region (TSDR) in the FOXP3 gene and found that post-infusion CAR Tregs are primarily derived from pre-existing Tregs. Of interest, flow cytometry analysis of baseline blood from a cohort of 53 patients treated with CD19-CAR for LBCL showed that Tregs are elevated in baseline blood relative to healthy donors. Removing Tregs from healthy donor T cells prior to manufacturing enhanced CAR T cell expansion and anti-tumor activity against JeKo-1 human lymphoma cell line, whereas adding 5% Tregs to mimic patient T cells rendered the resulting CAR T cells less active. These analyses pinpoint the identities of apheresis T cells and infusion CAR T cells with properties impacting efficacy, and also identify pre-existing Tregs as limiting efficacy of CAR T cell immunotherapies. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Moksha H. Desai, Zachary J. Ehlinger, Patrick J. Quinn, Yiyun Chen, Snehit Prabhu, Shin-Heng Chiou, Sreevidya Kurra, Eric Yang, Michael G. Ozawa, Matthew J. Frank, Lori Muffly, Gursharan K. Claire, Sushma Bharadwaj, Saurabh Dahiya, Katherine A. Kong, Mark M. Davis, Sylvia K. Plevritis, Elena Sotillo, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Lineage tracing defines responding CAR T cells in patients with B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6325.

Strain specific differences in vitamin D3 response: impact on gut homeostasis.

Vitamin D3 regulates a variety of biological processes irrespective of its well-known importance for calcium metabolism. Epidemiological and animal studies indicate a role in immune regulation, intestinal barrier function and microbiome diversity. Here, we analyzed the impact of different vitamin D3- containing diets on C57BL/6 and BALB/c mice, with a particular focus on gut homeostasis and also investigated effects on immune cells in vitro. Weak regulatory effects were detected on murine T cells. By trend, the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 suppressed IFN, GM-CSF and IL-10 cytokine secretion in T cells of C57BL/6 but not BALB/c mice, respectively. Using different vitamin D3-fortified diets, we found a tissue-specific enrichment of mainly CD11b+ myeloid cells but not T cells in both mouse strains e.g. in spleen and Peyer's Patches. Mucin Reg3γ and Batf expression, as well as important proteins for gut homeostasis, were significantly suppressed in the small intestine of C57BL76 but not BALB/c mice fed with a high-vitamin D3 containing diet. Differences between both mouse stains were not completely explained by differences in vitamin D3 receptor expression which was strongly expressed in epithelial cells of both strains. Finally, we analyzed gut microbiome and again an impact of vitamin D3 was detected in C57BL76 but not BALB/c. Our data suggest strain-specific differences in vitamin D3 responsiveness under steady state conditions which may have important implications when choosing a murine disease model to study vitamin D3 effects.

Epigenetic Scarring Leads to Irreversible NK Cell Dysfunction in Myeloid Malignancies

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) belong to the same spectrum of myeloid malignancies and have extremely poor outcomes in the relapsed/refractory setting. Despite advances in the understanding of the pathogenesis and molecular mechanisms of these disorders, and incremental improvements in treatment regimens, patients with MDS and AML often relapse and fail to achieve cure. These and other factors underscore the urgent need for new therapeutic alternatives that will improve the clinical outcomes of these patients. Immunotherapy using checkpoint molecule inhibitors and adoptive cell therapy using autologous immune effector cells have been mostly unsuccessful in patients with MDS and AML. This could be due to the immunosuppressive tumor microenvironment in the bone marrow niche or to intrinsic dysfunction in the immune effector cells of these patients. Natural killer (NK) cells are innate lymphocytes that play an important role in cancer immune surveillance and have distinct advantages over T cells as candidates for immunotherapy. Myeloid blasts are inherently susceptible to NK cell-mediated killing as they express many of the ligands recognized by NK cell activating receptors. However, malignant myeloid blasts are capable of adapting and developing defense mechanisms that allow them to evade NK cell-mediated cytotoxicity. Little is known about the mechanisms of NK cell immune evasion developed by myeloid blasts. Here, we show that NK cells from patients with myeloid malignancies display a global dysfunction with severely impaired secretory function and killing capacity. Single cell RNAseq and mass cytometry experiments revealed that these NK cells from MDS and AML patients display an exhausted phenotype at the transcriptomic and proteomic levels. We also show that these NK cells have an altered metabolism compared with age matched healthy control NK cells. We show that this dysfunction is mediated by a crosstalk between myeloid blasts and NK cells and cell-cell contact dependent release of transforming growth factor beta (TGF-β). This crosstalk leads to a profound epigenetic reprogramming of NK cells driven by transcription factors known to mediate exhaustion and immune suppression. Myeloid blast-induced NK dysfunction and epigenetic state of exhaustion can be prevented by pharmacologically inhibiting the TGF-β pathway or knockout of TGFBR2 in NK cells. However, our data reveal that once this dysfunction occurs it is irreversible owing to epigenetic scarring driven by the transcription factor BATF. In fact, we show that TGF-β induces the expression of BATF in NK cells, which in turn mediates a gene regulatory program leading to NK cell dysfunction by driving the expression of exhaustion and inhibitory genes (e.g. HAVCR2, ENTPD1, CTLA4, TGFBR2). Collectively, our findings reveal a novel mechanism of NK cell immune evasion manifested by stable epigenetic rewiring and inactivation of NK cells by myeloid blasts. Our data support the use of allogeneic sources for adoptive NK cell therapy in combination with strategies aiming at preventing immune suppression to treat myeloid malignancies rather than therapies aiming at reversing or rescuing the function of autologous NK cells.

Malaria-specific Type 1 regulatory T cells are more abundant in first pregnancies and associated with placental malaria

Malaria in pregnancy (MIP) causes higher morbidity in primigravid compared to multigravid women; however, the correlates and mechanisms underlying this gravidity-dependent protection remain incompletely understood. We aimed to compare the cellular immune response between primigravid and multigravid women living in a malaria-endemic region and assess for correlates of protection against MIP. We characterised the second trimester cellular immune response among 203 primigravid and multigravid pregnant women enrolled in two clinical trials of chemoprevention in eastern Uganda, utilizing RNA sequencing, flow cytometry, and functional assays. We compared responses across gravidity and determined associations with parasitaemia during pregnancy and placental malaria. Using whole blood RNA sequencing, no significant differentially expressed genes were identified between primigravid (n=12) and multigravid (n=11) women overall (log 2(FC)>2, FDR < 0.1). However, primigravid (n=49) women had higher percentages of malaria-specific, non-naïve CD4+ T cells that co-expressed IL-10 and IFNγ compared with multigravid (n=85) women (p=0.000023), and higher percentages of these CD4+ T cells were associated with greater risks of parasitaemia in pregnancy (Rs=0.49, p=0.001) and placental malaria (p=0.0073). These IL-10 and IFNγ co-producing CD4+ T cells had a genomic signature of Tr1 cells, including expression of transcription factors cMAF and BATF and cell surface makers CTLA4 and LAG-3. Malaria-specific Tr1 cells were highly prevalent in primigravid Ugandan women, and their presence correlated with a higher risk of malaria in pregnancy. Understanding whether suppression of Tr1 cells plays a role in naturally acquired gravidity-dependent immunity may aid the development of new vaccines or treatments for MIP. This work was funded by NIH (PO1 HD059454, U01 AI141308, U19 AI089674, U01 AI155325, U01 AI150741), the March of Dimes (Basil O'Connor award), and the Bill and Melinda Gates Foundation (OPP 1113682).

BATF is Required for Treg Homeostasis and Stability to Prevent Autoimmune Pathology.

Regulatory T (Treg) cells are inevitable to prevent deleterious immune responses to self and commensal microorganisms. Treg function requires continuous expression of the transcription factor (TF) FOXP3 and is divided into two major subsets: resting (rTregs) and activated (aTregs). Continuous T cell receptor (TCR) signaling plays a vital role in the differentiation of aTregs from their resting state, and in their immune homeostasis. The process by which Tregs differentiate, adapt tissue specificity, and maintain stable phenotypic expression at the transcriptional level is still inconclusivei. In this work, the role of BATF is investigated, which is induced in response to TCR stimulation in naïve T cells and during aTreg differentiation. Mice lacking BATF in Tregs developed multiorgan autoimmune pathology. As a transcriptional regulator, BATF is required for Treg differentiation, homeostasis, and stabilization of FOXP3 expression in different lymphoid and non-lymphoid tissues. Epigenetically, BATF showed direct regulation of Treg-specific genes involved in differentiation, maturation, and tissue accumulation. Most importantly, FOXP3 expression and Treg stability require continuous BATF expression in Tregs, as it regulates demethylation and accessibility of the CNS2 region of the Foxp3 locus. Considering its role in Treg stability, BATF should be considered an important therapeutic target in autoimmune disease.

Relative Expression of BATF and CD112 in PBMC of Patients with Chronic Lymphocytic Leukemia.

BATF, as a transcription factor, and CD112, as a receptor for TIGIT, are involved in T-cell exhaustion. We investigated BATF and CD112 gene expression in the peripheral blood mononuclear cells from CLL patients and healthy subjects. In a case-control study, 33 patients with CLL and 20 sex- and age-matched healthy individual were enrolled. Diagnosis and classification of patients was done according to immunophenotyping via flow cytometry and RAI staging system, respectively. Relative mRNA expression of BATF and CD112 was measured using qRT-PCR. Our results showed that the expression of BATF and CD112 in CLL samples were significantly decreased in comparison those of the healthy controls (P = 0.0236 and P = 0.0002, respectively). These findings suggest the role of BATF and CD112 not only as a role in T cell exhaustion, but in effector differentiation program in CLL, which warrants further studies in future.

Elevated exhausted effector tissue resident CD8+ T-cells in Chronic Graft versus Host Disease patient Oral mucosa unveiled by spatial transcriptomics and scRNA seq analysis

Abstract Chronic graft-versus-host disease (cGVHD), an autoimmune-like disease following allogeneic hematopoietic stem cell transplantation where T cells can attack host tissue. Oral mucosa (OM) is a most common cGVHD effector site in the oral cavity. We studied T cell populations and tissue-specific signals that regulate their differentiation and functionality. Patient OM biopsies from ongoing clinical trials (NCT03602599, NCT00331968) were taken at 6 months post-transplant. We found enrichment of immune cells in the OM mainly in the CD8 T-cell cluster and identified two unique populations in the OM of cGVHD patients: one characterized by expression of proliferative markers and the other more prominent, characterized by exhaustion makers (TIM3, LAG3, TIGIT). The latter had low expression of CD69 but expressed markers of tissue residency; CD103, BLIMP1, PD1, FABP5. Interestingly, this population showed high expression of BATF, IRF4, and RUNX3, transcription factors that induce sustained effector CD8 T-cell programming. These exhausted cells displayed higher expression of effector molecules IFNG, GZMB, PRF1 inducing sustained CD8 T-cell effector function. Ligand-receptor analysis revealed strong interactions between CXCL9-CXCR3 in CD8 T-cells and myeloid cells. Spatial transcriptomic analysis confirmed the CD8 T-cell infiltration in the submucosal region of OM in cGVHD colocalizing with the elevated markers of exhaustion, surrounded by myeloid cells, thus confirming the striking interaction of CXCL9 and CXCR3. These exhausted CD8 T-cells may play an important role in cGVHD pathogenesis causing by driving sustained tissue damage and could serve as a might be used as a novel target for cGVHD therapy. This work was supported by the intramural programs of the NIDCR and NCI.

The Enrichment of T-Cell Proliferation and Memory Gene Signatures of CD79A/CD40 Co-Stimulatory Domain Enhances CD19CAR-T Cell Efficacy

Introduction: CD28 or 4-1BB co-stimulated CD19 chimeric antigen receptor (CAR) T-cell has demonstrated remarkable outcomes in B-cell malignancies. Recently, we reported that a novel CD19CAR-T cell incorporated B-cell co-stimulatory molecules; CD79A/CD40 (CD19.79a.40z), exhibited superior anti-tumor activity in B-cell lymphoma model compared to CD28 or 4-1BB co-stimulated CAR-T cell. Despite these promising results, the molecular signatures of this novel CAR-T cell and its effects on the overall cellular state are not well understood. This study assessed mRNA expression using RNA-sequencing (RNA-seq) to interrogate the intrinsic transcriptional gene underlying CD19.79a.40z CAR-T cell response and identify the differentially expressed genes (DEGs) compared to CD19.28z or CD19.BBz CAR-T cell upon CD19-specific antigen exposure. Methods: We generated three CD19CAR structures contained different co-stimulatory domain (CD19.28z, CD19.BBz, CD19.79a.40z).The CAR genes were transfected into retroviral packaging cells and transduced into human CD3+ cells. We first screened the functions of each CAR-T cell structure in in vitro assay including cytotoxicity assay, T-cell proliferation assay, and T-cell phenotype assay to confirm whether each CAR-T cell structure contained proper functions. Total CAR-T 3 - 6 x 106 cells were harvested and extracted for RNA at post-stimulation with the target cell line on day seven. Nine samples were RNA-seq using Illumina Hiseq 4000. The short sequence reads were mapped to reference genome from GENCODE (GRCh38) and the expression count matrix was then computed using HTSeq. EdgeR program was used to perform DEGs analysis between three groups: CD19.79a.40z vs. CD19.28z, CD19.79a.40z vs. CD19.BBz and CD19.28z vs. CD19.BBz. The DEGs were selected at FDR <0.05 and fold change ≥ 1.5-fold difference. To identify the regulation of functionally enriched pathways, we performed both ranked gene list analyses on the whole gene expression level using the gene set enrichment analysis (GSEA) software from the Broad Institute and DEGs list separately analyzed for up- and down-regulated genes using g:Profiler. Results: A total of 374 DEGs (232 up-regulated and 142 down-regulated DEGs) and one up-regulated DEG were identified in CD19.79a.40z vs. CD19.28z CAR-T cell groups and CD19.79a.40z vs. CD19.BBz CAR-T cell groups respectively. We found 104 DEGs in which 32 DEGs were up-regulated and 72 DEGs were down-regulated in CD19.28z CAR-T cell compared to CD19.BBz CAR-T cell.Functional enrichment analysis using g;Profiler illustrated that CD19.79a.40z CAR-T cells consisted of down-regulated genes mediating apoptosis and exhaustion while up-regulated genes correlated with T-cell activation, proliferation, positive regulation of interferon production, and NF-κB pathway. Compared to either CD19.28z or CD19.BBz CAR-T cells, CD19.79a.40z CAR-T cells strongly enriched in genes associated with T-cell proliferation at the transcription level supported by the expression of BATF3, IL2, ZP3, TFRC,IRF1 and CD70. In addition, CD19.79a.40z CAR-T cell contained many down-regulated apoptosis-related genes, including ID3, PDCD1 and EOMES (Figure). GSEA revealed that CD19.79a.40z CAR-T cells enriched in both glycolysis and fatty acid metabolism, which are the underlying metabolic pathway closely linked to T-cell activation and proliferation. Regarding T-cell differentiation, CD19.79a.40z CAR-T cells were particularly enriched in naïve and memory-related genes compared to CD19.BBz CAR-T cell. Conclusions: This study provides a comprehensive insight into the understanding of gene expression and its related pathways that potentiates the superior anti-tumor functions by CD19CAR-T cell incorporated B-cell co-stimulatory domain. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TMIC-55. IMMUNE DIGITAL-SPATIAL PROFILING AND CHARACTERIZATION OF GLIOMA MICROENVIRONMENT AFTER ADOPTIVE CELLULAR THERAPY

Abstract INTRODUCTION Our group has previously shown that Adoptive Cellular Therapy (ACT) is efficacious against CNS malignancies including medulloblastoma, brain stem glioma, and glioblastoma. In this study, we characterized the spatial distribution of immune cells within the tumor microenvironment after ACT and conducted regional genomic analysis in situ to obtain insights into the underlying cellular dynamics. This method enabled us to define specific differences in gene expression within the T cells in the treated versus untreated tumors including changes in gene expression of regulatory T cell (Treg) associated genes, such as Runx1 and TGF-β. We also found a significant increase in Batf3 expression associated with therapy. METHODS We performed in situ GeoMx Digital Spatial Profiling (DSP) and whole genome transcriptomics (RNA) of murine glioma KR-158B-luciferase tumors after adoptive cellular therapy. ACT was conducted in orthotopic KR158B tumor-bearing mice. Histological slides were sent to be stained for CD45, CD3, GFP (to mark hematopoietic stem cell-derived cells) and nuclei, and processed following GeoMx DSP workflow for RNA. Overall gene expression clustering profiles were conducted using a Principal Component Analysis (PCA), and relative gene expression differences were analyzed by unpaired t-tests. RESULTS/CONCLUSIONS We observed differential expression of a substantial number of genes in the ACT-treated group compared to control. Our findings also indicate that tumors treated with ACT cluster separately from control tumors in PCA analysis. Furthermore, we found downregulation of genes associated with the suppressive properties of regulatory T cells (Runx1 and TGF-β), and upregulation of dendritic cell characteristic genes (Batf3) and antigen presentation genes (H2-Ab1) in ACT-treated tumors vs control. These findings provide insights into the immune cell dynamics and specific genes that are differentially expressed within the tumor microenvironment. This could help improve current adoptive cellular therapies to treat brain tumors.

BATF epigenetically and transcriptionally controls the activation program of regulatory T cells in human tumors.

Regulatory T (Treg) cells suppress effective antitumor immunity in tumor-bearing hosts, thereby becoming promising targets in cancer immunotherapy. Despite the importance of Treg cells in tumor immunity, little is known about their differentiation process and epigenetic profiles in the tumor microenvironment (TME). Here, we showed that Treg cells in the TME of human lung cancers harbored a completely different open chromatin profile compared with CD8+ T cells, conventional CD4+ T cells in the TME, and peripheral Treg cells. The integrative sequencing analyses including ATAC, single-cell RNA, and single-cell ATAC sequencing revealed that BATF, IRF4, NF-κB, and NR4A were important transcription factors for Treg cell differentiation in the TME. In particular, BATF was identified as a key regulator, which leveraged Treg cell differentiation through epigenetically controlling activation-associated gene expression, resulting in the robustness of Treg cells in the TME. The single-cell sequencing approaches also revealed that tissue-resident and tumor-infiltrating Treg cells followed a common pathway for differentiation and activation in a BATF-dependent manner heading toward Treg cells with the most differentiated and activated phenotypes in tissues and tumors. BATF deficiency in Treg cells remarkably inhibited tumor growth, and high BATF expression was associated with poor prognosis in lung cancer, kidney cancer, and melanoma. These findings indicate one of the specific chromatin remodeling and differentiation programs of Treg cells in the TME, which can be applied in the development of Treg cell-targeted therapies.

Evidence of Omics, Immune Infiltration, and Pharmacogenomics for BATF in a Pan-Cancer Cohort.

Background: Cytotoxic CD8+ T-cell exhaustion is the major barrier for immunotherapy in tumors. Recent studies have reported that the basic leucine zipper activating transcription factor–like transcription factor (BATF) is responsible for countering cytotoxic CD8+ T-cell exhaustion. Nevertheless, the expression and roles of BATF in tumors have been poorly explored. Methods: In the present study, we conducted a multi-omics analysis, including gene expression, methylation status, DNA alterations, pharmacogenomics, and survival status based on data from the Cancer Genome Atlas (TCGA) database to discern expression patterns and prognostic roles of BATF in tumors. We also explored potential roles of BATF in a pan-cancer cohort by performing immune infiltration, Gene Ontology (GO) enrichment, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In vitro assay was also performed to explore roles of BATF in tumor cells. Results: We found that BATF was aberrantly upregulated in 27 types of tumors with respect to the corresponding normal tissues. Abnormal BATF expression in tumors predicted survival times of patients in a tissue-dependent manner. The results of GO, immune infiltration, and KEGG analysis revealed that increased BATF expression in tumors participated in modulating immune cell infiltration via immune-related pathways. BATF expression could also predict immunotherapeutic and chemotherapy responses in cancers. Moreover, knockdown of BATF suppresses tumor cell viability. Conclusion: Our present study reports the vital roles of BATF in tumors and provides a theoretical basis for targeting BATF therapy.