CD19-Cre Mice Research Articles

The capicua-ataxin-1-like complex regulates Notch-driven marginal zone B cell development and sepsis progression

Follicular B (FOB) and marginal zone B (MZB) cells are pivotal in humoral immune responses against pathogenic infections. MZB cells can exacerbate endotoxic shock via interleukin-6 secretion. Here we show that the transcriptional repressor capicua (CIC) and its binding partner, ataxin-1-like (ATXN1L), play important roles in FOB and MZB cell development. CIC deficiency reduces the size of both FOB and MZB cell populations, whereas ATXN1L deficiency specifically affects MZB cells. B cell receptor signaling is impaired only in Cic-deficient FOB cells, whereas Notch signaling is disrupted in both Cic-deficient and Atxn1l-deficient MZB cells. Mechanistically, ETV4 de-repression leads to inhibition of Notch1 and Notch2 transcription, thereby inhibiting MZB cell development in B cell-specific Cic-deficient (Cicf/f;Cd19-Cre) and Atxn1l-deficient (Atxn1lf/f;Cd19-Cre) mice. In Cicf/f;Cd19-Cre and Atxn1lf/f; Cd19-Cre mice, humoral immune responses and lipopolysaccharide-induced sepsis progression are attenuated but are restored upon Etv4-deletion. These findings highlight the importance of the CIC-ATXN1L complex in MZB cell development and may provide proof of principle for therapeutic targeting in sepsis.

Abstract 36: Ablation of T-Lymphocyte β-Arrestins Reduces Inflammation and Increases Salt Retention in the Absence of Blood Pressure Changes Following Angiotensin II Infusion

Introduction: T-cells contribute to hypertension (HT) development. Among several G-protein coupled receptors regulating T-cell function, it has been hypothesized that some subpopulations use the angiotensin II (ANG II) type 1 receptor (AT 1 R) to modulate immune responses in HT. Selective ablation of AT 1 Rs in T-cells paradoxically increases hypertensive end-organ damage (EOD). While AT 1 Rs typically induces classical G-protein signaling, recent studies have uncovered a protective signaling cascade via β-arrestin ( ARRB ) proteins. We hypothesize that T-cell ARRB s mediate an anti-hypertensive and anti-inflammatory role in response to ANG II, decreasing EOD in HT. Methods: Breeding CD4-Cre mice with Arrb1 -Flox or Arrb2 -Flox mice generated lines with T-cell-specific deficiency of Arrb1 or Arrb2 genes. Hypertensive conditions modeling kidney damage consisted of unilateral nephrectomy, 4% salt diet, and continuous infusion of 1 mg/kg/min ANG II (UNX/HSD/ANG). Age/sex-matched Cre - littermates were used as controls (WT). Blood pressure (BP) was measured at baseline and for the following three weeks using radiotelemetry. Results: At baseline, there were no significant differences in systolic BP between groups (WT:124.1±8.0 mmHg; CD4 Arrb1-null :125.4±8.1; CD4 Arrb2-null :128.7±3.1; p =0.37; n=12/10/8). Three weeks of UNX/HSD/ANG equally increased systolic BP in all groups (WT:178.0±14.0 mmHg; CD4 Arrb1-null :174.8±26.3; CD4 Arrb2-null :179.9±11.1; p =0.86; n=7/9/7). Despite similar BPs, CD4 Arrb2-null mice demonstrated less renal inflammation than WT after four weeks of UNX/HSD/ANG, with a reduction in interleukin-1α protein (3.8±3.2 vs. 8.3±2.8 p <0.05; n=4/6) and a trend towards decreased tumor necrosis factor-α transcript (0.63-fold change vs. 1.9, p =0.051; n=10/8). Furthermore, CD4 Arrb1-null mice increased 24-hour voluntary water intake compared to WT (15.7±3.4 mL vs. 12.2±1.6; p <0.05; n=12/7), and 24-hour urine output (10.0±3.7 mL/d vs. 6.8±1.5; p <0.05; n=11/8). However, there was no difference in sodium excretion during this period (1.9±0.5 eq/d vs. 1.7±0.1; p =0.14; n=11/7). Interestingly, 4 hours after intraperitoneal saline injection, CD4 Arrb1-null mice excreted significantly less sodium compared to WT (5.6±3.5% vs . 31.4±10.6%; p <0.05; n=4/5). Conclusion: Contrary to our hypothesis, these data suggest that T-cell Arrb s do not protect against the hypertensive response to ANG II, but instead play a complicated role in the modulation of hydromineral balance and inflammation.

Mitochondrial Fatty Acid Synthesis and Mecr Regulate CD4+ T Cell Function and Oxidative Metabolism.

Lipid metabolism is fundamental to CD4+ T cell metabolism yet remains poorly understood across subsets. Therefore, we performed targeted in vivo CRISPR/Cas9 screens to identify lipid-associated genes essential for T cell subset functions. These screens established mitochondrial fatty acid synthesis (mtFAS) genes Mecr, Mcat and Oxsm as highly impactful. Of these, the inborn error of metabolism gene Mecr was most dynamically regulated. Effector and memory T cells were reduced in Mecrfl/fl; Cd4cre mice, and MECR was required for activated CD4+ T cells to efficiently proliferate, differentiate, and survive. Mecr-deficient T cells also had decreased mitochondrial respiration, reduced TCA intermediates, and accumulated intracellular iron, which contributed to cell death and sensitivity to ferroptosis. Importantly, Mecr-deficient T cells exhibited fitness disadvantages in inflammatory, tumor, and infection models. mtFAS and MECR thus play important roles in activated T cells and may provide targets to modulate immune functions in inflammatory diseases. The immunological state of MECR- and mtFAS-deficient patients may also be compromised.

The Role of Invariant Natural Killer T (iNKT) Cells in Early Responses to Influenza A Virus (IAV)

Abstract iNKT cells are a CD1d-restricted innate-like lineage of ⍺ßT cells that are prevalent in mucosal tissues, where they influence early immune responses. iNKT cells differentiate in the thymus into—iNKT1, iNKT2, and iNKT17 analogous to Th1, Th2, and Th17 cells. We have shown in ET-2;CD4cre mice, that sustained E protein activity results in changes in iNKT subset composition, with a shift from iNKT1s towards iNKT2/NKT17 cells and other subsets. To understand the physiological relevance of these changes, we tested the response of ET2 mice to IAV. ET2 mice showed less weight loss, reduction in lung-infected areas, decreased neutrophil infiltration, lower activation markers in inflammatory monocytes, reduced levels of CCL2 and interferon-responsive genes in ET2. We observed increased levels of IFNλ in infected ET2 mice vs. elevated IFNα in WT mice 24 hours post-infection. scRNA-seq analysis of sorted iNKT from WT and ET2 mice showed that ET2 profiles are closer to conventional naïve T cells, and that, after infection, their response is marked by overexpression of Ccr7, -Lselectin, IL1R1. WT response is predominantly type 1. Gene Ontology pathway analysis of infected WT show enrichment of cytokine production and immune cell activation pathways, while pathways enriched in infected ET-2 are associated with adhesion and repair. These experiments suggest that early type 1 responses driven by NKT cells during IAV infection can be deleterious, intensifying inflammation, and neutrophilia.

Deletion of MOF in CD4 T cells results in mitochondrial dysfunction, Th17 polarization, and colonic inflammation.

Abstract Introduction: The enzyme, MOF (males absent of the first), is a histone acetyltransferase that regulates the transcription of nuclear and mitochondrial genes. The role of MOF in acquired immunity has not been established. Hypothesis: Expression of MOF controls CD4 T cell phenotype by regulating cellular metabolism. Materials, Methods, and Results: We generated mice selectively deficient in MOF in CD4 T cells (Moff/f CD4cre). After ex vivo stimulation with CD3 and CD28, MOF-deficient splenic CD4 T cells displayed lower proliferation rate and viability, associated with altered mitochondrial morphology by electron microscopy, impaired oxidative phosphorylation, and higher glycolytic capacity, lactate production, and higher ATP production. In vivo, Moff/f CD4cre mice developed spontaneous rectal prolapse and colonic cryptitis. Intraepithelial and lamina propria CD4 T cell from these animals were disproportionately activated and displayed a Th17 phenotype. Treating these animals with a mitochondrion-targeted antioxidant, mitotempo, for a month resulted in normalization of T cell ATP production and lactate production. Conclusion: The histone acetyltransferase MOF moderates T cell activation and polarization by controlling mitochondrial respiration, protecting against the spontaneous development of colitis.

Abstract 6595: HDAC8 targeting impairs Foxp3+Treg cell function and promotes anti-tumor immunity

Abstract HDAC8 is an evolutionarily distinct, X-linked, zinc-dependent class I histone/protein deacetylase implicated in developmental disorders, parasitic infections, myopathy, and cancers such as neuroblastoma, gastric cancer, hepatocellular carcinoma, oral squamous cell carcinoma, and childhood acute lymphoblastic leukemia. Here, we highlight a hitherto unknown role of HDAC8 in immune cells by presenting data on conditional targeting of HDAC8 in T cells and the effects of selective HDAC8 inhibitors (HDAC8i) on T cell-dependent immune responses. First, as a genetic approach, we produced C57BL/6 mice with floxed exon 3 of the HDAC8 gene suitable for targeted deletion of HDAC8 and, after crossing with syngeneic CD4cre mice, demonstrated conditional deletion of HDAC8 and enhanced acetylation of SMC3, a classic HDAC8 substrate, in their T-cells. Next, in a pharmacologic approach, we tested isoform-selective inhibitors of HDAC8 (OJ-1, PCI-34051) and assessed effects on Foxp3+ T-regulatory (Treg) cell suppressive function. Finally, we performed flow cytometry, RNAseq, CHIP-seq and mass spectrometry to investigate the underlying mechanisms by which HDAC8 knockout affected Treg function. Our data demonstrate that knocking down or inhibiting HDAC8: (i) impaired murine Treg suppressive function in vitro and in vivo, including inhibiting the ability of Tregs to promote mouse cardiac allograft survival (p<0.01); (ii) decreased Treg function in homeostatic proliferation assays (p<0.01), e.g., involving adoptive transfer of WT or HDAC8-/- Tregs plus WT conventional T cells into immunodeficient hosts; (iii) decreased intratumoral Tregs, increased CD4 and CD8 T cells, increased production of effectors such as IFN-g and granzyme-B, and limited growth of syngeneic mouse non-small cell lung cancer cell lines (p<0.01) (whereas no effect of Hdac8i on tumor growth was seen in immunodeficient hosts, indicating the key action was via immune cell targeting). Mechanistically, HDAC8 co-precipitated with Foxp3 and NFAT5 and promoted the deacetylation of Foxp3 and NFAT5, and knockout of HDAC8 enhanced the acetylation level of NFAT5 and promoted cytokine expression. Hence, our combined genetic and pharmacologic studies establish the importance of HDAC8 in Foxp3+ Treg cells and show that therapy with selective HDAC8 inhibitors can be a novel therapeutic approach in immuno-oncology. Citation Format: Fanhua Kong, Liqing Wang, Aaron Beeler, Wayne W. Hancock. HDAC8 targeting impairs Foxp3+Treg cell function and promotes anti-tumor immunity [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 6595.

Mitigation of Osteoclast-Mediated Arthritic Bone Remodeling By Short Chain Fatty Acids.

The objective for this study was to evaluate the effects of short chain fatty acids (SCFAs) on arthritic bone remodeling. We treated a recently described preclinical murine model of psoriatic arthritis (PsA), R26STAT3Cstopfl/fl CD4Cre mice, with SCFA-supplemented water. We also performed in vitro osteoclast differentiation assays in the presence of serum-level SCFAs to evaluate the direct impact of these microbial metabolites on maturation and function of osteoclasts. We further characterized the molecular mechanism of SCFAs by transcriptional analysis. The osteoporosis condition in R26STAT3Cstopfl/fl CD4Cre animals is attributed primarily to robust osteoclast differentiation driven by an expansion of osteoclast progenitor cells (OCPs), accompanied by impaired osteoblast development. We show that SCFA supplementation can rescue the osteoporosis phenotype in this model of PsA. Our in vitro experiments revealed an inhibitory effect of the SCFAs on osteoclast differentiation, even at very low serum concentrations. This suppression of osteoclast differentiation enabled SCFAs to impede osteoporosis development in R26STAT3Cstopfl/fl CD4Cre mice. Further interrogation revealed that bone marrow-derived OCPs from diseased mice expressed a higher level of SCFA receptors than those of control mice and that the progenitor cells in the bone marrow of SCFA-treated mice presented a modified transcriptomic landscape, suggesting a direct impact of SCFAs on bone marrow progenitors in the context of osteoporosis. We demonstrated how gut microbiota-derived SCFAs can regulate distal pathology (ie, osteoporosis) and identified a potential therapeutic option for restoring bone density in rheumatic disease, further highlighting the critical role of the gut-bone axis in these disorders.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=<0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=<0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

Transcription Factor GATA2 Is Inevitable for the Survival and Proper Differentiation of Thymic Multipotent Progenitor Cells By Gene Expression Orchestration

Proper differentiation of T cells in the thymus is inevitable for the establishment of acquired immunity. Lymphoid-primed multipotent progenitors (LMPPs) emigrate from the bone marrow (BM) to the thymus for becoming earliest T cell progenitors, so-called early T cell progenitors (ETPs). Previous studies have shown that ETPs retain not only T cell but also innate immune cell (i.e., myeloid cells) differentiation potentials. Therefore, ETPs might be the bifurcation point of innate and adaptive immunity in thymus. However, still little is known about the mechanism by which the differentiation fates are determined in ETPs and the cellular heterogeneity in ETP subset. Since transcription factors that possess important roles in hematopoietic stem and progenitor cells (HSPCs) may play significant roles in the early differentiation of T cells as legacy stem cell genes, we sought to investigate the roles for GATA2, which is known to regulate the HSPC maintenance and myeloid differentiation, in ETPs. First, we revealed the GATA2 expression is preserved in ETPs and sharply disappear with their differentiation (GATA2 expressions were almost undetectable from double-negative 2 cells even by qPCR). To reveal the roles for GATA2 in T cell hematopoiesis, we analyzed Gata2 fl/fl; Mx1-Cre mice in which GATA2 was deletable in hematopoietic cells by polyI:C administration ( Gata2 KO). Gata2 KO mice showed a smaller thymus and a lower number of thymic T cells, including ETPs and double-positive (DP; CD4 +CD8 +) cells, than control. To rule out contributions of potential GATA2 deletions in non-hematopoietic cells, CD45.1 + wild-type unfractionated BM cells were transplanted into Gata2 KO or control mice. There were no particular differences in T cell hematopoiesis from CD45.1 + donor cells between them, supporting the notion that hematopoietic cell intrinsic GATA2 are inevitable for the proper T cell hematopoiesis. To further reveal the roles for GATA2 in ETPs, we performed global transcriptomic analysis of ETPs (RNA-seq; Gata2 KO vs control). Gene set enrichment analysis (GSEA) revealed that signatures associated with cell cycle regulation, hematopoietic progenitor cells and myeloid cells were down-regulated in Gata2 KO ETPs, suggesting that GATA2 is involved in the survival and myeloid differentiation potentials in ETPs. By taking the advantage of novel GATA2 reporter mice (GATA2-VENUS mice) in which GATA2 protein levels are detectable by flow cytometry, we revealed that the ETP fraction contained a mixture of cells with high or low GATA2 expression. To be noted, the protein expression intensity of GATA2 in GATA2-high ETPs was almost equivalent to that of LMPPs. These observations suggest that GATA2-high ETPs might be a thymic multipotent progenitors (T-MPPs), the earliest thymic progenitors in which T or myeloid cell differentiation commitment is defined. To elucidate the mechanism by which GATA2 regulates the survival and differentiation of T-MPPs, we integrated publicly available chromatin immunoprecipitation followed by sequencing data targeting GATA2 (GATA2 ChIP-seq) and our ETP RNA-seq data. We found several key genes in HSPCs maintenance (i.e. Lyl1) or myeloid differentiation (i.e. Itga2b) as potential targets of GATA2. Furthermore, we sought to reveal the effect of GATA2 deletion in progenitor cells to their progenies. To this end, we performed RNA-seq of DP cells ( Gata2 KO vs control). GSEA revealed that cell cycle related gene signature was repressed in Gata2 KO DP cells compared to control. To be noted, in CD4-Cre dependent GATA2 deletion model ( Gata2 fl/fl:CD4-Cre mice), there were almost no particular cell number and transcriptomic differences, which was also analyzed by RNA-seq, in DP cells compared to control. These results suggest that GATA2 in GATA2-high ETPs, but not in DP cells, may affect the feature of DP cells, potentially by epigenetic memory. Collectively, this study revealed that GATA2 contributes to T cell hematopoiesis. GATA2 expression was maintained in a part of the ETP subset (GATA2-high ETPs) which might be equivalent to T-MPPs. GATA2 may affect the differentiation trajectory and function from GATA2-high ETPs to their progenies. Further study of this mechanism will help us to understand how the acquired and innate immune bifurcation is defined, which might pave the way to overcome T cell development related disorders, such as T-cell/myeloid mixed-phenotype acute leukemia.

The Roles of Immune Cells Derived from Clonal Hematopoiesis in Colorectal Cancer Metastasis

[Backgrounds] Clonal hematopoiesis (CH) has been observed in over 20% of patients diagnosed with solid cancers, and it has been associated with a poor prognosis in most cases, with the exception of colorectal cancer (CRC) patients who tend to have a favorable prognosis. Among the various gene mutations detected in CH, TET2 mutations are particularly prevalent. Previous research has demonstrated that the impact of TET2-mutated CH immune cells depends on the specific types of cancer tissues involved. Specifically, studies have shown that Tet2-KO myeloid cells can inhibit the progression of melanoma while promoting the growth of hepatoma and lung cancers. However, there is a limited understanding of the roles of TET2-mutated CH immune cells on CRC progression. [ Methods] We conducted an experiment focusing on CRC and CH using CRC organoid cells transplanted into the spleens of different types of Tet2 conditional knockout mice: VAV1Cre (with Tet2 gene deletion in all hematopoietic cells), LysMCre (myeloid cells), CD19Cre (B lymphocytes), and CD4Cre (T lymphocytes). After a period of 30 days, we collected livers for analysis. The extent of liver metastasis tumor burden (LMTB) was determined by counting the number of tumor foci on 10 hematoxylin-stained slides, with an interval of 80 μm between each slide. We utilized immunohistochemistry (IHC) and flow cytometry (FC) to analyze immune cells. The number of positive cells was automatically counted in 10 fields at 20x magnification. Furthermore, we performed whole transcriptome analysis (WTA) on sorted CD4+, CD8+, CD11b+, and CD19+ cells, respectively. [Results] LMTB was significantly lower in VAV1Cre and CD4Cre mice compared to the control mice ( VAV1Cre vs. control: 55.65±35.62 vs. 89.93±34.57 foci/1000 mm², p<0.05; CD4Cre vs. control: 50.19±30.39 vs. 85.98±7.92 foci/1000 mm², p<0.05). However, the LMTB of CD19Cre and LysMCre mice was comparable to that of control mice. The differential gene expression of WTA of CD8+ cells revealed that 243 genes were up-regulated, while 189 genes were down-regulated in both of VAV1Cre and CD4Cre mice compared to the control mice. Among the down-regulated genes, a gene group consisting of Pdcd1, Tigit, Lag3 and Havcr2, which encode inhibitory molecules associated with exhausted T cells exhibited enrichment (Enrichment score =3.77) based on the analysis using DAVID. Ingenuity Pathway Analysis further indicated that these molecules were downregulated by the upstream regulator TOX. Furthermore, T cell exhaustion signaling pathways were downregulated in both VAV1Cre and CD4Cre mice using gene set enrichment analysis. The immunofluorescence results demonstrated that the number of CD8-T cells expressing PDCD1 and HAVCR2 was decreased in VAV1Cre mice and CD4Cre compared to the control mice ( VAV1Cre and CD4Cre vs control; 5.91±2.50 and 12.86±5.3 vs. 35.15±8.21 cells/100 CD8-T cells, p<0.01 for PDCD1; 4.51±4.06 and 13.65±7.84 vs. 24.51±9.94 cells/100 CD8-T cells, p<0.01 for HAVCR2). Moreover, the number of CD8-T cells expressing TOX was also significantly decreased in VAV1Cre mice and CD4Cre compared to the control mice ( VAV1Cre and CD4Cre vs control; 5.19±7.48 and 11.31±14.43 vs. 35.15±16.50 cells/100 CD8-T cells; p<0.01). [ Conclusions] The depletion of the Tet2 gene in hematopoietic cells resulted in a reduction of exhausted CD8+ T cells and suppressed the liver metastasis of CRC organoid cells. These findings provide insights into the favorable prognosis observed in CRC patients with CH. Considering the high prevalence of CH in cancer patients, our results suggest that studying the roles of CH-immune cells could shed new light on understanding the cancer microenvironment from a novel perspective.

A Novel Transgenic Mouse Model of Down Syndrome Acute Lymphoblastic Leukemia Identifies Targetable Vulnerabilities

Background: Children with Down syndrome (DS) have a 10-fold increased risk of developing B-cell acute lymphoblastic leukemia (B-ALL), and they have poorer survival due to increased relapses and treatment-related mortality (TRM). Targeted therapies for DS-ALL are needed to improve anti-leukemic efficacy and reduce the risk of TRM. Mouse models and cell lines recapitulating DS-ALL are lacking, and may aid in identifying new targets for DS-ALL. Methods: We used the Dp(16)1Yey (Dp16) mouse model of DS, which has a triplication of ~115 human chromosome 21 (Hsa21) orthologues. We introduced Kras G12D and Pax5 heterozygosity, both driven in B cells by CD19-Cre, in Dp16 and non-DS wild-type (WT) mice. We performed RNA-Sequencing (RNA-Seq) and gene set enrichment analysis (GSEA) to identify differentially regulated signaling pathways in Dp16 and WT B-ALL blasts. We cultured B-ALL blasts from mice to generate immortal cell lines. We tested the chemosensitivity of Dp16 and WT B-ALL cell lines with 35 agents with known efficacy in hematologic malignancies, and with 481 anti-cancer compounds used in the Cancer Therapeutics Response Portal project, to screen for drugs effective in DS-ALL. We screened top candidate drugs in DS-ALL and non-DS ALL patient samples in vitro, and tested FK866 and cucurbitacin I in vivo in mice xenografted with a DS-ALL patient sample. Results: Kras G12D.Pax5 +/- mice developed B-ALL with complete penetrance, with significantly shorter median survival in the Dp16 versus WT background (Figure 1A; 80 versus 114 days, p<0.0001). GSEA demonstrated upregulation of DNA repair signaling pathways in Dp16 B-ALL, recapitulating a signature observed in human DS-ALL. Growth of Dp16 and WT B-ALL cell lines, and DS-ALL and non-DS ALL patient samples, was inhibited at low nanomolar concentrations by novel therapies targeting NAMPT, DNA damage responses, autophagy, and JAK and PI3K/mTOR signaling. In mice xenografted with a DS-ALL patient sample, the NAMPT inhibitor FK866 significantly reduced the leukemic burden compared to vehicle (Figure 1B; 14.6% vs 22.4% after 4 weeks of treatment, p<0.005). The effect of FK866 was also significant after weeks 2 and 3 of treatment. Conclusions: We have generated the first de novo mouse model and cell lines recapitulating DS-ALL, which we have employed in drug screens to identify novel therapeutic approaches. These studies suggest promising candidates for further study in DS-ALL and other high-risk ALL subtypes to reduce toxicity and improve outcomes.

PHRF1 promotes the class switch recombination of IgA in CH12F3-2A cells.

PHRF1 is an E3 ligase that promotes TGF-β signaling by ubiquitinating a homeodomain repressor TG-interacting factor (TGIF). The suppression of PHRF1 activity by PML-RARα facilitates the progression of acute promyelocytic leukemia (APL). PHRF1 also contributes to non-homologous end-joining in response to DNA damage by linking H3K36me3 and NBS1 with DNA repair machinery. However, its role in class switch recombination (CSR) is not well understood. In this study, we report the importance of PHRF1 in IgA switching in CH12F3-2A cells and CD19-Cre mice. Our studies revealed that Crispr-Cas9 mediated PHRF1 knockout and shRNA-silenced CH12F3-2A cells reduced IgA production, as well as decreased the amounts of PARP1, NELF-A, and NELF-D. The introduction of PARP1 could partially restore IgA production in PHRF1 knockout cells. Intriguingly, IgA, as well as IgG1, IgG2a, and IgG3, switchings were not significantly decreased in PHRF1 deficient splenic B lymphocytes isolated from CD19-Cre mice. The levels of PARP1 and NELF-D were not decreased in PHRF1-depleted primary splenic B cells. Overall, our findings suggest that PHRF1 may modulate IgA switching in CH12F3-2A cells.

1671-P: T Cell Deficiency of STAT1 Reduces Obesity in Mice with High-Fat Diet or Aging

T cells accumulate in adipose tissue (AT) and change phenotypes, with increased polarization to Th1, in obesity, but the mechanisms for these changes and their contribution to metabolic functions remain incompletely understood. We hypothesize that STAT1, a transcription factor, plays a role in AT T cell polarization, thus impacting obesity and related metabolism. Mice with Stat1 knockout in αβT cells (tStat1-KO) were generated by crossbreeding Stat1fl/fl mice and CD4-cre mice. Strikingly, with high-fat diet (HFD) feeding or aging, tStat1-KO, compared to cre noncarrier wild type (WT) littermates, developed less obesity, with lower body weight and fat mass (body weight: 43 ± 0.5 g in KO [n=33] vs 48 ± 0.8 g in WT [n=32] at 12 weeks on HFD and 32 ± 0.7 g in KO [n=27] vs 37 ± 0.6 g in WT [n=25] at the age of 50 weeks [on normal chow], P<0.001; fat mass: 14 ± 0.5 g in KO [n=6] vs 17 ± 0.7 g in WT [n=4] at 12 weeks on HFD, P=0.02 and 4.6 ± 0.3 g in KO [n=9] vs 8.6 ± 1.5 g in WT [n=4] at the age of 50 weeks, P=0.002). Compared to WT, tStat1-KO had similar food intake, but increased energy expenditure with increased white AT browning indicated by elevated UCP1 and PRDM16 levels in inguinal AT. tStat1-KO vs WT with HFD or aging had improved insulin sensitivity and glucose tolerance. Analyses of AT showed a dramatic phenotypic switch from IFNγ+ Th1 to IL-5+ Th2 cells and increased eosinophils in perigonadal and inguinal AT of tStat1-KO vs WT fed HFD. Antibody neutralization of IL-5 reduced eosinophils but did not reverse less obesity in tStat1-KO fed HFD, suggesting that increased IL-5 and eosinophils may not account for reduced obesity in tStat1-KO. Adoptive transfer of Th1 cells in tStat1-KO and tissue culture studies showed that Th1 cells repressed white AT browning and reduced UCP1 in adipocytes. In summary, T cell ablation of STAT1 led to phenotypic switch from Th1 to Th2 cells in AT, correlated with reduced obesity, in mice with HFD or aging. These data support a role of STAT1 in AT T cell polarization, which may affect obesity development induced by HFD or aging. Disclosure Z.Lian: None. V.O'brien: None. X.D.Perrard: None. P.Saha: None. S.M.Hartig: None. C.M.Ballantyne: Consultant; Alnylam Pharmaceuticals, Inc., Althera Pharmaceuticals, Amarin Corporation, Amgen Inc., Arrowhead Pharmaceuticals, Inc., AstraZeneca, ESPERION Therapeutics, Inc., 89bio, Inc., Abbott Diagnostics, Denka Seiken, Genentech, Inc., Gilead Sciences, Inc., Illumina, Matinas BioPharma Inc, Merck & Co., Inc., New Amsterdam Pharma, Novartis, Novo Nordisk, Pfizer Inc., Regeneron, Roche Diagnostics, Research Support; Abbott Diagnostics, Akcea, Amgen Inc., Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Ionis Pharmaceuticals, Merck & Co., Inc., Novartis, Novo Nordisk, Regeneron, Roche Diagnostics. H.Wu: None. Funding American Diabetes Association (1-17-IBS-082 to H.W.); National Institutes of Health (R01DK121348, R01AG065197); American Heart Association (968367)

Ezh2 Knockout in B Cells Impairs Plasmablast Differentiation and Ameliorates Lupus-like Disease in MRL/lpr Mice.

EZH2 regulates B cell development and differentiation. We previously demonstrated increased EZH2 expression in peripheral blood mononuclear cells from lupus patients. The goal of this study was to evaluate the role of EZH2 expression in B cells in the pathogenesis of lupus. We generated an MRL/lpr mouse with floxed Ezh2, which was crossed with CD19-Cre mice to examine the effect of B cell EZH2 deficiency in MRL/lpr lupus-prone mice. Differentiation of B cells was assessed using flow cytometry. Single-cell RNA sequencing and single-cell B cell receptor sequencing were performed. In vitro B cell culture with an X-box binding protein 1 (XBP1) inhibitor was performed. EZH2 and XBP1 messenger RNA levels in CD19+ B cells isolated from lupus patients and healthy controls were analyzed. We show that Ezh2 deletion in B cells significantly decreased autoantibody production and improved glomerulonephritis. B cell development was altered in the bone marrow and spleen of EZH2-deficient mice. Differentiation of germinal center B cells and plasmablasts was impaired. Single-cell RNA sequencing showed that XBP1, a key transcription factor in B cell development, is down-regulated in the absence of EZH2. Inhibiting XBP1 in vitro impairs plasmablast development similar to EZH2 deficiency in mice. Single-cell B cell receptor RNA sequencing revealed defective immunoglobulin class-switch recombination in EZH2-deficient mice. In human lupus B cells, we observed a strong correlation between EZH2 and XBP1 messenger RNA expression levels. EZH2 overexpression in B cells contributes to disease pathogenesis in lupus.

Decreased memory persistence in Piezo1 deficient T cells ameliorates disease progression in autoimmune mouse models

Abstract To mount a successful immune response, T cells must successfully (1) migrate from the bloodstream into secondary lymphoid organs (naïve) or inflamed tissue (effector), and (2) form an effective immunological synapse for activation. Although the molecular interactions of these processes have been well described, the emergence of novel biophysical approaches has revealed that both critical processes require mechanosensing. Previously we have demonstrated that the mechanosensitive cation channel, Piezo1, forms a complex with activated integrin LFA-1, an important regulator of T cell migration and T cell receptor-triggered T cell activation. Therefore, we hypothesized that Piezo1 deficiency in T cells would diminish T cell activation and homing.To investigate Piezo1’s role on T cell function, we have generated T cell-specific Piezo1 knockout mice (P1cKO) by crossing Piezo1-floxed mice to CD4-Cre mice. Initial studies in vitro showed that under polarizing conditions P1cKO T cells produce more Th1 and Th17 inflammatory cytokines. However, when challenged with experimental autoimmune encephalomyelitis (EAE) and Graft-vs-Host Disease (GVHD), P1cKO mice showed a significant decrease in disease phenotype. Moreover, adoptive transfer mouse models of EAE show that activated P1cKO T cell transfer fails to establish disease phenotype. Upon further investigation, we found similar numbers of P1cKO T cells at sites of inflammation early in disease, but there was a decreased persistence of effector and central memory P1cKO T cells. Taken together, our data suggests that Piezo1 plays an important in role in the development and maintenance of T cell memory pools, while potentially contributing to T cell activation and migration. This project was supported by grants from the NIH (TL1 TR002549, T32 GM007250, T32 AI089474), the St. Baldrick's Foundation Innovation Fund, the Theresia G. & Stuart F. Kline Family Foundation, and the Karen & Alan Krause Pediatric Immunotherapy Innovation and Education Fund.

IKK1 aggravates ischemia–reperfusion kidney injury by promoting the differentiation of effector T cells

Ischemia–reperfusion injury (IRI) is one of the major causes of acute kidney injury (AKI), and experimental work has revealed detailed insight into the inflammatory response in the kidney. T cells and NFκB pathway play an important role in IRI. Therefore, we examined the regulatory role and mechanisms of IkappaB kinase 1 (IKK1) in CD4+T lymphocytes in an experimental model of IRI. IRI was induced in CD4cre and CD4IKK1Δ mice. Compared to control mice, conditional deficiency of IKK1 in CD4+T lymphocyte significantly decreased serum creatinine, blood urea nitrogen (BUN) level, and renal tubular injury score. Mechanistically, lack in IKK1 in CD4+T lymphocytes reduced the ability of CD4 lymphocytes to differentiate into Th1/Th17 cells. Similar to IKK1 gene ablation, pharmacological inhibition of IKK also protected mice from IRI. Together, lymphocyte IKK1 plays a pivotal role in IRI by promoting T cells differentiation into Th1/Th17 and targeting lymphocyte IKK1 may be a novel therapeutic strategy for IRI.

Cell-Autonomous Constitutive gp130 Signaling in T Cells Amplifies TH17 Cell Responses and Causes Severe Lung Inflammation.

IL-6 plays a fundamental role in T cell differentiation and is strictly controlled by surface expression and shedding of IL-6R. IL-6 also acts on other cells that might affect T cell maturation. To study the impact of cell-autonomous and uncontrolled IL-6 signaling in T cells, we generated mice with a constitutively active IL-6R gp130 chain (Lgp130) expressed either in all T cells (Lgp130 × CD4Cre mice) or inducible in CD4+ T cells (Lgp130 × CD4CreERT2 mice). Lgp130 × CD4Cre mice accumulated activated T cells, including TH17 cells, in the lung, resulting in severe inflammation. Tamoxifen treatment of Lgp130 × CD4CreERT2 mice caused Lgp130 expression in 40-50% of CD4+ T cells, but mice developed lung disease only after several months. Lgp130+ CD4+ T cells were also enriched for TH17 cells; however, there was concomitant expansion of Lgp130- regulatory T cells, which likely restricted pathologic Lgp130+ T cells. In vitro, constitutive gp130 signaling in T cells enhanced but was not sufficient for TH17 cell differentiation. Augmented TH17 cell development of Lgp130+ T cells was also observed in Lgp130 × CD4CreERT2 mice infected with Staphylococcus aureus, but gp130 activation did not interfere with formation of TH1 cells against Listeria monocytogenes. Lgp130+ CD4+ T cells acquired a memory T cell phenotype and persisted in high numbers as a polyclonal T cell population in lymphoid and peripheral tissues, but we did not observe T cell lymphoma formation. In conclusion, cell-autonomous gp130 signaling alters T cell differentiation. Although gp130 signaling is not sufficient for TH17 cell differentiation, it still promotes accumulation of activated T cells in the lung that cause tissue inflammation.

Loss of RPA1 Impairs Peripheral T Cell Homeostasis and Exacerbates Inflammatory Damage through Triggering T Cell Necroptosis.

The peripheral T cell pool is maintained at dynamic homeostasis through fine-tuning of thymic output and self-renewal of naïve T cells. Lymphopenia or reduced lymphocyte number is implicated in autoimmune diseases, yet little is known about the homeostatic mechanisms. Here, it is reported that the replication protein A1 (RPA1) plays a critical role in T cell homeostasis. Utilizing T cell-specific Rpa1-deficient (Rpa1fl/fl Cd4-cre) mice, loss of Rpa1 results in lymphopenia through restraining peripheral T cell population and limiting TCR repertoire diversity. Moreover, Rpa1fl/fl Cd4-cre mice exhibit increased susceptibility to inflammatory diseases, including colitis and hepatitis. Clinical analysis reveals that the expression of RPA1 is reduced in patients with ulcerative colitis or other autoinflammatory diseases. Mechanistically, depletion of RPA1 activates ZBP1-RIPK3 signaling through triggering the genomic DNA leakage into cytosol, consequently resulting in T cell necroptosis. This necroptotic T cell death induced by RPA1 deficiency allows the release of damage-associated molecular patterns (DAMPs), which in turn recruits leukocytes and exacerbates inflammatory response. Reciprocally, chemical or genetic inhibition of necroptosis signaling can ameliorate the Rpa1 deficiency-induced inflammatory damage. The studies thus uncover the importance of RPA1-ZBP1-RIPK3 axis in T cell homeostasis and provide a promising strategy for autoinflammatory disease treatment.

GPX4 aggravates experimental autoimmune encephalomyelitis by inhibiting the functions of CD4+ T cells

Multiple sclerosis (MS) is a common autoimmunity disease of the central nervous system (CNS) that mostly happens in young adults. The chronic clinical features of MS include inflammatory demyelination, infiltration of immune cells, and secretion of inflammatory cytokines, which have been proved to be associated with CD4+ T cells. Ferroptosis is a newly discovered programmed cell death mediated by the massive lipid peroxidation and more sensitive to CD4+ T cells. However, the effect of ferroptosis of CD4+ T cells on the occurrence and progression of MS retains unclear. Here, the experimental autoimmune encephalomyelitis (EAE) model was used to investigate the role of GPX4, a leading inhibitor of ferroptosis, which plays in the function of CD4+ T cells. Our results showed that GPX4 was highly expressed in CD4+ T cells of MS patients based on existing databases. Strikingly, conditional knockout of GPX4 in CD4cre mice (cKO mice) significantly alleviated the average symptom scores and immunopathology of EAE. The infiltration of immune cells, including CD4+ T and CD8+ T cells, and the generation of GM-CSF, TNF-α, and IL-17A, were remarkably reduced in the CNS from cKO mice compared with WT mice. These findings further revealed the vital role of GPX4 in the expansion and function of CD4+ T cells. Moreover, GPX4-deficient CD4+ T cells were susceptible to ferroptosis in EAE model. Overall, this study provided novel insights into therapeutic strategies targeting GPX4 in CD4+ T cells for inhibiting CNS inflammation and treating MS.

CARD11 mutation and HBZ expression induce lymphoproliferative disease and adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). In addition to HTLV-1 bZIP factor (HBZ), a leukemogenic antisense transcript of HTLV-1, abnormalities of genes involved in TCR-NF-κB signaling, such as CARD11, are detected in about 90% of patients. Utilizing mice expressing CD4+ T cell-specific CARD11(E626K) and/or CD4+ T cell-specific HBZ, namely CARD11(E626K)CD4-Cre mice, HBZ transgenic (Tg) mice, and CARD11(E626K)CD4-Cre;HBZ Tg double transgenic mice, we clarify these genes’ pathogenetic effects. CARD11(E626K)CD4-Cre and HBZ Tg mice exhibit lymphocytic invasion to many organs, including the lungs, and double transgenic mice develop lymphoproliferative disease and increase CD4+ T cells in vivo. CARD11(E626K) and HBZ cooperatively activate the non-canonical NF-κB pathway, IRF4 targets, BATF3/IRF4/HBZ transcriptional network, MYC targets, and E2F targets. Most KEGG and HALLMARK gene sets enriched in acute-type ATL are also enriched in double transgenic mice, indicating that these genes cooperatively contribute to ATL development.