Martinez, Kodi

Abstract Introduction Generation of T cells requires dynamic epigenetic alteration of histones to control gene expression at each developmental stage. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) facilitate addition or removal of various acyl groups from lysine residues, including acetylation (KAc) and crotonylation (KCr). The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have 3 functions: adaptor, decrotonylase, and deacetylase activity. The relative importance of each function in T cells is not known. Genetic knockout of HDAC3 in mice using CD2icre results in a severe block in T cell development and spontaneous development of severe colitis. Our lab determined that HDAC3 is required for three different aspects of this process: CD4+CD8 + (DP) cell survival, positive selection, and CD4+ lineage commitment. We generated a novel mouse model with CD2icre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 “VRPP” mutant mice, which maintained adaptor function but lacks enzymatic activity. Strikingly, these mice do not develop spontaneous colitis even up to one year of age. Methods We used flow cytometry to assess T cell development and maturation in the thymus, spleen, mLN, and colon of HDAC3 cKO vs. HDAC3 VRPP mice. CUT&RUN was utilized to determine differences in H3K9Ac, H3K27Ac, and H3K18Cr across CD8 and CD4 lineage genes. H&E was utilized to assess structural changes in thymus and colon. Results Similar to CD2icre HDAC3 cKO mice, HDAC3 VRPP mice have a block in positive selection due to failure to down regulate RORγt and decreased DP cell survival due to increased expression of P2RX7. Although fewer T cells are produced in HDAC3 VRPP mice, CD4+ lineage commitment is restored, likely mitigating the development of spontaneous colitis. Conclusion Our results demonstrate that HDAC3 enzymatic activity is necessary for several aspects of T cell development, while HDAC3 adaptor function is sufficient to restore CD4+ lineage commitment. Funding Source NIAID RO1 AI150100, T32 AI170478 Topic Categories Hematopoiesis and Immune System Development (HEM)

Abstract Introduction The mitochondrial transporter ABCB7 is essential for exporting iron-sulfur (Fe-S) clusters, a function known to be critical in B cells. This study was conducted to investigate the broader, previously unknown roles of ABCB7 in regulating the overall lymphocyte development and homeostasis. Methods The study utilized conditional knockout (cKO) mouse models (CD2-iCre and Mb1-Cre) to create a specific deficiency of ABCB7 in certain immune populations. To determine if the observed effects were due to ABCB7 deletion in myeloid cells, two further models (CD2i-Cre ABCB7 cKO/C/EBPα and CD2-iCre LSL TMT ABCB7 cKO) were developed. The effects of blocking the cytokine G-CSF were also examined to understand its role in the observed myelopoiesis. Results ABCB7 deficiency was found to dramatically impair erythropoiesis while simultaneously driving emergency myelopoiesis. We confirmed that the myeloid expansion was not a direct result of ABCB7 loss in myeloid cells. A significant autoimmune phenotype also developed, characterized by hyperactivated T cells and a sharp decrease in peripheral regulatory T cells (Tregs). This Treg instability was linked to decreased thymic expression of key stability genes (like CD28 and PD-1) and reduced O-GlcNAc modification in T cells, a process crucial for maintaining Treg lineage. Conclusion The findings establish ABCB7 as a pivotal regulator of hematopoietic balance, linking mitochondrial function to both myeloid expansion and Treg stability. Funding Source T32AI170478 Topic Categories Hematopoiesis and Immune System Development (HEM)

Abstract Description The lifetime risk for colorectal cancer in the United States is approximately 4%. Individuals with Inflammatory Bowel Disease, including Ulcerative Colitis and Crohn’s Disease, have a substantially increased risk of developing colorectal cancer. The mechanisms for accelerated tumorigenesis due to enhanced inflammation are not fully characterized. Siglecs (sialic acid immunoglobulin lectin-like proteins) are a family of inhibitory receptors that are negative regulators of the immune response. Siglec-E is an inhibitory receptor that is expressed by innate immune cells, including monocytes, macrophages, neutrophils, and dendritic cells. Interestingly, mice deficient in Siglec-E have accelerated the development of tumors and reduced survival in a spontaneous mouse model of colorectal cancer (TS4-cre LSL-KRasG12D APClox468/wt). Siglec-E knockout mice also have accelerated gut inflammation using the DSS colitis model as compared to WT mice. Using the DSS/AOM model of colitis-associated colorectal cancer, we found that Siglec-E KO mice develop more tumors as compared to wild-type controls. Thus, Siglec-E is an important regulator of gut homeostasis, and the loss of Siglec-E accelerates colon cancer in two murine models. Funding Sources Mayo Clinic Center for Biomedical Discovery NIH R01 CA243545-01A1 to VSS T32 AI170478 Mayo Clinic Graduate School of Biomedical Sciences Deans fellowship: Initiative for maximizing student development (IMSD) R25 GM055252-26 Doyon Foundation Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description Generation of T cells requires dynamic epigenetic alteration of histones to control gene expression at each developmental stage. The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have 3 functions: adaptor, decrotonylase, and deacetylase activity. The relative importance of each function in T cells is not known. CD2icre HDAC3 cKO mice develop severe colitis and demonstrate that HDAC3 is required during thymocyte development for positive selection, DP survival, and CD4 lineage commitment. Recently, a mutation was identified in HDAC3 that eradicated deacetylase activity (“VRPP”). We generated a novel mouse model with Cre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 VRPP, which maintained adaptor function but lacks both deacetylase and decrotonylase enzymatic activity. Similar to CD2icre HDAC3 cKO mice, HDAC3 VRPP mice have a block in positive selection due to failure to down regulate RORγt and decreased DP cell survival due to increased expression of P2RX7, demonstrating that HDAC3 enzymatic activity is required for these functions. However, when the positive selection defect is bypassed in HDAC3 VRPP mice, CD4+ lineage commitment is restored. Strikingly, this mouse does not develop spontaneous colitis even up to one year of age, although T cell development is still impaired. Thus, there are different roles for HDAC3 enzymatic vs. adaptor function during T cell development which are made evident in HDAC3 VRPP mice. Funding Sources NIAID RO1 AI150100; T32 AI170478; Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract Background Hypomagnesemia has been correlated with inferior outcomes in patients with large B cell lymphoma (LBCL) undergoing stem cell transplants. As T-cell and myeloid cell dysfunction have been associated with low magnesium conditions, we investigated whether serum magnesium (Mg) levels could predict clinical outcomes in LBCL patients who received chimeric antigen receptor T-cell therapy. Methods Patients with LBCL who received axi-cel under the ZUMA-1 trial or as FDA approved therapy at Mayo Clinic were examined. Serum samples were obtained at specified time points and cytokine analysis was performed. Single cell RNA sequencing was performed on peripheral blood mononuclear cells. The Student T-test, Kruskal Wallis, or Fisher's Exact Tests were used to compare differences in demographics across Mg levels. Survival curves were plotted using the Kaplan-Meier methodology and compared using the Wilcoxon test. Results We found that hypomagnesemia before lymphodepletion chemotherapy predicted for inferior progression-free and overall survival in the pivotal study ZUMA-1 (NCT02348216). These results were validated in an independent cohort of LBCL patients receiving axicabtagene ciloleucel (axi-cel) at Mayo Clinic. Hypomagnesemia correlated with increased inflammatory serum markers and cytokine levels including ferritin, IL-6, IL1Ra, IL-8, and MIP1a. scRNAseq analysis unveiled altered immune interactions between monocytes and T cells with a concordant immune suppressive transcriptome. Conclusions Hypomagnesemia at the time of CAR-T infusion associates with an unfavorable inflammatory profile and decreased response and survival in LBCL patients receiving axi-cel. These findings suggest a potentially actionable prognostic factor for patients with large cell lymphoma undergoing CAR-T.

Abstract Histone acetyltransferases (HATs) and histone deacetylases (HDACs) facilitate addition or removal of various acyl groups from lysine residues, including acetylation (KAc) and crotonylation (KCr). Recently, mutations in p300 and CBP were identified that abrogate their function as an acetyltransferase but not the ability to mediate crotonylation. These mutants can activate transcription independent of their ability to acetylate histones, demonstrating that crotonylation can also lead to transcriptional activation. The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have both decrotonylase and deacetylase activity, but the relative importance of each function in T cells is not known. Genetic knockout of HDAC3 using CD2 icre mice results in a severe block in T cell development and spontaneous development of severe colitis. Examination of HDAC3 cKO mice has shown an aberrant ratio of Treg:Th17 cells within the gut, driving inflammation and loss of mucosal barrier integrity. Our lab has also determined that HDAC3 is required for at least three aspects of T cell development: CD4+ CD8+ (DP) cell survival, positive selection, and CD4+ lineage development. Recently, a mutation was identified in either HDAC1 or HDAC3 that eradicated deacetylase activity but retained decrotonylase activity (“VRPP”). Through this mutation we were able to generate a novel mouse model with Cre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 VRPP. Strikingly, this mouse does not develop colitis, although T cell development is still impaired. Thus, HDAC3 decrotonylase activity alone is sufficient to impede the IBD phenotype. Support by grants from National Institute of Allergy and Infectious Diseases (R01 AI150100)

Abstract Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti–PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell–mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti–PD-1 or anti–PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen–specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell–intrinsic therapeutic target for enhancing cancer immunotherapy. See related article by Li et al., p. 154.

7558 Background: A low absolute lymphocyte to monocyte ratio (ALC/AMC) has been found to predict decreased survival in lymphoma patients receiving chemotherapy and stem cell transplant. We report its clinical significance and additional cellular phenotype changes in patients receiving chimeric antigen receptor T-cell (CART) therapy. Methods: Records were reviewed for patients (pts) who received axicabtagene ciloleucel between 6/2016 and 12/2020. Receiver operator curve was generated using nominal logistic regression to predict CR as best response. Survivals were calculated using Kaplan- Meier method. Blood immune phenotype were assayed by multiparametric flow. Principle component analysis (PCA) was performed using ClusterVis. Results: Low ALC/AMC (≤0.8) prior to lymphodepletion (LD) chemotherapy on day -5 was associated with lower CR rate (AUC=0.68, Table). Our cohort of 81 pts had similar baseline characteristics except that noted in Table. Low ALC/AMC ratio is associated with shorter EFS and OS (EFS: 2.6 vs. 6.4 months, P<0.0001; OS: 5.3 months vs. not reached, P=0.0006), respectively. Prognostic association remained significant in multivariate analysis including ASCT, bridging therapy and CRP. Interestingly, compared to the high ALC/AMC group, the low ALC/AMC group had decreased CD8 Tem, increased CD16+CCR2+ monocytes and increased monocytes’ producing IL12, IL-10, and IL-1β (n=26). Unsupervised PCA identified 3 clusters: 1. Low ALC/AMC, all non-CR; 2. High ALC/AMC, some non-CR; 3. High ALC/AMC, all CR. Compared to cluster 1 and 2, cluster 3 had increased CD4 Tnaive, CD8 Tcm and IL-17 producing CD4 T and NK cells. Conclusions: ALC/AMC is a clinically accessible test that is strongly associated with CAR-T response and survival. Immune characterization revealed that the biologic effect is not just associated with cell ratio. Increased inflammation has been found to negatively impact CAR-T response, with some cytokines known to be from the myeloid lineage. We show that CRP is elevated in the low ALC/AMC group with increased cytokine production by monocytes. In addition, presence of T cell subset and IL-17 producing cells, before LD, are associated with clinical response. Further investigation on optimizing host immunity may help improve clinical outcome with CAR-T.[Table: see text]