Signal transducer and activator of transcription 5 (STAT5) plays an important role in regulating gene expression in response to cytokines of the common (γc) chain family. In this capacity, STAT5 promotes CD8+ effector and memory T cell survival and regulatory T cell development. However, its function in conventional CD4+ T cells is less clear. In this study, the requirement of intact STAT5 signaling for CD4+ effector and memory T cell generation and maintenance was investigated by using DO11.10 TCR transgenic T cells that are genetically deficient in STAT5A or B, as well as by transducing DO11 T cells with a dominant-negative STAT5 to temporally block STAT5 function. We found that the presence of STAT5A or B alone was sufficient for primary CD4+ effector T cell generation, but not for establishing a long-lived memory cell population. Similarly, blocking STAT5 signaling during priming did not prevent initial T cell activation, but inhibited the generation of memory cells. Surprisingly, blocking STAT5 post-priming did not impact the long-term survival of CD4+ memory T cells in vivo. Mechanistically, intact STAT5B, but not STAT5A, was required for IL-7Rα re-expression in activated T cells, which is an important cytokine receptor for CD4+ memory generation. These data show that fully functional STAT5 is essential to deliver an early, non-redundant signal for memory programming during the primary CD4+ T cell response, while partial STAT5 signaling is sufficient for effector differentiation. Our results have implications for the precise use of STAT5 inhibitors to timely inhibit memory T cell responses.

Abstract Engineered T cell therapies have been remarkably successful in the treatment of B cell malignancies, yet lack of control over these “living drugs” can lead to significant toxicities or limited efficacy. One particular challenge is the achievement of durable anti-tumor responses because the reduction of tumor burden results in reduced antigen stimulation and therefore reduced antigen-dependent T cell expansion. Interleukin-15 (IL15) drives T and NK cell expansion and persistence in an antigen-independent manner, however unregulated expression of this cytokine may compromise the safety and efficacy of cellular immunotherapy. To address these issues, we engineered T cells with a pharmacologically controllable, membrane-bound IL15 that supports antigen-independent T cell expansion and has the potential to reduce the safety risks associated with continuous exposure to soluble IL15. Our approach utilizes Drug Responsive Domains (DRDs) which are fully human protein domains that are inherently unstable in the cell but are reversibly stabilized when bound to specific FDA-approved drugs. Fusion of a DRD to a protein of interest confers drug-dependent, reversible regulation of protein expression and function. We developed a DRD based on the carbonic anhydrase 2 (CA2) protein, which is stabilized in the presence of the FDA-approved drug acetazolamide (ACZ). Upon gene transfer of membrane-bound IL15 fused to a CA2 DRD, regulated IL15 expression on T cells was detected only in the presence of ACZ. In the absence of ACZ, the level of IL15 detected on the surface of gene-modified T cells is not substantially different from the level detected on untransduced T cells. ACZ treatment of gene-modified T cells increases surface IL15 expression in a dose-dependent manner. We observed prolonged survival and up to 15-fold expansion of IL15-CA2 DRD-transduced T cells in the absence of supplemental cytokines or antigen stimulation. In contrast, vehicle-treated IL15-CA2 DRD modified T cells and untransduced T cells did not survive or expand in vitro. Importantly, both IL15-CA2-transduced T cells and unengineered, co-infused NK cells survived and persisted significantly more in vivo in ACZ-treated but not vehicle-treated non-tumor-bearing NSG mice. Our results demonstrate that a novel membrane-bound IL15-CA2 fusion protein coupled with ACZ treatment induces antigen-independent T cell expansion and augments bystander NK cell persistence. Regulatable IL15 expression has significant implications for both T and NK cell therapies by providing more durable cell expansion and cytolytic activity in vivo, and thus the potential to significantly reduce cell dosing while maintaining clinical efficacy in patients. Citation Format: Steven Shamah, Kutlu Elpek, Tucker Ezell, Michelle Fleury, Michael Gallo, Jennifer Gori, Scott Heller, Mara Inniss, Meghan Langley, Grace Olinger, Celeste Richardson, Karen Tran, Dhruv Sethi, Dexue Sun, Vipin Suri. Drug responsive domain regulation of IL15-engineered T cells provides pharmacological control over antigen-independent cell expansion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6604.

Adoptive cell therapy with chimeric antigen receptor (CAR) modified T cells has demonstrated remarkable clinical efficacy in the treatment of certain B cell malignancies, and more recently in multiple myeloma. However, CAR-T therapy has been less successful in treating solid tumors due to multiple obstacles, including the lack of robust CAR-T cell expansion, the immunosuppressive tumor microenvironment, and tumor escape due to the loss of targeted antigen. Engineering CAR-T cells to produce immunomodulatory factors such as Interleukin 12 (IL12) and Cluster of Differentiation 40 Ligand (CD40L) has been shown to enhance functional activity by driving T cell expansion, conferring resistance to immunosuppression, improving antigen presentation, and inducing antigen spread. However, the clinical utility of both IL12 and activators of the CD40 signaling pathway have been limited by systemic toxicity associated with their potent pharmacological activities. Providing precise tuning of the timing and level of expression of these immunomodulatory factors in CAR-T cells could significantly enhance safety and therapeutic efficacy, in particular against solid tumor malignancies. We describe here the implementation of ligand-controlled regulation of IL12 and CD40L in vitro and in vivo in engineered primary human T cells via the use of destabilizing domain (DD) technology. DDs are small protein domains that are misfolded and inherently unstable in the cell, but which can be reversibly stabilized by the binding of approved pharmacologic agents. This conditional stability of DDs can be readily conferred to any protein of interest by fusing it to the DD, thus providing fine-tuned, exogenous regulation of protein expression and function. We have identified mutations in several human protein substrates, including phosphodiesterase 5 (PDE5), dihydrofolate reductase (DHFR), and estrogen receptor (ER), which convey DD activity and can be regulated by FDA-approved drugs. We show that transduction of human T cells with either DD-IL12 or DD-CD40L fusion constructs yields low expression levels in the basal state and a rapid, dose-dependent induction of IL12 or CD40L protein in the presence of the corresponding stabilizing ligand. Moreover, kinetically precise, on-demand production of either factor from CAR-T cells can be achieved in mice by oral dosing of stabilizing drugs. A CD19 CAR-T Nalm6 mouse model has been established that measures potent CAR-T expansion and enhanced anti-tumor efficacy with armored immunomodulatory constructs. Studies are underway in this model, as well as in solid tumor models, to test for enhanced CAR-T activity via drug-induced activation of IL12 and CD40L towards the development of next generation cell therapies with more favorable efficacy and safety profiles. Citation Format: Michelle Ols, Michael Schebesta, Emily Brideau, Kutlu Elpek, Michelle Fleury, Jennifer Gori, Scott Heller, Dan Jun Li, Benjamin Primack, Christopher Reardon, Dhruv Sethi, Alex Storer, Dexue Sun, Karen Tran, Elizabeth Weisman, Michael Briskin, Celeste Richardson, Vipin Suri, Steven Shamah. CAR-Ts armored with small molecule-regulated IL12 or CD40L cassettes for enhanced activity against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-013.

OBJECTIVE:Immune dysfunction is an important component of the disease process underlying systemic sclerosis (SSc), but the mechanisms contributing to altered immune cell function in SSc remain poorly defined. This study was undertaken to measure the expression and function of the coinhibitory receptors (co-IRs) programmed cell death 1 (PD-1), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin and mucin domain 3 (TIM-3), and lymphocyte activation gene 3 (LAG-3) in lymphocyte subsets from the peripheral blood of patients with SSc.METHODS:Co-IR expression levels on subsets of immune cells were analyzed using a 16-color flow cytometry panel. The functional role of co-IRs was determined by measuring cytokine production after in vitro stimulation of SSc and healthy control peripheral blood mononuclear cells (PBMCs) in the presence of co-IR-blocking antibodies. Supernatants from cultures of stimulated PBMCs were added to SSc fibroblasts, and their impact on fibroblast gene expression was measured. Mathematical modeling was used to reveal differences between co-IR functions in SSc patients and healthy controls.RESULTS:Levels of the co-IRs PD-1 and TIGIT were increased, and each was coexpressed, in distinct T cell subsets from SSc patients compared to healthy controls. Levels of TIM-3 were increased in SSc natural killer cells. PD-1, TIGIT, and TIM-3 antibody blockade revealed patient-specific roles of each of these co-IRs in modulating activation-induced T cell cytokine production. In contrast to healthy subjects, blockade of TIGIT and TIM-3, but not PD-1, failed to reverse inhibited cytokine production in SSc patients, indicating that enhanced T cell exhaustion is present in SSc. Finally, cytokines secreted in anti-TIM-3-treated PBMC cultures distinctly changed the gene expression profile in SSc fibroblasts.CONCLUSION:The altered expression and regulatory capacity of co-IRs in SSc lymphocytes may contribute to disease pathophysiology by modulating the cytokine-mediated cross-talk of immune cells and fibroblasts at sites of inflammation and/or fibrosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder associated with aberrant fibrosis of the skin and internal organs that can lead to organ failure and death in patients. With few treatment options many patients succumb to fibrosis of the lungs or kidneys, or due to vascular damage culminating in pulmonary disease; the 50 percent survival rate is only about 10 years. We collected PBMCs from 36 SSc patients that were not on immune-suppressants, along with 25 age-matched healthy controls. Using a 15 color flow cytometry panel our lab has observed increases in the inhibitory receptors PD-1, TIGIT, and Tim-3 on PBMCs from SSc patients. This increase in expression is cell type specific with PD-1 and TIGIT shown to be increased on defined T cell types including CD4+ T cells, both Treg and non-Treg subsets, as well as CD8+ T cells. Tim-3 was only statistically significantly increased on a mature subset of CD16+ CD56med natural killer cells. This increase was not seen for all inhibitory receptors. The expression of LAG-3 remained low across all cell types observed. This increase in inhibitory receptor expression was enhanced in patients with a high percent of Tregs within the CD4+ gate. In vitro blockade of these inhibitory receptors provided differential cytokine secretion in patients versus healthy subjects, suggesting a possible role in disease pathogenesis. The observation of increased inhibitory receptors in SSc as well as the ability to modulate cytokines by blocking the interaction of these receptors with their ligands could provide insights into possible immune modulation in patients.

Inhibitory Receptors (IRs) such as PD-1, TIM-3, CD160, and TIGIT are upregulated during activated, exhausted, and pathological immune cell states. How the combinational expression of multiple IRs on individual cells tracks with functional status and/or fate is largely unknown. Due to paucity of human samples, large panels for immune cell characterization are of a high value, and combining them with automated platforms of multi-dimensional single cell analysis would allow objective and comprehensive population characterization. We report an 18-parameter (16-color) fluorescent panel that allows IR signature analysis as well as cell sorting for functional studies, and adopt algorithmic methods previously used for mass cytometry into a novel platform to analyze fluorescent datasets in an unbiased analytical workflow. PBMC from diseased and healthy control subjects as well as tumor specimens were stained and analyzed on a FACSARIA cell sorter. For the automated analysis, the datasets were processed with t-SNE algorithm into a 2D map of phenotypic space that was subsequently clustered using SPADE. From the samples, a 32-plex combinational IR signature was determined for the CD4+ T, CD8+ T, NK, iNKT, and γδ T cell lineages. The results indicate novel combinational IR ‘hits’ for particular immune subsets and some immune subset IR signatures correlated with disease status. In conclusion, 18-parameter flow cytometry with automated analysis provides robust results from human blood and solid tissue samples. In combination with functional and other readouts, measurement of single immune cell IR signatures could provide new insight into activation, exhaustion, and pathogenic processes and potentially reveal novel therapeutic targets.

Type 1 diabetes is an autoimmune disease caused by T cell-mediated destruction of the insulin-producing β-cells in the pancreas. Therefore, approaches that effectively halt the pathogenic T cell response are predicted to have preventive or therapeutic benefit for type 1 diabetes patients. We previously demonstrated that long-term blocking of IL-7 signaling, which is critical for the survival and function of T cells, prevented and reversed type 1 diabetes in non-obese diabetic mice. However, such persistent inhibition of T cell responses raises concerns about causing immunodeficiency. Here, we asked whether a reduced duration of the treatment with anti-IL-7Rα antibodies retained efficacy in preventing diabetes. Moreover, we sought to identify immunoregulatory mechanisms induced by anti-IL-7Rα administration.

c‐Myb is a transcription factor and proto‐oncogene that is important for hematopoietic cell differentiation. The regulation of the c‐Myb protein and its down stream targets is well studied while the regulation of expression of the c‐myb gene is not well understood. c‐myb expression is controlled by a conditional block in transcriptional elongation in the first intron of the gene. Previous studies have shown that NF‐kB family members are involved in regulating the activation of c‐myb by binding to Rel‐Related Proteins Binding Elements (RRBE) in the first intron. This leads to the hypothesis that NF‐kB regulates expression of c‐myb during development of hematopoietic cells. Here we show using chromatin immunoprecipitations (ChIPs) and real‐time PCR in murine erythroleukemia cells that the down regulation of c‐myb expression via hexamethylene bisacetamide‐induced differentiation can be over‐come by activation of adenyl cyclase by forskolin. ChIP results demonstrate a correlation between occupancy of the RRBE sites by the p65 subunit of NF‐kB with elevated c‐myb mRNA levels. This study demonstrates for the first time a correlation between NF‐kB occupancy of RRBE sites and c‐myb expression in vivo in hematopoietic cells. Research support provided by a Providence College Committee on Aid to Faculty Research Grant.