In the autoimmune disease multiple sclerosis (MS) and its animal model Experimental Autoimmune Encephalomyelitis (EAE), expansion of pathogenic, myelin-specific Th1 cell populations drives active disease; selectively targeting this process may be the basis for a new therapeutic approach. Previous studies have hinted a role for protein arginine methylation in immune responses, including T cell-mediated autoimmunity and EAE. However, a conclusive role for the Protein Arginine Methyl Transferase (PRMT) enzymes that catalyze these reactions has been lacking. PRMT5 is the main PRMT responsible for symmetric dimethylation of arginine residues of histones and other proteins. PRMT5 drives embryonic development and cancer, but its role in T cells, if any, has not been investigated. Here, we show that PRMT5 is an important modulator of CD4+ T cell expansion. PRMT5 was transiently up-regulated during maximal proliferation of both mouse and human memory Th cells. PRMT5 expression was regulated upstream by the NF-κB pathway, and it promoted IL-2 production and proliferation. Blocking PRMT5 with novel, highly selective small molecule PRMT5 inhibitors severely blunted memory Th expansion, with preferential suppression of Th1 over Th2 cells. In vivo, PRMT5 blockade efficiently suppressed recall T cell responses and reduced inflammation in Delayed Type Hypersensitivity (DTH) and clinical disease in Experimental Autoimmune Encephalomyelitis (EAE) mouse models. These data implicate PRMT5 in regulation of adaptive memory T helper cell responses and suggest PRMT5 inhibitors may be a novel therapeutic approach for T cell-mediated inflammatory disease.

Background While combinations of immune checkpoint (ICP) inhibitors and neo-adjuvant chemotherapy (NAC) have begun testing in patients with breast cancer (BC), the effects of chemotherapy on ICP expression in circulating T cells and within the tumor microenvironment are still unclear. This information could help with the design of future clinical trials by permitting the selection of the most appropriate ICP inhibitors for incorporation into NAC. Methods Peripheral blood samples and/or tumor specimens before and after NAC were obtained from 24 women with operable BC. The expression of CTLA4, PD-1, Lag3, OX40, and Tim3 on circulating T lymphocytes before and at the end of NAC were measured using flow cytometry. Furthermore, using multi-color immunohistochemistry (IHC), the expression of immune checkpoint molecules by stromal tumor-infiltrating lymphocytes (TILs), CD8+ T cells, and tumor cells was determined before and after NAC. Differences in the percentage of CD4+ and CD8+ T cells expressing various checkpoint receptors were determined by a paired Student’s t-test. Results This analysis showed decreased ICP expression by circulating CD4+ T cells after NAC, including significant decreases in CTLA4, Lag3, OX40, and PD-1 (all p values < 0.01). In comparison, circulating CD8+ T cells showed a significant increase in CTLA4, Lag3, and OX40 (all p values < 0.01). Within tumor samples, TILs, CD8+ T cells, and PD-L1/PD-1 expression decreased after NAC. Additionally, fewer tumor specimens were considered to be PD-L1/PD-1 positive post-NAC as compared to pre-NAC biopsy samples using a cutoff of 1% expression. Conclusions This work revealed that NAC treatment can substantially downregulate CD4+ and upregulate CD8+ T cell ICP expression as well as deplete the amount of TILs and CD8+ T cells found in breast tumor samples. These findings provide a starting point to study the biological significance of these changes in BC patients. Trial registration NCT04022616.

IntroductionMyeloid-derived suppressor cells (MDSC) are a subset of immature myeloid cells that inhibit anti-tumor immunity and contribute to immune therapy resistance. MDSC populations were measured in melanoma patients receiving immune checkpoint inhibitors (ICI).MethodsPatients with melanoma (n=128) provided blood samples at baseline (BL), and before cycles 2 and 3 (BC2, BC3). Peripheral blood mononuclear cells (PBMC) were analyzed for MDSC (CD33+/CD11b+/HLA- DRlo/-) and MDSC subsets, monocytic (CD14+, M-MDSC), granulocytic (CD15+, PMN-MDSC), and early (CD14-/CD15-, E-MDSC) via flow cytometry. Statistical analysis employed unpaired and paired t-tests across and within patient cohorts.ResultsLevels of MDSC as a percentage of PBMC increased during ICI (BL: 9.2 ± 1.0% to BC3: 23.6 ± 1.9%, p&lt;0.0001), and patients who developed progressive disease (PD) had higher baseline MDSC. In patients who had a complete or partial response (CR, PR), total MDSC levels rose dramatically and plateaued (BL: 6.4 ± 1.4%, BC2: 26.2 ± 4.2%, BC3: 27.5 ± 4.4%; p&lt;0.0001), whereas MDSC rose less sharply in PD patients (BL: 11.7 ± 2.1%, BC2: 18.3 ± 3.1%, BC3: 19.0 ± 3.2%; p=0.1952). Subset analysis showed that within the expanding MDSC population, PMN-MDSC and E-MDSC levels decreased, while the proportion of M-MDSC remained constant during ICI. In PD patients, the proportion of PMN-MDSC (as a percentage of total MDSC) decreased (BL: 25.1 ± 4.7%, BC2: 16.1 ± 5.2%, BC3: 8.6 ± 1.8%; p=0.0105), whereas a heretofore under-characterized CD14+/CD15+ double positive MDSC subpopulation increased significantly (BL: 8.7 ± 1.4% to BC3: 26.9 ± 4.9%; p=0.0425).ConclusionsMDSC levels initially increased significantly in responders. PMN-MDSC decreased and CD14+CD15+ MDSC increased significantly in PD patients. Changes in MDSC levels may have prognostic value in ICI.

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Purpose: Treatment options are limited in patients with metastatic neuroendocrine neoplasms (NENs). We present the results for a phase 2 trial of combination nivolumab and temozolomide in patients with advanced NEN along with results of immune changes in peripheral blood. Experimental Design: NCT03728361 is a nonrandomized, phase 2 study of nivolumab and temozolomide in patients with NEN. The primary endpoint was response rate using RECIST 1.1. Secondary endpoints included progression free survival (PFS), overall survival (OS), and safety. Immune profiling was performed by mass cytometry to evaluate the effect on peripheral blood immune cell subsets. Results: Among all 28 patients with NEN, the confirmed response rate was 9/28 (32.1%, 95% CI: 15.9%-52.4%). Of 11 patients with lung NEN, the response rate was 64% (n=7); there was a significant difference in responses by primary tumor location (lung vs others, p=0.020). The median PFS was 8.8 months (95% CI: 3.9 – 11.1 months), and median OS was 32.3 months (95% CI: 20.7 – NR months). Exploratory blood immune cell profiling revealed an increase in circulating CD8+ T cells (27.9±13.4% vs. 31.7±14.6%, p = 0.03) and decrease in CD4+ T cells (59.6±13.1% vs. 56.5±13.0%, p = 0.001) after 2 weeks of treatment. LAG-3 expressing total T cells were lower in patients experiencing a partial response (0.18±0.24% vs 0.83±0.55%, p = 0.028). MDSC levels increased during study and did not correlate with response. Conclusions: Combination nivolumab and temozolomide demonstrated promising activity in NEN.

3111 Background: Myeloid-derived suppressor cells (MDSC) are expanded in cancer and promote immune suppression. We have shown that ibrutinib inhibits migration and immunosuppressive function of MDSC. Moreover, the combination of ibrutinib and a PD-L1 inhibitor has been found to have synergistic anti-tumor effects in a multiple solid tumor mouse models. Therefore, we conducted a pilot study testing the combination of ibrutinib and nivolumab in patients with metastatic solid tumors. Methods: Sixteen patients with advanced solid tumors were recruited to this trial. Ibrutinib was dosed as an oral single agent, starting 7 days prior to cycle 1 of nivolumab and given until cycle 1, day 8 of nivolumab. Nivolumab was administered intravenously on days 1 and 15 on 28-day cycles. Patients had blood samples collected prior to initiation of ibrutinib, day 1 of cycle 1, day 8 of cycle 1, day 1 of cycle 2, and at the time of disease progression. From these specimens, we measured circulating MDSC levels, other circulating immune subsets, T cell proliferation, and cytokines/chemokines levels. Circulating MDSC levels were measured by mass spectrometry. T cell function was evaluated by CFSE to monitor proliferating cells by dye dilution and cytokine/chemokine levels were measured with a U-PLEX assay. Data were analyzed using two-tailed, paired Student's t-tests to assess statistical significance. Results: An increase in circulating MDSC (22% to 28%; SD 9.158) levels was observed following 7 days of single-agent ibrutinib compared to baseline. However, in combination therapy, MDSC levels decreased (19%; SD 13.17) prior to cycle 2. Despite increasing levels of circulating MDSC, T cell function improved throughout the study. Furthermore, plasma levels of chemokines associated with MDSC recruitment and migration significantly decreased with ibrutinib treatment (IL-12, CCL2, CCL3, and CCL4). Of the 16 patients, four achieved a partial response and four achieved stable disease. Median progression free survival was 3.5 months and median overall survival was 11.5 months. Conclusions: The combination of ibrutinib and nivolumab was well tolerated, demonstrated early signs of immune modulation, and showed preliminary signs of promising clinical activity in patients with metastatic solid tumors. Clinical trial information: NCT03525925 .

BackgroundMyeloid-derived suppressor cells (MDSC) are immature immune cells that suppress immunity and mediate resistance to immune–based cancer therapies. MDSC exert their immunosuppressive effects partly through the production of reactive nitrogen and oxygen species, which combine to form peroxynitrite (PNT). PNT reacts with the tyrosine residues of key immune cell signaling proteins and inactivates them via nitration. Targeting MDSC via PNT inhibitors is an attractive avenue to improve the response to immunotherapy. The Peterson and Carson Labs have collaborated to develop a novel inhibitor of PNT and have explored its use in murine tumor models and human patients with cancer.MethodsSplenocytes (comprised of 12% MDSC) were isolated from mice bearing tumors derived from the EMT6 breast cancer cell line and cultured with 10 µm beads labelled with polyclonal antibodies (immunoglobulin-G or IgG). Fluorescence emitted upon MDSC recognition and reaction with IgG was detected with a previously reported fluorescent sensor compound termed PS3. Cells were mixed with PS3 and IgG beads (or controls: IgG without beads and beads without IgG) and treated for 4 hours with the following agents: (1) BRP0112233, a novel biaryl furan discovered via high-throughput screening using PNT depletion as the readout (6 or 12 µM); (2) Ibrutinib, an FDA-approved Bruton's tyrosine kinase inhibitor shown by the Carson Lab to inhibit the activity of nitric oxide synthase in MDSC, (2, 10 µM); and (3) PBS control. Fluorescence produced by reaction of PS3 with PNT was measured in triplicate wells using a Clariostar plate reader.ResultsSplenocytes from tumor-bearing mice produced significantly greater levels of PNT than normal splenocytes (24-fold vs 8-fold increase over plain beads, p<0.0001). Differences in fluorescence were confirmed via confocal microscopy. BRP0112233 inhibited PNT levels by 40% and 85% for the 6 and 12 µM doses, respectively. Ibrutinib inhibited PNT output by 90% and 100% at 2 and 10 µM. Cell viability was >90% except for the higher BRP dose (60% viability). In humans, peripheral blood mononuclear cells (PBMC) isolated from patients with cancer produced more PNT than healthy donor PBMC.ConclusionsPNT output could be reproducibly quantified via this assay and BRP0112233 and ibrutinib greatly inhibited MDSC PNT production. Using the EMT6 model, these compounds are being tested in combination with anti-PD-1 antibodies approved for patients with cancer. This assay has shown similar results in human peripheral blood mononuclear cells isolated from patients with cancer.AcknowledgementsWe thank the NIH (NCI UM1 CA186712, R01CA211720), a OSUCCC Translational Therapeutics Seed Grant, and the Pelotonia Fellowship Program for financial support.

Inhibition of methylation reactions suppresses T cell proliferation and inflammatory responses but the specific methylation enzyme target responsible for these effects is unknown. Protein Arginine Methyltranferases (PRMT), enzymes that catalyze dimethylation of arginine in histones and other proteins, have been proposed to mediate these effects. Among PRMTs, PRMT5 symmetrically dimethylates protein arginine and is associated with embryonic development and cancer but has no known role in T cells. Here, we used myelin-specific memory Th cells that model autoimmune responses in Multiple Sclerosis to investigate the role of PRMT5 in memory Th1 cell responses. PRMT5 was up-regulated as Th1 cells undergoing activation, with kinetics that closely matched the cycle of T cell proliferation. PRMT5 protein up-regulation correlated with loss of miR-96, an inhibitor of PRMT5 translation. Further, a highly selective, small molecule PRMT5 inhibitor blunted memory T cell proliferation, an effect that could be partially rescued by IL-2. These data implicate PRMT5 in regulation of adaptive memory T helper cell responses and open novel therapeutic opportunities for T cell-mediated autoimmune disease.