Reduction Of Tregs Research Articles

Atovaquone Suppresses Triple-Negative Breast Tumor Growth by Reducing Immune-Suppressive Cells.

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-β and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-β, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-β and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.

Early Life Imprinting of a Th2-Stromal Cell Niche in Skin

Abstract Tissue inflammation early in life can be imprinted on the immune system, causing lasting changes in immunologic tone that confer disease protection or susceptibility in adults. The cellular and molecular mechanisms responsible for immune imprinting in many nonlymphoid tissues remain largely unknown. We find that time-limited neonatal inflammation induced by transient reduction of regulatory T cells (Tregs) causes a dramatic dysregulation of skin stromal cells, accompanied by the selective accumulation of Th2 cells within a distinct microanatomic tissue niche. Th2 cells are maintained into adulthood through interactions with a previously uncharacterized stromal population in skin fascia that we refer to as Th2-interacting fascial fibroblasts (TIFFs), which expand following Treg reduction, respond to Th2 cytokines, and produce IL-33. Formation of the Th2-TIFF niche imprints skin with increased reparative capacity after wounding. Taken together, these data define a novel Th2 niche in skin and suggest a mechanism of immunologic imprinting whereby inflammation early in life creates networks between adaptive immune cells and parenchymal cells, establishing an immunological set-point in tissues that is maintained throughout life.

CHOLECALCIFEROL SUPPLEMENTATION AMELIORATES IMMUNE DYSFUNCTION IN OBESE HYPERTENSIVE PATIENTS

Objective: Reduction of T regulatory cells (Treg) and upregulation of T helper 17 cells (Th17) contributes to obesity-associated hypertension. Since vitamin D is a modulator of the immune system, we hypothesized that supplementation with cholecalciferol in hypertensive patients may lead to a shift to a more anti-inflammatory immune status. Design and method: a 24-week randomized, open, parallel groups clinical trial including 36 patients with hypertension and overweight or obesity was conducted to evaluate the effects of cholecalciferol in peripheral Treg and Th17 cells. Patients were randomized to receive cholecalciferol according serum vitamin D (VD) levels plus usual therapy or usual therapy alone. The levels of Treg and Th17 on week 0 and 24 were obtained as well as body mass index (BMI), waist circumference, serum free VD, lipids, HbA1c, T helper 1 (Th1) cells and monocytes. Results: 18 men, average age 57.1 ± 5.5 years, BMI 34.5 ± 5.1 Kg/m2 and waist circumference 113.8 ± 10.7 cm. Sixteen patients (44.4%) had grade 1 obesity, seven (19.4%) grade 2 and six (1.6%) grade 3 obesity. The remaining had 7 had 25 < BMI < 30 Kg/m2. All patients were on antihypertensive drugs and mean value of vitamin D at the beginning of the trial was 15.7 ± 5.1 ng/mL. After supplementation serum VD levels rose to 26.9 ± 5.6 ng/mL (p = 0.000) and serum free VD levels from 5.2 ± 1.2 to 6.2 ± 3.3 ng/mL (p = 0.044). At 24 weeks the average ± SD values of the different cells between active and control groups were: Tregs 6.1 ± 1.5 and 6.8 ± 1.8; p = 0.226, respectively, Th17 cells 1.8 ± 0.8 and 2.7 ± 1.2; p = 0.017, Th1 cells 35.4 ± 18.1 and 30.3 ± 9.2; p = 0.299 and monocytes 5.7 ± 1.3 and 6.4 ± 2; p = 0.215. Conclusions: in overweight and obese hypertensive patients supplementation with cholecalciferol at regular doses reduces the levels of Th17 cells. Although statistically non-significant a decrease in monocytes was also observed. The reduction of Treg was unexpected, the significance of which should be clarified in future studies.

IFNα Augments Clinical Efficacy of Regulatory T-cell Depletion with Denileukin Diftitox in Ovarian Cancer

AbstractPurpose:Immunotherapy treats some cancers, but not ovarian cancer. Regulatory T cells (Tregs) impede anti-ovarian cancer immunity but effective human Treg-directed treatments are lacking. We tested Treg depletion with denileukin diftitox (DD) ± IFNα as ovarian cancer immunotherapy.Patients and Methods:Mice with syngeneic ID8 ovarian cancer challenge were treated with DD, IFNα, or both. The phase 0/I trial tested one dose-escalated DD infusion for functional Treg reduction, safety, and tolerability. The phase II trial added IFNα2a to DD if DD alone failed clinically.Results:DD depleted Tregs, and improved antitumor immunity and survival in mice. IFNα significantly improved antitumor immunity and survival with DD. IFNα did not alter Treg numbers or function but boosted tumor-specific immunity and reduced tumor Treg function with DD by inducing dendritic cell IL6. DD alone was well tolerated, depleted functional blood Tregs and improved immunity in patients with various malignancies in phase 0/I. A patient with ovarian cancer in phase 0/I experienced partial clinical response prompting a phase II ovarian cancer trial, but DD alone failed phase II. Another phase II trial added pegylated IFNα2a to failed DD, producing immunologic and clinical benefit in two of two patients before a DD shortage halt. DD alone was well tolerated. Adding IFNα increased toxicities but was tolerable, and reduced human Treg numbers in blood, and function through dendritic cell–induced IL6 in vitro.Conclusions:Treg depletion is clinically useful but unlikely alone to cure ovarian cancer. Rational treatment agent combinations can salvage clinical failure of Treg depletion alone, even when neither single agent provides meaningful clinical benefit.

A Phase 2 Randomized Placebo-Controlled Adjuvant Trial of GI-4000, a Recombinant Yeast Expressing Mutated RAS Proteins in Patients with Resected Pancreas Cancer.

Purpose: GI-4000, a series of recombinant yeast expressing four different mutated RAS proteins, was evaluated in subjects with resected ras-mutated pancreas cancer.Methods: Subjects (n = 176) received GI-4000 or placebo plus gemcitabine. Subjects' tumors were genotyped to identify which matched GI-4000 product to administer. Immune responses were measured by interferon-γ (IFNγ) ELISpot assay and by regulatory T cell (Treg) frequencies on treatment. Pretreatment plasma was retrospectively analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectrometry for proteomic signatures predictive of GI-4000 responsiveness.Results: GI-4000 was well tolerated, with comparable safety findings between treatment groups. The GI-4000 group showed a similar pattern of median recurrence-free and overall survival (OS) compared with placebo. For the prospectively defined and stratified R1 resection subgroup, there was a trend in 1 year OS (72% vs. 56%), an improvement in OS (523.5 vs. 443.5 days [hazard ratio (HR) = 1.06 [confidence interval (CI): 0.53–2.13], p = 0.872), and increased frequency of immune responders (40% vs. 8%; p = 0.062) for GI-4000 versus placebo and a 159-day improvement in OS for R1 GI-4000 immune responders versus placebo (p = 0.810). For R0 resection subjects, no increases in IFNγ responses in GI-4000–treated subjects were observed. A higher frequency of R0/R1 subjects with a reduction in Tregs (CD4+/CD45RA+/Foxp3low) was observed in GI-4000–treated subjects versus placebo (p = 0.033). A proteomic signature was identified that predicted response to GI-4000/gemcitabine regardless of resection status.Conclusion: These results justify continued investigation of GI-4000 in studies stratified for likely responders or in combination with immune check-point inhibitors or other immunomodulators, which may provide optimal reactivation of antitumor immunity.ClinicalTrials.gov Number: NCT00300950.

Tumour angiogenesis normalized by myo-inositol trispyrophosphate alleviates hypoxia in the microenvironment and promotes antitumor immune response.

Pathologic angiogenesis directly responds to tumour hypoxia and controls the molecular/cellular composition of the tumour microenvironment, increasing both immune tolerance and stromal cooperation with tumour growth. Myo‐inositol‐trispyrophosphate (ITPP) provides a means to achieve stable normalization of angiogenesis. ITPP increases intratumour oxygen tension (pO2) and stabilizes vessel normalization through activation of endothelial Phosphatase‐and‐Tensin‐homologue (PTEN). Here, we show that the tumour reduction due to the ITPP‐induced modification of the tumour microenvironment by elevating pO2 affects the phenotype and properties of the immune infiltrate. Our main observations are as follows: a relative change in the M1 and M2 macrophage‐type proportions, increased proportions of NK and CD8+T cells, and a reduction in Tregs and Th2 cells. We also found, in vivo and in vitro, that the impaired access of PD1+NK cells to tumour cells is due to their adhesion to PD‐L1+/PD‐L2+ endothelial cells in hypoxia. ITPP treatment strongly reduced PD‐L1/PD‐L2 expression on CD45+/CD31+ cells, and PD1+ cells were more numerous in the tumour mass. CTLA‐4+ cell numbers were stable, but level of expression decreased. Similarly, CD47+ cells and expression were reduced. Consequently, angiogenesis normalization induced by ITPP is the mean to revert immunosuppression into an antitumor immune response. This brings a key adjuvant effect to improve the efficacy of chemo/radio/immunotherapeutic strategies for cancer treatment.

T Regulatory Cells in Relapsed/Refractory Chronic Lymphocytic Leukemia Treated with High Dose Methylprednisolone and Rituximab

Higher circulating T regulatory cell (Treg) numbers have been found in untreated patients with chronic lymphocytic leukemia (CLL) compared to healthy subjects and correlated with progressive disease as well as time to first treatment in low-risk patients [1]. Some agents can reduce Treg numbers in CLL patients, but there are no data on the prognostic role of Treg dynamics and patient outcome. We present data from the LT-CLL-001 study, in which the clinical benefit of dose-dense high dose methylprednisolone (HDMP) and rituximab (Rtx) combination in relapsed or refractory high-risk patients with CLL was evaluated [2]. During the study, the change of T regulatory cell frequencies was measured in relation to overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Twenty-nine CLL patients with clinically or biologically high-risk disease were included. Treg frequency was evaluated at screening, after three treatment courses, and at the end of therapy. Significant reduction of the median frequencies of Treg during treatment was observed: median (range) of Treg0-3 after three treatment courses was 2.14% (-1.84%- 9.42%), p < 0.001 and median (range) of Treg0-6 was 1.01% (-2.95%- 8.35%, p = 0.004). Patients with deeper Treg reduction between screening and three treatment courses had significantly better PFS and OS (Table 1 & 2). Our data for the first time show that HDMP and Rtx combination reduces Treg frequency in pretreated CLL patients. Early and deeper Treg reduction is an independent prognostic factor for longer PFS and OS. (ClinicalTrials.gov identifier: NCT005 58181).

IMMU-10. INTERIM ANALYSIS OF THE HIT-HGG REZ IMMUNVAC STUDY - DENDRITIC CELL VACCINATION WITH PARTIAL TREG DEPLETION IN CHILDREN, ADOLESCENTS, AND ADULTS WITH RELAPSED HIGH-GRADE GLIOMAS

Efficacy of therapeutic dendritic cell vaccines (DCV) can be limited by immunosuppressive mechanisms in the micromilieu of high-grade gliomas. In the HIT-HGG-Rez Immunovac trial (Eudra-CT 2013-000419-26), we investigate whether a reduction of Treg with metronomic cyclophosphamide (metrCyc) might be a feasible option to improve vaccine efficacy. 10 pediatric (mean age 11.4±4.2y) and 5 adult patients (mean age 39.5±19.9y) with relapsed glioblastoma were treated according to the HIT-HGG-Rez Immunovac protocol so far. 2 children were treated within the trial, the other 13 in the pilot phase. Patients received upfront oral metrCyc for 2–4 weeks. After reoperation and monocyte-apheresis, patients received 4 weekly intradermal doses of autologous, TNFa/IL-1ß matured DCs pulsed with tumor lysate in imiquimod-prepared skin. Thereafter, tumor lysate boosts were given. All patients received at least 5 vaccines (4xDCs, 1xlysate boosts). MetrCyc was well tolerated and led to a reduction in Treg-frequency of 35.6±17.8% followed by a rebound after cessation of metrCyc. Importantly, 13/14 analyzed patients showed a positive IFNg-T-cell response against autologous tumor lysate with a tendency to decrease over time. 6-month overall survival was 100%, compared to 65% in a historical control. Mean PFS and OS were 5.7 and 21.1 months with no difference between adults and children. We conclude that DCV in combination with partial Treg depletion is feasible, safe, and related with a high rate of tumor-specific IFNg-responses. As the clinically and immunologically beneficial effects seem to diminish over time, we aim to combine our approach with checkpoint inhibition in the next amendment.

IMMU-50. BBB CROSSING NANO-IMMUNOMEDICINE COMBINATION THERAPY TO TREAT BRAIN PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA

Abstract INTRODUCTION Lack of standard treatment of Primary Central Nervous System Lymphoma (PCNSL) was acknowledged in phase III intergroup study (HOVON 105/ALLG NHL 24). PCNSL incidence is increasing for unknown reasons, particularly among persons over 65 years. One of the major limitations of successful treatment of PCNSL is the blood-brain barrier (BBB), which prevents drug delivery into the brain. METHODS Polymer nanoconjugate based on biodegradable poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA) was used for treatment of BALB/C mice with intracranially inoculated murine A20 lymphoma. PHPMA-based nanoconjugates have defined synthesis strategy for BBB transcytosis, tumor targeting, and cancer cell killing. Nano immunodrug PHPMA/AP-2/Fab’/c-Myc inhibitor/H6 conjugate contains multiple AP-2 peptides for efficient delivery across the BBB and Fab’ fragments of αCD20 mouse Ab for CD20 receptor crosslinking on PCNSL cells. c-Myc inhibitor bound via a disulfide bond is transferred to the cytoplasm followed by cleavage. The therapeutic efficacy and survival were evaluated together with spectral flow cytometry and RNA-seq bioinformatic analysis of tumor tissues. RESULTS Nanoconjugates were able to penetrate BBB and accumulate in brain parenchyma. The best survival results were obtained for the group where nanoconjugate with αCD20 Fab’ causing tumor cell apoptosis and c-Myc antisense inhibitor was combined with αPD-1 checkpoint inhibitor. Survival compared to PBS-treated controls was significant (P=0.0006 by ANOVA). Ex vivo analysis of A20 brain lymphoma tissue after treatment with nanoconjugates demonstrated reduction of Tregs, associated with downregulation of IL-10 and IL-1β in plasma. Spectral flow cytometry revealed activation of tumor infiltrating T lymphocytes and M1 macrophages in lead nanodrug treated groups. RNA-seq data correlated with flow cytometry results indicating the activation of genes linked to IFN-γ pathway important for anti-tumor response and M1 macrophage activation. Immunostaining confirmed a marked increase of M1-specific iNOS expression in the nanodrug-treated tumors. Support: NIH grants: R01 CA188743, R01 CA206220, R01 CA209921

A Cross-Species Reactive TIGIT-Blocking Antibody Fc Dependently Confers Potent Antitumor Effects.

The T cell immunoreceptor with Ig and ITIM domains (TIGIT) has been shown to exert inhibitory roles in antitumor immune responses. In this study, we report the development of a human mAb, T4, which recognizes both human and mouse TIGIT and blocks the interaction of TIGIT with its ligand CD155 in both species. The T4 Ab targets the segment connecting F and G strands of TIGIT's extracellular IgV domain, and we show in studies with mouse tumor models that the T4 Ab exerts strong antitumor activity and induces durable immune memory against various tumor types. Mechanistically, we demonstrate that the T4 Ab's antitumor effects are mediated via multiple immunological impacts, including a CD8+ T immune response and Fc-mediated effector functions, through NK cells that cause significant reduction in the frequency of intratumoral T regulatory cells (Tregs). Notably, this Treg reduction apparently activates additional antitumor CD8+ T cell responses, targeting tumor-shared Ags that are normally cryptic or suppressed by Tregs, thus conferring cross-tumor immune memory. Subsequent engineering for Fc variants of the T4 Ab with enhanced Fc-mediated effector functions yielded yet further improvements in antitumor efficacy. Thus, beyond demonstrating the T4 Ab as a promising candidate for the development of cancer immunotherapies, our study illustrates how the therapeutic efficacy of an anti-TIGIT Ab can be improved by enhancing Fc-mediated immune effector functions. Our insights about the multiple mechanisms of action of the T4 Ab and its Fc variants should help in developing new strategies that can realize the full clinical potential of anti-TIGIT Ab therapies.

Tolerance mechanisms in allergen immunotherapy.

Allergen immunotherapy is the only treatment modality which alters the natural course of allergic diseases by restoring immune tolerance against allergens. Deeper understanding of tolerance mechanisms will lead to the development of new vaccines, which target immune responses and promote tolerance. Successful allergen immunotherapy (AIT) induces allergen-specific peripheral tolerance, characterized mainly by the generation of allergen-specific Treg cells and reduction of Th2 cells. At the early phase, AIT leads to a decrease in the activity and degranulation of mast cells and basophils and a decrease in inflammatory responses of eosinophils in inflamed tissues. Treg cells show their effects by secreting inhibitory cytokines including interleukin (IL)-10, transforming growth factor-β, interfering with cellular metabolisms, suppressing antigen presenting cells and innate lymphoid cells (ILCs) and by cytolysis. AIT induces the development of regulatory B cells producing IL-10 and B cells expressing allergen-specific IgG4. Recent investigations have demonstrated that AIT is also associated with the formation of ILC2reg and DCreg cells which contribute to tolerance induction. Research done so far, has shown that multiple molecular and cellular factors are dysregulated in allergic diseases and modified by AIT. Studies should now focus on finding the best target and ideal biomarkers to identify ideal candidates for AIT.

Abstract CT045: Synergistic anti-tumor efficacy of the dual PI3K-δ/PI3K-γ inhibitor duvelisib with PD-1 blockade in solid tumor and lymphoma models

Abstract Blockade of immune checkpoints, such as PD-1, has provided meaningful clinical benefit for patients with a broad range of malignancies. However, limited response rates and resistance after initial response suggest a need for additional strategies. Duvelisib is an oral, dual PI3K-δ,γ inhibitor that is FDA-approved as monotherapy for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and follicular lymphoma (FL) after ≥2 prior systemic therapies. In addition to targeting malignant B cells, duvelisib regulates key immune cells in the tumor microenvironment, making it a promising candidate for combination with PD-1 blockade. Using T cells from human donors or CLL patients, we now show that duvelisib induced a 3 to 4-fold increase in T cell expansion ex vivo, and that dual PI3K-δ,γ inhibition was necessary for maximal effect. Furthermore, duvelisib reduced expression of T cell exhaustion markers (Tim-3, Lag-3) and enriched early memory T cell populations. We have previously shown that duvelisib is more effective than inhibitors of only PI3K-δ or PI3K-γ in reducing Tregs and immunosuppressive myeloid cells when combined with an OX40 agonist antibody in a syngeneic tumor model. Accordingly, dual PI3K-δ,γ inhibition may represent an advantage over agents that inhibit only PI3K-δ or PI3K-γ for combination with PD-1 blockade. We next investigated whether duvelisib augments the efficacy of a PD-1 antibody (anti-PD-1) in solid tumor and lymphoma syngeneic models. In mice bearing CT26 colorectal tumors, duvelisib or anti-PD-1 alone induced tumor growth inhibition (TGI) of 59 and 49%, respectively, whereas duvelisib plus anti-PD-1 resulted in enhanced TGI (81%) and survival. Sixty percent of mice treated with duvelisib plus anti-PD-1 were tumor free, whereas only 10% of the mice treated with anti-PD-1 alone were tumor free by 60 days after tumor inoculation. In the A20 B-cell lymphoma model, reductions in tumor volume were observed with duvelisib or anti-PD-1 alone (TGI = 50 and 54%, respectively). Duvelisib plus anti-PD-1 was associated with enhanced antitumor activity (TGI = 85%) and survival (median survival of 37 days for the combination group compared to 16.5 and 18 days for duvelisib or anti-PD-1 monotherapy groups, respectively). Reductions in Tregs, M2 macrophages, and M-MDSCs in tumors were observed in the duvelisib plus anti-PD-1 combination group compared to anti-PD-1 alone. Taken together, these data indicate the value of dual PI3K-δ,γ inhibition for combination with PD-1 blockade and support clinical evaluation of duvelisib with anti-PD-1 in patients with solid tumors or hematologic malignancies. Clinical studies are underway investigating the combination of duvelisib plus nivolumab in aggressive lymphomas (Richter's transformation or transformed FL; NCT03892044) and duvelisib plus pembrolizumab in head and neck squamous cell carcinoma. Citation Format: Silvia Coma, Christopher R. Funk, Shuhua Wang, Edmund K. Waller, David T. Weaver, Jonathan A. Pachter. Synergistic anti-tumor efficacy of the dual PI3K-δ/PI3K-γ inhibitor duvelisib with PD-1 blockade in solid tumor and lymphoma models [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 CT045.

Abstract 2754: Inhibition of nitric oxide synthase and cyclooxygenase potentiate immune responses that control aggressive mammary tumor in a murine model

Abstract Progression of aggressive, triple negative breast cancers (TNBC) is driven by inflammation and the presence of tumor infiltrating lymphocytes (TIL) improves survival. Recent studies show that TNBCs show high levels of TILs but these cells are exhausted as evident from decreased IFNγ, Ki67 and granzyme B. High expression of the inflammation-associated protein, inducible nitric oxide synthase (NOS2) and co-expression with high cyclooxygenase 2 (COX2) means poor prognosis in ER- breast cancer. Interestingly, NOS2 and COX2 are upregulated in different cells in the tumor microenvironment (TME). Thus, we hypothesized that the tandem use of NOS2 and COX2 inhibitors would curtail tumor progression by targeting different cell types within the TME simultaneously. As COX2 has also been reported to regulate tumor infiltration of myeloid cells, this could regulate both the lymphoid and myeloid arms of the immune system for better response. We investigated the effects of NOS2 and COX2 on tumorigenesis and pulmonary metastasis in a murine model of TNBC by injecting 4T1 cells into the fourth mammary fat pad of wild type (WT) and NOS2 knockout (NOS2-/-) BALB/c mice followed by treatment with COX inhibitor, indomethacin. Changes in infiltrating immune cells was analyzed using flow cytometry and confocal microscopy. We measured the levels of serum cytokines using bead-based, LegendPlex assay. We found that NOS2-/- mice developed fewer pulmonary metastatic lesions than WT mice while indomethacin directly reduced primary tumor growth in both WT and NOS2-/- mice. Indomethacin treated NOS2-/- mice showed better median survival (60 days compared 51 in WT mice). Two NOS2-/-, indomethacin treated mice showed complete remission and long term (5-months) tumor growth delay after 4T1 re-challenge, indicating immune activation in mice lacking NOS2 and COX2 function. Flow cytometry of primary tumors showed higher infiltration of activated CD4, CD8 cells, macrophages and reduction of Tregs in treated mice. Microscopy revealed a physical ‘walling -off' of TILs by CD11b+ cells in untreated tumors that was abrogated upon indomethacin treatment. As many TNBC patients succumb to pulmonary metastasis and because we found reduced metastasis in NOS2-/- mice, we studied the immune cells in metastatic lungs. Lung niche of NOS2-/- mice had more activated T-cells, bone marrow-derived macrophages and neutrophils that are not conducive to establishment of metastasis. Serum of NOS2-/- mice had elevated IL-6 that drove increased IL17A subsequently contributing to reduced metastasis and this effect was improved upon indomethacin treatment. IL6 is also a potential inhibitor of Tregs thus further improving immune surveillance. COX inhibition can treat aggressive TNBC directly by reducing cancer cell proliferation and indirectly by immune activation. Tandem NOS2 inhibition can render the pulmonary microenvironment unfavorable for metastatic spread and induce a favorable systemic cytokine storm that further improves outcome. Citation Format: Veena Somasundaram, Debashree Basudhar, Christopher McGinity, Robert Y. Cheng, Lisa A. Ridnour, Stephen J. Lockett, David A. Wink. Inhibition of nitric oxide synthase and cyclooxygenase potentiate immune responses that control aggressive mammary tumor in a murine model [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 2754.

Abstract 1552: Single cell expression analysis of PIK3 genes to direct solid tumor treatment with the dual PI3K-δ,γ inhibitor duvelisib

Abstract Duvelisib is a dual PI3K-δ,γ inhibitor that targets malignant B and T cells in hematologic malignancies and modulates key non-malignant immune cells in the tumor microenvironment. PI3K-δ and PI3K-γ are thought to govern tumor immune modulation through reduction of immunosuppressive Tregs and myeloid cells in solid tumors (Ali, 2014, Kaneda, 2016; De Henau, 2016). Yet, a potential role of these isoforms in directly targeting cancer cells of solid tumors is not understood. Expression levels of PI3K-δ (PIK3CD) and PI3K-γ (PIK3CG) were analyzed in 9 major tumor types (BRCA, HNSCC, LUSC, PAAD, STAD, COAD, LUAD, SKCM and THCA), and it was found that PIK3CD reproducibly had higher expression levels than PIK3CG. For example, in head and neck squamous cell carcinoma (HNSCC), PIK3CD had a median expression of 9.51 RNA-Seq Expectation-Maximization (RSEM) log2 and PIK3CG had a median of 5.56 RSEM log2; each of which are higher than the levels in normal cells (8.40 RSEM log2 & 5.38 RSEM log2, respectively) (FireBrowse). By clustering solid tumors into ′Hot′ (inflamed) and ′Cold′ subgroups with an inflammatory/immunosuppressive gene signature (Givechian, 2018; TCGA, n=1687); a correlation was observed between higher PIK3CD and PIK3CG expression in ′Hot′ (median ≈11 log2 Transcripts Per Million (TPM) & ≈9.2 log2 TPM) compared to ′Cold′ tumors (median ≈9.4 log2 TPM & ≈6.6 log 2 TPM). Using CIBERSORT deconvolution, most ′Hot′ cluster tumors contained higher fractions of CD8+ T cells, M1-type macrophages, and lower M2-type macrophages; with Tregs at similar abundances in both ′Hot′ and ′Cold′ clusters. To evaluate cellular distribution, single-cell RNA expression of PIK3C genes in a HNSCC dataset (Puram, 2017) was examined at >0.3 log2 TPM. PIK3CD and PIK3CG expression was observed in T cells (32% & 11%, respectively), B cells (13% & 56%), dendritic cells (15% & 40%) and macrophages (24% & 29%) in HNSCC tumors. PIK3A and PIK3B were less frequently expressed in macrophages (10% & 17%) and T cells (3% & 4%) than PIK3D. A significant proportion of the HNSCC cancer cells expressed PIK3CD (20%) and PIK3CB (27%), whereas PIK3CA (14%) and PIK3CG (0%) were less frequently expressed. Most individual tumors had cancer cells expressing PIK3CD (6%-63%; >50 cells/tumor). Importantly, PIK3CD expression was mostly mutually exclusive with PIK3CG, PIK3CA, and PIK3CB in the cancer cells. Cancer cells expressing PIK3CD showed striking co-associated differentially expressed genes (p<0.009). These results indicate that PI3Kδ expression is observed in both the immune cells which are fashioning the suppressive microenvironment and in cancer cells. These findings suggest an approach to identify patients for treatment guided by cell type expression of PI3K isoforms, and provide strategies for duvelisib use as monotherapy and in combinations with PD-1 checkpoint inhibitors and other agents. Citation Format: Samantha Hidy, Jonathan A. Pachter, David T. Weaver. Single cell expression analysis of PIK3 genes to direct solid tumor treatment with the dual PI3K-δ,γ inhibitor duvelisib [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 1552.

Abstract 6050: Transient and selective depletion of MDSCs and Tregs as an effective immunotherapy against triple-negative breast cancer

Abstract Background: Checkpoint inhibitors (CPI) have revolutionized the field of cancer immunotherapy. However, CPIs have shown only modest efficacy in clinical trials for TNBC which may be attributed to the presence of immunosuppressive cells both myeloid-derived suppressor (MDSCs) and T regulatory (Treg) cells in the tumor microenvironment (TME). We hypothesized that transient depletion of Tregs and MDSCs along with the administration of checkpoint inhibitors would markedly reduce immunosuppressive signals to the effector arm of the immune system and thereby lead to a more robust anti-tumor response causing tumor regression and a better disease outcome. To target Treg/MDSCs cells, we have developed two diphtheria-toxin based fusion protein toxins and are currently evaluating the activity of these molecules both as monotherapies and dual therapies in combination with CPIs. Methods: To target MDSCs and TNBC tumor cells, we genetically constructed, expressed and purified a diphtheria toxin-based fusion protein, s-DAB389mIL-4 that targets the IL-4 receptor (IL-4Rα) on these cell populations. This fusion protein toxin was tested in vitro against murine TNBC 4T1 cells and in vivo in a syngeneic murine adenocarcinoma model both as a monotherapy and in combination with s-DABIL-2(V6A) which targets Tregs cells through high and intermediate affinity IL-2 receptors. Results: We found that s-DAB389mIL-4 is highly potent against 4T1 cells in vitro (IC50 ranging from 10 pM to ~3 nM), and adversely affects 4T1 cell survival, proliferation, invasion, and migratory abilities. Recombinant IL-4 and anti-IL-4 antibody block the cytotoxic action of s-DAB389mIL-4 confirming that the fusion protein's activity is mediated through the IL-4Rα. s-DAB389mIL-4 treatment in a 4T1 cell-based murine adenocarcinoma model resulted in a significant reduction of (a) tumor volume and weight, (b) MDSCs, (c) splenomegaly, (d) 4T1 metastasis to the lung and, (d) increased infiltration of IFNϒ+ effector T-cells. Sequential combination therapy with both s-DABIL-4 and s-DABIL-2(V6A) resulted in (1) reduction in both tumor volume and weight, (2) transient depletion of activated Tregs, and (3) increased infiltration of T-effector cells in the TME. NanoString analysis of RNA isolated from tumor tissues treated with these two fusion protein toxins show enrichment of gene signatures which enhance the infiltration of anti-tumor immune cells in the TME and concomitant inhibition of processes which encourage tumor growth and metastasis. Conclusions: These findings suggest that s-DABmIL-4 both as a monotherapy and in combination with s-DABIL-2(V6A) results in transient reduction of both MDSCs and Tregs. These observations suggest that these novel fusion protein toxins may serve as an effective immunotherapeutic regimen for the treatment of TNBC. Based upon these results, we are currently testing these fusion protein toxins in combination with checkpoint blockade therapy. Citation Format: Sadiya Parveen, Sumit Siddharth, Laurene Cheung, John R. Murphy, Dipali Sharma, William R. Bishai. Transient and selective depletion of MDSCs and Tregs as an effective immunotherapy against triple-negative breast cancer [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 6050.

Abstract 6675: Basal NK activity and early Tregs inhibition predicts nivolumab responsiveness in metastatic renal cancer patients (REVOLUTION) trial

Abstract Background: Regulatory T cells (Tregs) are responsible for maintaining self-tolerance, and inhibit antitumor immune responses. PD-1 blockade was reported to increase resistance of effector T cells to Tregs suppression and directly reduces in vitro Tregs suppressive function. Natural killer cells (NKs) expressed PD-1 and engagement of PD-1 reduces their cytolytic potential. To identify biomarkers informative and predictive of Nivolumab efficacy Tregs and NKs phenotype and function was determined in Nivolumab treated metastatic renal cancer (mRCC) patients (REV). Effect of CXCR4 antagonism are evaluated on ex vivo Tregs and NKs. Methods: 48 REV, 26 other than ICI treated-mRCC (CTR) and 23 Healthy Donors (HD) were enrolled. To date, the clinical evaluation is available for 42 REV [14 with objective response (OR) and 28 in progression (PD)] and 18 CTR (11 OR and 7 PD). 31 patients (pts) underwent first clinical evaluation at 3 months resulting in 25 pts with OR and 6 in PD. Tregs (CD4+CD25+CD127lowFOXP3+) and NKs (CD3-CD56+CD107a+) phenotype and function were evaluated at day 0, 14, 28, 90, and 180. CFSE-T-effector proliferation-Tregs dependent and CD107a externalization toward K562 as NKs function were evaluated. Results: Higher NKG2D was reported on CD3-CD56dim cells from OR as compared to PD pts (p=0.0054). Statistically significant low NKs basal activity was detected at T0 in 3 months PD- Nivolumab treated pts (p=0.032). CD107a+/CD53+CD56+ is higher in OR compared to PD pts over time. Percent Tregs CD4+CD25+CD127lowFOXP3+ and CD8/Tregs ratio were respectively low (p<0.001) and high (p<0.001) in 71 mRCC as compared to 23 HD. Significant lower PD-1 was observed in Tregs at 3 months of treatment in OR pts (p= 0.048). Moreover, higher % of CD4+CD25+CD127lowFoxp3+ Tregs were observed in PD compared to OR pts over time (p<0.001) with higher CTLA4 expression in PD group. Tregs function, as evaluated at 2 weeks post treatment, was inhibited in responder and potentiated in PD patients. Moreover, CXCR4 antagonism dramatically reversed Tregs function ex vivo as early as at day 14 of treatment in OR pts (p=0.0024). A statistically significant reduction in peripheral Tregs (Effector/Naïve and nonTregs) was revealed in 14 Long Responder compared to 23 PD pts (> 12 months) (p<0.05; p<0.01 and p<0.05, respectively). In 18 CTR no modification in Treg phenotype and function were detected but a common decrease in NK activity unrelated to the clinical outcome. Conclusions: Basal NK activity and 2 weeks Tregs evaluation discriminates mRCC Nivolumab responding patients. Ex vivo CXCR4 antagonism inhibits Tregs function in responding patients at early treatment time point (2 weeks). These evidence are not detected in non-immune treated mRCC patients. Citation Format: Sara Santagata, Anna Maria Trotta, Giuseppina Rea, Maria Napolitano, Anna Capiluongo, Crescenzo D'Alterio, Marilena Di Napoli, Sabrina Rossetti, Sandro Pignata, Stefania Scala. Basal NK activity and early Tregs inhibition predicts nivolumab responsiveness in metastatic renal cancer patients (REVOLUTION) trial [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 6675.

Abstract 2869: Nano-immuno combination therapy to treat brain primary central nervous system lymphoma

Abstract Purpose: Lack of standard treatment of Primary Central Nervous System Lymphoma (PCNSL) was acknowledged in phase III intergroup study (HOVON 105/ALLG NHL 24). PCNSL incidence is increasing for unknown reasons, particularly among persons over 65 years of age. The major limitation of successful treatment of PCNSL is the blood-brain barrier (BBB), which prevents drug delivery into the brain. Our novel treatment employs nano immunoconjugates (NICs) that cross BBB and activate brain lymphoma immunity. These NICs specifically block c-Myc expression and induce CD20 crosslinking. The NICs, in combination with αPD1, were systemically delivered to the lymphoma mice and this treatment led to a local and systemic broad-spectrum immune response by activation of IFN-γ that is an important anti-tumor M1 macrophage activator. Methods: Polymer nanoconjugate based on biodegradable poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA)-P was used for treatment of BALB/C mice with intracranially inoculated murine A20 lymphoma. PHPMA-based nanoconjugates have defined synthesis strategy for BBB transcytosis, tumor targeting, and cancer cell killing. Nano-immunodrug P/AP-2/CD20 Fab'or c-Myc inhibitor (anti-sense oligonucleotides)/H6 conjugate contains multiple AP-2 peptides for efficient delivery across the BBB and Fab' fragments of αCD20 Ab (mouse specific) for CD20 receptor crosslinking on PCNSL cells. c-Myc inhibitor bound via a disulfide bond is transferred to the cytoplasm followed by cleavage; this is facilitated by (His)6 peptide-mediated endosome disruption. The nanoconjugate was examined for the ability to cross BBB in vivo by live imaging. The therapeutic efficacy and survival were evaluated together with spectral flow cytometry (FC) and RNA-seq bioinformatic analysis of tumor tissues. Results: NIC was able to penetrate BBB and accumulate in brain parenchyma. The best survival results were obtained from the combination groups with CD20 Fab' crosslinking CD20 receptors resulting in apoptosis and c-Myc antisense inhibition in combination with αPD-1 checkpoint inhibitor. Survival compared to PBS-treated controls was significant (P=0.0006, P=0.0034, respectively, by ANOVA). FC analysis of A20 brain lymphoma tissue after treatment with NICs demonstrated reduction of Tregs, associated with downregulation of IL-10 and IL-1β in plasma. Spectral flow cytometry revealed activation of tumor infiltrating T lymphocytes and the M1 macrophages in lead treatment groups. RNA-seq data correlated with flow cytometry results indicating the activation of genes linked to IFN-γ pathway important for anti-tumor response and M1 macrophage activation. Immunostaining confirmed a marked increase of M1-specific iNOS expression in the nanodrug-treated tumors. Conclusion: The PCNSL treatment results demonstrate for the first time that the BBB delivery of NICs modified brain tumor microenvironment and activated brain local immune system though modulation of T cells and IFN-γ pathway. Support: NIH grants: R01 CA188743, R01 CA206220, R01 CA209921 Citation Format: Tao Sun, Ekaterina Shatalova, Oliver Braubach, Dmytro Klymyshyn, Eggehard Holler, Jiawei Wang, Lian Li, Jiyuan Yang, Jindrich Kopecek, Julia Y. Ljubimova. Nano-immuno combination therapy to treat brain primary central nervous system lymphoma [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 2869.

Treg sensitivity to FasL and relative IL-2 deprivation drive idiopathic aplastic anemia immune dysfunction

AbstractIdiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Abstract 4553: The CD25 antibody RG6292 selectively depletes Tregs while preserving IL-2 signaling and CTL activity for tumor control

Abstract Despite the advances in cancer immunotherapy, in particular in the field of checkpoint inhibitors (CPI), many patients fail to respond (primary resistance) or initially benefit but then progress upon treatment (secondary resistance). High regulatory T-cell (Treg) counts correlate with poor prognosis and reduced responsiveness to CPI therapy in humans, underscoring their potential as an immunotherapy target. Clinical attempts aiming to lower Treg counts, however, either failed to deliver convincing Treg reduction or lacked specificity for Treg over tumor antigen specific cytotoxic T cells (CTL). CD25 (the interleukin-2 receptor alpha (IL-2Ra) chain) is a recently revisited target for Treg depletion. For privileged access to IL-2, activated CTL up-regulate CD25 expression only transiently during clonal expansion. Further confirmation of an > 20 fold higher cell surface expression of CD25 on Tregs versus CTLs in human malignancies is provided. The novel compound RG6292 was developed as an ADCC and ADCP competent monoclonal antibody of human IgG1 isotype with afucosylated glycans in the Fc region. RG6292 binds with low monovalent affinity (KD 250 nM) to the extracellular domain of CD25 antigen. A high density of CD25 receptors promotes bivalent avidity of RG6292 increasing its binding strength to CD25 by at least 100 fold (KD 2-3 nM). RG6292 selectively favors the depletion of CD25 high Tregs over CD25 low activated CTLs, here shown in comparison to ipilimumab and mogamulizumab in human αCD3 activated PBMC, human tumor explants and immunopharmacodynamic studies in tumor bearing (BxPC-3), stem cell humanized mice and cynomolgus monkeys. IL-2 is an essential prerequisite for clonal expansion of CTLs, which is necessary to generate effective anti-tumor responses. Earlier immunosuppressant anti-CD25 antibodies (e.g. daclizumab and basiliximab) interfered with the formation of the high affinity IL-2R complex. Their evidenced lack of therapeutic activity in immunoncology tempered enthusiasm and highlights the pivotal role of IL-2. RG6292 is the first anti-human CD25 antibody developed to deplete Tregs selectively while fully preserving IL-2 signaling and CTL activity. Pre-clinically, a single administration of the RG6292 surrogate effectively promoted eradication of established tumors in several tumor mouse models and synergized with CPI in models of CPI resistance. RG6292 is expected to unleash the potential of selective Treg depletion while allowing for unrestricted access of IL 2 to CTLs and could therefore result in clinically superiority compared to other Treg depleting antibodies. RG6292 provides a novel therapeutic approach to alleviate a major mechanism of immune suppression in the tumor microenvironment. Clinical testing is currently ongoing to evaluate the safety and tolerability of RG6292 in patients with advanced solid tumors (NCT04158583). Citation Format: Maria Amann, Gabriel Schnetzler, Kolben Theresa, Isabelle Solomon, Christophe Boetsch, Estelle Marrer-Berger, Reto Flury, Claudio Murgia, Vaios Karanikas, Johannes Sam, Roger Sutmuller, Jan Eckmann, Hans Koll, Sara Belli, Frederic Arce Vargas, Dimitrios Zervas, Chen Qing, Mark A. Brown, Josephine Salimu, Anne Goubier, Sebastian Neumann, Karl S. Peggs, Sergio A. Quezada. The CD25 antibody RG6292 selectively depletes Tregs while preserving IL-2 signaling and CTL activity for tumor control [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 4553.

294-OR: Discovery of a Novel Secretory Protein from the Gut Microbiome That Promotes Immune Tolerance and Ameliorates Autoimmunity via Dendritic Cell Induction of IL-10

The human gut microbiota has been associated with diverse autoimmune diseases; however, the underlying mechanisms are largely unknown. To identify gut microbes and derived bioactive molecules that can promote immune tolerance, we systematically investigated the immunoregulatory potential of each member in the Altered Schaedler Flora (ASF) that has demonstrated diverse immune functions in mice. We observed that the eight ASF members presented a division of labor in promoting distinct immunophenotypes of bone marrow-derived dendritic cells, such as the production of IL-10, TNF-a, and TGF-b, and the induction of regulatory T cells. Interestingly, Parabacteroides goldsteinii ASF519 induced strong immune tolerance via novel mechanisms as evidenced by non-pan T cells-mediated promotion of Foxp3+ Treg proliferation and reduction of cytotoxic CD8 T cells, as well as TNF-a signaling-independent IL-10 production. Via bioactivity-guided fractionation, ASF519-derived molecules responsible for inducing immune tolerance were identified to be heat-labile secretory proteins with yet-uncharacterized functions. Genome-wide screening additionally identified a complex regulatory network that controls the immunoregulatory potency of ASF519. ASF519 colonization in germ-free mice significantly reduced IL-4 and IL-5 in sera and increased the transcript abundance of ll-10 and ll-17A in colon, suggesting potential roles of ASF519 in modulating both intestinal and systemic immune functions. In mouse immune disease models, oral administration of ASF519 demonstrated a protective role in the development of dextran sodium sulfate-induced colitis and collagen-induced arthritis. Relevant mechanisms are now under investigation. This study has the potential to provide novel therapeutic avenues for the prevention and treatment of autoimmune diseases. Disclosure A. Kostic: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-17-INI-13)