Reduction Of Tregs Research Articles

Phase Ib trial of selinexor (SEL) in combination with nivolumab (NIVO) alone or nivolumab plus ipilimumab (NIVO+IPI) in patients (pts) with advanced malignancies: The renal cell carcinoma (RCC) experience.

4551 Background: SEL is a first-in-class nuclear exportin 1 inhibitor, which blocks the transport of several proteins involved in cancer-cell growth from the nucleus to the cytoplasm. A syngeneic RENCA mouse model showed that SEL combined with checkpoint blockade resulted in increased effector and activated T cells, and NKT cells and reductions in myeloid cells and Tregs. We hypothesized that SEL+NIVO and SEL+NIVO+IPI are well tolerated and improve the overall response rate (ORR) observed with anti-PD1/CTLA-4. Methods: NCT02419495 is an open label, single center phase IB study of SEL with multiple standard chemotherapy or immunotherapy regimens in pts with advanced malignancies using 3 + 3 design in dose escalation. For SEL+NIVO+IPI, SEL was 60 mg (N = 4) or 80 mg (N = 6) given P.O. weekly with NIVO 3 mg/kg x 4 cycles then 480 mg Q4W plus IPI 1 mg/kg Q3W for 4 cycles only. For SEL+NIVO, SEL was 40 mg twice a week (N = 8), or 60 mg twice a week (N = 11) given with NIVO 240 mg Q2W or 480 mg Q4W. Primary objective was to establish the safety and tolerability of SEL+NIVO and SEL+NIVO+IPI. Secondary objectives included ORR and progression free survival (PFS). Results: 29 pts with RCC (25 males) were enrolled (table), including 3 pts with nonclear cell RCC (nccRCC), with a median age of 64 years (IQR 56-69). 21 patients (72%) had prior systemic therapies. Most common SEL or NIVO/IPI treatment-related ≥ grade (G) 3 adverse events (AEs) included endocrine disorders (N = 3), hyponatremia (N = 3), leukopenia (N = 3), transaminitis (N = 3), thromboemobolic events (N = 2), asymptomatic lipase increased (N = 2), anemia (N = 1), pneumonitis (N = 1), nephritis (N = 1), colitis (N = 1), fatigue (N = 1). Two pts discontinued therapy due to AEs (G3 pneumonitis from SEL+NIVO+IPI and G3 transaminitis from SEL+NIVO, respectively). At a median follow up of 17.5 months, 5 (17%) pts achieved a PR, and 17 (59%) achieved a stable disease (SD). The median PFS for the entire cohort was 17.5 months (SEL+NIVO+IPI: 7.2 months, SEL+NIVO: 17.5 months). Among the patients with clear cell RCC who received SEL+NIVO+IPI as first line (1L) therapy (N = 4), all (100%) achieved SD as best overall response. Among the patients with ccRCC who received SEL+NIVO as 1L (N = 2), one patient achieved confirmed PR and the other achieved SD. The median overall survival (OS) was 27.8 months (SEL+NIVO+IPI: unreached, SEL+NIVO: 21.3 months). Conclusions: Treatment with SEL in combination with NIVO or NIVO+IPI is well-tolerated and shows modest clinical activity as compared to historic NIVO or NIVO+IPI activity. Clinical trial information: NCT02419495.

Tumor-associated Tregs obstruct antitumor immunity by promoting T cell dysfunction and restricting clonal diversity in tumor-infiltrating CD8+ T cells

BackgroundAccumulation of regulatory T cells (Treg) has been described to often correlate with poor prognosis in many solid tumors. How Treg presence impinges on limited functionality and clonal composition of...

Combination therapy for treating drug-resistant multiple myeloma

Abstract Proteasome inhibitors (PIs) have remarkably improved the survival of multiple myeloma (MM) patients but dose-limiting toxicities and the development of drug resistance limit their long-term utility. Elevated TGF-β levels in MM patient sera correlate with chemoresistance, disease progression, metastasis and poor prognosis. Therefore, we evaluated the anti-MM therapeutic potential of a TGF-β type I receptor kinase inhibitor, Vactosertib. In vitro treatment of Vactosertib synergistically inhibited the growth of PI-resistant MM cells in combination with the ixazomib by suppressing TGF-β activation of Smad2/3 and expression of PSMB5, which encodes the proteasome 5 catalytic subunit targeted by PIs. Oral administration of Vactosertib as a single agent inhibited MM progression and induced a decrease in mortality and an increase in body weight of the mice bearing MM. Vactosertib alone also attenuated TGF-β activation of Smad2/3, reduced the expansion of CD11b+Gr-1+ myeloid derived suppressor cells (MDSCs) in bone marrow tumor microenvironment, and diminished the population of Foxp3+ regulatory T cells (Treg) in the spleen. Combination therapy of Vactosertib plus ixazomib was exhibited a synergistic anti-tumor effect when compared to either Vactosertib or ixazomib alone by greatly prolonged survival and significant a reduction in both MDSCs and Tregs. Taken together, our data provide the rationale for clinical evaluation of Vactosertib in MM and demonstrate proof-of-concept that combination of Vactosertib and PIs may overcome chemoresistance and enhance durable patient responses. Supported by 1R03CA259901-01A1

An Fc-Competent Anti-Human TIGIT Blocking Antibody Ociperlimab (BGB-A1217) Elicits Strong Immune Responses and Potent Anti-Tumor Efficacy in Pre-Clinical Models.

TIGIT (T-cell immunoglobulin and ITIM domain) has emerged as a promising target in cancer immunotherapy. It is an immune “checkpoint” inhibitor primarily expressed on activated T cells, NK cells and Tregs. Engagement of TIGIT to its ligands PVR and PVR-L2 leads to inhibitory signaling in T cells, promoting functional exhaustion of tumor-infiltrating T lymphocytes. Here, we described the pre-clinical characterization of Ociperlimab (BGB-A1217), a novel humanized IgG1 anti-TIGIT antibody (mAb), and systemically evaluated the contribution of Fc functions in the TIGIT mAb-mediated anti-tumor activities. BGB-A1217 binds to the extracellular domain of human TIGIT with high affinity (KD = 0.135 nM) and specificity, and efficiently blocks the interaction between TIGIT and its ligands PVR or PVR-L2. Cell-based assays show that BGB-A1217 significantly enhances T-cell functions. In addition, BGB-A1217 induces antibody dependent cellular cytotoxicity (ADCC) against Treg cells, activates NK cells and monocytes, and removes TIGIT from T cell surfaces in an Fc-dependent manner, In vivo, BGB-A1217, either alone or in combination with an anti-PD-1 mAb elicits strong immune responses and potent anti-tumor efficacy in pre-clinical models. Moreover, the Fc effector function is critical for the anti-tumor activity of BGB-A1217 in a syngeneic human TIGIT-knock-in mouse model. The observed anti-tumor efficacy is associated with a pharmacodynamic change of TIGIT down-regulation and Treg reduction. These data support the selection of BGB-A1217 with an effector function competent Fc region for clinical development for the treatment of human cancers.

The absence of B cells disrupts splenic and myocardial Treg homeostasis in coxsackievirus B3-induced myocarditis.

Although B cells are essential for humoral immunity and show noteworthy immunomodulatory activity through antibody-independent functions, the role of B cells in regulating Treg cell responses remains controversial. Tregs (CD4+CD25+Foxp3+) are considered to play an immunoprotective role in viral myocarditis (VMC) by controlling autoimmune effector T cells. Here, we proved that B-cell knockout can not only lead to significant reductions in Tregs in the spleen, blood, and heart of VMC mice but also decrease the activation and immune function of splenic Tregs, which was reversed by adoptive transfer of B cells; the transcription levels of TGF-β and Foxp3 in the myocardium were also significantly reduced. B-cell depletion by anti-CD20 impaired the anti-inflammatory function of splenic Tregs and the homeostasis of myocardial Tregs population. Moreover, B cells can convert CD4+CD25- T cells into Foxp3+ and Foxp3-, two functionally suppressive Treg subgroups. Although the reduction in myocardial inflammation in BKO mice indicates that B cells may play a proinflammatory role, the beneficial side of B cells cannot be ignored, that is, to control autoimmunity by maintaining Treg numbers. The results observed in the animal model of VMC highlight the potential harm of rituximab in the nonselective depletion of B cells in clinical applications.

An Early Wave of Macrophage Infiltration Intertwined with Antigen-Specific Proinflammatory T Cells and Browning of Adipose Tissue Characterizes the Onset of Orbital Inflammation in a Mouse Model of Graves' Orbitopathy.

Background: Graves' orbitopathy (GO) is an autoimmune-driven manifestation of Graves' disease (GD) where pathogenic autoantibodies to the thyrotropin receptor (TSHR) activate orbital fibroblasts/preadipocytes in the orbital tissue to induce inflammation and extracellular matrix deposition. Since there are significant limitations to study immunological and proinflammatory mediator expression in early and during disease progression in GO patients, we used our experimental mouse model to elucidate early pathogenic processes. Methods: We have developed a robust mouse model of GD/GO induced by electroporation immunization of plasmid encoding human TSHR A-subunit, comprising multiple injections over a course of 15 weeks to fully recapitulate the orbital pathology. In this study, we investigated kinetics of GO development in the model by serial analyses of immunological and cellular parameters during course of orbital inflammation. Results: Pathogenic anti-TSHR antibodies with thyroid-stimulating properties developed early after the second immunization step with concomitant induction of hyperthyroidism. Examination of orbital tissue showed an early wave of macrophage infiltration followed subsequently by CD3+ T cells into the orbital tissue. Examination of antigen-specific T cell activity using recombinant human A-subunit protein showed high CD8+ T cell proliferation during this early phase of disease onset, whereas effector CD4+ T cells and CD25+FOXP3+ regulatory T cells (Tregs) were downregulated. The early phase of disease was also characterized by abundant presence of proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Moreover, as the disease progressed, there was significant increase in browning of orbital fat tissue, which may be dependent on the proinflammatory milieu and/or the increased thyroid hormone levels during the established hyperthyroid status. Conclusions: This work revealed early infiltration of macrophages in the orbital region and induction of pathogenic anti-TSHR antibodies during disease onset in the model. This was followed subsequently by influx of CD8+ T cells specific for TSHR coupled with reduction in Tregs and substantial increase in brown adipose tissue. These new insights into the development of orbital inflammation in the model have implications for testing new therapeutic regimens by targeting macrophage function during early phases of orbital inflammation in the model.

Maximizing cancer therapy via complementary mechanisms of immune activation: PD-1 blockade, neoantigen vaccination, and Tregs depletion

BackgroundA number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the ‘Cancer Immunity Cycle’. Anti-PD-1 checkpoint blockade (CPB) therapy is active on...

Radiation, Adenosine Signaling, and Immunosuppressive Changes to the Tumor Microenvironment

Tumor irradiation causes pro-inflammatory changes in the tumor microenvironment (TME). We have shown that these changes may be countered by the induction of immunosuppressive changes that drive therapy resistance. Here, we test the hypothesis that radiation activates adenosine signaling and induces immunosuppressive myeloid cells in the TME.Using the 4T1 orthotopic breast cancer mouse model, we tested immunophenotypic changes to the TME by spectral flow cytometry and by bulk RNASeq of tumor infiltrating leukocytes (CD45+, TILs) 72-hours after tumor irradiation (8 Gy x 1) using the small animal radiation research platform (SARRP, Xstrahl; 220 kV). Human peripheral blood mononuclear cells (PBMCs) were cultured in vitro with 0.1, 1, and 10uM of adenosine receptor agonist, 5'-N-ethylcarboxamindoadenosine (NECA), in the presence of CD3/CD28 beads. Cytokine quantification of cell-free supernatant was performed 48-hours later using the Meso Scale Discovery platform.Radiation increases the abundance of CD11b+ myeloid cells (77.4% vs. 84.4%) and decreases TCRb+ lymphocytes (6.3% vs. 3.6%) in the TME. The reduction of lymphocytes was driven by CD4+ T-cells (5.2% vs 2.7%), including a proportional reduction in Tregs (1.4% vs 0.63%), with no change in abundance of CD8+ T-cells (0.53% vs 0.43%). The increase in myeloid cells was predominantly influenced by granulocytic- and monocytic-myeloid-derived suppressor cells (PMN-MDSCs: 22% vs 34.5%; M-MSCSs: 3.0% vs 10.0%). By bulk RNASeq of CD45+ TILs, radiation increased the expression of adenosine 2A receptor (A2AR, log2 fold change 1.13) and A2BR (log2 fold change 0.92), but not CD73 - an ecto-nucleotidase that metabolizes AMP to adenosine. By flow cytometry, radiation increased expression of both CD73 and A2AR in the myeloid population, with CD73 expression significantly increased in the monocytic compartment (Ly6C+, 63% vs 70%) and A2AR expression increased in the granulocytic compartment (Ly6G, 89% vs 97%). The addition of adenosine analog, NECA, to human PBMCs resulted in significant reduction in effector T-cell cytokines (IL-2, IFN- γ, granzyme B) across doses, an increase in IL-6 and MCP-1/CCL2 at the intermediate dose tested, and increased IFN-β and TGF- β while decreasing IL-1 β, IL-8 only at the highest dose.Radiation induces an immunosuppressive TME, increasing the abundance of PMN-MDSCs and M-MDSCs and activating adenosine signaling. Radiation increases the expression of adenosine receptors on CD45+ TILs at the transcriptomic level and cell surface expression of CD73 on monocytes and A2AR on granulocytes. Treatment of human PBMCs with adenosine receptor agonist resulted in loss of effector T-cell function and suppressive changes to cytokine milieu. These data suggest a mechanistic link between radiation, adenosine signaling and the myeloid compartment.

IL-35 promotes CD4+Foxp3+ Tregs and inhibits atherosclerosis via maintaining CCR5-amplified Treg-suppressive mechanisms.

Tregs play vital roles in suppressing atherogenesis. Pathological conditions reshape Tregs and increase Treg-weakening plasticity. It remains unclear how Tregs preserve their function and how Tregs switch into alternative phenotypes in the environment of atherosclerosis. In this study, we observed a great induction of CD4+Foxp3+ Tregs in the spleen and aorta of ApoE–/– mice, accompanied by a significant increase of plasma IL-35 levels. To determine if IL-35 devotes its role in the rise of Tregs, we generated IL-35 subunit P35–deficient (IL-35P35–deficient) mice on an ApoE–/– background and found Treg reduction in the spleen and aorta compared with ApoE–/– controls. In addition, our RNA sequencing data show the elevation of a set of chemokine receptor transcripts in the ApoE–/– Tregs, and we have validated higher CCR5 expression in ApoE–/– Tregs in the presence of IL-35 than in the absence of IL-35. Furthermore, we observed that CCR5+ Tregs in ApoE–/– have lower Treg-weakening AKT-mTOR signaling, higher expression of inhibitory checkpoint receptors TIGIT and PD-1, and higher expression of IL-10 compared with WT CCR5+ Tregs. In conclusion, IL-35 counteracts hyperlipidemia in maintaining Treg-suppressive function by increasing 3 CCR5-amplified mechanisms, including Treg migration, inhibition of Treg weakening AKT-mTOR signaling, and promotion of TIGIT and PD-1 signaling.

RNA sequencing of blood in coronary artery disease: involvement of regulatory T cell imbalance

BackgroundCardiovascular disease had a global prevalence of 523 million cases and 18.6 million deaths in 2019. The current standard for diagnosing coronary artery disease (CAD) is coronary angiography. Surprisingly, despite well-established clinical indications, up to 40% of the one million invasive cardiac catheterizations return a result of ‘no blockage’. The present studies employed RNA sequencing of whole blood to identify an RNA signature in patients with angiographically confirmed CAD.MethodsWhole blood RNA was depleted of ribosomal RNA (rRNA) and analyzed by single-molecule sequencing of RNA (RNAseq) to identify transcripts associated with CAD (TRACs) in a discovery group of 96 patients presenting for elective coronary catheterization. The resulting transcript counts were compared between groups to identify differentially expressed genes (DEGs).ResultsSurprisingly, 98% of DEGs/TRACs were down-regulated ~ 1.7-fold in patients with mild to severe CAD (> 20% stenosis). The TRACs were independent of comorbid risk factors for CAD, such as sex, hypertension, and smoking. Bioinformatic analysis identified an enrichment in transcripts such as FoxP1, ICOSLG, IKZF4/Eos, SMYD3, TRIM28, and TCF3/E2A that are likely markers of regulatory T cells (Treg), consistent with known reductions in Tregs in CAD. A validation cohort of 80 patients confirmed the overall pattern (92% down-regulation) and supported many of the Treg-related changes. TRACs were enriched for transcripts associated with stress granules, which sequester RNAs, and ciliary and synaptic transcripts, possibly consistent with changes in the immune synapse of developing T cells.ConclusionsThese studies identify a novel mRNA signature of a Treg-like defect in CAD patients and provides a blueprint for a diagnostic test for CAD. The pattern of changes is consistent with stress-related changes in the maturation of T and Treg cells, possibly due to changes in the immune synapse.

The long and winding road to the etiology of idiopathic nephrotic syndrome in children: Focusing on abnormalities in the gut microbiota.

Childhood nephrotic syndrome is idiopathic in 90% of cases. Despite its relatively high prevalence (30-35 per 100000 individuals under 15years old), the etiology of the disease remains elusive. It has become clear that oxidants are elevated, and antioxidants are decreased, at onset of idiopathic nephrotic syndrome (INS). It was suggested that overexpression of podocyte CD80 induced by abnormalities of Tregs was involved in the pathogenesis of INS. Subsequently, it became clear that quantitative or qualitative reduction of Tregs has a profound impact on the development of INS. To address why Tregs are decreased at onset of INS, it was hypothesized that a decrease in Tregs may be associated with dysbiosis. Given the critical role of butyrate-producing bacteria in the differentiation of Tregs, the gut microbiota was analyzed with a particular focus on the abundance of butyrate-producing bacteria, and it was found that pediatric patients with INS had low levels of butyrate in their stool and a low percentage of butyrate-producing bacteria. Interestingly, it was recently reported that gut dysbiosis increases oxidative stress in the intestinal tract. Taken together, we currently hypothesize that gut dysbiosis is associated with a predisposition to INS because of immunological abnormalities characterized by abnormal Tregs with increased oxidative stress.

Abstract CT107: A PHASE 1B/2 OPEN-LABEL STUDY WITH RANDOMIZATION IN PHASE 2 OF IMU-131 HER2/NEU PEPTIDE VACCINE PLUS STANDARD OF CARE CHEMOTHERAPY IN PATIENTS WITH HER2/NEU OVEREXPRESSING METASTATIC OR ADVANCED ADENOCARCINOMA OF THE STOMACH OR GASTROESOPHAGEAL JUNCTION

Abstract Background: HER2/neu is overexpressed in up to 30% of gastroesophageal adenocarcinoma (GEA) patients and associated with poor prognosis. Recombinant mAbs to treat HER2/neu-amplified malignancies are effective with limitations so alternatives are needed. Therefore, we developed a B-cell epitope immunotherapy vaccine (IMU-131/HER-Vaxx) consisting of 3 fused B-cell epitopes (P467) from the HER2/neu extracellular domain coupled to CRM197 and adjuvanted with Montanide. This open-label multicenter Phase 1b study aimed to evaluate the optimal/safe IMU-131 dose leading to immunogenicity and clinical responses.Patients & Methods: Fourteen patients with HER2/neu overexpressing GEA were enrolled and dose escalation with 10, 30, and 50µg was performed in 3 cohorts. Immunogenicity was evaluated by P467- and HER2-specific Ab levels and cellular responses, and clinical response rates by CT/MRI scan evaluated according to RECISTv1.1. Results: IMU-131 was safe with no vaccine-related significant local or systemic reactions or SAEs. 11/14 patients were evaluable for changes in tumor size and vaccine-specific immune responses. One patient showed complete response, 5 showed partial response and 4 stable disease as their best response. HER2-specific IgG levels were dose-dependent. While in cohort 1 and 2 only 1/3 and 2/5 patients respectively responded with moderate to high Ab titers, all patients in cohort 3 mounted moderate to high HER2-specific IgGs. Additionally, cellular vaccine responses were induced, e. g. Th1-biased cytokine ratios and reduction of Tregs. The clinical response (progression free survival) was significantly prolonged in cohort 3 and showed a clear association with the magnitude of humoral and cellular vaccine responses. Conclusions: IMU-131 was well tolerated and safe. The vaccine-induced HER2-specific Abs and cellular responses were dose-dependent and correlated with clinical responses. The highest dose (50 µg) was recommended for further evaluation in a Phase II trial with two arms, chemotherapy with IMU-131 vaccine or chemotherapy alone, which is currently ongoing. https://clinicaltrials.gov/ct2/show/NCT02795988 Citation Format: Marina Maglakelidze, Dinara Ryspayeva, Iurie Bulat, Zoran Andric, Ivan Nikolic, Tanuj Chawla, Rajnish Nagarkar, Vaibhav Chourdhary, Giri Venkata, Rajesh Kumar Singh, Davorin Radosavljevic, Zoran Petrovic, Ursula Wiedermann, Leslie Chong, Nicholas J. Ede, Rita Laeufle, Anthony Good. A PHASE 1B/2 OPEN-LABEL STUDY WITH RANDOMIZATION IN PHASE 2 OF IMU-131 HER2/NEU PEPTIDE VACCINE PLUS STANDARD OF CARE CHEMOTHERAPY IN PATIENTS WITH HER2/NEU OVEREXPRESSING METASTATIC OR ADVANCED ADENOCARCINOMA OF THE STOMACH OR GASTROESOPHAGEAL JUNCTION [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT107.

Abstract 1798: CTLA-4 photoimmunotherapy depletes intratumoral regulatory T cells (Tregs) to augment local and systemic immunity for anticancer activity

Abstract Introduction: Photoimmunotherapy is an investigational cancer treatment platform that utilizes an antibody conjugated to a light activated dye IRDye® 700DX (IR700) combined with illumination with a non-thermal, red light for selective cell killing. CTLA-4 is a lymphocyte exhaustion marker targeted by anti-CTLA-4 checkpoint inhibitors to induce anticancer immune response and is highly expressed on intratumoral regulatory T cells (Tregs). In this study we used anti-CTLA-4-IR700 photoimmunotherapy (CTLA-4 photoimmunotherapy), which involves intratumoral Treg depletion with checkpoint inhibition, in syngeneic mouse models, including models refractory to immune checkpoint inhibition, to assess anticancer activity and immune response. Methods: Mice inoculated with CT26, LL/2, MCA205, or 4T1 tumors were treated with CTLA-4 photoimmunotherapy. Tumor volume was determined by caliper measurements in target (illuminated) and distal (non-illuminated) tumors. Intratumoral immune responses were characterized by flow cytometry. To evaluate the activation of systemic immune responses, cytotoxic T lymphocyte assays were conducted with splenocytes derived from mice treated with CTLA-4 photoimmunotherapy. Tumor re-challenge studies were conducted to assess for tumor-specific immune memory responses. Results: CTLA-4 photoimmunotherapy induced notable anticancer responses including complete responses (CRs), and enhanced survival of mice in multiple tumor models, including tumors resistant to anti-PD-1 or anti-CTLA-4 treatment. CTLA-4 photoimmunotherapy resulted in a reduction of intratumoral Tregs, an increase of the CD8/Treg ratio, and an increase of intratumoral NK and CD8 T cell activation in comparison to control groups. All CR mice (n=7) rejected new tumor inoculations, suggesting enhancement of systemic immune memory. Splenocytes derived from CTLA-4 photoimmunotherapy-treated mice displayed an expansion of tumor antigen-specific lymphocytes in comparison to control groups. In mice bearing bilateral CT26 tumors, tumor regression was observed in target and distal tumors after CTLA-4 photoimmunotherapy, demonstrating systemic anticancer immunity and potential abscopal effects. Conclusion: CTLA-4 photoimmunotherapy elicited anticancer responses in a variety of tumor models, including tumors resistant to anti-PD-1 or anti-CTLA-4 treatment. Depletion of intratumoral Tregs by CTLA-4 photoimmunotherapy resulted in the local activation of CD8 T cell-mediated anticancer activity and systemic tumor specific immune response. These results indicate that treatment with CTLA-4 photoimmunotherapy can induce anticancer responses in checkpoint resistant mouse tumor models by combining rapid Treg depletion with anti-CTLA-4 checkpoint inhibition into a single method of treatment. Citation Format: C. Daniel de Magalhaes Filho, Stephanie M. Okamura, Daniele M. Bergeron, Ahiram Rodriguez, Abram Lozano, Jerry J. Fong, Miguel Garcia-Guzman. CTLA-4 photoimmunotherapy depletes intratumoral regulatory T cells (Tregs) to augment local and systemic immunity for anticancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1798.

Abstract 55: TT-702, a selective and potent A2B receptor antagonist for the treatment of cancer

Abstract Adenosine signaling via the A2A and A2B receptors is emerging as an important regulatory mechanism of immune responses. While A2AR has high affinity for adenosine, A2BR is a low-affinity receptor that becomes activated only under pathological conditions when adenosine concentration reaches micromolar ranges. Besides high levels of adenosine (up to 100 μM), the tumor microenvironment (TME) is characterized by hypoxia. Hypoxia upregulates A2BR on tumors and on immune cells. TT-702 is a novel, selective and highly potent, orally active A2BR antagonist. Here we show that TT-702 inhibits tumor growth through multiple mechanisms including enhancement of dendritic cell (DC) function and T-cell activation, reduction of MDSCs and Tregs, and suppression of cancer cell growth, fibrosis and angiogenesis. The cellular potency of TT-702 was assessed in NECA induced cAMP production. The ability of TT-702 to reverse NECA-mediated immune suppression was evaluated using standard human DC differentiation and MLR assays. The effects of TT-702 on tumor growth were determined in B16F10 melanoma and MC38 and CT26 colorectal cancers in mice treated with TT-702 at 0.3, 1, 3 or 10 mg/kg (BID), anti-PD-1 (RMP1-14, 5 mg/kg, Q2D) or TT-702+RMP1-14 intraperitoneally. TT-702 binds to human A2BR with Ki value of 8.3 nM and exhibits greater than 100x selectivity for A1, A2A and A3 receptors. It potently inhibited NECA-stimulated cAMP production (IC50=2.8 nM). TT-702 dose-dependently inhibited the growth of human prostate cancer cells LNCaP and PC-3 (IC50=1.4 μM). Combination of TT-702 with enzalutamide showed an additive and synergistic effects to inhibit LNCaP and PC-3 growth, respectively. TT-702 prevented NECA-induced suppression of human DC differentiation and completely reversed NECA-induced T cell inactivation (EC50=0.45 μM). By contrast, preladenant, an A2AR antagonist, had no effect on cancer cell growth and DC differentiation. Moreover, TT-702 increased MC38 tumor-infiltrating immune stimulatory cells (DCs, CD4+ and CD8+ T cells) and reduced immunosuppressive cells (MDSCs and Tregs). TT-702 dose-dependently inhibited the growth of mouse MC38 and CT26 colorectal cancers. Interestingly, TT-702 also inhibited the growth of mouse B16F10 melanoma cold tumor. Furthermore, combination of TT-702 with anti-PD-1 synergistically inhibited both B16F10 and CT26 tumor growth. Most importantly, TT-702 significantly reduced tumor fibrosis and angiogenesis, suggesting that TT-702 may improve the TME for T cell penetration, cancer killing and prevention of metastasis. TT-702, a novel, selective and potent A2B receptor antagonist, has the potential to treat a diverse range of hard-to-treat and aggressive tumors (including cold ones) as a monotherapy, and in combination with anti-PD-1, enzalutamide and other anticancer therapies. TT-702 exhibits excellent safety and PK profiles in animals and is slated to enter clinical development in 2021. Citation Format: Peidong Fan, Filomena Housley, Helena Haberstock, Ken Horne, Jim Liu, Elfatih Elzein, Lina Yao. TT-702, a selective and potent A2B receptor antagonist for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 55.

Taking AIM2 at regulatory T cells to modulate autoimmune diseases

AIM2 safeguards Treg stability by suppressing the Akt-mTOR pathway via the protein phosphatase PP2A. Chou, WC, Guo, Z, Guo, H, et al. AIM2 in regulatory T cells restrains autoimmune diseases. Nature. 2021; 591: 300– 305. Regulatory T cells that express the transcription factor Foxp3 (Tregs) are a centerpiece in immune tolerance, including tolerance to transplants. However, their suppressive activities are often abrogated in an inflammatory milieu, conditions under which tolerance is destabilized or even lost. Thus, a better understanding of the interface between Tregs and inflammatory responses is therapeutically important. One of the key triggers in inflammation is activation of the inflammasome, a large molecular complex consisting of a cytosolic sensor, an adaptor molecule called ASC and pro-caspase-1/11, which traditionally results in the production of inflammatory cytokines and pyroptotic death of inflammatory cells. In a recent study in Nature, Chou et al. made the surprising finding that besides myeloid cells, Tregs also express AIM2, a cytosolic sensor for double-stranded (ds)DNA. Instead of activating the inflammasome, AIM2 instead safeguards Tregs, protecting them from losing their suppressive functions even under inflammatory conditions that usually subvert Treg functions. Importantly, Treg protection by AIM2 is completely independent of the inflammasome pathway. Thus, besides its classical role in inflammasome activation, AIM2 is also critical in immune tolerance that relies on Tregs. The authors initially took a genetic approach, comparing the induction of experimental autoimmune encephalitis (EAE) in mice deficient for AIM2 (Aim2−/−) and ASC (Asc−/−), and in mice deficient for both caspase-1 and caspase-11 (Casp-1/11 double knockouts). They found that the Aim2−/− mice developed more severe EAE than did the wild-type mice, while the Asc−/− and Casp-1/11 double knockouts were protected from the induction of EAE, suggesting that AIM2 has additional roles other than inflammasome activation. Adoptive transfer experiments involving transfer of wild type and Aim2−/− CD4+ T cells into Rag1−/− hosts recapitulated the enhanced induction of EAE by Aim2−/− CD4+ T cells, which was accompanied by prominent Th17 cells and a marked reduction of Tregs in the spinal cord. In a colitis model, where mucosal injury induced by naïve CD4+ T cells is susceptible to suppression by wild-type Tregs, they showed that the Aim2−/− Tregs failed to suppress the induction of colitis, thus identifying a functional defect of Tregs deficient in AIM2. In a series of ChIP-seq experiments, the authors further showed that the transcription factors RUNX1, EST1, BCL11B and CREB, which are known to regulate Foxp3 expression and Treg stability, also bound to the Aim2 promoter, thus demonstrating a Treg-intrinsic transcriptional network that also favors AIM2 expression in Tregs. The authors then performed sophisticated lineage-tracing experiments, in which Tregs are genetically marked by the expression of a red fluorescent protein (i.e., Foxp3Cre-Rosa26tdTomato mice), and demonstrated that, upon EAE induction, Aim2−/− Tregs readily lost the expression of Foxp3 and became interferon (IFN)-γ–producing T effector cells in the spinal cord (i.e., inflammatory sites), but not in host spleen and lymph nodes, demonstrating a key role for AIM2 in Treg stability. They also found that Aim2−/− Tregs displayed much higher glycolic activities compared with wild-type Tregs, with a concurrent reduction in oxygen consumption rate and fatty acid oxidation, suggesting drastically altered metabolic pathways in Aim2−/− Tregs. Moreover, global transcriptional profiling (RNA-seq) of wild-type and Aim2−/− Tregs, together with gene set enrichment analysis, revealed that the Aim2−/− Tregs showed a prominent IFN-responsive gene signature, which is known to antagonize Foxp3 expression as well as selective enrichment of genes in the Akt-mTOR pathway. In fact, Akt and mTOR were highly activated in Aim2−/− Tregs, as shown by increased protein phosphorylation of Akt, S6 and 4E-BP1 (targets of mTOR), a process that could be blocked by the mTOR inhibitor rapamycin. This heightened activation of Akt and mTOR also prevented the conversion of Aim2−/− T effector cells to inducible Tregs in vitro by transforming growth factor (TGF)-β. In a series of unbiased mass spectrometric experiments, the authors identified and validated RACK1 as a binding partner of AIM2. They further demonstrated that RACK1 recruited the protein phosphatase PP2A, which inhibits the Akt-mTOR pathway by dephosphorylating Akt and reducing mTOR activation. Apparently, in Aim2−/− Tregs, this feedback inhibitory loop is impaired, leading to hyperactivation of the Akt–mTOR pathway, and consequently destabilizing Tregs. Clearly, this study provides novel insights into the role of AIM2, a cytosolic sensor traditionally linked to inflammasome activation, in regulating Treg stability and peripheral tolerance. Xian C. Li, MD, PhD is professor and director at the Immunobiology and Transplant Science Center and Department of Surgery at Houston Methodist Hospital in Texas.

The Dual PI3K-δ,γ Inhibitor Duvelisib Stimulates Anti-Tumor Immunity and Enhances Efficacy of Immune Checkpoint and Co-Stimulatory Antibodies in a B Cell Lymphoma Model

Duvelisib is an orally active, dual inhibitor of phosphoinositide 3-kinase (PI3K)-δ and PI3K-γ which has shown clinical activity as a monotherapy in chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), and T cell lymphoma (O'Brien ASH 2014; Flinn ASH 2014; Horwitz ASH 2014) and is being developed broadly for the treatment of B cell malignancies. Recent publications have demonstrated that PI3K-δ inhibition decreases immunosuppressive regulatory T cells (Tregs) in the tumor microenvironment (Ali, Nature, 2014; Ahmad, Cancer Res 2017) whereas PI3K-γ inhibition decreases immunosuppressive myeloid cells (MDSCs and M2 tumor-associated macrophages) (Kaneda, Nature, 2016; De Henau, Nature, 2016). Accordingly, duvelisib has recently been shown to reduce tumor and spleen MDSCs in a preclinical tumor model (Davis, Cancer Res 2017). These data collectively suggest that reduction of Tregs and immunosuppressive myeloid cells in the tumor microenvironment may contribute to the activity of duvelisib as a monotherapy, and may also augment the efficacy of immune checkpoint or co-stimulatory antibodies.These findings led us to investigate the efficacy of duvelisib (50 mg/kg po, BID) in combination with immune checkpoint (anti-PD-1, RMP1-14) or co-stimulatory (anti-OX40, clone OX86) antibodies. In the murine syngeneic A20 B cell lymphoma model, duvelisib and anti-PD-1 treatments each induced tumor growth delay. When duvelisib and anti-PD-1 were combined in mice bearing pre-existing A20 tumors, strong synergy was observed in the anti-tumor response (see figure). Similarly, duvelisib and anti-OX40 each induced tumor growth delay as single agents in the A20 model. When anti-OX40 and duvelisib were combined, striking tumor regression was observed (see figure). To assess immune memory, tumor-free mice following anti-OX40 alone or anti-OX40 + duvelisib combination were injected with A20 lymphoma cells in the contralateral flank with no further treatment. Whereas the mice that had received anti-OX40 alone grew new tumors upon A20 re-challenge, all tumor-free mice that had received the anti-OX40 + duvelisib combination did not grow A20 tumors upon re-challenge and showed elevated memory T cells in the blood and spleen. These findings demonstrate that the anti-OX40 + duvelisib treatment established immune memory, potentially contributing to the observed tumor regression.These data indicate that duvelisib treatment stimulates anti-tumor immunity. Furthermore, the unique dual inhibition of PI3K-δ and PI3K-γ may make duvelisib especially effective in enhancing the anti-tumor efficacy of immune checkpoint and co-stimulatory antibodies. These data support further exploration of duvelisib in combination with anti-PD-1/PD-L1 or co-stimulatory antibodies in patients with B cell malignancies. [Display omitted] DisclosuresPachter:Verastem, Inc.: Employment. Weaver:Verastem, Inc.: Employment.

Myeloid-Derived Suppressor Cell Subtypes Are Responsible for the Th2 Phenotype of T Cells in CLL

In chronic lymphocytic leukemia (CLL), monoclonal B cells expand and accumulate in the blood, bone marrow, spleen and secondary lymphoid organs in response to incompletely defined cellular and molecular interactions involving multiple cell types within the tumor microenvironment (TME). Bidirectional interactions of CLL cells with components of the TME - mesenchymal stromal cells, myeloid cells, T cells, and matrix - are critical for leukemic cell survival and proliferation. Here, we focused on understanding how two non-leukemic cell types within the TME, T lymphocytes and myeloid-derived suppressor cells (MDSCs), interact to create a TME supportive of a CLL clone.In CLL, it has been suggested alterations in CD4+ and CD8+ T cell ratios and functions are responsible for a non-inflammatory and immunosuppressive cytokine TME, with expansions of Th2s and Tregs. Conversely, higher numbers of Th17 and CD8+ cells are associated with longer survival. MDSCs are not well studied in CLL, although existing reports suggest that their expansion suppresses T cell proliferation in vitro. Here we studied the effects of MDSCs and CLL B cells on T cell numbers and function.First, we identified a correlation between blood T cells and MDSCs in 56 CLL patients. Absolute numbers of MDSCs correlated with total numbers of CD3+, CD4+ and CD8+ cells (P= 0.002, < 0.001, < 0.001 and Spearman r = 0.44, 0.646, = 0.61, respectively). However, the correlation of MDSC subtypes with CD4+ and CD8+ cells differed: monocyte-like MDSCs (mMDSCs) associated with CD4+ cells (P < 0.001, Spearman r = 0.73) whereas granulocyte-like MDSCs (gMDSCs) associated with CD8+ cells (P= 0.008; Spearman r = 0.45).Moreover, in a subset of 18 CLL patients tested, MDSC numbers directly correlated with the levels of Th2 (P= 0.049, Spearman r = 0.470), Th17 (P= 0.040, Spearman r = 0.488), Tc17 (P= 0.018, Spearman r = 0.550) and Tc22 (P= 0.010, Spearman r = 0.587) cells. When dividing MDSCs into subsets, gMDSCs exhibited a similar correlation except for the absence of Th2 and the presence of a correlation with Th22 (P= 0.033, Spearman r = 0.505). Conversely, mMDSCs positively associated with Th1 (P= 0.024, Spearman r = 0.549) and Tc1 (P= 0.019, Spearman r = 0.569) cells.We next addressed the functional effect of MDSCs on naïve CD4+ cell differentiation using samples from 16 CLL patients and 10 healthy, age-matched controls (HCs). CD3+CD45RO-CD62L+ CD4+ lymphocytes were FACS-sorted and stimulated in vitro with anti-CD3/CD28 beads plus IL-2, and the percentages of CD4+ T cells producing various cytokines were quantified by intracellular flow cytometry. These results were compared with those obtained in the presence of mMDSCs (HLA-DRlo/CD11b+/CD33+/CD14+), gMDSCs (HLA-DRlo/CD11b+/CD33+/CD15+), monocytes (HLA-DRhi/CD11b+/CD14+) or CLL B lymphocytes (CD19+). On day 7, CLL T cells, cultured in the absence of other cells, generated a higher percentage of cytokine-producing cells than those from HCs (22% vs. 8%; P < 0.001). This difference was due to an increase in all cytokine-producing cells except classical Th17s producing IL-17A. When CLL T cells were co-cultured with gMDSCs, a significant expansion of Th2 (IL-4+) (P= 0.044) and a trend in growth of IL-17F+ Th17cells (P= 0.101) was found. In contrast, co-culture of CLL T cells with mMDSCs led to a reduction in Th22 (IL-22+) (P= 0.005) and Treg (FoxP3+) (P= 0.006) cells and to a trend towards expansion of Th2 (IL-4+) cells. Co-cultures of CLL T cells with monocytes, similarly to mMDSCs, led to an expansion of Th2 (IL-4+) cells (P= 0.01) and a reduction in Treg (FoxP3+) cells (P= 0.002). Finally, co-culturing naïve CD4+ lymphocytes with CLL B cells had a different and unexpected effect, promoting the expansion of Th1 (IFNγ+) cells (P= 0.033) and Th22 (IL-22+) T cells (P= 0.052).In summary, the absolute number of blood T cells, CD4+ and CD8+ subsets, and MDSCs are linked, although associations of T cell subsets differ between mMDSCs and gMDSCs. MDSCs and their subsets influence naïve T cell differentiation differently, in general leading to expansion of Th2 (IL-4+) cells. In contrast, CLL B cells had a different effect on T cell polarization, preferentially expanding Th1 (IFNγ+) cells. Thus, it appears that myeloid cells and especially MDSCs are responsible for polarization of the T cell compartment to Th2 which is said to be dominant in CLL patients. DisclosuresKolitz:Gilead, Magellan, and Novartis: Honoraria; Gilead, Novartis, and Seattle Genetics: Other: Travel Support; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Celgene, Jazz: Equity Ownership; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy. Barrientos:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy; Gilead: Consultancy, Research Funding. Stamatopoulos:Novartis SA: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

Functional Characteristics of FAS-L Resistant and IL-2 Deprived Regulatory T Cells (Treg A) in Idiopathic Aplastic Anaemia

IntroductionAplastic anemia (AA) is an immune mediated bone marrow failure syndrome characterized by reduced numbers and dysfunctional regulatory T cells (Tregs). Using deep phenotyping, we have previously identified an immune signature that predicts clinical response to immunosuppression (IST) (Kordasti et al., Blood 2016). The so-called Treg A (CD4+CD25hiCD127loCD45RAhiCD95-CCR4lo) were higher in IST non-responders, whereas responder patients had a higher proportion of Treg B (CD4+CD25hiCD127loCD45RAloCD95+CCR4hi). The aims of the current study were to investigate the reasons for the reduction of specific Treg subset, the ex vivo expandability of the remaining Tregs in response to IL-2, their function and stability following expansion. The efficacy and longevity of these cells were tested in an animal model, with the aim of using autologous ex vivo expanded Tregs as a potential treatment for AA.Methods and ResultsFAS-L-induced apoptosis leads to reduction of Treg B, this effect can be reduced by low-dose IL-2Given the role of FAS-L positive T cells in the pathophysiology of AA pathophysiology and the fact that CD95 (FAS) is one of the significant non-redundant markers that defines Treg B, we tested the FAS-L sensitivity of these cells. After 5 hours culture with FAS-L (5 mg/ml), the number of apoptotic cells was significantly higher in Treg B compared to Treg A (3 replicates, p < 0.02). The effect was prevented by the addition of low-dose IL-2 (5 U/mL) to the culture.Treg expansion and their functionTotal Tregs from AA and healthy donors (HDs) are sensitive to low dose IL-2 as shown by STAT5 phosphorylation (p < 0.001, n = 6 AA and 2 HDs). Moreover, Tregs from AA and HDs can be expanded polyclonally at a comparable rate (fold increase 33x vs 21x, p = n.s. n = 6 AA and 8 HDs) in a Treg promoting culture (stimulation with anti CD3/CD28 beads and IL-2 1,000 IU/ml for 4 weeks with all-trans retinoic acid and rapamycin). Although Treg A had lower level of p-STAT5 in the presence of low-dose IL-2, this reached to a similar level to Treg B following culture with higher doses of IL-2. To investigate the potential differences between Treg A and B in terms of function and expandability, Treg A and B were sorted from 6 HDs and 4 AA patients, and then expanded for 4 weeks (as above). Treg A and B expanded in HDs (Treg A 45x, and B 33x fold increase, p = n.s.), whereas Treg A expanded at a higher rate compared to Treg B in AA (Treg A 2120x, and B 90x fold increase, p = 0.03). The in vitro expanded Treg A and B both suppress proliferation of conventional T cells (Tconv) (n = 6, p < 0.001, no difference between A and B, figure a). Treg stability was assessed by DNA methylation analysis using deep amplicon bisulfite sequencing of the Treg-specific demethylated region (TSDR) . TSDR CpG sites in the expanded Treg A and Treg B populations from HD and AA patients are 2% and 13% methylated respectively, confirming their stability.Treg function in vivoTo test the suppressive ability of these Tregs, we transplanted NSG-SGM3 mice with either HDs PBMCs alone (n = 3), or HDs ex vivo expanded Tregs alone (n = 3) or HDs PBMCs co-injected with HDs ex vivo expanded Tregs (n = 3). Mice injected with both PBMCs and Tregs received a further dose of Tregs at day 16 and then weekly injections until day 28. Mice injected with PBMCs only had worst overall survival compared to the mice with PBMCs + Tregs or Tregs only (13 versus 35 days, p < 0.0001) and developed acute GVHD during the first 10 days following the injection (day 13 PBMCs mice 17.36 g versus PBMCs + Tregs mice 22.7 g, p = 0.0052, figure b). There was no weight difference between PBCs + Tregs mice and Tregs only mice.ConclusionsIn summary, although the FAS-L sensitive Tregs, which are the most functional Tregs, are reduced in AA, the remaining Tregs in these patients are IL-2 responsive and can be expanded in vitro . We were able to sort Treg subpopulations (Treg A and B) from AA patients and show that they are expandable, stable and functional up to 8:1 ratio with Tconv. We have also tested the efficacy of ex vivo expanded Tregs in preventing GVHD in a mouse model. Overall, the current study suggests that in AA there may be an increased exposure to FAS-L and an insufficient level of IL-2, which selectively induce apoptosis of Treg B. Moreover, the ex vivo expansion of stable and functional AA Treg subsets is feasible. The expanded Tregs are likely to be able to control the inflammatory response in these patients, hence pave the way toward a novel cellular therapy for AA. [Display omitted] DisclosuresKordasti:Celgene: Research Funding.

Features in genomics and tumor immune microenvironment in NSCLC treated with neoadjuvant PD-1 blockade.

9063 Background: Results from several clinical trials have preliminarily demonstrated the safety and effectiveness of single PD-1 inhibitors in neoadjuvant setting for resectable non-small cell lung cancer (NSCLC). However, only around 40% patients could achieve Major Pathological Response. How to select patients who could benefit from single PD-1 blockade remains elusive. Methods: In this study, we aimed to assess the association of PD-L1 expression, tumor mutation burden (TMB), copy number alteration (CNA, including copy number gain and loss) burden with the pathological response to neoadjuvant PD-1 blockade. We also evaluated the dynamic changes of tumor immune microenvironment (TIME) by analyzing pre-immunotherapy treatment tumor biopsy samples from twenty-nine NSCLC patients as well as the matched post-surgery samples after neoadjuvant sintilimab treatment and resection. Targeted DNA sequencing (543 genes), PD-L1 immunochemistry staining (22C3) and multiplex immunofluorescence (CD4, CD8, CD9) were applied. Results: The degree of pathological regression and major pathological response (MPR), were positively correlated with tumor proportion score (TPS) of PD-L1 (R = 0.40, p = 0.04) and negatively correlated with copy number gain (CNgain) burden (R = -0.44, p = 0.04). Of note, the combination of CNgain burden and TPS can better stratify MPR patients compared to CNgain or TPS alone. Whereas, TMB only had a marginal association with pathological response (R = 0.32, p = 0.15). Additionally, PD-1 blockade led to an increase in CD8+PD-1-T cells in the tumor region (p = 0.04, Mann-Whitney U test for paired samples) and a reduction in Tregs and M2 macrophages in the stromal region (p &lt; 0.05, Mann-Whitney U test for paired samples). Further investigation showed that the degree of CD8+PD-1-T cell increase was significantly associated with MPR (p &lt; 0.05, Mann-Whitney U test). Intriguingly, we also observed a substantial reduction in CD19+ cells in the non-MPR group but not in the MPR group, indicating the involvement of B cells in improving neoadjuvant immunotherapy response in NSCLC patients. Conclusions: TPS and CNgain burden were correlated with pathological response to neoadjuvant immunotherapy in NSCLC patients. This may provide potential selective indicators for future clinical trials of neoadjuvant immunotherapy. The dynamic changes of components in the tumor immune microenvironment may provide novel insight into the immune responses induced by neoadjuvant PD-1 blockade therapy.

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.