Suppression Of Cell-mediated Immunity Research Articles

Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression

Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-β RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-β RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-β RII-inactivated cancers.

Focal haemorrhage as atypical feature on MRI in progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a severe progressive demyelinating disorder caused by the suppression of cell-mediated immunity and is diagnosed by typical imaging features on MRI and confirmed by the identification of the JC virus on CSF. We present a case of HIV positive adult male who presented with progressive hemiparesis and spastic dysarthria over 3 months duration, and MRI showed white matter T2 hyper, T1 isointense and peripherally diffusion restricting lesion in the right frontoparietal region with an additional focal haemorrhage within the lesion, which misled us away from considering PML. However, repeat imaging showed progression of the lesion and CSF analysis showed John Cunningham (JC) virus, confirming the diagnosis.

Disruption of IFNγ, GZMB, PRF1, or LYST Results in Reduced Suppressive Function in Human CD8+ T Cells.

An imbalance between proinflammatory and regulatory processes underlies autoimmune disease pathogenesis. We have shown that acute relapses of multiple sclerosis are characterized by a deficit in the immune suppressive ability of CD8+ T cells. These cells play an important immune regulatory role, mediated in part through cytotoxicity (perforin [PRF]/granzyme [GZM]) and IFNγ secretion. In this study, we further investigated the importance of IFNγ-, GZMB-, PRF1-, and LYST-associated pathways in CD8+ T cell-mediated suppression. Using the CRISPR-Cas9 ribonucleoprotein transfection system, we first optimized efficient gene knockout while maintaining high viability in primary bulk human CD8+ T cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry where appropriate, as well as confirmation of insertions and/or deletions at genomic target sites. We observed that the knockout of IFNγ, GZMB, PRF1, or LYST, but not the knockout of IL4 or IL5, resulted in significantly diminished invitro suppressive ability in these cells. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T cell-mediated suppression that could be targeted for immunotherapeutic intervention.

Cytomegalovirus Occurrence and Time-to-onset Analysis Under Bendamustine With Anti-CD20 Antibodies Using the JADER Database.

Patients with malignant lymphoma, in a latent state of weakened immune function, are at risk of chemotherapy-induced immunosuppression and cytomegalovirus (CMV) infection. Concomitant therapy with bendamustine and rituximab or obinutuzumab intensifies immunosuppression, potentially affecting CMV onset. This study aimed to assess CMV onset differences between bendamustine monotherapy and combination therapy with rituximab or obinutuzumab using the Japanese Adverse Drug Event Report database (JADER). A JADER analysis dataset (April 2004 to September 2022) defined CMV infection using 31 preferred term (PT) words from MedDRA 25.1J HLT "Cytomegalovirus infection (10011827)". Reporting odds ratios (ROR) calculated CMV infection signals for bendamustine monotherapy, rituximab, obinutuzumab, bendamustine+rituximab (BR), and bendamustine+obinutuzumab (GB). ROR confidence intervals exceeding 1 indicated a CMV signal. Days of CMV infection were calculated based on adverse event onset and administration start. CMV signals were confirmed for monotherapy and combination therapies. CMV infection durations (median, interquartile range) were 41.0 days (23.5-69.5) for bendamustine monotherapy, 63.5 days (35.2-95.0) for BR, and 61.0 days (33.0-102.5) for GB, with cases exceeding 200 days. JADER analysis detected significant CMV signals for rituximab, obinutuzumab, and bendamustine. Caution may be warranted 7-9 months post-bendamustine administration, necessitating further investigation, including cell-mediated immunity suppression assessment.

Vitamin D Levels and Microvascular Complications in Type 2 Diabetes Mellitus Patients: A Study from North Keral

Introduction: Vitamin D plays an important role in glucose metabolism. Several pleiotropic effects of Vitamin D have been studied like regulation of cell proliferation, suppression of cell mediated immunity, Glial cell line-derived neurotrophic factor, stimulation of nerve growth factor, suppression of Renin-angiotensin-aldosterone system (RAAS), reduction of albuminuria, immunomodulatory effects, and anti-inflammatory effects.(1)Thus, vitamin D is implicated in the pathogenesis of retinopathy, neuropathy and nephropathy.
 Materials and Methods: A Cross-sectional case-control study of 206 patients (>30 years), who met the American Diabetes Association, 2011 criteria (2) for type 2 DM, was conducted. Subjects were evaluated for the presence of microvascular complications by clinical evaluation, urine examination, fundus examination, clinical neuropathy scoring, and various biochemical tests. 25-OH cholecalciferol levels were done and cut off level for vitamin D deficiency was 30 ng/ml. 72 Vitamin D sufficient and 134 Vitamin D deficient cases were studied.
 Results: A total of 134 T2DM cases with Vitamin D deficiency were studied. 72 age matched T2DM patients with sufficient Vitamin D levels served as controls. The mean age of cases under study was 47.58 ± 9.63 years compared to 51.24 ± 8.75 years of controls. 61.2% were females, whereas 38.8% were males in the case group while in the control group, 69.4% were males, and 30.5% were females. The mean duration of diabetes in the cases studied was 57.08 ± 18.13 months. It was 52.69 ± 17.98 months in the Vitamin D sufficient controls. Significant statistical associations were established using Chi square test between Vitamin D status and development of microvascular complications like Diabetic nephropathy, retinopathy and neuropathy in Type 2 Diabetes Mellitus at p < 0.05.
 Conclusion and Implication: Vitamin D is an important factor in modifying the risk of Type 2 DM and its microvascular complications, especially Diabetic retinopathy, neuropathy and nephropathy. Administration of vitamin D supplements may prove to be a beneficial adjuvant therapy in mitigating microvascular complications. Frequent Monitoring of Vitamin D status can decrease the burden of co-morbidities in T2DM and thus can decrease the mortality rate associated with T2DM. However; the exact role of vitamin D in these processes requires further investigations.
 Keywords:
 Diabetes, Nephropathy, Neuropathy, Retinopathy, Vitamin D

Abstract 2936: INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1

Abstract Transforming growth factor-β (TGFβ) signaling is common in many solid tumors and is initiated by binding of the high affinity canonical ligands TGFβ1, 2, οr 3 to TGFβR2, which forms a heteromeric receptor complex with TGFβR1 (Derynck et al, Nature Review Clinical Oncology 2020). Activation of the pathway results in potent suppression of immune cell-mediated anti-tumor immunity and has been reported to predict poor response to PD-(L)1 targeted therapy in patients (Mariathasan et al, Nature 2018; Kieffer et al, Cancer Discovery 2020). However, TGFβ drug development has been hampered by the occurrence of adverse events. INCA33890 is a dual PD-1 and TGFβR2 binding bispecific Biclonics® antibody, developed to antagonize the TGFβ signaling pathway specifically in cells co-expressing PD-1 and TGFBR2. Additionally, it potently antagonizes the PD-1 axis independently of TGFβR2 co-expression. The cell-selective action of INCA33890 was designed to mitigate risks of the known adverse effects associated with TGFβ-pathway inhibition in tissues requiring active TGFβ signaling. ΙΝCΑ33890 mediates its specificity through a PD-1 binding arm with a >10-fold higher affinity relative to the TGFβR2 binding arm. Consistent with this profile, in isogenic Jurkat cells expressing TGFβR2 ± PD-1, INCA33890 potently inhibits TGFβ1-induced pSMAD activation in a PD-1-correlated manner. Additionally, in two independent PD-1 reporter assays, INCA33890 inhibited SHP recruitment and enhanced NFAT activation with a potency within an order of magnitude to that of pembrolizumab. In mixed lymphocyte reaction assays with exhausted primary human T-cells, INCA33890 was found to induce a similar level of anti-tumor cytokine production as the combination of pembrolizumab and an anti-TGFβR2 antagonist mAb. Treatment of primary ovarian ascites with INCA33890 ex vivo induced IFNγ production in all donors tested, while pembrolizumab had no activity. Similarly, in human CD34+ cell-engrafted NSG mice, INCA33890 significantly inhibited the growth of human MDA-MB-231 and A375 subcutaneous xenograft tumors, whereas pembrolizumab or an anti-TGFβR2 antibody had little or no monotherapy activity. INCA33890 had a balanced pharmacokinetic and potency profile and was well tolerated in NHPs at exposures required for pharmacodynamic and tumor growth inhibiting activity in rodents. Encouragingly, there was no evidence of adverse effects in NHPs due to TGFβ-pathway blockade. Collectively, these results provide compelling data for an effective and specific approach to simultaneously antagonizing TGFβ and PD-1 signaling in tumors. Clinical development of INCA33890 in checkpoint inhibitor-resistant and other cancers has been initiated. Citation Format: Liang-Chuan S. Wang, Rinse Klooster, Ashwini Kulkarni, Amaya Garcia de Vinuesa, Maxim Soloviev, Linda JA Hendriks, Lu Huo, Michael Weber, Arpita Mondal, Yonghong Zhao, Shane Harvey, Xin He, Hong Chang, April Horsey, Alla Volgina, Yue Zhang, Veethika Pandey, Yan-Ou Yang, Jonathan Rios-Doria, Evgeniy Eruslanov, Daniel J. Powell, Steven M. Albelda, John de Kruif, Horacio Nastri, Cecile Geuijen, Patrick A. Mayes. INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2936.

Disruption of IFNγ, PRF1, GZMB, or LYST results in reduced suppressive function in human CD8+ T cells

Abstract Human CD8+ T-cells play an important immune regulatory role during autoimmune diseases, characterized by suppressing the activation and proliferation of pathogenic effector cells, mediated in part through cytotoxicity (PRF/GZM), IFNγ secretion or by Fas/FasL interactions. Recent studies from our group demonstrated that IL-12 exposure during in vitro differentiation of naïve CD8+ T-cells induced an immune-suppressive deficit, with Tc1 cells showing significant loss of suppressive ability against CD4+ T-cells. RNAseq analysis revealed that various cytokines, transcription factors, and other regulatory molecules were differentially regulated in these cells. In this study, we further investigated the importance of IFNγ-, PRF1-, GZMB- and LYST-associated pathways in the suppressive ability of CD8+ T-cells. Using the CRISPR-Cas9 RNP optimized transfection system in primary bulk human CD8+ T-cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry when possible, as well confirmation of indels at genomic target sites. We observed that knockout of each of the four molecules, but not the knockout of IL-4 or IL-5, resulted in significantly diminished suppressive ability, as measured through in vitro immune suppression assays. Interestingly, in some of the knockouts, we observed differential expression of non-target molecules indicating potential functional links between these pathways. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T-cell-mediated suppression that could be targeted for immunotherapeutic intervention. This work was supported, in part, by grant awards to NJK from the NIH (R01 AI121567) and VA.

Global evolution of the tumor microenvironment associated with progression from preinvasive invasive to invasive human lung adenocarcinoma.

SUMMARYTo investigate changes in the tumor microenvironment (TME) during lung cancer progression, we interrogate tumors from two chest computed tomography (CT)-defined groups. Pure non-solid (pNS) CT density nodules contain preinvasive/minimally invasive cancers, and solid density nodules contain invasive cancers. Profiling data reveal a dynamic interaction between the tumor and its TME throughout progression. Alterations in genes regulating the extracellular matrix and genes regulating fibroblasts are central at the preinvasive state. T cell-mediated immune suppression is initiated in preinvasive nodules and sustained with rising intensity through progression to invasive tumors. Reduced T cell infiltration of the cancer cell nests is more frequently associated with preinvasive cancers, possibly until tumor evolution leads to a durable, viable invasive phenotype accompanied by more varied and robust immune suppression. Upregulation of immune checkpoints occurs only in the invasive nodules. Throughout progression, an effector immune response is present but is effectively thwarted by the immune-suppressive elements.

Combination of ultrasound-based mechanical disruption of tumor with immune checkpoint blockade modifies tumor microenvironment and augments systemic antitumor immunity

BackgroundDespite multimodal adjuvant management with radiotherapy, chemotherapy and hormonal therapies, most surgically resected primary breast cancers relapse or metastasize. A potential solution to late and distant recurrence is to augment...

Abstract 2680: SERPINB3 promotes myeloid cell chemotaxis and associated chemokine production in cervical cancer

Abstract Squamous cell carcinoma antigen (SERPINB3) is a widely used biomarker for epithelial cancers including uterine cervix, head and neck, and esophagus. We have previously shown that SERPINB3 contributes to radiation resistance, mainly due to its predominant function as a protease inhibitor that interfered with lysosomal cell death. In addition, elevated SERPINB3 is also observed in various inflammatory conditions related to increased cytokine expression such as, atopic dermatitis, psoriasis, asthma and chronic obstructive pulmonary disease, indicative of potential immunoregulatory function of SERPINB3. We hypothesized that elevated SERPINB3 may also contribute to altered tumor immunity, leading to reduced sensitivity and therapeutic effects of radiation treatment. Thus, we sought evidence of SERPINB3 expression in relation to immune regulation using RNA sequencing analysis of cervical tumor biopsies from 66 patients, and identified a positive correlation of SERPINB3 with chemokines CXCL1, CXCL8, S100A8, and S100A9. These chemokines are known to induce myeloid cell accumulation in the tumor microenvironment, and the presence of myeloid cells have been shown to reduce the efficacy of radiotherapy in cervical cancer. Indeed we found enrichment of myeloid cell subsets on immune cell prediction analysis with xCell in SERPINB3-high tumors. We then examined expression and secretion of myeloid-attractant chemokines via quantitative PCR and ELISA in SERPINB3-overexpressing cervical cancer cells, and confirmed the upregulation of CXCL1/8 and S100A8/A9. Supernatants collected from SERPINB3-overexpressing cells contained chemoattractant to myeloid cells as evidenced by transwell assay using human peripheral blood mononuclear cells from patients with cervical cancer. In the mouse models, tumors formed by SERPINB3-expressing cells induced an immunosuppressive microenvironment with the increased infiltration of M2 macrophages and MDSCs in tumors and spleens. Furthermore, we observed the elevated phosphorylation of STAT1/3/5 transcription factors, known to regulate inflammatory chemokine expression, in SERPINB3-high cells. In our patient cohort, RNA-seq analysis of biopsies collected from 20 patients both before and 3 weeks into chemoradiation therapy (CRT) were analyzed for changes of SERPINB3 in comparison to chemokine expression. We found that patients with decreased SERPINB3 expression in the mid-CRT biopsy (n=7) showed downregulated S100A8/A9 levels in tumors while patients with increased SERPINB3 (n=13) had upregulated S100A8/A9 expression. Our findings suggest that SERPINB3 promotes chemokine secretion involved in myeloid cell-mediated immune suppression which may contribute to negative impacts on radiation treatment in patients with cervical cancer. Citation Format: Liyun Chen, Songyan Wang, Victoria Shi, Fiona Ruiz, Kay Jayachandran, Jin Zhang, Pippa Cosper, Perry Grigsby, Julie Schwarz, Stephanie Markovina. SERPINB3 promotes myeloid cell chemotaxis and associated chemokine production in cervical 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 2680.

Abstract 1719: OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages

Abstract Background: OR2805 is a fully human IgG1 antibody that binds to CD163, an immune-suppressive receptor highly expressed on tumor associated macrophages (TAMs). High numbers of CD163-expressing TAMs generally predict an unfavorable prognosis in solid tumors. CD163-expressing TAMs contribute to an immune-suppressive tumor microenvironment (TME) and inhibit an anti-tumor T-cell response by engaging immune checkpoints, producing immune-suppressive cytokines, and promoting T-cell skewing towards a pro-cancer Th2 phenotype. Relieving the immune suppression of CD163-expressing TAMs in the TME to improve T-cell-mediated responses is a rational adjunct to immune checkpoint inhibitor (CPI) therapy. Methods: OR2805 was discovered using OncoResponse's discovery platform by cell-based functional and phenotypic assays. B cells derived from CPI elite responders were cultured at clonal density, and IgG antibodies in supernatants were evaluated for binding to myeloid-derived suppressor cells. Variable-regions from positive hits were sequenced, cloned, and expressed as recombinant IgG1. OR2805 was identified as one of the top hits that relieved myeloid cell-mediated immune suppression. Co-cultures of immunosuppressive primary human M2 macrophages and autologous T cells were used to interrogate OR2805-dependent immunomodulatory responses in vitro. The anti-tumor activity of OR2805 in vivo was evaluated in a humanized mouse model. Results: OR2805 binds to CD163 expressed on TAMs and M2-like macrophages. OR2805 does not bind to other hematopoietic cells nor to a panel of human primary non-immune cells. OR2805 treatment reduces expression of cell-surface markers associated with tumor-promoting M2c-like macrophages. In co-culture assays, OR2805 relieves the suppressive effect of M2 macrophages and results in increased T-cell activation and proliferation, upregulation of T-cell activation markers, and enhanced T-cell-mediated tumor cell killing. Administration of OR2805 in humanized NSG-SGM3 mouse tumor models resulted in approximately 55% and 75% reduction in A549 tumor growth and NCI-H1975 tumor growth, respectively. In this model, OR2805 treatment significantly increased the proportions of human CD8+ T cells and human CD11b+ myeloid cells, as well as significantly enhanced expression of activation markers by human CD8+ T cells. Conclusions: OR2805 reduces M2 macrophage-mediated immunosuppression and enhances anti-tumor immune responses. OR2805 treatment induces anti-tumor activity in lung cancer xenograft models in humanized mice. These data support further development of OR2805 as an anti-cancer therapy, both as a monotherapy and an addition to current CPI therapy. Citation Format: Peter Probst, Randi Simmons, Valerie Wall, Meghan Zuck, Myriam Bouchlaka, Sam Lam, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri. OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages [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 1719.

Targeted delivery of regulatory macrophages to lymph nodes interferes with Tcell priming by preventing the formation of stable immune synapses.

Immunosuppressive myeloid cells are frequently induced in tumors and attenuate anti-tumor effector functions. In this study, we differentiate immunosuppressive regulatory macrophages (Mregs) from hematopoietic progenitors and test their potential to suppress adaptive immune responses in lymph nodes. Targeted delivery of Mregs to lymph nodes is facilitated by retroviral overexpression of the chemokine receptor CCR7 and intra-lymphatic cell application. Delivery of Mregs completely abolishes the priming of cognate CD8 cells and strongly reduces delayed-type hypersensitivity reactions. Mreg-mediated Tcell suppression requires cell-cell contact-regulated nitric oxide production. Two-photon microscopy reveals that nitric oxide produced by Mregs reduces the interaction duration between dendritic cells and Tcells. Exposure of activated Tcells to nitric oxide strongly reduces their binding to ICAM-1, indicating that nitrosylation of proteins involved in cell adhesion affects synapse formation. Thus, this study identifies a mechanism of myeloid cell-mediated immune suppression and provides an approach for its therapeutic use.

Enzalutamide, an Androgen Receptor Antagonist, Enhances Myeloid Cell-Mediated Immune Suppression and Tumor Progression.

Androgen receptor (AR) antagonism increases overall survival in prostate cancer; however, treatment failure leads to tumor progression and patient mortality. The effect of AR modulation on AR+ nontumor cells that participate in the resistance to AR antagonism is poorly understood. Tumor-infiltrating myeloid cells, including macrophages and myeloid-derived suppressor cells (MDSC), express AR and promote prostate cancer progression. We investigated how AR antagonism affects myeloid cell function and metabolism in an AR-independent murine colon tumor model. Systemic blockade of AR with enzalutamide resulted in increased MC-38 tumor growth in vivo even when AR was knocked out of MC-38 tumor cells. MC-38 tumor growth was also increased when immunocompetent, but not immunodeficient, mice were coinjected with tumor cells and MDSCs treated with enzalutamide or lacking AR, suggesting that AR regulated the ability of MDSCs to suppress adaptive immunity. Myeloid AR-knockout male mice also displayed increased growth of TRAMP C2 prostate tumors when compared with wild type. Inhibition of AR signaling suppressed mitochondrial respiration in myeloid cells via MPC/AMPK signaling pathways; suppression of mitochondrial respiration increased MDSC tumor-promoting functions. Our work showed that AR regulates a tumor-promoting myeloid cell phenotype and influences myeloid cell metabolism. These findings suggest that tumor resistance to AR antagonism is due, in part, to changes in myeloid cell function and metabolism.

Abstract 937: Quantitative assessment of CD200 and CD200R expression in lung cancer

Abstract Background: CD200 is a membrane-bound glycoprotein expressed on normal tissue, tumor and immune cells. The interaction of CD200 with its receptor leads to attenuation of the inflammatory process, resulting in tumor cell mediated immune suppression. CD200 has been characterized in hematologic malignancies and consists of an independent negative prognostic factor for chronic lymphocytic leukemia, acute myeloid leukemia and multiple myeloma patients. Here we assess both CD200 and CD200R expression in lung cancer and evaluate its association with clinicopathologic characteristics, mutation status, outcome and PD-L1 expression. Methods: We used quantitative multiplexed immunofluorescence (QIF) to measure the expression of CD200 and CD200R in 287 non-small cell lung cancer (NSCLC) patients and 30 patients with large cell neuroendocrine carcinomas (LCNEC) of the lung. We performed target measurement with tyramide-based QIF panels and analyzed the data using the PM2000 microscope and AQUA software. Targets were measured in cytokeratin-positive (CK+) tumor compartment and cytokeratin-negative (CK-) stromal compartment. Results: CD200 tumor positivity was found in 29.7% of NSCLC patients and 33.3% of LCNEC patients. CD200 demonstrated notable intratumoral heterogeneity (R2=0.09-0.31 between different TMA blocks versus 0.46-0.60 for CD200R). CD200R was expressed in immune cells in 25% of NSCLC and 41.3% of LCNEC patients. While CD200R is predominantly expressed in immune cells, rare tumor cell staining was seen in a highly heterogeneous pattern. CD200R expression in the stromal compartment was significantly higher in patients with squamous differentiation (p<0.0001). Neither CD200 nor CD200R were associated with other clinicopathologic characteristics or mutation status. Both CD200 and CD200R were not prognostic for disease-free or overall survival in NSCLC. Since these biomarkers are associated with immune modulation, both markers were assessed for correlation with PD-L1. CD200 showed moderate correlation with PD-L1 (R2=0.28 in the CK+ compartment and 0.40 in the CK- compartment); CD200R was less correlated with PD-L1 (R2=0.07 in the CK+ compartment and 0.10 in the CK- compartment). Conclusions: CD200 and CD200R pathway represents a novel immune checkpoint, frequently expressed in lung cancer patients. Co-expression patterns of CD200 and CD200R with PD-L1 suggest a potential role for targeting this pathway alone or in combination with PD-1/PD-L1 axis inhibitors. Citation Format: Ioannis A. Vathiotis, Tyler MacNeil, Jon Zugazagoitia, Sandra Martinez-Morilla, Fahad Ahmed, Konstantinos N. Syrigos, Aaron M. Gruver, Kyla Driscoll, David L. Rimm. Quantitative assessment of CD200 and CD200R expression in lung 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 937.

Abstract LB-088: A STAT3 selective targeted protein degrader decreases the immunesuppressive tumor microenvironment and drives antitumor activity in preclinical models

Abstract Signal transducer and activator of transcription 3 (STAT3) has been implicated in multiple aspects of tumorigenesis. In addition to increasing cancer cell proliferation and survival, constitutively activated STAT3 is proposed to regulate cross-talk between tumor, stroma and immune cells to promote immune-evasion. STAT3 activity in tumors promotes the production of immune-suppressive factors that activate STAT3 in diverse immune-cell subsets. Mechanistically, genetic studies support a direct role of activated STAT3 in regulating myeloid cell differentiation to contribute to an immune-suppressed tumor microenvironment (TME) (Kortylewski et al.; Nat. Med. 2005 and Curr. Opin. Immunol. 2008) Therefore, STAT3 is a highly attractive target for immune-oncology. Here, we demonstrate that the degradation of STAT3 with a potent and selective STAT3 degrader reverses immune suppression in preclinical models. KYM-003 is a heterobifunctional molecule that hijacks the ubiquitin-proteasome system (UPS) for rapid STAT3 degradation. KYM-003 robustly degraded STAT3 in both human peripheral blood mononuclear cells (hPBMC)-derived monocytes and lymphocytes with DC50 < 100 nM. Degradation in hPBMCs was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) as evaluated by deep tandem mass tag proteomics. IL-6 treatment of hPBMCs upregulates STAT3 phosphorylation, resulting in transcription of genes involved in myeloid cell-mediated immune suppression, such as IL-10 and CD163, a marker of M2 macrophage differentiation. Treatment of cells with KYM-003 at DC90 for 6 hours abrogated the IL-6 induced up-regulation of immune-suppressive gene signatures consistent with a role of STAT3 in mediating an immune suppressive environment by regulating macrophages and other myeloid cells. In several tumor cell lines, degradation of STAT3 by KYM-003 led to strong downregulation of PD-L1. Importantly, in the BioMap StroNSCLC (DiscoverRx), a co-culture system composed of NCI-H1299 lung cancer cells, hPBMCs and primary human fibroblasts that models immune-suppressed TME biology, KYM-003 treatment decreased angiogenic and immune-suppressive cytokines, including VEGF and IL-10, and promoted the pro-inflammatory anti-tumor cytokines IL-2, IFNg and TNFa. Lastly, administration of KYM-003 to mice bearing syngeneic tumors exhibited anti-tumor activity as monotherapy in the CT26 syngeneic model. We have shown that targeting STAT3 for degradation may have a role in restoring an immune-permissive environment in tumors by both rescuing the suppressed immunologic microenvironment and directly downregulating immune checkpoint signals in tumor cells. These data support STAT3 degraders as a promising new therapeutic modality as immune-oncology agents.Citations: Kortylewski et al (2005) Nat. Med, 11(12):1314-21. Kortylewski et al (2008) Curr. Opin. Immunol, 20(2):228-33 Citation Format: Alfredo Csibi, Bin Yang, Yogesh Chutake, Karen Yuan, Michele Mayo, Veronica Campbell, Alice McDonald, Scott Rusin, Kirti Sharma, Hari Kamadurai, Henry Li, Mike Sintchak, Sean Zhu, Sharon Townson, Anthony Slavin, Haojing Rong, Phillip Liu, Chris De Savi, Jared Gollob, Duncan Walker, Nan Ji, Nello Mainolfi. A STAT3 selective targeted protein degrader decreases the immunesuppressive tumor microenvironment and drives antitumor activity in preclinical 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 LB-088.

Connexin-Mediated Signaling at the Immunological Synapse.

The immunological synapse (IS) is an intercellular communication platform, organized at the contact site of two adjacent cells, where at least one is an immune cell. Functional IS formation is fundamental for the modulation of the most relevant immune system activities, such as T cell activation by antigen presenting cells and T cell/natural killer (NK) cell-mediated target cell (infected or cancer) killing. Extensive evidence suggests that connexins, in particular connexin-43 (Cx43) hemichannels and/or gap junctions, regulate signaling events in different types of IS. Although the underlying mechanisms are not fully understood, the current evidence suggests that Cx43 channels could act as facilitators for calcium ions, cyclic adenosine monophosphate, and/or adenosine triphosphate uptake and/or release at the interface of interacting cells. These second messengers have relevant roles in the IS signaling during dendritic cell-mediated T and NK cell activation, regulatory T cell-mediated immune suppression, and cytotoxic T lymphocyte or NK cell-mediated target tumor cell killing. Additionally, as the cytoplasmic C-terminus domain of Cx43 interacts with a plethora of proteins, Cx43 may act as scaffolds for integration of various regulatory proteins at the IS, as suggested by the high number of Cx43-interacting proteins that translocate at these cell-cell interface domains. In this review, we provide an updated overview and analysis on the role and possible underlying mechanisms of Cx43 in IS signaling.

Hypofractionated Irradiation Suppressed the Off-Target Mouse Hepatocarcinoma Growth by Inhibiting Myeloid-Derived Suppressor Cell-Mediated Immune Suppression.

Background: Stereotactic radiotherapy treats hepatocellular carcinoma (HCC) at different stages effectively and safely. Besides its direct killing of cancer cells, radiotherapy stimulates host immunity against hepatoma. However, the role of myeloid-derived suppressor cells (MDSCs) in on-target and off-target anti-HCC effects induced by hypofractionated irradiation (IR) is unclear.Methods and Materials: Hepa1-6 and H22 allogeneic transplanted tumors on hind limbs of C57BL/6 and Institute of Cancer Research (ICR) mice, respectively, were irradiated with 0, 2.5, 4, 6, or 8 Gy/fraction until the total dose reached 40 Gy. The off-target effect induced by the IR was investigated by subsequently inoculating the same HCC cells subcutaneously on the abdomen. MDSCs in peripheral blood and tumor tissues were measured by flow cytometry or immunofluorescence microscopy analysis. IL-6, regulated on activation normal T cell expressed and secreted (RANTES), and granulocyte colony-stimulating factor (G-CSF) in irradiated mouse plasma and hepatoma cell cultures were measured with ELISA kits. Conditioned media (CM) from irradiated HCC cell cultures on bone marrow cell differentiation and MDSC proliferation were examined by co-culture and flow cytometry.Results: Our study showed that the IR of primarily inoculated HCC on hind limbs created an “in situ tumor vaccine” and triggered the antitumor immunity. The immunity was capable of suppressing the growth of the same type of HCC subcutaneously implanted on the abdomen, accompanied with reduced MDSCs in both blood and tumors. The decreased MDSCs were associated with low plasma levels of IL-6, RANTES, and G-CSF. The cytokines IL-6 and RANTES in the CM were lower in the high single IR dose group than in the control groups, but G-CSF was higher. The CM from high single-dose IR-Hepa1-6 cell culture reduced the differentiation of C57BL/6 mouse bone marrow cells into MDSCs, whereas CM from high single-dose IR-H22 cells reduced the proliferation of MDSCs, which might be due to the decreased p-STAT3 in bone marrow cells.Conclusions: The hypofractionated IR on transplanted tumors at the primary location exerted a strong antitumor effect on the same tumor at a different location (off target). This abscopal effect is most likely through the reduction of MDSCs and decrease of IL-6, RANTES, and G-CSF.

Epimedium polysaccharides attenuates hematotoxicity by reducing oxidative stress and enhancing immune function in mice model of benzene-induced bone marrow failure

Chronic benzene (BZ) exposure is associated with multiple adverse health effects and leads to progressive bone marrow failure (BMF). BZ-induced BMF is an acquired aplastic anemia characterized by severe anemia, neutropenia and thrombocytopenia, which is likely caused by immunotoxicity and oxidative stress. Previous studies showed that Epimedium polysaccharides (EPS), a natural and major herbal compound derived from Epimedium, has immunomodulatory and antioxidant potential. The purpose of this study was to evaluate the potential efficacy of EPS against BZ-induced BMF. BMF mouse model was established by subcutaneous injection of 2 ml/kg BZ in CD1 mice. Mice received daily oral treatment with 100 mg/kg high-dose EPS and 20 mg/kg low-dose EPS for four weeks. Our data showed that EPS treatment alleviated BZ-associated weight loss and increased the number of whole blood cells in peripheral blood and nucleated cells in bone marrow. Furthermore, EPS treatment decreased apoptotic rate and reactive oxygen species production, S-phase arrest in bone marrow cells. Finally, EPS treatment improved T cell-mediated immune suppression by increasing CD3+, CD4 + T-cell counts, and CD4+/CD8+ ratio. and modulated hematopoietic cytokines including EPO, IL-11, and IL-2 in peripheral blood. Our study suggests that EPS is a potential therapeutic target to attenuate hematotoxicity induced by BZ.

The perioperative period: a critical yet neglected time window for reducing the recurrence risk of endometriosis?

While surgery is commonly the management of symptomatic endometriosis when patients do not respond to medical or supportive therapy, recurrence after surgery poses a serious challenge, and repeat surgery increases the risk of premature ovarian failure, adhesion and organ injury. Conceivably, the recurrent endometriotic lesions could arise from minimal residual lesions (MRLs) or from de novo lesions. However, several lines of evidence suggest that the former is more likely. So far, most, if not all, efforts to combat recurrence have been focused on postoperative medication of hormonal drugs to reduce recurrence risk through lesional dormancy and possibly atrophy. However, the perioperative period may exert a disproportionally high impact on the risk of recurrence; it is likely to be amendable for possible intervention but has been generally neglected. Indeed, many perioperative factors are known to or conceivably could facilitate the recurrence of endometriosis through the suppression of cell-mediated immunity due to the activation of adrenergic signaling and the release of prostaglandins. Perioperative use of β-blockers and/or nuclear factor κB/jCycloxygenase 2 (NF-κB/COX-2) inhibitors may boost the cell-mediated immunity suppressed by surgery, resulting in the partial or even complete removal of MRLs and reduced recurrence risk. This is both biologically plausible and supported by a recent experimental study. We call for more research on possible perioperative interventions to reduce the recurrence risk of endometriosis. The potential payoff might be a substantial reduction in the risk of recurrence and cost when compared with the traditional approach of postoperative intervention.

Circulating markers of cellular immune activation in prediagnostic blood sample and lung cancer risk in the Lung Cancer Cohort Consortium (LC3).

Cell-mediated immune suppression may play an important role in lung carcinogenesis. We investigated the associations for circulating levels of tryptophan, kynurenine, kynurenine:tryptophan ratio (KTR), quinolinic acid (QA) and neopterin as markers of immune regulation and inflammation with lung cancer risk in 5,364 smoking-matched case-control pairs from 20 prospective cohorts included in the international Lung Cancer Cohort Consortium. All biomarkers were quantified by mass spectrometry-based methods in serum/plasma samples collected on average 6 years before lung cancer diagnosis. Odds ratios (ORs) and 95% confidence intervals (CIs) for lung cancer associated with individual biomarkers were calculated using conditional logistic regression with adjustment for circulating cotinine. Compared to the lowest quintile, the highest quintiles of kynurenine, KTR, QA and neopterin were associated with a 20-30% higher risk, and tryptophan with a 15% lower risk of lung cancer (all ptrend < 0.05). The strongest associations were seen for current smokers, where the adjusted ORs (95% CIs) of lung cancer for the highest quintile of KTR, QA and neopterin were 1.42 (1.15-1.75), 1.42 (1.14-1.76) and 1.45 (1.13-1.86), respectively. A stronger association was also seen for KTR and QA with risk of lung squamous cell carcinoma followed by adenocarcinoma, and for lung cancer diagnosed within the first 2 years after blood draw. This study demonstrated that components of the tryptophan-kynurenine pathway with immunomodulatory effects are associated with risk of lung cancer overall, especially for current smokers. Further research is needed to evaluate the role of these biomarkers in lung carcinogenesis and progression.