Increased Expression Of PD-1 Research Articles

Assessment of anti-CD69 antibody therapy alone or in combination with anti-PD-1 in murine GBM

ABSTRACT Background Glioblastoma (GBM) is an aggressive cancer with limited treatment options. Immunotherapy targeting CD69, an early activation marker on T cells, has shown promise in preclinical models of non-CNS malignancies. This study investigates anti-CD69 therapy alone or in combination with anti-PD-1 in a preclinical GBM model. Research design and methods CD69 expression in GBM patient tissues was analyzed using the TCGA database. Therapeutic efficacy of anti-CD69 was tested in a murine GBM model with different regimens. Immune cell populations in the tumor microenvironment (TME) were assessed by flow cytometry. Results Increased CD69 expression was observed in GBM patients compared to normal brain tissue and was associated with worse prognosis. Anti-CD69 treatment reduced percentages of CD69+ immune cells but did not improve survival in GBM-bearing mice. Increased PD-1 expression on NK cells was observed following anti-CD69 treatment. Anti-CD69 treatment was not improved by the addition of anti-PD-1 in vivo. Conclusions This is the first study evaluating anti-CD69 therapy in a preclinical GBM model. Despite promising preclinical data in other cancers, anti-CD69 monotherapy or combination therapy with anti-PD-1 did not improve survival in this GBM model.

Immunological responses to brain metastasis stereotactic radiosurgery in patient-matched longitudinal blood and tumour samples

BackgroundStereotactic radiosurgery (SRS) is highly effective as focal treatment for brain metastases (BrMs), but whether it can promote anti-tumour immune responses that synergise with immunotherapy remains unclear. We investigated this by examining blood samples from a clinical trial for HER2-amplified breast cancer (HER2-BC) BrMs, matched with longitudinal HER2-BC BrM samples resected from the same location in the same patient. MethodsBlood samples from 10 patients taken pre- and 7–14 days post-SRS were analysed by mass and flow cytometry. One patient received pre-operative SRS for a BrM that recurred 7 months after resection, followed by planned re-resection 8 days post-SRS. Pre- and post-SRS tumours from this patient were analysed by bulk RNAseq, multiplex immunohistochemistry (mIHC), and TCR sequencing. ResultsMonocytes, central memory CD8+ T and regulatory T cells were enriched in blood post-SRS, together with increased MHC-II expression on monocytes, conventional DCs, and monocytic MDSCs. In tumour, SRS upregulated antigen presentation, T cell proliferation and T cell co-stimulation signatures, alongside an influx of tumour-associated macrophages (TAMs) and CD4+ T cells. Specifically, TAMs and CD4+ T cells, but not CD8+ T cells, demonstrated spatial co-localisation post-SRS. These TAMs were lowly PD-L1 expressing, but CD4+ T cells showed increased PD-1 expression. A sizeable proportion of T cell clonotypes were retained post-SRS, and four clones demonstrated significant, non-stochastic expansion. ConclusionSystemic and local immunological changes in this homogenous patient cohort suggest that SRS may facilitate MHC-II-restricted T cell priming responses involving the monocyte-macrophage lineage and CD4+ T cells, which should be further explored.

450. TISSUE RESIDENT MEMORY CELLS: CORRELATING THE PHENOTYPIC AND FUNCTIONAL CAPABILITIES OF A POTENTIAL TUMOUR REACTIVE CELL POPULATION IN OESOPHAGEAL ADENOCARCINOMA

Abstract Background Oesophageal adenocarcinoma (OAC) is a disease with a high mortality and morbidity rate. Immunotherapy has recently demonstrated progress in the treatment of OAC but its impact on long term survival is currently uncertain. Tissue resident memory (TRM) T cells are a subset of lymphocytes within the tumour/tissue infiltrating lymphocyte (TIL) milieu that has attracted considerable interest within cancer immunology. Although TRM cells have been associated with better outcomes in multiple cancer types there is a lack of knowledge of their role in OAC. This study aims to critically correlate the phenotype of TRM with their functional capacity in this disease. Methods 33 patients undergoing surgical resection for OAC were recruited and consented for collection of fresh tumour (T), adjacent normal tissue (AN), and peripheral blood. Peripheral blood mononuclear cell (PBMC) from blood and infiltrating lymphocytes from T and AN were isolated using standard density gradient and enzymatic digestion protocols. Multiparametric flow cytometry was performed on 12 patients using matched PBMC, T and AN infiltrating lymphocytes for phenotypic analysis and 21 patients for functional analysis following cell stimulation assay. BD FACSymphony was used for data acquisition and data analysed on FlowJo version 10.10. Statistical analysis was carried out using GraphPad Prism. Results TRM (CD103+CD69+) dominated the CD8+ T cell population of T and AN TIL. Expression of checkpoint proteins PD1+ and CD39+, a marker of tumour reactivity, was identified on CD8+ TRM and CD8+ non TRM in TIL. The differentiation status of TRM within memory lineages demonstrated that TRM express a higher proportion of effector memory cells (CCR7-CD45RA) compared to those in blood. TRM cells also showed lower expression of the transcription factors EOMES, TBET and TCF compared to CD8+ cells within PBMC. Moreover, tumour CD8+ TRM populations had reduced ability to produce IL-2 following stimulation compared to adjacent normal TRM populations. Conclusion TRM make up a large proportion of CD8+ T cell within the tumour microenvironment (TME) of OAC. TRM generation is associated with the downregulation of T-Box transcription factors EOMES and TBET and expression of an effector memory phenotype with reduction of the stem-like transcription factor, TCF. High levels of CD39+ indicate a strong level of activation but the increased PD1 expression and reduced capability to produce IL2 indicate functional impairment suppressed within the TME. An opportunity may exist to reverse this with novel immunotherapeutic agents.

Activated PI3Kδ Specifically Perturbs Mouse Regulatory T Cell Homeostasis and Function Leading to Immune Dysregulation.

FOXP3+ regulatory T cells (Treg) are required for maintaining immune tolerance and preventing systemic autoimmunity. PI3Kδ is required for normal Treg development and function. However, the impacts of dysregulated PI3Kδ signaling on Treg function remain incompletely understood. In this study, we used a conditional mouse model of activated PI3Kδ syndrome to investigate the role of altered PI3Kδ signaling specifically within the Treg compartment. Activated mice expressing a PIK3CD gain-of-function mutation (aPIK3CD) specifically within the Treg compartment exhibited weight loss and evidence for chronic inflammation, as demonstrated by increased memory/effector CD4+ and CD8+ T cells with enhanced IFN-γ secretion, spontaneous germinal center responses, and production of broad-spectrum autoantibodies. Intriguingly, aPIK3CD facilitated Treg precursor development within the thymus and an increase in peripheral Treg numbers. Peripheral Treg, however, exhibited an altered phenotype, including increased PD-1 expression and reduced competitive fitness. Consistent with these findings, Treg-specific aPIK3CD mice mounted an elevated humoral response following immunization with a T cell-dependent Ag, which correlated with a decrease in follicular Treg. Taken together, these findings demonstrate that an optimal threshold of PI3Kδ activity is critical for Treg homeostasis and function, suggesting that PI3Kδ signaling in Treg might be therapeutically targeted to either augment or inhibit immune responses.

Abstract 1190: NK cell subsets as potential correlates of antitumor immunity in HPV+ cancers

Abstract High-risk type human papillomaviruses (HPV) are associated with genital and oral cancers, and the incidence of head and neck squamous cell cancers is fast increasing in the USA and worldwide. Survival rates for patients with locally advanced disease are poor and variable after standard of care (SOC) chemoradiation treatment (CRT). Identifying the antitumor host immune mediators important for treatment response and designing strategies to promote them are essential for improving clinical outcome. Antitumor immunity comprises of adaptive and innate immune effectors. The immediate innate immune response by natural killer (NK) cells is a critical component for antitumor efficacy. The effector function of NK cells is modulated by various receptors via a wide array of activation or inhibitory signals. Among these receptors is HLA-DR, a recognized activation marker on CD8 T and NK cells. Literature studies refer to HLA-DR+ NK cells as pre-mNK. Preclinical data demonstrated therapeutic vaccine mediated induction of pre-mNK equivalent to contribute to tumor-free long-term survival. In our studies with clinical samples from HPV+ cancer patients, we’ve observed a dysfunctional phenotype of circulating pre-mNK when compared to healthy donors, demonstrated by flow cytometry methods. The correlates for dysfunction include increased PD-1 expression, a known inhibitory receptor, and reduced CD16, an NK receptor for Antibody Dependent Cellular Cytotoxicity (ADCC). Further, we observed circulating pre-mNK cells in HPV+ head and neck patients gain functionality post-treatment, as shown by significant increases in granzyme B and CD16, both known contributors for NK cytotoxicity. In cervical cancer patients, we had the opportunity to analyze the tumor microenvironment (TME). Interestingly, we found a significant increased frequency of cytotoxic pre-mNK cells, shown by granzyme B, in the TME that was not present in circulation. Overall, pre-mNK represent a uniquely activated immune subset for antiviral and antitumor immunity. Citation Format: Madison O'Hara, Jagannadha Sastry. NK cell subsets as potential correlates of antitumor immunity in HPV+ cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1190.

Increased PD-1 Expression on Circulating T Cells Correlates with Inferior Outcome after Autologous Stem Cell Transplantation

High-dose chemotherapy followed by autologous stem cell transplantation (auto-SCT) is a well-established treatment option for multiple myeloma and malignant lymphoma patients. It is able to induce long-term progression-free survival (PFS) in both patient groups and even provide a cure in patients with aggressive lymphoma. However, relapse is common and has been associated with the pace and quality of immunologic reconstitution after transplantation, as well as with immune cell exhaustion and immunometabolic defects. We aimed to analyze the dynamics of the prototypical exhaustion marker PD-1 on immune cells during reconstitution on high-dose chemotherapy followed by auto-SCT and its impact on PFS. We performed a comprehensive analysis of exhaustion and metabolic markers on immune cells from myeloma and lymphoma patients undergoing auto-SCT using flow cytometry and NanoString technologies. The expression levels of PD-1 were increased during early reconstitution after transplantation on T cells and natural killer (NK) cells, as well as on monocytes. However, while PD-1 expression in NK cells and monocytes normalized over time, PD-1 expression on T cells demonstrated a variable course. Of note, lymphoma patients with continuously increasing PD-1 expression on T cells after auto-SCT had an inferior median PFS of only 146 days, whereas the median PFS was not reached in the lymphoma patients without such a PD-1 expression pattern. T cells from patients with increased PD-1 expression after auto-SCT exhibited an immunometabolic (over)activation and exhausted phenotype compared to T cells from patients with a low PD-1 expression after transplantation, including higher levels of the glycolytic pacemaker enzyme hexokinase 2 and the inhibitory receptor CTLA-4. In addition, proliferating Ki-67+ T cells were more abundant in patients with high PD-1 expression on T cells compared to those with low expression after auto-SCT (11.9% versus 4.2%). PD-1 expression on T cells might serve as an adverse biomarker for lymphoma patients undergoing auto-SCT; however, further validation by larger prospective studies is required.

Abstract A008: Intermittent fasting in combination with PD-inhibitor treatment as an innovative treatment strategy in a pre-clinical model of obesity-driven endometrial cancer

Abstract Objective: Endometrial cancer (EC) incidence and mortality continue to rise, with obesity a well-known driver of this disease. Obesity is associated with excess estrogen, hyperinsulinemia, activation of the Akt/mTOR pathway, chronic inflammation and a heightened capacity to harvest dietary energy, which all contribute to the development and progression of EC. Intermittent energy restriction (IER) is thought to have anti-tumorigenic benefits and has been shown to have synergistic effects with chemotherapy and hormonal therapy in various cancer types. IER may also promote anti-tumor immune responses by stimulating CD4+ and CD8+ cells, making this dietary strategy an innovative therapeutic partner with PD-1 inhibitors, which are commonly used in EC. Thus, we evaluated the anti-tumorigenic effects of a switch from high fat diet (HFD) to either IER or low-fat diet (LFD) with PD-1 inhibitor treatment in a mouse model of obesity-driven endometrioid EC. Methods: Lkb1fl/flp53fl/fl mice were fed either HFD or LFD starting at 4 weeks of age. At 12 weeks of age, the HFD-fed mice were randomized to three treatment groups: HFD, HFD-LFD switch or HFD-IER switch. The LFD-fed mice were maintained on LFD. At 16 weeks of age, all mice were injected with AdCre into the left uterine horn to induce EC development. At 26 weeks of age, mice were treated with placebo or a mouse PD-1 inhibitor (BioXCell) (10mg/kg, 2 times/wk, intraperitoneal) for 4 wks (N=8-10 mice per group). RNAseq was performed to determine differences in immune pathways between ECs of obese and lean mice. CD4+ and CD8+ T cells in tumors of obese and lean mice were evaluated by flow cytometry. Results: Obesity resulted in a 52% increase in tumor size, comparing obese versus lean mice (p<0.05). Expression of genes related to T-cell/B-cell receptors, NF-kappa B and TNF pathways were upregulated in the ECs of obese versus lean mice. PD-1 inhibitor therapy increased PD-1 expression on CD4+ and CD8+ T cells in both obese and lean ECs. In addition, PD-1 inhibitor treatment led to an increase in percentage of CD4+ T cells in obese and lean ECs but increased the percentage of CD8+ T cells in only lean ECs. At sacrifice and after PD-1 inhibitor treatment, mean body weights were significantly higher (p<0.05) in the HFD group (42g) than groups receiving LFD (29g), HFD-LFD switch (34g) or HFD-IER switch (23g). EC incidence was lowest in the HFD-IER group (~61%) and highest in the HFD group (~80%); rates were similar in the other diet groups (~75%). In placebo-treated groups, mean tumor weights were significantly lower in mice fed LFD (0.45g), HFD-LFD switch (0.41g) and HFD-IER switch (0.29g) relative to continuous HFD-fed mice (0.94g; p=0.001-0.05). PD-1 inhibitor treatment reduced EC size 24-68% across all diet groups (p=0.05-0.001); size reduction was greatest in the HFD-IER group (0.18g) relative to mice receiving continuous HFD (0.30g), HFD-LFD (0.31g) or LFD (0.31g). Conclusion: IER with PD-1 inhibitor treatment may be an innovative strategy in the management of EC, a highly obesity-driven cancer. Citation Format: Jennifer Haag, Olivia Lara, Chunxiao Zhou, Stephen D. Hursting, Victoria Bae-Jump. Intermittent fasting in combination with PD-inhibitor treatment as an innovative treatment strategy in a pre-clinical model of obesity-driven endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A008.

Targeted Sequencing in Gastric Cancer: Association with Tumor Molecular Characteristics and FLOT Therapy Effectiveness.

Heterogeneity of gastric cancer (GC) is the main trigger of the disease's relapse. The aim of this study was to investigate the connections between targeted genes, cancer clinical features, and the effectiveness of FLOT chemotherapy. Twenty-one patients with gastric cancers (GCs) were included in this study. Tumor-targeted sequencing was conducted, and real-time PCR was used to assess the expression of molecular markers in tumors. Seven patients with stabilization had mutations that were related to their response to therapy and were relevant to the tumor phenotype. Two patients had two mutations. The number of patients with TP53 mutations increased in HER2-positive tumor status. PD-L1-positive cancers had mutations in KRAS, TP53, PIK3CA, PTEN, and ERBB, which resulted in an increase in PD-1 expression. TP53 mutation and PTEN mutation are associated with changes in factors associated with neoangiogenesis. In concusion, patients who did not have aggressive growth markers that were verified by molecular features had the best response to treatment, including complete morphologic regression.

Inhibition of PTPN3 Expressed in Activated Lymphocytes Enhances the Antitumor Effects of Anti-PD-1 Therapy in Head and Neck Cancer, Especially in Hypoxic Environments.

In the tumor microenvironment, wherein cytotoxic lymphocytes interact with cancer cells, lymphocyte exhaustion, an immune checkpoint inhibitor target, is promoted. However, the efficacy of these inhibitors is limited, and improving response rates remains challenging. We previously reported that protein tyrosine phosphatase nonreceptor type (PTPN) 3 is a potential immune checkpoint molecule for activated lymphocytes and that PTPN3 inhibition should be a focus area for cancer immunotherapy development. Therefore, in this study, we focused on PTPN3-suppressive therapy in terms of lymphocyte exhaustion under hypoxic conditions, which are a cancer microenvironment, and investigated measures for improving the response to anti-programmed death receptor (PD)-1 antibody drugs. We found that PTPN3 expression was upregulated in activated lymphocytes under hypoxic conditions, similar to the findings for other immune checkpoint molecules, such as PD-1, T cell immunoglobulin mucin-3, and lymphocyte-activation gene-3; furthermore, it functioned as a lymphocyte exhaustion marker. In addition, PTPN3-suppressed activated lymphocytes promoted the mammalian target of rapamycin (mTOR)-Akt signaling pathway activation and enhanced proliferation, migration, and cytotoxic activities under hypoxic conditions. Furthermore, PTPN3 suppression in activated lymphocytes increased PD-1 expression and enhanced the antitumor effects of anti-PD-1 antibody drugs against head and neck cancer in vitro and in vivo. These results suggest that the suppression of PTPN3 expression in activated lymphocytes enhances the therapeutic effect of anti-PD-1 antibody drugs in head and neck cancer, especially under hypoxic conditions that cause lymphocyte exhaustion.

Implications of Senescent T Cells for Cancer Immunotherapy.

T-cell senescence is thought to result from the age-related loss of the ability to mount effective responses to pathogens and tumor cells. In addition to aging, T-cell senescence is caused by repeated antigenic stimulation and chronic inflammation. Moreover, we demonstrated that T-cell senescence was induced by treatment with DNA-damaging chemotherapeutic agents. The characteristics of therapy-induced senescent T (TIS-T) cells and general senescent T cells are largely similar. Senescent T cells demonstrate an increase in the senescence-associated beta-galactosidase-positive population, cell cycle arrest, secretion of senescence-associated secretory phenotypic factors, and metabolic reprogramming. Furthermore, senescent T cells downregulate the expression of the co-stimulatory molecules CD27 and CD28 and upregulate natural killer cell-related molecules. Moreover, TIS-T cells showed increased PD-1 expression. However, the loss of proliferative capacity and decreased expression of co-stimulatory molecules associated with T-cell senescence cause a decrease in T-cell immunocompetence. In this review, we discuss the characteristics of senescent T-cells, including therapy-induced senescent T cells.

Alterations in Immune Cell Composition during First-Line Therapy with Mosunetuzumab for Follicular or Marginal Zone Lymphoma

Background: Bispecific CD20xCD3 antibodies (BiAbs) that engage T-cells to eliminate CD20+ B-cells have emerged as a potent immunotherapy for the treatment of follicular (FL) and marginal zone (MZL) lymphomas. BiAbs have primarily been studied in patients with extensive prior immunosuppressive and lymphodepleting therapies, so their effects on the immune system without such exposures are not well understood. Unlike cellular therapies, BiAbs are administered continuously over time, spanning from 6 months to an indefinite period, and recent studies have raised concerns about T-cell exhaustion due to continuous BiAb exposure in B-lymphoblastic leukemia (Philipp et al, Blood 2022). Mosunetuzumab is a BiAb approved for treatment of relapsed/refractory FL. The ongoing phase 2 BrUOG-401 trial (NCT04792502) is investigating mosunetuzumab as first-line therapy for FL or MZL. Here, we present preliminary results focusing on treatment-induced changes in circulating immune cells. Methods: Patients with previously untreated, high-burden or symptomatic FL or MZL received subcutaneous mosunetuzumab for 8 planned cycles (C) with step-up dosing in C1 (day 1: 5mg, day 8 & 15: 45 mg; then 45mg on day 1 of C4-C8). Patients without a complete response (CR) at interim assessment after C4 also received lenalidomide augmentation (10mg daily) during C5-8. Immune cell subsets were examined using multicolor flow cytometry (BD LSRII Analyzer). Peripheral blood samples were collected longitudinally before C1, on day 1 of C2 and C5, and 4-6 weeks after the end of therapy (EOT). Median cell abundance and marker expression were compared using generalized linear mixed models with patient and sample-level random effects. Analysis was performed in R (v.4.3.1) using the flowCore, flowStats and diffcyt packages; uncorrected P<0.05 was considered statistically significant. Results: As of July 2023, 13 patients (median age 62, 77% women, 77% FL, 23% MZL) were accrued in the trial. At data cutoff, 7 have completed therapy, 11 were evaluable for response, and 8 attained CR ( Fig. A). So far, only 1 patient started lenalidomide; samples collected while on lenalidomide were excluded from analysis. No instances of cytokine release syndrome (CRS) or neurotoxicity were observed. CR's occurred early (by C5). Mosunetuzumab resulted in a rapid, deep, and sustained depletion of circulating CD19+ B-cells. A significant decrease in circulating CD8+ T-cells was noted at cycle 2 (from median 20% to 12%, P<0.001; Fig. B), followed by recovery (19% at C5). Among the CD8+ subsets, the naïve CD8+ T-cells (CD45RA+CCR7+CD27+) decreased relatively at C5 & EOT, while effector memory T-cells (CD45RA-CD45RO+CD27-CD62L-) increased. Notably, we observed a significant early, temporary, increase in the expression of LAG3 at C2 on CD8+ T-cells ( P=0.033), and a more pronounced increase in PD1 expression ( P=0.001), which persisted through the EOT ( Fig. B). No significant changes were observed in total abundance of CD4+ T-cells. However, CD4+CD25+CD127- Tregs notably increased at C2 ( P=0.008). This increase specifically driven by CD45RA+ “naïve” Tregs ( P<0.001 at C2), which remained elevated relative to CD45RO+ Tregs at C5 and EOT. All Tregs showed a significant increase in LAG3 expression at C2 ( P<0.001), which then subsided. In contrast to CD8+ T-cells, there was no significant increase in PD1 expression on CD4+ T-cells ( P=0.20 at C2). Throughout the therapy, there was a progressive increase in NK cells ( P=0.001 at EOT), with no significant shift in the relative proportions of CD16high or CD161high NK cells. Conclusions: In this initial investigation of immune cell changes in patients who received mosunetuzumab without prior chemotherapy exposure, we observed B-cell depletion and a temporary decrease in CD8+ cytotoxic T-cells (implying their migration out of the bloodstream), aligning with mosunetuzumab's mechanism of action. Additional data from this trial will aid in understanding whether the persistent increase in PD1+CD8+ T-cells and expansion of Tregs are linked to a functionally exhausted phenotype and whether lenalidomide could potentially reverse it. Although our observation is limited by sample size, it can still provide insights into optimizing the administration schedules of BiAb therapy in future trials (e.g., intermittent vs. continuous) or determining the most suitable timing for checkpoint inhibitor combinations.

T-Cell Exhaustion Signature Predicts Early Relapse after Autologous Stem Cell Transplant for Multiple Myeloma: BMT CTN 0702 Secondary Immune Analysis

Introduction: Autologous stem-cell transplant (ASCT) is standard-of-care for the treatment of multiple myeloma (MM), but most patients eventually relapse. Patients with early relapse, within 12-24 months of ASCT, have a lower overall survival that is not fully explained by conventional high-risk disease features, such as frailty, extramedullary disease, and cytogenetics. In this study, we performed high-dimensional flow cytometry-based immune phenotyping of the T-cell compartment of MM patients to identify features predictive of early relapse. Methods: Peripheral blood samples collected at 90 days post-ASCT from participants in BMT CTN 0702 comprised two cohorts: an early relapse cohort (ER), defined as relapse within 24 months from ASCT, and a long-term responder cohort (LR) defined as not relapsed at >4 years from ASCT. We performed 28-color flow cytometry to explore differences in T-cell subset frequency and activation state between cohorts. A total of 96 patients from the ER (n=51) and LR (n=45) cohorts were analyzed. We manually gated on live, CD3+ events and integrated all patient samples. High-dimensional flow cytometry analysis was conducted using the FlowSOM algorithm, which identified 20 cell clusters. We manually annotated each cluster based on CD45RA and CCR7 expression into separate CD4 and CD8 effector and memory subsets. Differential expression analysis was performed with empirical Bayesian testing, controlling for batch effect and treatment modality, followed by effect size estimates with Cohen's d. Results: We observed 2 out of 20 cell clusters with a significant difference in proportion between groups. For ER patients, there were two CD45RA -, CCR7 +, CD25 +, CCR4 + CD4 + clusters with a 29% (log fold change (LFC) = -0.628, p-value < 0.001) and 54% relative decrease in cell number (LFC = -1.25, p-value < 0.001), suggesting an absence of activated central memory CD4 T cells with early relapse. Analysis of positive and negative costimulatory molecules revealed upregulation of PD1, CD38, and TIGIT, with corresponding decreased expression of granzyme B, CD28, and CD27 within multiple CD4 and CD8 clusters among the ER group (Figure 1A). By differential expression testing, early relapse was most strongly associated with CD28 downregulation by effector memory (CD45RA -, CCR7 -) CD8 T cells (LFC = -0.016, p-value < 0.001), increased PD1 expression by effector memory CD8 T cells (LFC = 0.022, p-value < 0.001), increased TIGIT expression by central memory (CD45RA -, CCR7 +) CD8 T cells (LFC = 0.038, p-value < 0.001), and decreased granzyme B among T regulatory (FoxP3 +, CD25 +) cells (LFC = -0.030, p-value < 0.001) (Figure 1B). Conclusion: Distinct signatures of T-cell exhaustion and depletion are present in the early post-ASCT period among individuals relapsing within 24 months. Peripheral blood immune phenotyping may identify functionally high-risk patient populations in need of alternative treatment approaches to maintain durable disease control.

Nivolumab in Lymphomatoid Granulomatosis (LYG) and Other Epstein-Barr Virus (EBV)-Positive Lymphoproliferative Disorders (LPDs) and Non-Hodgkin Lymphomas (NHLs) with Systemic and/or CNS Involvement: Preliminary Analysis of Efficacy and Safety from an Ongoing Phase II Study

Background: Programmed cell death protein-1 (PD-1) is a signaling molecule on the surface of T-cells that suppresses the cytotoxic effects of T-cells on tumor cells. We previously demonstrated increased PD-1 expression on CD8+ T-cells compared to healthy controls in pts with LYG, which could be reversed with immunotherapy using interferon alpha-2b (Melani et al. Lancet Haematol. 2023). Additionally, elevated expression of programmed death-ligand 1 (PD-L1) has been observed in other EBV-LPDs and NHLs as well as in chronic viral infections, such as EBV. Given this likely shared disease pathobiology, we hypothesized that treatment with the anti-PD-1 antibody, nivolumab, may reverse T-cell exhaustion and result in anti-tumor responses in pts with LYG and other EBV-LPDs and NHLs, including those with systemic and/or CNS involvement. Methods: Relapsed/refractory (R/R) EBV-LPD and NHL pts age ≥ 12y with adequate organ function were eligible. Untreated pts were eligible if EBV-LPD. Exclusions included prior use of PD-1/PD-L1/PD-L2/CD137/CTLA-4 antibodies, solid organ transplant, and HIV infection. Pts with a known immunodeficiency or autoimmune illness were eligible if not requiring steroids or immunosuppression within 14d of study. CNS involvement was permitted if no seizure activity within 4w of study. Nivolumab 480 mg IV was given q4w for up to 2y. Pts who achieved CR discontinued nivolumab after 1y of treatment. Responding patients with relapse or progression within 1y of discontinuing treatment were eligible for retreatment. Baseline evaluation included CT, PET, MRI brain, flow cytometry of peripheral blood and CSF, BM biopsy, and optional tumor biopsy. Restaging CT scans were performed after cycles 3, 6, 13, and 19, and end of treatment (EoT). PET was performed after cycles 1, 3, and EoT. Surveillance CT was performed q3m for 1y, q6m for yrs 2-5, and annually thereafter. Results: 11 pts were enrolled and treated; 6 (55%) with EBV-LPD and 5 (45%) with EBV-NHL (all DLBCL). EBV-LPD subtypes included 4 pts with G1-2 LYG and 1 pt each with EBV B-LPD of the CNS and CAEBV. Median age was 48y (range 20-67), and all pts had stage III/IV disease with extranodal involvement of the lung most common in 64% (7/11) and CNS disease in 18% (2/11). Median baseline absolute CD8 count (cells/mcL) was 86 (range 9-1237; normal: 178-853), CD4 count (cells/mcL) was 255 (range 99-1304; normal: 359-1565), and EBV VL in plasma (Log10 IU/mL) was undetected (range undetected-6.78; normal: <2.3). Median prior therapies were 1 (range 0-6) with 3 previously untreated LPD pts and 50% of previously tx pts were refractory to last tx. In 10 pts evaluable for response(1 came off tx prior to restaging), ORR and CR rate were both 60% (6/10). CR rate was 67% (4/6) in EBV-LPD, including 75% (3/4) with LYG, and 50% (2/4) in EBV-NHL (Fig 1A). One (50%) of 2 EBV-LPD pts with CNS involvement achieved CR after 3C of therapy and remains in ongoing response. Median TTR was 3.0m with 50% (3/6) responses ongoing from 3-56m after first response (Fig 1B). With a median follow up of 32m, 2y PFS and OS was 33% and 72% overall. By cohort, median PFS was 36m in EBV-LPD and 1m in EBV-NHL (p=0.003), with a 2y OS of 100% in EBV-LPD and 40% in EBV-NHL (p=0.04). Three EBV-NHL pts died, all of disease progression. One EBV-NHL pt stopped therapy after 2C due to radiographic disease progression in the kidney, but later developed CR without further therapy and remains in remission 56m after stopping therapy consistent with initial pseudoprogression. Adverse events (AEs) were as expected for nivolumab therapy with the most common AEs (% pts) including maculopapular rash (27%), diarrhea (18%), hypothyroidism (18%), and fatigue (18%). One pt developed an immune-mediated G2 myositis and G3 hepatitis following 3C of therapy which resolved after drug discontinuation and corticosteroids. Conclusion: Nivolumab is well tolerated in pts with LYG and other EBV-LPDs and NHLs without unexpected toxicities. Preliminary activity, including durable CRs, is noted across all pt cohorts, including 1 EBV-LPD pt with CNS involvement. With small numbers, statistically longer PFS and OS was observed with nivolumab in LYG and EBV-LPD compared to EBV-NHL, indicating that monotherapy may be sufficient for LYG and other EBV-LPDs, but combination therapy is likely needed for pts with EBV-NHL. Study enrollment continues to better assess the activity and durability of nivolumab in LYG and other EBV-LPDs and NHLs.

Targeting TREM1 augments antitumor T cell immunity by inhibiting myeloid-derived suppressor cells and restraining anti-PD-1 resistance.

The triggering receptor expressed on myeloid cell 1 (TREM1) plays a critical role in development of chronic inflammatory disorders and the inflamed tumor microenvironment (TME) associated with most solid tumors. We examined whether loss of TREM1 signaling can abrogate the immunosuppressive TME and enhance cancer immunity. To investigate the therapeutic potential of TREM1 in cancer, we used mice deficient in Trem1 and developed a novel small molecule TREM1 inhibitor, VJDT. We demonstrated that genetic or pharmacological TREM1 silencing significantly delayed tumor growth in murine melanoma (B16F10) and fibrosarcoma (MCA205) models. Single-cell RNA-Seq combined with functional assays during TREM1 deficiency revealed decreased immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs) accompanied by expansion in cytotoxic CD8+ T cells and increased PD-1 expression. Furthermore, TREM1 inhibition enhanced the antitumorigenic effect of anti-PD-1 treatment, in part, by limiting MDSC frequency and abrogating T cell exhaustion. In patient-derived melanoma xenograft tumors, treatment with VJDT downregulated key oncogenic signaling pathways involved in cell proliferation, migration, and survival. Our work highlights the role of TREM1 in cancer progression, both intrinsically expressed in cancer cells and extrinsically in the TME. Thus, targeting TREM1 to modify an immunosuppressive TME and improve efficacy of immune checkpoint therapy represents what we believe to be a promising therapeutic approach to cancer.

High risk clear cell renal cell carcinoma microenvironments contain protumour immunophenotypes lacking specific immune checkpoints

Perioperative immune checkpoint inhibitor (ICI) trials for intermediate high-risk clear cell renal cell carcinoma (ccRCC) have failed to consistently demonstrate improved patient outcomes. These unsuccessful ICI trials suggest that the tumour infiltrating immunophenotypes, termed here as the immune cell types, states and their spatial location within the tumour microenvironment (TME), were unfavourable for ICI treatment. Defining the tumour infiltrating immune cells may assist with the identification of predictive immunophenotypes within the TME that are favourable for ICI treatment. To define the immunophenotypes within the ccRCC TME, fresh para-tumour (pTME, n = 2), low-grade (LG, n = 4, G1-G2) and high-grade (HG, n = 4, G3-G4) tissue samples from six patients with ccRCC presenting at a tertiary referral hospital underwent spatial transcriptomics sequencing (ST-seq). Within the generated ST-seq datasets, immune cell types and states, termed here as exhausted/pro-tumour state or non-exhausted/anti-tumour state, were identified using multiple publicly available single-cell RNA and T-cell receptor sequencing datasets as references. HG TMEs revealed abundant exhausted/pro-tumour immune cells with no consistent increase in expression of PD-1, PD-L1 and CTLA4 checkpoints and angiogenic genes. Additional HG TME immunophenotype characteristics included: pro-tumour tissue-resident monocytes with consistently increased expression of HAVCR2 and LAG3 checkpoints; an exhausted CD8+ T cells sub-population with stem-like progenitor gene expression; and pro-tumour tumour-associated macrophages and monocytes within the recurrent TME with the expression of TREM2. Whilst limited by a modest sample size, this study represents the largest ST-seq dataset on human ccRCC. Our study reveals that high-risk ccRCC TMEs are infiltrated by exhausted/pro-tumour immunophenotypes lacking specific checkpoint gene expression confirming that HG ccRCC TME are immunogenic but not ICI favourable.

CXCL10 Recruitment of γδ T Cells into the Hypoxic Bone Marrow Environment Leads to IL17 Expression and Multiple Myeloma Progression.

In multiple myeloma (MM), bone marrow stromal cells (BMSC) shape a unique niche within the bone marrow, promoting T-cell dysfunction and driving MM progression; however, the precise underlying mechanisms remain elusive. Here, we show that BMSC-mediated reprogramming of MM cells led to heightened production of CXCL10. CXCL10 orchestrated the recruitment of γδ T cells into the bone marrow, and this was observed in both the Vk*MYC and 5TGM1 mouse models of MM, as well as in patients experiencing refractory or relapsed MM. Furthermore, the dysfunctional γδ T cells in the MM bone marrow niche exhibited increased PD-1 expression and IL17 production. In the Vk*MYC mouse model, MM-associated bone lesions and mortality were markedly alleviated in Tcrd-/- mice, and MM disease progression could be rescued in these mice upon transplantation of γδ T cells expanded from wild-type mice, but not from Il17-/- mice. Mechanistically, the hypoxic microenvironment prevailing in the MM bone marrow niche stimulated the expression of steroid receptor coactivator 3 (SRC-3) in γδ T cells, which in turn interacted with the transcriptional factor RORγt, promoting Il17 transcription. Pharmacologic inhibition of SRC-3 utilizing SI-2 effectively suppressed Il17A expression in γδ T cells, leading to alleviation of MM progression in the murine models and enhancing the anti-multiple myeloma efficacy of bortezomib. Our results illuminated the bone marrow microenvironment's involvement in provoking γδ T-cell dysfunction throughout MM progression and suggest SRC-3 inhibition as a promising strategy to enhance the effectiveness of immunotherapies targeting γδ T cells.

Abstract 4237: Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: Do they explain enhanced mammary tumorigenesis

Abstract Social isolation (SI), are associated with increased mortality from many diseases, including breast cancer. Up to 41% of breast cancer patients are estimated to feel socially isolated when assessed between 6 months and 2 years following their cancer diagnosis. Since socially isolated breast cancer survivors are at a 64% higher risk of breast cancer-specific mortality than socially integrated survivors, the biological mechanisms which mediate the effects of SI need to be identified to prevent increased mortality. In a preclinical model, we found that SI caused recurrence of mammary cancers which had been treated with hormone therapy and thus led cancer cells to emerge from dormancy. We further found that mammary glands of SI rats exhibited upregulation of IL6/JAK/STAT3 signaling pathway and impaired oxidative phosphorylation (OXPHOS), compared with group housed (GH) rats. We investigated if these changes could be mediated through the gut-brain-axis. Stress can affect the gut microbiota, and the gut microbiota is critical for regulating the host immunity. In four separate experiments, the effect of SI on the gut microbiota composition was assessed in C57BL/6 mice that were either group housed or housed singly in SI for 4 weeks. In each study replicate, several differences in the gut microbiota composition were seen, but these differences were mostly unique to each experiment. Beta-diversity was increased in three of the four experiments in SI mice. Nevertheless, at the genus level, SI suppressed the abundance of Akkermansia in all four replicates and increased Acetatifactor in three of the four replicates. These two bacterial changes are expected to disrupt OXPHOS, perhaps by resulting a suppression in short-chain fatty acid production. Further, low Akkermansia and high Acetatifactor are expected to increase inflammation. Thus, it is possible that the changes in the composition of the gut microbiota of SI animals explain an increase in IL6 signaling and a suppression in OXPHOS in their mammary gland. We also have studied immune cells in the spleen in SI and GH mice. SI increased the frequency of pro-inflammatory CD4+RORy+ cells, and the immunosuppressive Treg cells (CD4+Foxp3+) and myeloid derived suppressor cells (PMN-MDSCs). SI also increased PD1 expression in Foxp3+ cells. In an on-going study, we found that fecal microbiota transplant (FMT) from SI mice increased E0771 mammary tumor growth in antibiotic treated GH recipient mice, compared with mice receiving FMT from GH mice. Whether the FMT also altered IL6 and OXPHOS signaling in the mammary glands and tumors of recipient mice is currently being investigated. Our findings suggest that social isolation may increase breast cancer through inducing gut dysbiosis. Citation Format: Fabia de Oliveira Andrade, Maddie McDermott, Lu Jin, Vivek Verma, Karla Andrade de Oliveira, Christopher Staley, Leena Hilakivi-Clarke. Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: Do they explain enhanced mammary tumorigenesis. [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 4237.

Abstract 2254: Immunotopography characterization after neoadjuvant therapy in the pancreatic cancer microenvironment

Abstract Pancreatic cancer is one of the most common cancers worldwide and its incidence is increasing globally. Treatment consists of surgical resection followed by adjuvant or neoadjuvant therapy. Neoadjuvant therapy (neoTx) has dramatically improved the prognosis of patients with locally advanced and borderline resectable pancreatic cancer. NeoTX has been shown to reverse the immunosuppression exerted by malignant cells and selectively deplete regulatory T cells and myeloid-derived suppressor cells in the peritumoral niche. In addition, neoTx mediates a significant decrease in the severity of neural invasion and stroma activation. However, the effect of neoTx on the topographical interactions between the different populations of tumor-infiltrating immune cells, the degree of intratumoral immune infiltration and distance to tumor cells as well as their spatial interactions with other key features of the tumor microenvironment (TME) remained unknown. In this study, we employed a multiplex immunofluorescence approach on the COMET™ platform from Lunaphore. COMET™ performs full-automated sequential immunofluorescence (seqIF™) assays, which consist of cycles of staining, imaging, and elution. We developed a customized 12-plex panel on COMET™ to characterize the TME of paraffin-embedded sections of pancreatic cancer samples. COMET™ outputs are OME.TIFF files that were analyzed and quantified with QuPath and CellProfiler for cell segmentation and cell phenotyping respectively. The optimized panel was used to analyze the immune architecture of neoadjuvant-treated pancreatic cancer patients compared to primary resected ones who underwent surgical resection with curative intention. We detected the following immune phenotypes: cytotoxic T cells (CD8+, CD3+), helper T cells (CD4+, CD3+), B cells (CD20+, CD3-), regulatory T cells (FoxP3+, CD4+), macrophages (CD68+), neutrophils (CD163+, CD11b+), and myeloid-derived suppressor cells (CD163, CD11b+, CD66b+). We also quantified the presence of both immune checkpoints (PD1+) cells and their apoptotic rate (Caspase-3), and how those correlate with the immunotopography after neoadjuvant therapy. Our optimized 12-plex panel was successfully transferred to an initial cohort of 10 patients. Our preliminary analysis shows interesting results on the effect of neoTx treatment on the immune microenvironment, such as decreased immune cell density, induction of apoptosis in lymphoid cells, and increased PD1 expression in T-killer cells compared to samples from primary resected patients. Our approach demonstrates the importance of a deep topographic characterization to understand the TME composition of pancreatic cancers, with or without neoTx treatment. Our preliminary findings highlight new differential cell identities in neoTx-treated patients, confirming their suitability for application in future clinical immunotherapy trials. Citation Format: Carmen Mota Reyes, Maximilian Kiessler, Pino Bordignon, Samuel Aubert, Pamela Pulimeno, Helmut Friess, Rouzanna Istvanffy, Ihsan Ekin Demir. Immunotopography characterization after neoadjuvant therapy in the pancreatic cancer microenvironment [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 2254.

Treatment of Hepatocellular Carcinoma by Multimodal In Situ Vaccination Using Cryoablation and a Plant Virus Immunostimulant

PurposeTo test the hypothesis that cryoablation combined with intratumoral immunomodulating nanoparticles from cowpea mosaic virus (CPMV) as an in situ vaccination approach induces systemic antitumoral immunity in a murine model of hepatocellular carcinoma (HCC). Materials and MethodsMice with bilateral, subcutaneous RIL-175 cell–derived HCCs were randomized to 4 groups: (a) phosphate-buffered saline (control), (b) cryoablation only (Cryo), (c) CPMV-treated only (CPMV), and (d) cryoablation plus CPMV-treated (Cryo + CPMV) (N = 11–14 per group). Intratumoral CPMV was administered every 3 days for 4 doses, with cryoablation performed on the third day. Contralateral tumors were monitored. Tumor growth and systemic chemokine/cytokine levels were measured. A subset of tumors and spleens were harvested for immunohistochemistry (IHC) and flow cytometry. One- or 2-way analysis of variance was performed for statistical comparisons. A P value of <.05 was used as the threshold for statistical significance. ResultsAt 2 weeks after treatment, the Cryo and CPMV groups, alone or combined, outperformed the control group in the treated tumor; however, the Cryo + CPMV group showed the strongest reduction and lowest variance (1.6-fold ± 0.9 vs 6.3-fold ± 0.5, P < .0001). For the untreated tumor, only Cryo + CPMV significantly reduced tumor growth compared with control (9.2-fold ± 0.9 vs 17.8-fold ± 2.1, P = .01). The Cryo + CPMV group exhibited a transient increase in interleukin-10 and persistently decreased CXCL1. Flow cytometry revealed natural killer cell enrichment in the untreated tumor and increased PD-1 expression in the spleen. Tumor-infiltrating lymphocytes increased in Cryo + CPMV–treated tumors by IHC. ConclusionsCryoablation and intratumoral CPMV, alone or combined, demonstrated potent efficacy against treated HCC tumors; however, only cryoablation combined with CPMV slowed the growth of untreated tumors, consistent with an abscopal effect.

Abstract P5-03-02: Adiposity and immune-related gene expression in the breast tumor microenvironment

Abstract Background: Extreme adiposity has been associated with tumor progression and increased mortality after a breast cancer diagnosis, but the underlying mechanisms remain unclear. Pre-clinical and in vitro analyses suggest that higher levels of adiposity impair anti-tumor immunity, but studies in human breast cancer patients are lacking. Previously, we found that higher levels of subcutaneous adiposity had stronger associations with breast cancer outcomes than did higher levels of visceral adiposity or overall obesity measured by BMI, underscoring the importance of measuring adipose tissue distribution as well as overall body size to understand the adiposity-cancer link. Methods: We identified women with a first-primary, stage 2 or 3 invasive breast cancer diagnosed and treated at Kaiser Permanente Northern California between 2005 and 2015. Using diagnostic computed tomography scans collected as part of routine clinical care, we measured subcutaneous (SAT) and visceral adipose tissue (VAT) areas in cm2 at the third lumbar vertebra. We calculated body mass index (BMI) from clinically-collected height and weight. We isolated RNA from 251 FFPE breast tumors collected at biopsy or excision; these were a preliminary, random sample within each immunohistochemical subtype groups from an ongoing study that will analyze 1400 breast tumors. We verified RNA quality prior to performing NanoString BC 360™ assays to calculate the PAM50 molecular intrinsic subtype and measure the expression levels of genes related to immune cell abundance and anti-tumor immune activity. Using linear regression models, we examined the mean change in log2 gene expression (dependent variables) associated with each adiposity exposure (BMI, SAT and VAT as independent variables). Results: Mean (SD) age at diagnosis was 56 (13); a majority of women were either overweight (BMI 25- &amp;lt; 30-kg/m2: 30%) or obese (BMI&amp;gt;30-kg/m2: 35%), and most were diagnosed with stage 2 (61%) vs. stage 3 (39%) breast cancer with representation from each PAM50 subtype: n (%) Luminal A, 46 (18%) Luminal B, 56 (22%), HER2-overexpressing 26 (27%), and 82 (33%) basal-like. In unadjusted analyses, expression of genes related to macrophages, PD-1 and TIGIT increased with increasing subcutaneous adiposity, whereas expression of genes related to mast cells decreased (see Table 1). We found a similar (though non-significant) pattern for BMI. Associations with increasing visceral adiposity were closer to the null. After adjusting for PAM50 subtype, age and stage at diagnosis, only the association of increasing subcutaneous adiposity with increasing PD-1 expression remained statistically significant. Conclusion: Excess subcutaneous adiposity was associated with increased PD-1 expression, whereas excess visceral adiposity or obesity defined by BMI were not. These results from the first 251 samples of an ongoing study of 1400 tumors provide evidence from human breast cancer patients to demonstrate the importance of measuring body composition to assess adipose tissue distribution and support the hypothesis that excess adiposity impairs anti-tumor immunity. Association of Adiposity Measures with Immune-Related Gene Expression in the Breast Tumor Microenvironment (n=251 patients with stage 2-3 breast cancer at Kaiser Permanente Northern California) 195% Confidence Interval 2A 1-unit increase represents a doubling in the log expression level of the genes in the signature Citation Format: Deborah A. Dillon, Elizabeth M. Cespedes Feliciano, Alexa Zimbalist, En Cheng, Bette J. Caan, Wendy Y. Chen, Elizabeth A. Mittendorf, Roberto Bonfim Pimenta Peixoto, Tabata Alves Domingos, Krishan Taneja, Ashka Patel. Adiposity and immune-related gene expression in the breast tumor microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-02.