Increase In CD4 Research Articles

Abstract A085: Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment

Abstract Breast cancer is the most common cancer diagnosis worldwide, and an unfortunately high number of women die every year due to a lack of effective therapeutic strategies for metastatic breast cancer. Patients with brain metastases in particular have a significantly poorer prognosis than women with metastasis to other sites (e.g., lung, liver, bone). Previous studies by our group identified a ligand-receptor complex in the tumor microenvironment (TME) that promotes metastasis of breast cancer to the brain. This interaction between platelet-derived growth factor-BB (PDGFB), produced by cancerous breast epithelial cells, and its receptor, platelet-derived growth factor receptor beta (PDGFRβ), expressed by mesenchymal cells, is also prognostic of breast cancer metastasis to the brain in patients. The mechanism of how the PDGFB-PDGFRβ pathway mediates brain metastasis is still unclear and is the focus of our current work. To further analyze the biological effects of PDGFB, our group evaluated gene expression changes between primary human breast tumors expressing high PDGFB versus those expressing low PDGFB and found significantly more immune signaling in the low-expressing tumors. These results led us to hypothesize that the PDGFB-PDGFRβ pathway creates an immunosuppressive microenvironment, allowing for increased metastasis of cancerous cells. To test this, flow cytometry was completed on the brains, spleens, lungs, and livers of control mice and mutant mice expressing stromal-specific hyperactive PDGFRβ following intracardiac injection of PDGFB-expressing mammary tumor cells. No significant changes in immune cell populations were observed in non-tumor-bearing mice. However, in the brains of tumor-bearing mutant mice, there are significant changes to the myeloid lineage. Specifically, analysis at day 10 post-injection (prior to detectable metastatic lesions) revealed diminished CD45+CD11b+ populations. Segregating by CD45hi/CD45lo confirmed a profound decrease in CD45loCD11b+F4/80+ microglia, likely a tumor-suppressive population. Looking at day 16 post-injection (detectable metastatic lesions), there is a shift towards an increase in CD45+ CD11b+GR1+ infiltrating myeloid-derived suppressor cells (MDSCs) in experimental mice, likely a tumor-promoting population. Minimal changes were observed in the lungs and livers at any time point. Confirmatory immunofluorescence staining showed a decrease in tumor-infiltrating microglia (Iba1+) and a significant increase in tumor-infiltrating peripheral macrophages (F4/80+), mirroring the flow data. This experiment was then repeated to test the contribution of the ligand (PDGFB), where a significant increase of infiltrating microglia (Iba1+) was observed upon tumor cell knockdown of PDGFB. Together, these results suggest that the PDGFB-PDGFRβ pathway creates an immunosuppressive environment in the brain TME at least in part through reorchestration of the myeloid populations. Citation Format: Alexis A Mossing, Johnathon G Schiebel, Rebecca L Packard, Amrendra Kumar, Sarah A Steck, Nathaniel S Grabinski, Katie A Thies, Bri Wasik, Tasneem Ariswala, Gary Tozbikian, Johnathan Godbout, Steven T Sizemore, Paul R Lockman, Anna E Vilgelm, Gina M Sizemore. Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A085.

Abstract B047: Epigenetic reprogramming by CBP/P300 bromodomain inhibition of triple-negative breast cancer and the immune microenvironment

Abstract Triple-negative breast cancer (TNBC) is a biologically heterogeneous and clinically important breast cancer subtype because if considered a distinct disease, TNBC would rank as the 5th leading cause of cancer deaths in women. TNBC patients resistant to standard-of-care therapies have poor survival. Tumor-associated immune cells such as neutrophils and macrophages can display a pro-tumoral phenotype and contribute to therapy resistance. Epigenetic enzymes have been shown to reprogram both the tumor cells and tumor-associated immune cells. Thus, targeting epigenetic enzymes is a potential strategy to improve the response of TNBC to standard-of-care therapies. Previously, our laboratory performed an epigenetic drug screen and identified several epigenetic inhibitors. We selected IACS-70654, a novel selective p300/CBP BRD inhibitor, to be the primary focus of this study. We determined the effects of IACS-70654 in our unique syngeneic TNBC mouse models. In a neutrophil-enriched luminal-like TNBC model 2208L, IACS-70654 as a single agent initially caused tumor regression and then durably prevented tumor growth, which is not usually observed for single agents in these aggressive TNBC models. IACS-70654 also substantially decreased infiltrated neutrophils. In the peripheral blood and bone marrow of 2208L tumor-bearing mice, IACS-70654 reduced neutrophils to the same level as non-tumor-bearing mice. Based on this finding, we treated 2208L tumors with IACS-70654 in combination with immune checkpoint blockade anti-PD1 and docetaxel. After 27 days, all treatments besides anti-PD1 were stopped, and the combination-treated tumors grew slower than the ones only treated with IACS-70654. More importantly, in tumors treated with IACS-70654 in combination with anti-PD1, there was a substantial increase in CD8+ T cell infiltration. In addition, B cell infiltration was induced in the tumor stroma, indicating that IACS-70654 stimulated immune response in the tumor microenvironment. In contrast, IACS-70654 failed to significantly reduce the growth of T12, a Claudin-low macrophage-enriched model. Therefore, we hypothesized that the infiltrated immune profiles, specifically myeloid cell infiltration, might partially account for this differential response. Accordingly, we studied the effects of IACS-70654 in two additional models, basal-like T6 and luminal-like 2250L tumors, which also have relatively high neutrophil infiltration. However, one of the principal differences in immune profiles between the two models and 2208L is that they also are infiltrated with twice as many macrophages. The response of infiltrated immune cells to IACS-70654 in those two models differed from those of 2208L tumors. IACS-70654 reduced monocytic myeloid-derived suppressor cells and pro-tumor macrophages. These results illustrate the immune heterogeneity among our tumor models. We are currently performing ChIP-seq and single-cell ATAC-seq to elucidate potential mechanisms and pathways employed by IACS-70654 in both tumor and tumor-associated immune cells. Citation Format: Xueying Yuan, Michael Soth, Na Zhao, Philip Jones, Jeffrey Rosen. Epigenetic reprogramming by CBP/P300 bromodomain inhibition of triple-negative breast cancer and the immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B047.

Presence of Epstein–Barr virus (EBV) antigens detected by sensitive methods has no influence on local immune environment in diffuse large B cell lymphoma

EBV+ diffuse large B cell lymphoma (DLBCL) not otherwise specified (NOS) is a new entity confirmed by the World Health Organization (WHO) in 2017. In this new entity, the virus may contribute to a tolerogenic microenvironment. Traces of the virus have been described in DLBCL with more sensitive methods, in cases that were originally diagnosed as negative. The aim of this study was to analyze the expression of immune response genes in the tumor microenvironment to disclose the role of the virus and its traces in DLBCL. In 48 DLBCL cases, the expression of immune response genes and the presence of molecules that induce tolerance, such as TIM3, LAG3 and PDL1 by immunohistochemistry (IHC), were studied. To broaden the study of the microenvironment, tumor-associated macrophages (TMAs) were also explored. No significant differences were observed in the expression of immune response genes in the EBV+ DLBCL and those cases that were EBV− DLBCL but that exhibited viral traces, assessed by ViewRNA assay. Only the EBV+ DLBCL cases displayed a significantly higher increase in the expression of CD8 and cytotoxic T cells detected by gene expression analysis, and of PDL1 in tumor cells and in the expression of CD68 in the tumor microenvironment detected by IHC, not observed in those cases with viral traces. The increase in CD8 and cytotoxic T cells, PDL1 and CD68 markers only in EBV+ DLBCL may indicate that traces of viral infection might not have influence in immune response markers.

Increase in CD8+ Effector Memory T Cells Re-Expressing CD45RA Correlates with Intractability of Severe Alopecia Areata

Increase in CD8+ Effector Memory T Cells Re-Expressing CD45RA Correlates with Intractability of Severe Alopecia Areata

Nivolumab Plus 5-Azacitidine in Pediatric Relapsed/Refractory Acute Myeloid Leukemia (AML): Phase I/II Trial Results from the Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) Consortium

Improvements in survival have been made over the past two decades for childhood acute myeloid leukemia (AML), but the approximately 40% of patients who relapse continue to have poor outcomes. A combination of checkpoint-inhibitor nivolumab and azacitidine has demonstrated improvements in median survival in adults with AML. This phase I/II study with nivolumab and azacitidine in children with relapsed/refractory AML (NCT03825367) was conducted through the Therapeutic Advances in Childhood Leukemia & Lymphoma consortium. Thirteen patients, median age 13.7 years, were enrolled. Patients had refractory disease with multiple reinduction attempts. Twelve evaluable patients were treated at the recommended phase II dose (established at dose level 1, 3 mg/kg/dose). Four patients (33%) maintained stable disease. This combination was well tolerated, with no dose-limiting toxicities observed. Grade 3–4 adverse events (AEs) were primarily hematological. Febrile neutropenia was the most common AE ≥ grade 3. A trend to improved quality of life was noted. Increases in CD8+ T cells and reductions in CD4+/CD8+ T cells and demethylation were observed. The combination was well tolerated and had an acceptable safety profile in pediatric patients with relapsed/refractory AML. Future studies might explore this combination for the maintenance of remission in children with AML at high risk of relapse.

A novel CpG ODN compound adjuvant enhances immune response to spike subunit vaccines of porcine epidemic diarrhea virus.

CpG oligodeoxynucleotides (CpG ODNs) boost the humoral and cellular immune responses to antigens through interaction with Toll-like receptor 9 (TLR9). These CpG ODNs have been extensively utilized in human vaccines. In our study, we evaluated five B-type CpG ODNs that have stimulatory effects on pigs by measuring the proliferation of porcine peripheral blood mononuclear cells (PBMCs) and assessing interferon gamma (IFN-γ) secretion. Furthermore, this study examined the immunoenhancing effects of the MF59 and CpG ODNs compound adjuvant in mouse and piglet models of porcine epidemic diarrhea virus (PEDV) subunit vaccine administration. The in vitro screening revealed that the CpG ODN named CpG5 significantly stimulated the proliferation of porcine PBMCs and elevated IFN-γ secretion levels. In the mouse vaccination model, CpG5 compound adjuvant significantly bolstered the humoral and cellular immune responses to the PEDV subunit vaccines, leading to Th1 immune responses characterized by increased IFN-γ and IgG2a levels. In piglets, the neutralizing antibody titer was significantly enhanced with CpG5 compound adjuvant, alongside a considerable increase in CD8+ T lymphocytes proportion. The combination of MF59 adjuvant and CpG5 exhibits a synergistic effect, resulting in an earlier, more intense, and long-lasting immune response in subunit vaccines for PEDV. This combination holds significant promise as a robust candidate for the development of vaccine adjuvant.

Serologic, radiographic, and tissue-based markers associated with major pathologic response after treatment with neoadjuvant immunotherapy in patients with resectable hepatocellular carcinoma.

561 Background: Neoadjuvant immunotherapy can induce a major pathologic response (MPR) in patients with resectable hepatocellular carcinoma (HCC), which may be associated with prolonged recurrence-free survival. This study aimed to understand which variables correlate with achieving an MPR. Methods: Patients with resectable HCC who received either neoadjuvant nivolumab plus ipilimumab or nivolumab alone and underwent surgery were included. 18 patients had baseline and post-treatment computed tomography, alpha-fetoprotein (AFP), and elastography. 17 of these patients had baseline and post-treatment tissue available for immunohistochemistry. Patients were classified into two groups: MPR, defined as necrosis of >70%, and no MPR. Data was summarized using descriptive statistics and compared using Wilcoxon rank sum test. P value <0.05 was considered statistically significant. Results: Statistically significant differences in objective response (OR) and changes in CD8, Granzyme B, and PD-1 expression were identified upon comparison of patients with an MPR to those without an MPR (Table 1). Additionally, numerical differences in baseline tumor size, change in AFP, and both non-tumor and tumor liver fibrosis (at baseline, after treatment, and interim change) were identified upon comparison of patients with an MPR to those without an MPR. Conclusions: Our data show that large baseline tumor size, greater OR, reduction in AFP, increase in liver and tumor fibrosis, and increase in CD8, Granzyme B, and PD-1 expression after receipt of neoadjuvant immunotherapy are associated with achieving an MPR in patients with resectable HCC. [Table: see text]

Extracellular Matrix-Trapped Bioinspired Lipoprotein Prolongs Tumor Retention to Potentiate Antitumor Immunity.

The immunomodulatory effects of many therapeutic agents are significantly challenged by their insufficient delivery efficiency and short retention time in tumors. Regarding the distinctively upregulated fibronectin (FN1) and tenascin C (TNC) in tumor stroma, herein a protease-activated FN1 and/or TNC binding peptide (FTF) is designed and an extracellular matrix (ECM)-trapped bioinspired lipoprotein (BL) (FTF-BL-CP) is proposed that can be preferentially captured by the TNC and/or FN1 for tumor retention, and then be responsively dissociated from the matrix to potentiate the antitumor immunity. The FTF-BL-CP treatment produces a 6.96-, 9.24-, 6.72-, 7.32-, and 6.73-fold increase of CD3+CD8+ T cells and their interferon-γ-, granzyme B-, perforin-, and Ki67-expressing subtypes versus the negative control, thereby profoundly eliciting the antitumor immunity. In orthotopic and lung metastatic breast cancer models, FTF-BL-CP produces notable therapeutic benefits of retarding tumor growth, extending survivals, and inhibiting lung metastasis. Therefore, this ECM-trapping strategy provides an encouraging possibility of prolonging tumor retention to potentiate the antitumor immunity for anticancer immunotherapy.

Abstract A051: PSGL-1-deficiency promotes pancreatic ductal adenocarcinoma tumor control and synergy with immune checkpoint blockade

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer with increasing incidence rates while patient prognosis remains poor with a 5-year survival rate of only 12%. PDAC tumors are refractory to all currently available treatments, including existing immune checkpoint blockade (ICB) therapies, and demonstrate limited T cell infiltration. There is a critical need to develop novel approaches to improve patient outcomes. We previously demonstrated that deficiency of P-selectin glycoprotein 1 (PSGL-1) promotes enhanced T cell responses that clear chronic LCMV Cl13 virus infection and inhibit growth of PD-1 blockade resistant melanoma. Here we evaluated if targeting PSGL-1 promotes T cell responses to PDAC tumors to inhibit growth and metastases. KPC.4662 tumor cells were injected orthotopically into the tail of the pancreas of C57BL/6 (wildtype control) and PSGL-1−/− (C57BL/6 background) male or female mice and tumor growth and immune infiltration was evaluated by histology and flow cytometry. At 28 days post-orthotopic tumor cell injection, KPC.4662 tumor burden was reduced by 50% on average (1.1 g vs 0.45g) in PSGL-1−/− mice compared to controls. Total immune infiltration (CD45+) was increased in tumors from PSGL-1−/- mice (10% vs 24%), with a significant increase in CD3+ T cell infiltration. Moreover, fewer mice developed metastases in the peritoneal cavity, liver, and lungs. To assess if PSGL-1−/− mice had the capability to reduce metastatic burden, KPC 4662 tumor cells were injected intravascularly and tumor development in the lungs was assessed on day 17. PSGL-1−/− mice reproducibly developed fewer tumors in the lungs compared to wildtype control mice as determined by histological examination. To address if enhanced control of PDAC tumors was mediated by T cells, we depleted CD4+ and CD8+ T cells immediately prior to orthotopic implantation of KPC.4662 tumor cells. Depletion of T cells resulted in the loss of PDAC tumor growth control in PSGL-1−/− mice. Given our previous studies demonstrating an intrinsic role for PSGL-1 in the development of T cell exhaustion, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 immune checkpoint blockade, a therapy known to be ineffective as a monotherapy in PDAC patients and mouse models. When PD-1 blocking antibody was therapeutically administered on days 10, 12, and 14 post-tumor implantation, 85% of PSGL-1−/− animals demonstrated complete responses. From these studies, we conclude that in the absence of PSGL-1, we observe significantly reduced growth of primary orthotopic PDAC tumors and decreased metastases. These findings are associated with increased CD45+ immune cell infiltration in the tumors and notably functions in a T cell-dependent manner. PSGL-1 therefore represents a novel target for promoting immune responses to PDAC tumors. Citation Format: Jennifer L. Hope, Yijuan Zhang, Hannah A. Faso, Sreeja Roy, Michelle Lin, Ashley B. Palete, Swetha Maganti, Dennis C. Otero, Cosimo Commisso, Linda M. Bradley. PSGL-1-deficiency promotes pancreatic ductal adenocarcinoma tumor control and synergy with immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A051.

Upregulation of CD8+ regulatory T cells following liver-directed AAV gene therapy

Liver-directed AAV gene therapy represents a unique treatment modality for a host of diseases. This is due, in part, to the induction of tolerance to transgene products. Despite the plethora of recognized regulatory cells in the body, there is currently a lack of literature supporting the induction of non-CD4+ regulatory cells following hepatic AAV gene transfer. In this work, we show that CD8+ regulatory T cells are up-regulated in PBMCs of mice following capsid only and therapeutic transgene AAV administration. Further, we demonstrate that hepatic AAV gene transfer results in a significant increase in CD8+ regulatory T cells following experimental autoimmune encephalomyelitis induction. Notably, this response occurred only in therapeutic vector treated animals, not capsid only controls. Understanding the role these cells play in treatment efficacy will result in the development of improved AAV vectors that take advantage of the full gamut of regulatory cells within the body.

A Novel Protozoa Parasite-Derived Protein Adjuvant Is Effective in Immunization with Cancer Cells to Activate the Cancer-Specific Protective Immunity and Inhibit the Cancer Growth in a Murine Model of Colorectal Cancer.

Cancer-specific CD8+ cytotoxic T cells play important roles in preventing cancer growth, and IFN-γ, in addition to IL-12 and type I interferon, is critical for activating CD8+ cytotoxic T cells. We recently identified the capability of the amino-terminus region of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii, an intracellular protozoan parasite, to activate IFN-γ production of microglia, a tissue-resident macrophage population. Therefore, in the present study, we examined whether recombinant GRA6Nt protein (rGRA6Nt) functions as an effective adjuvant to potently activate cancer-specific protective immunity using a murine model of MC38 colorectal cancer (CRC). When mice were immunized with non-replicable (either treated with mitomycin C or irradiated by X-ray) MC38 CRC cells in combination with rGRA6Nt adjuvant and received a challenge implantation of replication-capable MC38 tumor cells, those mice markedly inhibited the growth of the implanted tumors in association with a two-fold increase in CD8+ T cell density within the tumors. In addition, CD8+ T cells of the immunized mice secreted significantly increased amounts of granzyme B, a key mediator of the cytotoxic activity of CD8+ T cells, and IFN-γ in response to MC38 CRC cells in vitro when compared to the T cells from unimmunized mice. Notably, the protective effects of the immunization were specific to MC38 CRC cells, as the immunized mice did not exhibit a significantly inhibited growth of EL4 lymphoma tumors. These results indicate that rGRA6Nt is a novel and effective protein adjuvant when used in immunizations with non-replicable cancer cells to potently activate the protective immunity specifically against the cancer cells employed in the immunization.

The Correlation Between Low-Dose Radiotherapy Area of the Mediastinum and CD8+T Cells and the Efficacy of Radiotherapy for Non-Small Cell Lung Cancer.

Radiation therapy (RT) can cause changes in peripheral blood immune cells. The relationship between the efficacy of radiation therapy for non-small cell lung cancer (NSCLC) and immune cell changes and the study of how mediastinal radiation dose parameters affect immune cell changes is still unclear. This study aims to analyze the relationship between immune cell changes induced by radiotherapy and the efficacy of NSCLC radiotherapy, as well as the relationship between radiotherapy dose parameters and immune cell changes. We retrospectively analyzed the data of NSCLC patients receiving mediastinal radiation therapy from 2020 to 2022. Collect lymphocytes and circulating immune cells within one week before and after radiotherapy and collect the dose-volume parameters of the whole mediastinum in the patient's RT planning system. Analyze the changes in lymphocytes and radiotherapy effects after radiotherapy, and explore the relationship between radiotherapy dose parameters and immune cell changes. A total of 72 patients were enrolled. Compared with before radiotherapy, the proportion of CD3+T cells, CD8+T cells, and CD8/Treg in peripheral blood significantly increased after radiotherapy (P<0.05). The increase in CD8+T cells and CD8/Treg after radiotherapy was correlated with Objective response rate (ORR) (P<0.05). Based on binary logistic univariate and multivariate regression analysis, an increase in CD8+T cells after radiotherapy is an independent predictor of objective tumor response after radiotherapy (OR=12.71, 95% CI=3.64-44.64, P=0.01), and Volume of 200 cGy irradiation (V2) is an independent positive predictor of an increase in CD8+T lymphocyte ratio after radiotherapy (high group, OR=3.40, 95% CI=1.13-10.36, P=0.03). The increase in CD8+T cells after radiotherapy can positively predict the short-term efficacy of radiotherapy. Mediastinal low-dose radiation therapy can increase CD8+T cells, thereby improving the short-term efficacy of radiotherapy. These potentially related mechanisms are worth further verification and exploration.

Investigation of Clinical Value of NKT-like Cells in Tumor Diseases.

The goal is to study the changes of natural killer T-like (NKT-like) cells with age and explore the value of NKT-like cell changes in the evaluation of immune function and prognosis of tumor patients. From January 2021 to December 2021, 19 patients with lung cancer, 37 patients with lymphoma, 16 patients with multiple myeloma (MM), 13 patients with acute lymphoblastic leukemia (ALL), 70 patients with acute myeloid leukemia (AML) treated in the Affiliated Hospital of Chengde Medical College and 141 healthy volunteers included in the healthy control group were recruited to study the change trend of NKT-like cells with age and changes in different tumor patients. With the increase of age, NKT-like cells in peripheral blood increased gradually in healthy people, mainly composed of CD8+NKT-like cells and CD8-CD4-NKT-like cells. The proportion of NKT-like cells in the lymphoma group and AML group was significantly higher than that in the control group (p < 0.05). The proportion of CD8+NKT-like cells decreased in the Lymphoma, AML, ALL, and lung cancer groups (p < 0.05), there was no statistical significance between MM group and control group (p > 0.05). With the increase of age, NKT-like cells in peripheral blood gradually increased in healthy people and were mainly composed of CD8+NKT-like cells and CD8-CD4-NKT-like cells. The increase of CD8+NKT-like cells in peripheral blood has important reference value for the evaluation of immune function and prognosis of patients with lymphoma, AML, ALL, and lung cancer and provides direction for immunotherapy.

The impact of cholesteryl ester transfer protein on the progression of cutaneous leishmaniasis.

Pathogenesis of cutaneous leishmaniases involves parasite growth, persistent inflammation, and likely participation of lipoproteins (LP). The cholesteryl ester transfer protein (CETP), involved in LP remodeling, has been shown to participate in the inflammatory response and the evolution of infectious conditions. We evaluated the impact of the presence of CETP on infection by Leishmania (L.) amazonensis in an experimental model of cutaneous leishmaniasis using C57BL6/J mice transgenic for human CETP (CETP), having as control their littermates that do not express the protein, wild-type (WT) mice. The progression of the lesion after infection in the footpad was monitored for 12 weeks. Two groups of animals were formed to collect the plantar pad in the 4th and 12th week post-infection. The lesion increased from the 3rd week onwards, in both groups, with a gradual decrease from the 10th week onwards in the CETP group compared to the WT group, showing a reduction in parasitism and an improvement in the healing process, a reduction in CD68+ cells, and an increase in CD163+ and CD206, characterizing a population of M2 macrophages. A reduction in ARG1+ cells and an increase in INOS+ cells were observed. During infection, the LP profile showed an increase in triglycerides in the VLDL fraction in the CETP group at 12 weeks. Gene expression revealed a decrease in the CD36 receptor in the CETP group at 12 weeks, correlating with healing and parasite reduction. In vitro, macrophages derived from bone marrow cells from CETP mice showed lower parasite load at 48 h and, a reduction in arginase activity at 4 h accompanied by increased NO production at 4 and 24 h compared to WT macrophages, corroborating the in vivo findings. The data indicate that the presence of CETP plays an important role in resolving Leishmania (L.) amazonensis infection, reducing parasitism, and modulating the inflammatory response in controlling infection and tissue repair.

Abstract C033: Preliminary pharmacodynamic evaluation of AU-007 in phase 1 dose escalation trial in patients with advanced solid tumors

Abstract AU-007 is a monoclonal antibody currently being evaluated in a Phase 1/2 clinical trial. In conjunction with evaluation of clinical activity, a pharmacodynamic program has been established to assess the biological activity of AU-007 alone or with low dose subcutaneous aldesleukin in patients with advanced solid cancers. AU-007 binds IL-2 on its CD25-binding epitope, thus inhibiting IL-2’s interaction with CD25 in the trimeric IL-2 receptor (IL-2R) but allowing for IL-2’s binding to the dimeric IL-2R. This effectively inhibits the trimeric IL-2R expressing Tregs without impacting dimer expressing memory T cells, NK cells, NKT cells and naïve T cells. We evaluated the effect of AU-007 +/- aldesleukin on peripheral blood cell populations, IFN-gamma, IL-2 and sCD25. Here we report the interim pharmacodynamic data from the ongoing dose escalation portion of the trial. Peripheral blood samples were collected in Cyto-Chex BCT tubes and phenotyped for T cells, NK cells, B cells, and monocytes. A TruCount system was used to obtain absolute cell numbers. In addition, eosinophils were assessed through standard CBC differentials. Serum collections were done to assess circulating markers using the MSD ECL platform. It was observed that, regardless of AU-007 dose with or without aldesleukin, patients had a reduction in the circulating levels of Treg cells (i.e., based on assay validation, a change greater than +/-10% was considered a real change). This is consistent with the mechanism of action of AU-007, by binding IL-2 such that it prevents the interaction with the trimeric IL-2R, Tregs are affected. There was an increase in the CD8+ to Treg ratio consistent with the overall decrease in Tregs. Trends for increases in NK cells and CD8+ T cells were observed in most patients. Eosinophils express the trimeric IL-2R and are known to increase after IL-2 therapy. To date, no drug related increases in eosinophils with AU-007 +/- aldesleukin were observed, and in most cases the levels of circulating eosinophils decreased. IFN-g is a cytokine released after activation of the inflammatory/cytotoxic arm of the immune system. Twelve of the initial 17 patients for whom data are available demonstrated at least a 2-fold or greater increase in the circulating levels of IFN-g, often occurring within 48 hours after dosing. This was observed regardless of dose given. Higher frequency and amounts of IFN-g expression were observed when subcutaneous low dose aldesleukin was also given (dosed 2 – 24h post AU-007 dosing). A dose dependent trend with single-agent AU-007 was observed, leading to more patients with a &amp;gt;2-fold IFN-g. All patients who received low dose aldesleukin had at least 1 timepoint post dose with a &amp;gt;2-fold increase. Overall, AU-007 has demonstrated pharmacodynamic responses consistent with the mechanism of action, with decreases in the regulatory population of cells and increases in inflammatory cells and markers. The initial pharmacodynamic data support continued dose escalation of AU-007 +/- aldesleukin in patients with advanced cancer. Citation Format: Timothy Wyant, Sophia Frentzas, Elizabeth Ahern, Andrew Weickhardt, Andrew Haydon, John Powderly, Drew Rasco, Meredith McKean, Paul De Souza, Jenny Tang, Lori Richards, Aron Knckerbocker, Yanay Ofran, Inbar Amit, James Vasselli. Preliminary pharmacodynamic evaluation of AU-007 in phase 1 dose escalation trial in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C033.

Abstract A070: Olaparib enhances radiation-induced Type I interferon and sensitizes pancreatic cancer to PD-L1 immune checkpoint inhibition

Abstract Inhibitors of PARP have been shown to sensitize pancreatic ductal adenocarcinoma (PDAC) to radiation therapy through mechanisms involving DNA damage, replication stress, and PARP trapping independent of BRCA1/2 mutation status. These mechanisms have the potential to activate innate immune signaling leading to Type I Interferon (T1IFN) production. Thus, we hypothesized that the clinical PARP1/2 inhibitor olaparib would potentiate radiation-induced T1IFN to activate an anti-tumoral immune response and sensitize resistant PDAC to immunotherapy. To test this hypothesis, we assessed the effects of olaparib and radiation on T1IFN production and signaling, tumor growth, sensitivity to PD-L1 immunotherapy, and the tumor immune microenvironment in mouse models of PDAC. We found that clinically achievable concentrations of olaparib significantly enhanced radiation-induced T1IFN production measured by both an IFNβ promoter-driven reporter and Ifnβ mRNA levels. Additionally, we found that the downstream effects of radiation-induced T1IFN signaling, including PD-L1 and MHC-I cell surface expression and induction of interferon stimulated genes including Cxcl9 and Cxcl10, were enhanced by olaparib. Co-treatment with a T1IFN receptor blocking antibody diminished these effects supporting the requirement for T1IFN-dependent signaling for the innate immune effects of radiation and olaparib. In vivo treatment with olaparib and radiation sensitized tumors to anti-PD-L1 therapy with a 40% complete response rate. These therapeutic responses led to an adaptive long term immunogenic memory evidenced by reduced engraftment following tumor rechallenge. Single-cell RNA sequencing revealed that treatment with olaparib, radiation and anti-PD-L1 augmented an anti-tumoral CD8+ T cell immune response, which is supported by an increase in CD8+ effector T cells and a decrease in CD8+ dysfunctional T cells. The necessity of functional CD8+ T cells to therapy efficacy in vivo was further reflected by a significantly reduced therapeutic combinational efficacy using a CD8+ T cell blocking antibody. Taken together, the results of this study show that the combination of olaparib, radiation and anti-PD-L1 induce an anti-tumoral immune response mediated by both T1IFN and CD8+ T cells. The results of this study provide the rationale for a phase 1 clinical trial (NCT05411094) combining dose escalated olaparib with durvalumab and radiotherapy in patients with locally advanced pancreatic cancer. Citation Format: Victoria M Valvo, Qiang Zhang, Long Jiang, Ashkay Tate, Michael D Green, Meredith A Morgan. Olaparib enhances radiation-induced Type I interferon and sensitizes pancreatic cancer to PD-L1 immune checkpoint inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A070.

B cell and T cell populations are altered in the brain‐draining lymph nodes of the senescence accelerated mouse prone 8 (SAMP8)

AbstractBackgroundRecent studies have (re)‐discovered lymphatic vessels in the meninges and other sites that flow from the brain to deep cervical lymph nodes (dcLNs). Like elsewhere in the body, these lymphatic vessels play a critical role in the clearance of fluid and macromolecules from the brain including Aβ (a toxic protein that accumulates in the brain in Alzheimer’s disease) and in trafficking immune cells to the dcLNs, where immune activation occurs. The current study examined whether the immune cell populations are altered in the dcLNs of SAMP8 mice (a mouse model of accelerated aging with spontaneous overproduction of amyloid precursor protein in the brain) compared to the healthy control mice (SAMR1).MethodIn this study, dcLNs were collected from 12‐month‐old SAMP8 and SAMR1 mice (n = 6). Cells were isolated from the dcLNs, and the B cell and T cell populations (CD19 for B cells, PNA for germinal centre B cells, CD93 for early B cells, CD138 for antibody secreting B cells; CD3 for T cells, CD8a for cytotoxic T cells, CD4 for helper T cell) were studied using flow cytometry.ResultA significant 1.4‐fold increase in CD3+ T cells (p = 0.04) and a trend toward a decrease in CD19+ B cells (p = 0.06) was observed in the SAMP8 dcLNs compared with SAMR1 dcLNs. For B cell sub‐populations, a 77.9 ± 26.9% decrease (p = 0.0078) in geminal center B cells was observed in SAMP8 dcLNs. The T/B cell ratio was significantly increased in SAMP8 dcLNs (2.74 ± 0.14) comparing to SAMR1 dcLNs (2.10 ± 0.16). Such alteration was not observed in mesenteric lymph nodes.ConclusionThe B cell and T cell populations were altered in the brain‐draining dcLNs of SAMP8 mice, suggesting that the dcLNs responded to pathological changes in the brain. The reduced B cells, especially the germinal centre B cells, indicate a possible impairment in the production of antibody‐secreting B cells in SAMP8 mice. We will confirm these observations in APP/PS1 and 5xFAD mice in future studies, and investigate whether such alteration in B cells contributes to Alzheimer’s disease pathology.

Altered immune cell phenotypes within chronically ischemic human kidneys distal to occlusive renal artery disease.

Renal artery stenosis (RAS) is a major cause of ischemic kidney disease, which is largely mediated by inflammation. Mapping the immune cell composition in ischemic kidneys might provide useful insight into the disease pathogenesis and uncover therapeutic targets. We used mass cytometry (CyTOF) to explore the single-cell composition in a unique data set of human kidneys nephrectomized due to chronic occlusive vascular disease (RAS, n = 3), relatively healthy donor kidneys (n = 6), and unaffected sections of kidneys with renal cell carcinoma (RCC, n = 3). Renal fibrosis and certain macrophage populations were also evaluated in renal sections. Cytobank analysis showed in RAS kidneys decreased cell populations expressing epithelial markers (CD45-/CD13+) and increased CD45+ inflammatory cells, whereas scattered tubular-progenitor-like cells (CD45-/CD133+/CD24+) increased compared with kidney donors. Macrophages switched to proinflammatory phenotypes in RAS, and the numbers of IL-10-producing dendritic cells (DC) were also lower. Compared with kidney donors, RAS kidneys had decreased overall DC populations but increased plasmacytoid DC. Furthermore, senescent active T cells (CD45+/CD28+/CD57+), aged neutrophils (CD45+/CD15+/CD24+/CD11c+), and regulatory B cells (CD45+/CD14-/CD24+/CD44+) were increased in RAS. RCC kidneys showed a distribution of cell phenotypes comparable with RAS but less pronounced, accompanied by an increase in CD34+, CD370+, CD103+, and CD11c+/CD103+ cells. Histologically, RAS kidneys showed significantly increased fibrosis and decreased CD163+/CD141+ cells. The single-cell platform CyTOF enables the detection of significant changes in renal cells, especially in subsets of immune cells in ischemic human kidneys. Endogenous pro-repair cell types in RAS warrant future study for potential immune therapy.NEW & NOTEWORTHY The single-cell platform mass cytometry (CyTOF) enables detection of significant changes in one million of renal cells, especially in subsets of immune cells in ischemic human kidneys distal to renal artery stenosis (RAS). We found that pro-repair cell types such as scattered tubular-progenitor-like cells, aged neutrophils, and regulatory B cells show a compensatory increase in RAS. Immune cell phenotype changes may reflect ongoing inflammation and impaired immune defense capability in the kidneys.

Prediction Modeling of CAR-T Cell Therapy for Diffuse Large B-Cell Lymphoma Using Artificial Neural Networks on Tumor Vascular Phenotype

Chimeric antigen receptor T (CAR-T) cell therapy targeting CD19 only results in complete remission in approximately 30-40% of relapsed/refractory disease patients with diffuse large B-cell lymphoma (DLBCL). Failure may result from lack of CAR-T entry into the tumor. Endogenous T cell homing is initiated by binding to post-capillary venular endothelial cells (ECs). Naïve and central memory T cells use L-selectin binding to peripheral node addressin (PNad) on high endothelial venules (HEVs) of the lymph node while circulating effector memory T cells, lacking L-selectin, use other molecules to bind cytokine-induced endothelial proteins E-selectin, ICAM-1 and VCAM-1 on flat venules of inflamed tissues. Both of these venular EC types are present in inflamed lymph nodes. We hypothesized that the expression patterns of adhesion molecules on venular ECs in pre-treatment tumor biopsies could predict CAR-T efficacy. To test this, we applied cyclic immunofluorescence to 18 archived pre-treatment DLBCL patient FFPE biopsies (8 responders and 10 non-responders, defined by relapse at 12 months). We detected venular ECs using a combination of anti-CD31 and UEA lectin on larger diameter vessels and assessed expression for PNad, E-selectin, ICAM-1, VCAM-1. We detected endogenous CD8+ T cells and evaluated their expression of L-selectin, alpha 4 integrin and PSGL-1. See table 1. DAPI nuclear staining was included in each cycle. Images of each cycle were combined in ImageJ and imported into QuPath for analysis. Using Warpy, affine transformations were applied to DAPI staining to create overlays between imaging cycles. After alignment, representative images of several samples were randomly chosen for image training using a combination of Artificial Neural Networks (ANNs) and classifiers to perform identification and mask tissue regions of vessels and T-cells within the tissue. Positive and negative cells for each marker of interest (EC PNad, ICAM-1, E-Selectin, VCAM-1, and T cell alpha 4 integrin, L-Selectin, and PSGL-1) were used to create classification algorithms to determine marker expression for ECs and T-cells within the tissue. Results were analyzed by unpaired t-test and one-way ANOVA conducted in Graph-Pad Prism 9 software. Preliminary results from quantitative analysis of pre-treatment biopsies show a significant increase in CD31+/PNAD- venular ECs compared to non-responders that were double positive for ICAM1 and VCAM-1 (p=0.0273). This analysis also revealed a significant increase in CD31+/PNAD+ HEV ECs from responders that were triple positive for E-Selectin, ICAM-1, and VCAM-1 (p=0.0242). See figure 1. Our initial semi-quantitative analysis showed a trend toward elevated numbers of tissue-infiltrating CD3 + CD8 + T cells in the pre-treatment biopsies of responders. T cells expressing PSGL-1, a glycoprotein that may carry carbohydrate moieties recognized by E-Selectin and linked to T-cell exhaustion, was increased in the non-responder cohort. These data suggest an increased extent and activation state of intra-tumoral venular ECs and increased endogenous T-cell infiltrates, which may predict the outcome of CAR-T therapy. Analysis of T-cells in the pre-treatment biopsies leveraging machine learning algorithms is in progress and both complete quantitative findings and prognostic value of this approach will be presented. While our findings require further validation thru additional biopsy analysis, they provide emerging evidence for a rationale involving vascular readiness in DLBCL responder differences to CART therapy.

A Single Bout of Exercise Enhances the Efficacy of Rituximab Against Autologous Chronic Lymphocytic Leukaemia B Cells Ex Vivo By Transiently Increasing Natural Killer Cell Frequency in Blood, and Simultaneously Mobilises CD5 +CD19 +CD20 + B Cells into Blood

Chronic Lymphocytic Leukaemia (CLL) is characterised by the proliferation and accumulation of clonal B cells (B-CLL cells) and is often treated with anti-CD20 monoclonal antibody immunotherapies, including rituximab. One of the mechanism-of-action of rituximab is antibody-dependent cellular cytotoxicity (ADCC) which occurs when natural killer (NK) cells detect rituximab bound to CD20 + target cells. Rituximab often fails to induce stringent disease eradication, due in part to the diffuse distribution of clonal cells across multiple lymphoid and non-lymphoid tissues where NK cell frequency can be low. It is well established that an individual bout of aerobic exercise induces a transient relocation of lymphocytes - including NK cells and B cells - into peripheral blood. We hypothesised that this exercise-induced lymphocytosis could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab into blood. In this pilot study n = 20 treatment naïve patients with CLL (mean ± SD: age = 62 ± 10 years; height = 174.0 ± 7.5 cm; body mass = 83.3 ± 16.8 kg; body fat = 31.7 ± 9.8 %; blood leukocytes = 30.70 ± 22.21 ×10 9/L; anaerobic threshold = 14.1 ± 2.9 mL.kg -1.min -1) participated. Participants cycled at a moderate intensity (15% above their anaerobic threshold) for ~30-minutes, with blood samples collected pre-, post-, and 1-hour post-exercise. Given the importance of CD16 + NK cells in evoking ADCC, we enumerated NK cell subsets in blood samples collected pre, post-, and 1-hour post-exercise by flow cytometry. As expected, exercise induced a preferential increase of CD56 +CD16 + (+255%, p &amp;lt; 0.001) and mature, cytotoxic CD56 +CD57 +CD16 + NK cells (+322%, p &amp;lt; 0.001) pre- to post-exercise. Next, using immunomagnetic negative separation, NK cells and primary B-CLL cells were isolated from blood pre- and post-exercise and incubated together with heat inactivated foetal calf serum, with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab mediated cell lysis increased by +129% following exercise ( p &amp;lt; 0.001), with no change in antibody independent NK cell lysis of B-CLL cells - independent of rituximab - following exercise ( p = 0.25). Blocking CD16 on NK cells - vital for ADCC - blunted the effects of exercise on B-CLL cell lysis ( p = 0.84). In a subset of patients ( n = 9) we also explored the effects of autologous time-point matched plasma (instead of heat inactivated foetal calf serum) on rituximab mediated ADCC, which evoked a +92% increase in lysis pre- to post-exercise ( p = 0.038). Collectively, our results suggest that augmented efficacy of rituximab mediated ADCC was driven by an increase in CD16 + NK cells. We posited that the enhancement to rituximab mediated ADCC would have greater, clinically relevant implications if there was also a concomitant exercise-induced mobilisation of B-CLL cells expressing CD20 into blood from different body tissues. Flow cytometry revealed a +63% increase in CD5 +CD19 +CD20 + B-CLL cells ( p = 0.002) in blood after exercise. Further analyses revealed that CD5 +CD19 +CD20 + B-CLL cells with a phenotype consistent with recent egress from lymphoid tissue (CD5 brightCXCR4 dim; 70%, p = 0.004) and B-CLL cells with a propensity to migrate to peripheral tissues (CD5 dimCXCR4 bright; 67%, p = 0.002) were mobilised, with no change to overall CD20 surface antigen density ( p = 1.0) - determined by median fluorescence intensity. Furthermore, exercise evoked a +69% ( p = 0.022) increase in CD5 +CD19 +CD20 + B-CLL cells expressing CD49d, which is considered one of the strongest predictors of CLL prognosis. Taken together, these data demonstrate that exercise increased the frequency of CD20 + B-CLL cells with lymphoid origins and prognostic relevance into the blood, therefore rendering them susceptible to rituximab mediated ADCC. Our results show that individual bouts of moderate intensity aerobic exercise temporarily increased the number of cytotoxic CD16 + NK cells, and CD20 + B-CLL cells in blood. Additionally, our ex vivo investigations demonstrated enhanced rituximab mediated ADCC following exercise. Thus, exercise could be explored as a means of improving clinical responses in patients receiving rituximab, and/or other anti-CD20 monoclonal antibodies such as, Obinutuzumab.