Frequency Of Cells Research Articles

Neoadjuvant chemotherapy is associated with suppression of the B cell-centered immune landscape in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at advanced stages and associated with early distant metastasis and poor survival. Besides clinical factors, the tumor microenvironment (TME) emerged as a crucial determinant of patient survival and therapy response in many tumors, including PDAC. Thus, the presence of tumor-infiltrating lymphocytes and the formation of tertiary lymphoid structures (TLS) is associated with longer survival in PDAC. Although neoadjuvant therapy (NeoTx) has improved the management of locally advanced tumors, detailed insight into its effect on various TME components is limited. While a remodeling towards a proinflammatory state was reported for PDAC-infiltrating T cells, the effect of NeoTx on B cell subsets, including plasma cells, and TLS formation is widely unclear. We thus investigated the frequency, composition, and spatial distribution of PDAC-infiltrating B cells in primary resected (PR) versus neoadjuvant-treated patients using a novel multiplex immunohistochemistry panel. The NeoTx group displayed significantly lower frequencies of pan B cells, GC B cells, plasmablasts, and plasma cells, accompanied by a reduced abundance of TLS. This finding was supported by bulk RNA-sequencing analysis of an independent fresh frozen tissue cohort, which revealed that major B cell pathways were downregulated in the NeoTx group. We further observed that plasma cells frequently formed aggregates that localized close to TLS and that TLS+ patients displayed significantly higher plasma cell frequencies compared to TLS- patients in the PR group. Additionally, high densities of CD20+ intratumoral B cells were significantly associated with longer overall survival in the PR group. While CD20+ B cells held no prognostic value for NeoTx patients, an increased frequency of proliferating CD20+Ki67+ B cells emerged as an independent prognostic factor for longer survival in the NeoTx group. These results indicate that NeoTx differentially affects PDAC-infiltrating immune cells and may have detrimental effects on the existing B cell landscape and the formation of TLS. Gaining further insight into the underlying molecular mechanisms is crucial to overcome the intrinsic immunotherapy resistance of PDAC and develop novel strategies to improve the long-term outcome of PDAC patients.

Spectrum of chronic lung allograft dysfunction pathology in human lung transplantation

BackgroundLong-term survival after lung transplantation (LTx) remains limited by Chronic Lung Allograft Dysfunction (CLAD), which includes two main phenotypes: bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), with possible overlap. We aimed to detail and quantify pathological features of these CLAD sub-types. MethodsPeripheral and central paraffin-embedded explanted lung samples were obtained from 20 consecutive patients undergoing a second LTx for CLAD, from 3 lobes. Thirteen lung samples, collected from non-transplant lobectomies or donor lungs, were used as controls. Blinded semi-quantitative grading was performed to assess airway fibrotic changes, parenchymal and pleural fibrosis, as well as epithelial and vascular abnormalities. ResultsCLAD lung samples had higher scores for all airway- and lung-related parameters compared to controls. There was a notable overlap in pathological scores between BOS and RAS, with a wide range of scores in both conditions. Parenchymal and vascular fibrosis scores were significantly higher in RAS compared to BOS (p=0.003 for both). We observed a significant positive correlation between the degree of inflammation around each airway, the severity of epithelial changes and airway fibrosis. Immunofluorescence staining demonstrated a trend towards a lower frequency of club cells in CLAD, and a higher frequency of apoptotic club cells in BOS samples (p=0.01). ConclusionsCLAD is a spectrum of airway, parenchymal, and pleural fibrosis, as well as epithelial, vascular, and inflammatory pathological changes, where BOS and RAS overlap significantly. Our semi-quantitative grading score showed a generally high inter-reader reliability and may be useful for future CLAD pathological assessments.

Peripheral T-cell responses of EphB2- and EphB3-deficient mice in a model of collagen-induced arthritis

Both EphB2- and EphB3-deficient mice exhibit profound histological alterations in the thymic epithelial network but few changes in T-cell differentiation, suggesting that this organization would be sufficient to produce functional T lymphocytes. Also, other antigen-presenting cells involved in immunological education could substitute the thymic epithelium. Accordingly, we found an increased frequency of plasmacytoid dendritic cells but not of conventional dendritic cells, medullary fibroblasts or intrathymic B lymphocytes. In addition, there are no lymphoid infiltrates in the organs of mutant mice nor do they contain circulating autoantibodies. Furthermore, attempts to induce arthritic lesions after chicken type II collagen administration fail totally in EphB2-deficient mice whereas all WT and half of the immunized EphB3−/− mice develop a typical collagen-induced arthritis. Our results point out that Th17 cells, IL4-producing Th2 cells and regulatory T cells are key for the induction of disease, but mutant mice appear to have deficits in T cell activation or cell migration properties. EphB2−/− T cells show reduced in vitro proliferative responses to anti-CD3/anti-CD28 antibodies, produce low levels of anti-type II collagen antibodies, and exhibit low proportions of T follicular helper cells. On the contrary, EphB3−/− lymph node cells respond accurately to the different immune stimuli although in lower levels than WT cells but show a significantly reduced migration in in vitro transwell assays, suggesting that no sufficient type II collagen-dependent activated lymphoid cells reached the joints, resulting in reduced arthritic lesions.

Natural killer cell subsets and their functional molecules in peripheral blood of the patients with breast cancer.

Natural killer (NK) cells, CD3- lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3- NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). Thirty untreated females with BC and 20 age-matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome-conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four-color flow cytometer. Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3-CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p = .02) and the conventional cytotoxic (CD3-CD56dim CD16+) NK cells (p = .03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3-CD56brightCD16- /+) NK cells and CD3-CD56-CD16+ NK cells (p = .03 and p = .004, respectively) in the patients was lower than healthy subjects. The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis-related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3-CD56-CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC.

1231 Inhibition of YAP With Vertoporfin Makes Chordomas Sensitive to Radiation in Vitro and in Vivo: New Therapies to Combat Radioresistance

INTRODUCTION: Chordomas are locally invasive tumors of notochordal origin with a propensity to resist radiotherapy and with no FDA approved therapies. Verteporfin is a photosensitizer with the ability to inhibit the yes-associated-protein (YAP) pathway which is upregulated in chordomas and enables their resistance to radiotherapy. METHODS: We used five primary chordoma cell lines (JHC7, UCH1, UCH2, MUG-CHOR1, UM-CHOR1) to perform in vitro and in vivo experiments with 64 mice using a xenograft subcutaneous model of chordoma. We then knocked down the YAP gene in chordoma cells and performed experiments to confirm our results were secondary to verteporfin effects on YAP. RESULTS: Verteporfin has additive and synergistic effects on chordoma cells when combined with radiation. Chordoma cells treated with verteporfin have significantly more double-stranded DNA damage after radiation therapy (p < 0.0001) and verteporfin prevents repair of that DNA damage. Verteporfin significantly reduces DNA damage repair proteins and genes in chordoma cells (ATM: p = 0.0007, ATR: p < 0.0001, RADS1: p = 0.0033, BRCA1: p < 0.0001, BRCA2: p = 0.0012, PPP2R4: p < 0.0001). Verteporfin leads to significantly increased frequency of cells in G2/M phase (MUG-CHOR1: p < 0.0001, UCH2: p = 0.0021). We then performed all the above experiments using chordoma cells with the YAP gene knocked down and found that YAP knockdown led to enhanced radiation induced DNA damage (p<0.0001), diminished yH2AX response (p = 0.0264), and increased frequency of cells in G2/M phase (p < 0.0001). In vivo verteporfin treatment significantly inhibited chordoma tumor growth (p < 0.0001) and microparticles were most effective at radiosensitization. CONCLUSIONS: Inhibition of YAP using verteporfin radiosensitizes chordoma via 1) inhibition of the DNA damage repair cascade and 2) increasing frequency of the G2/M cell cycle phase when cells are most sensitive to radiation.

Radiotherapy Enhances Metastasis Through Immune Suppression by Inducing PD-L1 and MDSC in Distal Sites.

Radiotherapy (RT) is a widely employed anticancer treatment. Emerging evidence suggests that RT can elicit both tumor-inhibiting and tumor-promoting immune effects. The purpose of this study is to investigate immune suppressive factors of radiotherapy. We used a heterologous two-tumor model in which adaptive concomitant immunity was eliminated. Through analysis of PD-L1 expression and myeloid-derived suppressor cells (MDSC) frequencies using patient peripheral blood mononuclear cells and murine two-tumor and metastasis models, we report that local irradiation can induce a systemic increase in MDSC, as well as PD-L1 expression on dendritic cells and myeloid cells, and thereby increase the potential for metastatic dissemination in distal, nonirradiated tissue. In a mouse model using two distinct tumors, we found that PD-L1 induction by ionizing radiation was dependent on elevated chemokine CXCL10 signaling. Inhibiting PD-L1 or MDSC can potentially abrogate RT-induced metastasis and improve clinical outcomes for patients receiving RT. Blockade of PD-L1/CXCL10 axis or MDSC infiltration during irradiation can enhance abscopal tumor control and reduce metastasis.

Local administration of myeloid-derived suppressor cells prevents progression of immune-mediated dry eye disease

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature hematopoietic precursors with known immunoregulatory functions. The immunosuppressive role of MDSCs has been highlighted in several inflammatory ophthalmic disorders; however, their therapeutic application in suppressing the immune-mediated changes in dry eye disease (DED) has not been studied. We observed significant reduction in antigen presenting cell (APC) frequencies and their maturation in the presence of MDSCs. Moreover, co-culturing MDSCs with T helper 17 cells (Th17) resulted in reduced Th17 frequencies and their IL-17 expression. On the contrary, MDSCs maintained regulatory T cell frequencies and enhanced their function in-vitro. Furthermore, we delineated the role of interleukin-10 (IL-10) secreted by MDSCs in their immunoregulatory functions. We confirmed these results by flow cytometry analysis and observed that treatment with MDSCs in DED mice effectively suppressed the maturation of APCs, pathogenic Th17 response, and maintained Treg function and significantly ameliorated the disease. The results in this study highlight the potential therapeutic application of MDSCs in treating refractory DED.

MyD88 exacerbates inflammation-induced bone loss by modulating dynamic equilibrium between Th17/Treg cells and subgingival microbiota dysbiosis.

This study aimed to investigate the contribution of myeloid differentiation primary-response gene 88 (MyD88) on the differentiation of T helper type 17 (Th17) and regulatory T (Treg) cells and the emerging subgingival microbiota dysbiosis in Porphyromonas gingivalis-induced experimental periodontitis. Alveolar bone loss, infiltrated inflammatory cells, immunostained cells for tartrate-resistant acid phosphatase (TRAP), the receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin (OPG) were quantified by microcomputerized tomography and histological staining between age- and sex-matched homozygous littermates (wild-type [WT, Myd88+/+] and Myd88-/- on C57BL/6 background). The frequencies of Th17 and Treg cells in cervical lymph nodes (CLNs) and spleen were determined by flow cytometry. Cytokine expression in gingival tissues, CLNs, and spleens were studied by quantitative polymerase chain reaction (qPCR). Analysis of the composition of the subgingival microbiome and functional annotation of prokaryotic taxa (FAPROTAX) analysis were performed. P. gingivalis-infected Myd88-/- mice showed alleviated bone loss, TRAP+ osteoclasts, and RANKL/OPG ratio compared to WT mice. A significantly higher percentage of Foxp3+CD4+ T cells in infected Myd88-/- CLNs and a higher frequency of RORγt+CD4+ T cells in infected WT mice was noted. Increased IL-10 and IL-17a expressions in gingival tissue at D14-D28 then declined in WT mice, whereas an opposite pattern was observed in Myd88-/- mice. The Myd88-/- mice exhibited characteristic increases in gram-positive species and species having probiotic properties, while gram-negative, anaerobic species were noted in WT mice. FAPROTAX analysis revealed increased aerobic chemoheterotrophy in Myd88-/- mice, whereas anaerobic chemoheterotrophy was noted in WT mice after P. gingivalis infection. MyD88 plays an important role in inflammation-induced bone loss by modulating the dynamic equilibrium between Th17/Treg cells and dysbiosis in P. gingivalis-induced experimental periodontitis.

Umbilical cord blood cell characteristics in very preterm neonates for autologous cell therapy of preterm-associated complications

BackgroundThere are emerging clinical evidence for umbilical cord blood mononuclear cells (UCBMNCs) intervention to improve preterm complications. The first critical step in cell therapy is to obtain high-quality cells. This retrospective study aimed to investigate the quantity and quality of UCBMNCs from very preterm infants (VPIs) for the purpose of autologous cell therapy in prevention and treatment of preterm complications.MethodsVery preterm infants (VPIs) born in Guangdong Women and Children Hospital from January 1, 2017, to December 8, 2022, from whom cord blood was successfully collected and separated for public or private banking, were enrolled. The UCBMNCs characters from route cord blood tests performed in cord blood bank, impact of perinatal factors on UCBMNCs, the relationship between UCBMNCs characteristics and preterm outcomes, and the correlation of UCBMNCs characteristics and peripheral blood cells in VPIs were analyzed.ResultsTotally, 89 VPIs underwent UCB collection and processing successfully. The median cell number post processing was 2.6 × 108. To infuse a dose of 5 × 107 cells/kg, only 3.4% of infants required a volume of more than 20 mL/kg, which exceeded the maximum safe volume limit for VPIs. However, when infusing 10 × 107 cells/kg, 25.8% of infants required a volume of more than 20 ml/kg volume. Antenatal glucocorticoids use and preeclampsia was associated with lower original UCBMNCs concentration. Both CD34+ hematopoietic stem cells (HSC) frequency and colony forming unit - granulocyte and macrophage (CFU-GM) number correlated negatively with gestational age (GA). UCBMNCs characters had no significant effect on preterm outcomes, whereas a significant positive correlation was observed between UCBMNCs concentration and total white blood cell, neutrophil, lymphocyte and PLT counts in peripheral blood.ConclusionUCBMNCs collected from VPIs was feasible for autologous cell therapy in improving preterm complications. Setting the infusion dose of 5 × 107 cells/kg guaranteed a safe infusion volume in more than 95% of the targeted infants. UCBMNCs characters did not affect preterm complications; however, the effect of UCBMNCs concentration on peripheral blood classification count should be considered when evaluating the immunomodulation of UCBMNCs transfusion.

Pathogenic Potential of Eomesodermin-Expressing T-Helper Cells in Neurodegenerative Diseases.

Eomesodermin-expressing (Eomes+) T-helper (Th) cells show cytotoxic characteristics in secondary progressive multiple sclerosis. We found that Eomes+ Th cell frequency was increased in the peripheral blood of amyotrophic lateral sclerosis and Alzheimer's disease patients. Furthermore, granzyme B production by Th cells from such patients was high compared with controls. A high frequency of Eomes+ Th cells was observed in the initial (acutely progressive) stage of amyotrophic lateral sclerosis, and a positive correlation between Eomes+ Th cell frequency and cognitive decline was observed in Alzheimer's disease patients. Therefore, Eomes+ Th cells may be involved in the pathology of amyotrophic lateral sclerosis and Alzheimer's disease. ANN NEUROL 2024.

Abstract 734: Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression

Abstract The profound benefit of immune checkpoint blockade for cancer therapy is restricted to limited subsets of patients with specific cancers. Many factors contribute to primary and acquired resistance to immune checkpoint blockade, including local accumulation of immunosuppressive metabolites such as the nucleoside adenosine and downstream adenosine receptor signaling. Pharmacological inhibition of adenosine generation and signaling are active areas of clinical investigation. Here we report a novel mechanism whereby adenosine suppresses anti-cancer immune responses by intracellular accumulation. We show that human T cells readily take up adenosine via equilibrative nucleoside transporter 1 (ENT1), suppressing proliferation and effector function by inhibition of de novo pyrimidine nucleotide synthesis in T cells. ENT1 expression increases upon T cell activation, suggesting uptake of extracellular adenosine is a mechanism to limit T cell responses in high adenosine environments. Quantitative mass spectrometry imaging reveals adenosine concentrations in human tumors up to the range of 100 µM - levels significantly greater than previously demonstrated and consistent with suppression of T cell responses. Deletion of ENT1 leads to potent control of tumor growth in syngeneic mouse models including KPC, a poorly immunogenic model of pancreatic ductal adenocarcinoma, and is associated with increased CD8+ T cell frequency, proliferation and cytokine production within tumors. Furthermore, ENT1 expression was observed by flow cytometry on human tumor-infiltrating CD8+ T cells. We have discovered and characterized EOS301984, a potent ENT1 antagonist that is currently being evaluated in patients with advanced solid tumors. EOS301984 blocks intracellular adenosine transport and restores pyrimidine levels in T cells activated in the presence of adenosine, resulting in enhanced tumor cell killing by memory T cells and increased ex vivo expansion of functional human tumor-infiltrating lymphocytes in adenosine-rich environments. Finally, in a humanized mouse model of triple negative breast cancer resistant to anti-PD-1 blockade (MDA-MB-231), combination of EOS301984 with nivolumab synergistically led to control of tumor growth. Thus, ENT1 inhibition represents a novel approach to augment anti-cancer immune responses through restoring pyrimidine nucleotide synthesis in T cells suppressed by adenosine. Citation Format: Theodore J. Sanders, Christopher S. Nabel, Margreet Brouwer, Annelise L. Hermant, Lucas Chaible, Jean-Philippe Deglasse, Angelique Pabois, Wilfried Cathou, Aurore Smets, Michael Deligny, Joao Marchante, Quentin Dubray, Marie-Claire Lettelier, Chiara Martinoli, Reece Marillier, Olivier De Henau, Yvonne McGrath, Matthew G. Vander Heiden, Erica Houthuys. Inhibition of equilibrative nucleoside transporter 1 relieves intracellular adenosine-mediated immune suppression [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 734.

Abstract 1530: Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice

Abstract The relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2−/−, IL2Rγ−/−, SIRPαNOD and Flt3−/−) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models overexpressing human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and test novel therapeutics. Effect of Flt3L-induced boost on myeloid compartment is seen in a non-small cell lung cancer (A549) tumor model. A single boost prior to tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the tumor microenvironment (TME). Human triple negative cancer cell (TNBC) MDA-MB-231 and human pancreatic adenocarcinoma HPAFII cell lines are widely used in cancer research and drug development. We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft (CDX) is CD34+ donor independent and the TME composition varies according to cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231 bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII bearing mice is mainly composed of T cells enabling T cells-based therapy. Treatment of BRGSF-HIS mice bearing HPAFII tumor cells with combotherapy based on CD3xTAA + CD28xTAA efficiently reduces tumor growth in vivo compared to vehicle-treated mice or mice injected with CD3xTAA alone. Induced systemic immunomodulation is observed in the TME where increased numbers of both CD4 and CD8 T cells are observed upon combotherapy. Remarkably, while no CD34+ donor effect is observed in CDX growth kinetic, response to treatment appears to be donor dependent with apparition of “weak” and “good” responders. This heterogeneity of response mimics what is observed in clinic and enables further investigations of treatment mode of action and immune escape mechanisms. Citation Format: Perrine Martin-Jeantet, Siham Hedir, Fabiane Sonego, Gaëlle Martin, Yacine Cherifi, Kader Thiam. Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice [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 1530.

Abstract 109: Identification of tumor-intrinsic and extrinsic mechanisms of T cell exclusion in estrogen receptor-positive (ER+) breast cancer through integration of bulk and single-cell transcriptome and spatial analysis

Abstract T cells are generally present in low numbers in estrogen receptor-positive (ER+) breast cancer (BC) potentially due to limited antigens or impaired antigen presentation, however defined mechanisms are not fully understood. This study explores the interplay between estrogen receptor signaling (ERS), antigen presentation machinery (APM), and T cell signaling (TC) using diverse datasets, including BC cell lines, TCGA, METABRIC, and clinical trial bulk transcriptomes, single-cell RNA-sequencing of ER+ primary BC, and single-cell spatial multi-plex cyclic immunofluorescence (CyCIF) of primary BC patients. Co-expression analysis of both computationally and manually curated genes in TCGA and METABRIC data unveiled distinct 29-gene ERS and 225-gene combined APM/TC modules with strong inverse correlations in ER+ BC. This inverse correlation was confirmed in independent cohorts. ERS and APM/TC modules were down- and up-regulated, respectively, in response to aromatase inhibitor therapy in ER+ BC patients from the ACOSOG Z1301B trial who demonstrated clinical benefit. Analysis of published single-cell RNA-sequencing data of 20 primary ER+ BC was performed to elucidate cell types that express individual genes in the modules. It confirmed that high ERS activity is associated with reduced T cells, and identified distinct cell types contributing to each module, showing higher ERS activity in tumor cells and higher APM/TC activity in stromal and immune cells. At the cell type level, we found distinct contributions and precise insights of individual genes. The prevalence of ERS activity in tumor cells prompted investigation to determine if the ERS and APM/TC modules were preserved as tumor-intrinsic signaling. Analysis of 54 BC cell lines indicated partially preserved module correlations in cancer cell lines and revealed that the APM/TC module is a contribution of both tumor-intrinsic (e.g., complement and type-I IFN-response), and tumor-extrinsic (e.g., lymphocyte activation) factors. CyCIF analysis of 29 ER+ BC samples was performed to bridge gene signatures with spatial biology and validated that higher ERS activity correlates with lower T cell infiltration. The spatial pattern of T cells varied from immune desert to stromally bound to fully inflamed tumors. Neighborhood analysis revealed increased frequency of CD8 T cells around a tumor cell is associated with elevated MHC I and II in broad immune cells and macrophage subsets, respectively. pTBK1+ macrophages inversely correlated with CD8 T cell frequency and MHC expression in neighboring cells, suggesting an immunosuppressive role. This study uncovers robust and tightly regulated ERS and APM/TC signaling in ER+ patients, unraveling molecular intricacies and offering potential insights for targeted therapeutic interventions. Citation Format: Kenichi Shimada, Yvonne X. Cui, Daniel Michaud, Kelly F. Zheng, Jonathan Goldberg, Ricardo Pastorello, Lukas Kania, Sandra S. McAllister, Sara M. Tolaney, Adrienne Waks, Rinath Jeselsohn, Peter K. Sorger, Judith Agudo, Jennifer L. Guerriero, Elizabeth A. Mittendorf. Identification of tumor-intrinsic and extrinsic mechanisms of T cell exclusion in estrogen receptor-positive (ER+) breast cancer through integration of bulk and single-cell transcriptome and spatial analysis [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 109.

Abstract 1175: Differential patterns of immune infiltration in the tumor immune microenvironment associate with therapeutic response in primary prostate cancer following chemohormonal therapy

Abstract There is a critical need to develop novel therapeutic strategies and diagnostic tools to precisely deliver treatments to improve survival for men with prostate cancer. To support this development, improved strategies are needed to better understand heterogenous tumor microenvironments and tumor biology that associate with variable treatment responses. We hypothesized that the tumor immune microenvironment (TIME) plays a critical role in treatment resistance. In this study we aimed to evaluate TIME signatures of treatment response and resistance utilizing a novel, integrated technological tool to identify response patterns and enable precision sampling for comparative cellular and molecular analysis. 30 patients with newly diagnosed, locally advanced, high-risk, primary prostate cancer underwent 18F-DCFPyL PSMA PET/MRI with multiparametric MRI scans followed by 3 cycles of chemohormonal therapy (NCT03358563). Repeat PSMA PET/MRI was performed prior to prostatectomy and scans were used to categorize lesions as complete response (CR), partial response (PR), no response (NR) or normal tissue. MRI scans were used to print a 3D mold of the prostate to allow microdissection of regions of interest from the resected prostate. Cellular infiltrates were analyzed by flow cytometry in 3 to 5 tissue specimens per patient. Statistical analysis was performed with One-way ANOVA with Tukey-correction. The frequency of CD8+ T cells in the total CD45+ infiltrate was highest in normal and CR areas and was significantly reduced in PR vs CR (p<0.01). CXCR3+CD8+ and CD103+CD8+ T cell frequencies were also reduced in PR vs CR foci (p<0.01, p<0.05, respectively). Meanwhile, the frequency of CXCR3+CD8+ T cells was highest and significantly elevated in CR vs normal tissue. A tendency of reduced CCR6+, CXCR5+, and CCR4+CD8+ T cells was observed in PR vs CR foci, while those frequencies remained higher in normal and CR areas. An increase in total CD8+ and CD103+CD8+ T cells associated with longer progression-free survival. Additionally, the analysis of EpCAM+ cells showed a significant increase in B7H3 expression in PR vs CR lesions (p<0.05) and a tendency of reduced HLA I expression in foci that associated with treatment resistance. We are currently integrating analysis of myeloid cells and transcriptomic analysis of sorted CD4+, CD8+ and CD11b/CD14+ cells to further dissect patterns of therapeutic response in our study cohort. In conclusion, PSMA/PET MRI based precision sampling of tumor tissue associated with differential therapeutic response patterns captured differences in the TIME infiltrates and these observations may provide hypothesis to test biological mechanisms to expedite discovery of targetable mechanisms to improve tumor stratification and targeting in high-risk prostate cancer. Citation Format: Erika Heninger, Jamie M. Sperger, Kristin Weinstein, Brian P. Johnson, Peter G. Geiger, Shane Wells, Steve Y. Cho, Wei Huang, Philippos Tsourkas, Sean McIlwain, Irene M. Ong, Sheena C. Kerr, David F. Jarrard, David J. Beebe, Joshua M. Lang. Differential patterns of immune infiltration in the tumor immune microenvironment associate with therapeutic response in primary prostate cancer following chemohormonal therapy [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 1175.

Abstract 4100: AMP-peptide vaccination against mutant BRAF epitopes promotes lymph node delivery to generate potent, functional T cell immunity

Abstract Background Mutations in BRAF cause ~8% of human cancers[1]. Nonetheless, therapies designed to promote immune responses against mutated BRAF (mBRAF) have led to limited clinical success[2], despite evidence confirming T cell recognition of these mutations[3,4]. The Amphiphile (AMP) platform improves the potency of vaccine immunotherapy by programming the delivery of vaccine components to the lymph nodes where efficient uptake by resident immune cells initiates tumor-targeted immune responses. AMP-modification of vaccine components including peptide antigens and molecular adjuvants results in covalent conjugation to albumin-binding lipids. Upon injection, AMP-vaccines associate with tissue resident albumin which efficiently distributes to draining lymph nodes. This approach was shown to promote activation of polyfunctional, cytotoxic T cells with promising safety, and improved clinical outcomes in a Phase 1 trial of ELI-002, an mKRAS AMP therapeutic vaccine (AMPLIFY-201 NCT04853017). Application of this strategy to prevalent BRAF mutations with ELI-007 offers the potential for improved therapeutic activity in a setting of significant unmet need, as BRAF inhibitors benefit only a fraction of patients, with near universal progression. Methods Following multi-dose immunization of C57BL/6J mice with AMP-modified or soluble comparator vaccines consisting of mBRAF V600E/K peptides and CpG adjuvant, immunological readouts were performed 7 days after dosing. Antigen-specific T cell responses were assessed after ex vivo re-stimulation of cells with mBRAF overlapping peptides. Assays included analysis of antigen-specific T cell frequencies, multiplexed proteomics determining polyfunctionality, and effector function and cytotoxic potential evaluation. Responses were determined in secondary lymphoid tissues (spleen), as well as lung, to which many BRAF driven cancers metastasize. Results AMP-immunization generated robust in vivo immune responses yielding strong T cell activation against mBRAF epitopes. Non-lymph node targeted vaccination using soluble comparators were inactive with no response above mock treated mice. Responses to AMP-vaccination were characterized by the generation of significantly increased frequency of Th1-skewed, polyfunctional T cells (IFNγ, TNFα, IL2) specific to mBRAF V600E and V600K. Induced T cells further exhibited mBRAF-specific cytotoxic function. Conclusions AMP-conjugation enhances efficient delivery directly to the lymph nodes and thus improves the immunogenicity of peptide vaccination. These enhanced immune responses provide an encouraging therapeutic opportunity to treat >90% of BRAF-mutated cancers[5] with the AMP-mBRAF vaccine, ELI-007. Moreover, the AMP-platform technology is simple, rapidly executable, and scalable, promising broad clinical off-the-shelf application for BRAF. Citation Format: Martin P. Steinbuck, Xavier Cabana-Puig, Erica Palmer, Mimi M. Jung, Thomas Williams, Kristen Osaer, Jeff Zhang, Christopher M. Haqq, Peter C. DeMuth. AMP-peptide vaccination against mutant BRAF epitopes promotes lymph node delivery to generate potent, functional T cell immunity [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 4100.

Abstract 6490: Pt1a and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma effect through RNA m6A methylation modification

Abstract Hepatocellular carcinoma (HCC) is the most common type of liver cancer and has a poor prognosis. The survival benefits of sorafenib in patients with advanced HCC are modest owing to acquired resistance. Accumulating evidence has demonstrated that cancer stemness is an important cause of drug resistance and the root cause of tumor recurrence. [PtII(C^N^N)(NHC2Bu)]PF6 (Pt1a) is a newly developed platinum-based drug containing N-heterocyclic carbene with proven anticancer properties against a range of tumor types. However, its role in targeting cancer stemness and its ability to sensitize cancer cells to overcome molecular-targeted therapy drug resistance in HCC remains elusive. Herein, we demonstrate the synergistic inhibitory effect of sorafenib and Pt1a combination treatment on HCC through RNA m6A methylation modification. In cultured HCC cells and patient-derived organoids, the drug combination synergistically reduced the cell viability and promoted cell apoptosis compared to single-drug treatment. Combination treatment also attenuated self-renewal, as evidenced by a marked decrease in tumor-initiating cancer cell frequency, as measured by limiting dilution spheroid formation assay. These findings were further validated in HCC patient-derived xenograft mouse models and a mouse HCC model induced by hydrodynamic tail vein delivery of NRAS+AKT coactivation plasmids. Notably, the combination treatment with sorafenib and Pt1a, but not a single treatment, led to a significant reduction in tumor burden. The combination treatment was also effective in decreasing tumor-initiating cell frequency, as measured by subjecting the harvested tumor cells for limiting dilution spheroid formation assay ex vivo. Mechanistically, integrative RNA-sequencing and thermal proteome profiling analyses both indicated the enrichment of m6A-related pathways in combination drug treatment. An increased global m6A level upon combination treatment was validated using m6A dot blotting, confirming that the synergistic effect may be mediated by the elevated global m6A level in HCC cells. Collectively, our study demonstrated that Pt1a and sorafenib combination treatment synergistically inhibited the viability and stemness of HCC cells by increasing global RNA m6A level. These findings supported the potential of Pt1a as a therapeutic option for HCC and overcoming sorafenib drug resistance. Ongoing studies are focused on identifying m6A regulators and downstream targets involved in combination treatment. Citation Format: Yinjia Huang, Yalu Cui, Ianto Bosheng Huang, Yanyan Wang, Yang Xuan, Pui Ki Wan, Chun Nam Lok, Chi Ming Che, Stephanie Ma. Pt1a and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma effect through RNA m6A methylation modification [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 6490.

Abstract 1193: Peripheral immune analyses from phase I trial of HPV vaccine PRGN-2009 in combination with bintrafusp alfa in patients with HPV-associated cancers

Abstract Background: PRGN-2009 is a novel gorilla adenovirus vaccine targeting HPV16/18, and bintrafusp alfa (BA) is a bifunctional anti-PD-L1/TGFβ fusion protein. Patients with pretreated advanced HPV-associated malignancies received PRGN-2009 vaccine (5 × 1011 particle units, subcutaneous administration) q2 weeks for 3 administrations and biweekly 1200mg BA followed by q4 weeks vaccine and biweekly BA (NCT04432597), with an overall response rate of 30%. We report here correlative analyses from this first-in-human Phase I study using peripheral blood from 10 patients with HPV16+ (n=8), HPV18+ (n=1), or HPV45+ (n=1) disease to identify immune correlates associated with clinical response. Methods: Peripheral HPV-specific T cell responses, HPV circulating tumor DNA (ctDNA) from plasma, serum cytokines and soluble factors, and peripheral immune cell subsets were assessed to evaluate changes induced with treatment and peripheral correlates associated with clinical activity. Patients with complete response (CR, n=1), partial response (PR, n=2; 1 confirmed) or stable disease (SD, n=1) (n=4 total) were compared to patients with progressive disease (PD, n=6). Results: Eighty percent (8/10) of patients developed either HPV16 or HPV18-specific T-cell responses after repeat administration of PRGN-2009 with BA. Eight of 10 patients had detectable HPV16 or 18 ctDNA at baseline and changes in ctDNA load with treatment generally correlated with outcomes. Early transient increases in IL-8, TNFα, and IFNγ occurred, with greater increases in IL-8 at 2 weeks and TNFα at 4 weeks in patients with PD compared to CR/PR/SD. The patient who achieved a durable CR had HPV45+ disease, and had a) a 50% increase in CD8+ T cell frequency at 2 weeks, b) increases in frequencies of CD8+ naïve T cells expressing Ki67+ and central and effector memory T cells expressing 4-1BB, PD-1, or TIM-3, and c) considerable increases in HPV18-specific T cell responses through 484 days after treatment start, indicating durable antigen-specific T cell activity. Conclusions: The majority of patients developed HPV16/18 specific T-cell responses after repeat administration of PRGN-2009 in combination with BA. Transient increases in IL-8 and TNFα 2-4 weeks after start of treatment were associated with worse outcomes, suggesting that early changes in cytokines may reflect disease course. Furthermore, the early expansion of CD8+ T cell subsets in the patient developing a CR with HPV45+ disease after receiving a vaccine targeting HPV16 and HPV18 epitopes suggest a broadening and boosting of T cell responses that enabled potent anti-tumor activity. A high degree of homology between HPV18 and HPV45 may have contributed to the expansion of HPV18 specific T cells in the patient with CR. These data highlight the importance of assessing peripheral blood in immunotherapy trials to reveal important immunological phenomena. Citation Format: Meghali Goswami, Charalampos S. Floudas, Julius Strauss, Douglas E. Brough, Amy R. Lankford, Caroline Jochems, James L. Gulley, Jeffrey Schlom, Renee N. Donahue. Peripheral immune analyses from phase I trial of HPV vaccine PRGN-2009 in combination with bintrafusp alfa in patients with HPV-associated 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 1193.

Abstract 6733: Multiomics analysis of impact of CD8+ and other cell populations in STING agonist treated ex vivo mesothelioma samples

Abstract Background: Collective evidence highlights that agonism of the host STING pathway plays a critical role in eliciting robust and durable anti-tumor immunity. Dazostinag (TAK-676) is a novel systemically delivered STING agonist that ignites the innate immune system and mobilizes adaptive immunity. Preclinical studies with dazostinag demonstrated a strong, persistent CD8+ T cell anti-tumor response driven by innate immune derived type I IFN and pro-inflammatory cytokines. The role of CD8+ T cells in response to dazostinag was further supported by a previously demonstrated correlation between response and CD8+ T cell frequency in a patient-derived organotypic tumor spheroids (PDOTS) model of mesothelioma. Within the current work, we sought to determine the extent to which response to dazostinag in mesothelioma depends on CD8+ T cells versus the contribution of other immune cells. Methods: Surgical mesothelioma cases were collected along with clinicopathological variables at Brigham and Women’s Hospital under an IRB-approved protocol. PDOTS were generated as described previously. Ex vivo response was determined by fluorescent life/dead imaging, and orthogonally evaluated by multiplexed cytokine array, immunofluorescence, and flow cytometry; subset of tumors was studied using single cell RNA sequencing (scRNAseq). Results: Dazostinag-driven tumor cell killing was evaluated in 20 mesothelioma PDOTS. 11 samples were characterized as responders (R) with a cut-off of >30% reduction in live cell area; 9 as non-responders (NR). In 12 out of 20 PDOTS, CD8 neutralization effect was assessed: four were TAK-676 NR, and out of 8 R 4 had at least 20% killing abrogated by aCD8. Effect did not depend on the baseline amount of CD8+ T cells in the sample. scRNAseq analysis showed that R and NR T cells have different activation/exhaustion phenotypes and varying levels of interferon stimulated genes (ISG) in response to STING agonism. Other cell populations emerged as candidates for dazostinag resistance: NR cases carried remodeled immunosuppressive myeloid cells or matrix metalloproteinases-expressing fibroblasts. In general, myeloid cell abundance at baseline characterized by flow had a trend in negatively correlating with dazostinag response. Intrinsic expression of type I interferons shown by multiplexed cytokine array was linked to dazostinag sensitivity (R vs. NR IFN-β p=0.028; IFNα2a p=0.016) and can potentially be used as a predictor for response. Conclusion: We evaluated immune modulations in mesothelioma after treatment with a systemic STING agonist dazostinag using human tumor ex vivo culture. Innate interferon type I expression was higher in responding samples. TAK-676 efficacy was partially reduced in absence of effector lymphocytes, however other cells, such as immunosuppressive myeloid cells and fibroblasts, can have a negative impact on dazostinag response in mesothelioma. Citation Format: Elena Ivanova, Sung R. Park, Ari P. Zlota, Nathaniel Spicer, Minh Ha, Sophie Kivlehan, Iliana Gjeci, Simona Innocenti, Lauren Zasadil, Patrick Lizotte, Michael Y. Tolstorukov, Kai Ding, Adnan O. Abu-Yousif, Jeffrey Raizer, Vicky A. Appleman, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Multiomics analysis of impact of CD8+ and other cell populations in STING agonist treated ex vivo mesothelioma samples [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 6733.

Abstract 7297: Inhibition of lung tumorigenesis and potentiation of cancer preventive vaccines by a small molecule CA170 targeting the immune checkpoint protein VISTA

Abstract The V-domain Ig Suppressor of T cell Activation (VISTA) is expressed on cells of the myeloid and lymphoid lineages and is an emerging target for cancer immunotherapy. Blocking VISTA activates both innate and adaptive immunity to eradicate tumors in mice. Furthermore, a tripeptide small molecule antagonist of VISTA, CA170, exhibited potent anticancer efficacy on carcinogen-induced mouse lung tumorigenesis. We have previously demonstrated that KRAS or EGFR vaccines are effective in preventing lung tumorigenesis driven by either KRAS or EGFR in a pure prevention setting but are less effective in mice carrying existing lesions. Here, we determined if combining an MHC class II-restricted multi-antigen vaccine targeting KRAS or EGFR with CA170 will provide enhanced efficacy in preventing lung cancer progression. Using tetracycline-inducible KRAS and EGFR transgenic mice, we found that lung tumor development was significantly suppressed when CA170, delivered by aerosol inhalation to minimize systemic toxicity, was combined with either KRAS or EGFR MHCII-directed peptide vaccines using a post-initiation model. Flow cytometry and single-cell RNA sequencing (scRNA-seq) revealed that CA170 significantly increases tumor infiltrating CD8+ T cells and enhances effector-memory T cell frequencies and functions of both CD4+ and CD8+ T cells. These changes coincide with significant reductions in G-MDSCs (granulocytic myeloid-derived suppressor cells) and Foxp3+ Treg populations within lung tumors from mice treated with CA170. We found that the KRAS and EGFR preventive vaccines primarily induced the expansion of CD4+ effector T cells. VISTA antagonism by CA170 revealed strong efficacy against lung tumorigenesis with broad immunoregulatory functions that influence effector, memory, and regulatory T cells, and drive an adaptive T cell tumor-specific immune response that enhances the efficacy of the KRAS and EGFR preventive vaccines in transgenic mouse models of lung cancer. Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Ming You. Inhibition of lung tumorigenesis and potentiation of cancer preventive vaccines by a small molecule CA170 targeting the immune checkpoint protein VISTA [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 7297.

Abstract 1543: 40-color spectral flow cytometry for comprehensive immunophenotyping of murine cancers

Abstract Introduction: High-dimensional immunoprofiling is essential for studying host response to cancer immunotherapy. Synergistic agents often induce systemic changes in leukocyte abundance/function due to targeting, dosage, or delivery, necessitating multi-tissue analyses to decipher immune response mechanisms. We set out to dissect leukocyte composition in lymphoid tissues and tumors by using spectral cytometry, a multiplexed, high-throughput technology. Methods: We engineered a 40-color spectral flow cytometry panel to immunophenotype all major leukocytes and their functional subsets. Naïve and tumor-burdened C57BL/6 mice treated with combinations of agonist CD40 (aCD40) and checkpoint inhibitors were sacrificed, and tissues were processed. Stained cells were acquired on a 5-laser CyTEK Aurora. Unmixing accuracy and resolution was validated via 780 NxN plots, and high-dimensional analysis was performed. Results: Spectral cytometry affords resolution of 43+ parameters compared to 26 or less in conventional systems. We first designed a 25-marker backbone panel to identify all immune lineages. Additional channels were assigned to functional markers, allowing for profiling of migration, maturation, and activation in tumor-infiltrating leukocytes (TILs). In naïve secondary lymphoid tissues (SLTs), 35 distinct CD45+ populations were resolved. Germinal center B cells (17% vs. ~0.3% of CD45+ in other SLTs) and T follicular cells (31% vs. ~1-5% of CD4+ T cells in other SLTs) were mostly found in the Peyer’s patches, representing a hypothesized model tissue for studying tertiary immune structures in tumors. Peripheral blood contained the highest frequency of NK cells (4%), monocytes (6%), basophils (0.3%), eosinophils (4%), and neutrophils (7%) as a percentage of CD45+ cells. The spleen held the largest reservoir of CD4/8 central/effector memory cells (5% of CD45+) while ~80% of all T cells in the lymph nodes were naïve. In primary lymphoid tissues (PLTs), 12 distinct thymocyte and 7 B cell populations were resolved. Wanderlust trajectory analysis revealed CD24 and CD127 as key developmental regulators in lymphocyte differentiation. Autofluorescence extraction was essential for myeloid phenotyping. With this approach, we found monocytes comprised ~60% of TILs following aCD40 treatment (~8% in non-treatment and checkpoint only controls) and had altered macrophage differentiation fates. aCD40-treated tumors also contained higher frequencies of activated CD40/80/86+ DCs (~80/86/60% vs. ~60/51/50% of DCs in NTCs). Conclusion: This work describes a novel tool that efficiently resolves leukocytes on the axes of time, tissue, and treatment. Applications range from serial monitoring of circulating blood, endpoint analysis, validation of multiplexed-IHC, and cell sorting. PLT and SLT data has been made publicly available to serve as a training tool and reference proteomic atlas in future work. Citation Format: Aris John Kare, Lisa Nichols, Ricardo Zermeno, Marina N. Raie, Spencer K. Tumbale, Katherine W. Ferrara. 40-color spectral flow cytometry for comprehensive immunophenotyping of murine 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 1543.