Natural CD8 Research Articles

State of the Field: Cytotoxic Immune Cell Responses in C. neoformans and C. deneoformans Infection.

Cryptococcus neoformans is an environmental pathogen that causes life-threatening disease in immunocompromised persons. The majority of immunological studies have centered on CD4+ T-cell dysfunction and associated cytokine signaling pathways, optimization of phagocytic cell function against fungal cells, and identification of robust antigens for vaccine development. However, a growing body of literature exists regarding cytotoxic cells, specifically CD8+ T-cells, Natural Killer cells, gamma/delta T-cells, NK T-cells, and Cytotoxic CD4+ T-cells, and their role in the innate and adaptive immune response during C. neoformans and C. deneoformans infection. In this review, we (1) provide a comprehensive report of data gathered from mouse and human studies on cytotoxic cell function and phenotype, (2) discuss harmonious and conflicting results on cellular responses in mice models and human infection, (3) identify gaps of knowledge in the field ripe for exploration, and (4) highlight how innovative immunological tools could enhance the study of cytotoxic cells and their potential immunomodulation during cryptococcosis.

Efficacy and safety of camrelizumab combined with chemotherapy in the treatment of advanced biliary malignancy and associations between peripheral blood lymphocyte subsets and clinical outcomes.

Biliary tract cancer (BTC) is a highly heterogeneous aggressive tumor, and advanced patients have poor prognosis. This work aimed to evaluate the efficacy and safety of camrelizumab combined with chemotherapy in treating advanced BTC, and to explore predictive biomarkers for distinguishing effective population. 183 advanced BTC patients admitted from September 2018 to September 2021 were retrospectively selected. 93 patients were treated with camrelizumab combined with chemotherapy (C+C group) and 90 patients were treated with chemotherapy alone (C group). Objective response rate (ORR), disease control rate (DCR), median progression-free survival (mPFS), and median overall survival (mOS) were analyzed between two groups. Peripheral blood lymphocyte subsets were assessed by flow cytometry pre- and post-treatment. The mPFS (6.9 months) and mOS (12.1 months) in the C+C group were significantly longer than those in the C group, which were 5.2 months and 9.8 months respectively (HR 0.46, 95% CI 0.38-0.54, p=0.017; HR 0.39, 95% CI 0.32-0.47, p=0.033). The percentage of Total T, CD4+T, natural killer (NK) cells, lymphocyte, and CD4+/CD8+ cell ratios were significantly increased in effective patients after C+C treatment, but didn't increase in progressive disease (PD) patients. Higher percentage of Total T, CD4+T, and higher CD4+/CD8+ cell ratios post-treatment were associated with longer OS. Camrelizumab combining chemotherapy significantly prolonged the mPFS and mOS of advanced BTC patients. Immunotherapy may improve the immune status of advanced patients, and immunotherapy efficacy might be predicted based on the peripheral blood lymphocyte subsets.

Dysfunction of type 1 and type 2 immune cells: a lesson from exhausted-like ILC2s and their activation-induced cell death.

The concept of immune cell exhaustion/dysfunction has developed mainly to understand impaired type 1 immune responses, especially by CD8 T-cells against tumors or virus-infected cells, and has been applied to other lymphocytes. Natural killer (NK) cells and CD4 T cells support the efficient activation of CD8 T cells but exhibit dysfunctional phenotypes in tumor microenvironments and in chronic viral infections. In contrast, the concept of type 2 immune cell exhaustion/dysfunction is poorly established. Group 2 innate lymphoid cells (ILC2s) and T-helper 2 (Th2) cells are the major lymphocyte subsets that initiate and expand type 2 immune responses for antiparasitic immunity or allergy. In mouse models of chronic parasitic worm infections, Th2 cells display impaired type 2 immune responses. Chronic airway allergy induces exhausted-like ILC2s that quickly fall into activation-induced cell death to suppress exaggerated inflammation. Thus, the modes of exhaustion/dysfunction are quite diverse and rely on the types of inflammation and the cells. In this review, we summarize current knowledge of lymphocyte exhaustion/dysfunction in the context of type 1 and type 2 immune responses and discuss ILC2-specific regulatory mechanisms during chronic allergy.

Vaccination with DC-SIGN-Targeting αGC Liposomes Leads to Tumor Control, Irrespective of Suboptimally Activated T-Cells.

Cancer vaccines have emerged as a potent strategy to improve cancer immunity, with or without the combination of checkpoint blockade. In our investigation, liposomal formulations containing synthetic long peptides and α-Galactosylceramide, along with a DC-SIGN-targeting ligand, Lewis Y (LeY), were studied for their anti-tumor potential. The formulated liposomes boosted with anti-CD40 adjuvant demonstrated robust invariant natural killer (iNKT), CD4+, and CD8+ T-cell activation in vivo. The incorporation of LeY facilitated the targeting of antigen-presenting cells expressing DC-SIGN in vitro and in vivo. Surprisingly, mice vaccinated with LeY-modified liposomes exhibited comparable tumor reduction and survival rates to those treated with untargeted counterparts despite a decrease in antigen-specific CD8+ T-cell responses. These results suggest that impaired induction of antigen-specific CD8+ T-cells via DC-SIGN targeting does not compromise anti-tumor potential, hinting at alternative immune activation routes beyond CD8+ T-cell activation.

Cordyceps sinensis relieves non-small cell lung cancer by inhibiting the MAPK pathway

ObjectiveTo determine the pharmacodynamic mechanism underlying Cordyceps sinensis relief in a murine model of non-small cell lung cancer (NSCLC).MethodsWe created a murine model of NSCLC and studied the potential molecular mechanism by which C. sinensis relieved NSCLC using a combination of transcriptomics, proteomics, and experimental validation.ResultsC. sinensis markedly suppressed the fluorescence values in mice with NSCLC, improved the pathologic morphology of lung tissue, ameliorated inflammatory cytokines (tumor necrosis factor-alpha, interleukin-6, interleukin-10, and the oxidative stress indicators superoxide dismutase, malondialdehyde, and glutathione peroxidase). Transcriptomics results showed that the therapeutic effect of C. sinensis was primarily involved in the differentiation and activation of T cells. Based on the proteomic results, C. sinensis likely exerted a protective effect by recruiting immune cells and suppressing tumor cell proliferation via the MAPK pathway. Finally, the experimental validation results indicated that C. sinensis significantly decreased the VEGF and Ki67 expression, downregulated RhoA, Raf-1, and c-fos expression, which are related to cell migration and invasion, increased the serum concentration of hematopoietic factors (EPO and GM-CSF), and improved the percentage of immune cells (natural killer cells, dendritic cells, and CD4+ and CD8+ lymphocytes), which enhanced immune function.ConclusionsBased on our preclinical study, C. sinensis was shown to exert a protective effect on NSCLC, primarily by inhibiting the MAPK pathway.

Abstract 4927: Combining single-cell ATAC and RNA sequencing for supervised cell annotation

Abstract Background: Analysis of samples at the single-cell level offers insights into cellular heterogeneity and cell function. Cell type annotation is the first critical step for performing such an analysis. While current methods primarily utilize single-cell RNA sequencing (scRNA-seq) for annotation, several studies have demonstrated improved classification accuracy by combining scRNA-seq with transposase-accessible chromatin sequencing (ATAC-seq) using unsupervised methods. However, the utility of ATAC-seq features for supervised cell-type annotation has not been explored. Aims/Objectives: The objective of this study was to evaluate the relative performance of supervised cell-type classification using scRNA-seq alone vs. in multimodal combination with ATAC-seq; and how these data interplay with choice of classification and dimensionality reduction methods. Methods: A peripheral-blood mononuclear cell multi-omic dataset from a single, healthy female donor wasanalysed in this study. Ground truth annotations were generated using unsupervised annotation with the weighted nearest neighbour clustering method. Two dimensionality reduction methods (principal component analysis (PCA), single-cell Variational Inference (scVI) autoencoder) and four classification models (logistic regression, random forest, support vector machine (SVM)) were implemented and performance metrics (F1 score, precision, and recall) were compared over 10 bootstrap samples. Results: ATAC-seq features improved annotation quality and prediction confidence when using scVI embeddings, independent of the classifier. The best-performing model (SVM with scVI embeddings) showed an increase from a median macro F1 score of 0.907 (IQR = [0.902, 0.910]) using scRNA-seq alone to 0.946 (IQR = [0.940, 0.949], p <0.05) with ATAC-seq added. For PCA embeddings, improvements in macro F1 score were insignificant. All cell types (B, T, monocytes, natural killer and dendritic cells) showed significant improvements when using ATAC-seq with scVI embeddings. CD4 T effector memory cells showed the largest gain in F1 score (0.112, p <0.01), whilst type-2 conventional dendritic cells showed the smallest improvement (0.006, p <0.05). Prediction confidence was improved in B cells, monocytes, natural killer cells, CD4 and CD8 naïve cells, CD4 T central memory cells and CD8 T effector memory cells. Improvements in F1 scores were lost when only classifying major cell types rather than subtypes. Conclusions: Employing ATAC-seq embeddings with scVI autoencoder enhances supervised annotation quality over scRNA-only methods. Further studies should explore the use of ATAC to improve the annotation of highly heterogeneous tissues such as tumours. Citation Format: Jaidip Gill, Abhijit Dasgupta, Brychan Manry, Natasha Markuzon. Combining single-cell ATAC and RNA sequencing for supervised cell annotation [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 4927.

Epstein-Barr virus: the mastermind of immune chaos.

The Epstein-Barr virus (EBV) is a ubiquitous human pathogen linked to various diseases, including infectious mononucleosis and multiple types of cancer. To control and eliminate EBV, the host's immune system deploys its most potent defenses, including pattern recognition receptors, Natural Killer cells, CD8+ and CD4+ T cells, among others. The interaction between EBV and the human immune system is complex and multifaceted. EBV employs a variety of strategies to evade detection and elimination by both the innate and adaptive immune systems. This demonstrates EBV's mastery of navigating the complexities of the immunological landscape. Further investigation into these complex mechanisms is imperative to advance the development of enhanced therapeutic approaches with heightened efficacy. This review provides a comprehensive overview of various mechanisms known to date, employed by the EBV to elude the immune response, while establishing enduring latent infections or instigate its lytic replication.

NFIL3 contributes to cytotoxic T lymphocyte-mediated killing.

Cytotoxic T lymphocytes (CTLs) are key effectors of the adaptive immune system that recognize and eliminate virally infected and cancerous cells. In naive CD8+ T cells, T-cell receptor (TCR) engagement drives a number of transcriptional, translational and proliferation changes over the course of hours and days leading to differentiation into CTLs. To gain a better insight into this mechanism, we compared the transcriptional profiles of naive CD8+ T cells to those of activated CTLs. To find new regulators of CTL function, we performed a selective clustered regularly interspaced short palindromic repeats (CRISPR) screen on upregulated genes and identified nuclear factor IL-3 (NFIL3) as a potential regulator of cytotoxicity. Although NFIL3 has established roles in several immune cells including natural killer, Treg, dendritic and CD4+ T cells, its function in CD8+ CTLs is less well understood. Using CRISPR/Cas9 editing, we found that removing NFIL3 in CTLs resulted in a marked decrease in cytotoxicity. We found that in CTLs lacking NFIL3 TCR-induced extracellular signal-regulated kinase phosphorylation, immune synapse formation and granule release were all intact while cytotoxicity was functionally impaired in vitro. Strikingly, NFIL3 controls the production of cytolytic proteins as well as effector cytokines. Thus, NFIL3 plays a cell intrinsic role in modulating cytolytic mechanisms in CTLs.

Multi-omics analysis reveals the landscape of tumor microenvironments in left-sided and right-sided colon cancer.

Distinct clinical features and molecular characteristics of left-sided colon cancer (LCC) and right-sided colon cancer (RCC) suggest significant variations in their tumor microenvironments (TME). These differences can impact the efficacy of immunotherapy, making it essential to investigate and understand these disparities. We conducted a multi-omics analysis, including bulk RNA sequencing (bulk RNA-seq), single-cell RNA sequencing (scRNA-seq), and whole-exome sequencing (WES), to investigate the constituents and characteristic differences of the tumor microenvironment (TME) in left-sided colon cancer (LCC) and right-sided colon cancer (RCC). Deconvolution algorithms revealed significant differences in infiltrated immune cells between left-sided colon cancer (LCC) and right-sided colon cancer (RCC), including dendritic cells, neutrophils, natural killer (NK) cells, CD4 and CD8 T cells, and M1 macrophages (P < 0.05). Notably, whole-exome sequencing (WES) data analysis showed a significantly higher mutation frequency in RCC compared to LCC (82,187/162 versus 18,726/115, P < 0.01). Single-cell analysis identified predominant tumor cell subclusters in RCC characterized by heightened proliferative potential and increased expression of major histocompatibility complex class I molecules. However, the main CD8 + T cell subpopulations in RCC exhibited a highly differentiated state, marked by T cell exhaustion and recent activation, defined as tumor-specific cytotoxic T lymphocytes (CTLs). Immunofluorescence and flow cytometry results confirmed this trend. Additionally, intercellular communication analysis demonstrated a greater quantity and intensity of interactions between tumor-specific CTLs and tumor cells in RCC. RCC patients with an abundance of tumor-specific cytotoxic T lymphocytes (CTLs) and increased immunogenicity of tumor cells in the TME may be better candidates for immune checkpoint inhibitor therapy.

IPSC-Derived CD4 T Cell Generation and Investigation of CD4/CD8 T Cell Lineage Choice

iPSC-derived functionally rejuvenated antigen-specific cytotoxic T lymphocytes generated from exhausted CTLs show promising antitumor effect towards refractory tumors. Currently, we are preparing for clinical trials using iPSC-derived CTLs for EBV-associated lymphomas. These iPSC-derived T cells (iPSC-Ts) all generated into CD8 T cells, and stable generation of CD4 single positive (SP) T cells from iPSCs has never been accomplished without enforced CD4 gene transduction even though many research groups have challenged to do so. Even when an iPSC is established from a CD4 T cell clone, differentiating this iPSC does not generate into CD4 T cells, but in fact it generates into CD8 T cells. Therefore, we aimed to investigate why iPSC-Ts only differentiate into CD8T cells, and not into CD4T cells. We focused on adult T cell leukemia (ATL), because HTLV-1 infected CD4 T cells clonally expand. We reprogrammed CD4T cells obtained from acute-type ATL patients into iPSCs (ATL-iPSCs), then differentiated them into T cells using our established method. Interestingly, ATL-iPSCs from different donors all successfully differentiated into CD4 SP T cells (ATL-iPSC-Ts). Upon characterization of ATL-iPSC-Ts we confirmed that they showed FOXP3 positive naive regulatory T cell (Treg) phenotype. To investigate the function of these cells, we performed 51Cr-release assay. The cytotoxicity of HTLV-1 Tax-specific CTLs (Tax-CTLs) with or without ATL-iPSC-Ts against primary ATL cells was measured. The percentages of ATL cells lysed by Tax-CTLs was 34 ± 3.2%, whereas by the addition of ATL-iPSC-Ts, the cytotoxicity decreased to 23.4 ± 1.0% revealing the functionally suppressive effect of ATL-iPSC-Ts. In order to further understand the mechanism of CD4/CD8 T cell lineage choice, and discover the key regulators needed for generation of CD4 SP T cells from iPSCs, we performed single cell RNA-sequencing (scRNA-seq) on ATL-iPSC-Ts. As a control we used healthy donor (HD) CD4 SP T cell clone-derived iPSCs (HD-iPSCs), which differentiated into CD8 T cells (HD-iPSC-Ts [CD8 +]). We identified several genes expressed at a significantly higher level on HD-iPSC-Ts (CD8 +) than on ATL-iPSC-Ts (CD4 +). We presumed these genes to be essential for CD8 T cell differentiation. Therefore, we used CRISPR/Cas9 technology to knock out the respective gene in HD-iPSCs and confirmed if these iPSCs could differentiate into CD4 SP T cells, instead of CD8 T cells. This resulted in successful generation of CD4 SP T cells. We confirmed that these CD4 SP T cells were FOXP3 negative and were not Tregs. On the other hand, overexpression of this gene on ATL-iPSCs differentiated into CD8 SP T cells, instead of CD4 T cells. In conclusion, our work represents the first successful generation of natural CD4 SP T cells from iPSCs. The gene we identified appears to be a pivotal regulator of CD4/CD8 T cell lineage choice in our iPSC-T differentiation system. While more investigation is required, our method shows promise towards facilitating stable generation of CD4 T cells for T cell therapy.

Dominant CD4+ T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

In people with HIV (PWH), the post-antiretroviral therapy (ART) window is critical for immune restoration and HIV reservoir stabilization. We employ deep immune profiling and Tcell receptor (TCR) sequencing and examine proliferation to assess how ART impacts Tcell homeostasis. In PWH on long-term ART, lymphocyte frequencies and phenotypes are mostly stable. By contrast, broad phenotypic changes in natural killer (NK) cells, γδ Tcells, B cells, and CD4+ and CD8+ Tcells are observed in the post-ART window. Whereas CD8+ Tcells mostly restore, memory CD4+ T subsets and cytolytic NK cells show incomplete restoration 1.4 years post ART. Surprisingly, the hierarchies and frequencies of dominant CD4 TCR clonotypes (0.1%-11% of all CD4+ Tcells) remain stable post ART, suggesting that clonal homeostasis can be independent of homeostatic processes regulating CD4+ Tcell absolute number, phenotypes, and function. The slow restoration of host immunity post ART also has implications for the design of ART interruption studies.

The Effects of Thymosin Alpha-1 on Macrophages: A Cytological and Anti-Inflammatory Study

Thymosin alpha 1 (Tα-1) is a naturally occurring substance synthesized by the thymus tissue, known to activate various immune system cells. It has been reported to increase the production of Natural Killer cells, CD4 and CD8 cells, and cytokines such as IL-2, IL-3, and IFNγ. Furthermore, it plays a vital role in regulating immunity, inflammation, and tolerance. Its effect on the immune system is exerted via its modulatory action on innate immune system cells, thereby functioning as an endogenous regulator for both inflammatory and adaptive immune systems. In this study, we sought to evaluate the cytological and anti-inflammatory effects of Tα-1 on RAW 264.7 cells. The cytological effects of Tα-1 were assessed using the MTT assay, with the IC50 value against RAW 264.7 cells determined to be 368.105 ug/ml. The morphological observations made at various concentrations of Tα-1 showed increased cytotoxicity and decreased cell density of RAW 264.7 cells with increasing test concentration. Furthermore, the anti-inflammatory effects of Tα-1 were evaluated by analyzing the nitric oxide (NO) production in RAW 264.7 cells. Treatment with the test items at concentrations ranging from 7.813 to 31.25 ug/ml showed a dose-dependent reduction in NO production compared with the control group. These findings suggest that Tα-1 may have the potential as an anti-inflammatory agent in the treatment of various diseases associated with inflammation.

Interleukin-21 Influences Glioblastoma Course: Biological Mechanisms and Therapeutic Potential.

Brain tumors represent a heterogeneous group of neoplasms involving the brain or nearby tissues, affecting populations of all ages with a high incidence worldwide. Among the primary brain tumors, the most aggressive and also the most common is glioblastoma (GB), a type of glioma that falls into the category of IV-grade astrocytoma. GB often leads to death within a few months after diagnosis, even if the patient is treated with available therapies; for this reason, it is important to continue to discover new therapeutic approaches to allow for a better survival rate of these patients. Immunotherapy, today, seems to be one of the most innovative types of treatment, based on the ability of the immune system to counteract various pathologies, including cancer. In this context, interleukin 21 (IL-21), a type I cytokine produced by natural killer (NK) cells and CD4+ T lymphocytes, appears to be a valid target for new therapies since this cytokine is involved in the activation of innate and adaptive immunity. To match this purpose, our review deeply evaluated how IL-21 could influence the progression of GB, analyzing its main biological processes and mechanisms while evaluating the potential use of the latest available therapies.

Adjuvant dendritic cell-based immunotherapy after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy in patients with malignant peritoneal mesothelioma: a phase II clinical trial

BackgroundMalignant peritoneal mesothelioma (MPM) is an aggressive malignancy with a poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) improves survival outcomes, but recurrence rates remain high. Dendritic cell-based...

MP28-04 CLINICAL AND IMMUNOGENOMIC OUTCOMES OF CONSOLIDATIVE CYTOREDUCTIVE NEPHRECTOMY IN METASTATIC RENAL CELL CARCINOMA PATIENTS TREATED WITH IMMUNE CHECKPOINT BLOCKADE

You have accessJournal of UrologyCME1 Apr 2023MP28-04 CLINICAL AND IMMUNOGENOMIC OUTCOMES OF CONSOLIDATIVE CYTOREDUCTIVE NEPHRECTOMY IN METASTATIC RENAL CELL CARCINOMA PATIENTS TREATED WITH IMMUNE CHECKPOINT BLOCKADE Stephen W. Reese, Hui Jiang, Fengshen Kuo, Chih-Ying Wu, Linda H. Vuong, Eduardo A. Mascareno, Robert Motzer, Chung-Han Lee, Martin H. Voss, Ed Reznik, Victor Reuter, Johnathan Coleman, Paul Russo, Maria Carlo, Ying-Bei Chen, Ritesh R. Kotecha, and Ari Hakimi Stephen W. ReeseStephen W. Reese More articles by this author , Hui JiangHui Jiang More articles by this author , Fengshen KuoFengshen Kuo More articles by this author , Chih-Ying WuChih-Ying Wu More articles by this author , Linda H. VuongLinda H. Vuong More articles by this author , Eduardo A. MascarenoEduardo A. Mascareno More articles by this author , Robert MotzerRobert Motzer More articles by this author , Chung-Han LeeChung-Han Lee More articles by this author , Martin H. VossMartin H. Voss More articles by this author , Ed ReznikEd Reznik More articles by this author , Victor ReuterVictor Reuter More articles by this author , Johnathan ColemanJohnathan Coleman More articles by this author , Paul RussoPaul Russo More articles by this author , Maria CarloMaria Carlo More articles by this author , Ying-Bei ChenYing-Bei Chen More articles by this author , Ritesh R. KotechaRitesh R. Kotecha More articles by this author , and Ari HakimiAri Hakimi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003256.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Immune checkpoint blockade (ICB) alone and in combination has improved outcomes in metastatic renal cell carcinoma (RCC). We performed in-depth clinical, histopathologic and immunogenomic analysis of a cohort of patients who were treated with upfront ICB followed by consolidative nephrectomy. This highly selected cohort included extreme responders defined by complete pathologic response and no evidence of disease at more than 25 months of follow-up. METHODS: Clinicopathologic data was collected from 32 patients who received ICB (alone or combined with anti-VEGF therapy) and then underwent resection of primary tumor. Immunogenomic analysis was performed on select pre-treatment and nephrectomy tissue using targeted next-generation sequencing (MSK-IMPACT) and immunohistochemical (IHC) staining analyses. We further performed single-cell (sc) RNA sequencing of both peripheral blood and residual tumor tissue (where available), including 4 patients who demonstrated complete pathologic response. The Kaplan-Meier method was used to calculate survival outcomes. RESULTS: All 32 patients underwent successful consolidative surgery and were further stratified based on evidence of radiographic progression, with 13 patients (40.6%) demonstrating no evidence of disease after surgery. The most common histopathology was clear cell RCC (n=29, 90.6%). Median follow-up for the cohort was 33.1 months and median OS was 45.2 months. Median OS for those who had disease progression after CN was 25.5 months and 24-month survival rate was 100% for those without progression. On pathologic evaluation, those without evidence of disease had a median of 8% (IQR: 0-35%) residual viable tumor. Mutational analysis revealed enrichment for BAP1 (n=3, 20.0% vs. n=0, 0%) and SETD2 (n=7, 46.7% vs. n=0, 0%) in patients with disease progression. scRNAseq and scTCRseq analysis of peripheral blood revealed distinct expansion of Natural Killer (NK), CD4 subsets, and unique T cell clonal expansions amongst long-term responders. Additionally, we identified an inverse relationship between NK cell populations and B cell infiltration in long-term responders. CONCLUSIONS: Delayed cytoreductive nephrectomy in select patients is associated with favorable outcomes, especially in those who demonstrate complete response after surgery. Examination of this population offers opportunities for the identification of unique tissue and peripheral-based biomarkers for long-term clinical responses in metastatic RCC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e370 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Stephen W. Reese More articles by this author Hui Jiang More articles by this author Fengshen Kuo More articles by this author Chih-Ying Wu More articles by this author Linda H. Vuong More articles by this author Eduardo A. Mascareno More articles by this author Robert Motzer More articles by this author Chung-Han Lee More articles by this author Martin H. Voss More articles by this author Ed Reznik More articles by this author Victor Reuter More articles by this author Johnathan Coleman More articles by this author Paul Russo More articles by this author Maria Carlo More articles by this author Ying-Bei Chen More articles by this author Ritesh R. Kotecha More articles by this author Ari Hakimi More articles by this author Expand All Advertisement PDF downloadLoading ...

Clinical and immunogenomic outcomes of consolidative cytoreductive nephrectomy in patients with metastatic renal cell carcinoma treated with immune checkpoint blockade.

720 Background: Immune checkpoint blockade (ICB) alone and in combination has dramatically improved outcomes in metastatic renal cell carcinoma (RCC). We performed in-depth clinical, histopathologic and immunogenomic analysis of a cohort of RCC patients treated with upfront ICB followed by delayed consolidative cytoreductive nephrectomy. This highly selected cohort included extreme responders defined by complete pathologic response and no evidence of disease at more than 25 months of follow-up. Methods: Clinicopathologic data was collected from 32 patients who received ICB (alone or combined with anti-VEGF therapy) and then subsequently underwent resection of primary tumor. Immunogenomic analysis was performed on select pre-treatment and nephrectomy tissue using targeted next-generation sequencing (MSK-IMPACT) and immunohistochemical (IHC) staining analyses. We further performed single-cell (sc)RNA sequencing of both peripheral blood and residual tumor tissue (where available), including 4 patients who demonstrated complete pathologic response. The Kaplan-Meier method was used to calculate survival outcomes. Results: All 32 patients underwent successful consolidative surgery and were further stratified based on evidence of radiographic progression, with 13 pts (40.6%) demonstrating no evidence of disease after surgery. The most common histopathology was clear cell RCC (n=29, 90.6%). Half the patients received dual ICB (CTLA-4/PD-1) (n=16, 50%), while the rest received an immunotherapy agent plus an anti-VEGF agent (n=13, 40.6%) or a single immunotherapy agent (n=3, 9.4%). Median follow-up for the cohort was 33.1 mo. (95%CI: 20.5-42.5) and median OS was 45.2 mo. (95%CI:23.9 – NR). Median OS for those who had progressive disease after CN was 25.5 (95%CI: 16.4- NR). At a median follow-up of 25.5 mo. in patients without evidence of disease after CN, 24-month survival rate was 100%. On pathologic evaluation, those without evidence of disease had a median of 8% (IQR: 0-35%) residual viable tumor. Mutational analysis revealed enrichment for BAP1 (n=3, 20.0% vs. n=0, 0%) and SETD2 (n=7, 46.7% vs. n=0, 0%) in progressive patients. scRNAseq and scTCRseq analysis of peripheral blood revealed distinct expansion of Natural Killer (NK), CD4 subsets and unique T cell clonal expansions amongst long term responders. Additionally, we identified an inverse relationship between NK cell populations and B cell infiltration in long term responders. Conclusions: Delayed cytoreductive nephrectomy in select patients is associated with favorable outcomes, especially in those who demonstrate complete response after surgery. Examination of this population offers opportunities for identification of unique tissue and peripheral based biomarkers for long term clinical responses in metastatic RCC.

Carbon ion irradiation induces DNA damage in melanoma and optimizes the tumor microenvironment based on the cGAS-STING pathway.

Increased number of studies reveal the crucial role of the Cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway in anti-tumor immunity. In this study, we aim to explore the effect of cGAS/STING on tumor immune microenvironment of melanoma after carbon ion radiotherapy (CIRT) and the underlying mechanism. C57BL/6 mouse tumor models were used to evaluate the efficacy of different treatments (X-ray, carbon ion, PD-L1 inhibitor and combination therapies) on tumor growth and process. Mass cytometry was performed to assess tumor-infiltrating lymphocytes (TILs). DNA damage response (DDR) and cGAS/STING pathway were investigated by immunofluorescence-co-localization assays, γ-H2AX, P53-binding protein 1 (53BP1), Breast Cancer 1 (BRCA1), and cGAS measurements. Carbon ion irradiation caused more DNA damages and cGAS-STING pathway activation compared with X-ray irradiation, and the former slowed the melanoma growth in syngeneic model. Although X-ray irradiation is not sensitive for melanoma treatment, carbon ion irradiation showed a significant anti-tumor effect for melanoma treatment. TILs analysis revealed that CIRT boosted the infiltration of natural killer (NK), CD4+, and CD8+ T cells, meanwhile increased the number of immune checkpoint (programmed death-1, PD-1, lymphocyte activation gene 3, LAG-3 and T-cell immunoglobulin and mucin domain-containing protein 3, TIM-3). Moreover, CIRT increased PD-L1 exposure on cell surface compared with X-ray group. Furthermore, CIRT combined with PD-L1 inhibitor therapy increased the number of T cells and NK cells in melanoma, and slowed the growth of melanoma compared with other therapies. Our findings showed that CIRT displayed biological effects by increasing DNA damages of tumor cells and improving immunity in melanoma, which indicated that CIRT might be a potential synergetic treatment for radiotherapy and radioimmunotherapy in melanoma patients. Our works put forward a new insight to provide an effective strategy for melanoma therapy. These findings may help in the design of strategies on melanoma in clinical studies.

Abstract C111: Spatial transcriptome profiling of tumor and tumor microenvironment of triple negative breast cancer in patients of African American and European American descent

Abstract Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that disproportionately affects women of African origin. AA women with TNBC have worse clinical outcomes than women of European descent as it presents with a more aggressive and progressive disease. Understanding the underlying biology of the tumor and its microenvironment is critical to address the disparity associated with racial differences in breast cancer. Bulk RNA-sequencing is the most common and useful method to analyze whole transcriptome. However, this method cannot deconvolute the individual components of the tumor and its associated stroma. The NanoString GeoMx digital spatial profiling is a multiplexed platform that spatially resolves and quantifies the abundance of genes present in tumor and stroma. In this study, we have utilized spatial RNA-seq technology to determine the differential gene expression in tumor epithelial cells and its associated stroma in AA and EA TNBC patients. Gene expression from spatially defined regions in tumor and stroma in each patient and in between races identified distinct molecular pathways. The tumor epithelial region of AA has high expression of fatty acid biosynthesis, nucleotide excision repair, RNA helicase like proteins and exhaustion signaling while EA tumor epithelial region had high expression of neuropilin 2, interferon signaling and TGF beta signaling. The pathways overexpressed in the AA stromal region included tryptophan metabolism, chemokine signaling pathway, interferon signaling and exhaustion signature while in the EA stromal region, the pathways pertinent to mTOR signaling, AURORA kinase, cysteine endopeptidases were more prominently expressed. AA tumors were significantly more inflamed than EA and exhibited high infiltration of immune cells. The stromal region of AA tumors has high population of macrophages, CD8 T memory, Treg’s and neutrophils more than EA tumors. The tumor epithelial region of AA has high infiltration of plasmacytoid dendritic cells (pDC), B memory, monocytes, natural killer cells (NK), CD4 memory, CD4 naïve, Treg’s and plasma cells. However, the exhaustion signature proteins, PDCD1 and LAG3 were also higher in tumor-infiltrated and stromal immune cells in AA tumor. The EA tumors were scarce in immune cells in the stroma as well as inside the tumor epithelial, though interferon signaling was substantially higher in EA tumor epithelial cells as compared to AA. Thus, using NanoString GeoMx digital spatial profiling platform, we have spatial profiled AA and EA TNBC and assessed the inter and intra-tumor heterogeneity within and in between races. This is one of the first study that describes not only expression variation of genes between races but also between stroma and tumor epithelial cells within the tissue of same patient and between different patients. These findings address the biological determinant of racial disparity by patient-centered investigation of factors associated with tumor and tumor microenvironment. Citation Format: Deepa Bedi, Balasubramanyam Karanam, Isra Elhussin, Chitra R. Nayak, Clayton Yates. Spatial transcriptome profiling of tumor and tumor microenvironment of triple negative breast cancer in patients of African American and European American descent [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C111.

Ageing Affects Thymopoiesis and Experimental Autoimmune Encephalomyelitis Development in a Strain-Dependent Manner

Introduction: Considering significance of mechanisms of central tolerance for development of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), and suppressive influence of circulating proinflammatory cytokines and alterations in brain-thymus communication, characteristic for the central nervous system (CNS) autoimmune diseases, on thymopoiesis, the study interogated putative strain-based thymus-related specificities relevant for the opposite effects of ageing on susceptibility of Dark Agouti (DA) and Albino Oxford (AO) rats to EAE. Methods: Quantitative and qualitative changes in thymopoiesis including underlying mechanisms were examined using flow cytometry and RT-qPCR quantification of mRNAs for molecules relevant for integrity of stroma and T-cell development, respectively. Results: With ageing, differently from DA rats, in AO rats the surface density of CD90, a negative regulator of selection threshold, on thymocytes undergoing lineage commitment was upregulated (consistent with TGF-β expression downregulation), whereas the generation of natural CD4+CD25+Foxp3+ regulatory T cells (nTregs) was impaired reflecting differences in thymic expression of cytokines supporting their development. Additionally, specifically in old AO rats, in whom EAE development depends on IL-17-producing CD8+ T cells, their thymic differentiation was augmented, reflecting augmented thymic IL-4 expression. In turn, differently from old DA rats developing self-limiting EAE, in age-matched AO rats developing EAE of prolonged duration, EAE development led to impaired generation of nTregs and accumulation of proinflammatory, cytotoxic CD28–CD4+ T cells in the periphery. Discussion: The study indicates that strain differences in age-related changes in the efficacy of central tolerance, in addition to enhanced thymic generation of CD8+ T cells prone to differentiate into IL-17-producing cells, could partly explain the opposite effect of ageing on DA and AO rat susceptibility to EAE induction. Additionally, it suggested that EAE development leading to a less efficient thymic output of CD4+ cells and nTregs in old AO rats than their DA counterparts could contribute to prolonged EAE duration in AO compared with DA rats. Conclusion: The study warns to caution when designing therapeutic interventions to enhance thymic activity in genetically diverse populations, e.g., humans, and interpreting their outcomes. Furthermore, it indicates that CNS autoimmune pathology may additionally worsen thymic involution and age-related immune changes.

A Highly Effective African Swine Fever Virus Vaccine Elicits a Memory T Cell Response in Vaccinated Swine.

African Swine Fever Virus (ASFV) is the causative agent of a highly contagious and lethal vector-borne disease in suids. Recently, a live attenuated virus strain, developed using the currently circulating, virulent Georgia strain (ASFV-G) with a single gene deletion (ASFV-G-ΔI177L), resulted in an effective vaccine. Nevertheless, protective immune response mechanisms induced by this candidate are poorly understood. In this study, Yorkshire crossbred swine intramuscularly vaccinated with 106 50% hemadsorption dose (HAD50) of ASFV-G-ΔI177L or a vehicle control were challenged at 28 days post-inoculation (dpi) with 102 HAD50 of ASFV-G. Analysis of purified peripheral blood mononuclear cells following inoculation and challenge revealed that CD4+, CD8+ and CD4+CD8+ central memory T cells (CD44+CD25-CD27-CD62L+CCR7+, Tcm) decreased significantly by 28 dpi in ASFV-G-ΔI177L-vaccinated swine compared to baseline and time-matched controls. Conversely, CD4+, CD8+ and CD4+CD8+ effector memory T cells (CD44+CD25-CD27-CD62-CCR7-, Tem) increased significantly among ASFV-G-ΔI177L-vaccined swine by 28 dpi compared to baseline and time-matched controls. Additionally, the percentage of natural killer (NK), CD4+ and CD4+CD8+ Tem and CD8+ Tcm and Tem positive for IFNγ increased significantly following inoculation, surpassing that of controls by 28 dpi or earlier. These results suggest that NK and memory T cells play a role in protective immunity and suggest that studying these cell populations may be a surrogate immunity marker in ASF vaccination.