Immune Cell Subsets Research Articles

The effect of exercise and disease status on mobilization of anti-tumorigenic and pro-tumorigenic immune cells in women with breast cancer.

Mobilization of certain immune cells may improve the ability of the immune system to combat tumor cells, but the effect of acute exercise on mobilizing immune cells has been sparsely investigated in cancer patients. Therefore, we examined how acute exercise influences circulating immune cells in breast cancer patients. Nineteen newly diagnosed breast cancer patients aged 36-68 performed 30 minutes of moderate-intensity exercise with a cycle ergometer. Blood samples were collected at various time points: at rest, at 15 (E15) and 30 minutes (E30) after onset of the exercise, and at 30 and 60 minutes post-exercise. We analyzed several immune cell subsets using flow cytometry. Acute exercise increased the number of total leukocytes, neutrophils, lymphocytes, monocytes, basophils, total T-cells, CD4+ T-cells, T helper (Th) 2-cells, Th 17-cells, CD8+ T-cells, CD4-CD8- T-cells, CD56+ natural killer (NK) cells, and CD14-CD16+ monocytes. Many of the changes were transient. Proportions of NK-cells and CD8+ T-cells increased, while the proportion of myeloid derived suppressor cells (MDSCs) reduced, and proportion of regulatory T-cells remained unchanged by exercise. Several associations were detected between cell mobilizations and disease state. For instance, tumor size correlated negatively with NK cell mobilization at E15, and progesterone receptor positivity correlated negatively with CD8+ T-cell mobilization. The findings show that the proportions of CD8+ T-cells and NK cells increased and the proportion of MDSCs proportion decreased in breast cancer patients after 30-minute exercise, suggesting a change in the profile of circulating immune cells towards more cytotoxic/anti-tumorigenic. The mobilization of some immune cells also appears to be related to the disease state.

Integrative multi-omics analysis reveals cellular and molecular insights into primary Sjögren's syndrome

ObjectiveThis study aims to comprehensively analyze genomic, transcriptomic, proteomic, and single-cell sequencing data to unravel the molecular basis of primary Sjögren's syndrome (pSS) and explore potential therapeutic targets. MethodsMendelian randomization and single-cell RNA sequencing were employed to analyze pSS data. Differentially expressed genes specific to different blood cell types were identified. Integration of multiomics data facilitated the exploration of genetic regulatory relationships. ResultsThe analysis revealed distinct cell clusters representing various immune cell subsets. Several genes, including cathepsin S (CTSS) and glutathione S-transferase omega 1 (GSTO1), were identified as potential biomarkers and therapeutic targets for pSS. Diagnostic utility analysis demonstrated the discriminatory power of CTSS and GSTO1 in distinguishing pSS patients from healthy controls. ConclusionThe findings highlight the importance of integrating multiomics data for understanding pSS pathogenesis. CTSS and GSTO1 show promise as diagnostic biomarkers and potential therapeutic targets for pSS. Further investigations are warranted to elucidate the underlying mechanisms and develop targeted therapies for this complex autoimmune disease.

Development and validation of a novel disulfidptosis-related lncRNAs signature in patients with HPV-negative oral squamous cell carcinoma

Disulfidptosis is a recently identified mode of regulated cell death. Regulating disulfidptosis in carcinoma is a promising therapeutic approach. Long non-coding RNAs (lncRNAs) have been reported to be related to the occurrence and development of many cancers. Disulfidptosis-related lncRNAs (DRLs) in HPV-negative oral squamous cell carcinoma (OSCC) have not been studied. Based on The Cancer Genome Atlas (TCGA) database, least absolute shrinkage selection operator (LASSO) analysis and Cox regression analysis were used to identify overall survival related DRLs and construct the signature. Kaplan–Meier, time-dependent receiver operating characteristics (ROC) and principal component analyses (PCA) were explored to demonstrate the prediction potential of the signature. Subgroup analysis stratified by different clinicopathological characteristics were conducted. Nomogram was established by DRLs signature and independent clinicopathological characteristics. The calibration plots were performed to reveal the accuracy of nomogram. Immune cell subset infiltration, immunotherapy response, drug sensitivity analysis, and tumor mutation burden (TMB) were conducted. Underlying functions and pathways were explored by Gene Set Enrichment Analysis (GSEA) analysis. Previous lncRNA signatures of OSCC were retrieved from PubMed for further validation. Gene expression omnibus (GEO) datasets (GSE41613 and GSE85446) were merged as an external validation for DRLs signature. Consensus clustering analysis of DRLs signature and experimental validation of DRLs were also explored. This research sheds light on the robust performance of DRLs signature in survival prediction, immune cell infiltration, immune escape, and immunotherapy of HPV-negative OSCC.

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry.

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically limited by the number of fluorescent parameters that can be imaged at a single time. Most immune cell subsets have been defined using flow cytometry by using complex combinations of protein markers, often requiring four or more parameters to conclusively identify, which is beyond the capabilities of most conventional microscopes. As flow cytometry dissociates tissues and loses spatial information, there is a need for techniques that can retain spatial information while interrogating the roles of complex cell types. These issues are addressed here by creating a method for expanding the number of fluorescent parameters that can be imaged by collecting the signals of spectrally overlapping fluorophores and using spectral unmixing to separate the signals of each individual fluorophore. These images are then processed using an analysis pipeline to take high-parameter histology images and extract single cells from these images so that the unique fluorescent properties of each cell can be analyzed at a single-cell level. Using flow cytometry-like gating strategies, cells can then be profiled into subsets and mapped back onto the histology sections to not only quantify their abundance, but also establish how they interact with the tissue environment. Overall, the simplicity and potential of using histoflow cytometry to study complex immune populations in histology sections is demonstrated.

A joint population pharmacokinetic model to assess the high variability of whole-blood and intracellular tacrolimus in early adult renal transplant recipients

Tacrolimus (TAC) has high pharmacokinetic (PK) variability during the early transplantation period. The relationships between whole-blood and intracellular TAC concentrations and clinical outcomes remain controversial. This study identifies the factors affecting the PK variability of TAC and characterizes the relationships between whole-blood and intracellular TAC concentrations. Data regarding whole-blood TAC concentrations of 1,787 samples from 215 renal transplant recipients (<90 days postoperative) across two centers and intracellular TAC concentrations (648 samples) digitized from previous studies were analyzed using nonlinear mixed-effects modeling. The effects of potential covariates were screened, and the distribution of whole-blood to intracellular TAC concentration ratios (RWB:IC) was estimated. The final model was evaluated using bootstrap, goodness of fit, and prediction-corrected visual predictive checks. The optimal dosing regimens and target ranges for each type of immune cell subsets were determined using Monte Carlo simulations. A two-compartment model adequately described the data, and the estimated mean TAC CL/F was 23.6 L·h−1 (relative standard error: 11.5 %). The hematocrit level, CYP3A5*3 carrier status, co-administration with Wuzhi capsules, and tapering prednisolone dose may contribute to the high variability of TAC PK variability during the early post-transplant period. The estimated RWB:IC of all TAC concentrations in peripheral blood mononuclear cells (PBMCs) was 4940, and inter-center variability of PBMCs was observed. The simulated TAC target range in PBMCs was 20.2–85.9 pg·million cells−1. Inter-center variability in intracellular concentrations should be taken into account in further analyses. TAC dosage adjustments can be guided based on PK/PD variability and simulated intracellular concentrations.

Identification of KRT80 as a Novel Prognostic and Predictive Biomarker of Human Lung Adenocarcinoma via Bioinformatics Approaches.

According to the 2022 Global Cancer Statistics, lung cancer is the leading cause of cancer-related mortality worldwide. Lung adenocarcinoma (LUAD), which is a histological subtype of Non- Small Cell Lung Cancer (NSCLC), accounts for 40% of primary lung cancer. Therefore, there is an urgent need to identify new prognostic markers as clinical predictive markers for LUAD. This study aimed to investigate the role of Keratin 80 (KRT80) in the prognosis of LUAD and its underlying mechanisms. Bioinformatics analysis was conducted using data retrieved from The Cancer Genome Atlas (TCGA) databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were employed to predict the involved biological processes and signaling pathways, respectively. The LinkedOmics database was utilized to identify differentially expressed genes (DEGs) correlated with KRT80. Nomograms and Kaplan-Meier plots were constructed to evaluate the survival outcomes of patients diagnosed with LUAD. Moreover, TIMER was employed to conduct correlation analyses between KRT80 expression and immune cell infiltration, shedding light on the intricate interplay between KRT80 and the tumor microenvironment in LUAD. To ascertain the RNA and protein expression levels of KRT80 in LUAD and adjacent normal tissues, Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) and immunohistochemistry techniques were employed, respectively. Scrutiny of the TCGA dataset revealed KRT80 up-regulation across pan-cancer tissues, notably elevated in LUAD compared to healthy lung tissues. This finding was validated in our clinical samples, where Kaplan-Meier survival curves indicated poorer survival rates for high KRT80 expression in LUAD. A positive correlation was found between the transcription level of KRT80 in LUAD samples and clinical parameters, such as lymph node metastasis stage, distant metastasis, and pathological stage. Survival, logistic regression, and Cox regression analyses emphasized the clinical prognostic significance of high KRT80 expression in LUAD. Nomogram results underscored the robust predictive potential of KRT80 for the survival of LUAD patients. Gene functional enrichment analyses mainly associated KRT80 with cytokine-cytokine receptor interactions, cell cycle, apoptosis, and chemokine signaling pathways. Based on the results of the immune infiltration analysis, it can be found that the expression of KRT80 is related to the immune cell subsets and survival rate of patients with LUAD. Our research revealed a significant upregulation of KRT80 in LUAD, with heightened KRT80 expression correlating with unfavorable prognosis. This study represents a comprehensive and systematic evaluation of KRT80 expression in LUAD, encompassing its prognostic and diagnostic significance, as well as underlying mechanisms. Our findings suggest that KRT80 may emerge as a novel prognostic and predictive biomarker in LUAD.

Extracellular matrix remodelling pathway in peripheral blood mononuclear cells from severe COVID-19 patients: an explorative study.

There is a reciprocal relationship between extracellular matrix (ECM) remodelling and inflammation that could be operating in the progression of severe COVID-19. To explore the immune-driven ECM remodelling in COVID-19, we in this explorative study analysed these interactions in hospitalised COVID-19 patients. RNA sequencing and flow analysis were performed on peripheral blood mononuclear cells. Inflammatory mediators in plasma were measured by ELISA and MSD, and clinical information from hospitalised COVID-19 patients (N=15) at admission was included in the analysis. Further, we reanalysed two publicly available datasets: (1) lung tissue RNA-sequencing dataset (N=5) and (2) proteomics dataset from PBCM. ECM remodelling pathways were enriched in PBMC from COVID-19 patients compared to healthy controls. Patients treated at the intensive care unit (ICU) expressed distinct ECM remodelling gene profiles compared to patients in the hospital ward. Several markers were strongly correlated to immune cell subsets, and the dysregulation in the ICU patients was positively associated with plasma levels of inflammatory cytokines and negatively associated with B-cell activating factors. Finally, our analysis of publicly accessible datasets revealed (i) an augmented ECM remodelling signature in inflamed lung tissue compared to non-inflamed tissue and (ii) proteomics analysis of PBMC from severe COVID-19 patients demonstrated an up-regulation in an ECM remodelling pathway. Our results may suggest the presence of an interaction between ECM remodelling, inflammation, and immune cells, potentially initiating or perpetuating pulmonary pathology in severe COVID-19.

Corticosteroid-depending effects on peripheral immune cell subsets vary according to disease modifying strategies in multiple sclerosis

Corticosteroid-depending effects on peripheral immune cell subsets vary according to disease modifying strategies in multiple sclerosis

Pathological changes in the spleen of mice subjected to different time courses of restraint stress

The objective of this study was to investigate spleen pathology and immune cell subset alterations in mice exposed to acute and chronic restraint stress over various timeframes. A deeper understanding of stress-induced spleen injuries can provide new insights into the mechanisms underlying stress-induced disorders. C57BL/6N mice were restrained for different durations (1, 3, 7, 14 and 21 days) for 6–8 h daily. The control mice were observed at the same time points. Post restraint, behavioural experiments were conducted to assess spleen weight, gross morphology and microscopic histological changes. Immunohistochemical staining was used to detect changes in glucocorticoid receptor (GR) expression, immune cell subsets and cell proliferation in response to stress. Our analysis revealed significant behavioural abnormalities in the stressed mice. In particular, there was an increase in the nuclear expression of GR beginning on Day 3, and it peaked on Day 14. The spleens of stressed mice displayed a reduction in size, disordered internal tissue structure and reduced cell proliferation. NK cells and M2-type macrophages exhibited immune cell subset alterations under stress, whereas T or B cells remained unaltered. Restraint stress can lead to pathomorphological alterations in spleen morphology, cell proliferation and immune cell counts in mice. These findings suggest that stress-induced pathological changes can disrupt immune regulation during stress.

Grooved Microneedle Patch Augments Adoptive T Cell Therapy Against Solid Tumors via Diverting Regulatory T Cells.

The efficacy of adoptive T cell therapy (ACT) for the treatment of solid tumors remains challenging. In addition to the poor infiltration of effector T (Teff) cells limited by the physical barrier surrounding the solid tumor, another major obstacle is the extensive infiltration of regulatory T (Treg) cells, a major immunosuppressive immune cell subset, in the tumor microenvironment. Here, this work develops a grooved microneedle patch for augmenting ACT, aiming to simultaneously overcome physical and immunosuppressive barriers. The microneedles are engineered through an ice-templated method to generate the grooved structure for sufficient T-cell loading. In addition, with the surface modification of chemokine CCL22, the MNs could not only directly deliver tumor-specific T cells into solid tumors through physical penetration, but also specifically divert Treg cells from the tumor microenvironment to the surface of the microneedles via a cytokine concentration gradient, leading to an increase in the ratio of Teff cells/Treg cells in a mouse melanoma model. Consequently, this local delivery strategy of both T cell receptor T cells and chimeric antigen receptor T cells via the CCL22-modified grooved microneedles as a local niche could significantly enhance the antitumor efficacy and reduce the on-target off-tumor toxicity of ACT.

Innate and adaptive effector immune drivers of cytomegalovirus disease in lung transplantation: a double-edged sword.

Up to 90% of the global population has been infected with cytomegalovirus (CMV), a herpesvirus that remains latent for the lifetime of the host and drives immune dysregulation. CMV is a critical risk factor for poor outcomes after solid organ transplant, though lung transplant recipients (LTR) carry the highest risk of CMV infection, and CMV-associated comorbidities compared to recipients of other solid organ transplants. Despite potent antivirals, CMV remains a significant driver of chronic lung allograft dysfunction (CLAD), re-transplantation, and death. Moreover, the extended utilization of CMV antiviral prophylaxis is not without adverse effects, often necessitating treatment discontinuation. Thus, there is a critical need to understand the immune response to CMV after lung transplantation. This review identifies key elements of each arm of the CMV immune response and highlights implications for lung allograft tolerance and injury. Specific attention is paid to cellular subsets of adaptive and innate immune cells that are important in the lung during CMV infection and reactivation. The concept of heterologous immune responses is reviewed in depth, including how they form and how they may drive tissue- and allograft-specific immunity. Other important objectives of this review are to detail the emerging role of NK cells in CMV-related outcomes, in addition to discussing perturbations in CMV immune function stemming from pre-existing lung disease. Finally, this review identifies potential mechanisms whereby CMV-directed treatments may alter the cellular immune response within the allograft.

Comprehensive characterization of B7 family members in breast cancer: B7-H5 switch reverses breast cancer from “immuno-cold” into “immuno-hot” status

The members of the classic B7 family regulate the immune microenvironment of several malignant tumors. However, the potential relationship between the B7 family and the breast cancer (BrCa) tumor immune microenvironment has remained elusive. In the present study, we provide a comprehensive explanation of the expression, clinical significance, mutation, and immune cell infiltration of B7 family molecules in BrCa. First, we recruited 10 patients with BrCa surgery from the Wuxi Maternal and Child Health Hospital and performed single-cell RNA sequencing (scRNA-seq) analysis to investigate the distribution of B7 family members in multiple immune cell subsets. We focused on B7-2, B7-H3, and B7-H5 molecules of the B7 family and constructed tumor microarrays by self-recruiting patients to perform multiple immunohistochemical (mIHC) analyses and study tumor expression of B7-2, B7-H3, B7-H5 and CD8+ immune cell infiltration. B7-H5 displayed a strong correlation with CD8+ immune cell infiltration. In summary, B7-H5 provides a new perspective for the identification of immunothermal subtypes of BrCa and could function as a switch to reverse BrCa from an “immunologically cold” state to an “immunologically hot” state.Graphical abstract

Central memory CD4+ T cells play a protective role against immune checkpoint inhibitor-associated myocarditis.

The widespread use of immune checkpoint inhibitors (ICIs) has demonstrated significant survival benefits for cancer patients and also carries the risk of immune-related adverse events. ICI-associated myocarditis is a rare and serious adverse event with a high mortality rate. Here, we explored the mechanism underlying ICI-associated myocarditis. Using the peripheral blood of patients with ICI therapy and of ICI-treated mice with transplanted tumours, we dissect the immune cell subsets and inflammatory factors associated with myocarditis. Compared to the control group, patients with myocarditis after ICI therapy showed an increase in NK cells and myeloid cells in the peripheral blood, while T cells significantly decreased. Among T cells, there was an imbalance of CD4/CD8 ratio in the peripheral blood of myocarditis patients, with a significant decrease in central memory CD4+ T (CD4+ TCM) cells. RNA sequencing revealed that CD4+ TCM cells in myocarditis patients were immunosuppressive cell subsets, which highly express the immunosuppressive factor IL-4I1. To elucidate the potential mechanism of the decrease in CD4+ TCM cells, protein array was performed and revealed that several inflammatory factors gradually increased with the severity of myocarditis in the myocarditis group, such as IL-1B/CXCL13/CXCL9, while the myocardial protective factor IL-15 decreased. Correlation analysis indicated a positive correlation between IL-15 and CD4+ TCM cells, with high expression of IL-15 receptor IL15RA. Furthermore, in vivo studies using an anti-PDL1 antibody in a mouse tumour model indicated a reduction in CD4+ TCM cells and an increase in effector memory-expressing CD45RA CD8+ T (TEMRA) cells, alongside evidence of cardiac fibrosis. Conversely, combining anti-PDL1 antibody treatment with IL-15 led to a resurgence of CD4+ TCM cells, a reduction in CD8+ TEMRA cells, and a mitigated risk of cardiac fibrosis. Our data highlight CD4+ TCM cells' crucial role in cardiac protection during ICI therapy. IL-15, IL-4I1, and CD4+ TCM cells can serve as therapeutic targets to reduce ICI-associated myocarditis in cancer patients.

Peripheral endotoxin exposure in mice activates crosstalk between phagocytes in the brain and periphery.

Inflammation is a central process of many neurological diseases, and a growing number of studies suggest that non-brain-resident immune cells may contribute to this neuroinflammation. However, the unique contributions of specific immune cell subsets to neuroinflammation are presently unknown, and it is unclear how communication between brain-resident and non-resident immune cells underlies peripheral immune cell involvement in neuroinflammation. In this study, we employed the well-established model of lipopolysaccharide (LPS)-induced neuroinflammation and captured brain-resident and non-resident immune cells from the brain and its vasculature by magnetically enriching cell suspensions from the non-perfused brain for CD45 + cells. Then, we identified immune subtype-specific neuroinflammatory processes using single-cell genomics and predicted the crosstalk between immune cell subtypes by analyzing the simultaneous expression of ligands and receptors. We observed a greater abundance of peripheral phagocytes associated with the brain in this model of neuroinflammation, and report that these professional phagocytes activated similar transcriptional profiles to microglia during LPS-induced neuroinflammation. And, we observed that the probable crosstalk between microglia and peripheral phagocytes was activated in this model while homotypic microglial communication was likely to be decreased. Our novel findings reveal that microglia signaling to non-brain-resident peripheral phagocytes is preferentially triggered by peripheral inflammation, which is associated with brain infiltration of peripheral cells. Overall, our study supports the involvement of peripheral immune cells in neuroinflammation and suggests several possible molecular signaling pathways between microglia and peripheral cells that may facilitate central-peripheral crosstalk during inflammation. Examining these molecular mediators in human disease and other rodent models may reveal novel targets that modify brain health, especially in comorbidities characterized by peripheral inflammation.

Translational PK/PD and the first-in-human dose selection of a PD1/IL15: an engineered recombinant targeted cytokine for cancer immunotherapy.

Interleukin 15 (IL-15) is a potential anticancer agent and numerous engineered IL-15 agonists are currently under clinical investigation. Selective targeting of IL-15 to specific lymphocytes may enhance therapeutic effects while helping to minimize toxicities. We designed and built a heterodimeric targeted cytokine (TaCk) that consists of an anti-programmed cell death 1 receptor antibody (anti-PD-1) and an engineered IL-15. This "PD1/IL15" selectively delivers IL-15 signaling to lymphocytes expressing PD-1. We then investigated the pharmacokinetic (PK) and pharmacodynamic (PD) effects of PD1/IL15 TaCk on immune cell subsets in cynomolgus monkeys after single and repeat intravenous dose administrations. We used these results to determine the first-in-human (FIH) dose and dosing frequency for early clinical trials. The PD1/IL15 TaCk exhibited a nonlinear multiphasic PK profile, while the untargeted isotype control TaCk, containing an anti-respiratory syncytial virus antibody (RSV/IL15), showed linear and dose proportional PK. The PD1/IL15 TaCk also displayed a considerably prolonged PK (half-life range ∼1.0-4.1 days) compared to wild-type IL-15 (half-life ∼1.1h), which led to an enhanced cell expansion PD response. The PD was dose-dependent, durable, and selective for PD-1+ lymphocytes. Notably, the dose- and time-dependent PK was attributed to dynamic TMDD resulting from test article-induced lymphocyte expansion upon repeat administration. The recommended first-in-human (FIH) dose of PD1/IL15 TaCk is 0.003mg/kg, determined based on a minimum anticipated biological effect level (MABEL) approach utilizing a combination of in vitro and preclinical in vivo data. This work provides insight into the complex PK/PD relationship of PD1/IL15 TaCk in monkeys and informs the recommended starting dose and dosing frequency selection to support clinical evaluation of this novel targeted cytokine.

Biomarker analysis of zanzalintinib in clear cell renal cell carcinoma from STELLAR-001.

4545 Background: Zanzalintinib (zanza) is a novel oral multi-targeted TKI that inhibits several receptors, including VEGFR, MET, and TAM kinases. In an expansion cohort of previously treated patients (pts) with clear cell renal cell carcinoma (ccRCC) from the phase 1b STELLAR-001 study (NCT03845166), single-agent zanza showed encouraging activity (ORR, 38%; disease control rate, 88%), with responses seen in VEGFR TKI–pretreated pts, and a manageable safety profile (Pal, IKCS NA 2023:Abs 1). Here, we investigate biomarkers associated with zanza from the STELLAR-001 ccRCC expansion cohort. Methods: Adult pts (N=32) with advanced ccRCC, ECOG ≤1, and 1–3 prior systemic anticancer therapies received zanza 100 mg once daily until unacceptable toxicity or no longer deriving clinical benefit. Circulating plasma biomarkers and immune cell populations in blood were assessed at baseline and on-study. IHC for AXL, c-Met, phosphorylated Met, and VEGFR2 was performed on archival tissue. Associations between response to zanza and biomarker levels (at baseline and treatment-related changes) were investigated by comparing zanza responders (CR/PR; n=10) and non-responders (SD/PD; n=19). The relation between baseline biomarker levels and prior exposure to VEGFR TKI was also evaluated. P values were determined using a Wilcoxon test ( P&lt;0.05 considered significant). Results: Zanza elicited significant changes in soluble biomarkers related to angiogenesis, tumor growth and metastasis, and immune modulation, including an increase in the circulating ligands VEGF ( P&lt;0.001) and GAS6 ( P&lt;0.001); decreases in ANG-1 ( P&lt;0.001) and ANG-2 ( P&lt;0.001), and the soluble angiogenic receptors, VEGFR2 ( P&lt;0.001) and TIE-2 ( P&lt;0.001). A decrease in levels of the chemokine, RANTES ( P&lt;0.001), and an increase in IFNγ ( P=0.039) and the pro-apoptotic protein, granzyme B ( P&lt;0.001) were also observed. An analysis of immune cell subsets showed that zanza raised activated cytotoxic T cell numbers ( P=0.002). Reductions in monocytes ( P=0.001), along with immunosuppressive subsets (total MDSCs [ P=0.013] and granulocytic MDSCs [ P=0.014]) were also observed. Response to zanza was associated with lower baseline levels of plasma biomarkers including CRP ( P=0.025) and HGF ( P=0.017), but not with any tissue biomarkers or zanza-mediated changes in immune cells or plasma biomarkers tested. Lower baseline levels of VEGFR2 were observed in pts with prior VEGFR TKI exposure ( P=0.042). Conclusions: Zanza in pts with advanced ccRCC leads to modulation of plasma biomarkers associated with angiogenesis and peripheral immune cell subsets consistent with its expected mechanism of action. The reduction in immunosuppressive immune cells and activation of effector immune cells by zanza support the rationale for combining zanza with immune checkpoint inhibitors. Given the small sample sizes, further investigation in larger studies is warranted. Clinical trial information: NCT03845166 .

KRAS mutation subtypes responded differently to combination immunotherapy via disturbing myeloid-derived suppressor cells in non-small cell lung cancer.

8635 Background: The combination of immune checkpoint inhibitors (ICIs) and chemotherapy has shown high efficacy in non-small cell lung cancer (NSCLC) patients, but further exploration of the predictive biomarkers is needed. KRAS is the most common driver oncogene in patients with NSCLC. However, the influence of different KRAS mutation subtypes on immunotherapy response remains controversial. We aimed to evaluate the value of KRAS mutation subtypes for predicting response to immunotherapy in NSCLC patients and to further analyze the related tumor immune microenvironment (TiME) phenotypes. Methods: In this retrospective study, clinical data were extracted from the medical records of patients with advanced NSCLC treated with ICIs and whose available molecular analysis included KRAS mutation information between January 2017 and December 2022. NSCLC tissue samples were collected for TiME analysis. Differences in immune cell subsets between different groups were analyzed using the Mann‒Whitney U test. Clinical characteristics, ICI treatment efficacy, progression-free survival (PFS), and overall survival (OS) were analyzed using multivariate Cox models. Results: A total of 1913 eligible KRAS-mutated NSCLC patients were screened, and 137 patients who received systemic immunotherapy were enrolled (108 males vs 29 females), with a median age of 62 (range, 39-84) years. Among them, 126 (92.0%) patients had adenocarcinoma, 99 (72.3%) patients were systemic therapy naïve, and 124 (90.5%) patients received combined immunotherapy. Among the patients with KRAS mutations, 48 (35.0%) had G12C, 20 (14.6%) had G12V, 27 (19.7%) had G12D and 42 (30.7%) had other subtypes. PFS was significantly longer in the G12C subgroup than in the other mutation subgroup (median PFS: 11.3 vs 6.8 months, P = 0.001). The median PFS times in the G12C/G12V subgroup and the other groups were 11.1 months and 6.5 months, respectively ( P= 0.0005). Multivariate analysis of the immunotherapy cohort revealed that G12C/G12V was an independent predictor of PFS ( P = 0.003, hazard ratio (HR): 2.021, 95% CI: 1.265-3.227). Further TiME analysis suggested that the infiltration of myeloid-derived suppressor cells (MDSCs, CD33+CD11b+) in the tumor and stromal areas in the G12C/G12V subgroup was significantly lower than that in the other subgroup (tumor area, P= 0.045; stroma area, P= 0.015). However, no significant difference in PD-L1 expression was found between the G12C/G12V group and the other groups. Conclusions: KRAS G12C and G12V were associated with favorable combined ICI treatment efficacy in patients with NSCLC. Patients with G12C or G12V mutations had longer PFS and lower infiltration of MDSCs. KRAS mutation subtypes are potential predictive biomarkers disturbed by MDSCs for combination therapy in NSCLC patients.

Roginolisib, an oral, highly selective and allosteric modulator of phosphoinositide 3-kinase inhibitor delta (PI3Kδ) in patients with uveal melanoma and advanced cancers.

9597 Background: The highly selective, oral allosteric modulator of PI3Kδ, roginolisib (IOA-244), blocks the activity of PI3Kδ-dependent signaling, in both tumor cells and Tregs. Methods: Continuous daily dosing of roginolisib at 10, 20, 40 and 80 mg in patients (pts) with pretreated solid tumours and Follicular Lymphoma (FL) were evaluated (Part A). Part B consists of a dose confirmation cohort in uveal melanoma (UM) pts of 2 parts, B.1 and B.2. Primary objective: safety of the anticipated biologically effective dose (BED). Secondary objectives: PK; PD (e.g., inhibition of CD63 expression on basophils, changes in immune cell subsets in peripheral blood); RECIST 1.1 or Lugano-based responses; PFS and OS. Exploratory studies: phenotypic changes in circulating immune cells by mass cytometry (i.e., CyTOF); response assessments by radiomics; analysis of circulating protein signatures via Olink proteomics. Results: Part A Solid Tumour (completed): Sixteen pts were treated in 4 cohorts of 4 pts each. Pts characteristics: uveal melanoma (9/16; 56%), cutaneous melanoma (5/16; 31%) and pleural mesothelioma (2/16; 13%). Only Grade 1 and 2 AEs by CTCAE v.5 were observed, including 2 cases of transient (lasting &lt; 24 hrs) diarrhoea and 2 of AST/ALT elevation. Part A FL Cohort (completed): 8 pts were treated at 20 mg (n=4) and 80 mg (n=4). Grade 3 toxicities were transient and observed in 2/8 (25%) pts. 2/4 pts (50%) at the 80 mg had PR as per Lugano criteria. Treatment-emergent adverse events (TEAEs) did not result in study drug discontinuation, immune-related toxicity, or dose-limiting toxicity in either the solid tumor or hematological patients. UM pts (Part A and Part B)&gt;: 29 pts (Part A: 9 pts; Part B.1: 7 pts, Part B.2: 13 pts), of which 23 were treated at 80 mg QD (RP2D). Mean time on treatment: 10.7 mo (range: 1.5-39.6 mo). ORR (RECIST 1.1): PR: 1/29 (3%); SD: 21/29 (72%). Median OS for Part A: 20.8 mo (5/9 with 4 pts alive); Part B.1 20.1 (4/7 with 3 pts alive); Part B.2 not determined; 1-year OS rate: Part A 66.7%; Part B.1 71%; Part B.2 immature. Exploratory Investigations: At 3.7 mo (Cycle 5), there were 2 groups: one with SD (13/29; 45%) and the other with PD (16/29; 55%). At 6 mo, this split continued with 38% (11/29) showing SD and 62% having PD (18/29). Volumetric changes matched durable SDs while having higher sensitivity in the 15 analyzed patients. Interestingly, tumor burden reduction in mediastinal lymph nodes was indicative of SD and 70% of pts had volumetric spleen size reduction. Volumetric and radiomic changes were matched with peripheral changes in plasma proteins and immune cell changes. Conclusions: Roginolisib is well tolerated at 80 mg, demonstrates efficacy, and induces phenotypic changes in circulating immune cells. At the RP2D, the most robust anti-tumour activity was observed in FL (50% PR) and UM pts (3% PR; 72% SD). Clinical trial information: NCT04328844 .

Effect of novel autologous immune training platform on end-stage patients with cancer.

2546 Background: SUPLEXA therapeutic cells are the initial autologous and non-engineered candidate to emerge from the novel ENLIST cells training platform. Manufacturing is robust, reproducible and with an acceptable cost of goods. The method requires 35 days to produce multiple doses. Multiple mechanisms of SUPLEXA therapeutic cells have been delineated and all appear to complement that of immune checkpoint inhibitors (ICIs) with SUPLEXA cells serving to enhance the number of primed anti-tumor host T cells while ICIs serve to increase the durability of primed T cells by blocking their premature down-regulation. Methods: A 35 patient Phase 1 single-agent study survey study is currently ongoing in Australia to test the safety and clinical activity of SUPLEXA cells in end-stage patients with various tumor types. No chemo preconditioning or concomitant cytokine treatment was employed. CYTOF analysis of longitudinal peripheral blood is used to monitor changes to the host immune system. Results: To date, SUPLEXA therapeutic cells have demonstrated safety and significant clinical benefit with an observed CR, PR, and many durable SD in approximately half the patients. Several solid tumor patients remain on study beyond 1 year and doing well. Longitudinal analysis of PBMCs from SUPLEXA cell treated cancer patients demonstrated durable changes in the composition of myeloid immune cell subsets in SUPLEXA treated patients, impacting the anti-tumor bias of the host immune system. Specifically, we identified a dramatic decrease in the number of peripheral myeloid derived suppressor cells (MDSCs), within 3 weeks of SUPLEXA treatment. In patients with CR and PR, we also identified stable increases in activated classical CD14+ blood monocytes. Conclusions: SUPLEXA therapeutic cells are a highly differentiated approach to cellular therapy. The first-in-human experience demonstrated a pristine safety profile and strong clinical benefit as a single-agent. The pharmacodynamic decrease of in the number of MDSCs, known to suppress the anti-tumor immune response and limit immune checkpoint inhibitors (ICIs) efficacy, supports the further clinical testing to test the hypothesis that the multiple mechanisms of SUPLEXA cells will enhance the clinical activity of ICIs. Clinical trial information: NCT05237206 .

Predicting immunotherapy toxicity through single-cell sequencing of peripheral immune cells: A pilot study.

e14538 Background: Immune checkpoint inhibitors are widely used in oncology. However, immune-related adverse events (iRAEs) are idiosyncratic and can be severe. The overall incidence is 27%, with severe iRAEs occurring in 6% of patients. We hypothesised that distinct immune cell phenotypes (gene expression) correlate with immune-related toxicity from checkpoint inhibitor immunotherapy. Single-cell RNA sequencing (scRNA-seq) is a powerful tool that can assay the gene expression of thousands of individual cells. RPS26 expression mediates CD4/CD8 T cell activation through a known trans-expressive quantitative trait locus at rs1131017 and is linked with autoimmunity. FOS and JUN encode for c-Fos and c-Jun, which dimerise to create the activator protein-1 complex, a well-established transcriptional factor of the immune system. c-Fos is an immunoregulator known to suppress pro-inflammatory cytokines, suggesting that c-FOS deficiency plays a crucial role in developing autoimmunity. We aimed to analyse the transcriptomic differences of peripheral immune cells between patients who experienced severe checkpoint inhibitor therapy iRAE and those who did not. Methods: We identified a pilot cohort of eight patients who developed grade three or above toxicity due to immune checkpoint inhibitors and eight controls who were matched for age, cancer and treatment type. Peripheral blood mononuclear cells were collected prospectively at baseline and two subsequent treatment cycles. Cells were captured on the 10x Chromium platform, and libraries were sequenced to 30,000 reads per cell. Samples were demultiplexed, and the data was analysed with the Seurat package on R statistical software (version 4.3.0). Results: Five patients had baseline samples only, two patients had two samples from two cycles, and one patient had samples from three cycles. After quality control filtering, we identified 36,288 cells from 16 individuals and 24 samples. Immune cells were annotated with the Azimuth package into 29 subtypes. We identified differences in cell proportions of immune cell subsets in the toxicity vs control group, including B naive cells (5.57% vs 3.71% p=0.01) and reduced cytotoxic CD4 T lymphocyte cells (0.66% vs 2.26%, p=0.005). Differential gene expression analysis identified a significantly high expression of ribosomal genes, particularly RPS26, in multiple cell types at baseline (p&lt;0.01) and reduced expression of FOS and JUN-related genes in CD14 and CD16 monocytes, CD4 and CD8 T cells in the toxicity cohort (p&lt;0.01). Conclusions: Overexpression of ribosomal genes and reduced expression of FOS/ JUN genes amongst PBMCs at baseline are potentially predictive biomarkers of severe checkpoint inhibitor immunotherapy toxicity. Validation of these findings in a larger cohort is in progress.