Analysis Of Cell Subsets Research Articles

An abnormal increase in CD26(-)CD28(-) cytotoxic effector CD4 and CD8 T cell populations in patients with systemic lupus erythematosus.

CD26 is a human T cell costimulatory molecule as well as a T cell subset marker, and increase of CD26+ T cells in inflamed tissues and peripheral blood has been reported in diverse autoimmune diseases. In contrast, our group has previously shown that levels of circulating CD26+ T cells are decreased in patients with systemic lupus erythematosus (SLE), although the role of reduced CD26 T cell surface expression in SLE pathology remains to be elucidated. In the present study, we conducted CD26-based T cell subset analyses utilizing peripheral blood mononuclear cells from 57 SLE patients and 31 healthy adult volunteers. We show that the increase in CD26(-) T cell population reflects the abnormal expansion of CD26(-)CD28(-) cytotoxic subsets of both CD8 T cells and CD4 T cells in SLE patients. Single cell RNA sequencing analysis of the CD26(-)CD28(-) CD4 and CD8 T cell populations reveals unique characteristics with similarities to natural killer T cells. In addition, the level of CD26(-)CD28(-) T cells is increased in some active stage SLE patients with renal manifestation. Meanwhile, effect of prednisolone treatment on these populations varies from patient to patient, with levels of these cytotoxic effector populations still being elevated in some inactive stage SLE patients. Taken together, our data suggest that analysis of these populations in SLE may be a useful tool to classify this markedly heterogeneous condition.

Evaluation of the clinical significance of lymphocyte subsets and myeloid suppressor cells in patients with renal carcinoma

PurposeThe purpose of this study was to analyze the expression patterns of immune cells in renal cancer patients, including myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), CD3 + /CD4 + T cells, CD3 + / CD8 + T cells, and CD3- CD16 + CD56 + cells. In addition, this study will explore the correlation between these immune markers and the progression of renal cell carcinoma and evaluate their potential application in predicting the therapeutic effect of renal cell carcinoma.MethodsIn this study, 80 renal cancer patients who received treatment in our hospital from October 2022 to December 2023 were selected as the research object and 50 healthy people who underwent a physical examination at the same time were selected as the control group. All participants had a 3 ml venous blood sample taken in the morning on an empty stomach. All patients with renal cell carcinoma have been confirmed by histopathological diagnosis. Clinicopathological data including age, gender, BMI, clinical stage, tumor size and pathological type were collected.MDSC, Treg, CD3 + /CD4 + T cells, CD3 + /CD8 + T cells, the ratio of CD3 + /CD4 + T cells/CD3 + /CD8 + T cell and the expression level of CD3-CD16 + CD56 + cells were detected by flow cytometry.ResultsThrough the detection of flow cytometry, we observed that there was no significant difference in gender, age, BMI and other baseline characteristics between renal cancer patients and healthy controls, and the P value was greater than 0.05. However, in the analysis of peripheral blood immune cell subsets, including CD3 + /CD4 + , CD3 + /CD8 + , CD3 + /CD4 + /CD3 + /CD8 + ratio, NK cells, regulatory T cells (T-reg), polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and mononuclear myeloid-derived suppressor cells (M-MDSC) were significantly different between renal cell carcinoma group and normal control group (P < 0.05). Specifically, the expression levels of CD3 + /CD4 + and CD3 + /CD8 + cells in renal cancer patients were lower than those in normal subjects, while the expression levels of T-reg, PMN-MDSC and M-MDSC were relatively high. (2) In the flow cytometry analysis, the expression level of immune cell subsets in the peripheral blood of renal cancer patients was detected.The results showed that there was no significant correlation between the expression of CD3 + /CD4 + , CD3 + /CD8 + , CD3 + /CD4 + /CD3 + /CD8 + ratio, NK cells, T-reg cells, PMN-MDSC and M-MDSC and the sex, age, BMI and pathological type of the patients. These differences were not statistically significant (P > 0.05).At the same time, CD3 + /CD8 + T cells, the ratio of CD3 + /CD4 + /CD3 + /CD8 + and the expression level of NK cells were not significantly correlated with tumor size and clinical stage (P > 0.05). However, the expression levels of CD3 + /CD4 + cells, M-MDSC, PMN-MDSC, and T-reg cells were statistically significantly different with tumor size and clinical stage (P < 0.05).There was a significant difference between these indexes and lymph node metastasis (P < 0.05). (3) The results of Logistic regression analysis showed that the low expression of CD3 + /CD4 + lymphocytes and the high expression of T-reg, PMN-MDSC and M-MDSC in peripheral blood may be related to the clinical stage of renal cell carcinoma.Conclusion(1) Compared with healthy individuals, patients with renal cell carcinoma showed a significant decrease in CD3 + /CD4 + T cells, CD3 + /CD8 + T cells and CD3-CD16 + CD56 + cells, while the CD4 + /CD8 + ratio increased. In addition, the number of PMN-MDSC, M-MDSC and T-reg cells was significantly increased compared with the normal population, indicating that the immune system function of patients was impaired. (2) The expression levels of CD3 + /CD4 + , PMN-MDSC, M-MDSC and T-reg were different in tumor size and clinical stage. Specifically, the expression levels of PMN-MDSC, M-MDSC, and T-reg increased correspondingly with the increase in tumor diameter and the progression of the clinical stage.

DEPICT-seq: Single-Cell Transcriptomic Analysis of Rare Cell Subsets Isolated via Nucleic Acid Cytometry.

The ability to dive deep into specific rare cell populations is critical for understanding tissue physiology and pathology across various biological domains. As single-cell RNA-seq flourishes, many newly discovered cell subtypes are defined by their transcriptomic markers. However, our ability to retrieve and analyze cells based on their nucleic acid markers remains underdeveloped. Here, we present Double Emulsion PCR-Initiated Cell sorting and Transcriptomic Sequencing (DEPICT-seq), a high-throughput droplet nucleic acid cytometry method that integrates batch cell fixation for cellular information preservation, double emulsion digital PCR-based cell sorting to target nucleic acid markers of interest, and in-depth full-length transcriptomic analyses at single-cell resolution. We utilize DEPICT-seq to isolate and characterize T cell receptor (TCR)-engineered T cells within a mixed population and also demonstrate a variation of the workflow by incorporating an RNase H-dependent PCR step to enrich full-length TCR sequences for paired single-cell TCR sequencing and transcriptomic profiling.

Prognostic and immunological significance of tertiary lymphoid structures in nasopharyngeal carcinoma.

220 Background: Tertiary lymphoid structures (TLSs) are ectopic lymphoid tissues found in non-lymphoid organs, characterized by the presence of CD20+ B cell follicles adjacent to a T cell zone. Their presence has been associated with improved survival in various tumor types. However, the role of TLSs in nasopharyngeal carcinoma (NPC) remains unclear. In this study, we investigated the prognostic and immunological significance of TLSs in NPC patients. Methods: The investigation involved a discovery cohort (n = 145) comprising NPC patients diagnosed at Sun Yat-sen University Cancer Center between July 2010 and December 2016, with pretreatment primary tumor samples collected. An independent validation cohort included 150 NPC patients with gene expression profiles, among whom Hematoxylin &amp; Eosin (H&amp;E)-stained slides were available for 95 patients. The primary endpoint of this study is overall survival, defined as the duration of time from diagnosis until death from any cause. The presence of TLSs was assessed on H&amp;E and CD20-stained slides. Analyses of tumor-infiltrating immune cell subsets, differentially expressed genes, and BCR/TCR clonotypes were performed to explore the association between TLSs and the anti-tumor immune response. A molecular definition of TLSs was proposed and assessed for its prognostic value across multiple cohorts. Results: The presence of TLSs was independently associated with a favorable overall survival (hazard ratio [HR], 0.26; 95% confidence interval [CI], 0.07-0.94; log-rank p = 0.04), surpassing the significance of B cell density in multivariate analysis (HR, 0.83; 95% CI, 0.38-1.79, log-rank p = 0.63). These findings were corroborated in the independent validation cohort. TLSs positively correlated with multiple immune-related pathways and a higher proportion of functionally mature immune cells. Notably, the counts of BCR and TCR clonotypes showed a significant positive correlation with the TLSs score. We proposed a TLSs gene signature and demonstrated its prognostic relevance across multiple cohorts, including patients treated with immune checkpoint blockade. Conclusions: TLSs exhibit heightened immune activity and indicate improved survival outcomes in NPC patients. A TLSs gene signature was proposed and demonstrated its prognostic relevance across multiple cohorts, indicating potential guidance for treatment decisions in cancer.

Mass cytometric single cell immune profiles of peripheral blood from acute myeloid leukemia patients in complete remission with measurable residual disease.

Measurable residual disease (MRD) is detected in approximately a quarter of AML chemotherapy responders, serving as a predictor for relapse and shorter survival. Immunological control of residual disease is suggested to prevent relapse, but the mechanisms involved are not fully understood. We present a peripheral blood single cell immune profiling by mass cytometry using a 42-antibody panel with particular emphasis on markers of cellular immune response. Six healthy donors were compared with four AML patients with MRD (MRD+) in first complete remission (CR1MRD+). Three of four patients demonstrated a favorable genetic risk profile, while the fourth patient had an unfavorable risk profile (complex karyotype, TP53-mutation) and a high level of MRD. Unsupervised clustering using self-organizing maps and dimensional reduction analysis was performed for visualization and analysis of immune cell subsets. CD57+ natural killer (NK)-cell subsets were found to be less abundant in patients than in healthy donors. Both T and NK cells demonstrated elevated expression of activity and maturation markers (CD44, granzyme B, and phosho-STAT5 Y694) in patients. Although mass cytometry remains an expensive method with limited scalability, our data suggest the utility for employing a 42-plex profiling for cellular immune surveillance in whole blood, and possibly as a biomarker platform in future clinical trials. The findings encourage further investigations of single cell immune profiling in CR1MRD+ AML-patients.

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 .

Analysis of Immune Cell Subsets in Peripheral Blood from Patients with Engineered Stone Silica-Induced Lung Inflammation.

Silicosis caused by engineered stone (ES-silicosis) is an emerging worldwide issue characterized by inflammation and fibrosis in the lungs. To our knowledge, only a few reports have investigated leukocyte/lymphocyte subsets in ES-silicosis patients. The present study was designed to explore the proportions of the main lymphocyte subsets in ES-silicosis patients stratified into two groups, one with simple silicosis (SS) and the other with a more advanced state of the disease, defined as progressive massive fibrosis (PMF). The proportions of B (memory and plasmablasts) cells, T (helper, cytotoxic, regulatory) cells, and natural killer (NK) (regulatory and cytotoxic) cells were investigated by multiparameter flow cytometry in 91 ES-silicosis patients (53 SS patients and 38 PMF patients) and 22 healthy controls (HC). Although the total number of leukocytes did not differ between the groups studied, lymphopenia was observed in patients compared to healthy controls. Compared with those in healthy controls, the proportions of memory B cells, naïve helper T cells, and the CD4+/CD8+ T cells' ratio in the peripheral blood of patients with silicosis were significantly decreased, while the percentages of plasma cells, memory helper T cells, and regulatory T cells were significantly increased. For the NK cell subsets, no significant differences were found between the groups studied. These results revealed altered cellular immune processes in the peripheral blood of patients with ES-silicosis and provided further insight into silicosis pathogenesis.

Bortezomib restrains M2 polarization and reduces CXCL16-associated CXCR6+CD4 T cell chemotaxis in bleomycin-induced pulmonary fibrosis

BackgroundThe development of pulmonary fibrosis involves a cascade of events, in which inflammation mediated by immune cells plays a pivotal role. Chemotherapeutic drugs have been shown to have dual effects on fibrosis, with bleomycin exacerbating pulmonary fibrosis and bortezomib alleviating tissue fibrotic processes. Understanding the intricate interplay between chemotherapeutic drugs, immune responses, and pulmonary fibrosis is likely to serve as the foundation for crafting tailored therapeutic strategies.MethodsA model of bleomycin-induced pulmonary fibrosis was established, followed by treatment with bortezomib. Tissue samples were collected for analysis of immune cell subsets and functional assessment by flow cytometry and in vitro cell experiments. Additionally, multi-omics analysis was conducted to further elucidate the expression of chemokines and chemokine receptors, as well as the characteristics of cell populations.ResultsHere, we observed that the expression of CXCL16 and CXCR6 was elevated in the lung tissue of a pulmonary fibrosis model. In the context of pulmonary fibrosis or TGF-β1 stimulation in vitro, macrophages exhibited an M2-polarized phenotype and secreted more CXCL16 than those of the control group. Moreover, flow cytometry revealed increased expression levels of CD69 and CXCR6 in pulmonary CD4 T cells during fibrosis progression. The administration of bortezomib alleviated bleomycin-induced pulmonary fibrosis, accompanied by reduced ratio of M2-polarized macrophages and decreased accumulation of CD4 T cells expressing CXCR6.ConclusionsOur findings provide insights into the key immune players involved in bleomycin-induced pulmonary fibrosis and offer preclinical evidence supporting the repurposing strategy and combination approaches to reduce lung fibrosis.

Exploratory mass cytometry analysis reveals immunophenotypes of cancer treatment-related pneumonitis.

Anticancer treatments can result in various adverse effects, including infections due to immune suppression/dysregulation and drug-induced toxicity in the lung. One of the major opportunistic infections is Pneumocystis jirovecii pneumonia (PCP), which can cause severe respiratory complications and high mortality rates. Cytotoxic drugs and immune-checkpoint inhibitors (ICIs) can induce interstitial lung diseases (ILDs). Nonetheless, the differentiation of these diseases can be difficult, and the pathogenic mechanisms of such diseases are not yet fully understood. To better comprehend the immunophenotypes, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid from patients with PCP, cytotoxic drug-induced ILD (DI-ILD), and ICI-associated ILD (ICI-ILD) using two panels containing 64 markers. In PCP, we observed an expansion of the CD16+ T cell population, with the highest CD16+ T proportion in a fatal case. In ICI-ILD, we found an increase in CD57+ CD8+ T cells expressing immune checkpoints (TIGIT+ LAG3+ TIM-3+ PD-1+), FCRL5+ B cells, and CCR2+ CCR5+ CD14+ monocytes. These findings uncover the diverse immunophenotypes and possible pathomechanisms of cancer treatment-related pneumonitis.

Exploratory mass cytometry analysis reveals immunophenotypes of cancer treatment-related pneumonitis

Anticancer treatments can result in various adverse effects, including infections due to immune suppression/dysregulation and drug-induced toxicity in the lung. One of the major opportunistic infections is Pneumocystis jirovecii pneumonia (PCP), which can cause severe respiratory complications and high mortality rates. Cytotoxic drugs and immune-checkpoint inhibitors (ICIs) can induce interstitial lung diseases (ILDs). Nonetheless, the differentiation of these diseases can be difficult, and the pathogenic mechanisms of such diseases are not yet fully understood. To better comprehend the immunophenotypes, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid from patients with PCP, cytotoxic drug-induced ILD (DI-ILD), and ICI-associated ILD (ICI-ILD) using two panels containing 64 markers. In PCP, we observed an expansion of the CD16+ T cell population, with the highest CD16+ T proportion in a fatal case. In ICI-ILD, we found an increase in CD57+ CD8+ T cells expressing immune checkpoints (TIGIT+ LAG3+ TIM-3+ PD-1+), FCRL5+ B cells, and CCR2+ CCR5+ CD14+ monocytes. These findings uncover the diverse immunophenotypes and possible pathomechanisms of cancer treatment-related pneumonitis.

Abstract LB346: Dynamic changes in the tumor microenvironment from initial diagnosis through sequential therapies in patients with large B cell lymphoma undergoing axicabtagene ciloleucel (axi-cel) treatment

Abstract Axicabtagene ciloleucel (axi-cel), marketed as YESCARTA®, is an approved autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory large B cell lymphoma patients following one prior therapeutic intervention. The pivotal ZUMA-1 trial (NCT02348216), a Phase 1/2 multicenter study, reported an impressive 83% objective response rate and a robust 58% complete response rate at 5 years (Locke et al., Lancet Oncol. 2019). Subsequently, ZUMA-7 (NCT03391466), a Phase 3 multicenter trial focusing on patients with one prior systemic therapy, demonstrated comparable outcomes with an 83% objective response rate and a remarkable 65% complete response rate at 2 years (Locke et al., Lancet Oncol. 2022). Previous studies have highlighted the crucial connection between the tumor microenvironment (TME) and CAR T-cell therapy outcomes. Notably, the pre-treatment tumor immune contexture has been proposed as a major determinant of clinical outcomes in ZUMA-1 and ZUMA-7 patients (Scholler et al., Nature Medicine 2022; Locke et al., Nature Medicine, in press; ASH2023 oral presentation #226). This study aims to provide novel insights into the dynamic changes in the TME from initial diagnosis through sequential therapeutic interventions in large B-cell lymphoma patients treated with axi-cel. Utilizing multiplex immunohistochemistry (Brightplex®), we analyzed 165 commercial tumor biopsies (at diagnosis), 100 ZUMA-7 samples (40 at diagnosis, 60 after 1 prior therapeutic line), and 35 ZUMA-1 samples (20 pre- and 15 post-CAR T infusion). Three Brightplex® assays were developed to assess T-cell (CD3 CD8 FOXP3 TIM3 PD1 LAG3 GZMB) and macrophages infiltration (CD68 CD64 CD163 CD204 CD206 PDL1), along with single staining for the NK cell marker, NKP46. Our findings revealed significant changes and evolution from diagnosis through post-axi-cel infusion: a significant decrease in lymphocyte levels (cytotoxic, helper, and regulatory, with p-values of 0.042, 1.3e-05, 0.024, respectively), a notable decrease in M1 macrophages proportion (p-value=5.8e-06), and a substantial increase in M2 macrophages proportion (p-value=4.5e-10) across different lines of standard care therapy. Importantly, high baseline proportions of protumor M2 macrophages may serve as predictors of disease relapse post-axi-cel treatment, while low baseline M2 proportions could be associated with a complete response. This comprehensive characterization of the immune contexture provides crucial insights to better highlight TME modification depending on the line of therapy, and further correlative analysis of cell subsets will be presented, contributing to a deeper understanding of treatment impact on the TME in large B cell lymphoma. Citation Format: Michael Mattie, Regis Perbost, Aurelie Auguste, Sarah Turcan, Jenny J. Kim, Christina To, Jenny Nater, Simone Filosto, Rhine R. Shen, Jérôme Galon. Dynamic changes in the tumor microenvironment from initial diagnosis through sequential therapies in patients with large B cell lymphoma undergoing axicabtagene ciloleucel (axi-cel) treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB346.

Long-Term Clinical Outcomes and B Cell Immune Reconstitution Following Allo-HCT With Prophylactic, Post-Transplant Rituximab

BackgroundChronic graft-versus-host disease (cGVHD) remains a significant source of morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Post-transplant, prophylactic rituximab has successfully decreased cGHVD rates in clinical trials, but the durability of this strategy is uncertain. The long-terms effect of post-HCT B cell depletion on immune reconstitution, B cell function, and infectious complications are also unknown. ObjectiveIn this study, we provide 10 year follow-up and correlative analyses on patients given post-HCT, prophylactic rituximab. The objective of the study is to examine the durability of cGVHD protection as well as the long-term effect of rituximab prophylaxis on protective immune reconstitution, B cell function, and alloantibody formation. Study DesignWe analyzed 35 patients given prophylactic rituximab on phase II clinical trial. Clinical outcomes included cGVHD development, relapse and survival outcomes, and infectious outcomes. Correlative analyses included B cell subset analysis, development of antibodies to infectious antigens, and, for male patients receiving female donor grafts, development of antibodies to HY antigens. To further investigate the effect of rituximab on immune reconstitution and function, we also analyzed 43 similarly transplanted patients who did not receive post- or peri-HCT rituximab as a comparator group. ResultsFor patients who received rituximab, the 8-year cumulative incidence of cGHVD and freedom from immunosuppression were 20.0% and 76.2%, respectively. Importantly, no late incidences of cGVHD developed beyond 14 months post-HCT. Relative to patients who did not receive rituximab, post-HCT rituximab was associated with increased B cell aplasia at 1 year post-HCT (42.9% vs 11% of patients, p = 0.037); by 3 years post-HCT, this aplasia resolved. Patients who received rituximab also had a significantly lower proportion of IgD+/CD38+ transitional B cells at 3 years post-HCT (78.8% vs 89.9%, p = 0.039); at 10 years post-HCT, this percentage remained markedly decreased at 50.7%. Rituximab prophylaxis altered B cell function. In male patients receiving female donor grafts, fewer patients developed HY antibodies at 3 years post-HCT (20% vs 78%, p = 0.04). At 10 years post-HCT, HY antibody production remained decreased at 33%. Rituximab prophylaxis was also associated with significantly lower antibody response to tetanus and EBV infectious antigens as well as lower IgG levels. Despite these changes, post-HCT was not associated with increased infections, although patients who received rituximab required intravenous immunoglobulin (IVIG) supplementation more frequently than those who did not (62.9% vs 32.6% of patients, p = 0.01). ConclusionsPrior data on the efficacy and feasibility of rituximab prophylaxis are durable, with persistent reduction in cGVHD. Rituximab prophylaxis also results in lasting B cell immunologic changes, with altered B cell subset composition and decreased alloantibody formation. Associated infectious risks were not increased, perhaps mitigated by high IVIG use.

Abstract P25: Identification of biological components and novel clinical subsets of NPM1-mutated AML using an integrated, multi-omics approach

Abstract P25: Identification of biological components and novel clinical subsets of <i>NPM1</i>-mutated AML using an integrated, multi-omics approach

Immunophenotyping of canine T cell activation and proliferation by combined protein and RNA flow cytometry

The limited availability of canine-reactive monoclonal antibodies restricts the analyses of immune cell subsets and their functions by flow cytometry. The PrimeFlow™ RNA Assay may serve as a potential solution to close this gap. Here we report a blood immunophenotyping method utilizing combined protein- and RNA-based flow cytometry to characterize canine T cell activation and proliferation within individual cells. In this assay, CD69 expression was detected by an RNA probe and CD25 and Ki67 were detected by antibodies. Canine peripheral blood mononuclear cells (PBMCs) were stimulated with three agents with different modes of action, anti-CD3/CD28 antibodies, phytohemagglutinin, or phorbol myristate acetate /ionomycin. Robust T cell activation (CD25+ and/or CD69+) and proliferation (Ki67+) were detected. Both CD69 and CD25 appear to be robust and sensitive T cell activation markers with early induction and low background expression. Upon stimulation, T cell proliferation occurred later than T cell activation and was associated with CD25 expression. This canine T cell activation and proliferation immunophenotyping method was evaluated in 5 independent experiments using PBMCs from 10 different beagle dogs with satisfactory assay performance. This method can greatly facilitate the evaluation of immune disease pathogenesis and immunotoxicity risk assessment in nonclinical drug development in canine.

DNA framework signal amplification platform-based high-throughput systemic immune monitoring

Systemic immune monitoring is a crucial clinical tool for disease early diagnosis, prognosis and treatment planning by quantitative analysis of immune cells. However, conventional immune monitoring using flow cytometry faces huge challenges in large-scale sample testing, especially in mass health screenings, because of time-consuming, technical-sensitive and high-cost features. However, the lack of high-performance detection platforms hinders the development of high-throughput immune monitoring technology. To address this bottleneck, we constructed a generally applicable DNA framework signal amplification platform (DSAP) based on post-systematic evolution of ligands by exponential enrichment and DNA tetrahedral framework-structured probe design to achieve high-sensitive detection for diverse immune cells, including CD4+, CD8+ T-lymphocytes, and monocytes (down to 1/100 μl). Based on this advanced detection platform, we present a novel high-throughput immune-cell phenotyping system, DSAP, achieving 30-min one-step immune-cell phenotyping without cell washing and subset analysis and showing comparable accuracy with flow cytometry while significantly reducing detection time and cost. As a proof-of-concept, DSAP demonstrates excellent diagnostic accuracy in immunodeficiency staging for 107 HIV patients (AUC > 0.97) within 30 min, which can be applied in HIV infection monitoring and screening. Therefore, we initially introduced promising DSAP to achieve high-throughput immune monitoring and open robust routes for point-of-care device development.

Anti-Inflammatory Effects of a Novel Nuclear Factor-κB Inhibitory Derivative Derived from Pyrazolo[3,4-d]Pyrimidine in Three Inflammation Models.

Nuclear factor-κB (NF-κB) plays a central role in inflammatory responses, and its physiologic functions are essential for cell survival and proliferation. Currently, drugs targeting NF-κB inhibition have not yet been applied in clinical practice. We investigated the physiologic effect of a novel NF-κB inhibitory compound, 1H-pyrazolo[3,4-d]pyrimidin-4-amine derivative (INH #1), on three inflammatory animal models. The pharmacokinetics were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Acute hepatitis was induced by administrating lipopolysaccharide (LPS) and D-(+)-galactosamine hydrochloride followed by the analysis of survival time and inflammatory mediators. Collagen-induced arthritis (CIA) was induced by immunization with type II collagen (CII), and serum-transfer arthritis (STA) was caused by injecting K/BxN mice serum. Clinical and histologic scores were evaluated in both arthritis models. Immune cell subset analysis, CII-induced interferon-gamma (IFN-γ) production and proliferation, and measurement of anti-CII IgG antibodies were performed in the CIA model. In the acute hepatitis model, INH #1 suppressed tumor necrosis factor-α (TNF-α) production and prevented early death in a dose-dependent manner. INH #1 significantly attenuated arthritis scores and joint inflammation in both arthritis models. Additionally, in the CIA model, dendritic cells (DCs) in the regional lymph nodes were decreased in the treated mice and antigen-induced IFN-γ production and cell proliferation in splenocytes were inhibited, whereas the titers of anti-CII IgG antibodies were comparable regardless of the treatment. Here we revealed that INH #1 exerted anti-inflammatory effects in vivo via inhibition of inflammatory mediators and suppression of cellular immune responses. This compound could be a novel candidate for inhibition of NF-κB in certain inflammatory diseases. SIGNIFICANCE STATEMENT: A novel nuclear factor-κB (NF-κB) inhibitory compound, 1H-pyrazolo[3,4-d]pyrimidin-4-amine derivative (INH #1), which retains physiologically essential NF-κB bioactivity, suppressed inflammation in three different mouse models: the acute hepatitis model, the collagen-induced arthritis model, and the K/BxN serum-transfer arthritis model. These results suggest that this compound could be a novel and potent anti-inflammatory agent.

Quantitative analysis of Tr1 lymphocytes in patients with type 2 diabetes mellitus

BackgroundType 2 diabetes mellitus (T2DM) is usually accompanied by a low-grade inflammatory phenomenon, which participates in the pathogenesis of different complications of this condition. The inflammatory response is under the regulation of different mechanisms, including T regulatory (Treg) lymphocytes. However, the possible role of type 1 T regulatory (Tr1) cells in T2DM has not been explored so far.AimTo carry out a quantitative analysis of Tr1 lymphocytes and other immune cell subsets in patients with T2DM and correlate these results with clinical findings and treatments.Materials and methodsSixty patients with T2DM and twenty-three healthy controls were included in the study. Biochemical and anthropometric variables were evaluated, and Tr1 lymphocytes (CD4+CD49+LAG-3+IL-10+) and other cell subsets (Th17, Th22 and Foxp3 + Treg cells) were analyzed in peripheral blood samples by multiparametric flow cytometry.ResultsSignificant increased levels of Tr1 cells were detected in patients with severe and mild disease, compared to healthy controls. In addition, CD4+IL-10+ lymphocytes were also increased in patients with T2DM. In contrast, similar levels of Foxp3+ Treg cells, Th17 and Th22 lymphocytes were observed in patients and controls. Likewise, no significant associations were detected between Tr1 cell levels and different clinical and laboratory parameters. However, those patients receiving glucagon-like peptide-1 receptor agonists (GLP-1-RA) showed similar levels of Tr1 cells than healthy controls, and significant lower numbers than untreated patients.ConclusionWe observed an increase in Tr1 and CD4+IL10+ lymphocyte levels in T2DM. Moreover, GLP1-RA treatment was significantly associated with normalization of the Tr1 levels. This highlights another potential immune dysfunction in patients with T2DM, which could participate in the pathogenesis of this condition.

Investigating the complex interplay between fibroblast activation protein α-positive cancer associated fibroblasts and the tumor microenvironment in the context of cancer immunotherapy.

This study investigates the role of Fibroblast Activation Protein (FAP)-positive cancer-associated fibroblasts (FAP+CAF) in shaping the tumor immune microenvironment, focusing on its association with immune cell functionality and cytokine expression patterns. Utilizing immunohistochemistry, we observed elevated FAP+CAF density in metastatic versus primary renal cell carcinoma (RCC) tumors, with higher FAP+CAF correlating with increased T cell infiltration in RCC, a unique phenomenon illustrating the complex interplay between tumor progression, FAP+CAF density, and immune response. Analysis of immune cell subsets in FAP+CAF-rich stromal areas further revealed significant correlations between FAP+ stroma and various T cell types, particularly in RCC and non-small cell lung cancer (NSCLC). This was complemented by transcriptomic analyses, expanding the range of stromal and immune cell subsets interrogated, as well as to additional tumor types. This enabled evaluating the association of these subsets with tumor infiltration, tumor vascularization and other components of the tumor microenvironment. Our comprehensive study also encompassed cytokine, angiogenesis, and inflammation gene signatures across different cancer types, revealing heterogeneous cellular composition, cytokine expressions and angiogenic profiles. Through cytokine pathway profiling, we explored the relationship between FAP+CAF density and immune cell states, uncovering potential immunosuppressive circuits that limit anti-tumor activity in tumor-resident immune cells. These findings underscore the complexity of tumor biology and the necessity for personalized therapeutic and patient enrichment approaches. The insights gathered from FAP+CAF prevalence, immune infiltration, and gene signatures provide valuable perspectives on tumor microenvironments, aiding in future research and clinical strategy development.

Gansui Banxia decoction modulates immune-inflammatory homeostasis to ameliorate malignant ascites in rats

Background“Gansui Banxia decoction” (GBD) is a classical traditional Chinese medicine formula for treating abnormal accumulation of fluid, such as malignant ascites (MA). Although GBD has shown definite water-expelling effects, its exact underlying mechanism has not been elucidated. PurposeThis study aimed to investigate the drug effects of GBD on MA rats and its underlying mechanisms. MethodsThe main chemical composition was determined by ultra-high performance liquid chromatography. The drug effects of GBD was evaluated in the established cancer cell-induced MA rat model. The symptoms were analyzed, and biological samples were collected for detecting immune and inflammation-related indicators by enzyme-linked immunosorbent assays, western blot, and flow cytometry. ResultsGBD increased urine discharge, decreased ascites production, and alleviated cachexia. After GBD treatment, the expression of TLR4, MyD88, and NF-кB and the release of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were reduced. In addition, GBD increased G1 phase arrest and inhibit excessive proliferation of cells in bone marrow while alleviating G1 phase arrest and increasing proliferation of cells in the thymus. Correspondingly, the development and maturation of T cells also changed. GBD increased the proportion of mature T-cells (CD4+CD8− and CD4−CD8+ single-positive (SP) T-cells), and decrease the proportion of immature cells (CD4+CD8+ double-positive (DP) T-cells and CD4−CD8− double-negative (DN) T-cells) in the blood or tumor microenvironment (TME, the ascites microenvironment). Finally, we further analysis of immune cell subsets, GBD decreased the proportion of immunosuppressive T-cells in the blood (CD4+CD25+Foxp3+T-cells) and TME (CD8+CD25+Foxp3+T-cells), and increased the proportion of anti-tumor immune cells (CD8+CD28+T-cells and NK cells) in the TME. ConclusionThese findings indicated that the drug effects of GBD were attributed to regulating the immune-inflammatory homeostasis, thereby mitigating the destruction of cancer cells and reducing the generation of ascites, which provided theoretical support for the clinical rational application and extended the scientific connotation of “water-expelling” of GBD.

Cluster analysis of flowcytometric immunophenotyping with extended T cell subsets in suspected immunodeficiency.

Patients with immunodeficiencies commonly experience diagnostic delays resulting in morbidity. There is an unmet need to identify patients earlier, especially those with high risk for complications. Compared to immunoglobulin quantification and flowcytometric B cell subset analysis, expanded T cell subset analysis is rarely performed in the initial evaluation of patients with suspected immunodeficiency. The simultaneous interpretation of multiple immune variables, including lymphocyte subsets, is challenging. To evaluate the diagnostic value of cluster analyses of immune variables in patients with suspected immunodeficiency. Retrospective analysis of 38 immune system variables, including seven B cell and sixteen T cell subpopulations, in 107 adult patients (73 with immunodeficiency, 34 without) evaluated at a tertiary outpatient immunology clinic. Correlation analyses of individual variables, k-means cluster analysis with evaluation of the classification into "no immunodeficiency" versus "immunodeficiency" and visual analyses of hierarchical heatmaps were performed. Binary classification of patients into groups with and without immunodeficiency was correct in 54% of cases with the full data set and increased to 69% and 75% of cases, respectively, when only 16 variables with moderate (p < .05) or 7 variables with strong evidence (p < .01) for a difference between groups were included. In a cluster heatmap with all patients but only moderately differing variables and a heatmap with only immunodeficient patients restricted to T cell variables alone, segregation of most patients with common variable immunodeficiency and combined immunodeficiency was observed. Cluster analyses of immune variables, including detailed lymphocyte flowcytometry with T cell subpopulations, may support clinical decision making for suspected immunodeficiency in daily practice.