Cell Subsets In Peripheral Blood Research Articles

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 < 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)>: 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 .

Pan‑immune‑inflammation value as a novel prognostic biomarker in nasopharyngeal carcinoma.

The pan-immune-inflammation-value (PIV) is a comprehensive biomarker that integrates different peripheral blood cell subsets. The present study aimed to evaluate the prognostic ability of PIV in patients with nasopharyngeal carcinoma (NPC) undergoing chemoradiotherapy. PIV was assessed using the following equation: (Neutrophil count × platelet count × monocyte count)/lymphocyte count. The Kaplan-Meier method and Cox hazards regression models were used for survival analyses. The optimal cut-off values for PIV and systemic immune-inflammation index (SII) were determined using receiver operating characteristic analysis to be 428.0 and 1032.7, respectively. A total of 319 patients were recruited. Patients with a low baseline PIV (≤428.0) accounted for 69.9% (n=223) and patients with a high baseline PIV (>428.0) accounted for 30.1% (n=96). Compared with patients with low PIV, patients with a high PIV had significantly worse 5-year progression-free survival [PFS; 66.8 vs. 77.1%; hazard ratio (HR), 1.97; 95% confidence interval (CI), 1.22-3.23); P=0.005] and 5-year overall survival (OS; 68.7 vs. 86.9%, HR, 2.71; 95% CI, 1.45-5.03; P=0.001). PIV was also a significant independent prognostic indicator for OS (HR, 2.19; 95% CI, 1.16-4.12; P=0.016) and PFS (HR, 1.86; 95% CI, 1.14-3.04; P=0.013) and outperformed the SII in multivariate analysis. In conclusion, the PIV was a powerful predictor of survival outcomes and outperformed the SII in patients with NPC treated with chemoradiotherapy. Prospective validation of the PIV should be performed to better stratify radical treatment of patients with NPC.

Immune cell phenotype and function patterns across the life course in individuals from rural Uganda.

To determine the pattern of immune cell subsets across the life span in rural sub-Saharan Africa (SSA), and to set a reference standard for cell subsets amongst Africans, we characterised the major immune cell subsets in peripheral blood including T cells, B cells, monocytes, NK cells, neutrophils and eosinophils, in individuals aged 3 to 89 years from Uganda. Immune phenotypes were measured using both conventional flow cytometry in 72 individuals, and full spectrum flow cytometry in 80 individuals. Epstein-Barr virus (EBV) IFN-γ T cell responses were quantified in 332 individuals using an ELISpot assay. Full blood counts of all study participants were also obtained. The percentages of central memory (TCM) and senescent CD4+ and CD8+ T cell subsets, effector memory (TEM) CD8+ T cells and neutrophils increased with increasing age. On the other hand, the percentages of naïve T (TN) and B (BN) cells, atypical B cells (BA), total lymphocytes, eosinophils and basophils decreased with increasing age. There was no change in CD4+ or CD8+ T effector memory RA (TEMRA) cells, exhausted T cells, NK cells and monocytes with age. Higher eosinophil and basophil percentages were observed in males compared to females. T cell function as measured by IFN-γ responses to EBV increased with increasing age, peaking at 31-55 years. The percentages of cell subsets differ between individuals from SSA compared to those elsewhere, perhaps reflecting a different antigenic milieu. These results serve as a reference for normal values in this population.

Imbalance of Tfollicular helper cell subsets trigger the differentiation of pathogenic B cells in idiopathic membranous nephropathy.

This study aims to elucidate the role of T follicular helper (Tfh) cells and their subsets in idiopathic membranous nephropathy (IMN). The frequencies of Tfh cell subsets and B cell subsets in peripheral blood (PB) were detected in both IMN patients and healthy controls (HCs). The involvement of Tfh cells in the disease pathogenesis was examined by coculturing human Tfh cells with B cells. The dynamic changes of Tfh cells in PB or spleen were monitored in passive Heymann nephritis (PHN) rats. The frequencies of circulating Tfh (cTfh) cells, cTfh2 cells, and plasmablasts were enriched in the PB of patients with IMN. cTfh cells expressed higher ICOS, and lower BTLA than healthy counterparts. The frequency of ICOS + cTfh2 was associated with the severity of IMN, including 24h urine protein, IgG4 concentration and the IgG4: IgG ratio. Positive correlations were also observed between the frequency of cTfh2 cells with plasmablasts, serum IL-21 and IL-4 levels. Importantly, cTfh cells isolated from IMN patients were able to induce the differentiation of B cells to memory B cells (MBC) and plasmablasts, this process could be substantially attenuated by blocking the IL-21. Similar increases of ICOS + cTfh cells were also detected in spleen of PHN rats, concomitant with elevated urine protein levels. Collectively, our results demonstrate that the imbalance of cTfh cell subsets play a crucial pathogenic role in IMN by inducing the differentiation of B cells through IL-21, and cTfh2 cells might serve as useful markers to evaluate the progression of IMN.

Sofosbuvir (+) daclatasvir (+) ribavirin in Egyptian patients with hepatitis C virus: Therapeutic outcomes and the prognostic role of natural killer cells

BackgroundOne of the prominent causes of chronic liver disease worldwide is the hepatitis C virus (HCV). HCV believed that innate immunity contributes to a sustained virological response (SVR) to the treatment of Sofosbuvir (SOF) (+) Daclatasvir (DCV) (+) Ribavirin (RBV). This study aimed to evaluate the impact of SOF (+) DCV (+) RBV therapy and persistent HCV infection on the subset of natural killer cells (NK) in HCV genotype four patients from Egypt. Materials and MethodsOne hundred and ten patients with persistent HCV infections requiring SOF (+) DCV (+) RBV therapy were grouped, and a flow cytometry (FCM) study of the NK cell subset in peripheral blood was performed. The assessment was performed before and after three and/or six months of the cessation of viral suppression therapy when a patient had a long-term viral response (SVR). One hundred and ten volunteers from the National Liver Institute’s (NLI) blood bank were selected as controls. ResultsPatients with chronic HCV infection before therapy had considerably lower CD16+ and CD3− CD56+ cells than controls. Their levels increase during SOF (+) DCV (+) RBV therapy. In patients with SVR during treatment, CD16+ and CD3– CD56+ cells increased significantly compared to those who did not get SVR. Furthermore, CD56+ cells were significantly higher in patients with persistent infection before treatment than controls but diminished with the response to treatment. Conclusion: NK cell activation following SOF (+) DCV (+) RBV therapy and polarization to cytotoxicity occurred early in HCV antiviral therapy and was elevated in the respondents. Our data illustrated that establishing an inhibitory cytotoxic NK profile is related to therapeutic outcomes.

Repeated iv anti-CD20 treatment in multiple sclerosis: Long-term effects on peripheral immune cell subsets.

Repeated intravenous administration of anti-CD20 depleting monoclonal antibodies 6 months apart is among the highly effective treatment options in multiple sclerosis (MS). Here, we aimed to investigate peripheral immune cell subset depletion kinetics following either rituximab (RTX) or ocrelizumab (OCR) infusions in people with MS (pwMS). We studied pwMS treated de-novo with either RTX (n = 7) or OCR (n = 8). The examinations were scheduled before the initiation of anti-CD20 therapy and every 12 weeks for up to 15 months. Immunophenotyping of immune cell subsets in peripheral blood was performed by multiparametric fluorescence cytometry. A significant, persistent decrease of CD19+ B cells was observed already with the first anti-CD20 infusion (p < 0.0001). A significant proportional reduction of memory B cells within the B-cell pool was achieved only after two treatment cycles (p = 0.005). We observed a proportional increase of immature (p = 0.04) and naive B cells (p = 0.004), again only after the second treatment cycle. As for the peripheral T-cell pool, we observed a continuous proportional increase of memory T helper (TH) cells/central memory TH cells (p = 0.02/p = 0.008), while the number of regulatory T cells (Treg) decreased (p = 0.007). The percentage of B-cell dependent TH17.1 central memory cells dropped after the second treatment cycle (p = 0.02). No significant differences in the depletion kinetics between RTX and OCR were found. Peripheral immune cell profiling revealed more differentiated insights into the prompt and delayed immunological effects of repeated intravenous anti-CD20 treatment. The observation of proportional changes of some pathogenetically relevant immune cell subsets only after two infusion cycles deserves further attention.

Roginolisib a Highly Selective Allosteric Modulator of the Phosphoinositide 3-Kinase Delta (PI3Kδ) in Patients with Refractory/Relapsed Follicular Lymphoma

Introduction PI3Kδ inhibitors are active in patients (pts) with lymphomas, but their clinical development has been hampered by toxicity (Brown et al, Lancet Oncol 2022). Roginolisib (IOA-244) exerts an allosteric modulation of the PI3Kδ C-terminus and functions as a non-ATP competitive PI3Kδ inhibitor, delivering a selective inhibition without off-target effects typically seen with other PI3K inhibitors (Johnson et al, Cancer Res Commun 2023). Using the recommended phase 2 dose (RP2D) of 80 mg QD established in solid tumors patients, patients with refractory/relapsed (r/r) follicular lymphoma (FL) were treated to investigate its safety profile in pts with hematologic malignancies (NCT04328844). Methods In Part A of the FiH dose escalation study, roginolisib was investigated at 20 and 80 mg QD in pts with r/r FL. Primary objective: safety of the RP2D established in solid tumor pts. Secondary objectives: PK; PD (e.g., changes in immune cell subsets in peripheral blood); Lugano-based responses; PFS and OS. Results In Part A for pts with r/r FL, there were two cohorts: (a) Cohort 1: 20 mg QD daily (4/4; 2 female, 2 male); (b) Cohort 2: 80 mg QD (4/4; 3 female, 1 male). There were no DLT observed and no dose modifications were needed. The mean time on treatment was 1.9 months (20 mg QD) and 4.3 months (80 mg QD), with one patient at the 80 mg receiving Roginolisib for 9 mo. More than 4 prior lines of treatment were in 3/8 pts. Transient (lasting less than 48 hrs) platelet reduction (G3) and AST/ALT elevation (G3) were observed in 2/8 pts, which improved while pts continued without a treatment intervention, such as dose modification or medical treatment with steroids. Lugano-based PRs were observed in 2/4 pts at the 80 mg QD dose and none at the 20 mg QD. At the 80 mg QD dose a third pt had a clinical response manifested by improvement in night sweats and fever, reduction in cervical palpable lymph nodes and relief from chronic pleural effusion. This clinical improvement lasted less than 5 weeks. While Overall Survival (OS) is still ongoing, the median has not been reached in this study and is currently at 14.3 mo. Subsequent to ending Roginolisib treatment, patients received at least one additional therapy (6/8), one patient was able to undergo autologous transplant with a complete metabolic response (CMR). Biomarker evaluation using flow cytometry showed low counts of T regulatory cells (Treg) in peripheral blood in all 8 r/r FL patients compared to solid tumor patients at baseline and throughout the treatment. Myeloid derived suppressor cells in peripheral blood remained unchanged during the course of treatment with Roginolisib. Conclusions The safety profile of roginolisib in pts with r/r FL appears to match that in pts with solid malignancies. In contrast to other PI3Kδ inhibitors, roginolisib is highly selective for binding to PI3Kδ, favoring an inactive confirmation of PI3Kd and functions as a non-ATP competitive inhibitor.

Baseline CD57 + and CD16 + NK-Cells Predict Treatment Outcome in Non-Transplant Eligible Newly Diagnosed Multiple Myeloma Patients Treated with Daratumumab-Ixazomib-Dexamethasone

Background Non-fit multiple myeloma (MM) patients have inferior survival, increased toxicity, and higher rates of treatment discontinuation compared with their fit peers. Effective and less toxic treatment strategies are therefore urgently needed for this vulnerable population. The HOVON-143 trial (EudraCT: 2016-002600-90) was specifically designed to assess the efficacy and safety of daratumumab-ixazomib-dexamethasone in non-transplant eligible (NTE), intermediate fit and frail newly diagnosed (ND)MM patients. Patients were treated with nine 28-day induction cycles comprising daratumumab 16 mg/kg intravenously (cycles 1-2: days 1, 8, 15, and 22; cycles 3-6: days 1 and 15; cycles 7-9: day 1), ixazomib 4 mg orally (days 1, 8, and 15) and dexamethasone orally (only on days that daratumumab was given; cycle 1-2: 20 mg; cycle 3-9: 10 mg), followed by 8-week maintenance cycles consisting of daratumumab (day 1) and ixazomib (days 1, 8, 15, 29, 36, 43) for two years or until disease progression. Aims Since daratumumab has well-known NK-cell-mediated effector mechanisms and T-cell-stimulating immunomodulatory effects, we monitored these cell subsets in bone marrow (BM) and peripheral blood (PB) samples obtained before and during treatment. We specifically aimed to better understand these cell subsets related to frailty status, their predictive value for treatment outcome, and their dynamics during treatment. Methods 126 NDMM patients, among which 64 intermediate fit and 62 frail, were monitored. BM was collected at study-entry (baseline) and at disease progression; PB was collected at baseline, before cycle 4, before maintenance, and at disease progression. Absolute numbers of total T-cells and NK-cells (in PB) or percentages of T-cell and NK-cell subsets (in PB and BM) were profiled by flow cytometry. Data were analyzed using FlowSOM and UMAP. Total cells and cell subsets at baseline were compared between intermediate fit and frail patients, between responders (≥ partial response) and non-responders (&amp;lt; partial response), and associated with progression-free survival (PFS) and overall survival (OS). Results At baseline, we found no difference in absolute numbers of total T-cells and NK-cells between intermediate fit and frail patients. Besides, NK-cell phenotype was similar between both groups. However, within the T-cell compartment frail patients had a decreased CD4 +:CD8 + T-cell ratio, reduced naïve CD4 + and CD8 + T cells, and increased PD1 + CD4 + T-cells compared with intermediate fit patients in both BM and PB, independent of age. Interestingly, differences in T-cell phenotype were more pronounced in female than in male patients. Response data were available for 124 patients, of which 92 (74%) responded to treatment. In both BM and PB, preliminary results showed no clear association between response and absolute numbers of total T-cells or certain T-cell phenotypes at baseline. However, responders had higher absolute numbers of total NK-cells and higher percentages of NK-cells positive for maturation marker CD57 or fc-gamma receptor CD16 than non-responders, while higher absolute numbers of total NK-cells and higher percentages of CD57 + NK-cells were also associated with superior PFS and OS (Figure 1). Conversely, higher proportions of baseline NK-cells positive for inhibitory markers TIGIT and PD1 were associated with non-response, while higher percentages of CD56 bright and PD1 + NK-cells were negatively associated with PFS and OS. As expected, absolute numbers of total NK-cells markedly declined during the treatment course. Interestingly, percentages of CD57 + and CD16 + NK-cells decreased during treatment and remained low at disease progression, while percentages of CD56 bright and NKG2A + NK-cells substantially increased compared with baseline. Conclusions Frail patients had a distinct T-cell profile compared with intermediate fit patients, while NK-cell profiles were similar. Treatment outcome of NTE-NDMM patients treated with daratumumab-ixazomib-dexamethasone was positively associated with higher absolute numbers of total NK-cells and higher percentages of CD57 + and CD16 + NK-cells at baseline, corresponding to a mature and cytotoxic phenotype capable of mediating potent antibody-dependent cellular cytotoxicity. This study highlights the importance of NK-cell phenotype for the effectivity of daratumumab-based combination therapies in NTE-NDMM.

Identification of cell-intrinsic effects of mutated circulating cells due to clonal hematopoiesis in patients with heart failure

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and is associated with worse prognosis in patients with heart failure with reduced ejection fraction (HFrEF). Circulating immune cells derived from patients with HFrEF harboring CHIP mutations show enhanced proinflammatory signatures. However, it is unclear, whether these signatures are derived from the relatively low number of cells harboring CHIP-driver mutations, or are indicators of a systemic pro-inflammatory activation associated with CHIP. Methods and Results We assessed the cell-intrinsic effects of the most prevalent CHIP-driver gene (DNMT3A) mutant-carrying circulating immune cells in a total of 5 patients with HFrEF. Using an improved protocol to detect mutant cells on a single cell level (MutDetect-Seq), as illustrated in figure 1A, we show that CHIP mutations can be detected in all major immune cell types (e.g. T cells, monocytes, B cells, NK cells). DNMT3A mutant monocytes demonstrated the highest number of significantly upregulated genes relative to other cell types. Specifically, DNMT3A mutant monocytes exhibit altered gene expression profiles associated with inflammation and phagolysosome function (figure 1B). In addition, DNMT3A mutant carrying CD4+, but not CD8+ T cells, showed significant changes in gene expression patterns towards effector cells and away from a naïve T cell state. Finally, increased paracrine signaling pathways involved in amplifying inflammatory circuits are predicted between mutated and non-mutated monocytes. Conclusion This is the first study to decipher the genetic signature of DNMT3A-CHIP-driver mutation carrying circulating immune cells, thereby providing first evidence for the cell intrinsic effects of DNMT3A mutations on peripheral blood cell subsets in patients with HFrEF. The altered gene expression profiles associated with inflammation and phagolysosome function of mutant monocyte as well as the skewing of mutated T cells towards effector cells provide novel insights into how CHIP might promote worse prognosis in heart failure patients.MutDetect-seq

A novel flow-cytometric based method to assess post-HSCT donorchimerism exploiting RNA hybridization.

Analysis of donor-recipient chimerism after hematopoietic stem cell transplantation (HSCT) is of pivotal importance for patient's clinical management, especially in the context of mixed chimerism. Patients are routinely monitored for chimerism in sorted subsets of peripheral blood cells. However, measurement of chimerism in sorted immune cell subsets is technically challenging and time consuming. We here propose a novel, flow cytometry-based approach to detect donor cell chimerism in sex-mismatched HSCT. We exploit RNA PrimeFlow™ system, based on RNA hybridization, to detect mRNA from a lysine demethylase encoded by Y chromosome, KDM5D. This approach allows to distinguish male and female derived cells with around 1% sensitivity. The procedure can be coupled with multiparametric immunophenotyping to assess chimerism in specific immune cell subsets without the need for prior FACS-sorting. We apply this method to a cohort of HSCT patients (n = 10) and we show that it is consistent with standard PCR-based method. We also show that different T lymphocyte subsets display variable degrees of donor chimerism, especially in CD8+ T cell compartment where we observe an enrichment for recipient chimerism in central memory T cells. This method can be exploited to advance current knowledge on immune reconstitution focusing on specific subsets avoiding prior FACS-sorting.

The consensus of Chinese experts on refined analysis of immune cell subsets in peripheral blood by multi-parameter flow cytometry

The detection of immune cell subsets plays a very important role in the clinical diagnosis and treatment of various benign and malignant diseases and health management. In order to better carry out in-depth research on different functional immune cell subsets, establish reference intervals for clonality related indicators, establish special reference intervals for immune aging, individualized dynamic monitoring and treatment recovery, and discover the clinical significance of immune cells other than lymphocytes, it is urgent to analyze the peripheral blood immune cell subsets in a refined way. Multiparameter flow cytometry is an important technical method to detect immune cell subsets and evaluate immune function. In order to standardize the refined detection methods and protocols of peripheral blood immune cell subsets by flow cytometry, and further promote its application in clinical diagnosis and treatment of diseases and health management, Laboratory Medicine Committee of Chinese Association of Integrative Medicine (LMC-CAIM) organized experts to formulate this expert consensus.

B-142 Development and Clinical Validation of Artificial Intelligence Assisted Flow Cytometry Diagnosis for Acuteleukemia

Abstract Background Flow cytometry is one of the most commonly used diagnostic tools in hematopathology. Manual flow cytometry data analysis is vulnerable to substantial inter-operator variation and typically takes a technologist 20 min to analyze a case. In the last decade, artificial intelligence (AI) technology has advanced dramatically and become widely used in image processing. Previously, we created an AI-assisted workflow for classifying white blood cell subsets in peripheral blood. The goal of this study is to evaluate the automated flow cytometry analysis system for diagnosing acute leukemia. Methods A total of 117 cases were retrieved from the archived diagnostic cases, consisting of three diagnostic categories: 20 normal cases, 37 acute lymphoblastic leukemia and 60 acute myeloid leukemia. Flow cytometry data manually analyzed by a pathologist is the gold standard. The same flow cytometry data was processed by the flow cytometry analysis software DeepFlow® using proprietary AI algorithm. The final diagnosis and blast cell count of AI and manual analysis are compared by Pearson correlation coefficient. Results The AI-assisted blast cell enumeration showed moderate correlation as compared to manual evaluation (r = 0.8, Fig. 1). Manual adjustment for enumeration of CD34 negative blast population with uncommon immunophenotype is required for 7 cases, including 4 CD34−/CD117+ acute myeloid leukemia and 3 CD34−/TDT+ lymphoblastic leukemia. The AI analysis effectively recognized cell populations and provided correct diagnoses on 94% of cases (110/117). With pathologists’ review, all cases were classified into the correct diagnostic categories. Conclusion Our study demonstrated that AI can accurately recognize the flow cytometry pattern of leukemia and make the proper diagnosis. Additional training for CD34 negative blast gating and other uncommon blast immunophenotypes is being conducted. Ultimately, AI will decrease the demand on manual analysis by technologists and pathologists, standardize the analysis process, and provide accurate diagnosis.

Alterations in Helios+ T cell subsets in peripheral blood of early-stage lung adenocarcinoma patients: Implications for early diagnosis

ObjectiveThis study aimed to investigate the changes and significance of circulating Helios-associated T cell subsets in patients with early-stage lung adenocarcinoma (LUAD). MethodsBlood samples were collected from 35 healthy controls and 34 patients with early-stage LUAD. Flow cytometry was used to analyze various CD4+ T cell subsets, including regulatory T(Treg) cells, follicular regulatory T(Tfr) cells, follicular helper T (Tfh) cells, and conventional T (con-T) cells. Correlation analysis was conducted to investigate the association of Helios-related subsets with clinical indicators. The ROC curve was used to explore the potential clinical value of Helios+ T cell subsets in the screening of patients with early LUAD. Fifteen of these patients were tracked after lung cancer resection and changes in Helios+ T cell subsets before and after treatment were analyzed. ResultsThe percentage and absolute number of Tregs were up-regulated in LUAD patients while Tfh and con-T cells expressing Helios were down-regulated. Absolute counts of Tfr and con-T cells and Helios expression in Tfr and Treg decreased significantly after resection. Helios+ Tfh and con-T were negatively correlated with certain tumor markers. Areas under the curve (AUCs) of percentages and absolute counts of Helios+ Tfh, Treg, Tfr and con-T cells to distinguish early LUAD from healthy individuals were 0.7277, 0.5697, 0.5718, 0.7210 (percentages), 0.7336, 0.7378, 0.5908 and 0.7445(absolute numbers), respectively. ConclusionHelios+ T cell subsets in peripheral blood of early-stage LUAD patients has changed significantly, which may be related to the pathogenesis of LUAD and could help for early diagnosis of LUAD.

Human immune phenotyping reveals accelerated aging in type 1 diabetes.

The proportions and phenotypes of immune cell subsets in peripheral blood undergo continual and dramatic remodeling throughout the human life span, which complicates efforts to identify disease-associated immune signatures in type 1 diabetes (T1D). We conducted cross-sectional flow cytometric immune profiling on peripheral blood from 826 individuals (stage 3 T1D, their first-degree relatives, those with ≥2 islet autoantibodies, and autoantibody-negative unaffected controls). We constructed an immune age predictive model in unaffected participants and observed accelerated immune aging in T1D. We used generalized additive models for location, shape, and scale to obtain age-corrected data for flow cytometry and complete blood count readouts, which can be visualized in our interactive portal (ImmScape); 46 parameters were significantly associated with age only, 25 with T1D only, and 23 with both age and T1D. Phenotypes associated with accelerated immunological aging in T1D included increased CXCR3+ and programmed cell death 1-positive (PD-1+) frequencies in naive and memory T cell subsets, despite reduced PD-1 expression levels on memory T cells. Phenotypes associated with T1D after age correction were predictive of T1D status. Our findings demonstrate advanced immune aging in T1D and highlight disease-associated phenotypes for biomarker monitoring and therapeutic interventions.

Abstract P3055: Identification Of Cell-intrinsic Effects Of Clonal Hematopoiesis In Heart Failure

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and associates with worse prognosis in patients with heart failure (HF). While, circulating immune cells derived from patients with HF harboring CHIP mutations show heightened proinflammatory signatures, it is unclear whether these signatures are derived from the relatively low number of cells harboring CHIP-driver mutations, or are indicators of a systemic pro-inflammatory activation associated with CHIP. Methods and Results: We assessed the cell-intrinsic effects of the most prevalent CHIP-driver gene (DNMT3A) mutant-carrying circulating immune cells in a total of 5 HF patients. Using a novel protocol to detect DNMT3A mutant cells on a single cell level (MutDetect-Seq), we show that CHIP mutations can be detected in all major immune cell types (e.g. T cells, monocytes, B cells, NK cells), with detected mutation similar to that predicted by traditional targeted DNA sequencing. DNMT3A mutant monocytes demonstrated the highest number of significantly upregulated genes relative to other cell types. DNMT3A mutant monocytes exhibit altered gene expression signatures associated with inflammation and phagolysosome function. Additionally, DNMT3A mutant carrying CD4+, but not CD8+ T cells, showed significant changes in gene expression patterns towards an activated and cytotoxic profile, which was validated in vitro. Finally, increased paracrine signaling pathways involved in amplifying inflammatory circuits are predicted between mutated and non-mutated monocytes, this was validated in vitro with further implications for cardiac fibroblast activation via aSMA and COL1A1 expression. Conclusion: This is the first study to decipher the genetic signature of DNMT3A-CHIP-driver mutation carrying circulating immune cells, thereby providing first evidence for the cell intrinsic effects of DNMT3A mutations on peripheral blood cell subsets in patients with HF. The altered gene expression profiles associated with inflammation and phagolysosome function of mutant monocyte, as well as skewing of mutated T cells towards effector cells provide novel insights into how CHIP may promote worse prognosis in heart failure patients.

Children with extended oligoarticular and polyarticular juvenile idiopathic arthritis have alterations in B and T follicular cell subsets in peripheral blood and a cytokine profile sustaining B cell activation

ObjectivesThe main goal of this study was to characterise the frequency and phenotype of B, T follicular helper (Tfh) and T follicular regulatory (Tfr) cells in peripheral blood and the...

Cytotoxic T cell subsets in peripheral blood and cerebrospinal fluid from patients with multiple sclerosis

Using multicolor flow cytometry, the main cytotoxic T lymphocytes (Tcyt) subsets were identified, based on the expression of CD45RA and CD62L in paired samples of peripheral blood and cerebrospinal fluid from the patients during the relapse (n = 32) and remission (n = 20) of multiple sclerosis (MS), as well as in the peripheral blood samples of healthy volunteers (n = 51). During the relapse of MS, we have observed a decreased relative number of CD3+CD4+ cells and CD4/CD8 ratio in cerebrospinal liquor. In peripheral blood taken from the relapsed MS patients, we have found significant correlations between EDSS score and absolute counts (r = -0,430, p = 0.014), and with relative numbers of CD45RA+CD62L+Tcyt (r = -0,502, p = 0.003). In remission state of MS, the relative numbers of blood CD45RA-CD62L-Tcyt cells exhibited a significant decrease (p = 0.005) to 8.70% (6.51-11.63) against control group with 12.18% (10.38-15.24), although it did not significantly differ (p = 0.114) from the relapsed patients with 11.31% (8.28-13.90). Studies of liquor samples have shown that, during MS relapse, the percentage of CD45RA-CD62L-Tcyt was increased (p = 0.027) up to 8.16% (6.40-11.40), while in remission state these cells comprised only 6.49% (4.51-8.39) from the total CD3+ cell number. During relapse of MS, some positive correlations were revealed between the relative number of nave, CM, EM and TEMRA Tcyt from liquor, and the percentages, as well as contents of similar T cell subsets in peripheral blood samples. The inverse relationship between the level of EM Tcyt from liquor and peripheral blood naive cells showed the close relationship between these two Tcyt subsets and clinical manifestations of MS (i.e., scores of EDSS scale). During the remission period, most of these correlations are disrupted. Further investigations of cytotoxic T cells dynamics in peripheral blood and cerebrospinal fluid will help to approach the understanding of MS pathogenesis by revealing novel markers for the clinical prognosis in this disorder.

Association of NK Cells with the Severity of Fibrosis in Patients with Chronic Hepatitis C.

Some NK cell subpopulations may be involved in the modulation of fibrogenesis in the liver. The aim of the study was to evaluate the relationship between the number and phenotype of NK cell subsets in peripheral blood (PB) and total NK cell percentage, population density and the degree of liver fibrosis of patients infected with hepatitis C virus (HCV+). The study group consisted of 56 HCV+ patients, divided into two subgroups: patients with mild or moderate fibrosis and patients with advanced liver fibrosis or cirrhosis (F ≥ 3 in METAVIR classification). The preparations were stained with H-E and AZAN staining. NK cells were targeted with anti-CD56 antibody and identified automatically in situ using the DakoVision system. Assessment of different NK cell subsets in PB was performed with the flow cytometry technique. In the PB of HCV+ patients with advanced liver fibrosis, there was a lower proportion of CD62L+; CD62L+/CD94++; CD27+; CD127+/CD27+ and CXCR3+/CD27+ NK subsets, as compared to patients with mild/moderate liver fibrosis. The results also showed no association between total PB NK cell level and total intrahepatic NK cell population density between patients with mild/moderate fibrosis and with advanced liver fibrosis. However, positive correlations between the PB levels of CD94+ and CD62L+ NK cell subsets and the intrahepatic total NK cell percentage and population density in the liver, irrespectively to the extent of fibrosis, were observed. Additionally, positive correlation was found between the PB CXCR3+/CD94+ NK cell percentages and intrahepatic NK cell percentages in patients with advanced hepatic fibrosis. Lower blood availability of specific NK subsets in patients with chronic type C hepatitis might be a cause of progression of liver fibrosis via insufficient control over hepatic stellate cells.

Phase II, Open-Label Clinical Trial of Urinary-Derived Human Chorionic Gonadotropin/Epidermal Growth Factor for Life-Threatening Acute Graft-versus-Host Disease

Treatments that aid inflammation resolution, immune tolerance, and epithelial repair may improve outcomes beyond high-dose corticosteroids and other broad immunosuppressants for life-threatening acute graft-versus-host disease (aGVHD). We studied the addition of urinary-derived human chorionic gonadotropin/epidermal growth factor (uhCG/EGF; Pregnyl; Organon, Jersey City, NJ) to standard aGVHD therapy in a prospective Phase II clinical trial (ClinicalTrials.gov identifier NCT02525029). Twenty-two patients with Minnesota (MN) high-risk aGVHD received methylprednisolone 48 mg/m2/day plus 2000 units/m2 of uhCG/EGF s.c. every other day for 1 week. Patients requiring second-line aGVHD therapy received uhCG/EGF 2000 to 5000 units/m2 s.c. every other day for 2 weeks plus standard of care immunosuppression (physician's choice). Responding patients were eligible to receive maintenance doses twice weekly for 5 weeks. Immune cell subsets in peripheral blood were evaluated by mass cytometry and correlated with plasma amphiregulin (AREG) level and response to therapy. Most patients had stage 3-4 lower gastrointestinal tract GVHD (52%) and overall grade III-IV aGVHD (75%) at time of enrollment. The overall proportion of patients with a response at day 28 (primary endpoint) was 68% (57% with complete response, 11% with partial response). Nonresponders had higher baseline counts of KLRG1+ CD8 cells and T cell subsets expressing TIM-3. Plasma AREG levels remained persistently elevated in nonresponders and correlated with AREG expression on peripheral blood T cells and plasmablasts. The addition of uhCG/EGF to standard therapy is a feasible supportive care measure for patients with life-threatening aGVHD. As a commercially available, safe, and inexpensive drug, uhCG/EGF added to standard therapy may reduce morbidity and mortality from severe aGVHD and merits further study.

First-in-human (FIH) phase I dose escalation study (part A) of the first oral allosteric modulator of phosphoinositide 3-kinase inhibitor delta (PI3Kδ) roginolisib in patients with advanced cancer and dose confirmation in uveal melanoma (part B).

3110 Background: The highly selective oral allosteric modulator of PI3Kδ, roginolisib (IOA-244), blocks the activity of PI3Kδ -dependent signalling, in both tumour cells and Tregs. Methods: Part A explored the continuous daily dosing of roginolisib at 10, 20, 40 and 80 mg in patients (pts) with solid tumours and Follicular Lymphoma (FL). Part B consists of dose confirmation cohorts, including uveal melanoma (UM) pts. Primary objective: safety of the anticipated biologically effective dose (BED), or the recommended phase 2 dose (RP2D). 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 Cytometry by Time of Flight (CyTOF); response assessments by radiomics; analysis of circulating protein signatures via Olink proteomic. Results: Part A Solid Tumour (completed): Sixteen pts were treated in 4 cohorts, each with 4 pts. 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 v5 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. Neither in the solid tumour nor in the haematology pts treatment-emergent adverse events (TEAE) led to study drug discontinuation, immune related toxicity, or a Dose Limiting Toxicity. Subgroup evaluation: UM pts (Part A and Part B): 24 pts (Part A: 9 pts; Part B ongoing: 15 pts), of which 18 pts treated at 80 mg QD (RP2D). Mean time on treatment: 8.6 mo (range: 1.0-27.9 mo). ORR (RECIST 1.1): PR: 1/20 (5%); SD: 16/20 (80%). Median OS for Part A: 20.8 mo (5/9 with 4 pts alive); Part B: not determined; 1-year OS rate: Part A 66.7%; Part B: ongoing. Exploratory Investigations: Radiomics-based evaluation of CT images in pts with solid tumors assessed each metastatic organ, detecting mixed responses; peripheral proteomic evaluation detected changes in several cytokines and chemokines; CyTOF detected reduction in circulating Tregs associated with increased activated effector CD8+ T cells. Conclusions: Roginolisib is well tolerated at the 80 mg dose, shows signs of efficacy and induces phenotypic changes in the circulating immune cells. As best response at the RP2D, the most robust anti-tumour activity was observed in FL (50% PR) and UM pts (5% PR; 80% SD). Clinical trial information: NCT04328844 .