Cell Subsets In Blood Research Articles

A Novel Selective BCL2 Inhibitor with Limited Immune Suppression and Improved Safety Compared to Venetoclax

BCL2 is a key pro-survival protein that is overexpressed in many cancers. The first generation BCL2 inhibitor venetoclax has proved highly effective in multiple hematological malignancies including CLL and AML. However, the pharmacokinetic half-life of venetoclax is 26 hours which prevents optimization of the therapeutic window. Additionally, venetoclax metabolism is influenced by Cyp3A/4 inhibitors making their use in diseases where anti-fungal prophylaxis is required challenging. Venetoclax treatment can also result in significant immunosuppression including dose-limiting neutropenia and thrombocytopenia, and the loss of multiple lymphocyte populations, due in part to its effect on BCL-xL. Herein we present the pre-clinical evaluation of a novel highly selective BCL2 inhibitor, ZE50-0134, with equivalent anti-tumor efficacy, improved safety profile, and limited impact on non-malignant immune populations. ZE50-0134 binds the P2 pocket of BCL2, thereby increasing selectivity toward BCL-2, ZE50-0134 showed a 4600-fold greater selectivity for BCL2 over BCL-xL compared to venetoclax with only an 84-fold greater selectivity. In vivo murine and canine pharmacokinetics showed that ZE50-0134 has a substantially shorter half-life than venetoclax suggesting feasibility for periodic pulse dosing to limit the on-target adverse effects of BCL2 inhibition. The ADME properties of ZE50-0134 were exceptional. An in vivo murine study examining the pharmacology of ZE50-0134 or venetoclax given with ketoconazole, a strong CYP3A inhibitor, was performed demonstrating a 0.84-fold decrease in AUC with ZE50-0134 vs. a 1.52-fold increase in AUC with venetoclax suggesting less Cyp3A influence on ZE50-0134. Toxicology in rats and dogs suggest a 19-fold margin between therapeutically effective dose and toxicity with ZE50-0134. Given this data, we sought to validate that ZE50-0134 has less impact on normal immune populations. Wild-type C57BL6/J mice were treated daily with 100 mg/kg ZE50-0134, 100 mg/kg venetoclax or vehicle. A comprehensive 36-color spectral flow cytometry immune profiling panel was used to evaluate the effect of BCL2 inhibitors on multiple immune populations including B, T and NK cells as well as monocyte/macrophage and dendritic cell subsets in the peripheral blood. Venetoclax treated mice demonstrated a significant loss of multiple lymphocyte populations, including B, T, and NK cells, in the peripheral blood. Particularly, from Day 14 venetoclax treatment significantly reduced the percentage and absolute count of CD8 T cells (Figure 1). Some reduction in CD8 T cells was also observed with ZE50-0134 treatment, however it was significantly less than with venetoclax. No changes in the frequency of any normal myeloid population were observed. We next sought to examine the effectiveness of ZE50-0134 in B-cell and myeloid malignancies. Primary CLL cells were treated for 4 and 24 hours, corresponding to the expected half-life of ZE50-0134 and venetoclax, followed by washout. Cytotoxicity at 72 hours for both ZE50-0134 and venetoclax were equivalent for both short and prolonged treatment. We then assessed ZE50-0134 and venetoclax in vivo at 100 mg/kg in the RSV4;11 subcutaneous lymphoid model where we observed similar tumor growth regression. In AML, venetoclax is typically not effective as monotherapy and if often used in combination with azacytidine. To assess the relative efficacy of venetoclax and ZE50-0134 in combination with Azacitidine (5-Aza), we used the MOLM-13 cell line disseminated xenograft model. NCG mice were engrafted with MOLM-13 cells via the tail vein. From day 3, mice were treated with daily oral gavage of venetoclax or ZE50-0134 (100 mg/kg) alone or in combination with weekly intravenous 5-Aza (2.5 mg/kg). ZE50-0134 and venetoclax performed equally as single agents (median survival 23.5 days vs 22.5 days) and in combination with 5-Aza (median survival 35 days vs 35 days) (Figure 2). Together, this data suggests that ZE50-0134 is a highly effective BCL2 inhibitor with substantially less impact on the normal immune cells responsible for both anti-tumor and anti-infectious immunity. The pharmacology of this agent makes it an ideal therapeutic to optimize tumor BCL2 dependence and avoid untoward toxicity that occurs through continuous BCL2 targeting. This data supports the further clinical development of ZE50-0134 in both B cell malignancies and AML.

Syntaxin-4 and SNAP23 are involved in neutrophil degranulation, but not in the release of mitochondrial DNA during NET formation.

Neutrophils are a specialized subset of white blood cells, which have the ability to store pre-formed mediators in their cytoplasmic granules. Neutrophils are well-known effector cells involved in host protection against pathogens through diverse mechanisms such as phagocytosis, degranulation, extracellular traps, and oxidative burst. In this study, we provide evidence highlighting the significance of the SNARE proteins syntaxin-4 and synaptosomal-associated protein (SNAP) 23 in the release of azurophilic granules, specific granules, and the production of reactive oxygen species in human neutrophils. In contrast, the specific blockade of either syntaxin-4 or SNAP23 did not prevent the release of mitochondrial dsDNA in the process of neutrophil extracellular trap (NET) formation. These findings imply that degranulation and the release of mitochondrial dsDNA involve at least partially distinct molecular pathways in neutrophils.

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.

Dysregulated Tfr/Tfh2 cells in patients with polycystic ovarian syndrome

Recent research revealed the pathogenic role of B cells in the pathogenesis of polycystic ovary syndrome (PCOS), while the Tfh cell plays a critical role in the B cell mediated autoantibody production and humoral immunity, but had not been investigated in PCOS patients.The frequency of Tfh and B cell subsets (Tfh1, Tfh2, Tfh17, naïve B, memory B, and plasma cells) in the peripheral blood of 21 PCOS patients and 15 healthy controls were investigated by flow cytometry. And the levels of follicle-stimulating hormone, luteinizing hormone, testosterone, prolactin and estradiol progesterone were measured by using the immunoluminescence method. Also, the associations between these hormone levels and Tfh cell subsets or B cell subsets were analyzed.No significant difference was observed in total Tfh cells between 21 PCOS patients and 15 healthy controls (p > 0.05). But the percentages of Tfh2 and plasma cells were significantly higher in 21 PCOS patients compared to 15 healthy controls (p < 0.05). In contrast, the frequency of Tfr cells and Tfr/Tfh2 ratio were significantly lower than healthy controls (p < 0.01). Importantly, among these cells, only the percentage of Tfh2 cells was positively correlated with the levels of testosterone (r = 0.513, p = 0.018). And the percentage of Tfr cells and Tfr/Tfh2 ratio were also positively correlated with the levels of testosterone (r = 0.567, p = 0.007; r = 0.434, p = 0.05) and prolactin (r = 0.511, p = 0.018; r = 0.490, p = 0.024).These new findings provide unique insights into dysregulated Tfh/Tfr cells in mediating the immunopathogenesis of PCOS patients.

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.

203-OR: Features of Human Th-Like Regulatory T Cells in the Pathogenesis of Type 1 Diabetes

Background: The imbalance between Tregs, Bregs and proinflammatory Th cells plays a vital role in the pathogenesis and development of T1D. Accumulating evidence has revealed that Treg has phenotypic plastic changes into other subsets under pathological conditions. However, whether Tregs convert into other immune cell types in T1D and the potential influencing factors remain unknown. This study aimed to examine the changes of Th-like Tregs in T1D patients and determine whether Th cells and IL-10+ Bregs play roles in Treg polarization. Methods: A total of 49 T1D patients and 20 healthy controls (HC) were recruited in this study. Flow cytometry was used to measure Treg plasticity (including CD4+Foxp3+IFN-γ+ Th1-like, CD4+Foxp3+IL-4+ Th2-like and CD4+Foxp3+IL-17+ Th17-like Tregs), the proportion of CD27+CD39hi Bregs, and Th cell subsets (including Th1, Th2 and Th17 cells) in the peripheral blood. Cytokines were detected by ELISA. Results: The frequencies of Th1-like Tregs and Th17-like Tregs in T1D were lower than those in HC, while the proportion of Th17 cells was significantly increased in T1D. The frequency of Bregs was markedly higher in T1D relative to HC, but IL-10 expression of Bregs was reduced. Interestingly, compared with lean and overweight patients, Th1-like Tregs were higher in patients with normal weight, but Treg plasticity was not affected by disease status (age at diagnosis, islet autoantibodies and β-cell function) or T1D risk loci (rs3118470 or rs2104286) in IL2RA. Additionally, positive correlations between Th1-like Tregs and Th17-like Tregs and between Bregs and Th2-like Tregs were observed in T1D. Meanwhile, the frequency of Th1-like Tregs was negatively related to IL-6 and the frequency of Th2-like Tregs was negatively associated with TNF-α. Conclusion: Our results firstly explored the features of human Th-like Tregs in the pathogenesis in T1D, which might contribute to improve our understanding of the pathogenic mechanisms of T1D. Disclosure Y.Qin: None. Y.Li: None. L.Dai: None. Q.Wei: None. M.Huang: None. M.Zhang: None. Funding National Natural Science Foundation of China (81974103, 82000747)

B cell subsets in adult-onset Still’s disease: potential candidates for disease pathogenesis and immunophenotyping

BackgroundAdult-onset Still’s disease (AOSD) is a systemic autoinflammatory disorder of unknown etiology. B cells are critical participants in different rheumatic diseases, and their roles in AOSD are rarely investigated. This study aimed to unveil the B cell subset features in AOSD and provide evidence for B cell-based diagnosis and targeted therapies of AOSD.MethodsB cell subsets in the peripheral blood of AOSD patients and healthy controls (HCs) were detected by flow cytometry. Firstly, the frequencies of B cell subsets were compared. Then, the correlation analysis was performed to explore the correlation between B cell subsets and clinical manifestations in AOSD. Finally, unbiased hierarchical clustering was performed to divide AOSD patients into three groups with different B cell subset features, and the clinical characteristics of the three groups were compared.ResultsThe frequencies of B cell subsets were altered in AOSD patients. Disease-promoting subsets (such as naïve B cells, double negative B cells (DN B cells), and plasmablasts) increased, and potential regulatory subsets (such as unswitched memory B cells (UM B cells) and CD24hiCD27+ B cells (B10 cells)) decreased in the peripheral blood of AOSD patients. In addition, the altered B cell subsets in AOSD correlated with the clinical and immunological features, such as immune cells, coagulation features, and liver enzymes. Intriguingly, AOSD patients could be divided into three groups with distinct B cell immunophenotyping: group 1 (naïve B cells-dominant), group 2 (CD27+ memory B cells-dominant), and group 3 (precursors of autoantibody-producing plasma cells-dominant). Moreover, these three group patients demonstrated differential manifestations, including immune cells, liver or myocardial enzymes, coagulation features, and systemic score.ConclusionsB cell subsets are significantly altered in AOSD patients, potentially contributing to the disease pathogenesis. These findings would inspire B cell-based diagnosis and targeted therapies for this refractory disease.

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 .

Immunologic profiles in patients with chronic limb-threatening ischemia undergoing endovascular revascularization.

Inflammation and immune dysregulation have been associated with adverse outcomes in cardiovascular disease. There is limited understanding of the association of different profiles of white blood cell (WBC) subsets and red cell distribution width (RDW) in patients with chronic limb-threatening ischemia (CLTI). Patients with CLTI undergoing endovascular revascularization in our single-center, tertiary care hospital from 2017 to 2019, who had a preceding complete blood count (CBC) with WBC differentials (n =213), were included in the analysis. Patient characteristics, laboratory values, and clinical outcomes were collected. Cox proportional hazards regression models were used to assess for associations between all-cause mortality and leukocyte subset; multivariate analysis was used to account for confounders. Kaplan-Meier curves were generated to depict survival censored at 1 year postrevascularization using baseline CBC indices. Adjusting for confounders, elevated RDW was associated with increased mortality (continuous per % increase, adjusted hazard ratio [HR] 1.33, p < 0.001). Baseline lymphopenia was associated with mortality in univariate analysis. Other leukocyte subtypes were not associated with mortality outcomes in our population. Exploratory analysis showed negative deflections in ∆WBC from pre- to postprocedure day 1 were affiliated with increased mortality when adjusted for age, sex, race, chronic kidney disease, and baseline hemoglobin (∆WBC HR 1.16, p = 0.004). Further exploratory analysis showed an association between RDW and all-comers readmission. The utilization of a periprocedural WBC subset differential can be a useful adjunct to risk-stratify patients with CLTI undergoing endovascular revascularization. Further studies are needed to understand potential ways to modulate immune dysregulation so as to improve mortality outcomes.

Characterization of antigen-specific CD8+ memory T cell subsets in peripheral blood of patients with multiple sclerosis.

Increasing evidence indicates the importance of CD8+ T cells in autoimmune attack against CNS myelin and axon in multiple sclerosis (MS). Previous research has also discovered that myelin-reactive T cells have memory phenotype functions in MS patients. However, limited evidence is available regarding the role of CD8+ memory T cell subsets in MS. This study aimed to explore potential antigen-specific memory T cell-related biomarkers and their association with disease activity. The myelin oligodendrocyte glycoprotein (MOG)-specific CD8+ memory T cell subsets and their related cytokines (perforin, granzyme B, interferon (IFN)-γ) and negative co-stimulatory molecules (programmed cell death protein 1 (PD-1), T- cell Ig and mucin domain 3 (Tim-3)) were analyzed by flow cytometry and real-time PCR in peripheral blood of patients with relapsing-remitting MS. We found that MS patients had elevated frequency of MOG-specific CD8+ T cells, MOG-specific central memory T cells (TCM), MOG-specific CD8+ effector memory T cells (TEM), and MOG-specific CD8+ terminally differentiated cells (TEMRA); elevated granzyme B expression on MOG-specific CD8+ TCM; and, on MOG-specific CD8+ TEM, elevated granzyme B and reduced PD-1 expression. The Expanded Disability Status Scale score (EDSS) in MS patients was correlated with the frequency of MOG-specific CD8+ TCM, granzyme B expression in CD8+ TCM, and granzyme B and perforin expression on CD8+ TEM, but with reduced PD-1 expression on CD8+ TEM. The dysregulation of antigen-specific CD8+ memory T cell subsets, along with the abnormal expression of their related cytokines and negative co-stimulatory molecules, may reflect an excessive or persistent inflammatory response induced during early stages of the illness. Our findings strongly suggest positive regulatory roles for memory T cell populations in MS pathogenesis, probably via molecular mimicry to trigger or promote abnormal peripheral immune responses. Furthermore, downregulated PD-1 expression may stimulate a positive feedback effect, promoting MS-related inflammatory responses via the interaction of PD-1 ligands. Therefore, these parameters are potential serological biomarkers for predicting disease development in MS.

Accurate enumeration of leukocyte subsets in peripheral blood with a 15-color immunoprofiling panel on a Cytek ®spectral flow cytometer

Abstract A white blood cell (WBC) differential is routinely performed on automated hematology analyzers as a general health check to evaluate immune status of patients. The recent advances in high parameter flow cytometry, especially full spectral flow cytometry, enable the analysis of more comprehensive leukocyte subsets to meet the increased demands in clinical research. We designed a 15-color, single-tube panel to identify and enumerate all major subsets of leukocytes on a Cytek full spectral flow cytometer. Human peripheral blood collected in K2EDTA, Heparin, ACD and Cyto-chex blood tubes were stained with 15 cFluor ®conjugated mAbs with a Lyse No Wash method and analyzed on a Cytek spectral flow cytometer. The performance of this assay was evaluated side by side with BD Multitest™ 6-color TBNK assay on BD FACSCanto II system on 13 healthy donor samples. Assay precision was examined on 12 donor samples in triplicates on three consecutive days. This 15-color panel identifies more than 20 leukocyte subpopulations: including neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells, monocyte subsets, T cell subsets, B cells, NK cell subsets. The results are comparable to that from BD 6-Color TBNK assay with R 2=0.961, 0.986, 0.940, 0.954, and 0.930 for CD3+, CD4+, CD8+ T, B and NK cells respectively. The correlations for all subsets are statistically significant with P &amp;lt; 0.001. The coefficient of variations (CV) of intra assay precision for all cell populations of interest are less than 25%. Cytek’s single-tube 15-color pan leukocyte panel demonstrated an effective, high sensitivity flow cytometry approach that can be used for monitoring immune cell subsets in peripheral blood in discovery and translational research.

Single-cell profiling of peripheral blood and muscle cells reveals inflammatory features of juvenile dermatomyositis.

Introduction: Juvenile dermatomyositis (JDM) is a rare yet serious childhood systemic autoimmune condition that primarily causes skin rashes and inflammatory myopathy of the proximal muscles. Although the associated immune response involves the innate and adaptive arms, a detailed analysis of the pertinent immune cells remains to be performed. This study aims to investigate the dynamic changes of cell type, cell composition and transcriptional profiles in peripheral blood and muscle tissues, and in order to clarify the involvement of immune cells in the pathogenesis of JDM and provide a theoretical reference for JDM. Methods: Single-cell RNA sequencing combined with bioinformatic analyses were used to investigate the dynamic changes in cell composition and transcriptional profiles. Results: Analysis of 45,859 cells revealed nine and seven distinct cell subsets in the peripheral blood and muscle tissues respectively. IFITM2+ and CYP4F3+ monocytes were largely produced, and CD74+ smooth muscle cells (SMCs) and CCL19+ fibroblasts were identified as inflammatory-related cell subtypes in JDM patients, exhibiting patient-specific cell population heterogeneity.The dynamic gene expression patterns presented an enhanced type I interferon response in peripheral blood monocytes and T-cells, and SMCs and fibroblasts in muscle of untreated JDM patients. EGR1 and IRF7 may play central roles in the inflammation in both CD74+ SMCs and CCL19+ fibroblasts. Moreover, inflammatory-related monocytes could regulate T-cells, and the interaction between immune cells and SMCs or fibroblasts in muscle was enhanced under the inflammatory state. Conclusions: Immune dysregulation is one of the key pathogenic factors of JDM, and type I interferon responses are significantly enhanced in peripheral blood Monos and T cells as well as SMCs and fibroblasts. EGR1 and IRF7 may play central roles in the inflammation and are considered as potential therapeutic targets for JDM.

Abstract LB338: A novel class of T cell-activators targeting germline encoded TCRβ chains promotes antitumor activity in PD1-refractory models through expansion of a clonally enriched effector memory T cell subset

Abstract Background: Here we describe a new class of selective T cell activator molecule that combines antibody targeting of the variable (V) β regions of the T cell receptor (TCR) with a costimulatory cytokine. STAR0602, a molecule that selectively targets T cells expressing Vβ6 and Vβ10 TCRs and delivers IL-2R co-stimulation, activates and expands human Vβ6 and Vβ10 T cells that are enriched in tumor infiltrating lymphocytes (TIL) relative to other Vβ subsets. Using this molecular design, STAR0602 and molecules targeting other Vβ T cell subsets simultaneously engage a novel, non-clonal mode of TCR activation with cytokine co-stimulation. Methods: The effects of STAR0602 on activation and expansion of primary human T cells were assessed in vitro by flow cytometry, homogeneous time-resolved fluorescence, TCRseq, and NanoString. Several murine surrogate molecules targeting different Vβ T cell subsets with a range of baseline frequencies were tested in murine syngeneic tumor models. Tumors were excised for phenotyping of TILs including tetramer staining of antigen specific T cells using flow cytometry, and scRNAseq/TCRseq. Results: In vitro, STAR0602 induced TCR signaling and IL-2R pathway activation in human T cells that preceded expansion of Vβ6/Vβ10 T cells that acquired an atypical central memory T cell (TCM) phenotype. Across PD1-refractory syngeneic murine tumor models, monotherapy with fusion molecules targeting different Vβ TCR and delivering IL-2R co-stimulation induced potent anti-tumor activity that was associated with expansion and accumulation of targeted Vβ T cell subsets in blood and tumors. In vivo anti-tumor activity was dependent on the accumulation of effector memory T or TCM Vβ T cells expressing a novel effector gene signature. Additional analysis of TILs showed increased clonality in de novo expanded Vβ T cells compared to controls in addition to increased numbers of tumor-specific T cells. Conclusions: To our knowledge, the use of therapeutic antibodies to selectively target and expand Vβ T cell subsets and thereby modulate the germline TCR repertoire for therapeutic benefit is novel. Here we show that STAR0602 selectively binds and activates human Vβ6/Vβ10 T cells and promotes a novel TCM phenotype. Murine surrogate molecules targeting different Vβ T cell subsets demonstrated potent and durable single-agent anti-tumor activity even when targeting low frequency Vβ subsets. These expanded Vβ T cells appear to drive enhanced anti-tumor activity due to their increased effector potentials, novel memory phenotype, and a striking enrichment in clonal diversity. In summary, STAR0602 and similar Vβ TCR-targeting constructs have the potential to remodel the adaptive immune response to solid tumors that are refractory to anti-PD1 therapy. A Phase 1/2 clinical trial of STAR0602 is ongoing in patients with advanced metastatic cancers. Citation Format: Jacques Moisan, Madan Katraggada, Jonathan Hsu, Jessica Lowry, Wei Huang, Jian Tang, Roya Servattalab, Gurkan Guntas, Raj Chopra, Zhen Su, Andrew Bayliffe. A novel class of T cell-activators targeting germline encoded TCRβ chains promotes antitumor activity in PD1-refractory models through expansion of a clonally enriched effector memory T cell subset [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB338.

Abstract 4420: Carbohydrate-CD11b engagement enhances differentiation of tumor-associated myeloid cells in immunotherapy of solid cancers

Abstract Background: Methods to perturb tumor-associated myeloid cell function are being actively pursued to overcome these challenges in immunotherapy and search for a cure. CD11b is a potential therapeutic target to modulate suppressive myeloid derived cells and induce tumor-reactive T-cell responses. However, CD11b can bind to multiple carbohydrate ligands that trigger a variety of myeloid cell functions like adhesion, migration, and even phagocytosis and proliferation. This has created a major challenge to understand the molecular basis by which CD11b converts differences in receptor-ligand binding into subsequent immune signaling responses, and to use this knowledge for therapeutic development. Methods: This study investigated a previously reported carbohydrate ligand, BG34-200, that showed anti-tumor effect through modulating CD11b+ cells. Applying modern approaches of peptide microarray, multi-parameter FACS analysis, cellular/molecular immunological technology, advanced microscopic imaging, and transgenic mouse models of solid cancers, we studied the BG34-200-CD11b carbohydrate-protein engagement and immunological changes it triggered in the context of solid cancers including osteosarcoma, melanoma and pancreatic cancer. Results: Our results show that BG34-200 can mediate direct, specific, multi-site and multivalent binding to integrin CD11b to trigger cellular differentiation in a suppressive tumor-associated myeloid cell subset. In melanoma, osteosarcoma, and pancreatic cancer, we detected this rare myeloid cell subset in blood, which displayed an inflammatory monocyte phenotype and exhibited immunosuppressive behavior. BG34-200-CD11b engagement could induce differentiation of these tumor-associated inflammatory monocytes into dendritic cells by inducing phagocytosis that effectively trigger F-actin cytoskeletal rearrangement and intrinsic ICAM-1 clustering important for antigen presentation and effector T cell stimulation. Conclusions: Our findings improve understanding of molecular basis by which CD11b converts difference in carbohydrate ligands into subsequent immune signaling responses, may lead to development of safe and novel therapies modulating myeloid derived cell function for immunotherapy of solid cancers. Citation Format: Mei Zhang. Carbohydrate-CD11b engagement enhances differentiation of tumor-associated myeloid cells in immunotherapy of solid cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4420.