High-dimensional Flow Cytometry Research Articles

OP05 High-dimensional single-cell analysis identifies cellular signatures associated with response to vedolizumab therapy in ulcerative colitis

Abstract Background The unpredictability of response to biological therapy in ulcerative colitis (UC) patients, including vedolizumab (VDZ), an anti-α4β7 integrin antibody, poses a serious challenge for medical care. This study aims to identify the relevant cellular and molecular signatures that mark response to VDZ by combining single-cell transcriptomics (scRNAseq) and high-dimensional flow cytometry (Cytek). Methods Gut biopsies and peripheral blood mononuclear cells (PBMCs) from patients with active UC were collected 2 weeks prior and 14 weeks after starting VDZ therapy. Response was evaluated at week 14 based on endoscopy and Physician Global Assessment. Transcriptome profiles of 159.188 and 95.057 high-quality single cells were generated from biopsies and PBMCs, respectively. Additional 797.670 CD45+ mucosal cells and 900.000 PBMCs were processed for validating scRNAseq results by Cytek. Results From a total of 25 UC patients (mean age 41±12.7 years, 52% female, 36% anti-TNFα naive), 15 patients (60%) reached response at week 14. Patients with no prior use of anti-TNFα were more likely to achieve remission with VDZ compared to anti-TNFα exposed patients (89% vs 44% response rate, respectively). Analysis of cell abundances revealed a partial recovery of the epithelial and stromal cell mucosal compartments in responders at week 14. Both scRNAseq and Cytek show that in responders VDZ prevented T cells (CD8+IL17+ and CD4+PD1+ subsets) and innate immune cells (NKs, DCs and inflammatory monocytes) from entering mucosa, resulting in their enrichment in PBMCs. In contrast, non-response to VDZ was marked by an expansion of inflammatory fibroblasts and activated endothelial cells, and depletion of most epithelial subsets. VDZ retained CD4 T cells in PBMCs in non-responders, but failed to halt active mucosal inflammation at week 14. Strikingly, involvement of the innate immune cells in UC inflammation prior to VDZ treatment was much more prominent in non-responders who showed higher baseline abundance of NKs, ILCs, γδ-T cells, DCs and inflammatory monocytes in their peripheral blood and mucosa compared to responders. While VDZ inhibited integrin β7 expression on circulating T cells in all patients, only in responders it coincided with the lower mucosal abundance of lymphocytes, suggesting that T cells in non-responders might employ alternative, β7-independent trafficking routes. Conclusion This is the largest prospective scRNAseq study addressing the response to biological therapy in UC. We showed that active, innate immune cell-mediated inflammation marks the primary non-response to VDZ that persists prior to and throughout the treatment, accompanied by β7-independent lymphocyte migration.

THE EFFECTS OF BIOLOGIC AND SMALL MOLECULE INHIBITOR THERAPY ON THE MUCOSAL IMMUNE SYSTEM IN ULCERATIVE COLITIS

Abstract Ulcerative colitis (UC) is a subtype of inflammatory bowel disease (IBD) that causes relapsing and remitting inflammation in the colon and rectum. Numerous treatment options are available for patients with UC including biologics and small molecule inhibitors that target the immune system. Although these therapies have dramatically improved options for patients with IBD, there remains a modest ceiling for their overall efficacy. Currently there are no biomarkers that can guide treatment decisions in patients with UC. Some studies have reported certain molecules or immune populations that are associated with treatment resistance or as predictors of treatment response. However, their clinical utility have yet to be fully determined because there is a paucity of longitudinal studies examining the direct effects of current biologics and small molecule inhibitors approved for UC on the immune biology of the colon mucosa. In this study, we perform longitudinal immune phenotyping using flow cytometry in patients with UC before and after therapy. Colon biopsy tissue was taken prior to treatment from 10 UC patients who were then followed and repeat biopsies were obtained from the same patients after 6-12 months of treatment. Tissue was analyzed using high dimensional flow cytometry with a panel of 24 markers of both lymphoid and myeloid lineages. Pre-treatment samples with inflammation correlated with higher populations of CD4+ T cells and CD4+ FOXP3 regulatory T cells also positive for TIGIT and GITR. Inflammation also corresponded with the presence of a CD33+ HLA-DR- population classically known as myeloid-derived suppressor cells (MDSC). Overall, therapy significantly decreased immune cell infiltration in the colon, most notably in B and T cell populations. However, certain treatments increased the MDSC population along with the stromal compartment of non-immune cells. A comprehensive analysis of the populations present during active inflammation and the treatment-induced changes in mucosal immunity will help identify biomarkers to predict response to therapy. In addition, examining the interplay between immune cells and the surrounding stroma can may lead to important findings that improve our understanding of each drug’s mechanism and efficacy. Future experiments with single cell RNA and T cell receptor sequencing on the same patients will identify effects of biologic therapy on the T cell repertoire and interactions between immune, epithelial, and stromal cells in the colonic mucosa. With the advent of novel sequencing and immune profiling assays, we now have the ability to harness the power of precision medicine to make more informed therapeutic decisions for IBD patients that improve clinical outcomes.

Cross-platform immunophenotyping of human peripheral blood mononuclear cells with four high-dimensional flow cytometry panels.

Immunophenotyping using high dimensional flow cytometry is a central component of human immune system multi-omic studies. We present four high parameter flow cytometry panels for deep immunophenotyping of human peripheral blood mononuclear cells (PBMC). This set of four 25+ color panels include 64 cell surface markers to resolve broad immune compartment populations, as well as activation and memory of specific T, B, natural killer (NK), and myeloid lineages. Common lineage bridging markers are integrated into each panel to allow for inter-panel quality control through major lineage frequency verification. These panels were developed using a five laser BD Symphony A5 conventional cytometer and successfully transferred to a five laser Cytek Aurora spectral cytometer capable of acquiring the panels. Nine representative PBMC samples were stained with the four phenotyping panels and acquired on both instruments to evaluate population frequency and visual staining patterns for gating between the systems. Both instruments produced comparable high quality flow cytometry data and supported our decision to acquire samples on the spectral cytometer moving forward. This modular set of panels and instrument performance metrics provide guidelines for designing flow cytometry experiments suitable for longitudinal or cross-sectional immune profiling.

AliGater: a framework for the development of bioinformatic pipelines for large-scale, high-dimensional cytometry data.

AliGater is an open-source framework to accelerate the development of bioinformatic pipelines for the analysis of large-scale, high-dimensional flow cytometry data. AliGater provides a Python package for automatic feature extraction workflows, as well as building blocks to construct analysis pipelines. We illustrate the use of AliGater in a high-resolution flow cytometry-based genome-wide association study on 46 immune cell populations in 14288 individuals. Source code and documentation at https://github.com/LudvigEk/aligater and https://aligater.readthedocs.io.

Technology meets TILs: Deciphering Tcell function in the -omics era.

T cells are at the center of cancer immunology because of their ability to recognize mutations in tumor cells and directly mediate cancer cell killing. Immunotherapies to rejuvenate exhausted Tcell responses have transformed the clinical management of several malignancies. In parallel, the development of novel multidimensional analysis platforms, such as single-cell RNA sequencing and high-dimensional flow cytometry, has yielded unprecedented insights into immune cell biology. This convergence has revealed substantial heterogeneity of tumor-infiltrating immune cells in single tumors, across tumor types, and among individuals with cancer. Here we discuss the opportunities and challenges of studying the complex tumor microenvironment with -omics technologies that generate vast amounts of data, highlighting the opportunities and limitations of these technologies with a particular focus on interpreting high-dimensional studies of CD8+ Tcells in the tumor microenvironment.

PS-P13-6: DEFINING THE RELATIONSHIP BETWEEN IMMUNE TRAITS AND LIPIDS IN TWINSUK

Objective: The primary aim of this work was to evaluate associations of distinct immune cell subtypes and circulating lipids in a general population setting. Methods: The associations between 3,855 immune traits and lipids (HDL-C, LDL-C, TC, TG, non-HDL-C) were examined in 1,491 individuals from the TwinsUK cohort. Immune traits were defined by high-dimensional flow-cytometry which enabled in-depth assessment of all major lymphocyte and myeloid subsets in peripheral blood. Information on immune cell subset frequency (FREQ) and mean fluorescence intensity (MFI) was available. A linear mixed model regression analysis adjusted for family structure, gender, age, BMI and batch effect was used to examine the associations between immune traits and lipids. A sub-analysis in 441 individuals and 122 lipid metabolites quantified using nuclear magnetic resonance spectroscopy was undertaken to investigate the associations of 40 significant immune traits and lipoprotein particles. Secondary analyses explored the associations of lipids and immune traits with pulse wave velocity (PWV). A mediation model was constructed to test mediation effects of an immune marker on the total effect of lipids on PWV. Results: Significant associations were observed between 46 MFI and 6 FREQ immune traits and lipids (Bonferroni P < 2.45x10–5). These included associations between circulating lipids and different myeloid or lymphoid subsets including monocytes, NK cells and T cells. The top associations involved the expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) cytokine on classical (cM) and intermediate (iM) monocyte subsets. Decreased levels of non-HDL-C [cMP = 6.98x10–10, iMP = 3.72x10–7], LDL-C (cMP = 3.28x10–7, iMP = 1.75x10–5) and TG [cMP = 1.18x10–9, iMP = 2.97x10–6], and increased levels of HDL-C (cMP = 3.64x10–12, iMP = 1.44x10–10) were associated with higher TACI expression on both cM and iM subsets. Significant associations were identified between TACI expression and 13 lipid metabolites (Bonferroni P < 1.36x10–4). All linked metabolites referred to HDL related measures. Increased apolipoprotein A1 levels, total cholesterol in HDL and HDL2 and large and medium HDL related particles were associated with increased TACI expression on cM and iM subsets. The strongest association found between total cholesterol in HDL2-C and TACI expression levels on cM (P = 3.57x10–6). Mediation analysis showed that TACI expression on monocytes partially mediates the effect (∼10%) of TG on PWV. Conclusions: This study provides the first comprehensive picture of lipid-immune cell associations in the circulatory system. Interesting novel immune cell subset alterations and insights into the relationship between lipids, PWV and immunity have extended our knowledge. Further work is required to dissect the role of immune responses in cardiovascular disease.

Design, optimisation and standardisation of a high-dimensional spectral flow cytometry workflow assessing T-cell immunophenotype in patients with melanoma.

Despite the success of immune checkpoint blockade, most metastatic melanoma patients fail to respond to therapy or experience severe toxicity. Assessment of biomarkers and immunophenotypes before or early into treatment will help to understand favourable responses and improve therapeutic outcomes. We present a high-dimensional approach for blood T-cell profiling using three multi-parameter cytometry panels: (1) a TruCount panel for absolute cell counts, (2) a 27-colour spectral panel assessing T-cell markers and (3) a 20-colour spectral panel evaluating intracellular cytokine expression. Pre-treatment blood mononuclear cells from patients and healthy controls were cryopreserved before staining across 11 batches. Batch effects were tracked using a single-donor control and the suitability of normalisation was assessed. The data were analysed using manual gating and high-dimensional strategies. Batch-to-batch variation was minimal, as demonstrated by the dimensionality reduction of batch-control samples, and normalisation did not improve manual or high-dimensional analysis. Application of the workflow demonstrated the capacity of the panels and showed that patients had fewer lymphocytes than controls (P = 0.0027), due to lower naive CD4+ (P = 0.015) and CD8+ (P = 0.011) T cells and follicular helper T cells (P = 0.00076). Patients showed trends for higher proportions of Ki67 and IL-2-expressing cells within CD4+ and CD8+ memory subsets, and increased CD57 and EOMES expression within TCRγδ+ T cells. Our optimised high-parameter spectral cytometry approach provided in-depth profiling of blood T cells and found differences in patient immunophenotype at baseline. The robustness of our workflow, as demonstrated by minimal batch effects, makes this approach highly suitable for the longitudinal evaluation of immunotherapy effects.

Tumor-Associated CD19+CD39- B Regulatory Cells Deregulate Class-Switch Recombination to Suppress Antibody Responses.

B cells are an essential component of humoral immunity. Their primary function is to mount antigen-specific antibody responses to eliminate pathogens. Despite an increase in B-cell number, we found that serum-IgG levels were low in patients with breast cancer. To solve this conundrum, we used high-dimensional flow cytometry to analyze the heterogeneity of B-cell populations and identified a tumor-specific CD19+CD24hiCD38hi IL10-producing B regulatory (Breg)-cell subset. Although IL10 is a Breg-cell marker, being an intracellular protein, it is of limited value for Breg-cell isolation. Highly expressed Breg-cell surface proteins CD24 and CD38 also impede the isolation of viable Breg cells. These are hurdles that limit understanding of Breg-cell functions. Our transcriptomic analysis identified, CD39-negativity as an exclusive, sorting-friendly surface marker for tumor-associated Breg cells. We found that the identified CD19+CD39‒IL10+ B-cell population was suppressive in nature as it limited T helper-cell proliferation, type-1 cytokine production, and T effector-cell survival, and augmented CD4+FOXP3+ regulatory T-cell generation. These tumor-associated Breg cells were also found to restrict autologous T follicular helper-cell expansion and IL21 secretion, thereby inhibiting germinal transcript formation and activation-induced cytidine deaminase expression involved in H-chain class-switch recombination (CSR). This isotype-switching abnormality was shown to hinder B-cell differentiation into class-switched memory B cells and subsequent high-affinity antibody-producing plasma B cells, which collectively led to the dampening of IgG-mediated antibody responses in patients with cancer. As low IgG is associated with poor prognosis in patients with cancer, Breg-cell depletion could be a promising future therapy for boosting plasma B cell-mediated antibody responses.

2141. Investigating a (Natural) Killer by Studying the Victims: Searching for Mechanisms to Selectively Inhibit NK-cell Immunosuppressive Function to Improve Vaccine Responses

Abstract Background Decades of effort have yet to deliver an efficacious vaccine for pathogens such as HIV, despite major revelations about HIV antigens and development of new vaccine platforms. Innovative outside-the-box approaches are needed to overcome immunological roadblocks to the success of past and current vaccine regimens. We discovered that natural killer (NK) cells are a potent obstacle to vaccine success through perforin-dependent suppression of activated CD4 T cell responses early after vaccination. This killing reduces the quantity and quality of antibody responses. Yet, the features of NK cells that enable this activity or determine susceptibility of specific subsets of T cells remain ill-defined. Methods We used transgenic virus-specific mouse CD4 T cells to detect very early changes in responding T cells immediately proximal to immunoregulatory killing by NK cells. Transgenic T cells were seeded in recipient mice, depleted or not of NK cells, and then infected with lymphocytic choriomeningitis virus. At early time points post-infection, these T cells were isolated for single-cell RNA-sequencing or high-dimensional flow cytometry analyses. These data revealed transcriptomic and proteomic features of subsets of activated CD4 T cells whose presence was enabled or enhanced by the absence of NK cells. Results Perforin-dependent killing of CD4 T cells by NK cells is measurable on days 2 and 3 post-infection. Analysis of the pool of responding T cells on day 3 revealed phenotypic skewing away from follicular helper T cell-type responses to Th1-type responses in the presence of NK cells. Single cell RNA sequencing and high-dimensional spectral flow cytometry reveal clusters of T cells that are markedly enriched in the absence of NK cells. Conclusion We created a model system permitting characterization of the population of T cells targeted by NK cells after infection or immunization. Elucidation of features of the targeted T cells will provide mechanistic insights in the receptor-ligand system enabling this biology, improve our understanding of how NK cells influence the pool of responding T cells, and facilitate discovery of the corresponding subset of NK cells responsible for this activity. These discoveries are likely to facilitate development of methods to enhance vaccine responses. Disclosures All Authors: No reported disclosures.

The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis.

The epididymis functions as transition zone for post-testicular sperm maturation and storage and faces contrasting immunological challenges, i.e. tolerance towards spermatozoa vs. reactivity against pathogens. Thus, normal organ function and integrity relies heavily on a tightly controlled immune balance. Previous studies described inflammation-associated tissue damage solely in the distal regions (corpus, cauda), but not in the proximal regions (initial segment, caput). To understand the observed region-specific immunity along the epididymal duct, we have used an acute bacterial epididymitis mouse model and analyzed the disease progression. Whole transcriptome analysis using RNAseq 10 days post infection showed a pro-inflammatory environment within the cauda, while the caput exhibited only minor transcriptional changes. High-dimensional flow cytometry analyses revealed drastic changes in the immune cell composition upon infection with uropathogenic Escherichia coli. A massive influx of neutrophils and monocytes was observed exclusively in distal regions and was associated with bacterial appearance and tissue alterations. In order to clarify the reasons for the region-specific differences in the intensity of immune responses, we investigated the heterogeneity of resident immune cell populations under physiological conditions by scRNASeq analysis of extravascular CD45+ cells. Twelve distinct immune cell subsets were identified, displaying substantial differences in distribution along the epididymis as further assessed by flow cytometry and immunofluorescence staining. Macrophages constituted the majority of resident immune cells and were further separated in distinct subgroups based on their transcriptional profile, tissue location and monocyte-dependence. Crucially, the proximal and distal regions showed striking differences in their immunological landscapes. These findings indicate that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for addressing the contrasting immunological challenges and thus, preserving tissue integrity and organ function.

Improving head and neck cancer therapies by immunomodulation of the tumour microenvironment.

Targeted immunotherapy has improved patient survival in head and neck squamous cell carcinoma (HNSCC), but less than 20% of patients produce a durable response to these treatments. Thus, new immunotherapies that consider all key players of the complex HNSCC tumour microenvironment (TME) are necessary to further enhance tumour-specific T cell responses in patients. HNSCC is an ideal tumour type in which to evaluate immune and non-immune cell differences because of two distinct TME aetiologies (human papillomavirus (HPV)-positive and HPV-negative disease), multiple anatomic sites for tumour growth, and clear distinctions between patients with locally advanced disease and those with recurrent and/or metastatic disease. Recent technological and scientific advancements have provided a more complete picture of all cellular constituents within this complex TME and have evaluated the interplay of both immune and non-immune cells within HNSCC. Here, we include a comprehensive analysis of the complete ecosystem of the HNSCC TME, performed utilizing data-rich resources such as The Cancer Genome Atlas, and cutting-edge techniques, such as single-cell RNA sequencing, high-dimensional flow cytometry and spatial multispectral imaging, to generate improved treatment strategies for this diverse disease.

Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer.

BackgroundDespite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear.MethodsTo better...

Chagasic cardiomyopathy is marked by a unique signature of activated CD4+ T cells

Chagas disease is a neglected tropical disease in Latin America and an imported emerging disease worldwide. Chronic Chagas disease cardiomyopathy (CCC) is the most prominent clinical form and can lead to heart failure, thromboembolism, and sudden death. While previous reports have supported a role for CD4+ T lymphocytes in the pathogenesis of CCC a comprehensive analysis of these cells during different clinical forms is lacking. Here, we used high-dimensional flow cytometry to assess the diversity of circulating CD4+ T cells in patients with distinct clinical forms. We found increased frequencies of CD4+CD69+ T cells in patients compared to controls. CD39+ regulatory T cells, represented by mesocluster 6 were reduced in mild CCC patients compared to controls. Cytotoxic CD4+ T cells co-expressing granzyme B and perforin were expanded in patients with Chagas disease and were higher in patients with mild CCC compared to controls. Furthermore, patients with mild CCC displayed higher frequencies of multifunctional effector memory CD4+ T cells. Our results demonstrate an expansion in activated CD4+ T cells and a decrease in a functional subset of regulatory T cells associated with the onset of Chagas cardiomyopathy, suggesting their role in the establishment of cardiac lesions and as potential biomarkers for disease aggravation.

Immune phenotypes that are associated with subsequent COVID-19 severity inferred from post-recovery samples

Severe COVID-19 causes profound immune perturbations, but pre-infection immune signatures contributing to severe COVID-19 remain unknown. Genome-wide association studies (GWAS) identified strong associations between severe disease and several chemokine receptors and molecules from the type I interferon pathway. Here, we define immune signatures associated with severe COVID-19 using high-dimensional flow cytometry. We measure the cells of the peripheral immune system from individuals who recovered from mild, moderate, severe or critical COVID-19 and focused only on those immune signatures returning to steady-state. Individuals that suffered from severe COVID-19 show reduced frequencies of T cell, mucosal-associated invariant T cell (MAIT) and dendritic cell (DC) subsets and altered chemokine receptor expression on several subsets, such as reduced levels of CCR1 and CCR2 on monocyte subsets. Furthermore, we find reduced frequencies of type I interferon-producing plasmacytoid DCs and altered IFNAR2 expression on several myeloid cells in individuals recovered from severe COVID-19. Thus, these data identify potential immune mechanisms contributing to severe COVID-19.

GeoWaVe: geometric median clustering with weighted voting for ensemble clustering of cytometry data.

Clustering is an unsupervised method for identifying structure in unlabelled data. In the context of cytometry, it is typically used to categorize cells into subpopulations of similar phenotypes. However, clustering is greatly dependent on hyperparameters and the data to which it is applied as each algorithm makes different assumptions and generates a different 'view' of the dataset. As such, the choice of clustering algorithm can significantly influence results, and there is often not one preferred method but different insights to be obtained from different methods. To overcome these limitations, consensus approaches are needed that directly address the effect of competing algorithms. To the best of our knowledge, consensus clustering algorithms designed specifically for the analysis of cytometry data are lacking. We present a novel ensemble clustering methodology based on geometric median clustering with weighted voting (GeoWaVe). Compared to graph ensemble clustering methods that have gained popularity in single-cell RNA sequencing analysis, GeoWaVe performed favourably on different sets of high-dimensional mass and flow cytometry data. Our findings provide proof of concept for the power of consensus methods to make the analysis, visualization and interpretation of cytometry data more robust and reproducible. The wide availability of ensemble clustering methods is likely to have a profound impact on our understanding of cellular responses, clinical conditions and therapeutic and diagnostic options. GeoWaVe is available as part of the CytoCluster package https://github.com/burtonrj/CytoCluster and published on the Python Package Index https://pypi.org/project/cytocluster. Benchmarking data described are available from https://doi.org/10.5281/zenodo.7134723. Supplementary data are available at Bioinformatics online.

CD25 Targeting Eliminates Regulatory T Cells and CD25+ Blasts in Acute Myeloid Leukemia

Background Acute Myeloid leukemia (AML) is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. CD25 Mab (also referred to as RG6292 and RO7296682) is an afucosylated, IL-2 non-blocking human IgG1 antibody, shown to efficiently deplete immunosuppressive regulatory T cells (Tregs) in humans and solid tumour models (Kolben 2021) whilst allowing binding of IL-2 to effector T cells and the induction of anti-tumour adaptive immune responses (Solomon, Amann et al. 2020). CD25 Mab binds to CD25+ target cells and its crystallisable fragment (Fc) to Fc receptors (FcR) expressed on the surface of effector cells, such as FcgRIIIa on Natural Killer (NK) cells, monocytes and macrophages. It mediates killing of target cells through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). CD25 Mab is currently being investigated in a phase I dose escalation monotherapy study (NCT04158583) or in combination with atezolizumab (NCT04642365). In AML patients, a higher frequency of immunosuppressive bone marrow Tregs was observed as compared to that of healthy donors and their abundance correlated with poorer outcome (Szczepanski, Szajnik et al. 2009, Han, Dong et al. 2018, Dong, Han et al. 2020). In addition, CD25 is also expressed on a subset of AML cells and may be restricted to leukemic stem cells (LSC) or cells with a progenitor phenotype (Angelini, Ottone et al. 2015, Bertolini, Kageyama et al. 2018, Aref, Azmy et al. 2020). Considering the critical role of LSC in the propagation of the disease and the high relapse rate in AML, it would be important to understand if CD25 targeting could deepen responses and help prevent relapse in patients. In this study, we hypothesized that CD25 Mab may have a dual mode of action whereby it depletes suppressive Tregs and has a direct cytotoxic effect on CD25+ AML cells. Methods We conducted an in-depth characterization of bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry (up to 39 cellular markers) and RNA sequencing using computational analysis. Moreover, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab on Tregs and CD25+ AML cells, as compared to isotype control antibody. Results We found that the abundance of Tregs and CD25+ AML cells correlated strongly with that of the blood in 11 patients with time-matched samples, indicating that blood samples could be used for the identification of predictive biomarkers. Moreover, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing internal tandem duplication mutations in the FLT3 gene (FLT3-ITD). Furthermore, all four patients treated with a hypomethylating agent in combination with venetoclax displayed detectable levels of CD25+ AML cells. We adopted a patient-centric approach (n=14 patients) to study AML clusters with CD25 expression, due to the high inter-patient heterogeneity. Interestingly, CD25+ clusters most commonly expressed CD34, an LSC marker indicating a preferential expression in immature AML cells. Finally, ex vivo treatment of primary AML patient samples with CD25 Mab led to the specific killing of CD25+ AML cells and Tregs by allogeneic NK cells, as compared to isotype control treatment. Taken together, these results provide a proof of concept of CD25 Mab's dual mode of action in AML patient samples. Conclusions Using high dimensional flow cytometry and computational analysis, we provide a deep characterization of AML patient samples and highlight the heterogeneity in the pattern of expression of common AML targets currently under clinical investigation. We dissected the expression of CD25 on healthy T cells and malignant cells and demonstrated that the blood offers a window into the bone marrow composition and could be used to ascertain Treg and CD25+ AML cells prevalence. Mode of action studies demonstrated that CD25 Mab depletes suppressive Tregs and has a direct cytotoxic effect on the CD25+ AML cells. CD25 targeting represents an attractive target for the treatment of AML, especially in patients where CD25 is expressed on LSC or immature AML cells. Our study warrants further exploration of CD25 Mab as combinatorial treatment with, for instance, FLT3 inhibitors or venetoclax.

Salvage with Sequential T Cell Redirection for Myeloma Patients Who Have Previously Progressed on T Cell Directed Therapy Is Associated with Significant Cellular Activation of the Immune Microenvironment

Background: With the recent approval of two anti-BCMA CAR-Ts and multiple anti-BCMA and anti-GPRC5D bispecific antibodies (BiAbs) in clinical trials, rational salvage strategies for patients who progress after these therapies is an area of unmet need in multiple myeloma. We have previously reported favorable outcomes for patients receiving sequential T cell directed therapies with depth of response and progression free survival (PFS) (Mouhieddine et al Blood (2021) 138 (Supplement 1: 821). However, the underlying immune mechanisms are not well known and would be essential to design sequential strategies combining different T cell directed therapies. Here we show that sequential T cell mediated therapies promote not only T cell engagement but also immune activation in natural killer (NK) and dendritic cells (DC) cellular subsets in patients with relapsed/refractory multiple myeloma (RRMM). Methods: We identified 15 RRMM patients who had progressed on a T cell directed therapy, either anti-BCMA CAR-T (n=10) or BiAbs (n=5) and then received another BiAb as a subsequent line of therapy. 70% of patients who progressed on anti-BCMA CAR-T (7/10) and 20% who progressed on BiAbs had interim therapies before receiving subsequent BiAbs therapy. The median time between the first T cell directed therapy and subsequent therapy was 5 months (range 0- 26 months). We profiled immune cell activation in peripheral blood (PB) by high dimensional flow cytometry and Olink Proteomics. Clinical data such as patient demographics, disease characteristics and post-clinical trial outcomes were collected retrospectively until July of 2022. Data and sample collection for this study was approved by the institutional review board (IRB) and follows the Declaration of Helsinki and International Conference on Harmonization Guidelines for Good Clinical Practice (IRB: GCO#: 11-1433 & GCO #: 18-00456). Results: We compared baseline immune cell populations of 15 RRMM patients in the PB post relapse on T cell mediated therapy and during subsequent BiAb treatment (Figure 1a). 93% of patients (14/15) received BiAbs therapy with a different target than their first T cell directed regimen. 73% of patients (11/15) received sequential BiAbs therapy in combination with an anti-CD38 monoclonal antibody. Patients had a median age of 63 years (range 43-89) at time of progression after the first T cell directed therapy. MM patients were heavily pre-treated with a median of 6 prior lines of therapy (range 3-16). All patients were triple-class refractory while 66% were penta-drug refractory. Immune profiling at time of progression on the first T cell directed therapy, was associated with increased expression of exhaustion markers on CD4+ and CD8+ T cells. Treatment with a second BiAb was associated with significant re-activation of endogenous T cells. Specifically, CD8+ T cells demonstrated increased expression of hallmark surface activation markers such as HLA-DR and CD69+ (p<0.05, Figure 1b), and this increase was consistent in CD4+ T cells as well (p<0.01, Figure 1b). Alongside T cell engagement we saw an increase in surface activation markers CD69+ and HLA-DR on NK and DC populations respectively (p<0.01). This indicates that the application of the second T cell directed therapy not only reactivates T cell compartment but potentiates widespread immune cell activation. Plasma cytokine analysis from these patients revealed elevated CXCL10, CXCL9, and TNFa during bispecific therapy; reinforcing the activation state of the patients during treatment. Although the CD4:CD8 ratios remain conserved, we observed a significant differentiation of CD8+ T cells through sequential therapy, with a reduction of naïve CD8+ T cells (p=0.022) and increase of effector memory CD8+ T cells (p<0.01). Conclusion: Our data suggests that patients who progressed on T cell directed therapy and went on to receive T cell mediated BiAbs, not only respond clinically, but also have hallmark expression of activation markers. T cells can be reactivated post relapse from T cell mediated therapies, indicating that T cell targeted treatments can be applied successfully in sequential order with favorable patient outcomes. We are currently performing immune profiling on more PB and bone marrow samples to further confirm these findings in a larger patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cellular Immune Composition in Multiple Myeloma Patients Associated with Variable Humoral Responses to Sars-Cov-2 Vaccination

Background: Multiple Myeloma (MM) patients are immune-deficient and mount suboptimal immune responses to SARS-CoV-2 mRNA vaccine, making them more susceptible to severe COVID-19 manifestations. We have previously described that MM patients undergoing certain therapies have compromised humoral immune responses even after receiving multiple doses of the SARS-CoV-2 mRNA vaccine (Aleman, et al. Cancer Cell 2022). However, the mechanisms underlying the suboptimal antibody responses observed in these patients are not understood. Emerging research suggests that specific cell populations within the immune milieu, such as Follicular helper (TFH) CD4+ T cells, help B cells mount a robust serological response (Cui, et al. 2021). In addition, Natural Killer (NK) cells have been known to play a protective role in SARS-CoV-2 infection (Fuentes-Villalobos, et al. 2022). We report here on the cellular immune composition highly associated with suboptimal humoral immune responses in MM patients. Methods: We longitudinally followed 31 MM patients with variable serological responses after receiving SARS-CoV-2 vaccination, as determined by quantification of anti-spike IgG antibody titers, and compared to 13 age matched healthy donors. We categorized patients based on their production of anti-spike IgG post full vaccination of two doses. Of our selected cohort, 14 patients had failed to produce detectable anti-spike IgG (MM sub-optimal), whereas the other 17 MM patients had detectable anti-spike IgG (MM within detectable range). We analyzed samples collected post second dose and post third dose of COVID-19 vaccine using high dimensional spectral flow cytometry to elucidate immune phenotypic differences. Data and sample collection for this study was approved by the institutional review board (IRB) and follows the Declaration of Helsinki and International Conference on Harmonization Guidelines for Good Clinical Practice (IRB: GCO#: 16-00791 (MM patients) & GCO #: 20-03374 (PARIS). Results: We compared spike binding antibody responses pre and post third vaccine dose in our MM and control cohorts. While the third dose of mRNA vaccine resulted in a robust increase in anti-spike IgG for the majority of patients, the serological response in MM patients remained significantly lower (p<0.05) as compared to healthy controls (Figure 1A). The sub-optimal MM patient group had the lowest serological response, with 28% (4/14) of the patients showing no detectable anti-spike IgG levels post third booster vaccination. MM patients with sub-optimal responses did not have an increased incidence of lymphopenia or difference of absolute B cell and T cell counts. Further examination, however, revealed noticeable differences in TFH and NK cell populations. Sub-optimal MM patients showed a significantly lower prevalence of PD1+CXCR5+ TFH cells (p<0.01), as compared to MM patients within detectable serological range both pre and post third vaccination (Figure 1B). PD1+CXCR5+ TFH cells are associated with higher levels of anti-spike IgG neutralizing antibodies (Cui, et al. 2021). In addition, sub-optimal MM patients showed significantly fewer NK cells (p<0.05) when compared to MM patients within detectable serological range and healthy donors (Figure 1B). NK cell subset analysis in the sub-optimal MM patient group revealed a high prevalence of immature NK cell subsets (CD16-/dimCD56hi) (p<0.01), whereas MM patients within serological range and healthy donors showed a dominant mature cytolytic NK cell subset (CD16hiCD56dim). Half of the sub-optimal MM patients were on anti-CD38 monoclonal antibody therapy (7/14) compared to those mounting efficient responses (11%, 2/17), which we have previously described as affecting anti-spike IgG production and depleting NK populations. Conclusion: We describe here that significantly reduced numbers of CD4+ TFH and NK cells is associated with sub-optimal serological responses to SARS-COV-2 mRNA vaccination in MM patients. The distinct phenotype observed in our sub-optimal MM patient cohort could be a product of their pre-existing immune composition, the nature of their disease progression, or in response to ongoing therapies. Our data suggests that longitudinal immunophenotyping may identify vulnerable MM patients that may need additional vaccinations or prophylactic intervention, such as monoclonal antibody therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Principles of Advanced Flow Cytometry: A Practical Guide.

Recent advances have revolutionized the oldest high-throughput single-cell analytical tool, flow cytometry. Fluorescent analyzers and sorters with up to seven lasers and the potential to detect up to 50 parameters are changing the way flow cytometry is used, but old school practices which are inadequate for new technologies remain alive. This chapter summarizes recent advances, explains the most salient new features and offers a step-by-step guide to develop and successfully execute high-dimensional fluorescent flow cytometry experiments.

IMMU-27. SIMULTANEOUS TARGETING OF THE PD-L1 AND THE TGF-Β PATHWAYS WITH BINTRAFUSP ALFA IN EXPERIMENTAL GLIOMAS

Abstract Currently, immunotherapy is one of the most promising research areas in oncology, but immunotherapeutic strategies do not have proven clinical activity against glioblastoma so far. The immunosuppressive microenvironment of gliomas limits an appropriate anti-tumor immune response and represents a major challenge in the context of immunotherapy. Therefore, novel therapeutic strategies that reactivate the immune system and allow for potent anti-glioma immune responses are needed. Bintrafusp alfa is a bifunctional fusion protein that is composed of the extracellular domain of human transforming growth factor-beta receptor type II (TGF-βRII) and a human IgG1 monoclonal antibody blocking programmed death-ligand 1 (PD-L1). It is designed to target the TGF-β and the PDL-1 pathways simultaneously by specifically localizing to the tumor via the anti-PD-L1 moiety and sequestering TGF-β within the tumor microenvironment. We analyzed interrelations between the immunosuppressive factors TGF-β and PD-L1 in glioblastoma samples from patients and observed a significant correlation that points towards a co-regulation of both targets as part of a gene expression network. Next, we assessed the activity of bintrafusp alfa in human and murine glioma cell lines in vitro and observed interference with TGF-β-induced Smad2 phosphorylation as a bona fide response marker of TGF-β pathway activation and an increase in immune cell-mediated glioma cell killing. In vivo, bintrafusp alfa prolonged the survival of glioma-bearing immunocompetent mice, whereas the survival benefit was absent in immunodeficient Rag1-/- mice lacking T and B cell populations. Characterization of the tumor microenvironment by immunohistochemistry and high-dimensional flow cytometry confirmed the immune-activating effects of bintrafusp alfa. In summary, these encouraging results demonstrate that novel treatment options such as bintrafusp alfa may have the potential to overcome the lack of immunogenicity and reinvigorate anti-tumor immune responses to efficiently combat glioblastoma.