High-dimensional Immune Profiling Research Articles

ImmCellTyper facilitates systematic mass cytometry data analysis for deep immune profiling.

Mass cytometry is a cutting-edge high-dimensional technology for profiling marker expression at the single-cell level, advancing clinical research in immune monitoring. Nevertheless, the vast data generated by cytometry by time-of-flight (CyTOF) poses a significant analytical challenge. To address this, we describe ImmCellTyper (https://github.com/JingAnyaSun/ImmCellTyper), a novel toolkit for CyTOF data analysis. This framework incorporates BinaryClust, an in-house developed semi-supervised clustering tool that automatically identifies main cell types. BinaryClust outperforms existing clustering tools in accuracy and speed, as shown in benchmarks with two datasets of approximately 4 million cells, matching the precision of manual gating by human experts. Furthermore, ImmCellTyper offers various visualisation and analytical tools, spanning from quality control to differential analysis, tailored to users' specific needs for a comprehensive CyTOF data analysis solution. The workflow includes five key steps: (1) batch effect evaluation and correction, (2) data quality control and pre-processing, (3) main cell lineage characterisation and quantification, (4) in-depth investigation of specific cell types; and (5) differential analysis of cell abundance and functional marker expression across study groups. Overall, ImmCellTyper combines expert biological knowledge in a semi-supervised approach to accurately deconvolute well-defined main cell lineages, while maintaining the potential of unsupervised methods to discover novel cell subsets, thus facilitating high-dimensional immune profiling.

Amplified STAT3 increases expression of the CD39 ectoenzyme and impairs function of CD8+ T cells.

Abstract Regulation of T cell signaling is paramount to mount an appropriate immune response and protect the host from pathophysiology. Here, we study the impact on CD8+ T cells in monogenic inborn error of immunity patients exhibiting dysregulated signal 3 (cytokine signaling) pathways resulting from STAT3 gain- or loss-of-function variants (STAT3 GOF and STAT3 LOF). Using high dimensional immune profiling, we demonstrate increased expression of the ectoenzyme, CD39, which hydrolyzes extracellular ATP, on STAT3 GOF patient CD8+ T cells. Moreover, CD8+ T cells from two mouse models of STAT3 GOF (STAT3+/G421R and NOD-STAT3+/K392R) recapitulated this increase in CD39. In vitro, STAT3-activating cytokines (e.g., IL-6, IL-21, IL-27, IL-10) augment CD39 induction in a TCR-dependent manner in healthy donor and in STAT3 GOF CD8+ T cells. In contrast, STAT3 LOF patients show diminished CD39 induction. We find a positive correlation between CD39 induction and phosphorylated STAT3 levels while STAT3 inhibition prevents this cytokine-driven augmentation of CD39. Finally, acutely activated, and sorted CD39+CD8+ T cells from healthy donors inhibit bystander CD8+ T cell cytokine production (e.g., IFN-g). Pharmacologic inhibition of CD39 enzymatic activity or genetic ablation via CRISPR/Cas9 editing reversed decreases in cytokine production. Together, our data demonstrate a mechanistic connection between amplified STAT3 signaling and CD39 levels which is capable of impairing CD8+ T cell function.

Abstract 3904: Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia

Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogenous hematological malignancy with a complex clonal architecture that requires in-depth immunophenotyping for disease characterization. Conventional AML ex vivo models for preclinical drug screening lack functional readouts that provide single cell level data to identify drug activity in defined AML subsets with unique immune signatures. Here, we present a novel platform that links Multiparameter Flow cytometry with high-throughput EX vivo drug screening (MFLEX) in AML patient-derived cell models to simultaneously detect phenotypic changes and predict cell-subset specific drug responses in AML. Method: We developed a 21-color high-dimensional immune profiling panel that captures AML heterogeneity by delineating primitive and mature AML blasts along with normal hematopoietic lineages. The panel also includes markers for functional measurement of apoptosis, proliferation, differentiation, and expression of BCL2 family proteins in lymphoid and myeloid cell subsets. Ex vivo culture conditions were optimized using physiologically relevant stroma-free cytokine-rich media that supports primary AML cells with minimal phenotypic shift, allowing for high throughput functional drug sensitivity testing. Timepoints and dosing conditions for AML standard of care (SoC) agents were optimized to support combination screens. Multi-parameter datasets were analysed for functional differences and visualized using dimension reduction methods. Results: We tested MFLEX on clinically annotated AML samples to understand sensitivity profiles to venetoclax and combination partners: 5-azacytidine, cytarabine, fludarabine and gilteritinib. We identified distinct drug sensitivity profiles for venetoclax in AML patients with varying leukemic differentiation states where M0/M1 subtypes were more sensitive to therapy than M4/M5. We also observed cell lineage specific sensitivities, with myeloid blast being more sensitive to venetoclax compared to autologous CD4+ and CD8+ T-cells and this observed difference was correlated with higher expression of BCL2. Our platform demonstrated that patients who previously failed venetoclax therapy could benefit from a combination with 5-azacytidine or fludarabine and this was patient-specific. We further evaluated MFLEX’s predictive value for patient clinical response by systematically comparing venetoclax sensitivity in patient-derived xenograft models in vivo vs ex vivo and found a high correlation between drug responses. Conclusion: We have established a unique, immune drug screening platform, MFLEX, that has high translational value and can be implemented in clinical trials to identify drug response patterns, novel combinations, and potential biomarkers of response for patient stratification in AML. Citation Format: Reecha Shah, Heba Wander, Michelle Hsueh, Muskan Floren, Wei Liu, Patricia Cheung, Courtney Andersen, Michael White, Nathan Kingston, Pablo Morentin Gutierrez, Lisa Drew, Giulia Fabbri, Elena Bibikova, Daniel Auclair. Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3904.

Lower frequencies of circulating suppressive regulatory T cells and higher frequencies of CD4+ naïve T cells at baseline are associated with severe immune-related adverse events in immune checkpoint inhibitor-treated melanoma

BackgroundImmune-related adverse events (irAEs) are major barriers of clinical management and further development of immune checkpoint inhibitors (ICIs) for cancer therapy. Therefore, biomarkers associated with the onset of severe irAEs...

BinaryClust2: An Integrated Computational Pipeline for Systematic Mining of Mass Cytometry Data to Assist Deep Immune Profiling in Haematological Research

Introduction: The key role of immune system in modulating treatment efficacy and clinical outcomes in the settings of haematological diseases has been well recognized through the application of flow cytometry technologies over years. Mass cytometry (MC) is a next generation cytometry platform to simultaneously profile over 40 protein markers on single-cell resolution, which facilitates clinical research on immune monitoring and biomarker discovery. Nonetheless, the high-dimensional data generated present a daunting challenge for clinical investigators. Therefore, we describe BinaryClust2 (https://github.com/JingAnyaSun/BinaryClust2), an R-based computational framework integrated with the state-of-the-art algorithms and novel cell lineage characterisation method to assist comprehensive exploration of liquid MC data. Method: Here, we present the implementation of BinaryClust2 to three example MC datasets: In-house dataset from peripheral blood mononuclear cells (PBMCs) of 9 baseline myeloproliferative neoplasm (MPN) patients (~4 million cells), previously published data from 11 MPN patients receiving influenza vaccine (~0.2million cells), and published dataset of 59 covid patients and 23 healthy donors (~2million cells). BinaryClust2 has a streamlined analytical workflow, which comprises the following steps (Figure 1A): Step 1: Quality control, batch effect evaluation and correction Data quality control is a separate step before downstream analysis which includes diagnostic plots and batch effect examination. Algorithms CytofRUV and CytoNorm are available in the pipeline to remove unwanted variations caused by batch effects. Step 2: Semi-supervised identification of main cell types BinaryClust2 adopts a knowledge-based semi-supervised approach to predict main cell types. Users are required to provide a simple marker expression matrix of pre-defined cell types along with fcs files and metadata to construct a SingleCellExperiment (SCE) object, then the embedded algorithm can automatically classify cell populations without manual annotation. Step 3: In-depth interrogation and differential testing Specific population can be further extracted from whole cells and subject to in-depth exploration using unsupervised algorithms for subpopulation discovery. Dimensionality reduction tools UMAP and TSNE, unsupervised clustering methods Phenograph and flowSOM, and various data visualization plotting functions are offered in BinaryClust2. For statistical analysis, multiple study group comparison (n>2) of cell abundance and functional marker expression is supported via Kruskal Wallis test with multiple testing correction and post hoc analysis. Results: The performance of the semi-supervised classification function was tested independently in the MPN and influenza PBMC datasets, 7 main cell lineages were identified with accuracy comparable to manual gating by human experts (Figure 1B): average F-measure reached 0.93 and 0.98 respectively. Moreover, taking manual gating as ground truth reference, BinaryClust2 outperformed the unsupervised approach flowSOM concerning accuracy (F-measure: 0.93 vs. 0.70) and speed (140s vs 339s) in MPN dataset, while remaining equivalent to the well-performing semi-supervised approach LDA in accuracy (F-measure: 0.93 vs. 0.93) but faster in runtime (140s vs 595s), as shown in Table 1. Application to covid-19 dataset by Chevrier et al. achieved reproducible results and additional discoveries. 13 main cell types were characterised, abundance of B cells, Basophils, cDCs, DN T cells, Monocytes, Neutrophils, NK cells, pDCs, CD8 T cells obtained statistical significance (all P<0.05) among study conditions (healthy, mild covid, severe covid). We also grouped markers reflecting functional status of immune cells and found Granzyme B expression was significantly increased in the majority of main immune cells of covid patients. Phenograph was further applied in neutrophils and monocytes and returned 14 and 16 subsets respectively. Conclusion: Overall, BinaryClust2 incorporates expert's prior biological knowledge in a semi-supervised fashion to accurately deconvolute well-defined main cell lineages, while also preserving the potential of unsupervised approaches to discover novel cell subsets and providing a user-friendly toolset to remove the analytical barrier for high-dimensional immune profiling.

High-dimensional immune profiling using mass cytometry reveals IL-17A-producing γδ T cells as biomarkers in patients with T-cell-activated idiopathic severe aplastic anemia

Severe aplastic anemia (SAA) is a bone marrow failure syndrome characterized by activated T cells. Features of T-cell activation in the pathophysiology of SAA remain unknown. To understand T cell activation states, we investigated the atlas of peripheral immune cells and the secreted cytokine network with single cell mass cytometry analysis. We found decreased γδ T-cell frequencies in all patients with SAA, together with a significantly increased proportion of interleukin (IL)-17A-producing cell subsets. Cytokine network analysis of immune cells showed significant positive relationship between IL and 17A production from immune cells and disease severity of severe aplastic anemia. On separating SAA into two distinct subgroups based on T-cell activation stage, the proportion of γδ T cells tended to decrease in the T-cell-activated SAA group compared with non-T-cell-activated group. And the proportion of IL-17A-producing γδ T cells (γδT17) within γδ T cells was newly found to be significantly higher in the T-cell-activated SAA group, implying that IL-17A production by γδ T cells was associated with T-cell activation. Overall, our study revealed a role of γδT17 cells in mediating autoreactive T-cell activation in SAA and provided a novel diagnostic indicator for monitoring autoreactive T-cell activation status during the progression of aplastic anemia in the clinic.

IgA deficiency destabilizes homeostasis toward intestinal microbes and increases systemic immune dysregulation.

The ability of most patients with selective immunoglobulin A (IgA) deficiency (SIgAD) to remain apparently healthy has been a persistent clinical conundrum. Compensatory mechanisms, including IgM, have been proposed, yet it remains unclear how secretory IgA and IgM work together in the mucosal system and, on a larger scale, whether the systemic and mucosal anti-commensal responses are redundant or have unique features. To address this gap in knowledge, we developed an integrated host-commensal approach combining microbial flow cytometry and metagenomic sequencing (mFLOW-Seq) to comprehensively define which microbes induce mucosal and systemic antibodies. We coupled this approach with high-dimensional immune profiling to study a cohort of pediatric patients with SIgAD and household control siblings. We found that mucosal and systemic antibody networks cooperate to maintain homeostasis by targeting a common subset of commensal microbes. In IgA-deficiency, we find increased translocation of specific bacterial taxa associated with elevated levels of systemic IgG targeting fecal microbiota. Associated features of immune system dysregulation in IgA-deficient mice and humans included elevated levels of inflammatory cytokines, enhanced follicular CD4 T helper cell frequency and activation, and an altered CD8 T cell activation state. Although SIgAD is clinically defined by the absence of serum IgA, the symptomatology and immune dysregulation were concentrated in the SIgAD participants who were also fecal IgA deficient. These findings reveal that mucosal IgA deficiency leads to aberrant systemic exposures and immune responses to commensal microbes, which increase the likelihood of humoral and cellular immune dysregulation and symptomatic disease in patients with IgA deficiency.

Elevated CD38 expression on STAT1 GOF CD8+T cells – a potential driver of CD8+ T cell dysregulation?

Background and AimsSTAT1 has a central role in relaying signals from type I, II and III interferons (IFN), thus contributing to the antiviral immune response. Working in tandem, CD8+ T cells (CD8T) are key mediators of the adaptive immune response to viral infections. In patients with STAT1 gain of function (GOF) syndrome there is an increase in viral infections, as part of their immune dysregulatory phenotype. However, there is currently a limited understanding of how increased STAT1 signaling might alter CD8T function, potentially impairing anti-viral mechanisms. We thus aim to define the extent and nature of CD8T dysfunction and underlying mechanisms in STAT1 GOF patients. MethodsWe collected multi-modal high-dimensional immune profiling and functional data including phosphoflow on peripheral blood mononuclear cells (PBMCs) from 24 STAT1GOF patients and age matched controls. 7 Aicardi-Goutières-Syndrome (AGS) patients were included as a comparison population. ResultsThese cohorts allow us to differentiate the effects of increased type I IFN signaling vs chronic STAT1 activity on CD8T function. STAT1 GOF CD8T showed signs of immune dysregulation including reduced production of IFN-γ and TNF-α upon phorbol-myristate-acetate (PMA) and ionomycin stimulation. High-dimensional immune profiling revealed many activation markers altered on STAT1 GOF CD8T. CD38 was overall significantly elevated, with CD4T and CD8T most affected in STAT1GOF patients. This is also seen to a lesser extent in AGS patients. CD38 was most efficiently induced by T cell receptor (TCR) stimulation in combination with type I interferon in both healthy control and STAT1GOF CD8 T in vitro. Upon further analysis it was the STAT1hi CD8T, known to be elevated in STAT1GOF, that most dynamically upregulated CD38.Conclusion and Outlook: In STAT1 GOF, a model of chronic STAT1 signaling, we found increased expression of CD38 on CD8T. CD38 acts as an ectoenzyme that consumes nicotinamide adenine dinucleotide (NAD+). Elevated CD38 and low NAD+ have been associated with states of immune dysregulation. We thus suggest CD38 as a promising candidate present on STAT1GOF CD8T that might alter NAD+ levels. This could be a potential novel mechanism underlying the immune dysregulation present in STAT1GOF patients.

Abstract 2138: Immune features of irAEs and aPD1 response in urothelial cancer patients of the RADIOHEAD study, as detected in blood by mass cytometry immune profiling

Abstract Checkpoint immunotherapies (CPI) have resulted in long lasting clinical responses; however, CPI treatment comes with the risk of immune-related adverse events (irAEs). Previous reports have established a correlation between irAE occurrence and better CPI patient outcomes, yet these correlations and their underlying biological mechanisms are poorly understood. To better understand the immunological mechanisms behind the correlation of irAE presentation and response, we performed immune profiling on 40 urothelial carcinoma (UC) patients receiving aPD-1 as part of the RADIOHEAD study (Lucas et al. SITC 2022, abstract #1256). A 44-marker mass cytometry panel was used to profile pretreatment and early on-treatment PBMCs to capture cell subsets and functional states across innate and adaptive immune cells. We performed analysis on gated immune cell populations and through unsupervised clustering. Group comparisons were made by adjusted Kruskal-Wallis test, whereas survival correlates were assessed through univariate Cox regression analysis. For clinical outcome analyses, we defined responders as patients who had overall survival greater than 12 months (n=26) and noted 8 patients developed irAEs. Several immune features early on-treatment were associated with overall survival. KLRG1+ naive and HLA-DR+ CD38+ central memory CD8 T cells were negatively correlated with survival, whereas Treg and TIM-3+ classical monocytes were positively associated (HR= 2.6, 3.2, 0.4, 0.05; p=0.0002, 0.00005, 0.01,0.009 respectively). Next, we classified patients into the following groups: responders with irAE (R-irAE, n=5), responders without irAE (n=21), non-responders with irAE (n=3) and non-responders without irAE (n=10). We found that several immune features were unique to R-irAE patients early on aPD-1 treatment. R-irAE patients had up to 30% more CD74+ cDCs, up to 4-fold more TIM-3+ classical monocytes, and had a greater fold decrease in CD16+ NK cells from baseline compared to other patients (p<0.005, 0.001,0.008 respectively). They also had up to 2-fold more Treg cells early on treatment, including a subset of activated Tregs marked by expression of CD38, CTLA-4, ICOS and TIGIT (p=0.05) which trended up to significance at pretreatment (p=0.075). In this study, we demonstrate that high-dimensional immune profiling by mass cytometry can detect novel blood-based biomarkers associated with clinical outcomes. Although CPI response and irAE incidence both require activation of a patient’s immune system, different mechanisms likely apply. Based on our findings, there are markers associated with responders broadly, as well as specific to R-irAE patients. We plan to validate these findings and identify additional markers and mechanisms of response and irAE with immune profiling on more RADIOHEAD indications. Citation Format: Connor P. Healy, Johanna M. Sweere, Samantha I. Liang, Natalia Sigal, Elizabeth Enrico, Li-Chun Cheng, Anastasia Lucas, Marshall A. Thompson, Diane M. Da Silva, Justin A. Jarrell, Ramji Srinivasan, Matthew H. Spitzer, Ngan Nguyen, Christine N. Spencer. Immune features of irAEs and aPD1 response in urothelial cancer patients of the RADIOHEAD study, as detected in blood by mass cytometry immune profiling [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 2138.

Heterologous SARS-CoV-2 spike protein booster elicits durable and broad antibody responses against the receptor-binding domain

The immunogenicity of mRNA vaccines has not been well studied when compared to different vaccine modalities in the context of additional boosters. Here we show that longitudinal analysis reveals more sustained SARS-CoV-2 spike receptor-binding domain (RBD)-binding IgG titers with the breadth to antigenically distinct variants by the S-268019-b spike protein booster compared to the BNT162b2 mRNA homologous booster. The durability and breadth of RBD-angiotensin-converting enzyme 2 (ACE2) binding inhibitory antibodies are pronounced in the group without systemic adverse events (AEs) after the S-268019-b booster, leading to the elevated neutralizing activities against Omicron BA.1 and BA.5 variants in the stratified group. In contrast, BNT162b2 homologous booster elicited antibodies to spike N-terminal domain in proportion to the AE scores. High-dimensional immune profiling identifies early CD16+ natural killer cell dynamics with CCR3 upregulation, as one of the correlates for the distinct anti-RBD antibody responses by the S-268019-b booster. Our results illustrate the combinational effects of heterologous booster on the immune dynamics and the durability and breadth of recalled anti-RBD antibody responses against emerging virus variants.

Tumor-Associated Macrophages and Related Myelomonocytic Cells in the Tumor Microenvironment of Multiple Myeloma.

Multiple myeloma (MM) is the second-most common hematologic malignancy and remains incurable despite potent plasma cell directed therapeutics. The tumor microenvironment (TME) is a key player in the pathogenesis and progression of MM and is an active focus of research with a view to targeting immune dysregulation. Tumor-associated macrophages (TAM), myeloid derived suppressor cells (MDSC), and dendritic cells (DC) are known to drive progression and treatment resistance in many cancers. They have also been shown to promote MM progression and immune suppression in vitro, and there is growing evidence of their impact on clinical outcomes. The heterogeneity and functional characteristics of myelomonocytic cells in MM are being unraveled through high-dimensional immune profiling techniques. We are also beginning to understand how they may affect and be modulated by current and future MM therapeutics. In this review, we provide an overview of the biology and clinical relevance of TAMs, MDSCs, and DCs in the MM TME. We also highlight key areas to be addressed in future research as well as our perspectives on how the myelomonocytic compartment of the TME may influence therapeutic strategies of the future.

High Dimensional Immune Profiling of Smoldering Multiple Myeloma Distinguishes Distinct Tumor Microenvironments

Multiple myeloma (MM) is a malignancy of plasma cells that arises from premalignant Monoclonal Gammopathy of Undetermined Significance (MGUS) and often progresses through an asymptomatic Smoldering (SMM) phase. Understanding the interactions between abnormal clonal plasma cells and the tumor microenvironment (TME) in the early disease states (MGUS, SMM) may inform risk assessment and therapy. We performed high dimensional immunologic analysis of bone marrow specimens from 73 subjects with SMM by mass cytometry and T cell receptor sequencing of CD138-depleted bone marrow (BM) mononuclear cells, and proteomics and seromic profiling of BM plasma. Analysis of individual assay data identified self-organizing subgroups of SMM patients. We then applied novel bioinformatic methods to integrate data from pairs, trios, and quartets of assays. Mass cytometry, TCRSeq and proteomics identified three taxa (sing. taxon) of subjects that shared common characteristics across all three assays. Differential levels of BM plasma pleiotropin (PTN) and BM T cells and their productive clonality emerged as strong distinguishing factors among these taxa. These results suggest that the continuum from MGUS to MM does not consist of a single pathway in the TME, and that complex interactions between myeloma cells and the TME may ultimately determine progression and inform clinical management.

P2.12-05 Cancer and Atopy: Parallel Drivers? IL-4 Blockade Synergizes with PD-L1 Blockade to Reverse Type-2Mediated Immunosuppression

Type-2 cytokines, such as IL-4, alter macrophage immunogenicity in vitro leading to the M1/M2 paradigm, however, the role of IL-4 in-vivo in cancer remains unclear. Using high dimensional immune profiling of human NSCLC lesions we previously described several functionally distinct populations of macrophages, and a tumor-enriched dendritic cell program of concomitant immunosuppression and activation which we termed the mregDC, and we identified analogous cells in a murine model of lung cancer (Maier et al, Nature 2020, Casanova-Acebes et al, Nature, 2021, Leader et al, Cancer Cell 2021).

High-dimensional immune profiling identifies a biomarker to monitor dimethyl fumarate response in multiple sclerosis

Dimethyl fumarate (DMF) is an immunomodulatory treatment for multiple sclerosis (MS). Despite its wide clinical use, the mechanisms underlying clinical response are not understood. This study aimed to reveal immune markers of therapeutic response to DMF treatment in MS. For this purpose, we prospectively collected peripheral blood mononuclear cells (PBMCs) from a highly characterized cohort of 44 individuals with MS before and at 12 and 48 wk of DMF treatment. Single cells were profiled using high-dimensional mass cytometry. To capture the heterogeneity of different immune subsets, we adopted a bioinformatic multipanel approach that allowed cell population-cluster assignment of more than 50 different parameters, including lineage and activation markers as well as chemokine receptors and cytokines. Data were further analyzed in a semiunbiased fashion implementing a supervised representation learning approach to capture subtle longitudinal immune changes characteristic for therapy response. With this approach, we identified a population of memory T helper cells expressing high levels of neuroinflammatory cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon γ [IFNγ]) as well as CXCR3, whose abundance correlated with treatment response. Using spectral flow cytometry, we confirmed these findings in a second cohort of patients. Serum neurofilament light-chain levels confirmed the correlation of this immune cell signature with axonal damage. The identified cell population is expanded in peripheral blood under natalizumab treatment, substantiating a specific role in treatment response. We propose that depletion of GM-CSF-, IFNγ-, and CXCR3-expressing T helper cells is the main mechanism of action of DMF and allows monitoring of treatment response.

High-Dimensional Immune Profiling by Mass Cytometry Revealed the Circulating Immune Cell Landscape in Patients With Intracranial Aneurysm.

BackgroundIncreasing evidence supports a critical role of chronic inflammation in intracranial aneurysm (IA). Understanding how the immunological alterations in IA provides opportunities for targeted treatment. However, there is a lack of comprehensive and detailed characterization of the changes in circulating immune cells in IA.ObjectiveTo perform a comprehensive and detailed characterization of the changes in circulating immune cells in patients with IA.MethodsPeripheral blood mononuclear cell samples from IA patients (n = 26) and age-and sex-matched healthy controls (HCs, n = 20) were analyzed using high dimensional mass cytometry, and the frequency and phenotype of immune cell subtypes were assessed.ResultsWe identified 28 cell clusters and found that the immune signature of IA consists of cluster changes. IA patients exhibited dysfunction of immunity, with dysregulation of CD4+ T-cell clusters, increased B cells and monocytes, and decreased CD8+ T cells, DNT cells, and DPT cells. Moreover, compared with findings in HC, IA was associated with enhanced lymphocyte and monocyte immune activation, with a higher expression of HLA-DR, CXCR3, and CX3CR1. In addition, the expression of TLR4, p-STAT3, and the exhaustion marker PD1 was increased in T cells, B cells, and NK cells in IA patients.ConclusionsOur data provide an overview of the circulating immune cell landscape of IA patients, and reveal that the dysfunction of circulating immunity may play a potential role in the development of IA.

Abstract 6290: Microenvironment begets phenotype: Tumor specific CD8+ T cells derived from bone marrow and other lymphoid sites exhibit differential exhaustion profiles

Abstract Current immunotherapy approaches aim to mitigate or reverse CD8+ T cell exhaustion to generate meaningful anti-tumor immune responses. However, the multi-step process of exhaustion fixes epigenetic programs that permanently scar T cells, limiting immunotherapeutic efficacy. Rather than reversing T cell exhaustion, we hypothesize that exploiting tumor-specific T cells from protected niches could yield therapies with improved anti-tumor benefit. A subset of TCF1-expressing precursor exhausted CD8+ T cells (TPEX) drives the anti-tumor immune response. The tumor draining lymph nodes (dLN) maintain a reservoir of TPEX cells which replenishes intra-tumoral effector T cells. Similarly, the bone marrow (BM) houses long-lived memory T cells with specificity against a broad spectrum of antigens, including tumor-associated antigens. Unlike T cells from the dLN or tumor infiltrating lymphocytes (TILs), BM T cells can be easily acquired from all patients at any disease stage. We used the B16.OVA-OT-I model to delineate how exhaustion affects tumor-specific T cells within the BM, dLN, spleen (SP), and tumor (TUM) during tumor progression. This model uses adoptively transferred transgenic, OVA-specific OT-I cells as a source of tumor-specific CD8+ T cells. High-dimensional immune profiling using spectral flow cytometry enabled us to characterize in vitro and in vivo anti-tumor functionality and the phenotype of OT-I cells derived from different compartments. Interestingly, upon adoptive transfer, OT-I cells resident in the TUM, dLN, BM, and SP or found in circulation were heterogenous in terms of function, persistence, and stemness. These findings suggest that discrete tissues impart location-specific T cell adaptations, and that tissue microenvironment markedly influences the exhaustion profile of tumor-specific T cells. OT-I cells derived from distinct lymphoid organs exhibited differential capacity for cytokine production and response to cognate antigen, especially compared to their TIL counterparts. Importantly, we identified BM- and dLN-derived CD8+ T cells as less exhausted and more functional than TILs. Moreover, BM resident T cells appear phenotypically more stem-like and similar to TPEX. Further studies to characterize this population are ongoing. This work implies that, in the context of localized disease, the kinetics of T cell exhaustion differ in lymphoid organs as compared to TILs. Leveraging a less-exhausted pool of tumor-specific cells may lead to greater anti-tumor benefit with new approaches to adoptive cell therapy. As the BM is accessible in all patients, understanding where tumor-specific BM T cells fall on the spectrum of exhaustion and memory could justify their use for such applications. Ongoing work aims to transcriptionally characterize tumor specific BM CD8+ T cells and evaluate their role in anti-tumor immunity. Citation Format: Elizabeth Maria Zawidzka, Luca Biavati, Amy Thomas, Megan Heimann, Ervin Griffin, Ivan Borrello. Microenvironment begets phenotype: Tumor specific CD8+ T cells derived from bone marrow and other lymphoid sites exhibit differential exhaustion profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6290.

Preoperative durvalumab (D) with or without tremelimumab (T) for resectable head and neck squamous cell carcinoma (HNSCC): Updated results with high dimensional profiling of circulating immune cells.

6072 Background: Although PD-1 blockade has improved survival in patients with recurrent and/or metastatic HNSCC, safety and efficacy of neoadjuvant immunotherapy with PD-L1 inhibitor with or without CTLA-4 inhibitor has not been investigated. Here, we report the updated results of the safety and efficacy of a preoperative D with or without T (D+/-T) in patients with resectable HNSCC, accompanied with high dimensional profiling of circulating immune cells. Methods: Patients with locally advanced but resectable HNSCC were eligible. Enrolled patients were randomized into D or D+T, stratified by primary site and human papilloma virus (HPV) infection status. A single dose of preoperative D (1500mg) or D+T (1500mg+75mg) was administered, with surgery planned 2 to 8 weeks later for curative resection. Postoperative (chemo) radiation was prescribed based on standard guidelines, followed by maintenance with D every 4 weeks for 1 year. Dynamic changes in circulating immune cells were tracked with mass cytometry. The primary objective was to determine the local recurrence rate. Secondary endpoints included pathologic response, safety, survival outcome, and exploration of immune dynamics. Results: As of January 25th 2022 for the interim analysis, a total of 45 patients were completely enrolled and received surgical resection (D: 21 patients, D+T: 24 patients). Oropharyngeal cancer was most common (n = 23; 51.1%) and HPV-mediated cancer was observed in 20 patients (44.4%). Neoadjuvant D+/-T had acceptable safety profiles and was not associated with delays in surgery or unexpected adverse events. Tumor shrinkage was observed in 31 patients (68.9%), with 15.6% of average tumor shrinkage (range; 100.0% to -80.0%). Major pathologic response (no more than 10% of viable tumor cells) was achieved in 3 patients (6.7%), including 2 cases with pathologic complete response (4.4%). During median follow-up duration of 407 days after surgery, local recurrence and systemic recurrence were documented in 9 patients (20.0%) and 7 patients (15.6%), respectively. Median disease-free survival and overall survival was 910 days and not reached, respectively. High dimensional immune profiling with mass cytometry revealed that D+T disproportionally increased the frequency of regulatory T cells accompanied with the upregulation of their functional markers, which was absent in patients treated with D monotherapy. Conclusions: These updated data suggested that preoperative D+/-T was safe and feasible and had the potential to provide clinical benefits for patients with resectable HNSCC. Distinct immunologic changes in circulating immune cells were induced by each treatment regimen, warranting further investigation. The trial is ongoing and the updated outcomes with immune correlates will be presented in this ASCO. Clinical trial information: NCT03737968.

Interrogating mechanisms of CD8 T cell dysfunction in obese asthma

Abstract Pediatric obese atopic asthma (OA) is a complex and poorly understood disease at the intersection of two of the most common chronic inflammatory diseases of childhood. Pediatric OA patients often experience severe asthma exacerbations caused by respiratory viral infections. However, the immunological mechanisms by which obesity modifies immune function in asthma remain poorly understood. Using high dimensional immune profiling, we have demonstrated CD8 T cells from pediatric OA patients have an exhausted-like immunophenotype compared to non-obese asthmatics. To more deeply characterize the role of CD8 T cell dysfunction in OA, we developed a mouse model of OA using high fat diet (HFD) to induce obesity and house dust mite (HDM) to induce asthma. We found that non-naïve CD8 T cells isolated from the lungs of OA mice demonstrate increased expression of multiple activation and inhibitory receptors (e.g., PD-1, CD39 and Tim3) as well as TOX, all consistent with T cell exhaustion, compared to healthy controls. We hypothesize the exhausted-like state of CD8 T cells in OA impairs anti-viral responses, thereby increasing severity of asthma exacerbation during respiratory viral infections. Ongoing work is testing antigen specific responses to influenza infection. Overall, this reveals a previously unrecognized role for exhausted-like CD8 T cell responses in mediating the immunopathogenesis of pediatric obese asthma. This work was supported by the Translational Research Training Program in Environmental Health Sciences Pre-Doctoral Fellowship to C.A.H. and K08-AI135091 to S.E.H.

Immune characterization in platinum resistant ovarian cancer patients treated with pembrolizumab and guadecitabine

Abstract Immune checkpoint inhibitors (ICI) have limited activity in ovarian cancer (OC). Accumulating preclinical and early clinical results support that interventions targeting the epigenome could elicit an inflamed tumor milieu to augment ICI. Here we explore the clinical and biological activity of guadecitabine, a second generation Hypomethylating agents (HMA), given in low dose as a priming strategy before pembrolizumab, a humanized anti-PD1 antibody in a clinical trial for patients with recurrent, platinum-resistant OC. Among 35 evaluable patients, there were 3 partial responses (8.6%) and 8 (22.9%) patients with stable disease, resulting in clinical benefit rate (CBR) of 31.4%. Median duration of clinical benefit was 6.8 months. High dimensional immune profiling of PBMCs showed higher frequency of naive and/or central memory CD4+ T cells and of classical monocytes in patients with durable CBR. Higher baseline density of CD8+ T and CD20+ B cells and presence of tertiary lymphoid structures in tumors were associated with durable CBR. The molecular features such as expression levels of PD-L1 or A2AR linked to therapeutic benefit for resistant OC. Our study provides an in-depth view of the immune milieu of platinum resistant OC and of the effects of the combination of HMAs and pembrolizumab on the interactions between immune cell populations and tumor cells. Supported by USAMRMC/CDMRP (W81XWH170141)

Distinct immune cell dynamics correlate with the immunogenicity and reactogenicity of SARS-CoV-2 mRNA vaccine

SummaryTwo doses of Pfizer/BioNTech BNT162b2 mRNA vaccine elicit robust severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing antibodies with frequent adverse events. Here, by applying a high-dimensional immune profiling on 92 vaccinees, we identify six vaccine-induced immune dynamics that correlate with the amounts of neutralizing antibodies, the severity of adverse events, or both. The early dynamics of natural killer (NK)/monocyte subsets (CD16+ NK cells, CD56high NK cells, and non-classical monocytes), dendritic cell (DC) subsets (DC3s and CD11c− Axl+ Siglec-6+ [AS]-DCs), and NKT-like cells are revealed as the distinct cell correlates for neutralizing-antibody titers, severity of adverse events, and both, respectively. The cell correlates for neutralizing antibodies or adverse events are consistently associated with elevation of interferon gamma (IFN-γ)-inducible chemokines, but the chemokine receptors CCR2 and CXCR3 are expressed in distinct manners between the two correlates: vaccine-induced expression on the neutralizing-antibody correlate and constitutive expression on the adverse-event correlate. The finding may guide vaccine strategies that balance immunogenicity and reactogenicity.