High-dimensional Flow Research Articles

Evaluating the efficacy of commercial teclistamab in relapsed refractory multiple myeloma patients with prior exposure to anti-BCMA therapies.

8049 Background: Teclistamab (Tec) is the first CD3 x BCMA bispecific antibody (BsAb) receiving accelerated FDA approval for treatment of relapsed or refractory multiple myeloma (RRMM) in patients who have received ≥4 prior lines of therapy, including a PI, IMiD and an anti-CD38 monoclonal antibody. The approval was based on the results of the MajesTec-1 study (Usmani S et al Lancet 2021, Moreau P et al NEJM 2022), demonstrating a 63% overall response rate in a heavily pretreated RRMM population. Patients with prior exposure to anti-BCMA therapies, such as BCMA targeted ADCs, CAR T-cell products and BsAbs were excluded from this study. Herein, we present our institutional experience with commercial Tec for RRMM including patients with prior BCMA and GPRC5D directed therapies. Methods: We have performed an IRB-approved, retrospective analysis of clinical outcomes of all patients who have received commercial Tec at MSKCC since its approval on 10/26/2022 using the PCD research database. Descriptive analyses were performed for baseline characteristics. The IMWG criteria (Kumar S et al, Lancet Oncol 2016) were used to assess response and define prior therapy refractoriness. Immune profile was assessed via high-dimensional flow cytometry using lineage, exhaustion, and activation markers. Serum soluble BCMA levels were assessed using an immunoassay. Results: As of 2/4/2023, 24 patients have received commercial Tec and 15 are response evaluable with ≥1 month of clinical follow-up. Median age was 66 (51-80), prior lines of therapy was 7 (4-13), time from diagnosis was 7 years (1.5-16), 53% had high-risk cytogenetics, and 40% had EMD. All patients were triple class refractory and 80% were penta-drug refractory. Ten had prior anti-BCMA therapy (7 Belamaf, 8 BCMA CART, 1 BCMA BsAb, 5 with ≥2 anti-BCMA therapies). With a median follow-up time of 1.3 months, the median time to response was 16 days. ORR was 60% (9/15) in all patients and 50% (5/10) in the prior anti-BCMA therapy group. Pts with ≥2 anti-BCMA therapies had a 40% (2/5) response rate to Tec. Clinical benefit rate (CBR) in all patients was 73% (11/15). None of the responders have progressed at this short follow-up time. Cytokine release syndrome was observed in 7/15 patients (41%) during step-up dosing (5/7 with g1 and 2/7 with g2 CRS) and CBR was 100% in patients with CRS (71% ORR). Other notable toxicities include 2 patients with grade 2 neurotoxicity that improved with therapy discontinuation. Conclusions: To our knowledge, this is the first report of commercial Tec in RRMM. Tec remains effective in RRMM despite prior exposure to anti-BCMA therapies, though exposure to multiple prior anti-BCMA therapies may be predictive of diminished efficacy. Clinical data on additional patients will be presented at the meeting. Ongoing translational investigations on soluble BCMA levels and patient-specific immune phenotype will also be presented at the meeting.

Physics-assisted reduced-order modeling for identifying dominant features of transonic buffet

Transonic buffet is a flow instability phenomenon that arises from the interaction between the shock wave and the separated boundary layer. This flow phenomenon is considered to be highly detrimental during flight and poses a significant risk to the structural strength and fatigue life of aircraft. Up to now, there has been a lack of an accurate, efficient, and intuitive metric to predict buffet and impose a feasible constraint on aerodynamic design. In this paper, a Physics-Assisted Variational Autoencoder is proposed to identify dominant features of transonic buffet, which combines unsupervised reduced-order modeling with additional physical information embedded via a buffet classifier. Specifically, four models with various weights adjusting the contribution of the classifier are trained so as to investigate the impact of buffet information on the latent space. Statistical results reveal that the buffet state can be determined exactly with just one latent space when a proper weight of classifier is chosen. The dominant latent space further reveals a strong relevance with the key flow features located in the boundary layers downstream of shock. Based on this identification, the displacement thickness at 80% chordwise location is proposed as a metric for buffet prediction. This metric achieves an accuracy of 98.5% in buffet state classification, which is more reliable than the existing separation metric used in design. The proposed method integrates the benefits of feature extraction, flow reconstruction, and buffet prediction into a unified framework, demonstrating its potential in low-dimensional representations of high-dimensional flow data and interpreting the “black box” neural network.

Full spectrum flow cytometry in the clinical laboratory.

Contemporary full spectrum or "spectral" flow cytometry is a recently developed technology that allows for high-dimensional flow cytometric analyses of cells and particles in suspension. This single-cell technology has gained popularity in research settings because it can conservatively detect 35 or more antigens simultaneously in a single-tube assay format. Recently, spectral flow cytometry has obtained regulatory approval for use as an in vitro diagnostic device in China and Europe, enabling use of this technology in some clinical flow cytometry laboratories. The purpose of this review is to describe the basic principles of conventional and spectral flow cytometry, contrasting these two technologies. To illustrate the analytic power of spectral flow cytometry, we provide an example of spectral flow cytometry data analyses and the use of a machine learning algorithm to harvest the vast amount of information contained within large spectral flow cytometry datasets. Finally, we discuss the advantages of spectral flow cytometry adoption in clinical laboratories and preliminary studies comparing the performance of this technology relative to conventional flow cytometers that are currently used in clinical laboratory environments.

The Effect of Lung Resection for NSCLC on Circulating Immune Cells: A Pilot Study.

This pilot study sought to evaluate the circulating levels of immune cells, particularly regulatory T-cell (Treg) subsets, before and after lung resection for non-small cell lung cancer. Twenty-five patients consented and had specimens collected. Initially, peripheral blood of 21 patients was collected for circulating immune cell studies. Two of these patients were excluded due to technical issues, leaving 19 patients for the analyses of circulating immune cells. Standard gating and high-dimensional unsupervised clustering flow cytometry analyses were performed. The blood, tumors and lymph nodes were analyzed via single-cell RNA and TCR sequencing for Treg analyses in a total of five patients (including four additional patients from the initial 21 patients). Standard gating flow cytometry revealed a transient increase in neutrophils immediately following surgery, with a variable neutrophil-lymphocyte ratio and a stable CD4-CD8 ratio. Unexpectedly, the total Treg and Treg subsets did not change with surgery with standard gating in short- or long-term follow-up. Similarly, unsupervised clustering of Tregs revealed a dominant cluster that was stable perioperatively and long-term. Two small FoxP3hi clusters slightly increased following surgery. In the longer-term follow-up, these small FoxP3hi Treg clusters were not identified, indicating that they were likely a response to surgery. Single-cell sequencing demonstrated six CD4+FoxP3+ clusters among the blood, tumors and lymph nodes. These clusters had a variable expression of FoxP3, and several were mainly, or only, present in tumor and lymph node tissue. As such, serial monitoring of circulating Tregs may be informative, but not completely reflective of the Tregs present in the tumor microenvironment.

CD25 targeting with the afucosylated human IgG1 antibody RG6292 eliminates regulatory T cells and CD25+ blasts in acute myeloid leukemia.

Acute Myeloid leukemia is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. We characterized bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry and RNA sequencing using computational analysis. In addition, 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 (also referred to as RG6292 and RO7296682) or isotype control antibody on regulatory T cells and CD25+ AML cells. Bone marrow composition, in particular the abundance of regulatory T cells and CD25 expressing AML cells, correlated strongly with that of the blood in patients with time-matched samples. In addition, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing a FLT3-ITD mutation or treated with a hypomethylating agent in combination with venetoclax. We adopted a patient-centric approach to study AML clusters with CD25 expression and found it most highly expressed on immature phenotypes. Ex vivo treatment of primary AML patient samples with CD25 Mab, a human CD25 specific glycoengineered IgG1 antibody led to the specific killing of two different cell types, CD25+ AML cells and regulatory T cells, by allogeneic Natural Killer cells. The in-depth characterization of patient samples by proteomic and genomic analyses supported the identification of a patient population that may benefit most by harnessing CD25 Mab's dual mode of action. In this pre-selected patient population, CD25 Mab could lead to the specific depletion of regulatory T cells, in addition to leukemic stem cells and progenitor-like AML cells that are responsible for disease progression or relapse.

Determining antigen-specific T cell phenotypes in cases of Hepatitis C virus clearance versus chronic infection.

Abstract Globally, approximately 1.5 million new Hepatitis C virus (HCV) infections occur annually. A vaccine to prevent chronic HCV infection would limit HCV transmission and pathogenesis of HCV. To understand immunity to HCV infection in high risk populations, we prospectively followed HCV antibody negative high-risk people who inject drugs (PWIDs) in a protocol designed for monthly follow-up. We demonstrated previously that HCV reinfection in PWIDs is associated with a reduction in the magnitude and duration of viremia versus the initial infection and broadened cellular immune responses, consistent with protective immunity against chronic disease. We have characterized CD8+ HCV-specific T cells from the acute phase of HCV using high dimensional spectral flow cytometry to define the simultaneous expression of 25 intracellular metabolic and cell surface molecules. We found that HCV-specific CD8+ T cells from those who progress to chronic infection have a unique acute phase phenotype that clusters separately from those who later successfully control HCV. Acute phase CD8+ HCV-specific T cells from those who subsequently clear an initial HCV infection show increased expression of CD127 and CXCR3 and decreased expression of GLUT1, PD-1 and CD39 compared to acute phase T cells in those who later progress to chronic infection. Understanding HCV specific T cell phenotypes in successful control of HCV infection and reinfection will guide goals for vaccine induced immunity to prevent chronic HCV infection. Supported by grants from the NIH (U19 AI159822)

Dendritic cell therapy augments antitumor immunity triggered by CDK4/6 inhibition and immune checkpoint blockade by unleashing systemic CD4 T-cell responses

BackgroundCyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy are a mainstay treatment for hormone receptor-positive breast cancer. While their principal mechanism is inhibition of cancer cell proliferation, preclinical and...

CD4 T cells from mice with alopecia areata express an effector like phenotype and can transfer disease

Abstract Alopecia areata (AA) is an autoimmune disease with a 2% lifetime incidence. AA presents as hair loss and is characterized histologically by the presence of CD4 and CD8 T cells surrounding the hair bulb. Although CD4 T cells comprise a majority of the cellular infiltrate, little is known about the contribution of this population to disease pathogenesis. An emerging animal model of AA involves adoptive transfer of in vitro activated and expanded bulk skin draining lymph node (SDLN) cells from AA mice, resulting in the induction of disease in recipient mice. The objective of this study was to investigate the mechanisms by which those cells transfer disease. Expanded bulk SDLN cells overwhelmingly consisted of CD3+ T cells, including both CD8 and CD4 T cells. To address whether these T cell populations could separately induce AA, we first sorted CD8 T cells from the SDLNs of AA mice and found these cells potently induced disease in recipient mice. Surprisingly, isolated CD4 T cells from AA mice also had a robust capacity to induce disease, and they did so more efficiently than CD4 T cells from unaffected (UA) mice. High dimensional flow cytometric analysis of the SDLNs indicated that an enhanced number of CD4 T cells in AA mice exhibited an effector-like phenotype. Development of AA following transfer of expanded CD4 T cells relied on the presence of endogenous CD8 T cells and host responsiveness to IFN-γ, but, in contrast, loss of IL-17 signaling appeared to have no effect. Together, these data suggest that activated CD4 T-helper type 1 effector cells contribute to the activation of CD8 T cells and the subsequent attack of the hair follicle. Further studies are needed to further define the specific mechanisms by which CD4 T cells contribute to the development of AA. Dept of VA (I01BX004907), NIH/NIAMS (R01AR077194, K08AR069111, T32AI007485)

Time Course scRNAseq Analysis Reveals Mechanisms of Impaired Lung Recovery Post Influenza Infection during Aging: Zooming into Macrophages

Abstract Aging is considered to be a major risk factor for host morbidity and mortality upon acute respiratory virus infections. To better elucidate the immune determinants of viral pathogenesis during aging, we chronological characterized lung cellular and molecular profiles following influenza virus (PR8) infection in young or aged mice till 60 days post infection. Time-course scRNAseq coupled with high dimensional flow cytometry enabled the description of changes in both immune and stromal compartment between young and aged hosts. We further zoomed into the myeloid compartment and focused on the dynamic responses of lung macrophages given their complex roles in viral pathogenesis, tissue repair and lung fibrosis. Enlarged Monocyte-derived Macrophage (MoM) population as well as decreased resident-Alveolar Macrophage (AM) population were observed in the aged mice when compared to those of the young mice. Despite the self-sustain nature of AMs during homeostasis, it has been reported that Ly6C himonocytes can gain AM identity following severe lung damage like influenza virus infection. We found that the gain of AM identity from monocytes was abrogated in the aged hosts, which likely contributed to the age-associated impairment in lung recovery after influenzas virus infection. Additionally, trajectory analysis with scRNAseq allowed us to identify IFNα/γ signaling to be potential pathways contributing to such defect. This notion is also supported by elevated IFNγ level in BAL from the aged host during lung recovery. Our results thus have improved our understanding on the cellular and molecular mechanisms by which aged hosts exhibit severe diseases and delayed recovery after respiratory viral infection.

Localized immune dysregulation is associated with lung injury in severe pediatric COVID-19

Abstract Acute respiratory distress syndrome (ARDS) is a common and devastating consequence of SARS-CoV-2 infection in adults, however severe lung injury is a rare manifestation of COVID-19 in children. Aberrant local immune responses are known to drive lung pathology in adults, but little is known about localized immune responses in children with respiratory failure due to COVID-19. We obtained blood and respiratory tract samples from children (n=35, aged 1 month to 18 years) admitted to the hospital for COVID-19, stratifying by presence of respiratory failure (n=15). We defined the cytokine and chemokine profile of severe disease using a 71 analyte Luminex panel. Children with respiratory failure were found to have increased circulating levels of leukemia inhibitory factor (LIF), a growth factor upregulated during pulmonary inflammation. The local environment revealed significant inflammation with increased levels of pro-inflammatory cytokines including IL-6, IL-8, and IL-33 among others. Application of high dimensional flow cytometry to define immune cell subsets revealed elevated proportions of monocytes in both circulating and local environments. Monocytes demonstrated decreased expression of HLA-DR and CD86, suggesting an immunoparalysis phenotype, previously associated with severe COVID-19 in adults. Further, effector memory T cells in the airway exhibited an exhausted phenotype with high levels of PD-1 expression. Together this analysis reveals a highly inflammatory and dysregulated immune response underlies SARS-CoV-2 induced pediatric lung injury. These findings suggest a shared pathway with adult COVID-19 ARDS and provide new insights into the immune mechanisms associated with severe COVID-19 in children. Supported by grants from NIH (K23AI141686)

Pre-existing T cell immune response specific to computationally optimized broadly reactive hemagglutinin antigens (COBRA-HA) in influenza vaccinees

Abstract The biggest challenge in developing a universal influenza vaccine is the extensive antigenic drift. In contrast to humoral immunity, cell-mediated immunity is more cross-reactive and can protect against various subtypes and drift variants. Our research group has described computationally optimized broadly reactive antigens (COBRA) to develop HA-based broadly protective influenza vaccine. However, studies that characterize the potential of COBRA-HA to elicit a functional T-cell response have yet to be developed. Cross-reactive response to conserved T cell epitopes from previous exposure can provide an efficient heterologous immunity. Thus, we aim to immune profile the pre-existing and cross-reactive COBRA-HA T cell response at the functionality and epitope conservation level to identify potential targets to be included in universal flu vaccine design. To achieve this goal, 50 donors who received the seasonal influenza vaccine (Fluzone, Sanofi) for two consecutive seasons were HLA typed, and the epitopes of COBRA-HA vaccine candidates Y2 (H1) and NG2 (H3) were predicted using IEDB software. Using the high dimensional flow cytometry, eight peptides for Y2 and 15 for NG2 were combined in pools to determine the phenotypes and cytokine secretion. We found that the vaccination induced a cross-reactive COBRA HA-specific CD4+ and CD8+ T cell response independent of antibody titers. Next, we will evaluate the Th helper subsets (Th1/Th2/Th17/cTfh) and the breadth of immune response by deconvoluting the pools in IFNγ Fluospot assay. Nonetheless, the next generation of the universal influenza vaccine recalled a functional CD4+ pre-existing response, demonstrating the potential efficacy of COBRA-HA in clinical trials. National Institute of Allergy and Infectious Diseases (NIAID), U.S. National Institutes of Health (NIH), Department of Health and Human Services contract 75N93019C00052 (TMR).

STAT1 gain of function mutation impairs immune response to viral infections

Abstract The coordinated effort of the immune system in host defense is dependent upon the proper release and interpretation of different cytokine signals. Several examples of monogenic diseases targeting the JAK-STAT pathway reveal there is still a critical need to understand basic principles of cytokine signaling and cytokine output. For example, patients with STAT1 gain-of-function (GOF) mutations exhibit a type 1/IFN-gamma bias that antagonizes a type 3/IL-17 immune response important for controlling fungal infection, yet patients also, paradoxically, exhibit chronic and sometimes lethal viral infections. Using a novel conditional knock in STAT1-GOF mouse model, we demonstrate that STAT1-GOF mice respond poorly to viral infections that are easily controlled by WT mice, such as MCMV and LCMV-Armstrong. High-dimensional flow cytometry and scRNA-seq reveal an impaired NK and CD8 T cell effector response in STAT1-GOF mice that is not simply due to an exhaustion phenotype. Instead, STAT1-GOF mice respond poorly to viral infections due to an unexpected defect in IFN-gamma production early during the innate immune response, leading to a cytokine storm, immunopathology, and an impaired adaptive immune response. scATAC/RNA-seq uncovers altered usage of different STAT complexes in STAT1-GOF mice, leading to a misinterpretation of early innate-stimulating cytokines. These results provide insight into why patients with interferonopathies or elevated interferons respond poorly to viral infections and highlight the importance of properly tuning STAT1 during an immune response. R.L.P. was supported by a Postdoctoral Research Associate (PRAT) fellowship from the National Institute of General Medical Sciences (NIGMS), award number 1Fi2GM137942-01

Identification of multiple sclerosis endophenotypes by high dimensional blood signatures associated with distinct disease trajectories (P1-3.008)

Identification of multiple sclerosis endophenotypes by high dimensional blood signatures associated with distinct disease trajectories (P1-3.008)

PyInfinityFlow: optimized imputation and analysis of high-dimensional flow cytometry data for millions of cells.

While conventional flow cytometry is limited to dozens of markers, new experimental and computational strategies, such as Infinity Flow, allow for the generation and imputation of hundreds of cell surface protein markers in millions of cells. Here, we describe an end-to-end analysis workflow for Infinity Flow data in Python. pyInfinityFlow enables the efficient analysis of millions of cells, without down-sampling, through direct integration with well-established Python packages for single-cell genomics analysis. pyInfinityFlow accurately identifies both common and extremely rare cell populations which are challenging to define from single-cell genomics studies alone. We demonstrate that this workflow can nominate novel markers to design new flow cytometry gating strategies for predicted cell populations. pyInfinityFlow can be extended to diverse cell discovery analyses with flexibility to adapt to diverse Infinity Flow experimental designs. pyInfinityFlow is freely available in GitHub (https://github.com/KyleFerchen/pyInfinityFlow) and on PyPI (https://pypi.org/project/pyInfinityFlow/). Package documentation with tutorials on a test dataset is available by Read the Docs (pyinfinityflow.readthedocs.io). The scripts and data for reproducing the results are available at https://github.com/KyleFerchen/pyInfinityFlow/tree/main/analysis_scripts, along with the raw flow cytometry input data.

Intranasal Delivery of TGFα – A Novel Therapeutic Approach for Lesion Resolution in Multiple Sclerosis? (P7-3.001)

<h3>Objective:</h3> We here evaluate the therapeutic potential of tissue-protective TGFα for the resolution of CNS inflammation. <h3>Background:</h3> After acute lesions in the central nervous system (CNS) such as in Multiple Sclerosis (MS), the interaction of microglia, astrocytes and infiltrating immune cells defines the inflammatory microenvironment in the CNS which can either induce resolution of inflammation or lead to its chronification. However, the interaction between microglia, astrocytes and infiltrating immune cells and their respective contribution to the resolution of tissue damage is poorly characterized. In this context, we have recently identified microglia-derived TGFα as a novel driver of recovery in MS and its preclinical animal model experimental autoimmune encephalomyelitis (EAE). Here, we describe its regulation over the course of the disease and investigate its translational potential during acute inflammation in the CNS. <h3>Design/Methods:</h3> We here use CRISPR–Cas9-based genetic perturbation models, high-dimensional flow cytometry, immunohistochemistry and magnetic resonance imaging (MRI), as well as fine-tuned <i>in vitro</i> assays to investigate cellular targets, net effects and therapeutic potential of microglia-derived TGFα for recovery in MS and its preclinical animal model experimental autoimmune encephalomyelitis (EAE). <h3>Results:</h3> During acute CNS inflammation, microglia secrete TGFα in a highly regulated temporospatial manner. We show that microglial TGFα is required for EAE recovery by decreasing the number of infiltrating T cells and pro-inflammatory myeloid cells, as well as reducing oligodendrocyte loss, axonal damage, and demyelination. In a therapeutic approach, intranasally delivered TGFα diminishes the pro-inflammatory profile of astrocytes and CNS infiltrating immune cells, while promoting neuronal survival and lesion resolution, therefore promoting clinical recovery. <h3>Conclusions:</h3> Together, we show that TGFα acts on glial and infiltrating cells to promote the resolution of CNS inflammation. TGFα signaling might represent a novel checkpoint of CNS recovery, which offers therapeutic potential to promote recovery in acute and chronic stages of MS. <b>Disclosure:</b> Mrs. Loesslein has nothing to disclose. Mr. Linnerbauer has nothing to disclose. Dr. Tsaktanis has nothing to disclose. Dr. Quintana has nothing to disclose. Prof. Rothhammer has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Novartis. Prof. Rothhammer has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Merck. The institution of Prof. Rothhammer has received research support from German Research Foundation. The institution of Prof. Rothhammer has received research support from European Research Council.

Pseudomonas-dominant microbiome elicits sustained IL-1β upregulation in alveolar macrophages from lung transplant recipients

Isolation of Pseudomonas aeruginosa (PsA) is associated with increased BAL (bronchoalveolar lavage) inflammation and lung allograft injury in lung transplant recipients (LTR). However, the effect of PsA on macrophage responses in this population is incompletely understood. We examined human alveolar macrophage (AMΦ) responses to PsA and Pseudomonas dominant microbiome in healthy LTR. We stimulated THP-1 derived macrophages (THP-1MΦ) and human AMΦ from LTR with different bacteria and LTR BAL derived microbiome characterized as Pseudomonas-dominant. Macrophage responses were assessed by high dimensional flow cytometry, including their intracellular production of cytokines (TNF-α, IL-6, IL-8, IL-1β, IL-10, IL-1RA, and TGF-β). Pharmacological inhibitors were utilized to evaluate the role of the inflammasome in PsA-macrophage interaction. We observed upregulation of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) following stimulation by PsA compared to other bacteria (Staphylococcus aureus (S.Aur), Prevotella melaninogenica, Streptococcus pneumoniae) in both THP-1MΦ and LTR AMΦ, predominated by IL-1β. IL-1β production from THP-1MΦ was sustained after PsA stimulation for up to 96 hours and 48 hours in LTR AMΦ. Treatment with the inflammasome inhibitor BAY11-7082 abrogated THP-1MΦ IL-1β production after PsA exposure. BAL Pseudomonas-dominant microbiota elicited an increased IL-1β, similar to PsA, an effect abrogated by the addition of antibiotics. PsA and PsA-dominant lung microbiota induce sustained IL-1β production in LTR AMΦ. Pharmacological targeting of the inflammasome reduces PsA-macrophage-IL-1β responses, underscoring their use in lung transplant recipients.

Transcriptome network analysis implicates CX3CR1-positive type 3 dendritic cells in non-infectious uveitis.

Type I interferons (IFNs) promote the expansion of subsets of CD1c+ conventional dendritic cells (CD1c+ DCs), but the molecular basis of CD1c+ DCs involvement in conditions not associated without elevated type I IFNs remains unclear. We analyzed CD1c+ DCs from two cohorts of non-infectious uveitis patients and healthy donors using RNA-sequencing followed by high-dimensional flow cytometry to characterize the CD1c+ DC populations. We report that the CD1c+ DCs pool from patients with non-infectious uveitis is skewed toward a gene module with the chemokine receptor CX3CR1 as the key hub gene. We confirmed these results in an independent case-control cohort and show that the disease-associated gene module is not mediated by type I IFNs. An analysis of peripheral blood using flow cytometry revealed that CX3CR1+ DC3s were diminished, whereas CX3CR1- DC3s were not. Stimulated CX3CR1+ DC3s secrete high levels of inflammatory cytokines, including TNF-alpha, and CX3CR1+ DC3 like cells can be detected in inflamed eyes of patients. These results show that CX3CR1+ DC3s are implicated in non-infectious uveitis and can secrete proinflammatory mediators implicated in its pathophysiology. The presented work is supported by UitZicht (project number #2014-4, #2019-10, and #2021-4). The funders had no role in the design, execution, interpretation, or writing of the study.

Metabolic dysregulation impairs lymphocyte function during severe SARS-CoV-2 infection

Cellular metabolic dysregulation is a consequence of SARS-CoV-2 infection that is a key determinant of disease severity. However, how metabolic perturbations influence immunological function during COVID-19 remains unclear. Here, using a combination of high-dimensional flow cytometry, cutting-edge single-cell metabolomics, and re-analysis of single-cell transcriptomic data, we demonstrate a global hypoxia-linked metabolic switch from fatty acid oxidation and mitochondrial respiration towards anaerobic, glucose-dependent metabolism in CD8+Tc, NKT, and epithelial cells. Consequently, we found that a strong dysregulation in immunometabolism was tied to increased cellular exhaustion, attenuated effector function, and impaired memory differentiation. Pharmacological inhibition of mitophagy with mdivi-1 reduced excess glucose metabolism, resulting in enhanced generation of SARS-CoV-2- specific CD8+Tc, increased cytokine secretion, and augmented memory cell proliferation. Taken together, our study provides critical insight regarding the cellular mechanisms underlying the effect of SARS-CoV-2 infection on host immune cell metabolism, and highlights immunometabolism as a promising therapeutic target for COVID-19 treatment.

Abstract 6676: Exploratory biomarker analyses in phase 1 study of regorafenib, ipilimumab, and nivolumab (RIN) in chemotherapy resistant microsatellite stable (MSS) metastatic colorectal cancer (mCRC)

Abstract Background: We recently reported an objective response rate of 36.4% in a phase I trial of RIN in MSS mCRC patients without liver metastases, while no response was noted in patients with liver metastases. Here we characterized kinetics in local and systemic immune responses associated with clinical outcomes, liver metastasis, and immune related toxicity during RIN treatment. Methods: Patients with chemotherapy-resistant MSS mCRC received RIN at (R) at 80 mg QD x 21 days in 4-week(w) cycle, (I) at 1mg/kg Q6w and (N) at 240 mg Q 2w (n=29). Needle biopsies were collected at baseline (n=8) and 4 weeks (n=6) after treatment. Blood samples were collected at baseline (n=29), 4 weeks (n=29) and 8 weeks (n=25) after treatment. Alterations in the tumor microenvironment (TME) were characterized by RNA sequencing. We analyzed the immune cell composition and activities in the blood, including 90 subpopulations of leukocytes and 29 activation/suppression/proliferation markers, with high-dimensional spectral flow cytometry. Results: The clinical responses are positively correlated with the induction of local anti-tumor immunity. Patients with better clinical outcomes exhibited higher levels of lymphocyte infiltration, chemokine production, and antigen-presentation potential in the local TME following one cycle of RIN treatment. The duration of progression-free survival (PFS) was correlated with a higher baseline STING signal in the TME. In the peripheral blood, the responders have more circulating CD4 T cells, B cells, and cDC1, while expressing lower immune activation markers, especially in T cells with type 1 immunity at baseline. At 4 weeks, immune activation and proliferation markers were higher in responders than non-responders. Interestingly, immune effectors (non-Tregs, CD45RA− CD8 T cells, NKT cells, and M1 monocytes) in the blood were proportionally reduced in responders at 4 weeks, coincident with their enrichment in the local TME. We also compared peripheral immune signatures between patients with and without liver metastases, liver metastases (n=7) were associated with a significantly lower ratio of CD4/CD8 T cells in the blood, a phenomenon of immunosenescence, both at baseline and after RIN treatment. Lastly, the occurrence of immune-related toxicities was closely correlated with the induction of immune activities in both acellular and cellular components in the circulation. Conclusions: Clinical benefit of RIN was correlated to more robust immune activation of the TME and peripheral blood following one cycle of treatment. A higher baseline STING signal in the TME was associated with longer PFS. Liver metastasis was associated with a lower ratio of peripheral CD4/CD8 T cells which may contribute to the resistance to immunotherapy in those patients. Citation Format: Jian Ye, Chongkai Wang, Colt Egelston, Weihua Guo, Peter P. Lee, Marwan Fakih. Exploratory biomarker analyses in phase 1 study of regorafenib, ipilimumab, and nivolumab (RIN) in chemotherapy resistant microsatellite stable (MSS) metastatic colorectal cancer (mCRC). [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 6676.

Abstract 4461: Optimized human immunophenotyping panels enhance the flexibility for high-dimensional flow cytometry analysis with CyTOF

Abstract The immune system plays a pivotal role in the pathogenesis of cancer. Beyond its importance in leukemia and lymphoma, scientists now understand that the immune system is involved in virtually all malignancies. Heterogeneity of cell phenotypes within a tumor microenvironment, including blood cell phenotypes, is a hallmark of cancer that is poorly understood. Resolution of these phenotypes is essential to direct therapeutic development. CyTOF® technology has pioneered the field of high-dimensional flow cytometry through the use of isotopically pure metal-tagged antibodies and a highly sensitive mass cytometer to enable 50-plus-parameter analysis. Easy panel design without the need for compensation controls or issues of autofluorescence allows for comprehensive single-cell analysis in complex biological samples and is particularly well suited for revealing the intricacies of oncogenesis. Maxpar® OnDemand reagents were recently introduced for CyTOF to increase flexibility and facilitate larger panel design in a short period of time. To this end, lineage markers that have strong, reliable expression were reassigned to optimal metal isotopes to reserve the high-sensitivity lanthanide channels for more difficult-to-detect markers with low endogenous expression, common in immuno-oncology studies. In this study, we aimed to test the newly released Maxpar OnDemand™ Antibodies to create an optimized immune phenotyping panel and maximize further customization for CyTOF. Individual antibody performance was compared between the new and existing antibodies with different metal tags. Human peripheral blood mononuclear cells (PBMC) and fresh whole blood (WB) were stained to confirm equivalent capabilities for population gating. Importantly, we demonstrated the power of these antibodies to generate reproducible, impactful data by creating functional human immunophenotyping panels. A 20-marker human phenotyping panel was tested in PBMC and WB. We identified major immune cell populations including T cells, B cells, granulocytes, natural killer (NK) cells, and monocytes. Furthermore, the addition of CD45RO to the panel enhanced the delineation of naive, memory and effector T cell subsets. Additional myeloid markers were included to resolve the complexity within this compartment. With the updated placement of key lineage markers, the optimized phenotyping panel is ideal for future immuno-oncology studies as it can be easily expanded to interrogate cell cycle proteins, cytokines, cell signaling proteins, and oncogenic transcription factors. This work demonstrates the capability of CyTOF for robust, high-parameter immunophenotyping and a high degree of flexibility to expand our understanding of the complex processes underlying carcinogenesis and response to therapy. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Lauren J. Tracey, Michael Cohen, Christina Loh. Optimized human immunophenotyping panels enhance the flexibility for high-dimensional flow cytometry analysis with CyTOF. [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 4461.