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To investigate the immune cell profiles of patients with systemic lupus erythematosus (SLE), and to identify longitudinal changes in those profiles over time.We employed mass cytometry with 3 different panels of 38-39 markers (an immunophenotyping panel, a T cell/monocyte panel, and a B cell panel) in cryopreserved peripheral blood mononuclear cells (PBMCs) from 9 patients with early SLE, 15 patients with established SLE, and 14 controls without autoimmune disease. We used machine learning-driven clustering, flow self-organizing maps, and dimensional reduction with t-distributed stochastic neighbor embedding to identify unique cell populations in early SLE and established SLE. We used mass cytometry data of PBMCs from 19 patients with early rheumatoid arthritis (RA) and 23 controls to compare levels of specific cell populations in early RA and SLE. For the 9 patients with early SLE, longitudinal mass cytometry analysis was applied to PBMCs at enrollment, 6 months after enrollment, and 1 year after enrollment. Serum samples were also assayed for 65 cytokines using Luminex multiplex assay, and associations between cell types and cytokines/chemokines were assessed.Levels of peripheral helper T cells, follicular helper T (Tfh) cells, and several Ki-67+ proliferating subsets (ICOS+Ki-67+ CD8 T cells, Ki-67+ regulatory T cells, CD19Two major helper T cell subsets and unique Ki-67+ proliferating immune cell subsets were expanded in patients in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

Myelodysplastic syndromes (MDS) are clonal neoplasms of the hematopoietic stem cell that result in aberrant differentiation of hematopoietic lineages caused by a wide range of underlying genetic, epigenetic, and other causes. Despite the myriad origins, a recognizable MDS phenotype has been associated with miRNA aberrant expression. A model of aberrant myeloid maturation that mimics MDS was generated using a stable knockdown of miR-378-3p. This model exhibited a transcriptional profile indicating aberrant maturation and function, immunophenotypic and morphologic dysplasia, and aberrant growth that characterizes MDS. Moreover, aberrant signal transduction in response to stimulation specific to the stage of myeloid maturation as indicated by CyTOF mass cytometry was similar to that found in samples from patients with MDS. The aberrant signaling, immunophenotypic changes, cellular growth, and colony formation ability seen in this myeloid model could be reversed with azacytidine, albeit without significant improvement of neutrophil function.

Immunotherapy has emerged as a promising approach to treat cancer, however, its efficacy in highly malignant brain-tumors, glioblastomas (GBM), is limited. Here, we generate distinct imageable syngeneic mouse GBM-tumor models and utilize RNA-sequencing, CyTOF and correlative immunohistochemistry to assess immune-profiles in these models. We identify immunologically-inert and -active syngeneic-tumor types and show that inert tumors have an immune-suppressive phenotype with numerous exhausted CD8 T cells and resident macrophages; fewer eosinophils and SiglecF+ macrophages. To mimic the clinical-settings of first line of GBM-treatment, we show that tumor-resection invigorates an anti-tumor response via increasing T cells, activated microglia and SiglecF+ macrophages and decreasing resident macrophages. A comparative CyTOF analysis of resected-tumor samples from GBM-patients and mouse GBM-tumors show stark similarities in one of the mouse GBM-tumors tested. These findings guide informed choices for use of GBM models for immunotherapeutic interventions and offer a potential to facilitate immune-therapies in GBM patients., Syngeneic mouse models for glioblastoma (GBM) cannot fully recapitulate clinical findings and response to therapy in patients. Here the authors perform a comprehensive immune profiling of different syngeneic GBM tumour models and compare it with the immune landscape of GBM patients to identify similarities and potential confounding differences.

CD4+ regulatory T cells (Tregs) are acutely activated by traumatic injury, which suggests that they may react to injury with similar kinetics as memory T cells. Here, we used a mouse burn trauma model to screen for memory-like T cell responses to injury by transferring T cells from sham or burn CD45.1 mice into CD45.2 mice and performing secondary injuries in recipient mice. Among all T cell subsets that were measured, only Tregs expanded in response to secondary injury. The expanded Tregs were a CD44high/CD62Llow subpopulation, markers indicative of memory T cells. CyTOF (cytometry by time-of-flight) mass cytometry was used to demonstrate that injury-expanded Tregs expressed higher levels of CD44, CTLA-4, ICOS, GITR, and Helios than Tregs from noninjured mice. Next, we tested whether a similar population of Tregs might react acutely to burn trauma. We observed that Tregs with a phenotype that matched the injury-expanded Tregs were activated by 6 h after injury. To test if Treg activation by trauma requires functional MHC class II, we measured trauma-induced Treg activation in MHC class II gene deficient (MHCII−/−) mice or in mice that were given Fab fragment of anti-MHC class II antibody to block TCR activation. Injury-induced Treg activation occurred in normal mice but only partial activation was detected in MHCII−/− mice or in mice that were given Fab anti-MHCII antibody. These findings demonstrate that trauma activates a memory-like Treg subpopulation and that Treg activation by injury is partially dependent on TCR signaling by an MHC class II dependent mechanism.

Background Trauma causes tissue injury that results in the release of damage associated molecular patterns (DAMPs) and other mediators at the site of injury and systemically. Such mediators disrupt immune system homeostasis and may activate multicellular immune responses with downstream complications such as the development of infections and sepsis. To characterize these alterations, we used time-of-flight mass cytometry to determine how trauma plasma affects normal peripheral blood mononuclear cell (PBMC) activation to gain insights into the kinetics and nature of trauma-induced circulating factors on human immune cell populations. A better understanding of the components that activate cells in trauma may aid in the discovery of therapeutic targets. Methods PBMCs from healthy volunteers were cultured with 5% plasma (healthy, trauma-1day, or trauma-3day) or known DAMPs for 24 h. Samples were stained with a broad immunophenotyping CyTOF antibody panel. Multiplex (Luminex) cytokine assays were used to measure differences in multiple cytokine levels in healthy and trauma plasma samples. Results Plasma from day 1, but not day 3 trauma patients induced the acute expansion of CD11c+ NK cells and CD73+/CCR7+ CD8 T cell subpopulations. Additionally, trauma plasma did not induce CD4+ T cell expansion but did cause a phenotypic shift towards CD38+/CCR7+ expressing CD4+ T cells. Multiplex analysis of cytokines by Luminex showed increased levels of IL-1RA, IL-6 and IL-15 in trauma-1day plasma. Similar to trauma day 1 plasma, PBMC stimulation with known DAMPs showed activation and expansion of CD11c+ NK cells. Conclusions We hypothesized that circulating factors in trauma plasma would induce phenotypic activation of normal human immune cell subsets. Using an unbiased approach, we identified specific changes in immune cell subsets that respond to trauma plasma. Additionally, CD11c+ NK cells expanded in response to DAMPs and LPS, suggesting they may also be responding to similar components in trauma plasma. Collectively, our data demonstrate that the normal PBMC response to trauma plasma involves marked changes in specific subsets of NK and CD8+ T cell populations. Future studies will target the function of these trauma plasma reactive immune cell subsets. These findings have important implications for the field of acute traumatic injuries.

BackgroundImmune checkpoint blockade (ICB) response in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is limited to 15%–20% of patients and underpinnings of resistance remain undefined.MethodsStarting with an anti-PD1 sensitive murine HNSCC cell line, we generated an isogenic anti-PD1 resistant model. Mass cytometry was used to delineate tumor microenvironments of both sensitive parental murine oral carcinoma (MOC1) and resistant MOC1esc1 tumors. To examine heterogeneity and clonal dynamics of tumor infiltrating lymphocytes (TILs), we applied paired single-cell RNA and TCR sequencing in three HNSCC models.ResultsAnti-PD1 resistant MOC1esc1 line displayed a conserved cell intrinsic immune evasion signature. Immunoprofiling showed distinct baseline tumor microenvironments of MOC1 and MOC1esc1, as well as the remodeling of immune compartments on ICB in MOC1esc1 tumors. Single cell sequencing analysis identified several CD8 +TIL subsets including Tcf7 +Pd1− (naïve/memory-like), Tcf7 +Pd1+ (progenitor), and Tcf7-Pd1+ (differentiated effector). Mapping TCR shared fractions identified that successful anti-PD1 or anti-CTLA4 therapy-induced higher post-treatment T cell lineage transitions.ConclusionsThese data highlight critical aspects of CD8 +TIL heterogeneity and differentiation and suggest facilitation of CD8 +TIL differentiation as a strategy to improve HNSCC ICB response.

Background and ObjectiveShared decision-making (SDM) aligns patient preferences with health care team treatment goals. This quality improvement initiative implemented a standardized SDM bundle within a neurocritical care unit (NCCU), where unique demands make existing, provider-driven SDM practices challenging.MethodsAn interprofessional team defined key issues, identified barriers, and created change ideas to drive implementation of an SDM bundle using the Institute for Healthcare Improvement Model for Improvement framework incorporating Plan-Do-Study-Act cycles. The SDM bundle included (1) a health care team huddle pre-SDM and post-SDM conversation; (2) a social worker–driven SDM conversation with the patient family, including core standardized communication elements to ensure consistency and quality; and (3) an SDM documentation tool within the electronic medical record to ensure the SDM conversation was accessible to all health care team members. The primary outcome measure was percentage of SDM conversations documented.ResultsDocumentation of SDM conversations improved by 56%, from 27% to 83% pre/postintervention. Average time to documentation decreased by 4 days, from day 9 preintervention to day 5 postintervention. There was no significant change in NCCU length of stay, nor did palliative care consultation rates increase. Postintervention, SDM team huddle compliance was 94.3%.DiscussionA team-driven, standardized SDM bundle that integrates with health care team workflows enabled SDM conversations to occur earlier and resulted in improved documentation of SDM conversations. Team-driven SDM bundles have the potential to improve communication and promote early alignment with patient family goals, preferences, and values.

ObjectiveTo investigate the immune cell profiling and their longitudinal changes in systemic lupus erythematosus (SLE).MethodsWe employed mass cytometry with three different 38-39 marker panels (Immunophenotyping, T cell/monocyte, and B cell) in cryopreserved peripheral blood mononuclear cells (PBMCs) from nine patients with early SLE, 15 patients with established SLE, and 14 non-inflammatory controls. We used machine learning-driven clustering, FlowSOM (Flow Self-Organizing Maps) and dimensional reduction with tSNE (t-distributed Stochastic Neighbor Embedding) to identify unique cell populations in early and established SLE. For the nine early SLE patients, longitudinal mass cytometry analysis was applied to PBMCs at three time points (at enrollment, six months post-enrollment, and one year post-enrollment). Serum samples were also assayed for 65 cytokines by Luminex multiplex assay, and associations between cell types and cytokines/chemokines assessed.ResultsT peripheral helper cells (Tph cells), T follicular helper cells (Tfh cells) and several Ki67+ proliferating subsets (ICOS+ Ki67+ CD8 T cells, Ki67+ regulatory T cells, CD19int Ki67hi plasmablasts, and Ki67hi PU.1hi monocytes) were increased in early SLE. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from early SLE patients revealed that Tfh cells and CXCL10 decreased at one year post-enrollment. CXCL13 correlated positively with several of the expanded cell populations in early SLE.ConclusionsTwo major helper T cell subsets and unique Ki67+ proliferating immune cell subsets were expanded in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

The differential diagnosis for the comatose patient is includes structural abnormality, seizure, encephalitis, metabolic derangements, and toxicologic etiologies. Identifying and treating the underlying pathology in a timely manner is critical for the patient's outcome. We provide a structured approach to taking a history and performing a physical examination for this patient population. We discuss diagnostic testing and treatment methodologies for each of the common causes of coma. Our current understanding of the mechanisms of coma is insufficient to accurately predict the patient's clinical trajectory and more work needs to be done to investigate potential treatments for this often fatal disorder.

Prolonged exposure to hyperoxia has deleterious effects on the lung, provoking both inflammation and alveolar injury. The elements of hyperoxic injury, which result in high rates of lethality in experimental models, are thought to include multicellular immune responses. To characterize these alterations in immune cell populations, we performed time-of-flight mass cytometry (CyTOF) analysis of CD45-expressing immune cells in whole lung parenchyma and the bronchoalveolar space of mice, exposed to 48 hours of hyperoxia together with normoxic controls. At the tested time point, hyperoxia exposure resulted in decreased abundance of immunoregulatory populations (regulatory B cells, myeloid regulatory cells) in lung parenchyma and markedly decreased proliferation rates of myeloid regulatory cells, monocytes and alveolar macrophages. Additionally, hyperoxia caused a shift in the phenotype of alveolar macrophages, increasing proportion of cells with elevated CD68, CD44, CD11c, PD-L1, and CD205 expression levels. These changes occurred in the absence of histologically evident alveolar damage and abundance of neutrophils in the parenchyma or alveolar space did not change at these time points. Collectively, these findings demonstrate that pulmonary response to hyperoxia involves marked changes in specific subsets of myeloid and lymphoid populations. These findings have important implications for therapeutic targeting in acute lung injury.

BackgroundAlloplex Biotherapeutics has developed a novel autologous cellular therapy for cancer that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand a heterogeneous population of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). The 2-week manufacturing process from PBMCs consistently results 300-fold expansion of NK cells, CD8+ T cells, gamma/delta T cells, NKT cells and some CD4+ T cells, collectively called SUPLEXA therapeutic cells. SUPLEXA cells will be delivered back to cancer patients via intravenous administrations on a weekly schedule as an autologous adoptive cellular immunotherapy for cancer. In this study, we tested SUPLEXA cells developed from normal healthy volunteer PBMCs for their ability to infiltrate and kill patient-derived tumor organoids (PDO) as a pre-clinical assessment for potency against 2 different types of tumor organoids.MethodsTumor organoids derived from colorectal cancer (CRC) or non-small cell lung carcinoma (NSCLC) patients were labeled with cell-trace red dye and plated at equal density in a 96-well plate. After 3 days culture, SUPLEXA cells were thawed (82.8% viable), labeled with cell-trace violet dye, and added to PDO at 1:2 serial diluted numbers ranging from 2 million to 7,800 cells per well. Fluorescent images were captured at 24 hours after adding SUPLEXA cells to PDO models to measure PDO size, tumor infiltration, and PDO killing.ResultsAdding SUPLEXA cells to PDO from CRC and NSCLC resulted in significant infiltration and killing of organoids by 24 hours as shown by the fluorescent images and the organoid size plot for the CRC PDO model (figure 1). Significant reduction in PDO size was observed by adding 31,240 SUPLEXA cells. Similar results were observed with the NSCLC PDO model with significant reduction in PDO size by adding 15,600 SUPLEXA cells. Obvious organoid infiltration was observed in both PDO models and organoid fluorescence was significantly reduced by addition of SUPLEXA cells in both PDO models to suggest that SUPLEXA cells were able to reduce tumor burden (figure 2).Abstract 164 Figure 1CRC organoid infiltration and killing by SUPLEXA. A representative fluorescent image of CRC organoid killing with addition of increasing SUPLEXA cell numbers and a plot showing statistical analysis of 6 replicate wells for changes in CRC organoid size in relation to SUPLEXA cell number additionsAbstract 164 Figure 2Dose-dependent killing in CRC and NSCLC PDO models. CRC and NSCLC organoids were detected by total red fluorescence at 24 hours after adding the indicated numbers of SUPLEXA cells. Loss of red fluorescence after adding SUPLEXA is a measure of overall tumor cell killing/burden in organoids. Data is plotted as mean ± SEM for n=6 replicates per group.ConclusionsSUPLEXA cells infiltrated and killed tumor cells in patient-derived organoids within 24 hours of culture at low cell concentrations indicating potent tumor killing activity. The observed activity in both colorectal and lung cancer organoid models support broad anti-tumor killing activity by SUPLEXA. These results provide further evidence that PBMCs from cancer patients can be activated and expanded by our approach as a novel autologous cellular immunotherapy for cancer.

Introduction: A unique autologous cellular therapeutic (SUPLEXA) has been developed from human PBMC. It is comprised of NK cells, γδ T cells and CD8+ T effector cells, capable of broadly lysing a variety of tumor cell lines in vitro. SUPLEXA cells are manufactured using an efficient 2 week xeno-free manufacturing procedure employing two proprietary engineered leukocyte stimulator cell lines (ENLIST) that express an array of immunomodulatory proteins. The SUPLEXA cell manufacturing process is highly reproducible and demonstrates low inter-subject variability in cellular composition. SUPLEXA cells are distinguished from many other cellular approaches in that they are derived from autologous PBMC that have only been stimulated with ENLIST cells through naturally occurring receptors without any genetic modification. Here, we report on the in vitro characterization of SUPLEXA cytolytic activity on a variety of target tumor cell lines. Results: We prepared SUPLEXA cells from the PBMC of a variety of solid tumor patients (5 melanoma and 5 prostate). Yields and cell characterization by mass cytometry (CyTOF) yielded similar profiles within the variability expected for all the major cell subtypes. SUPLEXA cells were comparatively assessed for cytolytic activity on 4 tumor cell lines; K562 (leukemia), PC3 (prostate), M14 (melanoma) and COLO205 (colorectal cancer). In all instances, cytotoxicity was observed in a quantitative flow cytometry and microscopic assay and was titratable with cell number. Dose-dependent cytolysis was observed for all tumor lines, even at low E:T ratios of 1:10 suggesting that SUPLEXA cells acquire efficient and broad tumor cell lysis activity. Supernatants from cytolysis assays were analyzed for cytokine levels by a 35-plex assay revealing a remarkably restricted cytokine profile with constitutively high levels of RANTES production by the SUPLEXA cells and titratable levels of induced IL-6, IFN-γ and IL-8 production. Conclusions: SUPLEXA cells represent an easy to manufacture autologous anti-tumor cell therapy that overcomes many of the limitations associated with previous autologous therapies. They possess a broad anti-tumor activity at exceedingly low E:T ratios with specific production of IFN-γ, IL-6 and IL-8 during tumor cell killing. Ongoing efforts are underway to understand the relative contributions of NK cells, γδ T cells and CD8+ T cells to broad tumor cytolysis and the antigen-specificities seen with SUPLEXA cells from multiple donors. Planning for a first in human clinical trial is currently underway. Citation Format: Frank Borriello, Joshua W. Keegan, James A. Lederer. Development of PBMC derived tumor effector cells with potent pan-cancer cytolytic activity for autologous cellular immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1531.

Introduction: We developed a unique autologous cellular therapeutic strategy using a proprietary engineered leukocyte stimulator cell lines (ENLIST cells) that express an array of immunomodulatory proteins to activate and expand tumor cell killing effector cells from human peripheral blood mononuclear cells (PBMCs). Our clinical aim is to use these expanded cells, called SUPLEXA therapy in cancer patients. Results of prior immunophenotyping of SUPLEXA cells by mass cytometry (CyTOF) reveals that they are comprised of NK cells, CD8+ T cells and γδ T cells that express markers indicative of cytolytic function, e.g. granzymes and perforin. Using an unbiased approach to better define the specificity of SUPLEXA, we performed TCR repertoire analysis using the iRepertoire and 10X Genomics RNA sequencing platforms. Results: SUPLEXA was prepared from healthy donor PBMCs. RNA was subsequently prepared from SUPLEXA cells and donor PBMCs to compare TCR αβ and γδ chain clonality and diversity using the iRepertoire DAM-PCR sequencing platform. SUPLEXA cells showed dramatic and significantly increased clonality among the 4 TCR chains that were analyzed as compared to normal PBMCs. We also observed a diversification of TCRαβ and TCRγδ sequences in SUPLEXA as compared to PBMCs. In addition, to the increased diversity, clonal expansion was also observed. Single-cell RNA sequencing of flow-sorted CD3+ T cells using the 10X Genomics platform with paired TCRαβ and TCRγδ PCR sequencing feature overlays revealed high frequencies of T cells with paired TCR clonotypes. We discovered that the top 5 paired TCRαβ clonotype sequences accounted for 32.7% of T cells, while CD8+ T cells from a normal donor contains only 3.0% in the top 5 clonotypes. Similar high frequency clonotypes were observed for γδ T cells by 10X single-cell RNA sequencing. Conclusions: The creation of SUPLEXA from healthy adult PBMCs results in dramatic expansion of αβ and γδ TCR T cell clonotypes. Given that SUPLEXA cells demonstrate broad, dose-dependent tumor cytolytic activity, these findings suggest that the expanded αβ and γδ T cell clonotypes may react to tumor neoantigens that are present during the activation process by ENLIST cells, which are derived from the SK-MEL-2 melanoma cell line. Accordingly, TCR reagents made using the paired αβ and γδ T cell sequences from the expanded clonotypes will be useful for future tumor neoantigen discovery. Plans for a phase 1 clinical trial are currently underway. Citation Format: Frank Borriello, Joshua W. Keegan, Brandon L. Hancock, Fei Guo, James R. Lederer. A novel approach for autologous pan-cancer cellular immunotherapy reveals dramatic expansion of ab and gd TCR T cell clonotypes indicative of an antigen-driven response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1772.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell–B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4(+) T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1(hi)CXCR5(–)CD4(+) T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1(hi)CXCR5(–)CD4(+) T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5(–) T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c(+) B cells in SLE patients. PD-1(hi)CD4(+) T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

Trauma induces a complex immune response, requiring a systems biology approach to capture multicellular changes. Using mass cytometry by time-of-flight (CyTOF), we evaluated time-dependent changes in peripheral blood in trauma patients to identify changes correlated with infection.Total leukocytes were prepared via red blood cell lysis using peripheral blood samples from trauma patients with an Injury Severity Score greater than 20 at Days 1, 3, and 5 after injury, and from age- and sex-matched uninjured controls. Cells were stained using a 33-marker immunophenotyping CyTOF panel. Statistics were calculated using one-way analysis of variance with multiple comparisons.The CyTOF staining demonstrated changes in many cell subsets. The mean expression intensity of CD86 on monocytes decreased significantly at all time points after injury. When the patients were stratified based on development of infection, there was a trend to decreased CD86 expression on monocytes of those patients that developed subsequent infection. Based on stratification, we identified significantly increased expression of CD39 on NK cells only in patients that developed an infection.This study used a systems biology approach to identify novel changes in circulating immune cell subsets in trauma patients correlating with post-traumatic infection. Decreased expression of CD86, a costimulatory molecule, on monocytes demonstrates that trauma affects the innate system's ability to control T-cell immunity. We also found that CD39 expression on NK cells increased significantly in patients with subsequent infection. CD39 is a protein that generates adenosine, which has immunosuppressive effects on several immune cell types including NK cells. In summary, our results point to pathways that may be central to second-hit infections and further study to delineate these pathways could be key to generating clinical biomarkers or targeted immune therapies for trauma patients.Prognostic study, level II.

7517 Background: Alloplex Biotherapeutics has developed a cellular therapeutic that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand populations of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). This process leads to a 300-fold expansion of NK cells, CD8+ T cells, NKT cells, and TCRγδ T cells that are called SUPLEXA cells, which will be cryopreserved and transferred back into patients as an autologous immune cell therapy for cancer. In this study, PBMCs from CLL patients were used to generate SUPLEXA cells as a first approach to comparatively profile SUPLEXA cells from cancer patients and normal healthy volunteers (NHVs). Methods: ENLIST cell lines were engineered by expressing curated immunomodulatory proteins in the SK-MEL-2 melanoma cell line. Two million (M) PBMCs from 10 CLL patients or 2 NHVs were incubated with 0.4 M freeze/thaw killed ENLIST cells for 5 days in XVIVO-15 medium with 2% heat-inactivated human AB serum (XAB2) and then split 1:15 in XAB2 containing IL-7 and IL-15 to expand. After 9 days, SUPLEXA cells were harvested and cryopreserved. Results: Original PBMCs and matched SUPLEXA cells from each donor were thawed and characterized by mass cytometry (CyTOF) using a 47-marker antibody panel. CyTOF staining results of PBMCs from CLL patients demonstrated approximately 95% leukemia cells and few T cells, NK cells, B cells, and monocytes. CyTOF staining of SUPLEXA cells from all 10 CLL patients showed expansion of NK cells (17%), CD8 T cells (11%), and CD4 T cells (7.5%) that were similar in phenotype to SUPLEXA cells from NHVs showing high expression of granzymes and perforin that are indicative of potent tumor cell killing activity. Cancer cells in the original CLL PBMC samples were reduced to 0.78%. However, a population of non-T/non-B cells (60% ± 9.5%) was detected in SUPLEXA cells from all CLL patients that require further characterization. Next, SUPLEXA cells from CLL and NHV patients were comparatively tested for tumor cell killing activity at 2:1, 1:1, and 1:2 effector to target cell (MEL-14 melanoma cells expressing RFP) ratios. Percent killing of tumor cells by SUPLEXA cells prepared from CLL patients (77.8% ± 2.6% at 2:1) and NHVs (81.5% ± 0.3% at 2:1) were nearly identical at all effector to target ratios. Conclusions: We demonstrate for the first time that PBMCs from CLL patients can be converted into SUPLEXA cells despite low numbers of normal immune cells at baseline and the known immunologic impairment present in CLL patients. Importantly, SUPLEXA cells derived from CLL patients acquire potent tumor killing activity that is indistinguishable from SUPLEXA cells prepared from NHVs. Taken together, these findings support the feasibility of converting PBMCs from CLL patients with low percentages of NK and T cells into an autologous cellular therapy for cancer.

Immunotherapy has emerged as a promising approach to treat cancer, however, its efficacy in highly malignant brain-tumors, glioblastomas (GBM), is limited. Pre-clinical syngeneic mouse models are at the forefront of testing and understanding emerging novel immune based therapies prior to their translation in patients. We generated distinct imageable syngeneic GBM-tumor models and assessed tumor infiltrating immune cells from end-stage tumors by utilizing RNA sequencing, CyTOF (mass Cytometry by Time of Flight) and correlative immunohistochemistry. CyTOF facilitates multiparametric analysis of up to 40 different markers which is useful especially in the case of GBM where number of TILs is limiting. We identified immunologically-inert and active syngeneic tumor types and show that inert tumors have an immune-suppressive phenotype with numerous MDSCs (Ly6C+ CD11b+), exhausted CD8 T cells and fewer eosinophils. To mimic the clinical settings of first line of GBM treatment, we performed tumor resection in an immunologically inert tumor model. We show that tumor-resection invigorates an anti-tumor response via significant increase in CD8+ T cells, monocyte/macrophages and decrease in Ly6G+ myeloid cells. A comparative CyTOF analysis of resected-tumor samples from GBM patients and mouse GBM tumors showed stark similarities in one of the mouse GBM tumors tested. These findings guide informed choices for use of GBM models for immunotherapeutic interventions and offer a potential to facilitate immune-therapies in GBM patients. Citation Format: Jasneet K. Khalsa, Joshua W. Keegan, Nina Cheng, Joseph Driver, Ameen Chaudry, Wenya Linda Bi, James A. Lederer, Khalid Shah. Immune phenotyping of diverse syngeneic brain tumors identifies critical tumor micro-environmental differences [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-354.

Trauma-induced inflammatory dysfunction is a lesser appreciated field of conventional immunology. In fact, traumatic injury is the leading cause of death in the US for people under 44 years old and resultantly disrupts immune homeostasis that puts injured patients at risk for developing opportunistic infections and succumbing to post-injury sepsis. Regulatory T cells (Tregs) are unique cells that contribute to immune balance but play a key role in the undesirable post-trauma immune suppression by repressing important Th-1 type immunity. Interestingly however, several reports demonstrate that Tregs confer a survival advantage in trauma. Therefore, it raises the question, do injured patients with quantitively enhanced Tregs fare better than those with less Tregs? Accordingly, we examined the role of increased Tregs on immune cell subsets in a murine burn-trauma model. C57BL/6 mice were treated with anti-DR3 to increase Tregs before burn trauma. Mice treated with anti-DR3 prior to burn and secondary pseudomonas aeruginosa infection showed improved survival compared with untreated mice. CyTOF analysis revealed burn-injured mice given anti-DR3 showed increased counts of Ki-67+ splenic PMN-MDSCs compared to sham and burn-injured control mice. This corresponded with higher levels of PMN chemoattractants by stimulated splenocytes from burn-injured mice given DR3. Sham and burn-injured mice with higher Tregs also had increased numbers of activated γδ T cells compared to untreated mice, suggesting that these cells are regulated by an increase in Tregs. This study indicates that in trauma, it may be necessary to maintain high Treg levels to alleviate trauma-induced immune dysregulation and restore immune homeostasis.

Delirium has high prevalence in the intensive care unit (ICU) and carries high morbidity. Delirium is often underrecognized, and implementation of validated screening strategies increases detection rates. Strategies to minimize delirium include limitation or avoidance of certain medications (especially benzodiazepines), frequent reorientation, maintenance of normal sleep/wake cycles, and provision of hearing and vision aids. Optimal treatment of delirium once it occurs is unclear, but antipsychotics may have beneficial effects. Sedative medications to manage pain and agitation are often necessary in the ICU; however, they may contribute to delirium. The agent used should be targeted to the patient’s specific situation. Daily sedation interruptions and targeting light rather than deep sedation help to minimize the doses used. In a subset of patients specific to the neurologic ICU, deeper sedation may be required to avoid intracranial pressure crises.

Background Pathologic T cell-B cell interactions and production of autoantibodies are hallmark features of SLE. T follicular helper (Tfh) cells are generally considered the principal T cell population capable of helping B cells. However, distinct T cell populations can augment B cell responses in chronically inflamed peripheral tissues. We recently described a dramatically expanded population of T peripheral helper (Tph) cells that promotes B cell responses in synovium of patients with seropositive RA. Here we evaluate the frequency, phenotype, and clinical associations of Tph cells in the circulation of patients with lupus. Methods Mass cytometry data from the Accelerating Medicines Partnership RA/SLE Network were used to quantify cell populations in PBMCs from 27 lupus nephritis patients, 27 RA patients, and 25 non-inflammatory controls. Frequencies of Tph cells (PD-1hi CXCR5- CD4+ T cells), Tfh cells (PD-1hi CXCR5+ CD4+ T cells), and CD21low CD19+ B cells were quantified by standardized gating, and associations with SLEDAI and dsDNA titers were assessed. For in vitro T cell-B cell co-cultures, sorted Tph cells, Tfh cells, or control T cell populations from SLE patients were co-cultured with memory B cells and stimulated with SEB +LPS, and CD38hi CD27+ plasmablasts were quantified at day 5. Results We first confirmed that Tph cells (PD-1hi CXCR5- CD4+ T cells) from SLE patients possess B cell helper function, as we previously observed in RA. Tph cells sorted from blood from 5 different lupus patients strongly induced B cell differentiation into CD38hi CD27+ plasmablasts in vitro (figure 1A, n=5 donors). By mass cytometry, Tph cells are markedly expanded in the circulation of SLE patients compared to non-inflammatory controls (4.3-fold increase, p 50 (p=0.017) and with SELENA-SLEDAI >10 (p=0.046) compared to patients with lower disease activity measures. Similar associations with disease activity were not observed for Tfh cells. Expression of surface receptors on Tph cells from SLE and RA patients was similar. A strong positive correlation emerged between the frequencies of Tph cells and CD21low B cells, an activated B cell population highly expanded in SLE (Spearman r=0.56, p=0.0026, figure 1C, gray=SLE patients, black=all other patients). In contrast, no correlation was seen between Tfh cells and CD21low B cells in SLE patients. Conclusions Tph cells are markedly expanded in the circulation of patients with SLE and demonstrate robust B cell helper function. The strong and specific positive correlation between Tph cell and CD21low B cell frequencies suggests that these cells may act coordinately in the pathologic autoimmune response in SLE. Acknowledgements We acknowledge the Accelerating Medicines Partnership RA/SLE Network and its members.

Background Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers. Electronic supplementary material The online version of this article (10.1186/s13075-018-1631-y) contains supplementary material, which is available to authorized users.

Trauma induces a complex immune response that requires a systems biology research approach. Here, we used a novel technology, mass cytometry by time-of-flight, to comprehensively characterize the multicellular response to trauma.Peripheral blood mononuclear cells samples were stained with a 38-marker immunophenotyping cytometry by time-of-flight panel. Separately, matched peripheral blood mononuclear cells were stimulated in vitro with heat-killed Streptococcus pneumoniae or CD3/CD28 antibodies and stained with a 38-marker cytokine panel. Monocytes were studied for phagocytosis and oxidative burst.Single-institution level 1 trauma center.Trauma patients with injury severity scores greater than 20 (n = 10) at days 1, 3, and 5 after injury, and age- and gender-matched controls.None.Trauma-induced expansion of Th17-type CD4 T cells was seen with increased expression of interleukin-17 and interleukin-22 by day 5 after injury. Natural killer cells showed reduced T-bet expression at day 1 with an associated decrease in tumor necrosis factor-β, interferon-γ, and monocyte chemoattractant protein-1. Monocytes showed robust expansion following trauma but displayed decreased stimulated proinflammatory cytokine production and significantly reduced human leukocyte antigen - antigen D related expression. Further analysis of trauma-induced monocytes indicated that phagocytosis was no different from controls. However, monocyte oxidative burst after stimulation increased significantly after injury.Using cytometry by time-of-flight, we were able to identify several major time-dependent phenotypic changes in blood immune cell subsets that occur following trauma, including induction of Th17-type CD4 T cells, reduced T-bet expression by natural killer cells, and expansion of blood monocytes with less proinflammatory cytokine response to bacterial stimulation and less human leukocyte antigen - antigen D related. We hypothesized that monocyte function might be suppressed after injury. However, monocyte phagocytosis was normal and oxidative burst was augmented, suggesting that their innate antimicrobial functions were preserved. Future studies will better characterize the cell subsets identified as being significantly altered by trauma using cytometry by time-of-flight, RNAseq technology, and functional studies.

The response rate of 15-20% with anti-PD1 in head and neck squamous cell carcinoma (HNSCC) highlights the urgent need for strategies to overcome resistance. Our lab has previously developed a carcinogen-induced immunocompetent murine oral carcinoma (MOC) model to study HNSCC immunobiology. Specifically, MOC1 is an immunogenic cell line that, despite sensitivity to anti-PD-1, exhibits occasional development of escape tumors (MOC1esc). MOC1esc escape tumors display a resistance phenotype similar to those observed in HNSCC patients undergoing anti-PD1 therapy. When independent escape tumors are harvested and re-transplanted into naïve mice, they grow progressively and are resistant to anti-PD1 therapy. Intriguingly, whileMOC1esc is resistant to anti-PD1, it is completely rejected in tumor bearing mice treated with anti-CTLA4. Therefore, the anti-PD1 responsive MOC1 and resistant MOC1esc mouse model is an isogenic system that provides an excellent opportunity to study mechanism(s) in adaptive resistance to anti-PD1 therapy of HNSCC. To gain a comprehensive insight into the tumor microenvironment (TME) as a contributor to adaptive resistance, we analyzed tumor infiltrating lymphocytes (TIL) in naïve MOC1 and MOC1esc tumors using mass cytometry time-of-flight (CyTOF) with a 38-cell marker panel.MOC1esc tumors were highly infiltrated by CD103+ effector/memory regulatory T cells (Tregs) and M2-like tumor associated macrophages (TAMs), while MOC1 tumors have more M1-like TAMs and neutrophils. Furthermore, we observed that both anti-PD1 and anti-CTLA4 treatment dramatically expanded CD8+ T cells and decreased neutrophils in MOC1esc tumors. In responding MOC1esc tumors, anti-CTLA4 treatment resulted in depleted Tregs, decreased M2-like TAMs and neutrophils, as well as a striking increase in M1-like TAMs compared with isotype control treated tumors. In contrast, anti-PD1 treated resistant MOC1esc tumors showed decreased M1-like TAMs, while M2-like TAMs were increased compared with controls. Tregs were not affected by anti-PD1 treatment. Therefore, the comparison between the TME of anti-PD1 treated resistant tumors and anti-CTLA4 treated responding tumors suggeststhat Tregs, neutrophils, and TAMs may contribute to the sensitivity (or resistance) to checkpoint blockade therapy. Ongoing studies will test the functional contribution of these distinct TME components in antitumor immunity including cytokine production, proliferative capacity, and their roles in immunotherapy resistance. In summary, this study identified immune modulators within TME involved in adaptive immunotherapy resistance of HNSCC. Findings from this study have advanced our understanding of HNSCC immunotherapy resistance and will accelerate the discovery of new therapeutic targets and biomarkers for adaptive resistance. Citation Format: Liye Zhou, Yasutaka Nakahori, Fei Guo, Joshua Keegan, Rachel Riley, James A. Lederer, Ravindra Uppaluri. Identification of immunotherapy resistance mechanisms within tumor microenvironment in a mouse model of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1509.

Rationale : Ischemic mitral regurgitation, a complication after myocardial infarction (MI), induces adaptive mitral valve (MV) responses that may be initially beneficial but eventually lead to leaflet fibrosis and MV dysfunction. We sought to examine the MV endothelial response and its potential contribution to ischemic mitral regurgitation. Objective : Endothelial, interstitial, and hematopoietic cells in MVs from post-MI sheep were quantified. MV endothelial CD45, found post MI, was analyzed in vitro. Methods and Results : Ovine MVs, harvested 6 months after inferior MI, showed CD45, a protein tyrosine phosphatase, colocalized with von Willebrand factor, an endothelial marker. Flow cytometry of MV cells revealed significant increases in CD45 + endothelial cells (VE-cadherin + /CD45 + /α-smooth muscle actin [SMA] + and VE-cadherin + /CD45 + /αSMA− cells) and possible fibrocytes (VE-cadherin − /CD45 + /αSMA + ) in inferior MI compared with sham-operated and normal sheep. CD45 + cells correlated with MV fibrosis and mitral regurgitation severity. VE-cadherin + /CD45 + /αSMA + cells suggested that CD45 may be linked to endothelial-to-mesenchymal transition (EndMT). MV endothelial cells treated with transforming growth factor-β1 to induce EndMT expressed CD45 and fibrosis markers collagen 1 and 3 and transforming growth factor-β1 to 3, not observed in transforming growth factor-β1–treated arterial endothelial cells. A CD45 protein tyrosine phosphatase inhibitor blocked induction of EndMT and fibrosis markers and inhibited EndMT-associated migration of MV endothelial cells. Conclusions : MV endothelial cells express CD45, both in vivo post MI and in vitro in response to transforming growth factor-β1. A CD45 phosphatase inhibitor blocked hallmarks of EndMT in MV endothelial cells. These results point to a novel, functional requirement for CD45 phosphatase activity in EndMT. The contribution of CD45 + endothelial cells to MV adaptation and fibrosis post MI warrants investigation.

T cell receptor γδ cells are known to be the primary effector T cells involved in the response to bacterial infections, yet their phenotypic characteristics are not as well established as other T cell subsets. In this study, we used cytometry by time-of-flight mass cytometry to better characterize the phenotypic response of T cell receptor γδ cells to Streptococcus pneumoniae lung infection. Mice were infected, and cells from lung washouts, spleen, and lymph nodes were stained to detect cell-surface, intracellular, and signaling markers. We observed that infection caused a significant increase in T cell receptor γδ cells, which expressed high interferon-γ and interleukin-17A levels. Profiling T cell receptor γδ cells by cytometry by time-of-flight revealed that activated γδ T cells uniquely coexpressed cell-surface Gr-1, cluster of differentiation 14, and cluster of differentiation 274 (programmed death-ligand 1). Further classification of Gr-1 expression patterns on T cell receptor γδ cells demonstrated that Gr-1+ T cell receptor γδ cells were the primary source of interferon-γ, whereas Gr-1− cells mostly expressed interleukin-17A. Gr-1+ T cell receptor γδ cells also showed higher ζ-chain–associated protein kinase 70, p38, and 4eBP1 signaling in response to infection as compared with Gr-1− T cell receptor γδ cells. Taken together, Gr-1 expression patterns on γδ T cells in the lung provide a robust marker to differentiate interferon-γ– and interleukin-17A–producing subsets involved in the early immune response to bacterial pneumonia.

Although Streptococcus pneumoniae is usually found as a commensal in healthy individuals, it can act as a pathogen in trauma patients, causing such complications as early-onset pneumonia and sepsis. We discovered that treating mice with an A-class CpG-oligodeoxynucleotide (ODN) at 2 h after traumatic injury significantly improved mouse survival following early-onset secondary lung infection with S. pneumoniae. This study used mass cytometry (cytometry by time-of-flight) and Luminex technologies to characterize the cellular immune response to secondary S. pneumoniae lung infection at 1 and 3 d postinfection. We found increased expression of CD14, CD64, and PD-L1 on F4-80+ and F4-80+CD11c+ macrophages, CD11c+ dendritic cells, and CD14+CD172a+ cells after burn-injury and infection, supporting previous reports of innate immune cell activation in sepsis. CpG-ODN treatment at 2 h after burn-injury reversed these effects; improved pathogen clearance; and led to an increased expression of CD25, CD27, MHCII, and IL-17 on or in TCRγδ cells at 1 d postinfection. At 3 d postinfection, CpG-ODN treatment increased the expression of PD-L1 on innate cell subsets. Furthermore, we analyzed cytokine levels in lung-washout samples of TCRγδ cell–depleted (TCRγδ−) mice to demonstrate that the effects of CpG-ODN on cytokine expression after burn-injury and S. pneumoniae infection rely on functional TCRγδ cells. In summary, we demonstrate that cytometry by time-of-flight provides an effective strategy to systematically identify specific cellular phenotypic responses to trauma and bacterial pneumonia and to discover changes in immune system phenotypes associated with beneficial immunotherapy.