Blood immunophenotyping identifies distinct kidney histopathology and outcomes in patients with lupus nephritis.

Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythematosus, and fewer than half of patients achieve complete renal response with standard immunosuppressants. Identifying non-invasive, blood-based pathologic immune alterations associated with renal injury could aid therapeutic decisions. Here, we used mass cytometry immunophenotyping of peripheral blood mononuclear cells in 145 patients with biopsy-proven LN and 40 healthy controls to evaluate the heterogeneity of immune activation in patients with LN and to identify correlates of renal parameters and treatment response. Unbiased analysis identified 3 immunologically distinct groups of patients with LN that were associated with different patterns of histopathology, renal cell infiltrates, urine proteomic profiles, and treatment response at one year. Patients with enriched circulating granzyme B+ T cells at baseline showed more severe disease and increased numbers of activated CD8 T cells in the kidney, yet they had the highest likelihood of treatment response. A second group characterized primarily by a high type I interferon signature had a lower likelihood of response to therapy, while a third group appeared immunologically inactive by immunophenotyping at enrollment but with chronic renal injuries. Main immune profiles could be distilled down to 5 simple cytometric parameters that recapitulate several of the associations, highlighting the potential for blood immune profiling to translate to clinically useful non-invasive metrics to assess immune-mediated disease in LN.

Alternative lengthening of telomeres: mechanism and the pathogenesis of cancer.

Telomere maintenance and elongation allows cells to gain replicative immortality and evade cellular senescence during cancer development. While most cancers use telomerase to maintain telomere lengths, a subset of cancers engage the alternative lengthening of telomeres (ALT) pathway for telomere maintenance. ALT is present in 5%-10% of all cancers, although the prevalence is dramatically higher in certain cancer types, including complex karyotype sarcomas, isocitrate dehydrogenase-mutant astrocytoma (WHO grade II-IV), pancreatic neuroendocrine tumours, neuroblastoma and chromophobe hepatocellular carcinomas. ALT is maintained through a homology-directed DNA repair mechanism. Resembling break-induced replication, this aberrant process results in dramatic cell-to-cell telomere length heterogeneity, widespread chromosomal instability and chronic replication stress. Additionally, ALT-positive cancers frequently harbour inactivating mutations in either chromatin remodelling proteins (ATRX, DAXX and H3F3A) or DNA damage repair factors (SMARCAL1 and SLX4IP). ALT can readily be detected in tissue by assessing the presence of unique molecular characteristics, such as large ultrabright nuclear telomeric foci or partially single-stranded telomeric DNA circles (C-circles). Importantly, ALT has been validated as a robust diagnostic and prognostic biomarker for certain cancer types and may even be exploited as a therapeutic target via small molecular inhibitors and/or synthetic lethality approaches.

Mesenchymal Stromal Cells Facilitate Neutrophil-Trained Immunity by Reprogramming Hematopoietic Stem Cells.

Novel therapeutics are urgently needed to prevent opportunistic infections in immunocompromised individuals undergoing cancer treatments or other immune-suppressive therapies. Trained immunity is a promising strategy to reduce this burden of disease. We previously demonstrated that mesenchymal stromal cells (MSCs) preconditioned with a class A CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) agonist, can augment emergency granulopoiesis in a murine model of neutropenic sepsis. Here, we used a chimeric mouse model to demonstrate that MSCs secrete paracrine factors that act on lineage-negative c-kit+ hematopoietic stem cells (HSCs), leaving them "poised" to enhance emergency granulopoiesis months after transplantation. Chimeric mice developed from HSCs exposed to conditioned media from MSCs and CpG-ODN-preconditioned MSCs showed significantly higher bacterial clearance and increased neutrophil granulopoiesis following lung infection than control mice. By Cleavage Under Targets and Release Using Nuclease (CUT&RUN) chromatin sequencing, we identified that MSC-conditioned media leaves H3K4me3 histone marks in HSCs at genes involved in myelopoiesis and in signaling persistence by the mTOR pathway. Both soluble factors and extracellular vesicles from MSCs mediated these effects on HSCs and proteomic analysis by mass spectrometry revealed soluble calreticulin as a potential mediator. In summary, this study demonstrates that trained immunity can be mediated by paracrine factors from MSCs to induce neutrophil-trained immunity by reprogramming HSCs for long-lasting functional changes in neutrophil-mediated antimicrobial immunity.

Deep immunophenotyping reveals circulating activated lymphocytes in individuals at risk for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease with currently no universally highly effective prevention strategies. Identifying pathogenic immune phenotypes in 'At-Risk' populations prior to clinical disease onset is crucial to establishing effective prevention strategies. Here, we applied mass cytometry to deeply characterize the immunophenotypes in blood from At-Risk individuals identified through the presence of serum antibodies to citrullinated protein antigens (ACPA) and/or first-degree relative (FDR) status (n=52), as compared to established RA (n=67), and healthy controls (n=48). We identified significant cell expansions in At-Risk individuals compared with controls, including CCR2+CD4+ T cells, T peripheral helper (Tph) cells, type 1 T helper cells, and CXCR5+CD8+ T cells. We also found that CD15+ classical monocytes were specifically expanded in ACPA-negative FDRs, and an activated PAX5 low naïve B cell population was expanded in ACPA-positive FDRs. Further, we developed an "RA immunophenotype score" classification method based on the degree of enrichment of cell states relevant to established RA patients. This score significantly distinguished At-Risk individuals from controls. In all, we systematically identified activated lymphocyte phenotypes in At-Risk individuals, along with immunophenotypic differences among both ACPA+ and ACPA-FDR At-Risk subpopulations. Our classification model provides a promising approach for understanding RA pathogenesis with the goal to further improve prevention strategies and identify novel therapeutic targets.

The exoribonuclease XRN2 mediates degradation of the long non-coding telomeric RNA TERRA.

The telomeric repeat-containing RNA, TERRA, associates with both telomeric DNA and telomeric proteins, often forming RNA:DNA hybrids (R-loops). TERRA is most abundant in cancer cells utilizing the alternative lengthening of telomeres (ALT) pathway for telomere maintenance, suggesting that persistent TERRA R-loops may contribute to activation of the ALT mechanism. Therefore, we sought to identify the enzyme(s) that regulate TERRA metabolism in mammalian cells. Here, we identify that the 5'-3' exoribonuclease XRN2 regulates the stability of TERRA RNA. Moreover, while stabilization of TERRA alone was insufficient to drive ALT, depletion of XRN2 in ALT-positive cells led to a significant increase in TERRA R-loops and exacerbated ALT activity. Together, our findings highlight XRN2 as a key determinant of TERRA metabolism and telomere stability in cancer cells that rely on the ALT pathway.

Implementation of a Standardized Shared Decision-making Bundle to Improve Communication Practices in the Neurocritical Care Unit.

Background and Objective: Shared 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. Methods: An 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. Results: Documentation 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%. Discussion: A 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.

MACROPHAGE SWITCHING: POLARIZATION AND MOBILIZATION AFTER TRAUMA.

ABSTRACT: Introduction: Trauma alters the immune response in numerous ways, affecting both the innate and adaptive responses. Macrophages play an important role in inflammation and wound healing following injury. We hypothesize that macrophages mobilize from the circulation to the site of injury and secondary sites after trauma, with a transition from proinflammatory (M1) shortly after trauma to anti-inflammatory (M2) at later time points. Methods: C57Bl6 mice (n = 6/group) underwent a polytrauma model using cardiac puncture/hemorrhage, pseudofemoral fracture, and liver crush injury. The animals were killed at several time points: uninjured, 24 h, and 7 days. Peripheral blood mononuclear cells, spleen, liver nonparenchymal cells, and lung were harvested, processed, and stained for flow cytometry. Macrophages were identified as CD68 + ; M1 macrophages were identified as iNOS + ; M2 macrophages as arginase 1 + . Results: We saw a slight presence of M1 macrophages at baseline in peripheral blood mononuclear cells (6.6%), with no significant change at 24 h and 7 days after polytrauma. In contrast, the spleen has a larger population of M1 macrophages at baseline (27.7%), with levels decreasing at 24 h and 7 days after trauma (20.6% and 12.6%, respectively). A similar trend is seen in the lung where at baseline 14.9% of CD68 + macrophages are M1, with subsequent continual decrease reaching 8.7% at 24 h and 4.4% at 7 days after polytrauma. M1 macrophages in the liver represent 14.3% of CD68 + population in the liver nonparenchymal cells at baseline. This percentage increases to 20.8% after trauma and decreases at 7 days after polytrauma (13.4%). There are few M2 macrophages in circulating peripheral blood mononuclear cells and in spleen at baseline and after trauma. The percentage of M2 macrophages in the lungs remains constant after trauma (7.2% at 24 h and 9.2% at 7 days). In contrast, a large proportion of M2 macrophages are seen in the liver at baseline (36.0%). This percentage trends upward and reaches 45.6% acutely after trauma and drops to 21.4% at 7 days. The phenotypic changes in macrophages seen in the lungs did not correlate with a functional change in the ability of the macrophages to perform oxidative burst, with an increase from 2.0% at baseline to 22.1% at 7 days after polytrauma ( P = 0.0258). Conclusion: Macrophage phenotypic changes after polytrauma are noted, especially with a decrease in the lung M1 phenotype and a short-term increase in the M2 phenotype in the liver. However, macrophage function as measured by oxidative burst increased over the time course of trauma, which may signify a change in subset polarization after injury not captured by the typical macrophage phenotypes.

Longitudinal Immune Cell Profiling in Patients With Early Systemic Lupus Erythematosus.

OBJECTIVES: To investigate the immune cell profiles of patients with systemic lupus erythematosus (SLE), and to identify longitudinal changes in those profiles over time. METHODS: 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. RESULTS: 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, CD19intermediate Ki-67high plasmablasts, and PU.1high Ki-67high monocytes) were increased in patients with early SLE, with more prominent alterations than were seen in patients with early RA. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from patients with early SLE revealed that levels of Tfh cells and CXCL10 had decreased 1 year after enrollment. Levels of CXCL13 were positively correlated with levels of several of the expanded cell populations in early SLE. CONCLUSION: Two 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.

Distinct Injury Responsive Regulatory T Cells Identified by Multi-Dimensional Phenotyping.

CD4+ regulatory T cells (Tregs) activate and expand in response to different types of injuries, suggesting that they play a critical role in controlling the immune response to tissue and cell damage. This project used multi-dimensional profiling techniques to comprehensively characterize injury responsive Tregs in mice. We show that CD44high Tregs expand in response to injury and were highly suppressive when compared to CD44low Tregs. T cell receptor (TCR) repertoire analysis revealed that the CD44high Treg population undergo TCRαß clonal expansion as well as increased TCR CDR3 diversity. Bulk RNA sequencing and single-cell RNA sequencing with paired TCR clonotype analysis identified unique differences between CD44high and CD44low Tregs and specific upregulation of genes in Tregs with expanded TCR clonotypes. Gene ontology analysis for molecular function of RNA sequencing data identified chemokine receptors and cell division as the most enriched functional terms in CD44high Tregs versus CD44low Tregs. Mass cytometry (CyTOF) analysis of Tregs from injured and uninjured mice verified protein expression of these genes on CD44high Tregs, with injury-induced increases in Helios, Galectin-3 and PYCARD expression. Taken together, these data indicate that injury triggers the expansion of a highly suppressive CD44high Treg population that is transcriptionally and phenotypically distinct from CD44low Tregs suggesting that they actively participate in controlling immune responses to injury and tissue damage.

Checkpoint blockade-induced CD8+ T cell differentiation in head and neck cancer responders.

BACKGROUND: Immune 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. METHODS: Starting 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. RESULTS: Anti-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. CONCLUSIONS: These 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.

miR-378-3p Knockdown Recapitulates Many of the Features of Myelodysplastic Syndromes.

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.

Trauma induces expansion and activation of a memory-like Treg population.

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.

Diagnosis of Coma.

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.

Augmenting emergency granulopoiesis with CpG conditioned mesenchymal stromal cells in murine neutropenic sepsis.

Patients with immune deficiencies from cancers and associated treatments represent a growing population within the intensive care unit with increased risk of morbidity and mortality from sepsis. Mesenchymal stromal cells (MSCs) are an integral part of the hematopoietic niche and express toll-like receptors, making them candidate cells to sense and translate pathogenic signals into an innate immune response. In this study, we demonstrate that MSCs administered therapeutically in a murine model of radiation-associated neutropenia have dual actions to confer a survival benefit in Pseudomonas aeruginosa pneumo-sepsis that is not from improved bacterial clearance. First, MSCs augment the neutrophil response to infection, an effect that is enhanced when MSCs are preconditioned with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist. Using cytometry by time of flight, we identified proliferating neutrophils (Ly6GlowKi-67+) as the main expanded cell population within the bone marrow. Further analysis revealed that CpG-MSCs expand a lineage restricted progenitor population (Lin-Sca1+C-kit+CD150-CD48+) in the bone marrow, which corresponded to a doubling in the myeloid proliferation and differentiation potential in response to infection compared with control. Despite increased neutrophils, no reduction in organ bacterial count was observed between experimental groups. However, the second effect exerted by CpG-MSCs is to attenuate organ damage, particularly in the lungs. Neutrophils obtained from irradiated mice and cocultured with CpG-MSCs had decreased neutrophil extracellular trap formation, which was associated with decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs in vivo. Thus, this preclinical study provides evidence for the therapeutic potential of MSCs in neutropenic sepsis.

Immune phenotyping of diverse syngeneic murine brain tumors identifies immunologically distinct types.

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.

Circulating Factors in Trauma Plasma Activate Specific Human Immune Cell Subsets.

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.

PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.

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-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-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-1hiCD4+ 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.

Altered monocyte and NK cell phenotypes correlate with posttrauma infection.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. LEVEL OF EVIDENCE: Prognostic study, level II.

Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue

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.