Distinct Immune Subsets Research Articles

CD66b+/CD68+ circulating extracellular vesicles, lactate dehydrogenase and neutrophil-to-lymphocyte ratio can differentiate coronavirus disease 2019 severity during and after infection.

Coronavirus disease 2019 (COVID-19) has been a major public health burden. We hypothesised that circulating extracellular vesicles (cEVs), key players in health and disease, could trace the cell changes during COVID-19 infection and recovery. Therefore, we studied the temporal trend of cEV and inflammatory marker levels in plasma samples of COVID-19 patients that were collected within 24h of patient admission (baseline, n=80) and after hospital discharge at day-90 post-admission (n=59). Inflammatory markers were measured by standard biochemical methods. cEVs were quantitatively and phenotypically characterized by high-sensitivity nano flow cytometry. In patients recovered from COVID-19 lower levels of inflammatory markers were detected. cEVs from vascular (endothelial cells) and blood (platelets, distinct immune subsets) cells were significantly reduced at day-90 compared to admission levels, a pattern also observed for cEVs from progenitor, perivascular and epithelial cells. The best discriminatory power for COVID-19 severity was found for inflammatory markers lactate dehydrogenase and neutrophil-to-lymphocyte ratio and for granulocyte/macrophage-released CD66b+/CD68+-cEVs. Albeit inflammatory markers were good indicators of systemic inflammatory response and discriminators of COVID-19 remission, they do not completely reveal cell stress and organ damage states. cEVs reaching baseline pre-infection levels at 90 days post-infection in recovered patients discriminate parental cells affected by disease.

Abstract 1196: Peripheral immune analyses correlate with clinical outcome: A phase II trial of nivolumab, capecitabine, or the combination in patients with triple negative breast cancer

Abstract Introduction: Patients with triple negative breast cancer (TNBC) with residual disease after neoadjuvant chemotherapy have a higher risk of recurrence and worse outcomes than patients achieving pathologic complete response. Here, we report on immune correlates from the OXEL study (NCT03487666), an open label randomized phase II trial in TNBC patients with residual disease following neoadjuvant chemotherapy, who were treated with post-neoadjuvant nivolumab (360 mg intravenously every 3 weeks, Arm A), capecitabine (1,250 mg/m2 orally twice daily on days 1-14 of each 3-week cycle, Arm B), or the combination of nivolumab and capecitabine (Arm C). The primary endpoint was to assess the immunologic effects of nivolumab, capecitabine, or the combination. Methods: Peripheral blood mononuclear cells were evaluated at baseline and after 6 and 12 weeks of therapy in patients from Arm A (n=15), B (n=14), and C (n=13) by flow cytometry to identify 158 immune cell subsets. Peripheral immunoscores reflective of enhanced immune cell function were calculated to evaluate changes in the immune profile induced by each therapy, and the association between the immune profile at baseline and disease recurrence. Results: At 6 weeks versus baseline, an increase in immune cell function was seen in patients on Arms A+C compared to Arm B. Additional distinct immune subsets showed statistical changes after 6 and 12 weeks of therapy that were specific to each arm. At baseline, a higher peripheral immunoscore was associated with a lack of eventual disease recurrence in patients on Arm A or Arms A+C, but not for patients on Arm B. Patients on Arm A (p=0.0003) or Arms A+C (p=0.0085) with a peripheral immunoscore above the median also had a longer interval of disease-free survival than patients at or below the median; this association was not seen in Arm B. Distinct immune subsets at baseline also associated with development of recurrence in each arm; higher levels of naïve CD8+ T cells and HLA-DR+ Tregs and lower levels of NKp30+ NK and PD1+ NKT cells associated with recurrence in Arm A, while in Arm B, greater levels of intermediate and nonclassical monocytes associated with recurrence. In Arm C, higher frequencies of multiple Treg subsets were associated with recurrence. Conclusions: Patients receiving nivolumab, with or without capecitabine, showed enhanced immune cell function. A peripheral immunoscore based on the immune profile at baseline was associated with disease recurrence only in patients receiving immunotherapy. These data highlight the importance of assessing peripheral blood to reveal important immunologic phenomenon and potential associations with clinical outcome. Citation Format: Nicole J. Toney, Filipa Lynce, Candace Mainor, Claudine Isaacs, Jeffrey Schlom, Renee N. Donahue. Peripheral immune analyses correlate with clinical outcome: A phase II trial of nivolumab, capecitabine, or the combination in patients with triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1196.

Practical 10-Color T-Cell Panel for Phenotyping Diverse Populations Using Spectral Flow Cytometry: A Beginner's Guide.

Flow cytometry stands as the most employed high-throughput single-cell analysis technique, facilitating the profiling of remarkably diverse samples, such as blood, bone marrow and body fluids. In addition, it allows for the discrimination of diverse immune cell subsets, including infrequently encountered types like T regulatory cells and exhausted CD28Null T cells. However, analyzing rare immune cell subsets with conventional flow cytometry poses challenges stemming from factors like fluorophore overlap, compensation issues, and limited flexibility in fluorophore selection. Therefore, spectral flow cytometry offers advantages over traditional flow cytometry. It measures the full emission spectrum and then separates it to identify different fluorochromes. This enables the use of fluorochromes with significant overlap in a single test, allowing for the analysis of more protein markers. Following this, spectral technology employs precise calculations to separate individual fluorochromes, thereby enabling the detection and elimination of autofluorescent signals originating from cells within the entire emission spectrum. This capability is pivotal in achieving deep phenotyping of immune cells with the requisite sensitivity and resolution essential for monitoring the immune systems of patients with compromised immunity, such as cancer and autoimmune disorders. Additionally, it allows for the exploration of interactions between distinct immune subsets. In this context, we introduce an optimized protocol utilizing spectral flow cytometry for precise T-cell characterization and differentiation, encompassing the assessment of their activation states. Furthermore, this protocol extends its applicability to the identification of less common circulating T-cell populations, notably T-regulatory and CD28Null T cells, following autofluorescence correction within the spectrum. This protocol provides a set of steps and reagents for the surface and intracellular staining of human T cells using whole peripheral blood. The spectral-based design of this panel allows for its applicability to other spectral machines, providing a versatile and efficient tool for T-cell analysis. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Achieving optimal staining through effective antibody titration Basic Protocol 2: Single-cell staining Basic Protocol 3: Comprehensive panel staining post-titration and spectral library integration.

Unique Serum Immune Phenotypes and Stratification of Oklahoma Native American Rheumatic Disease Patients.

Native American (NA) populations have higher rates of rheumatic disease and present with overlapping disease symptoms and nontraditional serologic features, thus presenting an urgent need for better biomarkers in NA diagnostics. This study used a machine learning approach to identify immune signatures that more effectively stratify NA patients with rheumatic disease. Adult NA patients with autoantibody-positive (AAB+) rheumatoid arthritis (RA; n=28), autoantibody negative (AAB-) RA (n=18), systemic autoimmune rheumatic disease (n=28), arthralgia/osteoarthritis (n=28), or polyarthritis/undifferentiated connective tissue disease (n=28), and control patients (n=28) provided serum samples for cytokine, chemokine, and AAB assessment. Random forest clustering and soluble mediator groups were used to identify patients and control patients with similar biologic signatures. The American College of Rheumatology criteria specific for systemic disease and RA identified differences in disease manifestations across clusters. Serum soluble mediators were not homogenous between different NA rheumatic disease diagnostic groups, reflecting the heterogeneity of autoimmune diseases. Clustering by serum biomarkers created 5 analogous immune phenotypes. Soluble mediators and pathways associated with chronic inflammation and involvement of the innate, B cell, T follicular helper cell, and interferon-associated pathways, along with regulatory signatures, distinguished the 5 immune signatures among patients. Select clinical features were associated with individual immune profiles. Patients with low inflammatory and higher regulatory signatures were more likely to have few clinical manifestations, whereas those with T cell pathway involvement had more arthritis. Serum protein signatures distinguished NA patients with rheumatic disease into distinct immune subsets. Following these immune profiles over time may assist with earlier diagnoses and help guide more personalized treatment approaches.

Increased Levels of Circulating Plasma Cells in Patients with Newly Diagnosed Multiple Myeloma Are Independently Associated with Poor Prognosis

The presence of circulating tumor cells (CTCs) has long been suggested as a valuable prognostic biomarker in Multiple Myeloma (MM). Recently, increased baseline CTC levels in patients with newly-diagnosed MM (NDMM) have been associated with adverse outcomes in the GEM2012MENOS65/GEM2014MAIN and FORTE trials. Since January 2017, we have been evaluating the presence of CTCs utilizing Next-generation Flow cytometry (NGF) with a median limit of detection (LOD) reaching 2x10-6. NGF assessment has been performed according to the EuroFlow guidelines for sample preparation, antibodies, cytometer settings and analysis of data. Currently, we have analyzed 525 matched BM and PB samples from NDMM patients and have found clonal plasma cells in 100% of BM and 89.1% (468/525) of PB samples respectively. Patients presented with ISS III/R-ISS III, high-risk cytogenetics, serum β2 microglobulin above median and hemoglobin levels ≤ 8.5 g/dL had increased CTC levels. Inversely, the absence of CTCs or their low prevalence (<10-5) was associated with an increased normal plasma cell compartment within the BM (R2=0.82, P < .0001). No association was detected between the CTC number and the therapeutic response to induction treatment in accordance with previous reports. Nevertheless, over a median follow-up monitoring of 42 months (range: 3-66 months), a running log-rank test with a step increase of 0.1% CTCs, highlighted an optimal cut-off point for PFS risk assessment at the level of 2x10-4. In the multivariable analysis, NDMM patients with CTCs above this cut-off showed a 2.2-fold higher risk of progression which was independent from ISS, induction scheme and baseline cytogenetics (HR: 2.2, 95% CI: 1.24 - 3.28, P< .001). Our large number of matched samples allowed for a phenotypic comparison between BM clonal cells and CTCs. The majority of patients with detectable CTCs (86%) showed a matched phenotypic profile of aberrant plasma cell in the two sites. However, 66 patients showed phenotypic discrepancies based on one of the following patterns: i) all phenotypic subsets were present in both BM and PB but with significantly altered ratios (≥20% of relative prevalence) for at least two concomitant subsets; ii) presence of ≥ 1 phenotypic subsets (with a minimum relative prevalence of 20% of all clonal cells) only in the BM; and iii) presence of ≥ 1 phenotypic subsets only in the PB. Remarkably, patients with phenotypic discrepancies had significantly higher CTC levels than those with a phenotypic agreement on the two sites (P < .001), together with signs of a more diffuse disease pattern, as evidenced by the imaging results. It is likely that these patients may share a patchy BM infiltration (and/or even extramedullary disease) and accumulate circulating clonal cells from various sites (thus leading to increased tumor burden) with a spatial clonal heterogeneity, which, as a total, may differ from a specific phenotype derived from a particular BM site. The biological differences among patients with differential baseline CTCs expand beyond the tumor cells. Performing a deep phenotypic approach for the identification of 32 distinct immune subsets, we analyzed the immune profile of both ΒΜ and PB in NDMM patients differing to their CTC levels by two log decimals. Patients with CTCs ≥ 5x10-3 had substantially increased levels of total T cells (especially CD8+), NK cells, tumor associated macrophages (TAMs) and an elevated ratio of memory to naïve B cells (P < .0001 for all subsets) in their BM microenvironment than those with less than 5x10-5 CTCs. The same differences, though with weaker statistical significance, applied also in PB, where, we could also identify a clear immune signature with characteristics of tumor suppression and T cell exhaustion for NDMM with the high CTC burden. Altogether, our results confirm the negative and independent prognostic impact of increased CTC levels in the NDMM and further imply its relevance in the real-world setting to establish improved risk-stratification models. Moreover, since the liquid biopsy is a better representative of the entire tumor load than a tissue biopsy sample, the analysis of CTCs may serve as the new hallmark for the real-time evaluation of a patient's disease and/or immune status.

Why responses to immune checkpoint inhibitors are heterogeneous in head and neck cancers: Contributions from tumor-intrinsic and host-intrinsic factors.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment including in head and neck squamous cell carcinomas (HNSCCs); however, only a fraction of HNSCC patients respond to ICI, whereas the majority fail to do so. The mechanisms underlying such variable responses remain incompletely understood. A better understanding of such mechanisms may broaden the spectrum of responding patients and enhance the rate of ICI response. HNSCCs exhibit a high level of genetic heterogeneity, manifested as mutations or amplifications of oncogenes (e.g., PIK3CA) and mutations of tumor suppressor genes (e.g., TP53). The immune tumor microenvironment (TME) of HNSCCs also varies significantly in composition and in relative abundance of distinct immune subsets such as CD8 tumor-infiltrating lymphocytes (TILs) or tumor-associated macrophages (TAMs), which represents a high degree of immunological heterogeneity. Here, we briefly discuss how heterogeneous ICI responses may be attributed to tumor-intrinsic factors, including genetic, transcriptional, and functional variations in tumor cells, and host-intrinsic factors, including cellular composition of the TME (e.g., CD8 TILs and TAMs), and host-intrinsic differences in the T cell receptor (TCR) repertoire of CD8 TILs. We also discuss the potential impact of these factors on designing strategies for personalized immunotherapy of HNSCCs.

Identification of a Disease-Associated Network of Intestinal Immune Cells in Treatment-Naive Inflammatory Bowel Disease.

Chronic intestinal inflammation underlies inflammatory bowel disease (IBD). Previous studies indicated alterations in the cellular immune system; however, it has been challenging to interrogate the role of all immune cell subsets simultaneously. Therefore, we aimed to identify immune cell types associated with inflammation in IBD using high-dimensional mass cytometry. We analyzed 188 intestinal biopsies and paired blood samples of newly-diagnosed, treatment-naive patients (n=42) and controls (n=26) in two independent cohorts. We applied mass cytometry (36-antibody panel) to resolve single cells and analyzed the data with unbiased Hierarchical-SNE. In addition, imaging-mass cytometry (IMC) was performed to reveal the spatial distribution of the immune subsets in the tissue. We identified 44 distinct immune subsets. Correlation network analysis identified a network of inflammation-associated subsets, including HLA-DR+CD38+ EM CD4+ T cells, T regulatory-like cells, PD1+ EM CD8+ T cells, neutrophils, CD27+ TCRγδ cells and NK cells. All disease-associated subsets were validated in a second cohort. This network was abundant in a subset of patients, independent of IBD subtype, severity or intestinal location. Putative disease-associated CD4+ T cells were detectable in blood. Finally, imaging-mass cytometry revealed the spatial colocalization of neutrophils, memory CD4+ T cells and myeloid cells in the inflamed intestine. Our study indicates that a cellular network of both innate and adaptive immune cells colocalizes in inflamed biopsies from a subset of patients. These results contribute to dissecting disease heterogeneity and may guide the development of targeted therapeutics in IBD.

Inducible T-cell co-stimulator: Signaling mechanisms in T follicular helper cells and beyond.

Human patients with homozygous null mutations in the ICOS gene suffer from recurrent infections due to humoral immune defects. Studies on human patients and mouse models have shown that inducible T-cell co-stimulator (ICOS)-deficient individuals cannot form T follicular helper (Tfh) cells, a group of CD4 T cells that migrate into B cell follicles and facilitate germinal center (GC) reactions. ICOS-induced phosphoinositide 3-kinase signaling pathways have been shown to play critical roles in Tfh programming, migration of Tfh cells into the GC, and delivery of T cell help during Tfh-GC B cell conjugation. These processes are also assisted by ICOS-mediated intracellular calcium mobilization and TANK-binding kinase 1 signaling. However, ICOS signaling also has stimulatory roles in T regulatory cells and innate lymphoid cells (ILCs), providing another layer of complexity. In this review, we discuss cell-type-specific signaling mechanisms utilized by ICOS in Tfh cells, T regulatory cells, and ILCs. Whenever relevant, we compare the roles and signaling pathways of ICOS and CD28. Understanding ICOS signal transduction mechanisms used by distinct immune subsets at different stages of immune responses or disease progression may help improve vaccination protocols, treat autoimmune diseases, and enhance cancer immunotherapy.

Examining the Immune Composition of the Intestinal Mucosa by Mass Cytometry

Abstract BACKGROUND The inflammatory bowel diseases (IBD) can affect various segments of the gastrointestinal tract, and the biology behind regional differences in disease remains poorly understood, in part due to a lack of understanding of regional variation of the particular cell subsets present. By utilizing mass cytometry, our objective was to identify regional differences in the immune composition of the digestive tract in healthy individuals. METHOD Intestinal pinch biopsies were obtained from the following sites of healthy individuals (n=10); duodenum, terminal ileum, ascending colon, transverse colon, descending colon, and rectum. Immune cells were isolated from biopsies and stained with a panel of 37 markers for analysis using mass cytometry. RESULTS Marked variations in immune cell composition were observed along the digestive tract, in particular T and B lymphocytes. Increased abundance of T lymphocytes (&amp;gt;30% of total CD45+ cells) was identified in the duodenum and terminal ileum, whereas B lymphocytes became more abundant (&amp;gt;30% of total CD45+ cells) towards the distal end of the tract. In addition, intestinal homing molecules displayed variation at each site; integrin α4β7, a target for IBD therapeutic intervention, was increasingly expressed in the terminal ileum and ascending colon, compared with the descending colon (Mean 40% vs 25% abundance, n=10, p&amp;lt;0.01). CONCLUSIONS Mass cytometry has the ability to identify and characterize numerous distinct immune subsets throughout the intestinal mucosa, allowing a deep understanding of regional differences in immune composition. This understanding may shed light on the pathogenesis of IBD and allow standardization of intestinal sampling during the evaluation of therapeutics.

RBC-Specific CD47 Pruning Confers Protection and Underlies the Transient Anemia in Patients Treated with Anti-CD47 Antibody 5F9

CD47 is an anti-phagocytic (i.e. “don't eat me”) signal and macrophage checkpoint that cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with SIRPα, its cognate inhibitory receptor.5F9 is undergoing investigation in several clinical trials and preliminary analyses have revealed encouraging therapeutic potential.We previously established that administration of 5F9 clears a subset of aged RBCs, resulting in a compensatory reticulocytosis. This occurs transiently, predominantly after the first dose, and does not worsen with subsequent doses. These findings, aligned with the observations of Oldenborg et al., which established that removal of aged RBCs, is dependent on the balance between the accumulating pro-phagocytic signals and loss of inhibitory anti-phagocytic signals. We therefore hypothesized that an initial lower 5F9 “priming dose”, sufficient to trigger clearance of aged RBCs, would yield an overall younger pool of RBCs that are less vulnerable to subsequent higher 5F9 therapeutic “maintenance doses”. Indeed, this priming and maintenance dosing strategy has substantially mitigated the on-target anemia and has allowed for 5F9 treatment in multiple clinical trials and indications.To further probe this observed selective RBC clearance, we employed a receptor occupancy assay to quantitate the CD47 expression on the cell surface of RBCs, leukocytes (WBCs), and acute myeloid leukemia (AML) blasts. We discovered that the priming dose of 5F9 not only triggered clearance of a subset of RBCs, but also resulted in a near complete loss of CD47 - a phenomenon we term CD47 pruning. This effect was RBC-specific, as WBCs and AML bone marrow (BM) blasts did not exhibit this same CD47 pruning (Fig 1).To explore the functional consequence of these unexpected clinical findings, we investigated whether we could recapitulate them in a preclinical mouse model. Similar to the clinical setting, anti-mouse CD47 blocking Ab treatments exhibited a similar pruning effect and transient anemia. Using this model, we asked whether, in the context of anti-CD47 treatment, these pruned RBCs were protected compared to unpruned RBCs. We transfused fluorescently labeled RBCs from CD47 Ab-untreated and CD47-treated mice into animals receiving continuous anti-CD47 treatment. We observed that CD47-pruned RBCs exhibited a significantly longer half-life compared to unpruned RBCs, suggesting that CD47 pruning is protective for RBCs.To distinguish whether this protection is cell-intrinsic or cell-extrinsic, we transfused fluorescently labeled RBCs into CD47 Ab-untreated and -treated mice and observed that CD47-pruned RBCs are rapidly cleared in untreated recipients, suggesting that this treatment-induced protection of RBCs is cell extrinsic and a host-based alteration. Through depletion of distinct immune subsets by chemical depletion, antibody depletion, splenectomy, and partial hepatectomies, we demonstrated that host immune effector cells (e.g. red-pulp macrophages, neutrophils, T cells etc.) were not necessary for this pruning phenomenon. Critically, we also found that this CD47 pruning and resulting tolerance was Fc-independent.In order to understand whether these findings were generalizable to other anti-human CD47 blocking agents, we conducted similar studies in double humanized huCD47 and huSIRPa micewith additional agents and observed this same CD47 pruning and tolerance phenomenon. Thereby suggesting this represents a class-effect of this emerging potential therapy class. Notably, these clinical and pre-clinical findings have been RBC-specific and have not precluded anti-tumor efficacy. The loss of CD47 on RBCs after the priming dose also suggests that the potential risk of CD47 Ab-mediated RBC agglutination following subsequent maintenance dosing is substantially reduced.Unlike prior RBC antigen modulation reports, to our knowledge, these findings represent a novel RBC antigen depletion phenomenon that is independent of the known RBC regulatory compartments, including the spleen, liver, major immune effectors cells, complement, and is critically Fc-independent. These findings provide fundamental insight into the mechanism underlying how anti-CD47 Abs are tolerated without impairing therapeutic efficacy. [Display omitted] DisclosuresChen:Forty Seven Inc: Consultancy, Equity Ownership. McKenna:Forty Seven Inc.: Equity Ownership. Choi:Forty Seven Inc: Employment, Equity Ownership. Duan:Forty Seven Inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Schrier:Forty Seven Inc.: Consultancy. Weissman:Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Elrod:Forty Seven Inc.: Employment, Equity Ownership. Chao:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties.

P056 IMPLEMENTING MASS CYTOMETRY AS A TOOL FOR INFLAMMATORY BOWEL DISEASE RESEARCH

An expanding range of therapeutics have been approved or are under development for IBD. However, objective readouts of treatment efficacy are still lacking, often relying on partially subjective clinical endpoints (CDAI). High-dimensional mass cytometry (CyTOF) combines flow cytometry with elemental mass spectrometry, allowing the simultaneous quantification of up to 40 surface and/or intracellular markers on a single cell basis. This technique presents a novel tool by which the therapeutic effects of drugs could be examined directly within the target tissue. Intestinal biopsies and peripheral blood were obtained from healthy individuals and from patients with active IBD undergoing various treatments. Immune cells were isolated from biopsies/blood and stained with a panel of 37 markers for analysis using CyTOF. Optimized protocols were developed to allow for efficient implementation of mass cytometry into IBD studies, including freezing of biopsy samples (yields of viable cells >70% as compared with fresh biopsies), to allow for storage and shipping of clinical samples. Yields of CD45+ cells derived from healthy/inflamed colonic biopsies were similar (6x104 2x104), with significant increases and decreases in the proportion of CD4+ T and B cell populations, respectively (n>8/group, P<0.001) in inflamed samples. t-SNE and ClusterX analysis of the CD4+ T cell lineage identified 22 CD4+ subsets with differential abundance between healthy and inflamed tissue. In particular, increases in CD4+integrin β7+ cells were observed in inflamed tissue, alongside expression of CD69, CD127, CD161, PD-1, and CD49d. CyTOF has the potential to identify distinct immune subsets involved in intestinal inflammation, allow for evaluation of the effects of therapeutic agents, and ultimately highlight distinct immune subsets in peripheral blood which represent cellular biomarkers of clinical response to therapeutics.

P056 IMPLEMENTING MASS CYTOMETRY AS A TOOL FOR INFLAMMATORY BOWEL DISEASE RESEARCH

An expanding range of therapeutics have been approved or are under development for IBD. However, objective readouts of treatment efficacy are still lacking, often relying on partially subjective clinical endpoints (CDAI). High-dimensional mass cytometry (CyTOF) combines flow cytometry with elemental mass spectrometry, allowing the simultaneous quantification of up to 40 surface and/or intracellular markers on a single cell basis. This technique presents a novel tool by which the therapeutic effects of drugs could be examined directly within the target tissue. Intestinal biopsies and peripheral blood were obtained from healthy individuals and from patients with active IBD undergoing various treatments. Immune cells were isolated from biopsies/blood and stained with a panel of 37 markers for analysis using CyTOF. Optimized protocols were developed to allow for efficient implementation of mass cytometry into IBD studies, including freezing of biopsy samples (yields of viable cells >70% as compared with fresh biopsies), to allow for storage and shipping of clinical samples. Yields of CD45+ cells derived from healthy/inflamed colonic biopsies were similar (6x104 2x104), with significant increases and decreases in the proportion of CD4+ T and B cell populations, respectively (n>8/group, P<0.001) in inflamed samples. t-SNE and ClusterX analysis of the CD4+ T cell lineage identified 22 CD4+ subsets with differential abundance between healthy and inflamed tissue. In particular, increases in CD4+integrin β7+ cells were observed in inflamed tissue, alongside expression of CD69, CD127, CD161, PD-1, and CD49d. CyTOF has the potential to identify distinct immune subsets involved in intestinal inflammation, allow for evaluation of the effects of therapeutic agents, and ultimately highlight distinct immune subsets in peripheral blood which represent cellular biomarkers of clinical response to therapeutics.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Stratified analysis reveals chemokine-like factor (CKLF) as a potential prognostic marker in the MSI-immune consensus molecular subtype CMS1 of colorectal cancer.

The Colorectal Cancer (CRC) Subtyping Consortium (CRCSC) recently published four consensus molecular subtypes (CMS's) representing the underlying biology in CRC. The Microsatellite Instable (MSI) immune group, CMS1, has a favorable prognosis in early stage disease, but paradoxically has the worst prognosis following relapse, suggesting the presence of factors enabling neoplastic cells to circumvent this immune response. To identify the genes influencing subsequent poor prognosis in CMS1, we analyzed this subtype, centered on risk of relapse.In a cohort of early stage colon cancer (n=460), we examined, in silico, changes in gene expression within the CMS1 subtype and demonstrated for the first time the favorable prognostic value of chemokine-like factor (CKLF) gene expression in the adjuvant disease setting [HR=0.18, CI=0.04-0.89]. In addition, using transcription profiles originating from cell sorted CRC tumors, we delineated the source of CKLF transcription within the colorectal tumor microenvironment to the leukocyte component of these tumors. Further to this, we confirmed that CKLF gene expression is confined to distinct immune subsets in whole blood samples and primary cell lines, highlighting CKLF as a potential immune cell-derived factor promoting tumor immune-surveillance of nascent neoplastic cells, particularly in CMS1 tumors. Building on the recently reported CRCSC data, we provide compelling evidence that leukocyte-infiltrate derived CKLF expression is a candidate biomarker of favorable prognosis, specifically in MSI-immune stage II/III disease.

Mass Cytometry of the Human Mucosal Immune System Identifies Tissue- and Disease-Associated Immune Subsets

Inflammatory intestinal diseases are characterized by abnormal immune responses and affect distinct locations of the gastrointestinal tract. Although the role of several immune subsets in driving intestinal pathology has been studied, a system-wide approach that simultaneously interrogates all major lineages on a single-cell basis is lacking. We used high-dimensional mass cytometry to generate a system-wide view of the human mucosal immune system in health and disease. We distinguished 142 immune subsetsand through computational applications found distinct immune subsets in peripheral blood mononuclear cells and intestinal biopsies that distinguished patients from controls. In addition, mucosal lymphoid malignancies were readily detected as well as precursors from which these likely derived. These findings indicate that an integrated high-dimensional analysis of the entire immune system can identify immune subsets associated with the pathogenesis of complex intestinal disorders. This might have implications for diagnostic procedures, immune-monitoring, and treatment of intestinal diseases and mucosal malignancies.

MicroRNA-155 regulates tumor myeloid-derived suppressive cells.

MicroRNA is small non-coding RNA and can lead to translational repression or target degradation by base-pairing with complementary sequences of mRNA molecules. MicroRNA-155 (miR-155), one of the most studied microRNA, is the first one to be reported as oncogenic [1]. miR-155 is over expressed in a long list of both hematological and solid tumors and is of paramount importance in cancer diagnosis and prognosis. However, how miR-155 particularly in host immune system regulates the tumor progression remains poorly understood. Our study underscores a contextual role of miR-155 in regulating tumor growth and tumor immunity via distinct immune subsets within tumors [2]. We conclude that the balance of different effects between those immune cell populations, which are regulated by miR-155, appears to determine whether miR-155 promotes or inhibits tumor growth [2]. We demonstrated that host miR-155 deficiency promoted antitumor T cell immunity in multiple transplanted tumor models. Further analysis of immune cell compartments revealed that miR-155 was required for the accumulation and suppressive function of myeloid-derived suppressive cells (MDSC) in the tumor microenvironment. Apart from the direct modulation on MDSC, miR-155 was also required for the MDSC-mediated CD4+Foxp3+ regulatory T cells (Treg) induction. On the other hand, miR-155 deficiency hampered the antitumor responses of both dendritic cells and T cells. Therefore, it appears that in our tumor models, miR-155 mediated a dominant immunosuppressive effect by MDSC, leading to the enhanced overall antitumor immunity in miR-155 deficient hosts. Reduced colon inflammation and decreased colorectal carcinogenesis were also found in miR-155 deficient mice when azoxymethane (AOM) and dextran sodium sulphate (DSS) were combined to induce colon lesions. Furthermore, miR-155 was upregulated in MDSC either from tumor-bearing hosts or generated from bone marrow progenitors by GM-CSF and IL-6. These results support the notion that miR-155 is a prototypical microRNA bridging inflammation and cancer development [3]. Although miR-155 may regulate tumor growth in an intrinsic manner, it is likely that inflammation promotes the accumulation of functional MDSC by increased miR-155 that dampens the immune surveillance and antitumor immunity, thereby facilitating tumor growth. To identify the molecular mechanisms by which miR-155 regulates MDSC (Figure ​(Figure1),1), we found that miR155 retained the suppressive activity of MDSCs through inhibiting SOCS1. Moreover, inverse correlations between miR-155 expression and SHIP-1/SOCS1 expression were established in MDSC. As SHIP-1 was recently reported as a target of miR155 specifically in MDSC expansion [4], these results suggest both SHIP-1 and SOCS1 as target genes of miR-155 during functional MDSC generation. SOCS1 also restricted arginase I activity [5], which otherwise would limit the efficiency of MDSC proinflammatory responses. Indeed, we showed that miR-155−/− MDSC has a lower level of arginase activity than WT counterparts, and inhibition of arginase-I with specific inhibitors completely abrogated the suppressive activity of WT MDSC and did not affect the miR-155−/− MDSC. Our data indicate that miR-155 may modulate arginase-dependent suppressive function of MDSC via targeting SOCS1. Figure 1 miR-155 regulates tumor MDSC More interestingly, we observed the decreased production of MMP-9 and VEGF from miR-155−/− MDSC, which would presumably limit the tumor angiogenesis. Given a contribution of miR-155 expression by cancer cells to tumor angiogenesis [6], further studies will determine whether miR-155 regulates tumor angiogenesis through both cancer cells and MDSC within tumors. It is notable that our results on host miR155 deficiency and tumor growth differ from other recent studies [7, 8]. Differences in the tumor cell lines used that could change the accumulation of individual immune cell subsets in the tumor microenvironment may explain this discrepancy. The extent and modulation of major immune populations could vary in different tumor types and/or tumor stages. Thus, increased miR-155 could be a key player in balancing anti-and pro-tumor immune components within the tumor. In our given tumor model system, we provide clear evidence that miR-155 promotes tumor growth in an MDSC-dependent manner, as manifested via both “depletion” and “transfer” strategy in vivo. Taken together, our study highlights the essence of evaluating the intrinsic role of miR-155 carefully in distinct immune cell subsets, where miR-155 could be either protective or deleterious to antitumor immunity. In this regard, it would be safe and important to develop the anti-miR-155 cancer therapy in cell-specific manner. It is becoming evident that miR-155 functions as “OncomiR” in concert with “ImmunomiR” in orchestrating cancer growth and progression.

Concomitant evaluation of PMA+ionomycin‐induced kinase phosphorylation and cytokine production in T cell subsets by flow cytometry

Methods to detect intracellular kinase signaling intermediates by flow cytometry have been recently developed. Termed "phospho-flow," these methods employ fluorescence-conjugated monoclonal antibodies that recognize phosphorylated epitopes of intracellular kinases, and may be combined with surface phenotypic markers to observe changes in kinase pathways by cellular subset. Effector functions, like cytokine production, are processes intrinsically linked to intracellular signaling and kinase activity within each cell. Methodologies that would simultaneously detect changes to signaling pathways as well as effector responses at the single-cell level would allow for mapping of the functional consequences induced by signaling pathway modifications. However, there are challenges to developing such a combined protocol, relating to the different kinetics of rapid signaling events and the more prolonged time required to induce and observe cytokine responses. In this report, we describe the development of an assay that accommodates differences in protocol conditions and response kinetics, merging phospho-flow cytometry, and intracellular cytokine staining methods into a single experimental protocol. We examined intracellular ERK1/2 phosphorylation and IFN-γ production by CD4+ and CD8+ T cells upon polyclonal stimulation with PMA and ionomycin, while monitoring expression of the cytolytic molecule perforin and the T cell activation marker CD38. We present a method that allows observation of kinase phosphorylation and cytokine production within the same cell after stimuli, while maintaining a stable cellular phenotype. Monitoring of signaling and effector functions in distinct immune subsets provides a platform to investigate and relate intracellular kinase signaling activity to immune cell effector function and phenotype in disease states.