Deep Immunophenotyping Research Articles

Mapping of γ/δ T cells Reveals Vδ2 T cells Resistance to Senescence

Studies in human aging mainly focus on αβ T cells while γδ T cells have been neglected despite their role in immunosurveillance. In this study, we investigated γδ T cells and its subsets (i.e. Vδ1 , Vδ2 and DN γδ (γδ Vδ1-Vδ2-)) by deep immunophenotyping. Our data shows that peripheral Vδ2 are differentially susceptible to life-long stresses against all other αβ and γδ T cells. Using aging and cytomegalovirus history (the main confounding factor in immune-aging studies) as immunological models and via functional assays, telomere length determination, epigenetic methylome profiling and DNA damage response capacity, we show that Vδ2 T cells are resistant from cellular senescence. Our results highlight the differential impact of stress on γδ T cells subsets, reveal surface markers that are functionally relevant for γδ T cells subsets and highlight possible mechanisms that enable Vδ2 to be resistant to cellular aging. This new finding reinforces the concept that Vδ2 have an “innate-like” behavior and are more resilient to the environment as compared to “adaptive-like” Vδ1 T cells. Funding Statement: Weili Xu is funded by A*STAR Graduate Academy (AGA). The work was funded by the Singapore Immunology Network, the Agency for Science Technology and Research (JCO grant #1434m00115) and the Skin Research Institute of Singapore (SRG grant #14018). Declaration of Interests: The authors have no conflict of interests. Ethics Approval Statement: Participants of the study were recruited under National University of Singapore Institutional Review Board (IRB code NUS-IRB 01-256). Cone blood was obtained from Health Science Authority (HSA) Singapore, approved under NUS-IRB 10-250.

Deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses.

Multicolor flow cytometry is a technology of choice for phenotyping of immune cells, and it can be used routinely for the follow up of patients in clinical trials. But it is challenging to define combinations of conjugated antibodies that efficiently allow the detailed analysis of major immune cell subsets and the identification of rare cell populations. In a collaborative work among the Immunology, Immunopathology, Immunotherapy (I3 ) laboratory, and the laboratory of immunomonitoring in oncology (L.I.O), we developed and validated 12 different 10-color flow cytometry panels that allow the deep immunophenotyping of cells from whole blood for the follow up of autoimmune and cancer patients. Here, we describe these optimized flow cytometry panels, showing that they provide the advanced analysis of T cells (including regulatory T cells), B cells, NK cells, MAIT cells, myeloid cells, monocytes, and dendritic cells. Most of the panels have been dried to improve standardization of the labeling and the entire procedure can be performed on less than 2 ml of whole blood. These deep immunophenotyping flow cytometry panels constitute a powerful tool for the monitoring of immune blood cells and will hopefully lead to the discovery of new biomarkers and potential therapeutic targets in autoimmune and cancer clinical trials. © 2018 International Society for Advancement of Cytometry.

Tu1819 - Deep Immunophenotyping Reveals a Role for Mucosal Associated Invariant T (MAIT) Cells in the Response to Antitnf Treatment in Crohn's Disease

Tu1819 - Deep Immunophenotyping Reveals a Role for Mucosal Associated Invariant T (MAIT) Cells in the Response to Antitnf Treatment in Crohn's Disease

FRI-226 - Deep immunophenotyping of liver infiltrating lymphocytes reveals distinct differences in the T cell compartment of patients with primary sclerosing cholangitis

FRI-226 - Deep immunophenotyping of liver infiltrating lymphocytes reveals distinct differences in the T cell compartment of patients with primary sclerosing cholangitis

Changes in natural killer cells and exhausted memory regulatory T Cells with corticosteroid therapy in acute autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an immune‐mediated liver disease currently treated by immunosuppressive medications with significant side effects. Thus, novel mechanistic treatments are greatly needed. We performed prospective deep immunophenotyping of blood immune cells in patients with acute AIH before and after corticosteroid therapy. Blood samples from 26 patients with acute AIH (United Kingdom‐AIH Consortium) were phenotyped by flow cytometry at baseline and 4 months after starting corticosteroids. Pretreatment liver tissues were stained for forkhead box P3‐positive (FOXP3POS) regulatory T cells (Tregs), clusters of differentiation (CD)56POS natural killer (NK) cells, and chemokine (C‐X‐C motif) ligand 10. Chemokine secretion by cultured primary hepatocyte and biliary epithelial cells was measured by enzyme‐linked immunosorbent assay. Functional coculture assays with stimulated NK cells and Tregs were performed. CD161 ligand, lectin‐like transcript‐1 expression by intrahepatic immune cells was demonstrated with flow cytometry. Frequencies of NKbright cells declined with therapy (P < 0.001) and correlated with levels of alanine aminotransferase (P = 0.023). The Treg:NKbright ratio was lower pretreatment, and Tregs had an activated memory phenotype with high levels of CD39, cytotoxic T lymphocyte antigen 4, and FOXP3 but also high programmed death ligand 1, indicating exhaustion. Coculture experiments suggested the Tregs could not efficiently suppress interferon‐γ secretion by NK cells. Both Tregs and NK cells had high expression of liver infiltration and T helper 17 plasticity‐associated marker CD161 (P = 0.04). Pretreatment and CD161pos NK cells expressed high levels of perforin and granzyme B, consistent with an activated effector phenotype (P < 0.05). Lectin‐like transcript 1, a ligand for CD161, is expressed on intrahepatic B cells, monocytes, and neutrophils. Conclusion: Activated effector NK cells, which correlate with biochemical measurements of hepatitis, and exhausted memory Tregs are increased in the blood of patients with treatment‐naive AIH and decline with corticosteroid therapy. Inadequate regulation of NK cells by exhausted FOXP3pos Tregs may play a role in AIH pathogenesis and contribute to liver injury. (Hepatology Communications 2018;2:421‐436)

DOP077 Deep immunophenotyping reveals a role for Mucosal Associated Invariant T (MAIT) cells in the response to anti-TNF treatment in Crohn’s disease

DOP077 Deep immunophenotyping reveals a role for Mucosal Associated Invariant T (MAIT) cells in the response to anti-TNF treatment in Crohn’s disease

Abstract 3949: Deep immunophenotyping using flow cytometry of dissociated cells from Barrett's esophagus and matched adjacent squamous epithelium defines distinct phenotypic clusters

Abstract Barrett's esophagus (BE) is defined as metaplasia of the squamous epithelium to a specialized columnar epithelium with risk factors of gastroesophageal reflux and obesity and a predilection for middle-age and older white males. BE progresses through stages of dysplasia (low-grade and high-grade) before developing into esophageal adenocarcinoma. Challenges remain in early detection and predicting which patients may progress to dysplasia. Here we describe a method by which we compare human clinical IRB-approved BE biopsies and adjacent normal squamous epithelium using tissue dissociation and deep immunophenotyping by flow cytometric collection and analysis. A cassette of canonical epithelial or tumor stem cell-associated targets (EpCAM, CD24, CD44, CD49f, Her2/neu, CD133, CD90, CD166, and CD29), immune cell markers (CD3, CD45, CD127, HLA-DR, CD16, CD56, CD4, CD8, CD25, and CD19), as well as targets associated with myeloid derived suppressor cells (CD14, CD15, CD33, CD11b, HLA-DR, CD31 and CD86) were used to discern differences across subjects and between cellular compartments in normal and BE tissue. The Barrett’s samples show a majority population with a characteristic phenotype (EpCAM+CD133lowCD49fhigh) when compared with normal squamous tissue samples (EpCAM-CD133-CD49flow). The samples separate into two discrete groups using hierarchical clustering based on differential surface marker expression of combined epithelial and immune cell markers, but also reveal unexpected, shared phenotypes for some normal and BE samples. Principal component analysis supports this grouping and was used to identify more compelling targets for categorization, such as CD133 and CD49f. The resulting expression and distribution of targets offer a phenotypic fingerprint characterizing both the epithelial cell and immune cell compartment. Besides providing the potential for revealing clinically relevant differences between BE and normal tissue, as well as across subjects, the discovered surface immunophenotypes can be used to target specific subpopulations from dysplastic tissue for further molecular investigation. A deeper understanding of the role of such specific subpopulations should increase the prospects for more complete understanding of BE and its progression. Citation Format: Friedrich G. Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, William S. Dillmore, Stephanie S. Yee, Taylor Black, Maureen DeMarshall, Aaron Middlebrook, Smita Ghanekar, Anil Rustgi, Erica L. Carpenter, Rainer Blaesius. Deep immunophenotyping using flow cytometry of dissociated cells from Barrett's esophagus and matched adjacent squamous epithelium defines distinct phenotypic clusters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3949. doi:10.1158/1538-7445.AM2017-3949

ELEVATED CENTRAL AND PERIPHERAL INFLAMMATORY PROFILES IN A POPULATION AT RISK FOR ALZHEIMER’S DISEASE

Mid-life obesity and hypertension, two interconnected healthcare epidemics, increase the risk of cognitive decline and age-related neurodegeneration. These vascular risk factors are most common in women and African Americans, who are also at higher risk for Alzheimer's disease (AD). Elevated inflammatory signaling cascades are a primary characteristic of obesity and hypertension. Peripheral inflammation has been linked to cognitive decline and neurodegeneration. However, the mechanistic links remain unknown. Here we tested the hypothesis that obesity and hypertension are mechanistically linked to AD through a multistage process that involves dysregulation of the renin angiotensin system (RAS), systemic inflammation and a heightened peripheral immune response, followed by increased immune cell trafficking into the brain thereby leading to chronic inflammation. Cognitively normal, middle aged participants with a parental history of Alzheimer's disease were enrolled. Blood and CSF were centrifuged for isolation of immune cells for immunophenotyping by multicolor flow cytometry; supernatant biofluids were collected for measurement of inflammatory factors by multiplexed immunoassays. Deep immunophenotyping analysis of CSF revealed that individuals with high blood pressure displayed altered brain immune cell profiles, including elevated central memory T cells, compared to those with normal blood pressure. This same T cell population was altered in obese (BMI >30) compared to non-obese individuals (BMI 18.5–23.9). Altered immune cell profiles were also detected in participants who suffered from disturbed sleep, a common complaint of those who suffer from Alzheimer's disease. Profiling immune cell populations in the CSF is a novel approach in the neurodegeneration field that may reveal opportunities for therapeutic intervention with targeted immunomodulatory therapies. AD risk factors associated with hypertension correlate with altered central and peripheral immune cell profiles in cognitively normal middle-aged participants. Our results demonstrate that immune cell dysfunction is detectable in cognitively intact hypertensive individuals at risk for AD and suggest that RAS-targeted interventions may afford therapeutic benefit to delay onset of AD.

Human cytokine-induced memory-like NK cells expand in patients with AML and display enhanced anti-leukemia responses.

Abstract NK cells are innate lymphoid cells that mediate anti-tumor responses. Relapsed/refractory AML patients have a poor prognosis and new therapy approaches are needed. Recently, memory-like properties of NK cells have been discovered, including cytokine-induced memory-like NK cells. Human NK cells briefly pre-activated with IL-12, IL-15, and IL-18 and then allowed to differentiate in vitro have greater re-stimulation responses compared to control cells. We hypothesized that such human memory-like NK cells possess enhanced anti-leukemia functionality. Indeed, memory-like NK cells produced increased IFN-γ in response to primary AML blasts in vitro, compared to controls. Multi-dimensional mass cytometry analyses revealed minimal alterations of memory-like NK cell inhibitory KIR repertoire, and both KIR-ligand matched and mismatched memory-like NK cells responded to primary AML blasts. In the context of a first-in-human clinical trial, allogeneic memory-like NK cells expanded to become the majority of the cells in the blood and bone marrow from AML patients 7–14 days after infusion. Furthermore, donor memory-like NK cells displayed enhanced leukemia-triggered IFN-γ production compared to recipient NK cells in the same samples, and clinical responses have been observed in 3 of 6 evaluable patients. In one patient, mass cytometry revealed expansion of CD3− CD56+ CD8+ NK cells with restricted KIR expression and multiple activating receptors. Deep NK cell immunophenotyping of in vivo expanded memory-like NK cells in additional patients is ongoing. Since memory-like NK cells expand in vivo and display enhanced anti-leukemia functionality, they represent a promising immunotherapy approach for patients with AML.

Impaired Response to Influenza Vaccination in AML Patients Post-Chemotherapy Associated with a Highly Atypical B-Cell Profile

The immunotherapy of cancer is arguably the most promising therapy under development, and vaccination against cancer antigens is a promising strategy to stimulate adaptive immune responses against malignant clones. However, the ability of patients treated with chemotherapy to respond appropriately to immune challenge may be suboptimal. This study was designed to determine the functional capacity of the immune system in adult acute myeloid leukemia (AML) patients who have completed chemotherapy and are potential candidates for immunotherapy. We used the response to influenza vaccination as a surrogate for the health of the immune system in AML patients in a complete remission (CR) post-chemotherapy.Ten adult AML patients in CR after completion of intensive chemotherapy were recruited to the clinical protocol J1293. They were on average 37 weeks post-treatment (range 4-148) when receiving the 2012-2013 inactivated seasonal influenza vaccine. Peripheral blood samples were collected at baseline and 30 days post-vaccination. Ten age and sex matched healthy donors (HD) served as baseline controls. Serological response to vaccination was assessed via microneutralization assays; multi-parameter flow cytometry was used to characterize lymphocyte subsets. ELISPOT assays were used to evaluate lymphocyte function, microarrays were used to assess gene expression, and deep sequencing of the B-cell receptor heavy chain (IGH) was performed to determine expansion and clonality of B-cells. Wilcoxon rank-sum tests were used to assess statistical significance.Only 2/10 (20%) patients seroconverted (AML responders, or AML-R) with a four-fold increase or greater in influenza-specific antibody. One responder was 148 weeks post-treatment; the other had acute promyelocytic leukemia (APL) and was 4 weeks post-treatment. Deep immunophenotyping revealed no striking differences in T-cell compartments between AML at baseline and HD, indicating rapid T-cell recovery after chemotherapy. In contrast, we observed a highly atypical B-cell profile. AML non-responders (AML-NR) at day 0 versus HD had significantly reduced frequencies of mature IgA+ (4.5% vs. 11%) and IgG+ (2.4% vs. 5.9%) B-cells (as a percentage of CD19+CD20+). Further dissection exposed markedly higher frequencies of CD10+CD27- transitional B-cells (36% vs. 16%, p<0.005) and dramatically fewer memory (resting, tissue-like, activated) B-cells (5% vs. 25%, p<0.005) (as a percentage of CD19+CD20+) in AML-NR at day 0 versus HD. There were no significant changes in any B-cell population at day 30 over baseline in any patient. Examining frequencies of transitional, naive, and memory B-cells in AML patients at day 0 when ranked by time since treatment showed a decrease in transitional B-cells with a corresponding increase in naive B-cells over time but no concurrent increase in memory B-cell frequencies (Fig 1). These data suggest B-cell deficiencies of several types: loss of the memory B-cell compartment due to chemotherapy, a subsequent excess of transitional B-cells, and a lack of naive B-cell development into specific, class-switched effectors of the antibody response, which together likely lead to humoral immune incompetence.Interestingly, functional T-cell assays revealed that of 5 evaluable patients, including the 2 AML-R and 3 AML-NR, 5/5 (100%) patients had an increase in influenza-specific cytokine production (1.24 - 4.40x higher on day 30 over baseline), suggesting a functional T-cell response even with deficient influenza-specific antibody production. Supervised clustering of microarray data identified many upregulated genes in AML-NR related to apoptosis, BCR, IL2, IL-4, IL-8, and IL-12 signaling pathways, indicative of developing B-cells. IGH sequencing demonstrated AML-NR had greater variability in CDR3 length than seen in HD, consistent with an antigen inexperienced B-cell repertoire.These data suggest that while some aspects of cellular immunity recover comparatively quickly, the humoral immune system is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. Abnormal frequencies of transitional and memory B-cells may explain the poor response to vaccination often seen in patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immune capacity observed here might have implications for the success of immunotherapies based on vaccination. [Display omitted] DisclosuresNoonan:Celgene: Speakers Bureau. Borrello:Celgene: Research Funding.

Ankylosing spondylitis patients display altered dendritic cell and T cell populations that implicate pathogenic roles for the IL-23 cytokine axis and intestinal inflammation.

Objective. AS is a systemic inflammatory disease of the SpA family. Polymorphisms at loci including HLA-B27, IL-23R and ERAP-1 directly implicate immune mechanisms in AS pathogenesis. Previously, in an SpA model, we identified HLA-B27–mediated effects on dendritic cells that promoted disease-associated Th17 cells. Here we extend these studies to AS patients using deep immunophenotyping of candidate pathogenic cell populations. The aim of our study was to functionally characterize the immune populations mediating AS pathology.Methods. Using 11-parameter flow cytometry, we characterized the phenotype and functions of lymphocyte and myeloid cells from peripheral blood, and the synovial phenotype of AS patients and age-matched healthy controls.Results. Significantly fewer circulating CD1c-expressing dendritic cells were observed in AS patients, offset by an increase in CD14− CD16+ mononuclear cells. Ex vivo functional analysis revealed that this latter population induced CCR6 expression and promoted secretion of IL-1β and IL-6 when co-cultured with naive CD4+ T cells. Additionally, systemic inflammation in AS patients significantly correlated with increased proportions of activated CCR9+ CD4+ T cells.Conclusion. CD14− CD16+ mononuclear cells may contribute to AS by promoting Th17 responses, and antigen-presenting cells of mucosal origin are likely to contribute to systemic inflammation in AS.

Characterization of Innate Lymphoid Cells in Human Skin and Blood Demonstrates Increase of NKp44+ ILC3 in Psoriasis

Innate lymphoid cells (ILC) are increasingly appreciated as key regulators of tissue immunity. However, their role in human tissue homeostasis and disease remains to be fully elucidated. Here we characterise the ILC in human skin from healthy individuals and from the inflammatory skin disease psoriasis. We show that a substantial proportion of IL-17A and IL-22 producing cells in skin and blood of normal individuals and psoriasis patients are CD3 negative innate lymphocytes. Deep immunophenotyping of human ILC subsets showed a statistically significant increase in the frequency of circulating NKp44+ ILC3 in blood of psoriasis patients compared to healthy individuals or atopic dermatitis patients. More than 50% of circulating NKp44+ ILC3 expressed cutaneous lymphocyte-associated antigen indicating their potential for skin homing. Analysis of skin tissue revealed a significantly increased frequency of total ILC in skin compared to blood. Moreover the frequency of NKp44+ ILC3 was significantly increased in non-lesional psoriatic skin compared to normal skin. A detailed time course of a psoriasis patient treated with anti-TNF showed a close association between therapeutic response, decrease in inflammatory skin lesions, and decrease of circulating NKp44+ ILC3. Overall, data from this initial observational study suggest a potential role for NKp44+ ILC3 in psoriasis pathogenesis.

A115: Deep Immunophenotyping in the Identification of Clinically Meaningful Immune Signatures in Juvenile Idiopathic Arthritis

Background/Purpose:The evolution of molecular immunology has made available high‐throughput tools which can provide an entirely new, comprehensive and multidimensional picture of the immune system. We have applied a combination of deep phenotyping by cytometry, multiplex expression and functional assays, to identify immunological signatures which could associate with, and perhaps contribute to, lack of responsiveness to treatment in juvenile idiopathic arthritis (JIA).Methods:We developed a platform of deep immunophenotyping to identify immune signatures on a variety of blood cell types with special focus given to CD4+ effector and regulatory T cells (Teff and Treg, respectively).Results:Analysis of samples from the NIH funded TREAT JIA study showed there were no significant changes between R and NR when Teff and Treg functions were analyzed as a whole. However, in‐depth characterization of the two cell subsets led to the identification of specific phenotypical differences at both time points. Strikingly, the combination of a restricted set of CD4+ T cell markers was able to segregate R and NR with up to 87.5% accuracy. Importantly, our analysis identified a population of cells significantly more represented in NR and characterized by an activated phenotype, expressing pro‐inflammatory markers and chemokine receptors targeting cells to inflammatory sites, and bearing signs of recent antigen recognition, possibly indicating their direct involvement in autoimmunity. Our approach will be greatly benefitted by the addition of recent advances in flow cytometry, with the development of the mass cytometer CyTOF, which dramatically improves single cell multiplexing. These methods are currently being applied to the analysis of samples from 137 subjects enrolled from 16 sites in a prospective multicenter NIH funded study who had Polyarticular forms of JIA (extended oligoarticular, Poly RF+, Poly RF/‐) who had reached clinical inactive disease (CID) for /G6 months at the time the anti‐TNF agent was withdrawn and followed for at least /G8 months to determine if they do or do not meet a validated definition of disease “flare”. Samples for immunophenotyping were obtained at baseline, time of stopping anti‐TNF therapy and last study visit (i.e. time of flare or at least 8 months of CID off anti‐TNF therapy). Duration of CID at baseline was mean/median/range of 1.2/0.5/1 day‐12.1 yrs. 106 (77%) subjects maintained CID for the first 6 months and stopped anti‐TNF as per protocol. 67/106 (63%) maintained CID for ≥8 mos off anti‐TNF while 39 (37%) flared. The mean/median/range for time to flare was 108/105/7–271 days.Conclusion:Immunophenotyping, when performed as an integrated analysis of immune signatures, has demonstrated effectiveness in identifying immune signatures that have excellent accuracy in segregating and predicting clinical disease states and responses. These methods are currently being analyzed in these study populations.

A versatile platform for comprehensive chip‐based explorative cytometry

Analysis of the immense complexity of the immune system is increasingly hampered by technical limitations of current methodologies, especially for multiparameter- and functional analysis of samples containing small numbers of cells. We here present a method, which is based on the stepwise functional manipulation and analysis of living immune cells that are self-immobilized within microfluidic chips using automated epifluorescence microscopy overcoming current limitations for comprehensive immunophenotyping. Crossvalidation with flow cytometry revealed a 10-fold increased sensitivity and a comparable specificity. By using small sample volumes and cell numbers (2-10 microl, down to 20,000 cells), we were able to analyze a virtually unlimited number of intracellular and surface markers even on living immune cells. We exemplify the scientific and diagnostic potential of this method by (1) identification and phenotyping of rare cells, (2) comprehensive analysis of very limited sample volume, and (3) deep immunophenotyping of human B-cells after in vitro differentiation. Finally, we propose an informatic model for annotation and comparison of cytometric data by using an ontology-based approach. The chip-based cytometry introduced here turned out to be a very useful tool to enable a stepwise exploration of precious, small cell-containing samples with an virtually unlimited number of surface- and intracellular markers.