Five-color Flow Cytometry Research Articles

Granulocytic and Monocytic Myeloid-Derived Suppressor Cells are Functionally and Prognostically Different in Patients with Chronic Myeloid Leukemia.

Myeloid-derived suppressor cells (MDSCs) represent phenotypically heterogeneous populations that suppress tumor-specific T-cell responses. MDSCs are produced from myeloid precursors in emergent states and are increased in several hematologic malignancies. We evaluated the differences in the levels and prognostic significance of MDSCs according to the clinical status of chronic myeloid leukemia (CML). The percentages and numbers of granulocytic (g)MDSCs and monocytic (m)MDSCs in peripheral blood (PB) and bone marrow (BM) aspirates were determined by five-color flow cytometry (HLA-DR/CD11b/CD15/CD33/CD14). The median BM-gMDSC% and PB-gMDSC% of the CML group were lower than those of the complete hematologic response (CHR) and control groups (P<0.001). In the CHR group, patients with major molecular response (MMR) showed higher median BM-gMDSC% than those without MMR (P=0.039). Conversely, the PB-mMDSC number of the CML group was higher than those of the CHR and control groups (P<0.001). Patients with high PB-gMDSC number exhibited superior survival to those with low PB-gMDSC number (P=0.021), and patients with high PB-mMDSC% showed inferior survival to those with low PB-mMDSC%, but there was no statistical significance (P=0.182). Increased gMDSCs at CHR may reflect non-leukemic granulopoiesis, and a high number of PB-gMDSCs suggests better prognosis in CML. However, mMDSCs may be associated with malignant conditions and poor prognosis.

Comparative Analysis of Immune Reconstitution in HIV-Positive Recipients of Allogeneic and Autologous Stem Cell Transplant on the BMT-CTN-0903/AMC-080 and BMT-CTN-0803/AMC-071 Trials

Introduction We report the results of a phenotypic and functional analysis of immune reconstitution in HIV(+) patients treated with allogeneic hematopoietic stem cell transplant (allo-HSCT), in comparison with HIV(+) autologous transplant (auto-HSCT) recipients, HIV(-) auto-HSCT recipients and healthy controls (HCs). Methods Blood samples were collected at days 56, 180 and 365 post-transplant from HIV(+) allo-ASCT recipients (n=17, BMT-CTN-0903/AMC-080 trial), HIV(+) auto-SCT recipients (n=36, BMT-CTN-0803/AMC-071 trial) and HIV(-) auto-SCT recipients (n=30, NCT00569309/OSU-07044 trial), and at 1 time point from 71 HCs. Five-color flow cytometry was performed on whole blood. Principal component analysis (PCA) examined global differences across 18 immune cell subsets between all subject cohorts. Wilcoxon rank-sum tests compared median absolute and proportionate cell counts. Feature importance score analysis (FIS) identified contributions of 100 immune cell populations to differences between HIV(+) cohorts and HCs. Functional responsiveness of HIV(+) allo-HSCT recipients' T cells to stimulation with CD3- and CD28-directed antibodies, NK cells to stimulation with IL-12+IL-18 and monocytes to stimulation with lipopolysaccharide (LPS) was assessed by mass cytometry on peripheral blood mononuclear cells (n=2) and compared to HCs (n=2). Results PCA showed that immunomes of HIV(+) allo-HSCT recipients and HIV(+) auto-HSCT recipients clustered together at all time points. HIV(-) auto-SCT recipients and HCs clustered together and away from the HIV(+) cohorts. FIS identified 13 cell subsets that defined the difference between HIV(+) allo-HSCT recipients (all visits) and HCs and 11 immune cell subsets that defined the difference between HIV(+) auto-HSCT recipients (all visits) and HCs; in each comparison, activated CD3+/HLA-DR+ T cells had the greatest impact on the difference between composite HIV(+) and HC immunomes. At 1 year, both HIV(+) transplant recipient cohorts had higher absolute numbers of activated T cells, effector T cells and CD8+ T cells than HCs and lower numbers of CD4+ T cells, naive T cells, and activated NK cells compared to HCs (p Conclusion Chronic HIV infection confers pro-inflammatory immune characteristics on phenotypic and functional profiling of the cellular immunome of stem cell transplant recipients, irrespective of allogeneic or autologous stem cell donor source.

Comparative Analysis of Immune Reconstitution in HIV-Positive Recipients of Allogeneic and Autologous Stem Cell Transplant on the BMT CTN 0903/AMC-080 and BMT CTN 0803/AMC-071 Trials

Introduction. Our previous flow cytometry-based comparison of HIV(+) and HIV(-) autologous hematopoietic stem cell transplant (auto-HSCT) recipient immunomes at 56, 180 and 365 days post-transplant to each other and to healthy controls (HCs) showed that both sets of auto-HSCT recipient immunomes approached HCs over time, but retained significant differences. HIV(+), but not HIV(-), auto-AHCT recipients retained pro-inflammatory features consistent with chronic HIV infection. Here, we report the results of a quantitative and functional analysis of immune reconstitution in HIV(+) patients treated with allogeneic hematopoietic stem cell transplant (allo-HSCT), in comparison with HIV(+) auto-HSCT recipients and HCs. Methods. Blood samples were collected for analysis at days 56, 180 and 365 post-transplant from HIV(+) transplant recipients and at 1 time point from HCs. Whole blood analysis was performed by five-color flow cytometry across 100 immune marker combinations. Comparisons were made between HIV(+) allo-HSCT recipients (n=17, acute myeloid leukemia, acute lymphocytic leukemia, myelodysplastic syndrome, Hodgkin and non-Hodgkin lymphoma that received myeloablative or reduced intensity conditioning on the BMT-CTN-0903/AMC-080 trial), HIV(+) auto-HSCT recipients (n=36, aggressive B cell non-Hodgkin lymphoma or Hodgkin lymphoma that received myeloablative conditioning on the BMT-CTN-0803/AMC-071 trial) and 71 HCs. Unsupervised principal component analysis (PCA) examined differences in immune cell proportions, identified by flow cytometry across 100 cell subsets at each time point. Wilcoxon rank-sum tests compared median absolute counts and median proportions of cell subsets. An independent feature importance score analysis (FIS) identified contributions of immune cell populations expressing specific immune marker combinations to the differences between HIV(+) auto-HSCT recipients, HIV(+) allo-HSCT recipients and HCs. Functional responsiveness of HIV(+) allo-HSCT recipients' T cells to stimulation with CD3- and CD28-directed antibodies, NK cells to stimulation with IL-12 and IL-18 and monocytes to stimulation with lipopolysaccharide (LPS) was assessed in a preliminary mass cytometry on peripheral blood mononuclear cells isolated at the same time points (n=2) and compared to HCs (n=2). Results. PCA showed that immunomes of HIV(+) allo-HSCT recipients and HIV(+) auto-HSCT recipients clustered together with each other, but away from HCs at all time points throughout the post-transplant year. FIS identified: 1) 13 cell subsets that defined the difference between HIV(+) allo-HSCT recipients (all visits) and HCs, and 2) 11 immune cell subsets that defined the difference between HIV(+) auto-HSCT recipients (all visits) and HCs; in both of these comparisons, activated CD3+/HLA-DR+ T cells had the greatest impact on the difference between HIV(+) and HC immunomes. At 1 year, both HIV(+) transplant recipient cohorts had higher absolute numbers of activated T cells, effector T cells and CD8+ T cells than HCs (Wilcoxon rank-sum test, p&lt;0.0031). HIV(+) autologous and allogeneic HSCT recipients also had lower numbers of CD4+ T cells, naïve T cells and activated NK cells compared to HCs (p&lt;0.0031). FIS also identified 20 immune cell subsets that defined the difference between HIV(+) autologous and allogeneic HSCT recipients immunomes at 1 year, with CD8+/CD27- effector T cell subset exerting the highest impact on the difference. Preliminary functional mass cytometry analysis of 2 HIV(+) allo-HSCT recipients and 2 HCs showed that: 1) IFNʏ production by CD8+ T cells was increased above that of HCs at all time points. 2) Expanded populations of CD4+/T-bet+ cytotoxic cells expressing granzyme B and perforin, and CD8+ cytotoxic T cells expressing granzyme B and perforin, persisted in HIV(+) allo-HSCT recipients at all time points, but not in HCs. 3) NK cells retained an ability to produce IFNʏ in response to stimulation with IL-12 and IL-18 in HIV(+) allo-SCT recipients. 4) Monocytes showed an enhanced production of TNFα in response to stimulation with LPS in HIV(+) allo-HSCT recipients compared to HCs at 1 year post-HSCT. Conclusion. Chronic HIV infection confers the pro-inflammatory immune features on the phenotypic and functional profiling of the T lymphocyte immunome of stem cell transplant recipients, irrespective of allogeneic or autologous stem cell donor source. Disclosures Devine: Bristol Myers: Other: Grant for monitoring support &amp; travel support; Kiadis Pharma: Other: Protocol development (via institution); Magenta Therapeutics: Other: Travel support for advisory board; My employer (National Marrow Donor Program) has equity interest in Magenta. Noy:Janssen: Consultancy; Medscape: Honoraria; Prime Oncology: Honoraria; NIH: Research Funding; Pharamcyclics: Research Funding; Raphael Pharma: Research Funding. Popat:Bayer: Research Funding; Incyte: Research Funding; Jazz: Consultancy. Hofmeister:Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Navarro:Atara Biotherapeutics: Employment, Equity Ownership. Behbehani:Fluidigm corporation: Other: Travel funding. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding. Baiocchi:Prelude: Consultancy.

Proportion of the CD19-Positive and CD19-Negative Lymphocytes and Monocytes within the Peripheral Blood Mononuclear Cell Set is Characteristic for Rheumatoid Arthritis.

Background and objectives: Composition of the peripheral blood (PB) cell populations and their activation state reflect the immune status of a patient. Rheumatoid arthritis (RA) is characterized by abnormal B- and T-cell functions. The objective of this study was to assess the profiles of the PB mononuclear cell (PBMC) populations in patients with rheumatoid and osteoarthritis (OA) in comparison with healthy control (HC) subjects in order to evaluate the PBMC profiles as a potential diagnostic characteristic in RA. The second aim was to assess the CCR1 and CCR2 expression on PB lymphocytes and correlate it with the plasma levels of matrix metallopeptidase 9 (MMP-9), IL-17F, TNF-α, IL-6, and IL-10. Materials and Methods: The frequency and phenotype, including CCR1 and CCR2, of the PBMC populations (monocytes, CD19+B cells, and T/NK lymphocytes) in RA (n = 15) and OA (n = 10) patients and HC (n = 12) were analyzed by five-color flow cytometry. DNA of the viruses, HHV-6, HHV-7, and B19, in the whole blood and cell-free plasma, were assessed by nested-polymerase chain reaction (PCR). Results: Active persistent or acute infections, caused by HHV-6, HHV-7, or B19, were not detected in patients of this study. Both CCR1 and CCR2 were determined on the PB B and T/NK lymphocytes in several RA and OA patients and HCs. However, in patients, the frequency of the CCR1-positive T/NK lymphocytes showed a weak negative correlation with the IL-10 level, while the frequency of the CCR2-positive B cells correlated positively with the level of IL-6. Statistically significant differences in the proportions of the CD19-positive and CD19-negative lymphocyte and monocyte subsets within the PBMC set were determined between RA and OA patients and HC adults. Conclusions: We have shown in our pilot study with rather small cohorts of patients that the PBMC-population profiles were very consistent, and statistically significantly differed between RA and OA patients and HC subjects.

Increased circulating plasma cells detected by flow cytometry predicts poor prognosis in patients with plasma cell myeloma.

Flow cytometry (FC) is a reliable tool for diagnosing and monitoring of plasma cell myeloma (PCM). Recent studies used FC for quantifying plasma cells (PCs) in peripheral blood (PB) using various panels, and an adverse prognostic effect of circulating PCs (cPCs) has been reported. We investigated the prognostic implication of cPCs quantified using a simple panel in patients with PCM. Bone marrow (BM) and PB of 85 patients with PCM were analyzed by five-color FC at time of diagnosis. A serial gating strategy for quantification used CD38/CD138 to gate PCs in 100,000-200,000 acquired events, with subsequent gating for CD19, CD56, and CD45, to identify aberrant immunophenotypes. cPCs were observed in 74.1% patients (63/85, median 0.067% leukocytes). Patients were grouped based on a cPC cut-off level of 0.02% derived using the receiver operating characteristic curves. Compared with patients with cPCs < 0.02% (n = 28), those with cPCs ≥ 0.02% (n = 57) showed lower hemoglobin (P = 0.003) and platelets (P = 0.014), but higher calcium, M-protein and BM PCs (P = 0.013, 0.029, and P < 0.001, respectively). Survival analysis of 74 patients showed that cPCs ≥ 0.02% predicted shorter progression-free and overall survival (P = 0.001 and 0.013, respectively), and this negative prognostic impact was retained in multivariate analysis (P = 0.023). Flow cytometric quantification of cPCs using five surface antigens (CD138, CD38, CD56, CD19, and CD45) is a sensitive and simple method that can be used for assessing PCM prognosis; it would allow clinical laboratories to readily adopt a risk stratification strategy based on cPC levels in PCM patients. © 2017 International Clinical Cytometry Society.

Immunoprofiling of leukemic stem cells CD34+/CD38-/CD123+ delineate FLT3/ITD-positive clones.

BackgroundAcute myeloid leukemia (AML) is a heterogeneous clonal disorder presenting with accumulation of proliferating undifferentiated blasts. Xenograft transplantation studies have demonstrated a rare population of leukemia-initiating cells called leukemic stem cells (LSCs) capable of propagating leukemia that are enriched in the CD34+/CD38− fraction. LSCs are quiescent, resistant to chemotherapy and likely responsible for relapse and therefore represent an ideal target for effective therapy. LSCs are reported to overexpress the alpha subunit of the IL-3 receptor (CD123) compared to normal CD34+/CD38− hematopoietic stem cells. It has not been demonstrated whether CD123-positive (CD34+/CD38−) subpopulation is enriched for any clonal markers of AML or any LSC properties. The aims of this study were to investigate whether FMS-like tyrosine kinase (FLT3)/internal tandem duplication (ITD) mutations are present at LSC level and whether FLT3/ITD mutation is confined to LSC as defined by CD34+/CD38−/CD123+ and not CD34+/CD38−/CD123−.MethodsThirty-four AML cases were analyzed by five-color flow cytometry and sequential gating strategy to characterize of CD34+/CD38−/CD123+ cells. These cells were sorted, analyzed by PCR, and sequenced for FLT3/ITD.ResultsIn this study, we confirm significant expression of CD123 in 32/34 cases in the total blast population (median expression = 86 %). CD123 was also expressed in the CD34+/CD38− cells (96 ± 2 % positive) from 28/32 for CD123+ AML. CD123 was not expressed/low in normal bone marrow CD34+/CD38− cells (median expression = 0 %, range (0–.004 %). AML samples were tested for FLT3/ITD (10 positive/25). FLT3/ITD+ AML cases were sorted into two putative LSC populations according to the expression of CD123 and analyzed for FLT3/ITD again in the stem cell fractions CD34+/CD38−/CD123+ and CD34+/CD38−/CD123−. Interestingly, FLT3/ITD was only detected in CD34+/CD38−/CD123+ (7/7) and not in CD34+/CD38−/CD123− subpopulation (6/7).ConclusionsThis finding shows that FLT3/ITD are present at LSC level and may be a primary and not secondary event in leukemogenesis, and the oncogenic events of FLT3/ITD happen at a cell stage possessing CD123. It shows that CD123 immunoprofiling provides further delineation of FLT3+ LSC clone. This novel finding provides a rationale for treatment involving CD123-targeting antibodies with intracellular FLT3 inhibitors directed against CD34+/CD38−/CD123+. This may result in more effective anti-LSC eradication.

Increased Circulating Plasma Cells Predicts Poor Overall Survival in Symptomatic Plasma Cell Myeloma Patients

Abstract Backgrounds Flow cytometry can rapidly determine immunophenotypes of neoplastic plasma cells (PCs) and quantify PCs in patients with plasma cell myeloma. Flow cytometric immunophenotyping and quantification of neoplastic plasma cells is sensitive and reliable tool for diagnosis and disease monitoring in patients with monoclonal gammopathy. Circulating PCs (cPCs) in peripheral blood (PB) after autologous hematopoietic stem cell transplantation is a marker of high-risk disease in patients with plasma cell myeloma. We assessed the utility of quantification of cPCs using flow cytometry for risk stratification in newly diagnosed plasma cell myeloma patients in the era of novel agents. Methods PB and bone marrow (BM) aspirates of 85 newly diagnosed patients with symptomatic plasma cell myeloma from August 2013 to July 2014 were analyzed by five-color flow cytometry using monoclonal antibodies against CD45, CD19, CD56, CD38, and CD138. The gating strategy employed first used the expression of CD38 and CD138 to identify plasma cells among 100,000 to 200,000 events. cPCs in PB was determined according to the patient's specific immunophenotype of neoplastic PCs in BM. Results The median age of the patient population was 68 years (45~87) and 58% were female. Median follow-up duration was 19.2 months. Six out of 85 patients (7%) did not show cPCs. Among 79 patient (93%) who had detectable cPCs, the median cPCs was 0.09% (0.006~3.612%). Patients without cPCs or cPCs under 0.05% were assigned to low cPCs group (n=32, 38%) and others to high cPCs group (n=53, 62%) according to receiver operating characteristics analysis. High cPCs group showed higher level of BM neoplastic PCs detected by both methodologys of morphology and flow cytometry (P=0.002, 0.033, respectively), higher BM cellularity (P=0.011), higher serum M protein level (P=0.013), lower hemoglobin (P=0.008), and lower platelet level (P=0.034) than low cPCs group. High cPCs group was associated with adverse cytogenetics such as t(4;14) and monosomy 13 (P=0.008), and CD45 negative immunophenotype (P=0.007). In survival analysis, high cPCs presented shorter overall survival (OS) than low cPCs group (P=0.013) (Fig. 1). It was independent with patient age and cytogenetic risks (P =0.011). Conclusion By flow cytometry cPCs was detected in most symptomatic plasma cell myeloma patients. Increased cPCs ≥0.05% among PB leukocytes could be an independent prognostic factor showing adverse effect in overall survival in symptomatic plasma cell myeloma patients. Figure 1. Kaplan-Meier survival curve of patients with plasma cell myeloma who showed 0.05% or more circulating plasma cells in peripheral blood and patients with circulating plasma cells less than 0.05%. Figure 1. Kaplan-Meier survival curve of patients with plasma cell myeloma who showed 0.05% or more circulating plasma cells in peripheral blood and patients with circulating plasma cells less than 0.05%. Disclosures No relevant conflicts of interest to declare.

Simultaneous changes in serum HMGB1 and IFN-α levels and in LAIR-1 expression on plasmatoid dendritic cells of patients with juvenile SLE.. New therapeutic options?

We investigated the simultaneous changes in serum levels of HMGB1 and IFN-α as well as in LAIR-1 expression on plasmatoid dendritic cells (pDCs) of juvenile systemic lupus erythematosus (jSLE) patients in order to explore their involvement in the disease pathogenesis and their correlation with disease activity and other characteristics. In total, 62 blood samples were studied from 26 jSLE patients (18 girls), aged 8-16 years. Twenty healthy subjects (16 girls) of comparable age were included as healthy controls (HCs). Concentrations of serum HMGB1 and IFN-α were assessed by ELISA and LAIR-1 expression on pDCs by five-color flow cytometry. The disease activity index was assessed by SLEDAI and ECLAM scores. It was found that mean serum levels both of HMGB1 and IFN-α were significantly increased in jSLE patients compared to HCs and in jSLE patients with active disease with or without active nephritis compared to those with inactive disease. Mean serum levels of HMGB1 were positively correlated with levels of IFN-α and both were positively correlated with the SLEDAI and ECLAM scores. The expression of LAIR -1 on pDCs of jSLE patients was significantly lower than that of HCs. In conclusion, our findings indicate that serum HMGB1 not only represents a potential marker of disease activity but together with the lack of LAIR-1 inhibitory function may contribute to the sustained inflammatory action of IFN-α in jSLE. In this regard, blocking the action of HMGB1 and its receptors or enhancing the expression/inhibitory function of LAIR-1 on pDCs should be included in future immune interventions for controlling jSLE.

Clinical evaluation of leukocyte differential count in peripheral blood by five-color flow cytometry

To explore the clinical application values of five-color flow cytometry for leukocyte differential count in peripheral blood. Leukocyte differentiation in 265 peripheral blood samples collected at Peking University First Hospital from September 2010 to December 2010 was detected by standard microscopic cytology as a reference method. Meanwhile, Beckman-Coulter LH750 hematology analyzer and FC500 flow cytometer were performed. Then the correlations were analyzed between microscopic cytology, hematology analyzer and flow cytometry. Forty blood samples collected at Peking University First Hospital, Beijing Daopei Hospital and General Hospital of Beijing Military Command from August 2010 to November 2010 were analyzed by standard microscopic cytology, Beckman-Coulter LH750 hematology analyzer and NAVIOS flow cytometer. Then the correlations between microscopy, hematology analyzer and flow cytometry were explored to analyze the clinical diagnostic efficiency of flow cytometry. Correlation of leukocyte differential count between FC500 flow cytometer and standard microscopic cytology was significant (all P < 0.01) . And it was superior in the detection of lymphocytes, neutrophils and eosinophils (r = 0.955, 0.928, 0.876). Moreover, the correlation of leukocyte differential count between NAVIOS flow cytometer via manual gate and standard microscopic cytology was significant (r > 0.700, all P < 0.01) except for basophils. And it was superior in the detection of neutrophils, lymphocytes and blasts (r = 0.950, 0.915, 0.852). When 1% was set as the cut-off value of immature granulocytes on standard microscopic cytology, the sensitivity and specificity of flow cytometry was 87% and 76% respectively. When 0.5% was set as the cut-off value of blasts on standard microscopic cytology, the sensitivity and specificity of flow cytometry stood at 100% and 92% respectively. Five-color flow cytometry is well-correlated with standard microscopic cytology for leukocyte differential count in peripheral blood with different flow cytometers, and the sensitivity of detecting blasts and immature granulocytes is very excellent.

Evaluation of hMICL As a Marker Associated with Disease Progression in BCR-ABL Negative Myeloproliferative Neoplasms

Abstract 1724 Introduction:BCR-ABL negative myeloproliferative neoplasms (MPNs) (polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF)) have preponderance to progress to hematopoietic insufficiency with fibrosis and/or blast transformation. This progression is often difficult to detect, but can be preceded by a stage of variable duration commonly termed acceleration, which is not homogeneously defined, but characterized by worsening cytopenias and constitutional symptoms. The monitoration of this process is clearly important, but at the present time biomarkers for this are lacking. We and others have described the human myeloid inhibitory C-type lectin-like receptor (hMICL) (also known as CLL-1) to be a very stable marker for the malignant cells in acute myeloid leukemia (AML), even in CD34- cases (van Rhenen et al. Leukemia 2007, Roug et al. Cytometry 2011). Given the need for new markers in MPN we hypothesized that determining the level of circulating hMICL expressing stem cells could be predictive of disease progression in MPN. Materials and Methods:Peripheral blood (PB) was obtained from 37 PV-, 19 ET-, and 8 PMF patients. A total of 57 (36 PV, 16 ET, and 5 PMF) patients were in stable phase while 7 (1 PV, 3 ET, and 3 PMF) were defined as being in an accelerated phase. Samples were prepared for five-color flow cytometry protocols using the antibodies anti-CD34, anti-CD38, anti-CD45, anti-CD14, and anti-hMICL. Acquisition was performed on a BD FACSCanto II and data analyses were carried out in FlowJo software. We focused on the following subsets: CD45lowSSClowCD14-CD34+hMICL+ and CD45lowSSClowCD14-CD34+CD38-hMICL+. Gates defining hMICL positivity were established on the basis of hMICL- lymphocytes as internal controls. One million cells were analyzed per sample and subsets of interest were calculated as percentage of these. Results:We found a percentage of CD34+hMICL+ (mean 1.176%, SD 0.908) and CD34+CD38-hMICL+ (mean 0.256%, SD 0.329) in PMF patients, which was statistically different from both PV- (mean 0.038%, SD 0.101, and mean 0.005%, SD 0.018, respectively) (p=0.0002 and p<0.0001, respectively) and ET patients (mean 0.116%, SD 0.252, and mean 0.009%, SD 0.024, respectively) (p=0.001 and p=0.0007, respectively). Somewhat surprisingly, compared to PV patients the CD34+hMICL+ compartment in ET patients was significantly higher (p=0.036). Importantly, the stem cell compartment in the accelerated phase patients was significantly higher than in the stable phase ones: CD34+hMICL+ (stable phase: mean 0.108%, SD 0.31, and accelerated phase: mean 0.983%, SD 0.917, respectively) (p=0.0002) and CD34+CD38-hMICL+ (stable phase: mean 0.017%, SD 0.081, and accelerated phase: mean 0.202%, SD 0.325, respectively)(p=0.0003). Conclusion:We have identified circulating CD34+hMICL+ and CD34+CD38-hMICL+ cells as an interesting biomarker for evaluating the disease state in MPN patients. The addition of hMICL discriminates between normal and neoplastic stem- and progenitor cells in MPN patients and reliably assigns disease stages in these patients superior to the mere evaluation of circulating CD34+ and CD34+CD38- cells. Importantly, hMICL+ cells, though rare, were dependably identified in PB. Thus, this study provides possible options for flow cytometric monitoring of MPN patients. Disclosures:No relevant conflicts of interest to declare.

CD4+CD25+Foxp3+IFNg+CD178+ Human Induced Treg (Itreg) Contribute to Suppression of Alloresponses by Apoptosis of Responder Cells

Recently, a new subset of induced immunoregulatory T cells (iTreg) that produced IFNg was reported. In vivo, iTreg with the phenotype CD4+CD25+Foxp3+IFNg+ were shown to be associated with good long-term graft outcome in renal transplant recipients. In vitro, CD4+CD25+Foxp3+IFNg+ were shown to inhibit allogeneic T cell responses antigen-unspecifically as well as antigen-specifically. Inhibition was characterized by decreased activation of responder T cells. Others reported that iTreg initiate immunosuppression by apoptosis induction in effector cells. In the present study, we investigated whether apoptosis and Fas/FasL-dependent pathways contribute to the inhibition of T cell activation mediated by IFNg-producing CD4+CD25+Foxp3+ iTreg. Early apoptosis and necrosis rates as well as co-expression of immunostimulatory and immunosuppressive proteins in/on CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+ and CD4+CD25+IFNg+ PBL were analyzed using cells from healthy controls and four-color flow cytometry, PMA/Ionomycin-stimulated PBL, and MLC. 16h PMA/Ionomycin stimulation induced iTreg subsets with the phenotype CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+, and CD4+CD25+IFNg+ co-expressing CD178, CD95, CD152, CD279, Granzyme A, Granzyme B, Perforin, IL-10, and TGFβ1 (all p< 0.05). Using, in addition, five-color fluorescence flow cytometry, approximately 1% of stimulated CD4+ PBL were CD25+Foxp3+IFNg+CD178+ (unstimulated 0%). CD178+ iTreg increased within 3h after PMA/Ionomycin stimulation in parallel to early apoptotic Annexin+/PI− PBL, suggesting CD178-mediated apoptosis of responder cells by CD4+CD25+Foxp3+IFNg+CD178+ iTreg. Apoptosis was inhibited dose-dependently in the presence of increasing concentrations of monoclonal antibody against CD178, CD95, or IFNg (all p< 0.05). CD4+CD25+IFNg+ and CD4+CD25+CD178+ PBL separated from primary cell cultures and added to autologous PMA/Ionomycin stimulated secondary cell cultures induced apoptosis immediately. Early apoptosis was not antigen-specific as shown with CD4+CD25+IFNg+ and CD4+CD25+CD178+ PBL separated from primary MLC and added to secondary MLC with autologous or third-party cells as stimulator. In summary, polyclonal or allogeneic stimulation of PBL induces an iTreg subpopulation with the phenotype CD4+CD25+Foxp3+IFNg+CD178+. These iTreg, when isolated from primary cell cultures, enable immediate apoptosis in stimulated responder and effector cells. We speculate that CD4+CD25+Foxp3+IFNg+ iTreg detected in the blood of renal transplant recipients with good long-term graft outcome induce immunosuppression in part by Fas/FasL-dependent mechanisms. Because CD4+CD25+Foxp3+IFNg+ iTreg express IFNg receptors and are induced by IFNg, they represent the first line of iTreg during an initiating immune response. IFNg-activated effector cells with strongly up-regulated CD95 expression might be the perfect target for this early apoptosis-inducing iTreg subset.

CD4+CD25+Foxp3+IFNg+CD178+ Human Induced Treg (Itreg) Contribute to Suppression of Alloresponses by Apoptosis of Responder Cells

Recently, a new subset of induced immunoregulatory T cells (iTreg) that produced IFNg was reported. In vivo, iTreg with the phenotype CD4+CD25+Foxp3+IFNg+ were shown to be associated with good long-term graft outcome in renal transplant recipients. In vitro, CD4+CD25+Foxp3+IFNg+ were shown to inhibit allogeneic T cell responses antigen-unspecifically as well as antigen-specifically. Inhibition was characterized by decreased activation of responder T cells. Others reported that iTreg initiate immunosuppression by apoptosis induction in effector cells. In the present study, we investigated whether apoptosis and Fas/FasL-dependent pathways contribute to the inhibition of T cell activation mediated by IFNg-producing CD4+CD25+Foxp3+ iTreg. Early apoptosis and necrosis rates as well as co-expression of immunostimulatory and immunosuppressive proteins in/on CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+ and CD4+CD25+IFNg+ PBL were analyzed using cells from healthy controls and four-color flow cytometry, PMA/Ionomycin-stimulated PBL, and MLC. 16h PMA/Ionomycin stimulation induced iTreg subsets with the phenotype CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+, and CD4+CD25+IFNg+ co-expressing CD178, CD95, CD152, CD279, Granzyme A, Granzyme B, Perforin, IL-10, and TGFβ1 (all p< 0.05). Using, in addition, five-color fluorescence flow cytometry, approximately 1% of stimulated CD4+ PBL were CD25+Foxp3+IFNg+CD178+ (unstimulated 0%). CD178+ iTreg increased within 3h after PMA/Ionomycin stimulation in parallel to early apoptotic Annexin+/PI- PBL, suggesting CD178-mediated apoptosis of responder cells by CD4+CD25+Foxp3+IFNg+CD178+ iTreg. Apoptosis was inhibited dose-dependently in the presence of increasing concentrations of monoclonal antibody against CD178, CD95, or IFNg (all p< 0.05). CD4+CD25+IFNg+ and CD4+CD25+CD178+ PBL separated from primary cell cultures and added to autologous PMA/Ionomycin stimulated secondary cell cultures induced apoptosis immediately. Early apoptosis was not antigen-specific as shown with CD4+CD25+IFNg+ and CD4+CD25+CD178+ PBL separated from primary MLC and added to secondary MLC with autologous or third-party cells as stimulator. In summary, polyclonal or allogeneic stimulation of PBL induces an iTreg subpopulation with the phenotype CD4+CD25+Foxp3+IFNg+CD178+. These iTreg, when isolated from primary cell cultures, enable immediate apoptosis in stimulated responder and effector cells. We speculate that CD4+CD25+Foxp3+IFNg+ iTreg detected in the blood of renal transplant recipients with good long-term graft outcome induce immunosuppression in part by Fas/FasL-dependent mechanisms. Because CD4+CD25+Foxp3+IFNg+ iTreg express IFNg receptors and are induced by IFNg, they represent the first line of iTreg during an initiating immune response. IFNg-activated effector cells with strongly up-regulated CD95 expression might be the perfect target for this early apoptosis-inducing iTreg subset.

Flow cytometry in myelodysplastic syndrome: analysis of diagnostic utility using maturation pattern-based and quantitative approaches

Flow cytometry (FCM) is being increasingly evaluated for the diagnosis of myelodysplastic syndrome (MDS). We employed multiple FCM approaches to assess MDS. Five-color FCM, morphology blind, was done on bone marrow aspirates of 57 suspected MDS and 31 normal controls. Maturation pattern, quantitative FCM for low-grade MDS that awards FCM score, and expression of selected antigens on erythroid cells and CD34(+) blasts were evaluated. FCM results were correlated with clinical and laboratory workup. Patients (n = 57) included proven MDS (n = 14), suspected MDS (n = 13), and non-MDS (n = 30). By pattern-based approach, all proven cases were FCM positive. In suspected MDS, 11 (84.61 %) were positive including morphology-negative cases, and two (15.38 %) were intermediate. In non-MDS cases, 27 of 30 (90 %) were FCM negative, 2 of 30 (6.67 %) intermediate, and 1 of 30 (3.33 %) a hematinic-responsive case, positive. Quantitative parameters that characterized MDS included FCM score of >3, percentage CD34(+) B cells, and expression of CD11b, CD15, and CD56 on myeloblasts. CD71 MFI on CD235a(+) erythroblasts and CD38 MFI on myeloblasts were significantly lower in MDS. The former was present in FCM-intermediate suspected MDS but not FCM-intermediate non-MDS cases. Used in the overall clinical context, both maturation pattern recognition and quantitative approaches, the latter for low-grade MDS, are sensitive methods of diagnosing MDS, including cases negative by morphology and cytogenetics, especially if combined with evaluation of selected antigens, CD71 on CD235a(+) cells and CD38 on CD34(+) cells. The value of FCM in morphology-negative cases needs better definition of specificity through more extensive evaluation of secondary dyspoiesis.

Expressions of IL-22 in circulating CD4+/CD8+ T cells and their correlation with disease activity in SLE patients

Recently, Th17 cell-associated responses have received growing attention; however, the role of IL-22 (a cytokines also produced by Th17 cells) in the pathogenesis of systemic lupus erythematosus (SLE) has not been widely explored. In this study, we analyze the frequencies of IL-22-positive CD4+/CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from patients with SLE and their correlations with disease activity and clinical data. Five-color flow cytometry (FCM) was used to assess IL-22 production of CD4+/CD8+ T cells in PBMCs from 31 patients with SLE and 22 healthy control subjects, following stimulation ex vivo with phorbol 12-myristate 13-acetate and ionomycin for 4h. Results showed that the percentages of IL-22-positive CD4+ T cells were increased in the PBMCs of patients with SLE compared with healthy control subjects, whereas there were no significant differences in the percentages of IL-22-positive CD8+ T cells. There was a strong positive correlation between the proportion of CD4+ T cells expressing IL-22 and SLEDAI score (r (s)=0.65, P<0.001). Furthermore, the frequencies of IL-22-positive CD4+ T cells were significantly higher in patients with SLE with nephritis than those without nephritis (Z=-2.72, P<0.01). In conclusion, increased frequencies of IL-22-positive CD4+ T cells in patients with SLE and positive correlation with SLEDAI score and lupus nephritis suggest that this cytokine may be implicated in the pathogenesis of the disease.

Abstract 1736: Skewed usage of TCRVbeta repertoire and predictive role of CD60 and CD49d expression in the survival rate of patients with Sézàry Syndrome

Abstract Background: Sézary Syndrome (SS) is a rare and aggressive leukemic variant of Cutaneous T-Cell Lymphoma (CTCL) characterised by erythroderma and the presence of a malignant circulating memory CD4+ T cell clone with skin homing characteristics, lack of CD26 and CD49d and over-expression of CD60. The availability of a large panel of monoclonal antibodies to CDR3 Vß epitopes, allows the study of the TCR repertoire by flow cytometry and a more precise identification of the malignant clone phenotypic characteristics. Objective: The aim of this study was to evaluate both the distribution of TCR-Vß family and CD60/CD49d expression within the clonal T cell population in patients affected by SS. Methods: We studied, by five-color flow cytometry analysis, TCR-Vß repertoire in 62 patients affected by SS, in 180 patients with MF, 6 cases of B cell Lymphomas (BCL), 19 with chronic Eczema (CE) recruited and followed from 1998 to 2008. A sequential gating strategy was used in order to examine the CD60 and CR49d expression within the neoplastic clonal population identified through the TCR-Vß repertoire analysis. Results and Conclusion: A skewed usage of only 4 out of 25 TCR-Vß subfamilies was observed in circulating T cell clone in 61.9% of patients affected by SS. TCR-Vß 2 and 5.1 were the most frequently represented (42.8% of TCR-Vß reactive patients), followed by TCR-Vß 12 and 13.1. In 20 cases the TCR-Vß panel was unable to identify the neoplastic clone, recognizing less than 5% of T cells, thus representing an indirect proof for the presence of a clonal population. CD60 was over expressed and CD49d down regulated within clonal T lymphocytes. This phenotype of the dominant T cell clone in the peripheral blood of patients with SS was significantly associated with a lower survival rate (chi-square 5.467, p &amp;lt;0.019). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1736.

Immunoregulatory profiles in liver transplant recipients on different immunosuppressive agents

We compared peripheral blood immunophenotyping in 31 adult liver transplant recipients on differing long-term immunosuppressive (IS) monotherapy with and without peri-transplantation alemtuzumab (AL) induction. All patients had been stable on monotherapy with either sirolimus (SRL) ( n = 10) or without SRL (tacrolimus (TAC) ( n = 10), mycophenolate mofetil (MMF) ( n = 11)) for more than 6 months. Five-color flow cytometry for putative “regulatory” T and dendritic cells as well as serum assays for soluble HLA-G (sHLA-G) were performed. The SRL monotherapy group had significantly higher percentages of CD4+CD25 high+Foxp3+ T cells (1.3 ± 1.0) compared with the non-SRL group (0.7 ± 0.6) ( p = 0.04). The SRL effect was even higher in a subset with prior AL induction and no prior hepatitis C or rejection (1.7 ± 0.2) compared with all other subgroups (0.7 ± 0.6) ( p = 0.02). TAC patients showed significantly higher “regulatory” DC2:DC1 ratios (10 ± 7.6) compared with non-TAC patients (4.1 ± 2.3) ( p = 0.04). Although sHLA-G levels appeared higher in TAC patients, the differences were not statistically significant. In conclusion, IS monotherapy provides an opportunity to investigate regulatory roles of individual agents. SRL maintenance and prior AL induction in subsets of patients appeared to show a regulatory T cell immunophenotype. However, TAC patients may have other regulatory characteristics, supporting the need for larger, prospective studies to clarify differences.

Flow Cytometry Analysis of Peripheral Blood CD34+ Cells in Patients with Primary Myelofibrosis

Primary myelofibrosis (PMF) is a clonal chronic myeloproliferative disorder characterized by the accumulation of megakaryocytes in the bone marrow (BM), variable degrees of BM fibrosis, tear-drop erythrocytes, increased numbers of CD34+ hematopoietic progenitors in the peripheral blood (PB), and extramedullary hematopoiesis. Since the antigenic properties of the circulating CD34 cells may yield clues to disease pathogenesis and have not been extensively studied, we used five-color flow cytometry to examine these cells from 20 well characterized patients with PMF and 10 normal controls. Bone marrow biopsies, molecular and cytogenetic studies were also reviewed. As expected, the percentages of peripheral-blood CD34 cells were significantly higher in the PMF patients (mean 1.4%, range, range 0.065–7.15) compared to the controls (mean 0.05%, range 0.01–0.57). The mean fluorescence intensity (MFI) values related to HLA-DR expression were increased (more than 3 fold) on the CD34+ cells in 12/20 (60%) PMF patients relative to normal control levels, while increased levels of CD13 were seen in 5/20 (25%) of PMF patients. CD33 and CD117 expression were similar on the CD34+ cells in both groups. Aberrant expression of lymphoid antigens was observed in 6/20 (30%) with CD7, 6/20 (30%) with CD4, and 3/20 (15%) with CD56 on CD34 positive cells in PMF. In the18 cases also studied with antibodies against CD45RA and CD123, the majority of CD34+ CD38 + cells phenotypically resembled megakaryocyte-erythroid precursors (CD45RA−, CD123−) in 5 cases, common myeloid progenitors (CD45RA−, CD123+) in 12 cases, and granulocyte-macrophage progenitors (CD45RA+, CD123 +) in 1 case. JAK2-V617F mutations were detected in 9 of 20 cases, but were present in only 1 of 5 cases showing predominately megakaryocyte-erythroid precursors. The percentage of CD34+ cells also expressing CXCR4 (CD184) appears to be increased in some patients relative to normal controls in contrast to other reported studies. In conclusion, the peripheral blood CD34+, progenitor cells in PMF patients are heterogeneous phenotypically resembling megakaryocyte-erythroid precursors in approximately 30% of cases, and common myeloid progenitors in approximately 70% of cases. In addition, these cells often show phenotypic abnormalities (increased intensity of HLA-DR and CD13 expression) that can be detected with flow cytometry relative to normal peripheral blood CD34+ cells. Patterns of antigen expression in PMF also appear to differ from those reported for CD34 positive cells in other myeloproliferative disorders which may help in early diagnosis and/or monitoring treatment responses.

Characterization of nTreg in patients with metastatic renal cell carcinoma (RCC) undergoing DC-vaccination and cytokine therapy

16038 Background: CD4+CD25+FoxP3+ Regulatory T cells (nTreg) play a pivotal role in suppression of anti-tumor immune responses. We analyzed the subpopulation of nTreg in patients enrolled in a Phase II autologous dendritic cell (DC) vaccine trial. Methods: Consented and eligible patients received up to 5 cycles of intranodal DC-autologous tumor vaccine (1 × 107 cells) followed by 5 days of continuous i.v. infusion of IL-2 (18 MiU/m2) and s.c. Interferon alpha 2a (6 MiU) every other day. Samples were collected via leukapheresis at baseline and after two cycles. Five-color flow cytometry was performed on lymphocytes utilizing a comprehensive panel of cell surface and intracellular markers. Treg function was assessed in a Treg assay. Cytokines in supernatants of this assay were quantified using Luminex® ELISAs. Patients were divided into responders (R: complete and partial, n=9 using WHO criteria) and non-responders (NR: stable and progressive disease, n=9) and compared with age matched healthy donors (HD, n=12). P-values were assesed using Student's t-test. Values are expressed as mean±StD. Results: At baseline, cancer patients had a significantly higher percentage of CD4+CD25+FoxP3+ cells than HD (% of CD3+: 2.7% ± 1.2 vs. 1.3% ± 0.7, p<0.001). After two induction cycles, an increase in this percentage was observed (% of CD3+ Pre: 2.7% ± 1.2 vs. Post: 6.6% ± 4.5, p=0.006). After induction, significantly higher levels of Treg were detected in NR vs. R (% of CD3+: 9.2% ± 5.4 vs. 4.4% ± 1.6, p=0.036). Treg assays demonstrated preserved suppressor function before and after therapy. Cytokine analysis demonstrated a treatment related increase in Th2-related cytokines IL-4 and IL-10 (46 pg/ml ± 28 vs. 143 pg/ml ± 77, p=0.001 and 212 pg/ml ± 133 vs. 902 pg/ml ± 386, p=0.005), and a decrease in the Th1-related cytokine IL-2 (5785 pg/ml ± 5824 vs. 381 pg/ml ± 510, p=0.005). Conclusions: RCC patients showed higher levels of Treg cells than healthy donors. Lower levels of Treg cells were detected in responding RCC patients treated with an immunotherapeutic regimen. This data implies a threshold of Treg cells in the peripheral blood, which may affect clinical outcome and has implications for the design of future immunotherapeutic approaches to RCC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Berlex Oncology, NIH

Monitoring responses by use of five-color flow cytometry in subsets of peripheral T cells obtained from cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis vaccine

To monitor by use of 5-color flow cytometry the antigen-specific responses of subsets of peripheral T cells in cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine and to compare results with those for 2 established cell-mediated immunity assays. 45 female Holstein cattle with negative results for MAP in skin tests conducted at time of inoculation with MAP. Cattle were allocated to 4 groups. Cattle of group 1 (n = 12) were 0 to 3 months old and inoculated with a killed MAP vaccine. The 10 cattle of group 2 were the same age as those in group 1 but were not inoculated with MAP vaccine. The 11 cattle of group 3 were 9 to 12 months old and inoculated with killed MAP vaccine. The 12 cattle of group 4 were the same age as those in group 3 but were not inoculated with MAP vaccine. Flow cytometry identified T-cell subsets that responded specifically to the recall antigen. Results of assays for CD25 expression and wholeblood interferon-gamma had the strongest correlation with results for skin tests as well as results with each other. Intracellular expression of interferon-gamma was not correlated as well with results for the other tests. Flow cytometry can be useful for characterizing the immune response after administration of MAP vaccine and should be evaluated with regard to its sensitivity and specificity when used in detecting cattle naturally infected with MAP.

Phenotypic comparison of multiple monocyte‐related populations in murine peripheral blood and bone marrow

Monocyte subsets are not well defined in murine peripheral blood (PB). Monocyte-related populations could also be located in bone marrow (BM), but studies correlating monocyte populations found in these two tissues are lacking. This study simultaneously analyzed PB and BM to phenotypically define multiple monocyte-related subsets in each location. Murine PB and BM cells were simultaneously stained for monocyte-related populations, using five-color flow cytometry. Relevant subsets were defined on the basis of Ly-6C, CD11b, and wheat germ agglutinin phenotype in addition to light-scatter characteristics. These populations were extensively characterized for the expression of other myeloid and dendritic cell markers, adhesion molecules, chemokine receptors, and growth factor receptors. Six monocyte-related populations were defined, three each in BM and PB. No identical populations were found between the two tissues. Two BM populations and one PB population have heterogeneous expression of many markers, suggesting additional complexity among monocyte-related subsets. The murine monocytic series comprises multiple subsets, differing between PB and BM. This study defines and extensively phenotypes six of these populations, providing preliminary information about possible developmental relationships and migratory capacities of these cells.