Two New Neutrophil Subsets Define a Discriminating Sepsis Signature.

Rationale: Sepsis is the leading cause of death in adult ICUs. At present, sepsis diagnosis relies on nonspecific clinical features. It could transform clinical care to have immune-cell biomarkers that could predict sepsis diagnosis and guide treatment. For decades, neutrophil phenotypes have been studied in sepsis, but a diagnostic cell subset has yet to be identified. Objectives: To identify an early, specific immune signature of sepsis severity that does not overlap with other inflammatory biomarkers and that distinguishes patients with sepsis from those with noninfectious inflammatory syndrome. Methods: Mass cytometry combined with computational high-dimensional data analysis was used to measure 42 markers on whole-blood immune cells from patients with sepsis and control subjects and to automatically and comprehensively characterize circulating immune cells, which enables identification of novel, disease-specific cellular signatures. Measurements and Main Results: Unsupervised analysis of high-dimensional mass cytometry data characterized previously unappreciated heterogeneity within the CD64+ immature neutrophils and revealed two new subsets distinguished by CD123 and PD-L1 (programmed death ligand 1) expression. These immature neutrophils exhibited diminished activation and phagocytosis functions. The proportion of CD123-expressing neutrophils correlated with clinical severity. Conclusions: This study showed that these two new neutrophil subsets were specific to sepsis and detectable through routine flow cytometry by using seven markers. The demonstration here that a simple blood test distinguishes sepsis from other inflammatory conditions represents a key biological milestone that can be immediately translated into improvements in patient care.

Significance of CD34/CD123 expression in detection of minimal residual disease in B-ACUTE lymphoblastic leukemia in children.

BACKGROUND: MRD is seen as the major cause of disease relapse. So, it gives important feedback about conventional treatment success and helps in selecting therapeutic alternatives. We aimed to compare the expression of CD34/CD123 on normal B-cell precursors in bone marrow ("hematogones") and on leukemic blasts in B-acute lymphoblastic leukemias (B-ALL) pediatric cases by flowcytometric analysis. Our study conducted on 20 children as a control and 30 B-ALL children cases at diagnosis and after 28days of induction therapy. We found that the less mature hematogones (dim CD45+) that express CD34 lack CD123 expression, whereas the more mature hematogones (moderate CD45+) lack CD34 but always express CD123. In contrast with this discordant pattern of CD34 and CD123 expression in hematogones, blasts in 24 of 30 cases (80%) of B-ALL showed concordant expression pattern of the 2 antigens: 63% (19 of 30) cases expressed both antigens, whereas 17% (5 of 30) expressed neither. Our study concluded that these distinct patterns of CD34/CD123 expression on hematogones (discordant) and B-ALL blasts (concordant) are useful in differentiating small populations of residual blasts from hematogones after induction therapy to detect MRD.

Basophil markers for identification and activation in the indirect basophil activation test by flow cytometry for diagnosis of autoimmune urticaria.

BACKGROUND: The indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c). METHODS: Donor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20). RESULTS: For basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity. CONCLUSIONS: For basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.

Basophil Markers for Identification and Activation in the Indirect Basophil Activation Test by Flow Cytometry for Diagnosis of Autoimmune Urticaria

BACKGROUND: The indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c). METHODS: Donor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20). RESULTS: For basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity. CONCLUSIONS: For basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.

High levels of CD34+CD38low/-CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study.

BACKGROUND: Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34(+)CD38(low/-) compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34(+)CD38(low/-)CD123(+) phenotype. DESIGN AND METHODS: The percentage of CD34(+)CD38(low/-)CD123(+) cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses. RESULTS: A proportion of CD34(+)CD38(low/-)CD123(+) cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34(+)CD38(low/-)CD123(+) higher than 15% retained significance with an odds ratio of 0.33 (0.1-0.97; P=0.044). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells retained prognostic significance for both parameters after multivariate analysis. CONCLUSIONS: The percentage of CD34(+)CD38(low/-)CD123(+) leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure.

Expression of dendritic cell markers CD11c/BDCA-1 and CD123/BDCA-2 in coronary artery disease upon activation in whole blood.

OBJECTIVES: Previous in vivo studies on dendritic cell (DC) enumeration in coronary artery disease (CAD) were not always consistent. Therefore, we investigated by flow cytometry whether this was due to CAD-related differences in expression of subset markers for myeloid (m)DCs (blood DC antigen (BDCA)-1, CD11c) and plasmacytoid (p)DCs (BDCA-2, CD123), before and after in vitro stimulation with Toll-like receptor ligands. RESULTS: Our data showed that circulating DCs decline in CAD, irrespective of the DC subset marker that was used for enumeration. Upon in vitro activation, BDCA-2 was downregulated, whereas CD11c and CD123 were upregulated. This implies that the expression ratios CD11c/BDCA-1 and CD123/BDCA-2 can assess DC activation. Comparing these ratios between controls and CAD patients showed no differences in blood DC activation in both groups. CONCLUSIONS: This study suggests that when different DC numbers are found between two study populations, the DC activation status from both groups always needs to be verified, since a decrease in BDCA-2(+) pDCs or an increase in CD11c(+) mDCs or CD123(+) pDCs can be due to the altered expression of these markers during activation. Given that CD11c, BDCA-1, CD123 and BDCA-2 are more abundantly expressed on blood DCs than typical activation markers like CD83, CD86 or CCR-7, the use of the ratios is an easy and reliable way to determine DC activation in whole blood assays.

Plasmacytoid dendritic cell count on day 28 in HLA-matched related allogeneic peripheral blood stem cell transplant predicts the incidence of acute and chronic GVHD.

Dendritic cells (DC) are antigen-presenting cells involved in induction and regulation of immune responses. We investigated the impact of the number of infused and day 28 dendritic cells on the development of acute and chronic GVHD (aGVHD, cGVHD). Monocytoid (MC) and plasmacytoid (PC) dendritic cells were characterized as lin(-)HLA-DR(+)CD11c(+) and lin(-)HLA-DR(+)CD123(+), respectively. Sixty-eight consecutive patients who underwent HLA matched related granuloyte-colony stimulating factor (G-CSF) mobilized allogeneic PBSCT, from February 2005 to May 2006, were included in the analysis. Twenty-three patients developed aGVHD (grade II-IV) and 21 patients had cGVHD. On a univariate analysis the day 28 total DC and the day 28 MC and PC dendritic cells as continuous variables were significantly associated with development of aGVHD and cGVHD. Using an ROC plot analysis a cutoff value for total DC = 10.7/microL, MC = 9.7/microL, and PC = 4.5/microL on day 28 gave the highest likelihood ratios for aGVHD (2.7, 2.14, and 3.29, respectively). On a multivariate analysis, a low day 28 PC (

Immunosuppressive dendritic and regulatory T cells are upregulated in melanoma patients.

BACKGROUND: Immunologic therapies for melanoma rarely succeed, suggesting a persistent counter-regulatory immune modulation. Regulatory T cells (T(regs)) and plasmacytoid subpopulations of dendritic cells (pDCs) inhibit the immune response. We hypothesize that melanoma upregulates T(regs )and subpopulations of immunosuppressive dendritic cells (DCs). METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy controls, stage I and stage IV melanoma patients. T(regs )were identified as CD4+ and CD25(hi). Dendritic cells were identified using a DC cocktail of antibodies including CD11c+ myeloid dendritic cells (mDCs) and CD123+ pDCs. Serum transforming growth factor-beta (TGF-beta), interleukin-4 (IL-4) and interleukin-10 (IL-10) levels were determined by enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using analysis of variance (ANOVA). RESULTS: Stage IV melanoma patients had a doubling of regulatory T cells compared to both normal subjects and stage I melanoma patients. There was a significantly higher number of DCs in all melanoma patients compared to normal subjects. Stage I melanoma patients had a significantly higher number of pDCs than normal subjects, and all melanoma patients had a higher concentration of mDCs than controls. Serum IL-4 and IL-10 were not detectable but serum TGF-beta levels were significantly higher in stage I and stage IV melanoma patients compared to normal controls. CONCLUSION: Advanced melanoma is associated with increased numbers of circulating dendritic cells and regulatory T cells. These data suggest that melanoma induces immunosuppressive DCs and regulatory T cells in the systemic circulation.

[Application of CD123 in detection of minimal residual disease in acute promyelocytic leukemia].

The study was aimed to investigate the role and significance of CD123 with other immunological markers in detecting minimal residual disease (MRD) of APL patients. The immunophenotypes of 186 newly diagnosed APL patients and the percentages of cells identical with APL cell immunophenotypes in 20 normal bone marrow samples were analyzed using four-color flow cytometry. MRD in 172 specimens were monitored by mainly using CD34/CD117/CD123/HLA-DR four-color antibody panels, meanwhile 18 specimens were analyzed with the second antibody combination: CD9/CD117/CD34/CD33, simultaneously and the results were compared with real-time PCR. One hundred and sixteen of 172 bone marrow (BM) or peripheral blood (PB) specimens were from follow-up 19 newly diagnosed APL patients and the rest 56 samples were from 47 patients treated 3 to 24 months later. Among them, 117 samples and 55 samples were collected after achieving morphologic complete remission (mCR) and before achieving mCR respectively. The results of immunophenotyping demonstrated that except CD9, CD33 and CD117 were high-expressed and CD34 and HLA-DR were rarely expressed, the CD123 was expressed in 30/30 (100%) APL patients. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) and CD117(+)CD34(-)CD9(+)CD33(+) cells in nucleated cells were 0.066% +/- 0.012% and 0.089% +/- 0.066% in 20 normal bone marrow samples. The median time of achieving morphology complete remission in 19 APL patients was 4 weeks (3 - 6 weeks). The median time of FCM and PCR results turned to be negative in 13 APL patients was 7.5 weeks (5 - 11) and the median time of PCR results turned to be negative in 11 APL patients was 8 weeks (5 - 12). 41/117 (35.04%) samples were MRD positive by FCM after achieving mCR. The ratio of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells was < 5% in 33 specimens, but > 5% in another 8 specimens, their median percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells were 0.48% (range 0.02% - 4.70%) and 9.02% (range 5.26% - 18.14%) respectively. The median relative percentages of CD123(+)HLA-DR(-) cells in CD117(+)CD34(-) population were 63.59% (range 15.11% - 98.36%) and 86.77% (range 63.29% - 92.62%) respectively. In FCM MRD positive samples, 95.9% (93/97) were PCR positive, the false positive rate of FCM and the false negative rate of PCR were 4.1% (4/97) and 8.75% (7/93) respectively. In FCM negative samples, 92% (69/75) were PCR negative and 8% (6/75) were PCR positive. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells in 116 consecutive specimens and 117 specimens of mCR were related to PML/RARalpha quantified by real-time PCR (r = 0.824, P < 0.001 and r = 0.754, P < 0.001 respectively). It is concluded that the detection of APL patients by means of two sets of antibody panels is simple and suitable, which is complementary to PCR in monitoring MRD of APL patients.