Three-color Flow Cytometry Research Articles

High Frequencies Cytomegalovirus pp65495–503-Specific CD8+ T Cells in Healthy Young and Elderly Chinese Donors: Characterization of Their Phenotypes and TCR Vβ Usage

Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus which persists lifelong after primary infection and can lead to a significant disease in the immunocompromised individuals. CD8(+) T cells are believed to play a crucial role in both the elimination of active infection and maintenance of HCMV latency. Large expansions of CD8(+) T cells specific for a single epitope of HCMV have been well documented in Caucasoid population. To date, no similar study has been performed in Chinese populations. Here we report the characteristics of HCMV-specific CD8(+) T cells in healthy young and elderly Chinese donors using pp65(495-503)-loaded HLA-A*0201 tetramers. Cells were stained with a combination of the tetramers and antibodies for CD28 and CD57 or a panel of TCR Vbeta and analyzed by three-color flow cytometry. The frequencies of pp65(495-503)-specific T cells within total CD8(+) T cell population were between 0.14 and 6.84% (mean 2.45%) in the young donors and were from 0.33 to 6.89% (mean 1.95%) in the elderly donors, respectively. There was no significant difference between the two groups. The expression of CD28 was decreased whereas CD57 expression was increased in tetramer-negative CD8(+) T cells in the elderly when compared with the young group. However, neither of these changes was found within tetramer-positive cell populations. Moreover, TCR Vbeta usage within tetramer-positive population was predominated by certain TCR Vbeta subsets. These results demonstrate that large expansions of HCMV-specific CD8(+) T cells with certain subsets TCR Vbeta exist both in the healthy young and in the elderly Chinese individuals, which may play a role in the maintenance of virus latency but have potential detrimental influence on the immune responses to other pathogens or vaccinations.

Rapid Whole Blood Flow Cytometric Test to Detect ICOS Deficiency in Patients with Common Variable Immunodeficiency

Background/Aims: Common variable immunodeficiency (CVID) is the most common primary immunodeficiency. With respect to underlying defects it comprises a heterogeneous group of deficiencies. For some patients, distinct phenotypical abnormalities have been described, e.g. partial CD40L deficiency or complete ICOS deficiency. For the diagnosis of CD40L deficiency, a rapid whole blood flow cytometric method has been described several years ago. We aimed to determine if the same method can be used to diagnose ICOS deficiency. Methods: Whole blood from 8 healthy volunteers was stimulated for 4 and 20 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Induction of ICOS expression was analyzed on CD8–CD3+ lymphocytes using three-color flow cytometry. Blood from a patient with diagnosed ICOS deficiency was also analyzed. Results: Whole-blood stimulation with PMA and ionomycin for 20 h resulted in a significant induction of ICOS expression on CD8–CD3+ lymphocytes in healthy volunteers. Four-hour incubation also demonstrated ICOS upregulation but to a much lower extent. In CD8–CD3+ lymphocytes from an ICOS-deficient patient, no ICOS expression could be induced following 20 h of stimulation. Conclusion: ICOS expression can be analyzed using a rapid whole blood flow cytometric test.

Naïve and central memory T-cell lymphopenia in end-stage renal disease

End-stage renal disease (ESRD) is associated with increased propensity to infections, diminished response to vaccination, impaired cell-mediated immunity, and reduced CD4+/CD8+ T-lymphocyte ratio. Four subsets of CD4+ and CD8+ T cells have been recently identified: naïve cells (as yet uncommitted), central memory (CM) cells (previously programmed), and CD45RA-positive and CD45RA-negative effector memory (EM) cells (programmed to perform specific effector functions). The effect of ESRD on subpopulations of T lymphocytes is unclear and was studied here. Twenty-one hemodialysis patients and 21 age-matched controls were studied. Pre- and post-dialysis blood samples were obtained and analyzed by three-color flow cytometry. CD4+/CD8+ ratio and the numbers of the naïve and CM CD4+ and CD8+ T cells were significantly reduced, whereas the numbers of EM CD4+ and CD8+ T cells were unchanged in the ESRD group. The reduction of the naïve and CM T-cell counts in the ESRD group was associated with increased apoptosis of these cells. Negative correlations were found between severity of azotemia, oxidative stress, and hyperphosphatemia with the number of naïve T cells. Comparison of diabetic with non-diabetic ESRD patients revealed higher numbers of total CD8+ cells and EM CD8+ T cells in the diabetic group. Dialysis did not significantly change the naïve and CM CD4+ or CD8+ cell counts, but significantly lowered CD8+ EM cell count. Thus, ESRD results in increased apoptosis and diminished populations of naïve and CM T lymphocytes. This phenomenon may, in part, contribute to the impaired immune response in this population.

Skewed expression of natural‐killer (NK)‐associated antigens on lymphoproliferations of large granular lymphocytes (LGL)

Killer-immunoglobulin-like receptors (KIR) or C-type lectin-like receptors are heterogeneously expressed on NK cells and small subsets of T cells and might provide a new diagnostic tool for LGL lymphoproliferations (LGLL). We investigated the diagnostic impact of these cell surface molecules in T- and NK-type LGLL. Using three-color flow cytometry we examined the expression patterns of KIR (CD158a/b/e/i), CD85j, lectin-like receptors (CD94, CD161, NKG2A/D) and natural cytotoxicity receptors (NKp30/44/46) in 13 patients with LGLL (10 T-, 3 NK-LGLL) and compared them to those of the corresponding lymphocyte subsets in 20 control subjects. The presence of clonal TCR-gamma rearrangements and of Epstein Barr virus- (EBV) DNA were evaluated by PCR. All patients exhibited an altered expression of NK-associated markers. KIR were either lacking (6/13) or overexpressed (7/13). CD94 expression was significantly higher in all LGLL. NKG2A expression was significantly higher in NK-LGLL. Absence or overexpression was observed for NKG2A in T-LGLL and CD161 in most T/NK-LGLL. In NK-LGLL expression of NKp30 and NKp46 was significantly decreased, whereas CD85j was overexpressed. We consistently found a skewed expression pattern of novel NK markers as a pathological feature of LGLL. These antigens should be included in the diagnostic workup of this rare disease.

Cytokine production by bronchoalveolar lavage T lymphocytes in chronic obstructive pulmonary disease

T lymphocytes (predominantly CD8+ cells) have previously been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). We sought to describe the profile of cytokine production by CD8+ and CD4+ cells isolated from bronchoalveolar lavage fluid. Bronchoalveolar lavage was performed in 11 patients with COPD (median FEV1, 63.3% of predicted value) and 9 healthy control subjects. CD8+ and CD4+ T cells were isolated by means of positive selection after macrophage depletion. CD8+ and CD4+ cells were activated with anti-CD3/CD28 antibodies for 60 hours before restimulation with phorbol 12-myristate 13-acetate-ionomycin and brefeldin. Three-color flow cytometry was used to simultaneously measure levels of intracellular cytokines. IL-4 was expressed by a higher percentage of stimulated CD8+ T cells (TC2) compared with CD4+ T cells (TH2) in patients with COPD (P = .01). In contrast, IFN-gamma was expressed in a significantly higher percentage of stimulated CD4+ T cells (TH1) than CD8+ T cells (TC1) in the COPD group (P = .04). TNF-alpha was expressed by almost all TC1 and TH1 cells, with virtually no expression by TC2 and TH2 cells. In addition, a small number of T cells expressing TNF-alpha alone without concomitant IFN-gamma or IL-4 expression were seen in the majority of subjects. There was a higher percentage of TC2 cells in subjects with COPD compared with that seen in the control group (P = .03). Stimulation with anti-CD3/CD28 antibodies increased the percentage of TC2 cells and decreased the percentage of TH2 cells. Our results suggest that there are increased numbers of TC2-like cytokine-expressing cells in the lungs of patients with COPD. These cells might be a source of TH2 cytokines, which might, at least in part, explain the lung eosinophilia associated with COPD exacerbations.

Characterization of Peripheral Blood Lymphocyte Subsets in Patients with AcutePlasmodium falciparumandP. vivaxMalaria Infections at Wonji Sugar Estate, Ethiopia

We investigated the absolute counts of CD4+, CD8+, B, NK, and CD3+ cells and total lymphocytes in patients with acute Plasmodium falciparum and Plasmodium vivax malaria. Three-color flow cytometry was used for enumerating the immune cells. After slide smears were stained with 3% Giemsa stain, parasite species were detected using light microscopy. Data were analyzed using STATA and SPSS software. A total of 204 adults of both sexes (age, >15 years) were included in the study. One hundred fifty-eight were acute malaria patients, of whom 79 (50%) were infected with P. falciparum, 76 (48.1%) were infected with P. vivax, and 3 (1.9%) were infected with both malaria parasites. The remaining 46 subjects were healthy controls. The leukocyte count in P. falciparum patients was lower than that in controls (P=0.015). Absolute counts of CD4+, CD8+, B, and CD3+ cells and total lymphocytes were decreased very significantly during both P. falciparum (P<0.0001) and P. vivax (P<0.0001) infections. However, the NK cell count was an exception in that it was not affected by either P. falciparum or P. vivax malaria. No difference was found in the percentages of CD4, CD8, and CD3 cells in P. falciparum or P. vivax patients compared to controls. In summary, acute malaria infection causes a depletion of lymphocyte populations in the peripheral blood. Thus, special steps should be taken in dealing with malaria patients, including enumeration of peripheral lymphocyte cells for diagnostic purposes and research on peripheral blood to evaluate the immune status of patients.

Correlation of T-lymphocyte subpopulations with immunological markers in HIV-1-infected Indian patients

Progressive HIV disease is characterized by CD4 T cell decline and activation of the immune system. We aimed to study the quantitative alterations in the naive (CD45RA+CD62L+), memory/effector (CD45RO+) and activated (HLA-DR+CD38+) T-lymphocyte subpopulations in antiretroviral treatment naive, HIV-1 infected Indian patients by three-color multi-parametric flow cytometry. The association of different CD4+ and CD8+ T cell subsets with the immunological markers- CD4+ and CD8+ T cell percentages was examined by calculating the partial correlation coefficients. We also observed significant differences in the expression of different CD4+ and CD8+ T-cell subsets among the two groups of patients formed using the median CD4+ T cell percentage value (15%) of the study population. The correlations of different CD4+ and CD8+ T cell subsets reflected the quantitative alterations in the T-lymphocyte subpopulations and activation of the immune system during HIV-infection. The study outcome also emphasizes the significance of the CD38+CD8+ T-lymphocyte subset as a prognostic marker for HIV management and ART monitoring in resource-limited settings of developing countries like India.

Increased Fas and Bcl‐2 Expression on Peripheral Blood T and B Lymphocytes from Juvenile‐Onset Systemic Lupus Erythematosus, but not from Juvenile Rheumatoid Arthritis and Juvenile Dermatomyositis

Defective regulation of apoptosis may play a role in the development of autoimmune diseases. Fas and Bcl-2 proteins are involved in the control of apoptosis. The aims of this study were to determine the expression of Fas antigen and Bcl-2 protein on peripheral blood T and B lymphocytes from patients with juvenile-onset systemic lupus erythematosus (JSLE), juvenile rheumatoid arthritis (JRA) and juvenile dermatomyositis (JDM). Thirty-eight patients with JSLE, 19 patients with JRA, 10 patients with JDM and 25 healthy controls entered the study. Freshly isolated peripheral blood mononuclear cells (PBMC) were stained for lymphocyte markers CD3, CD4, CD8, CD19 and for Fas and Bcl-2 molecules. Expressions were measured by three-color flow cytometry. Statistical analysis was performed using Kruskal–Wallis test. Percentages of freshly isolated T lymphocytes positively stained for Fas protein from JSLE patients were significantly increased compared to healthy controls, patients with JRA and patients with JDM. Percentages of B lymphocytes positive for Fas from JSLE patients were higher than healthy controls and JRA patients. In addition, Fas expression on T cells from patients with JRA was increased compared to JDM patients. Otherwise, Fas expression on T and B cells from JRA and JDM patients were similar to healthy controls. MFI of Bcl-2 positive T lymphocytes from JSLE patients were significantly increased compared to healthy controls and JRA patients. MFI of Bcl-2 protein on B lymphocytes from JSLE patients was similar to healthy controls and patients with JRA and JDM. Bcl-2 expression did not differ between JRA and JDM patients and healthy controls. In conclusion, increased expression of Fas and Bcl-2 proteins observed in circulating T and B lymphocytes from patients with JSLE, but not from patients with JRA and JDM, suggests that abnormalities of apoptosis may be related to the pathogenesis of JSLE and probably are not a result of chronic inflammation.

Expression of CD27–CD70 on Early B Cell Progenitors in the Bone Marrow: Implication for Diagnosis and Therapy of Childhood ALL

CD27, a member of the TNF receptor family, plays an important role in lymphoid proliferation, differentiation and apoptosis. This study addresses the expression of CD27 and its ligand, CD70, in children with acute lymphoblastic leukemia (ALL) and the possible role of this receptor-ligand pair in the pathogenesis of ALL. Expression of CD27 and CD70 was evaluated with three-color flow cytometry in blood and bone marrow (BM) samples in children with ALL and controls. The biological role of these molecules on leukemic cell proliferation was studied in an in vitro culture system. The expression of the membrane bound CD27, as well as membrane bound CD70, on CD19(+) cells in the BM was significantly increased in ALL children compared to the expression found in the controls. Importantly, a substantial reduction in the in vitro proliferation of leukemic cells could be observed when the leukemic cells were cultured in presence of a blocking anti-human CD70 monoclonal antibody. The level of soluble CD27 (sCD27) in serum was also investigated and found to be significantly elevated in leukemic children as compared to healthy children. The high expression of CD27 and CD70 on ALL cells may represent an amplification of the normal CD27-CD70 expression present on early B cell progenitors. Our finding suggests that interference with CD27-CD70 interaction may represent novel treatment opportunities in ALL. Further studies are required to pin-point the role of this receptor-ligand pair in normal and malignant hematopoiesis.

The Characteristics of Immunophenotype in Acute Lymphoblastic Leukemia and Its Clinical Significance

Objective: To study the characteristics of immunophenotype in acute lymphoblastic leukemia (ALL) and its clinical significance. Methods: Immunophenotyping was performed on 81 ALL patients by three-color flow cytometry analysis using CD45/SSC gating, meanwhile the cytogenetic analysis was performed on 45 cases out of 81 ALL patients. Results: (1) CD19 was the most commonly expressed of all B-lineage antigens detected with the positive rate being 100%. In T-ALL, the positive expression rate of CD5 and CD7 was the highest, being 90%. Both B-ALL and T-ALL overlapped in expression of lineage antigens. There was no significant difference in the complete remission rate (CR rate) between T-ALL and B-ALL. (2) The incidence of ALL with myeloid antigens expression (My+ALL) was 39.5%. CD13 was most often seen among the myeloid markers. My+ALL always involved in B-lineage antigens and the CR rate in children and adults was 72.2% and 78.6% respectively. (3) The incidence of HAL was 19.8%. Coexpression of B-lineage and myeloid-associated antigens was the commonest subtype in HAL. The expression of CD34 was commonly seen in HAL patients (81.3%). The CR rate was low in HAL, 50% for children and 40% for adults. (4) Compared to T-ALL, B-ALL, My+ALL, and HAL had a higher positive rate of CD34 expression with the difference being significant (P<0.025). Conclusion: Immunophenotyping had remarkable predominance in diagnosing special category of ALL (such as HAL and My+ALL); CD19 and CD5 were highly sensitive in diagnosing B-ALL and T-ALL, but less special, and overlapping was found in expression. No significant association was found between the expression of CD34 or myeloid antigens and CR rate, while low CR rate was found in HAL patients, especially for those coexpressing CD34 antigen.

Alterations in the Umbilical Cord Blood NK Cells Related to the Mode and the Time of Delivery.

Natural killer (NK) cells take part in the early immunological response to infection. Their lower cytotoxic activity in the neonates, especially premature ones, compared to children and adults, is assumed to be one of the factors responsible for high susceptibility to infections. Moreover, alterations in every components of immune response during anesthesia and surgery have been suggested. The numbers of natural killer cells are decreased postoperatively. The aim of the study was to estimate the influence of the mode and time of delivery on the number of leukocytes, number and percentage of lymphocytes and natural killer (NK) cells. The NK cells were examined by the three-color flow cytometry with the use of monoclonal antibodies of Becton Dickinson in the following study groups: (1) full-term neonates born by normal spontaneous vaginal delivery (n=19); (2) preterm neonates born by normal spontaneous vaginal delivery (n=15); (3) full-term neonates born by elective cesarean section under epidural anesthesia (n=23); (4) preterm neonates born by cesarean section under epidural anesthesia (n=22). The number of leukocytes was similar in all examined neonates. The numbers of leukocytes were lower albeit not significantly in preterm neonates born by cesarean section. The numbers of lymphocytes were also similar in all examined neonates but the percentage of lymphocytes was higher in the preterm neonates than in the full-term ones (p<0,05). The number and percentage of natural killer (NK) cells were higher in the neonates born by normal spontaneous vaginal delivery both full-term and preterm ones. The significant lowest value of NK cells was in the preterm neonates born by cesarean section under epidural anesthesia. These results suggest that either mode of delivery or time of delivery might influence the NK cell numbers in the umbilical cord blood of the neonates.

JAK2 (V617F) Mutation Status and Neutrophil Apoptotic Resistance in Myelofibrosis with Myeloid Metaplasia (MMM): Correlation and Potential Identification through Phosphorylation Status of STAT3.

Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood 2003; 102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM.Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes.Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026).Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft.Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls.Results:The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P <0.05). The difference between cord blood and mobilized peripheral blood was not significant (P>0.05).The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P<0.01).The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P<0.01).Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P <0.01);The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P<0.01).Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.

Evaluation of Immunophenotypes and Activities of Cell Membrane Microparticles in Apheresis Platelets: Impact of Seven Day Storage.

Seven day storage of platelets has become a reality in transfusion management. Cell membrane microparticles (MP), 0.2–1.0 μm in size are released from stimulated platelets, blood- and endothelial cells. The pathogenic potential of MP has been documented. Since a portion of platelet transfusion adverse effects is likely not antibody related, MP are good candidates to study. We have previously demonstrated the presence of MP derived from platelets, blood-, and endothelial cells in apheresis platelet units (APU) on day 6 of storage. Here we investigate the pathophysiological activities of MP in APU and the impact of extended storage on MP immunophenotype and activity profiles. We analyzed MP in non-frozen APU supernatants (n=28) on day 6 and 8 of storage. A three-color flow cytometry assay of MP (Simak et al, British J Haematol, 2004) was used, and several functional assays were performed. Plasma from healthy volunteers (HVP) (n=38) was used for comparison. On day 6, the median platelet count in APU was 1514x103/μL (range: 1144x103–2248x103) and pH 7.28 (6.67–7.51). The pH decreased significantly to 6.97 (6.19–7.40) on day 8. All APU bacterial cultures were negative. When comparing APU on day 6 and 8 of storage, platelet MP (CD41+), endothelial MP (CD144+), red blood cell MP (CD235a+), and proinflammatory CD154+MP did not change significantly. In contrast, an increase of leukocyte MP, including leukocyte MP exposing tissue factor (TF, CD142) (CD142+CD45+MP; p=0.001) was found. Since both platelet CD41a+CD142+ and endothelial CD144+CD142+ MP tend to decrease during storage, the total CD142+MP (detected by V1C7 and HFT-1 Mab) showed only a slight increase. All studied MP phenotypes were significantly higher in APU on day 6 and 8 when compared to HVP. TF activity (using FVIIa, FX and FXa substrate) of washed MP was 619 nM TF (35–393) in APU on day 6 vs. 629 nM TF (38–2443) on day 8. MP associated TF activity correlated with counts of CD142+MP (r=0.76; p=0.02) and was in some individual APU over 10x the median value of HVP MP, 190 nM TF (20–440). The effect of APU MP on intracellular free Ca2+ concentration [Ca2+]i was also studied, using a ratio fluorometry in GT1-7 cells loaded with a Ca2+-sensitive probe FURA-2AM. Washed APU MP induced a rapid MP concentration dependent peak of [Ca2+]i, followed by a sustained slow increase of [Ca2+]i. The activity was significantly higher in day 6 vs. day 8 APU MP (p=0.001) and could be inhibited by EGTA, suggesting that extracellular Ca2+ predominate in APU MP stimulated calcium flux. No significant [Ca2+]i increase was achieved even with 3x concentrated MP from HVP. To test apoptotic activity (TUNEL), washed APU MP were incubated overnight with endothelial cells (HUVEC). About 25% of APU MP induced significant apoptotic activity which correlated with CD154+CD45−CD41a+MP (r=0.81; p=0.002) and CD45+MP (r=0.75; p=0.009) and was higher in day 6 vs. day 8 APU MP vs. HVP MP (p=0.03). In addition, cell cycle analysis showed that APU MP induced G1 arrest. In conclusion, APU MP exhibited various pathophysiological activities in vitro. Some individual units showed a high MP content, and correlations were found between certain MP phenotypes and APU MP activities. The 7-day storage of effectively leukoreduced APU should not have a dramatic impact on a potentially pathogenic MP content. Studies investigating an association of high MP counts in some APU with clinical adverse effects of APU transfusions are warranted. The views of the authors should not be construed as FDA policy.

Relationship between CTLA-4 and CD28 Molecule Expression on T Lymphocytes and Stimulating and Blocking Autoantibodies to the TSH-Receptor in Children with Graves’ Disease

The present study was performed to elucidate the relationship between CTLA-4/CD28 molecules and stimulating (TSAb) and blocking (TBAb) antibodies to the TSH-receptor (TSH-R) in Graves’ disease. CD28 and CD152 (CTLA-4) are glycoprotein molecules which provide a potent costimulatory signal for T-cell activation and proliferation via interactions with their ligands, B7.1/B7.2 molecules, which are present on the surface of antigen-presenting cells. The aim of the study was to estimate the expression of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152), CD28, B7.1 (CD80), and B7.2 (CD86) molecules on peripheral blood cells in patients with Graves’ disease (GD) (n = 55, mean age 15.5 ± 5.1 years) and nontoxic nodular goiter (NTNG) (n = 55, mean age 15.2 ± 4.5 years), in comparison with sex and age-matched healthy control subjects (n = 55, mean age 15.2 ± 3.9 years). The expression of the costimulatory molecules on mononuclear cells was analyzed by three-color flow cytometry using a Coulter EPICS XL cytometer. Detection of TSAb and TBAb to the TSH-R using JPO9 CHO cells in unfractionated serum was measured by a highly sensitive commercial radioimmunoassay. When compared with healthy control subjects and euthyroid patients with GD, untreated patients with GD showed a significant increase of CD152+ (p < 0.001, p < 0.001) and CD28+ (p < 0.01, NS) T lymphocytes, respectively. After 6–12 months of methimazole therapy, the percentage of these cells in the peripheral blood of hyperthyroid patients returned to normal values. In addition, patients with GD showed an increase in the percentage of both B7.1 (3.8%) and B7.2 (18.4%) molecules on activated monocytes, compared to patients with NTNG (0.5% p < 0.05, 2.5% p < 0.01, respectively) and healthy control subjects (0.2% p < 0.05, 0.8% p < 0.003, respectively). In patients with untreated GD there was a statistically significant positive correlation between the expression of CTLA-4 on the surface of peripheral blood T cells and the index of TSAb antibodies (R = 0.54, p < 0.001) as well as a negative correlation with TBAb antibody titer (R = –0.58, p < 0.001). However, no such correlations were noted with regard to CD28 and anti-TPO, anti-TG, and TRAb antibodies. We conclude that changes in the expression of costimulatory molecules on the surface of peripheral blood T cells and their significant relationship with the level of antithyroid antibodies indicate an involvement of these molecules in the pathogenesis of GD.

A differential assay of NK-cell-mediated cytotoxicity in K562 cells revealing three sequential membrane impairment steps using three-color flow-cytometry

Annexin V and propidium iodide (PI) staining is a general technique for detecting apoptosis by flow-cytometry (FCM). The release of 2′,7′-bis-(2-carboxyethyl)-5- (and-6)-carboxyfluorescein (BCECF), a non-lipophilic membrane-impermeable labeling dye, from the cytoplasm of target cells is an indicator of increased membrane permeability. This study aimed to devise a three-color FCM technique involving the BCECF-release parameter in addition to conventional Annexin V and PI staining for the analysis of target K562 cells undergoing cytotoxic/apoptotic processes mediated by natural killer (NK) cells. The results demonstrated the following step-wise process of membrane impairment: (1) initiation of Annexin V staining accompanied by increasing forward scatter (FSC) before BCECF-release, indicating membrane impairment without permeabilization by necrosis; (2) BCECF-release with decreasing FSC before PI influx; and (3) PI staining with the lowest FSC state. Therefore, the early stage of cytotoxicity/apoptosis conventionally defined by the flow-cytometric criteria of Annexin V staining before PI staining could be sub-divided into two stages before and after BCECF-release. Annexin-V staining in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was also initiated without BCECF-release. Although the underlying mechanism of the transition process from stage 1 to stage 2 is still unknown, this FCM technique should be a useful tool for differential assays of target cells regarding the sequential processes of NK-induced cytotoxicity.

Profiles of NK, NKT cell activation and cytokine production following vaccination against hepatitis B

Human natural killer (NK) cells (CD56+ CD3-) represent crucial components of the innate immune system especially against viral infections and because their activation can modulate the outcome of the adaptive immune response. NKT cells (CD56+CD3+), a lymphocyte T population characterized by expression of surface markers of NK cells, are known to be abundant in the liver and their activation could be associated with hepatic injury. Using three-color flow cytometry to measure surface receptors and intracellular cytokines, we have explored early activation signals and cytokine production in NK and NKT cells within a group of hepatitis B vaccinated and non-vaccinated individuals. A specific increase of the CD56bright cell population, the activation receptor CD69 and IFN-gamma, was observed in NK cells following incubation with recombinant HBsAg in responders to vaccination. Comparable results were observed in NKT cells showing an increment of CD69, CD25, IL-2 and IFN-gamma expression in responder subjects. These parameters were statistically diminished in non-responder individuals (p<0.05) in both groups of cells. These results demonstrate a diminished activation of these cells in non-responders to the vaccine, suggesting that NK and NKT cells play an important role in the immune response following hepatitis B vaccination.

Lymphocyte Subpopulations in Middle Ear Effusions: Flow Cytometry Analysis

The aim of this study was to identify lymphocyte subpopulations in middle ear effusions, peripheral blood, and adenoids in children suffering from otitis media with effusion. Tertiary referral center. Thirty-three children (55 ears) undergoing myringotomy for otitis media with effusion. CD3, CD4, CD8, CD19, and natural killer cell populations were investigated in middle ear effusion, peripheral blood, and adenoids using a three-color monoclonal antibody and flow cytometry method for quantitative estimation. T cells (CD3) are dominating lymphocytes in middle ear effusion. Among T lymphocytes, the majority are those of the helper type (CD4). The dominating isoform among CD4 lymphocytes are memory cells (CD4CD45RO); among CD8 lymphocytes, naive cells (CD8CD45RA). The percentage of CD4 cells, CD8 cells, and the CD4/CD8 ratio was significantly higher in middle ear effusions than in blood. The percentage of memory CD4 lymphocytes and naive CD8 lymphocytes was significantly lower in the middle ear effusion. Lymphocyte subsets were compared between 22 pairs of effusions from each patient. The percentage of each type of cell did not differ significantly. The results of this study indicate local regulation of the lymphocyte profile in middle ear effusions and the same phase of immune response in two ears of the same patient.

Detection of minimal residual disease (MRD) in bone marrow (BM) and peripheral blood (PB) in childhood acute lymphoblastic leukemia (ALL) by flow cytometry (FC)

8544 Background: MRD is an important prognostic factor for children with ALL and it provides crucial information on the response to treatment and risk of relapse. However, the frequency which MRD can be monitored is limited due the discomfort and practical difficulties posed by BM aspiration. Since there are few reported studies on the value of MRD in PB, we conducted a prospective study to evaluate and to compare FC MRD measurements in BM with PB and correlate with BM morphology in children with ALL. Methods: 76 BM aspirates and 64 PB samples obtained from 40 Brazilian children with ALL were analysed during days 14(D14) and 28(D28) of remission induction therapy with a three-color FC assay. Data acquisition and analysis were performed using BD-FACSCalibur flow cytometer with Cell-Quest and Paint-a-gate softwares, respectively. Detectable MRD was defined as ≥ 0,01% cells expressing an aberrant immunophenotype(detected at diagnosis) among total events in the sample. BM morphology was analysed at the same time points. Results: in 20/36 (55,6%) BM samples, MRD was detected at D14 and in 18/40 (45%) at D28. MRD was detected in PB in 12/28(42,9%) at D14 and in 11/36(30,6%) at D28. The concordance between MRD in BM and PB was 85,7%(P<.001) at D14 and a significant level of agreement was found in 71% of samples(P<.001) at D28,although MRD levels may differ. In patients in complete morphological remission,44% had detectable MRD at D14 and 38,9% at D28. MRD was not signicantly related to sex,age,WBC count or cell lineage. Conclusions: FC is a sensitive,fast and reproducible technique for detection of MRD in children with ALL. Our findings suggest that PB could be useful in monitoring MRD,particularly in T-lineage. This study reinforces the discrepancy between traditional morphology and MRD,reaffirming its prognostic importance in childhood ALL. No significant financial relationships to disclose.

The Measurement of Dendritic Cells in Umbilical Cord Blood: A Novel Technique Using Small Volumes of Whole Blood

To develop a technique that would permit the identification of dendritic cells (DC) in small volumes of preterm neonatal cord blood in order to investigate neonatal immune response in relation to infection and preterm labor. We used three-color flow cytometry and a fixed quantity of fluorospheres to determine absolute cell numbers. Four red cell lysis techniques, sample dilution, time delay experiments, and a comparison with traditional Ficoll cell separation techniques were performed. Absolute numbers of DC recovered using each technique were calculated and compared. With increasing time delay, there was a statistically significant reduction in the numbers of leukocytes in adult blood. In contrast, there was a significant increase in leukocytes in umbilical cord blood. Sample dilution did not significantly affect the total number of leukocytes or DC. The use of the reagent Optilyse B (Immunotech, Oxford, UK) combined with an additional washing step produced the best discrimination of all populations based on light scatter properties. For the same blood sample, antibody labeling of whole blood resulted in a greater recovery of DC when compared to prior cell purification using a Ficoll density gradient. Using traditional Ficoll cell separation, cell manipulation requires large volumes of blood and leads to cell loss and alteration in phenotype. We have validated a novel method using small volumes of whole blood, diluted if necessary, and using red cell lysis to enable analysis of small volumes of preterm neonatal cord blood. This may permit further analysis of the contribution of the fetal immune response in the development of spontaneous preterm labor and preterm birth due to infection.