Variation of T-Reg and CD 200+ T- Lymphocytes After in Vitro Treatment with Active Drugs against CLL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1239-1239
Author(s):  
Nunziatina Parrinello ◽  
Piera La Cava ◽  
Daniele Tibullo ◽  
Cesarina Giallongo ◽  
Annalisa Chiarenza ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Normal Subjects ◽  
Purine Analogues ◽  
Peripheral Blood Mononuclear ◽  
Regulatory Systems ◽  
Haemolytic Anemia ◽  
In Vitro Treatment

Abstract Abstract 1239 Poster Board I-261 Background Purine analogues, in particular fludarabine, are considered the gold standard of treatment of CLL. However, fludarabine therapy is sometimes complicated by autoimmune haemolytic anemia (AHA). The mechanism of this side effect is not clear but it is conceivable that a fludarabine-induced suppression of some regulatory systems, including T-reg, is responsible for this phenomenon. In addition, we have observed that patients affected by autoimmune diseases such as AHA or PTI have a reduced number of T lymphocytes bearing the CD200 antigen that is considered a tolerogenic molecule. In this perspective, we evaluated the variation of T-reg and CD200+ T lymphocytes induced by incubating in vitro peripheral blood mononuclear cells (PBMC) of CLL patients and normal subjects with purine analogues and other drugs active against CLL. Method PBMC obtained from patients with chronic lymphocytic leukaemia (CLL) (n=9) and from normal adult (n=6) were isolated by density gradient and cultured in RPMI supplemented with 10% FBS and 1% of penicillin streptomicyn. Cells were then incubated for 24 hours with drugs at two concentrations: bendamustine (1 and 50 μg/ml) campath (1 and 5 μg/ml) prednisone ( 1 and 10 nM), fludarabine (0,25 and 10 μg/ml) pentostatin (3 and 60 μg/ml). The cytotoxicity was evalutated after 24 hours by trypan Blue and flow cytometry. T-reg cells were identified as CD4+/CD25+/FoxP3+ T cells and expressed as a percentage of the CD4+T-cell population. Results Although all of these drugs induced lymphocytes cytotoxicity, fludarabine, prednisone, and campath reduced also the percentage of T-reg and CD200+ T –lymphocytes, while bendamustin and especially pentostatin induced the same cytotoxicity but spared T-reg populations and CD200+ T-lymphocytes. Table I indicates results obtained with the highest concentration of drugs. In conclusion, pentostatin and bendamustine seems to be active drugs against CLL and their usage shouldn't be complicated by autoimmune phenomena. Disclosures No relevant conflicts of interest to declare.

scholarly journals In vitro regulation of immunoglobulin synthesis after human marrow transplantation. II. Deficient T and non-T lymphocyte function within 3- 4 months of allogeneic, syngeneic, or autologous marrow grafting for hematologic malignancy

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 844-850 ◽  
Author(s):  
RP Witherspoon ◽  
LG Lum ◽  
R Storb ◽  
ED Thomas
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Hematologic Malignancy ◽  
Plaque Assay ◽  
Pokeweed Mitogen ◽  
Lymphocyte Function ◽  
Peripheral Blood Mononuclear ◽  
Immunoglobulin Secretion

Abstract Immunoglobulin secretion was studied in 37 patients between 19 and 106 days after allogeneic HLA-identical (30 patients), allogeneic one HLA- haplotype-identical (three patients), syngeneic (three patients), or autologous (one patient) marrow grafting. E rosette-positive (T) and E rosette-negative (non-T) peripheral blood mononuclear cells were cocultured with pokeweed mitogen for 6 days. Polyvalent immunoglobulin secretion was determined by counting plaque forming cells in a reverse hemolytic plaque assay. The number of antibody secreting cells in cocultures of autologous T and non-T lymphocytes was low in 40 of 44 tests conducted on samples from the 37 patients. Mononuclear or non-T cells from 38 of 40 tests failed to produce antibody when cultured with normal helper T cells. T cells from 23 of 37 tests failed to help normal non-T cells secrete antibody. T lymphocytes from 23 of 41 tests suppressed antibody production greater than 80% by normal T and non-T cells. The suppressor cells were radiosensitive in 17 of the 25 tests. The abnormal function of lymphocyte subpopulations in patients during the first 3 mo after syngeneic, allogeneic or autologous marrow grafting was similar regardless of the type of graft or the presence of acute graft versus host disease.

Characterization of Receptors Involved in Heparin Antibody Complex Mediated Induction of Tissue Factor Expression in Monocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 224-224 ◽  
Author(s):  
Sam Glover ◽  
Nigel S. Key ◽  
Gowthami M Arepally ◽  
Nigel Mackman ◽  
Raj S. Kasthuri
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Drug Induced ◽  
Peripheral Blood Mononuclear ◽  
Platelet Factor ◽  
Antibody Complex

Abstract Abstract 224 Introduction: Heparin-induced thrombocytopenia (HIT) is a major cause of drug-induced thrombocytopenia and occurs in 1–5% of individuals exposed to heparin. Paradoxically, 30–50% of individuals with HIT develop thrombosis. The mechanism of thrombosis in HIT is poorly understood. We recently reported that HIT antibody complexes induce tissue factor (TF) expression in monocytes and result in the release of TF-positive microparticles (MPs). The mechanism by which HIT antibody complexes induce monocyte TF has not been established. The objective of this study is to characterize the receptors involved in HIT antibody complex mediated induction of TF expression in monocytes. As HIT antibody complex mediated activation of platelets is dependent on the FcgRIIA receptor, we evaluated the role of the FcgRII receptor in the induction of monocyte TF by HIT antibody complexes. We also evaluated the role of toll like receptor-4 (TLR4) and the platelet factor 4 (PF4) chemokine receptor CXCR3 in this process. Methods: The combination of heparin, PF4 and the murine monoclonal PF4/heparin-specific antibody KKO has been shown to cause activation of platelets and monocytes, and mimic HIT in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were pre-incubated for 30 min at 37°C with an inhibitory antibody to the FcgRII receptor (IV.3); anti-CXCR2, 3, or 4 antibodies; anti-TLR4 antibody; or mouse-IgG (mIgG) control. Following pre-incubation with antibodies for 30 minutes, heparin (1U/mL), PF4 (10μg/mL), and KKO (100μg/mL) – together referred to as the HIT antibody complex – were added. Heat-aggregated mIgG and LPS were used as positive controls for the FcgRII and TLR4 receptors, respectively. Following a 6-hour incubation, PBMCs were pelleted by centrifugation and MPs were isolated from the supernatant. The procoagulant activity (PCA) of PBMCs and MPs was measured using clotting assays performed in the presence of the anti-TF antibody HTF-1 or control antibody. TF dependent PCA was calculated by reference to a standard curve generated using relipidated recombinant TF. Results: Incubation of PBMCs with heat aggregated mIgG for 6 hours resulted in significant induction of cellular TF (345 +/− 36 pg/106 cells) which was blocked by 30 min pre-incubation with the antibody IV.3 (146 +/− 17 pg/106 cells, N=3, p<.003). However, pre-incubation with IV.3 had no significant effect on TF induction (140 +/− 5 pg/106 cells) associated with the HIT antibody complex when compared to control mIgG (110 +/− 18 pg/106 cells, N=3, p<0.11). PBMCs incubated with HIT antibody complexes in the presence of a TLR-4 antibody showed less TF activity (52 +/− 4 pg/106 cells) compared to control mIgG (80 +/− 10 pg/106 cells N=3, p<0.025). A similar, partial inhibition of TF activity was also observed in PBMCs incubated with LPS in the presence of an anti-TLR4 antibody (121 +/− 3 pg/106) compared with a control antibody (89 +/− 2 pg/106, N=3, p<.0013). Experiments with a more effective inhibitor of TLR4 are in progress. PBMCs incubated with the HIT antibody complexes in the presence of an anti-CXCR3 antibody showed less TF activity (36 +/− 7 pg/mL) compared to control mIgG (118 +/− 15 pg/106 cells, N=3, p<0.004). Antibodies against CXCR2 and CXCR4 did not have any significant effect on TF induction. Measurement of MP TF activity mirrored the results described above. Using flow cytometry and an anti-CXCR3 antibody labeled with FITC, we found that 5% (± 0.5%) of monocytes expressed CXCR3 (N=3), which is consistent with the reported literature. Conclusions: These data suggest that induction of TF in monocytes by HIT antibody complexes is not mediated by the FcgRII receptor. This is contrary to the mechanism of platelet activation by these antibody complexes, which is an FcgRIIa dependent process. We found that TLR4 plays a role in HIT antibody complex mediated induction of TF in monocytes and blocking TLR4 led to a 30% decrease in TF activity. On the other hand, CXCR3 appeared to play a more significant role with blockade of CXCR3 leading to a 70% decrease in TF activity. Further characterization of the role of these receptors in HIT antibody complex mediated induction of TF expression in monocytes is required. We speculate that the extent of CXCR3 and TLR4 expression in monocytes may influence the susceptibility to developing thrombotic complications in HIT. Disclosures: No relevant conflicts of interest to declare.

CD70 Expression on HTLV-1 Infected T Cells of Carriers and ATL Patients and Its Clinical Significance

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1730-1730
Author(s):  
Izumi Masamoto ◽  
Sawako Horai ◽  
Tomohiro Kozako ◽  
Makoto Yoshimitsu ◽  
Junko Niimoto ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Surveillance Systems ◽  
Peripheral Blood Mononuclear ◽  
Tax Protein ◽  
Infected Cells

Abstract Abstract 1730 Human T-lymphotropic virus type-1(HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL). HTLV-1 infected T cell growth or leukemogenesis in ATL is controlled by various host immune surveillance systems. Among them, CD70 on HTLV-1 infected T cells coupled with CD27 on virus specific cytotoxic T cells has been suggested to play an important role in ATL leukemogenesis. The CD70 molecule is the only known ligand for CD27, a member of the tumor necrosis factor (TNF) receptor superfamily 7. This negative immunoregulatory pathway downregulates cytotoxic T lymphocyte activity against CD70-expressing virus infected cells. In the present study, we examined CD70 expression on primary lymphocytes of HTLV-1 carriers and ATL patients, its relationship with HTLV-1 Tax protein expression, and the effect on CTL induction. CD70 expression was higher on peripheral blood mononuclear cells (PBMCs) of HTLV-1 infected carriers compared with healthy donors (p = 0.021, n = 21, Mann-Whitney U test), and higher in ATL patients compared to carriers (p = 0.045, n = 38, Mann-Whitney U test). CD70 expression may be observed in CD4 T cells, as well as B cells, but not in CD8 T cells or monocytes. CD70 expression in CD4 T cells is related to HTLV-1 infection, because of increased detection of HTLV-1 Tax protein during over night culture of CD70-expressing cells. Experiments using an ATL cell line, in which Tax expression is inducible by doxycycline stimulation, demonstrated enhanced CD70 expression when Tax protein was induced in HTLV-1 infected cells. Anti-CD70 antibody enhanced CD107a mobilization, a marker of recent cytotoxic degranulation, in HTLV-1 Tax specific CTLs in PBMCs from asymptomatic carriers in vitro, suggesting that the CD70/CD27 pathway plays an important role in the immune response to HTLV-1 infection in carriers, as well as ATL patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lymphocyte Apoptosis during Classical Swine Fever: Implication of Activation-Induced Cell Death

1998 ◽  
Vol 72 (3) ◽  
pp. 1853-1861 ◽  
Author(s):  
Artur Summerfield ◽  
Sonja M. Knötig ◽  
Kenneth C. McCullough
Keyword(s):  
Cell Death ◽  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Classical Swine Fever ◽  
Observation Time ◽  
Peripheral Blood Mononuclear ◽  
Activation Induced Cell Death ◽  
Leukocyte Depletion ◽  
First Time

ABSTRACT Infection of pigs with classical swine fever virus (CSFV), a member of the Flaviviridae family, causes a severe leukopenia, particularly notable with the lymphocytes. The goal of this study was to analyze mechanisms behind this CSFV-induced lymphopenia. To this end, the kinetics of leukocyte depletion, the appearance of apoptotic cells, and virus infection of leukocytes after infection of pigs with the virulent CSFV strain Brescia were analyzed. Depletion of B and T lymphocytes was noted as early as 1 day postinfection (p.i.). Circulating viable lymphocytes with reduced mitochondrial transmembrane potential—a particular early marker for apoptosis—were also detectable as early as 1 day p.i. When isolated peripheral blood mononuclear cells were cultured for 6 h, significantly more sub-G1 cells with reduced DNA content were detected among the lymphocytes from CSFV-infected animals, again as early as 1 to 3 days p.i. The first time virus was first found in the plasma, as well as infection of leukocytes, was 3 days p.i. However, throughout the observation time of 1 week, <3% of the circulating leukocytes and no lymphocytes contained virus or viral antigen. Further analysis of the T lymphocytes from infected animals demonstrated an increase in CD49d, major histocompatibility complex class II, and Fas expression. An increased susceptibility to apoptosis in vitro was also observed, particularly after addition of concanavalin A as well as apoptosis-inducing anti-Fas antibody to the cultures. Taken together, these results imply that activation-induced programmed cell death was the mechanism behind lymphopenia during classical swine fever.

scholarly journals Polyfunctional T Lymphocytes Are in the Peripheral Blood of Donors Naturally Immune to Coccidioidomycosis and Are Not Induced by Dendritic Cells

2009 ◽  
Vol 78 (1) ◽  
pp. 309-315 ◽  
Author(s):  
Lance Nesbit ◽  
Suzanne M. Johnson ◽  
Demosthenes Pappagianis ◽  
Neil M. Ampel
Keyword(s):  
Dendritic Cells ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Interleukin 2 ◽  
High Expression ◽  
Peripheral Blood Mononuclear ◽  
Necrosis Factor Alpha ◽  
Ifn Γ

ABSTRACT Coccidioidomycosis is a fungal infection endemic in the southwestern United States that is increasing in incidence. While cellular immunity correlates with protection from clinical illness, the precise elements of that response are undefined. Using the coccidioidal antigen preparation T27K and multiparametric flow cytometry, the in vitro frequency of polyfunctional T lymphocytes in the peripheral blood of naturally immune healthy donors and those who were nonimmune was determined. Polyfunctional CD4 lymphocytes, defined as producing intracellular interleukin 2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor alpha simultaneously, had a frequency of 137 per 400,000 events among peripheral blood mononuclear cells (PBMC) of immune donors compared to 11 per 400,000 PBMC from nonimmune donors (P = 0.03). When monocyte-derived mature dendritic cells pulsed with T27K (mDCT27K) were used for antigen presentation, the frequency of polyfunctional CD4 T lymphocytes did not significantly increase for either group, although mDCT27K did significantly increase the concentrations of IL-2 and IFN-γ released by PBMC from nonimmune donors (P = 0.02). After in vitro stimulation with T27K, polyfunctional CD4 and CD8 lymphocytes of PBMC from immune donors had a mixture of low- and high-expression CCR7 cells, suggesting both effector and central memory, compared with predominantly high-expression CCR7 cells when PBMC were incubated with the mitogen phytohemagglutinin (P = 0.03). These data demonstrate the presence of polyfunctional T lymphocytes in the peripheral blood of individuals with coccidioidal immunity and suggest a model for the in vitro testing of vaccine candidates for coccidioidomycosis.

scholarly journals In vitro regulation of immunoglobulin synthesis after human marrow transplantation. II. Deficient T and non-T lymphocyte function within 3- 4 months of allogeneic, syngeneic, or autologous marrow grafting for hematologic malignancy

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 844-850 ◽  
Author(s):  
RP Witherspoon ◽  
LG Lum ◽  
R Storb ◽  
ED Thomas
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Hematologic Malignancy ◽  
Plaque Assay ◽  
Pokeweed Mitogen ◽  
Lymphocyte Function ◽  
Peripheral Blood Mononuclear ◽  
Immunoglobulin Secretion

Immunoglobulin secretion was studied in 37 patients between 19 and 106 days after allogeneic HLA-identical (30 patients), allogeneic one HLA- haplotype-identical (three patients), syngeneic (three patients), or autologous (one patient) marrow grafting. E rosette-positive (T) and E rosette-negative (non-T) peripheral blood mononuclear cells were cocultured with pokeweed mitogen for 6 days. Polyvalent immunoglobulin secretion was determined by counting plaque forming cells in a reverse hemolytic plaque assay. The number of antibody secreting cells in cocultures of autologous T and non-T lymphocytes was low in 40 of 44 tests conducted on samples from the 37 patients. Mononuclear or non-T cells from 38 of 40 tests failed to produce antibody when cultured with normal helper T cells. T cells from 23 of 37 tests failed to help normal non-T cells secrete antibody. T lymphocytes from 23 of 41 tests suppressed antibody production greater than 80% by normal T and non-T cells. The suppressor cells were radiosensitive in 17 of the 25 tests. The abnormal function of lymphocyte subpopulations in patients during the first 3 mo after syngeneic, allogeneic or autologous marrow grafting was similar regardless of the type of graft or the presence of acute graft versus host disease.

Differential Expression of Adhesion Molecules and Chemokine Receptors Confers Increased Migrative Capacity to ZAP-70-Positive Subclones in CLL Primary Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3889-3889
Author(s):  
Marta Crespo ◽  
Cecilia del Carmen Carpio ◽  
Eva Calpe ◽  
Pau Abrisqueta ◽  
Carlos Palacio ◽  
...  
Keyword(s):  
Differential Expression ◽  
Adhesion Molecules ◽  
Chemokine Receptors ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Peripheral Blood Mononuclear ◽  
Expression Levels

Abstract Abstract 3889 Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disease characterized by the accumulation and proliferation of mature B-lymphocytes. CLL clinical course is extremely heterogeneous; patients with worse prognosis can be identified by the presence of high ZAP-70 expression. Increasing evidence indicates that the microenvironment plays a critical role providing survival and proliferative signals to CLL cells. In this sense, ZAP-70 protein expression has been related to increased capability of the cells to respond to several survival and migration signals provided by the cellular microenvironment through chemokines and cell-to-cell direct contact. We aimed to analyze the expression levels of several adhesion molecules and chemokine receptors potentially involved in CLL pathogenesis and progression in subclones of CLL cells with high or low ZAP-70 expression within the same patient. For this we obtained peripheral blood mononuclear cells from 40 patients diagnosed with CLL at our institution after informed consent. We then performed a flow cytometry analysis with 7 parameters that allowed for the measurement of the expression of different molecules in CD19+/CD5+ CLL cells with high or low ZAP-70 expression. The expression level of ZAP-70 protein in CD3+ T lymphocytes was used to set the threshold between CLL cells with low or high ZAP-70 expression (Figure 1). Using this approach we analyzed the differential expression of CCR7, CXCR4, CXCR5, CD44, CD49d and CD62L. Interestingly, we found that the expression levels of all the adhesion molecules and cytokine receptors analyzed were significantly higher in those CLL subclones with high ZAP-70 expression compared to CLL cells with low ZAP-70 expression within the same patient (Table 1), suggesting that the relationship with the microenvironment is not uniform across the CLL clone, but those CLL cells with higher expression of ZAP-70 have increased potential to receive signals from other cells. In order to analyze if this could translate into an increased capacity of the CLL cells with high ZAP-70 expression to migrate towards chemokines, we performed an in vitro transmigration assay across bare polycarbonate filters using primary CLL cells from 7 of the patients. We allowed the cells to migrate for 6 hours towards a media containing 1 μg/ml of CCL21 (the ligand of CCR7) and proceed to measure the percentage of CD19+/CD5+ CLL cells with ZAP-70 expression among those cells that had transmigrated and to compare it with the percentage of ZAP-70-positive cells within the ones that did not migrate. Of note, for all the cases analyzed, we observed that the percentage of ZAP-70-positive cells was significantly higher in the cells that had migrated compared to the cells present in the upper chamber (p=0.018) indicating that ZAP-70-positive CLL cells have an enhanced ability to respond to and to migrate towards CCL21. In conclusion, the differential expression of adhesion molecules and chemokine receptors in primary CLL cells with higher expression of ZAP-70 can influence their relationship with the microenvironment and confers them a higher migratory potential towards CCL21.Figure 1:Figure 1:. Table 1:MFI: mean fluorescence intensity SEM: standard error of the meanMean MFI ± SEM Low ZAP-70 CLL cellsMean MFI ± SEM High ZAP-70 CLL cellsPaired samples t-test p valueCCR7163.56 ± 11.3178.20 ± 13.40.007CXCR4263.01 ± 36308.95 ± 45.30.004CXCR5604.02 ± 62.6652.89 ± 67.50.001CD441081.11 ± 76.91210.8 ± 82.5>0.001CD49d58.43 ± 16.169.6 ± 17.6>0.001CD62L33.81 ± 8.947.63 ± 12.1>0.001 Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document