The Immune Regulatory Functions Of Circulating CD5+ B Cells Are Impaired In Adult Patients With Primary Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3526-3526
Author(s):  
Fanli Hua ◽  
Feng Li ◽  
Shanhua Zou ◽  
Weiguang Wang ◽  
Yunfeng Cheng
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Healthy Subjects ◽  
Immune Thrombocytopenia ◽  
Ifn Γ ◽  
Regulatory Functions

Abstract Background and Objective Immune thrombocytopenia (ITP) is a typical autoimmune-mediated bleeding disorder in which isolated thrombocytopenia is resulted from increased platelet clearance and/or impaired thrombocytogenesis. In addition to producing antibodies and acting as antigen presenting cells, B cells have been proven to play an important role in regulating immune response mainly via production of interleukin 10 (IL-10). CD5+ B cells are able to produce IL-10, and CD5 is deemed as a hallmark of regulatory B cells. We have previously demonstrated insufficient IL-10 secretion by CD5+ B cells in patients with ITP. The objective of the current study was to investigate the alteration in regulatory functions of peripheral blood CD5+B cells in ITP patients. Methods Peripheral blood CD5+ B cells, CD5- B cells and CD4+ T cells were magnetically isolated from 7 adult patients with newly diagnosed ITP and 10 healthy controls. CD4+ T cells were labeled with CFSE (5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester) before cultured in RPMI1640 in the presence of CD3mAb/CD3mAb/IL-2 for 72 hours with or without CD5+/CD5- B cells at gradient B/T ratios. The proliferation of CD4+ T cells was assessed by the dilution of CFSE expression on a flow cytometry. After 72-hour culture, cells were stained with APC-CD4/FITC-Annexin-V/PI to evaluate the apoptosis of CD4+T cells. The concentrations of IL-4 and IFN-γ in cell culture supernatants were determined by ELISA. Results Purified CD5+ B cells from healthy subjects were capable of suppressing the proliferation of CD4+ T cells in a dose-dependent manner when co-cultured for 72 hours. The percentage of CD4+ T cells that underwent proliferation showed a statistically significant reduction (72.8 ± 9.4% vs 61.6 ± 12.8%; p = 0.039) in the presence of CD5+ B cells at 1:3 B/T ratio. With the B/T ratio increased to 1:1, the proportion of proliferating CD4+ T cells decreased to 43.7 ± 16.4% (p < 0.001). The apoptosis of CD4+ T cells was promoted by CD5+ B cells as the percentage of apoptotic CD4+ T cells was increased when co-cultured with CD5+ B cells at 1:1 B/T ratio (3.75 ± 0.82% vs 6.03 ± 2.51%; p = 0.014). In contrast, CD5- B cells did not suppress the proliferation of CD4+ T cells, quite contrary, the proportion of proliferating CD4+ T cells was slightly increased, though not statistically significant, when co-cultured at 1:1 with CD5- B cells for 72 hours as compared with CD4+ T cell cultured alone (72.8 ± 9.4% vs 78.9 ± 8.5%; p = 0.145). No effect of CD5- B cells on the apoptosis of CD4+T cells was observed. In ITP patients, CD5+ B cells showed compromised regulatory functions as the proliferation of CD4+ T cells was not altered by CD5+ B cells until the B/T ratio increased to 3:1 (1:1 B/T: 74.6 ± 12.5% vs 72.1 ± 16.2%, p = 0.752; 3:1 B/T: 74.6 ± 12.5% vs 60.7 ± 10.3%, p = 0.042, respectively). The ability of CD5+ B cells to promote the apoptosis of CD4+ T cells was also impaired in ITP patients: no difference in the percentage of apoptotic CD4+ T cells was found when CD5+ B cells (1:1 ratio of B to T) were added to the T cell culture system (2.97 ± 1.07% vs 3.24 ± 1.45%; p = 0.699). We also evaluated the ability of CD5+ B cells to suppress the secretion of IFN-γ, one of the Th1-type cytokines. In healthy subjects, the IFN-γ secretion by CD4+ T cells was decreased by CD5+ B cells at a minimal B/T ratio of 1:5 (2427.99 ± 632.62 pg/mL vs 1734.41 ± 507.16 pg/mL; p = 0.014) and dramatically dropped to 395.22 ± 136.54 pg/mL when the ratio of B/T increased to 1:1 (p < 0.001). The secretion of IL-4, a representative cytokine of Th2 subset, was not influenced by CD5+ B cells. CD5+ B cells from ITP patients showed no capacity for suppressing the IFN-γ secretion until the ratio of B/T increased as high as to 1:1 (3060.02 ± 493.98 pg/mL vs 2394.57 ± 417.42 pg/mL; p = 0.019) and, even under the condition of 5:1 B/T ratio, CD4+ T cells from ITP patients retained about 30% of the ability of IFN-γ secretion (1067.37 ± 499.70 pg/mL; p < 0.001 ). Conclusions CD5+ B cells from normal controls are competent for inhibiting the proliferation of CD4+ T cells and suppressing the secretion of IFN-γ by CD4+ T cells, suggesting potent immune regulatory functions of CD5+ B cells. While in patients with ITP, CD5+ B cells are severely functionally impaired, indicating that the immune regulatory dysfunctions in CD5+ B cells may participate in the pathogenesis of ITP. Disclosures: No relevant conflicts of interest to declare.

CD8+CD28− T cells: key cytotoxic players impacting disease pathogenesis in chronic HBV infection

2019 ◽  
Vol 133 (17) ◽  
pp. 1917-1934
Author(s):  
Madhuparna Nandi ◽  
Sourina Pal ◽  
Sumantra Ghosh ◽  
Bidhan Chandra Chakraborty ◽  
Debangana Dey ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Hepatitis ◽  
T Cell ◽  
Hepatitis B ◽  
Chronic Hepatitis B ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Hepatitis B E Antigen ◽  
Tnf Α ◽  
Ifn Γ

Abstract During chronic hepatitis B (CHB), CD8+ T cells down-regulate CD28, the primary co-stimulation molecule for T-cell activation. Diverse functional attributes of CD8+CD28− T cells are suggested in various disease contexts. The present study aimed to characterize CD8+CD28− T cells in different phases of chronic Hepatitis B virus (HBV) infection (CHI)- Immune-tolerance (IT), Hepatitis B e-antigen-positive CHB (EP-CHB), Inactive carriers (IC) and Hepatitis B e-antigen-negative CHB (EN-CHB), to appraise their contribution in HBV-related disease pathophysiology. Flow cytometry analysis of T cells in peripheral blood of study subjects revealed enhanced CD8+CD28− T-cell accumulation in EP-/EN-CHB, compared with IT/IC and they expanded equivalently in HBV-specific and non-specific CD8+ T-cell compartments. Profound increase in CD8+CD28− T cells expressing perforin/granzyme-B/CD57/IFN-γ/TNF-α and markers of terminal differentiation were observed exclusively in EP-/EN-CHB. Further, activation with anti-NKG2D resulted in heightened IFN-γ/TNF-α production selectively from CD8+CD28− T cells, suggesting NKG2D-mediated alternative co-stimulation. CD8+CD28− T cells sorted from CHB patients induced enhanced apoptosis of peripheral blood mononuclear cells (PBMC), including CD4+ T cells. However, NKG2D-ligand (major histocompatibility complex class I chain-related molecule A/B (MICA/B)) was preferentially expressed by HBV-specific CD4+ T cells of CHB patients, making these cells a potential target to NKG2D-dependent CD8+CD28− T-cell killing. Both CD28+ and CD28− T cells in CHB expressed CXCR3 at similar levels and thus capable of homing to the liver. A positive correlation was seen between CD8+CD28− T-cell frequency and serum-alanine transaminase (ALT) levels and CHB-derived CD8+CD28− T cells caused pronounced cell death in HBV-transfected Huh7 cells. Immunofluorescence staining identified greater intrahepatic incidence of CD8+CD28− T cells but decline in CD4+ T cells in CHB than IC. Collectively, CD8+CD28− T cells demonstrated differential distribution and phenotypic/functional skewing in different CHI phases and contribute to disease progression by Perforin-Granzyme- or IFN-γ-TNF-α-mediated cytotoxicity while restraining antiviral immunity through NKG2D-dependent HBV-specific CD4+ T-cell depletion.

Cytokine Production Profile of CD4+ T Cells From Patients with Immune Thrombocytopenia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4460-4460
Author(s):  
Shanhua Zou ◽  
Feng Li ◽  
Weiguang Wang ◽  
Yunfeng Cheng
Keyword(s):  
T Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Cd4 T Cells ◽  
Healthy Subjects ◽  
Immune Thrombocytopenia ◽  
Plasma Concentrations ◽  
Platelet Destruction ◽  
Expression Levels ◽  
Ifn Γ

Abstract Abstract 4460 Introduction To balance self-tolerance and immunity, the immune system depends on both activation mechanisms and down regulatory mechanisms. CD4+ T cells are important for the regulation of immune responses and they exert their effects through the secretion of cytokines. Immune thrombocytopenia(ITP) is a common hematologic disorder manifested by immune-mediated platelet destruction. ITP in adults often has a persistent course and frequently requires treatment to prevent bleeding. The mechanism of ITP has historically been attributed to platelet autoantibody production and the resultant platelet destruction. However, more recent evidence suggests that cell immunity pathogenesis plays an important role in ITP. Methods Real-time PCR assays was used for the quantification of mRNA of cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, IL-17, IFN-γ, TNF-α and TGF-β in CD4+ T cells from adult ITP patients and healthy subjects. The mRNA of T-bet, GATA-3, RORγt, and FOXP3 were also analyzed using Real-time PCR. The plasma concentrations of IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, IFN-γ, TNF and IL-17 were determined by enzyme-linked immunosorbent assay at the same time. Results The plasma concentrations of IL-2, IL-17 and IFN-γ in CD4+T cells were increased, while levels of IL-4, IL-6, IL-10 and TGF-β were decreased, comparing adult patients with ITP with healthy subjects. Patients with ITP presented significant increased mRNA expression levels on IL-2,IL-17,T-bet and RORγt. Conclusions In adult ITP patients, the expression levels of IL-2 and IL-17 were up-regulated, expression levels of TGF-β and IL-10 were down-regulated, suggesting that ITP is a Th1 and Th17 predominant disease although the precise mechanisms await further study. Disclosures: No relevant conflicts of interest to declare.

The Comparison of the Cytobiological Role of Bone Marrow CD4+ T Cells in Aplastic Anemia Patients and Healthy Subjects.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3758-3758
Author(s):  
Miao Zheng ◽  
Wenli Liu ◽  
Jianfeng Zhou ◽  
Hanying Sun ◽  
Yicheng Zhang
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Healthy Subjects ◽  
Culture Supernatant ◽  
Normal Group ◽  
Cd4 T Cell ◽  
Cell Culture Supernatant ◽  
Cord Blood Cd34

Abstract Recent studies indicate that immune-associated aplastic anemia (AA) resembles such autoimmune diseases as insulin-dependent diabetes and chronic autoimmune thyroiditis that belong to organ-specific autoimmune diseases. Many independent investigation groups have successfully isolated the pathopoiesis-associated T cell clone causing hematopoiesis failure with a CD4 phenotype from AA patients peripheral blood and bone marrow (BM). In the current study, BM CD4+T cells were isolated from both AA patients and healthy subjects with immunomagnetic beads sorting and were compared with regard to proliferation capability, apoptosis features and the impacts of their secreted cytokines on hematopoiesis stem/progenitor cells. With the improved MTT method, BM CD4+T cells of AA group presented 1.6 times more enhanced reproductive activity than those of normal group. Induced by high concentrational CD3 monoclonal antibody for 18h, evident apoptosis cells could be seen under the electron microscope in both normal group and AA group. Quantified by flow cytometry using Annexin-V FITC/PI dual staining method, apoptosis rates in the early and advanced stages of AA group were more than those of normal group (P&lt;0.01). Apoptosis rate in early stage: normal group(7.03±0.86)%, AA group(16.11±1.37)%; apoptosis rate in advanced stage: normal group(2.07±0.42)%, AA group(8.05±0.36)%. The influence of BM CD4+T cell culture supernatant on CFU-GM formation of cord blood CD34+ hematopoiesis stem/progenitor cells: the CFU-GM count in AA group was: (74.50±9.50)/104 cells; the CFU-GM count in normal group was: (124.25±19.80)/104 cells, observing under an inverted microscope. A significant difference was present in the comparison between the two groups (P&lt;0.01). CyclinD3 mRNA and protein expression levels of cord blood CD34+ cells were both down-regulated induced by BM CD4+ T cell culture supernatant of AA patients. These results indicate a strong likelihood of an abnormally proliferative and activated state of BM CD4+T cells among AA patients and its intimate correlation with AA hematopoiesis failure by secreting some soluble humoral agents that are able to inhibit CyclinD3 and consequently suppress hematopoietic stem cell in proliferation.

Dysfunctional Tregs with Absence of Cytokine-Secreting CD4+ T-Cell Subsets and Marked Expansion of Th1 Cells Is a Hallmark of Idiopathic Aplastic Anaemia (AA)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2233-2233
Author(s):  
Shahram Kordasti ◽  
Judith C. W. Marsh ◽  
Pilar Perez Abellan ◽  
Sufyan Alkhan ◽  
Janet Hayden ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cd4 T Cells ◽  
Cytokine Secretion ◽  
T Cell Subsets ◽  
Cd4 T Cell ◽  
Th1 Cells ◽  
Healthy Controls ◽  
Ifn Γ

Abstract Abstract 2233 Introduction: Autoimmunity is an important contributor in the aetiology of AA. Although the expansion of oligoclonal CD8+ T-cells and their correlation with response to immunosuppressive therapy (IST) has been reported previously, the role of CD4+ in the pathogenesis is not elucidated. The focus of this study was to investigate the role of different CD4+ T-cell subsets, including regulatory T-cells (Tregs) and T helpers (Th1, Th2 and Th17) in the pathobiology of idiopathic AA. Patients and Methods: The percentage and absolute numbers of CD4+ and CD8+ T-cell subsets, NK & B cells and dendritic cells (DCs) in peripheral blood were assessed in 42 patients with idiopathic AA prior to any IST and 8 healthy age matched controls. T-cells were stimulated first and stained intracellularly for IFN-γ and TNF-a (Th1), IL-4 (Th2) and IL-17 (Th17). Serum levels of 30 cytokines were measured by 30 Plex bead analysis (Luminex). NK cells were defined as CD3– CD56+. B cells were defined as CD3-CD19+. CD3+ CD4+.T-cell subsets were defined as CD45RO–CD27+ naïve, CD45RO+ CD27+ CD62L+ central memory, CD45RO+ CD27+ CD62L– effector memory, CD45RO+CD27– effectors and CD45RO– CD27– terminal effectors. DCs were defined based on their BDCA 1,2, 3 & CD16 expression. CD4 Tregs were defined as CD3+CD4+ CD25high CD27+Foxp3+. Treg subsets were defined as (1) CD45RA+CD25lo resting Tregs, (2) CD45RA-CD25hi activated Tregs, and (3) cytokine-secreting CD45RA-CD25lo non-Tregs1. Treg function was evaluated by cytokine secretion of T effector cells (Te) with and without Tregs. IFN-γ secreting CD4+ T-cells (Th1) were enriched by magnetic beads followed by FACS sorting. The clonality of Th1 cells was evaluated based on the diversity of T-cell receptors by spectratyping as well as sequencing. Transcription factor expression was measured by qPCR. Results: There were no significant differences in the number or percentage of different CD8 T-cells compared to healthy controls. Surprisingly, despite a borderline decrease in the absolute number of naïve (p=0.19) and central memory (p=0.20) CD4+T-cells the number and percentage of Tregs were no different from healthy controls (1.36×107/L v 1.34×107/L, p=0.57). Although the ratio of Tregs to CD4+ T-effectors (Te) was higher than in healthy controls, the difference was not significant (0.49 v 0.12, p=0.86). The absolute numbers and percentages of Th1 cells and TNF-α + CD4+ T-cells were significantly higher in AA patients compared to healthy controls (4.2 × 107/L v 0.9 × 107/L & 2.44 × 108 v 1.26 × 108(p=0.001, p=0.004)). The diversity of T-cell receptor on Th1 cells was significantly lower compared to healthy age matched controls (on average 21 & 52 peaks). Amongst AA patients, the numbers of Th2, Th17, NK and B cells were not significantly different from healthy controls, whereas the absolute numbers of all DCs were reduced(p<0.01). The serum levels of proliferative cytokines, EGF (p=0.01), HGF (p=0.01), VEGF (p=0.01) and pro-inflammatory cytokines IL-13 (p=0.02), IL-8 (p<0.001) were significantly higher in AA patients. The percentage of cytokine secreting CD4+ CD25+ T-cells was markedly decreased in AA patients and the activated Treg subsets were predominantly of CD45RA+ phenotype, which was significantly different from healthy controls. Sorted Tregs from AA patients were unable to suppress cytokine secretion by Te cells in a 1:1 co-culture. However, IL-2, IFN-γ and TNF-α secretion of Te from AA patients was suppressible by allogeneic Tregs from healthy controls (on average 11 time suppression), whereas Tregs from AA patients were unable to suppress healthy Te cells. However, dysfunctional Tregs were not associated with abnormality of transcription factors, as judged by the levels of STAT1, 3, 4, 5 & 6, FoxP3 & T-bet of Tregs that were not significantly different from healthy age matched controls. Conclusion: Our data show that although FoxP3+ Tregs are normal in AA, a subset of these cells is markedly reduced and the activated Tregs aberrantly express CD45RA. Furthermore, unlike normal Tregs, the Tregs from AA patients do not suppress the inflammatory cytokine secretion by Te cells. The absence of DCs in the peripheral blood suggests their immigration to the inflammation site (e.g. bone marrow), which may play a role in the polarisation of T helpers toward a Th1 phenotype. Clonal expansion of Th1 cells may suggest potential antigen specificity that may lead to AA phenotype. 1. Miyara M, et al. Immunity. 2009. Disclosures: No relevant conflicts of interest to declare.

Increased Peripheral T Cell Responses to EBV-Infected Cells with Frequent Detection of EBV-DNA In Plasma and Viral mRNA In Peripheral B-Cells In Immunocompetent EBV-Positive Diffuse Large B-Cell Lymphoma Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4163-4163 ◽  
Author(s):  
Satoko Morishima ◽  
Kazuhito Yamamoto ◽  
Hiroshi Kimura ◽  
Seiko Iwata ◽  
Tomohiro Kinoshita ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
B Cell Lymphoma ◽  
Viral Mrna ◽  
Ebv Dna ◽  
Ifn Γ

Abstract Abstract 4163 Introduction: It is well known that immunocompromised patients(pts) such as post-transplantation or HIV infection are at increased risk of EBV-associated B-cell lymphoproliferaitive disorders. However, the immunological status of immunocompetent EBV-positive diffuse large B-cell Lymphoma (DLBCL) has not been well elucidated. For example, EBV-positive DLBCL in the elderly is defined as an EBV-positive clonal B-cell lymphoid proliferation occurring in pts more than 50 years of age and without any known immunodeficiency in WHO classification 2008, accounting for 5 – 10% of DLBCL in Japan. This disease is speculated to be related to the immunological deterioration accompanying the aging process. We conducted a multi-center prospective study to assess EBV status and the T cell response to EBV of peripheral blood (PB) in EBV-positive DLBCL in comparison with EBV-negative DLBCL pts and normal old-healthy persons (old-HPs). Patients and Methods: Fifteen newly-diagnosed pts with EBV-positive DLBCL, 8 pts with EBV-negative DLBCL, and 16 old-HPs were enrolled. Median ages of pts with EBV-positive DLBCL, pts with EBV-negative DLBCL, and old-HPs were 74.5 years (range 29–82 years), 71 years (48-79), and 65 years (60-74), respectively. Among the 15 pts with EBV-positive DLBCL, 2 were pyothorax-associated lymphoma, 2 were treated with methotrexate for rheumatoid arthritis, and the remaining 11 pts were without past histories predisposing to immunodeficiency. The diagnosis of EBV-positive DLBCL was made by positive signals for in situ hybridization using EBV-encoded small RNA (EBER) on paraffin section. To analyze T cell reactivity to autologous EBV-infected B-cell lymphoblastoid cell lines (LCL), CFSE-labeled peripheral blood mononuclear cells (PBMCs) were co-cultured with irradiated autologous LCL, and the division indices (DI) of CD4+ and CD8+ T cells were determined on day 5 (Cytometry 34:143, 1998). Percentage of proliferating CFSElow IFN-γ+CD4+ T cells and CFSElow IFN-γ+CD8+ T cells was assessed on day 7. EBV DNA load in PBMCs and plasma was determined by quantitative real-time PCR. CD19+ B cells were separated from of PBMCs by immunomagnetic sorting, and viral mRNA expression of B cells was quantified by one-step multiplex real-time RT-PCR. Results: (1) Plasma EBV-DNA was detectable in 13 of the 15 EBV-positive DLBCL pts but in none of the 8 EBV-negative DLBCL pts or the 16 old-HPs. Copy number of EBV-positive DLBCL was significantly higher than EBV-negative DLBCL (p<0.00001) or old-HPs (p=0.0001). EBV-DNA in PBMC was positive in 10 of the 15 EBV-positive DLBCL pts, 2 of 8 EBV-negative DLBCL pts, and 2 of 15 old-HPs. (2) EBER1 of PB B cells was detected in all 10 EBV-positive DLBCL pts, 4 of 7 EBV-negative DLBCL pts, and 7 of 14 old-HPs. BamHI A rightward transcripts (BARTs) of B-cells was detected in 8 of 10 EBV-positive DLBCL pts, 3 of 10 EBV-negative DLBCL pts, and 2 of 14 old-HPs. Latent membrane protein 2 (LMP2) of B cells was detected in 2 of 10 EBV-positive DLBCL pts, 2 of 14 old-HPs, and none of 5 EBV-negative DLBCL pts. LMP1 of B cells was detected in 1 of 10 EBV-positive DLBCL pts, none of 7 EBV-negative DLBCL pts, and none of 14 old-HPs. EBV-encoded nuclear antigen 1 (EBNA1) or EBNA2 were not detected in any of the pts nor old-HPs. (3) DI of CD4+ T cells for 5 days in EBV-positive DLBCL (median 1.41) was significantly higher than in old-HPs (median 0.53) (p=0.0009), and DI of CD8+ T cells of EBV-positive DLBCL (median 1.94) showed a higher tendency than old-HP (median 1.35). (4) Percentage of CFSE-low IFN-γ+CD4+ T cells in EBV-positive DLBCL (median 9.8%) was significantly higher than in old-HPs (median 2.2%) (p=0.002), and percentage of CFSE-low IFN-γ+CD8+ T cells in EBV-positive DLBCL (median 12.0%) was significantly higher than in old-HPs (median 6.6%)(p=0.025). Conclusions: Measurement of the plasma EBV-DNA copy number is a good indicator for the diagnosis of EBV-positive DLBCL, and the frequent detection of EBV viral mRNA of peripheral B-cells in EBV-positive DLBCL pts might be associated with greater viral load. Proliferative and INF-γ secreting responses of both peripheral CD4+ T cells and CD8+ T cells to EBV-infected cells were increased in EBV-positive DLBCL compared to old-HPs, which might be driven by an elevated viral load. These findings suggest that systemic immunological reaction to EBV might be intact in EBV-positive DLBCL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 2081

2017 ◽  
Vol 1 (S1) ◽  
pp. 55-56
Author(s):  
Farha Sherani ◽  
Duane Moogk ◽  
Anuj Bapodra ◽  
Karolina Malecek ◽  
Una Moran ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Expression Profiles ◽  
Cytokine Expression ◽  
T Cell Response ◽  
Tumor Rejection ◽  
Cytokine Profiles ◽  
Ifn Γ

OBJECTIVES/SPECIFIC AIMS: To characterize the CD4+ T-cell response during CTLA-4 blockade immunotherapy with ipilimumab in patients with metastatic melanoma by correlating cytokine profiles with phenotypic changes in the intratumoral lymphocyte compartment of tumor biopsies obtained before and after treatment. METHODS/STUDY POPULATION: Peripheral blood mononuclear cell samples were obtained from patients with metastatic melanoma undergoing monotherapy with ipilimumab via the Interdisciplinary Melanoma Cooperative Group at New York University Langone Medical Center. We isolated CD4+ T-cells and used a cytometric bead array assay following in vitro activation with anti-CD3, anti-CD28 antibodies to characterize cytokine expression profiles by quantifying IFN gamma, IL-2, IL-4, IL-6, IL-10, IL-17, and TNF-α at 5 time points during therapy. In total, 53 peripheral blood samples were included from 12 patients. To correlate cytokine profiles with CD4+ T-cell phenotypes in the intratumoral lymphocyte compartment, multiplex immunofluorescence was performed using CD4, CD8, CCR7, CD45RO, and FOXP3 antibodies on tumors before and after treatment with ipilimumab. RESULTS/ANTICIPATED RESULTS: Patients with evidence of clinical benefit (CB), as defined by having achieved partial response or stable disease, were compared with nonresponders (NR). All patients had an increase in IFN-γ, IL-2, and IL-10 secretion by CD4+ T-cells during ipilimumab therapy. NR had a statistically higher increase in all 3 cytokines. Mean IL-10 secretion was 22.3-fold higher compared with patients with CB (p value 0.0458; 95% CI=0.6676–43.89). Mean IFN-γ secretion was 12.4-fold higher from baseline levels in NR compared with CB (p value 0.046; 95% CI=0.3589–24.35). Mean IL-2 secretion was 6.9-fold higher in NR compared with CB (p value 0.032; 95% CI=0.9688–12.75). There were no statistically significant differences seen in the secretion of IL-4, IL-6, IL-17, or TNF-α. Multiplex immunofluorescence for immune profiling of 20 pre and post treatment tumor biopsies is ongoing. We expect to see distinct intratumoral lymphocyte compartment changes which correlate with clinical response and the above described differential cytokine profiles. Specifically, we anticipate CB patients will have increased intratumoral effector T-cells and decreased regulatory T-cells when compared with their NR counterparts. DISCUSSION/SIGNIFICANCE OF IMPACT: Cytokine expression profiles of peripheral blood CD4+ T-cells have not been previously correlated with patient response in patients undergoing treatment with ipilimumab. We describe distinct secretion profiles for IFN-γ, IL-2, and IL-10 for CB Versus NR patients. NR had a statistically higher increase in IL-10, an inhibitory cytokine which typically indicates upregulation of regulatory T-cells and consequent immune escape. Increased secretion of IL-2 and IFN-γ suggests skewing towards a Th1 type, anti-tumor effector T-cell response; these cytokines increased with ipilimumab treatment in both patient groups. However, the mean increase was several fold higher in NR. Recent evidence suggests loss of the interferon gamma pathway in tumor cells confers resistance to anti-CTLA4 therapy. Chronic IFN-γ secretion is associated with an exhausted T-cell phenotype and impaired tumor rejection. Therefore, higher increases in IFN-γ secretion by CD4+ T-cells in NR suggest impaired IFN-γ dependent tumor rejection in these patients. Our findings suggest IFN-γ, IL-2, and IL-10 cytokine expression profiles can be useful as biomarkers for response to ipilimumab treatment.

Human Bone Marrow as a Source of Multifunctional CMV-Specific CD4+ T Cells for Adoptive Cell Therapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2973-2973
Author(s):  
Il-Kang Na ◽  
Anne Letsch ◽  
Ines Noack ◽  
Sandra Bauer ◽  
Jens Geginat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Human Bone ◽  
Ifn Γ

Abstract Introduction Adoptive cell transfer of ex vivo primed and expanded human cytotoxic T lymphocytes (CTLs) has emerged as a promising approach to treat both infectious and malignant diseases in humans. First clinical studies have shown that transfer of Cytomegalovirus (CMV)-specific CD8+ T cells is safe and effective in reconstitution of cellular immunity against CMV disease. Efficacy of adoptive T cell therapy is limited by the numbers of CTLs in vitro and the survival and function after infusion. CD4+ T cells may enhance activity via direct or indirect effector functions (Matloubian 1994 [1]). In this study we have analysed whether bone marrow is superior to peripheral blood for expansion of CMV-specific T cells. Experimental design Paired peripheral blood and bone marrow samples were obtained from patients who underwent total hip arthroplasty. By using two different protocols T cells were expanded in the presence of IL-2 and IL-7 either from bulk culture with exposure of two different peptide pools (IE1 and pp65) or after selection via IFN-γ secretion by stimulation with pp65. CMV specific immune responses were assessed by using multiparameter flow cytometry staining cells for CD3, CD4, CD8, CCR7 and CD45RA and for the cytokines IFN-γ IL-2 and TNF at day 0 and after 10 days of in vitro expansion. Results Similar frequencies of cytokine-producing pp65– and IE1-specific CD4+ and CD8+ T cells were found in unmanipulated paired PB and BM samples. Expansion of CMV-specific T cells from BM resulted in significantly higher frequencies of specific CD4+ T cells than from PB, whereas no difference in frequencies of CMV-specific CD8+ T cells was observed. Interestingly, significantly higher frequencies of BM pp65 and IE1-specific CD4+ T cells were multifunctional, characterized by producing simultaneously IFN-γ, TNF and IL-2 (IE1: BM mean 0.44% ± 0.16; PB mean 0.09% ± 0.05, p=0.031; pp65: BM mean 3.87% ± 2.46; PB mean 1.24% ± 0.90, p=0.031). Expansion of multi-functional CD4+ T cells from BM was observed with both the bulk and selection assay protocol. Both PB and BM CMV-specific CD4+ and CD8+ T cell lines had a predominant CD45RA-CCR7- effector memory phenotype. Conclusions This study implicates the use of human bone marrow as a source for expansion of multifunctional CMV-specific CD4+ T cells. Recent studies in HIV and Leishmania support the crucial role of multifunctional T cells in disease control (Darrah 2007 [2]).

Reprogramming Aberrant B Cell-Subsets to Improve Immune Function in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3986-3986
Author(s):  
Rao H. Prabhala ◽  
Andreea Negroiu ◽  
Saem Lee ◽  
Mariateresa Fulciniti ◽  
Puru Nanjappa ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Advisory Committees ◽  
Healthy Donors ◽  
Ifn Γ ◽  
B Cell Subsets

Abstract Abstract 3986 B cell-malignancies exhibit considerable immune dysfunction particularly in multiple myeloma (MM). We have previously demonstrated that in T cell-compartment, regulatory T helper cells are dysfunctional in multiple myeloma (MM) while Th17 cells are significantly elevated and IL-17 produced by them is associated with MM cell growth and survival as well as suppressed immune responses and bone disease. We have here investigated the B cell-subsets and their ability to re-program anti-tumor immunity in MM. We have first characterised four different B cell-subsets (B1a, B1b, B2 and regulatory B cells) using 10-color flow cytometric analysis in both peripheral blood and bone-marrow (BM) samples from MM patients compared with normal healthy donors. We observe that CD5+ B1a-B cells are significantly elevated in peripheral blood of MM patients (N=7) compared to healthy donor (N=15) (42±8% vs 13±3%, respectively, p<0.05); while normal B cells (B2 cells) are significantly reduced in peripheral blood (29.8±6.5, p<0.05) and in the BM samples (11±4.8, N=4, p<0.05) of MM patients compared to healthy donors (59±3, and 60.2±2, N=10, respectively). We also observed that both B1b (47.9±18 vs. 22.8±4) and regulatory B cells (7.1±4.5 vs. 1.54±0.3) are elevated in BM samples of MM compared to healthy donors, however there were no differences in B1b and regulatory B cells in the peripheral blood of MM compared to healthy donor samples. Interestingly, in myeloma we observe higher levels of activated B cell subsets but lower levels of memory B cell subsets compared to healthy donors. These results, particularly very low levels of normal B cells in MM patients, may explain the decreased levels of uninvolved immunoglobulin in MM. As removal of B cell population has been shown to re-program T helper cell populations, we next investigated impact of B cell population on T cell activation. We activated normal PBMC via the anti-CD3 antibody, in the presence or absence of B or CD25+ cells and measured intra-cellular IFN-γ levels in CD69+ cells. We found that the absence of B cells significantly inhibited interferon-producing T cells compared to PBMC (by 43%; p<0.05). Importantly, following removal of CD25+ cells, which consists of both Tregs and activated memory T cells, with or without B cells, we did not observe any difference in the inhibition of IFN-γ, indicating that B cells are influencing memory T cells rather than naïve T cells for the production of IFN-γ. This prompted us to identify the phenotypic signature of regulatory T cell populations when purified memory T cells are polarized with the regulatory T cell cocktail in presence or absence of B cells. We observed that B cells reduce FOXP3 expression by 18 %(N=5) and establish cognitive interactions with T cells. This occurred by increasing the expression of GITR (154%) and CTLA4 (54%); while reducing PD1 (−24%) and OX40 (−21%) expression on T cells without affecting HLA expression. We have also observed these improvements by B cell modulation on T cells in MM. Our results indicate that targeting these re-programmable capabilities of B cells to modulate T helper cell populations may enable us to improve T cell function in MM; and may improve immune function in MM and also allow us to enhance responses to vaccinations. Disclosures: Ghobrial: Millennium: Advisory Board Other; Novartis: Advisory Board, Advisory Board Other. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Treon:Onyx: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Cephalon: Consultancy; Avila: Consultancy. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

Immune Cell Phenotype and Function After Treatment With Blinatumomab For Childhood Relapsed B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2668-2668 ◽  
Author(s):  
Alice Bertaina ◽  
Perla Filippini ◽  
Valentina Bertaina ◽  
Barbarella Lucarelli ◽  
Aurelie Bauquet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Lymphoblastic Leukemia ◽  
Γδ T Cells ◽  
Cell Phenotype ◽  
Ifn Γ

Abstract Background Blinatumomab is a bi-specific monoclonal antibody designed to engage and tether cytotoxic T-cells (CTL) to CD19-expressing target B cells. An ongoing phase I multicenter study in pediatric patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has shown that blinatumomab induces morphological and molecular remissions, defined as minimal residual disease (MRD) levels <10-4, in 47% of patients [Gore L, et al. J Clin Oncol 31, 2013 (suppl; abstr 10007)]. It is presently unknown whether and to what extent blinatumomab affects T-cell phenotype and function in pediatric patients with BCP-ALL. Patients and Methods Eight children diagnosed with relapsed/refractory BCP-ALL at the Bambino Gesù Children’s Hospital in Rome (median age at diagnosis 5.8 years, range 0.5-14.6) received blinatumomab as continuous intravenous infusion for 28 consecutive days, followed by a 2-week drug-free period. Four out of 8 patients were given repeated treatment courses. Peripheral blood samples were collected before treatment (day 0) and weekly thereafter, for 4 consecutive weeks. Bone marrow (BM) aspirates were available on days 0 and +29 of each drug course. Peripheral blood mononuclear cells (PBMC) were labeled with appropriate combinations of fluorochrome-conjugated monoclonal antibodies to quantitate naïve/memory T cells, αβ/γδ-expressing T cells and other immune effectors with potential anti-leukemia activity, such as CD3+CD56+ natural killer (NK) T cells and CD3-CD56+ NK cells. T-cell production of interferon (IFN)-γ, interleukin (IL)-4 and IL-17 was measured at the single-cell level, after short-term (4-hour) stimulation with phorbol myristate acetate (PMA) and ionomycin. The TCR-Vβ Repertoire Kit® (Beckman Coulter, Milan, Italy) allowed the flow cytometry analysis of 24 different Vβ specificities on T cells, thus covering approximately 70% of the normal human TCR-Vβ repertoire. Results Peripheral blood lymphocytes reached their nadir on day +1 (median 300/µL of blood [inter-quartile range 40-380] compared with 1,080/µL of blood at baseline [inter-quartile range 360-2,310]; p=0.0037 by Mann-Whitney U test for paired data), expanded within 7 days up to 3.5-fold above baseline, and included both CD4+ and CD8+ T cells. By contrast, the frequency of both CD3+CD56+ NK T cells and CD3-CD56+ NK cells remained unchanged compared to baseline. IFN-γ production by patient-derived CD4+ T cells exceeded that observed in CD4+ T cells from healthy controls by 2-fold, indicating robust T helper type 1 (Th1) polarization. The frequency of Th2/Th17 cells, defined as CD4+IL-4+ and CD4+IL-17+ cells, respectively, was not different after treatment compared to baseline. CD31 expression on recovering CD45RA+ naïve T cells, a surrogate phenotypic feature for recent thymic emigrants (RTEs), suggested that thymic output may contribute to T-cell expansion after blinatumomab administration. Non-significant changes in the relative proportion of TCR-αβ and TCR-γδ-expressing CD3+ T cells were detected after treatment (median 79.5% TCR-αβ+ T cells and 19.3% TCR-γδ+ T cells among total CD3+ cells) compared with baseline (median 87.4% TCR-αβ+ T cells and 12.2% TCR-γδ+ T cells among total CD3+ cells). Importantly, both CD3+CD8bright T cells and NK cells expressed lytic granule proteins, such as perforin and granzyme-B, at levels that increased during treatment. The analysis of Vβ TCR repertoire revealed a restricted usage of single Vβ domains by BM-resident CD8+ T cells, but not by CD4+ T cells. Specifically, the sum of Vβ within CD8+ T cells in the BM averaged 56.7±6.2% after blinatumomab, compared with 78±5.1% in healthy controls (p=0.04; Mann-Whitney U test for unpaired data). Conclusions Blinatumomab expands both CD31+CD45RA+ thymic-naïve and memory T cells with heightened IFN-γ production and is highly effective at clearing MRD in children with BCP-ALL. Skewing of the Vβ repertoire within BM-resident CD8+ T cells may be consistent with clonal expansions. Disclosures: Zugmaier: Amgen: Employment.

scholarly journals CRTAM determines the CD4+ cytotoxic T lymphocyte lineage

2015 ◽  
Vol 213 (1) ◽  
pp. 123-138 ◽  
Author(s):  
Arata Takeuchi ◽  
Mohamed El Sherif Gadelhaq Badr ◽  
Kosuke Miyauchi ◽  
Chitose Ishihara ◽  
Reiko Onishi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic Activity ◽  
Cd4 T Cells ◽  
Intracellular Signaling ◽  
Ectopic Expression ◽  
T Cell Subsets ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Ifn Γ

Naive T cells differentiate into various effector T cells, including CD4+ helper T cell subsets and CD8+ cytotoxic T cells (CTL). Although cytotoxic CD4+ T cells (CD4+CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4+ T cells that express class I–restricted T cell–associated molecule (CRTAM) upon activation possesses the characteristics of both CD4+ and CD8+ T cells. CRTAM+ CD4+ T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM+ T cells are the precursor of CD4+CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4+CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM+ T cells traffic to mucosal tissues and inflammatory sites and developed into CD4+CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4+CTL through the induction of Eomes and CTL-related gene.

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