Reduction of Tregs with Expansion of Th1 Cells and Lack of IFN-γ Secretion by GPI Negative T-Cells In PNH

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2240-2240
Author(s):  
Shahram Kordasti ◽  
Modupe Elebute ◽  
Pilar Perez Abellan ◽  
Austin G Kulasekararaj ◽  
Janet Hayden ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
High Resolution ◽  
Peripheral Blood ◽  
Clonal Expansion ◽  
T Cell Subsets ◽  
Th1 Cells ◽  
Healthy Controls ◽  
Significant Difference ◽  
Ifn Γ

Abstract Abstract 2240 Introduction and Aim: Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haematopoietic stem cell disorder characterized by intravascular haemolysis and thrombosis. The pathogenetic link with bone marrow failure syndromes is well recognized, however the process of clonal expansion of the glycosylphosphatidylinositol (GPI)-deficient cells over normal haemotopoiesis remains unclear. To further elucidate mechanisms leading to clonal expansion in PNH, we investigated the immunological profile and performed high-resolution genome-wide karyotyping using Affymetrix SNP6 microarrays. Patients and Methods: The percentage and absolute numbers of CD4+ and CD8+ T-cell subsets, NK cells and B cells in peripheral blood were assessed in 8 patients with PNH prior to any therapy and 8 healthy age matched controls. High resolution SNP6 karyotyping was performed on bone marrow (n=15) and peripheral blood (n=12) of these patients. Bone marrow from an additional 8 patients was enriched for CD34+59- and CD34+59+ cell fractions for SNP array karyotyping. Abberations that overlapped by >50% with variations found in the Database of Genomic Variants, as well as an internal series of 91 normal subjects were excluded from further analysis. T-cells were stimulated and then stained intracellularly for TNF-α and IFN-γ (Th1), IL-4 (Th2) and IL-17 (Th17). 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. CD4+ Tregs were defined as CD3+CD4+ CD25high CD27+Foxp3+. Results: There were no significant differences in the number or percentage of different CD8+ and CD4+ T-cells compared to healthy controls except for the number of Tregs and Th1 cells. In our cohort of patients, the number of Th1 cells was significantly higher than healthy controls (4.1×107/L v 0.93 × 107/L, p=0.039), whereas the number of Tregs cells was lower (0.75 × 107/L v 1.36 × 107/L, p=0.028). There was no significant difference in the number of Th2 and Th17 cells between patient and healthy subjects. Within CD4+ T-cells two distinct CD59+ and CD59- populations were identified, of which the CD59- cells were unable to secrete IFN-γ in response to stimulation compared to CD59+ population. On average 48% of CD4+ CD59+ T-cells secrete IFN-γ compared to 2% in the CD59- population. There was no significant difference in IL17 and IL4 secretion between CD59+ and CD59- T-cells. SNP karyotyping revealed three regions of uniparental disomy (UPD); UPD1p26.11-p34.3, UPD1p13.3-p13.1in one peripheral blood sample and UPD7q32.1-q34 in one bone marrow sample. There were no additional somatic genomic aberrations detected in any of the samples. Of note, purified CD34+59- cells did not reveal any clonal copy number changes or regions of UPD. Conclusion: Specific analysis of Xp22.1 did not reveal any aberrations of the PIGA gene, suggesting aberrations of the PIGA gene may be restricted to mutations or epigenetic abnormalities. Our immunological profiling revealed an expansion of Th1 cells and diminished Tregs in the peripheral blood, which is in contrast to our published data from both MDS and AA patients. The lack of IFN-γ secretion by GPI deficient T-cells also suggests an additional immunological defect in these patients, which may contribute in disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bone Marrow NK1.1− and NK1.1+ T Cells Reciprocally Regulate Acute Graft versus Host Disease

1999 ◽  
Vol 189 (7) ◽  
pp. 1073-1081 ◽  
Author(s):  
Defu Zeng ◽  
David Lewis ◽  
Sussan Dejbakhsh-Jones ◽  
Fengshuo Lan ◽  
Marcos García-Ojeda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
T Cell Subsets ◽  
Host Disease ◽  
Graft Versus Host ◽  
Ifn Γ

Sorted CD4+ and CD8+ T cells from the peripheral blood or bone marrow of donor C57BL/6 (H-2b) mice were tested for their capacity to induce graft-versus-host disease (GVHD) by injecting the cells, along with stringently T cell–depleted donor marrow cells, into lethally irradiated BALB/c (H-2d) host mice. The peripheral blood T cells were at least 30 times more potent than the marrow T cells in inducing lethal GVHD. As NK1.1+ T cells represented <1% of all T cells in the blood and ∼30% of T cells in the marrow, the capacity of sorted marrow NK1.1− CD4+ and CD8+ T cells to induce GVHD was tested. The latter cells had markedly increased potency, and adding back marrow NK1.1+ T cells suppressed GVHD. The marrow NK1.1+ T cells secreted high levels of both interferon γ (IFN-γ) and interleukin 4 (IL-4), and the NK1.1− T cells secreted high levels of IFN-γ with little IL-4. Marrow NK1.1+ T cells obtained from IL-4−/− rather than wild-type C57BL/6 donors not only failed to prevent GVHD but actually increased its severity. Together, these results demonstrate that GVHD is reciprocally regulated by the NK1.1− and NK1.1+ T cell subsets via their differential production of cytokines.

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.

scholarly journals Elevated Expression of the Long Noncoding RNA MAFTRR in Patients with Hashimoto’s Thyroiditis

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Huiyong Peng ◽  
Xiangmei Ding ◽  
Juan Xu ◽  
Yue Han ◽  
Jun Yang ◽  
...  
Keyword(s):  
T Cells ◽  
Roc Curve ◽  
Peripheral Blood ◽  
Hashimoto’S Thyroiditis ◽  
Noncoding Rna ◽  
Hashimoto's Thyroiditis ◽  
Th1 Cells ◽  
Healthy Controls ◽  
Transcript Levels ◽  
Ifn Γ

Background. Long noncoding RNAs (lncRNAs) represent an important novel class of noncoding RNA molecule greater than 200 nucleotides that play a key role in the regulation of autoimmune diseases. Previous studies have demonstrated that MAFTRR (MAF transcriptional regulator RNA) regulated Th1 cells differentiation by inhibiting the expression of MAF in activated CD4+ T cells. However, the effect of MAFTRR on the pathogenesis of Hashimoto’s thyroiditis (HT) remains unclear. This research was aimed at investigating the expression of MAFTRR in Hashimoto’s thyroiditis (HT) as well as the correlation between MAFTRR and Th1 cells. Methods. Thirty-eight HT patients and thirty-eight healthy controls were enrolled in the study. The proportion of Th1 cells and CD8+IFN-γ+ T cells in peripheral blood mononuclear cells (PBMCs) from these specimens was determined by flow cytometric analysis. The transcript levels of MAFTRR, MAF, and IFNG in PBMCs and thyroid glands were detected by quantitative real-time PCR. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the potential value of MAFTRR in the HT patients. Results. We found that the proportion of circulating Th1 cells and the transcript levels of IFNG were increased in peripheral blood of the HT patients. The transcript levels of MAFTRR were significantly increased in the HT patients and positively correlated with the percentage of Th1 cells and serum levels of antithyroglobulin antibody and antithyroperoxidase antibody. The transcript levels of MAF, a transcription factor that inhibits Th1 cells activity and IFN-γ production, were attenuated in PBMCs from the HT patients. The transcript levels of IFNG had positive and inverse correlations with MAFTRR and MAF expression in PBMCs from the HT patients, respectively. Additionally, a significantly positive correlation between upregulated MAFTRR expression and augmented IFNG expression was revealed in thyroid tissues from the HT patients. ROC curve suggested that MAFTRR could potentially differentiate the HT patients from healthy controls. Conclusion. MAFTRR is significantly augmented in the HT patients and may contribute to the pathogenic role of the Th1 cells response in HT.

scholarly journals Imbalances within the peripheral blood T-helper (CD4+) and T-suppressor (CD8+) cell populations in the reconstitution phase after human bone marrow transplantation

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1196-1200 ◽  
Author(s):  
A Velardi ◽  
A Terenzi ◽  
S Cucciaioni ◽  
R Millo ◽  
CE Grossi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Peripheral Blood ◽  
Marrow Transplantation ◽  
Allogeneic Bone Marrow Transplantation ◽  
T Cell Subsets ◽  
Allogeneic Bone ◽  
T Helper

Abstract Peripheral blood T cell subsets were evaluated in 11 patients during the reconstitution phase after allogeneic bone marrow transplantation and compared with 11 age-matched controls. The proportion of cells coexpressing Leu7 and CD11b (C3bi receptor) markers was determined within the CD4+ (T-helper) and the CD8+ (T-suppressor) subsets by two- color immunofluorescence analysis. CD4+ and CD8+ T cells reached normal or near-normal values within the first year posttransplant. In contrast to normal controls, however, most of the cells in both subsets coexpressed the Leu7 and CD11b markers. T cells with such phenotype display the morphological features of granular lymphocytes (GLs) and a functional inability to produce interleukin 2 (IL 2). These T cell imbalances were not related to graft v host disease (GvHD) or to clinically detectable virus infections and may account for some defects of cellular and humoral immunity that occur after bone marrow transplantation./

Differential Expression Of TREM-1 In Myelogenous Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4951-4951 ◽  
Author(s):  
Huiyu Li ◽  
Wenying Li ◽  
Xiaoling Yi ◽  
Shiang Huang ◽  
Wei Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Complete Remission ◽  
Patient Group ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Control Group ◽  
Healthy Controls ◽  
Significant Difference ◽  
Patient Groups

Abstract Objectives Triggering receptor expressed on myeloid cells (TREM) -1 is a receptor as a member of the immunoglobulin superfamily expressed on the cell-surface of neutrophils, monocytes and macrophages. This receptor amplifies the inflammatory response, activating the signaling pathway. TREM-1 expression is associated with mature myeloid cell development. TREM-1 is shed from the membrane of activated macrophages without the transmembrane and intracellular domains, and can be found as soluble TREM (sTREM)-1. Soluble TREM-1 is thought to negatively regulate TREM receptor signaling. Some studies currently reported that TREM-1 regulates the malignant behavior of cancer cells in lung cancer and HCC. However, no related studies about the role of TREM-1 in leukemia have been carried out. The aims of this study was investigated the TREM-1 expression in myelogenous leukemia cells. Methods Thirty-five patients with AML, twenty-five patients with CML and a control group of eleven healthy people were subjected to the study. TREM-1 expressions on the surfaces of leukemia cells were measured by flow cytometry. Plasma sTREM-1 levels were measured by ELISA. Results In this study, our results provide the first evidence that TREM-1 was differentially expressed in myelogenous leukemia cells. The TREM-1 mean ratio of median fluorescence intensity (mean ratio of MFI) was 3.13±0.88 and 2.52±0.40 in CML and AML patients, respectively. The TREM-1 mean ratio of MFI was 3.03±1.40 in myelogenous leukemia cell lines (K562, HL60, THP-1). The TREM-1 mean ratio of MFI was 5.37±0.88 in healthy controls. Compared to healthy controls, myelogenous leukemia cells had decreased TREM-1 expressions (P<0.001). The TREM-1 mean ratio of MFI was 4.89±0.60 in patients who are in complete remission after Novartis's Gleevec therapy. Compared with CML patient groups, patients who are in complete remission after Gleevec therapy had rising TREM-1 expressions (P<0.01). TREM-1 expressions of patients who are in complete remission after Gleevec therapy were slightly lower than the healthy controls, but this did not reach significance. No significant difference in TREM-1 expressions was seen between AML and CML patient groups, male and female patient groups, and cells derived from peripheral blood and bone marrow of the same leukemia patients (p>0.1). In addition, the plasma sTREM-1 levels were measured by ELISA. sTREM-1 levels was 48.54±57.63pg/mL for AML group and 43.72±23.93pg/mL for CML group. Results indicated that plasma sTREM-1 levels significantly higher in AML and CML patients than that in healthy controls (P<0.01). However, there was no significant difference in plasma sTREM-1 levels observed in AML patient group compared with CML patient group, male patients group compared with female patients group, and plasma from peripheral blood compared with plasma from bone marrow of the same leukemia patients (p>0.1). An ongoing project focuses on the relationship between the function of TREM-1 and occurrence, progression and prognosis of myelogenous leukemia, advances will be reported in time. Conclusion TREM-1 expression on leukemia cells was significantly lower in patients with AML and CML than those in healthy controls and patients in complete remission had increased TREM-1 expression. Patients with AML and CML had increased plasma soluble TREM-1. The TREM-1 expression on leukemia cells had an inverse correlation with plasma sTREM-1 level in AML and CML patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals SLAM/SAP Decreased Follicular Regulatory T Cells in Patients with Graves’ Disease

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Lina Geng ◽  
Jun Yang ◽  
Xinyi Tang ◽  
Huiyong Peng ◽  
Jie Tian ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Regulatory T Cells ◽  
Autoimmune Diseases ◽  
Negative Correlation ◽  
Graves Disease ◽  
Healthy Controls ◽  
Significant Difference ◽  
Ifn Γ ◽  
Signaling Lymphocytic Activation Molecule

Signaling lymphocytic activation molecule (SLAM) and SLAM-associated protein (SAP) play important role in inflammatory and autoimmune diseases. Our study is aimed at detecting the expression of SLAM and SAP in patients with Graves’ disease (GD) and analyzing the effect of SLAM/SAP on circulating blood CD4+CXCR5+Foxp3+ follicular regulatory T (Tfr) cells. The level of SAP in CD4+CXCR5+ T cells and the level of SLAM on CD19+ B cells were significantly increased in the patients with GD, but no significant difference in the level of SLAM on CD4+CXCR5+ T cells was observed between the patients with GD and the healthy controls. A decrease in the percentage of Foxp3+ cells in CD4+CXCR5+ T cells was observed following anti-SLAM treatment, but the percentages of IFN-γ+ cells, IL-4+ cells, and IL-17+ cells showed no obvious differences. The proportion of circulating Tfr cells was decreased in the patients with GD, and the proportion of circulating Tfr cells had a negative correlation with the level of SAP in CD4+CXCR5+ T cells and the levels of autoantibodies in the serum of the patients with GD. Our results suggested that the SLAM/SAP signaling pathway is involved in the decrease of circulating Tfr cells in Graves’ disease.

scholarly journals SLAM/SAP Decreased Follicular Regulatory T Cells in Patients With Graves’ Disease 

2021 ◽  
Author(s):  
Lina Geng ◽  
Jun Yang ◽  
Xinyi Tang ◽  
Huiyong Peng ◽  
Jie Tian ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Regulatory T Cells ◽  
Negative Correlation ◽  
Real Time Pcr ◽  
Graves Disease ◽  
Healthy Controls ◽  
Significant Difference ◽  
Ifn Γ ◽  
Signaling Lymphocytic Activation Molecule

Abstract Background: Signaling lymphocytic activation molecule (SLAM) and SLAM-associated protein (SAP) play important role in inflammatory and autoimmune diseases. Our study aimed to detect the expression of SLAM and SAP in patients with Graves’ disease (GD) and analyze the effect of SLAM/SAP on circulating blood CD4+CXCR5+ Foxp3+ follicular regulatory T (Tfr) cells.Methods: The expression of SLAM and SAP was assessed by flow cytometry and real-time PCR. The percentages of IFN-γ+ cells, IL-4+ cells, IL-17+ cells and Foxp3+ cells in CD4+CXCR5+ T cells and circulating CD4+CXCR5+ Foxp3+ Tfr cells after treatment with anti-SLAM and anti-CD3 antibodies were also assessed by flow cytometry. The correlations between the percentages of Tfr cells and the levels of autoantibodies as well as SAP were analyzed.Results: The level of SAP in CD4+CXCR5+ T cells and the level of SLAM on CD19+ B cells were significantly increased in the patients with GD, but no significant difference in the level of SLAM on CD4+CXCR5+ T cells was observed between the patients with GD and the healthy controls. A decrease in the percentage of Foxp3+ cells in CD4+CXCR5+ T cells was observed following anti-SLAM treatment, but the percentages of IFN-γ+ cells, IL-4+ cells and IL-17+ cells showed no obvious differences. The proportion of circulating Tfr cells was decreased in the patients with GD, and the proportion of circulating Tfr cells had a negative correlation with the level of SAP in CD4+CXCR5+ T cells and the levels of autoantibodies in the serum of the patients with GD.Conclusions: Our results indicate that the SLAM/SAP signaling pathway regulates Tfr cells, which may be involved in the pathogenesis of Graves’ disease.

Rag1 Deficiency Does Not Affect Myelodysplasia but Leads to More Rapid Leukemic Transformation in a Mouse Model of MDS.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2784-2784
Author(s):  
Sheryl M Gough ◽  
Yang Jo Chung ◽  
Peter D. Aplan
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
Cytotoxic T Cells ◽  
Immune Surveillance ◽  
Severe Anemia ◽  
Control Groups ◽  
Leukemic Transformation ◽  
Significant Difference

Abstract Abstract 2784 Poster Board II-760 MDS comprises a premalignant heterogeneous group of clonal stem cell disorders that also show bone marrow dysplasia and which often evolve to acute myeloid leukemia (AML). Aplastic anemia (AA) patients also share the bone marrow failure, anemia and resulting peripheral blood cytopenias of MDS. AA is thought to be caused by an oligoclonal expansion of cytotoxic T-cells that target haematopoietic stem and progenitor cells. The severe anemia and leucopenia characteristic of both diseases is relieved in AA patients and some MDS patients by immunosuppressive therapy, supporting the role of cytotoxic T-cells in the etiology of AA. However, the role of the lymphocytes in progressive MDS remains unclear. MDS has been associated with a number of genetic aberrations, including chromosomal translocations involving the NUP98 gene. Using mice that express a NUP98-HOXD13 (NHD13) transgene, previously shown to manifest the same clinical symptoms as those of MDS patients, we have followed a cohort of NHD13/Rag1−/− mice to determine if the absence of lymphocytes, especially T cells, might 1) diminish the severity of the MDS, or 2) effect transformation and/or survival in the NHD13 mice, as would be predicted by an “immune surveillance” hypothesis of malignant transformation. Serial CBCs at two month time intervals were used to evaluate the extent of anemia and leucopenia in NHD13+ /Rag1+/+ and NHD13/Rag1−/−, as well as WT/Rag1+/− and WT/Rag1−/− control groups over a 15 month period. NHD13/Rag1−/− mice were generated by crossing the NHD13+ (C57BL/6) with the B6;129S7-Rag1tm1Mom/J mouse, and housed in a Specific Pathogen-Free (SPF) environment. Mice were euthanized and analyzed when CBCs indicated severe anemia/leucopenia or leukemic transformation, or when determined to be unwell (hunched, immobile, dyspnea) by observation. Flow cytometry, histology and genomic analyses further determined leukemia subtype, extent of infiltration and leukemia clonality. NHD13+ /Rag1+/+ and NHD13/Rag1−/− mice showed no significant differences at any two month time-point in hemoglobin (Hg), mean corpuscular volume (MCV), or platelet levels, and progressive MDS occurred in both groups. Consistent with previous studies, and excluding cases that showed evident transformation to acute leukemia, NHD13+ /Rag1+/+ mice showed low WBC, neutrophil and lymphocyte numbers, which were not significantly different from the NHD13/Rag1−/− mice. NHD13/Rag1−/− mice did however show a significantly reduced survival when compared with the NHD13+ /Rag1+/+ mice (Log-rank test, p = 0.0135), and survival medians of 11 and 13 months, respectively. Incidence of leukemic transformation was increased in the NHD13/Rag1−/− compared with the NHD13+ /Rag1+/+ mice (p=0.0079). A range of leukemia subtypes was observed in both the NHD13+ /Rag1+/+ and NHD13/Rag1−/− mice, including myeloid, B-cell, T-cell, and erythroid leukemias. In the SPF environment provided, the WT/Rag1+/− and WT/Rag1−/− control groups showed no significant difference in survival rates. Serial CBC data indicated that there was no significant difference in the timing or degree of peripheral blood cytopenias between the NHD13+ /Rag1+/+ and NHD13/Rag1−/− mice, supporting the conclusion that absence of lymphocytes does not lead to improvement in the peripheral blood cytopenias caused by the NHD13 transgene. This observation suggests that the NHD13 transgene does not produce MDS caused by an autoimmune phenomenon. The poorer survival and increased frequency of leukemic transformation in the NHD13/Rag1−/− mice suggests that lymphocytes might play a role in the evolution of MDS to AML in the NHD13 mouse model, and supports the ‘immune surveillance' hypothesis. Disclosures: No relevant conflicts of interest to declare.

Different Pattern of CD154 and CD40 Expression On B-CLL and T Lymphocytes In Peripheral Blood, Bone Marrow and Lymph Node Microenvironment In B-Cell Chronic Lymphocytic Leukemia (B-CLL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3607-3607
Author(s):  
Ozren Jaksic ◽  
Branimir Gizdic ◽  
Tajana Stoos Veic ◽  
Vlatka Pandzic Jaksic ◽  
Rajko Kusec ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Lymph Node ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Significant Difference ◽  
Cd40 Expression ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abstract 3607 Background: In B-cell chronic lymphocytic leukemia (B-CLL) there is a well documented intraclonal and interclonal variability of B-CLL cells in different lymphoid compartments with respect to the expression of a number of surface and intracellular molecules (for example CD38 and ZAP-70). This variability in part may reflect a number of interactions of malignant B-CLL clone with supporting microenviroment including cells (T-cells, nurse-like cells, etc.), cytokines, chemokines and stroma. One of the key interactions of B-CLL clone is with T-cells, through CD154/CD40 system. It is important pathway modulating survival, drug resistance and immunity. It is known that CD154 is transiently expressed on CD4+ T cells, as well as that CD154 can be coexpressed on B-CLL cells with CD40 in a subpopulation of B-CLL patients. Its expression on B-CLL cells can be induced by gene therapy and lenalidomide, being in part responsible for their therapeutic effects. Aim of this study was to determine the level of expression of CD154 and CD40 in vivo on B-CLL cells and T lymphocytes and to evaluate intra and interclonal differences due to different microenvironment, i.e. peripheral blood, bone marrow and lymph nodes. Methods: peripheral blood, (PB), bone marrow (BM) and lymph node (LN) samples were taken by conventional techniques (venepuncture and fine needle aspiration) on the same day. The expression level of CD154 and CD40 molecules on CD19+CD5+ B-CLL cells and CD19-CD5+ T cells was analyzed by flow cytometry. Results were expressed as mean fluorescence intensity (MFI) and analyzed by paired tests. Results: samples taken from 21 typical B-CLL patients with median age of 72 years were analyzed. There were 9 males and 12 females. Mean beta-2 microglobuin was 4.3mg/l, mean Total Tumor Mass size was 8.9 and mean Tumor Distribution pattern was 0.75. There were 2, 14 and 5 patients in Rai stage 0, I+II and III+IV, respectively. There were 6 previously treated patients (but off therapy 3 months before sampling). The expression level of CD154 was absent/low on T-cells and in 14/21 patients on B-CLL cells. However in 7/21 patients B-CLL cells had higher CD154 expression (“CD154 positive” patients). There was no detectible difference in CD154 expression on T cells between compartments, while on B-CLL cells there was highest expression in lymph nodes and lowest in peripheral blood (p<0.01). CD40 expression on B-CLL cells was significantly higher than CD154, i.e. all cases were positive, and there was no significant difference between lymphoid compartments. There was no significant difference between CD154 positive and negative patients in measured disease parameters. Conclusions: our results show that CD154 expression on T-cells is absent/low and not significantly different between lymphoid compartments regardless of different microenvironment milieu. CD40 expression on B-CLL cells is high and comparable through compartments. In subset of patients there is CD154 positivity on B-CLL cells and shows strong association with lymphoid compartments possibly indicating microenviroment influence on CD154/CD40 system in B-CLL in vivo. These results warrant further studies to indentify the role of CD154 expression on B-CLL cells in pathologic process and its regulation and may eventually uncover novel or modulate existing innovative therapeutic approaches (like gene therapy or immunomodulatory agents like lenalidomide). Disclosures: No relevant conflicts of interest to declare.

Novel Double Labeled Mixed Lymphocyte in Vitro Assay to Predict the Dominant Graft in 2 Unit UCB Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4699-4699
Author(s):  
Shicheng Yang ◽  
Xiao Huang ◽  
Hongyan Lu ◽  
Amandeep Salhotra ◽  
Alexander Wendling ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
In Vitro Assay ◽  
Proliferation Index ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Ifn Γ

Abstract Abstract 4699 Introduction: Umbilical cord blood cells (UCB) from allogeneic donors have been established as an alternative source for HSC transplantation in patients who lack suitably HLA matched bone marrow or peripheral blood stem cells from adult donors. Transplantation using 2 unit UCB has been shown to compensate the low engraftment and slow hematopoietic recovery resulting from 1 unit UCB transplantation in full stature adult patients. At present, there are no unit specific factors that reliably predicts for the “winning unit” in 2 unit UCB transplantation, e.g. cell viability, number of infused total nucleated cells, CD34+ or CD3+ cells, sex mismatch, ABO blood group, and degree of HLA mismatch. In vivo mouse models suggest that CD34 negative subsets play an important role. Among CD34 negative subsets, CD8 T subset accounts for approximately 34.0+/−23.3% of T lymphocytes from UCB. In bone marrow transplantation CD8 T cells have been found to facilitate donor hematopoietic cell engraftment. Moreover, it has been reported that 1 dominant unit coincides with a specific CD8 T cell response against the non-engrafted unit which was not observed from CD4 or NK cells. Methods: In this study, we used volunteer donated UCB research units (kindly provided by P. Rubinstein, MD, New York Blood Center). Mononuclear cells (MNC) were purified by Ficoll gradient centrifugation, and CD3 T cells were isolated with CD3 MicroBeads (Miltenyi Biotec; autoMACS). The purified CD3 (confirmed by FACS >95% purity) cells were labeled with CFSE and DDAO-SE. After labeling, the cells from two different donors were mixed in 96-well U-bottom plates for continued culture in 37 °C 5% CO2. The expansion from each labeled donor cells was evaluated using flow cytometry; the dead cells were gated out using propidium iodide, and the data was analyzed using FlowJo software. For proper T cells activation, we also compared different activation conditions using i.) anti-CD3/CD28 Beads, ii.) anti-CD3 antibody plus anti-CD28 antibody, and iii.) cytokine IL-2. The schematic illustration of methods is shown in Figure 1. Results and discussion: We noted that T cells from UCB are primarily at naïve stage as determined by CD45RA (93.8 +/− 7.11%) and CCR7 (84.9 +/− 12.0%) expression. We also determined the optimal activation condition using a modified mixed lymphocyte reaction from 2 UCB units. Four days after incubation, the proliferation from 2 units labeled with CFSE and DDAO-SE could be reproducibly distinguished using FL1 channel for CFSE and FL4 channel for DDAO-SE (Figure 1). The optimal concentration for labeling using CFSE (1 mM) and DDAO (1 μM or 3 mM) was determined by titration. To avoid cell toxicity resulting from CFSE and DDAO-SE labeling, as well as self-crossing from each donor using two dyes, we examined additional mixed lymphocyte analyses in which each donor was labeled with CFSE or DDAO-SE respectively and vice versa. As shown in Figure 1, we found consistently that the predicated dominant unit accounted for the majority of culture (73.2% stained with DDAO; 63.5% stained with CFSE) after 4 days co-culture. The dominance was not correlated with cell proliferation indicated by the proliferation index (1.12 for dominant and 1.48 for another unit). After confirmation of this in vitro assay, further studies were conducted to evaluate the IFN-γ release of 2 UCB units in this optimized mixed lymphocyte assay in the condition using cytokine IL-2. Interestingly, we could only detect IFN-γ by intracellular staining in one unit when co-culture was set-up using CD3 T cells from each unit; the expression of IFN-γ was not detected when we used CD3 T cells from 1 unit. The correlation between dominance and the expression of IFN-γ is currently under investigation. Conclusion: UCB Transplantation is an important alternative for patients lacking bone marrow or peripheral blood stem cell donors. With the establishment of this novel modified mixed lymphocyte in vitro assay for prediction of the “winning” immune dominant unit, routine analyses can be performed to guide unit selection. Further interventions can be exploited to preferentially treat the expected dominant unit with glycosylation, cytokines, prostaglandins, or C3a compliments to further enhance hematopoietic stem cells trafficking and engraftment to the marrow. Disclosures: No relevant conflicts of interest to declare.

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