Cyclosporine A Effect the CD28 Expression on CD8+T Cells of Patients with Aplastic Anemia through down Regulate FLIP.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3767-3767
Author(s):  
Haiwen Huang ◽  
De Pei Wu ◽  
Guanghua Chen ◽  
Jinxiang Fu
Keyword(s):  
T Cells ◽  
Aplastic Anemia ◽  
Effective Treatment ◽  
Cd8 T Cells ◽  
Cyclosporine A ◽  
Peripheral Blood ◽  
Caspase 8 ◽  
Healthy Controls ◽  
Rt Pcr ◽  
Normal Controls

Abstract Objective The exact reason of aplastic anemia is still unknown. Recently more and more researchers realized that immune factors play an important role in aplastic anemia. In this study, we detected the expression of CD28 on different subgroups of T cells in patients with aplastic anemia and furthermore studied the effect of cyclosporine A to CD28 and FLIP on these cells. Method 21 patients and 15 healthy controls were investigated. The Expression of CD4CD28 and CD8CD28 on patients’ T cells was detected with FCM; patients’ CD8+T cells were purified with Mini MACR system, then the expression of FLIP, caspase-8 in CD8+T cells were analyzed by RT-PCR; we also detected the expression of CD28, FLIP and caspase-8 in patients’ CD8+T cells after incubation with cyclosporine A. Result: The percentage of CD8+CD28−T cells in the patients is significantly higher than that of normal controls (30.45±5.26% versus 19.32±4.72%, p<0.05) and it can be induced to return to normal after effective treatment with Cyclosporine A; the expression of FLIP in patients’ purified CD8+ T cells is higher than that in healthy controls’ CD8+ T cells, but there is no difference in the expression of caspase-8; after being cultured with Cyclosporine A, the expression of FLIP in patient’s CD8+T cells was down regulated and the quantity of CD28 on the surface of patient’s CD8+T cells increased significantly (28.3±5.1% versus 40.6±7.71%40.6±7.71%). Conclusion: The increased expression of FLIP in patients’ CD8+T cells is related to the increased percentage of CD8+CD28−T cells in patients’ peripheral blood. Cyclosporine A can down regulate the FLIP expression and correct the abnormal percentage of CD8+CD28−T cells in patients with aplastic anemia.

Study on the Pathways to Damage Hematopoiesis by CD8+ Effector T Cells of the Patients with Severe Aplastic Anemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4371-4371
Author(s):  
Zonghong Shao ◽  
Le Feng ◽  
Rong Fu ◽  
Jun Wang ◽  
Chunyan Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Aplastic Anemia ◽  
Cd8 T Cells ◽  
Bone Marrow Failure ◽  
Granzyme B ◽  
Effector T Cells ◽  
Severe Aplastic Anemia ◽  
Control Group ◽  
Hla Dr ◽  
Normal Controls

Abstract Abstract 4371 Objective To investigate the quantity and their pathways to damage hematopoietic cells of CD8+CD25+ and CD8+HLA-DR+ effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia(SAA) and explore the heterogeneous immunopathogenesis of SAA further. Methods The quantity of CD8+CD25+and CD8+HLA-DR+ cells in PB and the expressions of perforin, granzyme B, tumor necrosis factor-β(TNF -β) and FasL of 29 SAA (14 untreated and 15 recovered) patients and 12 normal controls were analyzed by flow cytometry. Results The ratio of CD8+CD25+T cells in CD8+ T cells was (3.67±2.58)% in untreated SAA patients, (5.19±4.29)% in recovered patients and (4.84±2.31)% in normal controls, and the ratios of CD8+CD25+T cells in CD3+ cells in three groups were (2.25±1.35)%, (2.98±1.35)% and (2.11±1.88)% respectively. There was no statistic difference among 3 groups(P>0.05). The ratio of CD8+HLA-DR+T cells in CD8+T cells was (39.30±8.13)% in untreated patients, which was significantly higher than that of recovered patients[(20.65±5.38%)] and controls [(18.34±6.68%)](P<0.001). There was no statistic difference between recovered patients and controls(P>0.05). CD8+HLA-DR+T cells in CD3+ cells was (27.81±7.10)% in untreated group, higher than that of recovered patients (12.02±3.03)% and controls(8.50±2.33)%(P<0.01). And the ratio in recovered group was higher than in control group(P<0.05). The expressions of perforin, granzyme B, TNF-β and FasL of CD8+HLA-DR+ T cells of untreated SAA patients were 8.51% A96.08% A72.11% and 94.25% respectively, higher than those of recovered patients(1.78% A85.20% A34.38% A51.20%)and controls(1.86% A82.09% A17.92% A32.91%). There was no statistic difference between recovered patients and controls(P>0.05). Conclusion There were elevated quantity of CD8+HLA-DR+ T cells and high expressions of perforin, granzyme B, TNF-β and FasL in SAA, which might contribute to the bone marrow failure of SAA. Disclosures: No relevant conflicts of interest to declare.

Elevated Expression of T-Cell Immunoglobulin and Mucin Domain 3 on T Cells from Peripheral Blood in Patients with Cervical Carcinoma

2020 ◽  
pp. 1-8
Author(s):  
Juan Li ◽  
Xiao-fei Sun ◽  
Ying Shen ◽  
Qing Yang ◽  
Shu-yan Dai
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cervical Carcinoma ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Control Group ◽  
Healthy Controls ◽  
Novel Therapy ◽  
Domain 3

<b><i>Objective:</i></b> To investigate the expression of T-cell immunoglobulin and mucin domain 3 (TIM-3) on peripheral T cells of cervical carcinoma patients. <b><i>Methods:</i></b> Peripheral blood samples from 15 high-grade cervical squamous intraepithelial lesion (HSIL) patients, 24 cervical carcinoma patients, and 21 healthy controls were collected. TIM-3 expressions on the surface of peripheral CD4+ T cells and CD8+ T cells were analyzed with flow cytometry. <b><i>Results:</i></b> There was significantly lower expression of CD4+ T cells and CD8+ T cells in HSIL patients and cervical carcinoma patients compared with healthy controls. We also found that TIM-3 expression on peripheral CD4+ T and CD8+ T cells of both HSIL patients and cervical carcinoma patients was significantly increased compared to the control group. Further analyses revealed that the expression of TIM-3 on peripheral CD4+ T and CD8+ T cells significantly increased in stage III–IV cervical carcinoma patients compared to stages I–II. <b><i>Conclusion:</i></b> The increased expression of TIM-3 on CD4+ T cells and CD8+ T cells of patients with cervical carcinoma and HSIL suggests the potential role of TIM-3 in the development and progression of cervical carcinoma, which may be a novel therapy target for cervical carcinoma.

Determination of CD4+ and CD8+ T cells in the peripheral blood of dogs with demodicosis

Parasitology ◽  
2010 ◽  
Vol 137 (13) ◽  
pp. 1921-1924 ◽  
Author(s):  
S. K. SINGH ◽  
U. DIMRI ◽  
M. C. SHARMA ◽  
B. SHARMA ◽  
M. SAXENA
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Significant Alteration ◽  
Healthy Controls ◽  
Healthy Dogs

SUMMARYThe aim of this study was to evaluate the CD4+/CD8+ ratio in peripheral blood of dogs with localized and generalized demodicosis. Sixteen dogs were examined, 8 with localized and 8 with generalized demodicosis, while 8 healthy dogs were used as controls. Peripheral blood was obtained and CD4+ and CD8+ T cells were determined by flow cytometry. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were found in dogs with generalized demodicosis compared to dogs with localized demodicosis and healthy controls. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were also found in dogs with localized demodicosis compared to healthy controls. The CD4+/CD8+ ratio was also found to be significantly lower in dogs with generalized demodicosis in comparison with dogs with localized demodicosis and healthy controls. It is concluded that significant alteration in the CD4+/CD8+ ratio may be implicated in the pathogenesis of generalized canine demodicosis.

scholarly journals T Cell Mediated Suppression of Hematopoiesis in Myeloproliferative Neoplasm

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1886-1886
Author(s):  
Sarah J Morse ◽  
Samuel B Luty ◽  
Hew Yeng Lai ◽  
Brian J. Druker ◽  
Angela G Fleischman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Normal Control ◽  
Peripheral Blood ◽  
Colony Formation ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Equal Distribution ◽  
Normal Controls

Abstract Background: Increased inflammation is an established feature of MPN, but its precise role in disease pathogenesis has yet to be clearly defined. T cell mediated insult on hematopoietic stem cells can drive expansion of clones that have mutated in such a way as to escape these suppressive and/or apoptotic cues (this mechanism is clearly illustrated in aplastic anemia). To investigate whether T cell mediated suppression of hematopoiesis occurs in MPN we interrogated the T lymphocyte compartment of MPN patients, guided by abnormalities known to be present in aplastic anemia. Methods and Results: We first asked how selective depletion of T cells from peripheral blood mononuclear cells (MNC) would affect myeloid colony formation in MPN as compared to normal controls. MNC were depleted of T cells using CD3 magnetic beads or mock depleted and plated side by side in methylcellulose (+SCF, IL-3, Epo). Removal of CD3+ T cells from bulk MNC reduced colony formation in all 29 normal control samples tested, consistent with the ability of normal lymphocytes to enhance colony formation. In contrast, removal of CD3+ T cells from MPN MNC resulted in an increase in colony formation in 20 out of 51 MPN patient samples (containing an equal distribution of polycythemia vera, essential thrombocythemia, and myelofibrosis). From JAK2V617F-positive MPN patients, colonies were plucked and genotyped for JAK2V617F mutational status. The fraction of JAK2WT colonies increased with removal of T cells, demonstrating that JAK2WT progenitors are more sensitive to T cell mediated suppression as compared to their JAK2V617F counterparts. The observation that removal of T cells enhances myeloid colony forming ability of some MPN patients suggests that T mediated suppression of hematopoiesis may be occurring in MPN. Next, we tested whether MPN T cells have the ability to suppress colony formation of normal hematopoietic progenitors. CD3+ T cells from MPN (n=2) and normal controls (n=2) were added to normal control mobilized peripheral blood CD34+ cells (n=3). Each CD34+ sample was plated in methylcellulose by itself, with the addition of 100,000 MPN T cells/1.1ml or with the addition of 100,000 normal control T cells/1.1ml. For each CD34+ sample a unique MPN patient and normal control was used as the source of T cells. Addition of MPN T cells reduced colony formation in one of three CD34+ samples, whereas normal control T cells enhanced colony formation of all three CD34+ samples. In the remaining two CD34+ samples MPN T cells had an attenuated ability to enhance colony formation as compared to the normal control T cells. Conclusion: T-cell mediated suppression of myelopoiesis in vitro is demonstrable in MPN and may be a contributor to the anemia associated with myelofibrosis. Taken together, these data provide the rationale for further investigation into T mediated suppression of hematopoietic progenitors in MPN patients. Disclosures No relevant conflicts of interest to declare.

The Expression and Mechanism of Tim3 and PD-1 in Patients with Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4948-4948
Author(s):  
Caixia Li ◽  
Xiao Yu ◽  
Caihong Gu ◽  
Chao Ma ◽  
Hong Liu ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Myeloid Leukemia ◽  
Immune Escape ◽  
Heparin Anticoagulation ◽  
Ifn Γ ◽  
Normal Controls ◽  
Acute Myeloid

Abstract Objective: To investigatethe expression and the role of Tim3 and PD-1 on T cells of peripheral blood in patients with acute myeloid leukemia(AML). Consistently discuss the clinical significance of Tim3 and PD-1 co-expression on these cells and the mechanism of Tim3-mediated immune escape. Methods: Collectedclinical data of 36 patients who had been diagnosed with AML by bone marrow MICM classification, and then gathered their heparin anticoagulation peripheral blood before any treatment. The heparin anticoagulation peripheral blood from 20 healthy volunteers was gathered as normal control. We used the methods of immune fluorescence labeling and flow cytometry to detect expression of Tim3 on T cells. Then detected co-expression of Tim3 and PD-1 on T cells, consistently with IFN-γ secreting level on T cells. Results: 1.The proportions of CD4+ T cells and CD8+ T cells on lymphocytes in AML patients were (15.28±10.99)% and (9.19±7.54)% respectively, which were significantly lower than the proportion of normal controls [(31.12±2.22)% and (21.59±4.22)% respectively] (P<0.05). 2.The levels of Tim3 expression on CD4+ T cells and CD8+ T cells in AML patients were (4.77±3.560)% and (5.90±4.91)% respectively, which were significantly higher than the levels of normal controls [(0.73±0.62)% and (0.96±0.54)% respectively] (P<0.05). 3.Tim3 and PD-1 were co-expression on CD4+ T cells and CD8+ T cells in AML patients. 4.The IFN-γ secreting level in Tim3+ CD8+ T cells was significantly decreased than Tim3- CD8+ T cells (P<0.05). Conclusion: 1.The high expression of Tim3 on peripheral blood T cells in AML patients mediate T cell exhaustion/dysfunction, which can be an important mechanism of immune escape in leukemia. 2.PD-1 and Tim3 co-expression On CD4+ T cells and CD8+ T cells in AML patients which were dificient in there ability to produce IFN-γ. Further investigations are needed to explore the correlation of co-expression PD1 and Tim3 with process of AML, thus probably making Tim3 become another new immunotherapy target. Disclosures No relevant conflicts of interest to declare.

Decreased TIM-3 Expression Level of Peripheral Blood Natural Killer Cells in Severe Aplastic Anemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5078-5078
Author(s):  
Rong Fu ◽  
Tian Zhang ◽  
Xin Yuan ◽  
Zonghong Shao
Keyword(s):  
T Cells ◽  
Aplastic Anemia ◽  
Nk Cells ◽  
Mrna Expression ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Statistical Significance ◽  
Severe Aplastic Anemia ◽  
Hematopoietic Stem ◽  
Normal Controls

Abstract Severe aplastic anemia (SAA) is a life threatening disease characterized by severe pancytopenia and bone marrow failure. Natural killer (NK) cells are large granular lymphocytes which are one important component of the innate immune system and play a core role in regulation of adaptive immunity. T cell immunoglobulin mucin-3 (TIM-3), a member of the TIM family, appears to play an important negative regulatory role in T cells initial. Now, TIM-3 is widely detected on NK cells, and may contribute as a marker for activation and maturation of NK cells. Our previous studies have confirmed that the decrease of total NK cells, and CD56bright, CD56dim NK cell subsets and the higher expressions of NKp46 and perforin on NK cells may cause the over-function of T lymphocytes and thus lead to hematopoiesis failure in SAA. But, the expression of TIM-3 on NK cells in patients with SAA was still unknown. In this study, we investigated the expression of TIM-3 and its mRNA on NK cells in peripheral blood of untreated and recovered SAA patients by flow cytometry and Real-time PCR. Results showed that the expression of TIM-3 on peripheral blood NK cells in SAA patients before IST was (63.57±12.14) %, which was significantly decreased than that in normal controls (85.62±9.03) % ( p<0.01). The expression of TIM-3 on CD56dim NK cells was (66.41±11.74) % and (83.83±1.59) % separately in SAA patients before IST and normal controls. TIM-3 expressed in SAA patients before IST was lower than that in normal controls ( p<0.01). We also measured TIM-3 expressed on the surface of CD56bright NK cells, but the result showed that there was no statistical difference between SAA patients before IST (61.11±24.99%) and normal controls (62.64±12.06%) (p>0.05). More interesting, the expression of TIM-3 on NK cells was (75.88±12.83) % in SAA patients after IST, which was significantly increased than that in SAA patients before IST, and was no difference with normal controls. As well, we found TIM-3 expression on both CD56dim NK cells and CD56bright NK cells in SAA patients after IST also has a rising trend compared with SAA patients before IST. However, these differences had no statistical significance. Further, we detected TIM-3 mRNA expression in NK cells isolated from peripheral blood. TIM-3 mRNA expression in peripheral blood NK cells from newly diagnosis SAA patients, recovering SAA patients and normal controls was evaluated, respectively. The relative TIM-3 mRNA expression was significantly increased in NK cells in SAA patients after IST compared with SAA patients before IST and normal controls, and this difference had statistical significance. Meanwhile, the relative TIM-3 mRNA expression was lower in NK cells in SAA patients before IST compared with normal controls. However, the difference had no statistical significance. In SAA patients, the expression of TIM-3 on NK cells was positively correlated with the level of WBC (r=0.685, p=0.000), proportion of neutrophil (r=0.825, p=0.000), and proportion of reticulocyte in peripheral blood (r=0.465, p=0.029). And it was negatively correlated with the proportion of lymphocyte in peripheral blood (r=-0.802, p=0.000). According to our series studies, we hypothesize that the lower numbers and dysfunction of NK cells induce the failure of suppressing the over function of DC cells and T cells, that lead damage to healthy hematopoietic stem cells and the onset of SAA. Disclosures No relevant conflicts of interest to declare.

scholarly journals Insufficient Phosphorylation of STAT5 in Tregs Inhibits the Expression of BLIMP-1, Leading to a Lack of Tregs in Pediatric Aplastic Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2183-2183
Author(s):  
Lifen Huang ◽  
Junbin Huang ◽  
Chengming Zhu ◽  
Hongman Xue ◽  
Chi Kong Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Inflammatory Cytokines ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Healthy Controls ◽  
Healthy Donors ◽  
Ifn Γ ◽  
Low Expression

Abstract Aplastic anemia (AA) is a group of bone marrow failure diseases characterized by three-line blood cell reduction and decreased myeloproliferation. It is believed that T cell immune disorder is the leading cause of the disease, especially the number and functional damage of regulatory T cells (Tregs). BLIMP-1 is a transcription factor encoded by PRDM1 gene, which is indispensable for Tregs. The expression of BLIMP-1 is mainly induced by the IL-2/STAT5 signaling pathway. However, the level of phosphorylation of STAT5 and the expression of BLIMP-1 in Tregs from patients with AA has not been revealed, and the mechanism of Tregs damage in AA has not yet been clarified. In the present study, we collected peripheral blood from 10 newly diagnosed AA children and 10 age-matched healthy donors. We observed that the ratio of Tregs/lymphocytes and Tregs/CD4 + T cells decreased significantly in AA patients, compared with healthy controls by flow cytometry. In addition, we found significantly elevated levels of inflammatory cytokines IL-2, IL-6, and IFN-γ, but decreased levels of anti-inflammatory cytokines IL-10 and TGF-β in plasma of children with AA, compared with healthy controls. Quantitative real-time PCR showed decreased transcriptional level of BLIMP-1 in peripheral blood mononuclear cells (PBMC) from children with AA, compared with healthy donors. We used flow cytometry to detect the protein level of BLIMP-1 in Tregs and found that the level of BLIMP-1 in Tregs in the peripheral blood of children with AA was significantly lower than that of healthy donors. The correlation analysis showed that the percentage of BLIMP-1 + Tregs was positively correlated with the ratio of Tregs/CD4 + T cells (r=0.829, p＜0.001), the plasma level of IL-10 (r=0.492, p=0.027), and TGF-β (r=0.482, p=0.030), suggesting that low expression level of BLIMP-1 in Tregs may lead to decreased number of Tregs in peripheral blood and declined levels of IL-10 and TGF-β in children with AA. When stimulated IL-2, the level of pSTAT5 in CD4 + T cells of children with AA was significantly reduced compared with that of healthy donors. The level of pSTAT5 in CD4 + T cells was also positively correlated with the ratio of Tregs/CD4 + T cells (r= 0.575, p= 0.008) and the expression of BLIMP-1 in Tregs (r=0.693, p＜0.0001)，suggesting that STAT5 signal is poorly activated in pediatric AA, and it may be an important cause for the low expression of BLIMP-1 in Tregs and the decrease in the number of Tregs in children with AA. Furthermore, we constructed an AA mouse model by co-administering IFN-γ and busulfan for 10 consecutive days. These mice exhibited decreased ratio of Tregs/CD4 +T cells in the spleen and lower BLIMP-1 in Tregs, compared with controls. Also, we isolated Tregs with immunomagnetic beads from spleens of mice and observed that the level of IL-2-stimulated pSTAT5 was lower in isolated Tregs from AA mice than controls. These phenotypes were partially rescued by intervention of IL-2-JES6-1, an antibody complex tends to promote the proliferation of Tregs in mice, while inhibiting the proliferation of effector T cells. IL-2-JES6-1 increased the level of pSTAT5 and the expression of BLIMP-1 in Tregs from spleen of the AA mice, and elevated the ratio of Tregs/CD4 + T cells in the spleen. In conclusion, we found that Tregs from AA patients have impaired phosphorylation of STAT5 and insufficient expression of BLIMP-1, which may contribute to the pathogenesis of AA. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circulating T Cells Exhibit Different TIM3/Galectin-9 Expression in Patients with Obesity and Obesity-Related Diabetes

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Lili Sun ◽  
Shengyi Zou ◽  
Sisi Ding ◽  
Xuan Du ◽  
Yu Shen ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Healthy Controls ◽  
Fasting Insulin ◽  
Blood Samples ◽  
Healthy Donors ◽  
Simple Obesity ◽  
Circulating T Cells

Aims. Obesity is highly associated with type 2 diabetes mellitus (T2DM). The TIM3/galectin-9 pathway plays an important role in immune tolerance. Herein, we aimed to investigate the expression of TIM3 and galectin-9 in peripheral blood and to evaluate their clinical significance in patients with obesity and obesity-related T2DM. Methods. We performed flow cytometry on peripheral blood samples from healthy donors (HC), patients with simple obesity (OB), and patients with obesity comorbid T2DM (OD). The expression of TIM3 on CD3+, CD4+, and CD8+ T cells was determined. The level of galectin-9 in plasma was detected by ELISA. Results. We demonstrated the enhancement of TIM3 on CD3+, CD4+, and CD8+ T cells in the OB group when compared with healthy controls, while it was decreased significantly in the OD group. The TIM3+CD8+ T cells of the OB group were positively correlated with risk factors including BMI, body fat rate, and hipline. The concentration of galectin-9 of the OD group in plasma was significantly higher than that of healthy donors and the OB group. Moreover, the level of galectin-9 of the OD group was positively correlated with fasting insulin and C-peptide, which were two clinical features that represented pancreatic islet function in T2DM. Conclusions. Our results suggested that TIM3 and galectin-9 may be potential biomarkers related to the pathogenesis of obesity-related T2DM.

Changes of Intracellular Signal Pathway of mTOR/S6 in T Cells of Refractory/Relapsed Aplastic Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4110-4110
Author(s):  
Guangsheng He ◽  
Xiang Zhang ◽  
Wu Depei ◽  
Mingqing Zhu ◽  
Aining Sun
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Aplastic Anemia ◽  
Cd8 T Cells ◽  
Signal Pathway ◽  
Deficiency Anemia ◽  
Newly Diagnosed ◽  
Ifn Γ ◽  
Intracellular Signal ◽  
Normal Controls

Abstract Objects: To explore the activation state of intracellular signal pathway of mTOR/S6 in refractory/relapsed aplastic anemia (AA); the effect of rapamycin (RAPA) and CTLA-4 immunoglobulin (CTLA-4Ig) on this signal pathway in refractory/relapsed AA. Methods: Samples were collected from 13 refractory/relapsed AA patients [male: 6, female: 7, media age: 27 years (from 7 to 66years)], 8 newly diagnosed patients with severe aplastic anemia (SAA) [male: 5, female: 3, media age: 21.5 years (from 12 to 58 years)]. The intracellular percentages of p-mTOR, p-S6 and IFN-γ of CD3+, CD3+CD8+ and CD3+CD8− T cells in bone marrow were detected by flow cytometry (FCM). 10 iron deficiency anemia (IDA) patients [male: 3, female: 7, media age: 44 years (from 31 to 72 years)] were determined as case controls and normal controls respectively. After exposure to RAPA and CTLA-4Ig respectively, samples were detected by FCM for the expression of p-mTOR, p-S6 and IFN-γ in CD3+, CD3+CD8+ and CD3+CD8− T cells in bone marrow, in order to estimate the effect of RAPA and CTLA-4Ig on the pathway of mTOR/S6 in refractory/relapsed AA. Results: In refractory/relapsed AA, measurement of p-mTOR, p-S6 and IFN-γ in CD3+, CD3+CD8− and CD3+CD8+ T cells were (42.42±26.44)%, (44.38±24.95)%, (51.89±27.00)%; (6.47±2.72)%, (9.16±2.89)%, (9.61±5.34)%; (18.87±10.05)%, (13.17±5.88)%, (20.07±15.16)%, respectively; and showed an increased level compared to normal controls [(1.54±1.51)%; P=0.000], [(1.94±1.08)%; P=0.000], [(2.04±2.03)%; P=0.000]; [(0.83±0.82)%; P=0.000], [(0.91±0.88)%; P=0.000], [(0.95±0.93)%; P=0.000]; [(4.42±3.55)%; P=0.000], [(2.35±1.69)%; P=0.000], [(4.73±4.43)%; P=0.004]. In newly diagnosed patients with SAA, the levels of p-mTOR and p-S6 in CD3+, CD3+CD8− and CD3+CD8+ T cells were (1.71±1.66)%, (2.28±2.15)%, (1.59±1.52)%; (1.23±1.13)%, (1.23±1.07)%, (1.76±1.68)% respectively, and they were similar to normal controls (P&gt;0.05), but significantly lower than those of refractory/relapsed AA (P&lt;0.01). Expression of IFN-γ in CD3+, CD3+CD8− and CD3+CD8+ T cells was higher than normal controls with (10.38±3.83)%, (6.11±1.91)%, (13.14±7.05)% (P&lt;0.01). The percentages of CD3+IFN-γ+ and CD3+CD8−IFN-γ+ were lower than refractory/relapsed AA (P&lt;0.05), while it was comparable for CD3+CD8+IFN-γ+ cells between the two groups (P&gt;0.05). Exposed to RAPA, the expression of p-mTOR, p-S6 and IFN-γ in CD3+, CD3+CD8− and CD3+CD8+ T cells decreased markedly (P&lt;0.05) to (12.44±12.41)% (12.60±12.57)%, (16.85±15.64)%; (1.49±1.45)%, (1.46±1.43)%, (1.55±1.54)%; and (4.29±4.23)%, (3.16±3.32)%, (10.70±10.63)% in refractory/relapsed AA. And treated with CTLA-4Ig could also cause a significant reduction of p-mTOR, p-S6 and IFN-γ in CD3+, CD3+CD8− and CD3+CD8+ T cells, which were (6.40±6.13)%, (8.32±7.76)%, (7.18±7.02)%; (1.08±1.08)%, (2.69±2.37)%, (1.60±1.56)%; (1.67±1.60)%, (2.39±2.12)%, (1.30±1.30)%, respectively, (P&lt;0.01). Conclusions: In refractory/relapsed AA, the signal pathway of mTOR/S6 was activated, and it was quiescent in normal controls and newly diagnosed patients with SAA. The expression of p-mTOR, p-S6 and IFN-γ of this signal pathway in refractory/relapsed AA could be suppressed by RAPA or CTLA-4Ig. The signal pathway of CD28/mTOR/S6/IFN-γ might take part in immune pathogenesis of refractory/relapsed AA, and was sensitive to RAPA and CTLA-4Ig. It was worth exploring the clinical values of the two drugs.

High Frequency of Circulating CD8+ Memory Stem T Cells in Acquired Aplastic Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3613-3613
Author(s):  
Kohei Hosokawa ◽  
Pawel Muranski ◽  
Xingmin Feng ◽  
Danielle M. Townsley ◽  
Baoying Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autoimmune Diseases ◽  
Aplastic Anemia ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Memory T Cells ◽  
Healthy Controls ◽  
Hematopoietic Stem ◽  
Ifn Γ

Abstract Background. Aplastic anemia (AA), the prototypical bone marrow (BM) failure syndrome, is caused by immune-mediated destruction of hematopoietic stem/progenitor cells (HSPCs). CD8+ cytotoxic T cells with restricted TCR diversity (oligoclonal T cells) are expanded in AA, leading to production of proinflammatory cytokines, such as IFN-γ, which induce apoptosis of HSPCs. Recent studies have identified a new subset of memory T cells with stem cell-like properties, TSCM, which are the least differentiated cells of all distinct memory populations. Functionally, TSCM possess an enhanced capacity for self-renewal and can generate multiple memory T cell populations, and they likely have an important role in controlling immunity. In autoimmune diseases, there is abnormal CD4+ and CD8+ T cell activation. We evaluated TSCM frequency in AA and its association with severity, treatment response, relapse, and changes after immunosuppressive therapy (IST). Further, to evaluate the TSCM in other autoimmune diseases, we examined CD4+ and CD8+ TSCM frequencies in uveitis, systemic lupus erythematosus (SLE), and sickle cell disease (SCD), as compared with healthy controls. Method. We retrospectively analyzed CD4+ and CD8+ TSCM populations by flow cytometry. PB specimens were collected from 55 AA samples and 41 age-matched healthy donor samples. Among 55 AA samples, 21 samples were analyzed at diagnosis and 34 after IST. For comparison, blood samples were obtained from 34 uveitis patients (27 inactive or 7 active cases), 43 SLE patients who met the American College of rheumatology (ACR) criteria for the disease [19 inactive SLE (SLE disease activity index-2K (SLEDAI-2K) score < 3; and 24 active SLE (SLEDAI-2K score > 3)], and 5 SCD patients who were receiving frequent transfusions. TSCM was defined as CD3+ CD4 (CD8)+ CD45RO- CD45RA+ CCR7+ CD27+ CD95+ population. Results and Discussion. In healthy controls, TSCM represented a relatively small percentage of circulating CD4+ or CD8+ T cells (median 2.4% CD4+ TSCM and 2.1% CD8+ TSCM, Fig. 1A). A significantly higher CD8+ TSCM frequency was detected in AA patients (4.2% vs. 2.1%, p < 0.05) while there was no difference in the CD4+ TSCM frequency (p > 0.05), compared to controls (Fig. 1B-C). In AA, high CD8+ TSCM frequency at diagnosis correlated with complete (CR) or partial response (PR) to IST [5.0 % in CR and PR vs 2.8 % in non-responders (NR), p < 0.05). In AA patients prior to IST (n=21), CD8+ TSCM frequency was not correlated with age, sex, absolute neutrophil count, platelet count, time from diagnosis to therapy, and serum ferritin levels. CD8+ TSCM were significantly increased in the two AA cohorts (with or without IST), relative to controls (p < 0.05, respectively). Higher CD8+ TSCM frequency after IST associated with treatment-failure (3.5 % in responders vs 5.5 % in NR or relapse, p < 0.05). Stimulation with anti-CD3/CD28 beads successfully induced cytokine production in CD4+ and CD8+ T cells from AA and healthy controls. Elevated IFN- γ and IL-2 levels were seen in CD4+ and CD8+ TSCM in AA compared to healthy controls. We next compared CD4+ or CD8+ TSCM frequency between each patient group (AA, uveitis, SLE, or SCD) and a healthy control group. Among the four patient groups, the uveitis group alone displayed a reduction in CD4+ TSCM frequency (1.8%) relative to the healthy controls (2.4 %; p < 0.05). An elevated CD8+ TSCM frequency was observed in AA (4.2 %), uveitis (3.6 %), and SCD (4.3 %), but not in SLE, compared to controls (2.1%; p < 0.05) (Fig. 2A). Positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and SLE (Fig. 2B). Evaluation of PD-1 expression revealed that TSCM were the least exhausted T cell compartment, as compared to other types of memory T cells. Immune therapies appeared to have negative effects on the TSCM population both in uveitis and SLE patient cohorts, as well as in AA. Conclusion. We provide evidence for increased circulating CD8+ TSCM in AA, underscoring the importance of this novel subset in regulation of immune responses and pathogenesis of autoimmunity. Our work described previously unknown potential roles of TSCM in AA, such as cytokine secretion correlated with effector functions. Understanding the CD8+ TSCM population may offer new therapeutic strategies and novel mechanistic insight into the various autoimmune diseases. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

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