Tian Zhang

CD8+HLA-DR+ T cells are increased in patients with severe aplastic anemia

The aim of the present study was to investigate the number and function of CD8+HLA-DR+ cells, which are considered to be activated cytotoxic T lymphocytes (CTLs), in peripheral blood to further examine the pathogenesis of severe aplastic anemia (SAA). Thirty-eight patients with SAA were included in the present study. Patients were screened for paroxysmal nocturnal hemoglobinuria by flow cytometry using anti-CD55 and anti-CD59 antibodies. The number of CD8+HLA-DR+ T cells was measured by three-color flow cytometry using anti-CD8-peridinin chlorophyll, anti-CD3-fluorescein isothiocyanate (FITC) and anti-HLA-DR-FITC antibodies. The expression of perforin, granzyme B, tumor necrosis factor-β (TNF-β) and FasL in CD8+HLA-DR+ T cells was detected by flow cytometry with the appropriate monoclonal antibodies. Total RNA was prepared from purified CD8+HLA-DR+ cells of healthy controls and SAA patients, and then polymerase chain reaction (PCR) was performed. Apoptosis of CD8+HLA-DR+ cells was detected by flow cytometry following staining with Annexin V. The proportion of CD8+HLA-DR+ T cells was analyzed by flow cytometry in peripheral blood and was identified to be significantly higher in untreated SAA than in remission patients and in the controls. The expression of perforin, granzyme B, TNF-β and FasL in CD8+HLA-DR+ T cells was analyzed by flow cytometry and PCR, which revealed increased expression in the untreated SAA group compared with that in the control group. Furthermore, the apoptosis of CD3- bone marrow cells from normal individuals was enhanced following co-culture with CD8+HLA-DR+ T cells from untreated SAA patients. In conclusion, the present study demonstrated that CD8+HLA-DR+ T cells may contribute to bone marrow failure in SAA.

Abnormalities of quantities and functions of natural killer cells in severe aplastic anemia

Severe aplastic anemia (SAA) is a rare disease characterized by severe pancytopenia and bone marrow failure. Natural killer (NK) cells are large granular lymphocytes derived from hematopoietic stem cells (HSCs) or common lymphoid progenitors (CLP). They play a key role in n the innate immunity and adaptive immune. In this study, the quantitative and functional changes of natural killer (NK) cell subsets in peripheral blood of severe aplastic anemia (SAA) patients before and after immunosuppressive therapy (IST) were investigated. Results showed that the percentage of NK cells and its subsets in peripheral blood lymphocytes was decreased in SAA patients. After IST, the percentage of NK cells and their subsets increased dramatically. The median expressions of CD158a, NKG2D and NKp46 on NK cells were higher in SAA patients compared to that in normal controls, and the expressions of perforin in newly diagnosed and recovery SAA patients were higher than that in controls. Therefore, we concluded 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.

The clinical characteristics and therapy response of patients with acquired pure red cell aplasia

ABSTRACT Objective: To summarize the clinical characteristics of acquired pure red cell aplasia (PRCA) patients diagnosed in our hospital in the last 10 years. Method: The clinical features, immune state and treatment response of acquired PRCA patients diagnosed in our hospital from January 2007 to January 2017 were retrospectively analyzed. Results: The results showed that thymoma (13.21%) and parvovirus B19 (11.32%) were the most common causes for secondary PRCA. Ferritin (Fer) levels and erythropoietin (EPO) levels were increased in PRCA patients. The total CR and PR rate of immunosuppressive therapy in our studies was 68.29% and 12.20%, respectively. Patients with EPO level >400 U/L and Fer level >200 ng/ml had significantly lower CR rate than others. The patients with EPO level >400 U/L also had longer hemoglobin recovery time than patients with EPO level ≤400 U/L. Patients treated with corticosteroids (CS) + cyclosporine A (CsA) had lower relapse rate compared to the CS group (29.17% vs. 80.00%, P < .05). Conclusion: Our data showed that patients with PRCA had high EPO and Fer levels. Thymoma and viral infections are the most common causes for secondary PRCA. The CS+ CsA group had lower relapse rate than CS group although response rate was similar. Increased EPO and Fer levels might be the negative factors for prognosis of acquired PRCA.

NK Cells Suppress CD8+ T Cell Immunity via NKG2D in Severe Aplastic Anemia

The roles of natural killer (NK) cells in shaping the immune system had raised wide interests. Here we intended to explore the regulatory functions of NK cells on CD8+ T cells in severe aplastic anemia (SAA) using human participants and lymphocyte infusion-induced bone marrow failure (BMF) mouse model. In SAA patients, NK cells had over-expressions of NKG2D and NKp46, under-expression of NKG2A and enhanced cytotoxicity. NK cells limited autologous CD8+ T cell immunity in an effector/target ratio manner. The suppression was dependent on the existence of NKG2D. We also observed upregulated MICA expression on activated CD8+ T cells, which were susceptible to NK cell mediated lysis in SAA. Animal model concurred with the data from patients. Infusion of NK cells suppressed the proliferation of CD8+ T cells and decreased IFN-γ production. In conclusion, NK cells served NKG2D-dependent immunoregulatory roles by attenuating autologous CD8+ T cell response in SAA.

Epstein Barr Virus Infection Affects Function of Cytotoxic T Lymphocytes in Patients with Severe Aplastic Anemia

Severe aplastic anemia (SAA) is characterized by pancytopenia and failure of hematopoietic function and is caused by excessive functioning of cytotoxic T lymphocytes (CTLs). EBNA-1, a nucleoprotein of the Epstein Barr virus (EBV), can influence the proliferation and function of lymphocytes. We therefore tested the number of EBV copies in the CD8+ T cells of 27 patients with SAA and 10 healthy control subjects and observed the influences of EBNA-1 upon the CD8+ T cells of patients with SAA. The results showed that more EBV copies were found in the CD8+ T cells of patients with untreated SAA than in patients with SAA in remission or in the healthy control subjects. Their copy number was positively correlated with the expression of granzyme B and perforin, the secretion level of interferon-γ in CD8+ T cells, and the viability of CD8+ T cells, whereas no correlation was seen between the copy number and the interleukin 4 secretion level or the apoptosis rate. Meanwhile, the expression of granzyme B and perforin was reduced after EBNA-1 gene knockdown, whereas the interferon-γ secretion level and cell viability declined. Therefore, we infer that EBV infection may be a factor in the activation of CTLs and in damaging the bone marrow hematopoietic function of patients with SAA.

TRAIL in CD8+ T cells from patients with severe aplastic anemia

Severe aplastic anemia (SAA) is an autoimmune disease caused mainly by activated T lymphocytes. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of TNF family, which can induce apoptosis and play a significant role in the pathogenesis of many autoimmune disorders. In this study, we sought to investigate the role of TRAIL in peripheral CD8+ T cells (CTLs) from SAA patients to clarify the autoimmune mechanisms of bone marrow failure in SAA. The expression of TRAIL and TRAIL-R2 in CTLs from SAA patients and normal controls were determined by flow cytometry, real-time PCR, and western blot. Expression of perforin and granzyme B and apoptosis in CTLs were evaluated by flow cytometry. The expression of TRAIL and TRAIL-R2 in SAA patients was significantly decreased compared with controls; however, there was no statistical difference in TRAIL mRNA expression between the two groups. TRAIL expression in CTLs was negatively correlated with the expression of perforin and granzyme B, and negatively correlated with CTLs apoptosis in SAA patients. The TRAIL pathway may be responsible for abnormal CTL activation in SAA patients. Further study of TRAIL and its receptors may elucidate the pathogenesis of SAA.

Decreased TIM-3 expression of peripheral blood natural killer cells in patients with severe aplastic anemia.

Severe aplastic anemia (SAA) is an autoimmune disease characterized by severe pancytopenia and bone marrow failure. In our previous studies, we found natural killer (NK) cells were aberrant in SAA patients. T cell immunoglobulin mucin-3 (TIM-3), an important regulator of immunity, is widely detected on NK cells and may contribute as a marker of activation and maturation of NK cells. In this study, we found that SAA untreated patients had lower TIM-3 expression on NK cells and CD56dim NK subsets compared with normal controls, and were correlated with the severity of pancytopenia of SAA. After immunosuppressive therapy (IST), TIM-3 expression recovered to normal level. Moreover, the TIM-3 mRNA levels in NK cells significantly increased in SAA remission patients after IST. We inferred that low expression of TIM-3 on NK cells might lead to NK cells dysfunction and involve in the progress of bone marrow failure in SAA.

Hematogones: a sensitive prognostic factor for Chinese adult patients with acute myeloid leukemia.

BACKGROUND Hematogones (hgs) are normal B-lymphocyte precursors that increase in some hematologic diseases. Many studies indicate that hgs might be a favourable prognostic factor. We thus considered it important to determine whether hgs are also a prognostic factor for Chinese adult patients with acute myeloid leukemia (aml) and whether the hg-positive and hg-negative groups show any serologic or phenotypic differences. METHODS Chinese adult aml patients (n = 177) who were all initially hg-negative underwent standard chemotherapy and were thereafter divided into hg-positive and hg-negative groups according to hg levels in bone marrow during their first remission. RESULTS The follow-up study confirmed that survival duration (both leukemia-free and overall) was significantly greater in the hg-positive group than in the hg-negative group and was accompanied by a lower relapse rate. A retrospective study of patient characteristics at the time of first diagnosis revealed some differences between the hg-positive and the hg-negative groups, including elevations in white blood cells, lactate dehydrogenase, and β2-microglobulin in the hg-negative group. Retrospective phenotypic analysis revealed a significantly lower proportion of abnormal chromosome karyotype and CD34 expression in hg-positive patients. Finally, we evaluated whether additional intensive chemotherapy after standard chemotherapy could further increase hgs. CONCLUSIONS The present work verified the validity of hgs as a prognostic factor for Chinese adult patients with aml. Compared with hg-negative patients, hg-positive patients not only experienced longer survival and a lower relapse rate, but they also had some serologic and phenotypic characteristics that are all considered indicators of better outcome. Additional intensive chemotherapy could further increase the level of hgs, which might imply better clinical results.

A case of lung cancer with first signs of hematological manifestations

A 48-year-old man presented with a one-week history of palpitation, fatigue, and epistaxis. Streaky hemoptysis occurred occasionally, and decreased muscular strength of the left extremities was found. The cranial computer tomography (CT) taken at a local hospital indicated infarction of the right basal ganglia and temporoparietal lobe. Physical examination found T37.5°C, HR112bpm, Bp120/80 mmHg, and R20bpm. The patient looked pale, with petechia on the lower limbs. The muscular strength of the left extremities was grade IV, with positive Babinski signs on both sides. A routine blood test showed decreased counts of hemoglobin (81 g/L) and platelets (13 ¥ 10/L). Coagulation function showed an increased level of D-Dimer and a decreased level of fibrinogen. An electrolytes test indicated hypercalcemia. The liver function examination indicated elevated levels of aminotransferases. Urine routine was normal, while fecal occult blood test was positive. Bone marrow puncture was performed in order to find the cause of cytopenia. A hypoplastic marrow was indicated with a decreased ratio of granulocytic series and normal percentage of erythroid series. Platelet counts were decreased and no megakaryocytes were seen. Piles of irregular shaped cells were seen throughout the smear, especially at the end of the smear. The piled cells were in different sizes with round or irregular nuclei, coarse chromatin, obscure nucleoli, and dusty blue cytoplasm with vacuoles. Peripheral blood smear showed decreased granulocytes with immature cells occasionally seen. Thirty erythroblasts were counted among a hundred white blood cells. The impression diagnosis of marrow smear was: (i) marrow metastasis of extramedullary neoplasms; and (ii) lymphoma to be excluded (see Fig 1). Further examinations were performed to seek the existence of primary tumors. Chest CT showed soft tissue density shadow in the area of the right hilus pulmonis. Parts of the bronchi became narrow. Patchy consolidation shadow was seen at the dorsal segment of the inferior lobe of right lung, and interstitial lung markings were increased. Multiple mediastinal lymph nodes were markedly enlarged, and low-density focuses were seen in the liver (see Fig 2). Tumor markers were subsequently detected showing elevated levels of carbohydrate antigen 19-9, neurons enolase enzymes and cell keratin 19 pieces. As a result of these laboratory findings, a diagnosis of small cell lung cancer (SCLC) with bone marrow metastatic tumor was made. The patient refused chemotherapy, therefore, supportive treatments were given. However, the patient failed to respond well to platelet transfusion, and the platelet count ranged between 7 ¥ 10/L and 10 ¥ 10/L. Hemorrhage symptoms were aggravated. He suffered from continuous low fever and ache all over. Finally the patient abandoned treatment and died two weeks later. Cancer patients, especially those in late stage, may display various hematological manifestations, such as cytopenia and coagulation dysfunction. Mechanisms of cytopenia include deficiency of hematopoietic raw materials, marrow infiltration of tumor cells, anemia of chronic disease, microangiopathic hemolytic anemia, bone marrow fibrosis, and inhibited hematopoiesis as a result of chemotherapy. Thrombophilia presented by cancer patients is considered to be associated with abnormal blood flow, impaired vascular integrity, and changes of blood components. Li Xiao et al. analyzed bone marrow biopsies of 10112 patients with hematological diseases, and found 101 of them (about 1%) were marrow metastasis of non-hematological tumors, among which lung cancer was most frequently seen. The patient in this case presented with a right pulmonary hilar mass with elevated neurons enolase enzymes, which supported the diagnosis of SCLC. The piles of irregular shaped cells in the marrow smear, as well as the resulting cytopenia, were typical signs of marrow infiltration of extramedullary neoplasms. Primary bronchogenic carcinoma (abbr. lung cancer) is one of the most commonly seen malignant tumors. Small cell lung cancer (SCLC) accounts for 15–20% of lung cancers, and displays a higher tendency of marrow metastasis in its early Correspondence Zonghong Shao, Hematology Department, General Hospital, Tianjin Medical University, 154 Anshandao, Tianjin 300052, China. Tel: +86 2260362085 Fax: +86 2260362086 E-mail: shaozonghong@sina.com