Repopulation of circulating T, B and NK lymphocytes following bone marrow and blood stem cell transplantation

Abstract. A variety of T, B and natural killer (NK) cell subsets defined by surface markers were analyzed by double immunofluorescence flow cytometry in the peripheral blood of patients following autologous bone marrow transplantation (ABMT, n=14), autologous peripheral blood stem cell transplantation (PBSCT, n=10) and allogeneic bone marrow transplantation (allo-BMT, n=6). Patients following ABMT were divided in 2 groups, those who did not received G-CSF post-transplant (ABMT, n=6) and those who did (ABMT+G, n=8). All patients following PBSCT or allo BMT received G-CSF. In all the groups prolonged significant decreases with respect to normal numbers were observed for the T CD3+, CD2+ and CD25+ subsets, more profound for the CD4+ subset but less for the CD8+ subset, especially following PBSCT (only decreased at 1 month). A significant expansion of the CD3+CD57+ and CD8+CD57+ phenotypes was noticed between 9 and 12 months following ABMT, the group of longer follow-up. Long-lasting expansion of the NK-like CD3+CD56+ and CD3+CD16+ subsets was also observed. The B CD19+ and CD20+ subsets had a significant overexpression from 4 months after ABMT, showing a normally balanced Igk+ : Ig1+ ratio. Concordantly, the HLA-DR+ and HLA-DQ+ subsets showed significant increases. The NK CD56+ and CD16+ subsets had a faster recovery than the T or B subsets in all the groups. However, the CD3-CD56+, CD3-CD16+, CD16+CD56+, CD3-CD8+, and especially the CD3-CD57+, CD16+CD57+, and CD56+CD57+ subsets had a slower recovery than the global CD56+, CD16+, or CD57+ subsets. The biological and clinical implications of these findings are discussed.     

Repopulation of circulating T, B and NK lymphocytes following bone marrow and blood stem cell transplantation

Abstract. A variety of T, B and natural killer (NK) cell subsets defined by surface markers were analyzed by double immunofluorescence flow cytometry in the peripheral blood of patients following autologous bone marrow transplantation (ABMT, n=14), autologous peripheral blood stem cell transplantation (PBSCT, n=10) and allogeneic bone marrow transplantation (allo-BMT, n=6). Patients following ABMT were divided in 2 groups, those who did not received G-CSF post-transplant (ABMT, n=6) and those who did (ABMT+G, n=8). All patients following PBSCT or allo BMT received G-CSF. In all the groups prolonged significant decreases with respect to normal numbers were observed for the T CD3+, CD2+ and CD25+ subsets, more profound for the CD4+ subset but less for the CD8+ subset, especially following PBSCT (only decreased at 1 month). A significant expansion of the CD3+CD57+ and CD8+CD57+ phenotypes was noticed between 9 and 12 months following ABMT, the group of longer follow-up. Long-lasting expansion of the NK-like CD3+CD56+ and CD3+CD16+ subsets was also observed. The B CD19+ and CD20+ subsets had a significant overexpression from 4 months after ABMT, showing a normally balanced Igk+ : Ig1+ ratio. Concordantly, the HLA-DR+ and HLA-DQ+ subsets showed significant increases. The NK CD56+ and CD16+ subsets had a faster recovery than the T or B subsets in all the groups. However, the CD3-CD56+, CD3-CD16+, CD16+CD56+, CD3-CD8+, and especially the CD3-CD57+, CD16+CD57+, and CD56+CD57+ subsets had a slower recovery than the global CD56+, CD16+, or CD57+ subsets. The biological and clinical implications of these findings are discussed.     

Monomorphic agranular natural killer cell lymphoma/leukemia with no Epstein-Barr virus association.

The conceptual view of natural killer (NK) cell malignancies has recently undergone a significant evolution. The majority of such diseases are associated with Epstein-Barr virus (EBV), while only a limited number of EBV-negative cases has been reported. We report an unusual case of NK cell lymphoma/leukemia showing a monomorphic histology, absence of intracytoplasmic azurophilic granules, and no EBV association. The patient was a 57-year-old woman who died 26 months after the diagnosis. Autopsy revealed tumor infiltration in the liver, spleen, lymph node, blood, and bone marrow. There was no involvement of the skin or nasal cavity throughout the clinical course. The tumor showed the monotonous proliferation of medium-sized cells without intracytoplasmic azurophilic granules. Phenotypic analysis showed CD2+, CD3/Leu4-, cytoplasmic CD3epsilon+, CD4-, CD5-, CD7+, CD8-, CD16-, CD38+, CD56+, CD57-, TdT-, granzyme B-, and TIA1+ phenotype. There were no detectable rearrangements of T cell receptor genes or immunoglobulin heavy chain genes. Furthermore, there were no EBV-encoded small RNAs. These findings provide information to improve the understanding of poorly defined entities, i.e. aggressive NK cell lymphoma/leukemia and blastic NK cell lymphoma/leukemia.     

Peripheral blood T,B, and NK cells in relation to histological hepatitis activity and fibrosis stage in chronic hepatitis C

BACKGROUND/AIMS: Many data on the pathogenesis of chronic hepatitis C have pointed to host's immune system disorders and a high variety of virus. However, there are no known criteria that could prognose the course of chronic hepatitis C infection. The analysis of T and B lymphocyte subpopulations in the peripheral blood was undertaken in patients with chronic hepatitis C of more than 6 months of duration. METHODOLOGY: Fluorescein isothiocyanate or phycoerythryne conjugated monoclonal antibodies for CD3+, CD4+, CD8+, CD19+, CD3++ HLA DR+, CD16++ CD56+ were used. The correlation between histological hepatitis activity and fibrosis (according Scheuer's scale) and the distribution of lymphocytes in the peripheral blood was sought. RESULTS: All patients with chronic hepatitis showed statistically significant increase in active lymphocytes CD3++ HLA DR+ and CD16++ CD56+ NK cells in peripheral blood. We observed the correlation between these cells and histological hepatitis activity and fibrosis. There was no correlation between the value of CD3+ and CD8+ cells and the stage of liver failure. In the early stage of chronic hepatitis C we noted decrease CD4+ cells with increase B cells CD19+. CD4+/CD8+ ratio was maintained as slightly decreased in chronic hepatitis C in favor of lymphocytes CD8+. CONCLUSIONS: The results show the correlation between peripheral blood value of activated T cell (HLA DR+) and NK cells with histological activity and fibrosis in chronic hepatitis C. Lymphocyte T (CD4+, CD8+) and B (CD19+) did not correlate with grade and stage of hepatitis C.     

Induction and characterization of cutaneous lymphocyte antigen on natural killer cells

Cutaneous lymphocyte antigen (CLA) has been reported to be expressed mainly by memory/effector T lymphocytes infiltrating inflammatory skin lesions and cutaneous T-cell lymphoma. It has been suggested that CLA is a specific homing receptor, facilitating the T-cell migration into skin lesions, and also an indicator of the skin-homing T-cell subset. In the present study, we investigated the expression of CLA in natural killer (NK) cells defined phenotypically as surface CD3- and CD56+ cells in peripheral blood. CLA was definitely expressed on CD3-CD56+ cells at a level comparable to CD3+ cells in peripheral blood of normal Japanese volunteers. After in vitro stimulation of peripheral blood mononuclear cells with interleukin 2 (IL-2) and IL-12, there was a significant increase in the number and percentage of CLA+ NK cells but not CLA+ T cells (P < 0.01). To analyse the characteristics of CLA expressed by NK cells, we investigated a CLA+ NK-leukaemia cell line, NK-YS, established from a patient with NK leukaemia/lymphoma with skin infiltration. In the in vitro study, the CLA-expressing NK-leukaemic cell line bound to E-selectin-transfected cells and was inhibited by HECA 452 antibody or neuraminidase treatment of leukaemic cells. These findings suggest that CLA expressed by NK cells is a homing receptor for the E-selectin molecule and may explain skin infiltration by NK cells and NK lymphoma cells analogous to T cells. An NK-cell subset expressing CLA must play an important role in host defence against microorganisms and neoplasms in skin lesions.     

中国乙型肝炎患者外周血淋巴细胞亚群频率参考值范围

目的 调查中国乙型肝炎患者外周血淋巴细胞亚群频率参考值范围.方法 利用流式细胞术检测2846例乙型肝炎患者和117例健康人群外周血淋巴细胞亚群数值,调查我国健康人群和乙型肝炎人群的参考值范围.结果 调查了16～60岁健康人群和HBV感染相关的急性肝炎、慢性肝炎、重型肝炎和肝硬化人群外周血CD3+T淋巴细胞、CD3+CD...     

Decrease in CD3-negative-CD8dim(+) and Vdelta2/Vgamma9 TcR+ peripheral blood lymphocyte counts,low perforin expression and the impairment of natural killer cell activity is associated with chronic hepatitis C virus infection

BACKGROUND/AIMS: As chronic hepatitis C virus (HCV) infection is associated with impaired natural killer (NK) cell cytotoxicity, we examined the phenotypes and perforin expression of peripheral blood lymphocytes, as well as the effect of interferon-alpha2b (IFN-alpha2b) therapy. METHODS: Thirty-three patients had chronic hepatitis C, and of them 12 had been on IFN-alpha2b treatment. Eleven individuals had been treated earlier with IFN-alpha2b and completely cured, and eight were HCV carriers with persistently normal serum alanine aminotransferase. Three-colour flow cytometry was used to measure the percentage of CD3(+/-)CD8+, CD3+CD4+, gammadeltaTcR+, Vdelta2 TcR+, Vgamma9 TcR+, Vdelta1 TcR+, CD3-CD16+, CD3-CD56+, CD19+ and perforin-positive cells. NK cell activity was assessed by single cell cytotoxic and flow cytometric assay. RESULTS: Patients with chronic hepatitis C showed an impaired NK cytotoxicity, decreased percentage of CD3-negative-CD8dim-positive (NK subtype) and Vgamma9/Vdelta2 TcR+ as well as perforin-positive T lymphocytes, compared to controls and to those who were cured from HCV infection. IFN-alpha2b increased NK cell cytotoxicity and the percentage of perforin-positive lymphocytes. CONCLUSIONS: Our findings suggest that in chronic HCV infection a decreased percentage of CD3(-)CD8+, Vgamma9/Vdelta2 TcR+ and perforin-positive T cells and simultaneous decreased peripheral NK activity may contribute to the impaired cellular immune response and the chronicity of the disease.     

Lymphocyte subpopulations in peripheral blood in primary sclerosing cholangitis

BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is a chronic disease of autoimmunological etiology, leading to inflammation, destruction and atrophy of the bile ducts. The aim of the study was to determine peripheral lymphocyte B, T, and NK cells in PSC. METHODOLOGY: The estimation of peripheral blood lymphocytes in 17 patients (54+/-12 years old) with PSC was carried out; the control group consisted of 27 subjects (38+/-11 years). The following T lymphocyte subpopulations were determined using duo color flow cytometry: CD3+, CD4+, CD8+, CD3++HLA DR+, B cells CD19+, and NK cells CD16+ +CD56+. RESULTS: In PSC we observed doubled increase in activated T lymphocytes of CD3+ +HLA DR+ phenotype as compared to healthy subjects (7.9% vs. 2.7%, p<0.01) and NK cells (12.6% vs. 10.3%, respectively, p<0.05). There were no significant differences in the composition of CD3+, CD4+, and CD8+. In peripheral blood we noted, in patients with PSC, a decrease in B lymphocytes (11.2% vs. 12.3%, p<0.19). CONCLUSIONS: The examinations showed that activated T (HLA DR+) lymphocytes and NK cells played an important role in development of PSC.     

Increased peripheral blood gamma delta+ T cells and natural killer cells in Beh?et's disease.

Peripheral blood cytotoxic lymphocytes, including gamma delta+ T cells, natural killer (NK) cells, and CD56+ T lymphocytes were investigated by using 2-color flow cytometry in patients with Beh?et's disease. The percentage of circulating gamma delta+ T cells among total lymphocytes or among total T cells was significantly increased in patients with Beh?et's disease, compared to healthy controls. An increase in circulating gamma delta+ T cells was related to mucocutaneous lesions, but not the HLA-B51. Among gamma delta+ T cells, the number of CD8+ or CD56+ subset was significantly increased in patients with Beh?et's disease. Moreover, significant increases of peripheral blood NK cells (CD16+CD56+CD3-, CD2+CD3-, CD8dim+CD3-) and CD56+ T lymphocytes were also observed. Our data suggest that increased circulating gamma delta+ T cells and NK cells may play an important role in the pathogenesis of Beh?et's disease.     

Immunologic effects in patients with psoriatic arthritis treated with cyclosporine A.

Twelve patients with psoriatic arthritis (PsA) and very active articular disease resistant to conventional second line therapy entered into a 6-month open study of cyclosporine A (CsA) at a starting dosage of 3 mg/kg/day. Comparisons of phenotypic characteristics of lymphocytes and response to mitogens of peripheral blood mononuclear cells (PBMC) were made between these patients with PsA before CsA therapy, 7 patients without prior 2nd line therapy, 14 untreated patients with psoriasis alone, and 61 healthy controls. We confirmed a significant reduction of the basal percentage of CD8+ cells and an increase in the CD4/CD8 ratio in patients with PsA before CsA therapy compared to controls. These abnormalities were not present in patients with PsA without prior 2nd line therapy and in patients with psoriasis alone. Peripheral blood activated T cells (CD3+, HLA-DR+), natural killer (NK) (CD3-, CD16+ and/or CD56+), total B and CD5+ B cells were decreased only in patients with PsA before CsA therapy. The reduction of non-MHC restricted cytotoxicity T (CD3+, CD16+ and/or CD56+) was observed in all the 3 groups of patients compared to controls. After the 6 months of CsA therapy we observed a significant increase of CD3+, HLA-DR+, CD3+, CD16+ and/or CD56+, total B, and CD20+, CD5+ cells in the 11 patients with PsA compared to pretreatment values. Contrary to azathioprine, CsA does not impair the NK cell population which has a protective role against cancer and viral infections.     

HIV/AIDS外周血T淋巴细胞活化与病情进展的临床研究

目的 探讨HIV/AIDS外周血T淋巴细胞活化水平与病情进展的关系.方法 检测105例HIV/AIDS患者外周血可溶性CD27(sCD27)水平,CD28+CD8+、CD28+CD4+、HLA-DRCD8+和CD38+CD8+比例以及CD4+T淋巴细胞计数,并在正常体检人群中随机抽取15名健康人作为对照组.分别用流式细胞技术和ELISA方法检测和分析T细胞活化指标(CD2+CD8+、CD28+CD4+、CD38+CD8+、HLA-DRCD8+、sCD27)与疾病分期、CD4+T淋巴细胞计数的相关性.结果 HIV/AIDS的sCD27水平和HLA-DRCD8+和CD38+CD8+比例较正常人显著升高(P＜0.05),sCD27在AIDS C3期患者中明显升高,与B3期患者比较有显著性差异,CD28+CD4+、CD28+CD8+、HLA-DRCD8+和CD38+CD8+比例在CD4+T淋巴细胞计数超过200/ul的患者中显著升高(P＜0.05).结论 T淋巴细胞活化水平与AIDS病情进展密切相关,HIV/AIDS的T淋巴细胞活化水平明显升高,CD4+T淋巴细胞计数超过200/μl患者的T淋巴细胞活化水平高于CD4+T淋巴细胞计数低于200/μl的患者.     

T cells bearing gamma/delta T cell receptor and their expression of activation antigen in peripheral blood from patients with Sj?gren's syndrome.

T cells bearing gamma/delta T cell receptor (gamma/delta + T cells) and their expression of activation antigen (HLA-DR) or the marker of natural killer (NK) cells (CD56), were examined in the peripheral blood lymphocytes (PBL) from twenty-two patients with Sj?gren's syndrome (SS) by three-color flowcytometry to elucidate possible pathological roles of the T cell subset in SS. The frequency of gamma/delta + T cells in PBL was not elevated in SS patients, while that of gamma/delta - T cells, which are T cells bearing the alpha/beta T cell receptor (alpha/beta + T cells), was significantly low in the patients, as compared with 22 healthy controls. We found that the proportions of activated cells (HLA-DR+) in both the gamma/delta + and alpha/beta+T cell subsets were significantly higher in the patients than in the controls. The proportions of HLA-DR+ cells in cells in both patients and controls. Furthermore, the frequency of activated cells in both T cell subsets correlated with the duration of disease in SS patients. However, no difference was found in the percentages of total CD56+ cells, CD56+CD3- cells (true NK cells), CD56+CD3+T cells, CD56+gamma/delta+T cells, or CD56-gamma/delta+T cells between the patients and controls. The above results indicate that immunologic activation in SS patients is progressive and involves both alpha/beta+ and gamma/delta+ T cell subsets.     

原发性肝癌和乙型肝炎肝硬化患者外周血淋巴细胞凋亡率、T细胞亚群和NK细胞的水平变化和临床意义

目的通过检测乙型肝炎肝硬化和合并乙型肝炎病毒感染的原发性肝细胞癌患者的外周血的粒细胞、单核细胞、NK细胞、T细胞及其亚群和淋巴细胞及其凋亡率,探讨两者在细胞免疫方面的差异.方法用Ficoll Hypaque离心法分离外周血单个核细胞(PBMNC),流式细胞仪检测外周血T细胞及其亚群、NK细胞和淋巴细胞、单核细胞和粒细胞,AnnexinV/FITCKit检测凋亡细胞.结果乙型肝炎肝硬化患者的外周血单核细胞、粒细胞、淋巴细胞、CD3-CD16 CD56 NK细胞、CD3 T细胞、CD3 CD4 T细胞、CD3 CD4 T细胞和CD4 CD8 T细胞比值,均与正常对照组无显著性差异(P＞0.05);但淋巴细胞凋亡率明显低于对照组(P＜0.01).原发性肝癌外周血CD3-CD16 CD56 NK细胞、单核细胞和CD4 /CD8 T细胞比值与肝硬化组和正常对照组比较,均无显著性差异(P＞0.05),而粒细胞明显升高(P＜0.05);CD3 T细胞、CD3 CD4 T细胞和CD3 CD8 T细胞均较另两组明显减少(P＜0.05),淋巴细胞及其凋亡率均明显低于另两组(P＜0.01).结论乙型肝炎肝硬化患者的外周血细胞免疫只发生不显著的变化,但淋巴细胞的凋亡率明显降低.原发性肝癌外周血的细胞免疫和淋巴细胞凋亡率均明显低下.     

抗病毒治疗对HIV/AIDS患者自然杀伤细胞和γδT细胞的重建作用研究

目的 研究高效联合抗逆转录病毒治疗(HAART)对HIV/AIDS患者NK细胞和γδT细胞的重建作用,比较HAART对获得性免疫和固有免疫重建规律的异同.方法 对59例接受规律HAART的HIV/AIDS患者于治疗前、治疗后1、3、6、9、12个月末进行随访,采用分支DNA(bDNA)法检测血浆病毒载量,经流式细胞仪检测患者外周血淋巴细胞亚群.以56例健康献血员作为正常对照.结果 基线时HIV/AIDS患者CD4+T细胞中位数为45(164,13)个/μl,NK细胞和γδT细胞中位数分别为96(135,56)个/μl和30(45,19)个/μl,与正常对照组相比均显著减低(P<0.001).HAART开始后1个月,血浆病毒载量降低了2.33 lg拷贝/ml(P<0.001),CD4+T细胞数增长了111个/μl(P<0.001),主要以记忆CD4+T细胞的快速增长为主,而NK细胞和γδT细胞与基线相比无显著变化.在HAART后3个月NK细胞和.γδT细胞数量出现增长,分别较基线增加26个/μl(P=0.007)和15个/μl(P<0.001).治疗12个月,CD4+T细胞、NK细胞及γδT细胞计数分别达到271(365,134)个/μl、166(281,108)个/μl和46(66,34)个/μl,但仍低于正常对照.结论 HAART可以重建HIV/AIDS患者固有免疫,但NK细胞和γδT细胞数量的增加晚于CD4+ T细胞,HAART对固有免疫的重建作用机制有待进一步研究.     

CD16- CD56+ natural killer cells after bone marrow transplantation.

Natural killer (NK) cells are phenotypically defined as lymphocytes expressing the antigens CD56 and mostly CD16 (Fc gamma RIII), but lacking CD3. A small CD3- CD16- CD56+ NK cell subset has been described in normal individuals representing less than 2% of peripheral blood lymphocytes. We analyzed here 70 patients for their reconstitution of the immune system during follow-up after autologous or allogeneic bone marrow transplantation. In 35% of these patients, two different NK cell subsets, namely CD56+dim and CD56+bright cells, were observed. The mean duration of these two subsets after transplant was 4 months. Sixty-five percent of the patients exhibited an increased number of NK cells, but only the typical CD16+ CD56+dim population. The CD56+bright subpopulation represented a particular CD3- CD16- NK subset, with posttransplant frequencies up to 70% of all NK cells and 40% of peripheral blood lymphocytes, respectively. In contrast to normal CD56+dim NK cells, CD56+bright cells coexpressed the activation antigens p75 beta-chain of interleukin-2 receptor (IL-2R), CD2R, and CD26, but were negative for CD16. NK and antibody-dependent cellular cytotoxicity activity of CD56+bright cells was low compared with CD56+dim NK cells. But using IL-2 and interferon gamma, their cytotoxicity could be enhanced even more than in CD56+dim lymphocytes. These different subsets may reflect distinct activation or differentiation steps of NK cells during reconstitution of the immune system. Their differential response to IL-2 may be of functional importance for posttransplant cytokine therapy.     

LYMPHOCYTE SUBPOPULATIONS ANALYSIS IN PERIPHERAL BLOOD IN POLYMYALGIA RHEUMATICA/GLANT CELL ARTERITIS

The phenotypic characteristics of peripheral blood lymphocyteswere investigated in 22 patients suffering from active polymyalgiarheumatica/giant cell arteritis (PMR/GCA) prior to steroid treatment.We observed a significant reduction in the absolute number andthe relative percentage of CD4+, CD8+ and CD3+, CD16+ and/orCD56+ cells compared to controls. Fifteen patients were investigatedprospectively over a 6-month period of prednisone therapy. Atthe end of the study CD4–CD8+ cells had increased significantlycompared to baselines, CD3+CD16+ and/or CD56+ cells remainedsignificantly lower when compared to controls. We did not observeany abnormalities in the absolute number and percentage of HLA–DR+T lymphocytes, CD5+ B cells and NK cell phenotypes before orduring steroid treatment. Our study confirms that there wassignificant increase in the absolute number of CD8+T cells duringsteroid treatment in the PMR/GCA patients, but indicates thepersistence of an immunological alteration despite the controlof disease manifestations. KEY WORDS: Polymyalgia rheumatica, Giant cell arteritis, Lymphocyte subpopulations, Steroid therapy     

Effect of granulocyte colony-stimulating factor mobilization on phenotypical and functional properties of immune cells

Some phenotypic and functional properties of lymphocytes from bone marrow or peripheral blood stem cell donors were compared in a randomized study.Lymphocyte subsets were analyzed by immunocytometry in blood harvested from bone marrow donors (n = 27) and from peripheral blood stem cell donors before and after granulocyte colony-stimulating factor mobilization (n = 23) and in bone marrow and peripheral blood stem cell grafts.Granulocyte colony-stimulating factor mobilization increased the blood T and B, but not NK, lymphocyte counts. All lymphocyte counts were approximately 10-fold higher in peripheral blood stem cell grafts than in bone marrow grafts. Analysis of CD25, CD95, HLA-DR, and CD45RA expression shows that T-cell activation level was lower after granulocyte colony-stimulating factor mobilization. Similarly, granulocyte colony-stimulating factor reduced by twofold to threefold the percentage of interferon-gamma, interleukin-2, and tumor necrosis factor-alpha-secreting cells within the NK, NK-T, and T-cell subsets and severely impaired the potential for interferon-gamma production at the single-cell level. mRNA levels of both type 1 (interferon-gamma, interleukin-2) and type 2 (interleukin-4, interleukin-13) cytokines were approximately 10-fold lower in peripheral blood stem cell grafts than in bone marrow grafts. This reduced potential of cytokine production was not associated with a preferential mobilization of so-called "suppressive" cells (CD3+CD4-CD8-, CD3+CD8+CD56+, or CD3+TCRVA24+CD161+), nor with a modulation of killer cell receptors CD161, NKB1, and CD94 expression by NK, NK-T, or T cells.Our data demonstrate in a randomized setting that quantitative as well as qualitative differences exist between a bone marrow and a peripheral blood stem cell graft, whose ability to produce type 1 and type 2 cytokines is impaired.     

Aggressive variant of morphologically typical T large granular lymphocyte leukemia/lymphoma lacking NK cell markers

A 46-year-old woman with a previous diagnosis of sarcoidosis presented with morphologically typical large granular lymphocyte (LGL) leukemia/lymphoma with an aggressive clinical course. Epstein-Barr virus DNA was detected in peripheral blood mononuclear cells by PCR. The phenotype was typical of the T cell lineage (CD2+ CD3+ CD5+ CD7+ CD8+ TCRalphabeta+) but with the absence of the CD16, CD56, CD57 NK cell markers. In addition, the LGLs expressed CD122 (p75) in the absence of CD25 which is characteristic of LGLs. These leukemic LGLs did not exhibit NK activity. The clonal nature of this proliferation was demonstrated by the rearrangement of the TCRgamma gene. This phenotypically unusual but morphologically typical LGL leukemia/lymphoma may represent the clonal expansion of a minor normal subset of T-LGLs which do not express any NK cell markers, probably corresponding to in vivo activated T cells.     

Distribution of Peripheral Blood Lymphoid Subsets in Alcoholic Liver Cirrhosis: Influence of Ethanol Intake

The aim of the present study was to investigate the effect of chronic ethanol (EtOH) consumption on the immune system in patients with alcoholic liver cirrhosis (ALC), as analyzed by the distribution of peripheral blood (PB-) T, B, and NK lymphoid subsets using multiple stainings with monoclonal antibodies and flow cytometry. For that purpose, we have analyzed a group of patients with ALC and active EtOH intake (ALCET group) which were re-evaluated 3 months after alcohol withdrawal. As controls, both ALC patients with at least 1 year of alcohol withdrawal (ALCAW group) and healthy subjects were used. Regarding the alcohol intake period, the most relevant findings were a significant activation of the PB T-cell compartment, and specifically of the TCR alpha beta+ subset, as reflected by an increased expression of both the HLA DR and CD11c antigens as well as a significant increase of both the PB NK cells (CD3-/CD56+) and the cytotoxic T cells coexpressing the CD3 and CD56 molecules. In addition, a decrease of both the numbers of total B cells and their CD5+/CD19+ subset were observed. After a relatively short withdrawal period (3 months), the abnormalities of T, P, and NK cells disappeared. These findings suggest the existence of a close relationship between EtOH consumption and the abnormalities of the immune system observed during active alcoholism. Nevertheless, ALCAW individuals displayed marked alterations on the immunophenotypic profile, as reflected by a significantly decreased number of total T cells, due to reduced levels of the CD3+/TCR alpha beta+, CD4+, CD8+, and CD4+/CD45RA+ T-cell subsets. In addition, a significantly decreased number of total PB B cells was observed in this group of patients. Our results show that in patients suffering from ALC, the abnormalities of the immune system due to a direct effect of EtOH intake (or its metabolites) should be distinguished from the immunological alterations related to the liver disease itself.     

CD16+ CD56- NK cells in the peripheral blood of cord blood transplant recipients: a unique subset of NK cells possibly associated with graft-versus-leukemia effect

A marked increase in CD16+ CD56- NK cells in the peripheral blood (PB) was observed in a cord blood transplant (CBT) recipient with refractory acute myeloid leukaemia (AML) in association with attaining molecular remission. CD16+ CD56- NK cells isolated from the patient became CD16+CD56+NKG2D+ when they were cultured in the presence of IL-2. Although cultured CD16+CD56- NK cells retained the killer-cell immunoglobulin receptor (KIR)-ligand (KIR-L) specificity and the patient's leukemic cells expressed corresponding KIR ligands, they killed patient's leukemic cells expressing ULBP2. The cytotoxicity by cultured CD16+CD56- NK cells was abrogated by anti-ULBP2 antibodies. When leukemic cells obtained at relapse after CBT were examined, both the ULBP2 expression and susceptibility to the cultured NK cells decreased in comparison to leukemic cells obtained before CBT. An increase in the CD16+CD56- NK cell count (0.5 x 10(9)/L or more) in PB was observed in seven of 11 (64%) CBT recipients but in none of 13 bone marrow (BM) and eight peripheral blood stem cell (PBSC) transplant recipients examined during the similar period after transplantation. These findings suggest an increase in CD16+CD56- NK cells to be a phenomenon unique to CBT recipients and that mature NK cells derived from this NK cell subset may contribute to the killing of leukemic cells expressing NKG2D ligands in vivo.