Expression of CD8β and alteration of cell surface phenotype in adult T-cell leukaemia cells

Typical adult T-cell leukaemia (ATL) cells have a CD4⁺CD8⁻ cell surface phenotype, but atypical phenotypes such as CD4⁺CD8⁺ and CD4⁻CD8⁺ have also been reported. The CD8 molecule is composed of α and β chains and commonly used monoclonal antibodies against CD8 molecule detect only CD8α. Since it has been reported that CD8α can be induced in mature CD4⁺ T cells by cell activation, but not CD8β, we studied whether ATL cells which express CD8α may also express CD8β. We found some cases of CD8α⁺ ATL were also positive for CD8β. Furthermore, we experienced a case whose ATL cell surface phenotype changed from CD4⁺CD8α⁺CD8β⁺ to CD4⁻CD8α⁺CD8β⁺ and finally to CD4⁺CD8α⁻CD8β⁻. Southern blot analysis revealed that the monoclonal integration of human T lymphotropic virus type I (HTLV-I) was identical throughout the course of the study, indicating that a single clone had demonstrated the alterations. These data suggest that peripheral CD4⁺CD8⁺ ATL cells can express not only CD8α, but also CD8β and that a single ATL cell clone has the potential to change its surface phenotype in vivo as well as in vitro .     

Composition of peripheral blood lymphocyte populations during different stages of chronic infection with hepatitis B virus

To characterize the immunological populations associated with different stages of chronic infection with hepatitis B virus (HBV), we performed flow cytometric analyses on the peripheral blood leucocytes of 29 patients with various forms of chronic hepatitis B. The clinical spectrum of the patients ranged from asymptomatic infections, in the presence of high virus production, to intermittent or recurrent exacerbations of liver injury alternating with relatively normal liver function. Patients with partial resolution of disease who experienced an initial acute flare followed by prolonged seroconversion showed decreased percentages of CD3⁺ cells during the seroconversion phase when levels of serum alanine transferase (ALT) had normalized. These CD3⁺ cells were predominantly CD4⁺ cells bearing the αβ⁺ T-cell receptor (TCR). In addition, we saw an increase in CD4⁺ and CD8⁺ cells bearing the γδTCR in those patients who had seroconverted. No significant differences were seen between any of the groups with respect to percentage of cells with a naive (CD45RA) or memory (CD45RO) phenotype, or of cells displaying the activation markers CD38, HLA-DR or CD57. Longitudinal analyses of 15 patients failed to show any consistent pattern of changes in the immunophenotypic profile during acute flares and their resolution. Our results indicate that the turnover of circulating T lymphocytes during the apparent quiescent phase of chronic infections is higher than that during acute exacerbations, suggesting an active immunosurveillance role of T-cell subpopulations in maintaining low virus levels during seroconversion.     

CD3+ CD8+ CD56− clonal large granular lymphocyte leukaemia and HIV infection

High-grade malignant lymphomas associated with HIV infection are usually derived from B lymphocytes. Although a broad spectrum of T-cell-derived malignancies has been described, no case of monoclonal T large granular lymphocyte leukaemia has been reported to date. We report a case of clonal T-LGL (CD3⁺, CD4⁻, CD8⁺, CD56⁻, CD57⁺) in an HIV-infected, HTLV1/2-negative individual. Large granular lymphocytes are thought to represent activated cytotoxic T lymphocytes. HIV infection, as previously reported for HTLV1/2, may represent a pathway of antigen activation and lead to clonal expansion of T large granular lymphocytes.     

Persistent lymphocytosis of natural killer cells in autoimmune thrombocytopenic purpura (ATP) patients after splenectomy

We report three patients with primary autoimmune thrombocytopenic purpura (ATP) who developed an absolute lymphocytosis (lymphocyte count > 5×10⁹/I) after splenectomy and with a lymphocyte count between 5.4 and 8.9×10⁹/I. An immunophenotype study showed that the peripheral blood lymphocytosis was a persistent NK cell expansion (CD2⁺, CD56⁺, CD3^-), and was characterized by a typical large granular lymphocytes (LGL) morphology. Two of these three ATP patients were refractory to splenectomy.     

Epinephrine test and plasma elastase as diagnostic tools in a patient with CD3+ large granular lymphocyte proliferation

Large granular lymphocytes (LGL) proliferation is characterized by expansion of cytotoxic lymphocytes and associated with neutropenia. In a case of CD3⁺ LGL-proliferation the epinephrine stimulation test (EST) induced a striking elevation of CD3⁺, CD8⁺, CD57⁺ LGL in peripheral blood from 2.7 x 10⁹/1 to 20 x 10⁹/1 and might be an additional diagnostic tool in patients with normal or low absolute numbers of circulating LGL. After treatment with steroids, plasma elastase - a marker of neutrophil destruction - decreased from 162 to 40μg/1 (normal < 47μg/1) which correlated well with a simultaneous increase in peripheral neutrophil counts from 0.14 to 1.0 x 10⁹/1. This finding supports the hypothesis that neutropenia in CD3⁺ LGL proliferation is due to neutrophil destruction, possibly mediated by LGL.     

Monoclonal proliferation of CD4+ large granular lymphocytes with cytolytic activity

Summary. A rare case of monoclonal proliferation of CD3⁺4⁺8∼ T-cell receptor-αβ⁺ large granular lymphocytes (LGL) is presented. CD4⁺ LGL in the present case showed spontaneous cytotoxicity against herpes simplex virus-infected cells and antibody- and lectin-dependent cytotoxicity. Perforin, which is one of the important cytolytic mediators of cytotoxic T cells (CTL) and natural killer cells, was abundantly expressed in CD4⁺ LGL of this case. The present case suggests that perforin-positive CD4⁺ CTL, which have recently been shown in the in vitro studies, certainly exist in vivo.     

Recovery of T-lymphocytes for adoptive immunotherapy by lymphapheresis of HIV-infected patients without alterations of virological, immunological or clinical parameters

Summary. We performed repeated continuous flow cytaphereses (CFC) on 13 asymptomatic HIV-1-infected patients to study the feasibility of cell separation procedures to recover high yields of peripheral blood T-lymphocytes for adoptive immunotherapy in HIV-infected patients and to determine immunological and virological alterations following such procedures.
A mean yield of 6.23 * 10⁹ lymphocytes could be obtained by each cytapheresis, containing 1.82 * 10⁹ CD4⁺, 3.23 * 10⁹ CD8⁺ T-lymphocytes and 8.39 * 10⁶ CD34⁺ peripheral progenitor cells. The CD4/CD8 ratio (mean 0.53, SD ± 0.15) in the cell samples reflected the distribution of the lymphocyte subsets in vivo . Absolute lymphocyte counts decreased at a mean of 404/mm³ (25%) immediately after CFC but were replaced from the extravascular pool within 1 h. The CD4/CD8 ratios, p24-antigenaemia. HLA-DR expression and neopterin levels did not change significantly after cell separation. No alteration of the number of T-cells with integrated proviral DNA copies (1/10³ to 1/10⁶) could be detected in peripheral T-helper cells by PCR after lymphapheresis.
We conclude that high yields of peripheral T-lymphocytes can be obtained by continuous flow lymphapheresis for cell mediated immunotherapy, withou deterioration of virological or immunological parameters in HIV-infected patients. The separated T-cells are fully replaced from extravascular pools after 1 h.     

CD4+ T lymphocytes contribute to protective immunity induced in sheep and goats by Haemonchus contortus gut antigens

Immunization with parasite antigens derived from the gut of adult Haemonchus contortus induces significant levels of protection against the parasite in sheep and goats. However, the mechanisms of immunity involved in this protection are not clear. Here, we investigate the requirement for CD4⁺ T lymphocytes in gut antigen-induced immunity against H. contortus. Gut antigen immunized animals were depleted (>98%) of their CD4⁺ T lymphocytes in peripheral blood by intravenous injection of an anti-CD4 MoAb. Depletion in peripheral blood persisted for at least eight days, after which there was gradual recovery of CD4⁺ T lymphocytes. Serum antibody levels in gut antigen-immunized animals correlated significantly with worm parameters, suggesting a contribution by antibody to the immunity observed. By covariate analysis, using ELISA OD as the covariate, CD4⁺ T lymphocyte depletion was shown to partially abrogate immunity induced by gut antigen immunization, against challenge infection with H. contortus. The greatest effect of CD4⁺ T lymphocyte depletion was observed at 14 days post-infection, with differences between CD4⁺ T lymphocyte depleted and intact animals less apparent between days 21 and 25. Collectively, our data indicate that CD4⁺ T lymphocytes contribute to immunity induced by gut antigens. Our results also suggest that antibody works synergistically with CD4⁺ T lymphocytes to confer this immunity.     

Abnormalities of Peripheral Blood T Lymphocytes and Natural Killer Cells in Alcoholic Hepatitis Persist after a 3-Month Withdrawal Period

Present information about the behavior of the different lymphoid subsets in alcoholic hepatitis (AH), including cells displaying cyto-toxic activity, is scanty and contradictory. The aim of this study was to gain further insight into knowledge of the immunological abnormalities involved in AH and the possible role of ethanol (EtOH) consumption in these changes. We analyzed the distribution of a wide range of peripheral blood (PB) lymphoid subsets, both during active EtOH intake and after a 3-month withdrawal period, using multiple stainings with monoclonal antibodies and flow cytometry, as well as natural killer (NK) cytotoxic activity. AH patients entering the study were selected strictly; only those undergoing their first episode of AH with no other lesions at liver biopsy were enrolled. Regarding the alcohol intake period, the most striking finding was a significant increase of the absolute number of PB T cells affecting both CD4⁺ and CD8⁺ lymphocytes. These changes were associated with a higher expression of T-cell activation antigens, such as HLA DR and CD11c. Simultaneously, a significant increase in both NK cells (CD3⁻-^/ CD56⁺) and the cytotoxic T cells coexpressing the CD3 and the CD56 molecules together with an increased NK cytotoxic activity were observed. By contrast, the CD19⁺/CD5⁺ B-cell subset was significantly decreased. No significant changes were observed with EtOH withdrawal except in CD5⁺ B lymphocytes, which returned to normal values. Our results show that, in AH patients, a significant expansion of both activated T cells and NK lymphocytes occurs in the PB, which is associated with an increased NK cytotoxic activity. Interestingly these abnormalities persist during the withdrawal period.     

IL-12 is involved in the activation of CD3+ granular lymphocytes in patients with lymphoproliferative disease of granular lymphocytes

We investigated the effects of IL-12 on functional properties of CD3⁺CD8⁺ granular lymphocytes (GL) of patients with lymphoproliferative disease of granular lymphocytes (LDGL). To this aim, in 10 cases with a clonal CD3⁺ GL proliferation (nine cases with an associated TCR α/β receptor and one case with a TCR γ/δ receptor) we studied the proliferative and cytotoxic activities of resting and αCD3 monoclonal antibody (mAb) activated cells in the presence of rIL-12 and anti-IL-12 blocking antibodies. Specific mRNA for IL-12 p40 subunit was also investigated.   Our results showed that rIL-12 increased the proliferation of αCD3 pre-stimulated GL (2 to 6 times). Further, anti- IL-12 antibodies partially inhibited αCD3-induced cell growth, suggesting a role for this cytokine in the αCD3-mediated GL activation. The addition of antibodies blocking the p55 and p75 chains of IL-2 receptor (IL-2R) did not inhibit the rIL-12-mediated cell proliferation, indicating that the activity of rIL-12 is independent of IL-2 in the in vivo expanded GL of patients under study. Concerning the cytotoxic activity, rIL-12 increased the αCD3-mediated NK activity against K-562 target cells and αCD3 redirected cytotoxicity against P815 target cells. Molecular analysis pointed out that, following αCD3 stimulation, patients' GL increased the expression of specific mRNA for the p40 subunit of IL-12 as compared to baseline conditions.   Our data indicate that IL-12 is involved in the mechanisms of activation of clonal CD3⁺ GL in patients with LDGL; these features are consistent with the possibility that this discrete subset of GL might represent in vivo primed cytotoxic T lymphocytes.     

Immunophenotypic and clinical features of T-cell receptor γδ+ T-lineage acute lymphoblastic leukaemia

The immunophenotypic and clinical features of TCR-γδ⁺ T-lineage acute lymphoblastic leukaemia (T-ALL) were prospectively analysed in 52 children with membrane CD3⁺ T-ALL. We observed a relatively high incidence of TCR-γδ⁺ T-ALL (26/52 patients). Leukaemic blasts from 22 children demonstrated TCR-αβ positivity, and simultaneous expression of the TCR-β and -δ chain was found in four children. Clinical and haematological features of TCR-αβ and γδ⁺ T-ALL did not differ significantly, except that haemoglobin levels were significantly lower in TCR-γδ⁺ cases. Event-free survival at 4 years was significantly better in TCR-γδ⁺ compared with TCR-αβ⁺ T-ALL, but expression of TCR molecules did not emerge as an independent prognostic factor in multivariate analysis.     

Modification of Lymphocyte Subsets in the Intestinal-Associated Immune System and Thymus by Chronic Ethanol Consumption

Modification of the mucosa-associated intestinal immune system of female C57BL/6 mice was studied during consumption of the Lieber-DeCarli diet supplemented with 5% v/v ethanol or laboratory chow with ethanol (20% w/v) in the drinking water. All groups received ethanol for 11 weeks. Mice fed the Lieber-DeCarli diet had fewer CD8⁺ cells/villus than the chow-fed controls. Mice that received ethanol in the drinking water had fewer IgA-containing cells and CDB⁺ cells than controls. There were no differences in the number of cells in the mesenteric lymph nodes between ethanol-treated mice and their respective controls. Nevertheless, chow-fed control mice had more cells than those fed the Lieber-DeCarli control diet. Although no differences were detected in the percentages of CD4⁺, CD8⁺, LECAM-1⁺, and LECAM-1⁺ CD4⁺ cells, there was a decrease in the percentage of LECAM-1⁺ CD8⁺ cells in ethanol-fed mice when compared with their Lieber-DeCarli controls. Mice receiving ethanol in the drinking water showed alterations in the CD4 CD45RC subsets and in the CD8 CD45RC subsets. Similar results were observed in mice receiving Lieber-DeCarli diets alone or supplemented with ethanol. The low dose, chronic exposure of dietary ethanol in the Lieber-DeCarli-fed mice did not significantly affect the numbers of various thymocyte subsets. But, a decrease in the percentage of CD4^- CD8⁺ cells was observed in the thymus of mice receiving ethanol in the drinking water. Chronic ethanol consumption caused significant decreases in the number of CD8⁺ and IgA⁺ cells in the intestinal lamina propria, important in mucosal immune defenses.     

Splenic lymphoma with villous lymphocytes: clinical presentation, biology and prognostic factors in a series of 100 patients

The diagnosis of splenic lymphoma with villous lymphocytes (SLVL) was assessed by a panel of cytologists in a series of 100 patients. Clinical and biological characteristics were analysed in relation to prognosis.
SLVL is a chronic B-cell lymphoproliferative disorder characterized by splenomegaly and the presence, in peripheral blood, of lymphocytes with 'villous' projections. The cytological diagnosis can be difficult in patients without an absolute lymphocytosis which was observed in 24% of cases. B-cells expressed CD19⁺, CD20⁺, CD22⁺, CD24⁺ and DBA44⁺, whereas the expression of CD5, CD10 and CD25 was usually negative.
SLVL is a disease of the elderly with a relatively benign clinical course. In the present series the 5-year overall survival was 78%. Deaths in 15 patients were related to disease progression or treatment (nine cases). Patients with a leucocyte count >30 × 10⁹/l or lymphocyte count <4 × 10⁹/l or initially treated with chemotherapy had significantly ( P  < 0.001) lower overall survival than other patients. From these findings, treatment abstention should be considered in patients with favourable prognostic factors; on the other hand, the efficacy of conventional chemotherapy remains to be evaluated in patients with unfavourable prognostic factors.     

Nematode infection induces Th2 cell-associated immune responses in LEC mutant rats with helper T cell immunodeficiency

Long-Evans Cinnamon (LEC) rats have maturational arrest of CD4⁺8⁻ T cells from CD4⁺8⁺ cells in the thymus. Despite this, CD4⁺8⁻ T cells are always present in peripheral lymphoid organs of LEC rats, suggesting that these CD4⁺8⁻ T cells are generated by an uncommon pathway. We investigated the role of LEC rat peripheral CD4⁺8⁻ T cells in Th2-associated responses to infection with the nematode Nippostrongylus brasiliensis . After infection, the numbers of CD4⁺8⁻ TCRαβ⁺ T cells significantly increased in mesenteric lymph nodes (MLN) and the spleen, while those in the thymus were still negligible. Infection also induced significant up-regulation of IL-4 gene expression in LEC rat MLN cells. Total serum IgE levels in LEC rats were markedly increased two weeks after infection. Mucosal mast cell responses in the gut and lungs of LEC rats were induced as prominently as in control Long-Evans Agouti (LEA) rats. Faecal egg count data indicated that LEC rats rejected nematodes faster than LEA rats. These results suggested that Th2-associated responses can be induced by nematode infection in LEC rats probably through the extrathymic recruitment and proliferation of CD4⁺8⁻ TCRαβ⁺ T cells .     

T-helper phenotypes in the blood of myeloma patients on ECOG phase III trials E9486/E3A93

T-helper blood populations are frequently altered in multiple myeloma (MM). We measured the numbers of naive and activated cell subsets in the blood of a cohort of both previously untreated and treated MM patients. Two-colour flow cytometry to detect total CD4⁺, CD4⁺, CD45RA⁺ (naive) and CD4⁺, CD45RO⁺ (activated) subsets was then used to quantify the T-cell subsets in controls and MM patients. Previously treated MM patients either on or off treatment ( n  = 105) had significantly reduced ( P  < 0.0001) total CD4 and naive/activated cells than controls. Previously treated MM patients sampled for naive/activated cells while currently off therapy ( n  = 45) had no difference in the levels of CD4 and naive/activated cells compared to the currently treated patients ( n  = 60). However, newly diagnosed patients ( n  = 58) had a significantly reduced total CD4 ( P  = 0.023) and activated CD4 ( P  = 0.004), but not naive CD4 subsets, compared to controls. CD19⁺ cell levels above 125/μl were positively associated with higher T-helper cell levels. There was a strong positive association for better overall survival for patients with > 395 CD4 cells/μl ( P  = 0.0001). These data indicate that MM patients at diagnosis have altered T-helper subsets, with a selective reduction in activated but not naive cells. Subsequent chemotherapy or the disease process contributes to a further reduction in CD4 cells. Importantly, the association of higher CD19⁺ cell levels with higher T helper cells indicates that certain myeloma patients can be identified with a more quantitatively intact immune system.     

Interferon-γ Production Specific for Hepatitis B Virus Antigen by Intrahepatic T Lymphocytes in Patients with Acute and Chronic Hepatitis B

Objectives : Studies were undertaken to determine whether intrahepatic lymphocytes have a greater cellular immune response specific for hepatitis B virus (HBV) antigen than peripheral blood lymphocytes in HBV- infected man. Methods : HB nucleocapsid antigen-stimulated interferon-γ (IFN-γ) production of lymphocytes was measured in acute self-limited hepatitis (AH) (eight cases) and chronic hepatitis (CH) (14 cases). Results : In both patient groups, basal IFN-γ levels without any stimulation were higher in the hepatic T cells than in the peripheral T cells. The values in cultures from blood and liver were larger in AH than in CH. Alternatively, IFN-γ response to HBcAg and HBeAg was less in hepatic T cells than in the corresponding blood cultures; T cell response was HLA class II restricted. Cell flow cytometry study showed that in all patient groups, percentages of CD3⁺, CD8⁺, and HLA-DR⁺ cells were significantly greater in liver than in peripheral blood, whereas the proportion of hepatic CD4⁺ cells was decreased, compared with that in peripheral CD4⁺ cells. Conclusions : These findings suggest that liver infiltrates are already activated in vivo to produce IFN-γ, particularly in patients with AH. The changes in the proportion of lymphocyte subsets may be responsible for the altered IFN-γ response of hepatic T cells.     

Thymus-independent T-cell differentiation in vitro

The generatation of large quantities of novel human T-cell clones ex vivo would make a wide range of gene- and immuno-therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal-neonatal and adult human CD34⁺ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co-cultivation of CD34⁺ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34⁺ cells into CD4⁺ or CD8⁺ naive T cells in serum-deprived cultures stimulated with stem cell factor and interleukin 7. CD4⁺ or CD8⁺ CD45RA⁺ TCRαβ⁺ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T-cell receptor β-chain rearrangments were generated over time. These results pave the way for the development of very simple culture conditions for ex-vivo production of naive helper or cytotoxic T cells which could be very useful for gene- and immuno-therapy of human diseases.     

Quantitative and functional differences in CD8+ lymphocyte responses in resolved acute and chronic hepatitis C virus infection

CD8⁺ T lymphocyte responses are important in the clearance of viral infections. In chronic infections they may contribute to pathogenesis. To investigate the role of CD8⁺ T lymphocyte responses in viral clearance and chronic hepatitis C we have compared hepatitis C virus (HCV) specific cytotoxicity and interferon-gamma (IFN-γ) production in patients with resolved-acute, and chronic HCV infection. CD8⁺ T cell responses to a panel of 13 HCV T cell peptide epitopes were studied using Elispot assays of IFN-γ production and chromium release cytotoxicity assays. Responses of seven patients with resolved acute HCV infection were compared with those of 14 chronically infected patients. HCV-specific cytotoxicity differentiated the two populations of patients. The majority (71%) of patients with resolved acute infection tested positive to 42% of relevant peptides compared with the minority (28%) of patients with chronic hepatitis C ( P =0.03) who responded to only 8% of relevant peptides ( P =0.0009). In contrast, HCV-specific IFN-γ production was detected in 86% of patients with either resolved or chronic infection in response to 42% and 35%, respectively, of relevant peptides tested (not significant). In patients with chronic infection the magnitude of the HCV-specific IFN-γ production was inversely correlated to viral load ( R ²=0.52; P =0.042). Failure to clear HCV infection may be attributable to the presence of noncytolytic IFN-γ producing CD8⁺ T lymphocytes in chronically infected patients. However these CD8⁺ T cells may play a beneficial role in contributing to the control of viral load in chronic hepatitis C.     

Progenitor cell subsets and engraftment kinetics in children undergoing autologous peripheral blood stem cell transplantation

The main objective of the present study was to determine the role of CD34⁺ cell subsets in the haemopoietic recovery of children undergoing peripheral blood stem cell transplantation. For this purpose, 38 leukaphereses from 33 children with malignancies mobilized with G-CSF were analysed. Using dual-colour flow cytometry, different subpopulations of CD34⁺ cells were quantified and the number of each reinfused subsets correlated with haemopoietic resurgence. Multivariate analysis showed that the number of CD34⁺CD38⁻ cells and CD34⁺CD38⁺ cells correlated better with time to neutrophil and platelet recovery, respectively, than the total number of CD34⁺ cells. Threshold values for rapid haemopoietic recovery, determined by the receiver operating characteristic analysis, were found to be 0.5 × 10⁶ CD34⁺CD38⁻ cells for neutrophil engraftment, and 2.0 × 10⁶ CD34⁺CD38⁺ cells for platelet recovery. It is suggested that the analysis of CD34⁺ cell subsets could increase understanding of the repopulation capacity of a given leukapheresis product in peripheral blood stem cell transplantation procedures in children. In particular, this procedure could be extremely useful when low numbers of CD34⁺ cells are collected.