Clinical significance of CD45RO expression on peripheral blood mononuclear cells in HTLV-I-infected individuals

The phenotype of peripheral blood mononuclear cells (PBMC) was examined in 13 healthy volunteers, 26 HTLV-I carriers, and 58 ATL patients (22 smouldering, five chronic, 24 acute, and seven lymphoma type). The percentage of CD4⁺, CD25⁺, CD28⁺ and CD45RO⁺ cells in the PBMC of the chronic and acute type patients was significantly higher than that of the volunteers, whereas the percentage of CD8⁺ and CD45RA⁺ cells in these patients was significantly low. The histogram for CD45RO fluorescence intensity (FI) revealed two patterns: pattern A consisted of CD45RO⁺ cells with high FI (CD45RO^high) and intermediate FI (CD45RO^int). Pattern B consisted exclusively of CD45RO^high. Pattern A was evident in all volunteers. The percentage of subjects showing pattern B was increased in an order that reflected disease progression. In the patients with pattern A, the CD45RO^int cells were CD4⁺ and CD8⁻, and the FI of CD2, CD3, and Fas within the CD45RO^int cells appeared to be lower than that within the CD45RO^high cells. The acute type patients with pattern A had a significantly longer survival curve than that of these patients with pattern B. These results suggest that the presence of CD45RO^int cells may be related to protection against disease progression in HTLV-I-infected individuals.     

T-cell telomere length maintained in HIV-infected long-term survivors

Background and methods We have used the erosion of telomeric DNA as a measure of cellular division to study the replicative history of isolated T-lymphocyte subpopulations from a group of HIV-infected long-term survivors and age-matched healthy controls.
Results In keeping with previous studies, we found that CD45RO⁺ (memory) T-cells showed greater telomere erosion than CD45RA⁺ (naive) T-cells. We did not, however, find any significant differences in the telomere lengths of isolated CD4⁺, CD8⁺, CD45RA⁺ or CD45RO⁺ T-cells between HIV-infected long-term survivors and age-matched controls. Further, we found no evidence of telomerase activation in
T-cells from the HIV-infected groups to account for the lack of telomere erosion.
Conclusions Our data show no evidence, through telomere shortening, of clonal exhaustion or replicative senescence due to an increased rate of immune cell turnover in HIV-infected long-term survivors.     

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.     

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.     

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 .     

Monitoring of CD95 and CD38 expression in peripheral blood T lymphocytes during active human cytomegalovirus infection after orthotopic liver transplantation

Aim:  The aim of the present study was to quantitatively monitor the response of CD95 molecules expressed on CD3⁺ T cells (CD95⁺CD3⁺ cells) and CD38 molecules expressed on CD8⁺ T cells (CD38⁺CD8⁺ cells) to ganciclovir treatment after orthotopic liver transplant (OLT) in recipients with active human cytomegalovirus (HCMV) infection.
Methods:  Blood samples were collected from 20 liver transplanted recipients with active HCMV infection and 24 recipients without HCMV infection. CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were quantitatively detected with QuantiBRITE bead methods by dual-color flow cytometry analysis during the post-transplantation period.
Results:  CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were not significantly different among different ages of healthy adults ( P  > 0.05). CD95⁺CD3⁺ cells and CD38⁺CD8⁺ cells were drastically increased in the active HCMV infection group compared with that in the stable group or in the healthy group ( P  < 0.001), and then they were gradually decreased within the next several weeks after ganciclovir treatment when compared with active HCMV infection recipients ( P  < 0.001).
Conclusions:  The present study showed that CD38⁺CD8⁺ T cells can be an appropriate immunological marker for early detection and antiviral therapeutic monitoring of HCMV infection. The evaluation of CD95 molecule levels may be used routinely in clinical practice to assess the level of immunosuppression.     

Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia

In this study we describe a fast and sensitive method using three-colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10⁺CD19⁺ cells and CD20⁻CD22⁺ cells in the CD34⁺ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10–28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM samples indicated that only a subpopulation of the CD34⁺CD10⁺CD19⁺ and CD34⁺CD20⁻CD22⁺ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three-colour immunofluorescence assay is warranted.     

Flow cytometric identification of candidate normal stem cell populations in CD45-negative B-cell precursor acute lymphoblastic leukaemia

CD45-negative B-cell precursor acute lymphoblastic leukaemia (ALL) provides a unique model to study the stem cell compartment in ALL as leukaemic CD34-positive cells, unlike their normal counterparts, do not express CD45. By increasing the number of events analysed to 10⁶, storing only the events in the region of interest (storage gate), using appropriate isotype controls and stringent washing procedures, a flow cytometric protocol was established to characterize rare CD34⁺CD19⁻ events. In eight of 12 patients (67%) with CD45-negative B-cell precursor ALL, a distinct CD34⁺CD19⁻CD45⁺ candidate normal stem cell population could be detected. In one patient analysed by four-colour staining, the CD34⁺CD19⁻CD45⁺ cells, unlike the CD45-negative leukaemic cells, expressed CD117 (c-kit), providing further evidence that these cells represent residual non-leukaemic normal cells. By multiparameter analysis, this population of candidate normal stem cells could be separated from contaminating leukaemic CD34⁺CD19⁻CD45⁻cells, which were detected in 11 of the 12 patients within the CD34⁺CD19⁻ compartment.     

Ethanol and Natural Killer Cells. I. Activity and Immunophenotype in Alcoholic Humans

The human lymphocyte fraction with the greatest fresh killing activity against K562 targets is phenotypically the CD3⁻CD19⁻CD56⁺ subset There have been reports of reduced natural killer (NK) activity in human alcoholics, but overall consistency is lacking and phenotypic monitoring has been inadequate to allow reliable estimates of changes in the active cell fractions. We have evaluated a range of cell surface markers and fresh NK activity in controls and alcoholics, and now report abnormalities in both phenotype and function in some alcoholics, but a normal profile in others. Patients without evidence of active liver disease (AWLDs) tend to have normal fresh basal activities and phenotypic profiles. Patients with alcoholic liver disease (ALDs) have fewer Lin⁻ lymphocytes that are CD56⁺. Three of 14 ALDs assayed in the present work had absent NK activity, whereas others were activated. In normal controls and in AWLDs, the presence of monocytes in the lytic assay consistently inhibits lysis; but, in some patients with ALD, the presence of monocytes is stimulatoty to NK activity. In alcoholics as one group, there is a statistically significant dative increase in a novel Lin⁻ subset of unknown function; this subset has a phenotype of Lin⁻CD56⁻CD45RO⁺.     

CD3−CD4−CD8−CD56−CD19−CD14− cells and CD3+CD8 dull-positive cells produce IL-4 in AIDS patients

We used flow cytometry to identify the presence of intracellular cytokines (cytoflow) and analyse the production of IL-4 in peripheral blood from AIDS patients who have practically no CD4⁺ T cells. We found that IL-4 was produced by CD3⁻CD4⁻CD8⁻CD56⁻CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells in AIDS patients. Moreover, CD3⁻CD4⁻ CD8⁻CD56⁻CD19⁻CD14⁻ cells had helper activity for immunoglobulin synthesis. These findings indicate that instead of CD4⁺ T helper cells, C3⁻CD4⁻CD8⁻CD56⁻ CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells may be an important source of IL-4 in a variety of immune responses for AIDS patients.     

CD3-induced T-cell activation in the bone marrow of myeloma patients: major role of CD4+ cells

Summary. A large expansion of activated T cells (CD3⁺CD25⁺) with the potential to act as anti-tumour effector cells is inducible in multiple myeloma (MM) patients by culturing bone marrow mononuclear cells (BMMCs) with the anti-CD3 monoclonal antibody (mAb) OKT3. The aim of this study was to provide a greater characterization of CD3-activated T cells. On day 6, most T cells coexpressed the CD1 la, CD18, CD54, CD45R0 antigens and consisted of activated (CD25⁺) CD4⁺ and CD8⁺ cells in nearly equal proportions. Kinetics studies showed that CD4⁺CD25⁺ cells proliferated more rapidly and peaked earlier than CD8⁺CD25⁺ cells. When experiments were performed with purified subpopulations by removing CD4⁺ cells (resulting in CD8⁺ BMMCs) or by removing CD8⁺ cells (resulting in CD4⁺ BMMCs), T-cell activation and autologous plasma cell decrease were observed in CD4⁺ BMMCs only. Transwell cultures showed that CD4 help was necessary to make CD8⁺ BMMCs susceptible to CD3 stimulation. Relevant amounts of IL-2 were found in the supernatants of CD4⁺ BMMCs cultures, whereas no secretion of IL-4 was detected, indicating a Thl-like profile of CD3-activated CD4⁺ cells.
These data indicate that CD4⁺ cells proliferate earlier and provide optimal help to induce the subsequent expansion of CD8+ cells after CD3 stimulation of MM BMMCs. Adequate stimulation of CD4⁺ cells is therefore essential in any strategy aiming to recover T-cell-mediated immunity in MM.     

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.     

G-CSF alone mobilizes sufficient peripheral blood CD34+ cells for positive selection in newly diagnosed patients with myeloma and lymphoma

We have evaluated CD34⁺ cell positive selection from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) in 26 patients with either multiple myeloma (MM, n  = 18) or follicular non-Hodgkin's lymphoma (NHL, n  = 8). 26 PBPC were collected with two leukaphereses: 16 contained sufficient numbers of CD34⁺ cells and were selected. The absolute number of CD34⁺ cells in the leukapheresis products was found to be significantly related to the duration of underlying disease and exposure to prior treatment. CD34⁺ cell positive selection allowed recovery of a median of 35% of CD34⁺ cells, the selected fraction containing a median number of 1.43 × 10⁶/kg CD34⁺ cells/kg (range 0.48–41.5). 10 patients were transplanted and received a median dose of 1.51 × 10⁶ CD34⁺ cells (range 0.48–4.2). The median time to granulocyte (>0.5 × 10⁹/l) and platelet (>20 × 10⁹/l) engraftment was 12 and 13 d respectively (ranges 10–13 and 0–95). Lymphoma cells were found by a sensitive polymerase chain reaction technique in four out of five CD34⁺ cell fractions tested.     

Foxp3 expression on normal and leukemic CD4+CD25+ T cells implicated in human T-cell leukemia virus type-1 is inconsistent with Treg cells

Foxp3 is a master gene of Treg cells, a novel subset of CD4⁺ T cells primarily expressing CD25. We describe here different features in Foxp3 expression profile between normal and leukemic CD4⁺CD25⁺ T cells, using peripheral blood samples from healthy controls (HCs), human T-cell leukemia virus type-1 (HTLV-1)-infected asymptomatic carriers (ACs), patients with adult T-cell leukemia (ATL), and various hematopoietic cell lines. The majority of CD4⁺CD25⁺ T cells in HCs were positive for Foxp3, but not all CD4⁺CD25⁺ T cells in ACs were positive, indicating that Foxp3 expression is not always linked to CD25 expression in normal T cells. Leukemic (ATL) T cells constitutively expressing CD25 were characteristic of heterogeneous Foxp3 expression, such as intra- and inter-case heterogeneity in intensity, inconsistency with CD25 expression, and a discrepancy in the mRNA and its protein expression. Surprisingly, a discernible amount of Foxp3 mRNA was detectable even in most cell lines without CD25 expression, a small fraction of which was positive for the Foxp3 proteins. The subcellular localization of Foxp3 in HTLV-1-infected cell lines was mainly cytoplasmic, different from that of primary ATL cells. These findings indicate that Foxp3 has two facets: essential Treg identity and molecular mimicry secondary to tumorigenesis. Conclusively, Foxp3 in normal T cells, but not mRNA, is basically potent at discriminating a subset of Treg cells from CD25⁺ T-cell populations, whereas the modulation of Foxp3 expression in leukemic T cells could be implicated in oncogenesis and has a potentially useful clinical role.     

In pregnant mice, the infection of Toxoplasma gondii causes the decrease of CD4+CD25+ -regulatory T cells

Acute maternal infection with Toxoplasma gondii during pregnancy is associated with adverse pregnancy outcomes. Although previous reports have indicated that T. gondii may result in abortion without direct transmission of the parasite to the foetus, the molecular mechanism remains unclear. CD4⁺CD25⁺-regulatory T cells are known to be involved in maternal tolerance toward the foetus-bearing alloantigens. With a model of pregnant mice infected with T. gondii , we found that Foxp3 mRNA expression levels in both splenocytes and placenta were reduced markedly during the process of infection. Furthermore, the numbers of splenic CD4⁺CD25⁺-regulatory T cells and placental Foxp3⁺ cells decreased synchronously in the infected mice, and the reduction of splenic CD4⁺CD25⁺-regulatory T cells were associated with apoptosis induced by the infection. Additionally, injection of pregnant mice with excretory–secretory antigens (ESA) of T. gondii also resulted in foetal loss, which could be partly prevented by adoptive transfer of CD4⁺CD25⁺-regulatory T cells from normal pregnant mice. These data suggest that foetal loss caused by T. gondii can be independent of vertical infection and that the decrease of CD4⁺CD25⁺-regulatory T cells during infection may represent a previously unrecognized mechanism for the pathogenesis of abortion caused by this parasite.     

Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells

We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34⁺CD33⁻ cells expressed P-gp strongly, CD34⁺CD33⁺ cells moderately, and CD34⁻CD33⁺ cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34⁻CD33⁺ but not CD34⁺CD33⁻ at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations.     

HIGH-DOSE THERAPY WITH PERIPHERAL BLOOD STEM CELL TRANSPLANTATION RESULTS IN A SIGNIFICANT REDUCTION OF THE HaEMOPOIETIC PROGENITOR CELL COMPARTMENT

In order to study the effect of high-dose therapy with peripheral blood stem cell transplantation (PBSCT) on the haemopoietic reserve in man, the number and composition of bone marrow (BM) and peripheral blood (PB)-derived progenitor cells were examined in 137 cancer patients. In 45 patients, paired samples from BM and PB were obtained before PBSC mobilization and 6–27 months after transplantation. Following PBSCT, the proportion of CD34⁺ cells was significantly smaller than before mobilization (BM 1.99±0.24 versus 0.8±0.09, P <0.001), and no change was observed at several follow-up visits thereafter.
The reduction was most pronounced for the primitive BM progenitor subsets such as the CD34⁺/DR⁻ and CD34⁺/Thy-1⁺ cells. The impairment of hematopoiesis was also reflected by a significant reduction in the plating efficiency of BM and PB samples.
No relationship was found between the decrease in the proportion of CD34⁺ cells and any particular patient characteristics, kind of high-dose therapy or the CD34⁺ cell content in the autograft.
In conclusion, high-dose therapy with PBSC transplantation is associated with a long-term impairment of the haemopoietic system. The reduction in the number of haemopoietic progenitor cells is not associated with a functional deficit, as peripheral blood counts post-transplantation were normal in the majority of patients.