Cytokine profile of protective anti-Trichinella spiralis CD4+ OX22− and non-protective CD4+ OX22+ thoracic duct cells in rats: secretion of IL-4 alone does not determine protective capacity

We analysed the cytokine profile of a T cell subset (CD4⁺ CD45 RC⁻) that confers protection against Trichinella spiralis infection in rats. These CD4⁺ cells are generated in the gut and appear in the thoracic duct lymph within 72 h after infection. Cytokine mRNA levels for IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-γ and functional cytokine secretion for IL-4, IL-5, IFN-γ, TNF-α and mast cell differentiation activity were tested in vitro following stimulation with T. spiralis antigens. Compared to a non-protective T cell population (CD4⁺ CD45 RC⁺ or CD8⁺), also isolated from the same thoracic lymph, no significant differences were observed in the levels of mRNA for IL-2, IL-3, IL-4, IL-5, IL-10 or IFN-γ in the protective CD4⁺ CD45 RC⁻ cells. However, analysis of the cytokine activities in culture supernatant of these T cell subsets following 24 h stimulation in vitro with T. spiralis antigens showed that significant IL-4 and IL-5 activity but little IFN-γ or TNF-α was secreted by the protective CD4⁺ CD45 RC⁻ cells. Whereas the non-protective CD4⁺ CD45 RC⁺ cells secreted significant levels of IL-4, IFN-γ, mast cell differentiating activity and TNF-α but little IL-5 activity. Nonprotective CD8⁺ cells were found to secrete IL-4 but not IL-5. Production of IL-4 was essentially equal for both protective and non-protective T cell subsets. These findings suggest that the presence or absence of IFN-γ secretion, rather than IL-4 alone, determines whether a T cell subset has protective activity against T. spiralis infection in rats.     

CD8+/CD57+ cells and apoptosis suppress T-cell functions in multiple myeloma

The aim of this study was to evaluate the role of CD8⁺/CD57⁺ lymphocytes in the immune dysregulation of multiple myeloma (MM). Cytofluorimetry of peripheral blood lymphocytes (PBL) purified from 39 MM patients showed an inverse relationship between the percentage of CD8⁺/CD57⁺ cells and CD4/CD8 ratio. Analysis of their activation antigens revealed that they were prevalently HLA-DR⁺ and Fas⁺. Removal of CD8⁺/CD57⁺ cells from MM PBL significantly improved cell proliferation and pokeweed mitogen (PWM)-induced polyclonal Ig production in vitro, whereas the addition of supernatants from patients' CD8⁺/CD57⁺ cell cultures to normal PBL suppressed both the PWM-driven Ig synthesis and the proliferative rate of stimulated PBL, supporting the contention that CD8⁺/CD57⁺ cells release in vitro an inhibitory factor that is directly involved in T-cell regulatory function. However, since the proliferative recovery of PWM- and phytohaemagglutinin (PHA)-stimulated MM PBL in the absence of CD8⁺/CD57⁺ lymphocytes was only partial, a dysregulated activation-induced apoptosis was anticipated. In fact, patients' PBL displayed an increased susceptibility to apoptosis and this was significantly enhanced after PWM and, even more, after PHA stimulation. Analysis of CD57 antigen expression on apoptotic or viable cells demonstrated a substantial defect of apoptosis in the CD8⁺/CD57⁺ population. Our results indicate that both the immunosuppressive effect of CD8⁺/CD57⁺ cells and the enhanced susceptibility to apoptosis of PBL could be involved in the pathogenesis of the immunodeficiency observed in this disease.     

Idiotype-specific T lymphocytes in monoclonal gammopathies: evidence for the presence of CD4+ and CD8+ subsets

Tumour-specific CD4⁺ T helper (Th) and CD8⁺ T cytotoxic (Tc) cells may participate in the control and eradication of tumour cells. In the present study, idiotype-specific stimulation of CD4⁺ and CD8⁺ blood T cells from patients with monoclonal gammopathy of undetermined significance and patients with untreated multiple myeloma stage I was examined. Activation was measured in the CD4⁺ and CD8⁺ subsets enriched by magnetic microbeads as the incorporation of ³H-thymidine and the secretion of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 by single cells using the enzyme-linked immunospot assay. Idiotype-specific T cells were found in four of seven patients. Stimulation was mainly confined to the CD4⁺ subset in three of the four responding patients. This type of response was major histocompatibility complex (MHC) class II restricted as it could be inhibited by monoclonal antibodies against MHC class II (HLA-DR), but not against class I (HLA-ABC) molecules. Idiotype-specific CD8⁺ T cells were also demonstrated in these patients although at a lower frequency. One patient showed a strong and dominating activation of CD8⁺ T cells which could be blocked by antibodies against HLA-ABC but not against HLA-DR. Idiotype-specific CD4⁺ or CD8⁺ T cells were mainly of the type-1 subsets as judged by their secretion of IFN-γ and IL-2. Thus, this study provides evidence for the presence of idiotype-specific and MHC-restricted CD4⁺ and CD8⁺ T cells of the type-1 subsets in patients with monoclonal gammopathies. Such T cells with the potential to control the growth of tumour B cells may be a suitable target for immunotherapeutic interventions in patients.     

Inhibition of Plasmodium falciparum growth in vitro by CD4+ and CD8+ T cells from non-exposed donors

T cells from most adult non-exposed donors, which express a memory phenotype (CD45RO+), can respond by proliferation to P. falciparum asexual stages in vitro. Such cells may have arisen from exposure to environmental organisms. To address the efficacy of such cells in eliminating parasites and investigate the mechanisms involved, we have used an in vitro assay where parasite growth can be precisely monitored in the presence of different cell preparations. Unfractionated peripheral blood mononuclear cells (PBMC) from both malaria-exposed and non-exposed donors inhibited parasite growth by up to 62% in a two day assay. Purified T cells in the presence of adherent cells had a similar effect, but purified T cells alone or adherent cells alone had minimal effect. Antigens released at the time of schizont rupture were maximally effective in stimulating interferon-γ (IFNγ) production. Neutralizing antibodies to IFNγ showed a partial reduction of growth inhibition in some individuals tested suggesting that different mechanisms may be operative. Neutralizing antibody to TNFα had a partial effect in combination with anti-IFNγ. Antibodies to IL-1 and IL-4 had no effect. T cell fractionation experiments showed that while purified CD4⁺ T cells from some donors produced IFNγ and inhibited parasite growth, purified CD8⁺ T cells could inhibit parasite growth to a greater extent without production of detectable IFNγ. Four parasitised red blood cell clones (CD4⁺, TCRαβ⁺, IFNγ producing) derived from non-exposed donors inhibited parasite growth to comparable levels, but one clone showed low production of IFNγ whilst the other three produced high levels. Our data show that T cells from non-exposed donors have the potential to eliminate malaria parasites via non-IFNγ dependent mechanisms. Such mechanisms may contribute to a degree of innate resistance to malaria in vivo, and may be able to be targeted by malaria vaccine programs.     

CD56+ NK lymphomas:clinicopathological features and prognosis

The surface molecule CD56 marks a category of malignant lymphoma of putative natural killer (NK) cell origin. We conducted a retrospective analysis of 24 cases of CD56⁺ NK lymphoma/leukaemia to define the clinicopathologic and prognostic features of this specific group of lymphomas. 56 cases of nasal lymphomas and 204 cases with an initial diagnosis of peripheral T-cell lymphoma were retrospectively analysed. To specifically examine lymphomas of putative NK origin, only those that were negative for surface expression of CD3 but positive for CD56 were analysed. 24 cases were identified. The initial predominant sites of involvement were nasal (n =18), palate (n = 1), nodal (n = 1) and multi-organ (n =4). Clinically, in patients with disease localized to one anatomical site (n = 20), most had symptoms confined to the nose, with a high percentage in early stage (I: 91%; IV: 9%). The marrow was not involved in any of these cases. However, patients with multi-organ involvement at presentation (n = 4) behaved differently. All presented acutely with pancytopenia, hepatosplenomegaly, and marrow infiltration with haemophagocytosis. A leukaemic phase was observed in one case. Anthracycline containing combination chemotherapy resulted in complete remission in 75% of patients with localized disease, but only in 25% with multi-organ involvement. The median survival of patients with localized disease was 12 months, compared with 2 months in the multi-organ group (P =0.06); the disease-free survival was significantly better in the former (P <0.01). The overall median survival of all patients was still poor at 11 months. We conclude that CD56⁺ NK lymphomas could be divided into two main patterns of disease presentations: localized (predominantly nasal), and multi-organ involvement. Each has different clinicopathologic and prognostic features. Conventional chemotherapy appeared ineffective for the majority of patients, and innovative treatment modalities are needed to improve outcome.     

Expansion of CD8+CD57+ T cells after allogeneic BMT is related with a low incidence of relapse and with cytomegalovirus infection

Summary. Peripheral blood lymphocytes of 46 recipients of lymphocyte-depleted bone marrow allografts were pheno-typically analysed over a period of 1 year. We investigated the repopulation of lymphocyte subpopulations and their relation with clinical parameters such as graft-versus-host disease (GVHD), graft-versus-leukaemia and cytomegalovirus (CMV) infection. The number of repopulated T cells varied strongly between the blood samples of the recipients. In 45% of the recipients the number of T cells recovered to or above normal levels within 3 months after bone marrow transplantation (BMT), whereas the other recipients remained below normal up to 1 year after BMT. In recipients with a high repopulation, the CD8⁺ T-cell subset contributed more to this high repopulation than the CD4⁺ T-cell subset. We showed that the majority of T cells of these recipients expressed the a/3 T-cell receptor, CD8, CD57 and CDllb. HLA-DR was also highly expressed reflecting the activation stage of T cells in these recipients. BMT recipients with a high repopulation of CD8⁺ T cells showed a lower incidence of leukaemic relapse than recipients with a low repopulation. The 3-year probability of relapse was 19% versus 64% (P=O03), respectively. The relative high number of CD8⁺ T cells at 3 months after BMT was not associated with the incidence of GVHD. In contrast, occurrence of CMV infection after BMT was significantly higher in these recipients. Our results indicate that CD8⁺ T cells, predominantly CD57⁺, of BMT recipients with an expansion of these cells represent an in vivo activated cell population. This CD8⁺ T-cell population may consist partially of cytotoxic cells with anti-leukaemic activity as suggested by a low relapse rate. The signal for the strong expansion of these CD8+CD57+ T cells after BMT is still unclear, but association with CMV infection suggests that viral antigens are involved.     

Action of human interleukin-4 and stem cell factor on erythroid and mixed colony formation by peripheral blood-derived CD34+c-kithigh or CD34+c-kitlow cells

We studied the interaction of interleukin (IL)-4 and other burst-promoting activity (BPA) factors, such as IL-3, granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-9 and stem cell factor (SCF), on erythroid burst-forming unit (BFU-E) and erythrocyte-containing mixed (CFU-Mix) colony formation in serum-free culture. IL-4 alone did not support mixed colony formation in the presence of erythropoietin (Epo). However, IL-4 showed weak but significant BPA when peripheral blood (PB)-derived CD34⁺c-kit^low cells were used as the target population. The BPA of IL-4 was much weaker than that of IL-3, which exerted the most potent activity, as previously reported. When CD34⁺c-kit^high cells were used as the target, four factors known to have BPA, IL-3, GM-CSF, IL-9 and SCF, could express BPA. In contrast, IL-4 alone failed to support erythroid burst formation. Interestingly, IL-4 showed a remarkable enhancing effect with SCF in promoting the development of erythroid burst and erythrocyte-containing mixed colonies from CD34⁺c-kit^low and CD34⁺c-kit^high cells. Delayed addition of SCF + Epo or IL-4+Epo to the cultures initiated with either IL-4 or SCF alone clearly demonstrated that SCF was a survival factor for both BFU-E and CFU-Mix progenitors. In contrast, the survival effect of IL-4 was much weaker than that of SCF, and appeared to be more important for progenitors derived from CD34⁺c-kit^low cells than for those derived from CD34⁺c-kit^high cells. It was recently reported that CD34⁺c-kit^low cells represent a more primitive population than CD34⁺c-kit^high cells. Taken together, these results suggest that IL-4 helps to recruit primitive progenitor cells in the presence of SCF.     

Hyperinsulinaemia and Na+, K+ -ATPase activity in thyrotoxic periodic paralysis

OBJECTIVE Thyrotoxic periodic paralysis (TPP) usually follows a heavy carbohydrate meal and this may be explained by hyperinsulinaemia stimulating Na⁺, K⁺ -ATPase activity. To clarify this the effect of glucose load on serum insulin concentration and platelet Na⁺, K⁺ -ATPase activity In thyrotoxic periodic paralysis (TPP) was examined. DESIGN In all subjects a standard 75-g glucose tolerance test was done and blood samples were taken at 0, 1 and 2 hours. SUBJECTS Twenty-five healthy controls (8 M and 17 F), 17 uncomplicated thyrotoxic patients (7M and 10 F), 15 TPP patients who presented with paralysis and 4 TPP patients after treatment with antithyrold drugs. MEASUREMENTS Plasma glucose was measured by the glucose oxidase method, serum insulin by radioimmunoassay and platelet Na⁺, K⁺ -ATPase by the release of phosphate from ATP. RESULTS TPP patients showed glucose intolerance (area under the curve (AUC) 16·5 ± 4·4 (mean ± SD) In TPP compared to 12·9 ± 4·5 In controls (P < 0·01) and hyperinsulinaemia (AUC 189·6 ±100·6 vs 98·5 ±53·4, P < 0·001). In uncomplicated thyrotoxicosis the results were similar to that in healthy controls. Platelet Na⁺, K⁺ -ATPase were significantly higher in thyrotoxic patients compared to controls and In TPP patients were even higher. Ingestion of glucose increased platelet Na⁺, K⁺ -ATPase in all groups. AUC for platelet Na⁺, K⁺ -ATPase in TPP patients were significantly higher than in uncomplicated thyrotoxicosis (601 ±99·3 vs 482 ± 109·4, P < 0·01) or healthy controls (320 ± 107·3). In the 4 TPP patients studied after antithyroid treatment the results were similar to healthy controls. CONCLUSION Patients with thyrotoxic periodic paralysis have hyperinsulinaemia and this is accompanied by higher Na⁺, K⁺-ATPase activity.     

Purified unfractionated G-CSF/chemotherapy mobilized CD34+ peripheral blood progenitors and not bone marrow CD34+ progenitors undergo selective erythroid differentiation in liquid culture in the presence of erythropoietin and stem cell factor

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G-CSF)/chemotherapy mobilized peripheral blood CD34⁺ cells to selective erythroid differentiation in liquid culture with an average 28-fold increase in the total cell number after 21 d. From day 6 of culture, cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34⁺ cells were cultured with EPO plus SCF in serum-free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34⁺ cells underwent granulocytic/monocytic differentiation in liquid culture in response to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and SCF, demonstrating that these CD34⁺ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34⁺ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34⁺ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G-CSF/chemotherapy mobilized CD34⁺ progenitor cells in liquid culture. The absence of cytokines such as GM-CSF and IL-3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.     

Generation and functional characterization of human dendritic cells derived from CD34+ cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34⁺ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34⁺ cells, compared to that of BM CD34⁺ precursors in response to GM-CSF and TNF-α with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34⁺ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34⁺ cells enhanced both the percentage of total CD1a⁺ cells and CD1a⁺CD14^? cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34⁺ DC precursors into PB and circulating CD34⁺ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediate-late-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.     

GM-CSF promotes differentiation of a precursor cell of monocytes and Langerhans-type dendritic cells from CD34+ haemopoietic progenitor cells

Epithelia-associated dendritic cells (DC) including Langerhans cells in the skin (LC) are precursors of lymph node located interdigitating DC (iDC). CD1a⁺ LC are known to be derived from CD34⁺ haemopoietic progenitor cells (HPC); however, cells of an intermediate differentiation state that are CD34^? and CD1a^? have not been identified. Monitoring the differentiation pathway of HPC in the presence of GM-CSF + IL-4, we observed the emergence of a distinct LC precursor population that was CD33⁺ CD13⁺ CD4⁺ CD38⁺ CD44⁺ CD34^? CD14^? CD1a^?. The cells could be separated by FACS due to a unique CD44/CD38 expression pattern or by CD44 expression in conjunction with the SSC profile. It was found that they were similarly generated in the presence of GM-CSF alone and were detectable in culture for at least a week. Irrespective of being generated in the presence of GM-CSF + IL-4 or GM-CSF alone, CD44/SSC-sorted precursor cells matured to MHC class II compartments (MIIC) and Birbeck granules (BG) expressing LC, when subsequently cultured in the presence of GM-CSF + IL-4. When IL-4 was omitted, however, the same cells matured to phagocytically active adherent macrophages (MΦ). These culture conditions were associated with a > 4-fold increase in the concentration of IL-6 when compared to those used for LC differentiation. The identification of a distinct oligopotent precursor cell population that can deliberately be induced to give rise to BG⁺ MIIC⁺ CD1a⁺ CD14^? LC or to adherent CD14⁺ MΦ further substantiates the close relationship of monocytes and DC and may help to identify its in vivo equivalent.     

The role of CD4+ and CD8+ T cells in protective immunity to the murine nematode parasite Trichuris muris

The role ofCD4⁺ and CD8⁺ T cells in protective immunity to Trichuris muris was studied in CD4⁺ or CD8⁺ or both CD4⁺ and CD8⁺ T cell-depleted BALB/c mice. To assess in vivo depletion of T-cell subsets, CD4⁺ and CD8⁺ T cells in the Peyer's patches, the mesenteric lymph nodes (MLN), and the spleens of mice treated with T cell-specific monoclonal antibodies (MoAbs) were analysed by FACS. CD4⁺ T cells were selectively depleted in mice injected with anti-CD4 MoAb i.p. and injection of anti-CD8 MoAb resulted in selective depletion ofCD8⁺ T cells. The expulsion ofl. muris was inhibited in CD4⁺ T cell-depleted mice and numerous worms were detected in the large intestine on days 14 and 21 after infection, although no suppression of protective immunity to T. muris was observed in CD8⁺ T cell-depleted mice. Moreover, there was no difference in suppression of protective immunity to T. muris between CD4⁺ T cell-depleted and both CD4⁺ and CD8⁺ T cell-depleted mice. These results indicate that CD4⁺ T cells play a central role in protective immunity to T. muris infection.     

Primordial role of CD34+38− cells in early and late trilineage haemopoietic engraftment after autologous blood cell transplantation

In order to better define which cell subset contained in graft products might be the most predictive of haemopoietic recovery following autologous blood cell transplantation (ABCT), the relationships between the amounts of reinfused mononuclear cells (MNC), CFU-GM, total CD34⁺ cells and their CD33 and CD38 subsets, and the successive stages of trilineage engraftment kinetics, were studied in 45 cancer patients, using the Spearman correlation test, a linear regression model and a log-inverse model. No relationship was found between the infused numbers of MNC, CD33⁺ and CD33^? subsets observed and the numbers of days to reach predetermined absolute neutrophil (ANC), platelet and reticulocyte counts. The infused numbers of CFU-GM, CD34⁺ and CD34⁺38⁺ cells correlated inconstantly with haemopoietic recovery parameters. The strongest and the most constant correlations were significantly observed between the infused numbers of CD34⁺38^? cells and each trilineage engraftment parameter. The log-inverse model determined a threshold dose of 0.05 × 10⁶ (= 5 × 10⁴) CD34⁺38^? cells/kg, below which the trilineage engraftment kinetics were significantly slower and unpredictable. Post-transplant TBI-conditioning regimens increased the low cell dose-related delay of engraftment kinetics whereas post-transplant administration of haemopoietic growth factors (HGF) seemed to abrogate this delay. This would justify clinical use of HGF only in patients transplanted with CD34⁺38^? cell amounts lower than the proposed threshold value. This study suggests that the CD34⁺38^? subpopulation, although essentially participating in late complete haemopoietic recovery, is also composed of committed progenitor cells involved in early trilineage engraftment.     

Heterogeneity of antigen-specific CD4+ T cell clones from a patient with Schistosomiasis mansoni

Two subsets of differentiated murine helper T cells, Th1 and Th2, based on secretion products in response to antigen have been described (Cher & Mosmann 1987, Coffman et al. 1988, Lopez et al. 1988, Paliard et al. 1988, Patel et al. 1988, Mosmann & Coffman 1989). To analyse immunological function of antigen-specific CD4⁺ T cells in human schistosomiasis, we produced schistosomal egg antigenspecific T cell clones from a former patient. We identified four different types of CD4⁺ T cell clones by analysis of cytokine production. Two of the four types of the clones corresponded to murine Th1 or Th2 subsets; a third type was of the Th0 subset (Th1 + 2) and a fourth type produced IL-5 dissociated from IL-4. Analysis of the antigen(s) recognized by these T cell clones showed that all of the clones proliferated in response to soluble egg antigen(s) (SEA) found within a pl fraction whose pH was 5·2. T cell Western blot analysis of the stimulatory pl fraction demonstrated that the apparent Mr of the relevant antigens recognized by the clones were 38 kDafor the Th2 homologue, and 45–55 kDa for the Th1 homologue.     

Lack of DNA synthesis among CD34+ cells in cord blood and in cytokine-mobilized blood

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5 μg per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Despite considerably increasing numbers of CD34⁺ PB MNC, the latter were not found to be in S/G₂M phase, whereas, among the CD34^? MNC, the proportion of cells in S/G₂M phase increased from <0.1% to 0.75 ± 0.4% (GM-CSF) and to 1.34 ± 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34⁺ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G₂M phase. The non-cycling status of PB and CB progenitor cells was confirmed by the analysis of CD34⁺ cells enriched from the two cell sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34⁺ cells were in S/G₂M phase. The fact that cycling CD34⁺ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized ‘stem cells’ which may require the BM micro-environment for adequate proliferation in vivo     

Clonotypic CD20+ and CD19+ B cells in peripheral blood of patients with multiple myeloma post high-dose therapy and peripheral blood stem cell transplantation

The number of circulating clonotypic B cells in patients with multiple myeloma (MM) after high-dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) was investigated. Peripheral CD19+ B cells have been reported to persist throughout conventional and HDT and might resemble a source of relapse in patients with MM. We assessed the proportion of malignant cells in CD20+ and CD19+ cell fractions of 14 peripheral blood (PB) samples from 12 patients after HDT and PBSCT. Nine samples were obtained from patients in continuous remission, and five patients were in progressive disease or beginning relapse. The CD20+ fractions obtained had a mean purity of 96.8%. The percentages of tumour cells were determined using a quantitative allele-specific oligonucleotide PCR assay based on the method of limiting dilutions. In the group of patients in continuous remission the median number of tumour cells in the CD20+ cell fractions was 1.9/ml (range 0-7.2 tumour cells/ml PB) higher than in the CD20- fractions (median 0; range 0-29 tumour cells/ml PB). Higher tumour cell numbers in both fractions, particularly pronounced in the negative ones, were found in patients with progressive disease or beginning relapse (CD20+: range 3.8-585; median 32 tumour cells/ml PB; CD20-: range 25-25527; median 334 tumour cells/ml PB). Enrichment with the anti-CD19 antibody as a second pan B-cell marker revealed comparable tumour cell numbers. In conclusion, an anti-CD20 antibody treatment could be a promising approach for the eradication of malignant cells in the PB of patients in continuous remission after HDT and PBSCT with low amounts of tumour cells in the B-cell compartment and an almost complete absence of tumour cells in the CD20- fractions.     

Generation of multinuclear tartrate-resistant acid phosphatase positive osteoclasts in liquid culture of purified human peripheral blood CD34+ progenitors

Circulating CD34⁺ cells were isolated from leukapheresis products collected from patients with ovarian cancer. CD34^? contaminating cells, identified immediately after immunoselection, ranged from 5% to 25% in five different experiments and were predominantly CD3⁺ T-lymphocytes (range 2–12%), CD3^?/CD16⁺/CD56⁺ natural killer cells (range 2–11%) and rare mature CD15⁺/CD11b⁺ granulocytes (range 1–2%). CD34⁺ cells were cultured in liquid medium in the presence of interleukin-3, granulocyte-macrophage colony stimulating factor, stem cell factor, granulocyte colony stimulating factor and a powerful proliferation with prevalent differentiation along the granulocytic/monocytic lineage was obtained. After 10 d of culture a small but consistent number of early multinucleated osteoclasts were identified with a frequency of one cell per 700 granulocytic/monocytic cells, as revealed by cytologic examination. This observation was confirmed by staining for tartrate-resistant acid phosphatase activity which revealed red multinucleated elements with a frequency comparable to that reported above. Conversely, no osteoclasts were observed in those cultures in which macrophage overgrowth was obtained by culturing CD34⁺ cells until day 35. These observations suggest that circulating progenitors have a multilineage potential in vitro and contribute to the clarification of osteoclast development in humans; additionally, they provide the basis for the future development of optimized osteoclast culture techniques in liquid medium and the basic culture system, to test the distinct activity of 1,25(OH)₂D₃, parathyroid hormone, interleukin-11 and of other cytokines on osteoclast development in humans.     

Induction of CD4+ and CD8+ T cell responses in efferent lymph responding to Toxoplasma gondii infection: analysis of phenotype and function

The kinetics of induction of T cell responses were examined in efferent lymph from a node draining the site of a primary inoculation of Toxoplasma gondii. The numbers of T cells increased after infection, due initially to an expansion of the CD4⁺ T cell population followed by an increase in the number of CD8⁺ T cells which coincided with the peak lymphoblast response. Proliferative responses of CD4⁺ T cells to T. gondii antigen were detectable from day six after infection and immune efferent lymph cells inhibited the intracellular multiplication of T. gondii in vitro. Optimum inhibition was achieved using CD8⁺ T cells restimulated in vitro, and the effector function appeared to be directed preferentially against the autologous rather than the allogeneic infected target cell. The results provide unique information on the induction of immune responses to T. gondii in vivo and provide evidence that both CD4⁺ and CD8⁺ T cells are necessary for the development of protective immunity induced by the S48 strain of T. gondii which is used as a live vaccine in sheep.