D. Niederwieser

Generation of mature dendritic cells from human blood An improved method with special regard to clinical applicability

Two methods to generate human dendritic cells from hematopoietic precursor cells in peripheral blood have recently been published. One approach utilizes the rare CD34+ precursors and GM-CSF plus TNF-α. The other method makes use of the more abundant CD34− precursor population and GM-CSF plus IL-4. Here we report a method that is based on the latter approach. However, the GM-CSF and IL-4 treated cells are not stable mature dendritic cells, e.g., the characteristic morphology and nonadherence of dendritic cells is lost if the cytokines are removed. We describe the need for a monocyte-conditioned medium to generate fully mature and stable dendritic cells. This is achieved by adding a 3 day ‘maturation culture’ to the initial 6–7 day culture in the presence of GM-CSF and IL-4. Macrophage-conditioned medium contains the critical maturation factors. Mature dendritic cells are defined by their pronounced display of motile cytoplasmic processes (‘veils’), their high capacity to induce proliferative responses in resting T cells, particularly in naive umbilical cord T cells, their down-regulated antigen processing ability, and their characteristic phenotype: expression of CD83, high levels of MHC molecules and CD86, lack of CD115 and perinuclear dot-like CD68 staining. These features are stable for at least 3 days upon withdrawal of cytokines and conditioned media. IL-4 can be replaced by IL-13. When CD34+ progenitors are depleted from blood, there is only a minor reduction in the yield of dendritic cells by this method. We have adapted the method to consider several variables that are pertinent to clinical use, including a change from fetal calf serum to human plasma and to media approved for clinical use like X-VIVO or AIM-V. 1% plasma and RPMI 1640 are currently optimal. Additional reagents used for cell culture (Ig, cytokines) and cell separation (immunomagnetic beads) are approved for or already used in clinical applications. For 40 ml blood, the yield is 0.8–3.3 × 106 mature dendritic cells as defined by the expression of the new dendritic cell-restricted marker CD83. CD83+ cells constitute between 30 and 80% of all cells recovered at the end of the culture period. Yields can be enhanced up to six-fold if the blood donors are pretreated with G-CSF. Stable, mature dendritic cells generated by this method should be a powerful tool for active immunotherapy.

Dendritic cells generated from blood precursors of chronic myelogenous leukemia patients carry the Philadelphia translocation and can induce a CML-specific primary cytotoxic T-cell response.

Dendritic cells (DC) are professional antigen‐presenting cells specialized in the initiation of primary immune responses. We were interested to know whether mature DC can be grown in vitro from peripheral blood mononuclear cells (PBMC) of patients with chronic myelogenous leukemia (CML), and whether they carry the Philadelphia (Ph) translocation. Using a method recently described, DC were generated from PBMC precursors of 12 patients with CML using GM‐CSF, IL‐4, and monocyte‐conditioned medium. DC exhibited the typical morphology with thin cytoplasmatic processes and expressed high levels of MHC class II, CD86, and CD83 typical for mature DC. After sorting with the monoclonal antibody CD83, a cell population of more than 95% CD83 positive cells was obtained. The presence of the Ph translocation was analyzed in these cells, in PBMC, lymphoblastoid cell lines (LCL), and in phytohemagglutinin (PHA)‐induced T blasts from the same patients by fluorescence in situ hybridization (FISH). In contrast to all other cells analyzed, the vast majority of DC (95.9 ± 0.7%) displayed the Ph translocation, irrespective of disease stage or therapy. PBMC were predominantly positive for the Ph chromosome (67.6 ± 7.3%), whereas only 11.4 ± 1% of the B cells and 4.4 ± 1.1% of the PHA blasts carried the Ph translocation. Using such leukemic DC as antigen‐presenting cells, a primary CML‐directed cytotoxic immune response in vitro was obtained, as shown by the specific recognition of Ph chromosome positive cells. We conclude that DC can be generated from blood progenitors of CML patients in vitro and exhibit, to a large extent, the Ph translocation. Such DC, which in a preliminary experiment have been able to induce a primary CML‐directed cytotoxic immune response in vitro, might be ideal candidates for adoptive immunotherapy either by direct transfer of DC for in vivo generation of a T‐cell response or by in vitro generation of CML‐specific cytotoxic autologous or HLA‐matched normal T‐cell clones for use in vivo. Genes Chromosomes Cancer 20:215–223, 1997.

Rapid reappearance of large granular lymphocytes (LGL) with concomitant reconstitution of natural killer (NK) activity after human bone marrow transplantation (BMT)

The frequency of large granular lymphocytes and their relationship to functional NK‐activity as assessed by the capacity to lyse the K562 tumour target was analysed in five allogenic and two autologous human bone marrow transplant recipients. Date revealed: (i) almost identical disappearence and reconstitution of both parameters further indicating that LGL represent effector cells of spontaneous lysis of K 562 targets; (ii) a long‐lasting suppression of the absolute numbers per ml of blood of both LGL and functional NK activity which we believe was not the consequence of reconstitution with immature effector cells but rather reflected immunosuppressive therapy; (iii) LGL exhibits the fastest reappearance rate subsequent to total body irradiation of all populations of circulating leucocytes.

CYCLOSPORIN BLOOD LEVELS DO CORRELATE WITH CLINICAL COMPLICATIONS

SIR,—In October, 1983, participants in the European Multicentre Studyl discussed the use of cyclosporin in renal transplantation, focusing on adults and not mentioning specific paediatric aspects. We participated in this study in the hope of achieving better growth in children without resorting to conventional2 therapy with azathioprine and steroids. We observed that under cyclosporin therapy all children showed normal growth velocity or catch-up growth and that during the first year, without monitoring of cyclosporin levels, the one-year survival of 56% in children with renal transplants was lower than survival in adult graft recipients. From January, 1981, to March, 1984, we did twelve renal transplants in ten children aged 4 months to 17 years (mean =11.2 years). Cyclosporin was given alone in four transplants and with low-dose methylprednisolone (8 mg/1 I 73 m2 body surface area) in eight. In six cases there was no rejection, two patients were successfully treated for four episodes of rejection, and irreversible rejection (confirmed by needle biopsy) occurred in four patients all of whom had been treated with cyclosporin and low-dose steroids. In three of these four children (aged 12, 13, and 14 years) irreversible rejection took place between the 5th and 7th month. In all of them clinical symptoms were slight. Differentiation between rejection and cyclosporin toxicity was only possible histologically, because cyclosporin levels could not be measured before 1983. We suspected that the reduced cyclosporin dosageof6 mg/kg body weight at 6 months, as described in the protocol of the European study, was too low. Our third case had a serum cyclosporin level below 100 ng/ml (on a dose, 7 months after transplantation, of9’ 5

Human interferon-? induces expression of HLA-DR on keratinocytes and melanocytes

SummaryPrimary human epidermal cell cultures composed of keratinocytes and melanocytes were exposed to supernatants of phytohaemagglutinin (PHA)-stimulated T cells, various lymphokines and interferon-β, and checked for the emergence of HLA-DR antigen using immunofluorescence and immunoelectron microscopy. HLA-DR expression was induced by the supernatants and human recombinant interferon-γ (rIFN-γ), whereas recombinant α2, interleukin-2 and non-recombinant human interferon-β had no such effect. The threshold concentration of rIFN-γ required to induce this phenomenon was 10 IU/ml; no further increase of reaction intensity was observed using doses of more than 100 IU/ml. Maximum reaction intensity was achieved after 72 h of incubation; a minimum of 3 h of incubation with rIFN-γ followed by 72 h incubation in rIFN-γ-free medium proved sufficient to induce HLA-DR expression. The inductive effect of the supernatants and rIFN-γ could be completely abrogated by pretreatment with excess doses of the monoclonal antibody GZ4 specific for human IFN-γ. Keratinocytes and melanocytes reacted in an identical fashion both qualitatively and quantitatively in all experiments. These data indicate that IFN-γ possesses specific signal functions in the induction of HLA-DR expression on epidermal cells.

Effect of interleukin-3 pretreatment on granulocyte/macrophage colony-stimulating factor induced mobilization of circulating haemopoietic progenitor cells

Summary. Recombinant human colony stimulating factors (CSFs) as single agents are increasingly used for mobilizing peripheral blood progenitor cells (PBPCs) for stem cell transplantation. We have shown in rhesus monkeys that interleukin‐3 (IL‐3) pretreatment markedly potentiated the increase in PBPC numbers of subsequent administration of granulocyte/macrophage‐CSF (GM‐CSF). Here we studied the effect of IL‐3 pretreatment on GM‐CSF‐induced mobilization of PB progenitors in patients who were potential candidates for autologous stem cell transplantation (n=16). Patients were treated with GM‐CSF at a dose of 5 μg/kg/d for 5 d and after a treatment free interval received another cycle of GM‐CSF immediately following pretreatment with IL‐3 at different doses and duration: 2.5 μg/kg/d (n = 4), 5μg/kg/d (n = 3) and 10μg/kg/d (n = 3) for 3d, 5μg/kg/d for 7d (n = 4) and 5μg/kg/d for 14 d (n = 2), respectively. Only 7d pretreatment with IL‐3 showed consistent effects. Although IL‐3 did not mobilize by itself, pretreatment with 5μg/kg/d of IL‐3 for 7d significantly potentiated GM‐CSF‐induced mobilization of PB CFU‐GM numbers, leading to a mean increase in PB CFU‐GM numbers over baseline by 18.5 +5.2 (SEM) fold by IL‐3/ GM‐CSF as compared to a 4.7 + 1.7‐fold increase by GM‐CSF alone. A significant enhancement by the 7d IL‐3 pretreatment was also observed for erythroid (BFU‐E) and multi‐potential progenitor cells (CFU‐mix) which were 3.3 + 1.3‐and 3.4 + 0.9‐fold, respectively, mobilized by GM‐CSF alone, as compared to 8.5 + 2.3‐ and 19.2 + 3.4‐fold, respectively, by the IL‐3/GM‐CSF combination. Our results suggest that 7 d pretreatment with IL‐3 may be a useful mean to augment mobilization of circulating progenitors by more lineage‐restricted CSFs. These findings may be important for the design of mobilization strategies that use growth factors without preceding chemotherapy.

Neopterin, ein neuer biochemischer Marker zur klinischen Erfassung zellulärer Immunreaktionen

SummaryActivated T-lymphocytes represent crucial effector cells. They are pathogenetically involved into various disease states such as allograft rejection or viral infection. So far their assessment is laborious and rarely possible in clinical routine. In this review article we present the compount neopterin as a new biochemical marker for the in vivo and in vitro detection of activated T-lymphocytes. Our main finding was that in vitro as well as in vivo stimulation of T-lymphocytes with foreign and chemically or virally modified autologous cells in invariably associated with increased neopterin production. It thus appeared that neopterin might represent a potential marker for biochemical monitoring of diseases caused by or associated with T-lymphocyte activation. Our clinical experience with neopterin determination in allograft rejection and in infectious or autoimmune states strongly support this view. We conclude that evaluation of neopterin represents a useful and simple tool for the biochemical monitoring of immunological and/or malignant states.Activated T-lymphocytes represent crucial effector cells. They are pathogenetically involved into various disease states such as allograft rejection or viral infection. So far their assessment is laborious and rarely possible in clinical routine. In this review article we present the compount neopterin as a new biochemical marker for the in vivo and in vitro detection of activated T-lymphocytes. Our main finding was that in vitro as well as in vivo stimulation of T-lymphocytes with foreign and chemically or virally modified autologous cells in invariably associated with increased neopterin production. It thus appeared that neopterin might represent a potential marker for biochemical monitoring of diseases caused by or associated with T-lymphocyte activation. Our clinical experience with neopterin determination in allograft rejection and in infectious or autoimmune states strongly support this view. We conclude that evaluation of neopterin represents a useful and simple tool for the biochemical monitoring of immunological and/or malignant states.

Interferons (IFNs) and tumor necrosis factors (TNFs) in T cell-mediated immune responses against alloantigens. I. influence on the activation of resting and antigenprimed T cells

The aim of this study was to investigate the influence that endogenous IFNs released in response to antigenic or viral stimuli has on recognition of alloantigens in MLC. Results indicated that both the magnitude and the kinetics of response can be modified by IFNs. Neutralizing antibodies with specificity for IFN-gamma inhibit early and enhance late proliferative responses in MLC. Addition of physiological concentrations of IFN-gamma enhanced both early and peak proliferation, whereas IFN-alpha markedly inhibited alloantigen-induced lymphocyte proliferation. Further experiments revealed that IFN effects in MLC are not caused by direct interaction with responder cells: pretreatment with IFNs neither failed to alter their subsequent proliferative reactivity, nor did it influence production of IL 2 in MLC. IFN-gamma mainly affected MLC responses by direct interaction with stimulator cells. These influences on hemopoietic and non-hemopoietic stimulator cells were complex and could not simply be explained on the basis of an altered expression of class II MHC antigens. When induced by IFN-gamma to maximally express class II antigens, pbmnc, LCL or homogeneous populations of macrophages showed a marked deficiency to induce primary or secondary proliferative T cell responses. Resting unsensitized or sensitized T cells were not stimulated by class II MHC antigens constitutively expressed or induced by IFN-gamma on cell types other than dendritic cells or LCL. Class II antigens on the former cells were, however, readily recognized by T helper blasts, and this process involved the T4 epitope of the T cell receptor. IFN-gamma treatment also influenced the intrinsic suppressive capacity of macrophages or keratinocytes without involving prostaglandin synthesis or inducing expression of IL 2 receptors on non T cells.

Role of cytokines and major histocompatibility complex antigens in graft-versus-host disease: in vitro studies using T-cell lines and keratinocytes or hemopoietic targets.

Allogeneic bone marrow transplantation (BMT) for most oncologists still represents a means by which more aggressive tumor therapies can be applied. Immunologists in contrast would consider this approach a special form of immunotherapy. This view in man is mainly supported by the finding of reduced leukemic relapse rates in the context of clinical manifestation of graft-versus-host disease (GvHD) [1]. In this article, we provide further experimental evidence that allogeneic BMT across minor histocompatibility antigen barriers leads to generation of specific killer cells, which lyse host hemopoietic cells, and to the induction of enhanced production of endogenous cytokines, which are meaningful with respect to antitumor defense.

Treatment of relapsed and refractory acute myelogenous leukaemia with aclacinomycin A (ACA) and etoposide (VP-16)

Ten patients with refractory and relapsed acute myelogenous leukaemia (AML) and one patient with CML in blast crisis were treated with aclacinomycin A (ACA, 60 mg/m2/day for 5 days) and etoposide (VP-16, 100 mg/m2/day for 5 days) and analysed retrospectively. Of 11 patients, seven (63%) achieved an objective response including four CRs (36%). Most impressively, two patients experienced consecutive CRs (second, third, and fourth) following relapses. Only two patients (18%) were primary resistant with persisting leukaemia. In conclusion, the combination of ACA and VP-16 is an active regimen in refractory and relapsed AML with a toxicity comparable with other salvage regimens.