Martine Guigon

High level of retrovirus-mediated gene transfer into dendritic cells derived from cord blood and mobilized peripheral blood CD34+ cells.

Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.

In vivo effect of platelet factor 4 (PF4) and tetrapeptide AcSDKP on haemopoiesis of mice treated with 5‐fluorouracil

In vivo effects of platelet factor 4 (PF4) and tetrapeptide N‐acetyl‐Ser‐Asp‐Lys‐Pro (AcSDKP) on haemopoietic progenitors were studied in mice treated with 5‐fluorouracil (5‐FU). The mice were injected with PF4 (40 μg/kg) or AcSDKP (4 μg/kg) twice at 6 h intervals, and 20 h after the second injection they were given one injection of 5‐FU (150 mg/kg). 6, 8 and 13 d later the high proliferative potential‐colony forming cell (HPP‐CFC), burst‐forming unit erythroid (BFU‐E), colony forming unit granulocyte‐macrophage (CFU‐GM) colony forming unit megakaryocyte (CFU‐MK), and megakaryocytes (MK) were examined. The results showed that the administration of PF4 or AcSDKP resulted in a significant increase in the number of HPP‐CFC on days 6–8 and BFU‐E and CFU‐GM on day 8 when compared to 5‐FU alone. Furthermore, PF4 was found to increase significantly the number of CFU‐MK and MK on day 8, which was not observed with AcSDKP. However, both molecules had no obvious effect on peripheral blood cells. These data indicate that PF4 or AcSDKP accelerate the recovery in vivo of HPP‐CFC, CFU‐GM and BFU‐E after 5‐FU treatment but their effect may be different on megakaryocytic progenitors and suggests that both molecules may have a haemoprotective effect against chemotherapeutic agents.

Optimization of the Cycling of Clonogenic and Primitive Cord Blood Progenitors by Various Growth Factors

The cycling status of cord blood progenitors and the culture conditions triggering their activation into S‐phase have been studied using flow cytometry and a 3H‐thymidine suicide assay. Mononuclear cells cultured either in Iscoves modified Dulbeccos medium (IMDM) ± 10% fetal calf serum ([FCS]; IMDM + FCS) or in Dulbeccos modified Eagles medium (DMEM) ± 10% newborn bovine serum ([NBS]; DMEM + NBS) were stimulated by various growth factors (GFs). Results showed that CD34+ cells, clonogenic progenitors (colony forming cells [CFCs]) and long‐term culture initiating cells (LTC‐IC) present in freshly harvested cord blood were quiescent. CFC numbers were maintained without cycling after 48‐h cultures in serum‐containing media without GFs. Addition of interleukin 3 (IL‐3) + IL‐6 + stem cell factor stimulated into S‐phase ∼40% of CFCs within 24‐48 h, without modifying their number except in DMEM + NBS where erythroid progenitors decreased. When cells were stimulated in IMDM + FCS by these three GFs + insulin‐like growth factor I and basic fibroblast growth factor used at high concentration, more than 50% of CFCs were in S‐phase and their total number was maintained. The latter culture conditions also recruited up to 66% of LTC‐IC into S‐phase. Our data underline the importance of the combination of GFs and culture media used for optimizing the cycling and maintenance of CFCs and LTC‐IC within two days.

Comparison of the Effects of AcSDKP, Thymosin β4, Macrophage Inflammatory Protein 1α and Transforming Growth Factor β on Human Leukemic Cells

We have compared the effects of AcSDKP, Thymosin β4 (Tβ4), MIP1α and TGFβ on acute myeloid leukemia (AML) and B-lineage acute lymphoid leukemia (B-ALL) cells using liquid cultures in the presence of GM-CSF, IL-3 and SCF for AML cells and IL-3 and IL-7 for ALL cells. Each molecule was added daily and cell proliferation was evaluated on day 3 by thymidine incorporation. Whereas TGFβ was found inhibitory in all the AML and B-ALL cases studied, MlPla was inhibitory in 6/12 AML cases and had no effect on B-ALL cells. AcSDKP and Tβ4 showed an inhibitory effect in a few cases but only at high doses which were inactive on normal cells. Thus, our study not only confirms the effect of TGFβ, MIP1α and AcSDKP on AML cells but also provides new data concerning their effect on B-ALL and the possible inhibitory effect of AcSDKP at high doses. Furthermore, we show for the first time the effect of Tβ4 on leukemic cells. Altogether, our data indicate differences of sensitivity of leukemic cells to negative regulators, some...

Inhibitors of hematopoiesis. Report on the 2nd International Conference on Negative Regulators of Hematopoiesis, Providence, R.I., 22-25 August 1990.

It has become increasingly clear that the regulation of cell proliferation involves stimulatory as well as inhibitory influences on the molecular machinery of growth-related processes. While recent years have seen an extraordinary increase of our knowledge about the properties and actions of stimulatory growth factors, the corresponding rise in the field of inhibitory regulators has come more slowly, although the idea of negative growth control has been around for many years. In the field of hemopoiesis the well-known stimulators, like the various colonystimulating factors (CSF) and interleukins, have now received well-characterized functional counterparts in various inhibitory factors, cytokines, and small peptides, acting by inhibiting the proliferation of hemopoietic cells at various levels during their multistep differentiation and maturation journey through the compartments of the hemopoietic system. This increase in our knowledge about the inhibitors of hemopoiesis is reflected by an increasing interest of the scientific community in this field. Proliferation inhibitors are now well-respected beasts in the factor zoo and in the special field of hemopoiesis the second international meeting solely devoted to this topic was recently held in Providence, R.I. The Proceedings of this conference will be published in the Annals of the New York Academy of Sciences. The Proceedings of the previous meeting, held in Paris in 1987, were published by Najman A. et al. (eds) The Inhibitors ofhematopoiesis, J. Libbey Eurotext, London. In his introductory lecture H. Broxmeyer (Indianapolis, Ind.) presented data on a variety of molecules known to be inhibitory for hemopoiesis. Lactoferrin decreased

Cellular but not humoral immune responses generated by vaccination with dendritic cells protect mice against leukaemia

Dendritic cells (DC) are extremely efficient at generating both prophylactic and therapeutic anti‐tumour immunity. We aimed to analyse the respective roles of humoral and cellular immune responses generated in mice vaccinated with bone marrow (BM)‐derived DC in terms of in vivo anti‐leukaemia effect. We used the murine L1210 B lymphocytic leukaemia genetically modified to express on the cell surface of human CD4 (hCD4) (L1210/hCD4) as a model tumour‐associated antigen (TAA). DC cultures were loaded with either purified soluble hCD4 (shCD4) protein or unfractionated L1210/hCD4 extracts and injected as vaccine into mice. The efficacy of these vaccinations was compared with that of vaccination with shCD4 protein emulsified in Freund’s adjuvant (FA). We evaluated the immune responses generated after these vaccinal protocols and the survival rate of vaccinated mice subsequently challenged with a lethal injection of L1210/hCD4 cells. Our results demonstrated that vaccination with shCD4 protein or tumour extract‐loaded DC mainly generated an hCD4 antigen‐specific cell‐mediated cytotoxic immune response that was associated with a specific protection against leukaemia. In contrast, vaccination with the protein emulsified in FA only generated potent humoral immune responses that were not protective against leukaemia. Altogether, our results indicate that the unique property of loaded DC to trigger an anti‐leukaemia protective effect is mainly associated with cellular immune responses.

The Role of Dendritic Cells in the Transport of HIV to Lymph Nodes Analysed in Mouse

The migration route of dendritic cells (DC) from peripheral tissues to lymph nodes (1) and their capacity to pass HIV infection to T cells (2–4) support a critical role for DC in the early events of HIV infection. It is assumed, but not yet demonstrated, that DC are the first HIV target cells in the mucosa and that their migration to draining lymph nodes will result in the transmission of HIV to T lymphocytes. Interestingly, this can be studied in a murine model, although murine cells are not susceptible to HIV infection. Indeed, (i) murine DC pre-incubated with HIV are able to transfer it to human T lymphocytes as efficiently as human DC (4); (ii) murine DC can be manipulated in vitro, re-injected, and their in vivo migration can be analysed.