In vitro generation of dendritic cells from human blood monocytes in experimental conditions compatible for in vivo cell therapy.

DC are professional APC that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34+ pluripotent HPC have been developed recently. However, the various steps of their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. Our study focused on setting up such a full procedure, including collection of mononuclear cells (MNC) by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + autologous serum (SAuto) in sterile Teflon bags. The procedure was first developed with apheresis products from 7 healthy donors. Its clinical feasibility was then tested on 7 patients with breast cancer. The characteristics of monocyte-derived DC grown with SAuto (or in some instances with a pooled AB serum) were compared with those obtained in the presence of FBS by evaluation of their phenotype, their morphology in confocal microscopy, and their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined SAuto-derived DC as efficient as those derived with FBS.

Activation of the Janus kinase 3-STAT5a pathway after CD40 triggering of human monocytes but not of resting B cells.

CD40/CD40 ligand interactions play a key role in the immune responses of B lymphocytes, monocytes, and dendritic cells. The signal transduction events triggered by cross-linking of the CD40 receptor have been widely studied in B cell lines, but little is known about signaling following CD40 stimulation of monocytes and resting tonsillar B cells. Therefore, we studied the CD40 pathway in highly purified human monocytes and resting B cells. After CD40 triggering, a similar activation of the NF-kappaB (but not of the AP-1) transcription factor complex occurred in both cell preparations. However, the components of the NF-kappaB complexes were different in monocytes and B cells, because p50 is part of the NF-kappaB complex induced by CD40 triggering in both monocytes and B cells, whereas p65 was only induced in B cells. In contrast, although the Janus kinase 3 tyrosine kinase was associated with CD40 molecules in both monocytes and resting B cells, Janus kinase 3 phosphorylation induction was observed only in CD40-activated monocytes, with subsequent induction of STAT5a DNA binding activity in the nucleus. These results suggest that the activation signals in human B cells and monocytes differ following CD40 stimulation. This observation is consistent with the detection of normal CD40-induced monocyte activation in patients with CD40 ligand+ hyper IgM syndrome in whom a defect in CD40-induced B cell activation has been reported.

Light microscopic evaluation of human donor corneal stroma during organ culture.

PURPOSE: To try to facilitate evaluation of corneal stroma during organ culture by means of light microscopy. METHODS: Corneal stroma of 53 consecutive organ-cultured corneas was studied by means of light microscopy during endothelial quality control. Out of 9 corneas with bad stromal evaluation, 2 were studied by means of transmission electron microscopy, and 7 were grafted. From the remaining 44 corneas with a normal light microscopic appearance, 35 were grafted. RESULTS: Stromal abnormalities consisted of bright visible structures with a cell-like shape corresponding to keratocyte injuries (i.e. cellular edema, light and dark vacuoles, cell membrane disruption and, finally, internal cytolysis) as observed by TEM. At 3 months postoperatively no clinical differences between the two groups of transplants were observed. CONCLUSION: Corneal stroma can be evaluated qualitatively and easily by means of light microscopy during organ culture. Further studies are needed to investigate whether the presence of lysed keratocytes in the graft's stroma actually influences the outcome of transplantation.

Tumoricidal potential of human macrophages grown in vitro from blood monocytes.

Adoptive transfer of activated autologous human macrophages obtained by in vitro differentiation of monocytes in culture has successfully undergone phase I clinical trials in patients with metastatic cancer. The efficacy of these autologous macrophages in the in vivo killing of human tumors has now to be demonstrated. GM-CSF was shown to increase the number of monocytes differentiating in culture into macrophages. These were then activated with IFN gamma which was reported as the best cytokine for tumoricidal activation of macrophages. The aim of this paper was to evaluate the in vitro tumoricidal activity of macrophages grown with GM-CSF and IFN gamma. This tumoricidal function was investigated by measurement of the cytolytic (chromium-51 release assay), cytostatic (anti-proliferative activity as measured by inhibition of [3H]-thymidine incorporation in two different assays) and cytotoxic (MTT assay) activities on two tumor cells lines, U937 and K562 (respectively highly sensitive and resistant to soluble TNF alpha). Our results demonstrated that GM-CSF-grown macrophages exhibited significant cytolytic, cytotoxic and cytostatic activities on U937 cells. These were partly enhanced by IFN gamma activation. In contrast, they had no lytic and lower cytotoxic and cytostatic activities on K562 cells, and these were not modified by IFN gamma activation. Our data provide valuable information for future in vivo studies using adoptively transferred autologous macrophages.

Infusion of large quantities of autologous blood monocyte-derived macrophages in two cancer patients did not induce increased concentration of IL-6, TNF-alpha, soluble CD14 and nitrate in blood plasma.

In an attempt to increase the number of macrophages available for reinfusion in immunotherapy trials, GM-CSF was injected in vivo to mobilize circulating blood monocytes in 2 cancer patients. Subsequently mononuclear cells were collected by apheresis, cultured in the presence of GM-CSF and activated with IFN-gamma. This procedure resulted in the harvesting of 1.3 to 3.1 x 10(9) (mean 2 x 10(9)) macrophages per apheresis, product which was very well tolerated at autologous reinfusion. These infusions did not induce increased levels of TNF-alpha, IL-6, soluble CD14 nor nitrates in blood plasma (or urine). The lack of TNF-alpha and IL-6 release in blood plasma could explain the good tolerance of these infusions. No in vivo anti-tumoural activity of these high numbers of infused macrophages could be observed.

Efficient adenovirus-mediated gene transfer into human blood monocyte-derived macrophages.

The efficiency of gene transfer into human blood monocyte-derived macrophages has been evaluated using a replication-defective adenovirus vector harboring a lac Z gene of E. coli as a reporter gene. Whereas, no beta-galactosidase activity was found in freshly infected purified monocytes, 40% to 80% of infected macrophages which derived from these monocytes showed a beta-galactosidase activity, 2 to 4 days after infection and lasted for at least 3 weeks. Moreover, beta-galactosidase activity was found in infected monocyte/macrophages 7 days after their injection into a human tumor preestablished in nude mice. These data indicate that it is possible to transfer and stably express a gene of potential therapeutical function into human monocyte-derived macrophages using an adenovirus vector.

Autologous lymphocytes prevent the death of monocytes in culture and promote, as do GM-CSF, IL-3 and M-CSF, their differentiation into macrophages.

Blood monocytes collected by apheresis from healthy donors were differentiated in vitro to macrophages which were subsequently activated with recombinant human interferon-gamma. 7 day cultures were established by seeding Ficoll-separated mononuclear cells or elutriation-purified monocytes under different culture conditions. The best macrophage yields required the seeding of mononuclear cells (instead of purified monocytes) in teflon bags with a high air-liquid surface interface. The effects of GM-CSF, IL-3 and M-CSF on the macrophage yield were then evaluated. GM-CSF increased the average yield by 3.6- and 2.3-fold when purified monocytes or total mononuclear cells were seeded respectively. The corresponding increases with IL-3 were 2.5- and 2.1-fold respectively and with M-CSF 1.2- and 1.4-fold respectively. Macrophages matured under these various conditions displayed similar CD14, CD64, CD71, HLA-DR and Max 1 antigen expression and similar in vitro anti-tumoral activity against U937 cells. Culturing in the presence of cytokines permits the large scale production of activated macrophages for adoptive immunotherapy trials.

Production of human macrophages with potent antitumor properties (MAK) by culture of monocytes in the presence of GM-CSF and 1,25-dihydroxy vitamin D3.

Antitumoral macrophages (MAK) were obtained by the culture of human mononuclear cells in hydrophobic bags. From one cytapheresis, up to 10(9) mature macrophages could be purified by elutriation after one week of culture in IMDM medium in the presence of 2% human AB serum. These MAK cells were used for adoptive treatment in metastatic cancer patient with no dose-limiting toxicity. The present study aimed to improve the average MAK yield by addition of GM-CSF and of dihydroxy-cholecalciferol. The differentiated macrophages obtained presented higher antitumoral functionality in response to rh-IFN gamma than in their absence. These MAK presented all the differentiation antigens of cytotoxic macrophages compared to MAK cells differentiated in standard medium. They killed human tumor targets effectively in vitro at a low (1/1) effector/tumor ratio; furthermore, the antitumoral activity reached by MAK cells after IFN gamma activation appeared to be stabilized for several days.

Adoptive immunotherapy with activated macrophages grown in vitro from blood monocytes in cancer patients: a pilot study.

Ninety-three collections of leucocytes by cytapheresis followed by separation of monocytes by centrifugal elutriation were undertaken in twelve metastatic cancer patients (four melanomas, six colon carcinomas, one ovarian carcinoma, and one lung cancer). The leucaphereses were performed aiming to collect a product, ready for introduction into the elutriation chamber, i.e., with low contamination by erythrocytes and granulocytes. The median collection of leucocytes was 7.3 x 10(9). After elutriation, purified monocytes (mean: 0.91 x 10(9)) were cultured with 3-5% autologous serum for 7 days in the presence of 250 IU/ml of recombinant human gamma-interferon (Rh-IFN gamma) for the last 18 h of culture. The median number of activated macrophages (MAK) available for reinfusion was 2.4 x 10(8) for each culture. The phenotypes and the antitumoral potentiality of MAK cells were documented. Reinfusions performed i.v. or i.p. were well tolerated with no major side effects. No complete tumor response was obtained. One partial response and two stabilizations of the disease were observed in one melanoma and two colon carcinomas.