Patrick Herve

Hemovigilance network in France: organization and analysis of immediate transfusion incident reports from 1994 to 1998

BACKGROUND : Hemovigilance networks have been introduced in several countries to improve knowledge of blood transfusion‐related morbidity and mortality. The general organization of the French network and its results from 1994 through March 1999 are presented here.

Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype.

Summary. Human bone marrow mesenchymal stem cells (MSC) generate, via a fibroblast colony‐forming unit (CFU‐F), osteo‐chondroblastic cells as well as adipocytes and stromacytes. To date, these stem cells are isolated indirectly using a cell culture method and phenotyped as CD45 negative while the in vivo counterparts are undetermined. Our aim was to develop a direct selection method and to determine the phenotype of the MSC isolated in this way. Mesenchymal cells were selected with anti‐CD49a and/or anti‐CD45 antibodies using either flow cytometry or a magnetic beads method. All CFU‐F were always detected in the small population of CD49a‐positive cells. These CFU retained their differentiation potential and gave rise to osteo‐chondroblastic cells, adipocytes and stromacytes. Phenotypic studies on uncultured cells revealed a CD45med,low, CD34low, HLA‐II– cell population. Flow cytometry cell sorting showed that MSC with CFU‐F potential were obtained only from a CD49a+/CD45med,low population. In addition, when cultured, they clearly became CD45–, CD34–, HLA‐II–, CD49a+. These results confirmed that MSC can be directly selected easily from human bone marrow using magnetic beads without altering their differentiation potential. These cells expressed mildly the haematopoietic marker CD45, which was dramatically downregulated by in vitro culture. The expression of CD45 coupled to CD49a thus enabled direct selection of the MSC.

Retrovirus-Mediated Gene Transfer in Primary T Lymphocytes: Influence of the Transduction/Selection Process and of ex Vivo Expansion on the T Cell Receptor β Chain Hypervariable Region Repertoire

We have initiated a phase I/II clinical trial, involving the use of herpes simplex thymidine kinase gene (HS-tk)-expressing donor primary T cells, in order to modulate the graft-versus-host disease (GvHD) occurring after allogeneic hematopoietic stem cell transplantation. The preparation of gene-modified T cells (TkTCs) required a 12-day ex vivo culture comprising an initial OKT3 and IL-2 stimulation, a retrovirus-mediated transduction, and a 7-day selection step in the presence of G418 and IL-2. The low transduction efficiency as well as the culture conditions may significantly alter the diversity of the T cell repertoire. We therefore examined the T cell repertoire of HS-tk-expressing T cell samples from 11 different donors by the Immunoscope method. This method analyzes the hypervariable region of the T cell receptor beta chain (TCRBV) by amplifying the complementarity-determining region 3 (CDR3) and determining size diversity. In all examined samples (four of which were infused into patients), all TCRBV subfamilies were represented with, however, a significant skewing within a minority of subfamilies. Kinetic studies demonstrated that this skewing appeared between day 7 and day 12, with dates of appearance variable from one subfamily to another. In addition, the repertoire analysis of two different culture products, harvested and produced at different times from the same donors, suggested that some repertoire abnormalities could be donor specific. Quantitative analysis revealed no major modifications in gene usage, even in skewed TCRBV subfamilies, with a few clonal expansions concerning a limited number of TCRBV subfamilies. Importantly, identical abnormalities were found in control cells grown in parallel under similar conditions but not transduced or selected, thus demonstrating that these abnormalities were not related to the transduction or the selection process, but rather to the ex vivo culture. The initial stimulus used for T cell activation is a major source of TCRBV perturbation, since replacing the OKT3 + IL-2 stimulus by CD3 + CD28 monoclonal antibody-coated beads prevented the occurrence of alterations. Overall, the HS-tk-expressing T cells used in our clinical trial exhibit limited TCR repertoire skewing that is not due to the transduction/selection procedure. However, future T cell gene transfer protocols for clinical trials should be designed to take into account or possibly prevent such T cell repertoire alterations.

The broad spectrum of cytokine gene expression by myoid cells from the human marrow microenvironment.

Nontransformed stromal colony‐derived cell lines (CDCLs) consist of a pure stromal cell population that differentiates following a vascular smooth muscle cell repertoire, and whose in vivo counterpart is that of myoid cells found in adult and fetal human bone marrow cords. We studied the cytokine expression by reverse‐transcriptase polymerase chain reaction (RT‐PCR) from pooled fast‐growing clones from 10 different bone marrow samples.

Treatment of acute graft-versus-host disease with methylprednisolone and cyclosporine with or without an anti-interleukin-2 receptor monoclonal antibody : a multicenter phase III study

A double-blind, placebo-controlled trial of BT563, including 13 European centers, was initiated in October 1989 to compare the efficacy of the combination of in vivo anti-CD25 mAb (BT 563), cyclosporine, and steroids versus placebo and CSA-steroids in the treatment of grade II and III acute graft-versus-host disease (GVHD). Sixty-nine patients participated in the study, which excluded non-genotypically identical allogeneic bone marrow transplant recipients. No statistically significant differences were observed, clinically or biologically, between the 2 groups before the onset of the treatment. Treatment responses were scored during and after the 3-week treatment period (mAb or placebo). Efficacy was evaluated on days 4, 10, 20, 30, and 60 or on any day the patients condition was found to be deteriorating. Preceding and systemically untreated GVHD of grade I was observed in 59% of the cases. No statistically clinically significant differences between the 2 groups were observed during or upon completion of treatment in GVHD grade. Nine patients in the placebo group and 6 in the active group were withdrawn of the study. Thirteen of these 15 patients were withdrawn because of failure of GVHD therapy (9 in the placebo group and 4 in the BT563 group). At day 20 after onset of the treatment, the response rate was 63% and 70% for the placebo and BT563 groups, respectively (NS). Probability of survival at 1 year was 59% and 66% (NS) for the placebo and active groups, respectively. In conclusion, despite preliminary promising results in the treatment of steroid-resistant acute GVHD, the role of first-line treatment with an in vivo anti-interleukin-2 receptor mAb remains to be determined.

Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection

Perforin (P), Granzyme B (GB) and Fas‐Ligand (FAS‐L) are cytotoxic molecules involved in acute rejection (AR) after renal transplantation. A noninvasive diagnostic test to monitor AR and other complications could improve clinical management. We investigated the predictive and diagnostic interest of target mRNA measurements, with a quantitative PCR assay, in AR, as well as in other clinical complications recurrent in kidney transplantation. One hundred and sixty‐two urine specimens from 37 allograft recipients were investigated. Clinical settings were AR, urinary tract infection (UTI), cytomegalovirus infection (CMVi) or disease (CMVd), chronic allograft nephropathy (CAN), delayed graft function (DGF) and stable graft course (controls). In the case of AR, mRNA levels of all three molecules were significantly higher than in recipients not showing any clinically evident signs of complication. Indeed, it was observed that expression levels of P, GB and Fas‐L mRNA also increase in other clinical situations such as UTI, CMV and DGF. Finally, kinetic studies in three patients with AR revealed that increased P, GB and Fas‐L mRNA levels could precede or were concomitant with increased serum creatinin levels. P, GB and Fas‐L gene expression in urine specimens were upregulated in AR episodes but also in UTI, CMV infection and DGF. Therefore, this technique would appear to be of limited clinical value as a noninvasive method of diagnosing AR.

Distinct hematopoietic support by two human stromal cell lines.

OBJECTIVE The hematopoietic microenvironment is complex, and the role of myofibroblast in its function is crucial. In order to obtain a stable model reflecting this particular cell type, we have previously established human bone marrow cell lines from primary myofibroblastic Stro1(+) population (pStro1(+)). We placed HPV16 E6 and E7 expression under the control of different promoters. Here, we have characterized and studied the hematopoietic support for two cell lines corresponding to the promoters alpha-SM (alphaSM-56 line) and SV40 (SV40-56 line). MATERIALS AND METHODS The expression profile was analyzed at the RNA level by gene array and at the protein level by Western blot, flow cytometry, and ELISA. Hematopoietic support determined using colony-forming unit (CFU) and stroma-adherent colony-forming cell (SA-CFC) assays. RESULTS The phenotype of cell lines was not significantly modified compared with primary myofibroblastic cells. They secreted a broad spectrum of hematopoietic cytokines and nonspecific mediators. The two lines allowed the growth of hematopoietic precursors and had different support capabilities. CONCLUSIONS We have extensively characterized two novel human bone marrow stromal cell lines. They retained a myofibroblastic phenotype and have substantial but different hematopoietic support capabilities. These lines provided a basis for determining stromal factors involved in stem-cell regulation.

Allogeneic peripheral blood stem cell transplantation results in less alteration of early T cell compartment homeostasis than bone marrow transplantation

Since low T cell counts evaluated 1 month after allogeneic bone marrow transplantation (BMT) are associated with an increased risk of leukemia relapse (Powles et al., Blood 1998; 91: 3481–3486), we compared, in a randomized multicentric clinical study, the peripheral blood cells obtained 30 days after allogeneic BMT vs allogeneic G-CSF-mobilized peripheral blood stem cell transplantation (BCT) in an HLA-identical setting. T cell counts were higher 30 days after BCT (718 ± 142 cells/μl, n = 20) than after BMT (271 ± 53 cells/μl, n = 26, P = 0.006). However, T cells were less activated after BCT than after BMT, as demonstrated by a lower expression level of CD25 and a lower percentage of HLA-DR+ and CD95+ T cells. Furthermore, CD4+, CD8+and CD45RA+ post-BCT T cell counts correlated with the number of cells infused with the PBSC graft, while such a correlation was not observed between post-BMT counts and BM graft cell numbers, suggesting that the intensity of post-transplant peripheral lymphoid expansion and/or deletion differed between BCT and BMT. A comparison of the input of T cells expressing different CD45 isoforms with the post-transplant cell recovery further confirmed that, within the CD4+ T cell subset, post-transplant expansions occurred at a higher level after BMT than after BCT, affecting mainly the CD4+ CD45RO+subset. Altogether, our data demonstrate for the first time in a randomized setting that homeostasis of the T cell pool is less altered early after BCT than after BMT. This may have a strong impact on the graft-versus-leukemia (GVL) effect and subsequent relapse rate. Bone Marrow Transplantation (2001) 27, 167–175.

Influence of Ex Vivo Expansion and Retrovirus-Mediated Gene Transfer on Primary T Lymphocyte Phenotype and Functions

To modulate alloreactivity after hematopoietic stem cell (HSC) transplantation, suicide gene-expressing donor T cells can be administered with an allogeneic T cell-depleted HSC graft. Immune competence of such cells is a critical issue. We have examined the impact of our ex vivo gene transfer protocol (12-day culture period including CD3/IL-2 activation, retrovirus-mediated gene transfer, and G418-based selection) on the phenotype and functional properties of gene-modified cells (GMC). GMC were compared with control cells that had been cultured in parallel with GMC, but nontransduced and nonselected, as well as with peripheral blood mononuclear cells (PBMC). Our data show that phenotypical modifications are similar in control cells and GMC, demonstrating that alterations result from the 12-day culture rather than from the transduction and/or selection process itself. Such modifications include a reversal of CD4/CD8 ratio, activated phenotype (increased expression of CD45RO, CD95, and HLA-DR), and acquisition or increased expression of co-stimulatory molecules (CD80, CD86, and CD40). This led to an enhanced allostimulating potential of GMC, as compared with resting T cells, when used as stimulating cells in mixed lymphocyte reactions. Conversely, when using them as responder cells in mixed lymphocyte reactions, GMC exhibited a rapid loss of alloreactivity that resulted both from culture-dependent and from transduction and/or selection-dependent events. In conclusion, the retrovirus-mediated gene transfer can be associated with major phenotypical and functional alterations that could have strong clinical implications (increased immunogenicity, reduced anti-leukemic effect). Thus, future T cell expansion protocols should try to improve not only cell expansion or gene transfer efficiency, but also T cell functions.

Detection of intracellular cytokines in citrated whole blood or marrow samples by flow cytometry.

We have used three-color flow cytometric analysis for the detection of intracellular cytokines (IFN-gamma and IL-4) in CD3(+) cells, after stimulation for 4 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin. We report in the present paper a validation study for analysing IFN-gamma and IL-4 production by bone marrow (BM)-derived T cells and peripheral blood T cells after BM transplantation. Using citrate as anticoagulant for blood and marrow sampling interfered with PMA+ionomycin-based cell stimulation. Indeed, removing this anticoagulant by two washes with 10% pooled human AB serum-supplemented RPMI 1640 before cell stimulation improved the percentages of IL-4(+) (0.02+/-0.01% to 0. 47+/-0.17% without and with washes, respectively; p<0.01) and IFN-gamma(+) (6.8+/-2.75% to 39.33+/-4.6%; p<0.01) cells to levels similar to those observed in heparin-based whole blood cultures (0.38+/-0.17% IL-4(+) and 34.27+/-4.96% IFN-gamma(+) cells; p>0.05). Delaying the cell cultures for 24 h did not significantly modify the detection of IFN-gamma in washed whole blood, but significantly altered IFN-gamma secretion in culture supernatants, as assessed by ELISA. Moreover, the percentage of IFN-gamma-producing cells within the CD3(+) lymphocyte population was stable, since similar results were obtained in two or three different independent experiments performed with the same healthy donors. This method was shown to be applicable for different kinds of citrated samples, such as blood or BM-derived cells. Overall, our data suggest that in addition to allowing for the identification of cytokine-producing cell phenotype, intracellular cytokines staining using flow cytometry is more reliable than ELISA for the biological follow-up of clinical samples.