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A novel series of methyl ester-terminated C8-linked pyrrolobenzodiazepine (PBD)−poly(N-methylpyrrole) conjugates (50a−f) has been synthesized and their DNA interaction evaluated by thermal denaturation, DNA footprinting, and in vitro transcription stop assays. The synergistic effect of attaching a PBD unit to a polypyrrole fragment is illustrated by the large increase in DNA binding affinity (up to 50-fold) compared to the individual PBD and pyrrole components. 50a−fwere found to bind mainly to identical DNA sequences but with apparent binding site widths increasing with molecular length and the majority of sites conforming to the consensus motif 5'-XGXWz(z 3 ± 1; W A or T; X any base but preferably a purine). They also provided robust sequence-selective blockade of transcription at sites corresponding approximately to their DNA footprints. 50a−f were shown to have good cellular/nuclear penetration properties, and a degree of correlation between cytotoxicity and DNA-binding affinity was observed.

Stable renal transplant recipients (RTR) display high rates of atherosclerotic events (AE). Innate immunity and especially vascular inflammation play a role in the pathogenesis of atherosclerosis. It is illustrated both by an increased occurrence of postrenal transplant cardiovascular events in patients with elevated levels of C-reactive protein and by a correlation between posttransplant AE and Toll-like receptor-4 Asp299Gly polymorphism. Here, we analyze the influence NOD2/CARD15 gene polymorphism since NOD2 can modulate macrophage pro-inflammatory activity and macrophage is present in early atherosclerotic lesions. The incidence of single nucleotide polymorphism (SNP) in the three major polymorphic region of NOD2 gene (SNP8, SNP12 and SNP13) was assessed in 182 RTR and the correlation between such polymorphism and the development of AE was analyzed. No correlation was observed between NOD2 gene polymorphism and the occurrence of AE after renal transplantation. NOD2 gene polymorphism thus does not appear to influence cardiovascular complications in RTR.

Granulocyte colony-stimulating factor (G-CSF) is often administered after hematopoietic-cell transplantation (HCT) to accelerate neutrophil recovery, but it is unclear what impact G-CSF has on long-term transplantation outcomes. We analyzed within the database of the Center for International Blood and Marrow Transplant Research the impact of giving posttransplantation G-CSF on the outcomes of allogeneic HCT for acute myelogenous leukemia and chronic myelogenous leukemia in 2719 patients who underwent transplantation between 1995 and 2000. These included 1435 recipients of HLA-identical sibling bone marrow (BM), 609 recipients of HLA-identical peripheral-blood stem cells (PBSCs), and 675 recipients of unrelated donor BM transplants. Outcomes were compared between patients receiving or not receiving G-CSF within 7 days of HCT according to graft type. Median follow-up was more than 30 months (range, 2-87 months). G-CSF shortened the posttransplantation neutropenic period, but did not affect days +30 and +100 treatment-related mortality (TRM). Probabilities of acute and chronic graft-versus-host disease (GVHD), leukemia-free survival (LFS), and overall survival were similar whether or not G-CSF was given. Multivariate analyses confirmed that giving G-CSF did not affect the risk of GVHD, TRM, LFS, or survival. In conclusion, results of this study found no long-term benefit or disadvantage of giving G-CSF after transplantation to promote hematopoietic recovery.

Use of a reduced-intensity conditioning regimen before an allogeneic hematopoietic cell transplantation is frequently associated with an early state of mixed hematopoietic chimerism. Such a coexistence of both host and donor hematopoietic cells may influence posttransplant alloreactivity and may affect the occurrence and severity of acute and chronic graft-versus-host disease (GVHD) as well as the intensity of the graft-versus-leukemia effect. Here we evaluated the relation between chimerism state after reduced-intensity conditioning transplantation (RICT), autoantibody production, and chronic GVHD (cGVHD)-related pathology.

Hepatic veno-occlusive disease is a major limiting factor of high-dose chemotherapy in children. The cells lining the hepatic vascular endothelium express blood group A and/or B antigens according to the patient’s blood group. We designed a study evaluating the impact of platelet concentrates containing ABO-incompatible plasma transfused to young children with a high risk of hepatic veno-occlusive disease.

The association of ethnicity with the incidence of graft-versus-host disease (GVHD) and other clinical outcomes after transplantation is controversial. We compared the results of HLA-identical sibling bone marrow transplantations for leukemia, performed between 1990 and 1999, among different ethnic populations, including 562 Japanese, 829 white Americans, 71 African Americans, 195 Scandinavians, and 95 Irish. Results for adults and children were analyzed separately. Multivariate analyses of adult patients showed that white Americans, African Americans, and Irish cohorts were at significantly higher risk for acute GVHD than Japanese or Scandinavian cohorts (relative risk [RR] = 1.77, P< .001; RR = 1.84, P< .006; RR = 2.22, P< .001, respectively). White Americans, African Americans, and Irish, but not Scandinavians, were at significantly higher risk for early (within 3 months of transplantation) transplant-related mortality (TRM) compared with Japanese (RR = 2.99, P< .001; RR = 5.88, P< .001; RR = 2.66, P< .009, respectively). No differences in the risk for chronic GVHD, relapse, and overall survival were noted. In the pediatric cohort (limited to Japanese and white Americans), white Americans were at significantly higher risk for acute (RR = 1.93; P= .04) and chronic (RR = 3.16; P= .002) GVHD. No differences in other clinical outcomes were noted. Our findings suggest that ethnicity may influence the risk for GVHD, though overall survival rates after transplantation remain similar.

A new entity of acute leukemia coexpressing CD4+CD56+markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory, cells from a patient with such CD4+CD56+lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression, we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4+CD56+pDC malignancies, we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry, reverse transcriptase-polymerase chain reaction, and immunoblot analysis. Furthermore, CD33 monoclonal antibody stimulation of purified CD4+CD56+leukemic cells led to cytokine secretion, thus confirming the presence of a functional CD33 on these leukemic cells. Moreover, we found that circulating pDCs in healthy individuals also weakly express CD33. Overall, our results demonstrate that the expression of CD33 on CD4+CD56+lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4+CD56+malignancies.

Intravenous infusion of apoptotic donor or third-party leukocytes simultaneously with an allogeneic donor bone marrow (BM) graft favors engraftment across major histocompatibility barriers. While verifying that such apoptotic cell infusion might not also be associated with antibody (Ab)-mediated allo-immune responses, we found, rather strikingly, that apoptotic cell infusion could in fact successfully prevent a humoral allo-immunization against a BM graft in mice. Indeed, among recipients having rejected their BM graft, prior apoptotic cell infusion was associated with a near absence of Ab-mediated allo-responses, while such an immunization was frequently observed in the absence of apoptotic cell infusion. This was also observed when infusing host apoptotic cells, thus showing that the prevention of immunization was linked to the apoptotic state of the cells rather than mediated by residual anti-recipient activity. In vivo anti-transforming growth factor-β (TGF-β) treatment resulted in the loss of this apoptotic cell infusion-associated protective effect on humoral allo-responses. Further studies will determine whether apoptotic cell infusion, in addition to hematopoietic graft facilitation might also contribute to preventing deleterious Ab-mediated allo-responses in various transplantation settings.

Intravenous infusion of apoptotic donor or third-party leukocytes simultaneously with an allogeneic donor bone marrow (BM) graft favors engraftment across major histocompatibility barriers. While verifying that such apoptotic cell infusion might not also be associated with antibody (Ab)-mediated allo-immune responses, we found, rather strikingly, that apoptotic cell infusion could in fact successfully prevent a humoral allo-immunization against a BM graft in mice. Indeed, among recipients having rejected their BM graft, prior apoptotic cell infusion was associated with a near absence of Ab-mediated allo-responses, while such an immunization was frequently observed in the absence of apoptotic cell infusion. This was also observed when infusing host apoptotic cells, thus showing that the prevention of immunization was linked to the apoptotic state of the cells rather than mediated by residual anti-recipient activity. In vivoanti-transforming growth factor-β (TGF-β) treatment resulted in the loss of this apoptotic cell infusion-associated protective effect on humoral allo-responses. Further studies will determine whether apoptotic cell infusion, in addition to hematopoietic graft facilitation might also contribute to preventing deleterious Ab-mediated allo-responses in various transplantation settings.

In a clinical trial that we recently reported, a suicide gene transfer in human primary T cells required 12 days of ex vivo culture, including activation of peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody (CD3 mAb), retrovirus-mediated transduction, and selection of gene-modified cells (GMC) by G418. The aim of the present study was to determine the impact of the initial T cell activation and of the transduction/selection on T cell receptor β variable chain (TCRBV) repertoire of GMC by using the spectratyping method. The TCRBV repertoires of nontransduced, nonselected control (Co) cells and of GMC generated after an initial stimulation with CD3 mAb, CD3/CD28 beads, or allogeneic PBMC or Epstein-Barr virus-transformed B (B-EBV) cells were compared to the ones of their corresponding PBMC. The TCRBV repertoires were skewed in Co cells generated after CD3 mAb or after allogeneic stimulation, and even more so in their corresponding GMC, demonstrating that both culture-dependent and transduction/selection-dependent events led to TCRBV repertoire alterations. However, TCRBV repertoires were not altered, or to a lesser extent, in Co cells or GMC produced after CD3/CD28 bead activation, demonstrating a protective effect on both culture-dependent and transduction/selection-dependent repertoire alterations. Thus, we suggest to replace the initial CD3 mAb stimulation by CD3/CD28 beads for the production of clinical-grade GMC in the setting of future gene therapy trials.

To modulate alloreactivity after hematopoietic stem cell transplantation, “suicide” gene-modified donor T cells (GMCs) have been administered with an allogeneic T-cell–depleted marrow graft. We previously demonstrated that such GMCs, generated after CD3 activation, retrovirusmediated transduction, and G418 selection, had an impaired Epstein-Barr virus (EBV) reactivity, likely to result in an altered control of EBV-induced lymphoproliferative disease. To further characterize the antiviral potential of GMCs, we compared the frequencies of cytomegalovirus (CMV)–specific CD8+ T (CMV-T) cells and EBV-specific CD8+ T (EBV-T) cells within GMCs from CMV- and EBV-double seropositive donors. Unlike anti-EBV responses, the anti-CMV responses were not altered by GMC preparation. During the first days of culture, CMV-T cells exhibited a lower level of CD3-induced apoptosis than did EBV-T cells. In addition, the CMV-T cells escaping initial apoptosis subsequently underwent a higher expansion rate than EBV-T cells. The differential early sensitivity to apoptosis could be in relation to the “recent activation” phenotype of EBV-T cells as evidenced by a higher level of CD69 expression. Furthermore, EBV-T cells were found to have a CD45RA–CD27+CCR7– effector memory phenotype, whereas CMV-T cells had a CD45RA+CD27–CCR7– terminal effector phenotype. Such differences could be contributive, because bulk CD8+CD27– cells had a higher expansion than did bulk CD8+CD27+ cells. Overall, ex vivo T-cell culture differentially affects apoptosis, long-term proliferation, and overall survival of CMV-T and EBV-T cells. Such functional differences need to be taken into account when designing cell and/or gene therapy protocols involving ex vivo T-cell manipulation.

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.

The human formylpeptide receptor (FPR) is a seven-transmembranous G-protein-coupled receptor (7TM-GPCR) for chemotactic peptides of bacterial origins, possibly involved in the recruitment and activation of neutrophils in various inflammatory diseases of mucosal epithelia. Mutational analyses suggest that interactions of formylated peptides with FPR occur on the outer exoplasmic leaflet/domains of the plasma membrane. The immunosuppressive and antifungal antibiotic cyclic undecapeptide cyclosporin A (CsA; cyclo-[MeBmt¹-Abu²-MeGly³-MeLeu⁴-Val⁵-MeLeu⁶-Ala⁷-d-Ala⁸-MeLeu⁹-MeLeu¹⁰-MeVal¹¹]) and some tested analogues such as [Ala²]-CsA, [Thr²]-CsA, [Val²]-CsA, and [Nva²]-CsA were able of inhibiting the binding of formylpeptides to the FPR, with [d-MeVal¹¹]-CsA (CsH) being much more active than the other analogues. CsH is devoid of immunosuppressive and antifungal activities, and its large potency for human FPR inhibition is of inverse agonism origin. Formylpeptide binding to FPR-expressing cells does not only induce chemotaxis; it also causes a rapid release of granule enzymes in the extracellular medium, allowing the easy monitoring of any inhibition of FPR function “in vivo” (with intact live cells). With such an assay, CsH was confirmed to be the most potent FPR inhibitory cyclosporin, although a far related immunosuppressive cyclosporin analogue, FR901459 ([Thr², Leu⁵, Leu¹⁰]-CsA), was found to display a high FPR inhibitory activity (FPR-InhA). To establish structure−activity relationships (SAR) for FPR function inhibition, 59 cyclosporins were now studied by this standardized assay (with differentiated human leukemic cell line HL-60 as FPR-expressing cells and with N-acetyl-β-d-glucosaminidase release as read-out). These SAR confirmed the low FPR-InhA of classical cyclosporins, where such activity was only seldom found:  the most active ones ([Thr², Ile⁵]-CsA, [aMeIle¹¹]-CsA, and [MeAla¹¹]-CsA) remained 3−10-fold less potent than CsH. In contrast, the SAR disclosed that N¹⁰-desmethylated cyclosporins were particularly prone to display a large FPR-InhA:  their most potent one was a [Thr², Gly³, Leu⁵, d-Hiv⁸, Leu¹⁰]-CsA, found to be only 2−4-fold less active than [d-MeVal¹¹]-CsA (CsH), with which it shows six differences out of 11 residues. Because the free conformations of both CsH and N¹⁰-desmethylated cyclosporins differ from those of “classical” (N¹⁰-methylated, [l-MeVal¹¹]-using) cyclosporins, these potent FPR inhibitory cyclosporins probably bind to FPR pharmacophores for which classical cyclosporins show little affinity. Moreover, because the conformations of the N¹⁰-desmethylated cyclosporins widely differ from the CsH one, they probably bind to different pharmacophores on the FPR molecules.

Cyclic undecapeptide cyclo-[MeBmt¹-Abu²-MeGly³-MeLeu⁴-Val⁵-MeLeu⁶-Ala⁷-d-Ala⁸-MeLeu⁹-MeLeu¹⁰-MeVal¹¹], the immunosuppressive and antifungal antibiotic cyclosporin A (CsA), was reported to interfere with the MDR1 P-glycoprotein (Pgp), a transmembranous adenosine 5‘-triphosphate binding cassette (ABC) transporter with phospholipid flippase or “hydrophobic vacuum cleaner” properties that mediate multidrug resistance (MDR) of cancer cells. By use of photoaffinity-labeled cyclosporins and membranes from Pgp-expressing cells, it was recently shown that in vitro, Pgp molecules could bind a large cyclosporin domain involving residues 4−9 as well as the side chain of residue 1. Tumor cell MDR can also be reversed by a product more distantly related to cyclosporin with the structure [Thr², Leu⁵, d-Hiv⁸, Leu¹⁰]-CsA (SDZ 214-103). In a standardized assay that measures Pgp function in vivo (on intact live cells) by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL¹⁰⁰) cells as highly resistant Pgp-expressing cells, SDZ 214-103 was found to be one of the most active Pgp inhibitors among naturally occurring cyclosporins, with an IC50 of 1.6 μM in an assay where CsA gives an IC50 of 3.4 μM. Using the in vivo assay, 60, mostly natural, cyclosporin analogues were analyzed to establish structure−activity relationships (SAR). Our SAR are compatible with the in vitro-defined Pgp binding domain model and further disclose that in vivo Pgp inhibition is favored by larger hydrophobic side chains on cyclosporin residues 1, 4, 6, and 8 and a smaller one on residue 7, although with no effect on the residue 5 side chain; moreover, larger hydrophobic side chains on other residues 2, 3, 10, and 11 (outside the in vitro-defined Pgp binding domain) also favor the eventual inhibition of Pgp function. The N-desmethylation of any of the seven N-methylated amides, as naturally occurring in numerous cyclosporins, regularly leads to a decreased Pgp inhibitory activity (Pgp-InhA), up to its abrogation if it occurs at residues 4 and 9. Nevertheless, despite unfavorable use of [Thr²] and [Leu¹⁰] residues, all [d-Hiv⁸] analogues whose lead is SDZ 214-103 show a large Pgp-InhA. The SAR for Pgp inhibition by cyclosporins are thus very complex. Because CsA and SDZ 214-103 show largely different conformations when free in solution, but remarkably similar ones when bound to the cytosolic cyclophilins, SAR for Pgp inhibition must similarly include requirements for occurrence of suitable conformers for insertion in the cell membrane, sufficient conformational plasticity for gaining access to Pgp binding sites, and an adequate conformer structure there to achieve such binding with a high enough affinity and possibly escape from sequestration on cyclophilins.

Abstract Renal transplant recipients have a well‐recognized increased risk of de novo neoplasia. In this study, we investigated whether lymphocyte subset count could predict the risk of developing noncutaneous neoplasia (NCSC) in renal transplant recipients (RTR). Between January 1995 and December 1995, lymphocyte subsets (CD4, CD8, CD19) were measured in 281 RTR. This population was studied until November 1999 for the development of NCSC. The mean follow‐up was 42±9 months. Neoplasm was diagnosed in 22 patients (7.9%). Patients who developed a cancer were significantly older (53.8±6 years vs 38±16 years, P<0.0001), had lower CD4 (234±126/mm3vs 543±214/mm3, P<0.005) and CD19 (19±9/mm3vs 51±22/mm3, P<0.0001) levels, and more frequently had past histories of skin cancer (24% vs 4%, P<0.01). Cox regression revealed that high CD4 levels (RR 0.73, 95% CI 0.62–0.89 for each 100/mm3increase in CD4 cell count) were associated with decreased risk of NCSC, whereas age (RR 2.49, 95% CI 1.12–5.92 for each 10‐year increase in age) was predictive of the subsequent development of NCSC. To conclude, CD4 cell depletion is associated with the development of solid cancers and lymphoma in RTR.

We have recently shown that the use of allogeneic granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood hematopoietic stem cell transplantation (PBHSCT), as compared with bone marrow transplantation (BMT), is associated with increased titers of antibodies (Abs) directed against red blood cell ABO antigens. To further evaluate the influence of a G-CSF–mobilized PBHSCT graft on alloimmune Ab responses, we examined the frequency of anti-HLA Abs after transplantation in the setting of the same randomized study, comparing PBHSCT with BMT in adults. Anti-HLA Ab presence was determined by complement-dependent cytotoxicity assay (CDC) and flow cytometry in the recipient before and 30 days after transplantation as well as in the donor before graft donation. The use of PBHSCT was significantly associated with increased detection of anti-HLA immunoglobulin G (IgG) Abs early after transplantation as evidenced by flow cytometry (11 of 24 versus 4 of 27 transplant recipients, P = .03) and, less so, by CDC (5 of 24 versus 1 of 27 transplant recipients,P = .09). The difference between PBHSCT and BMT was further heightened when analysis was restricted to anti-HLA IgG Ab–negative donor/recipient pairs. In such a setting, early anti-HLA Ab was never detected after BMT but was repeatedly detected after PBHSCT (flow cytometry, 6 of 18 versus 0 of 17 transplant recipients, P = .02; CDC, 4 of 23 versus 0 of 26 transplant recipients, P = .04). Importantly, the PBHSCT-associated increase in anti-HLA Ab detection was observed despite a reduction in the median number of platelet-transfusion episodes per patient in PBHSC transplant versus BM transplant recipients (3 platelet-transfusion episodes [range, 1-21] in PBHSCT group vs 6 platelet-transfusion episodes [range, 3-33] in the BMT group; P = .02). In conclusion, this study strongly suggests that G-CSF–mobilized PBHSCT results in an increased incidence of circulating anti-HLA Abs and further confirms that the use of such a graft alters alloimmune Ab responses.

Mutations in the ZAP-70 protein tyrosine kinase gene result in a severe combined immunodeficiency (SCID) characterized by a selective inability to produce CD8+ T cells and a signal transduction defect in peripheral CD4+ cells. Transplantation of genetically modified hematopoietic progenitor cells that express the wild-type ZAP-70 gene may provide significant benefit to some of these infants. The feasibility of stem cell gene correction for human ZAP-70 deficiency was assessed using a ZAP-70 knock-out model. ZAP-70–deficient murine bone marrow progenitor cells were transduced with a retroviral vector expressing the human ZAP-70 gene. Engraftment of these cells in irradiated ZAP-70–deficient animals resulted in the development of mature CD4+ and CD8+ T cells. In marked contrast, both populations were absent in ZAP-70−/− mice undergoing transplantation with bone marrow progenitor cells transduced with a control vector. Importantly, ZAP-70–reconstituted T cells proliferated in response to T-cell receptor stimulation. Moreover, these ZAP-70–expressing T cells demonstrated a diverse T-cell receptor repertoire as monitored by the relative usage of each T-cell receptor β chain hypervariable region subfamily. The presence of ZAP-70 in B cells did not affect either lipopolysaccharide- or lipopolysaccharide/interleukin-4–mediated immunoglobulin isotype switching. Altogether, these data indicate that retroviral-mediated gene transfer of the ZAP-70 gene may prove to have a therapeutic benefit for patients with ZAP-70–SCID.

Pregnancy-induced alloimmunization (PIA) may decrease to a level that becomes undetectable by complement-dependent cytotoxicity (CDC). Nevertheless, such alloimmunization may provoke acute rejections after kidney transplantation and lead to broad-spectrum immunizations after transfusion. Flow-cytometry (FC) was used to estimate the frequency of low-level PIA and to evaluate its influence on posttransfusion alloimmunization profiles.

Cell-based therapies are promising approaches to treat uncontrolled pathologies, such as tumours. Apoptotic tumour cells have recently been proposed as a source of tumour-associated antigens to stimulate an efficient immune response. However, a complex relationship exists between apoptosis and the immune system. In this review, the different factors that may influence immune responses against apoptotic cells are detailed and discussed in the light of recent publications. These factors include the nature of the phagocytes and the receptors involved in apoptotic cell uptake, as well as the environment in which cells are dying. A possible distinction between apoptosis and necrosis by immune system sentinels adds a further level of complexity. The potential use of the immunomodulatory properties associated with apoptosis to favour engraftment and induce tolerance in transplantation is then discussed. In conclusion, this review will suggest appropriate conditions to efficiently and safely use apoptotic cells as a new cell therapy product.

To modulate alloreactivity after hematopoietic stem cell transplantation, suicide gene–expressing donor T cells can be administered with an allogeneic T-cell–depleted bone marrow graft. Immune competence of such cells is a critical issue. The impact of the ex vivo gene transfer protocol (12-day culture period including CD3/interleukin-2 [IL-2] activation, retroviral-mediated gene transfer, and G418-based selection) on the anti–Epstein-Barr virus (EBV) potential of gene-modified cells has been examined. Cytotoxic (pCTL) and helper (pTh) cell precursor limiting dilution assays, interferon-γ enzyme-linked immunospot, or fluorescence-activated cell sorter analysis after tetrameric HLA-A2/EBV peptide complexes revealed that the frequency of anti-EBV T cells was lower in gene-modified cells (GMCs) than in similarly cultured but untransduced T cells and was even lower than in fresh peripheral blood mononuclear cells, demonstrating both an effect of the culture and of the transduction or selection. The culture-dependent loss of EBV-reactive cells resulted from the preferential induction of activation-induced cell death in tetramer+ cells. Replacing the initial CD3/IL-2 activation by CD3/CD28/IL-2 partially restored the anti-EBV response of GMCs by reducing the initial activation-induced cell death and enhancing the proliferation of EBV-tetramer+cells. Moreover, the G418 selection, and not the transduction, was directly toxic to transduced tetramer+ cells. Replacing the G418 selection by an immunomagnetic selection significantly prevented the selection-dependent loss of EBV-specific cells. Overall, ex vivo gene modification of primary T cells can result in a significant reduction in EBV-reactive T cells through both culture-dependent and selection-dependent mechanisms. Improving immune functions of GMCs through modifications of the cell culture conditions and transduction/selection processes is critical for further clinical studies.

Dendritic cells are a heterogeneous population of bone marrow-derived cells present in most peripheral tissues. These cells are able to capture and present antigens to T cells. Such presentation can lead to two opposite outcomes potent activation (immunogenicity) or inhibition (tolerance) of the immune response. The fine regulation of these two distinct functions is not completely understood to date. In this review, we discuss three potential variables that may influence dendritic cell function the origin of dendritic cells, their maturation state, and their capture properties. Each hypothesis is illustrated with examples in the field of transplantation. Lastly, the criteria necessary for proposing tolerogenic dendritic cells to promote engraftment and long-term allograft survival are discussed.

Gene-transfer techniques are commonly employed for both in vitro and in vivo studies. However, modifications of the target cell following the introduction of the gene of interest are not often examined. These modifications can alter the immunogenicity and/or the susceptibility of the target cell to apoptosis and may produce unwanted consequences in vivo. Gene transfer into the murine fibroblastic Ψ-CRIP packaging cellline was performed using calcium phosphate precipitation, cationic liposome–DNA complexes or a retroviral RNA-mediated method. After gene transfer, Fas expression, cytokine production, and sensitivity to Fas ligand (FasL)-mediated death were assessed. Following transfection of a FasL expression vector by calcium phosphate precipitation, an unexpected increase was observed in apoptotic cell death in previously Fas-resistant Ψ-CRIP cells. This apoptosis was due to Fas upregulation and an increase of sensitivity to FasL-mediated death. Other plasmids coding non-cytotoxic factors also modulated this apoptotic pathway. Theco-stimulatory molecule CD80 was also upregulated. Exposure to naked DNA alone elicited the same response. The effect was not dependent on the methylation status of exogenous DNA, but was found to be dependent on the target cell type and might be avoided by the use of an RNA-mediated retroviral system. Plasmid transfection or simple exposure to naked DNA can increase sensitivity to apoptosis. The generation of FasL packaging cell lines is therefore limited by an increase in FasL/Fas-mediated apoptosis. These findings should be considered when using genetically modified transplantable cells in order to prevent elimination by host cytotoxic cells and in particular when cells are engineered using FasL. Copyright © 2001 John Wiley & Sons, Ltd.

A common view in face recognition research holds that there is a stored representation specific to each known face. It is also posited that semantic or memory-based information cannot influence low-level face processing. The two experiments reported in this article investigate the nature of this representation and the flow of face information processing. Participants had to search for a particular primed face among other faces. In Experiment 1, the search was done in a context where distractors had either a different degree of fame or the same degree of fame. In Experiment 2, the target face was primed either with semantic information or without any information. Both experiments demonstrated that increasing the display set size lengthened face detection time. However, the lengthening was a function of face fame. The search context also had an effect on the slope of the famous face detection. The results are explained in terms of the idea that face representations are reconstructed and that high- and low-level information are integrated into the processing. The integration process is not a conscious one.

Immortalized hepatocytes are an attractive cell source for hepatocyte transplantation and gene transfer. We compared the phenotype and immunogenicity of freshly isolated (FIH) and immortalized (IMH) rat hepatocytes. Effect of culture and proinflammatory cytokines (TNF-α, IFN-γ) was studied on phenotype. FIH were isolated by collagenase digestion. Two SV40 immortalized hepatocyte cell lines were tested (RH1 and P9). Immunophenotyping was performed by FACS analysis using anti-rat-specific antibodies. Immunogenicity was evaluated by a mixed lymphocyte hepatocyte reaction (MLHR). FIH suspension was an almost homogeneous parenchymal cell population with few (1–2%) CD8+ cells. FIH showed a positive staining for ICAM-1 (20–35%) and for Class I (RT1A, 30–60%) but no staining for Class II (RT1B). After 48 h of culture, the already ICAM-1-positive cells were more strongly stained and additionally 3.6% of the cells (possibly endothelial cells) were Class II positive. IMH showed a consistent expression of Class I (93–97%) and ICAM-1 (95–97%) but no expression of Class II. Culture of IMH for 48 h had no effect on Class II expression but increased ICAM-1 expression. Addition of TNF-α at 1000 UI/ml to cultures of FIH or IMH increased Class I and ICAM-1 expression whereas IFN-γ (50 or 1000 UI/ml) had no evident effect. Hepatocyte immunogenicity, assessed in MLHR and appreciated by the stimulation index (SI) test/SI syngeneic control, was similar for IMH (RH1: 2.68 ± 0.89; P9: 2.37 ± 0.78) and FIH (2.52 ± 0.18). In conclusion, despite some quantitative immunophenotypic differences, FIH and IMH induced the same proliferation rate of allogeneic T lymphocytes. Thus, immortalized hepatocytes may constitute an appropriate cellular model to study the prevention of hepatocyte rejection by gene transfer.

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Cross-tolerization of T lymphocytes after apoptotic cell uptake by dendritic cells may be involved in self-tolerance maintenance. Furthermore, immunosuppressive properties are attributed to apoptotic cells. This study evaluated the consequences of apoptotic leukocyte administration in a restrictive engraftment model of murine bone marrow (BM) transplantation. Sublethally irradiated recipients received a limited number of allogeneic BM, with or without irradiated apoptotic leukocytes of different origins. No graft-versus-host disease was observed. Whereas only a low proportion of mice receiving BM cells alone engrafted, addition of apoptotic irradiated leukocytes, independently of the origin (donor, recipient, third-party mice, as well as xenogeneic peripheral blood mononuclear cells), significantly enhanced engraftment. Similar results were obtained after infusion of leukocytes rendered apoptotic by UVB irradiation or by anti-Fas monoclonal antibody stimulation, thus confirming the role of apoptotic cells in engraftment facilitation. Overall, these results suggest that apoptotic leukocytes can nonspecifically facilitate allogeneic BM engraftment. Such a simple approach could be of interest in BM transplantation settings involving an important HLA donor/recipient disparity, a T-cell–depleted graft, or reduced conditioning regimen intensity.

Several acute hemolysis episodes, sometimes lethal, have been recently described after transplantation of allogeneic peripheral blood hematopoietic stem cells (PBHSCs). Hemolysis resulted from the production of donor-derived antibodies (Abs) directed at ABO antigens (Ags) present on recipient red blood cells (RBCs). A multicenter randomized phase III clinical study comparing allogeneic PBHSC transplantation (PBHSCT) versus bone marrow hematopoietic stem cell transplantation (BMHSCT) has been conducted in France. In the course of this study, serum anti-A and/or anti-B Ab titers were compared before the conditioning regimen and on day +30 after transplantation in 49 consecutive evaluable PBHSCT (n?=?21) or BMHSCT (n?=?28) recipients. PBHSCT resulted in a higher frequency of increased anti-A and/or anti-B Ab titers 30 days after transplantation as compared to BMHSCT: 8 (38%) of 21 versus 3 (11%) of 28 (P?=?.04). In PBHSCT recipients, increased titers were observed mostly after receiving a minor ABO mismatch transplant: 5 of 7 versus 3 of 14 in the absence of any minor ABO mismatch (P?=?.05), whereas this was not the case after BMHSCT: 1 of 8 versus 2 of 20. Anti-A and/or anti-B serum Abs detectable at day +30 after PBHSCT were always directed against A and/or B Ags absent both on donor and recipient RBCs. Finally, 3 of 21 PBHSCT versus 0 of 28 BMHSCT recipients developed anti-allogeneic RBC Abs other than ABO (P?=?.07). Overall, the data strongly suggest that immunohematologic reconstitution differs significantly after granulocyte colony-stimulating factor–mobilized PBHSCT when compared to BMHSCT. Such a difference could contribute to the acute hemolysis described after PBHSCT as well as to distinct alloreactivity after PBHSCT.

Event-related potentials (ERP) were recorded during a task involving the short-term recognition of unfamiliar faces. The purpose was to study the effects of changing the intrinsic context (facial expression) and/or the extrinsic context (background) between the encoding and recognition of a face. The new face caused an increase in the parietal N170 amplitude, but this component was not affected by contextual modifications. In contrast, the frontal N200 was very sensitive to context changes. There was also a well-defined, late parietal component modulated by the processing of information relevant to the face recognition decision. This late positive component reached its amplitude peak when the decision criterion was the strictest. The results obtain showed that ERP can be modulated by these context variations even though they are irrelevant to the task at hand.

Most patients with rheumatoid arthritis (RA) express the shared epitope (SE). It is not known whether SE‐negative HLA–DRB1 alleles influence the development of RA. This study examined the influence of SE‐negative HLA–DR alleles (DRB1*X) on the development of RA in 3 different French populations.HLA–DRB1 alleles were defined by polymerase chain reaction with sequence‐specific oligonucleotide hybridization or sequence‐specific primers. SE‐negative alleles were classified according to the electric charge of their P4 pocket. HLA–DRB1 alleles *0103, *0402, *07, *08, *11 (except *1107), *12, and *13 have a neutral or negative P4 charge and are called DRB1*XP4n. HLA–DRB1*03, *0403, *0406, *0407, *0901, *1107, *14, *15, and *16 have a positive P4 charge and are called DRB1*XP4p.Among the SE‐negative subjects, DRB1 genotypes with 1 or 2 DRB1*XP4n alleles were significantly overrepresented in the control subjects compared with the RA patients, whereas DRB1*XP4p/XP4p genotypes were equally represented in the patients and controls. In single‐dose SE–positive subjects, SE/XP4n genotypes were equally represented in the patients and controls. However, SE/XP4p genotypes were significantly overrepresented in the RA patients.The DRB1*X allele polymorphism influences susceptibility to RA. Alleles that have a neutral or negative electric charge in their P4 pocket (DRB1*XP4n), such as DRB1*0103, *0402, *07, *08, *11 (except *1107), *12, and *13, protect against RA. Alleles that have a positive electric charge in their P4 pocket (DRB1*XP4p), such as DRB1*03, *0403, *0406, *0407, *0901, *1107, *14, *15, and *16, have no influence on the predisposition to RA.

The herpes simplex virus thymidine kinase gene type 1 (HSV-Tk) ganciclovir (GCV) system is a novel therapeutic strategy for the modulation of graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation (allo-SCT). Retroviral-mediated gene transfer of the HSV-Tkgene into donor T lymphocytes before allo-SCT may allow their in vivo selective depletion after treatment with GCV. The expression of theHSV-Tkgene was analyzed in vitro in CEM cells, a human lymphoblastoid cell line, transduced with 2 different vectors, each containing the HSV-Tkgene and a selectable marker gene. GCV-resistant clones were identified within the clones expressing the marker gene. Characterization of the molecular events leading to this resistance revealed a 227-bp deletion in the HSV-Tkgene due to the presence of cryptic splice donor and acceptor sites within the HSV-Tkgene sequence. Furthermore, it was confirmed that this deletion was present in human primary T cells transduced with either vector and in 12 patients who received transduced donor T cells, together with a T-cell–depleted allo-SCT. In vivo circulating transduced T cells containing the truncated HSV-Tkgene were identified in all patients immediately after infusion and up to 800 days after transplantation. In patients who received GCV as treatment for GVHD, a progressive increase in the proportion of transduced donor T cells carrying the deleted HSV-Tkgene was observed. These results suggest that the limitations within the HSV-Tk/GCV system can be improved by developing optimized retroviral vectors to ensure maximal killing ofHSV-Tk–transduced cells.

Retroviral vectors based on the Moloney murine leukemia virus (MuLV) have become the primary tool for gene delivery into hematopoietic cells, but clinical trials have been hampered by low transduction efficiencies. Recently, we and others have shown that gene transfer of MuLV-based vectors into T cells can be significantly augmented using a fibronectin-facilitated protocol. Nevertheless, the relative abilities of naive (CD45RA+) and memory (CD45RO+) lymphocyte subsets to be transduced has not been assessed. Although naive T cells demonstrate a restricted cytokine profile following antigen stimulation and a decreased susceptibility to infection with human immunodeficiency virus, it was not clear whether they could be efficiently infected with a MuLV vector. This study describes conditions that permitted gene transfer of an enhanced green fluorescent protein-expressing retroviral vector in more than 50% of naive umbilical cord (UC) blood and peripheral blood (PB) T cells following CD3/CD28 ligation. Moreover, treatment of naive T cells with interleukin-7 resulted in the maintenance of a CD45RA phenotype and gene transfer levels approached 20%. Finally, it was determined that parameters for optimal transduction of CD45RA+ T cells isolated from PB and UC blood differed: transduction of the UC cells was significantly increased by the presence of autologous mononuclear cells (24.5% versus 56.5%). Because naive T cells harbor a receptor repertoire that allows them to respond to novel antigens, the development of protocols targeting their transduction is crucial for gene therapy applications. This approach will also allow the functions of exogenous genes to be evaluated in primary nontransformed naive T cells.

Cyclic depsipeptide cyclo-[d-Hmp¹-l-MeVal²-l-Phe³-l-MePhe⁴-l-Pro⁵-l-aIle⁶-l-MeVal⁷-l-Leu⁸-l-βHOMeVal⁹], the antifungal antibiotic aureobasidin A (AbA), was reported to interfere with ATP-binding cassette (ABC) transporters in yeast and mammalian cells, particularly the MDR1 P-glycoprotein (Pgp), a transmembrane phospholipid flippase or “hydrophobic vacuum cleaner” that mediates multidrug resistance (MDR) of cancer cells. In a standardized assay that measures Pgp function by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL¹⁰⁰) cells as highly resistant Pgp-expressing cells and the cyclic undecapeptide cyclosporin A (CsA) as a reference MDR-reversing agent (IC50 of 3.4 μM), AbA was found to be a more active Pgp inhibitor (IC50 of 2.3 μM). Out of seven natural analogues and 18 chemical derivatives of AbA, several were shown to display even more potent Pgp-inhibitory activity. The Pgp-inhibitory activity was increased about 2-fold by some minor modifications such as those found in the naturally occurring aureobasidins AbB ([d-Hiv¹]-AbA), AbC ([Val⁶]-AbA), and AbD [γHOMeVal⁹]-AbA). The replacement of the [Phe³-MePhe⁴-Pro⁵] tripeptide by an 8-aminocaprylic acid or the N⁷-desmethylation of MeVal⁷ led to only a 3.3-fold decreased capacity to inhibit Pgp function, suggesting that the Pgp inhibitory potential of aureobasidins, though favored by the establishment of an antiparallel β-sheet between the [d-Hmp¹-l-MeVal²-l-Phe³] and [l-aIle⁶-l-MeVal⁷-l-Leu⁸-] tripeptides, does not critically depend on the occurrence of the [l-Phe³-l-MePhe⁴-l-Pro⁵-l-aIle⁶] type II‘ β-turn secondary structure. In contrast, the most potent Pgp inhibitors were found among AbA analogues with [βHO-MeVal⁹] residue alterations, with some data suggesting a negative impact of the [l-Leu⁸-l-βHOMeVal⁹-d-Hmp¹] γ-turn secondary structure on Pgp inhibitory potential. The [2,3-dehydro-MeVal⁹]-AbA was the most potent Pgp inhibitory aureobasidin, being 13-fold more potent than AbA and 19-fold more potent (on a molar basis) than CsA. Finally, there was no correlation between the SAR for the human MDR1 Pgp inhibition and the SAR for Saccharomyces cerevisiae antifungal activity, which is mediated by an inositol phosphoceramide synthase activity.

The impact of cell dose (number of nucleated donor cells per kilogram recipient weight) on transplantation outcome is controversial and may differ for allogeneic and identical twin (syngeneic) bone marrow transplants. We studied the association between cell dose and outcome in 100 unmanipulated identical twin bone marrow transplantations for leukemia, reported to the International Bone Marrow Transplant Registry between 1985 and 1994, using Cox proportional hazards regression for multivariate analyses. Cell doses ranged from 0.3 to 7.4?×?108 nucleated cells/kg (median, 3.0?×?108cells/kg). Median follow-up was 75 months. Five-year cumulative incidences of transplant-related mortality with high (more than 3?×?108 cells/kg) versus low (less than or equal to 3?×?108 cells/kg) cell doses were 2% (95% confidence interval [CI], 0% to 8%) versus 10% (95% CI, 4% to 20%), respectively. Five-year probabilities of leukemia-free survival were 53% (95% CI, 39% to 67%) and 37% (95% CI, 23% to 52%), respectively. In multivariate analysis, among patients surviving in remission at least 9 months after transplantation, those receiving high cell doses were at significantly lower risk for treatment failure (relapse or death) than those receiving low cell doses (RR, 0.27; 95% CI, 0.12 to 0.6;P?=?.001). Lower treatment failure resulted from fewer relapses in the high cell dose group (RR for relapse, 0.28; 95% CI, 1.2 to 0.66; P?=?.003). These findings suggest that outcomes after syngeneic bone marrow transplantation could be improved by transplanting more than 3?×?108 nucleated cells per kilogram. The benefit of high cell dose on relapse may represent a delayed graft-versus-leukemia effect.

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 β 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 aim of this study was to clarify the relationship between accessing the identity of a face and making decisions about its expression. Three experiments are reported in which undergraduate subjects made expression decisions about familiar and unfamiliar faces. The decision was slowed either by concealing the mouth region with a black rectangle (experiment 1) or by using a short presentation time (experiments 2 and 3). Results of experiment 1 showed that subjects recognized the displayed expression of celebrities better than those of unknown persons when information from the mouth was not available. Results of experiment 2 showed that they recognized the expression displayed by celebrities more easily when the presentation time was short. Experiment 3, using familiarized faces, replicated the results of experiments 1 and 2 and ruled out a possible explanation of these results by the use of some identity specific representations that are expressive. Implications for face recognition models are discussed.

The aim of this study was to set up a sensitive and specific method to quantify the number of genemodified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (Delta TK, Delta NeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% ± 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK - expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.

The use of donor T cells expressing the herpes simplex thymidine kinase (HSV-TK) gene followed by ganciclovir (GCV) treatment could allow for specific modulation of the alloreactivity occurring after bone marrow transplantation. We are presently exploring such an approach in a phase I clinical trial.

Granulocytes for transfusion (GTX) continue to be administered, mostly to treat refractory infection 1. Single center series continue to be reported, but evidence of GTX effectiveness remains uncertain. Recent attempts at randomized trials of GTX have not been completed to target although the largest trial, RING 2, identified potentially promising results in a sub-group analysis by dose.