Evidence of premature immune aging in patients thymectomized during early childhood.

While the thymus is known to be essential for the initial production of T cells during early life, its contribution to immune development remains a matter of debate. In fact, during cardiac surgery in newborns, the thymus is completely resected to enable better access to the heart to correct congenital heart defects, suggesting that it may be dispensable during childhood and adulthood. Here, we show that young adults thymectomized during early childhood exhibit an altered T cell compartment. Specifically, absolute CD4+ and CD8+ T cell counts were decreased, and these T cell populations showed substantial loss of naive cells and accumulation of oligoclonal memory cells. A subgroup of these young patients (22 years old) exhibited a particularly altered T cell profile that is usually seen in elderly individuals (more than 75 years old). This condition was directly related to CMV infection and the induction of strong CMV-specific T cell responses, which may exhaust the naive T cell pool in the absence of adequate T cell renewal from the thymus. Together, these marked immunological alterations are reminiscent of the immune risk phenotype, which is defined by a cluster of immune markers predictive of increased mortality in the elderly. Overall, our data highlight the importance of the thymus in maintaining the integrity of T cell immunity during adult life.

Abnormal expression of only the CD34 part of a transgenic CD34/herpes simplex virus-thymidine kinase fusion protein is associated with ganciclovir resistance.

Donor T cell alloreactivity can be efficiently controlled by retrovirus-mediated ex vivo transfer of a "suicide" gene encoding the wild-type herpes simplex virus thymidine kinase (wtHSV-tk) gene, allowing gene-modified cells (GMCs) to be sensitive to ganciclovir (GCV). A limitation to this approach was related to the presence of an inactive form of the wtHSV-tk gene, resulting from alternative splicing. A corrected HSV-tk (cHSV-tk) gene was developed in order to circumvent this problem and was fused to a truncated splice variant of the human CD34 molecule (tCD34) suitable for the selection of retrovirally transduced GMCs. We demonstrate now that, despite this correction, CD34-positive, but GCV-resistant, HUT and primary GMCs can still be generated after transduction with a retroviral vector encoding a tCD34/cHSV-tk fusion protein (FuProtein). Deletions in the HSV-tk part of the transgene account in part for this resistance. However, an additional mechanism involving proteolytic-dependent "breakage" of the FuProtein has been observed: the CD34 part of the FuProtein can be detected by Western blot, separated from its HSV-tk part. Although the HSV-tk protein alone is not detectable in GCV-resistant tCD34/cHSV-tk-transduced HUT cells, it can be detected in 293T cells transduced with another tCD34/HSVTK fusion vector, demonstrating that a posttranslational effect leads to the breakage of the FuProtein. This is to our knowledge the first example of a loss of function of a FuProtein, of which one part is still expressed while the other one, suffering a selection pressure, is no longer detectable.

Regulatory T-cell expansion and function do not account for the impaired alloreactivity of ex vivo-expanded T cells.

CD3- and CD28-activated T cells expanded for 12 days ex vivo to produce suicide gene-modified T cells are hyporesponsive to alloantigens. To investigate whether this impaired alloreactivity is a result of preferential expansion of regulatory T (Treg) cells, we compared peripheral blood mononuclear cells (PBMC) activated with CD3 and CD28 antibodies co-immobilized on beads and expanded for 12 days with interleukin (IL)-2 (Co(CD3/CD28) cells) to the respective unactivated PBMC in terms of proliferation, cytokine production, and expression of Treg markers [cytotoxic T-lymphocyte antigen 4 (CTLA4), glucocorticoid-induced tumour necrosis factor receptor (GITR) and forkhead box P3 (FoxP3)] after allostimulation. Alloreactive cells were identified by carboxyfluoresceine succinimidyl ester staining dilution. Alloreactive cells in Co(CD3/CD28) cells had a lower proliferative response and a lower potential for IL-2 and interferon-gamma secretion than did those in PBMC, demonstrating a functional impairment of alloreactive cells during ex vivo expansion. Expression of Treg markers transiently increased during ex vivo expansion and was unaffected by depletion of CD25(+) cells (containing Treg cells) before ex vivo PBMC expansion. Such prior CD25(+) depletion did not restore the alloreactivity of Co(CD3/CD28) cells. After allostimulation, expression of Treg markers was restricted to proliferative (alloreactive) cells among PBMC or Co(CD3/CD28) cells. Lastly, CD4(+) CD25(+) cells purified from Co(CD3/CD28) cells lacked suppressive activity when used as a third party, in contrast to CD4(+) CD25(+) cells purified from PBMC. In conclusion, the impaired alloreactivity of T cells expanded ex vivo is not a result of preferential Treg cell expansion and/or enhanced suppressive Treg activity.

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.

No evidence of association between NOD2/CARD15 gene polymorphism and atherosclerotic events after renal transplantation.

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.

Retrovirus-mediated gene transfer in human primary T lymphocytes induces an activation- and transduction/selection-dependent TCR-B variable chain repertoire skewing of gene-modified cells.

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 beta 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.

Retrovirus-mediated gene transfer in polyclonal T cells results in lower apoptosis and enhanced ex vivo cell expansion of CMV-reactive CD8 T cells as compared with EBV-reactive CD8 T cells.

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, retrovirus-mediated 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.

Exposure to exogenous DNA can modify the sensitivity of the Fas apoptotic pathway.

BACKGROUND: 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. METHODS: Gene transfer into the murine fibroblastic Psi-CRIP packaging cell line 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. RESULTS: Following transfection of a FasL expression vector by calcium phosphate precipitation, an unexpected increase was observed in apoptotic cell death in previously Fas-resistant Psi-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. The co-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. CONCLUSIONS: 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.

Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein-Barr virus potential through both culture-dependent and selection process-dependent mechanisms.

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-gamma 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.

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.