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Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and B-cell development. The only curative treatment is allogeneic hematopoietic stem cell transplantation, which gives a high survival rate (90%) when an HLA-genoidentical donor exists but unsatisfactory results when only partially compatible donors are available. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG1 cDNA. We show here that this approach applied to RAG-1-deficient mice restores normal B- and T-cell function even in the presence of a reduced number of mature B cells. The reconstitution is stable over time, attesting to a selective advantage of transduced progenitors. Notably, a high transgene copy number was detected in all lymphoid organs, and this was associated with a risk of lymphoproliferation as observed in one mouse. Altogether, these results demonstrate that correction of RAG-1 deficiency can be achieved by gene therapy in immunodeficient mice but that human application would require the use of self-inactivated vector to decrease the risk of lymphoproliferative diseases.

We report the detection of an interleukin-4 (IL-4) variant whose expression is tightly associated with deprivation apoptosis. It is detected with the 8D4 anti–IL-4 monoclonal antibody (mAb) not only in T helper-2 (Th2) but also in Th1 clones, and primary T cells, and it is a nonsecreted molecule. It is not expressed during primary necrosis. Our data suggest that de novo IL-4 transcription of an alternative IL-4 mRNA (IL-4δ13) is induced during deprivation apoptosis. In HIV-infected patients, increased expression of IL-4 in T cells is highly correlated to increased apoptosis, restricted to 8D4 reactivity (r2 = 0.84 between % 8D4-8+ and % 7- amino-actinomycin D–positive [7-AAD+] peripheral T cells, P

Highly-active antiretroviral therapy (HAART) has lead to a dramatic decrease in the morbidity of patients infected with the human immunodeficiency virus (HIV). However, metabolic side effects, including lipodystrophy-associated (LD-associated) dyslipidemia, have been reported in patients treated with antiretroviral therapy. This study was designed to determine whether successful HAART was responsible for a dysregulation in the homeostasis of tumor necrosis factor- (TNF-), a cytokine involved in lipid metabolism. Cytokine production was assessed at the single cell level by flow cytometry after a short-term stimulation of peripheral blood T cells from HIV-infected (HIV+) patients who were followed during 18 months of HAART. A dramatic polarization to TNF- synthesis of both CD4 and CD8 T cells was observed in all patients. Because it was previously shown that TNF- synthesis by T cells was highly controlled by apoptosis, concomitant synthesis of TNF- and priming for apoptosis were also analyzed. The accumulation of T cells primed for TNF- synthesis is related to their escape from activation-induced apoptosis, partly due to the cosynthesis of interleukin-2 (IL-2) and TNF-. Interestingly, we observed that LD is associated with a more dramatic TNF- dysregulation, and positive correlations were found between the absolute number of TNF- CD8 T-cell precursors and lipid parameters usually altered in LD including cholesterol, triglycerides, and the atherogenic ratio apolipoprotein B (apoB)/apoA1. Observations from the study indicate that HAART dysregulates homeostasis of TNF- synthesis and suggest that this proinflammatory response induced by efficient antiretroviral therapy is a risk factor of LD development in HIV+ patients.

A recent study in the human-peripheral blood lymphocytes-severe combined immunodeficiency (hu-PBL-SCID) model, analyzing the specificity of the engrafted human T cells, showed that human T-cell lines and clones derived from engrafted cells presented a xenoreactivity toward murine host molecules. This observation raised the question of the influence of the SCID environment on the ex vivo repertoire and function on the human T cells reconstituting the murine host. We have characterized the human Vβ repertoire in the spleen of hu-PBL-SCID mice 1 to 3 months after their engraftment. Fluorescence-activated cell sorting (FACS) analysis of human Vβ T-cell representation showed that, for all chimeras, all tested Vβ subsets were submitted to underrepresentation and/or expansion upon engraftment. Importantly, these quantitative modifications of the T-cell repertoire were associated with a severe restriction in both the CDR3 size distribution pattern of the Vβ transcripts and the number of Jβ segments used by these transcripts. In addition, ex vivo phenotypic characterization of engrafted cells showed that 70% to 100% expressed the activation markers HLA-DR, CD45RO, and CD38. Taken together, these results suggest that, following their engraftment, human T cells were submitted to a massive antigenic selection. Moreover, we found that these activated T cells were unresponsive to in vitro mitogenic and superantigenic activation. The consequences of the skewed repertoire and altered function of engrafted human T cells on the validity of this humanized murine model are discussed.

In a previous study, we reported the existence of a specific anergy affecting selectively the V beta 8 subset in both CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected persons. Because this observation gives evidence for a previous in vivo activation of this subset by a superantigen, we further characterize, in the present study, this V beta 8-anergy associated with HIV infection. Molecular T cell receptor analysis indicates that the V beta 8-anergized T cells are polyclonal. Furthermore, we show the dependence of this anergy on the expression of allelic forms of HLA class II DRB1 molecules. These observations explain the frequency of anergic persons among HIV- infected donors (56%) and are consistent with a previous in vivo superantigenic activity. Comparative analyses of disease evolution between V beta 8 responder and anergic persons do not show any clear relation between the V beta 8 status and acquired immunodeficiency syndrome pathogenesis. However, the stability of the V beta 8 status, the absence of correlation with previous microbial infections, and the previously reported precocity of V beta 8 anergization are in favor of a strong association between the in vivo existence of a V beta 8- specific superantigen and HIV infection. Finally, the functional dichotomy we observe for all anergized donors between blood and lymph node T cells raises the question of the in vivo localization of the superantigenic activity.

The destruction of CD4 T cells in human immunodeficiency virus (HIV) infection is associated with activation of apoptotic programs, partly mediated by death receptors. The role of CD95L/CD95 in depletion of patients' CD4 T cells is well documented, but the possible contribution of the tumor necrosis factor/tumor necrosis factor receptor (TNF/TNFR) pathway has not been examined. In this study, we found that both TNFR1 and TNFR2 induced marked apoptosis in peripheral T cells from HIV-infected persons, involving both CD4 and CD8 T cells. Longitudinal follow-up of HIV+ patients suggests an association between the in vivo evolution of CD4 T-cell numbers and variations in susceptibility to TNFR-induced apoptosis. Analysis of molecular mechanisms involved showed that it was not related to altered ex vivo expression of TNFR1-associated death domain, receptor interacting protein, or TNFR-associated factor 2. Susceptibility to TNFR-mediated apoptosis was rather related to Bcl-2 expression, because patients' T cells expressing high levels of Bcl-2 were completely protected from TNFR1- and TNFR2-induced cell death, whereas T cells expressing normal levels of Bcl-2 were not protected in patients in contrast to controls. Early recruitment of caspase-8 and caspase-3 is needed to transduce the apoptotic signals, and expression of both caspases in their active form was detected in blood T cells from HIV+ patients, whereas it was hardly detected in controls. Moreover, ligation of TNFRs induced increased activation of both caspases in patients' T cells. Together these data demonstrate that exacerbated TNFR-mediated cell death of T cells from HIV-infected individuals is associated with both alteration of Bcl-2 expression and activation of caspase-8 and caspase-3 and may contribute to the pathogenesis of acquired immunodeficiency syndrome.

We have previously reported that ex vivo retroviraly-mediated gc gene transfer into CD34 (+) bone marrow precursor cells led to the correction of the immunodeficiency in 9 out of 10 patients with X-linked severe combined immunodeficiency. Follow-up now reaches more that 6 years for the first 2 treated patients. Patients’immune function has been restored. The distribution of both TCR Vb family usage and TCR Vb CDR3 length still reveals a broadly diversified T cell repertoire. Moreover 6 years after treatment the thymus is still seeded by transduced progenitor cells as attested by the presence of TRECS in peripheral blood RTE. Among these patients, three (P4, P5 and P10) developed at 30 to 34 months after gene therapy a monoclonal T cell proliferation requiring a chemotherapy. P4 received also an allogenic HSCT from a MUD but died 26 months after the occurence of the lymphoproliferation. For P5 and P10, chemotherapy has led to an overall control of the clonal proliferation. These two patients are doing well and P5 is off treatment with a good immunological recovery. Genetic analysis of the blastic cells showed that in the two first cases the vector had integrated within or upstream of the LMO2 locus causing an insertional activation of LMO2 transcription. The last case revealed the involvement of several targeted sites, but their exact contribution to the lymphoproliferation is still under investigation. The repeated involvment of LMO2 as a site of vector integration in the proliferating T-cells points to an insertional activation of this gene as at least one of the causes of the oncogenic process. However, the long latency observed in all cases (> 30 months) suggests that additional “hits” have been required for overt desease. Synergy with gc expression and thereby induced proliferative signals (explaining occurrence in SCID-X1 patients only) is the most obvious hypothesis which we are trying to analyse in a mouse model. A deep analysis of retroviral integration patterns has been performed on patients’PBMCS by LAM-PCR to estimate the frequency of potentially harmful integration events and to assess the risk factors associated with the LTR’s strong enhancer effect of the MLV-based retroviral vector. 708 unique integration sites (IS) have been obtained from all analysed patients post-gene therapy and among them, 577 could be mapped unequivocally to the human genome. *Most of these insertions (63%) are located in the vicinity of 10kb or within the coding sequence of a known gene*. A significant peak of insertion frequency is related closely to the transcription strart site *among the 577 IS, 43 are common integration sites. Among the latter, we found out a high selection of genes involved in human oncogenic process.