Search

Chronic granulomatous disease (CGD) is a primary immunodeficiency due to a deficiency in one of the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. CGD patients are characterized by an increased susceptibility to bacterial and fungal infections, and to granuloma formation due to the excessive inflammatory responses. Several gene therapy approaches with lentiviral vectors have been proposed but there is a lack of in vivo data on the ability to control infections and inflammation. We set up a mouse model of acute infection that closely mimic the airway infection in CGD patients. It involved an intratracheal injection of a methicillin-sensitive reference strain of S. aureus . Gene therapy, with hematopoietic stem cells transduced with regulated lentiviral vectors, restored the functional activity of NADPH oxidase complex (with 20–98% of dihydrorhodamine positive granulocytes and monocytes) and saved mice from death caused by S. aureus , significantly reducing the bacterial load and lung damage, similarly to WT mice even at low vector copy number. When challenged, gene therapy-treated XCGD mice showed correction of proinflammatory cytokines and chemokine imbalance at levels that were comparable to WT. Examined together, our results support the clinical development of gene therapy protocols using lentiviral vectors for the protection against infections and inflammation.

International audience; Ex vivo transduction of human CD34+ hematopoietic stem/progenitor cells (hCD34+ HSPCs) and T lymphocytes is a key process that requires high efficiency and low toxicity to achieve effective clinical results. So far, several enhancers have been used to improve this process. Among them, Retronectin highly meliorates VSV-G and RD114-TR pseudotyped lentiviral vector delivery in hCD34+ HSPCs and T lymphocytes. However, Retronectin is expensive and requires pre-coating of culture dishes or bags before cell seeding, resulting in a cumbersome procedure. Recently, an alternative transduction adjuvant has been developed, named Vectofusin-1, whose effect has been demonstrated on gene delivery to cell lines and primary hCD34+ HSPCs by lentiviral vectors pseudotyped with different envelope glycoproteins. In this study, we have focused our analysis on the effect of Vectofusin-1 on the transduction of hCD34+ HSPCs and T lymphocytes by using mostly RD114-TR pseudotyped lentivectors and clinical transduction protocols. Here, we have proved that Vectofusin-1 reproducibly enhances gene delivery to hCD34+ HSPCs and activated T cells without cell toxicity and with efficacy comparable to that of Retronectin. The use of Vectofusin-1 will therefore help to shorten and simplify clinical cell manipulation, especially if automated systems are planned for transducing large-scale clinical lots.

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.

Hereditary genetic disorders have been shown to be effectively treated by re-introduction of the wild-type gene product via viral modification of autologous CD34+ progenitor cells. However, current manufacturing processes are performed manually or at best by using semi-automated procedures. A standardized production of cellular therapeutic agents and their genetic modification is a major requirement to move cellular and gene therapies from their current translational setting into routine clinical use. A closed and highly automated manufacturing procedure would lead to large improvements in product performance, product safety and operator safety. We have recently developed a functionally closed and fully automated cell processing device, the CliniMACS Prodigy®, which enables complex cellular products to be manufactured.In this functionally closed system, the following steps have been automated: concentration and washing of blood products, magnetic labeling and enrichment of CD34+ cells, cultivation and pre-activation of the enriched CD34+ cells, transduction with lentiviral vectors and resuspension of the final cell product in buffer for infusion.Automated cell preparation, cell labeling and magnetic CD34+ cell separation was achieved from both mobilized and non-mobilized apheresis samples as well as bone marrow aspirates. CD34+ cells were enriched to purities exceeding 90%. Small scale experiments (10e5-10e7 purified CD34+ cells) performed with a newly developed automated transduction process, incorporating a single transduction cycle, resulted in high transduction efficiencies (up to 90%). A scale-up of the procedure was performed using larger numbers of cells (>10e7) enriched from mobilized apheresis material. CD34+ cells were transduced with a clinical-grade lentiviral vector using a two hit protocol already described in a clinical setting and used as standard for a control experiment run in parallel (Scaramuzza et al. 2013, Mol. Ther). Equivalent gene marking efficiencies were obtained. Moreover, gene modified cells maintained full engraftment potential as demonstrated by biodistribution studies in NSG mice.These preliminary data demonstrate that automated cell processing and genetic manipulation of haematopoetic progenitor cells can be efficiently performed in a closed system. Work in progress aims to further optimize this procedure and improve the efficiency of CD34+ cell transduction to levels superior to those currently achieved by manual protocols.

Deletion of the nei gene results in viral attenuation and confers protection against challenge with wild-type simian immunodeficiency virus in macaques. Regarding HIV-1 infection, a few long-term nonprogressors (LTNP) with nef deletions have been described. In this study, the nef genes of a group of seven LTNP and eight progressors, all belonging to the same cohort of infected hemophiliacs, were analyzed by cloning and sequencing from both virion RNA and peripheral blood mononuclear cell-associated proviral DNA. Defective nef sequences coexisted with full-length nef open reading frames in five of seven LTNP and two of eight progressors. The proportion of disrupted nef sequences within each individual was significantly higher in LTNP (ranging from 10 to 63%) than in progressors (ranging from 9 to 21%) (P = 0.013). Moreover, in-frame small deletions predicting to encode Nef were found in all RNA- and DNA-derived crones from one LTNP and four progressors. A chimeric virus in which the nef gene of NL4.3 was substituted with the nef allele containing the deletion of two alanines at position 49-50 found in two progressors showed a defective replicative capacity compared to NL4.3 virus. In summary, hemophiliacs with either progressing or nonprogressing HIV-1 infection are characterized by the presence of defective nef variants. (C) 1999 Academic Press.

The efficacy of several clinical approaches of adoptive T cell therapy (ACT) can be compromised by the insurgence of severe unfavorable events as graft-versus host disease (GvHD) secondary to bone marrow transplantation unless ex-vivo manipulation endow transplantable T cells with safety system. Introduction of drug-induced suicide genes, as for example the herpes simplex virus thymidine kinase (HSV-TK) transgene in T cells during bone marrow transplantation for leukemia and lymphoma treatment, represents a valid approach to control gene-modified cell proliferation and survival overtime. Transplanted modified cells are selectively deleted in vivo by specific activation of an otherwise inactive prodrug in those cells. Despite the well-documented utility of this approach, some limitations arose in terms of poor prodrug activation kinetics, escape from drug selection and immunological rejection.Engineered variants targeting the active site of the human deoxyCytidine Kinase (dCK) suicide enzyme were described as a powerful alternative to current strategies being able to activate a large variety of nucleoside analogue-based prodrugs, having low immunogenicity and permitting in vivo imaging of genetically modified cells. Lentiviral vector (LV)-mediated delivery of the triple mutant dCK.R104M.D133A.S74E (dCK.DM.S74E) fused to the truncated low density nerve growth factor receptor (LNGFR) selection marker renders human cells highly sensitive to the prodrugs bromovinyl-deoxyuridine (brivudine or BVdU), l-deoxythymidine (LdT), and l-deoxyuridine (LdU) via induction of apoptosis. MolMed has developed the proprietary RD-MolPack technology for the stable production of both second and third generation LVs, pseudotyped with the RD114-TR envelope that, in contrast to inducible systems based on VSV-G envelope, permits the generation of a simple, constitutive, no-toxic LV packaging system. We thus exploited the RD-MolPack technology to obtain several colonies of RD-MolPack-trLNGFR/dCK.DM.S74E producer cells by stable transfection of a SIN transfer vector plasmid carrying the trLNGFR/dCK.DM.S74E transgene followed by zeocin selection. The average titer calculated on CEM A3.01 target cells of several colonies is 1.9 × 105 ± 8.15 × 104 TU/106 cells/day, in line with the previous results obtained with the previously established RD2-MolPack-Chim3 and RD3-MolPack-GFP producer cells. These preliminary data suggest that the human suicide gene trLNGFR/dCK.DM.S74E can be constitutively produced in LV stable packaging cells without toxicity and its use can be therefore considered a valid option in the scenario of prodrug convertase enzymes.

To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.

To study the effects of the regulatory phosphoprotein ICP4 of the Herpes simplex virus, (HSV), a DNA tumor virus, on the induction of gene expression by the epidermal growth factor (EGF), we have cons

The multiple therapeutic approaches developed so far to cope HIV-1 infection, such as anti-retroviral drugs, germicides and several attempts of therapeutic vaccination have provided significant amelioration in terms of life-quality and survival rate of AIDS patients. Nevertheless, no approach has demonstrated efficacy in eradicating this lethal, if untreated, infection. The curative power of gene therapy has been proven for the treatment of monogenic immunodeficiensies, where permanent gene modification of host cells is sufficient to correct the defect for life-time. No doubt, a similar concept is not applicable for gene therapy of infectious immunodeficiensies as AIDS, where there is not a single gene to be corrected; rather engineered cells must gain immunotherapeutic or antiviral features to grant either short- or long-term efficacy mostly by acquisition of antiviral genes or payloads. Anti-HIV/AIDS gene therapy is one of the most promising strategy, although challenging, to eradicate HIV-1 infection. In fact, genetic modification of hematopoietic stem cells with one or multiple therapeutic genes is expected to originate blood cell progenies resistant to viral infection and thereby able to prevail on infected unprotected cells. Ultimately, protected cells will re-establish a functional immune system able to control HIV-1 replication. More than hundred gene therapy clinical trials against AIDS employing different viral vectors and transgenes have been approved or are currently ongoing worldwide. This review will overview anti-HIV-1 infection gene therapy field evaluating strength and weakness of the transgenes and payloads used in the past and of those potentially exploitable in the future.

The long-term expression and the ability of a therapeutic gene to confer survival advantage to transduced cells are mandatory requirements for successful anti-HIV gene therapy. In this context, we developed lentiviral vectors (LVs) expressing the F12–viral infectivity factor (Vif) derivative Chim3. We recently showed that Chim3 inhibits HIV-1 replication in primary cells by both blocking the accumulation of retrotranscripts, independently of either human APOBEC3G (hA3G) or Vif, and by preserving the antiviral function of hA3G. These results were predictive of long-lasting survival of Chim3+ cells after HIV-1 infection. Furthermore, Vif, like Vpr, deregulates cell-cycle progression by inducing a delay in G2 phase. Thus, the aim of this study was to investigate the role of Chim3 on both cell survival and cell-cycle regulation after HIV-1 infection. Here, we provide evidence that infected Chim3+ T cells prevail over either mock- or empty-LV engineered cells, show reduced G2 accumulation, and, as a consequence, ultimately extend their lifespan. Based on these findings, Chim3 rightly belongs to the most efficacious class of antiviral genes. In conclusion, Chim3 usage in anti-HIV gene therapy based on hematopoietic stem cell (HSC) modification has to be considered as a promising therapeutic intervention to eventually cope with HIV-1 infection.

Our previous results have demonstrated that T-cell lines and primary T lymphocytes transduced with a Tat-dependent HIV-based lentiviral vectors expressing the mutant isoform of the vif gene, F12-vif, are protected from HIV-1 infection. F12-Vif is a 192-aa natural variant polypeptide owing 14 unique amino acid substitutions. The substitutions are randomly scattered along the entire sequence with the exception of a 5-aa cluster located at positions 127, 128, and 130|[ndash]|132. None of the 14 aa substitutions is present in the SOCS box that recruits the E3 ubiquitin ligase responsible of APOBEC3G (AP3G) degradation during HIV infection. In line with this notion, we have shown that the antiviral function of F12-Vif is not due to a dominant negative feature of the mutant in regards to the Vif-mediated degradation of AP3G rather to some other unknown means. Therefore, in the effort to elucidate the F12-Vif mechanism of action, we started to identify the protein domain of F12-Vif responsible of HIV-1 inhibition. To this end, we have constructed three chimeric genes (Chim1, Chim2 and Chim3) composed by wild-type and F12-vif regions. T cell lines and cord blood derived CD4+T lymphocytes were transduced with the lentiviral vectors expressing the chimeric genes and then challenged with both X4 and R5 HIV-1 strains. We show that 45 amino acids in the C-terminal domain of the F12-Vif mutant are sufficient to exert anti-viral effect in transduced cells. In contrast to F12-Vif, Chim3 does not allow the rescue of the replication of a vif-deficient HIV-1 in the context of either X4 or R5 tropism in non permissive cells. This specific feature renders Chim3 a truly dominant negative protein more suitable than F12-Vif for an anti-HIV gene therapy approach.

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function.

AIDS has become the greatest pandemic in the human history counting approximately 40 millions people worldwide. To purge HIV-1 infection, new therapeutic approaches need to be searched in alternative and/or in addition to the current pharmacological ones. Recently, several independent laboratories have unveiled a non-immune intracellular anti-HIV-1 defense strategy based on the cytidine deaminase APOBEC3G, which restricts HIV-1 production by directly mutating the proviral DNA in infected cells. To counteract this defense pathway, HIV-1 has developed an evasion strategy by acquiring the accessory protein Vif, which blocks the action of APOBEC3G by inducing its proteasome-mediated degradation.

Human T cell leukemia virus (HTLV) type-2 is a human retrovirus whose infection has not been tightly linked to human diseases. However, the fairly high prevalence of this infection among HIV-1-positive individuals indicates the importance of better understanding the potential interference of HTLV-2 infection on HIV-1 infection and AIDS. We previously demonstrated that one signature of PBMC freshly derived from HIV-1-infected individuals is the constitutive activation of a C-terminal truncated STAT5 (STAT5Δ). Therefore, we analyzed the potential activation of STATs in HTLV-2 monoinfected and HTLV-2/HIV-1 dually infected individuals. We observed that PBMC of HTLV-2-infected individuals do not show STAT activation unless they are cultivated ex vivo, in the absence of any mitogenic stimuli, for at least 8 h. The emergence of STAT activation, namely of STAT1, in culture was mostly related to the secretion of IFN-γ. Of note, this phenomenon is not only a characteristic feature of HTLV-2-infected individuals but also occurred with PBMC of HIV-1+ individuals. Surprisingly, HTLV-2/HIV-1 coinfection resulted in low/absent STAT activation in vivo that paralleled a diminished secretion of IFN-γ after ex vivo cultivation. Our findings indicate that both HTLV-2 and HIV-1 infection prime T lymphocytes for STAT1 activation, but they also highlight an interference exerted by HTLV-2 on HIV-1-induced STAT1 activation. Although the nature of such a phenomenon is unclear at the present, these findings support the hypothesis that HTLV-2 may interfere with HIV-1 infection at multiple levels.

Human T-cell leukemia–lymphoma virus (HTLV) type-2 can induce the survival and proliferation of CD34+ TF-1 cells deprived of interleukin (IL)-3. This effect did not require productive infection and occurred when HTLV-2 was produced from T cells (CMo), but not from B cells (BMo), unless the latter virus was complexed with anti–HLA-DR monoclonal antibodies (mAbs). Cellular and molecular mechanisms triggered by HTLV-2 interaction with TF-1 cells were here investigated. Activation of signal transducer and activator of transcription (STAT) 5 protein occurred in TF-1 cells incubated either with IL-3 or with HTLV-2/CMo; in addition the virus, but not IL-3, activated STAT1. The effect of HTLV-2 required several hours, suggesting dependence on the induction of cellular factors. By screening a panel of secreted factors, granulocyte macrophage–colony-stimulating factor (GM-CSF), interferon (IFN)-γ, and stem cell factor (SCF) were found induced by HTLV-2 in TF-1 cells. Of note is the fact that these molecules induce a variety of biologic effects through the activation of STAT proteins, including STAT1 and STAT5. Neutralization experiments indicated that GM-CSF and IFN-γ, but not SCF, were responsible for HTLV-2–induced STAT activation, whereas anti–GM-CSF antibodies greatly inhibited TF-1 cell proliferation. Finally, incubation of BMo virus with anti–HLA-DR mAb rescued TF-1 cell survival in the absence of IL-3. Thus, HTLV-2 interaction with CD34+ precursor cells may lead to the expression of cytokines that, by inducing autocrine activation of STATs, may influence the host's regenerative capacity and immune response to HTLV-2 and to other infectious agents.

Intermittent administration of recombinant interleukin-a (rIL-2) to individuals infected with human immunodeficiency virus (HIV) has been shown to raise and maintain the absolute number of circulating CD4(+) T cells to normal or near normal levels. One of the signaling pathways triggered by IL-2 is the Janus kinase-signal transducer and activator of transcription (JAK-STAT). In particular, IL-2 activates the tyrosine kinases JAK1 and JAK3 and the transcription factors STAT3 and STAT5. We have previously observed that most HIV+ individuals, unlike healthy seronegative controls, show a constitutive activation of STAT1 and a C-terminal truncated isoform of STAT5 (STAT5 Delta). In the present study, we have analyzed the protein level and activation state of STAT5 isoforms expressed in peripheral blood mononuclear cells of two HIV-infected individuals who showed a good or a poor response to intermittent IL-2 administration, respectively, and of a single individual before and after initiation of Zidovudine monotherapy. We provide evidence that both therapeutic interventions enhanced the expression and activation of the C-terminal truncated isoform of STAT5 (STAT5 Delta) in vivo. (C) 2001 Academic Press.

We have constructed a fibroblast cell line (EL alpha 4-2) which constitutively expresses the alpha 4 gene of herpes simplex virus type 1. We studied the induction of the alpha 4 gene, in the absence of the viral activator VP16, by stimulating EL alpha 4-2 cells with different growth factors. Here we report that a rapid, transient induction of the alpha 4 gene occurs only when EL alpha 4-2 fibroblasts are stimulated with purified epidermal growth factor (EGF). Such an induction does not require de novo protein synthesis. The role of cellular factors on the EGF-mediated induction of the alpha 4 gene has been analyzed by gel mobility shift assays using nuclear extracts from EL alpha 4-2 cells stimulated or not with EGF. The results obtained show that two factors bind to TAATGARAT (R = purine) regardless of EGF-stimulation. We conclude that a mechanism, different from the one involving VP16, is responsible for alpha 4 gene activation in EL alpha 4-2 cells and that the DNA-protein architecture is maintained at the TAATGARAT regulatory site regardless of changes in the transcriptional state induced by EGF.

We have described a constitutive activation of naturally C-terminus truncated STAT5 (STAT5Δ) in PBMC of most HIV+ individuals (C. Bovolenta, Blood, 1999). We here report that the chronically HIV-infected promonocytic cell line U1 expresses exclusively a STAT5Δ isoform similar to that of PBMC of HIV+ individuals in virtual absence of STAT5FL. Consistently with the presence of functional STAT5 DNA binding sites in the HIV-1 LTR (Selliah et al., Virology, 2006), GM-CSF stimulation of U1 cells led to a modest induction of HIV expression as well as to the activation of both STAT5Δ and of an ERK-1/2-AP-1 pathway. Inhibition of ERKs/AP-1 by PD98059 abolished GM-CSF induced HIV expression in U1 cells, whereas inhibition of STAT5Δ expression by either AG490 or anti-STAT5 siRNA resulted in a significant enhancement of GM-CSF induced HIV expression in U1 cells. Finally, ex vivo IL-2 stimulation of PBMC from most HIV+ individuals in the presence of anti-STAT5 siRNA resulted in the enhancement of HIV-1 p24 Gag antigen expression. Thus, STAT5Δ is likely a novel natural inhibitor of HIV expression both in vitro and in vivo.

Viral infectivity factor (Vif) is required for high HIV-1 infectivity in non-permissive cells, which include the natural targets of HIV-1, whereas it is dispensable in several cell lines called permissive. This requirement depends on the ability of Vif to counteract the action of the cellular inhibitor APOBEC3G, whose expression is restricted to non-permissive cells. APOBEC3G is a cytidine deaminase which is incorporated, in the absence of Vif, into viral particles and induces massive G-to-A hyper mutation in the nascent plus strand cDNA during reverse transcription in new target cells. Vif overcomes the action of APOBEC3G by inducing, without the need of any other viral components, its proteasome-dependent degradation. We have constructed, in the context of a potential new approach of anti-HIV-1 gene therapy, a second generation, VSV-G pseudo-typed lentiviral vector carrying the F12-vif gene, which is transcriptionally regulated by the HIV 5 LTR. F12-Vif derives from the natural variant F12-HIV provirus, which confers homologous interference to HIV-1 super-infection. We have transduced several T cell lines and primary T lymphocytes with F12-vif and WT-vif vectors and then challenged them with either CCR5 or CXCR4 HIV-1 strains. Here, we show that the level of p24 Ag released in the culture medium of infected cells is 5 logs lower in cells expressing F12-Vif, compared to the mock transduced and the control WT-Vif expressing cells. To understand whether F12-Vif inhibits HIV-1 replication in a dominant negative fashion, we have infected with Δvif HIV-1 non-permissive cells, transduced with the Vif vectors. Of note, similarly to WT-Vif, F12-Vif rescues viral replication, suggesting that in the absence of WT-Vif, F12-Vif vicariates its function. Finally, to investigate whether F12-Vif interferes with APOBEC3G function, we have monitored the expression of endogenous APOBEC3G in transduced non-permissive cells infected with either wild type or Δvif HIV-1 strains. Results of this analysis will be presented.

The Southern blot hybridization technique has been applied to study the configuration of immunoglobulin and T-cell receptor genes in 6 cases of the so called mediastinal large cell lymphoma with sclerosis. This lymphoma has been recently recognized as a separate entity among non-Hodgkin lymphomas mainly affecting young adult patients. The B-cell origin of this neoplasm was suggested by means of immunohistochemical analysis. However, the immunophenotypical B-cell related markers used do not always exhibit lineage fidelity. The Southern blot analysis demonstrated the presence of unique heavy and k-light chain immunoglobulin gene rearrangements, establishing genotypically their B-cell origin.

The human immunodeficiency virus (HIV) replicates in activated CD4(+) T lymphocytes. However, only CD4(+) Th2 and Th0, but not Th1, CD4(+) T-cell clones have been reported to efficiently support HIV-1 replication. This dichotomous pattern was further investigated in the present study in Th1, Th2, or Th0 cell lines derived from umbilical human cord blood and in T-cell clones obtained from the peripheral blood mononuclear cells (PBMC) of healthy adults. Both primary and laboratory-adapted HIV-1 strains with CCR5 as the exclusive entry coreceptor (R5 viruses) efficiently replicated in Th1, Th2, and Th0 cells, In sharp contrast, CXCR4-dependent (X4) viruses poorly replicated in both polarized and unpolarized CD4(+) T cells, including adults' PBMC infected several days after mitogenic stimulation. Unlike the X4 HIV-1(NL4-3), a chimera in which the env gene had been replaced with that of the R5 HIV-1(NL(AD8)), efficiently replicated in both Th1 and Th2 cells. This X4-dependent restriction of HIV replication was not explained by either the absence of functional CXCR4 on the cell surface or by the inefficient viral entry and reverse transcription. T-cell receptor stimulation by anti-CD3 monoclonal antibodies fully rescued X4 HIV-1 replication in both Th1 and Th2 cells, whereas it did not alter the extent and kinetics of R5 HIV-1 spreading. Thus, R5 HIVs show a replicative advantage in comparison to X4 viruses in their ability to efficiently propagate among suboptimally activated T lymphocytes, regardless of their polarized or unpolarized functional profiles. This observation may help to explain the absolute predominance of R5 HIVs over X4 viruses observed after viral transmission and during early-stage disease. The human immunodeficiency virus (HIV) replicates in activated CD4+ T lymphocytes. However, only CD4+ Th2 and Th0, but not Th1, CD4+ T-cell clones have been reported to efficiently support HIV-1 replication. This dichotomous pattern was further investigated in the present study in Th1, Th2, or Th0 cell lines derived from umbilical human cord blood and in T-cell clones obtained from the peripheral blood mononuclear cells (PBMC) of healthy adults. Both primary and laboratory-adapted HIV-1 strains with CCR5 as the exclusive entry coreceptor (R5 viruses) efficiently replicated in Th1, Th2, and Th0 cells. In sharp contrast, CXCR4-dependent (X4) viruses poorly replicated in both polarized and unpolarized CD4+ T cells, including adults' PBMC infected several days after mitogenic stimulation. Unlike the X4 HIV- 1(NL)4-3, a chimera in which the env gene had been replaced with that of the R5 HIV-1(NL(AD)8), efficiently replicated in both Th1 and Th2 cells. This X4-dependent restriction of HIV replication was not explained by either the absence of functional CXCR4 on the cell surface or by the inefficient vital entry and reverse transcription. T-cell receptor stimulation by anti- CD3 monoclonal antibodies fully rescued X4 HIV-1 replication in both Th1 and Th2 cells, whereas it did not alter the extent and kinetics of R5 HIV-1 spreading. Thus, R5 HIVs show a replicative advantage in comparison to X4 viruses in their ability to efficiently propagate among suboptimally activated T lymphocytes, regardless of their polarized or unpolarized functional profiles. This observation may help to explain the absolute predominance of R5 HIVs over X4 viruses observed after vital transmission and during early-stage disease.