Gene transfer of two entry inhibitors protects CD4⁺ T cell from HIV-1 infection in humanized mice.

Targeting viral entry is the most likely gene therapy strategy to succeed in protecting the immune system from pathogenic HIV-1 infection. Here, we evaluated the efficacy of a gene transfer lentiviral vector expressing a combination of viral entry inhibitors, the C46 peptide (an inhibitor of viral fusion) and the P2-CCL5 intrakine (a modulator of CCR5 expression), to prevent CD4⁺ T-cell infection in vivo. For this, we used two different models of HIV-1-infected mice, one in which ex vivo genetically modified human T cells were grafted into immunodeficient NOD.SCID.γc⁻/⁻mice before infection and one in which genetically modified T cells were derived from CD34⁺ hematopoietic progenitors grafted few days after birth. Expression of the transgenes conferred a major selective advantage to genetically modified CD4⁺ T cells, the frequency of which could increase from 10 to 90% in the blood following HIV-1 infection. Moreover, these cells resisted HIV-1-induced depletion, contrary to non-modified cells that were depleted in the same mice. Finally, we report lower normalized viral loads in mice having received genetically modified progenitors. Altogether, our study documents that targeting viral entry in vivo is a promising avenue for the future of HIV-1 gene therapy in humans.

C-terminal cleavage of human Foxp3 at a proprotein convertase motif abrogates its suppressive function.

Foxp3 plays a critical role in the development and function of regulatory T cells (Tregs). Differences in translational and post-translational processing of murine and human Foxp3 have been recently reported. Human Foxp3 exists as four isoforms generated by alternative splicing. Mouse Foxp3 only exists as one isoform, but can be proteolytically cleaved by N-terminal and/or C-terminal proprotein convertase subtilisin/kexins (PCSKs). Here, we show by transcriptome analysis that the proprotein convertases PCSK7, PCSK5 and Furin are present in human CD4(+) T cells with different expression patterns. Notably, after in vitro activation, only PCSK7 and Furin are expressed in Tregs and T effector cells (Teffs), with overexpression of PCSK7 in Tregs compared to Teffs. Human Foxp3 protein displays specific motifs that can be potentially cleaved by convertases. Consequently, we transduced human CD4(+) cells with Foxp3-expressing lentiviral vectors and assessed the generation of proteolytically cleaved Foxp3 forms by Western blot. Three different Foxp3 forms were detected, indicating that human Foxp3 can also be subjected to proteolytic cleavage at the N-terminal and C-terminal ends. These results prompted us to assess the suppressive activity associated with each forms. We observed that full length and N-cleaved Foxp3-transduced CD4(+) T cells similarly suppressed the in vitro proliferation of Teffs. However, the C-cleaved or N&C-cleaved Foxp3 forms afforded almost no suppressive function, indicating a crucial role of the human Foxp3 C-terminal region in Tregs suppressive activity, in marked contrast with the report of a superior suppressive activity for the C-cleaved murine Foxp3 compared to the full length.

Antigen quality determines the efficiency of antitumor immune responses generated in the absence of regulatory T cells.

The observation that depletion or inhibition of regulatory T cells (Tregs) unleashes efficient antitumor effector immune responses that can lead to tumor eradication in mice has opened new perspectives for the development of cancer immunotherapy. The quality and overall efficiency of the effector immune responses induced in the absence of Tregs seem to depend on multiple factors that determine the result of a battle involving effector T cells (Teffs), Tregs and tumor cells. In this study, we investigated the quality of tumor-associated antigens (TAAs) as one such factor. We show that the presence of a strong dominant antigen is required for the induction of effector responses capable of tumor eradication in the absence of Tregs. The sole addition of a dominant antigen on tumor cells does not change tumor growth in unmanipulated mice, but improves tumor eradication rate from a few to almost 100% in the absence of Tregs. This eradication can be shown to result from the recruitment and activation of specific Teffs recognizing this antigen. We also show that the presence of such dominant antigens has the side effect of restricting the breadth of the immune response to other TAAs, which could favor the generation of escape mutant by tumor editing. Taken together, our results highlight the potential, and some requirements for cancer immunotherapy based on Treg depletion. They also show that, ultimately, tumor fate depends on multiple factors that should all be taken into consideration for the design of more efficient immunotherapy.

Identification and isolation of a full-length clone of mouse GMFB (Gmfb), a putative intracellular kinase regulator, differentially expressed in telencephalon.

We identified new transcribed sequences, using a differential display paradigm to select genes expressed in proliferating neuroblasts from mouse telencephalon at 10 days of embryonic development. In this systematic search, we isolated a 361-bp partial 3' untranslated region (3' UTR) homologous to the 3' UTR of the human gene encoding a putative intracellular kinase regulator, glia maturation factor beta (GMFB). We cloned a full-length, 4,311-bp mouse cDNA containing a 270-bp 5' UTR, a 3,615-bp 3' UTR, and an open reading frame of 426 nucleotides encoding a putative 142 amino-acid protein, identical to human GMFB, with the exception of two amino acids. This 4.3-kb transcript is present in a variety of adult tissues and is developmentally regulated as shown by Northern blot analysis. Differential expression in telencephalon was demonstrated by quantification of radioactive relative RT-PCR and confirmed by in situ hybridization. The isolation of this full-length clone of mouse Gmfb should facilitate investigation of the intracellular mechanisms involved in the development of telencephalon.

Impaired ventilatory responses to hypoxia in mice deficient in endothelin-converting-enzyme-1.

Endothelin-converting-enzyme (ECE-1) catalyzes the proteolytic activation of big endothelin-1 to mature endothelin-1. Most homozygous ECE-1-/- embryos die in utero and show severe craniofacial, enteric, and cardiac malformations precluding ventilatory function assessment. In contrast, heterozygous ECE-1+/- embryos develop normally. Their respiratory function at birth has not been studied. Taking into account previous respiratory investigations in mice with endothelin-1 gene disruption, we hypothesized that ECE-1-deficient mice may have impaired ventilatory control. We analyzed ventilatory responses to hypercapnia (8% CO(2)) and hypoxia (10% O(2)) in newborn and adult mice heterozygous for ECE-1 deficiency (ECE-1+/-) and in their wild-type littermates (ECE-1+/+). Ventilation, breath duration, and tidal volume were measured using whole-body plethysmography. Ventilatory responses to hypoxia were significantly weaker in ECE-1+/- than in ECE-1+/+ newborn mice (percentage ventilation increase: 1 +/- 25% versus 33 +/- 29%, p = 0.010). Baseline breathing variables and ventilatory responses to hypercapnia were normal in the ECE-1+/- newborn mice. No differences were observed between adult ECE-1+/- and ECE-1+/+ mice. We conclude that ECE-1 is required for normal ventilatory response to hypoxia at birth.

MASH-1/RET pathway involvement in development of brain stem control of respiratory frequency in newborn mice.

Respiratory abnormalities have been described in MASH-1 (mammalian achaete-scute homologous gene) and c-RET ("rearranged during transfection") mutant newborn mice. However, the neural mechanisms underlying these abnormalities have not been studied. We tested the hypothesis that the MASH-1 mutation may impair c-RET expression in brain stem neurons involved in the control of breathing. To do this, we analyzed brain stem c-RET expression and respiratory phenotype in MASH-1 +/+ wild-type, MASH-1 +/- heterozygous, and MASH-1 -/- knock-out newborn mice during the first 2 h of life. In MASH-1 -/- newborns, c-RET gene expression was absent in the noradrenergic nuclei (A2, A5, A6, A7) that contribute to modulate respiratory frequency and in scattered cells of the rostral ventrolateral medulla. The c-RET transcript levels measured by quantitative RT-PCR were lower in MASH-1 -/- and MASH-1 +/- than in MASH-1 +/+ brain stems (P = 0.001 and P = 0.003, respectively). Breath durations were shorter in MASH-1 -/- and MASH-1 +/- than in MASH-1 +/+ mice (P = 0.022) and were weakly correlated with c-RET transcript levels (P = 0.032). Taken together, these results provide evidence that MASH-1 is upstream of c-RET in noradrenergic brain stem neurons important for respiratory rhythm modulation.

Cloning and expression analysis of a novel gene, RP42, mapping to an autism susceptibility locus on 6q16.

We isolated a novel mouse gene, RP42, in a systematic search for genes expressed in proliferating neuroblasts whose human orthologs map to susceptibility loci for autism. This gene is intronless and encodes a putative 259-amino-acid protein that exhibits 30-36% overall sequence identity to a fission yeast and a nematode protein (GenPept Accession Nos. CAA17006 and CAB54261). Nevertheless, no homology to any known gene was found. RP42 has developmentally regulated expression, particularly in proliferating neuroblasts from which neocortical neurons originate. Its human ortholog is located in a cluster of embryonic neuronally expressed genes on the 6q16 chromosome, making it a positional candidate susceptibility gene for autism.

Developmental regulation of replication fork pausing in Xenopus laevis ribosomal RNA genes.

In early Xenopus embryos, replication forks move along the rRNA genes (rDNA) at a uniform rate and terminate at multiple, apparently random sites. In contrast, a polar replication fork barrier (RFB) is found at the 3' end of the rRNA genes in Xenopus cultured cells. We have now analysed the replication intermediates of Xenopus rDNA from a wide range of developmental stages by 2D gel electrophoresis. Surprisingly, up to 15 different replication fork pausing sites (RFPs) simultaneously appear in the rDNA at the midgastrula stage, when rRNA transcription abruptly increases. They disappear during the neurula stage, except for a polar RFP at the 3' end of Xthe transcription unit, which persists to the tadpole stage. The latter RFP is found at the same location as the RFB in cultured cells; however the arrest of replication forks at this RFP is not absolute, since termination occurs at multiple positions throughout the rDNA repeat. The efficiency of fork arrest at this RFP remains constant from midgastrula to early tadpole, and decreases around hatching. The transient appearance of multiple RFPs at midgastrula may reflect some chromatin remodeling associated with developmental activation of rRNA transcription.