HIV-infected T cells are migratory vehicles for viral dissemination

After host entry through mucosal surfaces, HIV-1 disseminates to lymphoid tissues to establish a generalized infection of the immune system. The mechanisms by which this virus spreads among permissive target cells locally during early stages of transmission, and systemically during subsequent dissemination are not known1. In vitro studies suggest that formation of virological synapses (VSs) during stable contacts between infected and uninfected T cells greatly increases the efficiency of viral transfer2. It is unclear, however, if T cell contacts are sufficiently stable in vivo to allow for functional synapse formation under the conditions of perpetual cell motility in epithelial3 and lymphoid tissues4. Here, using multiphoton intravital microscopy (MP-IVM), we examined the dynamic behavior of HIV-infected T cells in lymph nodes (LNs) of humanized mice. We found that most productively infected T cells migrated robustly, resulting in their even distribution throughout the LN cortex. A subset of infected cells formed multinucleated syncytia through HIV envelope (Env)-dependent cell fusion. Both uncoordinated motility of syncytia as well as adhesion to CD4+ LN cells led to the formation of long membrane tethers, increasing cell lengths to up to 10 times that of migrating uninfected T cells. Blocking the egress of migratory T cells from LNs into efferent lymph, and thus interrupting T cell recirculation, limited HIV dissemination and strongly reduced plasma viremia. Thus, we have found that HIV-infected T cells are motile, form syncytia, and establish tethering interactions that may facilitate cell-to-cell transmission through VSs. While their migration in LNs spreads infection locally, T cell recirculation through tissues is important for efficient systemic viral spread, suggesting new molecular targets to antagonize HIV infection

Rectal Transmission of Transmitted/Founder HIV-1 Is Efficiently Prevented by Topical 1% Tenofovir in BLT Humanized Mice

Rectal microbicides are being developed to prevent new HIV infections in both men and women. We focused our in vivo preclinical efficacy study on rectally-applied tenofovir. BLT humanized mice (n = 43) were rectally inoculated with either the primary isolate HIV-1(JRCSF) or the MSM-derived transmitted/founder (T/F) virus HIV-1(THRO) within 30 minutes following treatment with topical 1% tenofovir or vehicle. Under our experimental conditions, in the absence of drug treatment we observed 50% and 60% rectal transmission by HIV-1(JRCSF) and HIV-1(THRO), respectively. Topical tenofovir reduced rectal transmission to 8% (1/12; log rank p = 0.03) for HIV-1(JRCSF) and 0% (0/6; log rank p = 0.02) for HIV-1(THRO). This is the first demonstration that any human T/F HIV-1 rectally infects humanized mice and that transmission of the T/F virus can be efficiently blocked by rectally applied 1% tenofovir. These results obtained in BLT mice, along with recent ex vivo, Phase 1 trial and non-human primate reports, provide a critically important step forward in the development of tenofovir-based rectal microbicides

Involvement of mTOR in CXCL12 Mediated T Cell Signaling and Migration

CXCL12 is a pleiotropic chemokine involved in multiple different processes such as immune regulation, inflammatory responses, and cancer development. CXCL12 is also a potent chemokine involved in chemoattraction of T cells to the site of infection or inflammation. Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that modulates different cellular processes, such as metabolism, nutrient sensing, protein translation, and cell growth. The role of mTOR in CXCL12-mediated resting T cell migration has yet to be elucidated.Rapamycin, an inhibitor of mTOR, significantly inhibits CXCL12 mediated migration of both primary human resting T cells and human T cell leukemia cell line CEM. p70(S6K1), an effector molecule of mTOR signaling pathway, was knocked down by shRNA in CEM cells using a lentiviral gene transfer system. Using p70(S6K1) knock down cells, we demonstrate the role of mTOR signaling in T cell migration both in vitro and in vivo.Our data demonstrate a new role for mTOR in CXCL12-induced T cell migration, and enrich the current knowledge regarding the clinical use of rapamycin

Altered DNA Methylation in Leukocytes with Trisomy 21

The primary abnormality in Down syndrome (DS), trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq) confirmed strong differences in methylation (p<0.0001) for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2′deoxycytidine (5aza-dC) plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells

3D Profile-Based Approach to Proteome-Wide Discovery of Novel Human Chemokines

Chemokines are small secreted proteins with important roles in immune responses. They consist of a conserved three-dimensional (3D) structure, so-called IL8-like chemokine fold, which is supported by disulfide bridges characteristic of this protein family. Sequence- and profile-based computational methods have been proficient in discovering novel chemokines by making use of their sequence-conserved cysteine patterns. However, it has been recently shown that some chemokines escaped annotation by these methods due to low sequence similarity to known chemokines and to different arrangement of cysteines in sequence and in 3D. Innovative methods overcoming the limitations of current techniques may allow the discovery of new remote homologs in the still functionally uncharacterized fraction of the human genome. We report a novel computational approach for proteome-wide identification of remote homologs of the chemokine family that uses fold recognition techniques in combination with a scaffold-based automatic mapping of disulfide bonds to define a 3D profile of the chemokine protein family. By applying our methodology to all currently uncharacterized human protein sequences, we have discovered two novel proteins that, without having significant sequence similarity to known chemokines or characteristic cysteine patterns, show strong structural resemblance to known anti-HIV chemokines. Detailed computational analysis and experimental structural investigations based on mass spectrometry and circular dichroism support our structural predictions and highlight several other chemokine-like features. The results obtained support their functional annotation as putative novel chemokines and encourage further experimental characterization. The identification of remote homologs of human chemokines may provide new insights into the molecular mechanisms causing pathologies such as cancer or AIDS, and may contribute to the development of novel treatments. Besides, the genome-wide applicability of our methodology based on 3D protein family profiles may open up new possibilities for improving and accelerating protein function annotation processes

They Might Be Giants: Does Syncytium Formation Sink or Spread HIV Infection?

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