CXCR4 is a chemokine receptor that plays key roles with its specific ligand, CXCL12, in stem cell homing and immune trafficking. It is also used as a coreceptor by some HIV-1 strains (X4 strains), whereas other strains (R5 strains) use an alternative coreceptor, CCR5. X4 strains mainly emerge at late stages of the infection and are linked to disease progression. Two isoforms of this coreceptor have been described in humans: CXCR4-A and CXCR4-B, corresponding to an unspliced and a spliced mRNA, respectively. In this study, we show that CXCR4-B, but not CXCR4-A, mediates an efficient HIV-1 X4 entry and productive infection. Yet, the chemotactic activity of CXCL12 on both isoforms was similar. Furthermore, HIV-R5 infection favored CXCR4-B expression over that of CXCR4-A. In vitro infection with an R5 strain increased CXCR4-B/CXCR4-A mRNA ratio in PBMCs, and this ratio correlated with HIV RNA plasma level in R5-infected individuals. In addition, the presence of the CXCR4-B isoform favored R5 to X4 switch more efficiently than did CXCR4-A in vitro. Hence, the predominance of CXCR4-B over CXCR4-A expression in PBMCs was linked to the ability of circulating HIV-1 strains to use CXCR4, as determined by genotyping. These data suggest that R5 to X4 switch could be favored by R5 infection–induced overexpression of CXCR4-B. Finally, we achieved a specific small interfering RNA–mediated knockdown of CXCR4-B. This represents a proof of concept for a possible gene-therapeutic approach aimed at blocking the HIV coreceptor activity of CXCR4 without knocking down its chemotactic activity.

Altogether these data indicate that the presence of S1P1 facilitates HIV-1 replicative cycle by boosting viral genome transcription, S1P1 antagonists have anti-HIV effects and S1P1 agonists are HIV latency reversing agents.

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In an article in this issue, Pilch-Cooper et al report an artifact they observed when they tried to detect intracellular CCR5 molecules by flow cytometry. 1 When they stained fixed and permeabilized cells with an anti-CCR5 antibody, they obtained a strong signal even in non-CCR5-expressing cells, with a significant off-target staining in the nucleus. They concluded that their procedure generated irrelevant antibody binding sites during the fixing and permeabilization resulting in a false-positive result and that T cells do not have large intracellular pools of CCR5. Whereas the first part of their statement is probably correct, the second one might not be.With an alternative procedure of intracellular labeling we have previously published, 2 we were able to detect CCR5 molecules in the peripheral blood CD4 ϩ T cells from a wild-type (WT)/WT CCR5 donor but not from a delta32(⌬32)/⌬32 CCR5 donor, the epitope recognized by the anti-CCR5 monoclonal antibody (mAb) 2D7 we used being disrupted in ⌬32 CCR5 molecules 3,4 ( Figure 1A). When we transduced the WT CCR5 gene into the ⌬32/⌬32 CD4 ϩ T cells, we detected a signal in permeabilized cells ( Figure 1B). To distinguish between surface and intracellular labeling, we first exposed these transduced CD4 ϩ T cells to a saturating concentration of the unlabeled anti-CCR5 mAb 2D7, permeabilized the cells or not, and thereafter stained them with the same mAb conjugated with a fluorescent dye. Under these conditions, the permeabilized cells were labeled, whereas the nonpermeabilized cells were not ( Figure 1C). Fluorescence microscopy confirmed that the staining was in the cytoplasm ( Figure 1D). Moreover, overnight treatment with brefeldin A, which inhibits transport of proteins from endoplasmic reticulum to Golgi, leading to protein accumulation inside the endoplasmic reticulum, increased the amount of intracellular CCR5 molecules that were stained (mean fluorescence intensity [MFI], 33 Ϯ 2 and 61 Ϯ 1, P Ͻ .001, without and with brefeldin A, respectively; n ϭ 3, Figure 1E). Finally, when we exposed circulating CD4 ϩ T cells to the CCR5 ligand MIP-1␤, we observed a decrease in the intensity of the labeling obtained with nonpermeabilized cells (MFI, 38 Ϯ 2 and 24 Ϯ 1, P ϭ .005, without and with MIP-1␤, respectively; n ϭ 3, Figure 1F), contrasting with an increase in the intensity of the labeling obtained with permeabilized cells pre-exposed to nonconjugated 2D7 (MFI, 121 Ϯ 8 and 190 Ϯ 3, P ϭ .008, without and with MIP-1␤, respectively; n ϭ 3, Figure 1F). These data are compatible with the ligand-induced internalization of cell surface CCR5 molecules.Our data argue for the presence of intracellular CCR5 in peripheral blood CD4 ϩ T cells. The reason we did not get the nonspecific binding reported by Pilch-Cooper et al may be because of differences in the method of permeabilization. Our duration of exposition to the detergent is shorter, and we used saponin instead of Triton X-100, which is known to affect proteins and to permeabilize nuclear membrane. 5 Moreover, we did not f...

NK cells play a major role in the antiviral immune response, including against HIV-1. HIV-1 patients have impaired NK cell activity with a decrease in CD56dim NK cells and an increase in the CD56−CD16+ subset, and recently it has been proposed that a population of CD56+NKG2C+KIR+CD57+ cells represents antiviral memory NK cells. Antiretroviral therapy (ART) partly restores the functional activity of this lymphocyte lineage. NK cells when interacting with their targets can gain antigens from them by the process of trogocytosis. Here we show that NK cells can obtain CCR5 and CXCR4, but barely CD4, from T cell lines by trogocytosis in vitro. By UMAP (Uniform Manifold Approximation and Projection), we show that aviremic HIV-1 patients have unique NK cell clusters that include cells expressing CCR5, NKG2C and KIRs, but lack CD57 expression. Viremic patients have a larger proportion of CXCR4+ and CCR5+ NK cells than healthy donors (HD) and this is largely increased in CD107+ cells, suggesting a link between degranulation and trogocytosis. In agreement, UMAP identified a specific NK cell cluster in viremic HIV-1 patients, which contains most of the CD107a+, CCR5+ and CXCR4+ cells. However, this cluster lacks NKG2C expression. Therefore, NK cells can gain CCR5 and CXCR4 by trogocytosis, which depends on degranulation.