Treatment with IL-7 Prevents the Decline of Circulating CD4+ T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

Although treatment with interleukin-7 (IL-7) was shown to transiently expand the naïve and memory T-cell pools in patients with chronic HIV-1 infection receiving antiretroviral therapy (ART), it is uncertain whether a full immunologic reconstitution can be achieved. Moreover, the effects of IL-7 have never been evaluated during acute HIV-1 (or SIV) infection, a critical phase of the disease in which the most dramatic depletion of CD4+ T cells is believed to occur. In the present study, recombinant, fully glycosylated simian IL-7 (50 µg/kg, s.c., once weekly for 7 weeks) was administered to 6 rhesus macaques throughout the acute phase of infection with a pathogenic SIV strain (mac251); 6 animals were infected at the same time and served as untreated controls. Treatment with IL-7 did not cause clinically detectable side effects and, despite the absence of concomitant ART, did not induce significant increases in the levels of SIV replication except at the earliest time point tested (day 4 post-infection). Strikingly, animals treated with IL-7 were protected from the dramatic decline of circulating naïve and memory CD4+ T cells that occurred in untreated animals. Treatment with IL-7 induced only transient T-cell proliferation, but it was associated with sustained increase in the expression of the anti-apoptotic protein Bcl-2 on both CD4+ and CD8+ T cells, persistent expansion of all circulating CD8+ T-cell subsets, and development of earlier and stronger SIV Tat-specific T-cell responses. However, the beneficial effects of IL-7 were not sustained after treatment interruption. These data demonstrate that IL-7 administration is effective in protecting the CD4+ T-cell pool during the acute phase of SIV infection in macaques, providing a rationale for the clinical evaluation of this cytokine in patients with acute HIV-1 infection

The CD8-derived chemokine XCL1/lymphotactin is a conformation-dependent, broad-spectrum inhibitor of HIV-1.

CD8+ T cells play a key role in the in vivo control of HIV-1 replication via their cytolytic activity as well as their ability to secrete non-lytic soluble suppressive factors. Although the chemokines that naturally bind CCR5 (CCL3/MIP-1α, CCL4/MIP- 1β, CCL5/RANTES) are major components of the CD8-derived anti-HIV activity, evidence indicates the existence of additional, still undefined, CD8-derived HIV-suppressive factors. Here, we report the characterization of a novel anti-HIV chemokine, XCL1/lymphotactin, a member of the C-chemokine family that is produced primarily by activated CD8+ T cells and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that XCL1 inhibits a broad spectrum of HIV-1 isolates, irrespective of their coreceptor-usage phenotype. Experiments with stabilized variants of XCL1 demonstrated that HIV-1 inhibition requires access to the alternative, all-β conformation, which interacts with proteoglycans but does not bind/activate the specific XCR1 receptor, while the classic XCL1 conformation is inactive. HIV-1 inhibition by XCL1 was shown to occur at an early stage of infection, via blockade of viral attachment and entry into host cells. Analogous to the recently described anti-HIV effect of the CXC chemokine CXCL4/PF4, XCL1-mediated inhibition is associated with direct interaction of the chemokine with the HIV-1 envelope. These results may open new perspectives for understanding the mechanisms of HIV-1 control and reveal new molecular targets for the design of effective therapeutic and preventive strategies against HIV-1

Identification of the platelet-derived chemokine CXCL4/PF-4 as a broad-spectrum HIV-1 inhibitor

The natural history of HIV-1 infection is highly variable in different individuals, spanning from a rapidly progressive course to a longterm asymptomatic infection. A major determinant of the pace of disease progression is the in vivo level of HIV-1 replication, which is regulated by a complex network of cytokines and chemokines expressed by immune and inflammatory cells. The chemokine system is critically involved in the control of HIV-1 replication by virtue of the role played by specific chemokine receptors, most notably CCR5 and CXCR4, as cell-surface coreceptors for HIV-1 entry; hence, the chemokines that naturally bind such coreceptors act as endogenous inhibitors of HIV-1. Here, we show that the CXC chemokine CXCL4 (PF-4), the most abundant protein contained within the α-granules of platelets, is a broad-spectrum inhibitor of HIV-1 infection. Unlike other known HIV-suppressive chemokines, CXCL4 inhibits infection by the majority of primary HIV-1 isolates regardless of their coreceptor-usage phenotype or genetic subtype. Consistent with the lack of viral phenotype specificity, blockade of HIV- 1 infection occurs at the level of virus attachment and entry via a unique mechanism that involves direct interaction of CXCL4 with the major viral envelope glycoprotein, gp120. The binding site for CXCL4 was mapped to a region of the gp120 outer domain proximal to the CD4-binding site. The identification of a platelet-derived chemokine as an endogenous antiviral factor may have relevance for the pathogenesis and treatment of HIV-1 infection

The inhibitory structure of XCL1 against HIV-1 is the all-β, alternatively-folded conformation.

<p>Dose-dependent inhibition of an XCL1-sensitive primary HIV-1 isolate (92HT599; X4/R5) by recombinant wild type (WT) XCL1 (green bars), and two stabilized structural variants: W55D, a variant preferentially adopting the all-β/alternatively-folded structure (purple bars), and CC3, a variant locked in the classical chemokine-folded structure (light blue bars). Virus replication was assessed by measuring the amount of p24 Gag antigen in PBMC supernatants via AlphaLISA immunoassay. Data were normalized to the amount of viral replication observed in control cultures (not treated with XCL1). Data represent the mean values (±SD) of replicate wells, representative of at least 3 independent experiments performed on separate PBMC donors.</p

XCL1 blocks HIV-1 attachment and entry into target cells.

<p>Both attachment and entry assays were performed on TZM-bl cells incubated with the XCL1-sensitive dual tropic HIV-1 strain, 92HT599. (A) Virus attachment was measured as the total amount of cell-associated p24 Gag protein after 4 h incubation with virus at 37°C and subtraction of background levels. Background attachment/entry was quantified as the cell-associated p24 Gag after incubation of cells with virus at 4°C and subsequent trypsin treatment. (B) Virus entry assay was performed in a similar manner except cells were incubated with virus for 2 additional hours (6 h total) and trypsinized to remove extracellular-associated virus. Cells were lysed and virus entry was determined as the total amount of trypsin-resistant, intracellular p24 protein. Recombinant cytokines were used at 15 µg/mL, the T-20 fusion inhibitor at 50 µg/mL, and anti-CD4 antibody at 20 µg/mL. The data represent mean values (±SD) of replicate wells from at least 3 independent experiments.</p

XCL1 captures native HIV-1 virions and interacts directly with the external viral envelope glycoprotein, gp120.

<p>(A) The virion capture assay was performed using immunomagnetic beads armed with 3 different XCL1 variants (WT in green bars, W55D in purple bars, and CC3 in light blue bars) as molecular baits to capture whole HIV-1 virions (strain IIIB; X4). The specificity of the interaction was demonstrated upon pre-incubation of XCL1-armed beads (prior to virus addition) with 20 µg/mL of monoclonal (mAb) and polyclonal (pAb) anti-XCL1 antibodies. (B) Co-immunoprecipitation of the HIV-1 envelope glycoprotein gp120 observed with XCL1 WT and W55D, but not CC3. From left to right: lane 1 showing a negative-control (unconjugated XCL1 WT plus gp120); lane 2 with biotinylated XCL1 WT and gp120 co-precipitated; lane 3 with biotinylated XCL1 W55D and gp120 co-precipitated; lane 4 with no co-precipitation of biotinylated XCL1 CC3 and gp120; lane 5 with complete inhibition of XCL1 WT-gp120 co-precipitation in the presence of anti-XCL1 pAb; and lane 6 with IgG control showing minimal inhibition on XCL1-gp120 co-precipitation.</p

XCL1 does not interact with or inhibit VSV-G-pseudotyped virus.

<p>(A) Virion capture was performed using anti-mouse immunomagnetic beads (M-Beads) armed with anti-VSV-G mAb (Anti-VSV) or anti-rabbit immunomagnetic beads (R-Beads) armed with XCL1 WT (XCL1). Equal amounts of VSV-G-pseudotyped virus were added to all bead reactions. (B) Infection of PBMC with GFP-expressing VSV-G-pseudotyped virus was not affected by a dose-response treatment of XCL1 WT. Virus infection was quantified by the number of infected cells (GFP-positive) counted from the total PBMCs harvested from each well.</p

XCL1 antiviral activity is equally effective before and after digestion of glycosaminoglycans on PBMC.

<p>PBMC were incubated in the presence (‘Heparitinase-treated’) or absence (‘Untreated’) of heparitinase to digest cell surface glycosaminoglycan expression, followed by infection with HIV-1 IIIB (A) or BaL (B) in the presence of XCL1 WT (green bars), W55D (purple bars) or CC3 (light blue bars) at 1 µM. Virus replication was assessed by measuring the amount of p24 Gag antigen in PBMC supernatants via AlphaLISA immunoassay. Data were normalized to the amount of viral replication observed in control cultures (not treated with XCL1). Results represent the mean values (±SD) of replicate wells.</p