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

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 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

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

Primary human CD8+ T cells secrete XCL1.

<p>CD8+ T cells were activated with three different stimuli for 3 days: PMA plus ionomycin, anti-CD3/CD28 loaded beads, or PHA. Cells were washed and culture supernatants were harvested after 5 and 7 days to quantify XCL1 secretion.</p