Cxcl10

Activated CD8⁺ T cells (2×10⁵ cells/200 μl) were suspended in RPMI supplemented with 0.1% BSA and placed in the upper chamber of a 6.5mm transwell insert displaying a 5 μm-pore size membrane (Corning, Corning, New-York). Conditioned medium recovered from tumor cells (LLC, B16K1, 4T1 cells) or from CD8⁺ T cells isolated from KO and WT spleens was added in the lower chamber. CXCL10 (100 nM, R×D Systems) present in the lower chamber of the Boyden Chamber was used as a positive control and migration of CD8⁺ T cells without chemoattractant was determined using serum-free RPMI medium. After 3 hours of incubation, CD8⁺ T cells, which migrated through the filter, were harvested and stained with anti-CD8a-PE antibody. CD8⁺ T cells were enumerated in each experimental condition during 1 minute by FACS CANTO II flow cytometer (BD Biosciences). Data were analyzed using BD FACSDiva software (BD Biosciences).

Proteins (20 μg) were separated on SDS-PAGE gels. Then, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). The PVDF membranes were then blocked with 5% BSA diluted in TBS for 1 h at room temperature. Primary antibodies against CXCL10 (R&D), RANKL (Abcam), Cadherin-11 (Invitrogen), mTOR (Abcam), phosphorylated mTOR (Abcam), CREB (Abcam), phosphorylated CREB (Abcam), AKT (Abcam), phosphorylated AKT (Abcam), PI3K (Abcam), phosphorylated PI3K (Abcam) and GAPDH (Abcam) were then added according to the manufacturers’ protocols. The samples were agitated at 4 °C overnight. HRP-conjugated rabbit anti-mouse IgG (Abcam), mouse anti-rabbit IgG (Abcam) and goat anti-mouse IgG (Abcam) secondary antibodies were added and incubated at room temperature for 2 h. Densitometric analysis was performed using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories).

Carrier-free, recombinant murine IFNβ (mIFNβ) produced in CHO cells was generously provided by PBL Assay Science (formerly PBL Interferon Source, Piscataway, NJ, cat# 12410). Recombinant human IFNβ (hIFNβ) originally from Avonex (Research Triangle Park, NC) was kindly provided by Dr. Sumit Chanda’s laboratory (Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA). Recombinant murine CCL4 was purchased from PeproTech (Rocky Hill, NJ, cat# 25032). Carrier-free, recombinant gp120 of the macrophage-tropic HIV-1 strain BaL was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, NIAID. All cytokines and recombinant gp120 were reconstituted in 0.1% BSA/PBS (at 100 x final concentration). Bacterial lipopolysaccharide (LPS) was purchased from Sigma (St. Louis, MO, cat# L6529) and was sonicated before use. Neutralizing antibodies against the following mouse proteins were purchased from R&D Systems (Minneapolis, MN) and reconstituted in sterile PBS: CCL3 (cat# AF450NA), CCL4 (cat# AF451NA), CCL5 (cat# AF478NA), CXCL10 (cat# AF466NA), IFNγ (cat# AF585NA). All antibodies were tested for neutralizing activity, function and endotoxin level by the manufacturer’s Quality Assurance program. All reagents were stored in aliquots according to the manufacturer’s recommendations at 4, −20 or −80 °C until use.

Secreted cytokines and chemokines from the stimulated cells were measured by ELISA kits (IL-6, eBiosciece™, 12364003; TNF-a, eBiosciece™, 155501117; CCL2, eBiosciece™, 15561137; CCL5, R&D system, DY478-05, CXCL10, R&D system, DY466-05) per manufacturers’ instructions. The culture media were stored at − 80 °C if they were not immediately used.

Cell-free supernatants from stimulated NHEKs (10⁶ cells per mL) were tested. The production of chemokines was detected using ELISA kits CXCL9 (Elabscience, Wuhan, China), and CXCL10 (R&D, USA) according to the corresponding manufacturer’s instructions.

We used ELISA kits (sandwich elisa) to measure the levels of mouse cytokines IL-6, IL12p70 (BD Biosciences, San Jose, CA, USA), and CXCL10 (R&D Systems, Minneapolis, MN, USA) in the supernatants of BMDC cultures stimulated for 24 h with TLR ligands, or medium alone, as per the manufacturer’s protocol.

Serum concentrations of the inflammation markers CRP, IL-6 and CXCL-10 and the microbial translocation and inflammation markers sCD14, LPS and sCD163 were assessed with ELISA. IL-6, LPS, CXCL-10 (R&D systems, Minneapolis, MN), sCD-14 and sCD-163 (Thermo Scientific, Waltham, MA) were measured according to the Dynex DS-2 system manufacturer’s protocol. High-sensitivity CRP levels were measured and determined using Cobas (Roche Diagnostics).
Missing data were completed from the patient chart and by searching the database of the national HIV-monitoring Foundation (SHM), which is the executive organization for the registration and monitoring of consenting participants with HIV-infection for care in the 27 Dutch HIV-treatment centres [42 ].