Recombinant il 2

HeLa and Caco-2 cells (ATCC, Rockville, MD) were cultured and/or differentiated as previously described [23] . Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors (New York Blood Center, Long Island City, NY) and the 3×3 stimulation was performed as previously described by Trkola et al [24] (link). After activation, PBMCs were grown in fresh stimulation media consisting of RPMI 1640 (Life Technologies), 10% FBS, antibiotics at a final concentration of 50 U/ml of penicillin, 50 µg/ml streptomycin and recombinant IL-2 at 20 U/ml (Roche, Indianapolis, IN).
L. jensenii, L. crispatus and C. albicans strain SC5413 (ATCC) were propagated as previously described [23] .

HSV-2 stocks were propagated in Vero cells (American Type Culture Collection [ATCC] Manassas, VA), titered by plaque formation on Vero cells, and aliquots stored at −80°C [41] . RMs vaginal biopsies were equalized in size using 3 mm skin biopsy punches (Fisher Scientific, Waltham, MA) and infected in duplicates or triplicates with 2×10⁷ pfu/mL of HSV-2 in absence or presence (uninfected control) of 125 µg/mL acyclovir (Calbiochem, Billerica, MA) for 3 hours in a 96 well plate. Tissues were extensively washed with PBS and placed into a hole in a 24-well 3 µm pore size and 6.5 mm diameter trans-well insert (Corning, NY) using 2 mm skin biopsy punches (Fisher Scientific), with the mucosa facing the upper chamber. After either 18 hours or 3 days the tissue was digested and the cellular phenotype was determined by multi-color flow cytometry (LSRII). After 18 hours (T cells) and 3 days (DCs), the cells that migrated to the bottom chamber were collected and the cellular phenotype was determined by multi-color flow cytometry. In the experiments with SHIV, each vaginal punch biopsy was infected with 3000 TCID₅₀ of SHIV_162p3 in the presence of 1 µg/mL PHA (Sigma-Aldrich, St. Louis, MO), and 50 U/mL recombinant IL-2 (Roche, South San Francisco, CA) after the 3 hour HSV-2 infection. After 18 hours, the tissues were extensively washed with PBS and cultured for an additional 48 hours.

Human NK cells were isolated from peripheral blood of healthy U.S. donors by negative selection (Stemcell Technologies). NK cells were resuspended in Iscove's modified Dulbecco's medium (IMDM; Gibco) supplemented with 10% human serum (Valley Biomedical) and used within 4 days. To obtain IL-2-activated NK cells, freshly isolated NK cells were co-cultured with irradiated autologous feeder cells in OpTimizer (Invitrogen) supplemented with 10% purified IL-2 (Hemagen), 100 units/mL recombinant IL-2 (Roche), and 5 μg/mL phytohemagglutinin (PHA, Sigma) and expanded in the same medium without PHA and feeder cells. The human erythroleukemia cell line K562 (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 supplemented with 2 mM L-glutamine and 10% fetal bovine serum (FBS; Atlanta Biologicals).

HEK293T cells and Jurkat T cells were obtained from American Type Culture Collection (ATCC). HeLa TZMbl were obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH, kindly provided by John C. Kappes. Primary CD4+ T cells were purified from freshly isolated PBMCs from healthy donors. PBMCs were isolated by density gradient using Lymphoprep (Axis-Shield) and CD4⁺ T cells purified by negative selection using the Dynabeads Untouched Human CD4⁺ T Cell Isolation kit (Invitrogen) according to the manufacturer’s instructions. CD4⁺ T cells were activated using Human T-Activator CD3/CD28 Dynabeads (Invitrogen) according to the manufacturer’s instructions and maintained in RPMI GlutaMax supplemented with 10% FCS and 30 U/mL recombinant IL-2 (Roche).