Healthy female participants were enrolled in the study at the Center for AIDS Research, Education, and Services clinic and at the Kaiser Permanente Clinic in Sacramento, CA. Male participants were enrolled from the same general geographical area as the female participants. The inclusion criteria included: age between 20 and 60, healthy, and able to participate in an upper GI endoscopy procedure with minimal risk. The exclusion criteria for all participants included: incidence of cardiovascular disease, diabetes, smoking, excessive alcohol consumption, use of illicit drugs or medications. Exclusion criteria specific to female participants was a peri- or post-menopausal state as defined by SWAN . Samples were obtained during the follicular phase of the menstrual cycle (4–7 days following last menstrual period). Menopause is associated with changes in mucosal epithelial function which can further augment observed gender based differences in immune function. Thus postmenopausal state was an exclusion criterion. Age also plays a role in immune responses and to minimize the effects of age all participants enrolled were under the age of 55. Over 40 potential participants were reviewed by the research team and 34 fulfilled the enrollment criteria. The study protocol was approved by the UC Davis Institutional Review Board. All participants provided written, signed informed consent. Twenty milliliters (mL) of blood was collected by venipuncture in EDTA-containing tubes and processed at each time point. Pinch biopsies were obtained by upper GI endoscopy from the jejunum under conscious anesthesia according to previously published protocols . All samples were collected between 7:00 AM and 9:00 AM at the UCD Gastroenterology Clinic, Sacramento, CA after overnight fasting.
Isolation of peripheral blood mononuclear cells (PBMC)
Whole blood was collected in EDTA-containing tubes and centrifuged for 20 minutes. The white blood cell buffy coat was collected, diluted in 1X PBS, layered over ficoll gradients (Atlanta Biological, Lawrenceville, GA), and centrifuged 20 minutes. The mononuclear cell layer was collected and pelleted by centrifugation. Cells were cultured overnight at 37°C, 5% C02 in culture media (RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, and 1% L-glutamine (Gibco) . Five to seven million PBMC were used for CD4+T cell isolation.
Isolation of intestinal lymphocytes
Jejunum biopsies were placed in isolation media (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin (Gibco, Grand Island, NY) and collagenase type IIb (1 mg/mL) (Sigma, St. Louis, MO)  and subjected to shaking at 37°C for four periods of 30 minutes. Cells were washed with RPMI media and rested overnight in culture media (RPMI 1640 supplemented with 15% FBS, 1% gentamycin, 100 U/mL penicillin, 100 U/mL streptomycin, 1% L-glutamine, 1% sodium pyruvate, 2.5% Hepes, and 0.5% amphotericin B (Gibco)) at 37°C with 5% CO2. Cells were filtered through a 40 μm filter the next day and counted. Three to four million lymphocytes were filtered the same day for further CD4+T cell isolation.
CD4+ T cell isolation
Isolated lymphocytes from the jejunum and peripheral blood were enriched for CD4+T cells by magnetic bead sort (Dynal, Carlsbad, CA). Magnetic beads pre-coated with CD8 antibody were incubated with isolated lymphocytes for 30 minutes at 4°C on a gentle rotating platform and CD8 lymphocytes were collected by magnet and incubated with CD13 coated magnetic beads. Pan-mouse IgG beads (Dynal) were first incubated for 30 minutes at 4°C on a rotating platform with monoclonal IgG CD13 antibody (BD, San Jose, CA), and then CD13 coated beads were incubated with CD8 depleted lymphocytes for 30 minutes at 4°C on a gentle rotating platform. The CD8- CD13- lymphocytes were collected by magnet, washed in culture media, and incubated overnight at 37°C/5%CO2 at a maximum concentration of 2 million cells/ml. Cells were pelleted the following day and stored in -80°C until use. Supernatant from overnight culture was also stored at -80°C until needed. Purity of PBMC (90-100%) and jejunal lymphocytes (65-85%) was determined by flow cytometry.
Isolated lymphocytes (1 million cells/tube) were incubated with Aqua (Amcyan) Live/Dead Dye (Invitrogen, Carlsbad, CA) (0.5 uL/mL 1XPBS + 1million cells). Lymphocytes were then washed with staining buffer (filter sterilized 1XPBS with 3% heat-inactivated FBS and 0.1% sodium azide). Cells were incubated with 10% normal mouse serum (Jackson Immunoresearch Laboratories, Inc) and then with fluorescent mouse anti-human monoclonal antibodies. CD3 (Beckton Dickinson, Mountain View, CA), CD45RA, CD45RO, CD8 (Invitrogen), CD95 (Biolegend, San Diego, CA), CD28, CD4, HLADR, Ki67 (E-Bioscience) antibodies were used. Cells were washed and fixed with 1% paraformaldehyde (PFA) (Sigma) prior to analysis on the LSR II flow cytometer (Beckton Dickinson at the UC Davis core facility). The cells were first gated based on negative staining with live dye, followed by a lymphocyte gate. These populations were further analyzed by specific staining. A minimum of 500,000 events was collected per sample and data was analyzed using Flow Jo (Tree Star, Inc. San Carlos, CA).
Isolated lymphocytes (~1 million cells) were stimulated with phorbol 12-myristate 13-acetate (PMA) (1 ng/mL) + Ionomycin (1 μM/mL) (Sigma), or negative control media alone at 37°C with 5% CO2 for 6 hours. Anti-CD28 pure monoclonal antibody (Ebioscience) was added to aid co-stimulation, followed by addition of Brefeldin A (Sigma) for the final 5 hours to prevent Golgi transport of cytokines out of cells. Cells were washed with 1X PBS/1%BSA, stained for surface markers, and fixed in 1% PFA. Fixative was washed out and cells were permeabilized with Caltag Permeabilization Solution B (Invitrogen) and stained with monoclonal antibodies IFNγ, IL-2, and TNF-α (BD, Ebioscience) for 20 minutes at room temperature. Samples were washed and fixed with 1% PFA before analysis with LSR II. A minimum of 500,000 events were collected for each sample and analyzed using Flow Jo software. Polyfunctional cell analysis was performed using SPICE (v4.1.6) and PESTLE (v1.5.4) software courtesy of Mario Roederer, Vaccine Research Center, NIAID/NIH, Bethesda, MD.
Gene expression profiles in gut biopsies from participants were analyzed using microarray technology and real-time PCR. In brief, total RNA was isolated from cryopreserved jejunal tissue utilizing protocols and reagents in the RNeasy RNA isolation kit (Qiagen). Messenger RNA amplification, labeling, hybridization to human whole-genome U95av2 GeneChips© (Affymetrix) staining and scanning were performed as previously described [21, 22] utilizing kits and protocols described in the Affymetrix Gene Expression Analysis Technical Manual. Microarray data were analyzed cross-sectionally amongst all groups utilizing the dChip (http://www.hsph.harvard.edu/cli/complab/dchip/) software program. A minimum fold-change of 1.5 (p-value ≤ 0.05) between groups was established as a cut-off criterion for statistical validation. Samples from male participants were used as baseline values for identifying differentially expressed genes (DEG) in samples from female participants. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) (http://www.ingenuity.com) software.
RNA was also isolated from CD4+ T cells enriched from PBMC as well as LPL using the same methods as in the microarray analysis. CDNA was synthesized using Superscript III (Invitrogen, CA) and used in Taqman© Real-Time PCR assay as previously described (Applied Biosystems, CA) [21, 22]. The data were normalized to the housekeeping gene GAPDH and calibrated to the average of the male samples from the same tissue source. The data were presented as a fold increase in RNA level compared to the calibrator .
Immunohistochemical analysis (IHC) was performed to detect and localize expression of IL17, TNFα, FoxP3 and IL1β in the small intestinal mucosa. Jejunal biopsies were embedded in paraffin blocks following fixation with 4% PFA. Tissue sections (5 μm) were incubated with polyclonal anti-IL17, anti-TNFα, anti-FoxP3 or anti-IL1β overnight at 4°C, followed by incubation with FITC-labeled secondary antibodies for 1 hour as previously described. Sections were mounted using SlowFade with Dapi (Invitrogen, Carlsbad, CA). Images were captured by confocal laser microscopy using LSM 5 and PASCAL software (Zeiss, New York) and then analyzed using Image J software, NIH, Maryland.
Data sets were analyzed using the two-tailed paired or unpaired t-test as applicable, taking into account the distribution of the data (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, http://www.graphpad.com). Due to the limited availability of samples to perform all the analyses, a selected number of samples were utilized in each assay over 75% overlap between most experiments. The real-time data were analyzed for statistically significant differences at the level of the calibrated values as well as at the level of the linearized fold change representing relative expression.