Subjects and preparation of immune cells
Eighteen patients (5 men, 13 women, age range 17-60 years) were recruited in the Hassan II University Hospital of Fes (Morocco) among patients with cervical lymphadenitis. Active LNTB was diagnosed by history, physical examination, and lab studies by experienced clinicians. The diagnosis of LNTB was based on a combination of clinical symptoms, pathology and response to TB drug therapy. Clinical symptoms associated with lymphadenitis included local lymphadenopathy, weight loss, fever, sweats, and anorexia. Histopathological evidence consisted of the presence of a granulomatous lesion with caseation in excisional biopsy specimens. Pulmonary radiography and HIV serology were performed to exclude pulmonary TB and HIV infection respectively. All LNTB cases were newly diagnosed and none had received anti-TB chemotherapy before sample collection. Tuberculin skin test results were positive (induration > 10 mm) for 15 out of 18 patients (83%). All patients were BCG vaccinated, and none reported contact with a case of pulmonary TB.
For all patients, the affected lymph node was in the neck and was surgically removed. In addition, 10 ml of peripheral blood was collected before starting anti-TB treatment. One portion of the lymph node was used for histological examination, and the other for isolation of lymph node mononuclear cells (LNMC) for immunologic studies.
Biopsy specimens were crushed gently in tissue culture medium. LNMC were spun and separated using Ficoll-Hypaque density centrifugation. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood under endotoxin-free conditions by Ficoll-Hypaque (SIGMA) density centrifugation. Cells were cryopreserved and stored in liquid nitrogen until shipment by a cryoshipper to Case Western Reserve University for immunological studies.
Phenotypic and functional study of T cells
PBMC and LNMC (106/ tube) were stimulated with a pool of 34 overlapping peptides from Mtb-antigen ESAT6/CFP10 at 6.25 ug/ml per peptide (New England peptide, Gardner, MA), M. tuberculosis CDC1551 whole cells lysate (Mtb lysate) (BEI Resources) or staphylococcal enterotoxin B (SEB, 2 μg/ml, Sigma) overnight at 37 °C in 5% CO2. Unstimulated PBMC and LNMC served as negative controls. Anti-CD28/CD49d (1 μg/ml each, eBioscience and Biolegend) was added to each tube during stimulation and brefeldin A (5 μg/ml, Sigma) was added 2 hr later. After stimulation, cells were washed with PBS and surface stained with anti-CCR7-PE-Cy7 (BD Bioscience) for 15 min at 37 °C, then live dead yellow (Invitrogen), anti-CD14-BV570, anti-CD4-APC/Cy7, anti-CD8-BV510 (all Biolegend) and anti-CD45RA-PE/TR (Invitrogen) were added and incubated at RT for 25 min. Afterward cells were washed, permeabilized (Cytofix/Cytoperm Kit, BD Pharmingen) according to the manufacturer’s instructions and stained for intracellular expression with anti-CD3-PerCP, anti-IFNγ-Alexa700, anti-IL2-APC, anti-TNFα- Pacific Blue, anti-IL17-BV711 (all Biolegend) and anti-MIP-1α-FITC (R&D). Cells were then washed, fixed in 1% paraformaldehyde and 1x106 total events collected from each sample on an LSR-II flow cytometer (BD). Net responding cells for each cytokine were calculated by subtracting the no antigen condition (medium only) from the antigen simulated result. The analysis of all functional markers expressed after stimulation were done on viable total memory CD4+ and CD8+ T cells. Memory phenotype was determined by CCR7 and CD45RA expression. Naïve T cells were CD45RA+/CCR7+, central memory (CM) cells were CD45RA-/CCR7+, effector memory (EM) cells were CD45RA-/CCR7-and effector cells (E) were CD45RA+/CCR7-.
To identify Tregs and activated T cells, PBMC and LNMC were surface stained with live dead yellow (Invitrogen), anti-CD3-APC/Cy7, anti-CD4-PerCP, anti-CD8-Alexa700, anti-CD25-APC, anti-HLA-DR-FITC, anti-CD38-PE/Cy7(all Biolegend), and anti-CD127-BV650(BD Bioscience), and incubated at RT for 25 min. After, cells were washed, permeabilized and intracellular stained with, anti-FoxP3-PE (eBioscience) or isotype-matched negative control for gating purposes.
Plots were analyzed using FlowJo software (version 6.1.1; Tree Star, Ashland, OR, USA). Boolean analysis on Flow Jo and SPICE (NIAID, NIH, USA) was used to assess cytokine polyfunctionality.
Statistical analysis
Statistical analysis was performed using the paired Student’s t test for comparisons of PBMC and LNMC. P value for comparison of polyfunctional profile was done using SPICE software. Significance of correlations was analyzed by the nonparametric Spearman test. A p value of < 0.05 was considered significant. Data were analyzed by Statistical Package for the Social Sciences (SPSS) software (IBM) and the GraphPad Prism software, version 5.00 for Windows (GraphPad Software, San Diego).