MAIT cell function is modulated by PD-1 signaling in patients with active tuberculosis Jing Jiang, M.D., Xinjing Wang, M.D., Hongjuan An, M.Sc., Bingfen Yang, Ph.D., Zhihong Cao, M.Sc., Yanhua Liu, Ph.D., Jinwen Su, M.D., Fei Zhai, B.S., Ruo Wang, M.Sc., Guangyu Zhang, M.D., Xiaoxing Cheng #, M.B., Ph.D. Online Data Supplement
Supplementary methods (1) Human subjects One hundred and twenty-three patients with active TB, including 93 patients with pulmonary TB, 26 with tuberculous pleurisy, and 4 with tuberculous peritonitis, were recruited (Table 1), and they were diagnosed according to the 1990 edition of Diagnostic Standards and Classification of Tuberculosis, published by the American Lung Association. All patients were HIV-negative. In the 93 patients with pulmonary TB, forty-nine were sputum smear/culture-positive and 44 were sputum smear/culture-negative (Table 1). For patients with negative sputum smear/culture results, diagnosis was established by clinical symptoms, chest computed tomography and pathological examination from biopsy samples obtained through bronchoscopy and was confirmed by symptomatic and radiographic improvement after anti-tb chemotherapy. In the cohort of 49 sputum-positive patients with pulmonary TB, 30 were defined as new cases and 19 as cases of treatment failure (Table 1). TB patients who were not treated for TB or had been treated for less than two weeks at the time of blood collection were considered new cases. Nineteen TB patients who were previously treated with a standard anti-tb chemotherapy regimen without success were defined as cases of treatment failure. Patients with tuberculous pleurisy and tuberculous peritonitis were diagnosed based on clinical manifestations, computed tomography, ultrasonography, and clinical E2
laboratory tests, including cellular and biochemistry examinations, bacterial culture, real-time PCR specific for M. tuberculosis complex, pleural effusion or ascitic fluid ELISPOT assay using a T-SPOT.TB kit (Oxford Immunotec, Oxfordshire, United Kingdom), adenosine deaminase (ADA), pathological examination from biopsy samples, and responses to anti-tb chemotherapy. Seventy-nine healthy controls were randomly recruited from individuals undergoing annual health check-ups at the clinics of 309 Hospital using following inclusion criteria: (1) no fever, cough, or other signs of active TB; (2) normal physical examination results and normal radiography; and (3) no HIV infection. (2) Antibodies and reagents Antibodies of anti-human TCR Vα7.2-FITC or -PE (clone 3C10), anti-cd161-apc or -PE-Cy7 (clone HP-3G10), anti-cd197 (CCR7)-PE-Cy7 (clone G043H7), anti-cd279 (PD-1)-FITC (clone EH12.2H7), anti-cd160-alexa Fluor 647 (clone BY55), anti-tim-3-pe (clone F38-2E2), LEAF purified anti-human CD279 (PD-1) (clone EH12.2H7), LEAF purified anti-human/mouse/rat MR1 (clone 26.5), and LEAF purified anti-human CD28 (clone CD28.2) were purchased from BioLegend (San Diego, CA, USA). Antibodies of anti-human CD3-PE-CF594 or PE-Cy7 (clone UCHT1), anti-cd45ra-pe-cy5 (clone HI100), anti-cd45ro-fitc (clone UCHL1), anti-cd69-fitc or -PE (clone FN50), anti-cd279 (PD-1)-FITC (clone MIH4), anti-tnf-α-fitc or -PE (clone MAb11), anti-cd244-pe (clone 2-69), anti-btla-pe (clone J168-540), anti-tcr γδ-fitc (clone B1), the E3
anti-ki-67-fitc-set (clone B56), the annexin V-FITC apoptosis detection kit I, and the cytofix/cytoperm fixation/permeabilization kit were obtained from BD Biosciences (San Diego, California, USA). Antibodies of anti-ifn-γ-fitc (clone 25723) and goat anti-lag-3-pe were obtained from R&D Systems (Minneapolis, MN, USA). Antibodies of anti-cd274-apc (PD-L1) (clone MIH1) and anti-cd273-apc (PD-L2) (clone MIH18) were from ebioscience (San Diego, CA, USA). Isotype-matched control antibodies were purchased from the corresponding suppliers mentioned above to determine the background level of staining. (3) Intracellular cytokine staining For intracellular cytokine staining, cells were collected, washed, and stained first with surface markers. After being permeabilized with cytofix/cytoperm fixation/permeabilization buffer (BD Biosciences), the cells were incubated with fluorochrome-labeled anti-ifn-γ or anti-tnf-α antibody for 30 minutes at 4 C. Appropriate isotype-matched control antibodies were used to determine background levels of staining. The expression of protein was analyzed by an FC-500 flow cytometer (Beckman Coulter, Brea, CA, USA). (4) Apoptosis assay PBMCs from both patients with active TB and healthy controls were incubated with 5 10 4 CFU/ml live BCG overnight. Cells were stained with FITC annexin V apoptosis detection kit I (BD Biosciences) by following the manufacturer s E4
instructions, and cell apoptosis was analyzed by flow cytometry. E5