Examination of Tregs
Total Tregs (CD4+CD25+FOXP3highcells) and activated Tregs (CD4+CD25+FOXP3highCD45RAcells) were analyzed by four-color flow cytometric analysis. We used fluorescein isothiocyanate (FITC) conjugated anti-CD4 antibody (Cat. 555346; BD Pharmingen), phycoerythrin (PE) conjugated anti-CD25 antibody (Cat. 555432; BD Pharmingen), Alexa Fluor 647 conjugated anti-FOXP3 antibody (Cat. 560045; BD Pharmingen), and VioBlue conjugated anti-CD45RA antibody (Cat. 130-113-360; Miltenyi Biotec) for cell staining. One million PBMCs were initially stained with the FITC-CD4 antibody, PE-CD25 antibody, and V450-CD45RA antibody for 20 min. Subsequently, intracellular detection of FOXP3 was performed on fixed and permeabilized cells using the Human FOXP3 Buffer Set (Cat. 560098; BD Pharmingen) according to the protocol provided by the manufacturer. After staining with Alexa Fluor 647 conjugated anti-FOXP3 antibody for 20 min, PBMCs were washed twice with PBS containing 2% fetal bovine serum (FBS). More than 20,000 CD4+ T cell events were analyzed using a FACS Verse flow cytometer and the FACSuite software (BD Biosciences, San Jose, CA, USA).
Analysis of the TCR- Vβ repertoire
We evaluated the usages of the TCR-Vβ family of T cell subsets through five-color flow cytometric analysis. For the separation of T cell subsets (CD4+ T cell, CD4+CD25 T cell, CD4+CD25+CD127lowT cell), we used PerCP/Cy5.5 conjugated anti-CD4 antibody (Cat. 217428; Biolegend), V450 conjugated anti-CD25 antibody (Cat. 560355; BD Horizon), and allophycocyanin (APC) conjugated anti-CD127 antibody (Cat. 351316; Biolegend). TCR Vβ staining was determined using the IOTest Beta Mark TCR Repertoire Kit® (Beckman Coulter, Marseille, France). This kit consists of monoclonal antibodies designed to identify 24 distinct TCR-Vβ families, covering approximately 70% of the normal human CD4+ T cells. Each set consisted of three different anti-Vβ family-specific monoclonal antibodies labeled with FITC, PE, or both. Thawed PBMCs obtained from each patient and control subject were washed with cold PBS containing 2% FBS, and 5 × 105PBMCs were stained for surface antigens in room temperature for 60 min in the dark. After staining, PBMCs were washed thrice with PBS containing 2% FBS. At least 5,000 CD4+CD25+CD127lowT cell events were collected for analysis.
Furthermore, we calculated the means and standard deviations (SD) of TCR-Vβ family usage using the control group as standard to compare difference in the usages of TCR-Vβ families between patients with AIN and control subjects (Table II ). The increased/decreased Vβ subfamilies were defined for each TCR-Vβ family as above the mean +2 SD or below the mean −2 SD. The increased or decreased numbers indicated the numbers of Vβ families exceeding the limits of normal values of the TCR-Vβ family (mean +2 SD or mean –2 SD). We created heat maps of TCR-Vβ usages of conventional T cells and Tregs among patients and control subjects. In those heat maps, pink/red and pale green/green colors indicate values exceeding the upper (> +2 SD / > +3 SD, respectively) and lower (< −2 SD / < −3 SD, respectively) limits of the normal value of the TCR-Vβ family.