Examination of Tregs
Total Tregs
(CD4+CD25+FOXP3highcells) and activated Tregs
(CD4+CD25+FOXP3highCD45RA−cells) 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.