Cd7 8h8.1

Flow cytometry was performed using the following panel of lymphoid-associated monoclonal
antibodies: CD4 (RPA-T4), (DAKO Denmark), CD2 (RPA-2.10), CD3 (UCHT1), CD5 (L17F12), CD8
(SK1), CD25 (M-A251), and CD7 (8H8.1) (Beckman Coulter, Inc. CA, USA).
Human T-cell leukemia virus-I (HTLV-I) proviral integration into cell lines was performed
by an inspection agency (Special Reference Laboratory, Japan).
The origin of cell lines was determined by the fragment length comparison of the V-N-J
rearrangement portion of the T-cell receptor γ-chain gene.¹⁵ (link) The high-molecular-weight DNA was extracted and amplified by PCR
using three sets of primer mixtures labeled with fluorescent dyes. The primers used in
this analysis were as follows: Vγ (1–8) II 5’ACCAGGAGGGGAAGGCCCCACAG, Vγ9
5’GGAAAGGAATCTGGCATTCCG, Vγ10 5’AATCCGCAGCTCGACGCAGCA, Vγ 11 5’GCTCAAGATTGCTCAGGTGGG, Vγ12
5’CCTCTTGGGCACTGCTCTAAA, Jγ1/2 5’ACCTGTGACAACAAGTGTTGTTC(NED), Jγ P1/2
5’AGTTACTATGAGCT(T/C)TAGTCCC(6-FAM), Jγ P 5’TGTAATGATAAGCTTTGTTCC(HEX). The three mixtures
were Mixture I Vγ (1–8) II: Jγ1/2, JγP1/2, JγP, Mixture II Vγ9: Jγ1/2, JγP1/2, JγP, and
Mixture III Vγ10–12: Jγ1/2, JγP1/2, JγP. The PCR products of each mixture were analyzed
using an ABI PRISM 310 Genetic Analyzer (Life Technologies Corporation, Carlsbad, CA).

PBMCs or lymphocytes isolated from the colon or rectal mucosa were utilized for flow cytometry. Monoclonal antibodies against CD45 (clone J33; Beckman Coulter, Pasadena, CA, USA), CD3 (UCHT1, Beckman Coulter), CD4 (13B8.2; Beckman Coulter), CD8 (B9.11; Beckman Coulter), CD7 (8H8.1; Beckman Coulter), CD25 (B1.49.9; Beckman Coulter), CD30 (HRS4; Beckman Coulter), CD45RA (2H4; Beckman Coulter), CD56 (N901; Beckman Coulter), CD62L (DREG56; Beckman Coulter), CD127 (R34.34; Beckman Coulter), CCR4 (i.e., CD194; L291H4; BioLegend), HLADR (Immu-357; Beckman Coulter), and PD1 (CD279; PD1.3; Beckman Coulter) were employed. The immunostained cells were analyzed using FACScan (Navios flow cytometer, Beckman Coulter) and Kaluza analysis software (version 1.3; Beckman Coulter). Lymphocytes were separated by flow cytometry based on high CD45 antigen expression and forward and side scatter properties. Subsequently, the flow cytometry data were analyzed according to the percentage of cell populations detected in each quadrant on two-dimensional scatterplots. We calculated the percentages of CD4⁺, CD8⁺, CD56⁺, CD7⁺, PD1⁺, CCR4⁺, CD30⁺, and HLADR⁺ cells among CD3⁺ cells. We also assessed the percentages of Treg, CD45RA⁺, and CD62L⁺ cells among CD3⁺CD4⁺ cells and percentages of CD45RA⁺ and CD62L⁺ cells among CD3⁺CD4⁻ cells. In this study, we defined CD3⁺CD4⁺CD25⁺CD127^low/- cells as Treg cells.