Detection of Foreign Antigen-specific CD4⁺Foxp3⁺Regulatory T Cells by MHC Class II Tetramer and Intracellular CD154 Staining

Abstract

The unrestricted population of CD4+Foxp3+ regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific CD4+Foxp3+ Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect CD4+Foxp3+ Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV GP66-77-specific CD4+ T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV GP66-77-specific CD4+ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV GP66-77-specific CD4+Foxp3+ Tregs using Foxp3GFP knock-in mouse, and found that LCMV GP66-77-specific CD4+Foxp3+ Tregs and polyclonal CD4+Foxp3+ Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific CD4+Foxp3+ Treg in various models.

Ginsenoside-Rp1-induced apolipoprotein A-1 expression in the LoVo human colon cancer cell line

“…Flow-cytometric analysis for PI staining was performed as described previously [14,15] . LoVo (10 6 ) cells were washed with PBS, fixed in ethanol, suspended in PI solution (1 mg/mL RNase A, 50 micro g/mL PI, and 0.1% Triton X-100 in 3.8mM sodium citrate) and incubated on ice for 30 minutes in the dark.…”

Major Histocompatibility Complex Class II Dextramers: New Tools for the Detection of antigen‐Specific, CD4 T Cells in Basic and Clinical Research

“…The creation of tetrameric reagents requires expression of soluble MHC molecules that represent the extracellular portions in each class of MHC molecule [7,8]. The presence of a single a-chain may offer some advantage in allowing MHC class I molecules to be expressed more [43][44][45][46][47][48][49][50][51][52][53][54][55][56] Detection of Myhc-a-specific T cells in EAM mice and CVB3-infected mice [11,15] IA s PLP 139-151 and TMEV [70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86] Detection of PLP-specific T cells ex vivo, in vitro and in situ in EAE mice [12,13,15] PLP 139-151 and ACA [83][84][85][86][87][88][89][90][91]…”