Immune Netw.  2013 Dec;13(6):264-274. 10.4110/in.2013.13.6.264.

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

• Choi JY
• Eo SK

• KMID: 2150787
• DOI: http://doi.org/10.4110/in.2013.13.6.264

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.

Keyword

CD4+Foxp3+ regulatory T cells; MHC class II tetramer; Intracellular CD154 staining; Exhausted T cell

MeSH Terms

Animals
Arm
Autoimmune Diseases
Cytokines
Lymphocytic choriomeningitis virus
Mice
Plastics
T-Lymphocytes
T-Lymphocytes, Regulatory*
Cytokines
Plastics

Figure

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  Figure 1 Detection of LCMV Ag-specific CD4+Foxp3+ iTreg by MHC class II tetramer staining. (A) Diagram of Foxp3GFP and iTreg detection. CD4+Foxp3- T cells sorted from Foxp3GFP knock-in mice were stimulated with anti-CD3/CD28 in the presence or absence of TGF-β for 72 h. The conversion of CD4+Foxp3- Th cells to CD4+Foxp3+ iTregs was evaluated by the expression of GFP fused by Foxp3 molecule. (B) Detection of LCMV-specific CD4+Foxp3+ iTreg specific for three epitopes. Foxp3GFP knock-in mice that had been previously infected with LCMV Armstrong (Arms) were sacrificed to prepare the splenocytes 7 days pi, and the levels of CD4+ T cells specific for different epitopes of LCMV Ag were detected by each MHC class II tetramer staining (I-Ab/DIY, I-Ab/TSA, and I-Ab/TMF). MHC class II tetramer (I-Ab/CLIP) was used for negative control. (C) Opimization of condition to detect LCMV GP66-77-specific CD4+Foxp3+ iTreg. Staining conditions for MHC class II tetramer included I-Ab/CLIP, I-Ab/DIY (42 µg/ml), and I-Ab/DIY (70 µg/ml). After staining with MHC class II tetramer, cells were fixed, stained with anti-CD40 and F4/80 antibody. Some cells were stained by anti-CD4 and F4/80 antibody without fixation. The values in dot-plot denote the average percentage of detected CD4+I-Ab/DIY+ T cells after gating on F4/80-negative cells. (D) Detection of LCMV GP66-77-specific CD4+Foxp3+ iTregs by MHC class II tetramer staining. The splenocytes from LCMV-infected Foxp3GFP knock-in mice were prepared 7 days pi and used for MHC class II tetramer (I-Ab/DIY, 42 and 70 µg/ml) staining to detect CD4+Foxp3+ Treg cells specific for LCMV GP66-77 epitope. The values in dot-plot denote the average percentage of Foxp3+I-Ab/DIY+ gated on CD4+ T cells derived from three independent experiments.

• 

  Figure 2 Detection of LCMV-specific CD4+ T cells by intracellular CD154 staining. (A) Detection of viable LCMV-specific CD4+ T cells by intracellular CD154 staining. The splenocytes from mice infected with LCMV Arms or Cl13 were prepared 7 days pi and simulated with each specific epitope peptide (LCMV GP66-77, GP126-140, and GP6-20) in presence of PE-conjugated anti-CD154 antibody for 12 h. Splenocytes that was not stimulated with peptide in the presence of PE-conjugated anti-CD154 antibody were used for negative control. The values in dot-plot represent the percentage of CD4+ T cells specific for each LCMV epitope peptide. (B) The profile of IFN-γ and TNF-α expression in LCMV Ag-specific CD4+ T cells detected by intracellular CD154 staining. Following 12 h-stimulation of each epitope peptide in the presence of PE-conjugated CD154 antibody, the cells were stained with anti-CD4 antibody and the expression of IFN-γ and TNF-α in CD154+CD4+ T cells was determined by intracellular cytokine staining. The values in dot-plot represent the average percentage of IFN-γ and TNF-α in CD154+CD4+ T cells.

• 

  Figure 3 Detection of LCMV GP66-77-specific CD4+CD154+Foxp3+ iTreg. (A) Detection rate of LCMV GP66-77-specific CD4+ T cells in acute LCMV infection phase, depending on stimulation period. C57BL/6 mice were infected with LCMV Armstrong (Arms) or clone 13 (Cl13), and the spelenocytes were prepared 7 days pi and used for stimulation with LCMV GP66-77 epitope peptide in the presence of PE-conjugated anti-CD154 antibody for various periods. The values in dot-plot denote the percentage of CD4+CD154+ T cells specific for LCMV GP66-77 epitope peptide. (B) The frequency and absolute number of CD4+CD154+ T cells specific for LCMV GP66-77 epitope peptide in acute LCMV infection phase. The graphs represent the average percentage and absolute number of LCMV GP66-77-specific CD4+CD154+ T cells at the indicated stimulation time point. (C) Detection rate of LCMV GP66-77-specific CD4+ T cells in chronic LCMV infection phase, depending on stimulation period. C57BL/6 mice were infected with LCMV Armstrong (Arms) or clone 13 (Cl13), and the splenocytes were prepared 44 days pi and used for stimulation with LCMV GP66-77 epitope peptide in the presence of PE-conjugated anti-CD154 antibody for various periods. The values in dot-plot denote the percentage of CD4+CD154+ T cells specific for LCMV GP66-77 epitope peptide. (D) The frequency and absolute number of CD4+CD154+ T cells specific for LCMV GP66-77 epitope peptide in chronic LCMV infection phase. The graphs represent the average percentage and absolute number of LCMV GP66-77-specific CD4+CD154+ T cells at the indicated stimulation time point.

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  Figure 4 Differential expansion of LCMV GP66-77-specific CD4+Foxp3+ iTreg and CD4+Foxp3+ Treg in acute and chronic infection. (A) Frequency of LCMV GP66-77-specific CD4+CD154+Foxp3+ iTreg in acute and chronic LCMV infection. Foxp3GFP knock-in mice were infected with LCMV Armstrong (Arms) or clone 13 (Cl13) and the splenocytes from LCMV-infected Foxp3GFP knock-in mice were prepared 8 and 35 days pi and used for 12 h-stimulation with LCMV GP66-77 epitope peptide in the presence of PE-conjugated anti-CD154 antibody. The values in dot-plot denote the average of CD154+Foxp3- Th, CD154-Foxp3+ Treg, CD154+Foxp3+ iTreg cells gated on CD4+ T cells (B and C) The frequency and absolute number of LCMV GP66-77-specific CD4+CD154+Foxp3+ iTreg and nonspecific CD4+Foxp3+ Treg cells. The splenocytes of infected Foxp3GFP knock-in mice were prepared 8, 13 and 35 days pi, stimulated with LCMV GP66-77 in the presence of the presence of PE-conjugated anti-CD154 antibody. The bars in graphs represent the average±SD of LCMV GP66-77-specific CD4+CD154+Foxp3+ iTregs (B) and nonspecific CD4+Foxp3+ Treg (C) detected by intracellular CD154 staining in acute and chronic phase.

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