Introduction

Antigen (Ag)-specific T cells can be recognized using cell surface expression of markers specifically induced upon T cell receptor interaction with peptides presented in the context of HLA molecules on Ag-presenting cells. CD154 is a member of the TNF-superfamily and its expression is specifically induced upon T cell activation. CD154 cell surface expression allows viable isolation of Ag-specific CD4⁺ T cells and detailed phenotypic as well as functional analysis. To use Tregs as cell therapy in kidney transplantation, it is desirable to use alloAg-specific Tregs to prevent unwanted suppression of immune responses. In this study, we investigated whether CD154 cell surface expression is able to detect Ag-specific regulatory T cells upon allogeneic stimulation.

Methods

Highly enriched fractions of Tregs and T cells depleted for Tregs (Teff) were FACS sorted. These Tregs and Teff were stimulated with HLA-mismatched (MM) PBMC in the presence of co-stimulatory antibodies (ΑCD28 and ΑCD49d) as well as ΑCD40 to maintain CD154 expression on the cell surface. AlloAg-specific, CD154⁺ Tregs were studied extensively using phenotypic as well as functional analyses, testing their suppressive capacity in a mixed lymphocyte reaction (MLR) either immediately upon isolation or following expansion.

Results

Maximal CD154 expression within Tregs was observed upon 24 hour stimulation. AlloAg-specific CD154⁺Tregs consisted of both naÏve (mean±SEM; 38±12%) as well as memory (62±12%) T cells and FOXP3 expression (>80% of the Tregs are FOXP3⁺, median fluorescence intensity: 4928) remained high. There was no association between percentages of CD154⁺ Tregs and the number of HLA-MM. Sorted CD154⁺Tregs were superior (P<0.05) in suppressing Ag-specific responses when compared to CD154^–Tregs and total Tregs, i.e. at a 1:5 (Treg:Teff) ratio the median percentages of inhibition (IH) in a MLR amounted to 57% versus 25% and 31%, respectively. CD154⁺Tregs could be efficiently expanded in an Ag-specific manner, which enhanced their suppressive capacity. At a 1:5 ratio, the median % of IH in a MLR increased to 98%.

Conclusions

These data show for the first time that alloAg-specific Tregs can be detected using CD154 expression. CD154⁺ alloAg-specific Tregs can be isolated and efficiently expanded increasing their Ag-specific suppressive capacity. These alloAg-specific CD154⁺ Tregs may be of potential benefit for cellular immunotherapy in kidney transplantation.

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