Regulatory T cells (Tregs) are important to control the expression of autoimmune diseases and protective immunity to inflammatory reactions in many tumors and infectious diseases (1). Therefore, initially described as critical for the control of autoimmunity, Treg were found upon adoptive transfer to prevent experimental autoimmune diseases (2,3). More recently, evidences has been accumulated that Treg cells suppress allogeneic immune responses and prevent transplant rejection (4). However, despite decades of research into the phenotypes and functions of different subsets of Tregs, only recently have they been sufficiently well characterized to begin considering how they might be targeted therapeutically. Thus, it has been reported that manipulating the numbers and/or function of Tregs in mice can decrease pathology in a wide range of context, such as autoimmunity, transplantation, and cancer (5-8).

Several different types of Tregs exist in human, including specialized subsets of CD4⁺, CD8⁺, double negative CD3⁺CD4^- CD8^-, γδ T cells, and natural killer T (NKT) cells. Although these different types of Tregs work together in a network to maintain immune homeostasis, the majority of the current studies focus on defining the normal function of CD4⁺ Tregs, because these cells mediate dominant, long-lasting, and transferable tolerance in experimental models (9). Research into how Treg can be manipulated therapeutically is most advanced for main types of CD4⁺ Tregs: forkhead box protein 3 (Foxp3)⁺ Tregs and interleukin-10 (IL-10)-producing type 1 Tregs (Tr1). CD4⁺ Tregs that express Foxp3 transcription factor were originally identified by their constitutive expression of the IL-2 receptor-α chain (CD25) (10), and for many years these cells have been referred to as CD4⁺CD25⁺ Tregs. CD25, however, is not a specific marker for these cells, because it is also expressed by all activated CD4⁺ T cells. Therefore, it has been believed that the Foxp3 transcription factor is considered as a crucial marker and a lineage commitment factor for CD4⁺CD25⁺ Tregs (11). In mice, expression of Foxp3 seems to be restricted to T cells with regulatory activity. CD4⁺CD25⁺ Treg cells expressing Foxp3 basically full into two groups: those that are pre-existent and those induced in response to the disease situations (9,10). The former CD4⁺Foxp3⁺ Treg cells arise both in the thymus [natural Treg (nTreg) cells], whereas the latter CD4⁺Foxp3⁺ Treg cells are believed to be differentiated at the periphery [inducible Treg (iTreg) cells] as a consequence of exposure to Ags (9,10). Numerous studies have proposed and demonstrated that nTreg cells are generated in the thymus through MHC class II-dependent T cell receptor (TCR) interactions resulting in high avidity selection (12,13), although additional selection mechanisms may take place. In recent years it has become evident that Foxp3⁺ Treg cells can also be generated outside the thymus under a variety of conditions (9,10). However, little is known about the response of Treg during exposure to foreign Ag. In organ-specific autoimmune disease, the tissue inflammation and immunopathology are driven by autoreactive effector T cells that infiltrate the target tissue. It is believed that Treg can also expand in an Ag-specific manner and traffic to the target tissue (14). Thus, understanding the functions and features of expanded CD4⁺Foxp3⁺ iTreg cells during exposure to foreign Ags may be crucial for manipulating immunopathologic diseases caused by acute and chronic infections. However, before characterization of CD4⁺Foxp3⁺ iTreg specific for foreign Ags, appropriate detection tools for foreign Ag-specific CD4⁺Foxp3⁺ iTreg cells must be developed.

Analysis of Ag-specific T cells by flow cytometry using fluorescent oligomers of major histocompatibility complex (MHC)-peptide complexes, a technique known as 'MHC tetramer staining', was introduced in 1996 by Altman et al. (15). The procedure, little changed since the original report, allows for the identification of T cells of interest on the basis of the binding specificity of their cell surface receptors for particular MHC-peptide complexes (16-18). Studies using MHC class I tetramers have demonstrated that current methods for enumerating Ag-reactive CD8⁺ T cells vastly underestimate the size of the Ag-specific, virally induced T cell repertoire (17,18). However, broad application of this powerful technology to CD4⁺ T cells beyond a few well-defined test systems has been slow because of the difficulty in producing class II MHC-based reagents, a low frequency of CD4⁺ T cells of interest in many biological contexts, and complications caused by low T-cell receptor (TCR)-MHC avidity.

While recently activated CD4⁺ T cells express CD154 (CD40L) which provides costimulatory signals to activate B cells and Ag presenting cells (APCs), resting CD4⁺ T cells do not express CD154. Therefore, de novo synthesized CD154 can be used to identify Ag-specific CD4⁺ T cells in response to Ag stimulation (19-21). The intracellular CD154 assay is also compatible with intracellular cytokine staining, by which this assay may provide additional information about Ag-elicited CD4 function by quantifying cells that are capable of stimulating APCs through CD40. Most importantly, the intracellular CD154 assay provides a simple means to isolate viable Ag-specific CD4⁺ T cells. Furthermore, while few number of peptide-MHC class II multimers are available, and knowledge of the subject's MHC haplotype is required, intracellular CD154 assay does not necessarily identify functionally responsive cells. Recent studies found that CD4⁺CD25⁺Foxp3⁺ Treg cells have feature of Th17 cells at the various conditions. Thus, the unrestricted population of CD4⁺CD25⁺Foxp3⁺ Treg cells has extended concept and has led to a greater appreciation of functional plasticity. Therefore, in this study we attempted to detect foreign Ag-specific CD4⁺Foxp3⁺ iTregs using LCMV infection model. To this end, we employed two tools for detecting foreign Ag-specific CD4⁺Foxp3⁺ iTreg cells, including MHC class II tetramer staining and intracellular CD154 staining methods, and compared the abilities of two tools to characterize foreign Ag-specific CD4⁺Foxp3⁺ iTreg cells.

In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining to detect CD4⁺Foxp3⁺ iTregs specific for LCMV GP_66-77 epitope in acute and chronic infections of LCMV. It was revealed that MHC class II tetramer (I-A^b/DIY) staining showed a lower detection rate than intracellular CD154 staining, and was not suitable to characterize the functionality of LCMV GP_66-77-specific CD4⁺ T cells because most of MHC class II tetramers were revealed to be unbound and unstable when intracellular cytokine staining was performed. In contrast, intracellular CD154 staining has been shown to become easier and simpler for detecting LCMV GP_66-77-specific CD4⁺ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV GP_66-77-specfic CD4⁺Foxp3⁺ iTreg using Foxp3^GFP knock-in mouse, and found that LCMV GP_66-77-specific CD4⁺Foxp3⁺ iTreg and polyclonal CD4⁺Foxp3⁺ Treg showed differential expansion in mice infected with LCMV Arms or Cl13. Our results provide an insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific CD4⁺Foxp3⁺ iTreg in various models including those of infection as well as autoimmune diseases.

Classically, MHC class I tetramer staining technology enabled us to characterize epitope peptide-specific CD8⁺ T cells at a single cell level in a variety of studies (18). Similarly, several groups have developed MHC class II tetramer staining to detect Ag-specific CD4⁺ T cells (17). However, the generation and use of MHC class II tetramer staining seemed more problematic those that of MHC class I tetramer staining. Moreover, to detect Ag-specific CD4⁺ T cells by MHC class II tetramer, the sequence of epitope peptide and character of MHC II haplotype should be revealed. Similarly, MHC class II teramer (I-A^b/DIY) staining seemed difficult to detect LCMV GP_66-77-specific CD4⁺ T cells by sensitivity and binding stability of tetramers under staining conditions. Moreover, since intracellular cytokine staining in responses to epitope peptide GP_66-77 showed a higher frequency of functional effector CD4⁺ T cells, it is likely that most MHC class II (I-A^b/DIY) tetramer did not bind to cognate TCR of CD4⁺ T cells. This may cause the underestimate of Ag-specific CD4⁺ T cells in vivo.

In recent years, de novo synthesized CD154 was shown to be a useful marker to identify Ag-specific CD4⁺ T cells in a stimulation assay (19-21). Indeed, CD154 staining was previously limited by the highly transient nature of CD154 expression (27,28), which is rapidly internalized and/or degraded after surface expression (28) or is sometimes secreted (29). To overcome this limitation, simple incorporation of fluorescently conjugated antibody to CD154 and monensin in the culture during Ag stimulation showed stable staining of CD154 molecule (21). Using this intracellular CD154 staining, we could stably detect CD4⁺ T cells specific for several LCMV epitope peptides. The advantage of intracellular CD154 staining is that the defined sequence of epitope peptide is not absolutely required for detecting Ag-specific CD4⁺ T cells. Also, this assay is compatible with intracellular cytokines staining (19-21), although it cannot be used with brefeldin A, which completely blocks surface expression of CD154. Using combined staining of intracellular CD154 with intracellular cytokine (IFN-γ and TNF-α), we found that most of LCMV GP_66-77-specific CD4⁺ T cells were dysfunctional in mice which were chronically infected with LCMV Cl13. This result is in accordance with the generation of exhausted CD8⁺ T cells specific for LCMV in chronic LCMV infection (24-26). Subsequently, this intracellular CD154 staining to detect Ag-specific CD4⁺ T cells was subjected to detect CD4⁺Foxp3⁺ iTregs specific for LCMV GP_66-77 epitope peptide in Foxp3^GFP knock-in mice infected with LCMV. Interestingly, we found the different expression of Foxp3 transcription molecule in polyclonal CD4⁺ and LCMV GP_66-77-specific CD4⁺CD154⁺ T cells at acute (8 and 13 days) and chronic (35 day) phase after infecting mice with LCMV Arms or Cl13. This result suggests that differential expansion of polyclonal and LCMV GP_66-77-specific CD4⁺Foxp3⁺ iTreg could affect viral persistence during process of acute and chronic LCMV infection.

In CD4⁺Foxp3⁺ Treg study, an unresolved issue that remains is whether foreign Ag-specific CD4⁺Foxp3⁺ Treg cells are present in peripheral tissues, because such cells cannot be directly detected. A few study groups identified foreign Ag-specific CD4⁺ T cells in peripheral lymphoid tissues, but failed to show their presence at a single cell level (30). It is speculated that these foreign Ag-specific Tregs may originate from activated CD4⁺ effector T cells and are iTregs (30-32). Otherwise, foreign Ag-specific Tregs could be derived from conversion of naïve Foxp3⁺ nTregs into memory nTregs, in a similar fashion to the development of Ag-reactive memory CD4⁺ effector T cells that develop from Foxp3^- naïve CD4⁺ T cells (33). According to a previous report, the unchanged highly demethylated TSDR Foxp3 within CD154⁺ Tregs is in favor of the development of foreign Ag-specific Tregs from nTregs in CMV infection, rather than the development from CD4⁺ effector T cells (34). However, it is uncertain whether LCMV GP_66-77-specific CD4⁺CD154⁺Foxp3⁺ Tregs were generated from nTreg or from CD4⁺ effector T cells in response to Ag stimulation, because the demethylation status of Foxp3 was not assessed. One intriguing finding in this study is that the differential ratio of LCMV GP_66-77-specific CD4⁺ Th to CD154⁺Foxp3⁺ Tregs was found in mice infected with between LCMV Arms and Cl13. i.e., approximately 0.029 and 0.059 ratio of LCMV GP_66-77-specific CD4⁺CD154⁺Foxp3⁺ Treg to CD4⁺CD154⁺ Th at acute phase (8 days pi of LCMV Arms vs Cl13) and approximately 0.048 and 0.083 ratio of LCMV GP_66-77-specific CD4⁺CD154⁺ Foxp3⁺ Treg to CD4⁺CD154⁺ Th at chronic phase (35 days pi of LCMV Arms vs Cl13). The proportion of LCMV GP_66-77-specific CD4⁺CD154⁺Foxp3⁺ Treg in Ag-specific CD154⁺CD4⁺ population appeared to be gradually increased, as the duration of infection passed. The formation of Ag-specific Treg to Th in a fixed ratio between acute and chronic phases indicates that despite acute and chronic infection, foreign Ag-specific CD4⁺CD154⁺Foxp3⁺ Tregs may be conversely increased at the late phase of infection, in order to break the exuberant effector response. Hence, LCMV GP_66-77-specific CD4⁺CD154⁺Foxp3⁺ Treg compartment was determined to be filled with Ag-specific CD4⁺CD154⁺ Th cells as a mirror image of the memory CD4⁺ effector population. Also, our results support the results from TCR repertoire analysis of effector CD4⁺ T and Treg cells, which did not show any evidence for affinity for autoreactive Ag (35), and the results from Vβ T cell analysis which showed that human CD4⁺CD25⁺⁺ Tregs equally share complex and comparable repertoires with CD4⁺CD25^- Th counterparts (36). Moreover, our results are strengthened by the result that CMV seropositivity resulted in a similar shift in Vβ repertoire in effector CD4⁺ Th and Treg cells (36). Direct identification of foreign Ag-specific CD4⁺Foxp3⁺ Treg at a single cell level using fine analytic tools will facilitate further Treg research and provide more complete understanding of the significance and interaction of Tregs in human disease and immune responses. Furthermore, isolation and propagation of viable foreign Ag-specific CD4⁺Foxp3⁺ Tregs are of potential benefit in future immunotherapy with CD4⁺Foxp3⁺ Treg cells.