The present work demonstrates that immune response to allergens in health and disease is the result of a balance between allergen-specific Tr1 cells and allergen-specific Th2 cells. Active regulation has emerged as a very essential mechanism for both inducing and equally importantly maintaining specific immunological nonresponsiveness. By analyzing Tr1 cells specific to various food or inhalant antigens, we demonstrated that similar mechanisms take place in healthy immune response to mucosal allergens. The IL-10–secreting allergen-specific T cells represented the predominant subset with significantly high frequency in comparison to IL-4– and IFN-γ–secreting T cells in healthy individuals. Both ELISPOT for cytokines and purification of cytokine-secreting T cells gave similar frequency numbers. Mechanisms that control the in vivo shift between allergen-specific T cell subsets remain to be elucidated. It can be hypothesized that a switch in cytokine profile and apoptosis of effector T cells may play a role in the generation of an atopic phenotype (29
). The stability of cytokine profile in differentiated effector and memory T cell subsets in humans is not fully known, and recent studies have demonstrated that lineage-committed memory T cell subsets are responsive to cytokine signals of the opposing lineage (30
). In addition, Tr1 cells do not appear to be anergic and may efficiently expand in vivo (32
) as they are shown to proliferate by IL-2, IL-4, IL-7, and IL-15 in the present work.
There is clear evidence from various animal models and human studies for an active mechanism of immune suppression, whereby a distinct subset of T cells inhibits the activation of conventional T cells in the periphery (7
). This Tr cell population has been determined as CD4+
T cells. They can prevent the development of autoimmunity, indicating that the normal immune system contains a population of professional regulatory T cells. Elimination of CD4+
T cells leads to spontaneous development of various autoimmune diseases, such as gastritis or thyroiditis, in genetically susceptible hosts. The frequency of CD4+
Tr cells is ~10–15% of CD4+
T cells, whereas the frequency of IL-10–secreting T cells of a single allergen specificity ranges between 0.1 and 0.007% of CD4+
T cells. This shows that the frequency of single allergen-specific Tr1 cells, which are also CD4+
, ranges between 1 in 1,000 and 1 in 20,000 of the whole CD4+
Tr cell population.
Although many aspects of the mechanisms by which suppressor cells exert their effects remain to be elucidated, it is well established that Tr cells suppress immune responses via cell-to-cell interactions and/or the production of IL-10 and TGF-β (10
). Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+
T cells in the presence of IL-10 in vitro (36
). The nonspecific T cell suppressor activity of IL-10 and TGF-β has been consistently reported in experiments with high amounts of exogenously added suppressor cytokines (21
). However, the present work demonstrates that Tr1 cells display antigen-specific suppressor activity in very low numbers. If the number of cells exceed a threshold that provides sufficient quantities of suppressor signals, apparently they show nonspecific suppression. Depending on their frequency, the first T cell that contacts the APC may be very critical in the subsequent decision to stimulate or suppress the specific immune response. If the first T cell to contact the APC is a Tr1 cell, it may silence or regulate the maturation of APC. IL-10 down-regulates the antigen-presenting capacity, such as HLA-DR expression, costimulatory molecules, and several cytokines in dendritic cells and monocytes/macrophages (37
). Recently, differentiation of a distinct dendritic cell subset in the presence of IL-10 has been demonstrated that induces tolerance through the generation of Tr1 cells (38
). In addition, exposure of mature pulmonary dendritic cells to respiratory allergens stimulated the development of Tr1-like cells, which was dependent on dendritic cell IL-10 production (39
). Together, these findings suggest that IL-10–secreting T cells may regulate the functional state of APCs in a way that these APCs can now promote the generation of Tr1 cells.
T cells are the only lymphocyte subpopulation in mice and humans that express CTLA-4 constitutively. The expression apparently correlates with the suppressor function of CTLA-4. As demonstrated in the present work, the blocking of CTLA-4 activity of Tr1 cells reverses suppression in cocultures of CD4+
T cells (40
). Similarly, the treatment of mice that were recipients of CD4+
T cells with CTLA-4–blocking agents, abrogated the suppression of inflammatory bowel disease (41
). These studies indicate that signals that result from the engagement of CTLA-4 by its ligands, CD80 or CD86, are required for the induction of suppressor activity. Under some circumstances, the engagement of CTLA-4 on the CD4+
T cells by antibody or by CD80/CD86 might lead to inhibition of the TCR-derived signals that are required for the induction of suppressor activity.
A subset of human Tr1 cells expressed functional PD-1 in the present work. PD-1 is an immunoreceptor tyrosine-based inhibitory motif–containing receptor expressed upon T cell activation. PD-1–deleted mice develop autoimmune diseases, suggesting an inhibitory role for PD-1 in immune responses (42
). Members of the B7 family, PD-ligand (L)1 and PD-L2, are ligands for PD-1. PD-1/PD-L engagement on murine CD4 and CD8 T cells results in inhibition of proliferation and cytokine production. T cells stimulated with anti-CD3/PD-L1Fc-coated beads display dramatically decreased proliferation and IL-2 production (43
Collectively, our results indicate that the control of Th2 immune response against naturally exposed harmless environmental antigens is mediated by Tr1 cells in humans. Effector (allergen-specific Th2) and suppressor (allergen-specific Tr1) T cells exist in both healthy and allergic individuals in certain amounts. Their ratio determines the development of a healthy or an allergic immune response. These data may explain the spontaneous development and spontaneous healing of allergic diseases. Although in low frequency, the existence of potential suppressive allergen-specific Tr1 cells in allergic individuals suggests a possible way of treatment. The knowledge of this cellular and molecular basis is pivotal in understanding the mechanisms of immune tolerance or allergy development against harmless environmental proteins.