Central role of ILT3 in the T suppressor cell cascade
Introduction
The immune system has developed several checkpoints and regulatory mechanisms to discriminate between self and non-self antigens and avoid autoimmunity [1]. Included among them are: (1) negative selection of auto-reactive T cells in the thymus [2]; (2) elimination of auto-reactive T cells via activation-induced cell death (AICD) [3], [4], [5], [6], [7]; (3) induction of anergy upon TCR triggering in the absence of co-stimulation [8], [9], [10], [11], [12]; (4) inhibition of immune function by T suppressor cells [13], [14], [15], [16], [17], [18], [19], [20].
Research on T suppressor cells re-emerged in the late 1990s when several subsets of T cells were shown to inhibit the proliferation of other T cells. Two broad categories of regulatory T (Treg) cells have been recognized. The first consists of naturally occurring and the second, of induced Treg [13], [14], [15], [16], [17], [18], [19], [20].
The naturally occurring CD4+CD25+ Treg subset is generated in the thymus as a functionally distinct subpopulation of T cells [13], [14], [15], [16], [17], [18], [19], [20]. These natural Treg cells play a major role in regulating self-reactive T cells and preventing autoimmune diseases. The transfer of T cell populations, from which the CD25+ subset has been depleted, into T cell-deficient mice caused severe autoimmune disorders, such as thyroiditis, gastritis, insulin-dependent diabetes mellitus and colitis [16], [19], [20]. Conversely, infusion of the Treg cells in these animals strongly suppressed autoimmunity. Recent evidence indicates that the transcription factor FOXP3 acts as a “master control gene” for the development and function of Treg [21], [22], [23]. A mutation in the gene encoding FOXP3 was identified as the genetic defect underlying autoimmune and inflammatory disease in scurfy mice and in humans with IPEX (immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome) and XLAAD (X-linked autoimmunity allergic dysregulation syndrome), emphasizing the importance of CD4+CD25+ Treg in the maintenance of normal immune homeostasis [21], [22], [23].
FOXP3 is a member of the forkhead–winged helix family of transcription factors. Retroviral mediated expression of FOXP3 converts naive T cells into CD25+ Treg. Thus, FOXP3 induces either directly or indirectly the expression of Treg-associated molecules. While in mice FOXP3 is stably expressed by CD4+CD25+ Treg and cannot be induced by activation of CD4+CD25− T cells, in humans it can be induced by activation and exposure to TGF-beta [24].
CD4+CD25+ T cells suppress activation and proliferation of naive CD4+ T cells in vitro through T-cell-to-T-cell contact [20], [21]. Treg cells are in an anergic state and do not proliferate in response to antigens (Ag) presented by MHC class II molecules [14], [15], [16], [17], [18], [19], [20], [21]. Triggering of Treg cells by anti-CD3 plus anti-CD28 monoclonal antibodies (mAbs) or exposure to IL-4 breaks the anergic state and abrogates the suppressor function of Treg [24], [25]. Also, triggering by an agonistic Ab to the cell surface receptor glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18), which is constitutively expressed on the surface of Treg cells [26], [27] abrogates the suppressor activity of Treg. IL-2 is mandatory for the in vitro activation of CD4+CD25+ Treg cells [28].
The mechanisms by which natural CD4+CD25+ Treg cells act in different autoimmune disease models seem to involve a discretionary requirement for the cytokines IL-10 and TGF-beta [17], and may depend on signals through CTLA-4 [29], [30], as well as regulation coordinated by GITR [26], [27].
Induced Treg cells are divided into two categories, one consisting of non-antigen-specific CD4+ and CD8+ Treg and the other of antigen-specific CD8+ T suppressor cells (TS) and CD4+CD25+ Treg. Non-antigen-specific regulatory CD4+CD25+ T cells can be generated from naïve peripheral CD25− precursors under certain culture conditions, such as by exposure to IL-10 and IFN-alpha or TGF-beta, or by stimulation with immature myeloid dendritic cells (DC) or mature plasmacytoid DC [11], [31], [32], [33], [34]. Activation, in vitro or in vivo, of human or mouse CD4+ T cells in the presence of IL-10 results in the generation of T-cell clones which produce significant amounts of IL-10, IFN-alpha, TFG-beta and IL-5. These T-cell clones, named TR1, inhibit Ag-induced activation of naïve autologous T cells via a mechanism which is partially mediated by IL-10 and TGF-beta [31], [32]. T cells with a TR1 cytokine profile have been described in several models of autoimmune diseases and transplantation [35], [36], [37], [38], [39]. In most cases, TR1 cells arise following repeated Ag stimulation either in vitro or in vivo[35]. However, because they cause bystander suppression, their inhibitory effect on other T cells is not antigen-specific.
The extent to which cell contact or soluble factors are required for TR1-mediated suppression of TH reactivity is still unknown. Experiments in which cytokine production was excluded, first by activating and then fixing naturally occurring CD4+CD25+ Treg cells, showed that these cells maintain their capacity to suppress normally responding CD4+CD25− T cell populations rendering them anergic. The newly anergized population further suppressed syngeneic CD4+ T cells via the production of inhibitory cytokines [40], [41]. It was suggested that suppression occurs in two sequential steps: the first one is a cell contact-dependent ‘transmission’ of anergy from a regulatory T cell to another T lymphocyte, while the second is a cell contact-independent, cytokine mediated suppression of other T helper cells [40], [41], [42].
Other non-antigen-specific regulatory T cell subtypes have been described including gamma–delta T cells, NK1.1 T cells, CD8+CD25+ and CD4−CD8− T cells, but they remain poorly characterized [14].
A distinct category of regulatory T cells characterized by their antigen-specific activity and mechanism of action are CD8+CD28− TS cells first described in humans by our group. We showed that CD8+CD28− TS specific for alloantigens, xenoantigens or nominal antigens could be generated in vitro by repeated antigenic stimulation [43], [44], [45], [46], [47], [48], [49], [50], [51]. Next, we provided in vivo evidence that CD8+CD28− T cells act as antigen-specific suppressors in patients with heart, kidney or liver allografts [52], [53], [54], [55], [56]. We further demonstrated that antigen-specific CD8+CD28− TS express FOXP3, derive from an oligoclonal population of CD8+ FOXP3− cells, are MHC class I restricted, have no killing capacity and do not produce cytokines [43], [55], [56]. Instead they act on professional (DC) and non-professional (endothelial cells) APC directly i.e., by cell-to-cell contact, inducing qualitative changes characteristic of an alternative pathway of maturation toward a tolerogenic rather than immunogenic phenotype. These changes include the downregulation of NF-κB-dependent costimulatory molecules such as CD40, CD58, CD80, CD86, and the upregulation of inhibitory receptors, immunoglobulin like transcript 3 (ILT3) and ILT4 [48], [49], [50], [55], [56]. CD4+ TH, which interact with tolerogenic APC, become anergic and acquire regulatory activity [13], [55]. These data support a model in which T cell mediated suppression results from the sequential interaction between first, CD8+ Ts and APC and next, “tolerized” APC and CD4+ TH. In turn, anergic TH acquire regulatory capacity, in conjunction with FOXP3 expression, further perpetuating tolerance [13], [56]. The central role of ILT3high, ILT4high APC in the induction of suppression was further demonstrated in experiments in which the upregulation of these inhibitory receptors in DC and endothelial cells (EC) was induced by treating the cells with IL-10 plus IFN-alpha or IL-10 plus vitamin D3 [13], [55], [56]. Such ILT3high ILT4high APC induced the in vitro generation of TS and Treg from unprimed populations of CD4 and CD8 T cells [13], [55], [56].
Progress in understanding the mechanisms of T-cell activation and inactivation is currently being translated into strategies, enabling induction of selective immunosuppression for treatment of autoimmune diseases, allergies and allograft rejection. There is an imperative need for antigen-specific immunosuppression, as systemic immunosuppression is associated with an increased risk of malignancies, infections and considerable toxicity. Progress in the generation and characterization of antigen-specific TS, Treg cells and tolerogenic APC may pave the way to the induction of immunologic tolerance.
Section snippets
Molecular characterization of CD8+CD28− Ts
To gain insight into the common denominators of antigen-specific and non-specific Treg cells we have analyzed some of their characteristics at the molecular level. We compared by RT-PCR the expression of genes, known to be upregulated in natural CD4+CD25+ Treg from fresh peripheral blood [21], [26], [27], [56], [57], [58], [59], [60], [61], [62], [63] with their expression in allospecific CD8+CD28− TS from T cell lines (TCL) [64], [65]. The following genes were studied: CD25, GITR, CTLA-4,
Molecular and functional events resulting from TS mediated suppression
Molecular changes induced by CD8+ TS in DC were analyzed using Affymetrix gene chips. The overall picture that emerged was that tolerogenic DC differ from both immature and mature DC with respect to molecules involved in signal transduction, chemokines, cytokines, transcription factors, apoptosis-related proteins and cell growth regulators [49], [50]. Most importantly, mRNA expression profiling showed that tolerogenic DC exhibit a high cell surface expression of the inhibitory molecules ILT3
Evaluation of the clinical significance and therapeutic potential of allospecific TS
We have explored the in vivo relevance of CD8+CD28− TS and of CD4+CD25+ Treg in recipients of heart, kidney or liver transplants [52], [53], [54], [55], [56]. Serial studies of the phenotype displayed by T cells from heart allograft recipients demonstrated a significant increase of the CD8+CD28−CD27+ perforin negative T-cell population in rejection-free patients [52], [53], [54], [55], [56]. This phenotype is characteristic of in vitro generated TS as demonstrated by flow cytometry and cDNA
CD8+ FOXP3+ T suppressor cells transfer allogeneic tolerance in rodents and induce PIR-B in donor APC
Recently, evidence has been provided that the paired immunoglobulin like receptor (PIR)-B expressed on B cells and myeloid cells of rodents shares structural and functional characteristics with ILT4 [72]. PIR-B, which belongs to a MHC class I recognition system, contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic domain and can inhibit receptor-mediated activation signaling similar to its human orthologue ILT4 [73]. Transfer of allogeneic splenocytes into PIR-B
Immunoregulatory activity of membrane and soluble ILT3
Effective priming of CD4+ T lymphocytes requires cell-to-cell interaction between APC and T cells. This interaction allows bi-directional costimulatory signals which activate both the APC and T cells with cognate-specific TCR. Triggering of TCR induces upregulation of CD40L expression on activated CD4+ T cells. The interaction of CD40L with CD40 expressed by APC induced the upregulation of costimulatory molecules that interact with their counter receptor on CD4+ Th cells, eliciting their
Acknowledgments
This work was supported by a grant from the NIH RO1 AI55234-04 and the Interuniversitary Organ Transplantation Consortium, Rome, Italy
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