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T Cell Biology and Immunological Tolerance
Summary: Alexander Rudensky is studying the development of T lymphocytes, their function, and their role in the regulation of immune responses to infection and in the prevention of autoimmunity. His studies include investigation of the control of immune homeostasis by regulatory T cells and investigation of the molecular mechanisms instructing commitment of specialized T cell lineages.
The adaptive immune system randomly generates antigen receptors in developing lymphocyte clones through a process of somatic cell gene rearrangement mediated by the recombination-activating gene recombinase. The unlimited specificities of this anticipatory recognition system counterbalance the short reproduction cycles and high mutation rates of infectious microorganisms. However, this diversity of antigen recognition also poses the threat of autoimmunity due to the generation of self-reactive receptors.
T cell receptors (TCRs) recognize short peptides derived from foreign and self-antigens bound to products of highly polymorphic major histocompatibility complex (MHC) molecules. MHC molecules sample peptide products of constitutive protein breakdown. CD4 T cells, which play a commanding role in regulation of both the adaptive and the innate immune systems, recognize MHC class II molecules bound to peptides generated in the endocytic compartment of antigen-presenting cells (APCs) during degradation of foreign and self-protein antigens.
The majority of MHC-bound peptides presented on the surface of APCs are derived from self-proteins, and these complexes are involved in shaping T cell repertoire in the thymus and the periphery. A TCR generated in the thymus is subjected to a stringent selection process. Thymocytes expressing TCRs with a low affinity for self-peptides bound to MHC molecules displayed on thymic cortical epithelial cells undergo further maturation or positive selection. In contrast, thymocytes with a high affinity for self-peptide–MHC complexes are subjected to negative selection as a result of apoptosis or functional inactivation. However, the latter process, collectively recognized as recessive tolerance, is not 100 percent efficient, and autoreactive T cells are normally found within mature peripheral T cell subsets. A unique mechanism—dominant tolerance—has been proposed to counter this threat. Dominant tolerance involves regulation of lymphocyte reactivity against self- and environmental antigens by specialized regulatory T cells that act in a dominant, suppressive fashion.
Genetic and Cellular Mechanisms of Dominant Tolerance
Efforts to define the cell type that mediates suppression of autoimmunity identified CD4 T cells that express the interleukin-2 receptor (IL-2R) α-chain (CD25) as being highly "enriched" in suppressor activity. These "naturally arising" CD4+CD25+ regulatory T cells (TR cells) became the best candidates for the cell population mediating dominant tolerance to self. IL-2R is not, however, a unique marker of TR cells, as all activated T cells transiently express CD25. This suggested that TR cells are simply activated CD4 T cells that down-modulate immune responses by competing for limiting growth factors. Our studies indicated, however, that CD4+CD25+ TR cells likely represent a dedicated highly specialized lineage and guided our efforts to discover specific genetic mechanisms governing their development and function.
Our studies in mice identified the transcription factor Foxp3 as a definitive molecular TR cell marker. Several years ago, mutations in the X-chromosome–encoded Foxp3 gene were identified as the cause of the early-onset fatal autoimmune disorder observed in human IPEX patients (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) and in a strain of mutant mice that spontaneously develop autoimmune disease. To understand Foxp3's role in TR cell biology, we generated mice harboring a conditional Foxp3 allele and a targeted disruption of Foxp3 gene in the germline. Examination of bone marrow chimeric mice containing a mixture of Foxp3-deficient and wild-type hematopoietic precursor cells demonstrated that TR cells fail to develop from Foxp3-deficient progenitors. We also found that retroviral Foxp3 gene transfer into peripheral non–TR cells results in acquisition of suppressive function. These studies reveal a principal role for Foxp3 in guiding the development and function of TR cells.
To obtain further insights into the function of Foxp3 we generated a reporter Foxp3 allele by "knocking" green fluorescent protein (GFP) into the Foxp3 gene. The observation that Foxp3 is expressed in a subset of peripheral and thymic T cells with potent suppressive activity suggested that Foxp3 is a factor that specifies TR cell lineage. These cells also share a transcriptional signature that is distinct from either CD25+Foxp3– or CD25–CD4+Foxp3– T cells. Thus, Foxp3 is a dedicated and highly specialized genetic mechanism for the generation of T cells that can promote dominant tolerance.
Further analyses of Foxp3-deficient mice demonstrated that lack of TR cells is the cause of the highly aggressive lymphoproliferative syndrome. Evidence for this was provided by substantial reduction in this syndrome in mutant animals upon neonatal transfer of a small number of TR cells. Furthermore, we found that antigen-specific responses of mouse nonregulatory T cells expressing or lacking the Foxp3 gene are indistinguishable. Similarly, sensitivity of non–TR cells to negative selection in the thymus or periphery was not affected by Foxp3 gene deficiency. These observations, combined with our finding of the identical onset and progression of autoimmune disease in mice with germline and T cell–specific ablation of the Foxp3 gene, provide further proof that TR cell deficiency results in fatal autoimmune pathology affecting multiple organs.
Signals Turning on the TR Developmental Program
Our studies revealed that Foxp3+ TR cells display TCRs with increased avidity for self-peptide–MHC ligands compared with non–TR TCRs. A noticeable skewing of CD4+ T cell development toward the TR cell lineage was observed after transduction of bone marrow stem cells with TR-derived (but not CD25– non–TR cell–derived) TCRs. This suggests that this self-reactivity instructs TR cell-ineage commitment. Our observation of an MHC dependence of Foxp3 expression in both immature and mature thymocytes agreed with these results, which strongly support an instructive role for TCR signaling in TR cell lineage commitment. Our studies indicate that Foxp3-dependent development of TR cells is not hardwired but is induced in response to increased strength of TCR signaling during thymic development.
Upon further investigation, we found that the TCR repertoire of thymic TR cells is diverse and is more similar to that of peripheral TR cells than that of nonregulatory T cells. This suggests that thymic TR cells contribute substantially to the peripheral TR cell population. Furthermore, activated T cells in Foxp3-deficient mice, which lack TR cells, "preferentially" used TCRs found in the TCR repertoire of TR cells in Foxp3-sufficient mice. This suggests that these self-reactive TCRs contribute to the pathology of Foxp3-deficient mice. Our analyses suggest that TR cells and potentially pathogenic autoimmune T cells use overlapping pools of self-reactive TCRs. Recently we observed that the development of Foxp3-expressing TR cells, localized to the medullary compartment of the thymus, is substantially delayed relative to nonregulatory thymocytes during ontogeny. This indicates that, in addition to TCR, a second unidentified signal is likely to be required for TR cell–lineage commitment. We propose that induction of Foxp3 in developing thymocytes and, thus, TCR-mediated commitment to the TR cell lineage are facilitated by a second signal largely associated with the thymic medulla.
Role of TGFβ Signaling in Controlling Immune Homeostasis
Transforming growth factor-β (TGFβ)-family cytokines serve as positive and negative regulators of differentiation and proliferative programs in numerous cell types. Investigation of the germline ablation of Tgfb1 gene expression revealed the biological relevance of TGFβ1-mediated control of immune homeostasis. Mice with a targeted disruption of the Tgfb1 gene exhibit embryonic lethality with a variable degree of penetrance (depending on particular genetic background) due to developmental defects in nonlymphoid organs. TGFβ1-deficient mice that are born develop severe autoimmune multiorgan pathology and die at 3–4 weeks of age. However, specific cellular mechanisms of the autoimmune disease observed in these mice have been difficult to decipher, due to pleiotropic effects of TGFβ signaling on multiple tissues and to the broad distribution of TGFβ receptors both within and outside the immune system. The prevalent interpretation of complex pathology in Tgfb1–/– mice was that an impairment in either development or homeostasis of nonlymphoid tissues provokes, directs, or greatly accelerates exuberant T cell reactivity in the absence of TGFβ receptor signaling.
Unexpectedly, we discovered that complete ablation of TGFβ signaling in T cells engenders aggressive early-onset, multiorgan, autoimmune-associated lesions with 100 percent mortality. Peripheral T cells with TGFβ–receptor II (TGFβRII) deficiency undergo cytolytic and T helper 1 (Th1) effector cell differentiation in a cell-intrinsic TCR-specific fashion. Furthermore, TGFβRII deficiency blocked the development of canonical natural killer T (NKT) cells. Instead, it facilitated the generation of a highly pathogenic T cell subset exhibiting multiple hallmarks of NK cells, and sharply elevated amounts of FasL, perforin, granzymes, and interferon-γ. Thus, TGFβ signaling in peripheral T cells is crucial in restraining a TCR activation-dependent Th1, cytotoxic, and NK cell–like differentiation program that, when left unchecked, rapidly progresses to fatal autoimmunity. These data establish a role for TGFβ signaling in T cells as the distinct, equally vital, cell-intrinsic mechanism of control of autoimmunity acting in cooperation with the Foxp3-mediated cell-extrinsic mechanism of dominant tolerance.
Grants from the National Institutes of Health provided support for some of these studies.
Last updated: November 9, 2006
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