Selection of CD4+CD25+ regulatory T cells by self-peptides.

Regulatory T cells have been shown to prevent the development of autoimmune disease, and can modulate immune responses during infections or following tissue transplantation. Recently, the processes by which CD4+CD25+ regulatory T cells are produced during immune repertoire formation have begun to be elucidated. This review focuses on the role of self-peptides in mediating CD4+CD25+ regulatory T cell selection in the thymus. How self-peptides continue to have an important influence on the accumulation of CD4+CD25+ regulatory T cells in the periphery is also discussed.

Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide.

Despite accumulating evidence that regulatory T cells play a crucial role in preventing autoimmunity, the processes underlying their generation during immune repertoire formation are unknown. We show here that interactions with a single self-peptide can induce thymocytes that bear an autoreactive T cell receptor (TCR) to undergo selection to become CD4+CD25+ regulatory T cells. Selection of CD4+CD25+ thymocytes appears to require a TCR with high affinity for a self peptide because thymocytes that bear TCRs with low affinity do not undergo selection into this pathway. Our findings indicate that specificity for self-peptides directs the selection of CD4+CD25+ regulatory thymocytes by a process that is distinct from positive selection and deletion.

Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes.

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.

Thermolabile H-2Kb molecules expressed by transporter associated with antigen processing-deficient RMA-S cells are occupied by low-affinity peptides.

RMA-S cells do not express functional TAP, yet they express MHC class I molecules at the cell surface, especially at reduced temperatures (26 degrees C). It is generally assumed that such class I molecules are "empty," devoid of any associated peptide. A radiochemical approach was used to label class I-associated peptides and to determine the extent to which Kb molecules in RMA-S cells are associated with peptides. These studies revealed that at 26 degrees C Kb molecules in RMA-S cells are occupied with self-peptides. Such peptides stably associate with Kb at 26 degrees C but easily dissociate from them at 37 degrees C, suggesting low-affinity interactions between Kb and the associated peptides. At 26 degrees C, at least some of these Kb molecules are stably expressed in a peptide-receptive state on the cell surface, whereas at 37 degrees C they are short lived and are only transiently capable of binding and presenting exogenously supplied OVA 257-264 peptide for presentation to CD8+ Kb-restricted T lymphocytes. Thus contrary to current models of class I assembly in TAP-deficient RMA-S cells, the presumably "empty" molecules are in fact associated with peptides at 26 degrees C. Together, our data support the existence of an alternative mechanism of peptide binding and display by MHC class I molecules in TAP-deficient cells that could explain their ability to present Ag.

A molecular basis for how a single TCR interfaces multiple ligands.

CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.

Natural ligand of mouse CD1d1: cellular glycosylphosphatidylinositol.

Mouse CD1d1, a member of the CD1 family of evolutionarily conserved major histocompatibility antigen-like molecules, controls the differentiation and function of a T lymphocyte subset, NK1+ natural T cells, proposed to regulate immune responses. The CD1d1 crystal structure revealed a large hydrophobic binding site occupied by a ligand of unknown chemical nature. Mass spectrometry and metabolic radiolabeling were used to identify cellular glycosylphosphatidylinositol as a major natural ligand of CD1d1. CD1d1 bound glycosylphosphatidylinositol through its phosphatidylinositol aspect with high affinity. Glycosylphosphatidylinositol or another glycolipid could be a candidate natural ligand for CD1d1-restricted T cells.

Distinct roles for signals relayed through the common cytokine receptor gamma chain and interleukin 7 receptor alpha chain in natural T cell development.

The commitment, differentiation, and expansion of mainstream alpha/beta T cells during ontogeny depend on the highly controlled interplay of signals relayed by cytokines through their receptors on progenitor cells. The role of cytokines in the development of natural killer (NK)1(+) natural T cells is less clearly understood. In an approach to define the role of cytokines in the commitment, differentiation, and expansion of NK1(+) T cells, their development was studied in common cytokine receptor gamma chain (gammac) and interleukin (IL)-7 receptor alpha (IL-7Ralpha)-deficient mice. These mutations block mainstream alpha/beta T cell ontogeny at an early prethymocyte stage. Natural T cells do not develop in gammac-deficient mice; they are absent in the thymus and peripheral lymphoid organs such as the liver and the spleen. In contrast, NK1(+) T cells develop in IL-7Ralpha-deficient mice in the thymus, and they are present in the liver and in the spleen. However, the absolute number of NK1(+) T cells in the thymus of IL-7Ralpha-deficient mice is reduced to approximately 10%, compared to natural T cell number in the wild-type thymus. Additional data revealed that NK1(+) T cell ontogeny is not impaired in IL-2- or IL-4-deficient mice, suggesting that neither IL-2, IL-4, nor IL-7 are required for their development. From these data, we conclude that commitment and/or differentiation to the NK1(+) natural T cell lineage requires signal transduction through the gammac, and once committed, their expansion requires signals relayed through the IL-7Ralpha.

CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4.

Murine CD1 has been implicated in the development and function of an unusual subset of T cells, termed natural T (NT) cells, that coexpress the T cell receptor (TCR) and the natural killer cell receptor NK1.1. Activated NT cells promptly produce large amounts of IL-4, suggesting that these cells can influence the differentiation of CD4+ effector T cell subsets. We have generated mice that carry a mutant CD1d1 gene. NT cell numbers in the thymus, spleen, and liver of these mice were dramatically reduced. Activated splenocytes from mutant mice did not produce IL-4, whereas similarly treated wild-type splenocytes secreted large amounts of this cytokine. These results demonstrate a critical role for CD1 in the positive selection and function of NT cells.

Expansion of natural (NK1+) T cells that express alpha beta T cell receptors in transporters associated with antigen presentation-1 null and thymus leukemia antigen positive mice.

Thymic selection of natural killer-1+ natural T cells that express alpha beta T cell receptors requires a conserved beta 2-microglobulin-associated molecule, presumably CD1d, displayed by CD4+8+ thymocytes. Here we demonstrate that positive selection of natural T cells occurs independent of transporters associated with antigen presentation-1 (TAP-1) function. Moreover, natural T cells in TAP-1o/o mice are numerically expanded. Several H-2 class Ib molecules function in a TAP-independent manner, suggesting that if expressed in TAP-1o/o thymocytes, they could play a role in natural T cell development. Of these class Ib molecules, H-2TL is expressed by TAP-1o/o thymocytes. Moreover, we find that thymi of TL+ mice congenic or transgenic for H-2T18 also have a numerically expanded natural T cell repertoire compared with TL- mice. This expansion, as in TAP-1o/o thymi, is evident in each of the limited T cell receptor V beta chains expressed by natural T cells, suggesting that TL and CD1d impact similar repertoires. Thus TL, in addition to CD1d, plays a role in natural T cell development.