The role of TCR specificity in naturally arising CD25+ CD4+ regulatory T cell biology.

CD25+ CD4+ T cells (TR) are a naturally arising subset of regulatory T cells important for the preservation of self-tolerance and the prevention of autoimmunity. Although there is substantial data that TCR specificity is important for TR development and function, relatively little is known about the antigen specificity of naturally arising TR. Here, we will review the available evidence regarding naturally arising TR TCR specificity in the context of TR development, function, and homeostasis.

Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells.

Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.

Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis.

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ealpha(52-68) yields an epitope that is similar to the one generated during constitutive presentation of I-Ealpha as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Ealpha is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.

In vivo MHC class II presentation of cytosolic proteins revealed by rapid automated tandem mass spectrometry and functional analyses.

We report a strategy for high through-put sequence analyses of large MHC class II-bound peptide repertoires which combines automated electrospray ionization tandem mass-spectrometry with computer-assisted interpretation of the tandem mass spectra using the algorithm SEQUEST. This powerful approach discerned 128 peptide sequences displayed by the murine MHC class II molecule I-Ab in activated B cells and macrophages, including a surprisingly large number of peptides derived from self cytosolic proteins. Mice lacking the chaperone molecule H-2M were used to generate T cells specific for selected self peptides. Functional T cell analyses of ex vivo antigen-presenting cells indicated that peptides originating from cytosolic proteins are efficiently presented by splenic and thymic dendritic cells, but less so by resting B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class II-bound self peptides derived from cytosolic proteins exists in vivo.

Dynamic tuning of T cell reactivity by self-peptide-major histocompatibility complex ligands.

Intrathymic self-peptide-major histocompatibility complex class II (MHC) molecules shape the T cell repertoire through positive and negative selection of immature CD4(+)CD8(+) thymocytes. By analyzing the development of MHC class II-restricted T cell receptor (TCR) transgenic T cells under conditions in which the endogenous peptide repertoire is altered, we show that self-peptide-MHC complexes are also involved in setting T cell activation thresholds. This occurs through changes in the expression level of molecules on thymocytes that influence the sensitivity of TCR signaling. Our results suggest that the endogenous peptide repertoire modulates T cell responsiveness in the thymus in order to enforce tolerance to self-antigens.

Survival and homeostatic proliferation of naive peripheral CD4+ T cells in the absence of self peptide:MHC complexes.

TCR-self peptide:MHC interactions play a critical role in thymic positive selection, yet relatively little is known of their function in the periphery. It has been suggested that continued contact with selecting MHC molecules is necessary for long-term peripheral maintenance of naive T cells. More recent studies have also demonstrated a role for specific self peptide:MHC complexes in the homeostatic expansion of naive T cells in lymphopenic mice. Our examination of these processes revealed that, whereas self class II MHC molecules do have a modest effect on long-term survival of individual CD4+ T cells, interactions with specific TCR ligands are not required for peripheral naive CD4+ T cell maintenance. In contrast, selective engagement of TCRs by self-peptide:MHC complexes does promote proliferation of CD4+ T cells under severe lymphopenic conditions, and this division is associated with an activation marker phenotype that is different from that induced by antigenic stimulation. Importantly, however, the ability of naive T cells to divide in response to homeostatic stimuli does not appear to be stringently dependent on TCR-self peptide:MHC interactions. Therefore, these results show that the factors regulating survival and homeostatic expansion of naive T cells in the periphery are not identical. In addition, we provide evidence for a novel form of T cell proliferation that can occur independently of TCR signaling and suggest that this reflects another mechanism regulating homeostatic T cell expansion.

Competition for specific intrathymic ligands limits positive selection in a TCR transgenic model of CD4+ T cell development.

Efficient positive selection of a broad repertoire of T cells is dependent on the presentation of a diverse array of endogenous peptides on MHC molecules in the thymus. It is unclear, however, whether the development of individual TCR specificities is influenced by the abundance of their selecting ligands. To examine this, we analyzed positive selection in a transgenic mouse carrying a TCR specific for the human CLIP:I-Ab class II complex. We found that these mice exhibit significantly reduced CD4+ T cell development compared with two other transgenic mice carrying TCRs selected on I-Ab. Moreover, many of the selected cells in these mice express endogenous and transgenic receptors as a consequence of dual TCRalpha expression. Dramatic enhancement of the selection efficiency is observed, however, when fewer transgenic cells populate the thymus in mixed bone marrow chimeras. These results suggest that positive selection is limited by the availability of selecting peptides in the thymus. This becomes apparent when large numbers of thymocytes compete for such peptides in TCR transgenic animals. Under such conditions, thymocytes appear to undergo further TCRalpha gene rearrangement to produce a receptor that may be selected more efficiently by other thymic self-peptides.

Impaired invariant chain degradation and antigen presentation and diminished collagen-induced arthritis in cathepsin S null mice.

Cathepsins have been implicated in the degradation of proteins destined for the MHC class II processing pathway and in the proteolytic removal of invariant chain (Ii), a critical regulator of MHC class II function. Mice lacking the lysosomal cysteine proteinase cathepsin S (catS) demonstrated a profound inhibition of Ii degradation in professional APC in vivo. A marked variation in the generation of MHC class II-bound Ii fragments and presentation of exogenous proteins was observed between B cells, dendritic cells, and macrophages lacking catS. CatS-deficient mice showed diminished susceptibility to collagen-induced arthritis, suggesting a potential therapeutic target for regulation of immune responsiveness.

Peptide loading in the endoplasmic reticulum accelerates trafficking of peptide:MHC class II complexes in B cells.

In a combination of biochemical and immunoelectron-microscopical approaches we studied intracellular trafficking and localization of the endoplasmic-reticulum (ER)-formed complexes of murine MHC class II molecule I-Ab and an antigenic peptide Ealpha52-68 covalently linked to its beta-chain. The association with the peptide in the ER leads to sharp acceleration of the intracellular trafficking of the complexes to the plasma membrane. Within the cells, Ealpha52-68:I-Ab complexes accumulate in the multivesicular MHC class II compartment (MIIC), but not in denser multilaminar or intermediate type MIICs. The changes in the trafficking of ER-formed complexes result solely from the presence of the tethered peptide, since wild-type class II molecules traffic similarly in bare lymphocyte syndrome cells and in wild-type antigen-presenting cells.

Requirement for diverse, low-abundance peptides in positive selection of T cells.

Whether a single major histocompatibility complex (MHC)-bound peptide can drive the positive selection of large numbers of T cells has been a controversial issue. A diverse population of self peptides was shown to be essential for the in vivo development of CD4 T cells. Mice in which all but 5 percent of MHC class II molecules were bound by a single peptide had wild-type numbers of CD4 T cells. However, when the diversity within this 5 percent was lost, CD4 T cell development was impaired. Blocking the major peptide-MHC complex in thymus organ culture had no effect on T cell development, indicating that positive selection occurred on the diverse peptides present at low levels. This requirement for peptide diversity indicates that the interaction between self peptides and T cell receptors during positive selection is highly specific.

Evaluating peptide repertoires within the context of thymocyte development.

The process of antigen presentation by MHC molecules allows T cells to sample the proteins expressed within a particular cell. This sampling is in the form of short peptides bound within the grooves of MHC molecules displayed on the surface of cells. In the context of immune surveillance, this presentation allows the identification of infected cells by displaying peptides originating from foreign proteins within the cell. However, MHC-bound peptides play additional roles beyond serving as antigenic stimuli during an immune response. In fact, it has become clear that MHC-bound peptides derived from self proteins are critically involved in the development of T cells during selective events in the thymus. In this review we will discuss the nature of the population of MHC-bound peptides as it relates to thymocyte development, with particular emphasis on the recent finding that peptide-MHC complexes present at low levels can drive the positive selection of thymocytes.

The role of lysosomal proteinases in MHC class II-mediated antigen processing and presentation.

The recent analysis of cathepsin-deficient mice has shed light upon the role of lysosomal proteinases in the MHC class II processing and presentation pathway. Ubiquitous expression and involvement in the terminal degradation of proteins that intersect the endocytic pathway were previously perceived to be the hallmarks of these proteinases. However, recent evidence has demonstrated that several cathepsins are expressed in a tissue-specific fashion and that partial proteolysis of specific biological targets is a key function of cathepsins in antigen processing. Our work has focused on the differential expression of the cysteine proteinases cathepsins L (CL) and S (CS) and its pertinence to the generation of MHC class II: peptide complexes. Analysis of CL-deficient mice revealed a profound defect in invariant chain degradation in thymic cortical epithelial cells but not in bone marrow-derived antigen-presenting cells (APCs) (B cells, dendritic cells, and macrophages). The tissue-specific deficiency reflected the restricted pattern of expression of CL and CS in these cell types--CL is expressed in thymic cortical epithelial cells but not in DC or B cells, while CS exhibits the opposite expression pattern. The differential expression of proteinases by distinct APCs may affect the types of peptides that are presented to T cells and thereby the immune responses that are ultimately generated.

An altered invariant chain protein with an antigenic peptide in place of CLIP forms SDS-stable complexes with class II alphabeta dimers and facilitates highly efficient peptide loading.

We report an experimental system for abundant expression of specific peptide-class II complexes in vivo and in vitro. We have constructed a cassette which allows for the replacement of the CLIP region of invariant chain (Ii) with an antigenic peptide. In fibroblasts expressing an altered Ii protein, in which CLIP has been replaced with peptide 52-68 from the class II I-E alpha chain (pEalpha), pEalpha-I-Ab complexes are formed with high efficiency. This peptide loading occurs in the endoplasmic reticulum (ER) when the Ii:pEalpha fusion protein associates with the I-Ab alpha and beta chains. The trimeric complexes of Ii:pEalpha and I-Ab molecules are stable in SDS and can be detected by the pEalpha-I-Ab-specific mAb, YAe, indicating that pEalpha is bound in the class II groove in the context of full-length Ii. These data strongly suggest that the CLIP region of intact Ii prevents peptide loading in the ER by binding in the peptide binding groove of newly synthesized class II alphabeta dimers.

Subtle conformational changes induced in major histocompatibility complex class II molecules by binding peptides.

Intracellular trafficking of major histocompatibility complex (MHC) class II molecules is characterized by passage through specialized endocytic compartment(s) where antigenic peptides replace invariant chain fragments in the presence of the DM protein. These changes are accompanied by structural transitions of the MHC molecules that can be visualized by formation of compact SDS-resistant dimers, by changes in binding of mAbs, and by changes in T cell responses. We have observed that a mAb (25-9-17) that is capable of staining I-Ab on the surface of normal B cells failed to interact with I-Ab complexes with a peptide derived from the Ealpha chain of the I-E molecule but bound a similar covalent complex of I-Ab with the class II binding fragment (class II-associated invariant chain peptides) of the invariant chain. Moreover, 25-9-17 blocked activation of several I-Ab-reactive T cell hybridomas but failed to block others, suggesting that numerous I-Ab-peptide complexes acquire the 25-9-17(+) or 25-9-17(-) conformation. Alloreactive T cells were also able to discriminate peptide-dependent variants of MHC class II molecules. Thus, peptides impose subtle structural transitions upon MHC class II molecules that affect T cell recognition and may thus be critical for T cell selection and autiommunity.

Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus.

Degradation of invariant chain (Ii) is a critical step in major histocompatibility complex class II-restricted antigen presentation. Cathepsin L was found to be necessary for Ii degradation in cortical thymic epithelial cells (cTECs), but not in bone marrow (BM)-derived antigen-presenting cells (APCs). Consequently, positive selection of CD4+ T cells was reduced. Because different cysteine proteinases are responsible for specific Ii degradation steps in cTECs and BM-derived APCs, the proteolytic environment in cells mediating positive and negative selection may be distinct. The identification of a protease involved in class II presentation in a tissue-specific manner suggests a potential means of manipulating CD4+ T cell responsiveness in vivo.

Mtv-1 superantigen trafficks independently of major histocompatibility complex class II directly to the B-cell surface by the exocytic pathway.

Presentation of the Mtv-1 superantigen (vSag1) to specific Vbeta-bearing T cells requires association with major histocompatibility complex class II molecules. The intracellular route by which vSag1 trafficks to the cell surface and the site of vSag1-class II complex assembly in antigen-presenting B lymphocytes have not been determined. Here, we show that vSag1 trafficks independently of class II to the plasma membrane by the exocytic secretory pathway. At the surface of B cells, vSag1 associates primarily with mature peptide-bound class II alphabeta dimers, which are stable in sodium dodecyl sulfate. vSag1 is unstable on the cell surface in the absence of class II, and reagents that alter the surface expression of vSag1 and the conformation of class II molecules affect vSag1 stimulation of superantigen reactive T cells.

Altered antigen presentation in mice lacking H2-O.

HLA-DM catalyzes the release of MHC class II-associated invariant chain-derived peptides (CLIP) from class II molecules. Recent evidence has suggested that HLA-DO is a negative regulator of HLA-DM in B cells, but the physiological function of HLA-DO remains unclear. Analysis of antigen presentation by B cells from mice lacking H2-O (the mouse equivalent of HLA-DO), together with biochemical analysis using purified HLA-DO and HLA-DM molecules, suggests that HLA-DO/H2-O influences the peptide loading of class II molecules by limiting the pH range in which HLA-DM is active. This effect may serve to decrease the presentation of antigens internalized by fluid-phase endocytosis, thus concentrating the B cell-mediated antigen presentation to antigens internalized by membrane immunoglobulin.

Invariant chain-independent function of H-2M in the formation of endogenous peptide-major histocompatibility complex class II complexes in vivo.

Efficient loading of major histocompatibility complex class II molecules with peptides requires the invariant chain (Ii) and the class II-like molecule H-2M. Recent in vitro biochemical studies suggest that H2-M may function as a chaperone to rescue empty class II dimers. To test this hypothesis in vivo, we generated mice lacking both Ii and H-2M (Ii-/-M-/-). Antigen presenting cells (APCs) from Ii-/-M-/- mice, as compared with APCs from Ii-/- mice, exhibit a significant reduction in their ability to present self-peptides to a panel of class II I-Ab-restricted T cells. As a consequence of this defect in the loading of self peptides, CD4(+) thymocyte development is profoundly impaired in Ii-/-M-/- mice, resulting in a peripheral CD4(+) T cell population with low levels of T cell receptor expression. These findings are consistent with the idea that H-2M functions as a chaperone in the peptide loading of class II molecules in vivo.

Major histocompatibility complex class II compartments in human and mouse B lymphoblasts represent conventional endocytic compartments.

In most human and mouse antigen-presenting cells, the majority of intracellular major histocompatibility complex (MHC) class II molecules resides in late endocytic MHC class II compartments (MIICs), thought to function in antigen processing and peptide loading. However, in mouse A20 B cells, early endocytic class II-containing vesicles (CIIVs) have been reported to contain most of the intracellular MHC class II molecules and have also been implicated in formation of MHC class II-peptide complexes. To address this discrepancy, we have studied in great detail the endocytic pathways of both a human (6H5.DM) and a mouse (A20.Ab) B cell line. Using quantitative immunoelectron microscopy on cryosections of cells that had been pulse-chased with transferrin-HRP or BSA-gold as endocytic tracers, we have identified up to six endocytic subcompartments including an early MIIC type enriched in invariant chain, suggesting that it serves as an important entrance to the endocytic pathway for newly synthesized MHC class II/invariant chain complexes. In addition, early MIICs represented the earliest endocytic compartment containing MHC class II- peptide complexes, as shown by using an antibody against an abundant endogenous class II-peptide complex. The early MIIC exhibited several though not all of the characteristics reported for the CIIV and was situated just downstream of early endosomes. We have not encountered any special class II-containing endocytic structures besides those normally present in nonantigen-presenting cells. Our results therefore suggest that B cells use conventional endocytic compartments rather than having developed a unique compartment to accomplish MHC class II presentation.

Deficient positive selection of CD4 T cells in mice displaying altered repertoires of MHC class II-bound self-peptides.

The role of self-peptides in positive selection of CD4+ T cells has been controversial. We show that some self-peptides are presented by the MHC class II molecule I-A(b) in mice lacking Ii or H-2M but not in mice expressing a transgene-encoded peptide fused to I-A(b). In experiments using specific antibodies to block selection, these low-abundance self-peptides were implicated in the positive selection of some CD4+ T cells in H-2M-/- mice. However, all three mutant backgrounds failed to positively select two class II-restricted transgenic T cell receptors. Our findings suggest that minor components of the self-peptide repertoire can contribute to positive selection of a significant number of CD4+ T cells. In addition, the data suggest that T cell receptor repertoires selected in wild-type mice and in mice displaying limited spectra of self-peptides are distinct.