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.

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.

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.

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.

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.

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.

Endogenous peptides associated with MHC class II and selection of CD4 T cells.

CD4 T cells undergo positive and negative selection during thymic development mediated by the interaction of T-cell receptors with MHC class II molecules expressed by thymic epithelial cells and bone marrow-derived antigen presenting cells in the thymus. The majority of MHC molecules are occupied with peptides derived from self proteins. Although the role of self peptides in the negative selection of CD4 cells is well established, the influence of peptides on positive selection of CD4 T cells remained elusive. However, some recent results suggest a role of peptides in the positive selection of CD4 T cells.

Immunoglobulin-specific T-B cell interaction. I. Presentation of self immunoglobulin determinants by B lymphocytes.

Immunoglobulin (Ig)-specific T-B cell interactions have been studied in the model of T cell recognition of the kappa chain Ig kappa-1b allotype in Ig kappa-1-congeneic rat strains. An efficient presentation of endogenous Ig allotypic determinants by irradiated spleen cells from (WAG.1b x August)F1 (RT-1u/c; Ig kappa-1b/1a) rats to Ig kappa-1b-specific lymph node T cells from Ig kappa-1-congeneic (WAG x August)F1 (RT-1u/c; Ig kappa-1a) rats was demonstrated. This presentation was found to be sensitive to high irradiation doses (greater than 1000 rad). By fractionation of Ig kappa-1b+ F1 spleen cells on Percoll density gradient we have shown that a radioresistant, low-density fraction, consisting mainly of macrophages (M phi) and dendritic cells, triggers only weak Ig kappa-1b-specific T cell response. The high level of response was observed against radiosensitive spleen cell fractions of intermediate and high density, suggesting that B cells were the main antigen-presenting cells (APC) of Ig kappa-1b determinants of endogenous Ig. This conclusion was confirmed in the experiments using purified B cells from Ig kappa-1b-bearing rats. Earlier we have shown that the responsiveness of August (RT-1c; Ig kappa-1a) and WAG (RT-1u; Ig kappa-1a) rats to Ig kappa-1b in vivo is controlled by the dominant allele of an RT-1-linked Ir gene. August and (August X WAG)F1 rats were found to be responders to Ig kappa-1b while WAG rats were nonresponders. The same pattern of Ir gene-controlled reactivity was demonstrated using an Ig kappa-1b-specific T cell proliferation assay. Ig kappa-1b-specific F1 T cell response was only observed when Ig kappa-1b+ B cells or IgG (Ig kappa-1b)-pulsed M phi-bearing responder major histocompatibility complex (MHC) haplotype were used as the APC. Anti-RT-1 monoclonal antibody inhibition studies suggested that the RT-1Bc molecule is the main restricting element of T cell recognition of Ig kappa-1b+ B cell as well as exogenous IgG (Ig kappa-1b). We have demonstrated allelic exclusion of Ig kappa-1b presenting function by negatively and positively selecting for Ig kappa-1b+ and Ig kappa-1a+ B cells from heterozygous F1(Ig kappa-1b/1a) rats. This clearly indicate that the B cells presented exclusively Ig kappa-1b allotypic determinants of their own Ig.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

Phenotype and function of CD4+ T cells in mice lacking invariant chain.

Mice bearing a targeted disruption of the gene encoding the invariant chain (Ii) have provided a deeper understanding of the critical role that Ii plays in the assembly, transport, and peptide occupancy of MHC class II molecules. In this study, we have investigated the consequence of the altered class II expression in Ii-deficient (Ii zero) mice on the phenotype and function of their CD4+ T cells. As seen by others, these mice show a sharp reduction in the CD4+8- subset in the thymus and periphery. Furthermore, a large proportion of the peripheral CD4+, but not CD8+, T cells in Ii zero mice exhibit decreased surface TCR-alpha beta levels and express markers of T cell activation. Although the CD4+ T cells respond to mitogens, anti-CD3 mAbs, and alloantigens, they respond poorly to the bacterial superantigen staphylococcal enterotoxin B and do not respond to peptide and protein Ags. Analysis of reciprocal radiation bone marrow chimeras constructed using Ii zero and wild-type mice indicate that the lack of Ii in the thymus is responsible for the phenotype of the CD4+ T cells in mutant mice. These results suggest that the altered thymic peptide repertoire displayed by class II molecules in the absence of Ii has a dramatic impact on the selection of CD4+ T cells and their phenotype in the periphery.

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.

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.

Immunoglobulin-specific T-B cell interaction. III. B cell activation by immunoglobulin-recognizing T cell clones.

Immunoglobulin (Ig)-specific T-B cell interactions were studied in the model of T cell recognition of Ig kappa chain Ig kappa-1b allotype in Ig kappa-1-congenic rats. Using Ig kappa-1b-recognizing major histocompatibility complex (MHC)-restricted T helper clones from August rats we have shown that Ig kappa-1b+ B cells from congenic August.1b rats presented Ig kappa-1b epitope of the processed self-synthesized Ig to T clones. This interaction was found to be a bidirectional regulatory event inducing specific MHC-dependent proliferation of both interacting T cell and B cell as well as Ig(Ig kappa-1b) synthesis. Small Ig kappa-1b+ B cells were capable of inducing T clone proliferation and becoming activated in response to the same T clone. Limiting dilution analysis suggested that every tenth cell in Ig kappa-1b+ B cell population is involved in this interaction. The bystander activation of Ig kappa-1a+ B cells by T clones in the presence of irradiated Ig kappa-1b+ spleen cells, if observed, was less than the level of specific Ig kappa-1b+ B cell proliferation. In contrast to a 20-fold increase of Ig(Ig kappa-1b) levels upon stimulation of Ig kappa-1b+/1a+ B cell population from heterozygous (August x August.1b)F1 rats by T clones, a "nonspecific" increase of Ig(Ig kappa-1a) was not observed. This result demonstrates the requirement for direct T-B contact for B cell activation to occur. The data suggest a great functional potency of T-B interactions mediated by T cell recognition of Ig-derived peptide/MHC class II complexes on the B cell surface. The implication of the data for idiotypic regulation enables us to propose the existence of putative idiopeptidic network T-B cell interactions.

Presentation of endogenous immunoglobulin determinant to immunoglobulin-recognizing T cell clones by the thymic cells.

Using immunoglobulin (Ig)-recognizing T helper clones the expression of Ig peptide/major histocompatibility complex class II complexes derived by the processing of endogeneous Ig molecules in the thymus was demonstrated. It was found that thymic B cells but not "classic" thymic antigen-presenting cells and macrophages represent the major antigen-presenting cell type of determinants of endogenously synthesized surface Ig (Ig kappa-1b) and anti-surface Ig antibodies (IdC3B9). The Ig kappa-1b-presenting activity in the thymus appears relatively late, only after 3 weeks of postnatal life, while in the spleen an efficient presentation of endogenous Ig kappa-1b epitope is observed very early after birth. This difference between thymic and peripheral presentation of endogeneous Ig determinant could be important for understanding the mechanisms of T cell tolerance to self Ig and the role of self Ig in negative and positive selection of T cell repertoire.

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.

Variation in HLA-DM expression influences conversion of MHC class II alpha beta:class II-associated invariant chain peptide complexes to mature peptide-bound class II alpha beta dimers in a normal B cell line.

The maturation of invariant chain (Ii):MHC class II complexes into peptide-loaded alpha beta dimers occurs by proteolytic removal of Ii chain and binding of antigenic peptides derived from exogenous and endogenous Ags. A fragment of the Ii chain (class II-associated invariant chain peptide (CLIP) remains associated with class II alpha beta and is an intermediate in this process. Conversion of alpha beta:CLIP complexes into alpha beta:peptide complexes is facilitated by HLA-DM. Two unique mAbs, specific for I-Ab bound to human CLIP and I-Ab bound to DR alpha peptide, were used to assess the formation of these peptide:class II complexes in a human B lymphoblastoid cell line (B-LCL) (Swei) transfected with I-A(b). In multiple independent Swei:I-Ab transfectants, the amount of human CLIP (hCLIP):I-Ab expressed was inversely proportional to the amount of DR alpha 52-68:I-Ab; quantitative differences in HLA-DM expression accounted for this phenotype. In the low DM transfectant, a substantial proportion of I-Ab, but not DR molecules, was altered structurally and unable to present native protein Ags. Addition of DM transgenes to the DM-low cells resulted in an increase in DR alpha 52-68:I-Ab coupled with a decrease in hCLIP:I-Ab complexes and restoration of exogenous protein Ag presentation. The DR5 molecules in Swei cells, which have a lower affinity for hCLIP than I-Ab, were not affected by low DM expression, suggesting that the amount of DM required for conversion of CLIP:class II to peptide:class II may depend on the affinity of the class II molecules for CLIP or DM.

Immunoglobulin-specific T-B cell interaction. IV. B cell presentation of idiotypic determinant(s) of monoclonal anti-surface immunoglobulin antibody to idiotope-recognizing helper T clones.

Previously, we have demonstrated T-B cell interactions mediated by T cell recognition of immunoglobulin (Ig) peptide/major histocompatibility complex (MHC) class II complexes derived by the B cell processing of endogenously synthesized Ig molecules. In this report Ig-specific T-B cell interaction mediated by B cell presentation of idiotopes (Id) of anti-sIg antibodies to Id-specific T cell clones has been studied in Ig kappa-1-congenic rat strains. A panel of August (RT-1c; Ig kappa-1a) rat T helper clones specific for Id of syngeneic anti-Ig kappa-1b C3B9 monoclonal antibody (mAb) has been developed to study IdC3B9 presentation by Ig kappa-1b-bearing B cells from congenic August.1b (RT-1c; Ig kappa-1b) rats. Five of seven IdC3B9-specific T clones responded even at very low concentrations (100-200 pg/ml) of C3B9 mAb presented by Ig kappa-1b+ B cells. In contrast, the presentation of intact C3B9 mAb by nonspecific antigen-presenting cells (macrophages, Ig kappa-1a+ B cells, etc.) to IdC3B9-specific T cells was of low efficiency. The IdC3B9-specific T cell response to idiotopes of anti-Ig kappa-1b C3B9 mAb was found to be restricted by RT-1B molecule and required the processing of intact C3B9 molecule. IdC3B9 epitope recognized by C31 and C5 clones was mapped to the heavy chain of C3B9 mAb. Thus, B cells are able to present peptides related to the V region of anti-sIg Ab, i.e. Id peptide/MHC class II complexes, to Id-recognizing T cells. IdC3B9-presenting B cells are specifically activated both to proliferation and Ig production upon interaction with IdC3B9-specific T clones. Based on the results of our studies on B cell presentation of Ig epitopes to T cells a hypothetical model of Ig peptide-driven T-B cell interaction has been proposed.

Immunoglobulin-specific T-B cell interaction. II. T cell clones recognize the processed form of B cells' own surface immunoglobulin in the context of the major histocompatibility complex class II molecule.

In the preceding report (Eur. J. Immunol. 1989. 19: 1677) we have demonstrated that normal B cells, including small B cells, are capable of presenting Ig kappa-1b allotypic determinants of their endogeneously synthesized Ig+ to Ig kappa-1b-immune major histocompatibility complex (MHC) class II-restricted T cells. A panel of Ig kappa-1b allotype-specific T cell clones from August rats has been developed to study further the presentation of self surface Ig by B cells from Ig kappa-1-congeneic August.1b rats. All the clones studied were of the T helper/inducer phenotype (W3/25+,OX8-) and restricted by the RT-1Bc molecule. These clones responded both to the serum IgG(Ig kappa-1b) in the presence of irradiated spleen cells (SC) from August rats and to the Ig kappa-1b-bearing irradiated B cells from August.1b rats. SC presentation of secreted IgG was much less effective than B cell presentation of membrane Ig. Using CNBr cleavage of isolated C kappa (Ig kappa-1b) domain followed by high-performance liquid chromatography fractionation of the derived antigenic peptides, the kappa chain sequence between amino acids 176 and 214 has been identified as the T cell epitope recognized by all T cell clones in association with RT-1Bc. The fragment 176-214 of the Ig kappa-1b allotype differs from that of Ig kappa-1a allotype by three amino acid substitutions at positions 184, 185, 188. T cell recognition of pL kappa-1b(176-214) required no additional processing by the antigen-presenting cell: the efficient presentation of the peptide but not of intact IgG(Ig kappa-1b) by the paraformaldehyde-fixed SC was observed. These data provide clear-cut evidence for an absolute requirement of the processing of Ig molecules for T cell recognition to occur in our experimental system. Although the fixation of B cells from August.1b rats diminished their Ig kappa-1b-presenting ability, fixed Ig kappa-1b+ B cells were still able to induce Ig kappa-1b-specific T cell clone responses. Our results suggest that B cells can express the processed form of self-synthesized surface Ig in addition to intact surface Ig molecules. The former can be recognized by MHC-restricted T cell.