Positive selection of CD4(+) T cells is induced in vivo by agonist and inhibited by antagonist peptides.

The nature of peptides that positively select T cells in the thymus remains poorly defined. Here we report an in vivo model to study the mechanisms of positive selection of CD4(+) T cells. We have restored positive selection of TCR transgenic CD4(+) thymocytes, arrested at the CD4(+)CD8(+) stage, due to the lack of the endogenously selecting peptide(s), in mice deficient for H2-M and invariant chain. A single injection of soluble agonist peptide(s) initiated positive selection of CD4(+) transgenic T cells that lasted for up to 14 days. Positively selected CD4(+) T cells repopulated peripheral lymphoid organs and could respond to the antigenic peptide. Furthermore, coinjection of the antagonist peptide significantly inhibited agonist-driven positive selection. Hence, contrary to the prevailing view, positive selection of CD4(+) thymocytes can be induced in vivo by agonist peptides and may be a result of accumulation of signals from TCR engaged by different peptides bound to major histocompatibility complex class II molecules. We have also identified a candidate natural agonist peptide that induces positive selection of CD4(+) TCR transgenic thymocytes.

Alpha beta TCRs differ in the degree of their specificity for the positively selecting MHC/peptide ligand.

We have tested the peptide specificity of positive selection using three transgenic alphabetaTCRs, originally selected on class II MHC (A(b)) covalently bound with one peptide Ealpha (52-68) (Ep). The transgenic TCR specific for the cytochrome c-derived (43-58) peptide was selected on A(b) bound with different arrays of endogenous peptides or the analogue of Ep covalently bound to A(b), but not on the original A(b)Ep complex. In contrast, transgenic TCRs specific for two different analogues of the Ep peptide and A(b) did not mature as CD4(+) T cells in various thymic environments, including the A(b)EpIi(-) mice. These results show that TCRs can be promiscuous or specific for the selecting MHC/peptide complex, and suggest that in mice described in this study transgenic expression of the TCR changes the original requirements for the positively selecting MHC/peptide complex. Future studies will determine whether the latter phenomenon is general or specific for this system.

An incremental increase in the complexity of peptides bound to class II MHC changes the diversity of positively selected alpha beta TCRs.

Positive selection of the normal repertoire of TCRs results from low-avidity interactions with a set of self-peptides bound to the MHC molecules expressed by thymic epithelial cells. The contribution of the individual peptide to positive selection remains a matter of debate. Here, for the first time, we show that two covalent class II MHC-peptide complexes positively select different TCRs expressing a common transgenic TCRbeta-chain and endogenous TCRalpha-chains. Simultaneous expression of both A(b)-peptide complexes changed the diversity of positively selected TCRs, indicating an additive and possibly synergistic effect of various peptides in this process.

Altered selection of CD4+ T cells by class II MHC bound with dominant and low abundance self-peptides.

We have investigated the development of CD4(+) T cells in mice expressing low levels of transgenic class II MHC molecules (A(b)) preoccupied with covalent peptide (Ep), which in the presence of invariant chain (Ii) is extensively cleaved and replaced with self-derived peptides. In these mice, the transgenic A(b) molecules, bound with predominant peptide (Ep) and with multiple self-peptides, selected more CD4(+) T cells than A(b)/self-peptide complexes expressed in wild-type mice. The enhanced outcome of thymic selection was a result of impaired negative selection, rather than more efficient positive selection by an overall lowered abundance of self-derived A(b)/peptide complexes. Peripheral CD4(+) T cells in the A(b)EpIi(+) mice had memory phenotype, often followed by polyclonal activation of B cells. The A(b)EpIi(+) mice preserved their good health and had a normal life span despite the profound number of activated CD4(+) T cells and B cells in peripheral lymphoid organs, moderate hypergammaglobulinemia, and deposited complexes in the kidneys. We propose that CD4(+) T cells positively selected due to low avidity for high abundant A(b)Ep complex avoid negative selection on A(b) molecules loaded with low abundant peptides and become self-reactive in the peripheral lymphoid organs.

The alloreactive and self-restricted CD4+ T cell response directed against a single MHC class II/peptide combination.

The cellular basis for allograft rejection derives from the strong T cell response to cells bearing foreign MHC. While it was originally assumed that alloreactive T cells focus their recognition on the polymorphic residues that differ between syngeneic and allogeneic MHC molecules, studies with MHC class I-restricted CTL have shown that MHC-bound peptides play a critical role in allorecognition. It has been suggested that alloreactive T cells depend more strongly on interactions with the MHC molecule than with the associated peptide, but there is little evidence to support this idea. Here we have studied the alloreactive and self-restricted response directed against the class II H2-Ab molecule bound with a single peptide, Ep, derived from the H2-Ealpha chain. This MHC class II-peptide combination was a poor target and stimulator of alloreactive CD4+ T cell responses, indicating that MHC-bound peptides are as important for alloreactive CD4+ T cells as they are for alloreactive CTL. We also generated alloreactive T cells with exquisite specificity for the Ab/Ep complex, and compared their reactivity with self-restricted T cells specific for the same Ab/Ep complex. Our results showed that peptide-specific alloreactive T cells, as compared with self-restricted T cells, were more sensitive to peptide stimulation, but equally sensitive to amino acid substitutions in the peptide. These findings indicate that alloreactive and self-restricted T cells interact similarly with their MHC/peptide ligand.

Mature CD4+ T cells perceive a positively selecting class II MHC/peptide complex in the periphery.

A repertoire of TCRs is selected in the thymus by interactions with MHC bound to self-derived peptides. Whether self peptides bound to MHC influence the survival of mature T cells in the periphery remains enigmatic. In this study, we show that the number of naive CD4+ T cells that developed in mice with class II MHC bound with endogenous peptides (Abwt) diminished when transferred into mice with Ab covalently bound with a single peptide (AbEp). Moreover, transfer of a mixture of naive CD4+ T cells derived from Abwt and from AbEp mice into AbEp mice resulted in the expansion of the latter and decline of the former. In contrast, when wild-type activated CD4+ T cells were transferred into AbEp or Abwt mice, these cells survived in both recipients for more than 4 wk, but further expanded in the Abwt host. We conclude that to survive, naive CD4+ T cells favor peripheral expression of the class II MHC/peptide complex(es) involved in their thymic selection, whereas some of activated CD4+ T cells may require them only for expansion.

On the role of high- and low-abundance class II MHC-peptide complexes in the thymic positive selection of CD4(+) T cells.

The role of self-peptides bound to MHC molecules in the selection of T cells in the thymus remains controversial. Here, we have tested whether a high-abundance single class II MHC-peptide complex has a dominant effect on the repertoire of CD4(+) T cells selected by low-abundance class II MHC-peptide complexes. For these studies, we have used H-2(b) mice that lack an invariant chain (Ii) (A(b)Ii(-)) and their transgenic variant (A(b)A(b)EpIi(-)) that co-expresses A(b) molecules covalently bound with a single peptide Ep(52-68). In these latter mice, close to 50% of all A(b) molecules are occupied by Ep(52-68) peptide. Although the A(b)Ep complex was abundantly expressed in the thymus under conditions excluding negative selection on bone marrow-derived cells, no striking quantitative difference between repertoires of TCR expressed on CD4(+) T cells in A(b)Ii(-) and A(b)A(b)EpIi(-) mice was noticed. Our results are consistent with the view that diverse, low-abundance self-peptides play an important role in thymic positive selection and do not support the notion that dominant, high-abundance peptides may be critical for shaping the TCR repertoire.

Class II-associated invariant chain peptide-independent binding of invariant chain to class II MHC molecules.

The class II-associated invariant chain peptide (CLIP) region of invariant chain (Ii) is believed to play a critical role in the assembly and transport of MHC class II alphabetaIi complexes through its interaction with the class II peptide-binding site. The role of the CLIP sequence was investigated by using mutant Ii molecules with altered affinity for the DR1 peptide-binding site. Both high- and low-affinity mutants were observed to efficiently assemble with DR1 and mediate transport to endosomal compartments in COS cell transfectants. Using N- and C-terminal truncations, a region adjacent to CLIP within Ii(103-118) was identified that can complement loss of affinity for the peptide-binding site in mediating efficient assembly of alphabetaIi. A C-terminal fragment completely lacking the CLIP region, Ii(103-216), was observed binding stably to class II molecules in immunoprecipitation studies and experiments with purified proteins. The Ii(103-118) region was required for this binding, which occurs through interactions outside of the alphabeta peptide-binding groove. We conclude that strong interactions involving Ii(103-118) and other regions of Ii cooperate in the assembly of functional alphabetaIi under conditions where CLIP has little or no affinity for the class II peptide-binding site. Our results support the hypothesis that the CLIP sequence has evolved to avoid high-stability interactions with the peptide-binding sites of MHC class II molecules rather than as a promiscuous binder with moderate affinity for all class II molecules.

In the normal repertoire of CD4+ T cells, a single class II MHC/peptide complex positively selects TCRs with various antigen specificities.

In the thymus, immature T cells are positively and negatively selected by multiple interactions between their Ag receptors (TCRs) and self MHC/peptide complexes expressed on thymic stromal cells. Here we show that in the milieu of negative selection on physiological self class II MHC/peptide complexes (Abwt), a single class II/peptide complex AbEp52-68 positively selects a number of TCRs with various Ag specificities. This TCR repertoire is semidiverse and not biased toward Ep-like Ags. Our finding implies that the degeneracy of positive selection for peptide ligands exceeds peptide-specific negative selection and is essential to increase the efficiency and diversity of the repertoire so that T cells with the same Ag specificity can be selected by different self MHC/ peptide complexes.

Invariant chain can bind MHC class II at a site other than the peptide binding groove.

Invariant chain binds to class II molecules and guides them to the cell surface via the endosomes. Class II-associated invariant chain peptide (CLIP), a conserved sequence in an unstructured region of invariant chain, binds in the peptide binding groove of class II and is thought to be the major contributor to the interaction between invariant chain and class II molecules. However, other interaction sites between the two proteins may exist. The published data on this subject are conflicting. We have studied the ability of invariant chain to interact with a class II molecule in which the peptide binding groove of the protein is already occupied by a covalently attached peptide. Precipitation of these class II/peptide complexes with an Ab specific for this particular combination also precipitates invariant chain. This binding between class II/peptide and invariant chain is weak, and coprecipitation is only apparent in mild detergents. Thus, when the class II peptide binding groove is occluded by peptide and is not free to interact with CLIP, invariant chain can still bind the class II molecule at other lower affinity sites.

T cells can be activated by peptides that are unrelated in sequence to their selecting peptide.

We tested the ability of CD4+ T cells, selected in the thymus by reaction with class II protein bound to a single peptide, to react with the same class II protein bound to other peptides. The T cells reacted with all peptides tested, including one that was quite unlike the selecting peptide in T cell receptor binding residues. The receptors on class II/peptide-reactive T cells from class II/single peptide mice were similar but not identical to some of those from normal animals. Thus, class II bound to a single peptide selects a subset of T cells that is related to that selected by class II bound to many peptides.

The responses of mature T cells are not necessarily antagonized by their positively selecting peptide.

Transgenic mice have been produced in which all detectable MHC class II proteins, IAb, are bound to a single peptide. Hence, all of the CD4+ class II-restricted T cells in these animals are positively selected in the thymus by reaction with this single class II/peptide combination. It has been suggested that the peptide involved in positive selection of a particular T cell might antagonize the responses of that T cell to other peptides, a phenomenon that might serve to inhibit autoimmune reactions. To test this idea, we took advantage of the fact that T cells from the class II single-peptide mice react strongly in primary mixed lymphocyte cultures with IAb molecules from wild-type H-2b mice, i.e., loaded with a heterogeneous collection of self peptides. The responses of the T cells to other class II-bound peptides were not inhibited by high concentrations of the selecting peptide. Therefore, peptides involved in positive selection of T cells do not necessarily inhibit or antagonize the responses of mature T cells to Ag.

The repertoire of T cells shaped by a single MHC/peptide ligand.

Although the thymus produces many immature thymocytes, few of these cells mature. Positive selection has been thought to limit thymocyte development. In thymuses expressing a single MHC/peptide combination, however, surprisingly large numbers of thymocytes are selected to mature. Many of these react with the selecting MHC, bound to other self-peptides. Therefore, the number of thymocytes that mature is limited by the fact that positively selected cells die because they react too well with MHC bound to self-peptides that are not identical to those involved in positive selection. T cells that mature in thymuses expressing a single MHC/peptide ligand react frequently with foreign MHC, suggesting that the repertoire of alpha beta receptors may be more biased toward reaction with MHC than was previously thought.

The use of mammary tumor virus (Mtv)-negative and single-Mtv mice to evaluate the effects of endogenous viral superantigens on the T cell repertoire.

Most laboratory strains of mice have between two and eight endogenous superantigens. These viral superantigens (vSAGs) are coded by genes in the 3' long terminal repeats of endogenous mammary tumor viruses (Mtv's). A line of Mtv-negative mice and several lines of mice containing single Mtv's were created by inbreeding the F2 progeny of CBA/CaJ and C58/J mice, which have no Mtv integrants in common. This allowed the T cell repertoire of H-2k mice, unaffected by Mtv superantigens, as well as the effects of vSAGs upon that repertoire, to be studied. Although each individual mouse had a different mix of C58/J and CBA/CaJ background genes, the T cell repertoires of different Mtv-negative mice were very similar and were reproducible. Since the background genes did not affect the V beta repertoire, there are no super-antigens, other than those encoded by Mtv's, that differ between CBA/CaJ and C58/J. CD4 and CD8 T cells had quite different repertoires in the Mtv-negative mice because of the effects of class I and class II major histocompatibility complex molecules on positive and negative selection. vSAG3 was found to delete V beta 5 T cells, while vSAG8 deleted V beta 7 T cells, and vSAG9 deleted V beta 13 T cells in addition to their previously reported specificities. vSAG17 deletes a small proportion of CD4+ T cells bearing V beta 11 and -12. vSAG14 and -30 have little effect on the T cell repertoire and are not expressed in thymocytes and splenocytes. An endogenous superantigen that has a low avidity for a particular V beta may positively select thymocytes, leading to an increased frequency of peripheral T cells bearing the relevant V beta s. We found evidence that vSAG11 may positively select T cells bearing V beta 8.2. Our data, which analyzed the effects of seven endogenous Mtv's, showed little evidence of positive selection by any other vSAGs on T cells bearing any V beta tested, despite published reports to the contrary.

Cell surface expression of class II MHC proteins bound by a single peptide.

On normal cells, the peptide-binding grooves of class II MHC proteins contain a wide spectrum of peptides. For some purposes, however, it would be helpful to have cells bearing class II proteins engaged by only one peptide species. In an attempt to make such cells we constructed a gene for a MHC class II beta-chain, IA beta b, covalently linked to a peptide, E alpha 52-68, which is known to bind to the peptide-binding groove of IAb. This gene, together with the gene for IA alpha b, was transfected into B lymphoma cells and fibroblasts. The IAb-E alpha complex was expressed on the surfaces of these cells where it could be recognized by a mAb and T cells specific for IAb plus E alpha 52-68. Most of the peptide on fibroblasts remained covalently attached to the IAb beta-chain, but the covalent linker and/or peptide were degraded to some extent on B lymphoma cells. Nearly all of the IAb expressed by transfected fibroblasts was occupied by the E alpha peptide. Of 16 IAb-reactive T cell hybridomas, only 3 could respond to the IAb-E alpha complex on fibroblasts, confirming the idea that recognition of MHC may often involve recognition of the peptides bound to the MHC as well.

Identification of two V beta 7-specific viral superantigens.

The commonly used strains of laboratory mice have mouse mammary tumor viruses (MTV) integrated at various locations in their DNA. The number and position of these integrants varies from one strain of mouse to another. It has recently been shown that the genomes of many of the MTV code for superantigens. The predicted amino acid sequences of these superantigens and their specificity for TCR V beta differs for each MTV integrant. This study contains the predicted amino acid sequence and V beta specificity of two MTV superantigens that had not previously been analyzed. The results show that both of these MTV superantigens are specific for TCR that bear V beta 7, but unlike the MTV7 superantigen not for receptors bearing V beta 6 or V beta 8.1. The data also support the conclusion of previous studies that the COOH-terminal sequence of these proteins is a major factor in controlling their V beta reactivity.

Comparison of peptides bound to spleen and thymus class II.

In the past we and others have suggested that positive selection of developing thymocytes may depend upon interaction between the alpha beta receptors on these cells and major histocompatibility complex (MHC) proteins bound to peptides found uniquely in the selecting tissue, thymus cortical epithelium. To test this hypothesis, peptides were isolated from MHC class II proteins of spleen, thymus cortical plus medullary epithelium, or thymus cortical epithelium alone. The results showed that the major peptides bound to class II on thymus cortical epithelium were also associated with spleen class II. Some peptides could only be detected in isolates from spleen, probably because of differences in the distribution or uptake of the donor proteins between spleen and thymus. Thus, although we found some tissue-specific distribution of self-peptides, our data suggest that there are no fundamental differences among these tissues in the occupancy of class II MHC by self-peptides. These results limit hypotheses which depend on a specialized mechanism of peptide generation and/or MHC class II loading to account for the positive selection of T cells on thymic cortical epithelium.

T cell tolerance. Giles F. Filley Lecture.

T cell tolerance to self is induced by several mechanisms. Immature T cells may die, and mature T cells may die or be inactivated by contact with self. While we do not understand completely what governs which of these courses will be adopted by an autoreactive T cell, 2 points are clear. Self is in part distinguished from nonself by the fact that the former is always present, while the latter is only intermittently in the body. Secondly, it seems that the default response for T cells is "off" rather than "on." That is, peripheral T cells will become tolerant to antigen unless some other phenomenon such as adjuvant triggers them to respond. One other point should be mentioned. In spite of the power of the mechanisms which causes self tolerance, tolerance is not complete. Autoreactive T cells specific for cryptic antigens, expressed in the brain or eye for example, can easily be demonstrated. Many autoimmune diseases may be caused by aberrant activation and exposure to target organ of these cells, rather than failure of the normal mechanisms of tolerance themselves.

The effects of chronic infection with a superantigen-producing virus.

C3H/HeJ mice transmit a mouse mammary tumor virus from mother to pup in milk. The retrovirus infects mice shortly after birth and, when expressed in recipient mice, produces a V beta 14-specific superantigen. The consequences of such expression on V beta 14-bearing T cells are examined in this paper. Most cells bearing V beta 14 and either CD4 or CD8 are eliminated in the thymus. Some V beta 14-bearing cells escape to the periphery, however. Those bearing CD8 are unaffected by expression of the viral superantigen. The percentage of peripheral CD4+ T cells bearing V beta 14 drops with time after birth. In large part this seems to be due to the fact that many of these cells become anergic because of exposure to the viral superantigen. Unlike normal T cells, these anergic cells cannot undergo peripheral postthymic expansion. Consequently, they drop in percentage even during a time when their total numbers are constant.