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.     

Cutting edge: dueling TCRs: peptide antagonism of CD4+ T cells with dual antigen specificities.

T cells expressing two different TCRs were generated by interbreeding 3A9 and AND CD4+ TCR transgenic mice specific for the hen egg lysozyme (HEL) peptide 48-62:I-Ak and moth cytochrome c (MCC) peptide 88-103:I-Ek peptide:MHC ligands, respectively. Peripheral T cells in the offspring express two TCR V beta-chains and respond to HEL and MCC. We observed minimal or no additive effects upon simultaneous suboptimal stimulation with both agonist peptides; however, an antagonist peptide for the 3A9 TCR was able to inhibit the response of the dual receptor T cells to MCC, the AND TCR agonist. This HEL antagonist peptide did not affect AND single transgenic T cells, indicating that the antagonism observed in the dual TCR cells is dependent on the presence of the HEL-specific 3A9 TCR. In contrast, anti-TCR Abs mediate receptor-specific antagonism. These results demonstrate that peptide antagonism exerts a dominant effect.     

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.     

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.     

How the T cell repertoire becomes peptide and MHC specific

T cells bearing alphabeta T cell receptors (TCRs) recognize antigens in the form of peptides bound to class I or class II major histocompatibility proteins (MHC). TCRs on mature T cells are usually very specific for both peptide and MHC class and allele. They are picked out from a precursor population in the thymus by MHC-driven positive and negative selection. Here we show that the pool of T cells initially positively selected in the thymus contains many T cells that are very crossreactive for peptide and MHC and that subsequent negative selection establishes the MHC-restriction and peptide specificity of peripheral T cells. Our results also suggest that germline-encoded TCR variable elements have an inherent predisposition to react with features shared by all MHC proteins.     

MHC allele-specific molecular features determine peptide/HLA-A2 conformations that are recognized by HLA-A2-restricted T cell receptors

The structures of alphabeta TCRs bound to complexes of class I MHC molecules and peptide show that the TCRs make multiple contacts with the alpha1 and alpha2 helixes of the MHC. Previously we have shown that the A6 TCR in complex with the HLA-A2/Tax peptide has 15 contact sites on HLA-A2. Single amino acid mutagenesis of these contact sites demonstrated that mutation of only three amino acids clustered on the alpha1 helix (R65, K66, A69) disrupted recognition by the A6 TCR. In the present study we have asked whether TCRs that recognize four other peptides presented by HLA-A2 interact with the MHC in identical, similar, or different patterns as the A6 TCR. Mutants K66A and Q155A had the highest frequency of negative effects on lysis. A subset of peptide-specific CTL also selectively recognized mutants K66A or Q155A in the absence of exogenous cognate peptides, indicating that these mutations affected the presentation of endogenous peptide/HLA-A2 complexes. These findings suggest that most HLA-A2-restricted TCRs recognize surfaces on the HLA-A2/peptide complex that are dependent upon the side chains of K66 and Q155 in the central portion of the peptide binding groove. Crystallographic structures of several peptide/HLA-A2 structures have shown that the side chains of these critical amino acids that make contact with the A6 TCR also contact the bound peptide. Collectively, our results indicate that the generalized effects of changes at these critical amino acids are probably due to the fact that they can be directly contacted by TCRs as well as influence the binding and presentation of the bound peptides.     

Mimotopes for Alloreactive and Conventional T Cells in a Peptide–MHC Display Library

The use of peptide libraries for the identification and characterization of T cell antigen peptide epitopes and mimotopes has been hampered by the need to form complexes between the peptides and an appropriate MHC molecule in order to construct a complete T cell ligand. We have developed a baculovirus-based peptide library method in which the sequence encoding the peptide is embedded within the genes for the MHC molecule in the viral DNA, such that insect cells infected with virus encoding a library of different peptides each displays a unique peptide–MHC complex on its surface. We have fished in such a library with two different fluorescent soluble T cell receptors (TCRs), one highly peptide specific and the other broadly allo-MHC specific and hypothesized to be much less focused on the peptide portion of the ligand. A single peptide sequence was selected by the former αβTCR that, not unexpectedly, was highly related to the immunizing peptide. As hypothesized, the other αβTCR selected a large family of peptides, related only by a similarity to the immunizing peptide at the p5 position. These findings have implications for the relative importance of peptide and MHC in TCR ligand recognition. This display method has broad applications in T cell epitope identification and manipulation and should be useful in general in studying interactions between complex proteins.     

Critical relationship between TCR signaling potential and TCR affinity during thymocyte selection

Whether a developing thymocyte becomes positively or negatively selected is thought to be determined by the affinity/avidity of its TCR for MHC/peptide ligands expressed in the thymus. Presumably, differences in affinity translate into differences in the potency of the ensuing TCR-mediated signals, and these differences in signal strength determine the outcome of thymocyte selection. However, there is little direct evidence establishing a relationship between TCR-ligand affinity and signal strength during positive and negative selection. The TCR complex contains multiple signaling motifs, known as immunoreceptor tyrosine-based activation motifs (ITAMs) that are required for T cell activation. To examine the effects of TCR signal strength on selection, the signaling potential of the TCR was modified by substituting transgenic TCR zeta-chains containing either three, one, or zero ITAMs for endogenous (3-ITAM) zeta-chain. These zeta-chain variants were then bred into different alphabetaTCR transgenic backgrounds. We report that reductions in TCR signaling potential have distinct effects on the selection of thymocytes expressing different TCRs, and that the requirement for zeta-chain ITAMs critically depends upon the specificity and apparently, affinity, of the TCR for its selecting ligand(s).     

Structure of a human autoimmune TCR bound to a myelin basic protein self-peptide and a multiple sclerosis-associated MHC class II molecule

Multiple sclerosis is mediated by T-cell responses to central nervous system antigens such as myelin basic protein (MBP). To investigate self-peptide/major histocompatibility complex (MHC) recognition and T-cell receptor (TCR) degeneracy, we determined the crystal structure, at 2.8 A resolution, of an autoimmune TCR (3A6) bound to an MBP self-peptide and the multiple sclerosis-associated MHC class II molecule, human leukocyte antigen (HLA)-DR2a. The complex reveals that 3A6 primarily recognizes the N-terminal portion of MBP, in contrast with antimicrobial and alloreactive TCRs, which focus on the peptide center. Moreover, this binding mode, which may be frequent among autoimmune TCRs, is compatible with a wide range of orientation angles of TCR to peptide/MHC. The interface is characterized by a scarcity of hydrogen bonds between TCR and peptide, and TCR-induced conformational changes in MBP/HLA-DR2a, which likely explain the low observed affinity. Degeneracy of 3A6, manifested by recognition of superagonist peptides bearing substitutions at nearly all TCR-contacting positions, results from the few specific interactions between 3A6 and MBP, allowing optimization of interface complementarity through variations in the peptide.     

Functional evidence that conserved TCR CDR alpha 3 loop docking governs the cross-recognition of closely related peptide:class I complexes.

The TCR recognizes its peptide:MHC (pMHC) ligand by assuming a diagonal orientation relative to the MHC helices, but it is unclear whether and to what degree individual TCRs exhibit docking variations when contacting similar pMHC complexes. We analyzed monospecific and cross-reactive recognition by diverse TCRs of an immunodominant HVH-1 glycoprotein B epitope (HSV-8p) bound to two closely related MHC class I molecules, H-2K(b) and H-2K(bm8). Previous studies indicated that the pMHC portion likely to vary in conformation between the two complexes resided at the N-terminal part of the complex, adjacent to peptide residues 2-4 and the neighboring MHC side chains. We found that CTL clones sharing TCR beta-chains exhibited disparate recognition patterns, whereas those with drastically different TCRbeta-chains but sharing identical TCRalpha CDR3 loops displayed identical functional specificity. This suggested that the CDRalpha3 loop determines the TCR specificity in our model, the conclusion supported by modeling of the TCR over the actual HSV-8:K(b) crystal structure. Importantly, these results indicate a remarkable conservation in CDRalpha3 positioning, and, therefore, in docking of diverse TCRalphabeta heterodimers onto variant peptide:class I complexes, implying a high degree of determinism in thymic selection and T cell activation.     

How a T cell receptor-like antibody recognizes major histocompatibility complex-bound peptide

We determined the crystal structures of the T cell receptor (TCR)-like antibody 25-D1.16 Fab fragment bound to a complex of SIINFEKL peptide from ovalbumin and the H-2K(b) molecule. Remarkably, this antibody directly "reads" the structure of the major histocompatibility complex (MHC)-bound peptide, employing the canonical diagonal binding mode utilized by most TCRs. This is in marked contrast with another TCR-like antibody, Hyb3, bound to melanoma peptide MAGE-A1 in association with HLA-A1 MHC class I. Hyb3 assumes a non-canonical orientation over its cognate peptide-MHC and appears to recognize a conformational epitope in which the MHC contribution is dominant. We conclude that TCR-like antibodies can recognize MHC-bound peptide via two different mechanisms: one is similar to that exploited by the preponderance of TCRs and the other requires a non-canonical antibody orientation over the peptide-MHC complex.     

Unraveling a hotspot for TCR recognition on HLA-A2: evidence against the existence of peptide-independent TCR binding determinants

T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 alpha1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.     

T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions

αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.     

Homology between an alloantigen and a self MHC allele calibrates the avidity of the alloreactive T cell repertoire independent of TCR affinity

The self-restricted T cell repertoire exhibits a high frequency of alloreactivity. Because these alloreactive T cells are derived from the pool of cells selected on several different self MHC alleles, it is unknown how development of the alloantigenic repertoire is influenced by homology between a self MHC allele and an alloantigen. To address this, we used the 2C transgenic TCR that is selected by K(b), is alloreactive for L(d), and cross-reacts with L(q). L(q) is highly homologous to L(d) and binds several of the same peptide ligands, including p2Ca, the peptide recognized by 2C. We find that L(d)/p2Ca is a high avidity agonist ligand, whereas L(q)/p2Ca is a low avidity agonist ligand for 2C T cells. When mice transgenic for the 2C TCR are bred to L(q)-expressing mice, 2C(+) T cells develop; however, they express lower levels of either the 2C TCR or CD8 and require a higher L(d)/p2Ca ligand density to be activated than 2C(+) T cells selected by K(b). Furthermore, the 2C T cells selected in the presence of L(q) fail to detect L(q)/p2Ca complexes even at high ligand density. Thus, despite possessing the identical TCR, there is a functional avidity difference between 2C(+) T cells selected in the presence of L(q) vs K(b). These data provide evidence that homology between the selecting ligand and an alloantigen can influence the avidity of the T cell repertoire for the alloantigen, and suggest that thymic selection can fine tune T cell avidity independent of intrinsic TCR affinity.     

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.     

Immunodominance is altered in T cell receptor (beta-chain) transgenic mice without the generation of a hole in the repertoire.

Despite the tremendous plasticity of the TCR repertoire, T cells recognize a limited number of antigenic sites (frequently a single site, or immunodominant epitope) on a complex protein Ag. Current models suggest that the immunodominant epitope of a complex protein is the processed peptide that binds to the MHC molecule with the highest affinity. Conversely, the inability of the T cell population to recognize a specific epitope, termed a "hole" in the repertoire, can prevent the immunodominance of a peptide despite efficient processing and MHC binding of the peptide. The role of specific TCR alpha- or beta-chains in determining MHC restriction and recognizing specific epitopes is complex and incompletely understood. To evaluate the contribution of each TCR chain to the functional diversity of the T cell repertoire, we investigated in vivo the T cell response to phage lambda-repressor protein in transgenic mice expressing a single rearranged beta-chain gene (C57L beta mice) in association with the complete germline alpha-chain repertoire. Our results demonstrate that expression of the TCR beta-chain transgene alters the immunodominant epitope recognized by T cells. However, after immunization with the appropriate peptide the transgenic mice can also respond to the nonimmunodominant epitope; thus, the expression of the TCR beta-chain transgene does not create a hole in the repertoire. These data indicate that the primary site, or immunodominant epitope, of an Ag recognized by T cells can be altered by the preimmune TCR repertoire independent of antigen processing and MHC affinity.     

Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct viral epitopes restricted by a single major histocompatibility complex molecule.

Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.     

Peptide recognition by two HLA-A2/Tax11-19-specific T cell clones in relationship to their MHC/peptide/TCR crystal structures

The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues are optimal at each peptide position. A prominent feature of the TCR contact surface is a deep pocket that accommodates a tyrosine at position 5 of the peptide. For one of these TCRs, this pocket is highly specific for aromatic residues. In the other TCR structure, this pocket is larger, allowing many different residues to be accommodated. The CTL clones also show major differences in the specificity for several other peptide residues, including side chains that are not directly contacted by the TCR. Despite the specificity of these clones, peptides that are distinct at five or six positions from Tax11-19 induce CTL activity, indicating that substantial changes of the peptide surface are tolerated. Human peptides with limited sequence homology to Tax11-19 represent partial TCR agonists for these CTL clones. The distinct functional properties of these CTL clones highlight structural features that determine TCR specificity and cross-reactivity for MHC-bound peptides.     

Maturation and function of mouse T-cells with a transgenic TCR positively selected by highly disparate xenogeneic porcine MHC.

Remarkably normal cellular immune function, along with specific T-cell tolerance to highly disparate xenogeneic donors, can be achieved by grafting fetal pig thymus (FP THY) tissue to T and NK cell-depleted, thymectomized (ATX) mice. Porcine MHC can mediate positive selection of mouse CD4+ T-cells with a mouse MHC-restricted TCR in FP THY-grafted, T- and NK cell-depleted, ATX TCR-transgenic "AND" mice. However, functional studies were not performed on transgenic mouse T-cells selected in a FP THY graft. We have now performed further studies to confirm the ability of porcine MHC to mediate the positive selection of mouse T-cells with a mouse MHC-restricted TCR, and to exclude the possibility that the maturation of mouse T-cells with a mouse MHC-restricted TCR in FP THY grafts in ATX "AND" mice is a special case. For this purpose, TCR-transgenic mice with an unrelated transgenic TCR ["3A9", specific for hen egg lysozyme (HEL) peptide 46-61 presented by I-Ak] were employed. Similar to FP THY-grafted ATX "AND" mice, large numbers of mouse CD4 single positive thymocytes expressing the transgenic TCR (Vbeta8.2) and expressing a mature phenotype (Qa-2high and heat stable antigen, HSAlow) were detected in FP THY grafts. Porcine thymus grafting led to a high level of peripheral repopulation with mouse naive-type (CD44low CD45RBhigh CD62Lhigh) CD4+ cells expressing the transgenic TCR in T and NK cell-depleted ATX "3A9" mice, regardless of whether the recipients had a positive selecting or a non-selecting, class II deficient MHC background. The mouse CD4+ T-cells expressing the "3A9" TCR showed efficient primary proliferative responses to the protein antigen (HEL) when it was presented by mouse class II+ antigen presenting cells (APC) in vitro. These results, collectively, support the general conclusion that discordant xenogeneic porcine MHC can mediate positive selection of mouse T-cells with mouse MHC-restricted TCR. This study has implications for the potential clinical use of xenogeneic thymus transplantation to reconstitute cellular immunity in the setting of thymic insufficiency or thymectomy, and hence for its applicability to the induction of xenograft tolerance and in the treatment of immunodeficiency diseases.