Thyroxine-binding antibodies inhibit T cell recognition of a pathogenic thyroglobulin epitope

Thyroid hormone-binding (THB) Abs are frequently detected in autoimmune thyroid disorders but it is unknown whether they can exert immunoregulatory effects. We report that a THB mAb recognizing the 5' iodine atom of the outer phenolic ring of thyroxine (T4) can block T cell recognition of the pathogenic thyroglobulin (Tg) peptide (2549-2560) that contains T4 at aa position 2553 (T4(2553)). Following peptide binding to the MHC groove, the THB mAb inhibited activation of the A(k)-restricted, T4(2553)-specific, mouse T cell hybridoma clone 3.47, which does not recognize other T4-containing epitopes or noniodinated peptide analogues. Addition of the same THB mAb to T4(2553)-pulsed splenocytes largely inhibited specific activation of T4(2553)-primed lymph node cells and significantly reduced their capacity to adoptively transfer thyroiditis to naive CBA/J mice. These data demonstrate that some THB Abs can block recognition of iodine-containing Tg epitopes by autoaggressive T cells and support the view that such Abs may influence the development or maintenance of thyroid disease.     

Chronic relapsing experimental allergic encephalomyelitis. Cytotoxicity effected by a class II restricted T cell line specific for an encephalitogenic epitope

We have examined the possibility that Ag-specific CTL responses may play a role in the pathogenesis of CREAE by using an effector T cell line (LN400) specifically reactive to the SJL encephalitogenic epitope defined by myelin basic protein MBP residues(90-101). The LN400 cell line was capable of adoptively transferring CREAE to naive SJL mice and proliferated specifically to synthetic peptides corresponding to MBP residues(90-101) and an N-acetylated analogue of this epitope, as well as MBP. Moreover, the cell line generated Ag-specific CTL responses only against syngeneic targets that had been pulsed with these Ag. Targets pulsed with irrelevant Ag were not lysed. These CTL responses were MHC restricted to H-2s and were inhibited if targets were preincubated with mAb specific for relevant class II Ag. No inhibition was seen if targets were preincubated with mAb specific for class I Ag, indicating that the CTL responses generated by this L3T4+ Lyt-2.2- cell lines were class II restricted. Studies designed to detect nonspecific CTL through a bystander mechanism failed to demonstrate significant lysis of bystander targets by this Ag-specific cell line. These findings have relevance in defining potential mechanisms of disease induction in this model autoimmune disease.     

T cell recognition of Mlsc. I. Influence of MHC gene products in Mlsc-specific T cell recognition

Monospecific T cell clones have been proven to be powerful tools for the characterization of T cell recognition in many Ag-specific as well as allo-specific T cell responses. In this report, in order to elucidate the mechanism of T cell recognition of minor stimulating locus Ag (Mlsc) determinants, Mlsc-specific cloned T cells were employed together with primary T cell responses to clarify the role of MHC-gene products in Mlsc-specific T cell recognition. The results indicated that T cells recognize Mlsc determinants in conjunction with I-region MHC gene products. Moreover, certain MHC haplotypes (e.g., H-2a and H-2k) appear to function efficiently in the "presentation" of Mlsc, whereas other haplotypes (e.g., H-2b and H-2q) function poorly if at all in presenting Mlsc. Experiments with the use of stimulators derived from F1 hybrids between the low stimulatory H-2b, Mlsc strain, C3H.SW, and a panel of Mlsb, H-2-different or intra-H-2 recombinant strains strongly suggested that expression of E alpha E beta molecules on stimulators plays a critical role for Mlsc stimulation. The functional importance of the E alpha E beta product in Mlsc recognition was further demonstrated by the ability of anti-E alpha monoclonal antibody to inhibit the response of cloned Mlsc-specific T cells. Inhibition of the same Mlsc-specific response by anti-A beta k antibody suggests that the A beta product may also play a role in T cell responses to Mlsc.     

A T cell receptor V beta segment that imparts reactivity to a class II major histocompatibility complex product

We have identified in mice an allele of a new T cell receptor V beta gene, V beta 17a, whose product is bound by the monoclonal antibody KJ23a. Over 90% of T cell hybridomas prepared from V beta 17a+ T cells of SWR mice respond to allogeneic forms of the IE class II MHC protein, indicating that V beta 17a has an appreciable affinity for IE regardless of the other components of the T cell receptor. These results suggest a bias in the germ-line T cell receptor repertoire toward recognition of MHC proteins and indicate that the V beta portion of the receptor may form the most important contact points with MHC ligands.     

Inhibition of EBV-induced lymphoproliferation by CD4(+) T cells specific for an MHC class II promiscuous epitope

Posttransplant lymphoproliferative disorder (PTLD) and B cell lymphomas induced by EBV continue to be a major life-threatening complication in transplant patients. The establishment and enhancement of T cell immunity to EBV before transplantation and immunosuppressive therapy could help diminish these complications, but the lack of an effective vaccine has limited this prophylactic approach. We describe here the identification of a peptide epitope from the EBV EBNA2 Ag that is capable of inducing in vitro CD4(+) T cell responses that inhibit the EBV-mediated B lymphocyte proliferation associated with PTLD. Most significantly, T cell responses to the EBNA2 epitope were found to be restricted by numerous MHC class II alleles (DR1, DR7, DR16, DR52, DQ2, and DQ7), indicating that this peptide is highly promiscuous and would be recognized by a large proportion (>50%) of the general population. These results are relevant for the design of a simple, inexpensive and widely applicable peptide-based vaccine to prevent PTLD in solid organ transplant patients.     

Cells expressing an H chain Ig gene carrying a viral T cell epitope are lysed by specific cytolytic T cells.

The epitope corresponding to amino acid residues 147-161 of the nucleoprotein (NP) of influenza A virus is recognized by CTL in association with H-2Kd class I Ag. Herein, we engineered an Ig molecule carrying this CTL epitope by replacing the diversity gene segment of the H chain V region of an anti-arsonate antibody with an oligonucleotide that encodes the CTL epitope. The chimeric H chain gene was expressed either alone or together with the parental L chain in the nonsecreting BALB/c myeloma B cell line, SP2/0. The Ig produced by cells transfected with both the chimeric H chain and parental L chains genes expressed the NP epitope but lost the original arsonate binding activity. In addition, SP2/0 cells expressing the chimeric H chain either alone or together with the parental L chain were lysed by class I restricted NP-epitope specific CTL. By contrast, SP2/0 cells pulsed with soluble chimeric Ig molecules were not lysed by the specific CTL. These observations indicate that: 1) this particular CTL epitope can be expressed on Ig molecules without altering the H and L chain pairing; 2) this CTL epitope can be generated from this chimeric Ig in which it is surrounded by flanking regions distinct from those of the viral NP; and 3) the generation of this CTL epitope from the Ig molecule requires the endogenous pathway as do viral proteins.     

The identification of an Onchocerca-specific recombinant antigen containing a T cell epitope.

Recombinant Onchocerca volvulus Ag have been derived from expression libraries and examined for their ability to stimulate PBMC from patients infected with O. volvulus. Ten clones producing recombinant Ag were selected and plaque purified; lysogens were produced and found to express beta-galactosidase fusion proteins ranging in molecular mass from 115 to 138 kDa. When ammonium sulfate-precipitated lysates of these recombinant phage clones were examined for their ability to stimulate PBMC from a patient with onchocerciasis, all 10 recombinants produced stimulation above that to nonrecombinant phage. When individual fusion proteins, affinity purified on anti-beta-galactosidase linked to agarose, were used to stimulate PBMC from patients with onchocerciasis, only one of the recombinant Ag induced PBMC proliferation (stimulation index greater than 4) above that to Ag from nonrecombinant phage. Characterization of the DNA coding for this Ag showed it to be 1.2 kb in length with a small (90 bp) open reading frame; furthermore, it appears to be Onchocerca specific (on genomic dot blots) and single copy. Using overlapping peptides encompassing the entire open reading frame, one T cell epitope has been localized.     

MHC class II epitope nesting modulates dendritic cell function and improves generation of antigen-specific CD4 helper T cells

CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.     

T cell recognition of an engineered MHC class I molecule: implications for peptide-independent alloreactivity

Previously, we described H-2K(bW9) (K(bW9)), an engineered variant of the murine MHC class I molecule H-2K(b) (K(b)), devoid of the central anchor ("C") pocket owing to a point mutation on the floor of the peptide binding site; this substitution drastically altered selection of bound peptides, such that the peptide repertoires of K(b) and K(bW9) are largely nonoverlapping in vivo. On the basis of these observations, we used K(bW9) and K(b) to revisit the role of peptides in alloreactive T cell recognition. We first compared Ab and TCR recognition of K(bW9) and K(b). Six of six K(b)-specific mAbs, directed against different parts of the molecule, recognized K(bW9) well, albeit at different levels than K(b). Furthermore, K(bW9) readily served as a restriction element for a peptide-specific syngeneic CTL response. Therefore, K(bW9) mutation did not result in gross distortions of the TCR-interacting surface of class I, which was comparable between K(b) and K(bW9). Interestingly, when K(bW9) was used to stimulate allogeneic T cells, it induced an infrequent CTL population that cross-reacted against K(b) and was specific for peptide-independent MHC epitopes. By contrast, K(b)-induced alloreactive CTLs recognized K(b) in a peptide-specific manner, did not cross-react on K(bW9), and were present at much higher frequencies than those induced by K(bW9). Thus, induction of rare peptide-independent CTLs depended on unique structural features of K(bW9), likely due to the elevated floor of the peptide-binding groove and the consequent protruding position of the peptide. These results shed new light on the relationship between TCR and peptide-MHC complex in peptide-independent allorecognition.     

Re-evaluating the generation of a "proteasome-independent" MHC class I-restricted CD8 T cell epitope

The proteasome is primarily responsible for the generation of MHC class I-restricted CTL epitopes. However, some epitopes, such as NP(147-155) of the influenza nucleoprotein (NP), are presented efficiently in the presence of proteasome inhibitors. The pathways used to generate such apparently "proteasome-independent" epitopes remain poorly defined. We have examined the generation of NP(147-155) and a second proteasome-dependent NP epitope, NP(50-57), using cells adapted to growth in the presence of proteasome inhibitors and also through protease overexpression. We observed that: 1) Ag processing and presentation proceeds in proteasome-inhibitor adapted cells but may become more dependent, at least in part, on nonproteasomal protease(s), 2) tripeptidyl peptidase II does not substitute for the proteasome in the generation of NP(147-155), 3) overexpression of leucine aminopeptidase, thymet oligopeptidase, puromycin-sensitive aminopeptidase, and bleomycin hydrolase, has little impact on the processing and presentation of NP(50-57) or NP(147-155), and 4) proteasome-inhibitor treatment altered the specificity of substrate cleavage by the proteasome using cell-free digests favoring NP(147-155) epitope preservation. Based on these results, we propose a central role for the proteasome in epitope generation even in the presence of proteasome inhibitors, although such inhibitors will likely alter cleavage patterns and may increase the dependence of the processing pathway on postproteasomal enzymes.     

Cysteinylation of MHC class II ligands: peptide endocytosis and reduction within APC influences T cell recognition

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4(+) T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC. Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH. However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited. Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.     

Evolution of MHC class II E beta diversity within the genus Peromyscus

Progress in understanding the evolution of variation at the MHC has been slowed by an inability to assess the relative roles of mutation vs. intragenic recombination in contributing to observed polymorphism. Recent theoretical advances now permit a quantitative treatment of the problem, with the result that the amount of recombination is at least an order of magnitude greater than that of mutation in the history of class II genes. We suggest that this insight allows progress in evaluating the importance of other factors affecting the evolution of the MHC. We investigated the evolution of MHC class II E beta sequence diversity in the genus Peromyscus. We find evidence for extensive recombination in the history of these sequences. Nevertheless, it appears that intragenic recombination alone is insufficient to account for evolution of MHC diversity in Peromyscus. Significant differences in silent variation among subgenera arose over a relatively short period of time, with little subsequent change. We argue that these observations are consistent with the effects of historical population bottleneck(s). Population restrictions may explain general features of MHC evolution, including the large amount of recombination in the history of MHC genes, because intragenic recombination may efficiently regenerate allelic polymorphism following a population constriction.     

Interactions between the amino-terminal domains of MHC class II molecules have a profound effect on serologic and T cell recognition: an analysis using recombinant HLA-DR/H-2E molecules.

A series of transfected L cell lines were generated expressing the products of wild-type or recombinant HLA-DR1/H-2Ek beta-chain-encoding genes paired to DR alpha or E alpha. The recombinant genes were created by reciprocal exchange of the gene segments encoding the amino (NH2)-terminal and carboxy (COOH)-terminal halves of the beta 1 domain and the beta 2 domain. The majority of the serologic determinants, predicted from the genetic composition of the class II dimers, were expressed indicating that no gross conformational changes were induced by the creation of the interspecies recombinant molecules. Subtle conformational variation was detected by the anti-H-2Eb,k,s mAb Y17. Epitope expression was dependent on the presence of the E alpha-chain and NH2-terminal sequence from the beta 1 domain of H-2Ek. Substitution of DR1 sequence in either region led to loss of recognition by Y17. This pattern of reactivity maps the Y17 epitope either to the E alpha-chain or to an exposed sequence on the fourth strand of the beta sheet of the beta 1 domain. If the Y17 epitope is located on the E alpha-chain this raises the interesting possibility that the conformation of this chain, which is invariant by sequence, may vary according to the beta-chain with which it is coexpressed. The ability of the recombinant class II dimers to present Ag to the pigeon cytochrome c-specific, H-2Ek-restricted T cell hybridoma 2B4 was assessed. Transfected L cells expressing E beta k paired to E alpha or DR alpha presented Ag with equal efficiency, and the beta 2 domain of H-2Ek could be substituted with the equivalent region from DR1 without any loss of response. Wild-type DR1 failed to function as a restriction element, however, substitution of the COOH-terminal portion of the beta 1 domain with the equivalent sequence from H-2Ek was sufficient to produce a partial recovery of Ag recognition. Cells expressing a recombinant beta 1 domain comprising the COOH-terminal sequence from H-2Ek and the NH2-terminal sequence from DR1 presented Ag when paired to DR alpha but failed to do so when paired to E alpha. This indicates that a subtle conformational disturbance caused by mismatching of the NH2-terminal region of the beta-chain and the alpha-chain can have pronounced effects on T cell recognition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Class II genes of the human major histocompatibility complex. The DO beta gene is a divergent member of the class II beta gene family

A novel class II beta chain gene is described. This gene, tentatively called DO beta, displays considerably less polymorphism than beta genes of the DP, DQ, and DR loci. The nucleotide sequence of the DO beta gene is strikingly similar to that of the previously identified murine A beta 2 gene. The DO beta gene displays the same exon/intron organization as other beta genes although the fifth exon and the translated portion of the sixth exon are longer than in other genes. A striking feature of the amino acid sequence deduced from the DO beta gene sequence is the pronounced hydrophobicity of the NH2-terminal region. This feature distinguishes the putative DO beta chain from other class II beta chains and raises the possibility that DO beta chains may interact with an alpha chain that is structurally different from those of the DP, DQ, and DR loci. It further suggests that the putative DO molecule may have a function different from those of other class II antigens.     

T cell specificity for class II (I-A) and the encephalitogenic N-terminal epitope of the autoantigen myelin basic protein

The role of class II restriction in T cell recognition of an epitope of the autoantigen myelin basic protein (MBP) has been investigated. Encephalitogenic PL/J(H-2u) and (PL/J X SJL/J(H-2s))F1 ((PLSJ)F1) clones, isolated after immunization with intact MBP, recognize the N-terminal 11 amino acid residues of MBP in association with I-Au class II molecules. The synthetic peptide MBP 1-11 has been tested in vivo for induction of EAE. Clinical and histological EAE occurs in PL/J and (PLSJ)F1 mice but not SJL/J. The class II restriction of T cells primed with MBP 1-11 has been examined in primary cultures in vitro. Similar to encephalitogenic T cell clones, isolated after continuous selection in vitro, the population of MBP 1-11-specific proliferative PL/J and (PLSJ)F1 T cells, recognize this epitope in association with I-Au class II molecules. Not all MBP-specific T cell clones which are restricted to I-Au class II molecules cause autoimmune encephalomyelitis. The specificity of these non-encephalitogenic clones has been examined in this report. These clones also recognize MBP 1-11. Thus recognition of an encephalitogenic T cell epitope is not sufficient for induction of EAE.