Defects in antigen presentation of mutant influenza haemagglutinins are reversed by mutations in the MHC class II molecule

A functional analysis was undertaken of the effects of mutating single amino acid residues in the alpha chain of the I-Ak molecule (to alanine; residues 50-79) on the ability of I-Ak transfectants to process and present influenza haemagglutinin to CD4+ T cell clones specific for two major antigenic sites of the HA1 subunit. In each instance, T cells were insensitive to a majority of substitutions in Ak with the exception of a few critical residues that differed for individual T cell clones. But more significantly, the failure of T cell clones to respond to mutant influenza viruses, containing drift substitutions within a T cell recognition site, in association with wild type I-Ak, could be reversed by single substitutions in Ak alpha. A T cell clone specific for HA1 120-139 failed to respond to a laboratory mutant virus (HA1 135 Gly----Arg) whereas optimal responses were observed with a mutant Ak transfectant (Ak alpha 56 Arg----Ala). Similarly, mutant transfectant 62 (Ak alpha 62 Gly----Ala) was able to present a natural variant virus A/TEX/77 to a T cell clone specific for HA1 48-67. We propose that Ak alpha 56 and Ak alpha 62 increase the affinity of association of mutant HA1 peptides for class II and therefore confer T cell recognition of variant viruses.     

Multispecific CD4+ T cell response to a single 12-mer epitope of the immunodominant heat-shock protein 60 of Yersinia enterocolitica in Yersinia-triggered reactive arthritis: overlap with the B27-restricted CD8 epitope, functional properties, and epitope presentation by multiple DR alleles

Yersinia heat-shock protein 60 (Ye-hsp60) has recently been found to be a dominant CD4 and CD8 T cell Ag in Yersinia-triggered reactive arthritis. The nature of this response with respect to the epitopes recognized and functional characteristics of the T cells is largely unknown. CD4+ T cell clones specific for Ye-hsp60 were raised from synovial fluid mononuclear cells from a patient with Yersinia-triggered reactive arthritis. and their specificity was determined using three recombinant Ye-hsp60 fragments, overlapping 18-mer synthetic peptides as well as truncated peptides. Functional characteristics were assessed by cytokine secretion analysis in culture supernatants after specific antigenic stimulation. Amino acid positions relevant for T cell activation were detected by single alanine substitutions within the epitopes. Fragment II comprising amino acid sequence 182-371 was recognized by the majority of clones. All these clones were specific for peptide 319-342. Th1 clones and IL-10-secreting clones occurred in parallel, sometimes with the same fine specificity. The 12-mer core epitope 322-333 is a degenerate MHC binder and is presented to some T cell clones in a "promiscuous" manner. This epitope is almost identical with a B27-restricted CTL epitope of Ye-hsp60. Cross-reactivity of Ye-hsp60-specific T cell clones with self-hsp60 was not observed. In conclusion, an interesting Ye-hsp60 T cell epitope has been identified and characterized. It remains to be determined whether this epitope is also relevant in other reactive arthritis patients.     

The role of the endoplasmic reticulum in antigen processing. N-glycosylation of influenza hemagglutinin abrogates CD4+ cytotoxic T cell recognition of endogenously processed antigen.

Evidence is presented for an endogenous route of Ag processing for CD4+ T cell recognition of influenza hemagglutinin that requires obligatory traffic of de novo synthesized hemagglutinin across the lumen of the endoplasmic reticulum for processing in a cytosolic compartment. I-Ad-restricted T cell clones that recognize synthetic peptides corresponding to two distinct antigenic regions of the HA1 subunit, HA1 56-76 and HA1 177-199, are cytotoxic and, dependent on epitope specificity can recognize endogenously processed Ag and lyse class II+ target cells infected with a recombinant vaccinia-X31 HA virus. HA1 56-76 specific T cell clones fail to recognize (target cells infected with) influenza X31 viruses, containing a single residue change, HA1 63 Asp----Asn that introduces an oligosaccharide attachment site: Asp63Cys64Thr65. Recognition is restored, however, by tunicamycin treatment of mutant virus infected target cells. Inasmuch as N-glycosylation of nascent hemagglutinin polypeptides occurs in the lumen of the endoplasmic reticulum, this indicates a route of endogenous processing for hemagglutinin, requiring transport across the endoplasmic reticulum, which has been confirmed by the failure of CD4+ T cells to recognize a recombinant VACC-hemagglutinin virus in which the same single residue change, HA1 63 Asp----Asn has been introduced by site directed mutagenesis.     

Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin.

A total of 14 I-Ad-restricted helper T-cell clones specific for the hemagglutinin (HA) molecule of influenza virus were isolated from spleens of BALB/c or (BALB/c X C57BL/10)F1 mice immunized with the H3 subtype influenza virus A/Memphis/71 (Mem 71) and from lymph nodes of BALB/c mice primed with purified HA. The specificity of these T-cell clones was assessed in proliferation assays by reactivity with naturally occurring strains of viruses that arose by antigenic drift and contain known amino acid sequence changes in HA and with a panel of monoclonal antibody (MAb)-selected mutants of Mem 71 with single amino acid substitutions in HA. The HA genes of those mutant viruses that failed to stimulate one or more of the T-cell clones were sequenced. The clones could be allocated to at least four groups, each group having a distinct pattern of reactivity with the panel of natural field strains. The epitopes recognized by the four groups of clones were found, by reactivity with MAb-selected mutants, to be in very close proximity to one another and probably overlapping. All of the distinct epitopes recognized by the T-cell clones were adversely affected by a single amino acid substitution, either at residue 60 or at residue 63 in the HA1 polypeptide chain, within the region known from antibody-binding studies as site E. Some, but not all, of the epitopes may be influenced by the addition of a carbohydrate side chain to the HA of a particular MAb-selected mutant and certain field strains containing an Asp----Asn substitution at residue 63. Site E is therefore a major site of H-2d helper T-cell recognition on the H3 HA.     

CD25-expressing CD8+ T cells are potent memory cells in old age

We have recently described an IL-2/IL-4-producing CD8+CD25+ non-regulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 alphabeta molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25- memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25- memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25- memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25- memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.     

Fine specificity of murine class II-restricted T cell clones for synthetic peptides of influenza virus hemagglutinin. Heterogeneity of antigen interaction with the T cell and the Ia molecule

We have previously demonstrated diversity in the specificity of murine, H-2k class II-restricted, T cell clones for the hemagglutinin (HA) molecule of H3N2 influenza viruses and have mapped two T cell determinants, defined by synthetic peptides, to residues 48-68 and 118-138 of HA1. In this study we examine the nature of the determinant recognized by six distinct P48-68-specific T cell clones by using a panel of truncated synthetic peptides and substituted peptide analogs. From the peptides tested, the shortest recognized were the decapeptides, P53-62 and P54-63, which suggests that the determinant was formed from the 9 amino acids within the sequence 54-62. Asn54 was critical for recognition since P49-68 (54S) was not recognized by the T cell clones. Furthermore this peptide analog was capable of competing with P48-68 for Ag presentation, thereby suggesting that residue 54 is not involved in Ia interaction and may therefore be important for TCR interaction. Residue substitutions at position 63 also affected T cell recognition, but in a more heterogeneous fashion. Peptide analogs or mutant viruses with a single amino acid substitution at position 63 (Asp to Asn or Tyr) reduced the responses of the T cell clones to variable extents, suggesting that Asp63 may form part of overlapping T cell determinants. However since the truncated peptide P53-62 was weakly recognized, then Asp63 may not form part of the TCR or Ia interaction site, but may affect recognition through a steric or charge effect when substituted by Asn or Tyr. Ag competition experiments with the two unrelated HA peptides, P48-68 and P118-138, recognized by distinct T cell clones in the context of the same restriction element (I-Ak), showed that the peptides did not compete for Ag presentation to the relevant T cell clones, whereas a structural analog of P48-68 was a potent inhibitor. This finding is discussed in relation to the nature of the binding site for peptide Ag on the class II molecule.     

Direct ex vivo analyses of HLA-DR1 transgenic mice reveal an exceptionally broad pattern of immunodominance in the primary HLA-DR1-restricted CD4 T-cell response to influenza virus hemagglutinin

The recent threat of an avian influenza pandemic has generated significant interest in enhancing our understanding of the events that dictate protective immunity to influenza and in generating vaccines that can induce heterosubtypic immunity. Although antigen-specific CD4 T cells are known to play a key role in protective immunity to influenza through the provision of help to B cells and CD8 T cells, little is known about the specificity and diversity of CD4 T cells elicited after infection, particularly those elicited in humans. In this study, we used HLA-DR transgenic mice to directly and comprehensively identify the specificities of hemagglutinin (HA)-specific CD4 T cells restricted to a human class II molecule that were elicited following intranasal infection with a strain of influenza virus that has been endemic in U.S. human populations for the last decade. Our results reveal a surprising degree of diversity among influenza virus-specific CD4 T cells. As many as 30 different peptides, spanning the entire HA protein, were recognized by CD4 T cells, including epitopes genetically conserved among H1, H2, and H5 influenza A viruses. We also compared three widely used major histocompatibility class II algorithms to predict HLA-DR binding peptides and found these as yet inadequate for identifying influenza virus-derived epitopes. The results of these studies offer key insights into the spectrum of peptides recognized by HLA-DR-restricted CD4 T cells that may be the focus of immune responses to infection or to experimental or clinical vaccines in humans.     

Genetic restriction and fine specificity of human T cell clones reactive with rabies virus

Rabies virus-specific T cell clones isolated from a human vaccine recipient were studied for their fine specificity and genetic restriction using synthetic peptides of the viral Ag and mouse fibroblasts transfected with human MHC genes. Two clones were found to react with an epitope present in the rabies glycoprotein, which was presented by the HLA-DR7 molecule. Other T cell clones recognized synthetic epitopes corresponding to the rabies nucleoprotein in association with the HLA-DR7 or HLA-DQw3 molecule, and one clone responded to the viral nucleocapsid Ag in the presence of HLA-DPw4. T cell clones that exhibited different cross-reactivity patterns among several virus strains were found to recognize closely situated epitopes (within 15 amino acid residues), which were presented in the context of the same MHC molecule. The lack of recognition of a particular virus strain by a T cell clone was attributable in some cases to amino acid variations of the Ag that appear to affect the T cell's receptor for Ag specificity and not the ability of that epitope to associate with the corresponding MHC molecule. Comparisons of the T cell cross-reactivity patterns with various rabies and rabies-related viruses, the fine antigenic specificity, and MHC restriction may aid in understanding the role of individual amino acid variations among virus strains in the induction of cross-protective immunity.     

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.     

Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3(243-258)), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.     

Human CD28-CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones.

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28-CD8+ T cells has not previously been defined, we studied the contribution of CD28-CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR beta-chains. We designed clonotypic oligonucleotides complementary to each beta-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28- cells than in the CD8+ population as a whole. Because CD28-CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28- CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28-CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28-CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.     

Human CD4+ T cells induced by synthetic peptide malaria vaccine are comparable to cells elicited by attenuated Plasmodium falciparum sporozoites

Peptide vaccines containing minimal epitopes of protective Ags provide the advantages of low cost, safety, and stability while focusing host responses on relevant targets of protective immunity. However, the limited complexity of malaria peptide vaccines raises questions regarding their equivalence to immune responses elicited by the irradiated sporozoite vaccine, the "gold standard" for protective immunity. A panel of CD4+ T cell clones was derived from volunteers immunized with a peptide vaccine containing minimal T and B cell epitopes of the Plasmodium falciparum circumsporozoite protein to compare these with previously defined CD4+ T cell clones from volunteers immunized with irradiated P. falciparum sporozoites. As found following sporozoite immunization, the majority of clones from the peptide-immunized volunteers recognized the T* epitope, a predicted universal T cell epitope, in the context of multiple HLA DR and DQ molecules. Peptide-induced T cell clones were of the Th0 subset, secreting high levels of IFN-gamma as well as variable levels of Th2-type cytokines (IL-4, IL-6). The T* epitope overlaps a polymorphic region of the circumsporozoite protein and strain cross-reactivity of the peptide-induced clones correlated with recognition of core epitopes overlapping the conserved regions of the T* epitope. Importantly, as found following sporozoite immunization, long-lived CD4+ memory cells specific for the T* epitope were detectable 10 mo after peptide immunization. These studies demonstrate that malaria peptides containing minimal epitopes can elicit human CD4+ T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites.     

Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens

The dominant T cell determinant on moth and pigeon cytochromes c in B10.A (E beta k:E alpha k) mice is located in the C-terminal portion of the protein, contained within residues 93-103 or 93-104. Thirty-seven antigen analogs, containing single amino acid substitutions at positions 98, 99, 101, 102, 103, and 104, were synthesized. The effects of the substitutions on in vitro antigenicity and in vivo immunogenicity were determined. Functional assays with T cell clones identified residues 99, 101, 102, and 103 as critical, based on their effect on antigenic potency. Peptides containing substitutions at residues 99, 101, and 102 were capable of eliciting unique clones upon immunization of B10.A mice. This was consistent with the identification of these residues as part of the epitope, the site on the antigen that interacts with the T cell receptor. Immunization with peptides substituted at residue 103, however, failed to elicit clones with unique specificity for the immunogen. When these peptides were tested for their ability to stimulate the T cell clones with antigen-presenting cells from B10.A(5R) mice expressing the E beta b:E alpha k Ia molecule, a consistent change in the relative antigenic potency was observed with 50% of the peptides. The effect of the Ia molecule on the antigenic potency ruled out the possibility that residue 103 nonspecifically affected antigen uptake or processing and identified residue 103 as part of the agretope, the site that interacts with the Ia molecule. The locations of the agretope and the epitope on this antigenic determinant appear to be fixed, even in the presence of large numbers of amino acid substitutions. However, some substitutions were found to affect both the agretope and the epitope, placing limits on the functional independence of the two sites. The results are discussed in terms of the trimolecular complex model of T cell activation and the implications of these data for antigen-Ia molecule interactions.     

Use of synthetic peptides to establish anti-human acetylcholine receptor CD4+ cell lines from myasthenia gravis patients

Acetylcholine receptor-(AcChR) specific T cell lines were propagated from the PBL of six myasthenia gravis (MG) patients by the use of a pool of synthetic peptides (alpha-pool) corresponding to the complete sequence of the alpha-subunit of the human AcChR. All the lines had CD4+ phenotype and strongly recognized the alpha-pool. Four lines cross-reacted with native Torpedo AcChR. Five lines showed, at certain stages of their propagation, some degree of reactivity to autologous or DR-matched APC. One of the CD4+ T lines was challenged with each one of the peptides present in the alpha-pool. Several peptides, corresponding to the sequence segments 48-67, 101-120, 304-322, 320-337, and 419-437 of the human alpha-subunit were recognized, indicating that different epitopes and multiple T cell clones are involved in the recognition of the autoantigen in MG. Human AcChR-specific CD4+ T cell lines will be useful to identify the repertoire of epitopes recognized by the autoreactive Th cells in MG, to investigate the TCR genes utilized by autoreactive Th cells and to develop specific immunosuppressive treatments using anti-T cell vaccination.     

A limited region within hen egg-white lysozyme serves as the focus for a diversity of T cell clones

The C57BL/6 (H-2b) mouse is a nonresponder to hen egg-white lysozyme (HEL) injected i.p., owing to a T suppressor cell-inducing determinant at the amino-terminal region. After immunization with a 93-amino acid fragment (a.a. 13-105) of HEL lacking this determinant, all clones from two independently derived C57BL/6 T cell lines were found to be specific for epitopes within a subregion of peptide 74-96. Three specificity patterns for the clones could be defined on the basis of cross-reactivities with only two other species variant lysozymes. Reactivities of all three specificity groups was consistent with the serine to threonine substitution at position 91, although reactivity of one of the groups could be affected by substitutions at position 84. The results confirm at the clonal level that even for distantly related antigens, only limited regions are recognized by T cells. They are consistent with the notion that specific sites on the antigen capable of interaction with Ia molecules lead to dominance of certain regions for T cell reactivity. Moreover, the diversity in specificity among clones suggests that the limiting feature of T cell responsiveness is not a lack of available T cells in the repertoire directed against a single antigenic site.     

Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites

Surveys of the antigenic properties of a wide range of variants of the H3N2 (Hong Kong) influenza virus subtype have revealed complex patterns of variants cocirculating during each of the main epidemic eras of the subtype. We determined hemagglutinin (HA) gene sequences for 14 isolates chosen to give the wildest possible spread of variant types. The addition of these data to existing HA gene sequence information for other variants provides a comprehensive picture of HA gene evolution during antigenic drift among H3N2 subtype viruses. The data reveal the existence of multiple evolutionary pathways during at least one period of development of the subtype and strikingly demonstrate that amino acid changes are limited to a small number of locations on the HA molecule during antigenic drift. The occurrence of sequential amino acid changes at key positions within these variable regions suggests that the HA structure has remained constant during subtype evolution so that only limited possibilities remain for further antigenic drift among H3N2 viruses.     

One I region restriction determinant can associate with multiple antigenic epitopes

Studies presented in this paper show that T cell clones recognizing different epitopes of multideterminant antigens can be restricted by the same I-A molecule. These data further support the concept that a single I-A restriction site can present more than one antigenic epitope. This concept was supported by data on the proliferation of T cell clones reactive with either poly(L-Glu60, L-Ala30, L-Tyr10)n(GAT) or poly(Tyr, Glu)-poly D,L-Ala--poly Lys [(T,G)-A--L] which recognized different epitopes on these multideterminant antigens. Two clones recognizing different epitopes on the same multideterminant antigen can be blocked by the same monoclonal anti-I-A antibody. Additionally, the mutation in the Abm12 chain utilized in [B6.C-H-2bm12(bm12) X B10.A(4R)]F1 mice can affect the restriction determinant of clones recognizing different antigenic epitopes. These results suggest that in the strictest sense, the determinant selection theory is not tenable and would support the concept that T cell specificity is controlled by the T cell repertoire.     

Immune receptor repertoire for influenza haemagglutinin

An extensive analysis was made of receptor specificity and gene usage in the neutralising antibody (mAb) and Class II-restricted T cell responses to influenza haemagglutinin (HA) following natural infection of MHC (H-2^k or H-2^d) congenic mice with X31 virus (H3N2 subtype). Despite the diversity of available antigenic sites on the HA1 subunit, there was strikingimmunodominance in the mAb response as deduced by sequencing the HA genes of escape mutants and the corresponding antibody H and L chain gene rearrangements. Similarly, Class II restricted T cell responses of individual donors focused on a single antigenic site, or immunodominant peptide; and PCR sequence analysis of T cell receptor () gene usage indicated that T cell memory was derived from asingle progenitor cell. Focusing of the immune repertoire to limited regions of the HA molecule during a primary viral infection may be a significant factor in immune pressure for antigenic variation.     

Opposite role of Ras in tumor necrosis factor-alpha-induced cell cycle regulation: competition for Raf kinase

We previously reported an epitope presenting vector, pCI, a derivative of a human invariant chain (Ii) expression vector, in which the class II associated invariant chain peptide (CLIP, Ii p89-101) could be substituted with antigenic peptides. In the current study, we used this vector to develop a new expression cloning system to identify CD4+ T cell epitopes. We inserted double-stranded oligo DNAs of randomized sequences into this vector and prepared an epitope-presenting library which loads randomized 13-mer peptides onto HLA class II molecules coexpressed in COS-7 cells. Utilizing this library, we isolated a cross-reactive epitope recognized by a glutamic acid decarboxylase (GAD) 65-autoreactive T cell clone established from a patient with insulin-dependent diabetes mellitus. Although the newly identified epitope (PVQLSNQWHVVGATF) was far different from the original epitope, GAD65 p116-128 (NILLQYVVKSFDR), it did have the capacity to stimulate the T cell clone comparable to that of the original GAD epitope. Our system may be applicable not only for identifying of cross-reactive epitopes for CD4+ T cells of known specificity, but also for detection of epitopes stimulatory for CD4+ T cells the epitopes of which are unknown.     

Human autoreactive CD4+ T cells from naive CD45RA+ and memory CD45RO+ subsets differ with respect to epitope specificity and functional antigen avidity

T cells with specificity for self-Ags are normally present in the peripheral blood, and, upon activation, may target tissue Ags and become involved in the pathogenesis of autoimmune processes. In multiple sclerosis, a demyelinating disease of the CNS, it is postulated that inflammatory damage is initiated by CD4+ T cells reactive to myelin Ags. To investigate the potential naive vs memory origin of circulating myelin-reactive cells, we have generated myelin basic protein (MBP)- and tetanus toxoid-specific T cell clones from CD45RA+/RO- and CD45RO+/RA- CD4+ T cell subsets from the peripheral blood of multiple sclerosis patients and controls. Our results show that 1) the response to MBP, different from that to TT, predominantly emerges from the CD45RA+ subset; 2) the reactivity to immunodominant MBP epitopes mostly resides in the CD45RA+ subset; 3) in each individual, the recognition of single MBP epitopes is skewed to either subset, with no overlap in the Ag fine specificity; and 4) in spite of a lower expression of costimulatory and adhesion molecules, CD45RA+ subset-derived clones recognize epitopes with higher functional Ag avidity. These findings point to a central role of the naive CD45RA+ T cell subset as the source for immunodominant, potentially pathogenic effector CD4+ T cell responses in humans.