Sharing of Four DR-β Sequence Motifs Between HLA-DRB1*1601 and DRB1*1101 Correlates with Frequent Degenerate T-Cell Recognition of HA306–320 Peptide Complexed to these Two Molecules

This paper shows that the seven HA306–320 specific T-cell clones isolated from one individual recognize the peptide complexed to both autologous HLA–DRB1*1101 and allogeneic HLA-DRB1*1601 (or DRB5*0201) molecules. For each T-cell clone, a single T-cell receptor (TCR) is involved in the recognition of these two different peptide-DR complexes as evidenced by cold target competition experiments. Yet, the seven T-cell clones express several different TCRs as judged by Vβ-Jβ usage and fine specificities. Furthermore, one representative clone has the same fine specificity for HA306–320 analogues mutated at epitopic residues irrespective of the use of DR1101 or DR1601 APC. These results suggest that structural differences between DRB1*1101 and DRB1*1601 (or DRB5*0201) do not dramatically influence the orientation of HA306–320 in the grooves such that most residues interacting with TCRs are conserved. In another individual, the same pattern of restriction, i.e. DR1101 + DR1601, was found for several HA306–320 specific clones. Two additional patterns, DR1101 + DR0801 and DR1101 + DR0801 + DR1601, were identified. By comparing DR sequences the authors found that DRB1*1101 and DRB1*1601 share four important motifs, i.e. β85–86, β67–71, β57 and β28–31 supposed to line three distinct HLA-DR pockets. Three of these motifs are also shared with DRB1*0801. All the results further support that the motif similarities allow the peptide to adopt very similar orientations in the cross-reacting DR molecules.     

Contribution of T-cell receptor-contacting and peptide-binding residues of the class II molecule HLA-DR4 Dw10 to serologic and antigen-specific T-cell recognition.

The relative contributions of putative T-cell receptor (TCR)-contacting and peptide-binding residues of a major histocompatibility complex (MHC) class II restriction element to serologic and antigen-specific T-cell recognition were investigated by site-specific mutagenesis. Amino acids 70 and 71 in the DR beta 1 domain of DR4 Dw10 are uniquely differnet from the other Dw subtypes of DR4. Residue 70 is predicted to be located at the membrane-distal surface of the class II molecule, where it may influence T-cell recognition by a direct interaction with a TCR. Residue 71 is predicted to form part of the antigen-binding groove where its influence on T-cell recognition may be mediated indirectly via an effect on peptide binding. Transfected murine L cells were produced expressing the products of DR4 Dw10B genes in which the codons for residues 70 and 71 had been mutated towards DR4 Dw14. Support for the predicted orientations of beta-chain residues 70 and 71 was lent by the observation that only residue 70 plays an important role in the formation of a serologic determinant. Mutation of this residue was sufficient to produce recovery of recognition by a human monoclonal antibody, NI, which has specificity for all the DR4 subtypes with the exception of DR4 Dw10. The human T-cell clone HA1.7, specific for influenza virus hemagglutinin (HA) peptide 307-319 and restricted by DR1 Dw1, exhibits degeneracy of MHC restriction on the DR4 Dw subtypes with the exception of DR4 Dw10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flexibility in T-cell receptor ligand repertoires depends on MHC and T-cell receptor clonotype.

T-cell receptors (TCR) recognize peptides complexed to self-major histocompatibility complex (MHC) molecules. Recognition of peptide/MHC ligands by the TCR is highly peptide specific. However, certain TCRs can also recognize sequence-related and -unrelated ('mimicry') epitopes presented by homologous MHC molecules. Using two human, human leucocyte antigen-DR1 (HLA-DR1)-restricted T-cell clones specific for HA p307-319, we identified several diverse combinations of peptide-MHC complexes that are functionally equivalent in their ability to trigger T-cell stimulation. These findings demonstrate that a single TCR can productively interact with different peptides complexed to self- as well as non-self-MHC molecules. This extended reactivity is human leucocyte antigen (HLA) allele and TCR clonotype dependent, as the peptide repertoire recognized depends on the presenting HLA-DR molecule and varies among different TCRs that both recognize the HA p307-319/DR1 complex. Importantly, certain peptide analogues can completely change the HLA-restriction pattern of the TCR: T-cell recognition of the wild-type peptide that was absent in the context of a non-self HLA-DR molecule, was restored by complementing substitutions in altered peptide ligands, that could not be presented by the original restriction element. This mechanism may play an important role in allorecognition.     

Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability,and allows identification of antigen-specific CD4+T cells in peripheral blood mononuclear cells

Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P. and Kappler, J., Immunity 1998. 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA(306-318)). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells. It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios.     

Comparison of TCR-alpha beta sequences of cross-reactive anti-DR alloreactive T-cell clones: identification of possible contact residues based on charge complementarity between TCR chains and DR determinants.

We analysed alloreactive T-cell clones selected for their differential recognition of DR variants differing in the third hypervariable region (hvr) of the DRB1 gene (amino acid positions 67-70-71). This polymorphism leads to two main hvr3 types: a basic form (Leu67-Gln70-Arg/Lys71) and an acidic form (Ile67-Asp70-Glu71) where residue 70 is probably directly accessible to the TCR on DR beta chains. The TCRs have been sequenced. Three DRw13-reactive clones use similar V alpha 2 and V beta 13 gene family members but differ mainly by their cross-reactivity towards acidic or basic DR4 variants and by the sequence of CDR3 on their TCR alpha and/or beta chains. One anti-DRw13 clone cross-reacts with most specificities sharing the DRw13 type of hvr3 and reciprocally one anti-DRBon (DRB1*0103) clone cross-reacts with DRw13. These two clones use similar V beta genes and share negative charges in CDR2 alpha at position 56. They also share these negative charges in CDR2 alpha with two other clones reacting specifically with DRBon, the acidic variant of DR1. We hypothesized that the selective recognition of positively or negatively charged residues on the DR beta chain would necessitate reciprocal charges on the TCR complementarity determining regions (CDRs) responsible for this interaction. This facilitated identification of those residues of the TCR that possibly interact with the hvr3 determinant of HLA-DR. From these observations the mechanisms allowing the recognition of alloantigens by these T-cell clones are discussed.     

中国猪种SLA-DR基因的克隆及序列分析

为探讨SLA DR基因结构及其在猪 人异种器官移植中的作用 ,本研究采用RT PCR扩增三个中国猪种SLA DRcDNA ,然后克隆入测序载体 ,进行双向测序 ,获得了具有完整阅读框架的三个结构和已有序列不同的新DRB等位基因 ,其长度为80 1个核苷酸 ,除末端终止密码外 ,编码 2 6 6个氨基酸残基 ;三个中国猪种DRA基因和已报告的NIH小型猪DRA基因结构相同 ,其长度均为 75 9个核苷酸 ,除末端终止密码外 ,编码 2 5 2个氨基酸残基。分析还揭示 ,(1)中国猪种SLA DR基因及其推导的氨基酸序列与人相应基因的同源性明显高于小鼠I E基因 ;(2 )中国猪DR分子 (DRα链和β链 )存在和人CD4分子相结合的HLA同源序列 ,但个别关键氨基酸残基发生改变 ,其改变幅度明显低于小鼠I E分子。人 猪MHC显示较高的同源性 ,在分子水平为人体T细胞同时采用直接和间接方式识别猪SLA这一免疫学现象提供了依据。     

Substitutions in the HLA-DRα chain differentially affect DR7-restricted T-cell recognition of rabies virus antigen

To investigate the functional roles of DRα residues in T-cell recognition, 20 mutants of the DRα chain were constructed by site-directed mutagenesis. These DRα mutants were expressed with WT DR(β1∗0701) on mouse L cells and used as APC for four DR7-restricted T-cell clones specific for rabies virus antigens. The results indicate that the DRα residues are differentially involved in recognition of rabies virus antigen by different T-cell clones. Mutations in the floor of the antigen-binding groove (positions 9, 11, 22, and 24), on the α-helix (47, 55, 65, 66, and 72), and surprisingly on the outer loop (15, 18, and 19), abrogated recognition by at least one T-cell clone. Most of these residues appear to be involved in either peptide or TCR contact, based on the DR1 crystal structure. The involvement in T-cell recognition of DRα residues located in the outer loop outside the binding groove suggests that these residues may directly contact TCR, or indirectly contribute to the conformation of peptide sitting in the groove.     

Altered peptide ligands and wild-type peptide induce indistinguishable responses of a human Th0 clone

The response of the human influenza hemagglutinin (HA)-specific T helper clone HA1.7 to its wild-type (wt) HA307 – 319 peptide ligand and related altered peptide ligands (APL) was examined over a wide range of antigen concentrations. The time course of cytokine production and surface expression of CD40 ligand and CD3 was followed at the single-cell level by flow cytometry and compared with the induction of proliferation. We observed that the APL induced responses that were indistinguishable from those induced by the wt HA ligand, albeit at higher antigen densities. Moreover, the activation parameters were induced with identical kinetics. Blocking of CD4 co-ligation inhibited the recognition of the weakly stimulatory APL, but not of the wt HA307 – 319 peptide. Finally, in all cases the response of the T cell clone correlated with down-regulation of surface TCR/CD3 complexes. Together, these observations support a quantitative model of T cell activation.     

T cell recognition motifs of an immunodominant peptide of myelin basic protein in patients with multiple sclerosis: structural requirements and clinical implications

Myelin basic protein (MBP)-reactive T cells may play an important role in the pathogenesis of multiple sclerosis (MS). The T cell response to the 83 – 99 region of MBP represents a dominant autoreactive response to MBP in MS patients of DR2 haplotype. In this study, a large panel of DR2- and DR4-restricted T cell clones specific for the MBP83 – 99 peptide were examined for the recognition motifs and structural requirements for antigen recognition using alanine-substituted peptides. Our study revealed that although the recognition motifs of the T cell clones were diverse, the TCR contact residues within the 83 – 99 region of MBP were highly conserved. Two central residues (Phe90 and Lys91) served as the critical TCR contact points for both DR2- and DR4-restricted T cell clones. Single alanine substitution at residue 90 or residue 91 abolished the responses of 81 – 95 % of the T cell clones while a double alanine substitution rendered all T cell clones unresponsive. It was also demonstrated in this study that the substituted peptides altered the cytokine profile of some, but not all, T cell clones. Some MBP83 – 99-specific T cell clones were able to sustain alanine substitutions and were susceptible to activation by microbial antigens. The study has an important implication in designing a peptide-based therapy for MS.     

Sequencing of a new HLA-DR4 allele with an unusual residue at position 88 that does not affect T cell allo recognition.

It is rather common to encounter new HLA alleles identified by unorthodox patterns observed during low resolution typing performed with sequence specific oligonucleotide probes (SSOP). One of the best examples is locus DRB1, where allelic subtypes are characterized by a combination of a limited number of residues located in three hypervariable regions of exon 2. HLA-DR oligotyping analysis of a female caucasoïd bone marrow donor of the Lyon registry led to the identification of an individual that typed as DRB1^* 11, DRB3^* 02, DRB4^* 01, DQB1^* 0301-0302. This donor was however typed by serology as DR11 DR4, DR52 DR53, DQ7 DQ8. PCR-SSP typing for DR4 subtype revealed an amplification pattern typical for DRB1^* 0404. After PCR amplification of genomic DNA and sequencing the entire exon 2, a new DRB1 allele was identified : DRB1^* 04var that is identical to DRB1^* 0404, except for one nucleotide at codon 88 resulting in a Ser ==> Arg exchange. This mutation had prevented amplification with the DR generic primers. The donor inherited this new DRB1^* 04var from her mother who presented the same molecular characteristics. The complete SSOP typing of the implicated haplotype is : A^*68011, Cw^*0704, B^*4402, DRB1^* 04var, DRB4^* 0103, DQA^* 0301, DQB1^* 0302, DPB1^* 0601.

Cellular typing by three HTCs-DRB1^*0404/DW14 from the 9th Workshop showed that this DRB1^* 04var typed exactly like a DW14 cell (the S.R.R. being 40%, 34%, 36%, respectively). This suggests that residue 88 does not affect T cell recognition possibly due to its location at the far end of the alpha helix flanking the peptide binding groove.     

The effect of a single amino acid substitution within the V3 loop of HIV-1 gp120 on HLA-DR1-restricted CD4 T-cell recognition.

Viral variation has been proposed to play a role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1) infection, and is an important consideration in vaccine design. During the course of an infection, isolates with sequence changes in CD8 T-cell and B-cell epitopes arise. To determine whether sequence variation within the V3 loop of HIV-1 gp120 affects HLA-DR beta 1*0101-restricted CD4 T-cell recognition, we have generated CD4 T-cell clones (TLC) specific to gp120 V3 loop peptides. Four HLA-DR beta 1*0101-restricted groups of TLC were defined by distinct patterns of responses to a panel of peptides, consistent with a highly diverse T-cell repertoire recognizing the 30 amino acid stretch (296-326) of the gp120 V3 loop. Nevertheless, a single residue change at position 311 was found to abolish the recognition of two of the four groups of TLC. This was not due to an effect of the residue at 311 on binding to major histocompatibility complex (MHC), because: (1) irrespective of the residue at 311, peptides competed well with the influenza haemagglutinin peptide 307-319 for binding to cell-bound DR1; and (2) R311-specific TLC were also HLA DR beta 1*0101 restricted. Instead, the substitution of arginine for serine at position 311 blocked the interaction of the peptide with the T-cell receptor. Thus, despite the diversity of the T-cell response to the V3 loop of HIV-1, a single amino acid change can have a considerable influence on the responding T-cell population. As residue 311 is one of the most variable of the V3 loop residues, these results suggest that CD4 recognition can also exert pressure on viral variation consistent with a role for these cells in antiviral immunity.     

HLA CLASS II POLYMORPHISM AND IDDM SUSCEPTIBILITY IN THE GREEK POPULATION

The frequencies of HLA-DQA1, DQB1 and DRB1 alleles were compared between 50 Insulin-Dependent Diabetes Melitus (IDDM) patients and 49 healthy controls in the Greek population. Statistically significant difference in the frequencies of HLA-DQA1*0501-DQB1*0201 (P = 10^-4), DQA1*0301-DQB1*0201 (P= 0.01) and DQA P0301-DQB 1*0302 (P= 0.001) were observed. The DRB1*0405-DQA1*0301-DQB 1*0201 was the only DR, DQ combination significantly associated with the disease. The unexpected increase of DRB1*0405 observed in the Greek IDDM may suggest as reported in Chinese and Japanese IDDM a contribution of DRβ and DQα in susceptibility. Moreover, in contrast to the Asians, in the Greek, the DRβ, DQα are found with the usual DQβ 57-ve.     

Degenerate TCR recognition and dual DR2 restriction of autoreactive T cells: implications for the initiation of the autoimmune response in multiple sclerosis

TCR degeneracy may facilitate self-reactive T cell activation and the initiation of an autoimmune response in multiple sclerosis (MS). MHC class II alleles of the DR2 haplotype DR2a (DRB5*0101) and DR2b (DRB1*1501) are associated with an increased risk for MS in Caucasian populations. In order to selectively expand and characterize T cells with a high degree of TCR degeneracy that recognize peptides in the context of disease-associated DR2 alleles, we developed DR2-anchored peptide mixtures (APM). We report here that DR2-APM have a high stimulatory potency and can selectively expand T cells with a degenerate TCR (TCR(deg)). Due to the low concentration of individual peptides in the mixtures, T cell clones' proliferative response to DR2-APM implies that multiple peptides stimulate the TCR, which is a characteristic of TCR(deg). The frequency of DR2-APM-reactive T cells is significantly higher in MS patients than in healthy controls, suggesting that they may play a role in the development of the autoimmune response in MS. DR2-APM-reactive cells have a dual DR2 restriction: they recognize DR2-APM in the context of both DR2a and DR2b molecules. The DR2-APM-reactive cells' IL-17 secretion, together with cross-reactivity against myelin peptides, may contribute to their role in the development of autoimmune response in MS.     

Identification of conserved T cell receptor CDR3 residues contacting known exposed peptide side chains from a major histocompatibility complex class I-bound determinant

We have analyzed the T cell receptor (TCR) repertoire found in the major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response to the protein ovalbumin (OVA). Despite skewing towards the expression of Vβ5.2⁺ TCR by OVA-specific CTL from C57BL/6 mice, we found a relatively high degree of diversity in V(D)J usage in both TCR α- and β-chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR α- and β-chain VJ or VDJ junctions. These junctions form the third complementarity-determining regions (CDR3) of the TCR polypeptides involved in the direct interaction with the class I-bound peptide. Crystallographic analyses of K^b-peptide complexes predict that the major determinant from OVA, peptide OVA_257–264 (SIINFEKL), contains two exposed charged side chains which can contact the TCR. These are the negatively charged glutamic acid at determinant position 6 (P6) and the positively charged lysine at P7. To examine whether the TCR α-chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR β-chain derived from the Vβ5.2⁺ clone B3. OVA-specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR α-chains that also contained negatively charged junctional residues despite some further variation in Vα and Jα sequences. Stimulation of naive TCR β-chain transgenic T cells with a P7 substitution peptide analogue induced a T cell response that was no longer cross-reactive with the wild-type OVA_257–264 determinant, sugesting that the TCR α-chain from the T cell clone B3 can determine the specificity for this residue. Consequently, these results reveal the existence of conserved residues in the CDR3 of TCR α- and β-chains specific for OVA_257–264 and identify their possible orientation over the peptide-class I complex.     

Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H

Although the importance of CD4+ T cell responses to human cytomegalovirus (HCMV) has recently been recognized in transplant and immunosuppressed patients, the precise specificity and nature of this response has remained largely unresolved. In the present study we have isolated CD4+ CTL which recognize epitopes from HCMV glycoproteins gB and gH in association with two different HLA-DR antigens, DRA1*0101/DRB1*0701 (DR7) and DRA1*0101/DRB1*1101 (DR11). Comparison of amino acid sequences of HCMV isolates revealed that the gB and gH epitope sequences recognized by human CD4+ T cells were not only conserved in clinical isolates from HCMV but also in CMV isolates from higher primates (chimpanzee, rhesus and baboon). Interestingly, these epitope sequences from chimpanzee, rhesus and baboon CMV are efficiently recognized by human CD4+ CTL. More importantly, we show that gB-specific T cells from humans can also efficiently lyse peptide-sensitized Patr-DR7+ cells from chimpanzees. These findings suggest that conserved gB and gH epitopes should be considered while designing a prophylactic vaccine against HCMV. In addition, they also provide a functional basis for the conservation of MHC class II lineages between humans and Old World primates and open the possibility for the use of such primate models in vaccine development against HCMV.     

Characterization of the HLA-restrictive elements of a rubella virus-specific cytotoxic T cell clone: influence of HLA-DR4{beta} chain residue 74 polymorphism on antigenic peptide-T cell interaction

The influence of glutamic acid (E)-alanine (A) dimorphism atposition 74 of the DR4ß chain on cytotoxic T cellrecognition of an antigenic rubella virus peptide, E1(273–284),was studied using a panel of B cell lines and B cell transfectantsexpressing different HLA-DRB1 alleles as antigen-presentingcells and targets in 51Cr-release assays. Only B cell linesexpressing the DRB1^*0403, DRB1^*0406 or DRB 1^*0407 subtypes whichshared a residue, E, at position 74 in the DR4ß chainwhen sensitized with E1(273–284) elicited strong cytotoxicT lymphocyte responses. However, in direct binding and antibodyinhibition assays, it was shown that biotinylated E1(272–285)could bind to DR molecules with residues other than E at position74, including DRB1^*0401, DRB1^*0404 and DRB1^*1101 expressed ontransfectants. E1(272–285) bound with similar affinityto the transfectant with DRB1^*0403, which has E at position74, as well as the transfectant with DRB1^*0404, which does not.When T-B cell engagement rates were compared in cell conjugateassays, the percentage of T-B conjugates was higher when peptide-pulsedtransfectants with DRB1^*0403 were used than with transfectantsexpressing DRB1^*0404. Hence, the HLA DRß1 polymorphismat position 74, while not critical for the binding affinityof E1(272–285) to the HLA molecule, appears to be a primarydeterminant of restricted recognition and subsequent activationof the peptide-specific T cells.     

Sequencing of two new HLA class II alleles: DRB3*0218 and DQB1*030202

Two novel human leukocyte antigen (HLA) class II alleles for DRB3 and DQB1 genes detected in Caucasoid Spanish individuals are described: DRB3*0218 and DQB1*030202. Both alleles have been found during routine high-resolution typing by sequencing. DRB3*0218 shows a novel DRB3 gene polymorphic position, located at amino acid residue 58, alanine to glutamic acid. This residue is shared by several DRB1 alleles, including all described DRB1*11 subtypes. DQB1*030202 differs from DQB1*030201 by a point mutation at position 319 (T to C). This nucleotide change generates a new codon at amino acid position 75 that is not shared by any other DQB1 allele.     

Evidences of conformational changes in class II Major Histocompatibility Complex molecules that affect the immunogenicity

The N-terminal part of class II-associated invariant chain peptide (CLIP) is assumed to interact with an accessory peptide-binding site on the class II Major Histocompatibility Complex (MHC) molecule, and promote a conformational modification. We have linked this immunoregulatory segment (residues 81-88) to the N-terminus of the influenza hemagglutinin (HA) 307-319 epitope in order to evaluate relationships between the MHC conformational changes and their implication in immune responses. Our chimeric peptide, named CLIP-HA, bind with the same affinity to class II HLA-DR1 molecules as the HA peptide, and is normally recognized by HA-specific T cells. Interestingly, the presence of the N-terminal CLIP region enhances the rate of association to soluble DR1 molecules but prevents the formation of SDS-resistant complexes. These features suggest the existence of HLA-DR1 conformational changes induced by the chimeric peptide. Furthermore, while in vitro HA and CLIP-HA peptides associated to DR1 could not be differentiated based on T-cell recognition, in vivo the CLIP residues strongly impaired the immunogenicity of HA epitope as assessed in HLA-DR1 transgenic mice. Our study demonstrates for the first time that MHC conformational changes, revealed at molecular level, may influence the immunogenicity.     

CD4+ T cells from type 1 diabetic and healthy subjects exhibit different thresholds of activation to a naturally processed proinsulin epitope

Recent studies suggest that insulin is a primary autoantigen for type 1 diabetes. Several studies have identified preproinsulin (PPI) 76-90 as an immunodominant CD4+ T cell epitope. We developed a class II tetramer reagent using a modified PPI peptide with a lysine to serine substitution at position 88 (PPI 78-90(88S)) that has high binding affinity to DRA1*0101/DRB1*0401 (DR0401). Using this tetramer, positive responses were observed from both DR0401 healthy and type 1 diabetic subjects when T cells were stimulated with the PPI 78-90(88S) peptide. Seventy percent of these T cells proliferated in response to both the wild type PPI 76-90 and PPI 78-90(88S) peptides. However, when T cells were stimulated with wild type peptide and assayed with DR0401/PPI 78-90(88S), positive responses were only detected in the diabetic group but not in healthy subjects. When highly purified CD4+CD25-CD45RA+ T cells were stimulated with PPI 78-90(88S) peptide in the absence of antigen-presenting cells, T cells from the diabetic group were able to respond to peptide stimulation, while T cells from healthy subjects were not. These data suggest that T cells from type 1 diabetic subjects have a lower threshold of activation in response to PPI peptide stimulation as compared to healthy subjects.     

Characterization of in vivo expanded OspA-specific human T-cell clones

A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from a patient with treatment-resistant Lyme arthritis using a DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with outer surface protein A (OspA) peptide164-175, an immunodominant epitope of Borrelia burgdorferi. Sequencing of the T-cell receptors of the OspA reactive clones showed significant skewing of the T-cell receptor repertoire. Of the 101 T-cell clones sequenced, 81 possessed TCR beta chains that were present in at least one other clone isolated. Complete sequencing of both alpha and beta chains of a subset of clones showed that at least two distinct T-cell clones were expanded in vivo. Binding studies using a panel of Ala-substituted peptide ligands were performed to determine potential MHC binding sites of the OspAp164-175 to DRB1*0401. In addition, T-cell clones were tested functionally for their reactivity to the wild-type peptide as well as to altered peptide ligands (APLs) and peptide libraries based on the OspA epitope in order to determine the TCR contact residues and the stringency in T cell recognition. We are among the first to define the characteristics of TCR usage of T cells isolated from an inflamed immune compartment in an individual with an autoimmune disease potentially triggered by a microbial antigen.