CD8+ T cell-mediated HLA-A*0201-restricted cytotoxicity to transaldolase peptide 168-176 in patients with multiple sclerosis

Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes and targeted by autoreactive T cells of patients with multiple sclerosis (MS). Among 14 TAL peptides with predicted HLA-A2 binding, TAL 168-176 (LLFSFAQAV, TALpep) exhibited high affinity for HLA-A2. Prevalence of HLA-A2-restricted CD8+ T cells specific for TALpep was increased in PBMC of HLA-A2+ MS patients, as compared with HLA-A2- MS patients, HLA-A2+ other neurological disease patients, and HLA-A2+ healthy donors. HLA-A*0201/TALpep tetramers detected increased frequency of TAL-specific CD8+ T cells, and precursor frequency of TAL-specific IFN-gamma-producing T cells was increased in each of seven HLA-A2+ MS patients tested. Stimulation by TALpep or rTAL of PBMC from HLA-A2+ MS patients elicited killing of TALpep-pulsed HLA-A2-transfected HmyA2.1 lymphoma cells, but not HLA-A3-transfected control HmyA3.1 targets. Without peptide pulsing of targets, HLA-A2-transfected, but not control MO3.13 oligodendroglial cells, expressing high levels of endogenous TAL, were also killed by CD8+ CTL of MS patients, indicating recognition of endogenously processed TAL. TCR Vbeta repertoire analysis revealed use of the TCR Vbeta14 gene by T cell lines (TCL) of MS patients generated via stimulation by TAL- or TALpep-pulsed APCs. All TAL-specific TCL-binding HLA-A*0201/TALpep tetramers expressed TCR Vbeta14 on the cell surface. Moreover, Ab to TCR Vbeta14 abrogated cytotoxicity by HLA-A2-restricted TAL-specific TCL. Therefore, TAL-specific CTL may serve as a novel target for therapeutic intervention in patients with MS.     

A novel mutation in PTPRC interferes with splicing and alters the structure of the human CD45 molecule

CD45, encoded by the protein tyrosine phosphatase receptor type C ( PTPRC) gene, is essentially involved in maturation, activation, and migration of immune cells. Lack of CD45 results in severe immunodeficiency, and alterations of the receptor may result in autoimmunity. Here, we describe a novel mutation in PTPRCas a cause of variant CD45 expression in humans. Several members of a multiple sclerosis multiplex family showed expression of CD45RA on memory T cells and monocytes. The variant expression pattern was linked to the PTPRCgene by DNA microsatellite studies. DNA analysis identified a novel point mutation in exon 4 (position 59 C-->A) in all family members with variant CD45 expression, but not in donors with normal CD45 expression. The mutation interferes with alternative splicing and alters amino acid sequence (H-->Q), interfering with antibody binding to the CD45RA domain. Overall, we describe the first mutation in PTPRCthat interferes with splicing and results in surface expression of a structurally altered CD45 molecule in humans.     

Transformation of T-lymphocyte subsets by Marek''s disease herpesvirus.

Marek's disease herpesvirus (MDV)-transformed lymphoblastoid tumor cell lines were characterized for the presence of the surface markers. Monoclonal antibodies were used for CD3 (T-cell receptor [TCR] complex), TCR1, TCR2, and TCR3, CD4, CD8, and Ia antigen by indirect fluorescence staining followed by microscopic examination or flow cytometry. The lymphoblastoid cell lines were obtained from tumors from chickens infected with MDV (n = 44) or from local lesions induced by inoculation of allogeneic, MDV-infected chick kidney cells (n = 56). Lymphocytes were harvested from these lesions between 4 and 16 days postinoculation and cultured in vitro to establish cell lines. All cell lines expressed Ia antigen and CD3 and/or TCR and thus are activated T cells. Most of the cell lines developed from tumors were CD4+ CD8-; only one cell line was negative for both markers. Sixteen percent of the cell lines were TCR3+, while the remainder were TCR2+. The cell lines developed from local lesions were much more heterogeneous: 45% were CD4- CD8+, 34% were CD4- CD8-, and only 21% were CD4+ CD8-. The number of TCR3+ cell lines was larger than expected for the CD4- CD8+ and CD4- CD8- cell lines, as judged from the presence of these cells in the blood. These results indicate that several subsets of T lymphocytes can be transformed by MDV, depending on the pathogenesis of infection. Activation of T cells as a consequence of the normal pathogenesis or by allogeneic stimulation seem to be a first important step in the process of transformation.     

Proliferating CD4+ T cells undergo immediate growth arrest upon cessation of TCR signaling in vivo

To investigate the role of TCR signaling in the exit of CD4+ T cells from cell cycle, we took advantage of a low frequency TEa T cell adoptive transfer technique as well as the Y-Ae mAb to interrupt Ag/MHC recognition before the completion of clonal expansion. Termination of TCR signaling after 36 h of Ag exposure caused an immediate reduction in cell size and deceleration of G1->SG2M phase cell cycle progression. As a consequence, clonal expansion in the absence of durable TCR signaling decreased by two-thirds. Thus, CD4+ T cells scan for the presence Ag throughout their clonal expansion response, and continuously adjust their rate of cell growth and G1->S phase transition to match their intensity of TCR signaling.     

The repertoire of CD4+ CD28- T cells in rheumatoid arthritis.

BACKGROUND: While oligoclonality of circulating CD4- CD8 and of CD8+ T cells is not uncommon, clonal dominance within the CD4 compartment is not frequently found in healthy individuals. In contrast, the majority of patients with rheumatoid arthritis (RA) have clonally expanded CD4+ T cell populations. Previous studies have demonstrated that these clonogenic CD4+ T cells do not express the CD28 molecule. To examine the correlation between CD28 expression and clonal proliferation, we have analyzed the T cell receptor (TCR) diversity of CD4+ CD28- T cells in normal individuals and in RA patients. MATERIAL AND METHODS: The size of the peripheral blood CD4+ CD28- compartment was determined in 30 healthy individuals and 30 RA patients by two-color FACS analysis. In 10 RA patients and five controls with more than 2.5% CD4+ CD28- T cells, TCR BV gene segment usage was analyzed with 19 BV-specific antibodies. Oligoclonality was assessed in sorted CD4+ CD28+ and CD28- T cells using TCR BV-BC-specific polymerase chain reaction and size fractionation. Clonal dominance was confirmed by direct sequencing. RESULTS: The CD4+ CD28- T cell compartment was expanded to more than 2.5% in 70% of the RA patients and 30% of the normal individuals. Compared with the CD4+ CD28+ T cells, the TCR BV gene segment usage among CD4+ CD28- cells was grossly skewed with the dominance of single BV elements. Molecular TCR analysis provided evidence for oligoclonality in 17 of 21 expanded BV elements. In two unrelated RA patients who shared both HLA-DRB1 alleles, the TCR beta-chain sequences of dominant clonotypes were highly conserved. CONCLUSIONS: Oligoclonality is a characteristic feature of CD4+ CD28- T cells which are expanded in some healthy individuals and in the majority of RA patients. The lack of CD28 expression is a common denominator of CD4+, CD8+, and CD4- CD8- T cells prone to develop clonal dominance. The limited TCR diversity of clonal CD4+ CD28- populations in RA patients suggests that these T cells recognize a limited spectrum of antigens. The fact that the majority of individuals with marked expansions and oligoclonality of CD4+ CD28- T cells are RA patients suggests a role for these unusual lymphocytes in the pathogenetic events leading to RA.     

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.     

The exon A (C77G) mutation is a common cause of abnormal CD45 splicing in humans

The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted.     

Long-term stable expanded human CD4+ T cell clones specific for human cytomegalovirus are distributed in both CD45RAhigh and CD45ROhigh populations

T cells play an important role in the control of human CMV (HCMV) infection. Peripheral blood CD4+ T cell proliferative responses to the HCMV lower tegument protein pp65 have been detected in most healthy HCMV carriers. To analyze the clonal composition of the CD4+ T cell response against HCMV pp65, we characterized three MHC class II-restricted peptide epitopes within pp65 in virus carriers. In limiting dilution analysis, we observed high frequencies of pp65 peptide-specific CD4+ T cells, many of which expressed peptide-specific cytotoxicity in addition to IFN-gamma secretion. We analyzed the clonal composition of CD4+ T cells specific for defined HCMV peptides by generating multiple independent peptide-specific CD4+ clones and sequencing the TCR beta-chain. In a given carrier, most of the CD4+ clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used clonotype oligonucleotide probing to quantify the size of individual peptide-specific CD4+ clones in whole PBMC and in purified subpopulations of CD45RAhighCD45ROlow and CD45RAlowCD45ROhigh cells. Individual CD4+ T cell clones could be large (0.3-1.5% of all CD4+ T cells in PBMC) and were stable over time. Cells of a single clone were distributed in both the CD45RAhigh and CD45ROhigh subpopulations. In one carrier, the virus-specific clone was especially abundant in the small CD28-CD45RAhigh CD4+ T cell subpopulation. Our study demonstrates marked clonal expansion and phenotypic heterogeneity within daughter cells of a single virus-specific CD4+ T cell clone, which resembles that seen in the CD8+ T cell response against HCMV pp65.     

Increased CD8+ cytotoxic T cell responses to myelin basic protein in multiple sclerosis

Autoreactive T cells of CD4 and CD8 subsets recognizing myelin basic protein (MBP), a candidate myelin autoantigen, are thought to contribute to and play distinct roles in the pathogenesis of multiple sclerosis (MS). In this study we identified four MBP-derived peptides that had high binding affinity to HLA-A2 and HLA-A24 and characterized the CD8(+) T cell responses and their functional properties in patients with MS. There were significantly increased CD8(+) T cell responses to 9-mer MBP peptides, in particular MBP(111-119) and MBP(87-95) peptides that had high binding affinity to HLA-A2, in patients with MS compared with healthy individuals. The resulting CD8(+) T cell lines were of the Th1 phenotype, producing TNF-alpha and IFN-gamma and belonged to a CD45RA(-)/CD45RO(+) memory T cell subset. Further characterization indicated that the CD8(+) T cell lines obtained were stained with MHC class I tetramer (HLA-A2/MBP(111-119)) and exhibited specific cytotoxicity toward autologous target cells pulsed with MBP-derived peptides in the context of MHC class I molecules. These cytotoxic CD8(+) T cell lines derived from MS patients recognized endogenously processed MBP and lysed COS cells transfected with genes encoding MBP and HLA-A2. These findings support the potential role of CD8(+) CTLs recognizing MBP in the injury of oligodendrocytes expressing both MHC class I molecules and MBP.     

Age-associated change in the frequency of memory CD4+ T cells impairs long term CD4+ T cell responses to influenza vaccine

We investigated the relationship of memory CD4+ T cells with the evolution of influenza virus-specific CD4+ T cell responses in healthy young and elderly people. Elderly individuals had a similar frequency of CD69+CD4+ T cells producing IFN-gamma and TNF-alpha at 1 wk, but a lower frequency of these CD4+ T cells at 3 mo after influenza vaccination. Although the elderly had a higher frequency of central memory (CM; CCR7+CD45RA-) CD4+ T cells, they had a significantly lower frequency of effector memory (EM; CCR7-CD45RA-) CD4+ T cells, and the frequency of the latter memory CD4+ T cells positively correlated with the frequency of influenza virus-specific CD69+CD4+ T cells producing IFN-gamma at 3 mo. These findings indicate that the elderly have an altered balance of memory CD4+ T cells, which potentially affects long term CD4+ T cell responses to the influenza vaccine. Compared with the young, the elderly had decreased serum IL-7 levels that positively correlated with the frequency of EM cells, which suggests a relation between IL-7 and decreased EM cells. Thus, although the healthy elderly mount a level of CD4+ T cell responses after vaccination comparable to that observed in younger individuals, they fail to maintain or expand these responses. This failure probably stems from the alteration in the frequency of CM and EM CD4+ T cells in the elderly that is related to alteration in IL-7 levels. These findings raise an important clinical question about whether the vaccination strategy in the elderly should be modified to improve cellular immune responses.     

A CD45 polymorphism associated with abnormal splicing is absent in African populations

The CD45 antigen is essential for normal antigen receptor-mediated signalling in lymphocytes, and different patterns of splicing of CD45 are associated with distinct functions in lymphocytes. Abnormal CD45 splicing has been recognized in humans, caused by a C77G transversion in the gene encoding CD45 (PTPRC). Recently the C77G polymorphism has been associated with multiple sclerosis and increased susceptibility to HIV-1 infection. These studies suggest that the regulation of CD45 splicing may be critical for the proper function of the immune system. Because of these data we examined the frequency of the C77G allele in African and Asian populations from countries with high or low prevalence of HIV infection. Here we report that the variant CD45 C77G allele is absent in African populations. We further show that populations living in the Pamir mountains of Central Asia have a very high prevalence of the C77G variant.     

Isolation of CD45RO+, memory T cells recognizing proteolipid protein from neurologically normal subjects.

Previous studies provide evidence for in vivo activation of MBP-reactive T cells in subjects with multiple sclerosis. In general, in vivo activation occurs less frequently in healthy control subjects. In the current study we examined the T cell response to proteolipid protein in PBMC isolated from 9 control subjects. We used CD45 isotypes as markers for memory and na?ve T cells to assess in vivo activation of CD4+ T cells reactive with PLP. In contrast to the results obtained using MBP, we found that approximately 50% of PLP-reactive T cells were derived from the CD45RO+ memory subpopulation of T cells isolated from these control subjects. These results indicate that some myelin-reactive T cells have undergone activation in vivo in neurologically intact individuals. This suggests that immunoregulatory mechanisms may be present that prevent overt disease in spite of in vivo activation of PLP-reactive T cells.     

Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3alpha and CDR2beta amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1(+)/HLA-A*02(+) tumor cell lines by TCR gene-modified CD4(+) T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3alpha chain was identified that enhanced Ag-specific reactivity in gene-modified CD4(+) and CD8(+) T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27-35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4(+) T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.     

Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis

Chronic progression of two T cell-mediated central nervous system (CNS) demyelinating models of multiple sclerosis, relapsing EAE (R-EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is dependent on the activation of T cells to endogenous myelin epitopes (epitope spreading). Using transfer of carboxyfluorescein succinyl ester (CFSE)-labeled T-cell receptor (TCR)-transgenic T cells and mixed bone marrow chimeras, we show that activation of naive proteolipid protein (PLP)139-151-specific T cells in SJL mice undergoing PLP178-191-induced R-EAE or TMEV-IDD occurs directly in the CNS and not in the cervical lymph nodes or other peripheral lymphoid organs. Examination of the antigen-presentation capacity of antigen-presenting cell (APC) populations purified from the CNS of mice with PLP178-191-induced R-EAE shows that only F4/80-CD11c+CD45hi dendritic cells (DCs) efficiently present endogenous antigen to activate naive PLP139-151-specific T cells in vitro. In contrast, DCs as well as F4/80+CD45hi macrophages and F4/80+CD45lo microglia activate a PLP139-151-specific helper T cell line. The data suggest that naive T cells enter the inflamed CNS and are activated by local APCs, possibly DCs, to initiate epitope spreading.     

Homeostasis of the naive CD4+ T cell compartment during aging

Despite thymic involution, the number of naive CD4(+) T cells diminishes slowly during aging, suggesting considerable peripheral homeostatic expansion of these cells. To investigate the mechanisms behind, and consequences of, naive CD4+ T cell homeostasis, we evaluated the age-dependent dynamics of the naive CD4+ T cell subsets CD45RA+CD31+ and CD45RA+CD31-. Using both a cross-sectional and longitudinal study design, we measured the relative proportion of both subsets in individuals ranging from 22 to 73 years of age and quantified TCR excision circle content within those subsets as an indicator of proliferative history. Our findings demonstrate that waning thymic output results in a decrease in CD45RA+CD31+ naive CD4+ T cells over time, although we noted considerable individual variability in the kinetics of this change. In contrast, there was no significant decline in the CD45RA+CD31- naive CD4+ T cell subset due to extensive peripheral proliferation. Our longitudinal data are the first to demonstrate that the CD45RA+CD31+CD4+ subset also undergoes some in vivo proliferation without immediate loss of CD31, resulting in an accumulation of CD45RA+CD31+ proliferative offspring. Aging was associated with telomere shortening within both subsets, raising the possibility that accumulation of proliferative offspring contributes to senescence of the naive CD4+ T cell compartment in the elderly. In contrast, we observed retention of clonal TCR diversity despite peripheral expansion, although this analysis did not include individuals over 65 years of age. Our results provide insight into naive CD4+ T cell homeostasis during aging that can be used to better understand the mechanisms that may contribute to immunosenescence within this compartment.     

T cell receptor alpha/beta expressing double-negative (CD4-/CD8-) and CD4+ T helper cells in humans augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis

It is generally accepted that human Th cells express the surface glycoproteins CD4 and alpha/beta-chain heterodimer of the TCR whereas cytotoxic/suppressor cells are usually CD8+ and alpha/beta TCR+. Another minor set of T cells found in the periphery are CD4-/CD8- (double negative) and express the gamma/delta TCR; these cells can manifest MHC-restricted or nonrestricted cytotoxicity but no helper function. Herein we describe the existence of an unusual Th population in the peripheral blood of humans that are CD4-/CD8- and alpha/beta TCR+. These double-negative Th were markedly expanded in patients with the autoimmune disease SLE and along with CD4+ Th, they induced production of the pathogenic variety of anti-DNA autoantibodies that are IgG in class and cationic in charge. The cationic anti-DNA antibodies induced by the Th were markedly restricted in spectrotype indicating that an oligoclonal population of B cells were committed to produce the pathogenic autoantibodies in active lupus. IL-2-dependent T cell lines were also derived from the patients with active lupus nephritis but the majority of those T cell lines lacked pathogenic autoantibody-inducing capability. Only 4 out of 42 T cell lines from a lupus patient could induce the production of cationic IgG class anti-DNA autoantibodies. The phenotypes of the pathogenic autoantibody-inducing Th lines were similar to the Th subsets: CD4+, alpha/beta TCR+ or CD4-/CD8-, alpha/beta TCR+. These studies suggest that production of pathogenic autoantibodies in human lupus is mediated by mechanisms that are distinct from the generalized, nonspecific polyclonal B cell hyperactivity that leads to excessive production of natural autoantibodies.     

Deficient nucleotide excision repair increases base-pair substitutions but decreases TGGC frameshifts induced by methylglyoxal in Escherichia coli.

To investigate the mutation spectrum of a well-known mutagen, methylglyoxal, and the influence of nucleotide excision repair (NER) on methylglyoxal-induced mutations, we treated wild-type and NER-deficient (uvrA or uvrC) Escherichia coli strains with methylglyoxal, and analyzed mutations in the chromosomal lacI gene. In the three strains, the cell death and the mutation frequency increased according to the dose of methylglyoxal added to the culture medium. The frequencies of methylglyoxal-induced base-pair substitutions were higher in the NER-deficient strains than in the wild-type strain, in the presence and absence of mucAB gene. Paradoxically, the frequency of methylglyoxal-induced TGGC frameshifts was higher in the wild-type strain than in the NER-deficient strains. When the methylglyoxal-induced mutation spectra in the presence and absence of mucAB gene are compared, the ratios of base-pair substitutions to frameshifts were increased by the effects of mucAB gene. In the three strains, more than 75% of the base-pair substitutions occurred at G:C sites, independent of the mucAB gene. When the mucAB gene was present, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. When the mucAB gene was absent, the predominant mutations differed in the three strains: in the wild-type and uvrC strains, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions, while in the uvrA strains, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. These results suggest that NER may be involved in both the repair and the fixation of methylglyoxal-induced mutations.     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.     

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.     

A common TCR V-D-J sequence in V beta 13.1 T cells recognizing an immunodominant peptide of myelin basic protein in multiple sclerosis.

T cell responses to the immunodominant peptide (residues 83-99) of myelin basic protein are potentially associated with multiple sclerosis (MS). This study was undertaken to examine whether a common sequence motif(s) exists within the TCR complementarity-determining region (CDR)-3 of T cells recognizing the MBP83-99 peptide. Twenty MBP83-99-reactive T cell clones derived from patients with MS were analyzed for CDR3 sequences, which revealed several shared motifs. Some V beta 13.1 T cell clones derived from different patients with MS were found to contain an identical CDR3 motif, V beta 13.1-LGRAGLTY. Oligonucleotides complementary to the shared CDR3 motifs were used as specific probes to detect identical target CDR3 sequences in a large panel of T cell lines reactive to MBP83-99 and unprimed PBMC. The results revealed that, in contrast to other CDR3 motifs examined, the LGRAGLTY motif was common to T cells recognizing the MBP83-99 peptide, as evident by its expression in the majority of MBP83-99-reactive T cell lines (36/44) and PBMC specimens (15/48) obtained from randomly selected MS patients. The motif was also detected in lower expression in some PBMC specimens from healthy individuals, suggesting the presence of low precursor frequency of T cells expressing this motif in healthy individuals. This study provides new evidence indicating that the identified LGRAGLTY motif is preferentially expressed in MBP83-99-reactive T cells. The findings have important implications in monitoring and targeting MBP83-99-reactive T cells in MS.