Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

Common intra-articular T cell expansions in patients with reactive arthritis: identical beta-chain junctional sequences and cytotoxicity toward HLA-B27

Spondyloarthropathies constitute a group of autoimmune diseases of special interest because of their tight association with the MHC class I molecule HLA-B27 and the bacterial triggering of some clinical forms called reactive arthritis (ReA). One current hypothesis is the presentation by HLA-B27 of a so-called arthritogenic peptide to T cells. To better focus on the relevant T cell populations within the joint, we performed an extensive beta-chain T cell repertoire analysis of synovial fluid compared with PBL in seven patients, four of whom were characterized as having ReA triggered by Yersinia enterocolitica, Chlamydia trachomatis, or Shigella sonnei. Analysis of the size diversity of the beta-chain complementarity-determining region 3 (CDR3) allowed us to evaluate the degree of T cell clonality in the samples. Oligoclonal T cell expansions were frequently observed in the joint. In one patient, CDR3 amino acid sequences of major expansions using two different BV genes were identical. One dominant T cell expansion and several CDR3 amino acid sequences were identical in two different patients. Furthermore, one sequence was identical with a sequence reported independently in a Salmonella-induced ReA patient. Together, these data indicate a surprisingly high degree of conservation in the T cell responses in recent-onset ReA triggered by different micro-organisms. A CD8+ synovial line expressing shared clonotypes was established and reacted toward several B*2705 lymphoblastoid cell lines, therefore supporting a molecular mimicry phenomenon at the T cell level in the disease mechanism.     

A clonotype nomenclature for T cell receptors

T cell receptor (TCR) nucleotide sequences are often generated during analyses of T cell responses to pathogens or autoantigens. The most important region of the TCR is the third complementarity-determining region (CDR3) whose nucleotide sequence is unique to each T cell clone. The CDR3 interacts with the peptide and thus is important for recognizing pathogen or autoantigen epitopes. While conventions exist for identifying the various TCR chains, there is a lack of a concise nomenclature that would identify both the amino acid translation and nucleotide sequence of the CDR3. This deficiency makes the comparison of published TCR genetic and proteomic information difficult. To enhance information sharing among different databases and to facilitate computational assessment of clonotypic T cell repertoires, we propose a clonotype nomenclature. The rules for generating a clonotype identifier are simple and easy to follow, and have a built-in error-checking system. The identifier includes the V and J region, the CDR3 length as well as its human or mouse origin. The framework of this naming system could also be expanded to the B cell receptor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of T cell receptors in highly purified rat and human large granular lymphocytes (LGL): lack of functional 1.3 kb beta-chain mRNA

Recent reports have suggested that the T cell receptor for antigen is somehow involved in the cytotoxicity of natural killer (NK) cells. However, we now report that highly purified, freshly isolated large granular lymphocytes (LGL) from both the human and rat, as well as LGL cultured in the presence of recombinant IL 2, express only the 1.0 kb beta-chain mRNA. The lack of a 1.3 kb mRNA, indicative of a functional beta-chain rearrangement, strongly suggests that a functional T cell receptor beta-chain is absent in freshly isolated LGL, thus making it extremely unlikely that this molecule is involved in target cell recognition by NK cells. These results also suggest that LGL are derived from a lineage distinct from T cells or develop before a functional rearrangement of the T cell receptor beta-chain.     

Reconstitution of CD8+ T cells by retroviral transfer of the TCR alpha beta-chain genes isolated from a clonally expanded P815-infiltrating lymphocyte

Gene transfer of TCR alphabeta-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and Vbeta subfamily usage of CD8(+) TIL in the tumor sites demonstrated that T cells using Vbeta10 efficiently infiltrated and expanded clonally. The TCR alpha- and beta-chain sequences derived from a tumor-infiltrating CD8(+)/Vbeta10(+) single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR alphabeta-chain genes were retrovirally introduced into CD8(+) T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2L(d) mAb. Furthermore, T cells containing both TCR alpha- and beta-chains, but not TCR beta-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8(+) T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.     

T cell receptor gene segment utilization by HLA-DR1-alloreactive T cell clones.

Transplantation of histoincompatible tissues leads to allograft rejection, which involves recognition of allogeneic MHC molecules by Ag-specific receptors expressed on T cells. The interaction of these molecules is highly specific yet poorly understood. We have investigated the relationship between TCR gene utilization and allo-MHC restriction patterns by using a one-way polymerase chain reaction to amplify the alpha- and beta-chain mRNA from a panel of 10 HLA-DR1-alloreactive T lymphocyte clones. Two previously unreported V alpha and five J alpha gene sequences were obtained. Although a few V alpha, V beta, and J alpha genes were utilized more than once, no correlation between TCR gene usage and DR1 alloreactivity was identified. At the sequence level, the presumed TCR alpha- and beta-chain CDR1 and CDR2 regions displayed limited diversity, whereas the CDR3 or junctional sequences were highly variable. Although most TCR probably interact with subtly different surface features of the DR1 alloantigen, we predict that TCR with similar CDR1 and CDR2 sequences would contact essentially identical regions of the DR1 molecule. The lack of sequence conservation in the junctional regions suggests that different endogenous peptides also may be recognized. Thus, alloreactive T cells may recognize not only allogeneic MHC molecules but perhaps also bound endogenous peptides.     

Thymocyte maturation: selection for in-frame TCR alpha-chain rearrangement is followed by selection for shorter TCR beta-chain complementarity-determining region 3

Thymocyte maturation consists of a number of stages, the goal of which is the production of functioning T cells that respond to foreign antigenic peptides using their clonotypic receptors. Selection of a productively rearranged TCR beta-chain is the first stage in the process and occurs at the double-negative to double-positive (DP) transition. Later maturation stages are based on changes in markers such as CD5, CD69, or IL-7R. A stage in which a-chains are selected has also been identified using beta-chain transgenic mice. Here we identify two additional selection stages in human thymocytes based on characteristics of the TCR. alpha selection is measured directly by identification of in-frame rearrangements and is associated with the appearance of CD3 on the DP thymocyte surface. The next stage has not yet been described and involves selection of thymocytes that express shorter TCR beta-chain complementarity-determining region 3 (CDR3). This stage is associated with the acquisition of high levels of CDR3 by DP cells and the transition to SP thymocytes. The extent of CDR3 length selection observed is a function of the TCR V and J genes. We propose that CDR3 length selection is based on recognition of the MHC. Thus, there exist limitations on the allowable length of that portion of the TCR most intimately in contact with MHC and peptide. This may be a physical representation of positive selection.     

Hapten addition to an MHC class I-binding peptide causes substantial adjustments of the TCR structure of the responding CD8(+) T cells

T cell responses against hapten-modified peptides play an important role in the pathogenesis of certain diseases, including contact dermatitis and allergy. However, the structural features of TCRs recognizing bulky, potentially mobile hapten groups remain poorly defined. To analyze the structural basis of TCR recognition of defined hapten-modified peptides, the immunodominant octapeptide derived from vesicular stomatitis virus nucleoprotein (VSV8) was modified with a trinitrophenyl (TNP) group at the primary TCR contact residues (position 4 or 6) and used for immunization of mice carrying either the TCR alpha- or beta-chain of a VSV8 (unmodified)/H-2K(b)-specific CTL clone as a transgene. Such mice allow independent analysis of one TCR chain by maintaining the other fixed. The TCR V gene usage of the responding T cell population was specifically altered depending upon the presence of the TNP group and its position on the peptide. The CDR3 sequences of the TNP-modified peptide-specific TCRs showed a preferential J region usage in both the CDR3alpha and beta loops, indicating that the J regions of both CDR3s are critical for recognition of TNP-modified peptides. In contrast to our previous observations showing the prime importance of CDR3beta residues encoded by D-segment or N-addition nucleotides for recognition of position 6 of unmodified VSV8, our studies of TNP-modified peptides demonstrate the importance of the Jbeta region, while the Jalpha region was crucial for recognizing both TNP-modified and unmodified peptides. These data suggest that different structural strategies are utilized by the CDR3alpha and beta loops to allow interaction with a haptenated peptide.     

Polysaccharide A from the Capsule of Bacteroides fragilis Induces Clonal CD4+ T Cell Expansion

For 3 decades, the view of MHCII-dependent antigen presentation has been completely dominated by peptide antigens despite our 2004 discovery in which MHCII was shown to present processed fragments of zwitterionic capsular polysaccharides to T cells. Published findings further demonstrate that polysaccharide A (PSA) from the capsule of Bacteroides fragilis is a potent activator of CD4⁺ T cells and that these T cells have important biological functions, especially in the maintenance of immunological homeostasis. However, little is known about the nature of T cell recognition of the polysaccharide-MHCII complex or the phenotype of the resulting activated cells. Here, we use next-generation sequencing of the αβT cell receptor of CD4⁺ T cells from mice stimulated with PSA in comparison with protein antigen simulation and non-immunized controls and found that PSA immunization induced clonal expansion of a small subset of suppressive CD4⁺CD45RB^low effector/memory T cells. Moreover, the sequences of the complementarity-determining region 3 (CDR3) loop from top clones indicate a lack of specific variable β and joining region use and average CDR3 loop length. There was also a preference for a zwitterionic motif within the CDR3 loop sequences, aligning well with the known requirement for a similar motif within PSA to enable T cell activation. These data support a model in which PSA, and possibly other T cell-dependent polysaccharide antigens, elicits a clonal and therefore specific CD4⁺ T cell response often characterized by pairing dual-charged CDR3 loop sequences with dual-charged PSA.     

Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR beta-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vbeta subsets (Vbeta2, Vbeta4, and Vbeta16 positive) with highly conserved TCRbeta complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR beta-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR alpha-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR alpha-chain vs TCR beta-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.     

Characterization of the γδ T cell response to acute leukemia

Background: Previous work from our center has suggested a correlation between increased donor-derived Vδ1+ γδ T cells and long-term relapse-free survival following bone marrow transplantation for leukemia. Questions remain, however, as to whether this observation can be explained by a γδ T cell-based immune response against primary leukemia. Methods: We examined γδ T cell receptor (TCR) phenotype, cell proliferation, and cytolytic activity following culture with irradiated primary leukemia blasts from a haploidentical first-degree relative. Subsequently, we also studied the γδ TCR phenotype and complimentarity determining region 3 (CDR3) cDNA sequences from 17 newly diagnosed leukemia patients. Results: In 17/28 (61%) of in vitro cultures, γδ T cells proliferated in culture with primary blasts. Vδ1+ T cells were proportionally increased in all cultures and were the predominant cell population in 6/17. In the 7 cultures where cytotoxicity could be assessed, 6 (86%) showed some degree of cytotoxicity to the primary leukemia. Vδ1+ T cells were also the predominant γδ T cell subtype in pre-treatment leukemia patients principally due to loss of Vδ2+ T cells rather than expansion of Vδ1+ cells. The Vδ1 CDR3-region cDNA sequence from these patients revealed exclusive use of the Jδ1 constant region and sequence conservation in 4/11 patients. Conclusions: γδ T cells exhibit an in vitro response to primary leukemia blasts that is manifested by proliferation, an increased proportion of Vδ1+ T cells, and cytotoxicity to the primary leukemia blasts. The Vδ1+ T cell population is also predominant in newly diagnosed leukemia patients likely due to a loss of circulating Vδ2+ T cells. A small proportion of newly diagnosed patients showed Vδ1 CDR3 region similarity. These findings suggest a role for γδ T cells in the immune response to leukemia.Paul F. Meeh and Michelle King are contributed equally to this work.     

Influence of the route of infection on development of T-cell receptor beta-chain repertoires of reovirus-specific cytotoxic T lymphocytes

It is well established that the route of infection affects the nature of the adaptive immune response. However, little is known about the effects of the route of exposure on development of cytotoxic T-lymphocyte (CTL) responses. Alternative antigen-presenting cell populations, tissue-restricted expression of class I major histocompatibility complex-encoded molecules, and unique T-cell receptor (TCR)-bearing cells in mucosal tissues could influence the selection and expansion of responder T cells. This study addresses the question of whether the route of virus infection affects the selection and expansion of subpopulations of virus-specific CTLs. Mice were infected orally or in the hind footpads with reovirus, and the repertoires of TCR beta-chains expressed on virus-specific CD8(+) T cells in Peyer's patches or lymph nodes and spleens were examined. CD8(+) cells expressing the variable gene segment of the TCR beta-chain 6 (Vbeta6) expanded in the spleens of mice infected by either route and in CTL lines established from the spleens and draining lymphoid tissues. Adoptively transferred Vbeta6(+) CD8(+) T cells from orally or parenterally infected donors expanded in reovirus-infected severe combined immunodeficient recipient mice and mediated cytotoxicity ex vivo. Furthermore, recovered Vbeta6(+) cells were enriched for clones utilizing uniform complementarity-determining region 3 (CDR3) lengths. However, sequencing of CDR3beta regions from Vbeta6(+) CD8(+) cells indicated that Jbeta gene segment usage is significantly more restricted in CTLs from orally infected mice, suggesting that the route of infection affects selection and/or subsequent expansion of virus-specific CTLs.     

Affinity enhancement and transmembrane signaling are associated with distinct epitopes on the CD8 alpha beta heterodimer.

CD8 is a heterodimeric membrane glycoprotein on MHC class I-restricted T lymphocytes that cooperates with the alpha beta CD3 TCR in the recognition of MHC class I molecules presenting antigenic peptides. Co-operation has two components: enhancement of the affinity of MHC/peptide-TCR interaction, and signal transduction through the T cell membrane. The cytolytic function of CTL is primarily dependent on the affinity-enhancement component of CD8-TCR cooperation whereas activation of resting CD8+ T cells is primarily dependent on transmembrane signaling. Using a panel of mAb, two to the alpha-chain and three to the beta-chain of CD8, we investigated the relationships between epitopes and functional regions of the CD8 molecule. Two of the antibodies, one to the alpha-chain and one to the beta-chain of CD8, inhibit the cytolytic function of CTL but not the generation of CTL from resting T cells. Another two antibodies, also one to the alpha- and one to the beta-chain, inhibited the generation of CTL while enhancing the cytolytic function of CTL. These results suggest that both the alpha- and beta-chain of CD8 possess two distinct regions, one involved in affinity enhancement and the other in transmembrane signaling. The former may be the MHC class I-binding region whereas the latter may associate with the alpha beta CD3 TCR. The data can explain the apparent functional equivalence of CD8 alpha alpha homodimers and alpha beta heterodimers.     

Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clones

The unique structure of the T cell receptor (TCR) enables molecular identification of individual T cell clones and provides an unique opportunity for the design of molecular diagnostic tests based on the structure of the rearranged TCR chain e.g., using the TCR CDR3 region. Initially, clonal T cell malignancies, including T cell large granular lymphocyte leukemia (T-LGL), mucosis fungoides and peripheral T cell lymphoma were targets for the TCR-based analytic assays such as detection of clonality by T-gamma rearrangement using y-chain-specific PCR or Southern Blotting. Study of these disorders facilitated further analytic concepts and application of rational methods of TCR analysis to investigations of polyclonal T cell-mediated diseases. In hematology, such conditions include graft versus host disease (GvHD) and immune-mediated bone marrow failure syndromes. In aplastic anemia (AA), myelodysplastic syndrome (MDS) or paroxysmal nocturnal hemoglobinuria (PNH), cytotoxic T cell responses may be directed against certain antigens located on stem or more lineage-restricted progenitor cells in single lineage cytopenias. The nature of the antigenic targets driving polyclonal CTL responses remains unclear. Novel methods of TCR repertoire analysis, include VB flow cytometry, peptide-specific tetramer staining, in vitro stimulation assays and TCR CDR3-specific PCR. Such PCR assay can be either VB family-specific or multiplexed for all VB families. Amplified products can be characterized and quantitated to facilitate detection of the most immunodominant clonotypes. Such clonotypes may serve as markers for the global polyclonal T cell response. Identification of these clonotypes can be performed in blood and tissue biopsy material by various methods. Once immunodominant clonotypes corresponding to pathogenic CTL clones are identified they can serve as surrogate markers for the activity of the pathophysiologic process or even indicate the presence of specific antigens. The relevance of the individual clonotypes can be ascertained from clinical correlations with the activity of the disease. Quantitative clonotypic assays such as sequencing of multiple CDR3 clones or clonotypic Taqman PCR can be applied for the monitoring of the immunosuppressive therapy and prediction of relapse. Future technologies may allow for the design of clonotypic microarrays or other more clinically applicable methods of clonotypic diagnostics. Similarly, identification of immunodominant clonotypes may facilitate targeting of autoimmune or malignant clones with vaccination and induction of anti-idiotypic responses.     

Chemotactic effect of human recombinant interleukin 2 on mouse activated large granular lymphocytes

Human recombinant interleukin 2 (hrIL-2) was demonstrated in vitro to be chemotactic for mouse large granular lymphocytes (LGL) activated in vivo by virus infection. Peritoneal exudate cells harvested from virus-infected mice were used as a source of LGL. LGL collected from mouse hepatitis virus-infected mice at 3 days postinfection were a source for NK 1.1 positive natural killer (NK)/LGL. LGL collected from mice treated with antiserum to gangliotetraosylceramide and infected with lymphocytic choriomeningitis virus for 7 days were used as a source for Lyt-2 positive cytotoxic T lymphocytes (CTL)/LGL. Both NK/LGL and CTL/LGL responded chemotactically to hrIL-2, purified IFN-beta, and to crude cell-free washout fluids collected from the peritoneal cavity of virus-infected mice. hrIL-2 had chemotactic activity for virus-elicited granular and agranular lymphocytes but did not attract the contaminating macrophages, in contrast to IFN-beta, which displayed chemotactic activity for virus-elicited granular and agranular lymphocytes as well as macrophages. The migration to hrIL-2 was inhibited by a monoclonal antibody (7D4) to the IL-2 receptor, but treatment with 7D4 did not affect migration in response to IFN-beta. Microscopic examination of Wright's-Giemsa-stained migrated NK/LGL and CTL/LGL revealed that the majority of migrated LGL in either LGL population had a blast cell morphology (enlarged cells with rich basophilic cytoplasm). The frequency of cells bearing the LGL morphology within the virus-elicited nonadherent peritoneal exudate cell population was on incubation in vitro, stabilized by either hrIL-2 or IFN-beta. These data suggest that another important immunomodulating function of IL-2 may be to attract activated NK/LGL and CTL/LGL to sites of inflammation.     

Age-related appearance of a CMV-specific high-avidity CD8<Superscript>+</Superscript> T cell clonotype which does not occur in young adults

Old age is associated with characteristic changes of the immune system contributing to higher incidence and severity of many infectious diseases. Particularly within the T cell compartment latent infection with human Cytomegalovirus (CMV) is contributing to and accelerating immunosenescence. However, latent CMV infection and reactivation usually does not cause overt symptoms in immunocompetent elderly persons indicating immunological control of disease. Little is still known about the clonal composition of CMV-specific T cell responses in donors of different age. We therefore analyzed CD8⁺ T cells specific for an immunodominant pp65-derived nonamer-peptide (NLVPMVATV; CMV_NLV) in different age-groups. Independent of donor age CMV_NLV-specific CD8⁺ T cells preferentially use the V beta family 8. This family has monoclonal expansions in the majority of donors after stimulation of CD8⁺ T cells with the peptide. By sequencing the CDR3 region of the T cell receptor we demonstrated that CMV_NLV-specific, BV8⁺ CD8⁺ T cells share the conserved CDR3-sequence motif SANYGYT in donors of all age groups. Interestingly, a second conserved clonotype with the CDR3-sequence motif SVNEAF appears in middle-aged and elderly donors. This clonotype is absent in young individuals. The age-related clonotype SVNEAF binds to the pMHC-complex with higher avidity than the clonotype SANYGYT, which is predominant in young adults. The dominance of this high avidity clonotype may explain the lack of overt CMV-disease in old age.     

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.