Clonal expansion of myelin basic protein-reactive T cells in patients with multiple sclerosis: Restricted T cell receptor V gene rearrangements and CDR3 sequence

Myelin basic protein (MBP)-reactive T cells are thought to play an important role in the pathogenesis of multiple sclerosis (MS). In some patients with MS, these autoreactive T cells display a limited heterogeneity in their epitope recognition and T cell receptor (TCR) variable (V) gene usage. These individual-dependent properties of MBP-reactive T cells have led to the speculation that they may represent clonal expansion in vivo in some MS patients. In the present study, 51 MBP-reactive T cell clones derived from patients with MS and healthy individuals were examined for their epitope recognition and the TCR Vα and Vβ gene rearrangements. The V gene junctional region sequences of identified α and β genes were further analyzed to probe their clonal origins, as the sequences are unique for individual clones. Our data showed that 26 clones derived from nine patients with MS shared a predominant reactivity to the immunodominant regions of MBP, 84–102, 110–129 and 143–168, and used various TCR Vα and Vβ rearrangements. The V gene usage of the clones was restricted to certain Vα Vβ combination(s) in a given MS patient, but varied among different patients. The sequence analysis revealed that the clones generated from a given patient shared a limited or a single junctional region sequence pattern(s), indicating their oligoclonal or monoclonal origin(s). In contrast, 25 MBP-reactive T cell clones derived from normal individuals exhibited unfocused epitope recognition and V gene usage. Thus, the limited heterogeneity of MBP-reactive T cells in their structural and functional charactertistics reflects their clonal expansion in vivo in some patients with MS.     

Clonal evolution of B cells in transformation from low- to high-grade lymphoma

An outcome of low-grade B cell non-Hodgkins's lymphomas is the transformation to high-grade diffuse large B cell lymphomas (DLBL). To investigate the mechanisms of clonal evolution in the transformation to DLBL, we performed longitudinal molecular analyses of immunoglobulin (Ig), V(H)DJ(H) gene sequences expressed in cases of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), and follicular lymphoma (FL) that transformed to DLBL. Among the neoplastic CLL and SLL cells and their respective high-grade transformants, there was no evidence for a clonotypic shift or acquired mutations in the expressed Ig V(H)DJ(H) gene segments, as further confirmed by a specific and sensitive PCR-single strand polymorphism analysis. In contrast, among the FL cells there was a high degree of intraclonal diversification with highly divergent V(H)DJ(H) gene sequences. Despite this intraclonal heterogeneity, the related DLBL expressed a collinear but unique V(H)DJ(H) gene sequence. The intraclonal genealogical tree for the FL case demonstrated that the DLBL emerged in association with unique V(H)DJ(H) gene mutational events. Among the intraclonal FL and related DLBL transformants, the nature and distribution of the Ig V(H)DJ(H) gene mutations were consistent with antigenic selection. Thus, clonal evolution in the transformation from low- to high-grade B cell lymphoma may involve distinct pathways which vary according to the cellular origin and the type of the progenitor B cell tumor.     

Shared amino acid sequences in the NDβN and Nα regions of the T cell receptors of tumor-infiltrating lymphocytes within malignant glioma

The purpose of this study was to assess the V-(D)-J junctional region of the T cell receptor (TCR), the CDR3 region, which is responsible for glioma-specific antigen contact in αβ TCR-mediated recognition. We sequenced the TCR α and β chians of Vα7, and Vβ13.1 cDNA derived from tumor-infiltrating lymphocytes (TIL) of 12 glioma patients and also the corresponding clones from the patients' peripheral blood lymphocytes (PBL). A shared Vβ13.1 DJ sequence of the CDR3 region, NDβN, was demonstrated in 49 of 66 Vβ13.1⁺ clones (74.2 %) from the glioma TIL, whereas only 4 of 33 clones (12.1 %) were observed in the Vβ13.1⁺ clones from the PBL (p < 0.001). A common VDJ sequence, FCASS (Vβ13.1)-YRLPWGTSDS (NDβN)-GELFF(Jβ2.2), was observed not only in the gliomas from each patient, but also among all the patients with a preference for Vβ13.1. In contrast, the amino acid sequences of the Vβ13.1⁺ PBL clones were diverse and random. Next, we sequenced subclones from other Vβ subfamilies randomly selected to compare their VDJ region rearrangements (Vβ3 and Vβ5.1). In contrast to Vβ13.1, the amino acid sequences of these junctional regions were completely different in these subclones. The V-J junctional region of the α chain is dominated by a few clones in some patients, and no shared amino acid sequences were detected in the TCR Vα junctional region. However, in the Nα region of the Vα7-bearing TIL clones, arginine was used in 27 of 44 clones (61.4%) compared to only 3 of 12 clones from the PBL (p < 0.05). These results are consistent with the hypothesis that a clonal expansion/accumulation of glioma lineage-specific T cells occurred in vivo at the tumor site and that these T cells may be recognizing glioma-specific antigens.     

Diversity of novel recombining elements suggests developmentally programmed expression of the T cell receptor alpha/delta locus.

During fetal ontogeny, the first wave of gamma delta T lymphocytes appears in the thymus at day 14 of gestation assembling predominantly T cell receptors (TcR) with V gamma 3 and V delta 1. To identify V delta gene segments that are transcribed at day 16, subsequent to the first wave of V delta 1 expression, delta chain cDNA was amplified by the anchored polymerase chain reaction with single-sided specificity for C delta. Unexpectedly, most of the cDNA clones do not contain V gene segments. In some cDNA clones an alternative splice from the leader exon to the C delta exon has deleted the whole variable region exon. In other cDNA clones, multiple non-V-like elements are juxtaposed to the D delta 2 and J delta 1 gene segments. A large number of these diverse elements appear to be rearranged in fetal thymocytes, bringing V alpha gene segments located upstream of the recombining element into proximity to the J alpha locus. It is proposed that these rearrangements make irreversible the commitment to the TcR alpha beta lineage and determine a programmed read out of different clusters of V alpha gene segments.     

The influence of CD40-CD154 interactions on the expressed human V(H) repertoire: analysis of V(H) genes expressed by individual B cells of a patient with X-linked hyper-IgM syndrome

Analysis of the V(H)DJ(H) repertoire of peripheral blood IgM(+) B cells from a patient with X-linked hyper-IgM syndrome (X-HIgM) was undertaken to determine whether the distribution of V(H) families in the productive repertoire might be regulated by in vivo CD40-CD154 interactions. The distribution of V(H) genes in the non-productive repertoire of IgM(+) B cells was comparable in X-HIgM and normals. Unlike the normal productive V(H) repertoire, however, in the X-HIgM patient the V(H)4 family was found at almost the same frequency as the V(H)3 family. This reflected a diminution in the positive selection of the V(H)3 family observed in normals and the imposition of positive selection of the V(H)4 family in the X-HIgM patient. Unique among the V(H)3 genes, V(H)3-23/DP-47 was positively selected in both normals and the X-HIgM patient. No major differences in the usage of J(H) or D segments or the complementarity-determining region (CDR) 3 were noted, although the foreshortening of the CDR3 noted in the mutated V(H) rearrangements of normals was absent in the X-HIgM patient. Finally, a minor degree of somatic hypermutation was noted in the X-HIgM patient. These results have suggested that specific influences on the composition of the V(H) repertoire in normals require CD40-CD154 interactions.     

Fractal Organization of the Human T Cell Repertoire in Health and after Stem Cell Transplantation

T cell repertoire diversity is generated in part by recombination of variable (V), diversity (D), and joining (J) segments in the T cell receptor β (TCR) locus. T cell clonal frequency distribution determined by high-throughput sequencing of TCR β in 10 stem cell transplantation (SCT) donors revealed a fractal, self-similar frequency distribution of unique TCR bearing clones with respect to V, D, and J segment usage in the T cell repertoire of these individuals. Further, ranking of T cell clones by frequency of gene segment usage in the observed sequences revealed an ordered distribution of dominant clones conforming to a power law, with a fractal dimension of 1.6 and 1.8 in TCR β DJ and VDJ containing clones in healthy stem cell donors. This self-similar distribution was perturbed in the recipients after SCT, with patients demonstrating a lower level of complexity in their TCR repertoire at day 100 followed by a modest improvement by 1 year post-SCT. A large shift was observed in the frequency distribution of the dominant T cell clones compared to the donor, with fewer than one third of the VDJ-containing clones shared in the top 4 ranks. In conclusion, the normal T cell repertoire is highly ordered with a TCR gene segment usage that results in a fractal self-similar motif of pattern repetition across levels of organization. Fractal analysis of high-throughput TCR β sequencing data provides a comprehensive measure of immune reconstitution after SCT.     

Diversity of immunoglobulin χ light chain gene rearrangements and evidence for somatic mutation in VχIV family gene segments in X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency disease in man, characterized by an arrest in B lymphocyte differentiation at the precursor B cell stage. The structure of expressed immunoglobulin (Ig) χ light (L) chain rearrangements of nine B lymphoblastoid cell lines from one XLA patient was investigated by amplification of cDNA by the polymerase chain reaction using 5′ Vχ family-specific primers and a 3′ χ constant region primer. Members of all four Vχ gene families were found to be utilized in Ig χ L chain rearrangements at frequencies that were consistent with random Vχ family usage. There was no preference for usage of any particular χ joining segment. Additional diversity was generated by deletions and random nucleotide insertions at the site of juxtaposition. Particular Vχ members seemed to be overrepresented in the sample. The observed homology of the VχI, VχII and VχIII region sequences, both to each other and to known germ-line Vχ sequence indicated the absence of somatic mutations in the majority of these expressed Ig genes. In contrast of the single-member VχIV family four different sequences were found to be expressed. That these sequences were mutated derivatives of a germ-line VχIV element was substantiated both by sequence analysis and oligonucleotide hybridization. This finding shows that the mutation process can occur in early stages of B cell development i.e. before H chain class switch has occurred. The presence of these mutations is probably independent of clonal expansion since XLA patients are unable to respond to antigen. We conclude that the differentiation arrest in XLA does not preclude early onset of somatic mutation events in Vχ gene segments.     

DJ‐1 is a Parkinson's disease susceptibility gene in southern Italy

De Marco EV, Annesi G, Tarantino P, Nicoletti G, Civitelli D, Messina D, Annesi F, Arabia G, Salsone M, Condino F, Novellino F, Provenzano G, Rocca FE, Colica C, Morelli M, Scornaienchi V, Greco V, Giofrè L, Quattrone A. DJ‐1 is a Parkinson's disease susceptibility gene in southern Italy. Mutations in the gene DJ‐1 have been shown to be a rare cause of early‐onset Parkinson's disease (EOPD). Since DJ‐1 mutations have been found in patients with Parkinson's disease (PD) from southern Italy, we aimed to investigate whether polymorphisms within the DJ‐1 gene could represent a risk factor for sporadic PD. First, we genotyped 294 patients with PD and 298 controls coming from southern Italy to assess the distribution of the insertion/deletion (Ins/Del) polymorphism. In a second phase, we identified five single‐nucleotide polymorphisms (SNPs) useful to delimit a region potentially involved and genotyped all patients and controls for these markers. All the markers analyzed were significantly associated with PD at both allelic and genotypic level. The most significant association with the disease was found at the Ins/Del polymorphism (p = 0.0001; adjusted odds ratio (OR ) = 2.05; confidence interval (CI ) = 1.36–3.08). When we considered a three‐marker sliding window, we found a highly significant association between the disease and the haplotypes including markers rs17523802, Ins/Del, and rs3766606 (p = 0.0007) and markers Ins/Del, rs3766606 and rs7517357 (p = 0.0054). Our results indicate that polymorphisms located in a region spanning 3535 bp from the promoter to the intron 2 of the DJ‐1 gene confer risk to sporadic PD in southern Italy.     

Human Fab fragments specific for the Haemophilus influenzae b polysaccharide isolated from a bacteriophage combinatorial library use variable region gene combinations and express an idiotype that mirrors in vivo expression.

To determine whether the human antibody (Ab) repertoire to the Haemophilus influenzae type b capsular polysaccharide (Hib PS) could be studied at the molecular level with phage display technology, we constructed a phage Fab library by using peripheral blood from a vaccinated adult. Phage were selected based on Hib PS binding. Two distinct Hib PS-specific phage clones were identified whose Fab fragments used the same V(H) region paired with two different V(L) regions. The V(L) regions were derived from two independent rearrangements of the A2c gene with Jkappa1, and both contained a nontemplated arginine codon at the V-Jkappa junction. The two A2 V gene segments differed from the A2c germ line sequence in 0 and 5 bases. The V(H) region consisted of the V(H)26 gene segment having 98% identity to the germline nucleotide sequence, a D region of 9 bases, and J(H)4b1. Usage of V(H)26 in combination with A2 V regions containing a junctional arginine is a predominant configuration of naturally occurring Hib PS-specific Abs. Liquid- and solid-phase assays showed that phage-derived Fab reacted with Hib PS and expressed HibId-1, an idiotype associated with the kappaII-A2 V region. These findings extend the database of V region polymorphisms that can contribute to the Hib PS repertoire and demonstrate that Hib PS-specific Fab fragments isolated from combinatorial phage libraries use V gene combinations which mirror the natural repertoire.     

Serum and monoclonal immunoglobulin E antibodies from NC/Nga mice with severe atopic-like dermatitis recognize an auto-antigen, histone H3

Background NC/Nga mice are known to show a spontaneous outbreak of atopic‐like dermatitis accompanied by a marked elevation in serum IgE levels when reared in a conventional environment. The specific effects of such a strong serum IgE response on the development of the dermatitis and specific antigens recognized by the IgE antibodies are still uncertain. Objective and methods To characterize the IgE of NC/Nga mice, we established IgE‐secreting hybridoma clones from spleen cells of NC/Nga mice spontaneously developing dermatitis and identified variable‐region genes and specific antigens of the IgE monoclonal antibodies (mAbs). Serum polyclonal IgE, as well as IgG1 and IgG2a, specific for the identified antigen were also analysed. Results Four IgE‐producing hybridoma clones were established. Variable‐region nucleotide sequences of the IgE mAbs showed that these clones did not necessarily share common germline gene segments (V, D or J) for each variable region, and several somatic mutations had occured in the V gene segments. Through antigen screening, histone H3 was identified to be an auto‐antigen recognized by three of the four IgE mAbs. Serum IgE as well as IgG1 specific for histone H3 were almost undetectable in 6‐week‐old mice, but rapidly increased by 10–12 weeks of age. This age‐dependent increase in the serum anti‐histone H3 IgE was roughly in parallel with the onset of dermatitis, and slightly preceding total IgE elevation. The serum‐specific IgE level correlated well with a dermatitis‐severity score of each mouse at 12–16 weeks of age, and weakly with the severity of ear erosion of each mouse over 28 weeks of age. Furthermore, immunologically detectable histone‐H3 antigens were observed in skin tissue sections from the dermatitis sites. Conclusion In NC/Nga mice, anti‐histone H3 auto‐antibodies may contribute, at least in part, to the considerably elevated serum IgE and might play some roles in the development and exacerbation of dermatitis.     

The germline repertoire of T-cell receptor beta-chain genes in patients with multiple sclerosis

The T-cell receptor (TCR) beta-chain gene repertoire of 40 multiple sclerosis (MS) patients was compared to that of 100 normal individuals. V-beta probes that represent 14 different V-beta subfamilies plus a C-beta probe were used to identify 53 separate beta-chain gene segments. No duplication or deletion of any of these 53 gene segments was found in the MS patients. Restriction fragment length polymorphism (RFLP) alleles detected by V-beta 8, V-beta 11 and C-beta probes defined 8 different beta-chain haplotypes. The distribution of these haplotypes in Caucasian MS patients and normal individuals was significantly different (p = 0.012). Comparison of the DR2+ subset of MS patients (n = 32) to a second group of 43 Caucasian DR2+ normal individuals revealed that the distribution of these beta-chain haplotypes was significantly different in these two populations (p = 0.015). These results suggest that an MS susceptibility gene(s) may be located in the region of the TCR/beta-chain gene complex.     

Characterization of monoclonal anti-beta2-glycoprotein-I and anti-prothrombin antibody fragments generated by phage display from a patient with primary antiphospholipid syndrome

The molecular structure of antibodies associated with autoimmune thrombosis is beginning to be understood. We describe the binding specificities and sequence analysis of anti-beta2-glycoprotein-I (anti-beta2GP-I) or anti-prothrombin (anti-PT) antibody fragments generated by phage display from a patient with primary antiphospholipid syndrome (APS). We obtained 39 positive clones, two that had the correct size reacted with beta2GP-I (Beta 1 and Beta 2). Ten clones with the same restrictive pattern recognized PT (Prot 1) and cross-reacted with beta2GP-I. All three clones recognized anionic and zwitterionic phospholipids. The V(H) regions of both anti-beta2GP-I clones are members of the VH4 family. Prot 1 has a V(H) segment of the VH3 family. The Beta 1 J(H) segments are J(H)5b and J(H)4b for Beta 2 and Prot 1. V(L) genes are V(lambda)1, 3 and 1, respectively. No J(L) was identified for Beta 1, while Beta 2 and Prot 1 carry J(lambda)3b genes. Beta 1 and Beta 2 carry highly conserved germ-line V(H) and V(L) genes. Mutations of the Prot 1 gene appear to be antigen-dependent, most are hotspot mutations located in the CDR 1 and 2 regions. Our work suggests that some anti-beta2GP-I from patients with primary APS are natural autoantibodies. Our work may also help to explain the frequent coexistence of anti-beta2GP-I and anti-PT in the same patient.     

Two V(H)5 family genes expressed by human peripheral B cells display differential mutational frequencies in the V(H) region

The heavy chain variable segment gene (V(H))5 family, one of the seven immunoglobulin (Ig) V(H) families, contains two functional genes, VH251 and VH32. To investigate functional differences between these V(H)5 family genes, V(H) segments expressed by human peripheral B cells were sequenced and analyzed. One hundred fifty-three sequences with unique V(H)DJ(H) recombinations were obtained from 17 adults. The mutational frequency of VH32 derived sequences (6.4%) was higher than that of VH251 derived sequences (4.4%), resulting in a significant difference (P<0.01). Significant differences in mutational frequencies between VH251 and VH32 derived sequences were observed in CDRs and FRs. No significant differences were found in CDR3 length distribution, D segment usage, or J(H) segment usage between VH251 and VH32 derived sequences. These results suggest that mutational frequency is affected, in part, by V(H) gene structure. The difference may occur after recombinational events in B cell development.     

Restricted TCR Valpha gene rearrangements in T cells recognizing an immunodominant peptide of myelin basic protein in DR2 patients with multiple sclerosis

T cell responses to myelin basic protein (MBP) are thought to play an important role in the pathogenesis of multiple sclerosis (MS). The response to the 83-99 region of MBP represents a dominant response to MBP in patients with MS and is associated with HLA-DR2 that is linked with susceptibility to MS. Although T cell clones reactive to various regions of MBP have been found to exhibit heterogeneous TCR Vbeta gene usage in patients with MS, it is unclear whether T cell clones uniformly recognizing the 83-99 peptide of MBP in the context of the same DR molecule would have restricted TCR V gene rearrangements and recognition motifs. In this study, a panel of DR2- or DR4-restricted T cell clones specific for the MBP83-99 peptide were derived from 11 patients with MS and examined for TCR V gene usage by PCR and the recognition motifs using analog peptides. Our study revealed that despite a few T cell clone pairs having similar recognition motifs and shared sequence homology in the CDR3, the overall recognition motifs of MBP83-99-specific T cells were considerably diverse. Interestingly, the DR2-restricted T cell clones displayed a biased V gene usage for Valpha3 and Valpha8, while Vbeta gene rearrangements were highly heterogeneous. This study provided experimental evidence suggesting a limited heterogeneity in TCR Valpha gene rearrangements of MBP-reactive T cells in DR2 patients with MS.      

Biased T cell receptor V alpha region repertoire in the synovial fluid of rheumatoid arthritis patients.

Synovial T lymphocytes seem to contribute to the pathogenesis of rheumatoid arthritis (RA). Since very little is known about the structural heterogeneity of their T cell antigen receptors (TcR), we analyzed TcR alpha chain mRNA of synovial fluid T cells from two RA patients. TcR alpha chain cDNA was amplified by the polymerase chain reaction with single-sided specificity for the alpha chain constant (C alpha) gene segment, and the nucleotide sequences of 51 functionally rearranged cDNA clones were determined. Twenty different V alpha genes and 26 different J alpha gene segments were utilized in these cDNA clones. Three of the V alpha gene segments which are frequently (8%-17% total) expressed in synovial fluid T cells have rarely been found in the TcR repertoire of peripheral blood T cells from healthy individuals. The T cell responses in the rheumatic synovia analyzed here are not oligoclonal, but the usage of TcR V alpha genes is biased.     

Detection of Parkin (PARK2) and DJ1 (PARK7) mutations in early-onset Parkinson disease: Parkin mutation frequency depends on ethnic origin of patients

Mutations in the Parkin (PARK2) and the DJ1 (PARK7) gene cause early-onset Parkinson disease (EOPD). We tested 75 Serbian EOPD patients for mutations in both genes by conventional mutational screening (SSCP/dHPLC/sequencing) to detect small sequence alterations and by gene dosage studies (quantitative PCR) to reveal deletions or multiplications of one or more exons. A compound heterozygous Parkin mutation (exon deletion and point mutation; [c.836_972del]+[c.1411C>T]; +1 is first nucleotide of GenBank AB009973.1) was identified in a patient who showed a relatively benign course after a disease onset at 41 years. Another case had a heterozygous exon deletion in DJ1 ([c.253_322del]+[?]) and presented with an age at onset of 45 years and a rapid disease course. In conclusion, Parkin mutations are surprisingly rare in our Serbian EOPD sample, suggesting that the mutation rate depends on the ethnic origin of the patients. Although DJ1 mutations appear to be rare, we confirm their role in EOPD and demonstrate the importance of gene dosage studies.