Immune-mediated destruction of melanocytes in halo nevi is associated with the local expansion of a limited number of T cell clones

The beta-chain repertoire of the T cells that infiltrate spontaneously regressing nevi (the halo nevus phenomenon) was studied. In addition to the infiltration of the halo nevi by cutaneous lymphocyte-associated Ag-positive lymphocytes, oligoclonal expansion of T cells was observed in all halo nevi of all patients. T cells using the same TCR beta-chain were observed in distinct halo nevi of the same patient but not in his peripheral blood, demonstrating a local expansion of common clones that are most likely activated by the Ag(s) shared by independent halo nevi of the same patient.     

Oligoclonal accumulation of T cells in peripheral blood from patients with idiopathic thrombocytopenic purpura

To determine whether clonal T cells accumulate in idiopathic thrombocytopenic purpura (ITP), we performed single-strand conformation polymorphism (SSCP) analysis to detect T-cell receptor (TCR) beta-chain usage of peripheral T cells. We detected significantly more oligoclonal T cells (15.5 +/- 8.9 bands representative for clonal T-cell expansions) in peripheral blood from ITP patients than from healthy donors (2.8 +/- 2.6 bands). Frequently used V beta genes in these accumulated T cells in ITP were V beta 3, 6, 10, 13.1 and 14. To determine whether these bands were derived from clonal T cells, presumably in a preactivated state, we established some T-cell clones (expressing CD4 and TCR V beta 6. 13.1. or 14) by nonspecific stimulation from patients peripheral mononuclear cells, and examined their clonotypes. Clonal identities for three out of seven clones tested were confirmed using SSCP analyses to compare the migration of their beta-chain complementarity determining region 3 (CDR3) cDNAs, expanded by polymerase chain reaction (PCR) with those from peripheral blood. Therefore, distinctive T-cell clones accumulated in the periphery in ITP and they may be related to the autoimmune-mediated destruction of platelets.     

Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetiology

Insulin-dependent diabetes mellitus (IDDM) is a T-cell-mediated autoimmune disease whose onset is believed to be triggered by unknown environmental factors acting on a predisposing genetic background. Islet-infiltrating T (IIT) cells from two IDDM patients, who had died at the onset of the disease from brain swelling as a complication of ketoacidosis, were analysed. The results provided evidence for the involvement of a pancreatic islet cell membrane-bound superantigen as a diabetes aetiopathogenetic factor. There was a selective expansion of a T-cell receptor (TCR) variable segment of the beta-chain (V beta 7) in these IIT cells in association with unselected V alpha-chain segments; extensive junctional diversity of the TCR V beta 7 chains; and evidence of positive selection, after exposure to diabetic islet cell membrane preparations, of V beta 7+ T-cell clones among peripheral blood lymphocytes from non-diabetic individuals.     

CDR3 size spectratyping and sequencing of spectratype-derived TCR of spinal cord T cells in autoimmune encephalomyelitis

To characterize the nature of autoimmune disease-inducing T cells in the target organ, oligoclonal expansion of spinal cord T cells of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined by complementarity-determining region 3 (CDR3) size spectratyping. It is known that TCR of in vitro-established myelin basic protein-specific T cell clones and lines have a short CDR3 and that the amino acid sequence in this region is highly preserved. On the basis of these findings, we analyzed 22 spectratypes of the TCR beta-chain (Vbeta1-20). Among them, only Vbeta8.2 and Vbeta17 showed oligoclonal expansion of TCR with a short CDR3 at the early stage of EAE. More interestingly, the spectratype profile of Vbeta8.2 seen at the early stage was preserved throughout the course of EAE, whereas that of Vbeta17 became more diverse at the peak stage of the disease. Analysis of nucleotide and predicted amino acid sequences of Vbeta8.2 CDR3 derived from the spectratypes revealed that the clones with CASSDSSYEQYFGPG, which is one of the representative sequences of encephalitogenic T cell clones, constituted the predominant population not only at the early stage but also at the peak and recovery stages (71, 71, and 60%, respectively). These findings imply that although the phenotype of T cells in the target organ diversifies as the autoimmune disease progresses, disease-associated TCR spectratype(s) are preserved throughout the course of the disease. Thus, CDR3 size spectratyping is a powerful tool for the screening of disease-inducing T cells in an autoimmune disease of unknown pathomechanism.     

Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions

We have analyzed the T-cell receptor (TCR) V beta repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT) from related and unrelated donors at the City of Hope. Results of PCR studies from graft-versus-host disease (GVHD) skin lesions show a bias in the usage of TCR V beta families, whereas examination of peripheral blood (PB) withdrawn at the same time did not reveal a similar phenomenon. In one such family, TCR V beta 2 is predominantly expressed in 7 of 7 biopsy specimens examined. V beta 2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single V beta 2-bearing TCRs in GVHD lesions, and in the PB of some patients after diagnosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used to probe Southern blots of amplified V beta 2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute to the disease pathology.     

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.     

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity

In this study we have analyzed the TCR V alpha and V beta regions at the DNA level in the CD4+CD45RO+ memory T cell population of synovial tissue infiltrating T lymphocytes of three rheumatoid arthritis (RA) patients and one patient with chronic arthritis. Cell lines of CD4+CD45RO+, CD4+CD45RO-, CD8+CD45RO+ and CD8+CD45RO- T lymphocyte populations were generated following FACS cell sorting of freshly isolated synovial tissue mononuclear cell infiltrates (STMC) and of freshly isolated peripheral blood mononuclear cells (PBMC) of these patients. The phenotypic and molecular analyses have revealed the following. (i) The TCR repertoires of tissue infiltrating T lymphocytes in the various subsets were extensive on the basis of TCR V gene family usage. (ii) Furthermore, each patient displayed individual specific TCR V gene expression patterns in the various STMC and PBMC derived T cell subsets. However, the majority of these arthritis patients manifested increased expression of multiple TCR V gene families in the synovial tissue derived CD4+CD45RO+ T cell population when compared with the peripheral blood derived CD4+CD45RO+ subset. Of these gene families, we found enhanced expression of the TCR V alpha 7 and V beta 11 gene segments in synovial tissue to be shared by all four patients analyzed. (iii) Nucleotide sequence analysis of the CDR3 regions of a number of TCR V regions in the CD4+CD45RO+ T cell subsets has revealed that the CDR3 regions comprised within synovial tissue derived TCR V regions differed from those found in peripheral blood derived TCR V regions. These differences in CDR3 diversity might be the consequence of a specific interaction with particular MHC-peptide complexes expressed at the site of inflammation. (iv) The CDR3 region analysis also showed individual specific amino acid motifs within the N-D-N regions of all analyzed TCR V beta genes derived from PBMC as well as STMC.     

The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo

Human cytomegalovirus (HCMV)-specific CD8(+) cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vbeta segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.     

Conserved sequence motifs of CDR3 loops of TCR specific for two major epitopes of the grass pollen allergen PhI p 1

We analyzed the cDNA sequence data of complementarity determining regions (CDR3) of epitope mapped alphabeta TCR of T cell clones (TCC). The TCC were specific for the major timothy grass (Phleum pratense) pollen allergen Phl p 1 and were derived from the peripheral blood of seven unrelated grass pollen-allergic individuals. Each TCR recognized one of two immunodominant T cell epitopes, PP73 or PP103, of Phl p 1. Although a diversity of recombined V and J segments was observed, amino acid motifs as long as five residues were conserved among CDR3 loops of TCR from TCC of different atopic individuals specific for the same peptide. The conserved sequences could comprise as much as 60% of the CDR3. All amino acid residues of the motifs of the CDR3beta and most of the CDR3alpha of all TCR used in this study were encoded by randomly added nucleotides. This indicates that they were specifically selected for by the peptide bound to the MHC class II molecule. For one selected patient, a larger number of TCC, specific for PP103, was analyzed. The TCR repertoire was limited to three different TCR. The same MHC class II molecule, DRB1*1301, was identified to present PP103 to each of the three TCR.     

Diagnosis and assessment of preclinical and clinical autoimmune encephalomyelitis using peripheral blood lymphocyte TCR

In organ-specific autoimmune diseases, T cells involved in the disease development bear a particular type of TCR and infiltrate the target organ predominantly. However, it is difficult to identify disease-inducing T cells in peripheral blood lymphocytes (PBL) because such T cells are very few in number in a large pool of unrelated T cells. In the present study, we demonstrate that CDR3 spectratyping can identify experimental autoimmune encephalomyelitis (EAE)-specific patterns (oligoclonal expansion of Vbeta8.2 with the shortest CDR3) in PBL at the preclinical and clinical stages of acute EAE. Analysis of nucleotide and predicted amino acid sequences of Vbeta8.2 CDR3 of spectratype-derived clones revealed that CASSDSSYEQYFGPG, which is one of the representative sequences of encephalitogenic T cell clones, constituted the predominant population in both PBL and spinal cord T cells. In chronic relapsing EAE, the EAE-specific spectratype pattern in PBL was observed during the 1 st and 2nd attacks, but not at the remission and full recovery stage. These findings indicate that the spectratyping pattern in PBL reflects the disease activity of acute and chronic relapsing EAE. Thus, CDR3 spectratyping using PBL can be used for diagnosis and assessment of T cell-mediated autoimmune diseases and is applicable to human autoimmune diseases.     

Identification of autoimmune T cells among in vivo expanded CD25+ T cells in multiple sclerosis

Although clonal expansion of autoimmune T cells has been reported in multiple sclerosis (MS), very limited information is available on specificities, clonal size, or activation state of the expanded clones. Here we address the issue of clonal expansion by using a novel technique demonstrating clonotypes defined by single-strand conformation polymorphism of TCR beta-chain cDNAs. Examination of activated T cells (CD3+CD25+) isolated from the peripheral blood of MS revealed limited numbers (20 approximately 82) of expanded clones defined by single-strand conformation polymorphism clonotype. To estimate the Ag specificities of dominant clonotypes in the activated T cells, these samples were examined in parallel with Th1 T cell clones specific for myelin basic protein or proteolipid protein (PLP) derived from the same patients. Analysis of two patients demonstrated that the dominant clonotypes would contain those specific for myelin basic protein or PLP. Although the majority of the clonotypes could be detected only transiently, a PLP95-116-specific clonotype was found to persist for over 1 yr. Thus, single-strand conformation polymorphism clonotype analysis allows us to monitor the kinetics of given T cell clones in vivo and could provide useful information for designing clonotype (Id)-specific manipulation of human diseases such as MS.     

Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations

To better characterize the cellular immune response taking place in the MS central nervous system, we investigated the blood and CSF T cell receptor (TCR) V beta 5 and V beta 17 repertoire in HLA-typed patients with recently diagnosed MS or other neurological diseases (OND). Using a RT-PCR based technique, we analysed directly ex vivo the CDR3 size of TCR beta chains utilizing V beta 5 (eight patients with MS and one with OND) or V beta 17 (eight patients with MS and six with OND) gene segments on paired blood-CSF samples. Globally, the analysis of V beta 5-J beta and V beta 17-J beta repertoire showed a less diverse pattern in the CSF samples than in the corresponding peripheral blood lymphocytes both in MS and in OND patients. However, we did not detect any recurrent clonal expansion within the V beta 5+ T cells in MS patients, underlining the potential limits of V beta 5-based immunotherapy in MS. We found an expanded T cell population using the same V beta 17-J beta 1.6 combination with identical CDR3 length in the CSF of three MS patients and none of the control patients. These results suggest selective expansion of T cells expressing this segment gene in the MS central nervous system.     

Diagnosis and management of paradoxical embolism and patent formen ovale

In order to analyse the diversity of T-cell receptors (TCRs) expressed by the T-cell population activated by allogeneic HLA-DR stimulation, TCR beta cDNA was synthesized from mRNA of human CD4+ T cells that had been stimulated in a primary mixed lymphocyte reaction (MLR). The TCR beta cDNA was amplified by the polymerase chain reaction (PCR), subjected to bacterial cloning, and sequenced from V beta through J beta. Twenty-six different V beta and 10 different J beta segments were detected among 56 randomly selected cDNA clones. Occurrences of V beta 17.1 and J beta 1.5 were higher than those found in the CD4+ T-cell population activated with a CD3-specific antibody. A total of 53 different CDR3 sequences, two of them occurring more than once, were detected among the 56 cDNA clones. In order to estimate the degree of CDR3 diversity, amino acid similarity in the CDR3 region of the cDNA was calculated and compared with those of the anti-CD3-activated T-cell sequences as well as those of various published T-cell clone sequences, each directed to either alloantigens or single antigenic peptides. It was found that the similarity score among CDR3 sequences obtained from the MLR (56.4 +/- 10.3) was comparable to those of anti-CD3-activated T cells (55.7 +/- 10.7) and those of T-cell clones directed toward alloantigens (range, 48.4 +/- 12.4-59.4 +/- 13.1), but significantly smaller than those of T-cell clones directed toward single antigenic peptides such as those derived from myelin basic protein (75.6 +/- 17.9) and cytochrome c (76.9 +/- 20.5). These results provide quantitative proof that TCRs of T cells activated by primary allogeneic HLA-DR stimulation have a larger diversity than those recognizing single antigenic peptides.