Skewing of TCR V genes to CD4+ and CD8+ subsets of T lymphocytes is not determined by amino acid composition of CDR3

TCR V genes show differing expression patterns, termed skewing, in CD4+ and CD8+ subsets of T lymphocytes. To determine which elements of the TCR V regions contribute to these observed TCR V gene skewing patterns, we have performed an in-depth analysis, taking advantage of RT-PCR and DNA sequencing, which was focused on the multi-member TCRBV6 gene family. These studies allowed us to evaluate the contributions of the various elements, that constitute the TCR beta chain variable region, to the observed TCR V gene skewing patterns. The results of these analyses revealed that within the TCRBV6 family individual members exhibited differing skewing patterns, i.e. TCRB6S7 was significantly skewed towards the CD4+ T cell subset, whereas TCRBV6S5 was significantly skewed towards the CD8+ subset. Scrutiny of the usage of TCRBV6 family members in combination with TCRBJ gene usage and amino acid composition of CDR3 did not reveal obvious structural characteristics which would explain the differing skewing patterns between TCRBV6S7 and TCRBV6S5. Further examination of these TCR V regions showed that the CDR1 and 2 regions within these TCRBV elements were composed of different amino acids. These observations suggests that these components contribute to the observed TCR V gene skewing patterns.     

Evidence for cooperation between TCR V region and junctional sequences in determining a dominant cytotoxic T lymphocyte response to herpes simplex virus glycoprotein B

TCR repertoire availability has the potential to influence the immune response to foreign antigens. Here we have analysed how changes in V region availability influence the H-2b-restricted cytotoxic T lymphocyte (CTL) response to a dominant peptide determinant derived from the herpes simplex virus glycoprotein B (gB). We have previously shown that C57BL/6 mice mount a gB-specific, Kb-restricted CTL response which is dominated by a TCRBV10+ population and a TCRBV8S1+ subpopulation, both containing highly conserved CDR3 elements. We find that this dominant gB-specific CTL pool is lost in C57/L mice which have a different TCRBV haplotype. A population of CTL with diverse TCRBV and junctional sequence usage, which otherwise represents a minor subset in the gB-specific response, appears to emerge as a consequence of this TCRBV gene variation. The loss of preferential V region-encoded complementarity determining regions (CDR) 1- and/or CDR2-ligand interactions in this emerging population also results in a change in CDR3 sequence usage and a corresponding focusing of an otherwise promiscuous pattern of cross-reactivity with a panel of gB498-505 substitution analogues. This suggests that the difference between the two distinct TCR populations is the relative contributions of the CDR towards ligand recognition. Therefore, preferential V region-ligand interaction, at the expense of CDR3 peptide recognition, appears to control the dominant TCR selection in the C57BL/6 response to this peptide determinant.     

T-cell receptor variable gene analysis of renal allograft-infiltrating cells in biopsy specimens using a nonradioisotopic micromethod

BACKGROUND: A sensitive micromethod for T-cell receptor (TCR) analysis is needed for clonality analysis of renal allograft-infiltrating T cells (RAITs) obtained by needle biopsy. METHODS: TCR cDNA was amplified by the anchored polymerase chain reaction and was hybridized with 28 different TCR beta variable (TCRBV) genes fixed on nylon membranes, and the percentage of each TCRBV gene was measured spectrophotometrically. RESULTS: The specificity and linearity of the hybridization technique and the constancy of the TCRBV percentages over a wide range of sample amounts were demonstrated by control experiments. Analysis of RAITs of biopsy specimens from four patients showed broad or skewed TCRBV usage, indicating the presence of polyclonal and oligoclonal RAIT populations, respectively. In one patient who received OKT3 immunosuppressive treatment, the TCRBV skewness was dramatically reduced after the treatment. CONCLUSION: We have established a powerful method for analyzing RAIT clonality, which is especially useful for monitoring RAIT dynamics after immunosuppression therapy.     

Different TCRBV genes generate biased patterns of V-D-J diversity in human T cells

The aim of this work was to assess whether each T-cell receptor (TCR) BV segment generates a random pattern of junctional diversity or if, alternatively, biased patterns of V-D-J rearrangements limit the number of available TCR specificities. Detailed molecular analysis of T-cell receptors expressed by lymphocytes was obtained by generating a large number of junctional regions sequences from TCRBV3, TCRBV4, TCRBV5S1, TCRBV12, TCRBV13S2, TCRBV17, TCRBV20, and TCRBV22 variable genes. The > 800 sequences analyzed have allowed the characterization of the recombination frequencies of each germline-encoded V, D, and J segments, as well as of the magnitude of exonucleolytic nibbling and of the number of N nucleotides inserted for each group of TCRB segments. The data obtained indicate that the extent of junctional diversity varies considerably depending on the TCRBV gene implicated in the recombination event, due to the occurrence of skewed patterns of J and D region usage. Furthermore, our results show that "illegitimate" rearrangements occur with unexpectedly high incidence, specifically at the level of TCRBD to TCRBJ joining. These findings provide additional information for a more accurate estimation of the size of the TCRBV repertoire and for understanding the well-established biased pattern of TCRBV expression in humans.     

Enhanced prevalence of T cells expressing TCRBV8S2 and TCRBV8S3 in hearts of chronically Trypanosoma cruzi-infected mice

We have analysed the relative T cell receptor (TCR) BV gene usage in T cells from hearts and spleens of CBA/HJ mice chronically infected with the Tulahuén strain of Trypanosoma cruzi. During chronic infection, CBA/HJ mice recruit T cells at the major site of inflammation (i.e. the heart), with over-representation of certain TCRBV gene subfamilies (TCRBV8S2 and TCRBV8S3). In contrast, no signal or a very weak message from a limited number of T cells was recorded from one heart of the control group. No alteration of TCRBV distribution was recorded in spleens of chronically infected CBA/HJ. Our findings indicate that there is a preferential TCRBV gene usage in the T cell response in the hearts of chronically infected mice. Furthermore, the pattern of CDR3 lengths in inflammatory T cells was altered.     

TCR alpha-chain usage can determine antigen-selected TCR beta-chain repertoire diversity

There is considerable variation in the TCR repertoire diversity selected by different peptide Ags. Certain responses show limited V region bias with minimal restrictions in the remainder of the sequence while others can be dominated by a single TCR clonotype repeatedly isolated from different individuals. CTL specific for a Kb-restricted determinant from the herpes simplex virus glycoprotein B (gB) preferentially express a dominant TCRBV10 beta-chain subset with extensive conservation located at the V-D junction. However, unlike some biased responses, no single beta-chain V-D-J combination appears to dominate these CTL. Different animals respond with a large array of unique or "private" beta-chain sequences with little J region preference. Here we examine the contribution of the TCR alpha-chain to the gB-specific CTL diversity. The TCR alpha-chains from different TCRBV10-positive gB-specific CTL clones were found to exhibit extensive sequence variation. However, when T cells were forced to use a single alpha-chain in TCR alpha-chain transgenic mice, gB-specific CTL showed limited variation in their beta-chain selection. These T cells retained the TCRBV10 bias but were now dominated by a single beta-chain sequence that could be repeatedly isolated from different transgenic animals. This "public" TCR consisted of the transgenic alpha-chain and a common TCRBV10D2J2S6 beta-chain. These results suggest that preferential use of one TCR subunit can restrict the level of diversity in the other chain due to interchain interactions involving J-derived sequences.     

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.     

Persistently biased T-cell receptor repertoires in HIV-1-infected combination antiretroviral therapy-treated patients despite sustained suppression of viral replication

In most HIV-1-infected patients, highly active antiretroviral therapy (HAART) reduces plasma viral load to <50 copies/mL and increases CD4+ T-cell number and function. However, it is still unclear whether alterations of T-cell receptor (TCR) beta-chain variable region (BV) repertoire, tightly related to disease progression, can be fully recovered by long-term treatment with HAART. This study analyzed the evolution of both T-cell subset composition and TCRBV perturbations in chronically HIV-1-infected patients with moderate immunodeficiency during 36 months of HAART. Despite persistently suppressed HIV replication, the rate of CD4+ T-cell repopulation, after an initial burst, progressively declined throughout the study period, resulting in a mean CD4+ T-cell count at the end of follow-up that was still significantly lower in HIV patients than in HIV-seronegative controls. This was seen in association with an incomplete restitution of both CD4 and CD8 TCRBV repertoire disruptions and was also demonstrated by the appearance of new TCRBV oligoclonal expansions occurring during HAART. In conclusion, these data indicate that 3 years of fully suppressive HAART may be not adequate to normalize CD4 counts and TCRBV repertoires in patients starting HAART with moderately advanced disease.     

T-cell receptor V region beta-chain gene expression in the autoimmune thyroiditis of non-obese diabetic mice

The non-obese diabetic (NOD) mouse develops both a spontaneous T-cell-mediated autoimmune insulitis and, in addition, a well characterized thyroiditis. We have examined the repertoire of murine T-cell receptor (TCR) variable (V) beta-chain genes used by intrathyroidal T cells with specific oligonucleotides that amplified 17 murine V beta gene families in cDNA samples prepared from intact NOD thyroid tissues. Normal NOD thyroid tissue contained only low levels of TCR V gene mRNA. In contrast, NOD mice with histologic thyroiditis showed the marked expression of up to 3 TCR V beta genes consistent with a restricted T-cell invasion. Sequencing of amplified TCR V beta cDNA showed that within each NOD thyroid sample at least one of the overexpressed V beta gene families was clonally expanded. However, the clonally expanded T-cell V gene family was not consistent in all animals. Even within the same TCR V beta gene families, various D and J segments had been rearranged with open reading frames and together with insertions and deletions gave no significant homology at the nucleotide or amino acid level. In summary, these data showed that the intrathyroidal T-cell infiltrate in NOD mice was markedly biased towards the use of a single, but variable, TCR V gene family within each animal. It also appeared that the choice of the TCR V beta chain determined the intrathyroidal infiltrative process rather than the choice of D and/or J regions. However, there was no consistent use of a single TCR V beta chain. As thyroiditis does not occur uniformly in apparently genetically homogeneous animals, reared under similar environmental conditions, it may not be surprising that different TCR V genes are involved in different animals.     

Rejection of cardiac allografts by T cells expressing a restricted repertoire of T-cell receptor V beta genes

We have recently shown that T cells infiltrating cardiac allografts early in graft rejection use a limited T-cell receptor (TCR) V beta repertoire. In this study we tested whether this limited repertoire of V beta genes is important for graft rejection. A cell line, AL2-L3, was established from LEW lymphocytes infiltrating ACI heart allografts 2 days after transplantation. This cell line is composed of CD4+ T cells that primarily recognize the class II RTI.B major histocompatibility complex (MHC) molecule expressed by the donor graft. This cell line precipitated acute rejection of donor hearts with a median survival time (MST) of 10.5 days following adoptive transfer to sublethally irradiated LEW recipients. This rate of graft rejection was significantly (P < 0.0007) accelerated when compared with a MST of 60 days for allografts in irradiated control recipients. The AL2-L3-mediated acceleration of graft rejection was donor specific as WF third-party heart allografts were rejected with a delayed tempo (MST = 28.5 days). The V beta repertoire of this cell line was primarily restricted to the expression of V beta 4, 15 and 19 genes. The nucleotide sequence analysis of the beta-chain cDNAs from this cell line demonstrated that the restricted use of the V gene repertoire was not shared with the N, D and J regions. A wide variety of CDR3 loops and J beta genes were used in association with selected V beta genes. These data provide evidence for the role a restricted repertoire of V beta genes plays in cardiac allograft rejection in this model. The restricted usage of the V beta repertoire in an early T-cell response to allografts may provide the opportunity to therapeutically disrupt the rejection reaction by targeting selected T-cell populations for elimination at the time of organ transplantation.     

The orientation of a T cell receptor to its MHC class II:peptide ligands

The TCR found on CD4 T cells recognizes peptides bound to self MHC class II molecules as well as non-self MHC class II molecules. We have used the receptor on a cloned T cell line called D10.G4.1 (D10) to perform a structure-function analysis of this interaction. The D10 T cell clone recognizes not only a peptide from conalbumin (CA-wt) bound to syngeneic I-Ak against which it was raised, but also the allogeneic MHC molecules I-A(b,v,p,q,d). In the present study, we show that residue 30 in complementarity-determining region 1 (CDR1) of the TCR alpha-chain interacts with the I-A alpha-chain at hvr2 (residues 52, 53, and 55). We also show that residue 51 in CDR2 of the TCR alpha-chain interacts with the peptide at peptide residue 2. Finally, we show that residue 29 in CDR1 of the TCR beta-chain affects recognition of the glutamic acid at residue 66 in the I-A beta-chain. These data suggest an orientation of TCR relative to its peptide:MHC class II ligands. We argue that this orientation will be shared by all CD4 TCRs, and that it is only subtly different from the common orientation proposed for receptors binding to MHC class I.     

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.     

Sleep apnea in end stage renal disease

Antibodies directed against the beta chain of the T-cell receptor (TCR) have been detected in animals and in humans in a number of distinct immune states that do not involve direct immunization with either T cells or TCR epitopes. When C57B1/6 mice are infected experimentally with the LP-BM5 retrovirus mixture they produce increased titres of autoantibodies directed against TCR V beta complementarity determining region 1 (CDR1) epitopes. Here, the authors utilized hybridoma technology to isolate monoclonal immunoglobulin (Ig)M antibodies (MoAbs) that arose at the peak of infection. The authors characterized the binding specificity tested using synthetic peptides modelling the CDR1 segments of 24 distinct V beta gene products and determined the VH gene usage by two such monoclonals. One binds to a restricted set of TCR V beta CDR1 peptides, and the second reacts with approximately half of the CDR1 peptide homologues. These MoAbs are specific for T-cell receptor beta chains and do not bind to immunoglobulin light chains or to unrelated protein molecules. Both MoAbs bind to intact T cells expressing the V beta domain (human V beta 8 and mouse V beta 11) from which selection peptides were derived, and costimulate a V beta specific in vitro T cell proliferative response induced by the staphylcoccal enterotoxin E (SEE) superantigen.