Germline TCR Research Articles

T Cell Receptor Genotype and Ubash3a Determine Susceptibility to Rat Autoimmune Diabetes.

Genetic analyses of human type 1 diabetes (T1D) have yet to reveal a complete pathophysiologic mechanism. Inbred rats with a high-risk class II major histocompatibility complex (MHC) haplotype (RT1B/Du) can illuminate such mechanisms. Using T1D-susceptible LEW.1WR1 rats that express RT1B/Du and a susceptible allele of the Ubd promoter, we demonstrate that germline knockout of Tcrb-V13S1A1, which encodes the Vβ13a T cell receptor β chain, completely prevents diabetes. Using the RT1B/Du-identical LEW.1W rat, which does not develop T1D despite also having the same Tcrb-V13S1A1 β chain gene but a different allele at the Ubd locus, we show that knockout of the Ubash3a regulatory gene renders these resistant rats relatively susceptible to diabetes. In silico structural modeling of the susceptible allele of the Vβ13a TCR and its class II RT1u ligand suggests a mechanism by which a germline TCR β chain gene could promote susceptibility to T1D in the absence of downstream immunoregulation like that provided by UBASH3A. Together these data demonstrate the critical contribution of the Vβ13a TCR to the autoimmune synapse in T1D and the regulation of the response by UBASH3A. These experiments dissect the mechanisms by which MHC class II heterodimers, TCR and regulatory element interact to induce autoimmunity.

Regulatory T cells and co-evolution of allele-specific MHC recognition by the TCR.

What is the evolutionary mechanism for the TCR‐MHC‐conserved interaction? We extend Dembic's model (Dembic Z. In, Scand J Immunol e12806, 2019) of thymus positive selection for high‐avidity anti–self‐MHC Tregs among double (CD4 + CD8+)‐positive (DP) developing thymocytes. This model is based on competition for self‐MHC (+ Pep) complexes presented on cortical epithelium. Such T cells exit as CD4 + CD25+FoxP3 + thymic‐derived Tregs (tTregs). The other positively selected DP T cells are then negatively selected on medulla epithelium removing high‐avidity anti–self‐MHC + Pep as T cells commit to CD4 + or CD8 + lineages. The process is likened to the competitive selection and affinity maturation in Germinal Centre for the somatic hypermutation (SHM) of rearranged immunoglobulin (Ig) variable region (V[D]Js) of centrocytes bearing antigen‐specific B cell receptors (BCR). We now argue that the same direct SHM processes for TCRs occur in post‐antigenic Germinal Centres, but now occurring in peripheral pTregs. This model provides a potential solution to a long‐standing problem previously recognized by Cohn and others (Cohn M, Anderson CC, Dembic Z. In, Scand J Immunol e12790, 2019) of how co‐evolution occurs of species‐specific MHC alleles with the repertoire of their germline TCR V counterparts. We suggest this is not by ‘blind’, slow, and random Darwinian natural selection events, but a rapid structured somatic selection vertical transmission process. The pTregs bearing somatic TCR V mutant genes then, on arrival in reproductive tissues, can donate their TCR V sequences via soma‐to‐germline feedback as discussed in this journal earlier. (Steele EJ, Lindley RA. In, Scand J Immunol e12670, 2018) The high‐avidity tTregs also participate in the same process to maintain a biased, high‐avidity anti–self‐MHC germline V repertoire.

Diagnostic Assessment of Immunological History by High-throughput TCR sequence Analyses.

Abstract T cell receptor (TCR) rearrangement results in germ-line editing of a T cell’s TCR loci. During infection, T cells with TCRs specific for the pathogen proliferate. Resulting pathogen-specific memory T cells retain the unique germ-line TCR DNA rearrangements, which we hypothesized may be used as molecular biomarkers of prior pathogen exposure. We performed high-throughput sequencing of the TCRβ repertoires of HLA-A2 transgenic mice pre- and post- inoculation with either the smallpox vaccine or highly-related monkeypox virus (MPXV). We generated a database of >2×106 unique TCRβ clonotypes from samples pre- (naïve) and post-exposure to identify TCRs associated with vaccinated/infected samples vs naïve in 58 individual mice. We computationally identified 315 TCRβ sequences statistically associated with post-vaccinated samples, which we used to develop a diagnostic assay to classify samples as naïve or previously-exposed with 97% accuracy up to 9-months post-vaccination. We validated this assay in an independent cohort of MPXV infected mice. The diagnostic platform was adapted to analyze human TCR sequences to distinguish smallpox vaccinated or non-vaccinated individuals. We identified 239 vaccine-associated TCRβ sequences significantly expanded in 100 smallpox vaccinated individuals. A machine learning algorithm was developed to determine individuals’ vaccination status based on the presence of these 239 TCR sequences. Overall, the diagnostic assay correctly identified 74% of non-vaccinated samples and 85% of vaccinated samples. Thus, the analyses support that computational identification of pathogen-associated TCRs is a highly sensitive and specific method for determining prior exposure to an agent or pathogen.

Comment on “A Database of Human Immune Receptor Alleles Recovered from Population Sequencing Data”

It was with great interest that we read the recently published article by Yu et al. ([1][1]), which proposes a solution to the problem of building complete and accurate databases of germline Ig (IG) and TCR genes and alleles. This highlights one of the most formidable challenges in the

Germline TCR contributions.

Germline TCR contributions.

Public TCR use by herpes simplex virus-2-specific human CD8 CTLs.

Recombination of germline TCR alpha and beta genes generates polypeptide receptors for MHC peptide. Ag exposure during long-term herpes simplex infections may shape the T cell repertoire over time. We investigated the CD8 T cell response to HSV-2 in chronically infected individuals by sequencing the hypervariable regions encoding TCR alpha and beta polypeptides from T cell clones recognizing virion protein 22 aa 49-57, an immunodominant epitope. The most commonly detected TCRBV gene segment, found in four of five subjects and in 12 of 50 independently derived T cell clones, was TCRBV12-4. Nineteen to seventy-two percent of tetramer-binding cells in PBMCs were stained ex vivo with a TCRBV12 mAb. Three alpha-chain and three beta-chain public TCR sequences were shared between individuals. Public heterodimers were also detected. Promiscuous pairing of a specific TCRVA1-1 sequence with several different TCRB polypeptides was observed, implying a dominant structural role for the TCRA chain for these clonotypes. Functional avidity for cytotoxicity and IFN-gamma release was relatively invariant, except for one subject with both high avidity and unique TCR sequences and lower HSV-2 shedding. These data indicate that the CD8 response to a dominant alpha-herpesvirus epitope converges on preferred TCR sequences with relatively constant functional avidity.

Terminal Deoxynucleotidyl Transferase Establishes and Broadens Antiviral CD8+ T Cell Immunodominance Hierarchies

The action of TdT on mouse TCR genes accounts for approximately 90% of T cell repertoire diversity. We report that in TdT-/- mice, total T(CD8+) responses to influenza and vaccinia viruses are reduced by approximately 30% relative to wild-type mice. We find that T(CD8+) responses to three subdominant influenza virus determinants are reduced to background values in TdT-/- mice while responses to three immunodominant determinants undergo a major reshuffling. A similar reshuffling occurs in T(CD8+) responses to immunodominant vaccinia virus determinants, and is clearly based on broad differences in TCR family usage and CDR3 length between wild-type and TdT-/- mice. These findings demonstrate that TdT plays a critical role in the magnitude and breadth of anti-viral T(CD8+) responses toward individual determinants and suggests that germline TCR repertoire bias toward the most dominant determinants is a major factor in establishing immunodominance hierarchies.

Unusual childhood extramedullary hematologic malignancy with natural killer cell properties that contains tropomyosin 4–anaplastic lymphoma kinase gene fusion

This report describes an unusual extramedullary hematologic malignancy in an 18-month-old child who presented with a capillary leak syndrome that evolved into hyperleukocytosis with malignant cells. The circulating tumor cells did not express an antigen profile typical of any subtype of leukemia commonly observed in children. Tumor cells were CD3(-)/CD56(+); had germline TCR genes; and strongly expressed CD30, epithelial membrane antigen, and anaplastic lymphoma kinase (ALK) consistent with a null cell anaplastic large cell lymphoma (ALCL). The malignant cells contained a t(2;19)(p23;p13.1) that interrupted ALK and translocated it to the der(19). Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis revealed fusion of ALK to tropomyosin 4, an ALK fusion partner not described previously in hematologic malignancies. The clinical presentation and phenotypic features of this malignancy were not typical for ALCL because tumor cells expressed both myeloid (CD13, CD33, HLA-DR) and natural killer (NK) cell antigens. The neoplastic cells most resembled NK cells because in addition to being CD3(-)/CD56(+) with germline TCR genes, these cells were CD25(+)/CD122(+)/granzyme B(+) and possessed the functional properties of immature NK cells. The unusual clinical presentation, immunophenotype, and functional properties of these neoplastic cells suggest that this malignancy may be derived from the putative myeloid-NK precursor cell. Furthermore co-expression of NK and ALCL features supports the concept that a minority of null-ALCL may be derived from NK cells and expands the spectrum of phenotypes that can be seen in tumors produced by ALK fusion proteins. (Blood. 2001;98:1209-1216)

Expression of a TCR V beta 8.2 polypeptide from the unrearranged gene in a murine lymphoid precursor cell line.

The TCR in a mature T cell is a multimeric complex of TCR alpha and beta chains, and CD3 subunits. Functional TCR alpha and beta chains are encoded by genes that result from developmentally controlled somatic rearrangement events. By FACS analysis, we have detected a TCR V beta 8 protein on the surface of an immature lymphoid cell line, C1-V13D, that has all of its TCR genes in germline (unrearranged) configuration. RNA blot analysis detected a 1.4 kb polydenylated V beta 8 RNA in C1-V13D cells, but no expression of C beta was detected. Rapid amplification of 3' cDNA ends was used to clone an RNA that was initiated from the leader exon of the V beta 5.1 gene and spliced to the V exon of the V beta 8.2 gene. The putative sequence of the mature 10.8 kDa protein was entirely encoded by the V beta 8.2 exon. RT-PCR analysis confirmed that 97% of the V beta 8 RNA detected in C1-V13D cells was encoded by the V beta 8.2 gene, and only 3% by V beta 8.1 and V beta 8.3 genes. Furthermore, most of the V beta 8.2 RNA was spliced to the leader exon of the V beta 5.1 gene and not to the leader exon of the V beta 8.2 gene. The implications of preferential transcription from particular germline TCR genes for repertoire diversity and possible functions for proteins translated from germline TCR V beta genes are discussed.

T-cell receptors in immune responses.

T lymphocytes play a central role in the generation of both humoral and cell-mediated immune responses. Although T-cells mediate diverse effector functions, most recognize antigenic peptide presented by molecules encoded within the major histocompatibility complex (MHC). The fine specificity of a T-cell is determined by a heterodimeric receptor for antigen (TCR) displayed on the cell surface. The TCR present on the majority of T-cells is composed of an alpha and a beta chain which are similar to immunoglobulins (Ig) in both sequence and gene organization. Genes for the alpha and beta chains of the TCR are encoded in germline DNA as discontinuous gene segments that rearrange specifically in T-cells during development. The variable regions of each of these chains are responsible for antigen recognition and are encoded by juxtaposed variable (V), diversity (D) (for beta chains), and joining (J) gene segments (reviewed in Toyonaga and Mak, 1987; Wilson et al., 1988; Davis and Bjorkman, 1988). A critical difference between TCR and Ig is that somatic mutation does not appear to play a role in the generation of TCR diversity (Ikuta et al., 1985), which emphasizes the importance of germline polymorphism of TCR gene segments. Knowledge of both the extent and variability of the germline TCR repertoire is critical to understanding the diversity of potential T-cell specificities.KeywordsMajor Histocompatibility ComplexGene SegmentMajor Histocompatibility Complex GeneBeta ChainMajor Histocompatibility Complex AntigenThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Polymorphism of T‐cell receptor genes in nasopharyngeal carcinoma

Germ-line TCR genes from 41 Singaporean Chinese patients with nasopharyngeal carcinoma and 54 Singapore Chinese normal subjects were investigated by restriction fragment length polymorphism (RFLP) analysis. Genomic DNA from BLCLs of patients and normal subjects was digested with the restriction endonucleases BamHI, EcoRI, HindIII, TaqI, transferred to nylon membrane by the Southern-blot technique and hybridized with radioactively labelled TCR cDNA probes. When the combination of BamHI/V beta II was used, NPC patients had a lower frequency of the 25/25-kb allelic pattern and a higher frequency of the 20-kb fragment compared to controls. With the combination of TaqI/V beta 8, the 4.3/3.2-kb fragment allelic frequency was lower in total NPC but particularly in patients with HLA B46. The result suggested that TCR restriction may be important in the pathogenesis of NPC.

A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by CD44 and CD25 expression.

We have subdivided mouse CD4-CD8-CD3- triple-negative (TN) thymocytes into four subsets based upon expression of CD44 and CD25, including CD44+CD25-, CD44+CD25+, CD44-CD25+ and CD44-CD25-. Characterization of these cells revealed several features distinct to each subset, in particular the expression of high levels of c-kit (the receptor for stem cell factor) by CD44+CD25-TN and CD44+CD25+TN but not by CD44-CD25+TN and CD44-CD25-TN. The CD44+CD25+TN subset also included the IL-7 and stem cell factor-responsive cells, whereas only minimal responsiveness was observed by the CD44- populations. These subsets also showed differential cytokine production potential (CD44+CD25- > CD44+CD25+ > CD44-CD25+ > CD44-CD25-) after stimulation with calcium ionophore, PMA and IL-1. The repopulation potential of these subsets in 2-deoxyguanosine-treated fetal thymic lobes supports the following maturation sequence: CD44+CD25- -->CD44+CD25+ -->CD44-CD25+ -->CD44-CD25-. Furthermore, the sequence of progression from CD44+CD25+ to CD44-CD25+ cells was confirmed by their TCR beta-chain gene configuration. The former population exhibits germ-line TCR beta-chain configuration, whereas the latter subset shows a rearranged pattern.

A distinct large granular lymphocyte (LGL)/NK‐associated (NKa) abnormality characterized by membrane CD4 and CD8 coexpression

In a study of 870 individual patients with either lymphocytosis (excluding known lymphoproliferative disease), increased proportions of blood lymphocytes with granular morphology (LGL), or neutropenia, 14 cases were found with abnormally increased CD3+CD4+CD8+ components. Eleven of these were further investigated and 10 shown in follow-up studies to be persistent in nature. Morphological assessments revealed increased LGL in 9/11 cases, and in seven of these > 50% lymphocytes had discernable cytoplasmic granulation. Immunophenotypic studies indicated that CD8 expression by CD4+ lymphocytes in these patients was of low density (CD8dim+), and that both the CD4+CD8- and CD4+CD8dim+ fractions in each patient was characterized by a CD11b+CD16-CD56+CD57+ composite NK-associated (NKa) phenotype (in contrast to normal CD4+CD8- blood lymphocytes and CD4+CD8+ thymocytes which were consistently CD11b-CD16-CD56-CD57-). TCR genotypic studies revealed rearranged components (beta plus gamma, or beta alone) in 5/11 cases, but there were no obvious relationships between TCR configuration (including rearranged band densities) and immunophenotypes, absolute lymphocyte or neutrophil numbers, the proportions of blood LGL, or the proportions of CD4+ cells coexpressing CD8. The occurrence of identical NKa phenotypic profiles in both germline and rearranged TCR cases does, however, suggest the possibility of an evolutionary process from a non-clonal expansion to a clonal state. Serum studies, including soluble CD4, CD8 and IL2-R concentrations and autoantibody investigations, of representative germline and rearranged TCR cases failed to indicate any consistent abnormalities, but there was some suggestion for the existence of a chronic reactive process in some of the patients with germline TCR. These findings suggest that expanded LGL/NKa+ components with phenotypic evidence of CD4/CD8 coexpression should be regarded as a distinct diagnostic category and that persistent CD4+CD8+ abnormalities with germline TCR should be monitored for possible clonal transition.

T cell receptor α chain polymorphisms in multiple sclerosis

Numerous studies have implicated the major histocompatibility complex (MHC) class II alleles, DR2 and DQw1, as multiple sclerosis (MS) susceptibility loci; however, the involvement of other loci is implied by twin studies and the relative lack of haplotype sharing for MHC. to evaluate the role that the TCR α chain genes may have in MS susceptibility, three variable (V) α polymorphisms were examined for associations in MS patients. Genotype and allele frequencies were compared to four different control groups: unaffected siblings and parents of the MS patients, patients with insulin-dependent diabetes mellitus (IDDM) and healthy unrelated Caucasians. No significant differences in allele and genotype frequencies at these three loci were observed in the MS population compared to the control groups. In addition, we analysed the distribution of haplotype sharing in affected sibling pairs. Among 30 informative families, there was no significant increase in haplotypes shared by affected siblings over that expected based on random segregation. Our results do not support suggestions that germline TCR α chain genes contribute to genetic susceptibility in MS.

悪性組織球症の１例

An autopsy case of malignant histiocytosis (MH) in a 16 y.o. male is reported. He was acutely ill at admission, complaining of high fever, diarrhea and generalized skin symptoms. Hepatosplenomegaly and an enlarged lymph node were noticed. Bone marrow aspirate showed increased numbers of histiocytoid cells with various degree of atypism. They had vacuoles and azurophilic granules, but rarely showed erythrophagocytosis. Biopsied lymph node microscopically showed proliferation of atypical cells with the distinct appearance of histiocyte. Immunohistochemically, they had positive marker stain for lysozyme, α1-antitrypsin and anti-macrophage. A diagnosis of MH was made. Genotypic analysis by Southern method showed germline TCR and JH genes. Ultrastructurally, mature tumor cells were rich in organallae and contained 10nm filaments. The patient died after development of marked pancytopenia. Total duration of illness was 4.5 months. Autopsy revealed systemic involvement of lymph nodes, bone marrow, liver, spleen and lungs by diffuse proliferation of atypical histiocytoid cells. They showed a diversity of cellular atypism, including mature and immature histiocytes, and pleomorphic and multinucleated giant cells. The present case is characterised clinically by prominent skin desquamation and progressive course, and pathologically by proliferation of a diversity of neoplastic histiocytoid and its precursor cells.

T cell receptor DJ but not VDJ rearrangement within a recombination substrate introduced into a pre-B cell line

To elucidate mechanisms that regulate ordered and tissue-specific assembly of Ig and TCR variable region gene segments, we have introduced a recombination substrate comprised of germline TCR beta V, D, and J gene segments into an Abelson murine leukemia virus-transformed pre-B cell line that actively rearranges endogenous Ig H chain variable region gene segments but does not rearrange endogenous light chain or TCR variable region gene segments. We find that these cells efficiently join D beta segments to J beta segments within the mini-locus, but that they do not make any detectable site-specific rearrangements of the introduced V beta segment even though it is closely linked in the same construct to the D beta. These findings suggest that factors necessary for V beta to (D beta)J beta joining may be absent in these pre-B cells and also imply that the order in which TCR V beta, D beta, and J beta segments are rearranged can be influenced by factors other than the 12/23 recombination rule. Furthermore, in agreement with the an accessibility model of VDJ recombinase control, the D beta region of the construct was found to be relatively more sensitive to DNAase I digestion in isolated nuclei when compared to the unrearranged V beta region.

B lymphoblastoid cell lines with rearranged T cell receptor beta-chain genes retain conventional B cell surface antigen and Ig expression.

Transformation of peripheral blood lymphocytes by co-incubation with EBV produces B lymphoblastoid cell lines, but rearrangement of TCR beta-chain genes was observed in three different cell lines derived from two individuals. Because rearrangement of TCR genes in B lymphocytes is considered a rare event, these B lymphoblastoid cell lines with rearranged TCR beta-genes were examined in detail to determine whether there were any additional characteristics to distinguish them from B lymphoblastoid cell lines with germ-line TCR beta-genes. All B lymphoblastoid cell lines contained rearranged Ig H and kappa L chain genes, secreted Ig, and expressed B and not T cell surface markers. Cell lines with rearranged TCR beta-genes had rearranged both IgH genes and had rearranged and subsequently deleted both kappa C region genes. Furthermore all three B lymphoblastoid cell lines with rearranged TCR beta-genes produced small amounts of Ig with lambda-L chains. Although the cellular mechanisms maintaining lineage-specific rearrangement events remain unknown, extensive Ig gene rearrangement and inefficient Ig production by B cells may be indicators of a cellular status where normally stringent lineage-specific control elements fail to function efficiently.