Origin and evolutionary malleability of T cell receptor α diversity.

Lymphocytes of vertebrate adaptive immune systems acquired the capability to assemble, from split genes in the germline, billions of functional antigen receptors1-3. These receptors show specificity; unlike the broadly tuned receptors of the innate system, antibodies (Ig) expressed by B cells, for instance, can accurately distinguish between the two enantiomers of organic acids4, whereas T cell receptors (TCRs) reliably recognize single amino acid replacements in their peptide antigens5. In developing lymphocytes, antigen receptor genes are assembled from a comparatively small set of germline-encoded genetic elements in a process referred to as V(D)J recombination6,7. Potential self-reactivity of some antigen receptors arising from the quasi-random somatic diversification is suppressed by several robust control mechanisms8-12. For decades, scientists have puzzled over the evolutionary origin of somatically diversifying antigen receptors13-16. It has remained unclear how, at the inception of this mechanism, immunologically beneficial expanded receptor diversity was traded against the emerging risk of destructive self-recognition. Here we explore the hypothesis that in early vertebrates, sequence microhomologies marking the ends of recombining elements became the crucial targets of selection determining the outcome of non-homologous end joining-based repair of DNA double-strand breaks generated during RAG-mediated recombination. We find that, across the main clades of jawed vertebrates, TCRα repertoire diversity is best explained by species-specific extents of such sequence microhomologies. Thus, selection of germline sequence composition of rearranging elements emerges as a major factor determining the degree of diversity of somatically generated antigen receptors.

Nurse shark T-cell receptors employ somatic hypermutation preferentially to alter alpha/delta variable segments associated with alpha constant region.

In addition to canonical TCR and BCR, cartilaginous fish assemble noncanonical TCR that employ various B-cell components. For example, shark T cells associate alpha (TCR-α) or delta (TCR-δ) constant (C) regions with Ig heavy chain (H) variable (V) segments or TCR-associated Ig-like V (TAILV) segments to form chimeric IgV-TCR, and combine TCRδC with both Ig-like and TCR-like V segments to form the doubly rearranging NAR-TCR. Activation-induced (cytidine) deaminase-catalyzed somatic hypermutation (SHM), typically used for B-cell affinity maturation, also is used by TCR-α during selection in the shark thymus presumably to salvage failing receptors. Here, we found that the use of SHM by nurse shark TCR varies depending on the particular V segment or C region used. First, SHM significantly alters alpha/delta V (TCRαδV) segments using TCR αC but not δC. Second, mutation to IgHV segments associated with TCR δC was reduced compared to mutation to TCR αδV associated with TCR αC. Mutation was present but limited in V segments of all other TCR chains including NAR-TCR. Unexpectedly, we found preferential rearrangement of the noncanonical IgHV-TCRδC over canonical TCR αδV-TCRδC receptors. The differential use of SHM may reveal how activation-induced (cytidine) deaminase targets V regions.

Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire.

Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells.

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRß locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications.

MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage.

Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire as a whole, especially for TCRß chain sequences. Moreover, different pathogen-specific responses were characterized by distinct TCR usage, both between and within individuals, suggesting that MAIT cell adaptation was a direct consequence of exposure to various exogenous MR1-restricted epitopes. In line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped by microbial exposure.

Distinct morphologic, phenotypic, and clinical-course characteristics of indolent peripheral T-cell lymphoma.

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) consists of a heterogeneous group of lymphomas. Patients generally show an aggressive clinical course and very poor outcome. Although the 2008 World Health Organization classification of PTCL-NOS includes 3 variants, low-grade lymphoma is not included. Of 277 PTCL-NOS cases recorded in our consultation files, we examined the clinicopathologic characteristics of 10 patients with T-cell lymphomas composed of small-sized cells with slight nuclear atypia. Eight patients showed extranodal involvement (5 patients, spleen; 3 patients, thyroid), and 5 patients were at clinical stage I or II. Histologically, all samples presented diffuse infiltrate of small lymphoid cells, with few mitotic figures. Immunohistologically, all samples were positive for CD3, and CD20 was detected in 5 samples. All samples showed a low Ki-67 labeling index (mean, 1.05%), and 7 samples were positive for central memory T-cell markers. Clonal T-cell receptor γ chain and/or α-ß chain gene rearrangements were detected in all 10 patients. Five patients received chemotherapy, whereas for 3 patients, treatment consisted only of observation following surgical resection of the spleen or thyroid. Nine patients were alive at a median follow-up time of 19.5 months, whereas 1 patient died of an unrelated disease. The present study strongly indicates that T-cell lymphoma with small-sized lymphoma cells and a low Ki-67 labeling index is a distinct variant. Recognition of this novel lymphoma subtype, which should not be defined merely as PTCL-NOS, should be seriously considered.

Extensive molecular mapping of TCRα/δ- and TCRß-involved chromosomal translocations reveals distinct mechanisms of oncogene activation in T-ALL.

Chromosomal translocations involving the TCR loci represent one of the most recurrent oncogenic hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) and are generally believed to result from illegitimate V(D)J recombination events. However, molecular characterization and evaluation of the extent of recombinase involvement at the TCR-oncogene junction has not been fully evaluated. In the present study, screening for TCRß and TCRα/δ translocations by FISH and ligation-mediated PCR in 280 T-ALLs allowed the identification of 4 previously unreported TCR-translocated oncogene partners: GNAG, LEF1, NKX2-4, and IL2RB. Molecular mapping of genomic junctions from TCR translocations showed that the majority of oncogenic partner breakpoints are not recombinase mediated and that the regulatory elements predominantly used to drive oncogene expression differ markedly in TCRß (which are exclusively enhancer driven) and TCRα/δ (which use an enhancer-independent cryptic internal promoter) translocations. Our data also imply that oncogene activation takes place at a very immature stage of thymic development, when Dδ2-Dδ3/Dδ3-Jδ1 and Dß-Jß rearrangements occur, whereas the bulk leukemic maturation arrest occurs at a much later (cortical) stage. These observations have implications for T-ALL therapy, because the preleukemic early thymic clonogenic population needs to be eradicated and its disappearance monitored.

Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation.

BACKGROUND: The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system. RESULTS: In this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3+TCRαß+DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vß repertoire. CONCLUSIONS: Our study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.

Regulation of TCRß allelic exclusion by gene segment proximity and accessibility.

Ag receptor loci are regulated to promote allelic exclusion, but the mechanisms are not well understood. Assembly of a functional TCR ß-chain gene triggers feedback inhibition of V(ß)-to-DJ(ß) recombination in double-positive (DP) thymocytes, which correlates with reduced V(ß) chromatin accessibility and a locus conformational change that separates V(ß) from DJ(ß) gene segments. We previously generated a Tcrb allele that maintained V(ß) accessibility but was still subject to feedback inhibition in DP thymocytes. We have now further analyzed the contributions of chromatin accessibility and locus conformation to feedback inhibition using two novel TCR alleles. We show that reduced V(ß) accessibility and increased distance between V(ß) and DJ(ß) gene segments both enforce feedback inhibition in DP thymocytes.

Evolutionarily conserved TCR binding sites, identification of T cells in primary lymphoid tissues, and surprising trans-rearrangements in nurse shark.

Cartilaginous fish are the oldest animals that generate RAG-based Ag receptor diversity. We have analyzed the genes and expressed transcripts of the four TCR chains for the first time in a cartilaginous fish, the nurse shark (Ginglymostoma cirratum). Northern blotting found TCR mRNA expression predominantly in lymphoid and mucosal tissues. Southern blotting suggested translocon-type loci encoding all four chains. Based on diversity of V and J segments, the expressed combinatorial diversity for gamma is similar to that of human, alpha and beta may be slightly lower, and delta diversity is the highest of any organism studied to date. Nurse shark TCRdelta have long CDR3 loops compared with the other three chains, creating binding site topologies comparable to those of mammalian TCR in basic paratope structure; additionally, nurse shark TCRdelta CDR3 are more similar to IgH CDR3 in length and heterogeneity than to other TCR chains. Most interestingly, several cDNAs were isolated that contained IgM or IgW V segments rearranged to other gene segments of TCRdelta and alpha. Finally, in situ hybridization experiments demonstrate a conservation of both alpha/beta and gamma/delta T cell localization in the thymus across 450 million years of vertebrate evolution, with gamma/delta TCR expression especially high in the subcapsular region. Collectively, these data make the first cellular identification of TCR-expressing lymphocytes in a cartilaginous fish.

Numerical modelling of the V-J combinations of the T cell receptor TRA/TRD locus.

T-Cell antigen Receptor (TR) repertoire is generated through rearrangements of V and J genes encoding alpha and beta chains. The quantification and frequency for every V-J combination during ontogeny and development of the immune system remain to be precisely established. We have addressed this issue by building a model able to account for Valpha-Jalpha gene rearrangements during thymus development of mice. So we developed a numerical model on the whole TRA/TRD locus, based on experimental data, to estimate how Valpha and Jalpha genes become accessible to rearrangements. The progressive opening of the locus to V-J gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. Furthermore, the possibility of successive secondary V-J rearrangements was included in the modelling. The model points out some unbalanced V-J associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the J genes, depending on their location in the locus. The model shows that 3 to 4 successive rearrangements are sufficient to explain the use of all the V and J genes of the locus. Finally, the model provides information on both the kinetics of rearrangements and frequencies of each V-J associations. The model accounts for the essential features of the observed rearrangements on the TRA/TRD locus and may provide a reference for the repertoire of the V-J combinatorial diversity.

T cell receptor repertoire in BALB/c mice varies according to tissue type, sex, age, and hydrocortisone treatment.

Diversity in T cell recognition of antigens is determined by diverse usage of T cell receptor (TCR) repertoire. TCR repertoire analysis provides fundamental information for understanding T cell immune responses in the pathogenesis of various diseases. In the present study, we examined the TCR repertoire in various tissues in normal BALB/c mice. The TCR alpha chain variable region repertoires were consistent among the spleen, lymph nodes, and the thymus. The TCR beta chain variable region (TCRBV) repertoires were consistent between the spleen and lymph nodes, but different in the thymus. The TCR repertoires also differed in the lungs and the intestinal tract. The TCR repertoires were consistent between male and female mice, except for TCRBV15-1. TCR repertoire was almost similar in 3- and 7-week-old mice, except for TCRBV1-1, 8-3, and 14-1. The present findings suggest that the TCR repertoire of mice varies according to tissue type, sex and age. Additional analysis of the TCR repertoire, i.e., the effect of hydrocortisone (HC), was carried out. After the HC treatment, although the thymic T cells decreased to one-tenth, only a small fraction of CD4(+)CD8(+) T cells survived the treatment. Furthermore, the percentages of thymic T cells bearing TCRBV3-1, 5-1, 5-2, and 16-1 substantially decreased, but the percentage of cells bearing TCRBV12-1 did not decrease. The present findings suggest that the HC susceptibility of immature thymic T cells is different between TCR families.

The magnitude of thymic output is genetically determined through controlled intrathymic precursor T cell proliferation.

The thymus plays a crucial role in providing the immune system with naive T cells showing a diverse TCR repertoire. Whereas the diversity of thymic production is mainly ensured by TCR rearrangement at both the TRA and TRB loci, the number of cells reaching the double-positive differentiation stage defines the extent of thymic output. A quantitative analysis of TCR excision circles (TREC; signal-joint TRECs and DJbetaTRECs) produced at different stages of thymopoiesis was performed in nine laboratory mouse strains. The results clearly demonstrate that the magnitude of thymic output is directly proportional to the extent of proliferation in the double-negative 4 thymocyte subset. Strikingly, intrathymic precursor T cell proliferation was found to be strain dependent, thus suggesting a genetic regulation of thymic output. The inherited character of thymic output was further confirmed by the transmission of the phenotype in a recessive fashion in F(1) progeny of the different parental strains. Our results provide the first demonstration of the genetic regulation of thymic output.

A novel mouse model for invariant NKT cell study.

We have generated a novel mouse model harboring the in-frame rearranged TCRValpha specific for invariant NKT (iNKT) cells (Valpha14-Jalpha18) on one allele by crossing the mouse cloned from NKT cells with wild-type mice. This genomic configuration would ensure further rearrangement and expression of TCRValpha14-Jalpha18 under the endogenous promoters and enhancers. Mice harboring such an in-frame rearranged TCRValpha (Valpha14-Jalpha18 mouse) possessed an increase in iNKT cells in the thymus, liver, spleen, and bone marrow. Intriguingly, both Th1- and Th2-type cytokines were produced upon stimulation with alphaGalactosylceramide, an agonist of iNKT cells, and the IgE level in the serum remained unaffected in the Valpha14-Jalpha18 mouse. These features markedly distinguish the nature of iNKT cells present in the Valpha14-Jalpha18 mouse from that of iNKT cells found in the Valpha14-Jalpha18 transgenic mouse. Besides these, the expression of TCRVgammadelta cells remained intact, and the use of the TCRVbeta repertoire in iNKT cells was highly biased to TCRVbeta8 in the Valpha14-Jalpha18 mouse. Furthermore, alphaGalactosylceramide-CD1d dimer-reactive immature iNKT cells expressed less Rag2 as compared with the conventional immature T cells at the positive selection stage. Cell cycle analysis on the thymocytes revealed that no particular subset proliferated more vigorously than the others. Crossing the Valpha14-Jalpha18 mouse with the CD1d knockout mouse revealed a novel population of iNKT cells whose coreceptor expression profile was similar to that assigned to iNKT precursor cells. These mice will be useful for the study on the development of iNKT cells as well as on their functions in the immune system.

Age-dependent TCR revision mediated by interaction between alphabeta TCR and self-antigens.

Interactions between TCR and self-peptide/MHC complex play an important role in homeostasis and Ag reactivity of mature peripheral T cells. In this report, we demonstrate that the interactions between mature peripheral T cells and endogenous Ags have a negative impact on the maintenance of foreign Ag-specific T cells in an age-dependent manner. This is mediated by RAG-dependent secondary rearrangement of the TCR alpha-chain (receptor revision). The TCR revision in mature T cells is readily observed in mouse expressing transgenic TCR alpha-chain inserted into the physiological locus (knockin mouse) but not in conventional transgenic mouse with an identical TCR alpha-chain. Thus, our results suggest that under physiological conditions in which all TCR alpha-chains are susceptible to deletion by secondary rearrangement, TCR revision in mature peripheral T cells is an ongoing process in adult animals and contributes to age-dependent changes in T cell function and repertoire.

Multiple constraints at the level of TCRalpha rearrangement impact Valpha14i NKT cell development.

CD1d-restricted NKT cells that express an invariant Valpha14 TCR represent a subset of T cells implicated in the regulation of several immune responses, including autoimmunity, infectious disease, and cancer. Proper rearrangement of Valpha14 with the Jalpha18 gene segment in immature thymocytes is a prerequisite to the production of a TCR that can be subsequently positively selected by CD1d/self-ligand complexes in the thymus and gives rise to the NKT cell population. We show here that Valpha14 to Jalpha rearrangements are temporally regulated during ontogeny providing a molecular explanation to their late appearance in the thymus. Using mice deficient for the transcription factor RORgamma and the germline promoters T early-alpha and Jalpha49, we show that developmental constraints on both Valpha and Jalpha usage impact NKT cell development. Finally, we demonstrate that rearrangements using Valpha14 and Jalpha18 occur normally in the absence of FynT, arguing that the effect of FynT on NKT cell development occurs subsequent to alpha-chain rearrangement. Altogether, this study provides evidence that there is no directed rearrangement of Valpha14 to Jalpha18 segments and supports the instructive selection model for NKT cell selection.

Impact of cellular lifespan on the T cell receptor repertoire.

Pro-survival members of the Bcl-2 family are potent inhibitors of cell death and determine the lifespan of immature thymocytes by counteracting the intrinsically active apoptotic program in these cells. BH3-only proteins are potent antagonists of Bcl-2-like molecules and regulate death and survival of lymphocytes during their development and homeostasis. The intrinsic lifespan of CD4(+)8(+) double-positive thymocytes was reported to actively shape the diversity of the immune repertoire, since mice overexpressing Bcl-x(L) were reported to show a bias towards the usage of distal 3' Jalpha elements 1. To gain support for this concept, we analyzed TCRalpha rearrangements in T lymphocytes that show an extended lifespan due to either loss of the BH3-only protein Bim or overexpression of Bcl-2. A minor but reproducible skewing towards the usage of the more distal 3' Jalpha elements was observed in developing thymocytes and mature T cells from bim(-/-) and vav-bcl-2 transgenic mice, indicating that prolonged survival of double-positive thymocytes does have a significant impact on the selected TCRalpha repertoire. However, the changes that we observed were less pronounced than those found in lck-bcl-x(L) transgenic mice, pointing towards qualitative differences between Bcl-2- and Bcl-x(L)-mediated cell death inhibition during T cell development.

Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination.

During the recombination of variable (V) and joining (J) gene segments at the T cell receptor alpha locus, a ValphaJalpha joint resulting from primary rearrangement can be replaced by subsequent rounds of secondary rearrangement that use progressively more 5' Valpha segments and progressively more 3' Jalpha segments. To understand the mechanisms that target secondary T cell receptor alpha recombination, we studied the behavior of a T cell receptor alpha allele (HYalpha) engineered to mimic a natural primary rearrangement of TRAV17 to Jalpha57. The introduced ValphaJalpha segment was shown to provide chromatin accessibility to Jalpha segments situated within several kilobases downstream and to suppress germ-line Jalpha promoter activity and accessibility at greater distances. As a consequence, the ValphaJalpha segment directed secondary recombination events to a subset of Jalpha segments immediately downstream from the primary rearrangement. The data provide the mechanistic basis for a model of primary and secondary T cell receptor alpha recombination in which recombination events progress in multiple small steps down the Jalpha array.

Gene-specific signal joint modifications during V(D)J recombination of TCRAD locus genes in murine and human thymocytes.

V(D)J recombination assembles functional T-cell receptor (TCR) genes from V, D and J components in developing thymocytes. Extensive processing of V, D and J extremities before they are ligated creates a high degree of junctional diversity which results in the generation of a large repertoire of different TCR chains. In contrast, the extremities of the intervening DNA segment, which bear the recombination signal sequences, are generally held to be monomorphic, so that signal joints (SJs) consist of the perfect head-to-head juxtaposition of recombination signal extremities. We analyzed the structure of SJs generated during the recombination of TCRAD locus genes in murine and human thymocytes. Junctional diversity resulting from N nucleotide additions or from N nucleotide additions and base loss was found for each type of SJ examined. Different patterns of processing/modification were found, suggesting that different enzymatic activities operate during recombination of TCRA and TCRD genes, although they are located within the same genetic locus. Recombination of the deltaRec-1 element generates a diverse repertoire of SJs exhibiting both combinatorial and junctional diversity in murine and human thymocytes. Therefore, SJ diversity appears to be an intrinsic feature of V(D)J recombination in unmanipulated thymocytes.