Characterization of TCR gene rearrangements during adult murine T cell development

Development of the alphabeta and gammadelta T cell lineages is dependent upon the rearrangement and expression of the TCRalpha and beta or gamma and delta genes, respectively. Although the timing and sequence of rearrangements of the TCRalpha and TCRbeta loci in adult murine thymic precursors has been characterized, no similar information is available for the TCRgamma and TCRdelta loci. In this report, we show that approximately half of the total TCRdelta alleles initiate rearrangements at the CD44highCD25+ stage, whereas the TCRbeta locus is mainly in germline configuration. In the subsequent CD44lowCD25+ stage, most TCRdelta alleles are fully recombined, whereas TCRbeta rearrangements are only complete on 10-30% of alleles. These results indicate that rearrangement at the TCRdelta locus can precede that of TCRbeta locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCRdelta and TCRgamma genes among CD44highCD25+ thymocytes, suggesting that functional gammadelta TCR complexes can be formed before the rearrangement of TCRbeta. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence alphabeta/gammadelta lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44highCD25+ stage of development.     

Rates of recombination and chain pair biases greatly influence the primary gammadelta TCR repertoire in the thymus of adult mice

Analyses of the rearrangement status of the TCRgamma and TCRdelta chain loci in progenies of individual gammadelta thymocytes showed a hierarchy of the different Vgamma and Vdelta gene segments to participate in a recombination reaction. Moreover, individual TCRgamma chains only pair efficiently with a variable number of TCRdelta chains. Interestingly, these two parameters are inversely correlated such that the TCRgamma and TCRdelta chains that rearrange more often show a higher level of restriction in their pairing capabilities. Our data suggest that these mechanisms, together with a natural variation affecting the expected frequencies at which rearrangement of different Vgamma gene segments give raise to functional TCRgamma chains, have coevolved to maximize the diversity of the gammadelta TCR repertoire minimizing the risk that a gammadelta T cell will express more than one TCR specificity at the cell surface, despite the fact that multiple TCRgamma rearrangements take place in the same progenitor cell.     

Early expression of a functional TCRbeta chain inhibits TCRgamma gene rearrangements without altering the frequency of TCRgammadelta lineage cells

To investigate the consequences of the simultaneous expression in progenitor cells of a TCRgammadelta and a pre-TCR on alphabeta/gammadelta lineage commitment, we have forced expression of functionally rearranged TCRbeta, TCRgamma, and TCRdelta chains by means of transgenes. Mice transgenic for the three TCR chains contain numbers of gammadelta thymocytes comparable to those of mice transgenic for both TCRgamma and TCRdelta chains, and numbers of alphabeta thymocytes similar to those found in mice solely transgenic for a rearranged TCRbeta chain gene. gammadelta T cells from the triple transgenic mice express the transgenic TCRbeta chain, but do not express a TCRalpha chain, and, by a number of phenotypic and molecular parameters, appear to be bona fide gammadelta thymocytes. Our results reveal a remarkable degree of independence in the generation of alphabeta and gammadelta lineage cells from progenitor cells that, in theory, could simultaneously express a TCRgammadelta and a pre-TCR.     

Developmental stage-specific regulation of TCR-alpha-chain gene assembly by intrinsic features of the TEA promoter

The TCR delta- and alpha-chain genes lie in a single complex locus, the TCRalpha/delta locus. TCRdelta-chain genes are assembled in CD4(-)CD8(-) (double negative (DN)) thymocytes and TCRalpha-chain genes are assembled in CD4(+)CD8(+) (double positive) thymocytes due, in part, to the developmental stage-specific activities of the TCRdelta and TCRalpha enhancers (Edelta and Ealpha), respectively. Edelta functions with TCRdelta promoters to mediate TCRdelta-chain gene assembly in DN thymocytes. However, Edelta is unable to function with TCRalpha promoters such as the TEA promoter to drive TCRalpha-chain gene assembly in these cells. This is important, because the premature assembly of TCRalpha-chain genes in DN thymocytes would disrupt alphabeta and gammadelta T cell development. The basis for TEA inactivity in DN thymocytes is unclear, because Edelta can activate the Vdelta5 gene segment promoter that lies only 4 kb upstream of TEA promoter. In this study, we use gene targeting to construct a modified TCRalpha/delta locus (TCRalpha/delta(5DeltaT)) in which the TEA promoter lies in the same location as the Vdelta5 gene segment on the wild-type TCRalpha/delta allele. Remarkably, the TEA promoter on this allele exhibits normal developmental stage-specific regulation, being active in double positive thymocytes but not in DN thymocytes as is the case with the Vdelta5 promoter. Thus, the inactivity of the TEA promoter in DN thymocytes is due primarily to intrinsic developmental stage-specific features of the promoter itself and not to its location relative to other cis-acting elements in the locus, such as Edelta.     

Human TCR that incorporate CD3zeta induce highly preferred pairing between TCRalpha and beta chains following gene transfer

TCR gene therapy is adversely affected by newly formed TCRalphabeta heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRalphabeta dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3zeta with specificities for three different Ags. Transfer of either TCRalpha:CD3zeta or beta:CD3zeta genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCRalphabeta:zeta does not compromise surface expression and functions of an endogenous TCRalphabeta. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRalphabeta:CD3zeta is the first strategy that results in highly preferred pairing between CD3zeta-modified TCRalpha and beta chains as well as absence of TCR mispairing between TCR:CD3zeta and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3zeta chains is independent of endogenous CD3gamma, delta, and epsilon. Taken together, our data support the use of TCRalphabeta:CD3zeta to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.     

Beyond the 12/23 rule of VDJ recombination independent of the Rag proteins

The combinatorial repertoire of AgRs is established through somatic recombination of V, D, and J gene segments during lymphocyte development. Incorporation of D segments into IgH, TCRbeta, and TCRdelta chains also contributes to junctional diversification by substantially extending the length of the third CDR. The V, D, and J gene segments are flanked by recombination signals (RS) of 12- or 23-mer spacer length that direct recombination according to the 12/23 rule. D genes in the TCRbeta and TCRdelta loci are flanked by a 12RS and 23RS, and their incorporation is controlled by mechanisms "beyond the 12/23 rule." In the TCRbeta locus, selective interactions between Rag proteins and the RS flanking the V-D and D-J genes, respectively, are sufficient to enforce D gene usage. In this article, we report that in the TCRdelta locus, the Rag proteins are not the major determinant of D gene incorporation. In developing mouse and human thymocytes, the two Ddelta genes rearrange predominantly to form D-D coding joints. In contrast, when tested in ex vivo transfection assays in a nonlymphoid cell line, the flanking RS mediate deletion, rather than incorporation, of the two D genes on both exogenous recombination substrates and the endogenous locus. These results suggest that selective Rag-RS interactions are not the sole regulators of D gene segment incorporation, and additional, perhaps lymphocyte-specific, mechanisms exist that allow proper shaping of the primary AgR repertoire.     

Limited pattern of TCR delta chain gene rearrangement on the RNA level in multiple sclerosis

Susceptibility to multiple sclerosis (MS) is most likely affected by a number of genes, including HLA and T-cell receptor (TCR) genes. T cells expressing gamma/delta receptors seem to contribute to autoagression in MS, as evidenced by their localization in the MS plaques in the brain. The aim of this study was to analyse the TCRdelta chain gene rearrangement at the RNA (cDNA) level and compare to the DNA pattern rearrangement. TCRdelta gene rearrangement was analysed in MS patients and healthy individuals with the use of primers specific for Vdelta1-6 and Jdelta1 genes (at the DNA level) and specific for Vdelta1-6 and Cdelta1 genes (at the cDNA level). The size of PCR products was analysed on agarose gel and by ALF-Express (Pharmacia). Additionally, the lymphocyte surface immunophenotype was studied with specific monoclonal antibodies. At the DNA level a restricted pattern of Vdelta3-Jdelta1 and Vdelta5-Jdelta1 was found only in MS patients. Contrary to DNA, mono-, oligoclonal RNA (cDNA) rearrangements were limited to Vdelta1-Cdelta1, Vdelta2-Cdelta1 and Vdelta3-Cdelta1 only in MS patients as well. Surface immunophenotype analysis revealed in MS a much higher frequency of activated gamma/delta T lymphocytes, i.e. expressing HLA-DR and CD25. An elevated level of CD56 positive cells in MS was recorded. Mono-oligoclonal pattern of TCRdelta gene rearrangement at the RNA level, along with increase in activated gamma/delta T cells, strongly argue for a significant role of gamma/delta T lymphocytes in the pathogenesis of MS.     

The role of multiubiquitination in dislocation and degradation of the alpha subunit of the T cell antigen receptor

Unassembled alpha subunits of the T cell receptor (TCRalpha) are degraded by proteasomes following their dislocation from the endoplasmic reticulum membrane. We previously demonstrated that a variant of TCRalpha lacking lysines (KalphaR) is degraded by this pathway with kinetics indistinguishable from those of the wild type protein (Yu, H., Kaung, G., Kobayashi, S., and Kopito, R. R. (1997) J. Biol. Chem. 272, 20800-20804), demonstrating that ubiquitination on lysines is not required for TCRalpha degradation by the proteasome. Here, we show that dislocation and degradation of TCRalpha and KalphaR are suppressed by dominant negative ubiquitin coexpression and by mutations in the ubiquitin activating enzyme, indicating that their degradation requires a functional ubiquitin pathway. A cytoplasmic TCRalpha variant that mimics a dislocated degradation intermediate was degraded 5 times more rapidly than full-length TCRalpha, suggesting that dislocation from the endoplasmic reticulum membrane is the rate-limiting step in TCRalpha degradation. We conclude that ubiquitination is required both for dislocation and for targeting TCRalpha chains to the proteasome.     

Exclusion and inclusion of TCR alpha proteins during T cell development in TCR-transgenic and normal mice

Allelic exclusion of immune receptor genes (and molecules) is incompletely understood. With regard to TCRalphabeta lineage T cells, exclusion at the tcr-b, but not tcr-a, locus seems to be strictly controlled at the locus rearrangement level. Consequently, while nearly all developing TCRalphabeta thymocytes express a single TCRbeta protein, many thymocytes rearrange and express two different TCRalpha chains and, thus, display two alphabetaTCRs on the cell surface. Of interest, the number of such dual TCR-expressing cells is appreciably lower among the mature T cells. To understand the details of TCR chain regulation at various stages of T cell development, we analyzed TCR expression in mice transgenic for two rearranged alphabetaTCR. We discovered that in such TCR double-transgenic (TCRdTg) mice peripheral T cells were functionally monospecific. Molecularly, this monospecificity was due to TCRalpha exclusion: one transgenic TCRalpha protein was selectively down-regulated from the thymocyte and T cell surface. In searching for the mechanism(s) governing this selective TCRalpha down-regulation, we present evidence for the role of protein tyrosine kinase signaling and coreceptor involvement. This mechanism may be operating in normal thymocytes.     

TCR beta chain that forms peptide-independent alloreactive TCR transfers reduced reactivity with irrelevant peptide/MHC complex

A major feature of the TCR repertoire is strong alloreactivity. Peptides presented by allogeneic MHC are irrelevant for recognition by a subset of alloreactive T cells. To characterize peptide-independent TCRs at the molecular level, we forced the expression of a TCRbeta chain isolated from a peptide-independent alloreactive CD8+ T cell line. The alloreactive TCR repertoire in the transgenic mouse was peptide dependent. However, analysis of essential TCR contacts formed during the recognition of self-MHC-restricted Ag showed that fewer contacts with peptide were established by the transgenic TCRbeta chain, and that this was compensated by additional contacts formed by endogenous TCRalpha chains. Thus, reduced interaction with the peptide appears to be a transferable feature of the peptide-independent TCRbeta chain. In addition, these findings demonstrate that reactivity to peptides is preferred over the reactivity to MHC during the formation of the TCR repertoire.     

The tight interallelic positional coincidence that distinguishes T-cell receptor Jalpha usage does not result from homologous chromosomal pairing during ValphaJalpha rearrangement

The T-cell receptor (TCR) alpha locus is thought to undergo multiple cycles of secondary rearrangements that maximize the generation of alphabeta T cells. Taking advantage of the nucleotide sequence of the human Valpha and Jalpha segments, we undertook a locus-wide analysis of TCRalpha gene rearrangements in human alphabeta T-cell clones. In most clones, ValphaJalpha rearrangements occurred on both homologous chromosomes and, remarkably, resulted in the use of two neighboring Jalpha segments. No such interallelic coincidence was found for the position of the two rearranged Valpha segments, and there was only a loose correlation between the 5' or 3' chromosomal position of the Valpha and Jalpha segments used in a given rearrangement. These observations question the occurrence of extensive rounds of secondary Valpha-->Jalpha rearrangements and of a coordinated and polarized usage of the Valpha and Jalpha libraries. Fluorescence in situ hybridization analysis of developing T cells in which TCRalpha rearrangements are taking place showed that the interallelic positional coincidence in Jalpha usage cannot be explained by the stable juxtaposition of homologous Jalpha clusters.     

A model for TCR gene segment use

The TCR alpha-chain is assembled by somatic recombination of variable (V) and joining (J) gene segments at the CD4+ CD8+ stage of development. In this study, we present the first analytical model for deletional rearrangement and show that it is consistent with almost all available data on Valpha Jalpha use in mice and humans. A key feature of the model is that both "local" and "express service" models of rearrangement can be obtained by varying a single parameter that describes the number of gene segments accessible at a time. We find that the window is much larger for Valpha segments than Jalpha segments, which reconciles seemingly conflicting data for the former. Implications for the properties of the repertoire as a whole and experiments that seek to probe them are discussed. Special considerations for allelic inclusion are treated in the Appendices.     

The TCRalpha locus control region specifies thymic, but not peripheral, patterns of TCRalpha gene expression

The molecular mechanisms ensuring the ordered expression of TCR genes are critical for proper T cell development. The mouse TCR alpha-chain gene locus contains a cis-acting locus control region (LCR) that has been shown to direct integration site-independent, lymphoid organ-specific expression of transgenes in vivo. However, the fine cell type specificity and developmental timing of TCRalpha LCR activity are both still unknown. To address these questions, we established a transgenic reporter model of TCRalpha LCR function that allows for analysis of LCR activity in individual cells by the use of flow cytometry. In this study we report the activation of TCRalpha LCR activity at the CD4-CD8-CD25-CD44- stage of thymocyte development that coincides with the onset of endogenous TCRalpha gene rearrangement and expression. Surprisingly, TCRalpha LCR activity appears to decrease in peripheral T cells where TCRalpha mRNA is normally up-regulated. Furthermore, LCR-linked transgene activity is evident in gammadelta T cells and B cells. These data show that the LCR has all the elements required to reliably reproduce a developmentally correct TCRalpha-like expression pattern during thymic development and unexpectedly indicate that separate gene regulatory mechanisms are acting on the TCRalpha gene in peripheral T cells to ensure its high level and fine cell type-specific expression.     

Human alpha beta and gamma delta thymocyte development: TCR gene rearrangements, intracellular TCR beta expression, and gamma delta developmental potential--differences between men and mice

To evaluate the role of the TCR in the alphabeta/gammadelta lineage choice during human thymocyte development, molecular analyses of the TCRbeta locus in gammadelta cells and the TCRgamma and delta loci in alphabeta cells were undertaken. TCRbeta variable gene segments remained largely in germline configuration in gammadelta cells, indicating that commitment to the gammadelta lineage occurred before complete TCRbeta rearrangements in most cases. The few TCRbeta rearrangements detected were primarily out-of-frame, suggesting that productive TCRbeta rearrangements diverted cells away from the gammadelta lineage. In contrast, in alphabeta cells, the TCRgamma locus was almost completely rearranged with a random productivity profile; the TCRdelta locus contained primarily nonproductive rearrangements. Productive gamma rearrangements were, however, depleted compared with preselected cells. Productive TCRgamma and delta rearrangements rarely occurred in the same cell, suggesting that alphabeta cells developed from cells unable to produce a functional gammadelta TCR. Intracellular TCRbeta expression correlated with the up-regulation of CD4 and concomitant down-regulation of CD34, and plateaued at the early double positive stage. Surprisingly, however, some early double positive thymocytes retained gammadelta potential in culture. We present a model for human thymopoiesis which includes gammadelta development as a default pathway, an instructional role for the TCR in the alphabeta/gammadelta lineage choice, and a prolonged developmental window for beta selection and gammadelta lineage commitment. Aspects that differ from the mouse are the status of TCR gene rearrangements at the nonexpressed loci, the timing of beta selection, and maintenance of gammadelta potential through the early double positive stage of development.