Evidence for recombinatorial hot spots at the T cell receptor Jα locus

The complex genomic organization of the murine T cell receptor (TcR) δ-α region has hindered detailed studies of α gene rearrangement and Jα gene usage in individual differentiating T cell precursors. We have isolated a novel set of Jα probes which, in combination with a few restriction enzyme digests, enable a reliable, simple and nearly complete analysis and location of any rearrangement at the Jα locus by conventional Southern blotting. The probes were used to analyze TcR α gene rearrangements in T cell hybridomas derived from an in vitro culture system that supports T cell differentiation of bone marrow cells. Our results indicate that Jα genes are unequally accessible for rearrangement and two hot spots for rearrangement could be demonstrated. In addition, only a restricted set of Jα genes was rearranged in each culture indicating that the slightly variable composition of factors can influence the recombinatorial accessibility of Jα genes. The hot spots for rearrangement were not only limited to T cells differentiating in vitro but could also be demonstrated among functional T cell clones based on the published sequence information from isolated TcR α gene rearrangements. The demonstration and the location of the hot spots for rearrangement in the T cell differentiation culture system opens up the possibility to study factors and mechanisms that regulate recombinatorial accessibility of TcR α genes.     

Early activation of TCR alpha gene rearrangement in fetal thymocytes.

We have previously demonstrated that the onset of TCR alpha gene rearrangement is mainly restricted to the J alpha50 gene in fetal day 1delta thymocyte hybridomas. Now, J alpha50 rearrangements from fetal thymocyte hybridomas and from day 15.5 fetal thymus have been isolated and sequenced. We demonstrate that J alpha50 is rearranged to the rearranged Vdelta1 Ddelta2 gene segment. This indicates that the TCR alpha rearrangement process is initiated in fetal thymocytes far earlier than previously thought. These thymocytes have their delta genes still accessible for rearrangement and therefore, are controlled by the TCR delta enhancer (Edelta) (and/or another TCR delta specific cis-acting element). Our results further suggest that both Edelta and the TCR alpha enhancer (Ealpha) are active at the onset of alpha rearrangements or alternatively, the initial activation of the J alpha locus is controlled by Edelta. In addition, Vdelta1 Ddelta2 J alpha50 gene segments are replaced by secondary alpha rearrangements, indicating that thymocytes with the early alpha rearrangement are committed to the alphabeta lineage.     

T cell receptor α gene rearrangement and transcription in adult thymic γδ cells

T cells belong to two separate lineages based on surface expression of αβ or γδ T cell receptors (TCR). Since during thymus development TCR β, γ, and δ genes rearrange before α genes, and γδ cells appear earlier than αβ cells, it has been assumed that αδ cells are devoid of TCR α rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic γδ cells undergo VJα rearrangements more frequently than immature αβ lineage thymic precursors. Sequence analysis shows VJα rearrangements in γδ cells to be mostly (70 %) nonproductive. Furthermore, VJα rearrangements in γδ cells are transcribed normally and, as shown by analysis of TCR β-/- mice, occur independently of productive VDJβ rearrangements. These data are interpreted in the context of a model in which precursors of αβ and γδ cells differ in their ability to express a functional pre-TCR complex.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

Lack of directed Vα14-Jα281 rearrangements in NK1+ T cells

NK1.1⁺ T cells are an unusual subset of TCRαβ cells distinguished by their highly restricted Vβ repertoire and predominant usage of an invariant Vα14-Jα281 chain. To assess whether a directed rearrangement mechanism could be responsible for this invariant α chain, we have analyzed Vα14 rearrangements by polymerase chain reaction and Southern blot in a panel of cloned T-T hybrids derived from thymic NK1.1⁺ T cells. As expected a high proportion (17/20) of the hybrids had rearranged Vα14 to Jα281. However, Vα14-Jα281 rearrangements always occurred on only one chromosome and were accompanied by other Vα-Ja rearrangements (not involving Vα14) on the homologous chromosome. These data argue that rigorous ligand selection rather than directed rearrangement is responsible for the high frequency of Vα14-Jα281 rearrangements in NK1.1⁺ T cells.     

A transgenic T cell receptor restores thymocyte differentiation in interleukin-7 receptor α chain-deficient mice

Interleukin-7 (IL-7) receptor α chain-deficient (IL-7Rα^-/-) mice have severely depleted lymphocyte populations and thymocyte development is arrested at the double-negative (DN) stage. We show that thymocyte development in these mice can be reconstituted by the introduction of a transgenic T cell receptor (TCR), implying that one function of the IL-7Rα chain is to initiate TCR gene rearrangement. Expression of the recombinase-activating genes RAG1 and RAG2 was greatly reduced in the IL-7Rα^-/- thymuses, and in DN thymocytes from the TCR transgenic IL-7Rα^-/- mice, but was restored in double-positive thymocytes from the TCR transgenic IL-7Rα^-/- mice. These data suggest that the IL-7Rα chain controls RAG expression and initiation of TCRβ chain VDJ rearrangement in DN cells. In contrast, once cells have progressed beyond the DN stage of development the IL-7Rα chain becomes no longer essential for RAG expression.     

Thymocyte differentiation in γ-irradiated severe-combined immunodeficient mice: characterization of intermediates and products of V(D)J recombination at the T cell receptor α locus

Treatment with DNA-damaging agents promotes rescue of V(D)J recombination, limited thymocyte differentiation, and development of thymic lymphomas in severe-combined immunodeficient (SCID) mice. One intriguing aspect of this system is that irradiation rescues rearrangements at the T cell receptor (TCR) β, γ and δ loci, but not at the TCR α locus. Current models posit that only those loci that are recombinationally active at the time of irradiation can be rescued. Here, we employ sensitive, semiquantitative ligation-mediated polymerase chain reaction assays to detect a specific class of recombination intermediates, hairpin coding ends, at the TCR α locus. We found that Jα-coding ends are undetectable in unirradiated SCID thymocytes, but accumulate after irradiation at times coincident with the emergence of a CD4⁺CD8⁺ thymocyte population. Coding joints produced by joining of these ends, however, are extremely rare. To test whether the presence of hairpin coding ends at TCR α is sufficient for irradiation-mediated rescue of coding joint formation, we administered a second dose of γ-irradiation after abundant CD4⁺ CD8⁺ thymocytes and hairpin TCR α coding ends had accumulated. This treatment failed to stimulate rescue of TCR α coding joints. Thus, the presence of hairpin coding ends at the time of irradiation, while perhaps necessary, is not sufficient for rescue of V(D)J rearrangements. These results support a refined model for irradiation-mediated rescue of TCR rearrangements in SCID mice.     

T cell receptor diversity in alloreactive responses against HLA-B27 (B*2705) is limited by multiple-level restrictions in both α and β chains

The T cell receptors (TCR) in HLA-B27 (B*2705) alloreactivity were analyzed in cytotoxic T lymphocytes (CTL) from two individuals. Non-random usage was found in Vβ, N+Dβ, Vα, and Jα, but not in Jβ segments or Nα-regions. Vβ segments from homology subgroup 4 were predominant and not associated to a particular donor or fine specificity, suggesting involvement in recognizing the HLA-B27 molecule. In contrast, preferential Vα usage was associated with particular individuals and fine specificities, indicating distinct Vβ and Vα recruitment and contribution to allorecognition. Recurrent N+Dβ motifs and Jα segments, even from different donors, limited junctional diversity, suggesting that CDR3 usage was determined by the alloantigenic epitope independently of individuals. TCR were selected differently at various levels, as indicated by the following findings. Four clonotypes with similar fine specificity had identical β and unrelated α chains. Similar α were associated with unrelated β chains, and vice versa. CTL using Vβ subgroup 4 did not globally show concomitant predominance of other TCR elements. Vα7, one of the preferred Vα segments, was always associated with Vβ subgroups other than 4. Sometimes, a TCR showed homology in elements of one chain to a second TCR or group of TCR, and to another in the other chain. These results are best explained by differential selection of TCR elements by different epitopes, providing a key to the inner structure of allospecific TCR repertoires.     

T cell regulation of collagen-induced arthritis in mice. III. Is T cell vaccination a valuable therapy?

Since T cells play a critical role in collagen-induced arthritis (CIA), CD4⁺ T cell hybridomas were derived from DBA/1 mice immunized with bovine type II collagen (CII). The hybrid clones selected were Thy-1-2⁺, CD4⁺, CD8^?, T cell receptor (TcR) αβ⁺ and produced interleukin-2 in response to CII peptides presented by I-A^q molecules. The clones were collagen type-specific and recognized CII from many species except the mouse. More precisely, the reactivity was directed against the immunodominant cyanogen bromide-cleaved fragment CB11(II). Analysis of the TcR carried by the T cell hybridomas showed that they used identical Vα and Jα (VαBMB, Jα20) gene segments and two distinct Vβ (Vβ1 and Vβ4) associated with the Jβ2.5 gene segment. Interestingly, the junctional regions were highly conserved in structure and length. These findings may indicate a strong in vivo selection by the antigen for a particular combination of both α and β chains of the TcR. Inoculation of irradiated anti-CII T cell hybrids into DBA/1 mice, before priming with CII, altered the course of the disease resulting in either a long-lasting suppression or an exacerbation of CIA whereas a control CD4⁺ hybridoma with an unrelated specificity did not influence the development of arthritis. However, the regulatory effect of anti-CII T cell clones was unpredictable, suggesting that the TcR structure may not solely account for the modulation of CIA and that T cell vaccination is not a reliable method for inducing suppression of CIA.     

Selection of dual Vα T cells

Incomplete allelic exclusion of TCRa gene rearrangement permits the generation of dual Vα T cells, though the issues of their frequency and whether both α β pairs participate in thymic selection have not been resolved. Both questions have been investigated using lymphocytes from mice hemizygous at the TCRa locus and consequently unable to express two rear ranged TCRa genes, as background controls. The data presented show that both the frequency of dual Vα T cells and the relative expression levels of co-expressed Vα chains are variable and are determined by thymic selection. Possession of a Vα chain which is inefficiently positively selected appears to increase the likelihood that a second Vα chain will be co-expressed, whilst the relative cell surface levels of a given pair of Vα chains differbetween CD4 and CD8 subsets. Further, for some but not all Vα pairs, dual Vα T cells appear to express elevated levels of surface TCR. Finally, contrary to previous claims, dual Vα T cells do not appear to be relatively frequent amongst immature thymocytes.     

The T cell receptor αβ V-J shuffling shows lack of autonomy between the combining site and the constant domain of the receptor chains

In order to assess the structural independence of the T cell receptor (TCR) combining site from the rest of the molecule we have generated two recombinant chains consisting of aTCR V-J α region linked to the Cβ and aTCR V-J β linked to the Cα. If the V and C domains of the TCR form independent domains, as has been shown for the Ig molecules, we would expect to obtain a functional chimeric TCR. Interestingly, it was found that the shuffled molecules are produced intracellularly in T cell hybridomas, but are not expressed on the cell surface. To explain this failure of the shuffled molecules we propose that the TCR has a more compact structure, compared to the Ig, and that it is indispensable to keep a longitudinal inter-domain contact between the V-J and C portion to have a functional molecule.     

A TCR α chain transgene induces maturation of CD4− CD8− α β+ T cells from γ δ T cell precursors

The proportion of CD4⁻ CD8⁻ double-negative (DN) α β T cells is increased both in the thymus and in peripheral lymphoid organs of TCR α chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to α β and γ δ T cells. We show that the transgenic DN cells are phenotypically similar to γ δ T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCRα genes nor been negatively selected by the Mls^a antigen, suggesting that they originate from a differentiation stage before the onset of TCR α chain rearrangements and CD4/CD8 gene expression. Neither in-frame VδDδJδ nor VγJγ rearrangements are over-represented in this population. However, since peripheral γ δ T cells with functional TCRβ gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to γ δ lineage-committed precursors can be delivered via TCR α β heterodimers.