Pablo Pereira

Most IL-4-Producing γδ Thymocytes of Adult Mice Originate from Fetal Precursors

Thy-1dull γδ T cells constitute a distinct adult γδ T cell subset characterized by the expression of a TCR composed of Vγ1Cγ4 and Vδ6Cδ chains with limited junctional sequence diversity. However, several features of the expressed Thy-1dull TCR-γδ genes, in particular the absence or minimal presence of N region diversity and the almost invariable Dδ2-Jδ1 junction, are typical of rearrangements often found in the fetal thymus. In this study, we have investigated the origin of these cells. Few Thy-1dull γδ thymocytes developed in syngeneic radiation adult chimeras, regardless of whether the recipient mice were given adult bone marrow or fetal liver cells as a source of hemopoietic precursors. In contrast, normal numbers of Thy-1dull γδ T cells developed in fetal thymi grafted into adult syngeneic recipients. Interestingly, the majority of Thy-1dull γδ thymocytes present in the grafts were of graft origin, even when most conventional γδ and αβ thymocytes in the grafted thymi originated from T cell precursors of recipient origin. Single-cell PCR analyses of the nonselected TCR-γ rearrangements present in adult Thy-1dull γδ thymocytes revealed that more than one-half of these cells represent the progenies of a limited number of clones that greatly expanded possibly during the first weeks of life. Finally, the second TCR-δ allele of a large number of Thy-1dull γδ Τ cells contained incomplete TCR-δ rearrangements, thus providing an explanation for the adult-type rearrangements previously found among nonfunctional V(D)J rearrangements present in Thy-1dull γδ thymocytes.

Rates of Recombination and Chain Pair Biases Greatly Influence the Primary γδ TCR Repertoire in the Thymus of Adult Mice

Analyses of the rearrangement status of the TCRγ and TCRδ chain loci in progenies of individual γδ thymocytes showed a hierarchy of the different Vγ and Vδ gene segments to participate in a recombination reaction. Moreover, individual TCRγ chains only pair efficiently with a variable number of TCRδ chains. Interestingly, these two parameters are inversely correlated such that the TCRγ and TCRδ 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 Vγ gene segments give raise to functional TCRγ chains, have coevolved to maximize the diversity of the γδ TCR repertoire minimizing the risk that a γδ T cell will express more than one TCR specificity at the cell surface, despite the fact that multiple TCRγ rearrangements take place in the same progenitor cell.

Early Expression of a Functional TCRβ Chain Inhibits TCRγ Gene Rearrangements without Altering the Frequency of TCRγδ Lineage Cells

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

Mechanisms controlling termination of V-J recombination at the TCRgamma locus: implications for allelic and isotypic exclusion of TCRgamma chains.

Analyses of Vγ-Jγ rearrangements producing the most commonly expressed TCRγ chains in over 200 γδ TCR+ thymocytes showed that assembly of TCRγ V-region genes display properties of allelic exclusion. Moreover, introduction of functionally rearranged TCRγ and δ transgenes results in a profound inhibition of endogenous TCRγ rearrangements in progenitor cells. The extent of TCRγ rearrangements in these cells is best explained by a model in which initiation of TCRγ rearrangements at both alleles is asymmetric, occurs at different frequencies depending on the V or J segments involved, and is terminated upon production of a functional γδ TCR. Approximately 10% of the cells studied contained two functional TCRγ chains involving different V and Jγ gene segments, thus defining a certain degree of isotypic inclusion. However, these cells are isotypically excluded at the level of cell surface expression possibly due to pairing restrictions between different TCRγ and δ chains.

Differential Requirement of RasGRP1 for γδ T Cell Development and Activation

γδ T (γδT) cells belong to a distinct T cell lineage that performs immune functions different from αβ T (αβT) cells. Previous studies established that Erk1/2 MAPKs are critical for positive selection of αβT cells. Additional evidence suggests that increased Erk1/2 activity promotes γδT cell generation. RasGRP1, a guanine nucleotide-releasing factor for Ras, plays an important role in positive selection of αβT cells by activating the Ras–Erk1/2 pathway. In this article, we demonstrate that RasGRP1 is critical for TCR-induced Erk1/2 activation in γδT cells, but it exerts different roles for γδT cell generation and activation. Deficiency of RasGRP1 does not obviously affect γδT cell numbers in the thymus, but it leads to increased γδT cells, particularly CD4−CD8+ γδT cells, in the peripheral lymphoid organs. The virtually unhindered γδT cell development in the RasGRP1−/− thymus proved to be cell intrinsic, whereas the increase in CD8+ γδT cells is caused by non–cell-intrinsic mechanisms. Our data provide genetic evidence that decreased Erk1/2 activation in the absence of RasGRP1 is compatible with γδT cell generation. Although RasGRP1 is dispensable for γδT cell generation, RasGRP1-deficient γδT cells are defective in proliferation following TCR stimulation. Additionally, RasGRP1-deficient γδT cells are impaired to produce IL-17 but not IFNγ. Together, these observations revealed that RasGRP1 plays differential roles for γδ and αβ T cell development but is critical for γδT cell proliferation and production of IL-17.

Genetic Mapping of Two Murine Loci that Influence the Development of IL-4-Producing Thy-1dull γδ Thymocytes

IL-4-producing γδ cells belong to a novel subset of γδ lymphocytes that expresses a very restricted repertoire of TCRs. To gain a deeper insight into the development and in vivo functions of these cells, we have analyzed the genetic control of their representation in the thymus. Using an intercross between C57BL/6 and DBA/2 mice we found two loci on chromosomes 13 and 17—named LadT1 and LadT2, respectively—with marked influence in their development. The LadT2 locus does not appear to be the MHC locus. The region identified on mouse chromosome 13 contains the structural genes for TCRγ as well as the IL-9 gene, which has been suggested as a candidate gene influencing the complex pathogenesis of asthma.