V delta repertoire during thymic ontogeny suggests three novel waves of gamma delta TCR expression.

Taking advantage of a PCR technique that allows amplification of all variable region genes with equal efficiency, we defined three novel waves of TCR delta-chain transcription during thymic ontogeny. The canonical DV101-D2-J2 rearrangement was confined to a narrow window from days 14 to 18 of gestation, indicating that the postulated two consecutive gamma delta precursor waves bearing this canonical DV101 rearrangement will coincide on day 16. Neonatal delta-chain transcripts used a second wave of diverse V alpha gene segments that are exclusively located in the delta locus-proximal gene cluster of intermingled single members of different V alpha subfamilies. In the adult, only expression of a clan of three homologous subfamilies, ADV7, DV104, and ADV17, persists. The members of the ADV7 subfamily are also scattered across the alpha locus, but their usage does not show the position-dependent bias of the other V alpha-to-delta rearrangements.

TCR-delta gene rearrangement and selection during fetal thymocyte development.

TCR delta-chain gene rearrangements were analyzed at different stages of thymic ontogeny. The VDJ delta junctional sequences of the dominantly expressed TCR delta-chain V7 subfamily are highly conserved in fetal and neonatal thymocytes. They are equally conserved in C delta-targeted mice lacking cell surface expression of gamma delta receptors, indicating evolutionary selection acting at the level of DNA rearrangement. Yet, in C delta-mutant mice, the frequency of in-frame transcripts is reduced to 61%, from 88% in wild-type mice, suggesting cellular selection of this DV7-overexpressing gamma delta thymocyte subset. In contrast, in genomic DNA rearrangements, no difference was found in the frequency of productive rearrangements between mutant (26%) and wild-type mice (31%). This in-frame rate, characteristic of random rearrangements, indicates that positive selection is not required for thymic maturation of gamma delta T cells. The results are discussed with regard to models of alpha beta/gamma delta lineage commitment.

Inverse PCR amplification of low-abundancy message of gamma delta T cell receptor genes.

The technique of inverse PCR permits the rapid amplification and identification of unknown DNA segments adjacent to well characterized core regions. In the field of immunology anchored PCR and inverse PCR are useful methods for examining junctional diversity and unknown variable gene segments of rearranged T cell receptor genes. We have applied an improved inverse PCR protocol to study the repertoire of gamma delta T cell receptor genes in the developing thymus of the mouse.

Antigen-induced death of alloreactive human T-lymphocytes occurs in the absence of low molecular weight DNA fragmentation.

Stimulation via the CD3/TCR molecular complex induces proliferation of resting T cells, but triggers programmed cell death (apoptosis) in immature thymocytes and preactivated mature T cells. Activation-induced cell death (AICD) triggered by anti-CD3/TCR mAb or by staphylococcus enterotoxin superantigen is associated with fragmentation of genomic DNA into oligonucleosomal fragments of 200 bp length, thus displaying the characteristic features of apoptosis. Here, we show that a fraction (20-50%) of cells in alloreactive CD8 human short-term T cell lines, generated by repeated restimulation with EBV-transformed B cell lines, undergo AICD when restimulated with the appropriate (but not with third party) stimulator cells. AICD of responder T cells is inhibited when stimulator cells are preincubated with anti-HLA class I mAb but not with anti-HLA class II mAb, indicating that T cell death is dependent on alloantigen (HLA class I) recognition by responding CD8 T cells. Importantly, alloantigen-induced T cell death occurs in the absence of detectable DNA fragmentation. Thus, several independent assay systems all failed to reveal low molecular weight DNA fragmentation, even though DNA fragmentation was readily detected in T cell lines exposed to PHA or gamma-irradiation. Alloantigen-induced T cell death was prevented by aurintricarboxylic acid, which has previously been shown to inhibit apoptosis in experimental systems where no DNA fragmentation occurs. Taken together, these results demonstrate that alloantigen can trigger AICD in mature responding T cells in the absence of low molecular weight DNA fragmentation.