TCRA Gene Rearrangement in Immature Thymocytes in Absence of CD3, Pre-TCR, and TCR Signaling

Abstract

During thymocyte differentiation, TCRA genes are massively rearranged only after productively rearranged TCRB genes are expressed in association with pTα and CD3 complex molecules within a pre-TCR. Signaling from the pre-TCR via the CD3 complex is thought to be required to promote TCRA gene accessibility and recombination. However, αβ+ thymocytes do develop in pTα-deficient mice, showing that TCRα-chain genes are rearranged, either in CD4−CD8− or CD4+CD8+ thymocytes, in the absence of pre-TCR expression. In this study, we analyzed the TCRA gene recombination status of early immature thymocytes in mutant mice with arrested thymocyte development, deficient for either CD3 or pTα and γc expression. ADV genes belonging to different families were found rearranged to multiple AJ segments in both cases. Thus, TCRA gene rearrangement is independent of CD3 and γc signaling. However, CD3 expression was found to play a role in transcription of rearranged TCRα-chain genes in CD4−CD8− thymocytes. Taken together, these results provide new insights into the molecular control of early T cell differentiation.

p53-dependent and p53-independent pathways for radiation-induced immature thymocyte differentiation

“…With four reactions with primers specific for ADV2 on the one hand and for four different AJ genes (AJ56, AJ48, AJ27, AJ23) on the other, we can amplify up to 16 ADV2/AJ combinations in C57BL/6 thymocytes. As expected, none are detected in V(D)J recombination-deficient RAG-2 À/À mice ( Figure 1c, see Mancini et al, 2001 for more details). The amplification of genomic DNA prepared from unirradiated and irradiated CD3e D5/D5 total thymocytes clearly shows that more bands are present after irradiation, and they are more intense than in control animals.…”

“…ADV2/ AJ rearrangements were amplified in four PCR reactions with primers hybridising downstream of AJ56, AJ48, AJ27 or AJ23 and an ADV2 gene family-specific primer, and PCR products were revealed by Southern blotting as described (Mancini et al, 2001). DNA input was controlled by the amplification of a nonrearranging segment of the TCRa chain constant region (AC).…”

“…RT-PCR analysis of rearranged TCRA gene transcription was performed as previously described (Mancini et al, 2001). Thymocyte cDNA prepared from control and irradiated mice were amplified with primers specific for rearranged ADV2 and ADV8 gene families.…”

“…Indeed, they are TCR-negative, because of the absence of CD3 complex expression, and they are induced into apoptosis following a second exposure to radiation, whereas mature SP thymocytes found in a wild-type (wt) thymus are TCR þ and are more resistant than DP thymocytes to IRinduced apoptosis (see below, Figures 4 and 5). TCRa chain gene transcription, which is massively induced during the DN/DP transition in wt mice, have never been found in CD3e D5/D5 mice ( Figure 1b) (Malissen et al, 1995;Gallagher et al, 1998;Mancini et al, 2001), despite the fact that the TCRA genes, coding for this chain, are rearranged at a low level in these mice (Mancini et al, 2001). To determine whether IR also rescues rearranged TCRA gene transcription, cDNA prepared from unirradiated and irradiated CD3e D5/D5 thymocytes was amplified with oligonucleotide primers specific for the ADV2 and ADV8 gene families and the AC region.…”

“…Irradiation first induces a five-to six-fold reduction of thymic cellularity, followed by a slow recovery during the subsequent weeks (Table 1) (Mancini et al, 2001). The right panel shows the amplification of a nonrearranging segment from the TCRA constant region gene (AC) from each sample, as a control for DNA input.…”