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. 2003 Mar 3;197(5):601-14.
doi: 10.1084/jem.20021945.

T cell receptor gene rearrangement lineage analysis reveals clues for the origin of highly restricted antigen-specific repertoires

Abdelbasset Hamrouni  1 , Anne Aublin, Philippe Guillaume, Janet L Maryanski
Affiliations

T cell receptor gene rearrangement lineage analysis reveals clues for the origin of highly restricted antigen-specific repertoires

Abdelbasset Hamrouni et al. J Exp Med. .

Abstract

Due to ordered, stage-specific T cell receptor (TCR)-beta and -alpha locus gene rearrangements and cell division during T cell development, a given, ancestral TCR-beta locus VDJ rearrangement might be selected into the mature T cell repertoire as a small cohort of "half-sibling" progeny expressing identical TCR-beta chains paired with different TCR-alpha chains. The low frequency of such a cohort relative to the total alphabeta TCR repertoire precludes their direct identification and characterization in normal mice. We considered it possible that positive selection constraints might limit the diversity of TCR-alpha chains selected to pair with beta chains encoded by an ancestral VDJ-beta rearrangement. If so, half-sibling T cells expressing structurally similar, but different TCR-alpha chains might recognize the same foreign antigen. By single cell polymerase chain reaction analysis of antigen-specific TCRs selected during a model anti-tumor response, we were able to identify clusters of T cells sharing identical VDJ-beta rearrangements but expressing different TCR-alpha chains. The amplification of residual DJ-beta rearrangements as clonal markers allowed us to track T cells expressing different TCR-alpha chains back to a common ancestral VDJ-beta rearrangement. Thus, the diversity of TCR-alpha's selected as partners for a given VDJ-beta rearrangement into the mature TCR repertoire may indeed be very limited.

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Figures

Figure 1.
Figure 1.
Sequences of oligonucleotide primers. Forward (A) and reverse (B) primers are named for gene segments with a laboratory code in parentheses. Primers used for the first or second rounds of RT-PCR or DNA-PCR or for sequencing (Seq) are indicated. Optional (opt) primers were used to obtain shorter PCR products for sequencing or for confirmation.
Figure 2.
Figure 2.
Single cell RT-PCR analysis reveals clusters of CW3-specific clones expressing β chains encoded by identical VDJ-β nucleotide sequences paired with different TCR-α chains. The cDNA from tubes containing single CW3-specific CD8 T cells sorted from PBL of M-2 and M-3 or M-33 was subjected to RT-PCR, and PCR products were sequenced and assigned a TCR sequence code (NS). The complete analysis is shown in Figs. S1, S2, and S3, available at http://www.jem.org/cgi/content/full/jem.20021945/DC1. Represented here are those cells for which both a CW3-like Vβ10 sequence and a CW3-like Vα3, Vα4, or Vα8 sequence were amplified. The deduced aa sequences of the TCR junctions are shown. All TCR-α sequences incorporate the Jα35 sequence. Also shown are the number (#) of cells found for each αβ TCR clone and its corresponding percentage (%) within the αβ TCR repertoire defined here for each mouse. Each cluster of αβ TCR clones sharing an identical Vβ10 TCR nucleotide sequence is framed. Cells for which an in-frame (IF) or out of frame (OF) second TCR-α rearrangement was also amplified are indicated.
Figure 3.
Figure 3.
Repertoire analysis by single cell DNA-PCR for coamplification of CW3-specific TCR VDJ-β and VJ-α sequences together with DJ-β rearrangements as potential clonal markers. For each of three mice (M-41, M-42, and M-43), single pCW3Kd+Vβ10+CD8+ splenocytes were sorted 2 wk after immunization with P815-CW3 tumor cells. The rearranged TCR-α and TCR-β nucleotide sequences were amplified by single cell DNA-PCR. PCR products from cells with successful amplifications for TCR-α and TCR-β rearrangements were sequenced to identify paired αβ TCRs. Other details are as described for Fig. 2.
Figure 4.
Figure 4.
Two clones expressing CW3-specific αβ TCRs encoded by an identical VDJ-β but different VαJα sequences share an identical Dβ1-Jβ1.4 rearrangement. Individual cells (identified by #) from M-42 or M-43 were amplified by DNA-PCR as described in Fig. 3 to identify their CW3-specific αβ TCRs and their potential DJ-β rearrangements. Shown for each cell are the codes for the TCR-α and TCR-β sequences, the amplified DJ rearrangements together with a code to indicate D and J gene segment usage, and the unrearranged D2-J2 GL sequences. Where indicated, there was no detectable PCR amplification (na) using the indicated primer combinations. No Dβ1 to Jβ2 rearrangements were found for any of these cells. Sequences in italics shown above each series represent the 3′ or 5′ ends of the relevant genomic D or J sequences, respectively. DJ nucleotide sequences are separated to indicate the D, N-nucleotide, and J portions.
Figure 5.
Figure 5.
Analysis of DJ-β rearrangements for clones that appear to share CW3-specific TCR-α chains encoded by identical nucleotide sequences. Note that the V gene portions were only partially sequenced, so identity of the TCR-α rearrangements with the same nucleotide sequence code might be only partial. The analysis and presentation of the data is the same as that described for Fig. 4. No Dβ1 to Jβ2 rearrangements were found for any of these cells.
Figure 6.
Figure 6.
A model linking two different CW3-specific αβ TCR clones to the same, ancestral VDJ-β precursor. At the top of the figure, T cell development in the thymus is schematized for the CD4−CD8− (DN) to CD4+CD8+ (DP) stages, showing the progressive changes in surface expression of CD44 and CD25 that define the DN1 to DN4 stages. The stages at which different TCR gene rearrangements occur are indicated, with potential stages for continued D to J recombination shown in parentheses. The lower part shows our model for the rearrangements at the different TCR loci on both chromosomes (arbitrarily designated a or b) at different stages of development for the two clones from M-42 that share the Vβ10Dβ1Jβ1.2-2i and Dβ1Jβ1.4 rearrangements shown in Fig 4. The cell making the ancestral VDJ-β rearrangement that is shared by all progeny is indicated with *. Potential (?) additional progeny expressing other TCR-α and/or Dβ2Jβ2 rearrangements are also shown.
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