Regulation of T cell receptor (TCR) beta gene expression by CD3 complex signaling in immature thymocytes: implications for TCRbeta allelic exclusion

Abstract

During alphabeta thymocyte development, clonotype-independent CD3 complexes are expressed at the cell surface before the pre-T cell receptor (TCR). Signaling through clonotype-independent CD3 complexes is required for expression of rearranged TCRbeta genes. On expression of a TCRbeta polypeptide chain, the pre-TCR is assembled, and TCRbeta locus allelic exclusion is established. We investigated the putative contribution of clonotype-independent CD3 complex signaling to TCRbeta locus allelic exclusion in mice single-deficient or double-deficient for CD3zeta/eta and/or p56(lck). These mice display defects in the expression of endogenous TCRbeta genes in immature thymocytes, proportional to the severity of CD3 complex malfunction. Exclusion of endogenous TCRbeta VDJ (variable, diversity, joining) rearrangements by a functional TCRbeta transgene was severely compromised in the single-deficient and double-deficient mutant mice. In contrast to wild-type mice, most of the CD25(+) double-negative (DN) thymocytes of the mutant mice failed to express the TCRbeta transgene, suggesting defective expression of the TCRbeta transgene similar to endogenous TCRbeta genes. In the mutant mice, a proportion of CD25(+) DN thymocytes that failed to express the transgene expressed endogenous TCRbeta polypeptide chains. Many double-positive cells of the mutant mice coexpressed endogenous and transgenic TCRbeta chains or more than one endogenous TCRbeta chain. The data suggest that signaling through clonotype-independent CD3 complexes may contribute to allelic exclusion of the TCRbeta locus by inducing the expression of rearranged TCRbeta genes in CD25(+) DN thymocytes.

Figure 1

Flow cytometric analysis of total thymocytes of adult wt, ζ-sd, Lck-sd, and ζ/Lck-dd mice, with and without the P14βtg, for CD4 and CD8. Absolute numbers of total thymocytes are given on top of each panel; percentages of cells in each subpopulation are given in the insets within each panel. Genotypes are given at the top.

Figure 2

Semiquantitative PCR of sorted DN cells of adult wt, ζ-sd, Lck-sd, and ζ/Lck-dd mice (A) and of sorted DP cells of wt, ζ-sd, and Lck-sd mice (B) with and without the P14β transgene. DNA was adjusted to similar concentrations according to insulin PCR signals from 5-fold dilutions. After adjustment, DJ (Dβ₂-Jβ₂) and VDJ (Vβ5-D-Jβ₂, Vβ8-D-Jβ₂) PCR reactions were done in 5-fold dilutions as indicated. The Dβ₂-Jβ₂ and Vβ8-D-Jβ₂ PCRs detect only the endogenous Vβ8 rearrangements, not the P14β transgene. In the DJ rearrangements, the top band represents the germline configuration. The six bands underneath and in the VDJ rearrangements represent the Jβ₂1–6 elements from top to bottom.

Figure 3

Single parameter histiograms of intracellular TCRβ expression (TCRβIC) and intracellular Vβ8 expression (Vβ8IC), in gated DN CD25⁺CD44⁻ thymocytes of wt, ζ-sd, Lck-sd, and ζ/Lck-dd mice, with and without the P14β transgene (shaded profiles). The mice were 4–5 weeks of age. Genotypes are given at the top. Relative numbers (percent) of TCRβIC⁺ cells and Vβ8IC⁺ cells are indicated in each frame. Negative controls for intracellular stainings used blocking with an excess of the same, unlabeled mAb (open profiles, bold).

Figure 4

Three color flow cytometric analyses of thymocytes of adult wt, ζ-sd, and Lck-sd mice, with and without the P14β transgene, for CD4+CD8 and intracellular (IC) expression of TCRVβ elements (shaded profiles). In P14βtg⁺ mice, Vβ8 staining detects endogenous and/or transgenic TCRVβ elements. Vβ10/11 staining detects endogenous TCRVβ elements only. Negative controls for intracellular stainings used blocking with an excess of the same, unlabeled mAb (open profiles, bold). The mice were 4–5 weeks of age. Genotypes are given at the top. Percentages of positive cells are given in each frame. Percentages of Vβ8^low and Vβ8^high cells are indicated separately where appropriate. (A) Single parameter histiograms of Vβ8 and Vβ10/11 staining in DP cells. (B) Analysis of intracellular expression of Vβ10/11 polypeptide chains in total P14βtg⁺ ζ-sd or Lck-sd DP thymocytes and in P14βtg⁺ ζ-sd or Lck-sd DP thymocytes gated for low or high levels of Vβ8 polypeptide chains. To facilitate comparison, the Vβ10/11 profiles of total DP cells from A are repeated. (C) Analysis of intracellular expression of Vβ8 polypeptide chains in total P14βtg⁺ ζ-sd or Lck-sd DP thymocytes, and in P14βtg⁺ ζ-sd or Lck-sd DP thymocytes gated positive for Vβ10/11 polypeptide chains. To facilitate comparison, the Vβ8 profiles of total DP cells from A are repeated.

Figure 5

RT-PCR for expression of the P14βtg in isolated CD25⁺ DN and DP thymocytes of wt, ζ-sd, and Lck-sd mice. To sort DP cells, thymocytes were stained for CD4 and CD8 in different colors. To sort CD25⁺, DN cells thymocytes were stained for CD4+CD8+CD44 in one color against CD25 in a second color. Sorted populations were subjected to RT-PCR as described (19) by using hypoxanthine phosphoryltransferase as a quantitative marker.