PreTCR and TCRγδ signal initiation in thymocyte progenitors does not require domains implicated in receptor oligomerization

Abstract

Whether thymocytes adopt an αβ or a γδ T cell fate in the thymus is determined at the β selection checkpoint by the relatively weak or strong signals that are delivered by either the pre-T cell receptor (preTCR) or the γδ TCR, respectively. Signal initiation at the β selection checkpoint is thought to be independent of ligand engagement of these receptors. Some reports have suggested that receptor oligomerization, which is thought to be mediated by either the immunoglobulin (Ig)-like domain of the preTCRα (pTα) chain or the variable domain of TCRδ, is a unifying mechanism that initiates signaling in early CD4(-)CD8(-) double-negative (DN) thymocyte progenitors. Here, we demonstrate that the extracellular regions of pTα and TCRδ that are implicated in mediating receptor oligomerization were not required for signal initiation from the preTCR or TCRγδ. Indeed, a truncated TCRγδ that lacked all of its extracellular Ig-like domains still formed a signaling-competent TCR that drove cells through the β selection checkpoint. These observations suggest that signal initiation in DN thymocytes is simply a consequence of the surface-pairing of TCR chains, with signal strength being a function of the abundances of surface TCRs. Thus, processes that regulate the surface abundances of TCR complexes in DN cells, such as oligomerization-induced endocytosis, would be predicted to have a major influence in determining whether cells adopt an αβ versus γδ T cell fate.

Fig. 1

Schematic representations of early thymocyte development and the pTα chains used in this study. (A) Schematic of αβ versus γδ T cell lineage fate during early thymocyte development. Cells that do not express TCR complexes are blocked at the β-selection checkpoint. Weak signaling from the preTCR or TCRγδ drives commitment to the αβ T cell lineage, whereas strong signaling from the TCRγδ drives the development of γδ cells. DN, CD4⁻CD8⁻ cells; DP, CD4⁺CD8⁺ cells. (B) Schematic showing the four exons of the gene that encodes pTα and the proteins that are generated from the two known alternative splice products: pTα^a and pTα^b. The amino acid residues Asp²² (D22), Arg²⁴ (R24), Arg¹⁰² (R102), and Arg¹¹⁷ (R117) have been implicated in preTCR oligomerization.

Fig. 2

pTα chains that lack regions implicated in preTCR oligomerization are able to initiate signaling that complements pTα deficiency. Bar charts showing (A) the absolute number of GFP⁺ cells after 10 days in FTOC (n = 8 experiments), (B) the percentage of γδ cells after 12 days in FTOC (n = 7 experiments), and (C) the ratio of DP cells to γδ cells after 12 days in FTOC (n = 7 experiments) for embryonic (E14) pTα^−/− thymocytes transduced with a GFP-expressing control vector or with GFP-expressing retroviral vectors encoding pTα^a, pTα^b, pTα^aDRRA (a mutant pTα^a containing alanines substituted at positions 22, 24, and 102), or pTα^bR117A (a mutant pTα^b with an alanine at position 117). Black bars represent GFP-negative (that is, untransduced) cells, whereas white bars represent GFP⁺ cells. (D) Graph showing the percentage of GFP⁺ pTα^−/− CD4⁻ CD8⁻TCRδ⁻ DN cells transduced with vector only, vector expressing pTα^a, or vector expressing pTα^b after 8 days in FTOC, with >2n DNA content as judged by FAC-staining for cellular DNA with the intercalating fluorescent dye 7-Aminoactinomycin D (7-AAD), (n = 5 experiments). ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns = not significant.

Fig. 3

Truncated TCRγδ receptors that lack Vδ or regions implicated in ligand binding initiate signaling in DN cells. (A) Schematic showing the TCRγ and TCRδ chains and receptors used in this study. CDRs, Ig-like domains (Vγ, Vδ, Cγ, and Cδ), and disulfide bonds are indicated. (B) Bar chart showing the percentage representation of γδ cells (white bars) or DP cells (black bars) and (C to E) representative flow cytometry plots from 8-day FTOC of E14 RAG-2^−/− thymocytes transduced with GFP-expressing retroviral vector alone (vector control) or with GFP-expressing retroviral vectors encoding TCRγ, TCRδ, TCRΔIγ, TCRΔIδ, TCRΔIIγ, TCRΔIIδ, or the indicated combinations thereof. Percentages of gated cells are indicated. Data in (B) are from at least n = 3 experiments, while figures (C to E) are representative of data summarized in (B).

Fig. 3

Truncated TCRγδ receptors that lack Vδ or regions implicated in ligand binding initiate signaling in DN cells. (A) Schematic showing the TCRγ and TCRδ chains and receptors used in this study. CDRs, Ig-like domains (Vγ, Vδ, Cγ, and Cδ), and disulfide bonds are indicated. (B) Bar chart showing the percentage representation of γδ cells (white bars) or DP cells (black bars) and (C to E) representative flow cytometry plots from 8-day FTOC of E14 RAG-2^−/− thymocytes transduced with GFP-expressing retroviral vector alone (vector control) or with GFP-expressing retroviral vectors encoding TCRγ, TCRδ, TCRΔIγ, TCRΔIδ, TCRΔIIγ, TCRΔIIδ, or the indicated combinations thereof. Percentages of gated cells are indicated. Data in (B) are from at least n = 3 experiments, while figures (C to E) are representative of data summarized in (B).

Fig. 4

A truncated TCRγ chain lacking both extracellular Ig-like domains can pair with pTα to initiate signaling. Graphical representation of the percentages of CD4⁺CD8⁺ DP cells generated when (A) E14 RAG-2^−/− (n = 3 experiments) or (B) [pTα^−/−.TCRδ^−/−] thymocytes (n = 3 experiments) were transduced with retroviral vector expressing GFP alone (vector control) or with retroviral vectors expressing GFP and TCRΔIIγ, TCRΔIIδ, or both, as indicated, and cultured on OP9-DL1 stromal cells for 7 days. Mock cells received no virus. Black bars represent GFP⁻ cells from the culture, whereas white bars represent GFP⁺ cells. **, P ≤ 0.002; ***, P ≤ 0.0005; ns = not significant.