Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals

Abstract

Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.

Keywords: Notch; T-cell development; β-selection; γδ T-lineage.

Fig. 1.

γδ-TCR–expressing DN3 cells predominantly differentiate along the γδ-lineage, irrespective of TCR-β coexpression and the availability of Notch signals. (A and B) Developmental progression of in vitro-derived Rag2^−/− DN3 cells retrovirally transduced to express TCR-β, γδ-TCR, neither, or both, and cultured for 6 d with OP9-DL or OP9-Ctrl cells. Flow cytometric analysis of cell surface expression for CD4 and CD8 (A), and CD24 along with forward size scatter (FSC) (B) are shown for GFP⁺ YFP⁺-gated cells; C shows the corresponding fold-expansion in cellularity. Fold-expansion was obtained from the total cellularity divided by the number of cells used at the start of the culture (input). (D) Bar graph showing percentage of DP from the indicated cocultures in relation to TCR-β/YFP–transduced cells set at 100. Data are derived from at least three independent experiments. GFP(G), MigR1-transduced; YFP(Y), MIY-transduced.

Fig. 2.

Loss of Id3 inhibits the maturation of γδ-TCR–expressing T cells as IFN-γ–producers. Sorted CD45⁺ GFP⁺ cells were obtained from in vitro-derived Rag2^−/− DN3 or Rag2^−/−Id3^−/− DN3 cells retrovirally transduced to express TCR-β, γδ-TCR, neither, or both, and cultured for 6 d with OP9-DL or OP9-Ctrl cells (as indicated) and were stimulated for 36 h (P+I, PMA+Iono) or placed in culture without stimulation (Unstim). Levels of IFN-γ in culture supernatants were quantified by an antibody-capture ELISA. Data are derived from at least three independent experiments. *P ≤ 0.05; **P ≤ 0.01.

Fig. 3.

Provision of T22^b expression and TCRβ enhances the development of γδ-TCR–expressing DN3 cells toward the γδ-lineage. (A) Development of in vitro-derived Rag2^−/− DN3 cells retrovirally transduced to express TCR-β, γδ-TCR, or both, and cultured for 5.5 d with B/c-DL cells expressing T22^b or MICherry alone. Flow cytometric analysis of cell surface expression for CD4 and CD8 (A) and CD73 and FSC (B). Data are derived from at least two independent experiments.

Fig. 4.

Provision of a weak γδ-TCR ligand with loss of Id3 does not promote αβ-lineage choice in γδ-TCR–expressing DN3 cells, both in the presence and absence of Notch signals. (A and B) Development of culture-derived Rag2^−/−Id3^−/− DN3 cells retrovirally transduced to express TCR-β, γδ-TCR, neither, or both, and cultured for 6 d with OP9-DL4 and B/c-DL4. Flow cytometric analysis of cell-surface expression for CD4 and CD8 (A), and CD24 and CD73 (B) are shown for GFP⁺ YFP⁺-gated cells; C shows the corresponding fold expansion in cellularity. (D) Quantitative RT-PCR analysis of γδ-biased genes: Crem, Nurr1, and Rgs1, and Trac, in transduced Rag2^−/−Id3^−/− DN3 cells cultured for 6 d on the indicated cells, with mRNA levels normalized to β-actin. β, γδ, and β/γδ represent TCR-β/MIY–, MigR1/TCR-γδ–, and TCR-β/TCR-γδ–transduced DN3 cells, respectively. Data are derived from at least three independent experiments. **P ≤ 0.01.