A novel T cell subset with trans-rearranged Vγ-Cβ TCRs shows Vβ expression is dispensable for lineage choice and MHC restriction

Abstract

αβ T cells, which express the α-β TCR heterodimer, express CD4 or CD8 coreceptors on cells that are MHC class I or MHC class II dependent. In contrast, γδ T cells do not express CD4 or CD8 and develop independently of MHC interaction. The factors that determine αβ and γδ lineage choice are not fully understood, and the determinants of MHC restriction of TCR specificity have been controversial. In this study we have identified a naturally occurring population of T cells expressing Vγ-Cβ receptor chains on the cell surface, the products of genomic trans-rearrangement between the Vγ2 gene and a variety of Dβ or Jβ genes, in place of an intact TCRβ-chain and in association with TCRα. Identification of this population allowed an analysis of the role of TCR variable regions in determining T cell lineage choice and MHC restriction. We found that Vγ2(+)Cβ(+) cells are positive for either CD4 or CD8 and are selected in an MHC class II- or MHC class I-dependent manner, respectively, thus following the differentiation pathway of αβ and not γδ cells and demonstrating that Vβ V region sequences are not required for selection of an MHC-restricted repertoire.

Figure 1. Vγ2-(Dβ)-Jβ trans-rearrangements are expressed as hybrid TCR chains on peripheral T cells

(A) Real-time PCR analysis of trans-rearranged Vγ2-Cβ transcripts in WT and ATM^−/− splenocytes using a forward primer specific for the Vγ2 gene and a reverse primer specific for the Cβ1 and Cβ2 genes. Experimental results were normalized to a control PCR for β-Actin. Results are a summary of 3 independent experiments. (B) WT and ATM^−/− splenocytes were negatively gated for B220, GL3, and a pool of Vβ antibodies. The remaining cells were analyzed for Vγ2 and Cβ (H57) double expression. Results are representative of 5 independent experiments. (C) Quantitation of the percentage of Vγ2⁺Cβ⁺ cells in WT and ATM^−/− splenocytes. Data are a summary of 5 independent experiments. (D) Vγ2⁺Cβ⁺ and Vγ2⁻Cβ⁻ cells were flow sorted from the spleens of 10–15 WT and ATM^−/− mice and real-time PCR was performed to verify the presence of a Vγ2-Cβ transcript. Experimental results were normalized to β-Actin. Data are representative of 2 independent experiments for WT and 3 independent experiments for ATM^−/−. (E) Vγ2⁺Cβ⁺ and Cβ+ cells that were not negatively gated for Vβ were analyzed for Vβ expression by flow cytometry.

Figure 2. Vγ2⁺Cβ⁺ cells express Vα and are developmentally dependent on TCRα expression

(A) Flow cytometric analysis of pooled Vα3.2 and Vα8 expression on Vβ11⁺, GL3⁺ and Vγ2⁺Cβ⁺ ATM^−/− splenocytes. Leu-4 isotype control is shown in grey. Representative of 3 independent experiments. (B,C) Splenocytes from WT, TCRα^−/−, ATM^−/− and ATM^−/− TCRα^−/− mice were analyzed for the presence and frequency of (B) Vγ2⁺Cβ⁺ cells and (C) Vγ2⁺GL3⁺ cells. Data are a summary of 3 independent experiments.

Figure 3. Vγ2⁺Cβ⁺ cells express CD4 or CD8 and contain αβ-like subsets

(A) CD4 and CD8 expression were analyzed by flow cytometry on Cβ⁺ (H57), γδ (GL3⁺), and Vγ2⁺Cβ⁺ splenocytes from WT and ATM^−/− mice. Data are representative of 4 independent experiments with 4–10 mice pooled for each experiment. (B) αβ (H57⁺), γδ (GL3⁺) and Vγ2⁺Cβ⁺ cells were analyzed for ThPOK expression in ThPOK-YFP reporter mice. Representative of 3 independent experiments with 2–6 mice pooled per experiment. (C) Naïve and memory T cell subsets were analyzed on ATM^−/− Cβ⁺ (H57) and Vγ2⁺Cβ⁺ T cells by double staining with CD44 and CD62L. Data are representative of 3 independent experiments (D) FoxP3-GFP reporter mice were used to analyze the presence of T regulatory cells within the Cβ (H57)⁺, γδ (GL3⁺) and Vγ2⁺Cβ⁺ populations of splenocytes. Data are representative of 3 independent experiments.

Figure 4. Vγ2⁺Cβ⁺ cells are responsive to TCR stimulation in vitro

(A) CFSE-labeled splenocytes from ATM^−/− mice were cultured for 3–4 days with 500ng/ml soluble anti-CD3 and cell division was analyzed by CFSE dilution. Cells were gated on Vβ11⁺, Vβ10⁺, GL3⁺, and Vγ2⁺Cβ⁺ populations. Unstimulated cells are shown in grey. (B) Alloreactivity was analyzed by a mixed lymphocyte reaction (MLR). ATM^−/− CFSE-labeled splenocytes were incubated with irradiated BALB/c antigen presenting cells for 3–4 days at a ratio of 1:2. Cell division was analyzed by CFSE dilution. Cells were gated on Vβ11⁺, Vβ10⁺, GL3⁺, and Vγ2⁺Cβ⁺ populations. CFSE in cells cultured with syngeneic stimulators are shown in grey. Data are representative of 3 independent experiments.

Figure 5. Selection of Vγ2⁺Cβ⁺ cells is highly MHC I- and MHC II-dependent

CD4 and CD8 expression were analyzed by flow cytometry on Cβ⁺ (H57), γδ(GL3⁺), and Vγ2⁺Cβ⁺ splenocytes from WT, ATM^−/−, β2M^−/− and MHC II^−/− mice. Data are representative of 4 independent experiments with 4–10 mice pooled for each experiment.