Defective development of gamma/delta T cells in interleukin 7 receptor-deficient mice is due to impaired expression of T cell receptor gamma genes

Abstract

Mice lacking the interleukin 7 receptor (IL-7R) generate alpha/beta T cells at a detectable but greatly reduced rate, but gamma/delta T cells are completely absent. The special role of IL-7R signaling in gamma/delta T cell development has remained unclear. IL-7Ralpha(-/-) mice exhibit a paucity of gamma gene rearrangements. This striking observation can be explained by a defect in T cell receptor (TCR)-gamma gene rearrangement, a defect in TCR-gamma gene transcription leading to death of gamma/delta lineage cells, and/or a requirement for IL-7R in commitment of cells to the gamma/delta lineage. To determine the role of IL-7R signaling in gamma/delta T cell development, we examined transcription of a prerearranged TCR-gamma transgene in IL-7Ralpha(-/-) mice, as well as the effects of IL-7 on transcription of endogenous, rearranged TCR-gamma genes in alpha/beta lineage cells. The results demonstrate that IL-7R-mediated signals are necessary for the normal expression of rearranged TCR-gamma genes. Equally significant, the results show that the poor expression of TCR-gamma genes in IL-7Ralpha(-/-) mice is responsible for the selective deficit in gamma/delta cells in these mice, since a high copy TCR-gamma transgene exhibited sufficient residual expression in IL-7Ralpha(-/-) mice to drive gamma/delta cell development. The results indicate that the absence of gamma/delta T cells in IL-7Ralpha(-/-) mice is due to insufficient TCR-gamma gene expression.

Figure 1

Lack of significant TCR-γ gene rearrangement in thymocytes of IL-7Rα^−/− mice. Southern blot analysis of EcoR1-digested genomic DNA probed with a Cγ-specific probe reveals a virtually undetectable level of TCR-γ gene rearrangement in thymocytes of IL-7Rα^−/− mice. Three germline bands at 13.4, 10.5, and 7.5 kb detected in the RAG-1^−/− lane represent unrearranged J-C gene segments of Cγ1, Cγ2, and Cγ3 loci, respectively. Cγ3 gene segment is a pseudogene and is found mostly in the germline configuration in normal thymocytes. Rearranged Cγ genes migrate at 16–20 kb, representing two predominant gene rearrangements, Vγ2-Jγ1-Cγ1 and Vγ1.2-Jγ2-Cγ2, found in adult thymocytes. The RAG-1^−/− lane contains approximately eightfold less digested genomic DNA than the other two lanes. Numbers on the right represent DNA sizes (in kb).

Figure 2

Expression of rearranged TCR-γ transgene in thymocyte precursors is dependent on IL-7R signaling. (A) A representative RNase protection assay of transgene-specific RNA in purified CD4⁻CD8⁻TCR-β⁻ thymocytes from high (>30) and low (<2) copy transgenic IL-7Rα^+/− (designated +) and IL-7Rα^−/− (−) mice using the transgene V-J junction–specific riboprobe. Labeled, undigested probes are shown on the right (γA, γ-actin; v2, G8 VJ junction–specific probe). Identities of the protected bands are indicated beside the arrows. The two protected bands marked as originating from G8 TCR-γ gene template (G8 Vγ2) at 273 and 249 bp are so designated based on the predicted sizes of the protected VJ region of the G8 mRNA and are only detected in transgenic RNA samples. Size markers were included in all of the gels. The 249-bp fragment corresponds to correctly spliced mRNA, and the 273-bp fragment represents an mRNA in which the correct J-C splice has not occurred. Numbers below the panel represent the degrees of reduction in transgene-specific RNA level (combined quantities of two bands marked as G8γ) in transgenic IL-7Rα^−/− mice compared with IL-7Rα^+/− mice as determined by the PhosphorImager densitometric analysis with γ-actin RNA level as a loading control. Numbers on the right represent RNA sizes (in bp). Sub-bands present in the high copy IL-7R⁺ lane are transgene transcription specific and may originate from incorrect splicing, aberrant transcription, and/or unstable mRNA, and are possibly enhanced by an abnormally elevated expression of the transgene. (B) Semiquantitative RT-PCR assay for transgene-specific RNA in purified CD4⁻CD8⁻TCR-β⁻ thymocytes from low copy transgenic IL-7Rα^+/− and IL-7Rα^−/− mice. Results from fourfold serial dilutions of the cDNA samples used for PCR are shown. Tubulin RT-PCR was used as a control. The highest initial amount used for tubulin PCR was fourfold diluted compared with the highest amount used for Vγ2-G8 junction–specific PCR. No significant level of product was detected in PCR reactions without the RT step, and no RT-PCR product was detectable in nontransgenic samples (data not shown). Results from two of three independent sorting experiments are shown.

Figure 3

Expression of a rearranged TCR-γ gene results in γ/δ T cell development in IL-7Rα^−/− mice. (A) Proportions of gated CD4⁻CD8⁻ DN thymocytes expressing TCR-γ/δ in representative nontransgenic B6, high copy transgenic B6, high copy transgenic B6 IL-7Rα^−/−, and nontransgenic B6 IL-7Rα^−/− littermate mice. Transgenic mice predominantly express TCR-γ/δ composed of G8 Vγ2 chain. Numbers in brackets represent total thymocyte number. (B) CD25/CD44 expression profiles of total and gated DN and DP thymocytes in high copy transgenic IL-7Rα^+/+ or IL-7Rα^−/− mice. No significant difference is seen in the CD25/CD44 subset distribution between the nontransgenic and transgenic IL-7R^+/+ mice. Tg, transgenic.

Figure 4

γ/δ T cells are not generated in low copy TCR-γ transgenic (loTg) IL-7Rα^−/− mice. Proportions of gated DN thymocytes expressing TCR-γ/δ in representative nontransgenic IL-7Rα^+/−, low copy TCR-γ transgenic IL-7Rα^+/−, and low copy TCR-γ transgenic IL-7Rα^−/− littermates. Fractions of TCR-γ/δ cells that express Vγ2 chain are also compared. Numbers in brackets represent total thymocyte number.

Figure 5

IL-7 induces transcription of endogenous, rearranged TCR-γ genes in purified CD4⁺ α/β thymocytes. (A) Semiquantitative RT-PCR assay of endogenous, rearranged Vγ2-Jγ1–specific RNA from sorted TCR-γ/δ⁺ or CD4⁺ or CD8⁺HSA^loTCR-β⁺ thymocytes cultured for 2 d in IL-2 alone (designated as –IL-7) or in medium supplemented with IL-2 and IL-7 (1% supernatant of transfected IL-7 cDNA–expressing J558 cells, designated as +IL-7). Addition of IL-7 did not significantly alter the level of TCR-γ gene expression in cultures of γ/δ thymocytes (data not shown). Results from threefold serial dilutions of the cDNA samples used for PCR are shown. The highest amount used for control tubulin PCR was threefold diluted compared with the highest amount used for Vγ2-Jγ1–specific PCR. No product was detected in PCR reactions without the RT step (data not shown). (B) Kinetics of endogenous Vγ2-Jγ1 gene expression in sorted mature α/β thymocytes cultured with 2 ng/ml rIL-7. RT-PCR products specific for rearranged Vγ2-Jγ1 and tubulin at various times after culture (numbers in the boxes refer to hours in culture) were resolved by gel electrophoresis and visualized by ethidium bromide staining. Sorted HSA^loTCR-β⁺CD4⁺ or CD8⁺ thymocytes (designated as CD4⁺ or CD8⁺) were cultured in IL-2 medium with (+) or without (−) IL-7. No significant TCR-γ gene expression was detectable in cultured CD8⁺ thymocytes or sorted CD4⁺ or CD8⁺ thymocytes at the beginning of culture (0h). Abundant TCR-γ transcripts were detected in DN (purified by complement kill of CD4⁺ and/or CD8⁺ thymocytes) and sorted CD25⁺CD44⁻ triple negative cells (pre-T) containing γ/δ lineage cells. No product was detected in PCR reactions without the RT step (data not shown).