The common cytokine receptor gamma chain and the pre-T cell receptor provide independent but critically overlapping signals in early alpha/beta T cell development

Abstract

Intracellular signals emanating from cytokine and antigen receptors are integrated during the process of intrathymic development. Still, the relative contributions of cytokine receptor signaling to pre-T cell receptor (TCR) and TCR-mediated differentiation remain undefined. Interleukin (IL)-7 interactions with its cognate receptor complex (IL-7Ralpha coupled to the common cytokine receptor gamma chain, gammac) play a dominant role in early thymopoiesis. However, alpha/beta T cell development in IL-7-, IL-7Ralpha-, and gammac-deficient mice is only partially compromised, suggesting that additional pathways can rescue alpha/beta T lineage cells in these mice. We have investigated the potential interdependence of gammac- and pre-TCR-dependent pathways during intrathymic alpha/beta T cell differentiation. We demonstrate that gammac-dependent cytokines do not appear to be required for normal pre-TCR function, and that the rate-limiting step in alpha/beta T cell development in gammac- mice does not involve TCR-beta chain rearrangements, but rather results from poor maintenance of early thymocytes. Moreover, mice double mutant for both gammac and pre-Talpha show vastly reduced thymic cellularity and a complete arrest of thymocyte differentiation at the CD44(+)CD25(+) cell stage. These observations demonstrate that the pre-TCR provides the gammac-independent signal which allows alpha/beta T cell development in gammac- mice. Thus, a series of overlapping signals derived from cytokine and T cell receptors guide the process of alpha/beta thymocyte development.

Figure 1

Early thymocyte development in control and γ_c ⁻ mice. (A) Thymocytes from 3–4-wk-old γ_c-deficient mice and their littermate controls were stained using CD4-PE and CD8β-FITC, and absolute numbers of DN, DP, CD4 SP, and CD8 SP cells were calculated (mean ± SD from 10 mice of each genotype). (B) Thymocytes were stained with a combination of FITC-conjugated antibodies (CD3, CD4, CD8β, TCR-α/β, TCR-γ/δ, B220, DX5, and Gr-1), CD44-PE, and CD25-biotin followed by streptavidin-TRI. CD44 versus CD25 expression is shown on electronically gated FITC⁻ TN thymocytes. Absolute numbers were calculated (mean ± SD) from six mice of each genotype.

Figure 2

Altered survival and proliferation of early thymocyte precursors in the absence of γ_c. (A) Abnormal BrdU incorporation in γ_c ⁻ thymocytes. Mice received a single pulse of BrdU before killing. Indicated thymocyte subsets were sorted, and percentages of cells incorporating BrdU were analyzed. (B) Annexin V staining of γ_c ⁺ or γ_c ⁻ thymocyte subsets. Cells were stained for TN cells (see Fig. 1), CD25, and Annexin V. Propidium iodide–negative CD25⁻ and CD25⁺ TN cells were electronically gated, and percentages of Annexin V–positive cells were calculated (mean ± SD) from four mice of each genotype. (C) Intracellular Bcl-2 staining of DN thymocyte precursors was performed; γ_c ⁻ thymocytes demonstrated severely reduced Bcl-2 levels (thick lines). Thin lines, isotype control staining.

Figure 3

Pre-TCR function in the absence of the γ_c chain. (A) CD4/CD8 profiles of thymocytes from 3–4-wk-old TCR-α–deficient and TCR-α/γ_c double- deficient mice. Total thymocyte numbers were calculated (mean ± SD) from six mice of each genotype. (B) Intracellular TCR-β chain expression in thymocytes from γ_c-deficient, pTα-deficient, or wild-type mice. (C) Thymocyte CD4/CD8 profiles from mice bearing a functional TCR Vβ8.2 Tg on γ_c-deficient or wild-type backgrounds. Absolute thymocyte cell numbers (mean ± SD) were derived from four to six mice of each genotype.

Figure 4

Intrathymic development in γ_c/pTα^−/− double mutant mice. (A) CD4/CD8 profiles of thymocytes from 3-wk-old mice. (B) CD44/CD25 expression on gated TN thymocytes (see Fig. 1). For these experiments, thymi from four to six γ_c/pTα^−/− double mutant mice were pooled. Absolute thymocyte cell numbers (mean ± SD) were calculated for the indicated mice (n = 6–10 for each genotype).

Figure 5

Intracellular expression of pTα and TCR-β chain in CD25⁺ thymocyte precursors. Thymocytes from γ_c ⁺ or γ_c ⁻ mice were surface stained for TN cells (see Fig. 1), CD44, and CD25, fixed, and permeabilized with saponin before detection of intracellular (IC) TCR-β or pTα chains. Gated CD44⁺CD25⁺ and CD44⁻CD25⁺ thymocyte subsets are boxed. Negative controls (dotted lines) are staining of thymocytes from RAG-2–deficient (for TCR-β) or pTα-deficient (for pTα) mice.

Figure 6

TCR-β rearrangements in thymus and spleen. Genomic DNA from the indicated mice were amplified by PCR using a combination of primers specific for TCR Vβ6 and Vβ8 (sense) and a primer specific for the 3′ region of TCR Jβ2.5 (antisense). Amplification products were detected by blot hybridization using a probe specific for the 5′ region of TCR Jβ2.5.

Figure 7

Cytokine and TCR signaling in intrathymic development. Hematopoietic stem cells (HSC) give rise to thymocytes via common lymphoid progenitor cells (CLP). Within the thymus, TN precursor cells can be further subdivided based on expression of CD44 and CD25. Immature SP cells (ISP) are the immediate precursors of DP thymocytes, which after tolerance induction via α/β TCR selection develop into CD4⁺ or CD8⁺ SP cells which exit the thymus to seed peripheral lymphoid organs. γ/δ T cells can derive from the various subsets of TN cells. Concerning the molecules required to generate and maintain α/β T cells, growth factor receptors including c-kit and the common γ chain (γ_c) in concert with the pre-TCR and TCR-α/β appear to provide overlapping signals which guide the developmental process. In the absence of either IL-7Rα, γ_c, or the pre-TCR, signals provided by adjacent pathway(s) permit a limited degree of α/β thymocyte development. In contrast, when two adjacent signals are absent (for example, in c-kit × γ_c or γ_c × pTα double mutants), α/β T cell development is abrogated. Moreover, α/β T cells require continual TCR triggering in the periphery. In contrast, γ/δ T cells appear strictly cytokine dependent, both intrathymically and in the periphery. Hatched circles, cycling cells.