The Common Cytokine Receptor γ Chain Plays an Essential Role in Regulating Lymphoid Homeostasis

Materials and Methods

Results and Discussion

Like humans with XSCID, γ_c-deficient mice have hypoplastic thymi and lack NK cells (5–8). However, peripheral T cells are generally not found in humans with XSCID (4), whereas peripheral CD4⁺ T cells are found in the γ_cdeficient mice (5–8). Although these cells are diminished in numbers in γ_c-deficient mice at 3 wk of age (6, 7), in most mice there is a dramatic age-dependent accumulation of these cells (7) (Fig. 1 A), even though the thymus remains small (7). Analysis using a series of different TCR Vβ-specific mAbs (Vβ2, Vβ3, Vβ4, Vβ5.1,5.2, Vβ6, Vβ8.1,8.2, Vβ10, and Vβ11) indicated that the CD4⁺ T cell accumulation was polyclonal (unpublished data). Interestingly, the splenic CD4⁺ T cells in γ_c-deficient mice were increased in size (Fig. 1 B). The large size of the cells, coupled with the finding of CD4⁺ T cell infiltrates, particularly in the gut in association with colitis (5, 7), suggested that the CD4⁺ T cells might be activated and involved in mediating the pathological changes found in these mice. Therefore, we investigated the basis for the CD4⁺ T cell expansion.

A striking difference between splenic CD4⁺ T cells from γ_c-deficient and wild type mice was that the cells from the γ_c-deficient mice exhibited a CD62L^lowCD69^high phenotype typical of memory/activated cells, whereas those from wild-type mice exhibited a CD62L^highCD69^low phenotype typical of naive cells (Fig. 1 C) (15). Interestingly, CD4⁺ T cells in mice deficient in IL-2 (16, 17), IL-2Rα (18), and IL-2Rβ (19) also exhibited activated phenotypes. Expression of CD49d (VLA-4 α chain) and CD44 was slightly increased in γ_c-deficient CD4⁺ T cells (data not shown), whereas no significant difference was seen in expression of IL-2Rα (CD25) (Fig. 1 C), or in CD40L, CD28, CTLA-4, or in Fas (data not shown). The lack of increase in these activation markers suggested that these cells were partially rather than fully activated and that γ_c-dependent signals are required for completing T cell activation. In contrast with the different phenotypes in splenic T cells, CD4⁺CD8⁻ thymocytes from γ_c-deficient mice and wild type mice expressed similar levels of CD62L, CD69, and Qa-2^a (data not shown).

The accumulation and activation of the CD4⁺ T cells appeared to result from a defect in T cells, because the defect was corrected in mice in which γ_c expression was selectively reconstituted in T cells (transgenic mice in which γ_c is under control of the CD3δ enhancer and promoter) but not in mice in which γ_c expression was selectively reconstituted in B cells (transgenic mice in which γ_c is under control of the mb-1 promoter and Eμ heavy chain enhancer) (data not shown). A similar activated phenotype in Jak3-deficient mice was corrected when these mice were reconstituted with Jak3 under control of the Lck promoter (19a), a finding consistent with the similar phenotypes in mice and humans deficient in either γ_c or Jak3 and with the conclusion that Jak3 is the major mediator of γ_c-dependent signals (4, 5).

Next, we investigated whether the CD62L^lowCD69^high phenotype of the γ_c-deficient splenic CD4⁺ T cells reflected active proliferation and found that bromodeoxyuridine (BrdU) uptake in these cells was greater than in wild-type mice (Fig. 1 D, left). In contrast, γ_c-deficient thymocytes exhibited lower BrdU uptake than wild-type thymocytes (Fig. 1 D, right). Thus, there was more active cell division in the spleens than in the thymi of adult γ_c-deficient mice, correlating with the activated phenotype of the γ_c-deficient splenocytes. It is important to recognize that because these mice were not subjected to thymectomy, some splenic BrdU uptake could have occurred in thymocytes before CD4⁺ T cell migration to the periphery; however, because the thymi in γ_c-deficient mice are very small (0.5–5% of littermate controls) and the BrdU uptake in thymocytes in these mice was lower than in wild-type mice, it is likely that the majority of BrdU uptake occurred in the periphery.

Based on the BrdU uptake studies, the γ_c-deficient mice had an apparent replication rate much greater than wildtype mice (21.43%/d for γ_c-deficient mice and 6.32%/d for wild-type mice; see Materials and Methods). Such an increased rate for the γ_c-deficient mice should have yielded a much greater accumulation of cells than actually seen (Fig. 1 A), suggesting that CD4⁺ T cells from the γ_c-deficient mice had an increased rate of cell death (see Materials and Methods). Indeed, when cultured without stimulation, purified splenic CD4⁺ T cells from γ_c-deficient mice exhibited a higher level of death than cells from wild-type mice, with the greatest difference occurring in the first 8 h (Fig. 2 A). Increased apoptosis was confirmed both by TUNEL analysis (data not shown) and by staining with annexin V (Fig. 2 B), which allows the detection of apoptotic cells at an early stage because of its selective affinity for phospholipids, especially phosphatidylserine (PS), which is exposed on the cell surface in apoptotic cells, while normally PS is restricted to the inner cell membrane (20–22). The rapid increase in death seen at 8 h in γ_c-deficient mice (Fig. 2 A) presumably is accounted for by the large number of cells that stained with annexin V at time 0 (Fig. 2 B), suggesting that splenic CD4⁺ T cells of γ_c-deficient mice may be primed to die by apoptosis in vivo. Bcl-2 levels correlate with the survival of lymphoid cells (23, 24) and are induced by γ_c-dependent cytokines that protect against apoptosis (25–28). Therefore, it was interesting that γ_c-deficient splenic CD4⁺ T cells expressed very low levels of Bcl-2 (Fig. 2 C) (including both CD62L^high and CD62L^low subpopulations; data not shown), suggesting that γ_c is required for maintaining the normal levels of Bcl-2 expression in both activated and naive peripheral CD4⁺ T cells.

There are at least two possible explanations for why peripheral CD4⁺ T cells in γ_c-deficient mice were already activated without exogenous stimulation. In the first model, the activated phenotype does not require TCR stimulation (TCR-independent activation model); instead, γ_c-dependent signals are required for keeping mature CD4⁺ T cells in a naive stage and, that without such signals, these cells are activated nonspecifically. In the second model, peripheral CD4⁺ T cells in γ_c-deficient mice respond to self- or environmental antigens (TCR-dependent activation model). We investigated these possibilities using mice expressing MHC class II–restricted transgenic TCRs specific for ovalbumin (DO10 mice) or cytochrome c (AND mice). In these experiments, the mice were not exposed to the specific antigens. In DO10 male mice deficient in γ_c (DO10⁺γ_c ^−/Y mice), far fewer splenocytes were found than in DO10⁻γ_c ^−/Y mice (6.4 × 10⁶ versus 79 × 10⁶ cells/spleen) (Fig. 3 A). Analogous differences were also seen between AND⁺γ_c ^−/Y and AND⁻γ_c ^−/Y mice (2.7 × 10⁶ versus 50 × 10⁶ cells/ spleen). These data suggested that endogenous TCR expression might be important in regulating CD4⁺ T cell accumulation. Using the DO10⁺γ_c ^−/Y mice, we compared the relative frequency of memory (CD62L^low) and naive (CD62L^high) phenotypes in splenic CD4⁺ T cells expressing transgenic (KJ1-26⁺) versus endogenous (KJ1-26⁻) TCRs. Approximately 70% of CD4⁺ T cells from DO10⁺γ_c ^−/Y mice express the transgenic TCR (Fig. 3 B, fourth histogram), analogous to DO10⁺γ_c ^+/Y mice (second histogram). Surprisingly, although relatively high numbers of KJ126⁺CD4⁺ T cells in DO10⁺γ_c ^−/Y mice were CD62L^high (naive phenotype), similar to the finding for DO10⁺γ_c ^+/Y mice (84.9% versus 84.6%) (Fig. 3 C, bottom), relatively few of the endogenous KJ1-26⁻ CD4⁺ T cells in DO10⁺γ_c ^−/Y mice were CD62L^high (10%), similar to the finding for DO10⁻γ_c ^−/Y mice (8.3%) (Fig. 3 C, top). Thus, the CD62L^low phenotype correlated with the presence of endogenous TCRs. Analogous results were found in AND TCR transgenic γ_c-deficient (AND⁺γ_c ^−/Y) mice. In AND⁺γ_c ^−/Y mice, 93% of transgenic TCR⁺ (Vα11⁺) CD4⁺ T cells were CD62L^high, similar to AND⁺γ_c ^+/Y mice (94%). These results support the TCR-dependent activation model and suggested that TCR(s) expressed on expanding peripheral CD4⁺ T cells in γ_c-deficient mice are specific for self-antigen or environmental antigens. The finding that over 80% of adult γ_c-deficient mice (C57BL/6 background) develop an inflammatory bowel disease (data not shown) is consistent with the possibility of self-reactive CD4⁺ T cells in these mice.

In the immune response, there is a careful balance between clonal expansion and clonal deletion (29–31). Clonal expansion is believed to be regulated by mitogenic cytokines, especially IL-2. In γ_c-deficient mice, it was therefore surprising that although splenic T cells exhibit diminished in vitro responsiveness to mitogens (5), there was TCRdependent activation and a marked expansion of CD4⁺ T cells in vivo. In addition to this in vivo proliferation, we also detected an augmented rate of apoptosis in γ_c-deficient CD4⁺ T cells, a finding consistent with the known action of γ_c-dependent cytokines as survival factors (25–28); nevertheless, it was evident that the overall rate of death was inadequate for the degree of proliferation. Although the CD4⁺ T cells were activated, they expressed only low levels of CD25, CTLA-4, and Fas. Therefore, it is conceivable that γ_c-dependent signals are required to achieve induction of pathways that are required to negatively regulate immune responses. Thus, γ_c-dependent signals are essential components of a carefully controlled homeostatic mechanism that regulates the fate of activated cells.