In the present report, we provide evidence that peptide concentration regulates the level of IL-4–independent early GATA-3 expression and the degree of responsiveness to IL-2, both of which are indispensable for TCR-induced, IL-4–independent early IL-4 production by naive CD4+
T cells. Thus, low concentrations of peptide allow naive CD4+
T cells to express early GATA-3 and to respond to IL-2. Therefore, these cells are capable of producing early IL-4 and undergoing subsequent differentiation into high-rate IL-4–producing (Th2) cells. By contrast, stimulation with high concentrations of peptide abrogates early GATA-3 expression and the sensitivity to IL-2, and results in the failure to produce early IL-4. The preferential capacity of low peptide concentrations to induce Th2 responses also was observed by other investigators who used naive CD4+
T cells from different TCR transgenic mice (14
). We also provide evidence that the degree of ERK activation accounts for peptide concentration–mediated early IL-4 transcription in naive CD4+
The requirement of GATA-3 for early IL-4 transcription is based on the evidence that naive (CD44low
T cells purified from spleen and lymph nodes of Gata3f/f
CD4-Cre mice failed to induce early IL-4 mRNA when stimulated with immobilized anti-CD3 and anti-CD28, whereas such T cells from Gata3f/f
mice expressed considerable amounts of early “IL-4–independent” IL-4 mRNA. Moreover, there is much indirect evidence that early GATA-3 expression is required for low concentration–mediated induction of IL-4. Thus, the dose response curve of early GATA-3 and early IL-4 mRNA expression were the same, and GATA-3 mRNA appeared slightly earlier than did IL-4 mRNA (unpublished data). Although GATA-3 has been reported to play an important role in Th2 differentiation (19
), our current work is the first to demonstrate clearly that GATA-3 is responsible for TCR-induced, IL-4–independent early IL-4 production by naive CD4+
We have not clarified the mechanism by which TCR-mediated signaling induces early GATA-3 expression. Das et al. (29
) reported that CD4+
T cells from NF-κB p50-deficient mice failed to express GATA-3 in the nucleus 96 h after stimulation with anti-CD3 and anti-CD28, and that these cells are impaired markedly in Th2 polarization. They also showed that CD4+
T cells that were pretreated with an inhibitor of NF-κB p50 did not express GATA-3 in the nucleus at 96 h. However, it remains unclear whether early GATA-3 induction is dependent on NF-κB p50. Previous reports have demonstrated that TCR-induced NF-κB activation is down-stream of protein kinase C θ activation (30
), and that the degree of protein kinase C θ activation is directly dependent on the strength of TCR signaling (31
). However, given the fact that early GATA-3 expression was suppressed in a peptide concentration–dependent manner, the degree of NF-κB activation may not account for TCR-induced early GATA-3 expression.
We showed in the present report that blockade of the ERK pathway allowed naive CD4+
T cells that were stimulated with high concentrations of peptide to induce GATA-3 mRNA and to express its protein in the nucleus. Komine et al. (32
) reported that retroviral infection with Runx1 cDNA dramatically diminished the frequency of IL-4–producing cells by inhibiting GATA-3 mRNA expression under neutral and Th2-skewing conditions. This raised the possibility that the peptide concentration effect might be mediated by preferential induction of Runx1 at high peptide concentration. However, our real-time PCR analysis showed no significant difference in Runx1 mRNA levels between cells that were stimulated with low and high concentrations of peptide at 12–24 h of stimulation (unpublished data). Moreover, U 0126 pretreatment did not alter the levels of Runx1 mRNA expression at high peptide concentration. Thus, it is unlikely that Runx1 is responsible for high peptide concentration–mediated suppression of early GATA-3 expression.
Jorritsma et al. (17
) showed that treatment of naive AND TCR transgenic CD4+
T cells with a MEK inhibitor (PD98059) during priming with relatively high concentrations (5 μg/ml) of moth cytochrome c
peptide resulted in an increased ratio of nuclear JunB homodimers to JunB/c-Fos heterodimers that was capable of binding to the IL-4 promoter upon recall challenge with moth cytochrome c
peptide. Therefore, they concluded that robust ERK activation inhibits IL-4 transcription by altering the pattern of distinct AP-1 complexes. However, we observed that 10 μM pPCC induced significantly higher nuclear JunB expression than did 0.01 μM pPCC at 12 to 24 h of priming, and that pretreatment with U 0126 did not alter the pattern of JunB expression (unpublished data). Moreover, 0.01 and 10 μM pPCC induced comparable levels of nuclear c-Fos expression, and pretreatment with U 0126 did not alter the pattern of c-Fos expression at low and high pPCC concentrations (unpublished data). Therefore, although it is possible that cells that have completed their differentiation to Th1 or Th2 phenotype have different AP-1 complexes that are capable of binding to the IL-4-promoter, it is unlikely that the formation of such different complexes is responsible for the peptide concentration–dependent early IL-4 production.
LeGros et al. (1
) originally demonstrated that IL-4 and IL-2 are required for priming of CD4+
T cells to develop into IL-4–producing cells; however, the precise role of IL-2 in Th2 priming was not clarified. Recently, Cote-Sierra et al. (5
) showed that IL-2 mediates its effect by stabilizing the accessibility of the Il4
gene after 48 h of priming. In the present report, we found that when naive line 94 CD4+
T cells were primed with 0.01 μM pPCC, induction of early IL-4 mRNA expression required IL-2, whereas early GATA-3 mRNA expression was independent of IL-2. We also observed that naive CD4+
T cells from IL-2−/−
5C.C7 TCR transgenic RAG2−/−
mice failed to express “24-h” IL-4 mRNA in response to 0.01 μM pPCC, and that addition of exogenous IL-2 restored IL-4 mRNA expression. By contrast, IL-2−/−
cells expressed levels of GATA-3 mRNA that were comparable to WT cells; addition of exogenous IL-2 did not enhance its expression (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20051304/DC1
). Although the importance of STAT5, especially STAT5a, in Th2 differentiation has been reported (33
), it remains to be elucidated whether IL-2–driven STAT5 activation is involved in early IL-4 production.
In conclusion, peptide concentration–mediated strength of TCR signaling regulates early IL-4 production by naive CD4+ T cells by controlling the levels of ERK activation which, in turn, influence early GATA-3 expression and responsiveness to IL-2. Weak and transient ERK activation that is induced by low concentrations of peptide allows naive CD4+ T cells to express early GATA-3 and to respond to endogenous IL-2, both of which are required for early IL-4 production. This endogenously produced early IL-4 is required for priming of CD4+ T cells to develop into high-rate IL-4–producing (Th2) cells. By contrast, intense and sustained ERK activation that is induced by high concentrations of peptide inhibits early GATA-3 expression and transiently desensitizes the IL-2R; this results in the failure of naive CD4+ T cells to produce early IL-4 and to undergo subsequent Th2 differentiation.
Biologically, these results imply that low concentration challenge of individuals without overt activation of Toll-like receptors would lead to preferential priming of naive CD4+
T cells to develop into a Th2 phenotype, and thus, predispose to allergic inflammatory response. Bottomly and colleagues argued that low concentrations of LPS may be required to allow mice to develop an antigen-stimulated airway hypersensitivity response in vivo (35
). Addition of LPS (10 ng/ml) did not alter the overall antigen concentration effects described here (unpublished data). This suggests that peptide concentration/strength of TCR signaling may be a central element in determining in vivo priming for Th2 responses, and thus, bias toward allergic inflammation.