To study physiologically relevant DC populations from two distinct lymphoid tissues, namely PP and SP, we used freshly isolated DCs purified on the basis of their surface expression of CD11c. These highly purified DC populations devoid of B cells, T cells, and macrophages from PP and SP were strikingly different in their ability to stimulate T cells in vitro. First, PP DCs were much more potent in stimulating proliferation of both allogeneic () and antigen-specific () T cells compared with SP DCs. A possible explanation for these findings is that PP DCs were found to express 5–10-fold higher levels of MHC class II antigens than SP DCs () and thus on a per cell basis are likely to provide a more potent signal to the responding T cell by engaging more TCRs. The enhanced ability to induce proliferation by PP DCs also suggested that these cells may be intrinsically more “mature” in their phenotype, since maturation has been shown to correlate with enhanced expression of MHC class II, as well as costimulatory molecules that could affect T cell proliferation. However, we found that CD80, CD86, and CD40 expression was low to modest, and similar for PP and SP DC populations. In addition, we found that levels of the adhesion molecules ICAM-1 and ICAM-2 were also similarly expressed by PP and SP DCs (). Thus, it appeared that neither population of cells was fully mature or differentiated, and that higher expression of these costimulatory and adhesion molecules could not explain the enhanced ability of PP DCs to induce primary T cell proliferation. The findings presented here are consistent with prior studies by Ruedl et al. that demonstrated that freshly isolated CD11c+
cells from PP are functionally immature 25
. Thus, it was shown that upon overnight culture in the presence of either GM-CSF and TNF-α or anti-CD40 antibody, freshly isolated PP DCs matured as determined by their higher levels of expression of MHC class II, CD80, and CD86, and their loss of the ability to process intact antigens 25
. Whether PP DCs express as yet unidentified costimulatory molecules or soluble factors responsible for the induction of increased T cell proliferation remains to be determined.
We next determined whether the tissue specificity of DCs influences the type of Th cell responses induced during antigen-specific stimulation. For these studies, we primed naive CD4+ T cells from OVA TCR transgenic mice in vitro with freshly isolated CD11c+/B220− DCs from PP or SP, and determined the phenotype of the primed cells by measuring cytokine production after secondary stimulation of T cells with anti-CD3 and anti-CD28 antibodies. Interestingly, we found that PP but not SP DCs primed T cells for the production of IL-4 and particularly IL-10. In addition, the level of IFN-γ produced by T cells primed with SP DCs was significantly higher than that produced by T cells primed with PP DCs. Furthermore, when cytokine production was normalized to T cell expansion, we found even greater differences in the amounts of cytokines produced by T cells primed with either PP or SP DCs.
In an effort to investigate the mechanisms underlying the particular ability of PP DCs to stimulate Th2 cytokine responses, we next determined whether TGF-β was preferentially expressed by PP DCs or generated in PP DC–T cell cultures, and whether this cytokine was affecting the differentiation of T cells into a Th2 pathway. This possibility was based on prior studies suggesting a role for TGF-β in directing Th2 responses in murine infection with Leishmania 26
as well as in driving Th2 immune deviation seen after antigen administration to the anterior chamber of the eye 27
. We initially determined the ability of neutralizing anti–TGF-β antibodies added to the primary culture to alter the T cell phenotype induced by PP and SP DCs. Interestingly, neutralization of TGF-β resulted in increased levels of IFN-γ, IL-4, and IL-10 production by PP DC–, but not by SP DC–, primed T cells (). This increase in cytokine production did not appear to be due to enhanced expansion of cytokine-producing T cells, as [3
H]thymidine incorporation during secondary stimulation was identical in cells treated and untreated with blocking antibodies (). We extended our finding of the inhibitory effects of TGF-β on IFN-γ production by PP DC–primed T cells to another TCR transgenic system of different genetic background (B10.A), confirming that the effect is not a BALB/c strain–dependent phenomenon (). Although the role of TGF-β in suppressing IFN-γ secretion has been well documented 2628
, its effect in modulating Th2 cytokine secretion from T cells is at best controversial. Our observations argue that TGF-β in the priming culture was suppressive to the induction of IFN-γ– as well as IL-4– and IL-10–producing T cells. Although there is evidence that the presence of TGF-β can suppress IL-4 and IL-5 secretion from purified T cells activated with anti-CD3 29
, further investigation is required to clarify the direct effect of TGF-β in the differentiation of Th2 cells.
The source of TGF-β responsible for the observed suppression was either PP DCs and/or T cells primed with PP DCs. To address this possibility, we attempted to directly measure TGF-β production from freshly isolated PP and SP DCs. Although we were unable to detect TGF-β by ELISA from overnight cultures of freshly isolated DCs stimulated with CD40L, we did find that the level of expression of TGF-β mRNA was found to be much higher in PP DCs compared with SP DCs in both strains of mice (). Moreover, there was no detectable mRNA for TGF-β in SP DCs in the B10.A mice. Although TGF-β mRNA levels do not always correlate with the level of protein secretion 3031
, our data suggested that only PP DCs had constitutive TGF-β mRNA expression, and that the level of TGF-β produced is able to suppress both Th1 and Th2 cytokine secretion by T cells in the PP DC–T cell, but not SP DC–T cell, coculture. In PP, the presence of high levels of TGF-β, in combination with the Th2-inducing phenotype of PP DCs, may play an important role in regulating the local Th cell responses. In other words, the cytokine environment in PP may modify DCs as well as T cells to ensure that unwanted Th1 responses towards noninfectious materials such as food antigens are prevented.
We next determined the role of IL-10 in T cell differentiation by PP and SP DCs. We found that the addition of a neutralizing anti–IL-10 during priming cultures resulted in significantly enhanced IFN-γ secretion from the T cells primed with PP DCs, almost to the levels secreted by T cells stimulated with SP DCs (). This result is consistent with IL-10 being a potent inhibitor of Th1 development 32
. It was also found that T cells primed by SP DCs in the presence of anti–IL-10 antibody secreted higher levels of IFN-γ. However, when cytokine secretion was normalized to the expansion of T cells during the priming culture, we found considerable increase in IFN-γ only from PP DC–primed T cells in the presence of anti–IL-10 antibody (). Thus, significant suppression of IFN-γ was mediated by IL-10 present in the PP DC–T coculture. We next explored whether IL-10 produced directly from PP DCs could be responsible for the differentiation of T cells that secrete low levels of IFN-γ and high levels of IL-4 and IL-10. When IL-10 production by purified DCs was assessed upon stimulation with CD40L trimer, we found that PP but not SP DCs secrete high levels of IL-10. Therefore, the exclusive ability of PP DCs to generate IL-10–secreting T cells may at least be partially due to the production of IL-10 by PP DCs themselves. However, the fact that blocking IL-10 during T cell priming by PP DCs only partially converts T cell phenotype towards Th1 suggests that other factors might be involved in the Th2 development by PP DCs.
One final possible explanation for why PP and not SP DCs induce Th2 differentiation is related to the enhanced ability of PP DCs to stimulate T cell proliferation. Recently, it has been shown that naive murine CD4+
T cells have an intrinsic program for the transcription of the IL-4 gene that is directly related to the number of times the T cell divides. Thus, it was shown that the expression of IL-4 begins to occur after the third cell division, and that this expression is independent of exogenous IL-4 signaling, as similar results were found with T cells from STAT-6–deficient mice 33
. This suggests that the enhanced proliferation of T cells stimulated by PP DCs may result in an intrinsic bias toward Th2 development, which, when occurring in an environment that has low levels of IL-12 due to the production of IL-10 by PP DCs, results in the selection or emergence of T cells with a Th2 phenotype.
Taken together, the data presented here provide the first demonstration of the ability of DCs from the murine PP to preferentially induce the differentiation of CD4+
T cells into a Th2 pathway. Previous studies have suggested that different APC types could direct Th responses to either Th1 or Th2 34353637
. In addition, a recent study by Stumbles et al. suggested a similar Th2-inducing phenotype of DCs derived from the rat airway mucosa 28
. In that study, repeated injections of OVA-pulsed rat respiratory tract DCs lead to increasing levels of IgG1 but not IgG2b in the rats known to be Th2- and Th1-dependent IgG subclasses, respectively 3839
. The data presented here supports the claim that resting tissue-resident DCs at mucosal surfaces have a unique ability to drive T cell responses towards the Th2 pathway. Finally, it has been reported recently that DCs that express the CD8α molecule (lymphoid-derived) drive predominant Th1 responses and that DCs lacking the CD8α molecule preferentially drive Th2 responses in vivo 4041
. Since we found that equivalent numbers of DCs from SP and PP express CD8α, a difference in DCs populations, per se, cannot offer an explanation for the differences in T cell differentiation shown here. We are currently investigating the potential differences in the functions of these DC subsets between PP and SP.
The mechanism by which tissue-specific DCs influence Th cell development after an oral antigenic challenge can be speculated as follows. Intestinal antigens transported via M cells are taken up by SED DCs, which migrate to the T cell region and become IFR DCs. During migration, SED DCs can undergo two distinct developmental pathways. If the antigen encountered is a noninfectious food antigen, the default pathway for IFR DCs is to generate Th2/Th3 responses. However, upon encounter with infectious agents, maturation of DCs is triggered by interaction with some components of the invading microorganism such as LPS 4243
. This maturation of DCs leads to secretion of high levels of IL-12, which induces T cells to secrete IFN-γ resulting in Th1 responses. In support of this hypothesis, IFN-γ secretion by PP T cells has been observed after gastrointestinal infection with microorganisms known to stimulate production of IL-12 by macrophages and DCs, such as Salmonella typhimurium 3456
and Toxoplasma gondii 7
. Efforts to test this hypothesis are currently underway by analyzing the phenotype of PP DCs in vivo after oral delivery of microbial stimuli. Such a default Th2/Th3 environment in the intestinal tract may be indispensable because aberrant Th1 induction in the intestine is strongly associated with pathogenesis of inflammatory bowel diseases such as Crohn's disease 44