Our findings confirm that TSLPr is preferentially expressed on rare actively dividing Th2 cells under Th2-promoting conditions. While a large proportion of cells expressing TSLPr did not co-express CD127, T cells that were pre-activated with TSLP+allergen alone did respond weakly to TSLP as judged by phosphorylation of STAT5. Alternate STATs implicated in TSLP signaling in human DCs and T cells, including STAT6, were not examined in our study.24, 25
Interestingly, sequential pre-activation of T cells with TSLP+allergen followed by anti-CD3+anti-CD28 resulted in secretion of TSLP-induced Th2 cytokines, while pre-activation with TSLP+allergen alone did not. Collectively, these findings suggest that Th2 cells induced in a “pro-allergic” milieu require re-activation by a potent TCR stimulus in order to respond optimally to TSLP. In terms of the biological relevance, it is tempting to speculate that Th2 cells respond to the direct actions of TSLP during the effector phase of the response, after Th2 differentiation has occurred. Such a scenario would equip Th2 cells primed in regional nodes with the capacity to respond to TSLP in inflamed tissues. Consistent with this theory, TSLPr expression on T cells, but not DCs, was required for enhanced Th2 cytokine secretion by skin-infiltrating CD4+
T cells in a mouse model of allergic skin inflammation.15
Analysis of cells isolated from AD skin supported the presence of T cells expressing TSLPr at inflamed sites and these cells constituted ~10% of IL-4+
T cells. While low expression of this receptor could reflect a transient event associated with Th2 differentiation or cell division in situ,
its presence in lesional but not non-lesional skin suggests a functional role at the site of allergic inflammation.
It has been reported that IL-4-expressing T cells infiltrating AD skin, a site where TSLP is present, represent a heterogeneous population.26
Our findings indicate that expression of TSLPr on T cells may not be unique to classical Th2 cells since IL-4-expressing cells induced by SEB co-expressed a variety of other cytokines. Thus, further studies are warranted to elucidate whether TSLPr expression is exclusive to T cells with an IL-4-restricted cytokine profile.
The lack of a functional TSLPr complex on some TSLPr+
T cells was a novel observation. The absence of CD127 on TSLPr+
T cells that were stimulated specifically with TSLP alone did not appear to be explained by downregulation of the TSLPr complex upon binding of TSLP since the ligand binding component (TSLPr chain) of the TSLPr complex12
was readily detectable on the cell surface. T cells were maintained in culture with TSLP in our system in order to reproduce a “pro-allergic” milieu. Several lines of evidence argue against receptor downregulation in the presence of TSLP. First, time course studies indicated a consistent signal for TSLPr following stimulation with TSLP. Second, TSLPr+
T cells were detected at low frequencies in SEB-stimulated cultures in the absence of TSLP. Third, work performed by other groups has demonstrated only low surface TSLPr and a modest increase in TSLPr mRNA in human T cells activated in the absence of TSLP.14, 27
In the present study, identification of TSLPr+
T cells using image-based technology was a labor intensive process requiring visual inspection of thousands of cells. This approach facilitated the enumeration of bona fide TSLPr+
T cells. The microscopy component of this platform also allowed us to elucidate the presence of a functional receptor by confirming whether CD127 was expressed on rare TSLPr+
cells at the single-cell level. Another advantage of the approach described was the ability to visualize mitotic cells based on a distinctive nuclear and surface morphology, thereby linking TSLPr to actively dividing T cells. The technology we describe allowed the simultaneous assessment of up to 5 cellular markers (CD127, CD25, IL-4, DNA, and TSLPr). In order to analyze the maximal number of markers related to TSLPr cells, the T cell marker, CD4, was not included. Thus, it could be argued that the rare TSLPr+
T cells identified in our system constituted a contaminating DC population. There are several key immunobiologic features of DCs that argue against this. Notably, expression of IL-4 is not an attribute of human DCs.28
To the contrary, secretion of the Th1-promoting cytokine IL-12, is a notable characteristic of DCs under Th2-promoting conditions.29, 30
Furthermore, DCs primed specifically with TSLP do not produce detectable amounts of IL-4.1
Initial studies performed using standard flow cytometry detected a TSLPr signal on CD4+
T cells under Th2-promoting conditions at a similar frequency to that obtained by image-based technology (data not shown). This provided further evidence that TSLPr expression was a feature of T cells in our system. Finally, when considered collectively, the morphologic attributes and staining properties of TSLPr+
cells were characteristic of a T cell phenotype.
In summary, our findings support the view that in a “pro-allergic” milieu, TSLPr is preferentially expressed on rare actively dividing Th2 cells. The capacity for these cells to respond directly to TSLP may contribute to amplification of Th2 responses at sites of allergic inflammation.
- TSLP receptor is expressed on rare actively dividing Th2 effectors.
- T cells induced in a “pro-allergic” milieu can respond directly to TSLP.