IL-22–expressing cells represent a tissue T cell subset.
To investigate the cytokine profile of circulating and skin-homing T lymphocytes, we analyzed freshly isolated PBMCs (n
= 4) and short-term cultures of skin biopsies obtained from patients with PS (n
= 3), AE (n
= 4), and ACD (n
= 4). T cells were permeabilized, and their cytokine profile was studied by flow cytometry and multicolor intracellular staining. As expected, obvious differences between the samples were observed. AE-derived T cells were characterized by a large proportion of IL-4–expressing T cells (30.5% ± 10.0%), whereas PS- and ACD-derived T cells showed a prevalence of IFN-γ–expressing T cells (PS, 34.3% ± 3.7%; ACD, 36.4% ± 4.8%; Figure F and Supplemental Table 1; supplemental material available online with this article; doi:
). The IL-17–expressing population was largest in PS (17.4% ± 4.2%), followed by ACD (13.1% ± 3.1%) and AE (9.0% ± 1.2%; Figure ). This IL-17–expressing population overlapped with IFN-γ–producing T cells and, to a lesser degree, with those expressing IL-4, whereas approximately 50% were pure Th17 cells (both IL-4 and IFN-γ negative). IL-22–producing T cells were observed in both PBMCs and inflammatory skin diseases. Although interindividual differences were high, the frequency of IL-22+
T cells was significantly higher in skin-derived T cell lines than in the circulation (PBMCs, 2.3% ± 0.4%; PS, 15.9% ± 2.3%; AE, 13.8% ± 4.0%; ACD, 12.9% ± 2.4%; Figure E). Thus, IL-22+
cells were enriched in inflamed skin.
Th22 cells represent a distinct T cell subset enriched in inflammatory skin diseases.
In addition to distinct IL-4, IFN-γ, and IL-17 populations, a subset of T cells secreting IL-22 alone was present as a robust single-positive entity. Depending on the disease, about one-third of total IL-22+ cells were clear-cut Th22 cells (Supplemental Figure 1), and another third were Th17 cells coproducing Il-22; thus, Th22 and Th17 cells were the major T cell sources of IL-22. The overlap of cytokine expression observed in T cells derived from inflammatory skin disorders is shown in Figure F. These data illustrate that the size and likely plasticity from Th1, Th2, Th17, and Th22 subsets toward neighboring phenotypes was distinct for the different diseases.
Because IL-22 is not released solely by T lymphocytes, we investigated IL-22+ cells from blood and skin for their expression of T cell markers. The majority of IL-22+ cells were found within the CD4+ subpopulation in circulating PBMCs and AE, whereas in both PS and ACD, a minor, but substantial, fraction was IL-22+CD4– (Figure G). Our data highlight that CD4+ Th cells represent the major source of IL-22 in the skin, although IL-22 was also secreted by other leukocyte subsets, such as NK cells and CD8+ T cells (data not shown).
Characteristics of Th22 clones.
T cell subsets are defined by an epigenetically imprinted cytokine profile that is assumed to be stable over multiple cell divisions, thereby allowing amplification of immune responses by clonal expansion. Starting from T cell lines, as analyzed in Figure , we generated 244 distinct T cell clones from 2 donors with AE (143 clones), 44 clones from a patient with ACD, and 57 clones from a patient with PS (Table ).
Characterization of skin-derived T cell clones
Consistently with the single-cell analysis from biopsies, multiple Th22 clones were characterized by solitary IL-22 expression, which did not show substantial protein secretion of IL-4, IFN-γ, or IL-17 (Figure , A–C, left). As observed for the large majority of T cell clones isolated from skin, Th22 cells were capable of secreting IL-10 and/or TNF-α. The cytokine secretion pattern of isolated Th22 clones was remarkably stable even after 4 restimulations over sequential 10-week cultures. Th22 clones released IL-22 within 6 hours, reaching a peak at 12 hours and persisting at this level for at least 48 hours (Figure , A–C, right). Surface expression profiling of Th22 clones confirmed the CD3+CD4+ phenotype of Th22 cells, whereas the CD8 and NK cell markers CD56, NKp44, and NKp46 were negative (Supplemental Figure 2).
Th22 clones show a stable phenotype.
Th22 cells represent an independent and stable lineage of Th cells.
Because Th cell are increasingly understood to differ in plasticity and differentiation status, we investigated the phenotype stability of Th22 cells under different polarizing conditions. Skin-derived memory Th22 clones did not lose the capacity to secrete IL-22 under Th1, Th2, Th17, and Treg conditions, nor did they gain the capacity to produce substantial amounts of an additional subset-defining cytokine, although IFN-γ was slightly induced under different conditions (Supplemental Figure 3). These findings were confirmed in freshly isolated CD45RA–
T cell lines from PBMCs of 3 independent donors (Figure ). CCR10+
sorting enriched IL-22–producing, antigen-experienced T cells (Supplemental Figure 4 and ref. 9
). The frequency of IL-22+
T cells and the secretion of IL-22 remained at high levels under all polarizing conditions (Figure , B–F, and Supplemental Figure 4), being slightly diminished only under Treg conditions. Importantly, induction of IFN-γ under Th1 conditions and IL-17 under Th17 conditions was not caused by a conversion of Th22 cells into Th1 or Th17 cells, as the number of Th22 cells within the CCR10+
T cell lines was not diminished under different polarizing conditions (Supplemental Figure 4). In contrast, both Th2 and Th22 conditions tended to increase the number of Th22 cells, whereas IL-13 and IL-4 were induced only marginally or not at all (Supplemental Figure 4). Thus, Th22 cells represent a stable T cell lineage, and memory Th22 cells do not convert into another subset.
Th22 cells are stable and cannot be deformed into other T cell phenotypes.
Transcriptome of Th22 cells.
Th22 clones were subjected to full transcriptome analysis to validate the phenotype compared with known T cell subsets, specifically Th17 cells. We compared 3 Th22 cell clones originating from different AE and PS patients with Th1, Th17, and Th2 clones (n = 5; Table ). To exclude the possibility that Th22 clones derive from Th17 cells, we chose IL-22–producing Th17 clones for gene chip comparison (Table ). Despite the genetic distance, the Th22 clones showed surprisingly low variance (Figure A). The transcriptome for Th22 cells demonstrated similar numbers of up- and downregulated transcripts, whereas the profile for Th22 cells was distinct and not closely related to those of other known T cell subtypes (Figure B). The gene chip analysis also confirmed the selectivity of IL-22 expression and the subset-independent nature of TNF-α (Figure C). IL-10 was produced substantially less by Th22 clones; however, substantial secretion was observed (Figure and Table ). Therefore, IL-10 can also be regarded as subset independent. Interestingly, Th22 cells upregulated a number of FGFs upon stimulation. This upregulation seemed to be exclusively limited to Th22 cells, as we observed substantial differences to other known T cell subsets. In contrast, SPRY1, an FGF antagonist, was substantially reduced in Th22 cells compared with Th1, Th2, and Th17 cells. The chemokine expression pattern of Th22 cells provides further evidence for tissue remodeling activity of this subset. Compared with other T cell populations, they produced substantially more transcripts of CCL7, involved in tissue fibrosis, and CCL15 and CCL23 splice variant 2; CCL23 splice variant 1 showed higher expression in Th1 cells (Figure C and Supplemental Table 3). In contrast to Th22 cells, Th17 cells strongly upregulated CCL20, a chemokine attracting CCR6+ cells. Furthermore, Th17 cells selectively expressed IL-23R. Transcription factors also underlined the individual expression pattern of Th22 cells, given the reduced RORC2, GATA3, and T-bet expression and the presence of BNC-2 and FOXO4 (Figure C). It remains to be demonstrated that these factors are also involved in the polarization process upon Th22 differentiation. The expression profile clearly identified Th22 cells as a separate subset, including subset-specific surface receptors and transcription factors (Figure D).
Whole genome transcriptome analysis of Th22 compared with Th1, Th2, and Th17 cells reveals unique functional profiling of Th22.
Epidermal location of Th22 cells.
Epidermis and dermis of skin biopsies were separated by dispase treatment, and infiltrated lymphocytes of each fraction were analyzed for the expression of lead cytokines. Figure A shows a representative 3-color FACS analysis, which revealed a higher proportion of IL-22+ T cells and Th22 cells in the epidermal compartment. On average, a 2.2-fold enrichment of IL-22+ cells was observed in epidermal compared with dermal isolations of inflamed skin of AE (n = 4) and ACD (n = 4) patients (median [of 8 total] dermis, 10.98; median epidermis, 24.03; P ≤ 0.05; Figure B; see Supplemental Figure 1B for disease-separated values).
Th22 cells are enriched in the epidermal compartment of the skin.
The increased number of IL-22–producing T cells in the epidermis was the result of an increase in Th22 cells and in IFN-γ/IL-22–coproducing T cells. The distribution of IL-22+ T cell subpopulations shifted toward IFN-γ/IL-22–coproducing T cells (dermis, 30.5%; epidermis, 42.0%), whereas Th22 cells were comparably distributed in the dermal (35.3%) and epidermal (31.0%) compartments. Accordingly, IL-4/IL-22–coproducing T cells were diminished in the epidermal compartment (dermis, 4.6%; epidermis, 2.8%).
Th22 cells orchestrate potentially novel innate immune responses by keratinocytes.
To investigate the potential activities of the Th22 resident within the epidermis on epidermal cells, human primary keratinocytes of healthy donors were exposed for 12 hours to medium or Th22 supernatants of 3 independent and activated Th22 clones. Whole transcriptome analysis of keratinocytes revealed alterations of multiple genes (Figure A), including genes involved in the intracellular transport of proteins, genes for chemokines regulating different T cell subsets and cytokines, and genes belonging to the innate immune system (Figure B and Supplemental Table 4). The Th1-attracting and antimicrobial chemokines CXCL9, CXCL10, and CXCL11 were induced up to 400-fold. A minor, 10-fold induction was also observed for monocyte-attracting CCL2, the antiviral and antibacterial Th1 chemokine CCL5, the CCR6+ cell–attracting CCL20, and the fibroblast- and eosinophil-attracting CCL26. Furthermore, Th22 supernatants induced T cell growth factors IL-7 and IL-15 and the macrophage differentiation factor IL-32. Surprisingly, Th22 supernatants also substantially induced C1s and C1r, inducible factors of the classical complement pathway, and the initial alternative pathway factor CFB, as well as its regulator, CFH. This Th22-induced pattern of antimicrobial defense also included the antimicrobial peptide S100A7 as well as TLR3 (recognizing double-stranded RNA) and TLR6 (recognizing bacterial lipoproteins).
Th22 cells induce a specific response pattern in primary human keratinocytes.
Th22 cells regulate keratinocyte defense mechanisms by an IL-22/TNF-α combination key.
The DNA array analysis was validated using real-time PCR (Figure C) and also involved the neutralization of IL-22 in these cultures. A clear reduction was observed in all induced genes, ranging 2- to 10-fold depending on the gene. Because Th22 cells also produce TNF-α, we further hypothesized a synergistic effect of IL-22 and TNF-α in stimulation of keratinocytes. This was tested by the addition of recombinant IL-22, TNF-α, or a combination of both cytokines to primary keratinocyte cultures. Whereas IL-22 alone only marginally induced production of chemokines, complement factors, and cytokines, TNF-α clearly upregulated the expression of all candidate immune genes (Figure ). However, the combination of IL-22 and TNF-α resulted in a substantially higher stimulation of keratinocytes, multiplying the effects of TNF-α 5- to 15-fold (Figure ).
Th22 cells influence keratinocyte functions by a combination of IL-22 and TNF-α.
Th22 cells enhance epidermal wound healing in an IL-22–dependent manner.
Besides inducing a defense armory against invading pathogens in keratinocytes, Th22 supernatants effectively enhanced rapid wound healing in a functional keratinocyte in vitro injury model (Figure ). As soon as 4 hours after wounding, Th22 supernatants had substantial and persistent effects on epithelial layer closure, whereas other supernatants of other T cell subsets were inefficient. Consistent with the data described above, these effects were dependent on IL-22, as IL-22–neutralizing antibodies reverted this effect and recombinant IL-22 restored rapid wound healing. Moreover, in contrast to Th22 supernatant–induced defense genes, IL-22 alone efficiently induced rapid wound healing, and no synergism with TNF-α was observed in that context. These data indicate that IL-22 may function as a biphasic cytokine: protective and regenerative in steady state while amplifying proinflammatory signals given by TNF-α.
Th22 supernatant enhances wound healing in a functional in vitro injury model.