IL 22 increases the proliferation of human cutaneous SCC in vitro.

<p>A431 cells were cultured in full media (10% FBS) or in serum starvation media (0.1% FBS) with or without the addition of the indicated cytokines for 72 hours. (a) Cells cultured in full media, and in starvation media supplemented with IL-22 (40 ng/ml and 100 ng/ml) show considerably greater proliferative behavior with increased colony formation when compared to those grown in starvation media alone or supplemented with IL-24 (40 ng/ml). (b) Representative images of IF staining using the proliferation marker Ki-67 (green) and the nuclear stain DAPI (blue). Cells grown in full media, as well as those treated with IL-22 (40 ng/ml and 100 ng/ml) show an increased number of proliferating nuclei when compared to those grown in starvation media alone or supplemented with IL-24 (40 ng/ml). Additionally, they demonstrate a more disorganized pattern of proliferation, with KI67+ cells no longer limited to the periphery, but rather seen throughout the tumor colonies. (c) Cell counts were performed after 72 hours of cultivation in the indicated conditions. The addition of 100 ng/ml IL-22 to the starvation media (0.1% FBS) resulted in a hyperproliferation of tumor cells, yielding significantly greater cell numbers when compared to those grown in serum starvation alone, or serum starvation supplemented with IL-22 (40 ng/ml) or IL-24 (40 ng/ml) (one-way ANOVA, p<0.001).</p

The proposed model of accelerated development of TSCC.

<p>An increased proportion of T regs, combined with decreased numbers of CD8⁺ and IFN-γ producing T cells, leads to decreased tumor surveillance. Greater percentage of IL-22 producing T cells suggests an increased proliferation stimulus. The overall imbalance could explain the rapidly proliferative nature of TSCC. IL-22 blockade may be an attractive candidate for targeted SCC therapy, especially in the transplant population.</p

TSCC shows increased Tc22 and decreased Th1 polarization.

<p>T cell “crawl outs” were activated and intracellular cytokines stained. Live CD3⁺CD4⁺ and CD3⁺CD8⁺ cells were gated, and then frequencies of the cells producing indicated cytokines were analyzed. (a) Representative dot plot analysis of IFN-γ, IL-4, IL-17, and IL-22 expression in CD4⁺ and CD8⁺ T cells from SCC specimens. Numbers indicate percent gated cells. (b) Summary results from 20 SCC and 12 TSCC patients. The Mann-Whitney U-test was used for the statistical comparison between two groups. Asterisks (*) indicate statistical significance (p<0.05).</p

TSCC shows fewer CD8+ T cells and increased Foxp3/CD8 ratio.

<p>Representative immunohistochemistry and summary data with median cell count values of (a) CD3+ T cells (b) CD8+ T cells (c) Foxp3+ T cells in normal skin (n = 5), SCC (n = 10) and TSCC (n = 10). Each dot represents one patient. Asterisks (*) indicate significance, where *P<0.05, **P<0.01 and ***P<0.005. Bar = 100 µm. Triple label immunofluorescence confirms the presence of CD4+CD25+Foxp3+ T regulatory cells (see arrows) in SCC (e) and TSCC (f). Images are presented in pseudo color: Foxp3 (red), CD4 (blue) and CD25 (green), located above merged image. Red and green overlapping cells appear yellow in color; red and blue appear purple; and green and blue appear aqua; cells labeled with all three stains appear white.</p

IL-22 expression is increased in SCC, TSCC, and juxtatumoral skin.

<p>The relative mRNA expression of IL-22 relative to <i>Human Acidic Ribosomal Protein (HARP)</i> in normal (n = 9), SCC (n = 9), SCC peritumoral (n = 9), TSCC (n = 7), and TSCC peritumoral tissue (n = 7). Data expressed as mean relative mRNA expression ± standard error. Asterisks (*) indicate statistical significance, where <i>*P<0.05</i>.</p

Transplant associated SCC (TSCC) shows diffuse Ki-67 staining and increased numbers of Ki-67+ cells.

<p>Representative immunohistochemistry at (a) 10X and (b) 20X, with (c) mean cell count values of Ki-67+ cells in SCC (n = 5) and TSCC (n = 5). T indicates tumor. Only cells along or within tumors were counted. Asterisks (*) indicate statistical significance, where *P<0.05. Bar = 100 µm. (d) Ki-67 (red) did not colocalize with CD3 (green) in SCC and TSCC. (e) Intracytoplasmic staining of CK5/6 (red) colocalizes with intranuclear staining of KI-67(green).</p