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. 2010 Jun;130(6):1726-36.
doi: 10.1038/jid.2009.362. Epub 2009 Nov 19.

Molecular analysis of tumor-promoting CD8+ T cells in two-stage cutaneous chemical carcinogenesis

Bernice Y Kwong  1 , Scott J Roberts, Tobias Silberzahn, Renata B Filler, Jason H Neustadter, Anjela Galan, Swapna Reddy, William M Lin, Peter D Ellis, Cordelia F Langford, Adrian C Hayday, Michael Girardi
Affiliations

Molecular analysis of tumor-promoting CD8+ T cells in two-stage cutaneous chemical carcinogenesis

Bernice Y Kwong et al. J Invest Dermatol. 2010 Jun.

Abstract

T-pro are tumor-infiltrating TCRalphabeta(+)CD8(+) cells of reduced cytotoxic potential that promote experimental two-stage chemical cutaneous carcinogenesis. Toward understanding their mechanism of action, this study uses whole-genome expression analysis to compare T-pro with systemic CD8(+) T cells from multiple groups of tumor-bearing mice. T-pro show an overt T helper 17-like profile (high retinoic acid-related orphan receptor-(ROR)gammat, IL-17A, IL-17F; low T-bet and eomesodermin), regulatory potential (high FoxP3, IL-10, Tim-3), and transcripts encoding epithelial growth factors (amphiregulin, Gro-1, Gro-2). Tricolor flow cytometry subsequently confirmed the presence of TCRbeta(+) CD8(+) IL-17(+) T cells among tumor-infiltrating lymphocytes (TILs). Moreover, a time-course analysis of independent TIL isolates from papillomas versus carcinomas exposed a clear association of the "T-pro phenotype" with malignant progression. This molecular characterization of T-pro builds a foundation for elucidating the contributions of inflammation to cutaneous carcinogenesis, and may provide useful biomarkers for cancer immunotherapy in which the widely advocated use of tumor-specific CD8(+) cytolytic T cells should perhaps accommodate the cells' potential corruption toward the T-pro phenotype. The data are also likely germane to psoriasis, in which the epidermis may be infiltrated by CD8(+) IL-17-producing T cells.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1
Figure 1. CD8+ T cells infiltrating cutaneous tumors induced by two-stage chemical carcinogenesis are associated with tumor promotion
Tumors were induced on the dorsal skin of FVB wild-type (wt), CD4−/−, and TCRβ−/− mice using low-dose (LD) and high-dose (HD) protocols of two-stage chemical carcinogenesis. Under both protocols, augmented tumor susceptibility was greatest in CD8-intact (CD4−/−; wt) mice. (a, week 12; b, week 15): At LD, wt controls produced greater (although not statistically distinguishable) tumor burdens than (TCRβ−/−) mice deficient in all TCRαβ+ T cells, whereas CD4−/− mice showed significantly higher tumor susceptibility (P<0.002) than TCRβ−/− mice. (c, week 12; d, week 15): At HD, tumor burdens were comparably high in both wt and CD4−/− strains compared with TCRβ−/− mice (P<0.001 for wt and P<0.00004 for CD4−/−). Error bars, ±SE. (e) Tumors that developed under two-stage chemical carcinogenesis were scored as clinically apparent papillomas or carcinomas. Tumor appearance of two representative mice from each of the wt, CD4−/−, and TCRβ−/− cohorts are shown. (f) Immunohistochemistry readily identified CD8+ cells within tumors (T), but less so in the stroma (S). CD4−/− mouse tumor sections shown; F denotes follicle. Bar = 500, 125, and 60 μm, respectively.
Figure 2
Figure 2. Illumina expression analysis of CD8+TCRβ+CD44HICD62LO TIL and PBL populations
mRNA from highly purified (>98%) CD44HICD62LLOTCRβ+CD8+ tumor-infiltrating lymphocyte (TIL) and peripheral blood lymphocyte (PBL) from tumor-bearing (Tum+) CD4−/− and Tum+ wild-type (wt) mice were analyzed by Illumina Sentrix bead chips (~47,000 genes). (a) Absolute probe hybridization values were consistent with high-fidelity purifications. (b) Relative to the naive PBL, a total of 800 (~1.7%) genes were found to be >10-fold differentially expressed by the TIL and PBL sorts in Tum+ mice. The vast majority of these (91%) were within the TIL sets. (c) Hierarchical clustering analysis showed that the PBL populations isolated from tumor-bearing (Tum+) mice were similar to phenotypically equivalent (CD44HICD62LLOTCRβ+CD8+) effector T cells purified from PBL of naive animals. The dendrogram shows that the primary clusters based on global gene expression differentiate between PBL and TIL, whereas the heatmap (red, high expression; blue, low expression) highlights some of the most differentially expressed genes defining these two major groups. (d) Analysis of the most differentially expressed genes unique to CD8+ T-pro (based on absolute probe values) showed differentiation along the RORγt pathway, poor cytotoxic potential, and production of regulatory mediators/growth factors.
Figure 3
Figure 3. Real-time RT-PCR validation of differentially expressed genes in CD8+ TIL/PBL
Quantitative reverse transcriptase-PCR of mRNA isolated from FVB mouse tumor-infiltrating lymphocyte (TIL) and peripheral blood lymphocyte (PBL) (stained and sorted to >99.5% purity for TCRαβ+CD8+CD44HICD62LLO) validated key findings from the Illumina expression analysis. Fold-expression differences of TIL/PBL confirmed (a, b) TH17 differentiation, (c) low cytotoxic potential, and (d) increased expression of regulatory mediators and growth factors. Error bars, ±SD. Independent isolates (n = 3) for all genes, except Gro-1 and Gro-2 (n = 2). *Ratios beyond designated axes limits.
Figure 4
Figure 4. Confirmation of IL-17 production by CD8+ TIL (T-pro)
(a) Representative Amnis multispectral imaging identified cells coexpressing TCRβ, IL-17A, and CD8β. (b) These analyses showed that IL-17-producing T cells represented a small minority of the CD8+ tumor-infiltrating lymphocyte (TIL) (mean 6.80%; 5 separate isolations).
Figure 5
Figure 5. CD8+ T-pro expression profile is associated with malignant progression
(a) TIL from week 16 carcinomas (malignant) versus papillomas (benign) were sorted by MoFlo to >99% purity for CD8+TCRβ+CD44HICD62LLO and each compared with tumor-infiltrating lymphocyte (TIL) from week 12 aggregate tumors (that is, preponderance of papillomas) by quantitative reverse transcriptase (qRT)-PCR. (b–e) The “T-pro profile” was more strongly associated with the CD8+ TIL of malignant tumors (red) than benign tumors (blue) as shown by qRT-PCR. Fold difference of TIL from week 16 carcinomas (malignant) or week 16 papillomas (benign) compared with week 12 aggregate tumors. n = 15 mice per group; error bars, SD of triplicate wells; n.d., nondetectable; *Ratios beyond designated axes limits.
Figure 6
Figure 6. CD8+ T-cell corruption hypothesis
Antitumor CD8+ cytolytic T cells (CTLs) may be redirected into CD8+ T-pro that inhibit T cell-mediated responses (for example, through IL-10), enhance chronic inflammation (for example, through IL-17A and IL-17F), and facilitate tumor proliferation (for example, through AREG and Gro-1).
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