Chronic UV radiation–induced RORγt+ IL-22–producing lymphoid cells are associated with mutant KC clonal expansion

Chronic ultraviolet (UV) radiation exposure is the greatest risk factor for cutaneous squamous cell carcinoma (cSCC) development, and compromised immunity accelerates this risk. Having previously identified that epidermal Langerhans cells (LC) facilitate the expansion of UV-induced mutant keratinocytes (KC), we sought to more fully elucidate the immune pathways critical to cutaneous carcinogenesis and to identify potential targets of intervention. Herein, we reveal that chronic UV induces and LC enhance a local immune shift toward RORγt+ interleukin (IL)-22/IL-17A–producing cells that occurs in the presence or absence of T cells while identifying a distinct RORγt+ Sca-1+ CD103+ ICOS+ CD2^+/− CCR6+ intracellular CD3+ cutaneous innate lymphoid cell type-3 (ILC3) population (uvILC3) that is associated with UV-induced mutant KC growth. We further show that mutant KC clone size is markedly reduced in the absence of RORγt+ lymphocytes or IL-22, both observed in association with expanding KC clones, and find that topical application of a RORγ/γt inhibitor during chronic UV exposure reduces local expression of IL-22 and IL-17A while markedly limiting mutant p53 KC clonal expansion. We implicate upstream Toll-like receptor signaling in driving this immune response to chronic UV exposure, as MyD88/Trif double-deficient mice also show substantially reduced p53 island number and size. These data elucidate key immune components of chronic UV–induced cutaneous carcinogenesis that might represent targets for skin cancer prevention.

The capacity to identify and therapeutically target the major relevant immune pathways driving disease pathogenesis has proven particularly advantageous for controlling inflammatory cutaneous disorders. This has occurred despite the complexity of lymphoid cell subtypes and cytokine receptor networks active within cutaneous lesions and draining lymph nodes. Marked durable responses are now possible with antibody-based therapeutic agents disrupting, for example, the interleukin (IL)-23/IL-17A (Th17) axis in psoriasis vulgaris (1) and the IL-4/IL-13 (Th2) axis in atopic dermatitis (2).Exposure to ultraviolet (UV) light results in extensive DNA damage to keratinocytes (KC) because of two major phenomena. UV exposure, mostly in the type B UV (UVB) bandwidths, directly targets genomic DNA as a chromophore in which the absorbed energy readily and immediately induces cyclobutane pyrimidine dimers. This is complimented by a more complex indirect (and relatively delayed) series of energy transfers initiated by epidermal molecules, for example, melanin degradation products, that absorb/transfer UV energy before giving rise to triplet state intermediates that may also eventuate into DNA damage (3). Possible fates for such UV-damaged KCs include enzymatic DNA repair, elimination via apoptosis induction, metabolic stress that induces NKG2D-ligand surface expression to mark the damaged cells for elimination, and acquisition of mutations within cell cycle regulators (e.g.TP53, CDKN2A) and stimulators (e.g., RAS, NOTCH-1,2,3) that endow apoptosis resistance and clonal persistence.Martincorena et al. (4) have definitively shown by genomic deep sequencing that such driver mutations persist within chronically sun-exposed skin and that mutant-associated KC clones accumulate as epidermal collections long before the emergence of clinically apparent skin tumors of premalignant actinic keratoses (AK) and cutaneous squamous cell carcinomas (cSCC). Currently, clinical efforts in preventing the outgrowth of these skin neoplasms have included the topical application of chemotherapeutic (e.g., 5-fluorouracil) or immunostimulatory (e.g., imiquimod) agents or the administration of systemic retinoids in attempt to minimize, eliminate, and/or differentiate the mutant clones (5). In this context, we hypothesize that elucidating the immunopathogenesis of chronic UV exposure might identify other potential targets and strategies for skin cancer prevention.It is in this setting of epithelial tissue harboring of persistent driver mutant clones that chronic inflammatory states have long been appreciated for their association with malignant development and progression, for example, inflammatory bowel disease and increased risk of colon cancer (6). Indeed, locally secreted IL-17A and IL-22, both known stimulators of epithelial proliferation, have been implicated in epithelial tumor promotion, including within the gut, lung, and skin (7–9). Nonetheless, IL-22–inhibiting antibodies have met with limited clinical success in preventing or treating intestinal tumors, and while anti–IL-22 antibodies injected into established cSCC might prevent progression (10), the role of IL-22–producing cells in mutant KC clonal expansion and photocarcinogenesis prior to tumor emergence has not been fully elucidated.Recognizing that UVR exposure induces an epidermal damage–driven inflammatory response, we sought to more fully characterize the effects of chronic UV exposure on the local cutaneous immune milieu relevant to the promotion of photocarcinogenesis. A cascade of KC-derived triggers of the local immune response are precipitated by acute UV exposure in the skin, including Toll-like receptor (TLR) ligands (e.g., U1 RNA), antimicrobial peptides (e.g., beta-defensins, S100A7), thymic stromal lymphopoietin (TSLP), and proinflammatory cytokines (e.g., IL-1, IL-36). In addition, chronic UVR has been reported to markedly decrease epidermal Langerhans cells (LC) and increase local and systemic levels of cytokines (e.g., IL-10, TGFβ) that down-regulate cellular antitumor immunity. However, little is known about if and how chronic UV alters the predominant local immune milieu to promote mutant KC clonal expansion so fundamental to cutaneous carcinogenesis. Having previously demonstrated that the absence of epidermal LC markedly decreases mutant KC clone growth during chronic UV exposure, we also considered whether targeting any identifiable associated dominant immune cell population and signals might also limit the expansion of mutant KC and thereby uncouple the tumor-promoting effects of UV-associated inflammation. Herein, we reveal that chronic UV exposure stimulates specific skin immune cell populations, including RORγt-differentiated innate lymphoid cells (ILC) and T cells, that shift the local cytokine milieu to an IL-22/IL-17A–rich state in association with mutant KC growth. Moreover, we identify and implicate a distinct cutaneous (CD4- NKp46- MHCII-) ILC type-3 (ILC3) population (that we term uvILC3) of phenotype RORγt+ Sca-1+ CD103+ ICOS+ CD2^+/− CCR6+ intracellular (ic)CD3+ that drives chronic UV–induced mutant KC clonal expansion in the absence of T cells. We believe that this deciphering of the immunopathogenesis of UV carcinogenesis identifies potential translational targets for cutaneous cancer prevention for patients with chronically photodamaged (mutant clone-harboring) skin and, in particular, potentially for those patients (e.g., organ transplant recipients) with iatrogenically compromised T cell function.