The skin of an adult human contains approximately 20 billion memory T cells. Epithelial barrier tissues are infiltrated by a combination of resident and recirculating T cells in mice but the relative proportions and functional activities of resident versus recirculating T cells have not been evaluated in human skin. We discriminated resident from recirculating T cells in human engrafted mice and lymphoma patients using alemtuzumab, a medication that depletes recirculating T cells from skin, and then analyzed these T cell populations in healthy human skin. All non-recirculating resident memory T cells (TRM) expressed CD69, but the majority were CD4+, CD103− and located in the dermis, in contrast to studies in mice. Both CD4+ and CD8+ CD103+ TRM were enriched in the epidermis, had potent effector functions and had a limited proliferative capacity compared to CD103− TRM. TRM of both types had more potent effector functions than recirculating T cells. Induction of CD103 on human T cells was enhanced by keratinocyte contact, depended on TGFβ and was independent of T cell keratinocyte adhesive interactions. We observed two distinct populations of recirculating T cells, CCR7+/L-selectin+ central memory T cells (TCM) and CCR7+/L-selectin− T cells, which we term migratory memory T cells (TMM). Circulating skin-tropic TMM were intermediate in cytokine production between TCM and effector memory T cells. In patients with cutaneous T cell lymphoma, malignant TCM and TMM induced distinct inflammatory skin lesions and TMM were depleted more slowly from skin after alemtuzumab, suggesting TMM may recirculate more slowly. In summary, human skin is protected by four functionally distinct populations of T cells, two resident and two recirculating, with differing territories of migration and distinct functional activities.

SUMMARY Therapeutic antibodies targeting programmed cell death-1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNA interference, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.

Interleukin-9 is a T cell cytokine that acts through a γC-family receptor on target cells. We determined that T cells from mice deficient in the TH17 pathway genes ROR-γ and IL-23R produced abundant IL-9, and observed significant growth inhibition of B16F10 melanoma tumor in these mice. IL-9 blocking antibodies reversed this tumor growth inhibition, and enhanced tumor growth in normal mice. IL9R−/− mice showed accelerated tumor growth, while administration of rIL-9 to tumor bearing mice inhibited tumor growth. Adoptive transfer of tumor antigen-specific TH9 cells blocked tumor growth; this was reversed by anti-IL-9. Exogenous rIL-9 inhibited tumor growth in Rag1−/− mice, but not in mast cell deficient mice, suggesting a T cell independent process. Finally, we found TH9 cells in normal human skin and blood, and low IL-9 production from melanoma tumor infiltrating lymphocytes. These results suggest a role for IL-9 in tumor immunity, and suggest therapeutic strategies.

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CTCL is a cancer of skin homing T cells with variants that include leukemic CTCL (L-CTCL), a malignancy of central memory T cells (TCM), and mycosis fungoides (MF), a malignancy of skin resident effector memory T cells (TEM). We report that low-dose alemtuzumab (αCD52) effectively treated patients with refractory L-CTCL but not MF. Alemtuzumab depleted all T cells in blood and depleted both benign and malignant TCM from skin, but a diverse population of skin resident TEM remained in skin after therapy. T-cell depletion with alemtuzumab required the presence of neutrophils, a cell type frequent in blood but rare in normal skin. These data suggest that TCM were depleted because they recirculate between the blood and skin whereas skin resident TEM were spared because they are sessile and non-recirculating. After alemtuzumab treatment, skin T cells produced lower amounts of IL-4 and higher amounts of IFNγ. Moreover, there was a marked lack of infections in alemtuzumab-treated L-CTCL patients despite the complete absence of T cells in blood, suggesting that skin resident TEM can protect the skin from pathogens even in the absence of T cell recruitment from the circulation. Together, these data suggest that alemtuzumab may treat refractory L-CTCL without severely compromising the immune response to infection by depleting circulating TCM but sparing the skin resident TEM that provide local immune protection of the skin.

T helper type 9 (TH9) cells can mediate tumor immunity and participate in autoimmune and allergic inflammation in mice but little is known about the TH9 cells that develop in vivo in humans. We isolated T cells from human blood and tissues and found that most memory TH9 cells were skin-tropic or skin-resident. Human TH9 cells co-expressed TNFα and granzyme B, lacked coproduction of TH1/TH2/TH17 cytokines and many were specific for C. albicans. IL-9 production was transient and preceded the up-regulation of other inflammatory cytokines. Blocking studies demonstrated that IL-9 was required for maximal production of IFN-γ, IL-9, IL-13, and IL-17 by skin tropic T cells. IL-9 producing T cells were increased in the skin lesions of psoriasis, suggesting these cells may contribute to human inflammatory skin disease. Our results indicate human TH9 cells are a discrete T cell subset, many are tropic for the skin, and although they may function normally to protect against extracellular pathogens, aberrant activation of these cells may contribute to inflammatory diseases of the skin.

Purpose In leukemic CTCL (L-CTCL) malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased IgE, decreased Th1 responses and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, Th2 and Th17 cells. Experimental design We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells and studied the immunomodulatory effects of treatment modalities in L-CTCL patients. Results 12/12 L-CTCL patients overproduced Th2 cytokines. Remaining benign T cells were also strongly Th2 biased, suggesting a global Th2 skewing of the T cell repertoire. Culture of benign T cells away from the malignant clone reduced Th2 and enhanced Th1 responses but separate culture had no effect on malignant T cells. Co-culture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to IL-4 and IL-13 restored Th1 responses. In patients, enhanced Th1 responses were observed following a variety of treatment modalities that reduced malignant T cell burden. Conclusions A global Th2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. Th2 cytokines from malignant cells strongly inhibited Th1 responses. Our results suggest therapies that inhibit Th2 cytokine activity, by virtue of their ability to improve Th1 responses, may have the potential to enhance both anti-cancer and anti-pathogen responses.

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