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.

Double-stranded RNA (dsRNA), a frequent byproduct of virus infection, is recognized by Toll-like receptor 3 (TLR3) to mediate innate immune response to virus infection. TLR3 signaling activates the transcription factor IRF-3 by its Ser/Thr phosphorylation, accompanied by its dimerization and nuclear translocation. It has been reported that the Ser/Thr kinase TBK-1 is essential for TLR3-mediated activation and phosphorylation of IRF-3. Here we report that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways. One involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells. Thus, the PI3K-Akt pathway plays an essential role in TLR3-mediated gene induction.

Early diagnosis of CTCL is difficult and takes on average six years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL but the TCRγ PCR analysis in current clinical use detect clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46/46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting a niche of finite size may exist for benign T cells in skin. Lastly, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases and provided insights into the cell of origin and location of malignant CTCL cells in skin.

Mycosis fungoides (MF), the most common cutaneous T cell lymphoma (CTCL) is a malignancy of skin-tropic memory T cells. Most MF cases present as early stage (stage I A/B, limited to the skin), and these patients typically have a chronic, indolent clinical course. However, a small subset of early-stage cases develop progressive and fatal disease. Because outcomes can be so different, early identification of this high-risk population is an urgent unmet clinical need. We evaluated the use of next-generation high-throughput DNA sequencing of the T cell receptor β gene () in lesional skin biopsies to predict progression and survival in a discovery cohort of 208 patients with CTCL (177 with MF) from a 15-year longitudinal observational clinical study. We compared these data to the results in an independent validation cohort of 101 CTCL patients (87 with MF). The tumor clone frequency (TCF) in lesional skin, measured by high-throughput sequencing of the gene, was an independent prognostic factor of both progression-free and overall survival in patients with CTCL and MF in particular. In early-stage patients, a TCF of>25% in the skin was a stronger predictor of progression than any other established prognostic factor (stage IB versus IA, presence of plaques, high blood lactate dehydrogenase concentration, large-cell transformation, or age). The TCF therefore may accurately predict disease progression in early-stage MF. Early identification of patients at high risk for progression could help identify candidates who may benefit from allogeneic hematopoietic stem cell transplantation before their disease becomes treatment-refractory.

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Innate immune response to viral infection is often triggered by Toll-like receptor 3 (TLR3)-mediated signaling by doublestranded (ds) RNA, which culminates in the activation of the transcription factor NF-B and induction of NF-B-driven genes. We demonstrated that dsRNA-induced phosphorylation of two specific tyrosine residues, 759 and 858, of TLR3 was necessary and sufficient for complete activation of the NF-B pathway. When Tyr-759 of TLR3 was mutated, gene induction was inhibited, although NF-B was partially activated. It was released from IB and translocated to the nucleus but failed to bind to the B site of the target A20 gene promoter. This defect could be attributed to incomplete phosphorylation of the RelA (p65) subunit of NF-B, as revealed by two-dimensional gel analyses of p65, isolated from dsRNA-treated cells expressing either wild type TLR3 or the Tyr-759 3 Phe mutant TLR3. Thus, two phosphotyrosine residues of TLR3 activate two distinct pathways, one leading to NF-B release and the other leading to its phosphorylation.Innate immune response to viral infection of mammalian cells is mediated by the products of viral stress inducible genes (1). Many of these genes are transcriptionally induced when cells are treated with double-stranded RNA (dsRNA), 2 a common by-product of virus infection. Several cellular proteins, that can recognize dsRNA and mediate gene induction, have been identified recently. They include the cytoplasmic RNAhelicases, RIG-I and Mda-5, and the membrane-bound protein Toll-like receptor 3 (TLR3) (2-4). The signaling pathways activated by these dsRNA receptors, converge to activate the same set of transcription factors, namely, NF-B, IRF-3/IRF-7, and members of the AP-1 family. Consequently, genes induced by the TLR3 and the RIG-I signaling pathways are largely overlapping.We have been analyzing the TLR3-mediated signaling pathways activated by dsRNA in a HEK293 cell based experimental system. TRIF is the unique adaptor protein that connects TLR3 to the IKK complex which activates NF-B. It also connects TLR3 to TBK-1, the enzyme that activates IRF-3 (5). We have studied, in detail, the characteristics of signaling to IRF-3 by TLR3 and demonstrated that phosphorylation of two separate tyrosine residues, located in the cytoplasmic domain of TLR3, is essential for complete activation of IRF-3 (6, 7). One phosphotyrosine, 858, triggers TBK-1 activation, leading to partial phosphorylation of IRF-3 causing its dimerization and nuclear translocation. However, this partially phosphorylated IRF-3 cannot bind efficiently to the cognate sites in the promoters of target genes to drive their transcription. For full activation of IRF-3, its further phosphorylation is needed. The second step of phosphorylation of IRF-3 requires the action of PI 3-kinase, although the proximal kinase that directly phosphorylates IRF-3 remains to be identified. Engagement of PI 3-kinase by TLR3 requires ligand-induced phosphorylation of another residue, Tyr-759. Thus, ligand-induced phosphorylation of Tyr-759 and Ty...