Tumor-specific T cells in human Merkel cell carcinomas: a possible role for Tregs and T cell exhaustion in reducing T cell responses

Merkel cell carcinomas (MCC) are rare but highly malignant skin cancers associated with a novel polyomavirus. MCC tumors were infiltrated by T cells, including effector, central memory and regulatory T cells. Infiltrating T cells showed markedly reduced activation as evidenced by reduced expression of CD69 and CD25. Treatment of MCC tumors in vitro with IL-2 and IL-15 led to T cell activation, proliferation, enhanced cytokine production and loss of viable tumor cells from cultures. Expanded tumor-infiltrating lymphocytes showed TCR repertoire skewing and upregulation of CD137. MCC tumors implanted into immunodeficient mice failed to grow unless human T cells in the tumor grafts were depleted with denileukin diftitox, suggesting tumor-specific T cells capable of controlling tumor growth were present in MCC. Both CD4+ and CD8+ FOXP3+ regulatory T cells were frequent in MCC. 50% of non-activated T cells in MCC expressed PD-1, a marker of T-cell exhaustion, and PD-L1 and PD-L2 were expressed by a subset of tumor dendritic cells and macrophages. In summary, we observed tumor-specific T cells with suppressed activity in MCC tumors. Agents that stimulate T cell activity, block Treg function or inhibit PD-1 signaling may be effective in the treatment of this highly malignant skin cancer

HIV-1 Neutralization Profile and Plant-Based Recombinant Expression of Actinohivin, an Env Glycan-Specific Lectin Devoid of T-Cell Mitogenic Activity

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH's anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1′s AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide

Skin resident T cells producing TNFalfa but not TH1, TH2, or Th17 cytokines: their posible role as immune sentinels in skin.

ABSTRACT BODY: The skin of a normal individual contains nearly 20 billion memory T cells, nearly twice as many as are present in the circulation. These T cells are polarized effector memory T cells that, from our current understanding, have arisen from recognition of their antigens within the skin. We previously reported that human skin contains significant numbers Th1, Th17 and Th2 cells. We report now that these T cell subsets are quiescent with respect to cytokine production when isolated from unstimulated skin and that additional signals (e.g., cellular proliferation) are required to unlock cytokine production. TNFα is a cytokine produced by both APC and subsets of effector T cells. We describe here a novel and previously undescribed population of skin resident T cells that produces TNFα alone, without concurrent production of IFNγ, IL-4 or IL-17. These cells comprise a mean 48% of the skin T cells resident in normal human skin. These T cells are CD45RO+ memory T cells, express CLA, CCR4 and CCR6 and have a diverse TCR repertoire. This T cell subset differs from the other cutaneous effector T cells in that half of these cells produce TNFα upon TCR triggering without a requirement for additional activating signals. TNFα T cells are the most numerous subset in normal human skin, outnumbering Th1, Th2 and Th17 T cells. TNFα has pleiotropic effects in the skin. TNFα induces maturation of dendritic cells, enhancing antigen presentation, and induces endothelial activation and chemokine production, leading to enhanced leukocyte recruitment. Skin TNFα T cells, specific for pathogens previously encountered in skin, may serve as antigen-specific immune sentinels. These T cells have the ability to rapidly produce TNFα upon re-exposure to cognate antigen, leading to activation and recruitment of both innate immune cells and other T cell subsets. Thus, they may serve to both initiate and amplify immune responses in skin

High-scatter T cells: a reliable biomarker for malignant T cells in cutaneous T-cell lymphoma

In early-stage cutaneous T-cell lymphoma (CTCL), malignant T cells are confined to skin and are difficult to isolate and discriminate from benign reactive cells. We found that T cells from CTCL skin lesions contained a population of large, high-scatter, activated skin homing T cells not observed in other inflammatory skin diseases. High-scatter T (THS) cells were CD4+ in CD4+ mycosis fungoides (MF), CD8+ in CD8+ MF, and contained only clonal T cells in patients with identifiable malignant Vβ clones. THS cells were present in the blood of patients with leukemic CTCL, absent in patients without blood involvement, and contained only clonal malignant T cells. The presence of clonal THS cells correlated with skin disease in patients followed longitudinally. Clonal THS cells underwent apoptosis in patients clearing on extracorporeal photopheresis but persisted in nonresponsive patients. Benign clonal T-cell proliferations mapped to the normal low-scatter T-cell population. Thus, the malignant T cells in both MF and leukemic CTCL can be conclusively identified by a unique scatter profile. This observation will allow selective study of malignant T cells, can be used to discriminate patients with MF from patients with other inflammatory skin diseases, to detect peripheral blood involvement, and to monitor responses to therapy