Human Langerhans cells use an IL-15R-α/IL-15/pSTAT5-dependent mechanism to break T-cell tolerance against the self-differentiation tumor antigen WT1

Abstract

IntroductionThe dendritic cell (DC) hematopoietic lineage comprises heterogeneous subsets that share the capacity for potent initiation and control of innate and adaptive immunity, 1-5 but with sufficient plasticity to orchestrate the quantity and quality of lymphocyte responses. Indeed, defined subsets of human DCs generated with cytokines in vitro also yield sufficient numbers with counterparts in vivo to discern distinct consequences to T cells interacting with one or another subtype. [6][7][8][9][10][11][12] Human Langerhans-type DCs derived from CD34 ϩ hematopoietic progenitor cells (HPCs) have consistently proven superior to other conventional DC subsets as stimulators of CD8 ϩ cytolytic T lymphocytes (CTLs) in vitro. 9,11 This has led some investigators to advocate inclusion of CD34 ϩ HPC-derived Langerhans cells (LCs) in DC vaccines 13,14 rather than relying solely on the more commonly used monocyte-derived DCs (moDCs). The mechanisms underlying LC potency have remained elusive, but IL-15 has emerged as an important cytokine mediator. 9,14 First, LCs' potent stimulation of CD8 ϩ CTLs occurs in the complete absence of IL-12p70, 9,15 which has proven more critical for the activation of NK cells 15 than for CD8 ϩ CTLs. 9 Despite several shared functions, IL-15 also has important contrasting roles with IL-2. [16][17][18] IL-2 controls autoreactive T cells through their activation-induced cell death, or apoptosis, and the maintenance and expansion of Tregs. In contrast, IL-15 is critical to the generation of durable, high-avidity, memory CD8 ϩ T cells, which could include autoimmune populations with specificity for self-differentiation tumor antigens. [16][17][18] LCs secrete more IL-15 than any other conventional DC subtype, 9,15 but investigators have not previously identified IL-15R-␣ on the surface of LCs. This ␣-subunit is required for transport of IL-15 to the cell membrane to bind ␤ (CD122) and ␥ (CD132) chains on responding lymphocytes. Only then is the biologically active, heterotrimeric receptor-cytokine complex complete, supporting subsequent lymphocyte signaling and activation. [16][17][18] We therefore undertook alternative approaches to define the expression of IL-15R-␣ by resident populations of primary human LCs emigrating from cultured human epidermal sheets, as well as LCs, dermal-interstitial DCs (DDC-IDCs), and moDCs generated with recombinant human cytokines in vitro. 9 Eliminating exposure to IL-15 during DC development allowed comparison of tumorspecific CTLs stimulated by LCs with those elicited by moDCs, as Methods Media and noncytokine supplementsComplete RPMI 1640 included 10mM HEPES, 1% penicillin/streptomycin (Media Lab, Memorial Sloan-Kettering Cancer Center [MSKCC]), 50M 2-mercaptoethanol (Invitrogen), 1% L-glutamine (Invitrogen), and 1% or 10% volume/volume heat-inactivated, pooled normal human serum (NHS; Atlanta Biologicals). X-VIVO 15 (BioWhittaker; Lonza Walkersville) was used as manufactured without additives. All media and reagents were endotoxin-free. Human cells, media...

Phenotypic and Functional Activation of Hyporesponsive KIRnegNKG2Aneg Human NK-Cell Precursors Requires IL12p70 Provided by Poly(I:C)-Matured Monocyte-Derived Dendritic Cells

“…LCs and moDCs (collectively termed DCs, except when the specific subtypes are specified) were generated respectively from either tissue culture plastic-adherent peripheral blood monocytes or CD34 pos hematopoietic progenitor cells, as published (8, 25). Both immature moDCs and LCs were terminally matured in 6-well tissue culture plates (Costar; Corning, NY) at a concentration of 1×10 6 cells /3 mL/well, by exposure for two days to either a combination of inflammatory cytokines (IL-1β (2 ng/mL), IL-6 (1000 IU/mL), TNF-α (10 ng/mL), and prostaglandin E2 (5 µM) (25)), termed LC cyto or moDC cyto ; TLR3 ligand (25 µg/ml poly(I:C); Invivogen; San Diego, CA), termed LC poly or moDC poly ; or TLR4 ligand (10 ng/ml LPS, Sigma-Aldrich), termed LC LPS or moDC LPS .…”

“…Monocyte-derived DCs (moDC), generated in vitro and corresponding to inflammatory DCs in vivo (1), are critical for activating resting mature NK cells (5–7), while Langerhans-type DCs (LC) are essential to sustaining activated NK-cell viability through their provision of IL-15 (5, 8). In contrast to prior studies emphasizing bulk NK cells, the DC-based mechanisms for the development of a functionally responsive NK-cell repertoire from hyporesponsive KIR neg NKG2A neg precursors, which could in turn be manipulated for more effective immunotherapy, remain important unknowns.…”

“…By exposing moDCs and LCs (5, 8, 25) to a variety of maturation stimuli, including a combination of inflammatory cytokines (general inflammation) (25); LPS (bacterial TLR4 ligand); or poly(I:C) (viral TLR3 ligand), we recapitulated the inflammatory scenarios in which other groups have reported functional NK maturation (12, 19, 24, 26–30), thereby ascertaining whether and how activated conventional DC subtypes support the generation of a functional NK-cell repertoire. Our findings have important implications for generating functional NK cells for immunotherapy, where activation by exogenous cytokines alone has not proven optimally effective and the resulting activation-induced cell death has compromised NK-cell expansion after administration in vivo .…”

Duality at the gate: Skin dendritic cells as mediators of vaccine immunity and tolerance

“…This co-stimulatory cytokine results in increased expansion of the T cell response through STAT5 signaling, even when presenting a normally tolerizing antigen, such as the tumor antigen Wilm's Tumor-1. 108 LCs may also be uniquely prone to tailoring the humoral response to antigens toward an IgG1 response, as the loss of LCs resulted specifically in the decrease of this antibody isotype in a gene gun vaccination model. 83 In the setting of infectious disease, LCs have a demonstrated role in adaptive immunity to C. Albicans.…”

Dendritic Cells as Pharmacological Tools for Cancer Immunotherapy

“…As discussed above, LCs, presumably via a mechanism that involves IL-15, are far more superior than dermal DCs and moDCs (Romano et al, 2012;Hutten et al, 2014) in terms of their capacity to stimulate antigen-specific CTL immunity. This explains the impetus behind the recent efforts to design new protocols for the generation of DCs with LC-like features (Hutten et al, 2014).…”