The differential production of cytokines by human Langerhans cells and dermal CD14+ DCs controls CTL priming

Abstract

IntroductionChronic diseases such as cancer and viral infections have long resisted the development of effective therapeutic vaccines. It is currently thought that such vaccines will need to effectively harness dendritic cells (DCs) to induce specific and long-lasting cellular immunity, in particular cytotoxic T cells of higher potency. 1,2 Therefore, understanding the combination of signals that promote activation, proliferation, differentiation, and survival of effector CD8 ϩ T cells is crucial for the development of such vaccines. [3][4][5] The immunologic synapse is formed between a T cell and an antigen presenting cell (APC) and delivers a unique combination of signals that ultimately shape the type and strength of the T-cell response. 6 Ag dose, costimulatory molecules, and cytokines can all direct the fate of naive T-cell differentiation to mature effector cells. 7-10 IL-12 and IFN-␣, as well as the costimulatory molecules CD70 and 4-1BBL, for example, regulate and fine-tune the magnitude and duration of the effector CD8 ϩ T-cell response, as well as the nature of the elicited memory T-cell population within the immunologic synapse. 11 The diverse subpopulations of DCs provide the differential composition of molecules and receptors that allows for a specialized immune response to occur. 12-15 Indeed, as we have reported, Langerhans cells (LCs) are potent activators of naive CD8 ϩ T cells compared with dermal CD14 ϩ DCs. 16 Dermal CD1a ϩ DCs show an intermediate activity and are able to induce effector CD8 ϩ T-cell differentiation, although not as robustly as do LCs. 16 We sought to examine the role of the epidermal and dermal DC subset-specific cytokines IL-15 and IL-10 in the differential capacity to induce CD8 ϩ T-cell responses. [16][17][18] In humans, IL-15-differentiated DCs are particularly efficient at priming melanomaspecific CD8 ϩ T cells into CTLs. 19,20 In contrast, IL-10-treated DCs were shown to induce anergic CD8 ϩ T-cell responses. 21,22 Given their unique cytokine expression profile and their differential ability to prime effector CD8 ϩ T cells, we assessed the direct contribution of skin DC-specific cytokines to the quality of a primary CD8 ϩ T-cell response. Methods DC subsetsIn vitro DC subsets were differentiated from CD34 ϩ hematopoietic progenitor cells that were isolated from the blood of G-CSF-mobilized healthy volunteers. Hematopoietic progenitor cells were cultured at 0.5 ϫ 10 6 cells/mL in Yssel medium (Irvine Scientific) supplemented with 5% autologous serum, 50M ␤-mercaptoethanol, 1% L-glutamine, 1% penicillin/streptomycin, GM-CSF (50 ng/mL; Genzyme), Flt3-L (100 ng/ mL; R&D Systems), and TNF-␣ (10 ng/mL; R&D Systems) for 9 days. Media and cytokines were refreshed at day 5 of culture. Subsets of DCs, CD1a ϩ CD14 Ϫ (in vitro LCs) and CD1a Ϫ CD14 ϩ DCs (in vitro CD14 ϩ DCs) were then purified by cell sorting after staining with anti-CD1a FITC (Dako) and anti-CD14 APC mAbs (Invitrogen), yielding a purity of 95%-99%.Epidermal LCs, dermal CD1a ϩ DCs, and dermal CD14 ϩ DCs were puri...

In situloading of skin dendritic cells with apoptotic bleb-derived antigens for the induction of tumor-directed immunity

“…It has recently been described that CD14 C DDCs are far less potent in effectively priming CD8 C T cells to differentiate into cytotoxic effector cells, as compared to CD1a C DDCs and LCs, and moreover induce regulatory T cells, a process driven by the production of IL-10 and the expression of Ig-like transcript inhibitory receptors. [32][33][34] Interestingly, next to the predominant migration of CD1a C DDCs and a significant increase in uptake by this potent DDC subset, injecting 4/GM greatly diminished the migration of CD14…”

“…The modest increased uptake of blebs compared to ACR (14.8% versus 9.2%, respectively), and thereby the higher number of MART-1 [26][27][28][29][30][31][32][33][34][35] presenting skin DCs, is unlikely to be solely responsible for the selective activation of the TAA-specific CD8 C CTLs. It is known that heat shock proteins (HSPs), previously described to be present at high levels in apoptotic blebs, 35 greatly facilitate cross-presentation.…”

“…injected in skin explants from HLA-A2 positive donors in the presence of 4/GM, and skin DC were harvested after 48-72 hours. After a subsequent 24 hours of co-culture with a MART-1 [26][27][28][29][30][31][32][33][34][35] recognizing CTL line (HLA-A2 restricted; >95% pure), the supernatant was analyzed for the presence of IFNg, as a measure of cross-presentation of the MART-1 [26][27][28][29][30][31][32][33][34][35] epitope by egressed DC. The MART-1 [26][27][28][29][30][31][32][33][34][35] specific IFNg production was assessed by deducting the IFNg production following a co-culture with DC from wild-type ACR or blebs injected skin, from that secreted after co-culturing with DC from MART-1-transduced ACR or blebs (D IFNg, Fig.…”

“…C and CD8 C T-cell proliferation (n D 7). (B) DCs that egressed from the skin biopsies after injecting either wild-type or MART-1 expressing apoptotic cell remnants (ACRs) or blebs, were co-cultured with a MART-1 [26][27][28][29][30][31][32][33][34][35] recognizing and HLA-A2 restricted cytotoxic T lymphocyte (CTL) line (> 95% pure) for 24 hours. Antigen-specific activation was determined by deducting the levels of interferon g (IFNg) produced following the injection of wild-type ACRs or blebs from the levels produced after injecting MART-1 expressing apoptotic fractions (n D 3).…”

“…12 This effect is most likely mediated by IL-10, as co-culture of IL-10-conditioned skin emigrated DC with T cells leads to a diminished T-cell proliferation, T H 2 skewing, an increase in regulatory T cells, and a reduced priming of functional cytotoxic T cells. 12,[32][33][34] The negative modulation of the skin surrounding the tumor area, or even systemically under advanced metastatic conditions, can thereby hamper i.d. vaccination strategies.…”