Antigen Presentation Capacity and Cytokine Production by Murine Splenic Dendritic Cell Subsets upon Salmonella Encounter 1 Ulf Yrlid and Mary Jo Wick 2 Salmonella typhimurium is an intracellular bacterium that replicates in the spleen and mesenteric lymph nodes (MLN) of orally infected mice. However, little is known about the Ag presentation and cytokine production capacity of dendritic cells (DC), particularly CD8  , CD8  CD4  , and CD8  CD4  DC, from these organs in response to Salmonella. Infection of purified splenic DC with S. typhimiurium expressing green fluorescent protein (GFP) and OVA revealed that all three splenic DC subsets internalize bacteria, and splenic as well as MLN DC process Salmonella for peptide presentation. Furthermore, presentation of Salmonella Ags on MHC-I and MHC-II was evident in both CD8  and CD8  splenic DC subsets. Direct ex vivo analysis of splenic DC from mice infected with GFP-expressing Salmonella showed that all three subsets harbored bacteria, and splenic DC purified from mice given Salmonella-expressing OVA presented OVA-derived peptides on MHC-I and MHC-II. Cytokine pro- duction analyzed by intracellular staining of splenic DC infected with GFP-expressing Salmonella revealed that TNF- was produced by a large percentage of CD8  DC, while only a minor proportion of CD8  DC produced this cytokine following bacterial exposure. In contrast, the greatest number of IL-12p40-producing DC were among CD8  DC. Experiments inhibiting bacterial uptake by cytochalasin D as well as use of a Transwell system revealed that bacterial contact, but not internalization, was required for cytokine production. Thus, DC in sites of Salmonella replication and T cell activation, spleen and MLN, respond to bacterial encounter by Ag presentation and produce cytokines in a subset-specific fashion. The Journal of Immunology, 2002, 169: 108 –116. D endritic cells (DC) 3 are bone marrow-derived cells of rare frequency but wide distribution in peripheral tis- sues. These migratory cells collect Ags in the periphery and transport them to draining lymph nodes for presentation to T cells (1, 2). In contrast to DC precursors in blood and peripheral tissues, DC in secondary lymphoid organs have, since their dis- covery almost 30 years ago, been considered to be nonphagocytic (3, 4). More recent studies, however, suggest that DC in lymphoid organs, particularly the spleen, have endocytic and phagocytic ca- pacity (5–11). Murine splenic DC were originally divided into two subsets based on surface expression of CD8 (12). The observation that CD8 - splenic DC could be further divided into CD4 + and CD4 - populations resulted in the definition of three major murine splenic DC subsets, CD8 + , CD8 - CD4 + (CD4 + ), and CD8 - CD4 - double-negative (DN) subsets (7, 13, 14), which appear to develop along independent pathways (7). The DC subsets have a differen- tial capacity to secrete cytokines upon microbial stimulation (15– 21). Recent studies have also suggested that the splenic DC subsets may differ in their capacity to take up and present soluble or par- ticulate Ags (5– 8). A critical feature of DC is their capacity to activate naive T cells (1). Despite this, the role of DC, particularly DC subsets, in trig- gering T cells during bacterial infection has not been established. Using a murine infection model with the Gram-negative bacterium Salmonella enterica serovar Typhimurium (Salmonella typhi- murium) it has been shown, however, that both splenic (22) and Peyer’s patch (23) DC harbor Salmonella during infection. Fur- thermore, splenic DC are activated in Salmonella-infected mice (22), and bone marrow-derived DC loaded with S. typhimurium can elicit bacteria-specific CD4 + and CD8 + T cells after transfer into naive animals (22). In addition, the three splenic DC subsets are differentially modulated with respect to distribution, number, and cytokine production in response to oral Salmonella infection (16). Despite these observations, and the finding that immature bone marrow-derived DC can process Salmonella for peptide pre- sentation on MHC-I and MHC-II (24 –26), the capacity of DC from lymphoid organs to process bacteria for peptide presentation is not known. Thus, the present study examined the capacity of DC from spleen and MLN, sites of Salmonella replication and T cell acti- vation, to present bacterial Ags. In addition, the capacity of splenic DC subsets to internalize bacteria and produce cytokines upon in- fection as well as the relationship between bacterial internalization and cytokine production were examined. Materials and Methods Mice BALB/c, C57BL/6, C3H/HeN, C3H/HeJ, and OT-I (27) mice were bred at the animal facilities of Lund University (Lund, Sweden) or purchased from Department of Cell and Molecular Biology, Section for Immunology, Lund Univer- sity, Lund, Sweden Received for publication December 13, 2001. Accepted for publication April 29, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by funds from the Swedish Research Council (Project 621-2001-1720, K2001-16X-14005), the Koch Foundation, the O ¨ sterlund Founda- tion, the Crafoord Foundation, and the Lund University medical faculty. 2 Address correspondence and reprint requests to Dr. Mary Jo Wick at the current address: Department of Clinical Immunology, University of Goteborg, Guldhedsgatan 10A, SE-413 46 Goteborg, Sweden. E-mail address: mary-jo.wick@immuno.gu.se 3 Abbreviations used in this paper: DC, dendritic cell; BFA, brefeldin A; CCD, cy- tochalasin D; DN, double negative; Flt3L, Flt3 ligand; GFP, green fluorescent protein; HEL, hen egg white lysozyme; LB, Luria-Bertani; MLN, mesenteric lymph node; PFA, paraformaldehyde. The Journal of Immunology Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00