Autocrine IL-12 Is Involved in Dendritic Cell Modulation Via CD40 Ligation 1 Roberta Bianchi, Ursula Grohmann, Carmine Vacca, Maria L. Belladonna, Maria C. Fioretti, and Paolo Puccetti 2 Ligation of CD40 on dendritic cells (DC) triggers production of IL-12. Using an adoptive transfer model we have previously shown that rIL-12 acts directly on DC to enhance presentation of an otherwise poorly immunogenic tumor peptide. Using the same experimental model, we now describe a similar adjuvanticity of CD40 ligation on peptide presentation by DC. We also explore the possibility that the IL-12 resulting from CD40 ligation directly affects the APC function of DC, mediating or contributing to the adjuvant effect of CD40 ligation. CD40 engagement in vitro and rIL-12 at concentrations in the range induced by CD40 ligation were equally effective in priming DC for presentation of the tumor peptide in vivo. Remarkably, the copresence in vitro of neutralizing Ab to IL-12, but not to TNF-a, IL-1b, or IFN-g, ablated the enhancing effect of CD40 engagement on the APC function of DC. These data suggest a major role for autocrine IL-12 in DC modulation via CD40 ligation. The Journal of Immunology, 1999, 163: 2517–2521. I t is well established that IL-12 drives the development of cell-mediated immune responses (1, 2). The identification of the cells and mechanisms responsible for IL-12 production and of the cellular targets at the site of T cell priming is a critical issue that still needs to be clarified. Previous evidence indicates that appropriate stimuli can induce APC to release IL-12 and that the cytokine will act on bystander T cells that recognize Ag on physically distinct APC (3). Thus, IL-12 appears to function in a paracrine fashion, because IL-12 production and Ag presentation can be carried out by different cells. In addition, IL-12 production by APC is induced by the interaction between CD40 on the APC and CD40 ligand expressed on T cells after activation (4). CD40 ligand is the most potent stimulus in up-regulating costimulatory molecules (5) and cytokines such as IL-12 (6), TNF-a (7), and IL-1b (8). We have previously shown that IL-12 primes dendritic cells (DC) 3 in vitro for effective presentation of a poorly immunogenic tumor peptide upon transfer of the DC into recipient hosts (9 –11). Nonameric P815AB represents a minimal core peptide recognized by CTL in vitro and is part of a protein encoded by gene P1A which, silent in most normal tissues except testis and placenta, is instead expressed by murine mastocytoma cells (11). We have previously shown that P815AB fails to initiate class I-restricted reactivity in vivo, presumably as a result of a poor ability to recruit CD4 1 cells to the afferent induction of the response initiated by host transfer with P815AB-pulsed DC (9). However, the poor im- munogenicity of the peptide under such priming conditions can be overcome by preexposure of the DC to rIL-12 (10). The aim of the present study was to investigate whether CD40 engagement on DC may also prime the cells for effective presen- tation of the tumor peptide. By comparing CD40 ligation and rIL- 12, we found that the former stimulus was highly effective in prim- ing DC, and that rIL-12 concentrations in the range of those induced by CD40 cross-linking would likewise be an effective stimulus for DC priming. Most interestingly, the presence of Ab to IL-12 during cross-linking in vitro blocked the adjuvant effect of CD40 modulation in the DC. In contrast, no effect was afforded by neutralization of TNF-a, IL-1b, or IFN-g. These data suggest an important role for autocrine IL-12 in the adjuvant effect of CD40 engagement on DC. Materials and Methods Mice and reagents Male DBA/2J (H-2 d ) mice were obtained from Charles River Laboratories (Calco, Milan, Italy) and were used at the age of 2– 4 mo. Murine rIL-12 was a generous gift from Dr. B. Hubbard (Genetics Institute, Cambridge, MA). IL-12 was 98.8% pure, as assessed by SDS-PAGE, and endotoxin contamination was ,0.9 EU/mg on Limulus amebocyte assay. The specific activity of the purified rIL-12 preparation, measured as ability to stimulate proliferation in human PHA-activated blasts, was 3.1 3 10 6 U/mg. Endo- toxin was removed from all solutions containing rIL-12 or anti-CD40 Abs with Detoxi-gel (Pierce Chemical, Rockford, IL), resulting in endotoxin contamination below the detection limit (0.05 EU/ml) of the assay (Coatest Endotoxin, Chromogenix, Mo ¨lndal, Sweden) (12). Rat anti-mouse IL-12 p40 mAb C17.8 and hamster anti-mouse IL-12 p35 (clone Red-T) were from Genzyme (Cambridge, MA). C17.8 mAb was conjugated to biotin using conventional methods. Hamster anti-murine CD40 (HM40-3) was from PharMingen (San Diego, CA) and goat anti-hamster IgG was from Pierce Chemical. Affinity-purified sheep anti-mouse IL-12 polyclonal Ab was generously provided by the Immunology Department of Genetics In- stitute, and the control Ab (sheep IgG) was purchased from Pierce Chem- ical. Neutralizing rat anti-mouse TNF-a (MP6-XT3, PharMingen), hamster anti-mouse IL-1b (Genzyme), and rat anti-mouse IFN-g (XMG1.2) (10) mAbs were also used. The nonameric P815AB peptide (LPYLGWLVF) was synthesized and purified as described (13). DC preparation and in vitro treatments DC were prepared from collagenase-treated spleens (collagenase type IV, Sigma Chemical, St. Louis, MO), as described (12). Briefly, DC were purified using a positive selection column and CD11c MicroBeads (Milten- yi Biotec, Bergisch Gladbach, Germany). The recovered cells were rou- tinely .98% N418 1 . For CD40 cross-linking (14), DC were incubated on Department of Experimental Medicine, University of Perugia, Perugia, Italy Received for publication April 29, 1999. Accepted for publication June 15, 1999. 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 the Italian Association for Cancer Research (AIRC). 2 Address correspondence and reprint requests to Dr. Paolo Puccetti, Department of Experimental Medicine, Pharmacology Section, University of Perugia, Via del Gio- chetto, I-06126 Perugia, Italy. E-mail address: pccplo@tin.it 3 Abbreviations used in this paper: DC, dendritic cell(s); DTH, delayed-type hypersensitivity. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00