Cooperation of TNF Family Members CD40 Ligand, Receptor Activator of NF- κB Ligand, and TNF- α in the Activation of Dendritic Cells and the Expansion of Viral This information is current as Specific CD8+ T Cell Memory Responses in of September 24, 2021. HIV-1-Infected and HIV-1-Uninfected Individuals Qigui Yu, Jenny X. Gu, Colin Kovacs, John Freedman, Elaine K. Thomas and Mario A. Ostrowski Downloaded from J Immunol 2003; 170:1797-1805; ; doi: 10.4049/jimmunol.170.4.1797 http://www.jimmunol.org/content/170/4/1797 http://www.jimmunol.org/ References This article cites 36 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/170/4/1797.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Cooperation of TNF Family Members CD40 Ligand, Receptor Activator of NF-␬B Ligand, and TNF-␣ in the Activation of Dendritic Cells and the Expansion of Viral Specific CD8؉ T Cell Memory Responses in HIV-1-Infected and HIV-1-Uninfected Individuals1 Qigui Yu,* Jenny X. Gu,* Colin Kovacs,* John Freedman,† Elaine K. Thomas,‡ and Mario A. Ostrowski2*† Members of the TNF superfamily have been shown to be instrumental in enhancing cell-mediated immune responses, primarily Downloaded from through their interactions with dendritic cells (DCs). We systematically evaluated the ability of three TNF superfamily molecules, CD40 ligand (CD40L), receptor activator of NF-␬B ligand (RANKL), and TNF-␣, to expand ex vivo EBV-specific CTL responses in healthy human individuals and ex vivo HIV-1-specific CTL responses in HIV-1-infected individuals. In both groups of indi- viduals, we found that all three TNF family molecules could expand CTL responses, albeit at differing degrees. CD40L treatment alone was better than RANKL or TNF-␣ alone to mature DCs and to expand CTL. In healthy volunteers, TNF-␣ or RANKL could cooperate with CD40L to maximize the ability of DCs to expand virus-specific CTL responses. In HIV-1 infection, cooperative http://www.jimmunol.org/ effects between TNF-␣ or RANKL in combination with CD40L were variable. TNF-␣ and RANKL cooperated with CD40L via differing mechanisms, i.e., TNF-␣ enhanced IL-12 production, whereas RANKL enhanced survival of CD40L-stimulated DCs. These findings demonstrate that optimal maturation of DCs requires multiple signals by TNF superfamily members that include CD40L. In HIV-1 infection, DCs may only require CD40L to maximally expand CTL. Finally, CTL responses were higher in CD4؉ T cell-containing conditions even in the presence of TNF family molecules, suggesting that CD4؉ T cells can provide help .to CD8؉ T cells independently of CD40L, RANKL, or TNF-␣. The Journal of Immunology, 2003, 170: 1797–1805 ϩ vigorous and sustained CD8 T cell cytotoxic (CTL) tion and activation (12, 13), as manifested by the increased surface by guest on September 24, 2021 response in the host is required to control infections expression of MHC class I and II molecules, the costimulatory A caused by persistent viruses such as lymphocytic cho- molecules CD80 (B7-1) and CD86 (B7-2), and the adhesion mol- riomeningitis virus, CMV, EBV, and possibly HIV-1 and SIV (1– ecules CD54 (ICAM-1) and CD58 (LFA-30). In addition, CD40 ϩ 7). In this regard, CD4 T cell help has been shown to be instru- ligation of mature DCs stimulates the production of proinflamma- mental in maintaining effective CTL memory responses, through at tory cytokines IL-1, IL-6, and TNF-␣, and T cell growth and dif- ϩ least two ways. In murine models, activated Ag-specific CD4 T ferentiation factors, IL-12 and IL-15, as well as increasing DC cells have been shown to help CTL by local IL-2 production and survival (12, 14–20). These phenotypic changes confer an en- more importantly through the expression of CD40 ligand hanced capacity of DCs to induce CD8ϩ T cell proliferation and 3 (CD40L) (8–11). CD40L is a type II membrane protein of the cytotoxicity. Thus, CD4ϩ T cells help CTL responses via their ϩ TNF superfamily predominantly expressed on activated CD4 T effects on DC activation and maturation. cells. CD40 is constitutively expressed on immature dendritic cells Other members of the TNF receptor/ligand superfamily have (DCs), and CD40 ligation of immature DCs induces their matura- also been proposed to play a role in either enhancing or bypassing CD4ϩ T cell help in eliciting potent antiviral CTL responses. The receptor activator of NF-␬B ligand (RANKL)-RANK (also called *Clinical Sciences Division, University of Toronto, Toronto, Canada; †St. Michael’s TRANCE (TNF-related activation-induced cytokine)-TRANCE Hospital, University of Toronto, Toronto, Canada; and ‡Immunex Corporation, Se- attle, WA 98101 receptor) pathway has recently been shown in murine models to Received for publication May 17, 2002. Accepted for publication December 6, 2002. have similar functions to that of the CD40L-CD40 pathway in the ϩ The costs of publication of this article were defrayed in part by the payment of page generation of antiviral CD4 T-specific immune responses (21– charges. This article must therefore be hereby marked advertisement in accordance 23). RANKL is a member of the TNF superfamily sharing greatest with 18 U.S.C. Section 1734 solely to indicate this fact. amino acid similarity (28%) with CD40L (CD154). RANKL is 1 This study was funded through grant support supplied by American Foundation for expressed on both activated CD4ϩ T and CD8ϩ T cells (24). AIDS Research and Aventis-Pasteur. Q.Y. and M.A.O. are career scientists of The Ontario HIV Treatment Network. RANK, the receptor for RANKL, is constitutively expressed on DCs (22); however, RANK expression is greater on mature DCs or 2 Address correspondence and reprint requests to Dr. Mario A. Ostrowski, Clinical Sciences Division, Room 6271, Medical Sciences Building, University of Toronto, on CD40-ligated DCs (24). Similar to CD40L, conditioning of Toronto, Ontario, Canada, M5S 1A8. E-mail address: [email protected] murine DCs with RANKL has been shown to elicit production of 3 Abbreviations used in this paper: CD40L, CD40 ligand; B-LCL, B-lymphoblastoid proinflammatory cytokines, IL-1 and IL-6, and T cell growth and cell line; CD40LT, trimeric CD40L; DC, dendritic cell; iMDDC, immature MDDC; MDDC, monocyte-derived DC; RANK, receptor activator of NF-␬B; RANKL, differentiation factors, IL-12 and IL-15. RANKL also dramatically RANK ligand; RANKLT, trimeric RANKL. inhibits DC apoptosis via increased Bcl-xL expression (22–25). Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 1798 COOPERATION BETWEEN CD40L, RANKL, AND TNF-␣ RANKL, however, does not markedly up-regulate DC surface ex- Generation of MDDCs, cell cultures, and stimulation pression of MHC molecules, costimulatory molecules (CD80 and MDDCs were generated by a modification of a method previously de- CD86), CD40, or adhesion molecules (CD54), which is observed scribed (19, 30). Briefly, PBMCs obtained by the Ficoll-Paque gradient with CD40L conditioning. It has been postulated that the primary centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden) were effect of RANKL on DCs is by enhancing DC survival in tissues, separated on multistep Percoll gradients (Sigma-Aldrich, St. Louis, MO). thus allowing greater opportunity for DCs to prime T cells (26). The recovered monocytes were depleted of contaminating B cells, T cells, NK cells, and granulocytes using Ab-conjugated magnetic beads from the The role of the RANKL-RANK pathway in induction of murine or Monocyte Negative Isolation Kit (Dynal, Oslo, Norway). Purified mono- human CTL responses, however, has not yet been evaluated. cytes were cultured at 1 ϫ 106 cells/ml in medium consisting of RPMI TNF-␣ is produced and secreted early in the inflammatory re- 1640 plus 10% FCS, 2 mM glutamine, 25 mM HEPES, and antibiotics in sponse by activated monocytes, NK cells, and Th1 T cells. TNF-␣ the presence of 50 ng/ml human rGM-CSF and 100 ng/ml human rIL-4 ϩ (PeproTech, Rocky Hill, NJ). GM-CSF and IL-4 were added again on days has been shown to differentiate CD34 progenitor cells into DCs. 3 and 5 with the fresh complete RPMI 1640 medium. After 7 days of In addition, TNF-␣ has been shown to up-regulate adhesion and culture, more than 50% of the cells were CD1ahigh, MHC class IIϩ, costimulatory molecules of immature monocyte-derived DCs CD80low, and CD14Ϫ, which represents an immature DC phenotype. The (iMDDCs), but not to the extent as that seen with CD40L (14). immature DCs were then aliquoted and cultured for 72 h in the following Thus, DC activation and maturational signals can originate from conditions: 1) medium alone; 2) soluble recombinant human trimeric CD40L (CD40LT) at 1 ␮g/ml; 3) soluble recombinant human trimeric multiple sources. The relative importance of each of these signals, RANKL (RANKLT) at a final concentration of 1 ␮g/ml (CD40LT and either alone or in combination, has not been systematically eval- RANKLT were obtained as a gift from Immunex, Seattle, WA); 4) soluble uated, although knowledge of this would be important for vaccine human rTNF-␣ at a final concentration of 10 ng/ml (PeproTech); 5) ϩ ϩ ␣ ϩ ␣ design.