HLA-DR-Mediated Apoptosis Susceptibility Discriminates Differentiation Stages of Dendritic/Monocytic APC1 Nicolas Bertho,* Bernard Dre´nou,* Be´atrice Laupeze,* Claudine Le Berre,† Laurence Amiot,* Jean-Marc Grosset,* Olivier Fardel,‡ Dominique Charron,§ Nuala Mooney,2§ and Rene´e Fauchet* Professional APC are characterized by their ability to present peptide via HLA class II in the presence of costimulatory molecules (CD40, CD80, and CD86). The efficiency of Ag presentation can be classed as follows: mature dendritic cells (DC) are most efficient, immature DC and macrophages are intermediate, and monocytes are considered poor APC. There is a large body of evidence demonstrating that HLA-DR transmits signals in the APC. In this study, we have addressed the question of the outcome of HLA-DR signals on APC of the monocyte/DC lineages throughout their differentiation from immature to mature APC. DC were .generated from both monocytes and CD34؉ cells of the same individual, macrophages were differentiated from monocytes Immunophenotypical analysis clearly distinguished these populations. HLA-DR-mediated signals led to marked apoptosis in mature DC of either CD34 or monocytic origin. Significantly less apoptosis was observed in immature DC of either origin. Nonetheless, even immature DC were more susceptible to HLA-DR-mediated apoptosis than macrophages, whereas monocytes were resistant to HLA-DR-mediated apoptosis. The mechanism of HLA-DR-mediated apoptosis was independent of caspase activation. Taken together, these data lead to the notion that signals generated via HLA-DR lead to the demise of mature professional APC, thereby providing a means of limiting the immune response. The Journal of Immunology, 2000, 164: 2379– 2385. rofessional APC represent a subset responsible for thymic membrane ligands (CD40-L), they migrate from tissues to periph- selection and peripheral T cell activation (1). Ag-specific eral lymph nodes. In the course of their migration, they undergo P immune responses of CD4 T cells depends on the success- maturation and are considered mature when localized in lymph ful presentation of processed peptide Ags by HLA class II mole- nodes. Mature DC express CD83 (6) and have enhanced HLA cules (HLA-DR, -DP, and -DQ) (2). These molecules are consti- class I, class II, CD80, and CD86 expression (7). Immature DC can tutively expressed on professional APC including dendritic cells be generated in vitro from either peripheral blood monocytes (8) 3 (DC) (3), macrophages, and B lymphocytes. using GM-CSF and IL-4 or from CD34ϩ hemopoietic stem cells DC are the most potent APC (reviewed in Ref. 4), with the (9) using GM-CSF and TNF-␣. Maturation of cultured immature unique ability to prime naive T cells (5). Tissular DC are consid- DC may be obtained by a 48-h incubation with TNF-␣, bacterial ered as immature and can be identified by their expression of HLA LPS (10), or CD40-L (11). DC differentiation and maturation can class I and II molecules, CD1a, CD40, CD80 (B7.1), and CD86 therefore be reproduced in vitro. The success of Ag presentation by (B7.2) but not CD83. They capture and process Ag with high DC has led to them being considered as promising tools for im- efficiency. When DC receive inflammation signals mediated by munotherapy (12, 13). chemokines (macrophage-inflammatory protein 3␣, macrophage- Macrophages express HLA class I and HLA class II Ags as well inflammatory protein 1␣, and RANTES) and cytokines (TNF-␣)or as CD80 and CD86 accessory molecules (14). They are character- ized by their high ability to phagocytose, which allows them *Laboratoire Universitaire d’He´matologie et de Biologie des Cellules Sanguines, In- to process and to present particulate Ags. However, macrophages stitut National de la Sante´ et de la Recherche Me´dicale CRI 9606-UPRES EA 22-33, are less effective than DC in Ag presentation to T lymphocytes † Rennes, France; Etablissement de Transfusion Sanguine de Bretagne, Rennes, (15, 16). France; ‡Institut National de la Sante´ et de la Recherche Me´dicale U456 Universite´de Rennes I, Rennes, France; and §Institut National de la Sante´ et de la Recherche B lymphocytes are considered to be professional APC (17) and Me´dicale U396 Institut des Cordeliers, Paris, France express the requisite accessory molecules for T lymphocyte acti- Received for publication July 12, 1999. Accepted for publication December 20, 1999. vation (such as CD86, CD80, and CD40 molecules (18)) as well as The costs of publication of this article were defrayed in part by the payment of page HLA class II molecules. There is ample evidence for HLA-DR- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mediated signal transduction in B lymphocytes (reviewed in Ref. ␤ 1 This work was supported by grants from the Agence Franc¸aise du Sang. N.B. is a 19). The intracytoplasmic region of the HLA-DR -chain is crit- recipient of a fellowship from Rennes I University. B.L. is a recipient of a fellowship ical for the translocation of protein kinase C ␣ and ␤II (20). Con- from la Ligue contre le cancer (Comite´ des Coˆtes d’Armor). We are grateful for the sequences of these signals are either proliferation and differentia- financial support of Association pour la Recherche sur le Cancer, La Ligue Contre le Cancer (LLCLC), and LLCLC (Comite´ de Paris), EC grants B70-4-98-0458 and tion (21), cell-cell adhesion (22), or cell death (23). Activated ESF-IGA. human B lymphocytes are more susceptible to HLA-DR-mediated 2 Address correspondence and reprint requests to Dr. Nuala Mooney, Laboratoire apoptosis than resting cells (24, 25). The mechanism of HLA-DR- d’Immunoge´ne´tique Humaine, Institut Biome´dical des Cordeliers, 15 rue de l’e´cole de me´decine/Bat A, 75006 Paris, France. mediated cell death has not been elucidated, although enhancement 3 Abbreviations used in this paper: DC, dendritic cell; SCF, stem cell factor; L, ligand; of sensitivity to CD95/Fas (26) and expression of Fas-L (27) has PI, propidium iodide. been described after HLA-DR cross-linking. Fas, in common with Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 2380 PROFESSIONAL APC ARE TARGETS FOR HLA-DR-MEDIATED APOPTOSIS other death receptors, initiates activation of cysteinyl aspartate- were composed of 84.5 Ϯ 5.8% CD14ϩ cells when analyzed by flow cy- ϩ specific proteinases (caspases) which are considered as the prin- tometry. Nonadherent cells were removed for isolation of CD34 cells. Nonadherent cells were allowed to adhere for another 30 min in 45-cm2 cipal executioners of Fas-induced apoptosis (28, 29). ϩ flasks to remove remaining monocytes before isolation of CD34 cells. Ag presentation needs TCR and CD4 interaction with HLA Hemopoietic progenitors were positively selected using CD34-conjugated class II molecules but also activation of cells by costimulation immunomagnetic beads (Dynabeads M-450 CD34; Dynal, Oslo, Norway) molecules (30). Numerous mechanisms to control the T cell re- according to the manufacturer’s instructions. The final purity of CD34ϩ sponse have been described (anergy, apoptosis). Mechanisms con- cells was 88.9% Ϯ 5.7% as demonstrated by flow cytometry analysis. trolling the APC response have not yet been elucidated. Since Generation of DC and macrophages HLA-DR mediated more cell death in activated than in resting B cells, it has been postulated to play a role in the termination of the Macrophages. Monocytes (adherent cells) were cultured for 7 days in immune response (23). If this were the case, HLA-DR-mediated IMDM (Life Technologies) and 10% FCS supplemented with 800 U/ml GM-CSF. Cultures were fed by replacing half of the medium at day 3. cell death could be also expected to occur in other APC. Tyrosine Monocyte-derived DC. Adherent cells were cultured in IMDM, 10% protein kinase activation via HLA-DR on DC has been reported FCS supplemented with 800 U/ml GM-CSF, and 1000 U/ml IL-4. Half of (31); however, the question of apoptosis has not been addressed. the medium was replaced at days 3, 5, and 7 by fresh medium with 800 We have examined the role of HLA-DR-mediated signals U/ml GM-CSF and 500 U/ml IL-4. At day 7, 100 U/ml TNF-␣ was added to culture to provided mature monocyte-derived DC. throughout differentiation of primary professional APC. Hemopoi- ؉ etic progenitors, monocytes, macrophages, as well as immature Hemopoietic progenitor (CD34 cells)-derived DC. Isolated CD34ϩ cells were cultured in IMDM, 10% FCS supplemented with 500 and mature DC were examined. We have compared DC derived ␣ ϩ U/ml GM-CSF, 2.5 U/ml SCF, 10 U/ml Flt3-L, and 200 U/ml TNF- . from both monocytes and CD34 cells of the same individual to Cultures were fed by replacing half of the culture medium by fresh medium ensure that the results obtained were representative of DC regard- at days 5, 9, and 14. At day 14, 100 ng/ml LPS was added to provide less of their origin. We report HLA-DR-mediated apoptosis of mature CD34-derived DC. mature differentiated APC which did not appear to depend on Phenotypic analysis of cells by flow cytometry caspase activation. An in vitro model of APC differentiation and maturation allowed us to demonstrate that the apoptotic function of Before labeling, cells were incubated for1hinhuman AB serum, at 4°C, MHC class II is differentially regulated in professional APC sub- to avoid nonspecific mAb binding. Several mAbs were used for immuno- labeling: FITC- or PE-conjugated mouse mAb against CD1a, CD14, CD34, sets. Finally, these results strongly suggest an “autoregulation” of and HLA-DR, and FITC-labeled isotype controls were purchased from MHC class II-mediated Ag presentation.