purified by M. Witmer, The Rockefeller University, by diethylaminoethyl cellulose ... AI 13013 from the National Institutes of Health (to R. M. Steinman) and BC-.
DENDRITIC
CELLS
INITIATE
T LYMPHOCYTE
A TWO-STAGE
MECHANISM
FOR
PROLIFERATION*
Be J O N A T H A N M. AUSTYN,~ RALPH M. STEINMAN,§ DAVID E. WEINSTEIN, ANGELA GRANELLI-PIPERNO, AND MICHAEL A. PALLADINO From the Rockefeller University, New York 10021; and the Memorial Sloan-Kettering Cancer Center, New York 10021
Previous studies of dendritic cells (DC)1 (reviewed in 1-3) have revealed the p o t e n t i m m u n o s t i m u l a t o r y capacities of this class of leukocyte. However, relatively little is k n o w n of their m e c h a n i s m of action. W e have used a polyclonal l y m p h o c y t e mitogenesis system to e x a m i n e this question. K l i n k e r t et al. (4) a n d Phillips et al. (5) showed that T cells, modified with sodium periodate, proliferate quickly a n d extensively in the presence of DC. This assay has been used to identify two stages in oxidative mitogenesis. I n the first stage, viable D C i n d u c e b o t h the release of T cell growth factor, or i n t e r l e u k m 2 (IL-2) (reviewed in 6-8), a n d T lymphocyte responsiveness to IL-2. I n the second stage, which can occur in the absence of DC, IL-2 alone mediates proliferation of these responsive T cells. Materials and Methods Animals. Mice were of either sex and 4-14 wk old. Swiss mice were from Taconic Farms, Germantown, NY, and the Laboratory Animal Research Center of The Rockefeller University; C3H/HeJ were from The Jackson Laboratory, Bar Harbor, ME; all other inbred strains were from The Trudeau Institute, Saranac Lake, NY. Antibodies. 33D1 (9) and B21-2 (10) are rat monoclonal antibodies specific for mouse DC and IAb'd antigens, respectively. The latter also binds to Swiss mouse cells. Both antibodies were purified by M. Witmer, The Rockefeller University, by diethylaminoethyl cellulose fractionation of ascites. Primary cells. Mice were killed in chloroform vapor or by cervical dislocation. Spleen cell suspensions were prepared by teasing with fine forceps and pressing through a stainless steel mesh in RPMI 1640 (Gibco Laboratories, Grand Island Biological Co., Grand Island, NY). For unfractionated spleen cells, the suspension was treated with ammonium chloride (0.88%: Mallinckrodt Inc., Science Products, Div., St. Louis, MO) to lyse erythrocytes. Spleen adherent cells, representing 1-2% of the total were obtained by culture in medium (see below) on 100mm petri dishes (3002; Falcon Labware, Div. of Becton, Dickinson & Co., Oxnard, CA) for 23 h at 37°C. Nonadherent cells were then removed by rigorous washing; the adherent cells remaining consisted primarily of DC and macrophages in a ratio of ~ 1:3 (11). After overnight * Supported by grants AI 13013 from the National Institutes of Health (to R. M. Steinman) and BC316 (to A. Granelli-Piperno), IM-310, and JFRA 39 (to M. A. Palladino) from the American Cancer Society. ~:Recipient of a NATO Postdoctoral Fellowship and a Postdoctoral Fellowship from the Cancer Research Institute, New York. § Established Investigator of the American Heart Association. 11Recipient of a Junior Faculty Research Award from the American Cancer Society. 1Abbreviations used in this paper: Con A, concanavalin A; CTLL, cytotoxic T lymphocyte line; DC, dendritic cell; IL-1, IL-2, interleukins 1 and 2; MHC, major histocompatibility complex; MLR, mixed leukocyte reaction. J. Exv. MEo. © The RockefellerUniversity Press • 0022-1007/83/04/1101/15 $1.00 Volume 157 April t983 1101-1115
1102
TWO STAGES IN DENDRITIC CELL-INITIATED T PROLIFERATION
culture the cells were harvested by gently pipetting over the tissue culture surface. Enriched populations of D C were derived from low density adherent cells that consist predominantly of DC and macrophages. After overnight culture, macrophages were depleted by readherence to 60-mm petri dishes (3002; Falcon Labware). T lymphocytes (>90% Thy-1 positive) were obtained from mixed spleen and mesenteric lymph node cell suspensions passed over nylon wool columns as described (12). M e d i u m for cell culture was R P M I 1640 supplemented with 2.5-10% heat-inactivated fetal bovine serum (KC Biologicals Inc., Lenexa, KS), 2-mercaptoethanol (5 X 10-5 M; Sigma Chemical Co., St. Louis, MO), gentamycin sulphate (20/~g/ml; Schering Corp., Kenilworth, N J) and, for experiments in Table II, indomethacin (1/~g/ml; Sigma Chemical Co.). Cells were irradiated with 1,500 rad from a laTCs source (Gamma Cell 1000; Atomic Energy of Canada Ltd., Ottawa, Canada). Oxidative Mitogenesis Assay. T cell mitogenesis was induced by modification of cells with periodate (13). In one experiment (see Fig. 1) stimulators a n d / o r responders were treated, but in all others only responder T cells were modified. T lymphocytes were washed twice in ice-cold phosphate-buffered saline with calcium and magnesium (Gibco Laboratories) and resuspended in the same to 2 × 10V/ml. An equal volume of a freshly prepared solution of sodium m-periodate (Sigma Chemical Co.) at 0.5 m g / m l was added to the cells and incubated on ice for 15 min. Control cells were treated with buffer alone. The cells were centrifuged, without further dilution, at 300 g for 10 min and washed once in medium (2.5% serum) before use. Graded numbers of irradiated stimulators were added to 4-5 × 105 periodate-treated T cells in a total volume of 200 #1 in flat-bottomed microtiter plates (3596; Costar, Data Packaging, Cambridge, MA). At 24-27 h, 0.5/~Ci/well of [3H]thymidine (Schwarz/Mann Div., Becton, Dickinson & Co., Orangeburg, NY) was added and cells were harvested 14-18 h later on a multisample harvester. Kinetic studies (not shown) demonstrated that irrespective of stimulator dose, the bulk of D N A synthesis occurred from ~24 h onwards. Unless otherwise stated, results are given as the means of triplicate cultures; standard deviations were generally 90% pure) were at least 100 times m o r e active as accessory cells t h a n u n f r a c t i o n a t e d spleen cells, which c o n t a i n e d < 1 % D C (Fig. 1). T o d o c u m e n t t h e c o n t r i b u t i o n o f D C further, selective d e p l e t i o n e x p e r i m e n t s were p e r f o r m e d using t h e specific a n t i - D C m o n o c l o n a l a n t i b o d y , 33D1. T r e a t m e n t with 33D1 a n d c o m p l e m e n t r e m o v e d 90% or m o r e o f the T cell s t i m u l a t o r y c a p a c i t y o f spleen a d h e r e n t cells. T h e s e were tested i m m e d i a t e l y after isolation from the a n i m a l ( T a b l e I) or following overnight c u l t u r e ( T a b l e II). A n t i - D C a n t i b o d y a n d complem e n t also r e m o v e d 50-75% o f the s t i m u l a t o r y c a p a c i t y of freshly p r e p a r e d , unfract i o n a t e d spleen cells ( T a b l e II). T h e effects o f 33D1 a n d c o m p l e m e n t were similar to those p r o d u c e d b y specific a n t i - I a a n t i b o d y a n d c o m p l e m e n t (e.g., T a b l e I, e x p e r i m e n t 2). Selective e l i m i n a t i o n o f D C h a d similar effects on accessory function in o x i d a t i v e mitogenesis a n d the p r i m a r y m i x e d leukocyte reaction ( M L R ) ( T a b l e II a n d ref. 11). T r e a t m e n t w i t h 33D1 a n t i b o d y alone, a n t i b o d y , a n d h e a t - i n a c t i v a t e d c o m p l e m e n t (Tables I a n d II), or the c o n t i n u o u s presence o f a n t i b o d y at 2 0 / t g / m l (not shown) d i d
1104
TWO
S T A G E S IN D E N D R I T I C
CELL-INITIATED T PROLIFERATION
160-
x ~ 120-
Z O Q. •
80
I~e
=- 40
~/
i
10 3
10 4 NUMBER
OF
10 5 STIMULATORS
10 6
F1o. l. T h e relative potency of D C c o m p a r e d with u n f r a c t i o n a t e d spleen cells as stimulators in o x i d a t i v e mitogenesis. G r a d e d n u m b e r s of i r r a d i a t e d Swiss mouse DC ( ) or u n f r a c t i o n a t e d spleen cells (- - -) were a d d e d to 5 × l0 s T cells in m e d i u m c o n t a i n i n g 5% serum, a n d proliferation was assessed. Periodation was carried out before mixing, on both stimulators a n d responders (O), responders only (~), s t i m u l a t o r s only (Lx), or neither (×).
not alter DC function. We conclude that the DC is the critical cell required to stimulate the proliferation of periodate-modified T cells. Release of Interleukins during Oxidative Mitogenesis. Interleukins, notably IL-1 (lymphocyte-activating factor, reviewed in 19) and IL-2 (T cell growth factor, reviewed in 6-8), can act as effector molecules for lymphocyte mitogenesis. We therefore tested supernatants from DC, T cells, and D C - T cell co-cultures for interleukin activity in two standard bioassays. These were costimulation with phytohemagglutinin of thymocyte mitogenesis, which detects both IL-1 and IL-2 (18), and proliferation of an IL-2-dependent T cell line, which detects only IL-2. T h e supernatants from D C - T cell co-cultures were active in stimulating both thymocyte mitogenesis and proliferation of the IL-2-dependent C T L L line (Fig. 2). T h e interleukin activity in both bioassays was parallel. Interleukins were not detected in cultures of DC alone (up to 106/ml; not shown). Low levels of IL-2 activity were found in supernatants of T cells cultured in the absence of DC, but these were always
