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Comparison of functional properties of thymic and splenic dendritic cells
CELLULAR
IMMUNOLOGY
102,152- 167 ( 1986)
Comparison of Functional Properties of Thymic and Splenic Dendritic Cells ROSEMARIE A. PEREIRA, NICHOLAS J. C. KING, AND ROBERT V. BLANDEN’ Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C. T. 2601, Australia Received January 27,1986; acceptedMay I,1986 Murine dendritic cells (DC) which are negative for surface immunoglobulin (sIg), Fc receptor (FcR), Thy- 1 antigen, and the mononuclear phagocyte-specific marker F4/80, but positive for the DC-specific marker 33Dl and major histocompatibility complex (MHC) Class 1 and Class II antigens were obtained from either the spleen (SDC) or thymus (TDC) and used to stimulate alloreactive cytotoxic T lymphocyte (Tc) generation in vitro, in 5-day mixed lymphocyte reaction (MLR)to investigate some of their functional properties. SDC were potent stimulators of Tc cells in the absenceof supematant of concanavalin-A-stimulated spleen cells (CSS)and using medium containing 1Zthioglycolate-stimulated mouse serum (TMS) to avoid potential artifactual helper T cell activation by fetal calf serum (FCS) antigens. TDC did not stimulate Tc cells under these conditions, but did so when mixed with small numbers of SDC, or in the presence of CSS. 33Dl and complement (C)-treated SDC and TDC populations failed to stimulate Tc cell responsesin the absence of CSS. In the presence of CSS, TDC treated in a similar manner did not stimulate Tc cell activity, while 33Dl and C-treated SIX stimulated a weak but detectable Tc cell response. These results indicate that genuine DC were the dominant cells presenting Class I MHC antigens in the TDC population and also that a minority of contaminating nonDC in the SDC population expressed sufficient MHC Class I antigens for Tc cell activation, but did not produce factor(s) required for Tc cell activation. The pretreatment of SDC and TDC with CSS before using them as stimulators did not improve their ability to stimulate Tc cell responses,indicating that the effect of CSS in improving the ability of TDC to stimulate Tc cell responseswas not solely due to MHC-elevating effects of components of CSS. Lipopolysaccharide (LPS)-treated SDC synthesized abundant interleukin 1 (IL-l), but TDC synthesized low levels of IL- I. Contact for 24 hr between TDC and splenic Tc cell precursors in medium containing 1%TMS but without CSS and FCS did not tolerize the T cells. o 1986 Academic press, Inc.
INTRODUCTION Lymphoid dendritic cells (DC) were first described morphologically as cells with pseudopods of varying lengths, dense granular cytoplasm and large irregular and refractive nuclei (1). Since then, the class of DC which populates thymus-dependent areas of the murine spleen has been extensively studied and characterized (2-l 1). Phenotypically, they lack surface immunoglobulin (sIg), receptors for immunoglobulin Fc receptor (FcR), Thy-l antigen, and the mononuclear phagocyte-specific ’ To whom correspondence should be addressed.
152 0008-8749/86 $3.00 Copylight 0 1986 by Academic PITS, Inc. All rights of repmdwtion in any form reserved.
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marker F4/80. They express a dendritic-cell-specific marker as defined by a monoclonal antibody 33Dl(l2) and high levels of Class I and Class II major histocompatibility complex (MHC)-encoded antigens (13, 14). Functionally, they are highly motile, and weakly phagocytic if at all. In vitro they have been shown to be strong stimulators of primary allogeneic and syngeneic mixed leukocyte reactions (MLR) (S-10) and of T helper cells (Th) required for both specific antibody responsesto sheep red blood cells and haptens coupled to polypeptide carriers (15, 16). They have been described as accessory cells necessary for mitogen-induced T cell proliferation (17, 18) and for the development of anti-trinitrophenol (TNP)-specific cytotoxic T-lymphocytes (Tc) to TNP-modified T cells (7). These properties of DC and their strategic location in thymus-dependent areas of spleen and lymph nodes and probably in afferent lymph (19, 20), as well as their high degree of membrane motility (which increases their chances of making contact with other cells), make them candidates for a major role in antigen presentation for MHC-restricted T cell responses. Dendritic-like cells which are nonphagocytic, lack FcR, sIg, and Thy-l antigen, and express Class I and Class II MHC antigens have been reported to be present in the thymus (21-24). However, in these published reports, the characterization of these cells is far from complete. It is believed that the thymus is the organ in the body where selection of the T cell repertoire and the primary imposition of T cell tolerance to self antigens take place (25). There is probably no requirement for foreign antigen presentation as such. What, therefore, is the role of dendritic cells in the thymus? This paper describesthe isolation of DC from the thymus (TDC) and comparison of some of their functional properties with those of splenic dendritic cells (SDC). MATERIALS AND METHODS Mice. C57BL/6 (B6), B6.C-H-2bm1 (bml), BlO.BR, BlO.A(2R), BALB/c, and C3H/HeJ strains maintained in the John Curtin School of Medicine were used at 4-7 weeks of age. Antibodies. Anti-Thy- I .2 monoclonal antibody produced by clone F7D5 was obtained from OLAC (Bicester, Oxon). Sheep anti-mouse immunoglobulin (SAMIg) produced by (NH&SO4 precipitation of serum from a sheep which had been hyperimmunized with mouse Ig was a kind gift from Dr. R. B. Ashman. 33Dl is a mouse-rat hybridoma which produces a rat anti-mouse DC-specific antibody ( 12). 33D 1 monoclonal antibody was kindly provided by Dr. R. M. Steinman. F4/80 is a mouse-rat hybridoma (26) which secretesa rat anti-mouse macrophagespecific antibody which does not crossreact on DC. F4/80 culture supernatant was kindly provided by Dr. D. A. Hume. Cell preparation. SDC and TDC were both isolated using the following method: Spleens and thymuses were minced in GIBCO Eagle’s minimum essential medium (EMEM) and lo8 cells/ml were treated with collagenase (Clostridium histolyticum, Boehringer Mannheim, GmbH, West Germany) at 2 mg/ml in EMEM for 15 min at 37°C. Cells were washed and resuspended in EMEM with 10m42-mercaptoethanol (2-ME) and 25 rruV Hepes and seededat 3 X 106/ml in 185-cm2 tissue culture flasks (Nunclon, Denmark) at 50 ml per flask. Cells were allowed to adhere for 2 hr at 37°C in an atmosphere of 5% CO2 in air after which as many nonadherent cells as possible
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were washed off, and remaining adherent cells recultured in EMEM supplemented with 5-10% heat-inactivated fetal calf serum (HIFCS), lop4 2-ME, 25 mM Hepes, and antibiotics (complete medium). After 16 hr of incubation at 37°C nonadherent or loosely adherent cells were harvested by gentle pipetting. These cells were then treated with anti-Thy- 1.2 and rabbit complement (C’) (Low Tox, Cedarlane, Ontario, Canada) to remove contaminating T cells. They were then rosetted with sheep erythrocytes coupled by CrC& with hyperimmune SAMIg (27) to remove Ig and FcR positive cells. Dead cells and rosetted cells were removed by separation on IsopaqueFicoll, giving a viable cell population rich in DC. The yields of DC from the spleen and thymus were OS-l% and 0.03-0.08%, respectively, of initial cell numbers. Mixed lymphocyte reactions. Primary one-way MLR were set up in flat-bottom 16mm (Linbro, Flow Labs., Virginia) or round-bottom 6-mm (Nunclon, Denmark) wells. Responder adherent cell-depleted or whole spleen cells were seeded at 4-5 X lo6 in 2-2.5 ml of culture medium per 16-mm well or 3-4 X lo5 in 0.15-0.2 ml of culture medium per 6-mm well, with varying numbers of stimulator cells irradiated with 2000 rads from a 6oCo source. Where adherent cell-depleted responders were used, adherent cells were depleted by incubating 1.5 X lo8 spleen cells suspended in 50 ml of EMEM containing lop4 2-ME in a 185-cm* tissue culture flask (Nunclon, Denmark), for 2 hr at 37°C to allow the cells to adhere. As many nonadherent cells as possible were removed, washed, and used as responders. Unless otherwise indicated, culture media consisted of EMEM with 1Op4MZME, antibiotics and 1%serum from responder strain mice treated with thioglycolate broth (TMS), as described elsewhere (28). Where indicated, 10%supernatant from concanavalin A-stimulated spleen cells (CSS) prepared as described (29) was added to the culture medium. Generation of Tc was determined after 5 days of culture, using appropriate “Crlabeled syngeneic and allogeneic peritoneal macrophage or tumor cell targets as described elsewhere (30, 3 1). Effecters and targets were incubated for 5 hr at 37°C and cytotoxic activity is expressedas mean percentage of specific 5’Cr releasegiven by the equation test release - spontaneous release x 100 total release - spontaneous release f standard error of the mean in triplicate assays. Interleukin-1 (IL-l) assay. IL-l secretion was assayedaccording to the method of Mizel et al. (32). To trigger IL- 1 release,DC were suspended in RPM1 1640 medium containing 10% HIFCS and 20 &ml lipopolysaccharide (LPS) and were seededat 1 X 106/ml in 16-mm or 6-mm flat-bottom wells in 2- and 0.2-ml aliquots, respectively. These cells were then incubated for 48 hr at 37°C in an atmosphere of 5% CO2 in air, after which cells were gently resuspended, transferred to centrifuge tubes, and spun at 1OOgfor 5 min. Supernatants were removed for assaying and the cell pellet was resuspended in its original volume of medium and frozen and thawed three times to release intracellular IL-l. Cell debris was eliminated by centrifugation. Both secreted (in supematants) and intracellular (from freeze-thawed cells) IL- 1 activity was assayed. Augmentation by IL-l of phytohemagglutinin (PHA) stimulation of C3H/HeJ mouse thymocytes was used as a measure of IL-l activity as described elsewhere (32). Briefly, serial twofold dilutions of 50 ~1of material to be assayed,were made in 96-well flat-bottom plates. To these wells were added 50 ~1 of thymocytes
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CELL FUNCTION
TABLE 1 Percent Contamination of SDC and TDC Preparation with Thy+ ’ and FcR+ and/Ig+ Cells b TDC
SIX Expt.
%Thy-1+
% FcR+/Ig+
% Thy+
% FcR+/Ig+
I II III
5 8 9
1 2 0
6 5 6
0 3 0
’ SDC and TDC (1 X 105)in 25 ~1of complete medium were incubated with 25 ~1of anti-Thy- 1.2 at a predetermined concentration and C’ (see Materials and Methods). Percentage of dead cells above a C’ control (approximately 3% dead) were scored as being Thy- 1+. ’ SDC and TDC (1 X 10’) in 25 ~1of complete medium were incubated with 25 pl of a 1%suspension of SAMIg-coupled sheep erythrocytes (see Materials and Methods). Cells forming rosettes were scored as being FcR+/Ig’.
(from 5- to 7-week-old C3H/HeJ mice) suspended at 107/ml in RPM1 1640 medium containing 10% HIFCS, 10M4M 2-ME, and 1 pg/ml PHA. A positive control consisted of serial dilutions of IL- 1-containing supernatants from freeze-thawed cell contents of the BALB/c macrophage cell line PU5-1.8 (33) which had been stimulated with LPS. Twelve replicates of thymocytes alone in medium containing 1 &ml PHA were a negative control. Plates were incubated for 48 hr at 37°C. During the last 6 hr of incubation, 1 &i of tritiated thymidine was added to each well and the cells were harvested onto glass fiber filter paper (Whatman GF/A) using a Titertek cell harvester. Tritiated thymidine uptake was assessedby counting in a Packard Tri-Carb scintillation counter. RESULTS
PhenotypicCharacterization of SDC and TDC By rosetting with SAMIg-coupled sheeperythrocytes and treating with anti-Thyl.2 and C’, we determined that the DC-rich populations obtained from both the thymus and spleen were >97% for FcR-/Ig-, and >9 1%Thyl.2- (Table 1). Fluorescence activated cell sorter (FACS) analysis and immunoelectron microscopy (IEM) were used to investigate morphology and expression of 33D 1, F4/80, and Class I and Class II MHC antigens on DC-rich cell populations from B6(H-2b) mice. Data are not given here and will be reported in detail elsewhere. Briefly, however, there were no detectable F4/80+ cells present in either SDC or TDC as compared with positive control peritoneal macrophages. Cells of classical DC morphology made up approximately 80% of the SDC populations and 50% of the TDC preparations. These cells were 33D l+, whereas control macrophages were 33D 1-. The DC in both splenic and thymic populations were positive for both Class I and Class II MHC antigens, whereas the contaminating non-DC were virtually all MHC. These findings correlate with the functional data presented below which indicate that 33Dl+ cells in the SDS and TDC populations were the predominant source of MHC antigens that triggered alloreactive Tc cell generation.
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STIMULATOR
CELL
NUMBER
(X 1 O-4)
FIG. 1. Curves showing generation of Tc activity using various doses of irradiated BALB/c SDC (A) or whole spleen cells (0) as stimulators of 5 X lo6 adherent cell-depleted B6 responder spleen cells in S-day MLR. Data given are mean percent specific lysis of 5’Cr-labeled BALB/c macrophage target cells in triplicate assaysof l/ 10 dilutions ofthe MLR. SE of mean were lessthan +40/oand have been omitted for clarity.
Capacity of SDC as Stimulators of Tc Cell Activity in MLR Other workers have reported the extraordinary potency of SDC as stimulators of alloreactive T cells (8, 10). Therefore, we first compared SDC prepared under our conditions with unfractionated spleen cells as stimulators of alloreactive Tc cells, under culture conditions in which artifactual activation of Th cells (and thus indirectly, of Tc cells) by foreign sera was prevented by the use of mouse serum from the responder mouse strain as a medium supplement. Various numbers of irradiated BALB/c SDC or spleen cells were used to stimulate 5 X lo6 adherent cell-depleted spleen cells in MLR. After 5 days, cultures were assayed for Tc cell activity (Fig. 1). SDC were superior stimulators to crude spleen cells over the entire dose range tested. The Tc cell response was maximal with doses of SDC between 8 X lo3 and 1 X 106;there was detectable Tc cell activity with I .6 X lo3 SDC. With spleen cells as stimulators, the Tc cell response generated was maximal at 1 X lo6 cells and declined sharply with lower stimulator numbers until it became undetectable below 2 X 105. The maximum response with 1 X 1O6spleen cells was at a level comparable to that stimulated by SDC at 8 X 103.From these results it is clear that SDC are approximately loo-fold more potent stimulators of primary Tc cell responses in MLR using our culture conditions than whole unfractionated spleen cells. Since SDC constitute up to 1%of splenocytes (2), these data are in accord with the idea that SDC are probably the most important stimulator cells in unfractionated spleen cell populations. We have also observed in this system that use of high numbers of SDC (e.g., 1 X lo6 in Fig. 1) consistently depressesdetectable Tc cell activity below maximal levels. This phenomenon is under further investigation.
Comparison of SDC with TDC as Stimulators of Tc Cell Generationin Primary MLR Irradiated BlO.BR (H-2k) SDC or TDC were used as stimulators at 1 X lo5 and 3 X 1O4per 5 X 1O6B6 (H-2b) spleen cell responders in a 5-day primary MLR. At both
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TABLE 2 Capacity of SDC and TDC to Stimulate Generation of Tc Cells in 5Day MLR”
Number and type of stimulators 1x 3x 1x 3x 1x
105 IO4 lo5 104 105+ 3 x 104
SIX SDC TDC TDC TDC SDC
Yield of responder cells @jb 64 42 12 26 56
% % release from BlO.BR macrophages caused by dilutions of 5day MLR’ l/l0 56.6 f 0.9 33.8 2 1.8 0.1 kO.5 9.3 + 2.5 54.1 + 0.9
l/30
l/90
51.7 f 1.1 17.5 + 0.5 2.2 + 0.6 4.5 + 1.0 42.0 + 2.5
32.7 + 0.3 5.3 + 0.7 1.1 + 1.3 1.6 f 1.6 22.1 * 2.0
a Adherent cell-depleted B6 spleen cell responders (5 X 106)were cultured with irradiated B lO.BR stimulators in 16-mm wells as described under Materials and Methods. b Percentageof viable cells recovered from the 5day MLR where original responder cell number is 100%. ‘Cells contained in l/IO, l/30, and l/90 aliquots from the 5&y MLR were assayed in triplicate on 5 X IO4 “Cr-labeled BlO.BR and B6 macrophage targets. Lysis of B6 targets was negligible and the data have been omitted for clarity. Data presented are the means of triplicates + SE of mean.
of these stimulator cell concentrations, SDC stimulated a good cytotoxic response, whereas TDC were either weakly stimulator-y or nonstimulatory (Table 2). When 1 X lo5 TDC and 3 X lo4 SDC were mixed together, they stimulated a Tc cell response similar in magnitude to that caused by 1 X lo5 SDC and significantly greater than that caused by 3 X lo4 SDC. These data indicate that the failure of TDC alone to stimulate was not due to a suppressive influence and suggested that they were stimulatory in the presence of SDC. The same phenomenon was observed in a B6 anti-bml MLR (data not shown) in which the difference between responder and stimulator was limited to two amino acids in the sequence of the H-2K Class I antigen (34).
Capacity of TDC to Stimulate Generation of Tc Cell Responsesin Primary MLR in the Presenceof CSS Data presented thus far suggestthat TDC display Class I MHC antigens and that they do not suppress Tc cell generation at the numbers used in this system when mixed with SDC. Therefore it seemed possible that the failure of TDC to stimulate Tc cell responseswas caused by a failure to secrete factor(s) (signal 2) known to be necessary for induction of Tc cell responses (35). A corollary of this proposition is that TDC should stimulate Tc cell responsesin the presence of CSSknown to contain factor(s) required for T cell activation (36). Various numbers of irradiated BALB/c TDC were used as stimulators with 4 X 1O6 cells from an adherent cell-depleted B6 spleen population as responders in 5-day MLR and culture media were either supplemented or not with 10% v/v CSS. In the absence of CSS, TDC were weak stimulators of generation of Tc cell activity in B6anti-BALB/c MLR at all numbers tested (Table 3), whereas SDC stimulated strongly with the typical dose response characteristics demonstrated before (Fig. 1). However, in the presence of CSS,TDC stimulated strong Tc cell activity with comparable dose-
158
PEREIRA, RING, AND BLANDEN TABLE 3 Capacity of TDC to Stimulate Generation of Tc Cells in B6 Anti-BALB/c MLR” in the Presenceor Absence of CSS
Number and type of stimulators
css added to MLR
Yield of responder cells (%I*
% “Cr release from BALB/c macrophage targets caused by dilutions of klay MLRC l/10
l/30
l/90
4x 105 8x 10“ 1.6x lo4 3.2 x lo3
TDC TDC TDC TDC
-
41.0 5.5 6.5 10.0
10.1 + 5.6 k 3.1 f 5.0 +
2.5 3.4 2.2 1.5
4.7 + 0.6 8.0 + 1.2 0.0 + 1.7 9.4 f 0.3
10.8 * 1.9 6.6 + 2.0 7.2 + 1.4 3.0 f 0.9
4 x 105 8x 10.’ 1.6 x lo4 3.2 x 10’
SDC SDC SDC SDC
-
71.0 70.0 50.0 13.5
43.7 zk3.8 49.1 f 1.1 50.8 f 0.3 10.3 + 2.2
13.8 + 3.8 23.3 + 2.8 11.9 f 1.8 11.6+0.4
12.5 f 14.4 5 11.9 k 10.6 -t
4x 105 8x lo4 1.6 x lo4 3.2 x 10’
TDC TDC TDC TDC
+ + + +
35.0 52.5 40.0 26.3
51.7 + 55.7 + 48.0 k 12.6 +
3.9 2.1 2.9 1.5
39.6 + 0.9 29.6 + 0.2 24.4 + 1.7 11.9kO.O
18.6 + 1.3 18.3 + 0.5 15.1 kO.2 12.4 + 0.3
4x 105 8x lo4 1.6 x lo4 3.2 x IO3
SDC SIX SDC SDC
+ + + +
85.0 80.0 90.0 55.0
50.8 k 69.5 k 70.2 + 47.2 f
4.0 1.2 2.0 1.5
22.8 + 0.3 45.6 + 0.3 40.4 k 0.3 25.2 f 1.9
14.3 * 1.3 20.3 f 2.2 20.0 + 3.5 8.3 f 1.2
0.4 0.7 3.7 1.5
’ Adherent cell-depleted B6 spleen cell responders (4 X 106)were cultured with various dosesofirradiated BALB/c stimulators in 16-mm wells as described under Materials and Methods. *scAs for Table 2.
response characteristics to SDC (in the absence of CSS). The response stimulated by SDC was also improved in the presence of CSS but this improvement was not as dramatic as that seen for TDC stimulators. As seen above, high numbers of SDC depressedTc activity. Thus SDC at 4 X lo5 apparently stimulated less activity than 8 X lo4 SDC, particularly in the presence of CSS. We also carried out B6 anti-bm 1 MLR comparing SDC with TDC as stimulators. Results obtained in this experiment (Table 4) were very similar to those in B6-antiBALB/c MLR, thus indicating that the degree of H-2 difference between responder and stimulator did not influence the outcome. Addition of 10% FCS to the culture medium also enabled bml TDC to stimulate generation of B6 Tc cells (Table 4), though not so efficiently as in the presence of CSS. B6 Tc cells stimulated by bml TDC in the presence of either CSS or FCS were as specific for bml macrophage target cells as Tc stimulated by bml SDC (e.g., lysis by l/l0 of the MLR of self B6 macrophages was negligible, and lysis of third-party CBA macrophages was approximately 10%).
Efect of 3301 and Complementon the Ability of TDC and SDC to Stimulate Tc Cell Generation in the Presenceor Absenceof CSS Data presented above (Table 3) indicate that cells in the TDC population are able to stimulate Tc cell generation in the presence of CSS.Electron microscopy (EM) has
THYMIC
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TABLE 4 Capacity of TDC to Stimulate Generation of Tc Cells in B6 Anti-bm 1 MLR’ in the Presenceor Absence of CSS
Number and type of stimulators
css added to MLR
10%FCS added to MLR
Yield of responder cells (%)*
% “Cr releasefrom bm 1 macrophage targets caused by dilutions of 5-day MLRC l/l0
l/30
l/90
1 x lo5 2.5 x 10“ 6.25 x lo3
TDC TDC TDC
55 40 8.5
1.1 kO.1 1.9 f 0.8 O.OkO.1
0.0 f 0.4 0.0 k 1.3 0.1 kO.5
0.0 + 1.0 0.0 f 1.7 0.0 f 0.9
1 x lo5 2.5 x 10“ 6.25 x 10’
SDC SDC SDC
92.5 65 35
43.0 f 2.0 39.4 * 3.3 16.2 + 2.8
13.1 kO.5 13.6 + 0.7 4.5 f 1.9
1.9 + 2.3 5.1 f 0.6 0.0 + 0.8
1 x lo5 2.5 x lo4 6.25 x 10’
TDC TDC TDC
+ + +
-
65 52.5 45
41.3 2 2.2 35.0 It 0.2 12.7 + 0.7
24.3 k 0.9 10.4 + 1.6 6.1 +0.7
6.7 + 1.2 7.4 * 0.7 5.0 k 2.5
1 x IO5 2.5 x lo4 6.25 x 10’
SDC SDC SDC
-
65 81.5 65
64.2 + 1.1 50.8 f 1.3 33.7 + 2.5
28.8 + 1.0 24.1 + 1.6 14.2 + 1.5
7.5 + 1.0 8.5 + 0.7 0.0 + 1.0
1 x 105 2.5 x lo4 6.25 x 10’
TDC TDC TDC
+ + + -
+ + +
70 45 30
59.0 + 0.9 0.5 f 1.7 0.4* 1.1
32.5 + 0.5 -2.0 f 0.6 0.7 + 1.9
12.1 + 1.3 -2.8 + 0.6 -1.7 + 1.9
1 x 105 2.5 x lo4 6.25 x IO3
SDC SDC SDC
-
+ + +
80 75 60
53.8 * 1.0 63.4 f 0.6 61.4k0.3
19.5 + 2.4 32.7 + 1.8 27.0 -t 0.5
5.5 + 1.8 13.5 f 1.6 13.1 + 1.7
’ Adherent cell-depleted B6 responders (4 X 106)were cultured with various doses of irradiated bml stimulators in 16-mm wells as described under Materials and Methods. *J As for Table 2.
shown that TDC and SDC populations isolated using the technique described under Materials and Methods contain approximately 50 and 80%, respectively, of cells with classical DC morphology (2). Therefore it was possible, particularly in the case of TDC populations, that in the presence of CSSother unidentified contaminating cells were responsible for stimulating Tc cell generation in MLR. To test this proposition, TDC and SDC populations from BALB/c mice were treated with 33Dl and C’ according to a protocol described elsewhere (lo), to lyse dendritic cells. Various numbers of irradiated treated and untreated TDC and SDC were used to stimulate 4 X 1O5 adherent cell-depleted B6 spleen cells in the presence and absence of CSS in 5-day MLR. As expected, TDC only stimulated Tc cell responses in the presence of CSS (Table 5), whereas SDC stimulated strongly both in the absenceand presence of CSS. C’-treated TDC and SDC populations showed some reduction in stimulator activity, probably due to nonspecific toxic effectsof the C. However, when a TDC population was treated with 33Dl and C’, the remaining cells failed to stimulate Tc cell generation in MLR even in the presence of CSS, thus indicating that TDC were the domi-
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TABLE 5 Effect of 33Dl and C’ on the Ability of TDC and SDC to Stimulate Tc Cell Generation in B6 Anti-BALB/c MLRa in the Presenceor Absence of CSS
Number and type of stimulators 2x 104 5x 10’ 1.25 x 10’ 2x 104 5x 10’ 1.25 X 10’ 2x 104 5x 10’ 1.25 x 10’ 2x 104 5x 10’ 1.25 X 10’ 2x 104 5x 103 1.25 x 10’ 2x 104 5x 10’ 1.25 x 10’ 2x 104 5x 10’ 1.25 x 10’ 2x 104 5 x 10’ 1.25 x 10’ 2x 104 5 x 10’ 1.25 x 10’ 2x 104 5 x 10’ 1.25 x 10’ 2x 104 5x 10” 1.25 X 10’ 2x 104 5x 10’ 1.25 x 10’
TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC SDC SDC SDC SIX SDC SDC SIX SDC SDC SIX SDC SDC SDC SDC SDC SDC SIX SDC
css added to MLR
+ + + + + + + + + + + + + + + + + +
% “Cr releasefrom P8 15 targets causedby dilutions of S-day MLRC Treatment of cells
33Dl+ C’ 33Dl+ C’ 33Dl+ C’ 33Dl+ C’ 33Dl+ C’ 33Dl+ C’ C’ only C’ only C’ only C’ only C’ only C’ only 33Dl + C’ 33Dl+ C’ 33Dl + C’ 33Dl+ C’ 33Dl+ C’ 33Dl+ C’ C’ only C’ only C only C’ only C’ only C’ only
213
219
2121
4.6 - 0.2 0.0 k 0.7 0.0 + 0.7 66.2 f 1.8 66.6 * 0.9 -0.9 f 0.7 -2.8 + 0.1 -2.6 k 0.4 -2.5 + 1.2 -3.2 + 0.6 -3.4 f 0.7 -3.0 + 0.8 -0.7 + 1.5 -1.450.5 -2.5 k 0.7 42.4 + 3.4 37.4 + 3.2 38.2 + 3.3 79.6 + 3.5 57.2 f 1.2 26.1 + 0.6 75.7 f 3.0 74.6 zk2.4 78.6 + 0.4 0.6 + 0.2 -0.5 k 0.4 -4.0 r 0.5 20.6 + 0.7 4.5 + 1.9 -2.0 + 0.3 85.2 + 0.4 41.5 + 1.8 24.6 + 0.3 73.1 f 1.7 58.9 + 3.5 13.1 + 2.8
-2.2 k 1.1 0.0 + 0.3 0.0 f 1.2 66.8 + 4.6 54.8 2 2.9 -3.2 -t 1.2 0.4 + 0.8 -1.5kO.6 -3.8 + 0.6 -5.1 -+ 1.0 -3.4 * 0.5 -2.9 + 0.5 -1.1 * 1.1 -0.7 * 1.0 -4.1 + 0.4 31.6 + 1.1 15.4 * 0.2 3.0 + 1.2 74.1 + 2.5 32.0 ?I 0.7 10.1 + 1.0 66.1 + 0.2 67.9 + 4.2 61.0 A 2.2 1.8 + 1.2 -3.8 zk0.8 -3.4 + 0.5 1.6 + 3.3 -1.8 f 1.3 -3.1 f 0.2 66.1 f 5.6 22.9 f 0.7 9.3k2.1 73.6 + 2.7 42.6 + 0.4 4.7 f 0.3
-2.3 f 0.9 0.0 + 0.2 0.0 f 1.0 28.3 + 1.7 37.5 k 1.4 -3.0 + 1.0 0.2 2 0.3 -1.7 kO.6 -3.2 + 0.2 -5.4 + 1.0 -4.4 + 0.6 -4.2 + 0.3 -0.1 + 0.8 -2.3 + 0.7 -3.1 -+0.9 8.2 + 1.5 1.3 + 0.9 -0.7 + 1.8 43.1 f 2.9 14.8 f 0.2 3.4 + 1.0 47.1 + 4.2 50.0 + 3.3 38.0 k 4.0 -1.3 kO.1 -4.2 + 0.8 -3.4 k 1.2 2.9 + 1.3 -6.8 + 0.3 -3.2 f 0.3 36.8 + 2.4 6.3 + 1.3 1.1 kO.6 44.2 f 3.3 19.3 + 2.8 0.8 k 1.4
’ Adherent cell-depleted B6 spleen cell responders (4 X 10’) were cultured in triplicate with various numbers of irradiated BALB/c stimulators in 6-mm wells as described under Materials and Methods. bCells contained in 213,219, and 2127 aliquots from the triplicate 5day MLR were assayedon 1 X lo4 “Cr-labeled P8 15 targets. Data presented are the means of triplicates + SE of mean.
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nant and perhaps the only cells in the population that presented Class I MHC antigens to Tc cell precursors. SDC which had been treated in a similar manner failed to stimulate Tc cell generation in the absence of CSS. In the presence of CSS, however, they stimulated a weak but detectable Tc cell response at 2 X lo4 but not at lower cell numbers, suggesting either that there were some cells remaining in this population which did not produce signal 2 but which expressed MHC Class I antigens (signal 1) and were thus capable of stimulating Tc cell generation in the presence of CSS, or that the presence of CSS increased the MHC antigen expression on some of these cells to a level sufficient for stimulation.
Efect of Treating DC with CSSprior to their Useas Stimulators in MLR As shown above (Tables 2-5), TDC do not stimulate T cells in the absenceof CSS, thus suggesting the possibility that TDC express MHC antigens (signal l), but do not provide additional factor(s) essential for T cell activation (35). Another formal possibility, even though it was not obvious from our IEM studies referred to above, is that TDC normally expresslevels of MHC antigens that are insufficient for efficient T cell activation. The effect of CSS,which is known to contain y-interferon (37), may therefore be to increase MHC antigen expression (38) on TDC, thus improving their ability to stimulate T cells. If this latter possibility applies, then pretreatment of TDC with CSS before their use as stimulators should improve their stimulating capability in the absence of CSS. Accordingly, 5% CSS was added to the culture medium of portions of a thymus cell and spleen cell population for a 16-hr time interval during the course of the DC preparation procedure. The interval chosen was during overnight incubation at 37°C immediately following the initial 2-hr incubation and nonadherent cell removal (seeMaterials and Methods). The rationale for this protocol is, first, that 16 hr is an ample time for MHC-elevating effectsof CSSon other cell classes (38); and second, that virtually all of the Tc cell activity measured at the end of 5-day MLR results from contact with stimulator cells in the first 24 hr after mixing (39). CSS-treated or untreated TDC and SDC were then used as stimulators in 5-day B6 anti-BALB/c MLR. CSS pretreatment of TDC or SDC did not improve their stimulatory capacity for Tc cell generation (Table 6). CSS-treated SDC were significantly inferior to untreated controls. However, as above, in the presence of CSSduring the 5-day MLR, TDC stimulated Tc cell activity. These data do not determine whether or not CSS treatment causes changes in MHC antigen expression or has other effects on TDC or SDC, a topic of ongoing investigation, but they are consistent with the view that the failure of TDC to stimulate T cells is caused by a lack of signal 2.
Comparison of the Ability of TDC and SDC to Produce IL-l upon Stimulation with LPS Data presented thus far strongly suggestthat the difference in the abilities of TDC and SDC to stimulate Tc cell generation stems from an inability of TDC to produce factor(s) necessaryfor T cell activation (35) such as IL-l. It has been shown (40) that upon stimulation with LPS, cells such as the macrophage cell line PU5- 1.8 secrete IL-l or at least synthesize IL-1 which may be detected intracellularly. Therefore, 8 X 10’ SDC and TDC were treated with LPS, as described under Materials and
162
PEREIRA, RING, AND BLANDEN TABLE 6 Effect of Exposure of TDC to CSS prior to B6 Anti-BALB/c Way MLR“ on their Capacity to Stimulate Tc Cell Activation
CSS addedb Number and type of stimulators 1 x 105 2.5 x lo4 6.25 X lo3 1 x 10s 2.5 x lo4 6.25 X lo3 1 x 105 2.5 X lo4 6.25 x 10’ 1 x 105 2.5x104 6.25 x 10’ 1 x 105 2.5 x lo4 6.25 X 10’ 1 x 10s 2.5 x lo4 6.25 x lo3
TDC TDC TDC SDC SDC SDC TDC TDC TDC SDC SDC SDC TDC TDC TDC SDC SDC SDC
% “Cr release from BALB/c macrophage targets causedby dilutions the 5-day MLRd
ToDC preparation
To 5day MLR
Yield of responder cells (%Y
l/10
l/30
l/90
+ + + + + +
+ + + + + + -
12 3 2 25 37.5 15 35 30 12 45 57.5 30 8.5 3.5 3 25 30 14
1.4 + 0.4 3.3 + 1.2 4.3 + 1.1 37.6 zk0.7 38.1 + 0.6 5.8 + 0.6 25.2 + 0.5 14.2 + 0.7 8.2 f 1.8 53.8 + 1.0 42.8 + 2.2 26.0 -c 0.3 6.2+ 1.1 3.1 kO.3 3.2 f 1.5 26.6 + 1.8 19.0 f 0.4 1.3 k 1.6
1.9 f 2.0 0.0 + 0.3 0.0 1- 0.6 14.9 f 1.1 16.0 + 0.7 2.1 +0.2 10.1 kO.6 2.4 k 0.3 -0.1 + 2.3 34.4 + 1.1 17.2 k 2.4 9.3 + 0.5 5.2 f 0.6 3.2 + 0.8 1.9 -+ 0.5 8.3 + 1.0 13.3 f 1.0 0.0 + 1.6
0.0 f 0.5 1.7 rt 0.5 0.3 lk 0.9 5.4 + 0.3 5.1 +0.3 0.9 + 0.2 1.7 + 3.1 1.3 k 0.6 1.4kO.6 12.3 k 1.4 6.7 k 0.5 3.8 L 0.8 3.6 AZ1.0 6.1 -t 1.6 3.0 + 0.5 2.6 + 0.7 0.0 -t 0.8 0.0 -t 1.0
’ Adherent cell-depleted B6 spleen cell responders (4 X 106)were cultured with various dosesof irradiated BALB/c stimulators in 16-mm wells as described under Materials and Methods. b Where indicated, 5% CSSwas added during DC preparation for 16 hr and then washed off (seeResults) or to 5-day MLR. csdAs for Table 2.
Methods. Supernatants, as well as the contents of freeze-thawed cells, were assayed for IL- 1 activity. Strong IL1 activity was detected in the contents of freeze-thawed SDC and significant IL- 1 activity was also found in supernatants of SDC (Table 7). However, only weak IL 1 activity was detected in supematants and in the contents of freeze-thawed TDC. The data therefore support the suggestion that the weak stimulation of Tc cells by TDC shown here is caused by the low levels of IL- 1 in TDC.
Examination of the Capacity of TDC to Tolerize Mature T Cells Data presented above indicate that TDC provide antigen (signal 1) but not signal 2 for the activation of Tc cells. Bretscher and Cohn have postulated that signal 1 alone should be tolerogenic (4 1). Therefore, we investigated whether TDC are tolerogenic for mature T cells. This was determined by examining the ability of B6 responder spleen cells to generate Tc cell responses upon optimal stimulation with irradiated bml or BlO.A(2R) spleen cells following preincubation for 24 hr at 37°C with bml
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TABLE 1 Capacity of BALB/c TDC and SDC to Produce IL- 1 upon Stimulation with LPS” Sampleb
IL- 1 titerc(logiO)
TDC (supematants) TDC (cell contents) SDC (supematants) SDC (cell contents) PUS-l.8 (cell contents)
0.56~0.131 0.7 1 k 0.085 1.58 -c 0.026 2.41 + 0.072 2.16?0.125
’ BALB/c SDC and TDC (5 X 10’) were cultured with LPS in 6-mm flat-bottom wells as described under Materials and Methods. b Samples consisted of supematants from cells cultured with LPS and also the contents of freeze-thawed cells (seeMaterials and Methods). ‘IL-1 titer is expressed as log,, endpoint dilution which was taken at 3 SD above background (374 rfr 100 cpm). Data presented are means of triplicate assays 2 SE of the mean. The IL-l titers between corresponding samples of TDC and SDC are significantly different (P < 0.005) and the difference between IL- 1 titers of SDC and PU5- 1.8 cell contents is not significant.
TDC. Controls consisted of B6 responder spleen cells preincubated with B6 TDC and SDC and bm 1 SIX. During the 24-hr preincubation step, two kinds of culture medium were used. The first consisted of EMEM with 5% HIFCS, 1%TMS, and 1O-4A4 2-ME, and the second, EMEM with 1%TMS and 10e4M2-ME. After preincubation for 24 hr with DC, restimulation with spleen cells was carried out for 4 days in medium with 10%HIFCS. When 5% HIFCS together with 1% TMS were used during preincubation with B6 and bm 1 TDC and SDC, subsequent restimulation with irradiated bm 1 spleen cells yielded a strong Tc cell response against bml targets (lines 1-4, Table 8). Likewise, in the same culture medium preincubation of B6 responder spleen cells with bml TDC and subsequent restimulation with irradiated B lO.A(2R) spleen cells yielded a strong anti-B lO.A(2R)-specific cytotoxic response (line 5, Table 8). Preincubation of B6 responder cells with B6 TDC and SDC and bml SDC, in culture medium supplemented with 1% TMS alone, followed by restimulation after 24 hr with irradiated bm 1 spleen cells, led to the generation of strong anti-bm 1 specific Tc cell responses (lines 6-8, Table 8). In the same culture medium, when B6 responder spleen cells were preincubated with bm 1 TDC, there was a threefold reduction in the anti-bml Tc cell response generated upon restimulation with irradiated bml spleen cells (line 9, Table 8). This reduction was not specific, however, as there was also a threefold reduction in the magnitude of the anti-BlO.A(2R) Tc cell responsegenerated after restimulation with BlO.A(2R) spleen cells (line 10, Table 8). These data suggestthat neither TDC nor SDC are tolerogenic for mature splenic T cells, but do not exclude a role for TDC in imposing self-tolerance upon immature intrathymic T cells. DISCUSSION Dendritic-like or interdigitating cells have been reported to be present in murine and rat thymuses (2 l-23). It was therefore of interest to isolate enriched populations
164
PEREIRA, KING, AND BLANDEN TABLE 8 Capacity of bm 1 TDC to Tolerize Mature B6 T cells’
Type of stimulator cells used in preincubation for first 24 lx
Medium supplement (tint 24 bry
B6 B6 bml bml bml B6 B6 bml bml bml
5% HIFCS 5% HIFCS 5% HIFCS 5% HIFCS 5% HIFCS 1% TMS l%TMS IWTMS l%TMS l%TMS
SDC TDC SDC TDC TDC SDC TDC SDC TDC TDC
+ + + + +
1%TMS 1% TMS 1% TMS I % TMS 1%TMS
strain of spleen cells used for lestimuIatioo over 4 days
Yield of reqander ceus(?a)=
Strain of m&mphage tarsets
bml bml bml bml BlO.A(ZR) bml bml bml bml BlO.A(ZR)
34 38 36 44 44 40 36 34 14.6 14.6
bml bml bml bml BlO.A(ZR) bml bml bml bml BlO.A(2R)
W “Cr releasefrom macropbage targets causedby dilution of 5day MLR’ l/IO
l/30
1190
42.6 + I .4 46.3 k 3.0 47.0 * 1.1 50.2 + 2.1 44.9* 1.9 45. I f 2.6 45.0 t 1.7 44.3 f 1.2 32.2 + 1.6 20.0 k 2.3
24.6 f 1.1 28.9 f 3.2 26.2 + 1.1 34.7 k 2.2 24.8 k 0.8 33.9 zt 2.8 29.0 k 2.4 27.6 f I.1 18.0 f 2.0 11.5* 1.7
15.2 f 1.5 14.3 f 2.2 10.1 k 2.2 14.3 + 3.0 9.1 f 1.6 20.9 f 1.3 8.1 + 1.0 9.7 f 3.8 5.8 t 1.8 4.6 f 0.5
’ B6 spleen cell responders (5 X lb) were preincubated for 24 hr with 1.2 X IO’ irradiated SDC or TDC in l6-mm weUs as descried under Materials and Methods After 24 hr, the cells were resuspended, removed into tubes, centrifuged, and the medium decanted and replaced with fresh medium containing 1.25 X ld irradiated bml or BlO.A(ZR) spleen &Is for restimulation for an additional 4 days in 16-mm wells. b During the first 24 lx, responder cells were preincubated with DC in medium containing either 5% HIFCS plus 1% TMS or 1% TMS alone. Aher preincubation, the replacement medium for all cultures was EMEM containing 10% HIFG?.. cd As for Table 2.
of murine TDC with classical DC morphology and cell surface marker expression, and to compare some of their functions with those of the well-characterized murine SDC. We wish to emphasize that mouse serum syngeneic with the responder T cells was used as a medium supplement in vitro. This was to ensure that the true ability (or inability) of DC to stimulate Tc cell generation in vitro was not masked by the artefactual activation, by FCS antigens, of Th cells capable of secreting interleukin 2 (IL2). DC were obtained from the thymus using the same method developed for SDC isolation (see Materials and Methods). IEM and FACS analysis (not shown in this paper) has determined that approximately 50% of the cells from the thymus and 80% of those from the spleen isolated according to this method are 33D l+ DC with typical dendritic morphology. About 5-10% of the cells in both populations are Thy-l+ or sIg+ and/FcR+, while the remaining contaminating cells are negative for these markers and remain unidentified. Therefore, we had to be rigorous in excluding the possibility that any functional differences that were observed were due to the different numbers of unidentified cells contaminating these DC populations. Taking into consideration the IEM observation that 50 and 80% of our isolated TDC and SDC populations, respectively, are genuine DC,a population of 1 X lo5 TDC would contain approximately 5 X 1O4genuine DC while a population of 1 X 1O5 SDC would contain 8 X lo4 genuine DC. Thus, according to data presented here, 3 X lo4 irradiated SDC (containing 2.4 X lo4 genuine DC) mixed with 4-5 X lo6 responder spleen cells stimulated strong Tc cell responseswithout added CSS, in all MLR investigated. However, under the same culture conditions, TDC at doses of l-4 X 1O5(containing 5-20 X 1O4genuine DC) stimulated very weak or undetectable Tc cell responses.The failure of TDC to stimulate Tc cell generation could not have been due to a suppressive effect of cells in the TDC population as a mixture of 1 X 10’
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TDC and 3 X 1O4SDC stimulated stronger Tc cell responsesthan 3 X 1O4SDC alone (Table 2). It is known that two signals are required for T cell activation (35). These are cell surface antigen patterns involving MHC encoded antigens (signal 1) and IL-l or IL-2 (signal 2). The experiments described by Tables 2-4 revealed that cells in the SDC population provided both signals 1 and 2 for Tc cell activation, whereas the TDC population was deficient in either one or both of these signals. Thus only when CSS was added to 5-day MLR did TDC stimulate strong Tc cell responses,whereas SDC stimulated strongly in the presence or absenceof CSS. Becausethere was substantial contamination, particularly of the TDC preparation, by unidentified cells, before further analysis of the functional difference between SDC and TDC it was essential to determine whether the contaminating cells were a significant stimulus for Tc cells. When 33Dl and C’ were employed to eliminate DC from TDC and SDC populations, the surviving cells were unable to stimulate Tc cell responses in the absence of CSS. Treated SDC but not TDC populations were able to stimulate very weak Tc cell responsesin the presence of CSS, indicating that there were some contaminating cells in the SDC populations deficient in either one or both signals for Tc cell activation. These results showed that in our isolated TDC and SDC populations, DC are the dominant, and in the case of TDC, the sole cell class responsible for Tc cell stimulation. Further analysis was undertaken to determine the nature of the functional difference between SDC and TDC in Tc cell stimulation. There were two possible explanations for the need for CSSin the caseof TDC. First, TDC might not produce signal 2 factors required for Tc cell activation; second, TDC might express insufficient Class I MHC antigens (signal 1) to stimulate a Tc cell response. Since it has been reported that gamma interferon (present in CSS) raises the level of MHC Class I and Class II antigens on cells (38), it was possible that the effect of CSS on TDC was to increase the quantity of MHC Class I antigens expressedon these cells to a sufficient level for efficient recognition by responder T cells. This second possibility seemsunlikely for two reasons. First, EM and FACS analysis (described in detail elsewhere) has shown that TDC and SDC express similar high levels of Class I and Class II MHC antigens. Second, exposing TDC to CSS for 16 hr did not improve their subsequent ability to stimulate Tc in the absence of CSS (Table 6). Further evidence that the functional defect in TDC is a lack of signal 2 has been provided by investigation of IL-1 levels in SDC and TDC. It was shown that upon stimulation by LPS TDC synthesize very low levels of IL-l, while SDC synthesize large amounts of IL-l comparable to the PU5-1.8 macrophage cell line used as a positive control. In contrast to our results, Wong et al. (24) isolated TDC from the rat using a modification ( 17) of the method developed by Steinman et al. (4), and presented evidence that these cells stimulated MLR and also played an accessoryrole in T cell responses to the mitogens concanavalin A (Con A) and sodium periodate. It is unlikely that contamination of TDC with macrophages could explain their ability to stimulate proliferative T cell responsesas Wong et al. were rigorous in excluding adherent and FcR+ and Ig+ cells from TDC preparations. However, heat-inactivated horse serum was used in their culture media and potential antigen-presenting cells (APC) were not
166
PEREIRA, RING, AND BLANDEN
removed from the responder cells of the MLR. Therefore, it is possible that there were APC in the responder population capable of presenting antigens from horse serum for MHC-restricted Th cell activation. Activated Th cells could release IL-2, thus providing signal 2 required for further activation of precursor T cells recognizing MHC antigens of the TDC. We have also found that if murine TDC are used as stimulators of MLR using HIFCS and spleen cell responders containing APC, then TDC are capable of stimulating Tc cell responses(data not shown). We reiterate that in all experiments described in this paper where the functional properties of TDC and SDC were compared, 1% TMS of responder strain, which has been shown to be an adequate medium supplement (28), was used in the culture media to avoid potential artefactual T cell activation by the processdescribed above. The experimental evidence presented here indicates that TDC differ from SDC in that they do not produce signal 2 and therefore they are not suited for a role as APC for mature T cells. However, in keeping with these findings, their role in the thymus is more likely to concern immature T cells. There are two potential functions that TDC could have in the thymus. They could be involved in positive selection of the repertoire of MHC-restricted T cells (42,43) and/or in the imposition of T cell selftolerance (4 1,44). In this context, Jenkinson et al. (44) have recently presented evidence consistent with a role for TDC in T cell tolerance induction. They constructed chimeric thymuses in vitro, using deoxyguanosine to eliminate lymphoreticular cells from thymuses while keeping the thymic epithelium intact. These thymuses were then recolonized with stem cells from fetal mice of a different MHC type. Lymphocytes maturing in such chimeric thymuses were tolerant only of the MHC antigens of the colonizing stem cells and not of MHC antigens of the thymic epithelium, thus raising the possibility that TDC derived from the stem cells were involved in inducing tolerance to MHC antigens. Bretscher and Cohn (43) have hypothesized that if T cells receive signal 1, they are inactivated, while both signals 1 and 2 lead to their activation. Our findings that TDC provide signal 1 and not signal 2 are compatible with both the Bretscher and Cohn hypothesis (43) and the Jenkinson et al. (44) suggestion that TDC are present in the thymus as tolerance inducers. However, if this is the case, TDC probably act on thymic T cells before they reach a similar state of maturity to extrathymic T cells, becausethe results of an experiment (Table 8) in which the ability of TDC to tolerize mature splenic T cells was investigated, suggestedthat TDC are not tolerogenic for mature T cells. Further investigations concerning interactions between TDC and immature T cells from the thymus are in progress. ACKNOWLEDGMENT We thank Ms. Geeta Chaudhri for performing the IL-l assaydescribed here.
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IMMUNOLOGY
102,152- 167 ( 1986)
Comparison of Functional Properties of Thymic and Splenic Dendritic Cells ROSEMARIE A. PEREIRA, NICHOLAS J. C. KING, AND ROBERT V. BLANDEN’ Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C. T. 2601, Australia Received January 27,1986; acceptedMay I,1986 Murine dendritic cells (DC) which are negative for surface immunoglobulin (sIg), Fc receptor (FcR), Thy- 1 antigen, and the mononuclear phagocyte-specific marker F4/80, but positive for the DC-specific marker 33Dl and major histocompatibility complex (MHC) Class 1 and Class II antigens were obtained from either the spleen (SDC) or thymus (TDC) and used to stimulate alloreactive cytotoxic T lymphocyte (Tc) generation in vitro, in 5-day mixed lymphocyte reaction (MLR)to investigate some of their functional properties. SDC were potent stimulators of Tc cells in the absenceof supematant of concanavalin-A-stimulated spleen cells (CSS)and using medium containing 1Zthioglycolate-stimulated mouse serum (TMS) to avoid potential artifactual helper T cell activation by fetal calf serum (FCS) antigens. TDC did not stimulate Tc cells under these conditions, but did so when mixed with small numbers of SDC, or in the presence of CSS. 33Dl and complement (C)-treated SDC and TDC populations failed to stimulate Tc cell responsesin the absence of CSS. In the presence of CSS, TDC treated in a similar manner did not stimulate Tc cell activity, while 33Dl and C-treated SIX stimulated a weak but detectable Tc cell response. These results indicate that genuine DC were the dominant cells presenting Class I MHC antigens in the TDC population and also that a minority of contaminating nonDC in the SDC population expressed sufficient MHC Class I antigens for Tc cell activation, but did not produce factor(s) required for Tc cell activation. The pretreatment of SDC and TDC with CSS before using them as stimulators did not improve their ability to stimulate Tc cell responses,indicating that the effect of CSS in improving the ability of TDC to stimulate Tc cell responseswas not solely due to MHC-elevating effects of components of CSS. Lipopolysaccharide (LPS)-treated SDC synthesized abundant interleukin 1 (IL-l), but TDC synthesized low levels of IL- I. Contact for 24 hr between TDC and splenic Tc cell precursors in medium containing 1%TMS but without CSS and FCS did not tolerize the T cells. o 1986 Academic press, Inc.
INTRODUCTION Lymphoid dendritic cells (DC) were first described morphologically as cells with pseudopods of varying lengths, dense granular cytoplasm and large irregular and refractive nuclei (1). Since then, the class of DC which populates thymus-dependent areas of the murine spleen has been extensively studied and characterized (2-l 1). Phenotypically, they lack surface immunoglobulin (sIg), receptors for immunoglobulin Fc receptor (FcR), Thy-l antigen, and the mononuclear phagocyte-specific ’ To whom correspondence should be addressed.
152 0008-8749/86 $3.00 Copylight 0 1986 by Academic PITS, Inc. All rights of repmdwtion in any form reserved.
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marker F4/80. They express a dendritic-cell-specific marker as defined by a monoclonal antibody 33Dl(l2) and high levels of Class I and Class II major histocompatibility complex (MHC)-encoded antigens (13, 14). Functionally, they are highly motile, and weakly phagocytic if at all. In vitro they have been shown to be strong stimulators of primary allogeneic and syngeneic mixed leukocyte reactions (MLR) (S-10) and of T helper cells (Th) required for both specific antibody responsesto sheep red blood cells and haptens coupled to polypeptide carriers (15, 16). They have been described as accessory cells necessary for mitogen-induced T cell proliferation (17, 18) and for the development of anti-trinitrophenol (TNP)-specific cytotoxic T-lymphocytes (Tc) to TNP-modified T cells (7). These properties of DC and their strategic location in thymus-dependent areas of spleen and lymph nodes and probably in afferent lymph (19, 20), as well as their high degree of membrane motility (which increases their chances of making contact with other cells), make them candidates for a major role in antigen presentation for MHC-restricted T cell responses. Dendritic-like cells which are nonphagocytic, lack FcR, sIg, and Thy-l antigen, and express Class I and Class II MHC antigens have been reported to be present in the thymus (21-24). However, in these published reports, the characterization of these cells is far from complete. It is believed that the thymus is the organ in the body where selection of the T cell repertoire and the primary imposition of T cell tolerance to self antigens take place (25). There is probably no requirement for foreign antigen presentation as such. What, therefore, is the role of dendritic cells in the thymus? This paper describesthe isolation of DC from the thymus (TDC) and comparison of some of their functional properties with those of splenic dendritic cells (SDC). MATERIALS AND METHODS Mice. C57BL/6 (B6), B6.C-H-2bm1 (bml), BlO.BR, BlO.A(2R), BALB/c, and C3H/HeJ strains maintained in the John Curtin School of Medicine were used at 4-7 weeks of age. Antibodies. Anti-Thy- I .2 monoclonal antibody produced by clone F7D5 was obtained from OLAC (Bicester, Oxon). Sheep anti-mouse immunoglobulin (SAMIg) produced by (NH&SO4 precipitation of serum from a sheep which had been hyperimmunized with mouse Ig was a kind gift from Dr. R. B. Ashman. 33Dl is a mouse-rat hybridoma which produces a rat anti-mouse DC-specific antibody ( 12). 33D 1 monoclonal antibody was kindly provided by Dr. R. M. Steinman. F4/80 is a mouse-rat hybridoma (26) which secretesa rat anti-mouse macrophagespecific antibody which does not crossreact on DC. F4/80 culture supernatant was kindly provided by Dr. D. A. Hume. Cell preparation. SDC and TDC were both isolated using the following method: Spleens and thymuses were minced in GIBCO Eagle’s minimum essential medium (EMEM) and lo8 cells/ml were treated with collagenase (Clostridium histolyticum, Boehringer Mannheim, GmbH, West Germany) at 2 mg/ml in EMEM for 15 min at 37°C. Cells were washed and resuspended in EMEM with 10m42-mercaptoethanol (2-ME) and 25 rruV Hepes and seededat 3 X 106/ml in 185-cm2 tissue culture flasks (Nunclon, Denmark) at 50 ml per flask. Cells were allowed to adhere for 2 hr at 37°C in an atmosphere of 5% CO2 in air after which as many nonadherent cells as possible
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were washed off, and remaining adherent cells recultured in EMEM supplemented with 5-10% heat-inactivated fetal calf serum (HIFCS), lop4 2-ME, 25 mM Hepes, and antibiotics (complete medium). After 16 hr of incubation at 37°C nonadherent or loosely adherent cells were harvested by gentle pipetting. These cells were then treated with anti-Thy- 1.2 and rabbit complement (C’) (Low Tox, Cedarlane, Ontario, Canada) to remove contaminating T cells. They were then rosetted with sheep erythrocytes coupled by CrC& with hyperimmune SAMIg (27) to remove Ig and FcR positive cells. Dead cells and rosetted cells were removed by separation on IsopaqueFicoll, giving a viable cell population rich in DC. The yields of DC from the spleen and thymus were OS-l% and 0.03-0.08%, respectively, of initial cell numbers. Mixed lymphocyte reactions. Primary one-way MLR were set up in flat-bottom 16mm (Linbro, Flow Labs., Virginia) or round-bottom 6-mm (Nunclon, Denmark) wells. Responder adherent cell-depleted or whole spleen cells were seeded at 4-5 X lo6 in 2-2.5 ml of culture medium per 16-mm well or 3-4 X lo5 in 0.15-0.2 ml of culture medium per 6-mm well, with varying numbers of stimulator cells irradiated with 2000 rads from a 6oCo source. Where adherent cell-depleted responders were used, adherent cells were depleted by incubating 1.5 X lo8 spleen cells suspended in 50 ml of EMEM containing lop4 2-ME in a 185-cm* tissue culture flask (Nunclon, Denmark), for 2 hr at 37°C to allow the cells to adhere. As many nonadherent cells as possible were removed, washed, and used as responders. Unless otherwise indicated, culture media consisted of EMEM with 1Op4MZME, antibiotics and 1%serum from responder strain mice treated with thioglycolate broth (TMS), as described elsewhere (28). Where indicated, 10%supernatant from concanavalin A-stimulated spleen cells (CSS) prepared as described (29) was added to the culture medium. Generation of Tc was determined after 5 days of culture, using appropriate “Crlabeled syngeneic and allogeneic peritoneal macrophage or tumor cell targets as described elsewhere (30, 3 1). Effecters and targets were incubated for 5 hr at 37°C and cytotoxic activity is expressedas mean percentage of specific 5’Cr releasegiven by the equation test release - spontaneous release x 100 total release - spontaneous release f standard error of the mean in triplicate assays. Interleukin-1 (IL-l) assay. IL-l secretion was assayedaccording to the method of Mizel et al. (32). To trigger IL- 1 release,DC were suspended in RPM1 1640 medium containing 10% HIFCS and 20 &ml lipopolysaccharide (LPS) and were seededat 1 X 106/ml in 16-mm or 6-mm flat-bottom wells in 2- and 0.2-ml aliquots, respectively. These cells were then incubated for 48 hr at 37°C in an atmosphere of 5% CO2 in air, after which cells were gently resuspended, transferred to centrifuge tubes, and spun at 1OOgfor 5 min. Supernatants were removed for assaying and the cell pellet was resuspended in its original volume of medium and frozen and thawed three times to release intracellular IL-l. Cell debris was eliminated by centrifugation. Both secreted (in supematants) and intracellular (from freeze-thawed cells) IL- 1 activity was assayed. Augmentation by IL-l of phytohemagglutinin (PHA) stimulation of C3H/HeJ mouse thymocytes was used as a measure of IL-l activity as described elsewhere (32). Briefly, serial twofold dilutions of 50 ~1of material to be assayed,were made in 96-well flat-bottom plates. To these wells were added 50 ~1 of thymocytes
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CELL FUNCTION
TABLE 1 Percent Contamination of SDC and TDC Preparation with Thy+ ’ and FcR+ and/Ig+ Cells b TDC
SIX Expt.
%Thy-1+
% FcR+/Ig+
% Thy+
% FcR+/Ig+
I II III
5 8 9
1 2 0
6 5 6
0 3 0
’ SDC and TDC (1 X 105)in 25 ~1of complete medium were incubated with 25 ~1of anti-Thy- 1.2 at a predetermined concentration and C’ (see Materials and Methods). Percentage of dead cells above a C’ control (approximately 3% dead) were scored as being Thy- 1+. ’ SDC and TDC (1 X 10’) in 25 ~1of complete medium were incubated with 25 pl of a 1%suspension of SAMIg-coupled sheep erythrocytes (see Materials and Methods). Cells forming rosettes were scored as being FcR+/Ig’.
(from 5- to 7-week-old C3H/HeJ mice) suspended at 107/ml in RPM1 1640 medium containing 10% HIFCS, 10M4M 2-ME, and 1 pg/ml PHA. A positive control consisted of serial dilutions of IL- 1-containing supernatants from freeze-thawed cell contents of the BALB/c macrophage cell line PU5-1.8 (33) which had been stimulated with LPS. Twelve replicates of thymocytes alone in medium containing 1 &ml PHA were a negative control. Plates were incubated for 48 hr at 37°C. During the last 6 hr of incubation, 1 &i of tritiated thymidine was added to each well and the cells were harvested onto glass fiber filter paper (Whatman GF/A) using a Titertek cell harvester. Tritiated thymidine uptake was assessedby counting in a Packard Tri-Carb scintillation counter. RESULTS
PhenotypicCharacterization of SDC and TDC By rosetting with SAMIg-coupled sheeperythrocytes and treating with anti-Thyl.2 and C’, we determined that the DC-rich populations obtained from both the thymus and spleen were >97% for FcR-/Ig-, and >9 1%Thyl.2- (Table 1). Fluorescence activated cell sorter (FACS) analysis and immunoelectron microscopy (IEM) were used to investigate morphology and expression of 33D 1, F4/80, and Class I and Class II MHC antigens on DC-rich cell populations from B6(H-2b) mice. Data are not given here and will be reported in detail elsewhere. Briefly, however, there were no detectable F4/80+ cells present in either SDC or TDC as compared with positive control peritoneal macrophages. Cells of classical DC morphology made up approximately 80% of the SDC populations and 50% of the TDC preparations. These cells were 33D l+, whereas control macrophages were 33D 1-. The DC in both splenic and thymic populations were positive for both Class I and Class II MHC antigens, whereas the contaminating non-DC were virtually all MHC. These findings correlate with the functional data presented below which indicate that 33Dl+ cells in the SDS and TDC populations were the predominant source of MHC antigens that triggered alloreactive Tc cell generation.
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STIMULATOR
CELL
NUMBER
(X 1 O-4)
FIG. 1. Curves showing generation of Tc activity using various doses of irradiated BALB/c SDC (A) or whole spleen cells (0) as stimulators of 5 X lo6 adherent cell-depleted B6 responder spleen cells in S-day MLR. Data given are mean percent specific lysis of 5’Cr-labeled BALB/c macrophage target cells in triplicate assaysof l/ 10 dilutions ofthe MLR. SE of mean were lessthan +40/oand have been omitted for clarity.
Capacity of SDC as Stimulators of Tc Cell Activity in MLR Other workers have reported the extraordinary potency of SDC as stimulators of alloreactive T cells (8, 10). Therefore, we first compared SDC prepared under our conditions with unfractionated spleen cells as stimulators of alloreactive Tc cells, under culture conditions in which artifactual activation of Th cells (and thus indirectly, of Tc cells) by foreign sera was prevented by the use of mouse serum from the responder mouse strain as a medium supplement. Various numbers of irradiated BALB/c SDC or spleen cells were used to stimulate 5 X lo6 adherent cell-depleted spleen cells in MLR. After 5 days, cultures were assayed for Tc cell activity (Fig. 1). SDC were superior stimulators to crude spleen cells over the entire dose range tested. The Tc cell response was maximal with doses of SDC between 8 X lo3 and 1 X 106;there was detectable Tc cell activity with I .6 X lo3 SDC. With spleen cells as stimulators, the Tc cell response generated was maximal at 1 X lo6 cells and declined sharply with lower stimulator numbers until it became undetectable below 2 X 105. The maximum response with 1 X 1O6spleen cells was at a level comparable to that stimulated by SDC at 8 X 103.From these results it is clear that SDC are approximately loo-fold more potent stimulators of primary Tc cell responses in MLR using our culture conditions than whole unfractionated spleen cells. Since SDC constitute up to 1%of splenocytes (2), these data are in accord with the idea that SDC are probably the most important stimulator cells in unfractionated spleen cell populations. We have also observed in this system that use of high numbers of SDC (e.g., 1 X lo6 in Fig. 1) consistently depressesdetectable Tc cell activity below maximal levels. This phenomenon is under further investigation.
Comparison of SDC with TDC as Stimulators of Tc Cell Generationin Primary MLR Irradiated BlO.BR (H-2k) SDC or TDC were used as stimulators at 1 X lo5 and 3 X 1O4per 5 X 1O6B6 (H-2b) spleen cell responders in a 5-day primary MLR. At both
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TABLE 2 Capacity of SDC and TDC to Stimulate Generation of Tc Cells in 5Day MLR”
Number and type of stimulators 1x 3x 1x 3x 1x
105 IO4 lo5 104 105+ 3 x 104
SIX SDC TDC TDC TDC SDC
Yield of responder cells @jb 64 42 12 26 56
% % release from BlO.BR macrophages caused by dilutions of 5day MLR’ l/l0 56.6 f 0.9 33.8 2 1.8 0.1 kO.5 9.3 + 2.5 54.1 + 0.9
l/30
l/90
51.7 f 1.1 17.5 + 0.5 2.2 + 0.6 4.5 + 1.0 42.0 + 2.5
32.7 + 0.3 5.3 + 0.7 1.1 + 1.3 1.6 f 1.6 22.1 * 2.0
a Adherent cell-depleted B6 spleen cell responders (5 X 106)were cultured with irradiated B lO.BR stimulators in 16-mm wells as described under Materials and Methods. b Percentageof viable cells recovered from the 5day MLR where original responder cell number is 100%. ‘Cells contained in l/IO, l/30, and l/90 aliquots from the 5&y MLR were assayed in triplicate on 5 X IO4 “Cr-labeled BlO.BR and B6 macrophage targets. Lysis of B6 targets was negligible and the data have been omitted for clarity. Data presented are the means of triplicates + SE of mean.
of these stimulator cell concentrations, SDC stimulated a good cytotoxic response, whereas TDC were either weakly stimulator-y or nonstimulatory (Table 2). When 1 X lo5 TDC and 3 X lo4 SDC were mixed together, they stimulated a Tc cell response similar in magnitude to that caused by 1 X lo5 SDC and significantly greater than that caused by 3 X lo4 SDC. These data indicate that the failure of TDC alone to stimulate was not due to a suppressive influence and suggested that they were stimulatory in the presence of SDC. The same phenomenon was observed in a B6 anti-bml MLR (data not shown) in which the difference between responder and stimulator was limited to two amino acids in the sequence of the H-2K Class I antigen (34).
Capacity of TDC to Stimulate Generation of Tc Cell Responsesin Primary MLR in the Presenceof CSS Data presented thus far suggestthat TDC display Class I MHC antigens and that they do not suppress Tc cell generation at the numbers used in this system when mixed with SDC. Therefore it seemed possible that the failure of TDC to stimulate Tc cell responseswas caused by a failure to secrete factor(s) (signal 2) known to be necessary for induction of Tc cell responses (35). A corollary of this proposition is that TDC should stimulate Tc cell responsesin the presence of CSSknown to contain factor(s) required for T cell activation (36). Various numbers of irradiated BALB/c TDC were used as stimulators with 4 X 1O6 cells from an adherent cell-depleted B6 spleen population as responders in 5-day MLR and culture media were either supplemented or not with 10% v/v CSS. In the absence of CSS, TDC were weak stimulators of generation of Tc cell activity in B6anti-BALB/c MLR at all numbers tested (Table 3), whereas SDC stimulated strongly with the typical dose response characteristics demonstrated before (Fig. 1). However, in the presence of CSS,TDC stimulated strong Tc cell activity with comparable dose-
158
PEREIRA, RING, AND BLANDEN TABLE 3 Capacity of TDC to Stimulate Generation of Tc Cells in B6 Anti-BALB/c MLR” in the Presenceor Absence of CSS
Number and type of stimulators
css added to MLR
Yield of responder cells (%I*
% “Cr release from BALB/c macrophage targets caused by dilutions of klay MLRC l/10
l/30
l/90
4x 105 8x 10“ 1.6x lo4 3.2 x lo3
TDC TDC TDC TDC
-
41.0 5.5 6.5 10.0
10.1 + 5.6 k 3.1 f 5.0 +
2.5 3.4 2.2 1.5
4.7 + 0.6 8.0 + 1.2 0.0 + 1.7 9.4 f 0.3
10.8 * 1.9 6.6 + 2.0 7.2 + 1.4 3.0 f 0.9
4 x 105 8x 10.’ 1.6 x lo4 3.2 x 10’
SDC SDC SDC SDC
-
71.0 70.0 50.0 13.5
43.7 zk3.8 49.1 f 1.1 50.8 f 0.3 10.3 + 2.2
13.8 + 3.8 23.3 + 2.8 11.9 f 1.8 11.6+0.4
12.5 f 14.4 5 11.9 k 10.6 -t
4x 105 8x lo4 1.6 x lo4 3.2 x 10’
TDC TDC TDC TDC
+ + + +
35.0 52.5 40.0 26.3
51.7 + 55.7 + 48.0 k 12.6 +
3.9 2.1 2.9 1.5
39.6 + 0.9 29.6 + 0.2 24.4 + 1.7 11.9kO.O
18.6 + 1.3 18.3 + 0.5 15.1 kO.2 12.4 + 0.3
4x 105 8x lo4 1.6 x lo4 3.2 x IO3
SDC SIX SDC SDC
+ + + +
85.0 80.0 90.0 55.0
50.8 k 69.5 k 70.2 + 47.2 f
4.0 1.2 2.0 1.5
22.8 + 0.3 45.6 + 0.3 40.4 k 0.3 25.2 f 1.9
14.3 * 1.3 20.3 f 2.2 20.0 + 3.5 8.3 f 1.2
0.4 0.7 3.7 1.5
’ Adherent cell-depleted B6 spleen cell responders (4 X 106)were cultured with various dosesofirradiated BALB/c stimulators in 16-mm wells as described under Materials and Methods. *scAs for Table 2.
response characteristics to SDC (in the absence of CSS). The response stimulated by SDC was also improved in the presence of CSS but this improvement was not as dramatic as that seen for TDC stimulators. As seen above, high numbers of SDC depressedTc activity. Thus SDC at 4 X lo5 apparently stimulated less activity than 8 X lo4 SDC, particularly in the presence of CSS. We also carried out B6 anti-bm 1 MLR comparing SDC with TDC as stimulators. Results obtained in this experiment (Table 4) were very similar to those in B6-antiBALB/c MLR, thus indicating that the degree of H-2 difference between responder and stimulator did not influence the outcome. Addition of 10% FCS to the culture medium also enabled bml TDC to stimulate generation of B6 Tc cells (Table 4), though not so efficiently as in the presence of CSS. B6 Tc cells stimulated by bml TDC in the presence of either CSS or FCS were as specific for bml macrophage target cells as Tc stimulated by bml SDC (e.g., lysis by l/l0 of the MLR of self B6 macrophages was negligible, and lysis of third-party CBA macrophages was approximately 10%).
Efect of 3301 and Complementon the Ability of TDC and SDC to Stimulate Tc Cell Generation in the Presenceor Absenceof CSS Data presented above (Table 3) indicate that cells in the TDC population are able to stimulate Tc cell generation in the presence of CSS.Electron microscopy (EM) has
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TABLE 4 Capacity of TDC to Stimulate Generation of Tc Cells in B6 Anti-bm 1 MLR’ in the Presenceor Absence of CSS
Number and type of stimulators
css added to MLR
10%FCS added to MLR
Yield of responder cells (%)*
% “Cr releasefrom bm 1 macrophage targets caused by dilutions of 5-day MLRC l/l0
l/30
l/90
1 x lo5 2.5 x 10“ 6.25 x lo3
TDC TDC TDC
55 40 8.5
1.1 kO.1 1.9 f 0.8 O.OkO.1
0.0 f 0.4 0.0 k 1.3 0.1 kO.5
0.0 + 1.0 0.0 f 1.7 0.0 f 0.9
1 x lo5 2.5 x 10“ 6.25 x 10’
SDC SDC SDC
92.5 65 35
43.0 f 2.0 39.4 * 3.3 16.2 + 2.8
13.1 kO.5 13.6 + 0.7 4.5 f 1.9
1.9 + 2.3 5.1 f 0.6 0.0 + 0.8
1 x lo5 2.5 x lo4 6.25 x 10’
TDC TDC TDC
+ + +
-
65 52.5 45
41.3 2 2.2 35.0 It 0.2 12.7 + 0.7
24.3 k 0.9 10.4 + 1.6 6.1 +0.7
6.7 + 1.2 7.4 * 0.7 5.0 k 2.5
1 x IO5 2.5 x lo4 6.25 x 10’
SDC SDC SDC
-
65 81.5 65
64.2 + 1.1 50.8 f 1.3 33.7 + 2.5
28.8 + 1.0 24.1 + 1.6 14.2 + 1.5
7.5 + 1.0 8.5 + 0.7 0.0 + 1.0
1 x 105 2.5 x lo4 6.25 x 10’
TDC TDC TDC
+ + + -
+ + +
70 45 30
59.0 + 0.9 0.5 f 1.7 0.4* 1.1
32.5 + 0.5 -2.0 f 0.6 0.7 + 1.9
12.1 + 1.3 -2.8 + 0.6 -1.7 + 1.9
1 x 105 2.5 x lo4 6.25 x IO3
SDC SDC SDC
-
+ + +
80 75 60
53.8 * 1.0 63.4 f 0.6 61.4k0.3
19.5 + 2.4 32.7 + 1.8 27.0 -t 0.5
5.5 + 1.8 13.5 f 1.6 13.1 + 1.7
’ Adherent cell-depleted B6 responders (4 X 106)were cultured with various doses of irradiated bml stimulators in 16-mm wells as described under Materials and Methods. *J As for Table 2.
shown that TDC and SDC populations isolated using the technique described under Materials and Methods contain approximately 50 and 80%, respectively, of cells with classical DC morphology (2). Therefore it was possible, particularly in the case of TDC populations, that in the presence of CSSother unidentified contaminating cells were responsible for stimulating Tc cell generation in MLR. To test this proposition, TDC and SDC populations from BALB/c mice were treated with 33Dl and C’ according to a protocol described elsewhere (lo), to lyse dendritic cells. Various numbers of irradiated treated and untreated TDC and SDC were used to stimulate 4 X 1O5 adherent cell-depleted B6 spleen cells in the presence and absence of CSS in 5-day MLR. As expected, TDC only stimulated Tc cell responses in the presence of CSS (Table 5), whereas SDC stimulated strongly both in the absenceand presence of CSS. C’-treated TDC and SDC populations showed some reduction in stimulator activity, probably due to nonspecific toxic effectsof the C. However, when a TDC population was treated with 33Dl and C’, the remaining cells failed to stimulate Tc cell generation in MLR even in the presence of CSS, thus indicating that TDC were the domi-
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TABLE 5 Effect of 33Dl and C’ on the Ability of TDC and SDC to Stimulate Tc Cell Generation in B6 Anti-BALB/c MLRa in the Presenceor Absence of CSS
Number and type of stimulators 2x 104 5x 10’ 1.25 x 10’ 2x 104 5x 10’ 1.25 X 10’ 2x 104 5x 10’ 1.25 x 10’ 2x 104 5x 10’ 1.25 X 10’ 2x 104 5x 103 1.25 x 10’ 2x 104 5x 10’ 1.25 x 10’ 2x 104 5x 10’ 1.25 x 10’ 2x 104 5 x 10’ 1.25 x 10’ 2x 104 5 x 10’ 1.25 x 10’ 2x 104 5 x 10’ 1.25 x 10’ 2x 104 5x 10” 1.25 X 10’ 2x 104 5x 10’ 1.25 x 10’
TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC TDC SDC SDC SDC SIX SDC SDC SIX SDC SDC SIX SDC SDC SDC SDC SDC SDC SIX SDC
css added to MLR
+ + + + + + + + + + + + + + + + + +
% “Cr releasefrom P8 15 targets causedby dilutions of S-day MLRC Treatment of cells
33Dl+ C’ 33Dl+ C’ 33Dl+ C’ 33Dl+ C’ 33Dl+ C’ 33Dl+ C’ C’ only C’ only C’ only C’ only C’ only C’ only 33Dl + C’ 33Dl+ C’ 33Dl + C’ 33Dl+ C’ 33Dl+ C’ 33Dl+ C’ C’ only C’ only C only C’ only C’ only C’ only
213
219
2121
4.6 - 0.2 0.0 k 0.7 0.0 + 0.7 66.2 f 1.8 66.6 * 0.9 -0.9 f 0.7 -2.8 + 0.1 -2.6 k 0.4 -2.5 + 1.2 -3.2 + 0.6 -3.4 f 0.7 -3.0 + 0.8 -0.7 + 1.5 -1.450.5 -2.5 k 0.7 42.4 + 3.4 37.4 + 3.2 38.2 + 3.3 79.6 + 3.5 57.2 f 1.2 26.1 + 0.6 75.7 f 3.0 74.6 zk2.4 78.6 + 0.4 0.6 + 0.2 -0.5 k 0.4 -4.0 r 0.5 20.6 + 0.7 4.5 + 1.9 -2.0 + 0.3 85.2 + 0.4 41.5 + 1.8 24.6 + 0.3 73.1 f 1.7 58.9 + 3.5 13.1 + 2.8
-2.2 k 1.1 0.0 + 0.3 0.0 f 1.2 66.8 + 4.6 54.8 2 2.9 -3.2 -t 1.2 0.4 + 0.8 -1.5kO.6 -3.8 + 0.6 -5.1 -+ 1.0 -3.4 * 0.5 -2.9 + 0.5 -1.1 * 1.1 -0.7 * 1.0 -4.1 + 0.4 31.6 + 1.1 15.4 * 0.2 3.0 + 1.2 74.1 + 2.5 32.0 ?I 0.7 10.1 + 1.0 66.1 + 0.2 67.9 + 4.2 61.0 A 2.2 1.8 + 1.2 -3.8 zk0.8 -3.4 + 0.5 1.6 + 3.3 -1.8 f 1.3 -3.1 f 0.2 66.1 f 5.6 22.9 f 0.7 9.3k2.1 73.6 + 2.7 42.6 + 0.4 4.7 f 0.3
-2.3 f 0.9 0.0 + 0.2 0.0 f 1.0 28.3 + 1.7 37.5 k 1.4 -3.0 + 1.0 0.2 2 0.3 -1.7 kO.6 -3.2 + 0.2 -5.4 + 1.0 -4.4 + 0.6 -4.2 + 0.3 -0.1 + 0.8 -2.3 + 0.7 -3.1 -+0.9 8.2 + 1.5 1.3 + 0.9 -0.7 + 1.8 43.1 f 2.9 14.8 f 0.2 3.4 + 1.0 47.1 + 4.2 50.0 + 3.3 38.0 k 4.0 -1.3 kO.1 -4.2 + 0.8 -3.4 k 1.2 2.9 + 1.3 -6.8 + 0.3 -3.2 f 0.3 36.8 + 2.4 6.3 + 1.3 1.1 kO.6 44.2 f 3.3 19.3 + 2.8 0.8 k 1.4
’ Adherent cell-depleted B6 spleen cell responders (4 X 10’) were cultured in triplicate with various numbers of irradiated BALB/c stimulators in 6-mm wells as described under Materials and Methods. bCells contained in 213,219, and 2127 aliquots from the triplicate 5day MLR were assayedon 1 X lo4 “Cr-labeled P8 15 targets. Data presented are the means of triplicates + SE of mean.
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nant and perhaps the only cells in the population that presented Class I MHC antigens to Tc cell precursors. SDC which had been treated in a similar manner failed to stimulate Tc cell generation in the absence of CSS. In the presence of CSS, however, they stimulated a weak but detectable Tc cell response at 2 X lo4 but not at lower cell numbers, suggesting either that there were some cells remaining in this population which did not produce signal 2 but which expressed MHC Class I antigens (signal 1) and were thus capable of stimulating Tc cell generation in the presence of CSS, or that the presence of CSS increased the MHC antigen expression on some of these cells to a level sufficient for stimulation.
Efect of Treating DC with CSSprior to their Useas Stimulators in MLR As shown above (Tables 2-5), TDC do not stimulate T cells in the absenceof CSS, thus suggesting the possibility that TDC express MHC antigens (signal l), but do not provide additional factor(s) essential for T cell activation (35). Another formal possibility, even though it was not obvious from our IEM studies referred to above, is that TDC normally expresslevels of MHC antigens that are insufficient for efficient T cell activation. The effect of CSS,which is known to contain y-interferon (37), may therefore be to increase MHC antigen expression (38) on TDC, thus improving their ability to stimulate T cells. If this latter possibility applies, then pretreatment of TDC with CSS before their use as stimulators should improve their stimulating capability in the absence of CSS. Accordingly, 5% CSS was added to the culture medium of portions of a thymus cell and spleen cell population for a 16-hr time interval during the course of the DC preparation procedure. The interval chosen was during overnight incubation at 37°C immediately following the initial 2-hr incubation and nonadherent cell removal (seeMaterials and Methods). The rationale for this protocol is, first, that 16 hr is an ample time for MHC-elevating effectsof CSSon other cell classes (38); and second, that virtually all of the Tc cell activity measured at the end of 5-day MLR results from contact with stimulator cells in the first 24 hr after mixing (39). CSS-treated or untreated TDC and SDC were then used as stimulators in 5-day B6 anti-BALB/c MLR. CSS pretreatment of TDC or SDC did not improve their stimulatory capacity for Tc cell generation (Table 6). CSS-treated SDC were significantly inferior to untreated controls. However, as above, in the presence of CSSduring the 5-day MLR, TDC stimulated Tc cell activity. These data do not determine whether or not CSS treatment causes changes in MHC antigen expression or has other effects on TDC or SDC, a topic of ongoing investigation, but they are consistent with the view that the failure of TDC to stimulate T cells is caused by a lack of signal 2.
Comparison of the Ability of TDC and SDC to Produce IL-l upon Stimulation with LPS Data presented thus far strongly suggestthat the difference in the abilities of TDC and SDC to stimulate Tc cell generation stems from an inability of TDC to produce factor(s) necessaryfor T cell activation (35) such as IL-l. It has been shown (40) that upon stimulation with LPS, cells such as the macrophage cell line PU5- 1.8 secrete IL-l or at least synthesize IL-1 which may be detected intracellularly. Therefore, 8 X 10’ SDC and TDC were treated with LPS, as described under Materials and
162
PEREIRA, RING, AND BLANDEN TABLE 6 Effect of Exposure of TDC to CSS prior to B6 Anti-BALB/c Way MLR“ on their Capacity to Stimulate Tc Cell Activation
CSS addedb Number and type of stimulators 1 x 105 2.5 x lo4 6.25 X lo3 1 x 10s 2.5 x lo4 6.25 X lo3 1 x 105 2.5 X lo4 6.25 x 10’ 1 x 105 2.5x104 6.25 x 10’ 1 x 105 2.5 x lo4 6.25 X 10’ 1 x 10s 2.5 x lo4 6.25 x lo3
TDC TDC TDC SDC SDC SDC TDC TDC TDC SDC SDC SDC TDC TDC TDC SDC SDC SDC
% “Cr release from BALB/c macrophage targets causedby dilutions the 5-day MLRd
ToDC preparation
To 5day MLR
Yield of responder cells (%Y
l/10
l/30
l/90
+ + + + + +
+ + + + + + -
12 3 2 25 37.5 15 35 30 12 45 57.5 30 8.5 3.5 3 25 30 14
1.4 + 0.4 3.3 + 1.2 4.3 + 1.1 37.6 zk0.7 38.1 + 0.6 5.8 + 0.6 25.2 + 0.5 14.2 + 0.7 8.2 f 1.8 53.8 + 1.0 42.8 + 2.2 26.0 -c 0.3 6.2+ 1.1 3.1 kO.3 3.2 f 1.5 26.6 + 1.8 19.0 f 0.4 1.3 k 1.6
1.9 f 2.0 0.0 + 0.3 0.0 1- 0.6 14.9 f 1.1 16.0 + 0.7 2.1 +0.2 10.1 kO.6 2.4 k 0.3 -0.1 + 2.3 34.4 + 1.1 17.2 k 2.4 9.3 + 0.5 5.2 f 0.6 3.2 + 0.8 1.9 -+ 0.5 8.3 + 1.0 13.3 f 1.0 0.0 + 1.6
0.0 f 0.5 1.7 rt 0.5 0.3 lk 0.9 5.4 + 0.3 5.1 +0.3 0.9 + 0.2 1.7 + 3.1 1.3 k 0.6 1.4kO.6 12.3 k 1.4 6.7 k 0.5 3.8 L 0.8 3.6 AZ1.0 6.1 -t 1.6 3.0 + 0.5 2.6 + 0.7 0.0 -t 0.8 0.0 -t 1.0
’ Adherent cell-depleted B6 spleen cell responders (4 X 106)were cultured with various dosesof irradiated BALB/c stimulators in 16-mm wells as described under Materials and Methods. b Where indicated, 5% CSSwas added during DC preparation for 16 hr and then washed off (seeResults) or to 5-day MLR. csdAs for Table 2.
Methods. Supernatants, as well as the contents of freeze-thawed cells, were assayed for IL- 1 activity. Strong IL1 activity was detected in the contents of freeze-thawed SDC and significant IL- 1 activity was also found in supernatants of SDC (Table 7). However, only weak IL 1 activity was detected in supematants and in the contents of freeze-thawed TDC. The data therefore support the suggestion that the weak stimulation of Tc cells by TDC shown here is caused by the low levels of IL- 1 in TDC.
Examination of the Capacity of TDC to Tolerize Mature T Cells Data presented above indicate that TDC provide antigen (signal 1) but not signal 2 for the activation of Tc cells. Bretscher and Cohn have postulated that signal 1 alone should be tolerogenic (4 1). Therefore, we investigated whether TDC are tolerogenic for mature T cells. This was determined by examining the ability of B6 responder spleen cells to generate Tc cell responses upon optimal stimulation with irradiated bml or BlO.A(2R) spleen cells following preincubation for 24 hr at 37°C with bml
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TABLE 1 Capacity of BALB/c TDC and SDC to Produce IL- 1 upon Stimulation with LPS” Sampleb
IL- 1 titerc(logiO)
TDC (supematants) TDC (cell contents) SDC (supematants) SDC (cell contents) PUS-l.8 (cell contents)
0.56~0.131 0.7 1 k 0.085 1.58 -c 0.026 2.41 + 0.072 2.16?0.125
’ BALB/c SDC and TDC (5 X 10’) were cultured with LPS in 6-mm flat-bottom wells as described under Materials and Methods. b Samples consisted of supematants from cells cultured with LPS and also the contents of freeze-thawed cells (seeMaterials and Methods). ‘IL-1 titer is expressed as log,, endpoint dilution which was taken at 3 SD above background (374 rfr 100 cpm). Data presented are means of triplicate assays 2 SE of the mean. The IL-l titers between corresponding samples of TDC and SDC are significantly different (P < 0.005) and the difference between IL- 1 titers of SDC and PU5- 1.8 cell contents is not significant.
TDC. Controls consisted of B6 responder spleen cells preincubated with B6 TDC and SDC and bm 1 SIX. During the 24-hr preincubation step, two kinds of culture medium were used. The first consisted of EMEM with 5% HIFCS, 1%TMS, and 1O-4A4 2-ME, and the second, EMEM with 1%TMS and 10e4M2-ME. After preincubation for 24 hr with DC, restimulation with spleen cells was carried out for 4 days in medium with 10%HIFCS. When 5% HIFCS together with 1% TMS were used during preincubation with B6 and bm 1 TDC and SDC, subsequent restimulation with irradiated bm 1 spleen cells yielded a strong Tc cell response against bml targets (lines 1-4, Table 8). Likewise, in the same culture medium preincubation of B6 responder spleen cells with bml TDC and subsequent restimulation with irradiated B lO.A(2R) spleen cells yielded a strong anti-B lO.A(2R)-specific cytotoxic response (line 5, Table 8). Preincubation of B6 responder cells with B6 TDC and SDC and bml SDC, in culture medium supplemented with 1% TMS alone, followed by restimulation after 24 hr with irradiated bm 1 spleen cells, led to the generation of strong anti-bm 1 specific Tc cell responses (lines 6-8, Table 8). In the same culture medium, when B6 responder spleen cells were preincubated with bm 1 TDC, there was a threefold reduction in the anti-bml Tc cell response generated upon restimulation with irradiated bml spleen cells (line 9, Table 8). This reduction was not specific, however, as there was also a threefold reduction in the magnitude of the anti-BlO.A(2R) Tc cell responsegenerated after restimulation with BlO.A(2R) spleen cells (line 10, Table 8). These data suggestthat neither TDC nor SDC are tolerogenic for mature splenic T cells, but do not exclude a role for TDC in imposing self-tolerance upon immature intrathymic T cells. DISCUSSION Dendritic-like or interdigitating cells have been reported to be present in murine and rat thymuses (2 l-23). It was therefore of interest to isolate enriched populations
164
PEREIRA, KING, AND BLANDEN TABLE 8 Capacity of bm 1 TDC to Tolerize Mature B6 T cells’
Type of stimulator cells used in preincubation for first 24 lx
Medium supplement (tint 24 bry
B6 B6 bml bml bml B6 B6 bml bml bml
5% HIFCS 5% HIFCS 5% HIFCS 5% HIFCS 5% HIFCS 1% TMS l%TMS IWTMS l%TMS l%TMS
SDC TDC SDC TDC TDC SDC TDC SDC TDC TDC
+ + + + +
1%TMS 1% TMS 1% TMS I % TMS 1%TMS
strain of spleen cells used for lestimuIatioo over 4 days
Yield of reqander ceus(?a)=
Strain of m&mphage tarsets
bml bml bml bml BlO.A(ZR) bml bml bml bml BlO.A(ZR)
34 38 36 44 44 40 36 34 14.6 14.6
bml bml bml bml BlO.A(ZR) bml bml bml bml BlO.A(2R)
W “Cr releasefrom macropbage targets causedby dilution of 5day MLR’ l/IO
l/30
1190
42.6 + I .4 46.3 k 3.0 47.0 * 1.1 50.2 + 2.1 44.9* 1.9 45. I f 2.6 45.0 t 1.7 44.3 f 1.2 32.2 + 1.6 20.0 k 2.3
24.6 f 1.1 28.9 f 3.2 26.2 + 1.1 34.7 k 2.2 24.8 k 0.8 33.9 zt 2.8 29.0 k 2.4 27.6 f I.1 18.0 f 2.0 11.5* 1.7
15.2 f 1.5 14.3 f 2.2 10.1 k 2.2 14.3 + 3.0 9.1 f 1.6 20.9 f 1.3 8.1 + 1.0 9.7 f 3.8 5.8 t 1.8 4.6 f 0.5
’ B6 spleen cell responders (5 X lb) were preincubated for 24 hr with 1.2 X IO’ irradiated SDC or TDC in l6-mm weUs as descried under Materials and Methods After 24 hr, the cells were resuspended, removed into tubes, centrifuged, and the medium decanted and replaced with fresh medium containing 1.25 X ld irradiated bml or BlO.A(ZR) spleen &Is for restimulation for an additional 4 days in 16-mm wells. b During the first 24 lx, responder cells were preincubated with DC in medium containing either 5% HIFCS plus 1% TMS or 1% TMS alone. Aher preincubation, the replacement medium for all cultures was EMEM containing 10% HIFG?.. cd As for Table 2.
of murine TDC with classical DC morphology and cell surface marker expression, and to compare some of their functions with those of the well-characterized murine SDC. We wish to emphasize that mouse serum syngeneic with the responder T cells was used as a medium supplement in vitro. This was to ensure that the true ability (or inability) of DC to stimulate Tc cell generation in vitro was not masked by the artefactual activation, by FCS antigens, of Th cells capable of secreting interleukin 2 (IL2). DC were obtained from the thymus using the same method developed for SDC isolation (see Materials and Methods). IEM and FACS analysis (not shown in this paper) has determined that approximately 50% of the cells from the thymus and 80% of those from the spleen isolated according to this method are 33D l+ DC with typical dendritic morphology. About 5-10% of the cells in both populations are Thy-l+ or sIg+ and/FcR+, while the remaining contaminating cells are negative for these markers and remain unidentified. Therefore, we had to be rigorous in excluding the possibility that any functional differences that were observed were due to the different numbers of unidentified cells contaminating these DC populations. Taking into consideration the IEM observation that 50 and 80% of our isolated TDC and SDC populations, respectively, are genuine DC,a population of 1 X lo5 TDC would contain approximately 5 X 1O4genuine DC while a population of 1 X 1O5 SDC would contain 8 X lo4 genuine DC. Thus, according to data presented here, 3 X lo4 irradiated SDC (containing 2.4 X lo4 genuine DC) mixed with 4-5 X lo6 responder spleen cells stimulated strong Tc cell responseswithout added CSS, in all MLR investigated. However, under the same culture conditions, TDC at doses of l-4 X 1O5(containing 5-20 X 1O4genuine DC) stimulated very weak or undetectable Tc cell responses.The failure of TDC to stimulate Tc cell generation could not have been due to a suppressive effect of cells in the TDC population as a mixture of 1 X 10’
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TDC and 3 X 1O4SDC stimulated stronger Tc cell responsesthan 3 X 1O4SDC alone (Table 2). It is known that two signals are required for T cell activation (35). These are cell surface antigen patterns involving MHC encoded antigens (signal 1) and IL-l or IL-2 (signal 2). The experiments described by Tables 2-4 revealed that cells in the SDC population provided both signals 1 and 2 for Tc cell activation, whereas the TDC population was deficient in either one or both of these signals. Thus only when CSS was added to 5-day MLR did TDC stimulate strong Tc cell responses,whereas SDC stimulated strongly in the presence or absenceof CSS. Becausethere was substantial contamination, particularly of the TDC preparation, by unidentified cells, before further analysis of the functional difference between SDC and TDC it was essential to determine whether the contaminating cells were a significant stimulus for Tc cells. When 33Dl and C’ were employed to eliminate DC from TDC and SDC populations, the surviving cells were unable to stimulate Tc cell responses in the absence of CSS. Treated SDC but not TDC populations were able to stimulate very weak Tc cell responsesin the presence of CSS, indicating that there were some contaminating cells in the SDC populations deficient in either one or both signals for Tc cell activation. These results showed that in our isolated TDC and SDC populations, DC are the dominant, and in the case of TDC, the sole cell class responsible for Tc cell stimulation. Further analysis was undertaken to determine the nature of the functional difference between SDC and TDC in Tc cell stimulation. There were two possible explanations for the need for CSSin the caseof TDC. First, TDC might not produce signal 2 factors required for Tc cell activation; second, TDC might express insufficient Class I MHC antigens (signal 1) to stimulate a Tc cell response. Since it has been reported that gamma interferon (present in CSS) raises the level of MHC Class I and Class II antigens on cells (38), it was possible that the effect of CSS on TDC was to increase the quantity of MHC Class I antigens expressedon these cells to a sufficient level for efficient recognition by responder T cells. This second possibility seemsunlikely for two reasons. First, EM and FACS analysis (described in detail elsewhere) has shown that TDC and SDC express similar high levels of Class I and Class II MHC antigens. Second, exposing TDC to CSS for 16 hr did not improve their subsequent ability to stimulate Tc in the absence of CSS (Table 6). Further evidence that the functional defect in TDC is a lack of signal 2 has been provided by investigation of IL-1 levels in SDC and TDC. It was shown that upon stimulation by LPS TDC synthesize very low levels of IL-l, while SDC synthesize large amounts of IL-l comparable to the PU5-1.8 macrophage cell line used as a positive control. In contrast to our results, Wong et al. (24) isolated TDC from the rat using a modification ( 17) of the method developed by Steinman et al. (4), and presented evidence that these cells stimulated MLR and also played an accessoryrole in T cell responses to the mitogens concanavalin A (Con A) and sodium periodate. It is unlikely that contamination of TDC with macrophages could explain their ability to stimulate proliferative T cell responsesas Wong et al. were rigorous in excluding adherent and FcR+ and Ig+ cells from TDC preparations. However, heat-inactivated horse serum was used in their culture media and potential antigen-presenting cells (APC) were not
166
PEREIRA, RING, AND BLANDEN
removed from the responder cells of the MLR. Therefore, it is possible that there were APC in the responder population capable of presenting antigens from horse serum for MHC-restricted Th cell activation. Activated Th cells could release IL-2, thus providing signal 2 required for further activation of precursor T cells recognizing MHC antigens of the TDC. We have also found that if murine TDC are used as stimulators of MLR using HIFCS and spleen cell responders containing APC, then TDC are capable of stimulating Tc cell responses(data not shown). We reiterate that in all experiments described in this paper where the functional properties of TDC and SDC were compared, 1% TMS of responder strain, which has been shown to be an adequate medium supplement (28), was used in the culture media to avoid potential artefactual T cell activation by the processdescribed above. The experimental evidence presented here indicates that TDC differ from SDC in that they do not produce signal 2 and therefore they are not suited for a role as APC for mature T cells. However, in keeping with these findings, their role in the thymus is more likely to concern immature T cells. There are two potential functions that TDC could have in the thymus. They could be involved in positive selection of the repertoire of MHC-restricted T cells (42,43) and/or in the imposition of T cell selftolerance (4 1,44). In this context, Jenkinson et al. (44) have recently presented evidence consistent with a role for TDC in T cell tolerance induction. They constructed chimeric thymuses in vitro, using deoxyguanosine to eliminate lymphoreticular cells from thymuses while keeping the thymic epithelium intact. These thymuses were then recolonized with stem cells from fetal mice of a different MHC type. Lymphocytes maturing in such chimeric thymuses were tolerant only of the MHC antigens of the colonizing stem cells and not of MHC antigens of the thymic epithelium, thus raising the possibility that TDC derived from the stem cells were involved in inducing tolerance to MHC antigens. Bretscher and Cohn (43) have hypothesized that if T cells receive signal 1, they are inactivated, while both signals 1 and 2 lead to their activation. Our findings that TDC provide signal 1 and not signal 2 are compatible with both the Bretscher and Cohn hypothesis (43) and the Jenkinson et al. (44) suggestion that TDC are present in the thymus as tolerance inducers. However, if this is the case, TDC probably act on thymic T cells before they reach a similar state of maturity to extrathymic T cells, becausethe results of an experiment (Table 8) in which the ability of TDC to tolerize mature splenic T cells was investigated, suggestedthat TDC are not tolerogenic for mature T cells. Further investigations concerning interactions between TDC and immature T cells from the thymus are in progress. ACKNOWLEDGMENT We thank Ms. Geeta Chaudhri for performing the IL-l assaydescribed here.
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