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The Journal of Immunology, 1998, 161: 3966-3973.
Copyright © 1998 by The American Association of Immunologists

Initiation of the Autologous Mixed Lymphocyte Reaction Requires the Expression of Costimulatory Molecules B7-1 and B7-2 on Human Peripheral Blood Dendritic Cells

Clemens Scheinecker1,*,{dagger}, Klaus P. Machold{dagger}, Otto Majdic{ddagger}, Paul Höcker§, Walter Knapp{ddagger} and Josef S. Smolen*,{dagger}

* II. Department of Medicine with Rheumatology, Lainz Hospital, Vienna, Austria; {dagger} Department of Rheumatology, University of Vienna, Vienna, Austria; {ddagger} Institute of Immunology, University of Vienna; Vienna, Austria; and § Department of Transfusion Medicine, University of Vienna, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human autologous mixed lymphocyte reaction (AMLR) consists of a proliferative response of primarily CD4+ T lymphocytes stimulated by autologous non-T cells expressing class II MHC-encoded gene products and is thought to represent a self-recognitive mechanism that might be important in regulating the cellular interactions involved in the generation of normal immune responses. To further define appropriate stimulator cell populations, as well as the molecular mechanism responsible for the initiation of AMLR, we compared the T cell-stimulatory capacity of highly purified populations of peripheral blood dendritic cells (DCs) and monocytes (Mos) under serum-free conditions, thus carefully avoiding the presence of xenogeneic Ags. Whereas both freshly isolated Mos and DCs were found to be poor stimulators of autologous T cell proliferation, preactivation of DCs, but not of Mos, for 48 h with granulocyte-macrophage CSF led to a 113-fold increase in DC stimulatory capacity. AMLR was inhibited by mAbs against HLA-DR and CD4 molecules, and, in addition, showed a higher dependence on the granulocyte-macrophage CSF-induced up-regulation and/or de novo expression of the costimulatory molecules B7-2 and, in particular, B7-1 as compared with an Ag-specific or allogeneic MLR. Thus, our data suggest that the high density of costimulatory molecules together with MHC class II molecules on competent APCs appear to be the major triggers for the initiation of AMLR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The in vitro proliferation of T cells in response to stimulation by autologous non-T cells has been termed the autologous mixed lymphocyte reaction (AMLR)2 (1, 2). The AMLR has been shown to bear specificity and memory (3, 4, 5, 6), and, as a genuine immunologic response, proliferation of T cells in the AMLR is triggered by MHC class I and II products via the T cell Ag receptor and seems to be regulated by the same mechanisms that control Ag-specific T cell activation (5, 6, 7, 8, 9, 10, 11, 12). In contrast, the role of the AMLR itself has not been finally clarified, although helper, suppressor, and general immunoregulatory effects have been attributed to it (13, 14, 15, 16, 17). Such possible roles have been supported by observations of an abnormal AMLR in diseases with associated dysfunction of the immune system (18, 19, 20, 21).

Whereas the predominant cell proliferating in the AMLR was found to be CD4+ (9) and to belong to the CD45RA+ memory Th cell subset (12, 17), several cell types, including monocyte/macrophages (Mos) (22, 23), dendritic cells (DCs) (24, 25), null cells (23), NK cells (2), and B cells (26, 27, 28), as well as Ag- and mitogen-activated T cells (29, 30, 31), have been considered to act as stimulator cells in the AMLR.

Nevertheless, comparative analyses have led to the suggestion that DCs bear the unique ability to stimulate autologous T cells in the AMLR (24, 25, 32, 33, 34). However, DCs constitute only a minor fraction (0.1–1%) of PBMCs, and the isolation procedure used until recently depended on physical separation with prolonged culture in serum. This process, however, resulted in low yields and purities and probably affected phenotypic and functional properties (35, 36). In addition, there is controversy in the literature as to whether or not the AMLR represented proliferation of T cells to xenoantigens encountered by APCs during the isolation and/or culture procedure (37, 38), rather than one to autologous Ags (39, 40).

New approaches using immunomagnetic separation techniques now allow rapid isolation of large numbers of highly purified DCs without concomitant alteration of their phenotypic or functional characteristics (41). In addition, by using serum-free media, the addition of FCS or human serum containing potentially stimulating xeno- or allogeneic Ags can be omitted. Therefore, it is now more easily and reliably possible to analyze and compare purified populations of peripheral blood DCs and Mos for their stimulatory capacities in the AMLR, and to try to define some of the molecular mechanisms that might be involved in the initiation of T cell proliferation in the AMLR.

In the present investigation, we demonstrate that under serum-free conditions only matured peripheral blood DCs are able to induce considerable proliferation of autologous T cells and that this proliferation depends on up-regulation of costimulation associated molecules on DCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

A variety of mAbs/fluorochrome conjugates were used in the study: mAbs to CD1a (NA1/34-HLK), CD14 (UCH-M1), CD32 (AT10), CD50 (101-ID2), CD58 (BRIC5), CD64 (10.1), and CD80 (BB-1) were obtained from Serotec (Oxford, U.K.); mAbs to CD3 (SK7), CD11a (G-25.2), CD11c (S-HCL-3), CD19 (4G7), CD25 (LA3), CD33 (P67.6), CD34 (HPCA-2), CD45RA (L48), CD56 (MY31), CD69 (L78), and HLA-DR (L243) were obtained from Becton Dickinson (San Jose, CA); mAbs to CD11b (44), CD40 (5C3), CD86 (IT2.2), HLA-A,B,C (G46-2.6), HLA-DP (HI43), and HLA-DQ (TÜ169) were obtained from PharMingen (San Diego, CA); mAbs to CD16 (3G8), CD45RO (UCHL1), CD54 (84H10), and CD71 (YDJ1.2.2) were obtained from Coulter (Hialeah, FL); and mAbs to CD68 (Ki-M7) were obtained from Behring (Marburg, Germany). The myeloperoxidase-specific mAb H-43-5 and the lysozyme-specific mAb LZ-1 were obtained from An der Grub (Kaumberg, Austria). CD4 (VIT4) and CD8 (VIT8) mAbs were from our own sources (O. Majdic). In all experiments, irrelevant control mAbs of the same IgG isotype were included.

Cell separation

PBMCs were isolated from heparinized whole blood of normal healthy donors by standard gradient centrifugation with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). PBMCs were harvested from the interface, washed twice, and resuspended in PBS supplemented with 5 mM EDTA and 0.5% human serum albumin. FCS supplementation of the buffers used during cell isolation, as recommended by the manufacturer, was replaced by heat-inactivated human serum to avoid the presence of potentially stimulatory xenogeneic Ags.

Monocytes. CD14+ Mos were separated by high-gradient magnetic sorting using the VARIOMACS technique (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). This method has been described in detail elsewhere (41). Briefly, PBMCs were incubated with colloidal superparamagnetic microbeads conjugated with anti-human CD14 mAbs for 15 min on ice, and thereafter passed over a column in a strong magnetic field (MACS; Miltenyi Biotec). Labeled and positively enriched cells were eluted from magnetic columns by removal of columns from the magnetic device. Purity of CD14+ Mos was determined by flow cytometry and was found to be 93 ± 3%.

T cells. T cells were subsequently prepared either from the Mo-depleted cell fraction or directly from PBMCs by incubation of the cells with saturating concentrations of purified mAbs against CD14, CD33, CD16, CD56, CD19, and HLA-DR molecules for 15 min on ice. After washing, the cells were incubated with rat anti-mouse Ig-conjugated superparamagnetic microbeads for 15 min and passed over a magnetic column as described above. The negatively selected T cells were collected and more than 98% expressed CD3 by flow cytometric analysis.

Dendritic cells. DCs were obtained either from the Mo-depleted cell fraction or directly from PBMCs by depletion of all Mo, NK, B, and T cells by the method described above using mAbs against CD14, CD16, CD56, CD19, and CD3. The negatively selected cells were subsequently incubated with anti-CD4-conjugated magnetic beads and the positively selected cells were collected.

Short term preactivation of DCs and Mos

Freshly isolated DCs or Mos were cultured for 48 h in 24-well plates (Costar, Cambridge, MA) (1 x 106 cells in 1 ml/well) in the serum-free medium X-VIVO15 (BioWhittaker, Walkersville, MD) supplemented with penicillin (125 IE/ml), streptomycin (125 mg/ml), and granulocyte-macrophage CSF (GM-CSF; 100 ng/ml; Novartis, Basel, Switzerland) in a humidified atmosphere in the presence of 5% CO2.

Immunofluorescence staining procedure

Membrane staining. For membrane staining, cells (1–10 x 107/ml) were incubated for 15 min at 4°C with fluorescein isothiocyanate or phycoerythrin-conjugated or -unconjugated mAbs. For triple stainings, peridine chlorophyll protein-conjugated mAbs were used. For stainings using unconjugated mAbs, fluorescein isothiocyanate-conjugated F(ab')2 of sheep anti-mouse Ig Abs (An der Grub) were used as a second step reagent, as described previously (42).

Intracellular staining. For suspension staining of intracellular Ags, we used the reagent combination Fix&Perm (An der Grub) as described previously (43). Briefly, cells were first fixed in fixation medium for 15 min at room temperature, and, after one washing step, resuspended and mixed with permeabilization medium plus fluorochrome (fluorescein isothiocyanate, phycoerythrin, or peridine chlorophyll protein)-labeled Abs. After a further incubation for 15 min at room temperature, cells were washed again and analyzed.

Flow cytometry

Membrane and intracellular fluorescence was analyzed on a standard FACScan flow cytometer supported by PC Lysis software (Becton Dickinson).

Determination of morphology of cells

Freshly isolated Mos and DCs and cells cultured for 48 h were directly analyzed from culture wells by invert light microscopy (Olympus, Tokyo, Japan).

Proliferation assays

For induction of AMLR (or allogeneic MLR (allo-MLR)), T cells were incubated with graded numbers of autologous (or allogeneic) irradiated (3000 rad, 137Cs source) stimulator cells. Stimulator cells were either freshly isolated (f) DCs and fMos or GM-CSF (100 ng/ml)-preactivated cultured (c) Mos and cDCs, respectively. For presentation of specific Ags, cultures were set up in the presence of 5LF tetanus toxoid (Connaught Laboratories, Willowdale, Ontario, Canada). Cultures were performed either in medium RPMI 1640 (Gibco/BRL, Bethesda, MD) supplemented with 10% FCS or in the serum-free medium X-VIVO15 (BioWhittaker). Proliferation of T cells was monitored by measuring [methyl-3H]TdR (Amersham, Buckinghamshire, U.K.) incorporation on day 7 of culture. All values of cpm were calculated from triplicates and indicated as mean ± SEM.

mAb blocking assays

For blocking experiments, cDCs were incubated with mAbs against CD4, CD8, CD80, CD86, or HLA-DR at varying final concentrations (as indicated in Results) for 15 min at 4°C before the addition of T cells (ratio 1:1), and the mAbs were left throughout the culture period. Irrelevant mAbs were used as isotype controls. Inhibitory effects on T cell proliferation were expressed as percent reactivity of control cultures without addition of mAbs performed in parallel.

Statistics

Student’s t test was used to determine whether the difference between control and sample was significant (p < 0.05 being significant).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotypic characterization of Mos, DCs, and T cells from human PBMCs

Monocytes. Isolation of Mos from human PBMCs using MACS sorting with anti-CD14 labeled superparamagnetic microbeads led to populations of 93 ± 3% purity, as determined by the expression of CD14 and MHC class II molecules by FACS analysis (Table IGo). Moreover, virtually all cells expressed intracellular myeloperoxidase and lysozyme.


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  		Table I. Expression of surface marker molecules on freshly isolated cell populaitons1

 
Dendritic cells. For the isolation of human peripheral blood DCs, the previously established criteria for identifying DCs were employed, i.e., MHC class II+ cells negative for lineage-specific Ags (25) while expressing relatively high levels of CD4 molecules (44). Thus, PBMCs were first depleted of cells that expressed lineage-specific Ags (CD3, CD14, CD16, CD19, CD56), and subsequently positively selected for CD4+ cells. As can be seen in Table IGo, fDCs virtually lacked expression of lineage marker molecules typical of T, B, and NK cells. fDCs had high levels of MHC class II molecules, and only few cells (14 ± 7%) weakly expressed CD14 molecules. As described by O’Doherty et al. (44), we also identified two distinct subsets concerning the expression of CD11c molecules. Both the CD11c-positive and -negative subset, however, were positive for the intracellular pan-macrophage marker molecule CD68, but lacked expression of intracellular myeloperoxidase.

T cells. To obtain highly purified populations of T cells without concomitant activation during the course of the isolation procedure, T cells were isolated from PBMCs by negative depletion of all non-T cells and HLA-DR-positive cells. Such isolated T cells virtually lacked expression of the activation-associated molecules CD69 and CD71 (Table IGo), and only low proportions (3 ± 3%) were found to be CD25 (IL-2R)-positive.

Morphology of freshly isolated and GM-CSF-preactivated DCs and Mos

fDCs were found to represent a homogenous cell population of nonadherent, uniformly sized, round shaped cells with the appearance of medium-sized lymphocytes lacking detectable cytoplasmic processes (Fig. 1GoA). In contrast, Mos, although having a generally similar appearance, spontaneously adhered to the plastic surface of culture plates (Fig. 1GoD).



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  		FIGURE 1. Morphologic appearance of cell preparations in liquid culture. Characteristic phase contrast morphology of freshly isolated DCs (A) and Mos (D), DCs (Band C) and Mos (E and F) after culture for 48 h with serum-free medium containing GM-CSF is shown (final magnification: A, B, D, and E, x250; C and F, x500).

 
Previous studies have shown that, compared with mature DCs, fDCs are immature and less immunostimulatory but attain the characteristic morphology, phenotype, and immunostimulatory function of mature DCs upon cytokine-mediated maturation (44). Therefore, DCs and Mos were preactivated by culturing both in the presence of GM-CSF (100 ng/ml) for 48 h.

After stimulation with GM-CSF, cDCs showed a strong tendency for aggregate formation (Fig. 1GoB) in liquid culture, and cytoplasmic processes or veils extended from the surfaces of the aggregates (Fig. 1GoC). In contrast, Mos treated under the same culture conditions (cMos) formed only small aggregates (Fig. 1GoE) and appeared as a rather heterogeneous population of adherent and nonadherent cells in liquid culture (Fig. 1GoF).

T cell-stimulatory capacity of freshly isolated and cultured Mos and DCs under serum-free conditions

Comparative analyses of fMos and fDCs in terms of their capacity to induce proliferation of highly purified autologous T cells revealed that under culture conditions with supplementation of 10% FCS only fDCs, but not fMos, were able to induce a weak proliferation of autologous T cells (data not shown).

Since the first description of the AMLR in 1976 by Opelz and Kuntz (1), various hypotheses for an in vivo role of the AMLR within the normal immune response have been proposed (reviewed in 45 . In addition, however, there have been suggestions that the AMLR observed in vitro did not truly represent an "autologous" phenomenon but might rather be initiated by the presence of xenogeneic Ags during either the cell isolation procedure or subsequent culture period (37, 38). This prompted us to further analyze autologous T cell proliferation under conditions that avoided the presence of potential stimulatory foreign Ags as much as possible. For this purpose, FCS supplementation of buffers used during cell isolation was replaced by heat-inactivated human serum. Control experiments using autologous serum instead of allogeneic serum for the preparation of the isolation buffer did not reveal differences in T cell proliferation (data not shown). In addition, instead of serum-supplemented RPMI 1640 medium, we used the serum-free medium X-VIVO15 for the following entire culture period. Under such conditions, (see Fig. 2Go) [3H]thymidine uptake in T cells cocultered with fDCs was still found to be on average 12 ± 8 times higher compared with cultures of T cells alone, although reactivity was low in absolute counts; again, fMos did not induce any proliferative response.



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  		FIGURE 2. T cell-stimulatory capacity in the AMLR of fDCs and fMos and of cDCs and cMos. Autologous (5 x 104), highly purified T cells were incubated with graded numbers of either irradiated fDCs and fMos or cDCs and cMos in serum-free medium. Proliferation of T cells was monitored in all instances by measuring [methyl-3H]TdR incorporation on day 7 of culture. Degree of proliferation is indicated as cpm on the ordinate. The experiments represent mean values ± SEM calculated from four independently performed experiments. The nature of stimulator cells is indicated in the upper left corner.

 
Comparative analyses of cDCs and fDCs in terms of their capacity to activate highly purified autologous T cells revealed that cDCs induced a 113 ± 48-fold higher proliferation of autologous T cells compared with fDCs. In contrast, no considerable increase in the stimulatory capacity of cMos as compared with fMos was observed under the same culture conditions (Fig. 2Go).

Comparative analysis of the T cell-stimulatory capacity of cDCs under serum-free conditions in the AMLR, Ag-MLR, and allo-MLR

To characterize some of the molecular mechanisms that might be operative in the initiation of autologous T cell proliferation, we next performed comparative analyses and evaluated the capacity of cDCs to activate autologous or allogeneic T cells and to present soluble Ags to autologous T cells. As shown in Figure 3Go, even under serum-free conditions cDCs were strong stimulators of allogeneic T cells and were equally highly efficient in presenting the soluble tetanus toxoid Ag to autologous T cells. At high cDC:T cell ratios (1:1), cDCs were also efficient as AMLR inducers. At these ratios, however, T cell proliferation was found to be on average 2 to 4 times higher in the Ag-MLR or allo-MLR as compared with the AMLR. This difference was even more pronounced at low cDC:T cell ratios (1:3), where T cell proliferation was found to be on average 34 ± 12 or 57 ± 31 times higher in the Ag-MLR or allo-MLR, respectively, than in the AMLR.



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  		FIGURE 3. T cell-stimulatory capacity of cDCs in the AMLR, Ag-MLR, and allo-MLR. For induction of AMLR (or allo-MLR), T cells (5 x 104) were incubated in serum-free medium with graded numbers of autologous (or allogeneic) irradiated cDCs; for presentation of specific Ags, cultures were set up in the presence of 5LF tetanus toxoid (Ag-MLR). Proliferation of T cells was monitored in all instances by measuring [methyl-3H]TdR incorporation on day 7 of culture. Degree of proliferation is indicated as cpm on the ordinate. The experiments represent mean values ± SEM calculated from four independently performed experiments.

 
As can be seen in Figure 4Go, not only cDCs but also fDCs were found to efficiently present the soluble tetanus toxoid Ag to autologous T cells (and to induce an allo-MLR, data not shown), but fDCs were not able to induce an AMLR reponse. Similar stimulation as with tetanus toxoid was also found with the neoantigen keyhole limpet hemocyanin (data not shown). It is particularly noteworthy that the stimulatory capacity of fDCs for Ag-specific responses (as shown in Fig. 4Go) exceeded that of cDCs significantly (p < 0.05) at low cDC:T cell ratios (1:3). Although hereby not formally proven, such differences might partly reflect the described loss of Ag-capturing and -processing capacity of DCs upon maturation (46, 47, 48, 49, 50, 51, 52). In contrast, considerable T cell proliferative response in the AMLR could only be observed upon preactivation of DCs with GM-CSF.



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  		FIGURE 4. T cell stimulatory-capacity of fDCs and cDCs in the AMLR and Ag-MLR. For induction of AMLR, T cells (5 x 104) were incubated in serum-free medium with graded numbers of autologous fDCs or cDCs; for presentation of specific Ags, cultures were set up in the presence of 5LF tetanus toxoid (Ag-MLR). Proliferation of T cells was monitored in all instances by measuring [methyl-3H]TdR incorporation on day 7 of culture. Degree of proliferation is indicated as cpm on the ordinate. The experiments represent mean values ± SEM calculated from three independently performed experiments.

 
Changes in surface marker molecule expression on Mos and DCs upon preactivation with GM-CSF

To define differences between fDCs and cDCs that might have accounted for the increased stimulatory capacity of cDCs in the AMLR, phenotypic analyses were performed before and after preactivation with GM-CSF of Mos and DCs.

Typical FACS analyses of surface marker molecule expression on Mos and DCs before and after short-term preactivation with GM-CSF are shown as overlay histograms in Figure 5Go. Both, cMos and cDCs had a higher expression of the costimulatory molecule CD86 (B7-2) as compared with fMos or fDCs, respectively. For the costimulatory molecule CD80 (B7-1), however, neoexpression on cytokine-activated cDCs, but not on cMos treated under the same conditions, was observed. An increased expression intensity was found for the HLA class II locus products HLA-DR, HLA-DP, and HLA-DQ on both cMos and cDCs. A large increase of expression intensity was observed for CD40 on cDCs and to a lesser extent on cMos. Only low proportions of both cell populations expressed CD1a molecules upon GM-CSF stimulation. An evident down-regulation of surface marker molecules was observed for CD14 and CD33 on cMos and to a lesser extent for CD33 on cDCs.



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  		FIGURE 5. Phenotypic changes of DCs and Mos upon culture with GM-CSF. Single histogram profiles of the indicated markers are shown of one of six representative experiments. Phenotypic analyses for the expression of the indicated marker molecules on DCs and Mos were performed before (open profiles) and after culture for 48 h with medium containing GM-CSF (gray profiles). The abscissa represents fluorescence intensity (log10 scale); the ordinate, the respective cell number.

 
Inhibitory effects of mAbs on T cell proliferation in the AMLR, Ag-MLR, and allo-MLR

To assess the effects of various surface molecules on cDC-induced T cell proliferation, cDCs were incubated with mAbs against HLA-DR, CD4, CD8, CD80 (B7-1), and CD86 (B7-2) at a final concentration of 5 µg/ml for 15 min at 4°C before onset of culture and subsequently during the entire culture period. As can be seen in Figure 6Go, almost no inhibitory effect on T cell proliferation in the AMLR, Ag-MLR, or allo-MLR was observed when mAbs to CD8 were present throughout the culture period. mAbs against CD4 and HLA-DR molecules efficiently inhibited T cell proliferation in the AMLR, Ag-MLR, and allo-MLR. Important differences, however, were observed concerning blocking effects of mAbs against the costimulatory molecules CD80 (B7-1) and CD86 (B7-2). Whereas AMLR-induced T cell proliferation was found to be strongly inhibited by the presence of mAbs against CD80 molecules, inhibition by anti-CD80 was found to be significantly lower in allo-MLR (p < 0.05) and Ag-induced (p < 0.01) T cell proliferation. Finally, anti-CD86 mAbs strongly inhibited T cell proliferation in the AMLR and allo-MLR, but to a lesser, although not significant, degree in the Ag-MLR.



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  		FIGURE 6. Effects of mAbs on T cell proliferation in the AMLR, Ag-MLR, and allo-MLR. cDCs were incubated with mAbs against the indicated surface marker molecules for 15 min at 4°C before onset of culture. Inhibitory effects on T cell proliferation in the AMLR, Ag-MLR, and allo-MLR (as indicated in the upper right corner) was expressed as percent reactivity of control cultures without addition of mAbs performed in parallel. Proliferation of T cells was monitored in all instances by measuring [methyl-3H]TdR incorporation on day 7 of culture. Bars represent mean values of percent inhibition ± SEM of four independently performed experiments.

 
This higher inhibitory activity of anti-CD80 upon the AMLR could have been due either to a higher sensitivity of the AMLR than the Ag-MLR for CD80-mediated costimulation or to a requirement of the Ag-MLR for higher concentrations of blocking mAb for optimal inhibition. Therefore, to define if higher amounts of mAbs against CD80 and CD86 would show similar inhibitory capacity in the Ag-MLR as in the AMLR, additional experiments were performed with mAbs against CD80 and CD86 at final concentrations of 20, 5, and 1 µg/ml. As shown in Table IIGo, inhibition of T cell proliferation in the Ag-MLR even at high mAb concentrations was significantly (p < 0.05) lower as compared with the AMLR. This difference was more pronounced at mAb concentrations of 5 µg/ml and even 1 µg/ml. In contrast to CD80 inhibition, application of mAbs against CD86 inhibited the AMLR and Ag-MLR to similar degrees at 20, 5, and 1 µg/ml.


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  		Table II. Inhibition of T cell proliferation in the AMLR and Ag-MLR with mAbs against B7-1 and B7-2 molecules1

 
These data indicate that the presence of costimulatory and MHC class II molecules seem to be essential for the initiation of T cell proliferation in the AMLR, and that the combined expression of both might be the triggers for activation of AMLR-induced T cell proliferation. Moreover, CD80 appeared more intimately involved in the AMLR than Ag-induced T cell responses.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show here that mature human peripheral blood DCs that have undergone maturation upon stimulation with GM-CSF (cDCs), in contrast to fDCs, are potent stimulators of autologous T cell proliferation in the AMLR. Moreover, the stimulatory capacity of cDCs in the AMLR appears to be dependent on the increased and/or de novo expression of costimulatory molecules, in addition to MHC class II Ags.

DCs have been considered to bear the unique ability to stimulate T cell proliferation in the AMLR (24, 25, 33, 34, 53, 54), but so far little is known about the molecular mechanisms that enable DCs to perform this function. In general, analyses of DCs function are hampered by the lack of selective surface marker molecules for DC characterization and low proportions of peripheral blood DCs among PBMCs. Isolation techniques for DC purification frequently include the depletion of unwanted cells such as Mos by adherence to plastic surfaces during overnight culture at 37°C or T cell depletion by rosetting with sheep RBCs. However, such treatment has been suggested to affect phenotype and/or functional properties of DCs (35, 36), and may to some extent elicit a maturation step from immature to mature DCs that is associated with the development of characteristic morphology, phenotype, and immunostimulatory function (35, 44, 49, 55).

The use of high-gradient magnetic cell sorting allowed rapid isolation of sufficient numbers of immature DCs that acquired typical DC morphology and phenotype only upon culture for 24 to 48 h in the presence of GM-CSF. Moreover, analyses of DC functional capacity were performed under serum-free conditions because there was controversy in the literature as to whether or not the AMLR represented proliferation of T cells to xenoantigens encountered by APCs during the isolation and/or culture procedure (37, 38) rather than one to autologous Ags (39, 40). In line with other researchers, we have shown that Mos lacked stimulatory capacity in the AMLR (24, 25). In this study, we were able to show that, even under serum-free conditions, DCs induced autologous T cell proliferation, but only upon cytokine-mediated maturation. Characterization of the phenotype of fDCs and cDCs led to the observation that, besides an up-regulation of MHC class I and II molecules on cDCs, fDCs almost lacked expression of the costimulatory molecule CD80 (B7-1), while only a mean of 30% expressed CD86 (B7-2) molecules. Upon stimulation of fDCs with GM-CSF, proportions of CD86+ DCs increased slightly, but a dramatic, approximately 70-fold increase of CD80+ DCs was observed. Because CD80 contributes an important costimulatory molecule (reviewed in 56 , the change in DC phenotype predicted that CD80 could have accounted for the functional differences observed between fDCs and cDCs. This indication of the involvement of CD80 in the stimulation of the AMLR was further strengthened by the observation that T cell proliferation in the AMLR, but to a lesser extent in the Ag-MLR or allo-MLR, was inhibited by addition of mAb against CD80, whereas with mAb against CD86 a similar degree of inhibition of T cell proliferation was observed in all three culture systems, AMLR, Ag-MLR, and allo-MLR.

In line with these findings, fDCs that lacked expression of CD80 but expressed CD86 molecules were good stimulators in the Ag-specific T cell response. This, however, does not exclude a functional role of CD80 in the Ag-MLR because up-regulation of costimulatory molecules might occur during culture of fDCs, and in line with this several microbial products have been shown to induce the up-regulation of costimulatory molecules on APCs (reviewed in Refs. 57 and 58).

Proliferation of T cells in the AMLR, however, was only observed upon the expression of CD80 molecules. Thus, both CD80 and CD86 molecules seem to be involved in the initiation of AMLR, Ag-MLR, and allo-MLR, but their relative contribution may differ, suggesting a quantitative rather than a qualitative difference in the requirement of costimulatory molecules.

This finding does not exclude that other, partly undefined interactions between T cells and cDCs might additionally contribute to this process. Whether autologous peptides that are constitutively expressed by class II molecules (59) also contribute to the stimulatory capacity of DCs in the AMLR needs to be further elucidated. However, a more relevant role for costimulatory compared with MHC class II molecules for the presentation of self-epitopes by DCs has recently been demonstrated (60).

It is now accepted that a major T cell costimulatory pathway involves the CD28 molecule (61, 62, 63). Its interactions with the B7 family of costimulatory ligands are essential for initiating Ag-specific T cell responses, up-regulating cytokine expression, and promoting T cell expansion and differentiation (reviewed in 56 . While both B7-1 and B7-2 can provide necessary costimulatory signals in vitro to induce T cell activation, recent reports have suggested that B7-1 and B7-2 molecules expressed by APCs may induce qualitatively different signals in T cells, although the nature of this difference is not well understood (64, 65, 66). Due to its constitutive expression on APCs, B7-2, but not B7-1, is thought to represent the primary costimulatory molecule responsible for initiating T cell responses and providing cognate help for B cells (56). However, B7-1 appears to be more potent in stimulating inflammatory responses and tumor immunity, as compared with B7-2 (56) and seems to be critically involved in certain autoimmune diseases (65, 67).

Among the many hypotheses about the in vivo biologic significance of the AMLR, it has been suggested that the AMLR represents a self-recognitive mechanism regulating the cellular interactions involved in the generation of normal immune responses (15, 68, 69). The existence of the AMLR might thus be evidence that self-tolerance in the periphery is not entirely a passive process, but rather an active, dynamic state (45, 70). However, some investigators have even raised doubts about its existence and have suggested that the AMLR merely reflects a specific response to Ags introduced during the isolation and/or culture period (37, 38). Aside from other indications against such assumptions (39, 40), additional evidence against the involvement of exogenous Ags in the AMLR has been presented here, because the AMLR was inducible even under serum-free conditions. Moreover, control experiments for remaining possible sources of exogenous Ags were negative: one possible (xenogeneic) source remained the murine mAbs, used for isolation of cell populations, but control murine Ig administered into cultures did not induce T cell proliferation. Supplementation of buffers with serum, used during the cell isolation procedure, might be an additional source of exogenous (allogeneic) Ags in our experimental system. Control experiments using autologous serum instead of allogeneic serum for the preparation of the isolation buffer, however, did not reveal differences in T cell proliferation (data not shown). Furthermore, the rather short isolation procedure that was strictly performed at 4°C, in our opinion, makes it rather unlikely that xeno- or alloantigens would interfere with our experimental system. Finally, as shown here, even under serum-free conditions, fDCs as opposed to cDCs can present nominal Ags to T cells; in contrast, fDCs were unable to stimulate the AMLR. Thus, it is rather unlikely that the AMLR response reflects proliferation to an exogenous Ag.

In summary, we have shown that highly purified cDCs can induce proliferation of autologous T cells under serum-free conditions and that expression of high levels of MHC class II and costimulatory molecules CD86 (B7-2) and especially CD80 (B7-1) substantially contribute to this effect.

We conclude that the "strength of signal" by which cDCs at high numbers encounter resting T cells under culture conditions leads to the observed proliferative response of an AMLR in vitro. One might speculate that these signals also occur under physiologic conditions, where cytokines such as GM-CSF are present. There, an AMLR might be permanently ongoing at low and therefore hardly detectable levels. Moreover, at sites of inflammation where GM-CSF is produced in highly increased amounts and causes an enhanced DCs activation (71, 72, 73), a locally deranged AMLR response may lead to loss of self-tolerance as observed in certain autoimmune diseases (6, 10, 19, 20, 74, 75, 76, 77, 78).


   Acknowledgments
 
We thank Irene Hartnett for carefully reading the manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Clemens Scheinecker, II. Department of Medicine with Rheumatology, Lainz Hospital, Wolkersbergenstrasse 1, A-1130 Vienna, Austria. E-mail: Back

2 Abbreviations used in this paper: AMLR, autologous mixed lymphocyte reaction; Ag-MLR, Ag-specific mixed lymphocyte reaction; allo-MLR, allogeneic mixed lymphocyte reaction; DC, dendritic cell; fDC, freshly isolated dendritic cell; cDC, cultured dendritic cell; Mo, monocyte; fMo, freshly isolated monocyte; cMo, cultured monocyte; GM-CSF, granulocyte-macrophage CSF. Back

Received for publication February 2, 1998. Accepted for publication June 15, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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