International Immunology

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International Immunology, Vol. 11, No. 11, 1739-1744, November 1999
© 1999 Japanese Society for Immunology

MHC class II molecules transferred between allogeneic dendritic cells stimulate primary mixed leukocyte reactions

Penelope Bedford, Keith Garner and Stella C. Knight

Antigen Presentation Research Group, Imperial College School of Medicine, Northwick Park Institute for Medical Research, Watford Road, Harrow, Middlesex HA1 3UJ, UK

Correspondence to: S. C. Knight

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Presentation of antigen to T cells is generally restricted by MHC type but the mixed leukocyte reaction (MLR) was thought to involve direct stimulation by dendritic cells (DC) of allogeneic T cells. However, here we showed that DC bearing allogeneic MHC class II acted synergistically with responder-type DC. Removal of residual DC from `purified' responder T cell populations was achieved using treatment with DC-specific antibody and complement. These DC-depleted cells showed a significantly reduced response to allogeneic DC which was restored by addition of DC syngeneic with responder T cells. The studies support the concept that a major component of the MLR is the secondary presentation of alloantigens acquired from stimulator DC by DC of responder type. To investigate the reasons why DC and not other cells stimulate an MLR, synergy between DC and other cell types was investigated. Synergy was found exclusively between DC; macrophages, B cells or L cells transfected with MHC class II molecules did not contribute. When allogeneic DC were mixed in culture, transfer of MHC molecules between DC was observed as assessed by flow cytometry. Freshly obtained cell-free supernatants from cultured DC contained MHC class II and stimulated primary allogeneic MLR. DC of responder type acquired allogeneic MHC molecules from the supernatants and stimulated proliferation in syngeneic T cells. The capacity of DC both to shed and to acquire MHC molecules may contribute to their potency in stimulating primary responses, and could explain why passenger DC within allografts provide a potent stimulus for graft rejection.

Keywords: dendritic cells, MHC, mixed leukocyte reaction

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Dendritic cells (DC) are uniquely potent at stimulating primary immune responses (1–4) and the stimulation of allogeneic T cells in the mixed leukocyte reaction (MLR) is used as a functional test to identify DC. No single marker for DC has been found which accounts for this potency and it is generally attributed to their high expression, not only of MHC class II molecules but also of many co-stimulatory molecules (5,6). DC secrete a number of chemokines and express some chemokine receptors, and it seems likely that these properties could also contribute to the capacity of DC to cause the aggregation and stimulation of naive T cells (7).

Stimulation of T cells normally requires restricted antigen presentation via MHC antigens shared between the antigen-presenting cell and the responding T cell. One apparent anomaly in the restricted presentation of antigen to T cells was the capacity of allogeneic DC to stimulate primary proliferative T cell responses in the MLR (1). The indirect presentation of allogeneic MHC class II to responding T cells by recipient DC is now considered to be the major pathway for initiating graft rejection (8,9). This leaves a question over the reasons for the potent stimulation of T cells by allogeneic DC; DC and not other cells are potent stimulators in the MLR. Since DC are associated with antigen presentation in primary responses, the potency of these cells in stimulating allogeneic MLR has been assumed to occur via direct presentation of alloantigens by the allogeneic DC to responder T cells. The current study examines the role of DC syngeneic with responder T cells in producing an MLR. DC syngeneic with responder T cells were required for optimal stimulation and we provide evidence that this occurs via transfer of MHC class II molecules between DC. DC have been shown to be efficient at exchanging acquired antigens such as haptenized protein or influenza virus (10). The capacity of DC to shed and acquire MHC molecules may mean that the major presenting events in the MLR occur via conventional syngeneic presentation of antigens. It also means that the MLR—the `in vitro homograph reaction'—like in vivo graft rejection occurs mainly via indirect presentation of alloantigens. The principal functional test for DC, the MLR, may be measuring largely the capacity of DC to transmit MHC molecules to other DC rather than their capacity to stimulate T cells directly.

	Methods

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Animals
Animals (CBA or C57/BL10), between 6 and 10 weeks of age, were obtained from the Specific Pathogen-Free Unit at Northwick Park Institute for Medical Research. Mice of the same sex were used within experiments.

Cell suspensions
Inguinal, brachial and axillary lymph nodes were taken from normal mice and single-cell suspensions prepared in medium [RPMI 1640, Dutch modification; Flow, Irvine, UK) with penicillin (100 U/ml), streptomycin (100 g/ml), L-glutamine (2 mM), 5x10^–5 M 2-mercaptoethanol and 10% FCS] by pressing the nodes through wire mesh. Enriched T cells (>90%) were obtained by passage of the cells over nylon wool columns (11). Lysis with the antibody 33D1 (12) plus complement was used to deplete cell preparations of DC; cells at 5x10⁵ to 5x10⁶/ml were mixed with the antibody 33D1 (as culture supernate) and rabbit serum as a source of complement (Buxted Rabbit, Sussex, UK) or with complement alone as a control for cytotoxicity, and incubated for 60 min at 37°C and washed twice. To separate DC from lymph nodes, cell suspensions prepared in medium (5–8 ml) at 5x10⁶/ml were layered on 2 ml gradients of metrizamide (Nygaard, Analytical Grade, 14.5 g added to 100 ml of medium to give a 13.7% w/v solution) and centrifuged for 10 min at 600 g (13). Cells at the interface were collected, washed once and resuspended in medium. In specific pathogen-free animals, the suspensions obtained in this manner were between 70 and 90% pure DC with small numbers of contaminating T cells and <1% F4/80-positive macrophages (13).

In vitro stimulation assay
Enriched T cells or enriched T cells with DC removed by antibody and complement treatment were cultured in triplicate at different concentrations of viable (dye-excluding) cells in 20 µl hanging drop cultures in Terasaki plates and stimulated by DC. After 3 days the cultures received 1 µl [³H]thymidine (Amersham International, Little Chalfont, UK) at a sp. act. of 2 Ci/mM, giving a final concentration of 1 µg/ml of thymidine. After 2 h the cultures were blotted onto filter disks, and washed with saline, trichloroacetic acid and methanol, and counted in a scintillation counter. These conditions for labeling resulted in `flooding' conditions throughout the pulse time and low radiation damage. They gave low counts but these accurately reflect the DNA synthesis (14). This system with a high density of cells on a well gassed meniscus was previously shown to allow the development of primary proliferative and cytotoxic T cell responses in vitro to protein and peptide antigens as well as to allogeneic DC (4,14). In this system maximal responses to the mitogen concanavalin A (Con A) were 2000–5000 c.p.m. and primary responses to antigens were 300–3500 c.p.m. Variability in triplicates was generally within 20% of the mean and analysis of variance of log-transformed data to assess significance of differences in c.p.m. showed that a trebling of counts indicated P < 0.01.

Shedding and acquisition of MHC class II molecules by DC
DC (1x10⁶) in 0.1 ml medium were cultured for 24 h at 37°C. Supernatants were collected after spinning at 1000 g; the supernatants were further spun at 13,000 g before use. In some experiments these were filtered through 0.22 µm filters or centrifuged at 160,000 g before use.

Antibodies
Antibodies directed against class II were chosen such that they were haplotype specific and did not cross-react with other haplotypes. All FACS staining was carried out using the following directly conjugated antibodies, anti-I-A^k clone 11-52-specific for A^k, I-A^d, AMS-32.1 (PharMingen, San Diego, CA). The same antibodies, unconjugated, were used for the ELISA studies. In addition anti-class I antibodies H-2D^k clone 15-5-5 and H-2K^k and anti-I-E^k clone 17-3.3 clone AF3-T2.1 were also used in the ELISA studies. The antibodies used for blocking in the functional studies were culture supernatants of ATCC clones HB-42 (I-A^k) and H-81 (I-E^k ).

FACS analysis
FACS analyses were performed using a Becton Dickinson (Mountain View, CA) FACScan and CellQuest analysis software. All of the analyses shown were carried out on a population of cells gated by forward and side scatter characteristics to include the majority of the DC population, and exclude small mononuclear cells, large granular cells or clusters of cells.

ELISA
Flat-bottom 96-well ELISA plates (Maxisorb; Nunc, Paisley, UK) were coated overnight with supernatants from DC culture made up to 200 µl in carbonate–bicarbonate buffer. Each well was then washed 3 times with PBS/Tween (0.05% v/v Tween 20). Antibodies directed against I-A^k, I-E^k, H-2K^k or H-2D^k (PharMingen) were added and the plates incubated for 2 h at room temperature. After a further three washes with PBS/Tween, alkaline phosphatase-conjugated anti-mouse Ig was added to each well. The plates were again washed 3 times and p-nitrophenyl phosphate solution added to each well as a substrate for the alkaline phosphatase enzyme and the absorbance of each well was measured at 405 nm on a multiplate reader (Titertek, Irvine, UK).

	Results

Top Abstract Introduction Methods Results Discussion References

 
The requirement for DC in proliferative T cell responses was shown initially using Con A (Fig. 1). Addition of the mitogen Con A stimulated proliferation in enriched T cell populations (Fig. 1a). This response was abrogated by removing DC from the responding T cell population using antibody and complement, and was restored by adding back syngeneic DC (Fig. 1b). These cell populations were also used in the MLR. Addition of allogeneic, but not syngeneic DC, stimulated a proliferative T cell response (Fig. 1c). Removal of the DC from the responding cell population resulted in a significantly reduced allogeneic response producing a >10-fold reduction in counts at the lower responder cell concentration (Fig. 1d). The MLR activity was never completely removed by the depletion of DC from the responder cell population, despite the lack of responses to Con A. This observation leaves open the possibility that some direct stimulation by allogeneic DC is still occurring. The reduced responses on removal of DC were reversed by addition of DC syngeneic with the responding T cells (Fig. 1d). The enhancement shown by adding responder-type DC was significant (P = 0.01) but was never complete and Fig. 1(d) shows a typical result. Thus, the loss of response could be produced by antibodies to 33D1, a DC-specific antibody, but DC purified from lymph nodes did not completely restore this deficit. The purified DC represent a myeloid population selected by the separation technique; this result suggests that the presence of a mixed population of DC, perhaps including those of the lymphoid type found in the germinal centres, may be required for complete reconstitution.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Effect of DC syngeneic with responder T cells in the MLR. Different numbers of nylon wool non-adherent, enriched T cells (H-2k) (a and c) or DC-depleted T cells (b and d) in 20 µl cultures received 1 µg/ml Con A + 1000 syngeneic DC (a and b) or 500 allogeneic DC (H-2b) or 500 allogeneic + 1000 syngeneic DC (c and d). In specific pathogen-free mice there is no syngeneic stimulation using this number of DC. The effect of small numbers of DC syngeneic with responder T cells in addition to the allogeneic DC is shown. •, T cells only; , 1 µg/ml Con A; , 1 µg/ml Con A + 1000 syngeneic DC; , 1000 syngeneic DC; , 500 allogeneic DC; , 500 allogeneic DC + 1000 syngeneic DC

 
In order to ascertain whether the requirement for responder type DC merely reflected a requirement for donor type, MHC experiments were carried out using I-A-transfected L cells as a source of MHC molecules. In contrast to the effect of DC the presence of up to 10,000 I-A^k transfected L cells had no effect on the proliferation of DC-depleted I-A^k T cells in response to allogeneic DC. The addition of up to 10,000 L cell transfectants expressing allogeneic (I-A^b) antigen caused a negligible increase in proliferation on addition to T cells (I-A^k). Adding DC syngeneic with the responder T cells did not alter this response (not shown). Similar experiments showed a lack of synergy also between B cells or macrophages and DC. The synergistic effect was only seen when both responder and stimulator 33D1-positive DC were present

DC from CBA (H-2^k) and BALB/c (H-2^d) mice were analysed by FACS using antibodies specific for I-A^k or I-A^d which were shown not to cross-react (Fig. 2a and b). Equal numbers of CBA and BALB/c DC were mixed and cultured for 2 h before labelling with anti-I-A^k and anti-I-A^d. This analysis showed transfer of significant levels of MHC molecules between the cells (Fig. 2c and d). Between 30 and 50% of the DC became double-positive.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Equal numbers of CBA DC and BALB/c DC were mixed and labelled after 0 or 2 h with phycoerythrin-conjugated anti-I-Ak, FITC-conjugated anti-I-Ad or double labelled with both antibodies and analysed by FACS: (a) 0 h mixture labelled with phycoerythrin-conjugated anti-I-Ak, (b) 0 h mixture labelled with FITC-conjugated anti-I-Ad, (c) 0 h mixture and (d) 2 h mixture dual labelled with phycoerythrin-conjugated anti-I-Ak and FITC-conjugated anti-I-Ad. All analyses shown are on DC populations gated on forward and side scatter characteristics. The same gate was used on all populations.

 
Allogeneic stimulation is known to require live metabolically active stimulator cells and contact between the allogeneic cell populations. Freshly prepared supernatants from DC cultures were used to investigate the possibility that the observed synergy between responder and stimulator DC was mediated by shed factors. Supernatants from overnight cultures of mature lymph node DC stimulated proliferation when added to allogeneic but not to syngeneic lymph node T cells (Fig. 3). The stimulation observed was less efficient than that seen by adding DC (Fig. 1). This stimulatory function was dependent on the presence of allogeneic MHC class II molecules from the supernatants since antibodies directed against MHC molecules of the supernatant, which did not cross-react with the MHC or the responding cell population, blocked the stimulation (Fig. 4). Antibodies either to I-E or I-A inhibited the responses. With two stimuli present the effects are likely to be additive and antibody to the more dominant stimulus should cause the greater inhibition. Antibodies to I-E were more effective than those to I-A molecules. Complete loss of activity with anti-I-E in some experiments (Fig. 4) suggests that the amount of I-A present was not sufficient to initiate detectable stimulation on its own. The stimulatory function of the supernatants was lost on holding the supernatant overnight at 4°C or by freezing and thawing. The supernatants could be filtered through a 0.2 µm filter indicating that the stimulatory effect was not dependent on the presence of small numbers of cells remaining within the supernatants.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Stimulation of lymph node cells with supernatant from allogeneic DC. Different numbers of lymph node cells were cultured with 1 µl of supernatant from DC cultured overnight. (a) •, BALB/c lymph node cells; , BALB/c lymph node cells + 1 µl supernatant from B10 DC. (b) •, B10 lymph node cells; , B10 lymph node cells + 1 µl supernatant from B10 DC.

 

View larger version (17K): [in this window] [in a new window]   Fig. 4. Stimulation by DC supernatant is blocked by antibodies to class II. Different numbers of lymph node cells were stimulated with supernatant from allogeneic DC. Antibodies directed against the class II molecules of the supernatant were added to the cultures. •, B10 lymph node cells alone; , B10 lymph node cells + 1 µl supernatant from CBA DC; , B10 lymph node cells + CBA supernatant plus anti-I-Ak; , B10 lymph node cells + CBA supernatant plus anti-I-Ek.

 
The presence of MHC molecules in the DC supernatants has been described (15) and was also indicated by the antibody blocking experiments described in Fig. 4. The presence of MHC molecules in the 24 h supernatant was confirmed in our study by blotting the protein onto filters and staining for MHC molecules. Blots of the MHC class II molecules in the supernatants gave staining levels similar to those in the lysates of cells from which they were derived (not shown). The relative amounts of MHC molecules were assessed using an ELISA assay. This showed the presence of I-A and H-2K, and lesser amounts of I-E and H-2D (Fig. 5).

View larger version (10K): [in this window] [in a new window]   Fig. 5. Supernatants from DC contain MHC molecules. Supernatants from DC cultured overnight were assayed for the presence of MHC class I and class II molecules by ELISA. , Supernatant from B10 DC (H-2b); , supernatant from CBA DC (H-2k).

 
DC incubated in supernatants of allogeneic but not syngeneic DC for 2 h showed significant labelling for allogeneic MHC class II molecules which could be visualized using flow cytometry (Fig. 6). These cells bearing allogeneic MHC molecules stimulated proliferation in syngeneic T cells (Fig. 7).

View larger version (9K): [in this window] [in a new window]   Fig. 6. DC incubated with allogeneic supernatant acquire allogeneic MHC molecules. B10 lymph node DC (I-Ab) were incubated with supernatant from CBA DC (I-Ak), labelled with FITC-conjugated anti-I-Ak and analysed by FACScan. There was no detectable labelling with an isotype control antibody (data not shown). Solid line, B10 lymph node DC labelled with anti-I-Ak; dashed line, B10 lymph node DC + CBA supernatant labelled with anti-I-Ak.

 

View larger version (13K): [in this window] [in a new window]   Fig. 7. DC pulsed with supernatant from allogeneic DC stimulate proliferation in syngeneic lymph node cells. CBA lymph node DC were pulsed with supernatants from overnight cultures of B10 DC or CBA DC and cultured with CBA lymph node cells. •, CBA lymph node cells alone; , CBA lymph node cells + 1000 CBA DC; , CBA lymph node cells + 1000 CBA DC pulsed with CBA supernatant; , CBA lymph node cells + 1000 CBA DC pulsed with B10 supernatant.

 

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrated the requirement for responder-type DC for the production of maximal mixed leukocyte responses. This requirement may be mediated via MHC molecules shed from stimulator DC and acquired by DC of the responder cell populations. Since some response to allogeneic cells was maintained even following stringent depletion of DC from responder cells, there may still be a role for direct stimulation by allogeneic DC. However, the effectiveness of small numbers of DC in restoring responses leaves a question mark over whether direct stimulation across an allogeneic barrier is occurring.

The presence of soluble MHC molecules has been widely reported (16–18). The evidence in this study suggests that DC may be major contributors to any pool of secreted histocompatibility antigens. MHC class II molecules are known to be unstable after secretion and the stimulatory capacity of the class II-rich supernatants from DC is short-lived. This may reflect the instability of shed histocompatibility antigens. Recent studies described how MHC class II molecules can be shed from B cell lines in small vesicles. When these vesicles were derived from cells exposed to antigens, they were able to cause secondary stimulation in T cell lines (19). Stimulation of tumour immunity with supernatants of DC has also been described (15) and our experiments would suggest that DC of the recipient are also required to initiate these responses. The MHC class II-bearing vesicles were removed from the supernatants by graded centrifugation procedures culminating with centrifugation at 60,000 g. In our study stimulatory molecules were shed from DC and were not lost by direct spinning at 160,000 g, but it is conceivable that the stimulatory MHC molecules are in small vesicles; more details of the configuration of the molecules in question is under study but direct analysis of the stimulatory materials is hampered by their liability which suggests they may generally operate during close cell contacts. We have recently demonstrated the transfer of other antigens between different DC in the generation of primary antigen responses (10), suggesting that transfer of antigens between DC may be a general mechanism for initiation of primary immune responses rather than an unusual allogeneic response mechanism.

As Langerhans' cells begin to mature from DC specialized at acquiring and processing antigens, they express higher levels of MHC class II molecules (20). Since DC are known to require very small numbers of molecules to stimulate T cell proliferation (e.g. just 200–300 molecules for some antigens) (21), the capacity to `share' antigens with other DC may be of great significance in their potency in targeting the small numbers of antigen specific T cells available, particularly for primary stimulation. The synergistic effects are not merely due to the increase in class II per se within the cultures since adding class II on L cells, B cells or activated macrophages did not contribute to these responses.

In transplantation two mechanisms for the presentation of alloantigens have been described; the first is the direct stimulation of the immune system by alloantigens on allogeneic DC, and the second is the acquisition and presentation of the antigens of the graft by DC of the recipient (8,9,22,23). In experimental transplantation systems, DC of the recipient acquire the alloantigens and cells expressing both types of MHC class II molecules have been detected in recipients (22). The importance of each of these pathways has been assumed since the presence of allogeneic DC in the transplant and of recipient DC have both been shown to contribute to the speed of graft rejection (8,9). The concept of a direct role of DC in stimulating allogeneic T cells was based on the fact that DC initiate host versus graft responses (13,24) and primary allogeneic T cell responses (1–3). The importance of the donor DC, as shown here, could be largely due to the capacity of these cells to secrete large quantities of MHC antigens which could be acquired by recipient DC.

If MHC molecules acquired from other DC are important in stimulating the proliferation of T cells, then DC may have specialized mechanisms for acquiring MHC molecules. Observations that different MHC molecules on the surface of DC may be internalized by different pathways (25,26) may, therefore, have more significance than merely reflecting the recycling of surface MHC molecules in DC. In studies of human DC, immunogold-labelled HLA-DR molecules were internalized in long channels which had some similarities with Birbeck granules, HLA-DQ was internalized in small vesicles in vacuoles and HLA-DP was seen going into small round vesicles. The significance of these observations was not understood at the time but it is tempting to speculate that they reflect different specialized mechanisms for acquiring and internalizing antigens which have been shed from other DC. Birbeck granules in Langerhans' cells of the skin were shown to be formed by invagination from the cell surface and to contain MHC class II molecules (27,28). Other recent studies also confirm and extend the observations of the presence of MHC class II in vesicles in DC (29,30). Whatever the precise mechanisms involved in the synergistic effects between DC, the evidence provided in this study is that shedding and acquisition of MHC molecules by DC is of importance in the stimulation of primary T cell proliferation in response to allogeneic histocompatibility antigens. The major functional test for DC may be measuring the transfer of MHC molecules and not the actual presentation of the antigen to stimulate T cells.

	Abbreviations

 

Con A concanavalin A

DC dendritic cell

MLR mixed leukocyte reaction

	Notes

 
Transmitting editor: A. McMichael

Received 7 December 1998, accepted 15 July 1999.

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Top Abstract Introduction Methods Results Discussion References

 

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