- Email: [email protected]
Enhanced proliferation of CD4+ T cells induced by dendritic cells following antigen uptake in the presence of specific antibody
ELSEVIER
Veterinary Immunology and Immunopathology 49 (1996) 321-330
Veterinary immunology and immunopathology
Enhanced proliferation of CD4+ T cells induced by dendritic cells following antigen uptake in the presence of specific antibody S. Coughlan, G.D. Harkiss
*,
J. Hopkins
Department of Veterinary Pathology, University of Edinburgh, Summerhall, Edinburgh EH9 lQH, UK
Accepted 18 April 1995
Abstract Afferent lymph dendritic cells bear an Fey receptor which binds antigen/ antibody complexes thereby enhancing uptake of antigen. In this report, we have addressed the question of whether the enhanced uptake of antigen results in augmented antigen presentation and T cell proliferation in in vitro secondary responses in sheep. Inclusion of affinity-purified IgG anti-ovalbumin antibody in cultures of afferent lymph dendritic cells, purified CD4’ T cells, and substimulating amounts of ovalbumin resulted in a five- to 169-fold enhancement of T cell proliferation. This effect was antigen-specific as replacement of the anti-ovalbumin antibody with an IgG anti-human serum albumin specific antibody did not cause enhanced T cell responses. The antigen-specific augmentation required intact antibody Fc portions as F(ab’), fragments of the anti-ovalbumin antibodies were ineffective. The enhanced antigen presentation was found to be maximal with immune complexes in moderate antibody excess (three- to 30-fold), but still occurred at antibody/ antigen ratios of 300. The augmented responses were inhibitable with anti-MHC Class II specific antibodies, indicating that at least some of the antigen taken in via Fey receptors entered a Class II processing pathway. The results thus show that antigen uptake via Fey receptors on dendritic cells results in functional augmentation of antigen presentation and T cell proliferation. Keywords: Antigen/antibody
complexes; Fey receptors; T cell proliferation
0. Abbreviations Con A, concanavalin A, DC, dendritic cells; ELISA, enzyme-linked FcyR, Fey receptor; FCS, fetal calf serum; HSA, human serum
* Corresponding
author. Tel: 0131 6506169;
Fax: 0131 6506511:
immunoassay; albumin; Ova,
Email: [email protected].
01652427/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0165.2427(95)05478-2
322
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ovalbumin; PBM, peripheral TCR, T cell receptor.
blood mononuclear
49 (1996) 321-330
cells; PBS, phosphate
buffered
saline;
1. Introduction Dendritic cells (DC) are unique in their ability to induce primary immune responses and are the most potent inducers of secondary responses (Steinman, 1991; Knight et al., 1992). The DC population is a minor one numerically, but it is ubiquitously distributed throughout the body in both lymphoid and non-lymphoid tissues. DC in different parts of the body seem to represent variant forms originating from a common precursor (Fossum, 1988). Afferent lymph DC are thought to arise from a bone marrow-derived dendritic stem cell via skin Langerhans cells and perhaps blood-borne precursors, and populate the draining lymph nodes as interdigitating reticular cells (Fossurn, 1988). Despite their universally recognised potency as antigen presenting cells, the mechanisms underlying their superior accessory function are poorly understood. The factors likely to influence these unique DC functions are the efficiency of antigen uptake and processing, and the expression of cytokines and surface costimulatoty molecules. Previous work has shown that antigen presentation by rat DC isolated from lymph nodes is augmented by the presence of specific antibody, though the mechanism operating was not investigated (Schalke et al., 1985). We have shown previously that sheep afferent lymph DC bear a functional Fc receptor for IgG (Fc~R) (Harkiss et al., 1990). Antigen uptake by DC is greatly augmented in the presence of specific IgG antibody in vitro and in vivo in primed animals. The existence of such an antigen concentrating mechanism might explain, at least partially, the increased accessory function displayed by these cells following antigen challenge in vivo reported previously (Hopkins et al., 1989). H ere, we have addressed the question of whether the enhanced uptake of antigen via the ovine DC FcyR results in functional enhancement of antigen presentation to T cells in secondary responses.
2. Materials and methods 2.1. Animals and surgery Sheep (2-4 years old) and the surgical procedures used to generate cannulated pseudoafferent prefemoral lymphatics were as described previously (Dutia et al., 1993). Antigenic priming was performed by injecting 1 mg of ovalbumin (Ova) or human serum albumin (HSA) subcutaneously in Freund’s complete adjuvant. The animals were boosted 10 days and 17 days later by two further injections of 0.2 mg of these antigens in incomplete Freund’s adjuvant respectively. The sera were tested for anti-Ova or anti-HSA antibodies by enzyme-linked immunosorbent assay (ELISA). Lymph was collected into sterile bottles containing 2.5 X lo3 units of heparin and 2.5 X lo4 units of penicillin and streptomycin.
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2.2, Cell purification DC were obtained from afferent lymph by centrifugation over 14% metrizamide (Nycomed, Oslo) as described previously (Knight et al., 1986; Hopkins et al., 1989). Peripheral blood mononuclear cells (PBM) were isolated using Lymphoprep (1.077 g ml-’ ) (Nycomed). Autologous CD4+ T cells were obtained from the purified PBM using a magnetic activated cell sorter (MACS) and biotinylated anti-CD4 monoclonal antibody; 10’ cells in 90 ml of phosphate buffered saline (PBS) were incubated with 1 ml of streptavidin-labelled magnetic MACS microbeads (Miltenyi Biotech, Bergisch Glasbach, Germany). Streptavidin-phycoerythrin (Sigma) was added to allow assessment of the purity of the selected population by flow cytometry. The purity of the CD4+ T cells assessed by flow cytometry was routinely > 93% using a live gate for lymphocytes based on the forward/side scatter profile. 2.3. Purification
of antibodies
Sera from sheep immunised to Ova or HSA were fractionated on DEAE-cellulose ion exchange columns (Whatman, Maidstone) equilibrated in 10 mM phosphate buffer pH 7.5. The fractions containing IgG were pooled and applied to Sepharose 4B affinity columns bearing Ova or HSA respectively. The bound antibodies were eluted in 0.1 M glycine HCl pH 2.5 and neutralised immediately. Both affinity purified antibodies were of the IgG, isotype as determined by ELISA using isotype-specific monoclonal antibodies. To prepare F(ab’), antibody fragments, affinity-purified IgG anti-Ova antibodies were digested with pepsin (Sigma) (3% w/w) in 0.1 M sodium acetate pH 4.5 overnight at 37°C. The F(ab’j2 antibody fragments were separated from residual whole IgG by DEAE-cellulose ion exchange chromatography. The antibody preparations were dialysed into PBS. The purity of the F(ab’), fragments was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The antibody activity was determined by ELISA. 2.4. Uptake of antigen/antibody
complexes
by DC
Ova (30 pg ml-’ 1 was preincubated with afferent lymph from two sheep containing IgG anti-Ova antibodies for 30 min at 37°C before adding to 1 X 10” autologous DC per
Table 1 Monoclonal
antibodies
used in the studv
Antibody specificity
Designation
lsotype a
Reference
Ovine Ovine Ovine Ovine Ovine
SBU.T4 SW73.2 VPM6 RShyl RShy 2
mIgG2a rIgG2a mIgG1 rIgG2a rIgG2b
Maddox et al. (198.5) Hopkins et al. (1986) Jones (1988) Hobbs h Hobbs ’
CD4 MHC Class II IgG IgGl IgG2
a m, murine; r, rat. b Kindly supplied by Dr. S. Hobbs, Royal Marsden Hospital,
Sutton. Surrey, UK.
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S. Coughlan et al. / Veterinary Immunology and immunopathology 49 (1996) 321-330
tube. After a further 30 min at 37°C the DC were washed three times in PBS containing 2% bovine serum albumin. Bound IgG was detected using a murine monoclonal antibody specific for sheep IgG (VPM6), and rat monoclonal antibodies specific for sheep IgG, (RShyl) or sheep IgGz (RShy2) (Table 1). After washing, the bound monoclonal antibodies were detected with fluorescein isothiocyanate-labelled sheep anti-mouse or anti-rat IgG conjugates as appropriate. The DC were analysed by flow cytometry. 2.5. Lymphocyte
proliferation
assays
Purified DC and CD4+ T cells from six sheep were resuspended in RPM1 1640 medium containing 10% fetal calf serum (FCS), 2 mM glutamine, 5 X 10d5 M 2-mercaptoethanol, 100 units benzylpenicillin ml- ’ and 100 units streptomycin ml _ ’ . DC were irradiated with 2500 rad. Antigen and antibody dilutions in 50 ~1 volumes of sterile PBS were added to 96 well flat bottomed culture plates (Nunclon, Denmark) followed by incubation for 30 min at 37°C to allow the formation of immune complexes. Each well contained 5 X lo4 DC and 1 X 10’ responding CD4+ T cells in RPM1 1640 medium containing 10% FCS final concentration in a total volume of 200 ~1. After 5 days, the cells were pulsed with 1 @Zi of 3[H]thymidine per well, harvested 5 h later, and counted by beta-spectroscopy. All assays were performed in triplicate, and concanavalin A (Con A; 5 pg ml-‘) was included as a positive control for proliferation. Inhibition of antigen-specific proliferation was assessed in two sheep by the inclusion of F(ab’), fragments of SW73.2, a rat monoclonal antibody (20 pg ml-‘) specific for ovine MHC Class II (Hopkins et al., 1989). A polyclonal rat F(ab’)z anti-mouse IgG control antibody (Pierce & Warriner, Chester, UK) was used at the same concentration. 2.6. Statistics The results were analysed
using the Student’s
two sample t-test.
3. Results 3.1. In vitro uptake of Oua/anti-Ova
antibody complexes
by DC
Metrizamide-purified DC were incubated with Ova in the presence of unfractionated autologous afferent lymph from two sheep containing anti-Ova antibodies, and the uptake of IgG subclasses was detected by flow cytometry. The results from one animal are shown in Fig. 1. About 5% of DC had IgG on their surface in the absence of specific anti-Ova antibodies, whereas with antibody present 34% of DC registered positive. The bound IgG was of the IgG, subclass. Similar results were obtained in the second animal. The results thus show that DC take up excess immunoglobulin, presumably specific antibody, when incubated with antigen/antibody complexes. 3.2. Enhancement
of T cell proliferation
by specific antibody
To test whether FcyR-mediated uptake of antigen/antibody complexes results in increased antigen presentation, purified DC were cultured with autologous CD4+ T cells
S. Coughlan et al. / Veterinary Immunology and Immunopatholom 49 (2996) 321-330
315
1000500 250 12562.5 31.3 15.6 7.8 3.9 0
Concentration
of OVA (ugml-‘1
Fig.
1. Uptake of sheep immunoglobulin by DC after in vitro incubation with Ova and autologous afferent lymph containing IgG anti-Ova antibodies. Bound immunoglobulin was detected by flow cytometry using monoclonal antibodies specific for sheep IgG, or IgG,.
from six Ova-primed animals with antigen in the presence or absence of specific affinity-purified IgG antibody. Typical results are shown in Fig. 2. Proliferation of CD4’ T cells was observed with Ova alone only at 10 pg ml-‘, whereas in the presence of specific antibody proliferation was observed at 1 and 0.1 pg ml -I. DC in
30 ? 0 X
E
OVA A
20
anubody
OVA and antiHSA anllbody
2 IO
10
I
Concentration
0.1
0.0 I
0
of OVA (pgml-I)
Fig. 2. Enhancement of CD4+ T cell proliferation induced by DC in the presence of specific antibody. Ova (O-10 /.~g ml-‘) and affinity-purified IgG anti-Ova antibodies (30 pg ml-’ ) in sterile PBS were incubated at 37°C for 30 min before addition of an equal volume of medium containing 5 X lo4 DC and 1 X 10s T cells to each well. The final concentration of FCS in RPM1 medium was 10%. After 5 days, T cell proliferation was measured as the mean counts of triplicate cultures + standard deviation. * P < 0.001.
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Table 2 Maximal enhancement
of CD4+
T cell proliferation
Sheep number
[jH]-Thymidine
incorporation
Ova a only
Ova + anti-Ova antibody
150 1.51 156 21 19 23
1538 +507 1459f 94 363k 30 1621+344 1216+327 3353+438
27669 + 2323 36139&3900 61505+6131’ 12261+ 541 11500+ 1652 16875+1616
and fmmunopathology
49 (19961 321-330
induced by DC in the presence of specific antibody
kpm) Anti-Ova antibody * (X 18) * (X25) (X169) * (x8) ’ (X 10) * (X5)
h
1479 f 420 188+ 29 104+ 23 215+ 51 669 f 572 2634k602
Medium only
Con A only
1037+360 521k 79 102& 83 1654,272 1354f602 1204k827
116054* 1508 128621k10.527 142488k 85.51 89723+_ 5247 81424+ 3997 102977i 5479
a Ova concentrations used were 1 pg ml -’ in Sheep 1.50, 151 and 19, and 0.1 pg ml -’ in Sheep 1.56,21 and 23. b IgG anti-Ova concentration used was 30 pg ml-‘. * P < 0.001 compared to counts obtained with Ova alone. Figures in parentheses represent enhancement in T cell proliferation over Ova atone counts.
the presence of substimulating amounts of Ova alone or in the presence of specific antibody alone did not induce T cell proliferation above background levels. Similarly, no significant T cell proliferation was obtained with Ova, antibody, Ova/anti-Ova complexes or Con A in any of the wells if DC were omitted (data not shown). Table 2 shows the maximal enhancement of T cell proliferation obtained from the six sheep after incubation of complexed Ova compared to Ova alone. The degree of enhancement ranged from five-fold up to 169-fold in individual sheep. In most cases, the greatest enhancement of T cell proliferation was observed with antibody/antigen ratios between 3 and 30, though significant enhancement was also found in some sheep at a ratio of 300 (data not shown). When F(ab), anti-Ova antibodies or IgG anti-HSA antibodies were no significant enhancement of T cell used in place of IgG anti-Ova antibodies, proliferation was observed (Fig. 2). Since the F(ab), anti-Ova antibodies had a similar antibody titre when tested against Ova by ELISA, the results indicate that the mechanism of enhancement requires the Fc portion of the IgG antibodies. 3.3. Effect of anti-MHC
Class II antibodies
on enhancement
of T cell proliferation
Antigen presentation by ovine DC in the absence of specific antibody is almost completely inhibited by anti-MHC Class II antibodies (Hopkins et al., 1989). To test whether antigen taken up via an FcyR is also processed and presented via the MHC Class II pathway, rat monoclonal antibodies with pan specificity for ovine MHC Class II were included in the DC/T cell cultures from two sheep. The results from one sheep are shown in Fig. 3. The anti-MHC Class II antibodies almost completely inhibited the T cell proliferation obtained when the antigen was given with specific antibodies. In contrast, the control rat monoclonal antibodies did not significantly inhibit the enhanced T cell proliferation obtained with complexed Ova. Similar results were obtained in the second animal. The results indicate that antigen taken up via the DC FcyR is processed and presented by MHC Class II molecules to the CD4+ T cells.
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49 (1996) 321-330
327
f f
lo 5
0
Concentration
of OVA (pgml- 1)
Fig. 3. Effect of anti-MHC Class II antibody on enhanced CD4+ T cell proliferation induced by DC in the presence of specific antibodies. Ova (O-10 /.~g ml-’ 1 and affinity-purified IgG anti-Ova antibodies (30 pg ml-’ I were incubated at 37°C for 30 min before addition of rat IgG anti-MHC Class II monoclonal antibody (20 pg ml-’ 1 or control rat IgG anti-mouse IgG (20 pg ml-’ 1, 5 X lo4 DC and 1 X 10’ T cells to each weil. After 5 days, T cell proliferation was measured as the mean counts of triplicate cultures f standard deviation. * P < 0.001; + P < 0.05.
3.4. Effect of decreasing the DC / T cell ratio on enhanced T cell proliferation To ascertain if the efficiency of antigen presentation by DC was increased following uptake of antigen in the form of antigen antibody complexes via the FcyR, the cultures
30
0
DC Number Fig. 4. Enhancement of CD4+ T cell proliferation DC. Ova (0.2 gg ml-‘) and affinity-purified IgG for 30 min before addition of 1 X lo5 T cells and After 5 days, T cell proliferation was measured as * P < 0.001: * P < 0.05.
OVA only
x 10-i
in the presence of specific antibody at limiting numbers of anti-Ova antibodies (30 @g ml-’ 1 were incubated at 37°C DC numbers ranging from 2 X lo6 to 1.66 X 10” per well. the mean counts of triplicate cultures + standard deviation.
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49 (1996) 321-330
were set up with constant numbers of CD4’ T cells and constant amounts of Ova and anti-Ova antibodies but with decreasing numbers of DC. Similar results were obtained in two sheep. The results from one animal are shown in Fig. 4. No proliferation above background was seen when the DC numbers were reduced to 3 X 10’ per well. When specific antibody was included in the cultures, T cell proliferation also decreased with decreasing numbers of DC but the degree of proliferation was considerably higher than with antigen alone from 50 X lo3 DC per well down. With 3 X lo3 and 1.6 X 10” DC, T cells showed no proliferation in the presence of Ova but were stimulated significantly with Ova/anti-Ova complexes. The results thus indicate that, on a per cell basis, DC are more efficient at stimulating T cell if the antigen is complexed with antibody than if it is given alone.
4. Discussion Previous studies have shown that ex vivo sheep afferent lymph DC are very potent activators of T cells in secondary responses (Hopkins et al., 19891. The mechanisms underlying this potency are not well understood. We recently demonstrated that these afferent lymph DC bear an FcR for IgG which functions to concentrate antigen on to the cell surface both in vitro and in vivo (Harkiss et al., 1990). Here we show that increased antigen uptake via the DC FcyR in sheep results in functional enhancement of CD4’ T cell proliferation. The results showed that the enhancement was antigen-specific and was dependent on the IgG antibodies bearing an intact Fc portion. The augmentation of T cell proliferation was maximal if the immune complexes were in moderate antibody excess, but still occurred to a lesser degree in 300-fold antibody excess in some sheep. Thus, addition of immune complexes in antibody excess to DC resulted in the conversion of a substimulating dose of antigen into a potent stimulating dose for CD4+ T cell proliferation. Similarly, addition of specific antibody to cultures containing substimulating numbers of DC resulted in substantial T cell proliferation, indicating that on a per cell basis the DC possessed more potent antigen presenting function. In conditions of antibody excess, no free antigen would be available for uptake by the DC. This indicates that all the antigen would be taken up via the DC FcyR. The augmented T cell proliferation was substantially inhibited by the inclusion of anti-MHC Class II antibodies in the cultures, indicating that some of the antigen was being routed into an MHC Class II processing pathway. The mechanisms underlying the enhanced T cell proliferation mediated via FcyR occupancy in DC are not known. The basis for such augmentation may simply be the concentration of antigen into the cell thereby resulting in increased amounts of antigenic peptides being presented to T cells. This, in combination with the expression of increased levels of MHC Class II (Hopkins et al., 1989), could result in an increased density of MHC Class II molecules bearing specific antigenic peptides. It is known that cross-linking of MHC Class II molecules by the T cell receptor (TCR) or the release of interferon-y by T cells results in upregulation of the costimulatory molecule B7 in B cells (Freedman et al., 1991; Nabavi et al., 1992). Such signals from T cells clearly
S. Coughlan
et al. / Veterinary
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and Immunopathology
49 (1996) 321-330
729
could induce DC to express B7 or other costimulatory molecules thereby enhancing their T cell stimulating function. It is also possible that engagement of the Fey R results in one or more signals being transduced which could result in the activation or upregulation of surface molecules such as MHC Class II and B7. The upregulation of MHC Class II on DC following secondary antigenic challenge in vivo (Hopkins et al., 1989) is consistent with this hypothesis. The ability of FcyR to transduce signals in humans has previously been thought to reside in the FcyRI and FcyRIII class of receptors, and to be mediated by homo- or heterodimers of the y and 5 chains usually found in the TCR/CD3 complex and high affinity FceR respectively (Kurosaki et al., 1991, 1992; Wirthmueller et al., 1992; Ernst et al., 1993; Van der Winkel and Capel, 1993). However, recent evidence suggests that all three types of human FcyR (CD64, CD32, CD16) have an associated y chain homodimer, suggesting that signals may be transduced by one or more isoforms of FcyRII as well (Masuda and ROOS, 1993). Although the FcyR expressed by ovine afferent lymph DC have not been fully characterised, we have preliminary evidence from Western blotting studies that these cells express both FcyRI and FcyRII (Coughlan, Harkiss and Hopkins, unpublished observations, 1995). This is consistent with the expression of both these receptors on human blood DC (Thomas et al., 1993), and the expression of FcyRII by Langerhans cells (Schmitt et al., 19901, a cell type thought to be a precursor for afferent lymph DC (Macatonia et al., 1987). Augmentation of T cell proliferation by specific antibodies has been demonstrated previously for macrophages (Manta et al., 1991) and DC isolated from rat lymph nodes (Schalke et al., 1985) or from human blood and maintained by cytokine activation (Sallustro and Lanzavecchia, 1994), although the mechanism of augmentation by DC was not addressed in these studies. In contrast to the studies in rats, sheep and humans, no functional enhancement of T cell proliferation was observed using bovine afferent lymph DC (McKeever et al., 1992). The reasons for this discrepancy are not known, but may have been due to the use of stimulating rather than substimulating amounts of antigen in the latter study. Consequently, no augmentation would have been observed. Increased antigen uptake and presentation to T cells via DC FcyR may thus be an important mechanism operating in chronic diseases with an immune complex aetiology.
Acknowledgements This work was supported by the Agricultural and Food Research Council, UK. (LRG 31). S. Coughlan was supported by an AFRC Research Training Scholarship.
References Dutia, B.M., McConnell, I., Bird, K., Keating, P. and Hopkins, J., 1993. Patterns of major histocompatibility class II expression by T cell subsets in different immunological compartments. 1. Expression on resting T cells. Eur. .I. Immunol., 23: 2882-2888. Ernst, L.K., Duchemin, A.-M. and Anderson, C.L., 1993. Association of the high-affinity receptor for IgG (FcyRI) with the y subunit of the IgE receptor. Proc. Natl. Acad. Sci.. 90: 6023-6027.
330
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Fossum, S., 1988. Lymph-borne dendritic leucocytes do not recirculate, but enter the lymph node to become interdigitating cells. Stand. J. lmmunol., 27: 97-105. Freedman, AS., Freeman, G.J., Rhynhart, K. and Nadler, L.M., 1991. Selective induction of B7/BB-I on interferon-y stimulated monocytes: a potential mechanism for amplification of T cell activation through the CD28 pathway. Cell. lmmunol., 137: 429-437. Harkiss, G.D., Hopkins, J. and McConnell, I., 1990. Uptake of antigen by afferent lymph dendritic cells mediated by antibody. Eur. J. lmmunol., 20: 2367-2373. Hopkins, J., Dutia, B.M. and McConnell, I., 1986. Monoclonal antibodies to sheep lymphocytes. I. ldentification of MHC class 11 molecules on lymphoid tissue and changes in the level of class 11 expression on lymph-borne cells following antigen stimulation in vivo. Immunology, 59: 433-438. Hopkins, J., Dutia, B.M., Bujdoso, R. and McConnell, I., 1989. In vivo modulation of CD1 and MHC class 11 expression by sheep afferent lymph dendritic cells. J. Exp. Med., 170: 1303-1318. Jones, P., 1988. B cell differentiation in sheep. Ph.D. Thesis, University of Edinburgh. Knight, S.C., Farrant, J., Bryant, A., Edwards, A.J., Burman, S., Lever, A., Clarke, J. and Webster, D.B., 1986. Non-adherent, low-density cells from human peripheral blood contain dendritic cells and monocytes, both with veiled morphology. Immunology, 57: 595-603. Knight, S.C., Stagg, A., Hill, S., Fryer, P. and Griffiths, S., 1992. Development and function of dendritic cells in health and disease. J. Invest. Dermatol., 99: 33s-38s. Kurosaki, T., Gander, I. and Ravetch, J.V., 1991. A subunit common to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions. Proc. Natl. Acad. Sci., 88: 3837-3841. Kurosaki, T., Gander, I., Wurthmueller, U. and Ravetch, J.V., 1992. The p subunit of the FceRI is associated with the FcyRlll on mast cells. J. Exp. Med., 175: 447-451. Macatonia, S.E., Knight, S.C., Edwards, A.J., Griffiths, S. and Fryer, P., 1987. Localisation of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. J. Exp. Med., 166: 1654-1667. Maddox, J.F., Mackay, CR. and Brandon, M.R., 1985. Surface antigens, SBU-T4 and SBU-T8, of sheep lymphocyte subsets defined by monoclonal antibodies. Immunology, 5.5: 739-748. McKeever, D., Awino, E. and Morrison, WI., 1992. Afferent lymph veiled cells prime CD4 + T cells in vivo. Eur. J. lmmunol., 22: 3057-3061. Manta, F., Fenoglio, D., Li Pira, G., Ku&l, A. and Celada, F., 1991. Effect of antigen/antibody ratio on macrophage uptake, processing and presentation to T cells of antigen complexed with polyclonal antibodies. J. Exp. Med., 173: 37-48. Masuda, M. and Roos, D., 1993. Association of all three types of FcyR (CD64, CD32, and CD16) with a y-chain homodimer in cultured human monocytes. J. lmmunol., 151: 7188-7195. Nabavi, N., Freeman, G.J., Gault, A., Godfrey, D., Nadler, L.M. and Glimcher, L.H., 1992. Signalling through the MHC class 11 cytoplasmic domain is required for antigen presentation and induces B7 expression. Nature, 360: 266. Sallustro, F. and Lanzavecchia, A., 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor (Y. J. Exp. Med., 179: 1109-1118. Schalke, B.C.G., Klinkert, W.E.F., Wekerle, H. and Dwyer, D.S., 1985. Enhanced activation of a T cell line specific for acetylcholine receptor (AChR) by using anti-AChR monoclonal antibodies and receptor. J. lmmunol., 134: 3643-3648. Schmitt, D.A., Hanau, D., Bieber, T., Dezutter-Dambuyant, C., Schmitt, D., Fabre, M., Pauly, G. and Cazenave, J.P., 1990. Human epidermal Langerhans cells express only the 40-kilodalton Fey receptor (FcRII). J. Immunol., 144: 4284-4290. Steinman, R.M., 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. lmmunol., 9: 271-296. Thomas, R., Davis, L.S. and Lipsky, P.E., 1993. Isolation and characterisation of human peripheral blood dendritic cells. J. lmmunol., 150: 821-834. Van der Winkel, J.G.J. and Capel, P.J.A., 1993. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol. Today, 14: 215-221. Wirthmueller, U., Kurosaki, T., Murakami, M.S. and Ravetch, J.V., 1992. Signal transduction by FcyRlIl (CD161 is mediated through the y chain. J. Exp. Med., 175: 1381-1390.
Veterinary Immunology and Immunopathology 49 (1996) 321-330
Veterinary immunology and immunopathology
Enhanced proliferation of CD4+ T cells induced by dendritic cells following antigen uptake in the presence of specific antibody S. Coughlan, G.D. Harkiss
*,
J. Hopkins
Department of Veterinary Pathology, University of Edinburgh, Summerhall, Edinburgh EH9 lQH, UK
Accepted 18 April 1995
Abstract Afferent lymph dendritic cells bear an Fey receptor which binds antigen/ antibody complexes thereby enhancing uptake of antigen. In this report, we have addressed the question of whether the enhanced uptake of antigen results in augmented antigen presentation and T cell proliferation in in vitro secondary responses in sheep. Inclusion of affinity-purified IgG anti-ovalbumin antibody in cultures of afferent lymph dendritic cells, purified CD4’ T cells, and substimulating amounts of ovalbumin resulted in a five- to 169-fold enhancement of T cell proliferation. This effect was antigen-specific as replacement of the anti-ovalbumin antibody with an IgG anti-human serum albumin specific antibody did not cause enhanced T cell responses. The antigen-specific augmentation required intact antibody Fc portions as F(ab’), fragments of the anti-ovalbumin antibodies were ineffective. The enhanced antigen presentation was found to be maximal with immune complexes in moderate antibody excess (three- to 30-fold), but still occurred at antibody/ antigen ratios of 300. The augmented responses were inhibitable with anti-MHC Class II specific antibodies, indicating that at least some of the antigen taken in via Fey receptors entered a Class II processing pathway. The results thus show that antigen uptake via Fey receptors on dendritic cells results in functional augmentation of antigen presentation and T cell proliferation. Keywords: Antigen/antibody
complexes; Fey receptors; T cell proliferation
0. Abbreviations Con A, concanavalin A, DC, dendritic cells; ELISA, enzyme-linked FcyR, Fey receptor; FCS, fetal calf serum; HSA, human serum
* Corresponding
author. Tel: 0131 6506169;
Fax: 0131 6506511:
immunoassay; albumin; Ova,
Email: [email protected].
01652427/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0165.2427(95)05478-2
322
S. Coughlan et al. / Veterinary Immunology and Immunopathology
ovalbumin; PBM, peripheral TCR, T cell receptor.
blood mononuclear
49 (1996) 321-330
cells; PBS, phosphate
buffered
saline;
1. Introduction Dendritic cells (DC) are unique in their ability to induce primary immune responses and are the most potent inducers of secondary responses (Steinman, 1991; Knight et al., 1992). The DC population is a minor one numerically, but it is ubiquitously distributed throughout the body in both lymphoid and non-lymphoid tissues. DC in different parts of the body seem to represent variant forms originating from a common precursor (Fossum, 1988). Afferent lymph DC are thought to arise from a bone marrow-derived dendritic stem cell via skin Langerhans cells and perhaps blood-borne precursors, and populate the draining lymph nodes as interdigitating reticular cells (Fossurn, 1988). Despite their universally recognised potency as antigen presenting cells, the mechanisms underlying their superior accessory function are poorly understood. The factors likely to influence these unique DC functions are the efficiency of antigen uptake and processing, and the expression of cytokines and surface costimulatoty molecules. Previous work has shown that antigen presentation by rat DC isolated from lymph nodes is augmented by the presence of specific antibody, though the mechanism operating was not investigated (Schalke et al., 1985). We have shown previously that sheep afferent lymph DC bear a functional Fc receptor for IgG (Fc~R) (Harkiss et al., 1990). Antigen uptake by DC is greatly augmented in the presence of specific IgG antibody in vitro and in vivo in primed animals. The existence of such an antigen concentrating mechanism might explain, at least partially, the increased accessory function displayed by these cells following antigen challenge in vivo reported previously (Hopkins et al., 1989). H ere, we have addressed the question of whether the enhanced uptake of antigen via the ovine DC FcyR results in functional enhancement of antigen presentation to T cells in secondary responses.
2. Materials and methods 2.1. Animals and surgery Sheep (2-4 years old) and the surgical procedures used to generate cannulated pseudoafferent prefemoral lymphatics were as described previously (Dutia et al., 1993). Antigenic priming was performed by injecting 1 mg of ovalbumin (Ova) or human serum albumin (HSA) subcutaneously in Freund’s complete adjuvant. The animals were boosted 10 days and 17 days later by two further injections of 0.2 mg of these antigens in incomplete Freund’s adjuvant respectively. The sera were tested for anti-Ova or anti-HSA antibodies by enzyme-linked immunosorbent assay (ELISA). Lymph was collected into sterile bottles containing 2.5 X lo3 units of heparin and 2.5 X lo4 units of penicillin and streptomycin.
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2.2, Cell purification DC were obtained from afferent lymph by centrifugation over 14% metrizamide (Nycomed, Oslo) as described previously (Knight et al., 1986; Hopkins et al., 1989). Peripheral blood mononuclear cells (PBM) were isolated using Lymphoprep (1.077 g ml-’ ) (Nycomed). Autologous CD4+ T cells were obtained from the purified PBM using a magnetic activated cell sorter (MACS) and biotinylated anti-CD4 monoclonal antibody; 10’ cells in 90 ml of phosphate buffered saline (PBS) were incubated with 1 ml of streptavidin-labelled magnetic MACS microbeads (Miltenyi Biotech, Bergisch Glasbach, Germany). Streptavidin-phycoerythrin (Sigma) was added to allow assessment of the purity of the selected population by flow cytometry. The purity of the CD4+ T cells assessed by flow cytometry was routinely > 93% using a live gate for lymphocytes based on the forward/side scatter profile. 2.3. Purification
of antibodies
Sera from sheep immunised to Ova or HSA were fractionated on DEAE-cellulose ion exchange columns (Whatman, Maidstone) equilibrated in 10 mM phosphate buffer pH 7.5. The fractions containing IgG were pooled and applied to Sepharose 4B affinity columns bearing Ova or HSA respectively. The bound antibodies were eluted in 0.1 M glycine HCl pH 2.5 and neutralised immediately. Both affinity purified antibodies were of the IgG, isotype as determined by ELISA using isotype-specific monoclonal antibodies. To prepare F(ab’), antibody fragments, affinity-purified IgG anti-Ova antibodies were digested with pepsin (Sigma) (3% w/w) in 0.1 M sodium acetate pH 4.5 overnight at 37°C. The F(ab’j2 antibody fragments were separated from residual whole IgG by DEAE-cellulose ion exchange chromatography. The antibody preparations were dialysed into PBS. The purity of the F(ab’), fragments was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The antibody activity was determined by ELISA. 2.4. Uptake of antigen/antibody
complexes
by DC
Ova (30 pg ml-’ 1 was preincubated with afferent lymph from two sheep containing IgG anti-Ova antibodies for 30 min at 37°C before adding to 1 X 10” autologous DC per
Table 1 Monoclonal
antibodies
used in the studv
Antibody specificity
Designation
lsotype a
Reference
Ovine Ovine Ovine Ovine Ovine
SBU.T4 SW73.2 VPM6 RShyl RShy 2
mIgG2a rIgG2a mIgG1 rIgG2a rIgG2b
Maddox et al. (198.5) Hopkins et al. (1986) Jones (1988) Hobbs h Hobbs ’
CD4 MHC Class II IgG IgGl IgG2
a m, murine; r, rat. b Kindly supplied by Dr. S. Hobbs, Royal Marsden Hospital,
Sutton. Surrey, UK.
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tube. After a further 30 min at 37°C the DC were washed three times in PBS containing 2% bovine serum albumin. Bound IgG was detected using a murine monoclonal antibody specific for sheep IgG (VPM6), and rat monoclonal antibodies specific for sheep IgG, (RShyl) or sheep IgGz (RShy2) (Table 1). After washing, the bound monoclonal antibodies were detected with fluorescein isothiocyanate-labelled sheep anti-mouse or anti-rat IgG conjugates as appropriate. The DC were analysed by flow cytometry. 2.5. Lymphocyte
proliferation
assays
Purified DC and CD4+ T cells from six sheep were resuspended in RPM1 1640 medium containing 10% fetal calf serum (FCS), 2 mM glutamine, 5 X 10d5 M 2-mercaptoethanol, 100 units benzylpenicillin ml- ’ and 100 units streptomycin ml _ ’ . DC were irradiated with 2500 rad. Antigen and antibody dilutions in 50 ~1 volumes of sterile PBS were added to 96 well flat bottomed culture plates (Nunclon, Denmark) followed by incubation for 30 min at 37°C to allow the formation of immune complexes. Each well contained 5 X lo4 DC and 1 X 10’ responding CD4+ T cells in RPM1 1640 medium containing 10% FCS final concentration in a total volume of 200 ~1. After 5 days, the cells were pulsed with 1 @Zi of 3[H]thymidine per well, harvested 5 h later, and counted by beta-spectroscopy. All assays were performed in triplicate, and concanavalin A (Con A; 5 pg ml-‘) was included as a positive control for proliferation. Inhibition of antigen-specific proliferation was assessed in two sheep by the inclusion of F(ab’), fragments of SW73.2, a rat monoclonal antibody (20 pg ml-‘) specific for ovine MHC Class II (Hopkins et al., 1989). A polyclonal rat F(ab’)z anti-mouse IgG control antibody (Pierce & Warriner, Chester, UK) was used at the same concentration. 2.6. Statistics The results were analysed
using the Student’s
two sample t-test.
3. Results 3.1. In vitro uptake of Oua/anti-Ova
antibody complexes
by DC
Metrizamide-purified DC were incubated with Ova in the presence of unfractionated autologous afferent lymph from two sheep containing anti-Ova antibodies, and the uptake of IgG subclasses was detected by flow cytometry. The results from one animal are shown in Fig. 1. About 5% of DC had IgG on their surface in the absence of specific anti-Ova antibodies, whereas with antibody present 34% of DC registered positive. The bound IgG was of the IgG, subclass. Similar results were obtained in the second animal. The results thus show that DC take up excess immunoglobulin, presumably specific antibody, when incubated with antigen/antibody complexes. 3.2. Enhancement
of T cell proliferation
by specific antibody
To test whether FcyR-mediated uptake of antigen/antibody complexes results in increased antigen presentation, purified DC were cultured with autologous CD4+ T cells
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315
1000500 250 12562.5 31.3 15.6 7.8 3.9 0
Concentration
of OVA (ugml-‘1
Fig.
1. Uptake of sheep immunoglobulin by DC after in vitro incubation with Ova and autologous afferent lymph containing IgG anti-Ova antibodies. Bound immunoglobulin was detected by flow cytometry using monoclonal antibodies specific for sheep IgG, or IgG,.
from six Ova-primed animals with antigen in the presence or absence of specific affinity-purified IgG antibody. Typical results are shown in Fig. 2. Proliferation of CD4’ T cells was observed with Ova alone only at 10 pg ml-‘, whereas in the presence of specific antibody proliferation was observed at 1 and 0.1 pg ml -I. DC in
30 ? 0 X
E
OVA A
20
anubody
OVA and antiHSA anllbody
2 IO
10
I
Concentration
0.1
0.0 I
0
of OVA (pgml-I)
Fig. 2. Enhancement of CD4+ T cell proliferation induced by DC in the presence of specific antibody. Ova (O-10 /.~g ml-‘) and affinity-purified IgG anti-Ova antibodies (30 pg ml-’ ) in sterile PBS were incubated at 37°C for 30 min before addition of an equal volume of medium containing 5 X lo4 DC and 1 X 10s T cells to each well. The final concentration of FCS in RPM1 medium was 10%. After 5 days, T cell proliferation was measured as the mean counts of triplicate cultures + standard deviation. * P < 0.001.
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Table 2 Maximal enhancement
of CD4+
T cell proliferation
Sheep number
[jH]-Thymidine
incorporation
Ova a only
Ova + anti-Ova antibody
150 1.51 156 21 19 23
1538 +507 1459f 94 363k 30 1621+344 1216+327 3353+438
27669 + 2323 36139&3900 61505+6131’ 12261+ 541 11500+ 1652 16875+1616
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49 (19961 321-330
induced by DC in the presence of specific antibody
kpm) Anti-Ova antibody * (X 18) * (X25) (X169) * (x8) ’ (X 10) * (X5)
h
1479 f 420 188+ 29 104+ 23 215+ 51 669 f 572 2634k602
Medium only
Con A only
1037+360 521k 79 102& 83 1654,272 1354f602 1204k827
116054* 1508 128621k10.527 142488k 85.51 89723+_ 5247 81424+ 3997 102977i 5479
a Ova concentrations used were 1 pg ml -’ in Sheep 1.50, 151 and 19, and 0.1 pg ml -’ in Sheep 1.56,21 and 23. b IgG anti-Ova concentration used was 30 pg ml-‘. * P < 0.001 compared to counts obtained with Ova alone. Figures in parentheses represent enhancement in T cell proliferation over Ova atone counts.
the presence of substimulating amounts of Ova alone or in the presence of specific antibody alone did not induce T cell proliferation above background levels. Similarly, no significant T cell proliferation was obtained with Ova, antibody, Ova/anti-Ova complexes or Con A in any of the wells if DC were omitted (data not shown). Table 2 shows the maximal enhancement of T cell proliferation obtained from the six sheep after incubation of complexed Ova compared to Ova alone. The degree of enhancement ranged from five-fold up to 169-fold in individual sheep. In most cases, the greatest enhancement of T cell proliferation was observed with antibody/antigen ratios between 3 and 30, though significant enhancement was also found in some sheep at a ratio of 300 (data not shown). When F(ab), anti-Ova antibodies or IgG anti-HSA antibodies were no significant enhancement of T cell used in place of IgG anti-Ova antibodies, proliferation was observed (Fig. 2). Since the F(ab), anti-Ova antibodies had a similar antibody titre when tested against Ova by ELISA, the results indicate that the mechanism of enhancement requires the Fc portion of the IgG antibodies. 3.3. Effect of anti-MHC
Class II antibodies
on enhancement
of T cell proliferation
Antigen presentation by ovine DC in the absence of specific antibody is almost completely inhibited by anti-MHC Class II antibodies (Hopkins et al., 1989). To test whether antigen taken up via an FcyR is also processed and presented via the MHC Class II pathway, rat monoclonal antibodies with pan specificity for ovine MHC Class II were included in the DC/T cell cultures from two sheep. The results from one sheep are shown in Fig. 3. The anti-MHC Class II antibodies almost completely inhibited the T cell proliferation obtained when the antigen was given with specific antibodies. In contrast, the control rat monoclonal antibodies did not significantly inhibit the enhanced T cell proliferation obtained with complexed Ova. Similar results were obtained in the second animal. The results indicate that antigen taken up via the DC FcyR is processed and presented by MHC Class II molecules to the CD4+ T cells.
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f f
lo 5
0
Concentration
of OVA (pgml- 1)
Fig. 3. Effect of anti-MHC Class II antibody on enhanced CD4+ T cell proliferation induced by DC in the presence of specific antibodies. Ova (O-10 /.~g ml-’ 1 and affinity-purified IgG anti-Ova antibodies (30 pg ml-’ I were incubated at 37°C for 30 min before addition of rat IgG anti-MHC Class II monoclonal antibody (20 pg ml-’ 1 or control rat IgG anti-mouse IgG (20 pg ml-’ 1, 5 X lo4 DC and 1 X 10’ T cells to each weil. After 5 days, T cell proliferation was measured as the mean counts of triplicate cultures f standard deviation. * P < 0.001; + P < 0.05.
3.4. Effect of decreasing the DC / T cell ratio on enhanced T cell proliferation To ascertain if the efficiency of antigen presentation by DC was increased following uptake of antigen in the form of antigen antibody complexes via the FcyR, the cultures
30
0
DC Number Fig. 4. Enhancement of CD4+ T cell proliferation DC. Ova (0.2 gg ml-‘) and affinity-purified IgG for 30 min before addition of 1 X lo5 T cells and After 5 days, T cell proliferation was measured as * P < 0.001: * P < 0.05.
OVA only
x 10-i
in the presence of specific antibody at limiting numbers of anti-Ova antibodies (30 @g ml-’ 1 were incubated at 37°C DC numbers ranging from 2 X lo6 to 1.66 X 10” per well. the mean counts of triplicate cultures + standard deviation.
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were set up with constant numbers of CD4’ T cells and constant amounts of Ova and anti-Ova antibodies but with decreasing numbers of DC. Similar results were obtained in two sheep. The results from one animal are shown in Fig. 4. No proliferation above background was seen when the DC numbers were reduced to 3 X 10’ per well. When specific antibody was included in the cultures, T cell proliferation also decreased with decreasing numbers of DC but the degree of proliferation was considerably higher than with antigen alone from 50 X lo3 DC per well down. With 3 X lo3 and 1.6 X 10” DC, T cells showed no proliferation in the presence of Ova but were stimulated significantly with Ova/anti-Ova complexes. The results thus indicate that, on a per cell basis, DC are more efficient at stimulating T cell if the antigen is complexed with antibody than if it is given alone.
4. Discussion Previous studies have shown that ex vivo sheep afferent lymph DC are very potent activators of T cells in secondary responses (Hopkins et al., 19891. The mechanisms underlying this potency are not well understood. We recently demonstrated that these afferent lymph DC bear an FcR for IgG which functions to concentrate antigen on to the cell surface both in vitro and in vivo (Harkiss et al., 1990). Here we show that increased antigen uptake via the DC FcyR in sheep results in functional enhancement of CD4’ T cell proliferation. The results showed that the enhancement was antigen-specific and was dependent on the IgG antibodies bearing an intact Fc portion. The augmentation of T cell proliferation was maximal if the immune complexes were in moderate antibody excess, but still occurred to a lesser degree in 300-fold antibody excess in some sheep. Thus, addition of immune complexes in antibody excess to DC resulted in the conversion of a substimulating dose of antigen into a potent stimulating dose for CD4+ T cell proliferation. Similarly, addition of specific antibody to cultures containing substimulating numbers of DC resulted in substantial T cell proliferation, indicating that on a per cell basis the DC possessed more potent antigen presenting function. In conditions of antibody excess, no free antigen would be available for uptake by the DC. This indicates that all the antigen would be taken up via the DC FcyR. The augmented T cell proliferation was substantially inhibited by the inclusion of anti-MHC Class II antibodies in the cultures, indicating that some of the antigen was being routed into an MHC Class II processing pathway. The mechanisms underlying the enhanced T cell proliferation mediated via FcyR occupancy in DC are not known. The basis for such augmentation may simply be the concentration of antigen into the cell thereby resulting in increased amounts of antigenic peptides being presented to T cells. This, in combination with the expression of increased levels of MHC Class II (Hopkins et al., 1989), could result in an increased density of MHC Class II molecules bearing specific antigenic peptides. It is known that cross-linking of MHC Class II molecules by the T cell receptor (TCR) or the release of interferon-y by T cells results in upregulation of the costimulatory molecule B7 in B cells (Freedman et al., 1991; Nabavi et al., 1992). Such signals from T cells clearly
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could induce DC to express B7 or other costimulatory molecules thereby enhancing their T cell stimulating function. It is also possible that engagement of the Fey R results in one or more signals being transduced which could result in the activation or upregulation of surface molecules such as MHC Class II and B7. The upregulation of MHC Class II on DC following secondary antigenic challenge in vivo (Hopkins et al., 1989) is consistent with this hypothesis. The ability of FcyR to transduce signals in humans has previously been thought to reside in the FcyRI and FcyRIII class of receptors, and to be mediated by homo- or heterodimers of the y and 5 chains usually found in the TCR/CD3 complex and high affinity FceR respectively (Kurosaki et al., 1991, 1992; Wirthmueller et al., 1992; Ernst et al., 1993; Van der Winkel and Capel, 1993). However, recent evidence suggests that all three types of human FcyR (CD64, CD32, CD16) have an associated y chain homodimer, suggesting that signals may be transduced by one or more isoforms of FcyRII as well (Masuda and ROOS, 1993). Although the FcyR expressed by ovine afferent lymph DC have not been fully characterised, we have preliminary evidence from Western blotting studies that these cells express both FcyRI and FcyRII (Coughlan, Harkiss and Hopkins, unpublished observations, 1995). This is consistent with the expression of both these receptors on human blood DC (Thomas et al., 1993), and the expression of FcyRII by Langerhans cells (Schmitt et al., 19901, a cell type thought to be a precursor for afferent lymph DC (Macatonia et al., 1987). Augmentation of T cell proliferation by specific antibodies has been demonstrated previously for macrophages (Manta et al., 1991) and DC isolated from rat lymph nodes (Schalke et al., 1985) or from human blood and maintained by cytokine activation (Sallustro and Lanzavecchia, 1994), although the mechanism of augmentation by DC was not addressed in these studies. In contrast to the studies in rats, sheep and humans, no functional enhancement of T cell proliferation was observed using bovine afferent lymph DC (McKeever et al., 1992). The reasons for this discrepancy are not known, but may have been due to the use of stimulating rather than substimulating amounts of antigen in the latter study. Consequently, no augmentation would have been observed. Increased antigen uptake and presentation to T cells via DC FcyR may thus be an important mechanism operating in chronic diseases with an immune complex aetiology.
Acknowledgements This work was supported by the Agricultural and Food Research Council, UK. (LRG 31). S. Coughlan was supported by an AFRC Research Training Scholarship.
References Dutia, B.M., McConnell, I., Bird, K., Keating, P. and Hopkins, J., 1993. Patterns of major histocompatibility class II expression by T cell subsets in different immunological compartments. 1. Expression on resting T cells. Eur. .I. Immunol., 23: 2882-2888. Ernst, L.K., Duchemin, A.-M. and Anderson, C.L., 1993. Association of the high-affinity receptor for IgG (FcyRI) with the y subunit of the IgE receptor. Proc. Natl. Acad. Sci.. 90: 6023-6027.
330
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Fossum, S., 1988. Lymph-borne dendritic leucocytes do not recirculate, but enter the lymph node to become interdigitating cells. Stand. J. lmmunol., 27: 97-105. Freedman, AS., Freeman, G.J., Rhynhart, K. and Nadler, L.M., 1991. Selective induction of B7/BB-I on interferon-y stimulated monocytes: a potential mechanism for amplification of T cell activation through the CD28 pathway. Cell. lmmunol., 137: 429-437. Harkiss, G.D., Hopkins, J. and McConnell, I., 1990. Uptake of antigen by afferent lymph dendritic cells mediated by antibody. Eur. J. lmmunol., 20: 2367-2373. Hopkins, J., Dutia, B.M. and McConnell, I., 1986. Monoclonal antibodies to sheep lymphocytes. I. ldentification of MHC class 11 molecules on lymphoid tissue and changes in the level of class 11 expression on lymph-borne cells following antigen stimulation in vivo. Immunology, 59: 433-438. Hopkins, J., Dutia, B.M., Bujdoso, R. and McConnell, I., 1989. In vivo modulation of CD1 and MHC class 11 expression by sheep afferent lymph dendritic cells. J. Exp. Med., 170: 1303-1318. Jones, P., 1988. B cell differentiation in sheep. Ph.D. Thesis, University of Edinburgh. Knight, S.C., Farrant, J., Bryant, A., Edwards, A.J., Burman, S., Lever, A., Clarke, J. and Webster, D.B., 1986. Non-adherent, low-density cells from human peripheral blood contain dendritic cells and monocytes, both with veiled morphology. Immunology, 57: 595-603. Knight, S.C., Stagg, A., Hill, S., Fryer, P. and Griffiths, S., 1992. Development and function of dendritic cells in health and disease. J. Invest. Dermatol., 99: 33s-38s. Kurosaki, T., Gander, I. and Ravetch, J.V., 1991. A subunit common to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions. Proc. Natl. Acad. Sci., 88: 3837-3841. Kurosaki, T., Gander, I., Wurthmueller, U. and Ravetch, J.V., 1992. The p subunit of the FceRI is associated with the FcyRlll on mast cells. J. Exp. Med., 175: 447-451. Macatonia, S.E., Knight, S.C., Edwards, A.J., Griffiths, S. and Fryer, P., 1987. Localisation of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. J. Exp. Med., 166: 1654-1667. Maddox, J.F., Mackay, CR. and Brandon, M.R., 1985. Surface antigens, SBU-T4 and SBU-T8, of sheep lymphocyte subsets defined by monoclonal antibodies. Immunology, 5.5: 739-748. McKeever, D., Awino, E. and Morrison, WI., 1992. Afferent lymph veiled cells prime CD4 + T cells in vivo. Eur. J. lmmunol., 22: 3057-3061. Manta, F., Fenoglio, D., Li Pira, G., Ku&l, A. and Celada, F., 1991. Effect of antigen/antibody ratio on macrophage uptake, processing and presentation to T cells of antigen complexed with polyclonal antibodies. J. Exp. Med., 173: 37-48. Masuda, M. and Roos, D., 1993. Association of all three types of FcyR (CD64, CD32, and CD16) with a y-chain homodimer in cultured human monocytes. J. lmmunol., 151: 7188-7195. Nabavi, N., Freeman, G.J., Gault, A., Godfrey, D., Nadler, L.M. and Glimcher, L.H., 1992. Signalling through the MHC class 11 cytoplasmic domain is required for antigen presentation and induces B7 expression. Nature, 360: 266. Sallustro, F. and Lanzavecchia, A., 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor (Y. J. Exp. Med., 179: 1109-1118. Schalke, B.C.G., Klinkert, W.E.F., Wekerle, H. and Dwyer, D.S., 1985. Enhanced activation of a T cell line specific for acetylcholine receptor (AChR) by using anti-AChR monoclonal antibodies and receptor. J. lmmunol., 134: 3643-3648. Schmitt, D.A., Hanau, D., Bieber, T., Dezutter-Dambuyant, C., Schmitt, D., Fabre, M., Pauly, G. and Cazenave, J.P., 1990. Human epidermal Langerhans cells express only the 40-kilodalton Fey receptor (FcRII). J. Immunol., 144: 4284-4290. Steinman, R.M., 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. lmmunol., 9: 271-296. Thomas, R., Davis, L.S. and Lipsky, P.E., 1993. Isolation and characterisation of human peripheral blood dendritic cells. J. lmmunol., 150: 821-834. Van der Winkel, J.G.J. and Capel, P.J.A., 1993. Human IgG Fc receptor heterogeneity: molecular aspects and clinical implications. Immunol. Today, 14: 215-221. Wirthmueller, U., Kurosaki, T., Murakami, M.S. and Ravetch, J.V., 1992. Signal transduction by FcyRlIl (CD161 is mediated through the y chain. J. Exp. Med., 175: 1381-1390.