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Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Spleens from 10- to 12-wk-old male SPF PVG rats were digested in
collagenase, dispase, and DNase (Boehringer Mannheim, Lewes, U.K.;
(23)). RBC were lysed and cells incubated with OX52 (pan T
cell), OX8 (T cells and NK cells), OX12 (anti-light chain), OX33
(pan-B cells), and biotinylated anti-µ and anti-
Abs
(Binding Site, Birmingham, U.K.) for 1 h at 4°C. Labeled cells
were depleted by rosetting with anti-mouse Ig-coated SRBCs and
layering over Histopaque (Sigma, St. Louis, MO). Contaminating cells
were depleted using the MACS system (Miltenyi Biotec, Bergisch
Gladbach, Germany). The final DC-enriched population was examined by
flow cytometry and immunocytochemistry and contained 
85% DC based
on morphology and expression of MHC class II, with less than 1% T
cells, B cells, or macrophages. The flow cytometry profile in Figure 1
A shows the absence of cells
expressing OX52 (T cells) or OX12 (B cells), with the majority of cells
expressing MHC class II. Immunocytochemistry showed less than 1%
macrophages or plasma/preplasma cells (Table I).
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B cells were isolated from rat spleens by disruption through a
cell filter, followed by depletion of RBC with Gey’s solution. The
remaining cells were incubated in petri dishes for 1 h at 37°C
to remove adherent cells (macrophages and fibroblasts), followed by
treatment with 10 µg/ml OX52 (pan-T cell), OX62 (DC specific marker),
anti--biotin, and OX41 (macrophages and DC subpopulation) for
1 h at 4°C. Labeled cells were depleted by rosetting with
anti-mouse Ig-coated SRBCs and layering over Histopaque (Sigma).
Contaminating cells were depleted using the MACS system (Miltenyi
Biotec). Flow cytometry profiles in Figure 1
B show that more
than 95% of cells are B cells that expressed IgM and low levels of MHC
class II. Less than 1% of cells expressed OX52 (T cells), or
moderately high MHC class II levels (DC). Immunocytochemistry showed
<1% of macrophages or plasma/preplasma cells (Table I).
Flow cytometry
Cells were incubated with 10 µg/ml OX52, OX6 (MHC class II), OX12 (Ig light chain), or anti-IgM-biotin for 1 h at 4°C. Labeled cells were detected by goat anti-mouse IgG (rat adsorbed)-phycoerythrin (Serotec, Oxford, U.K.) or Streptavidin-Quantum red (Sigma).
Detection of macrophages
Opsonized SRBC were prepared by incubation of SRBC with rabbit anti-SRBC for 1 h at 37°C and washed in PBS three times. To detect macrophages in cell preparations, the opsonized SRBC were mixed with a sample of these cells for 1 h at 37°C, the red cells lysed, and the cells cytocentrifuged onto slides. The slides were stained with Giemsa stain and the number of cells that had ingested opsonized SRBC were scored as macrophages. DC show no evidence of uptake of opsonized SRBC.
Detection of preplasma and plasma cells
Samples of cell preparations were cytocentrifuged onto slides and fixed in cold ethanol. The cells were treated with OX12 for 1 h at room temperature followed by HRP-anti-Ig and substrate (diaminobenzidine and hydrogen peroxide, both from Sigma). While B cells were very weakly stained, plasma and preplasma cells had large quantities of cytoplasmic Ig.
In vitroca Ag pulsing
DC were pulsed with 100 µg/ml keyhole limpet hemocyanin (KLH, Sigma) or DNP-KLH (Calbiochem, La Jolla, CA) for 1 h at 37°C and washed in PBS. To remove noninternalized Ag, cells were incubated in 0.02 M EDTA/PBS (24) for 5 min and given three washes in PBS before use in cultures or administration to rats. To confirm that EDTA removed surface Ag, we measured the mean flurocence intensity (MFI) of DC pulsed with fluoresceinated DNP-KLH at 4°C and 37°C, before and after treatment with EDTA. EDTA reduced the MFI of cells pulsed with Ag at 4°C to background levels, with negligible effects on the MFI of DC pulsed at 37°C, i.e., EDTA did not affect endocytosed Ag.
In vivo Ag pulsing
Rats were given 500 µg DNP-KLH i.v. and the DC isolated from their spleens after 4, 12, and 24 h.
In vivo immunization
A total of 1 x 106 DC pulsed in vitro with DNP-KLH (see above) were administered i.v. to groups of KLH-primed or unprimed PVG rats. To prime rats for KLH, approximately 5 x 105 KLH-pulsed DC were administered i.v. 24 to 48 h before immunization. Positive control rats were given 500 µg DNP-KLH i.v. Each group contained four rats and the Ab titers were assayed by ELISA at the time of immunization and after 7 and 14 days.
In vitro cell culture
Cells were cultured in Iscove’s DMEM with 5% FCS (Life Technologies, Paisley, Scotland, U.K.), 2 mM glutamine (Life Technologies), 25 mM 2-ME and 45 µg/ml penicillin/streptomycin (Life Technologies). B cells and Ag-pulsed DC were cultured at a ratio of 10 B cells/DC at a cell density of 2.5 x 106 cells/ml in 24-well plates. After 20 h, DC were depleted by rosetting or MACS and B cells incubated with a DC-activated, KLH-specific T cell line. The isolated B cell population did not contain detectable numbers of DC as assessed by FACS or immunocytochemistry.
Confocal microscopy
To localize Ag in DC, purified cells were pulsed with FITC-human
serum albumin (HSA), treated with EDTA, and cultured. After 6, 12, 24,
and 48 h, the cells were examined by confocal microscopy. To show
retention of native Ag, cells were pulsed with 1 mg/ml horseradish
peroxidase (HRP) for 1 h, cultured, and enzyme activity was
detected by diaminobenzidine (Sigma) and HRP (Sigma). Images were
obtained using a Bio-Rad (Richmond, CA) MRC 1000 confocal
laser-scanning microscope. Images were collected sequentially (zoom
x8.0). For Figure 2L, a phase
contrast image was obtained using the Bio-Rad transmission
detector.
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Ab titers were determined as described by van Essen et al., using 100 µg/ml (for serum) and 500 µg/ml (for culture supernatants) DNP-HSA to coat plates (25). Curves of absorbance against serum dilution were plotted and the Ab titers were determined as the reciprocal dilution that gave an absorbance of 30% of the maximum absorbance reading for that particular assay. Ab titers shown are mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To show that DC can retain native Ag, ex vivo splenic DC were
incubated with FITC-HSA or HRP, and prepared for confocal microscopy
immediately or after 6, 12, 24, or 48 h in culture. No fluorescent
Ag could be detected in DC immediately after pulsing, but by 6 h
Ag was located in cytoplasmic vesicles dispersed through the cell.
Figure 2 (A–F) shows equatorial optical sections of DC
pulsed with FITC-Ag after 12 h and stained for MHC class I (Fig. 2, A–C) and MHC class II (Fig. 2, D–F) to
determine the localization of Ag and the markers. Spleen DC varied in
their morphology and distribution of class II molecules (Fig. 2, D–K). Some DC were large, very irregular cells with most
class II molecules expressed on their plasma membrane (Fig. 2, F, J, and K) whereas others were less
irregular with a lower level of class II expression on the cell surface
and the majority of class II molecules contained in cytoplasmic
vesicles (Fig. 2, G–I). These cells represent DC in
different stages of maturation (26, 27). Those DC that
contained large amounts of cytoplasmic MHC class II appeared to have
endocytosed more Ag than those with much membrane class II expression.
These figures show that although some degree of colocalization occurs
between vesicles containing Ag and MHC class I or class II molecules,
in all cells Ag was also present in vesicles not expressing these
molecules. The uptake and retention of HRP enzyme activity confirmed
that unprocessed Ag was retained in DC for at least 48 h. Figure 2L shows retention of HRP after 12 h.
Ag-pulsed DC can initiate a primary Ag-specific Ab response and induce class switching in vivo
To investigate the functional significance of Ag retention by DC,
ex vivo splenic DC were pulsed with DNP-KLH, treated with EDTA to
remove surface-bound Ag, and injected i.v. into naive or KLH-primed
rats. The preimmune sera of these rats did not contain detectable
anti-DNP or anti-KLH Ab. Both naive and KLH-primed animals
produced similar titers of IgG anti-DNP Ab with 4- to 10-fold less
IgM anti-DNP (Fig. 3A).
Similarly, the anti-KLH response in naive rats was also
predominantly IgG (Fig. 3B). KLH-primed animals produced
better responses to KLH than naive animals, suggesting a secondary
response to the carrier protein. KLH-primed rats were also given DC
that had been lysed after pulsing with DNP-KLH, to provide an
equivalent quantity of free Ag that would control for nonspecific
release of Ag. These animals produced a little IgM anti-DNP Ab but
no detectable total IgG anti-DNP, indicating a need for intact DC
to transport Ag and skew the response toward IgG (Fig. 3A).
Moreover, Ag-pulsed BMDC provoked IgG responses with no detectable IgM
in naive animals (data not shown). In comparison, naive animals given
500 µg free DNP-KLH i.v. gave much higher but similar titers of IgM
and IgG of anti-DNP Ab (Fig. 3C), indicating the skew
toward IgG was not dependent on the Ag.
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D). IgG1 and IgG2a titers were similar in primed and naive
animals given intact DC, while IgG2b and IgG2c levels were
significantly higher in primed animals suggesting that switching to the
latter two subclasses is enhanced by T cell priming. Moreover, very low
titers of IgG2b, trace amounts of IgG2c, but no IgG1 or IgG2a, was
detected in animals given lysed DC. DC pulsed with Ag in vivo can retain Ag and initiate an immune response
To show that Ag retention and transfer to B cells was not an
artifact of pulsing DC in vitro, we loaded DC with Ag in vivo. DC were
isolated 4, 12, and 24 h after rats were given soluble DNP-KLH and
injected i.v. into naive rats. These DC that had captured Ag in vivo
initiated an anti-DNP Ab response in naive animals, which was again
predominantly IgG with 4- to 13-fold less, or no, IgM (Fig. 4). The predominance of IgG anti-DNP
Ab suggested that DC may have a direct role in class switching. These
observations are not peculiar to KLH, since they could be reproduced
with human serum albumin (Fig. 5).
Furthermore, the IgG response observed in recipient rats could not be
due to the transfer of B cells or plasma cells since such cells were
not detected in significant numbers in the preparations transferred
(Fig. 1 and Table I).
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To show that DC have a direct role in class switching, in vitro
cultures were prepared as described in Figure 6. DC pulsed with DNP-KLH were cultured
with naive B cells for 20 h, the DC depleted, and the recovered B
cells cultured with KLH-primed T cells (Fig. 6a). To provide
optimal help, the T cell line was cultured with KLH-pulsed DC before
the addition of B cells. After 7 days, both IgG and IgM anti-DNP Ab
was detected in the culture supernatants (Fig. 6a). To show
that the class switch was dependent on early interaction with DC, B
cells were cultured with soluble DNP-KLH for 20 h and then added
to KLH-primed T cells (Fig. 6b). These cultures produced
only IgM anti-DNP Ab (Fig. 6b). To show that contact
between DC and B cells was essential, DC pulsed with DNP-KLH were
cultured in transwells with B cells in a separate compartment (Fig. 6c). When these B cells were subsequently cultured with
primed T cells, no IgG and only low titers of IgM anti-DNP Ab were
detected (Fig. 6c). B cells cultured with DNP-KLH-pulsed DC
in the absence of T cell help did not produce either IgM or IgG. These
in vitro experiments demonstrate that contact between DC and B cells is
essential for efficient transfer of Ag to B cells and that DC provide B
cells with an early signal that is essential for subsequent switching
to IgG. However, B cells will not produce Ab without T cell
help.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Functional significance of Ag retention by DC
It is well recognized that DC acquire Ag in vivo and subsequently present peptides to naive T cells (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). Moreover, it has been widely assumed, by analogy with macrophages (28), that all native Ag would be rapidly broken down following endocytosis. However, if DC can present Ag to B cells, they must retain it in its native form for long enough to permit its transport from the periphery to secondary lymphoid tissues. This study shows that i.v. injection of Ag-pulsed splenic DC into naive rats stimulates the secretion of Ag-specific Ab within 7 days, showing that such DC can retain Ag long enough for it to be presented to B cells. The titers of the anti-DNP Ab resulting from the administration of DNP-KLH-pulsed DC were much lower than rats given soluble DNP-KLH. This reflects the minute quantity of Ag transported by DC and that only B cells that meet the DC will encounter Ag. When rats were injected with the same number of lysed Ag-pulsed DC, the titers were barely above background, demonstrating that DC can act as adjutants for small quantities of Ag.
We estimated the kinetics of Ag retention by DC in two ways. First, DC were pulsed in vitro with HRP, and active enzyme could be detected for at least 48 h, showing that some native Ag is retained for this period. To show that this was not just an in vitro phenomenon, we assessed Ag retention by DC in vivo. We reasoned that if we could load splenic DC in vivo, we could test Ag retention in vivo by transferring the DC to naive recipients and assess Ab synthesis. If the recipients made specific Ab this would show unequivocally that DC had retained Ag in a native form and that transfer to B cells was physiologically relevant. The results show that DC retain native Ag for 24 h in vivo (maximum period tested), as well as the time taken to transport Ag from the site of injection to the spleen and to interact with B cells. Furthermore, confocal microscopy showed that some vesicles containing Ag did not contain MHC class II or class I molecules. These vesicles could be detected for at least 48 h, and although the presence of fluorescence cannot be directly equated with the retention of native Ag, this observation correlates well with HRP retention. Indeed, immature DC have been shown to possess mildly acidic compartments that store Ag for 24 h (29).
We do not know how Ag is released from DC to be recognized by B cells, but it has been shown that human DC can retain HIV in an internal compartment and, subsequently, this HIV can infect T cells adhering to the DC (30). We suggest that HIV may occupy the compartment used for Ag retention, and that the virus may have "hijacked" this mechanism to escape lysosomal enzymes and be subsequently released.
A role for DC in isotype switching by B cells
The regulation of B cell isotype switching in T-dependent responses is complex and requires both cell-to-cell signaling and cytokine recognition by B cells. CD40-CD40L interactions are essential for switching as shown by the absence of switching in patients with the X-linked hyper-IgM syndrome, in whom the CD40L gene is mutated (31), and in CD40 knockout mice (25). Studies in vitro have shown that, as well as CD40-CD40L interactions, T cell cytokines are also essential for switching (13, 14, 15, 16, 17, 18, 19, 20, 25, 32, 33, 34, 35, 36, 37, 38, 39, 40). However, some observations suggest that this picture is still incomplete. For example, a study of the dynamics of a primary response found that switching from IgM to IgG was initiated by day 2 of the response and was maximal by days 6 to 8 (41). It was assumed that cytokines induce isotype switching very early in the immune response. Moreover, during B cell activation, transcription of unarranged heavy chain C region genes precedes switch recombination and is independent of cytokines (42). These observations suggest that B cells receive as yet unidentified signals that occur very early in the response, possibly before interaction with T cells.
In a primary Ab response, IgM secretion precedes IgG. We observed that
rats immunized with Ag-pulsed BMDC or splenic DC consistently produced
much higher levels of specific IgG than IgM. Indeed, with BMDC it was
frequently impossible to detect an IgM response. To show that the skew
toward IgG was not dependent on T cell priming, we compared the
response of naive and KLH-primed rats to DNP-KLH-pulsed DC. KLH-pulsed
DC were used for priming T cells to minimize free Ag in the rats, which
could complicate the interpretation. In both naive and KLH-primed
animals, the anti-DNP response was predominantly IgG. However,
whereas anti-KLH responses in naive rats were predominantly IgG,
KLH-primed animals produced higher total levels of anti-KLH Ab with
relatively higher levels of IgM. To explain this apparent anomaly of
higher IgM levels, we propose that the first injection of KLH-pulsed DC
recruited Ag-specific recirculating T and B cells to the spleen, and
initiated Ab synthesis with a skew toward IgG (as for naive rats in
Fig. 3B). When the recruited lymphocyte population was given
a second dose of KLH on DNP-KLH-pulsed DC, they gave a better Ab
response. The IgM response was also higher since T cell help for IgM
production was readily available and, as soon as the DC provided Ag,
IgM anti-KLH could be synthesized. Previous recruitment of
KLH-specific T cells and B cells may also explain why the small amount
of DNP-KLH released from lysed DC was able to stimulate significant
levels of IgM anti-KLH Ab but very little IgM anti-DNP Ab (Fig. 3, A and B).
The consistent observation that rats given Ag pulsed-DC produced mostly IgG suggested that DC might have direct involvement in isotype switching. Since this switch occurred without additional soluble Ag, we proposed that DC might provide such a signal soon after Ag transfer, before Th cells had been recruited/activated. To investigate this possibility, we developed an in vitro model of Ab synthesis in which B cells were first cultured with Ag-pulsed DC, purified, and subsequently cultured with activated Th cells. We showed that B cells that had acquired DNP-KLH from DC secreted both IgM and IgG anti-DNP when given T cell help. In contrast, B cells cultured with free DNP-KLH (without DC) secreted only IgM when subsequently cultured with T cells under identical conditions. This shows clearly that Ag-bearing DC can transmit signals involved in isotype switching to Ag-specific B cells via direct contact. Contact is essential because when DC and B cells were separated by a transwell, no switching was seen when the B cells subsequently received T cell help. The Ab titers in these in vitro microcultures were low compared with in vivo titers, but we consider that this reflects the small proportion of Ag-specific B cells present in in vitro cultures, where specific recruitment is not possible. Moreover, Ag-specific B cells may not expand to the same levels in vitro, in the absence of in vivo microenvironment.
The molecular basis of DC-B cell signaling is not known. Direct CD40 signals to B cells are required for class switching since B cells from CD40 knockout mice do not switch (25). Since human DC have been shown to express CD40 ligand (43), it is possible that DC may provide CD40 signals to B cells. However, a study by Banchereau and colleagues showed that human B cells given CD40 signals (to replace T cells) and cytokines (IL-10 and TGF-ß) did not switch to IgA without dendritic cells (44). This study shows that a CD40 signal was insufficient to isotype switch B cells and that another signal was provided by DC. Furthermore, a murine CD40 fusion protein did not inhibit subsequent switching when added to rat DC-B cell cultures (data not shown). This could signify that CD40 plays no role in the DC-B cell interaction, or that murine CD40 does not cross-react with rat.
DC also influence the subclass of IgG secreted after immunization. Moser and colleagues (45, 46) have shown that the type of APC (B cells, macrophages, or DC) used to immunize mice could affect the subclass of Ab produced. These differences were thought to reflect differences in T cell priming since the animals were subsequently given soluble Ag. Here we show that intact DC are essential for IgG1 and IgG2a production, and previous T cell priming is required for IgG2b and IgG2c. In rats, IgG1 and IgG2a represent Th2 responses whereas IgG2b is a Th1 Ab (47, 48, 49, 50, 51). Since all four subtypes are secreted after immunization with Ag-pulsed DC, we suggest that DC provide "switch" signals, but particular cytokines drive the expansion of switched B cells. DC may prime B cells to solicit Th2 cytokines from T cells to drive the switched phenotype. Studies have found that while T cells primed by DC produce Th1 cytokines, the addition of B cells results in Th2 cytokine production (52, 53, 54).
In conclusion, our results show that DC have important roles in the initiation and regulation of Ab synthesis, quite apart from their function in T cell activation. They acquire and retain protein Ags in an intact form for long enough to permit their migration to secondary lymphoid tissues and delivery to naive, recirculating, Ag-specific B cells. At the same time, they give signals to these B cells that modulate isotype switching and that can influence the subclass of IgG that is secreted. These results suggest that DC have complex roles in the regulation of humoral immune responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michelle Wykes, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cells; B cells, B lymphocytes; KLH, keyhole limpet hemocyanin; HSA, human serum albumin; HRP, horseradish peroxidase; MACS, magnetic cell separation system; MFI, mean fluorescence intensity; SPF, specific pathogen-free; BMDC, bone marrow dendritic cells.
Received for publication November 26, 1997. Accepted for publication April 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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germline transcription: role of B-cell-specific activator protein. Immunoregulatory role of CD40 in human B cell differentiation. Int. Arch. Allergy Immunol. 113:35.[Medline]
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T lymphocytes express CD40 ligand and induce isotype switching in B lymphocytes. J. Exp. Med. 181:1239.-dependent switching of IgG alloantibody subclass. Eur. J. Immunol. 26:1217.[Medline]
expression correlates with increased IgG1 alloantibody response following intrathymic immunomodulation of sensitized rat recipients. Transplantation 60:1516.[Medline]
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