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*
National Institute of Allergy and Infectious Diseases, Laboratory of Immunogenetics, National Institutes of Health, Rockville, MD 20852; and
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, the study of the influence of the CD19/CD21 complex on B cell activation has focused primarily on the effect of the CD19/CD21 complex on BCR signaling. However, the BCR plays a second essential role in the response to T cell-dependent Ags, namely to transport bound Ag to the intracellular compartments in which peptide/MHC class II complexes are assembled (reviewed in Ref. 13). Indeed, Ag-specific B cells process and present Ags to Th cells at greatly reduced concentrations of Ag as compared with those required for nonspecific B cells, suggesting that in vivo where Ag concentrations may be limiting, BCR-mediated Ag processing is essential. Current evidence indicates that the Ag targeting and signaling functions of the BCR are interrelated, and that signaling through the BCR influences and may be necessary for the correct targeting of Ag to the class II peptide-loading compartment (14). BCR-mediated Ag processing is enhanced by BCR cross-linking (15, 16), and BCR, which are signaling deficient, are impaired in their ability to transport Ag for processing (17). Thus, the CD19/CD21 complex has the potential to influence BCR-mediated Ag processing by enhancing BCR signaling. In addition, the CD19/CD21 complex is an Ag-binding receptor for complement-tagged Ags, and as such has the potential to augment B cell Ag processing by independently targeting complement-tagged Ags for processing. The phenotype of CD19- and CD21-deficient mice, namely, reduced responses to T cell-dependent but not T cell-independent Ags (8, 9), may be attributable in part to a requirement for a role for the CD19/CD21 complex in B cell Ag processing. Indeed, Boackle et al. (18, 19) recently provided evidence that immune complexes were efficiently presented by nonspecific human B cells, indicating that complement-tagged Ags enhance B cell processing, and Baiu et al. (20) showed that Ags covalently coupled to Abs specific for CD21 were more effectively processed as compared with Ag alone. These results indicate that engagement of CD21 augments Ag processing; however, the effects of the CD19/CD21 complex on BCR-mediated processing and the Ag-targeting function of the CD19/CD21 complex remain to be elucidated.
Using the complement-tagged Ag construct HEL-C3d described above, we explore the role of the CD19/CD21 complex in B cell Ag processing in vitro. Evidence is provided that the CD19/CD21 complex significantly augments B cell Ag processing of HEL-C3d. The enhancement of processing of complement-tagged Ags mediated by the CD19/CD21 complex may play an important role in vivo, ensuring efficient B cell responses to T cell-dependent Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male MD4 H chain transgenic (Tg) mice (H-2b) hemizygous for the HyHEL 10 H and L chain genes encoding the anti-HEL-specific BCR were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred to female CBA/J (H-2k) mice. F1 mice (H-2b x H-2k) expressing the HEL-specific BCR transgenes were identified by PCR analysis for the HEL-Ig sequences. The primers (The Jackson Laboratory) used to identify the HEL-specific Ig transgenes in the PCR were: 5'-gCg ACT CCA TCA CCA gCg AT-3'; 5'-CTg gAg CCC TAg CCA Agg AT-3'; and 5'-ACC ACA gAC Cag Cag gCA gCA gA-3' (21). The F1 litters typically segregated in a 1:1 (anti-HEL-Tg:non-Tg) ratio, and the 430-bp Ig band amplified using the above primers was detected only in the HEL-Ig heterozygous offspring (anti-HEL-Tg) of F1 litters.
The mouse B cell lymphoma CH27 (H-2k,
IgM+, Fc
RIIB1-) was
maintained in DMEM supplemented with 15% FCS (15% complete medium
(CM)). The mouse T cell hybrid TPc 9.1 specific for pigeon cytochrome
c (Pcyt) presented by I-Ek-expressing
APC was generated in this laboratory and maintained in 10% CM. The
mouse T cell hybrid 3A9 also maintained in 10% CM is specific for HEL
presented by I-Ak-expressing APC and was kindly
provided by E. Unanue (Washington University, St. Louis, MO). The Sf9
insect cell line was cultured at 25oC in
Grace’s Insect Medium (Life Technologies, Grand Island, NY)
supplemented with 10% FCS and antibiotics. The IL-2-dependent CTLL-2
cell line was maintained in 10% CM supplemented with rat IL-2 and
additional growth factors.
Pcyt was prepared as detailed previously (22). HEL was
purchased from Sigma (St. Louis, MO). The HEL-C3d DNA construct
pCMV.C3d3 that encodes aa 1–129 of HEL fused to three copies encoding
aa 1024–1320 of the C3d region of the complement component C3 (kindly
provided by D. Fearon, University of Cambridge, Cambridge, U.K.) was
cloned into a baculovirus vector and expressed in Sf9 insect cells. The
fusion cassette containing one copy of HEL and three tandemly arranged
copies of C3d was linked to the 
-chain sequence of tubulin at the 3'
end. The HEL-C3d protein secreted into the culture supernatant was
purified by affinity chromatography on a tubulin-specific Ab (YL1/2)
column using the C-terminal tubulin sequence of the construct. Briefly,
the clarified insect cell supernatant containing HEL-C3d was passed
over a YL1/2-Sepharose 4B column. The column was washed in 50 mM Tris,
pH 7.5, 150 mM NaCl, 0.1 mM EDTA (buffer 1), followed by washes in
buffer 2 consisting of buffer 1 with 0.2% Nonidet P-40. The column was
washed again in buffer 1, and bound HEL-C3d was eluted using 50 mM
triethylamine, pH 11.5, 150 mM NaCl, and 0.1 mM EDTA. Fractions were
pooled, concentrated, and loaded onto a HR-300 (Pharmacia, Piscataway,
NJ) sizing column. Protein fractions eluted from the sizing column were
pooled and dialyzed against PBS, and protein concentration was
determined relative to a HEL standard using the NanoOrange Protein
Quantitation Kit purchased from Molecular Probes (Eugene, OR). HEL-C3d
migrates as a 120-kDa protein in SDS-PAGE, indicating that the three
copies of C3d are expressed in the protein. Phosphorylcholine (PC) was
coupled to HEL (PC-HEL) and to HEL-C3d (PC-HEL-C3d) using
diazo-phosphatidylcholine, as previously described (23).
The number of moles of PC per mole of HEL and HEL-C3d was determined to
be 4.8 ± 0.5 and 5.1 ± 0.4, respectively, based on the PC
excitation maximum and the molar extinction coefficient. Human rC3dg
(24) that binds to mouse CD21 and CD35 was kindly provided
by D. Isenman (University of Toronto, Toronto, Canada).
The rat hybridoma 1D3 producing an IgG2a mAb specific for the extracellular domain of mouse CD19 and the mouse hybridoma 10-2.16 secreting an IgG2b specific for mouse class II Ak molecules were purchased from the American Type Culture Collection (Manassas, VA) and maintained in this laboratory. The rat hybridoma 7G6 producing an IgG2b specific for mouse CD35/CD21 was a generous gift from M. Holers (University of Colorado, Denver, CO). The rat hybridoma C4H3 secreting an IgG2b mAb specific for the HEL peptide 46–61 bound to the I-Ak molecule was generously provided by R. Germain (National Institutes of Health, Bethesda, MD). The rat hybridoma YL1/2 secreting an IgG mAb specific for mouse tubulin was provided by D. Fearon. The mouse hybridoma HyHEL 10 specific for HEL sequences was generously provided by F. Finkelman (University of Cincinnati, Cincinnati, OH). The mAbs produced by the above cell lines were purified from culture supernatants by protein G affinity chromatography. Rat mAbs specific for mouse CD35 (8C12), CD81, B7-1, B7-2, LFA-1, FITC-conjugated secondary goat Abs, as well as the OPT EIA mouse IL-2 ELISA kit were purchased from PharMingen (San Diego, CA).
Flow cytometry
Flow cytometry was conducted on CH27 cells using C4H3 or rat IgG2b for primary labeling and FITC-conjugated goat Abs specific for rat IgG2b for secondary labeling following protocols previously described (25). CH27 and splenic B cells were labeled for the expression of surface molecules, including CD19, CD21, CD81, B7-1, B7-2, and LFA-1 using specific mAbs. To measure the binding of HEL-C3d to CD21, CH27 and non-Tg splenic B cells were incubated with HEL-C3d for 1 h at 4°C, washed, and incubated with the HEL-specific mAb, HyHEL10, or isotype-matched control. Where indicated, cells were preincubated for 1 h at 4°C in the presence of the mAbs 7G6 or 8C12 or C3dg. Cells were washed and incubated with FITC-conjugated goat anti-mouse IgG1 before performing flow cytometry on the cells. Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). The fluorescence intensity measurements for all flow cytometry experiments shown in this study were subjected to the z test for statistical variance, and the values reported were found to be in the 99% confidence interval.
APC assays
CH27 cells (5 x 104), or splenic B cells obtained from anti-HEL-Tg or non-Tg mice (2 x 105) were cocultured with 3A9 or TPc 9.1 cells (5 x 104) in 96-well tissue culture plates for 24 h at 37oC with graded concentrations of Ag in 5% CM. Where indicated, CH27 cells (2 x 106/ml) were incubated with specified concentrations of HEL Ags for 5, 10, or 24 h, washed, fixed in 1% formaldehyde, and serially diluted 1/2 in 5% CM for incubation with 3A9 cells for 24 h at 37oC. The IL-2 content of the culture supernatant was determined by its ability to maintain the growth of the IL-2-dependent CTLL-2 cell line, as measured by the incorporation of [3H]TdR. Alternatively, the IL-2 content of the supernatants was measured using a PharMingen mouse IL-2 ELISA kit that allows measurement of the absorbance of a chemiluminescent substrate at 450 nm. Radioactive counts and 450-nm absorbance measurements were converted into IL-2 units, respectively, based on a standard curve. All APC assays were done in triplicate, and the average value for each time point reported in this work with SE bars shown. Data points were also subjected to the Student t test for statistical significance and determined to be in the 99% confidence interval.
Measurement of CD19/CD21 internalization
The CD19-specific mAb, 1D3 (125I-labeled anti-CD19 (125I-anti-CD19)), and the CD21-specific mAb, 7G6 (125I-labeled anti-CD21 (125I-anti-CD21)), were iodinated using the iodine monochloride method, as described (26), to a sp. act. of 0.5–1 x 107 cpm/µg. Unlabeled mAb competed with 125I-labeled mAb for binding to the cell surface, indicating that iodination did not affect the binding properties of radiolabeled Abs. To measure internalization of the CD19/CD21 complex, CH27 cells (6 x 107) were incubated at 4oC for 1 h with 1 µg 125I-anti-CD19 or 125I-anti-CD21 IgG in the absence or presence of 10 µg/ml of the Ags, HEL-C3d, PC-HEL-C3d, or HEL-C3d + anti-Ig. Cells were washed four times in DMEM containing 10 mg/ml BSA, resuspended at 5 x 106 cells/ml, and incubated at 37°C for varying lengths of time. The radioactivity released from the cells, on the cell surface, and internalized by cells was measured as previously described (27). Briefly, following incubation, cells were pelleted and the supernatants collected. The radioactivity in the supernatant represented the released fraction. The cell pellets were resuspended in a low pH solution (20 mM HCl, 150 mM NaCl) at 4°C for 15 min to strip 125I-labeled Abs from the cell surface, and the radioactivity released was taken as the surface fraction. The radioactivity associated with cells after acid stripping was taken as the internal fraction. Only radioactivity associated with the internal fractions is reported in this work. Data represent the mean of triplicate experiments with error bars shown.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the CD19/CD21 complex plays a role in the
processing of complement-tagged Ags by B cells either by augmenting
BCR-mediated processing and/or by internalizing Ag for processing, the
ability of B cells to process and present Ags that bind to the BCR or
to the CD19/CD21 complex alone or that coligate the CD19/CD21 complex
to the BCR was determined. The ability of the PC-specific B lymphoma,
CH27, to process and present the Ag, HEL, in the forms depicted in Fig. 1
was determined. These included: PC
coupled to HEL (PC-HEL), HEL-C3d, PC coupled to HEL-C3d (PC-HEL-C3d),
and HEL alone. PC-HEL is predicted to cross-link the BCR on CH27 cells;
unmodified HEL-C3d to cross-link the CD19/CD21 complex to itself;
PC-HEL-C3d to coligate the CD19/CD21 complex to the BCR; and HEL,
for which the B cell has no receptor, to enter the CH27 cells by
fluid-phase pinocytosis.
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). CH27 cells were also shown to
specifically bind HEL-C3d, detected using an HEL-specific mAb, HyHEL-10
(Fig. 2). The binding of HEL-C3d to the CH27 cells was blocked by the
CD21-specific mAb 7G6, which binds to the C3d binding site on CD21
(11). Blocking was specific in that the mAb 8C12 that
binds to CD35, but outside the C3d binding site, had no effect. Binding
of HEL-C3d to CH27 cells was also blocked by the monomeric precursor of
C3d, C3dg. Taken together, these results indicate that CH27 cells
specifically bind HEL-C3d through CD21.
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in the presence of the
HEL-specific I-Ak-restricted T cell hybrid, 3A9,
and activation of the T cell hybrid was measured by the secretion of
IL-2 into the culture supernatant (Fig. 3). The concentration of Ag required to
stimulate the T cell hybrid to secrete 10 pg/ml IL-2 and the maximal
IL-2 response achieved given 10 µM HEL were compared (Table I). CH27 cells processed and presented
HEL-C3d 
10-fold more efficiently than HEL, as judged by the
concentration of Ag required to activate the T cell hybrid to secrete
10 pg/ml IL-2 (0.31 vs 3 µM, respectively) (Fig. 3A and
Table I). The maximal IL-2 response achieved for HEL-C3d was also over
3-fold higher than that for HEL. The presentation of PC-HEL that
cross-links the BCR was 150-fold more efficient as compared with HEL
(Fig. 3A and Table I). PC-HEL-C3d that coligated the BCR to
the CD19/CD21 complex was processed 5-fold more efficiently as compared
with PC-HEL that bound to the BCR alone, although the maximal responses
achieved were similar (Fig. 3A). Thus, for CH27 cells, the
most efficient presentation was achieved by PC-HEL-C3d that coligated
the BCR and the CD19/CD21 complex, followed by, in order, PC-HEL that
bound the BCR alone, HEL-C3d that bound the CD19/CD21 complex alone,
and HEL that was taken up by fluid-phase pinocytosis.
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), blocks the binding of
HEL-C3d to CD21, reduced the presentation of HEL-C3d to the levels
achieved by HEL (Fig. 3B). Blocking was specific in that the
CD35-specific mAb 8C12 that has no effect on HEL-C3d binding, had no
effect on presentation. Moreover, monomeric C3dg that blocks HEL-C3d
binding to CD21 also reduced presentation of HEL-C3d to the levels
achieved by HEL. Similarly, both 7G6 and C3dg reduced the presentation
of PC-HEL-C3d to levels of PC-HEL (Fig. 3C). These results
indicate that the efficiency of presentation of HEL-C3d and PC-HEL-C3d
as compared with HEL and PC-HEL, respectively, is attributable to their
specific recognition by CD21.
The same order of efficiency of the presentation of C3d-containing Ags
was observed for splenic B cells (Fig. 3D). Splenic B cells
from MD4 Tg mice expressing the HyHEL 10 H and L chain transgenes
encoding a HEL-specific BCR (anti-HEL-Tg) were analyzed and
compared with B cells from non-Tg littermates for their ability to
process and present the Ags HEL and HEL-C3d to the HEL-specific T cell
hybrid, 3A9. As shown in Fig. 1, for Ag-specific B cells from
anti-HEL-Tg mice, HEL is predicted to ligate the BCR and HEL-C3d to
coligate the BCR to the CD19/CD21 complex. For B cells from non-Tg
littermates, HEL-C3d is predicted to cross-link the CD19/CD21 complex
and HEL, for which the B cells have no receptor, to enter the cell via
fluid-phase pinocytosis. Flow cytometry was used to verify that splenic
B cells express the CD19/CD21 complex components CD19, CD21, and CD81,
and bind HEL-C3d (Fig. 2). CD19, CD21, and CD81 were detected using
specific Abs, and the binding of HEL-C3d to CD21 was detected using an
HEL-specific mAb, HyHEL10.
To assess the ability of B cells from Tg and non-Tg mice to process and
present HEL and HEL-C3d, B cells were incubated with graded
concentrations of the Ags and the HEL-specific,
I-Ak-restricted T cell hybrid, 3A9, and the
activation of the T cell hybrid was measured by the secretion of IL-2
into the culture supernatant (Fig. 3D). B cells from non-Tg
mice processed HEL-C3d more efficiently as compared with HEL, as judged
either by the amount of Ag required to activate the T cell hybrid or
the maximal IL-2 response achieved (Fig. 3D and Table I).
The B cells from non-Tg mice require 3-fold more HEL as compared with
HEL-C3d to induce equivalent secretion of IL-2, and the maximal IL-2
response induced by HEL was less than that induced by HEL-C3d. Thus,
binding complement-tagged Ags to the CD19/CD21 complex stimulated B
cell processing of the Ag.
HEL was processed and presented by B cells from anti-HEL-Tg mice
50-fold more efficiently than by B cells from non-Tg mice (Fig. 3D and Table I). Significantly, HEL-C3d was processed
24-fold more efficiently as compared with HEL by B cells from
anti-HEL-Tg mice (Fig. 3D and Table I). In addition, the
maximal T cell response achieved to HEL-C3d presented by
anti-HEL-Tg B cells was twice that of HEL (Fig. 3D and
Table I). Thus, the C3d-containing Ags enhance processing by
Ag-specific B cell as well as by nonspecific B cells.
Thus, the order of efficiency of the processing of the Ags was the same for CH27 cells and normal splenic B cells. Ags that coligate the BCR and the CD19/CD21 complex were most efficiently presented, followed, in order, by Ags that: cross-link the BCR; cross-link the CD19/CD21 complex; and enter the cell by fluid-phase pinocytosis.
A comparison of coligation vs independent engagement of the BCR and the CD19/CD21 complex
It was of interest to determine whether the BCR and the CD19/CD21
complex need to be physically coligated for maximal presentation, or if
simultaneous, independent cross-linking of the BCR and the CD19/CD21
complex would be sufficient to achieve the same effect. To do so, CH27
cells were incubated with both PC-HEL and HEL-C3d, and the presentation
compared with that of PC-HEL-C3d or PC-HEL alone. The results showed
that at limiting Ag concentrations, presentation of
PC-HEL plus HEL-C3d was slightly more efficient than that of PC-HEL
alone, showing a 2-fold difference in the amount of Ag required to
activate the T cell hybrid to secrete 10 pg/ml IL-2 (Fig. 4A, Table I). However,
coligation of the BCR and the CD19/CD21 complex by
PC-HEL-C3d was most efficient, requiring one-eighth the Ag
concentration as compared with PC-HEL plus HEL-C3d. Thus, the
independent ligation of the BCR and the CD19/CD21 complex was not as
effective as the coligation of the BCR and the CD19/CD21 complex in
augmenting Ag processing at low Ag concentrations.
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To determine whether the enhancement of processing observed for
C3d-containing Ags is due to an effect of the CD19/CD21 complex
signaling on B cell Ag processing, CH27 cells were tested for the
ability to process and present the Ag, Pcyt, that enters CH27 cells by
fluid-phase pinocytosis, in the presence of PC-HEL, HEL-C3d, or
PC-HEL-C3d (Fig. 4B). CH27 cells treated with HEL-C3d
processed Pcyt slightly better than untreated cells. Cross-linking the
BCR using PC-HEL also augmented the processing of Pcyt, reducing the
amount of Ag required by untreated cells to achieve the same level of
activation (Fig. 4B). The coligation of the BCR and the
CD19/CD21 complex further increased the efficiency of processing,
requiring less Ag as compared with cells treated with PC-HEL or HEL-C3d
to achieve the same response. Thus, ligand binding to either the
CD19/CD21 complex or the BCR independently enhanced processing of Ag
taken up by fluid-phase pinocytosis and coligation of the CD19/CD21
complex and the BCR further reduced the amount of Ag required for T
cell activation.
The CD19/CD21 complex fails to target Ag intracellularly
To determine whether the CD19/CD21 complex, in addition to
signaling, directly targeted bound Ag for intracellular processing, the
internalization of the CD19/CD21 complex following binding of
C3d-tagged Ags was analyzed. CH27 cells were incubated at 4°C with
125I-anti-CD19 to label the CD19/CD21 complex
in the presence or absence of HEL-C3d to cross-link CD19/CD21, washed,
and warmed to 37°C for varying lengths of time. At the end of each
time point, the radioactivity in the supernatant was measured and taken
as the released fraction. The cells were treated with acid to remove
cell surface-bound 125I-anti-CD19. The
radioactivity in the acid wash was taken as the surface fraction, and
the radioactivity associated with the cells after acid stripping was
taken as the internal fraction. Only the internal fractions are shown
(Fig. 5). Over the 60-min course of the
experiment, there was negligible internalization of the
125I-anti-CD19 in untreated cells or in cells
treated with HEL-C3d to cross-link the CD19/CD21 complex (Fig. 5A). There was a decrease of 10% of
125I-anti-CD19 from the internalized fraction
over the 60-min incubation, which was accounted for by release of the
125I-anti-CD19 from the cell surface into the
supernatant (data not shown). In control experiments, CH27 cells
labeled with 125I-labeled Fab anti-Ig
internalized 30–35% of the label following BCR cross-linking by
PC-HEL (data not shown). The internalization of CD21 was also followed
using 125I-anti-CD21 (Fig. 5B),
and the results showed that CD21 was not internalized on CH27 cells
even when cross-linked by the addition of HEL-C3d. Moreover, coligating
the CD19/CD21 complex and the BCR had no effect on the internalization
of CD21 on CH27 cells (Fig. 5B). Similarly, CD19 was not
internalized on splenic B cells from non-Tg (Fig. 5C) or
anti-HEL-Tg (Fig. 5D) mice upon cross-linking the
CD19/CD21 complex alone, coligating the CD19/CD21 complex and the BCR,
or independently ligating the BCR and the CD19/CD21 complex. These
results indicate that the CD19/CD21 complex does not have the potential
to independently target complement-tagged Ag for processing. Thus, the
CD19/CD21 complex does not appear to be an Ag-targeting receptor, but
rather primarily influences Ag processing through signaling.
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Earlier studies showed that the targeting of Ag for processing was
accelerated by BCR cross-linking (27, 28). Consequently,
it was of interest to determine the time course of Ag processing and
presentation for the Ags analyzed above. To do so, concentrations of
Ags were chosen that stimulated equivalent T cell responses at 24
h (Fig. 3). Thus, graded numbers of CH27 cells were incubated with HEL
(4 µM), HEL-C3d (0.4 µM), PC-HEL (0.02 µM), or PC-HEL-C3d (0.008
µM) for 5, 10, or 24 h. At the end of each time point, the cells
were washed and fixed, and their ability to stimulate a HEL-specific T
cell hybrid was measured. As predicted, by 24 h all Ags were
presented equivalently (Fig. 6). However,
maximal presentation was reached earliest, at 5 h, by cells that
processed PC-HEL-C3d, coligating the BCR to the CD19/CD21 complex (Fig. 6). PC-HEL was presented significantly earlier than HEL-C3d or HEL, and
HEL-C3d was processed more rapidly than HEL, as shown after 5 and
10 h of incubation (Fig. 6). Thus, the efficiency of processing
and presentation of the various HEL-containing Ags was reflected both
in the absolute concentration of Ag required for maximal T cell
response as well as in the time required for presentation.
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Signaling through the BCR and the CD19/CD21 complex initiates a
variety of activation events in the B cell that have the potential to
influence Ag presentation, including increased cell surface expression
of the adhesion molecules, LFA-1 and ICAM-1 (29), and
costimulatory molecules, B7-1 and B7-2 (30). Although T
cell hybrids are, in general, insensitive to the costimulatory and
adhesion properties of APCs, it was of interest to determine whether
the observed enhancement of Ag presentation by the ligation of the
CD19/CD21 complex was the result of an absolute increase in the number
of antigenic peptide/class II complexes produced. To do so, the mAb
C4H3, specific for complexes of the HEL peptide, residues 46–61, bound
to I-Ak (31), was used to quantify
by flow cytometry the number of HEL peptide/I-Ak
complexes expressed on the surfaces of CH27 cells that had processed
either HEL, PC-HEL, or PC-HEL-C3d at the concentrations specified above
to yield equivalent presentation at 24 h. The ability of the CH27
cells to present these Ags directly correlated with the number of
HEL/I-Ak complexes produced. The CH27 cells
showed equivalent staining with the C4H3 mAb when presentation was
equivalent at 24 h of incubation with either HEL, PC-HEL, HEL-C3d,
or PC-HEL-C3d (Fig. 7). The histograms
showing staining of HEL/I-Ak complexes 24 h
after incubation with the different HEL Ags are shown (Fig. 7A) as well as a summary of the mean fluorescence
intensities (Fig. 7B). The staining of cells that did not
process HEL was equivalent to the background staining using an
isotype-matched control mAb. Incubation with the various Ags had no
effect on the overall expression of I-Ak
molecules, as measured by flow cytometry using an
I-Ak-specific mAb, 10-2.16 (data not shown).
Thus, the ability to activate the T cell hybrid correlated with the
number of HEL/I-Ak complexes expressed on the
cell surface. Maximal numbers of HEL/I-Ak
complexes were achieved the earliest for cells processing PC-HEL-C3d,
followed by PC-HEL, HEL-C3d, and HEL, respectively (Fig. 7B). Thus, the enhancement in Ag presentation observed for
Ags that engage the BCR and/or the CD19/CD21 complex was accounted for
by an increase in the absolute number of HEL/I-Ak
complexes assembled and presented on the surface.
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). However, inclusion of
mAbs specific for B7-1, B7-2, or LFA-1 had little effect on the
processing and presentation of HEL-C3d to the HEL-specific T cell
hybrid by either splenic B cells or CH27 cells at the Ab concentrations
tested (Fig. 8). Thus, these costimulatory and adhesion molecules did
not appear to contribute significantly to the enhancement of
presentation of the T cell hybrid studied in this work, although we
would not rule out a contribution of costimulatory molecules in other
systems.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we use a rC3d-tagged Ag, HEL-C3d, that has proven to be highly efficacious as an immunogen in vivo and provide evidence that the CD19/CD21 complex functions to augment B cell Ag processing and presentation. The CD19/CD21 complex has the potential to influence Ag processing directly through its signaling function or indirectly through its effect on BCR signaling. Alternatively, the CD19/CD21 complex binds complement-tagged Ags, and consequently has the potential to target Ags for processing, thus augmenting B cell Ag processing. The evidence presented in this work rules out an Ag-targeting function for the CD19/CD21 complex, and indicates that signaling via the CD19/CD21 complex augments both the processing of BCR-bound Ag by augmenting BCR signaling as well as the processing of Ag taken up by fluid-phase pinocytosis. The effects of the CD19/CD21 complex on Ag processing did not require that the complex be cocross-linked to the BCR. Cross-linking the CD19/CD21 complex to itself by the binding of complement-tagged Ags enhanced the processing of Ags entering the cell by fluid-phase pinocytosis. These results suggest that complement-tagged Ags may have an adjuvant-like effect on Ag processing for B cells binding unmodified Ags.
The molecular mechanisms by which signaling via the CD19/CD21 complex influences Ag processing remain to be elucidated and will most likely await a more complete description of the mechanisms by which the BCR signals and targets Ag for processing. Recent studies have elucidated more clearly the earliest steps in the BCR-mediated Ag processing pathway initiated by the binding of Ag to the BCR. These results indicate that cholesterol- and sphingolipid-rich membrane microdomains or lipid rafts play a central role in both BCR signaling and Ag targeting (34). Thus, in resting B cells, the BCR is excluded from rafts that concentrate the Src family kinase, Lyn. Following cross-linking, the BCR rapidly translocates into rafts in which the BCR and Lyn become phosphorylated and signaling is initiated. The BCR is subsequently transported from the rafts to the intracellular compartment, where peptide/class II complexes are assembled. We recently showed that the CD19/CD21 complex is also excluded from lipid rafts in resting cells, but upon cross-linking or coligation using the Ags described in this work, the CD19/CD21 complex translocates to the rafts and initiates signaling from rafts. The CD19/CD21 when present in the rafts prolongs BCR signaling from the rafts (6). Thus, the effect of the CD19/CD21 complex on B cell Ag processing may be on events initiated from within the lipid rafts. The class II peptide-loading compartment itself appears to be a target of BCR signal cascades. Indeed, BCR signaling has been shown to result in biochemical changes associated with the subcellular compartments in which peptide/class II complexes are assembled, including changes in the phosphoprotein patterns and low m.w. GTPases (35), and to result in morphological changes in the class II-containing compartments as a result of membrane fusion events (36). How the CD19/CD21 complex influences these BCR-induced events remains to be determined.
In summary, the results presented in this work indicate that the CD19/CD21 complex in addition to augmenting BCR signaling enhances B cell Ag processing to Th cells. The complement-containing Ag complex, HEL-C3d, evaluated in this work has been shown to function as a potent immunogen in vivo in the absence of adjuvants (5), and to prolong BCR signaling from within membrane microdomains in vitro (6). The results presented in this study suggest that the augmented presentation of HEL-C3d may contribute to its efficacy as an immunogen in vivo.
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2 Abbreviations used in this paper: BCR, B cell Ag receptor; CM, complete medium; HEL, hen egg lysozyme; 125I-anti-CD19, 125I-labeled anti-CD19; 125I-anti-CD21, 125I-labeled anti-CD21; PC, phosphorylcholine; Pcyt, pigeon cytochrome c; Tg, transgenic.
Received for publication August 1, 2000. Accepted for publication April 25, 2001.
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