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The Journal of Immunology, 2008, 181, 948 -953
Copyright © 2008 by The American Association of Immunologists, Inc.
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Transfusion of IgG-Opsonized Foreign Red Blood Cells Mediates Reduction of Antigen-Specific B Cell Priming in a Murine Model1

Davor Brinc*,{dagger}, Hoang Le-Tien*,{dagger}, Andrew R. Crow*,{dagger}, Vinayakumar Siragam*,{dagger}, John Freedman{dagger},{ddagger} and Alan H. Lazarus2,*,{dagger},{ddagger}

* Canadian Blood Services, {dagger} Department of Laboratory Medicine of St. Michael’s Hospital, Keenan Research Centre in the Li Ka Shing Knowledge Institute of St. Michael’s Hospital, Toronto, and {ddagger} Department of Medicine, University of Toronto, and the Toronto Platelet Immunobiology Group, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hemolytic disease of the fetus and newborn can be effectively prevented by administration of anti-D to the mother. The administered IgG results in the attenuation of RBC-specific Ab production, a process termed Ab-mediated immune suppression (AMIS). Because in animal models of AMIS no major effect on T cell priming occurs, we hypothesized that the effect of the IgG on the immune system under AMIS conditions may involve a deficiency in B cell priming. We therefore challenged mice with either untreated RBCs or IgG-opsonized RBCs (AMIS) and assessed B cell priming. B cells from mice transfused with untreated RBCs, but not from mice treated under AMIS conditions, were primed as assessed by their ability to function as Ag-specific APCs to appropriate T cells. To our knowledge, this is the first report demonstrating that AMIS inhibits the appearance of Ag-primed RBC-specific B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The routine administration of anti-D for the prevention of the hemolytic disease of the fetus and newborn to rhesus D-negative mothers pregnant with a D-positive child is one of the most powerful applications of Ab-based immunotherapy for a human disease (1). The suppressive effect of IgG on the Ab response in the recipient has been termed Ab-mediated immune suppression (AMIS)3 (2). The effectiveness of anti-D commonly exceeds that of the vaccine preparations, such as measles-mumps-rubella or diphtheria-tetanus toxoids-pertussis vaccines (3, 4). The inhibitory AMIS effect is also observed with some vaccines; e.g., the Ab response in vaccinated infants may be down-modulated by the presence of maternal vaccine-specific IgG Ab that crosses the placenta during pregnancy (5). Although infrequent, lower Ab responses can be observed upon revaccination compared with the response following initial vaccination (6), as there is a period after first Ag challenge during which subsequent boosting may lead to poor immune responses (7); this has been considered to be due to pre-existing IgG binding to the re-introduced Ag, mediating an AMIS effect.

The humoral immune response involves a series of events leading to Ab production (8). Ag uptake, presentation, and maturation of innate APCs, particularly dendritic cells (9), leads to recruitment, activation, and clonal expansion of Ag-specific T cells. Concurrently, B cell activation by the Ag occurs and leads to Ag internalization, followed by B cell Ag processing and relocation of Ag-activated B cells to the T/B border (8). The interaction between the primed effector Th cells and Ag-activated B cells with processed Ag is followed by a branching of the B cell activation pathway: one subset of B cells undergoes clonal expansion and differentiates into short-lived Ab-secreting cells (ASC), whereas the other subset begins the germinal center reaction involving clonal expansion, somatic hypermutation, and affinity-based maturation, resulting in differentiation of long-lived plasma cells and memory B cells (8, 10, 11, 12, 13, 14, 15, 16). In contrast to these events under normal conditions (see Fig. 1, boxes 1–7), under AMIS conditions, serum IgM and IgG Abs (17), as well as Ag-specific ASC (18, 19) are decreased (see Fig. 1, boxes 6 and 7).


Figure 1
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  		FIGURE 1. AMIS and the humoral immune response to RBCs. Under normal conditions, B cells engage an Ag (e.g., RBC) and become activated (box 2), they then migrate toward T cell areas (box 1), followed by B cell-T cell interaction. These events lead to the appearance of primed B cells which expand in number (box 3) and differentiate into ASC (box 7) or germinal center (GC) B cells (box 4), giving rise to IgM and IgG (boxes 6 and 7), respectively, as well as memory B cells (box 5). Previously known AMIS defects are depicted in boxes 6 and 7, whereas normal immune functions have been observed in box 1. This study tests the AMIS effects on the appearance of primed B cells (box 3).

 
In murine models of AMIS, it is clear that T cells are primed against SRBCs following transfusion of IgG-opsonized SRBCs to mice (17, 18, 19), indicating that sufficient Ag processing and presentation to T cells likely occurs under AMIS conditions (see Fig. 1, box 1). APCs exposed to IgG-opsonized SRBC in vitro and transfused into recipient mice can also mediate an enhanced Ab response (17), again indicating the lack of a measurable suppressive IgG effect on innate APCs.

These results collectively suggest that the AMIS alteration may be occurring upstream of ASC differentiation (see Fig. 1, boxes 6 and 7) and possibly upstream of the development of germinal centers (see Fig. 1, box 4). The appearance of Ag-primed B cells (see Fig. 1, box 3) is an event upstream of a germinal center formation, isotype class switching, somatic hypermutation, and differentiation of ASCs (see Fig. 1, boxes 4–7), yet downstream of MHC class II-restricted B-T cell interaction (see Fig. 1, box 2). We investigated the effect of AMIS on B cell priming against foreign RBCs using a standard model in which mice are transfused with SRBCs vs SRBCs opsonized with IgG (20, 21, 22, 23). Because it is difficult to detect low frequency Ag-specific T cells and B cells directly, we used an in vitro model of B cell Ag presentation to T cells to assess the degree of B cell priming (see Fig. 1, box 3) under normal vs AMIS conditions (24, 25). The appearance of primed B cells was markedly reduced in mice immunized under AMIS conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and reagents

C57BL/6 mice (females, 6 to 8 wk of age) were purchased from The Jackson Laboratory. SRBCs were supplied in Alseviers solution (Colorado Serum). SRBCs were washed three times in PBS (pH 7.22) before use. IgG anti-SRBC was purified from sera from mice hyperimmunized against SRBCs using protein G-agarose column chromatography (Pierce). Con A and LPS were purchased from Sigma-Aldrich. Abs used in flow cytometry: FITC-conjugated anti-mouse CD3 (Caltag Laboratories), FITC-conjugated anti-mouse CD4 (Caltag Laboratories), PE-conjugated anti-mouse CD19 (1D3; BD Biosciences), PE-conjugated anti-mouse CD8{alpha} (Ly-2) (53-6.7; BD Biosciences), PerCP-conjugated anti-mouse CD45R/B220 (RA3–6B2; BD Biosciences), FITC-conjugated F(ab')2 goat anti-mouse IgG Fc{gamma}-chain-specific (Jackson ImmunoResearch Laboratories), and PE-conjugated goat F(ab')2 anti-mouse IgM (Caltag Laboratories).

Immunizations

Mice were challenged i.v. with 107 washed SRBCs in PBS (pH 7.22) or with 107 SRBCs preincubated for 1 h at 37°C with 10 µg of IgG anti-SRBC, without further washing, and both the SRBCs and IgG were injected (19).

Detection of the SRBC-specific Ab response

To detect the presence of anti-SRBC IgM and IgG from mouse serum, SRBCs (5 x 107/ml in PBS) were incubated with serum (1/100 dilution) from appropriate mice for 1 h at 22°C, washed, and resuspended in 8 µg/ml FITC-conjugated goat F(ab')2 anti-mouse IgG or 2 µg/ml PE-conjugated goat F(ab')2 anti-mouse IgM. SRBCs were acquired using a FACScan flow cytometer (BD Biosciences).

Cell purifications

To prepare B cell-depleted spleen cells, B cells were removed by a positive selection strategy using a Dynabeads mouse pan B (B220) cell kit (Dynal Biotec) or CD19 Microbeads (Miltenyi Biotec) according to the manufacturer’s directions. In brief, spleen cells were first depleted of RBCs using ACK buffer (ammonium chloride/potassium lysing buffer: 0.15 M ammonium chloride, 0.1 mM sodium EDTA, 1.0 mM potassium carbonate (pH 7.22)) and resuspended at 9 x 106/ml in PBS containing 1% BSA for B220+ cell depletion or at 108/mL cells in PBS, 0.5% BSA, 2 mM EDTA for CD19+ cell depletion. Spleen cells were then incubated for 30 min at 4°C with B220 Dynabeads or for 15 min with CD19 Microbeads. Cells binding B220 Dynabeads were magnetically removed. For CD19+ cell depletion, cells were first washed and up to 108 cells were resuspended in 500 µl of PBS, 0.5% BSA, 2 mM EDTA, and added to LS column (Miltenyi Biotec) and placed in the MACS Separator (Miltenyi Biotec). The column was washed three times and effluent (i.e., depleted cells) was collected. Spleen cells depleted of B cells were washed and resuspended in complete RPMI 1640 (cRPMI; RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 80 µg/ml streptomycin sulfate, 0.2 µg/ml amphotericin B, 80 U/ml penicillin G, and 1.6 mM L-glutamine) before use. To confirm B cell depletion, spleen cells were incubated for 15 min with anti-CD16/32 (2.4G2) and then for 30 min with FITC-conjugated anti-mouse CD3 (7 µg/ml) and PE-conjugated anti-mouse CD19 (3 µg/ml) or PerCP-conjugated anti-mouse B220 (6 µg/ml), washed once in PBS at 22°C, resuspended in 500 µl of PBS, acquired by a FACScan flow cytometer, and analyzed using CellQuest software (data not shown).

Purified T cell or B cell populations were prepared by a negative selection strategy from the spleen by magnetic separation using the T cell or B cell negative selection kit, respectively (StemCell Technologies), according to the manufacturer’s directions. In brief, single cell suspensions of spleen cells were prepared at 108 nucleated cells/ml in Ca2+- and Mg2+-free PBS containing 2% heat-inactivated FCS and 5% normal rat serum. Spleen cells were then incubated for 15 min at 4°C with the T cell negative selection mixture (containing Abs to CD11b, CD19, CD45R, CD49b, and TER 119) or B cell negative selection mixture (containing Abs to CD4, CD8, CD11b, Ly-6G(Gr-1), CD43, CD49b, CD16/32, and TER 119), followed by the biotin selection mixture and magnetic nanoparticles. To test the purity of T and B cells, cells were incubated for 15 min with anti-CD16/32 (2.4G2) and then for 30 min with FITC-conjugated anti-mouse CD3 (7 µg/ml) and PE-conjugated anti-mouse CD19 (3 µg/ml), washed once in PBS at 22°C, resuspended in 500 µl of PBS, and analyzed by flow cytometry (data not shown).

Proliferation assay

Spleen cells (5 x 106/ml) or spleen cells depleted of B cells (2.5 x 106/ml) were cultured with 5 x 106/ml SRBC in 96-well flat-bottom plates in 200 µl of cRPMI. Spleen cells were also cultured with Con A (1 µg/ml) or LPS (10 µg/ml). In addition, spleen cells depleted of B cells (2.5 x 106/ml) were cultured with B cells (2.5 x 106/ml) and SRBCs (5 x 106/ml). Purified T cells (2.5 x 106/ml) were cocultured with B cells (2.5 x 106/ml) and SRBC (5 x 106/ml). All cells were cultured in 96-well flat-bottom plates at 37°C and 5% CO2 for 4 days. Cultured cells were pulsed with 1 µCi of [3H]thymidine (MP Biomedicals) 18–24 h before harvesting, and [3H]thymidine incorporation was measured using a liquid scintillation counter.

CFSE labeling

CFSE (Molecular Probes division of Invitrogen) was dissolved in 5 mM DMSO and added to spleen cells suspended in PBS containing 0.1% BSA for a final concentration of 0.25 µM. Cells were incubated for 10 min at 37°C in CFSE, followed by addition of ice-cold cRPMI and further incubation for 5 min on ice. Spleen cells were then washed three times in cRPMI and cultured (5 x 106/ml) with a 1:1 ratio of SRBCs in 96-well flat-bottom plates in 200 µl of cRPMI. After 4 days, cells were resuspended at 2 x 107/ml in PBS containing 0.1% BSA, incubated for 15 min on ice with anti-CD16/32 (2.4G2), and then for 30 min on ice with FITC-conjugated anti-mouse CD4 (1.4 µg/ml), PE-conjugated anti-mouse CD8{alpha} (2.8 µg/ml), and PerCP-conjugated anti-mouse CD45R/B220 (6 µg/ml), washed once in PBS at 22°C, and resuspended in 500 µl of PBS containing 0.1% BSA. Cells were acquired by a FACScan flow cytometry and data analyzed using CellQuest software. Unstimulated cultured CFSE-labeled cells were used to determine the position of non-dividing cells. To analyze the proliferation of CD4+ or CD8+ cells, CD4+ or CD8+ cells were separately analyzed for a decrease in CFSE fluorescence. To analyze B cell proliferation, CD4+ and CD8+ cells were excluded from the acquired events and the remaining lymphocyte population was gated on B220+ cells.

Statistical analysis

Data were analyzed using the Student’s t test, Mann-Whitney + Dunns, or one-way ANOVA + Bonferroni test as stated. The level of significance was set at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IgG down-regulates the anti-RBC response in mice

To verify that the Ab response against foreign RBCs is attenuated using RBCs opsonized with anti-RBC IgG, mice were transfused with SRBCs or SRBCs opsonized with anti-SRBC IgG (AMIS). Transfusion of SRBCs into mice induced an Ab response against SRBCs (SRBC, Fig. 2). In contrast, transfusion of opsonized SRBCs induced a significantly lower Ab response (SRBC vs AMIS, p < 0.001, Fig. 2). This effect was specific to SRBCs because the Ab response against HRBCs was not impaired (data not shown).


Figure 2
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  		FIGURE 2. IgG prevents the Ab response to SRBCs in vivo. Mice were left untreated (naive), transfused with SRBCs, or with IgG anti-SRBC and SRBCs (AMIS). After 5 days, mice were bled for sera which were analyzed for SRBC-specific IgM by flow cytometry. The x-axis indicates the treatment groups; the y-axis indicates mean fluorescence intensity (MFI). Data represent the mean ± SEM; n = 64 mice/group. ***, p < 0.001 SRBC vs AMIS (ANOVA+Bonferroni).

 
AMIS impairs B cell priming

In vivo, an important early event subsequent to interaction with an Ag involves the priming of B cells, necessary for the ensuing Ab response (24, 25). B cells that have been successfully primed with SRBCs in vivo have the ability to subsequently present fresh SRBCs to SRBC-specific T cells in vitro. To test whether B cells from SRBC- vs AMIS-treated mice were primed, we incubated the isolated B cells with fresh SRBCs plus SRBC-specific T cells in vitro and measured the resulting cellular proliferation. B cells derived from mice primed with SRBCs elicited a significant T cell response using T cells from SRBC-primed mice (Fig. 3B). In contrast, B cells from AMIS-treated mice could not stimulate SRBC-specific T cell responses (SRBC vs AMIS, p < 0.01, Fig. 3B). Control cultures did not respond (Fig. 3, A and B, and data not shown). Thus, AMIS appears to inhibit the appearance of primed functional Ag-specific B cells.


Figure 3
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  		FIGURE 3. Down-regulation of B cell priming following transfusion of IgG-opsonized RBCs. Mice were left untreated (naive), transfused with SRBCs (SRBC), or with IgG anti-SRBC and SRBCs (AMIS). After 5 days, T cells from naive (A) or SRBC-primed mice (B) were cocultured with B cells from naive, SRBC-immunized or AMIS-treated mice, as indicated in the graph, in the presence of SRBC as Ag. No response was detected in wells without SRBCs added (not shown). B cell-depleted spleen cells from naive (C) or day 5 SRBC-primed mice (D) were also incubated in vitro with SRBCs or with SRBCs + B cells from naive (naive-B), SRBC-primed (SRBC-B), or AMIS-treated (AMIS-B) mice, as indicated. SRBCs were added as Ag as indicated. Cell proliferation was measured by [3H]thymidine incorporation. The x-axis represents the treatment groups (A and B) or the culture conditions in vitro (C and D); the y-axis indicates cpm. Data represent the mean ± SEM; n = 8 (A), n = 11 (B), n = 3 (C and D) mice/data point from 11 (A and B) or 3 (C and D) independent experiments. **, p < 0.01; ***, p < 0.001 SRBC vs AMIS ANOVA+Bonferroni (A), Mann-Whitney+Dunns (B–D).

 
To confirm that AMIS conditions impair B cell priming and their ability to drive proliferation with SRBC-specific T cells, B cells from the SRBC-primed vs AMIS mice were added back to B cell-depleted spleen cells from SRBC-immunized mice. B cells from SRBC-primed mice, but not those from AMIS mice, stimulated an increased cell proliferative response (Fig. 3D), confirming that B cells from AMIS mice lack Ag-primed B cells.

Primed B cells are the major APCs for T cell-dependent responses in splenic bulk culture

The spleen is the major secondary lymphoid organ for the induction of the immune response against i.v. transfused SRBCs (26). Although B cells have been observed to be major APCs using cells from Ag-primed mice (24, 25), other APCs may also contribute to the SRBC-specific T cell proliferative responses in vitro. To more specifically address the role of B cells vs innate APCs in stimulating SRBC-specific T cells, splenic cells vs B cell-depleted splenic cells from SRBC-primed mice were cultured with SRBCs in vitro. When cells from SRBC-primed mice were used, the splenic cell proliferative response to SRBCs in vitro was clearly detected (Fig. 4, A and B, column 2). When B cell-depleted spleen cells were used, this response was attenuated by ~80% (Fig. 4, A and B, column 4 vs column 2). The reduced response of B cell-depleted spleen cells was observed following depletion of either B220+ (Fig. 4A) or CD19+ cells (Fig. 4B). This suggested that B cells were the major, but not the only, APC in splenic bulk cultures. As expected, spleen cell populations derived from naive mice failed to proliferate in response to SRBCs in vitro (data not shown).


Figure 4
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  		FIGURE 4. The proliferative response to SRBCs in vitro is largely B cell dependent. Mice were treated as described in Fig. 2. After 5 days, spleen cells (unfractionated spleen cells) or spleen cells depleted of B220+ cells (A) or CD19+ (B) (B cell-depleted spleen cells) from SRBC-primed mice were incubated with medium only (Nil) or SRBCs (SRBC). Cells were analyzed for proliferation by [3H]thymidine incorporation. The x-axis represents the treatment groups and the type of cells added in vitro; the y-axis indicates [3H]thymidine radioactivity measured in cpm. Data represent the mean ± SEM; n = 6 mice/data point from three independent experiments.

 
AMIS impairs B cell priming in splenic bulk culture

Because the above data suggest that innate APC function can contribute to a small but significant T cell response under the above conditions, we further tested SRBC-specific proliferative responses using bulk cultures, where these other APCs are present at their naturally abundant levels. Spleen cells were removed from mice immunized with SRBCs under normal or AMIS conditions and rechallenged with SRBCs in vitro. Proliferative responses from splenic cells derived from AMIS mice were significantly reduced relative to the response from SRBC-challenged mice (SRBC vs AMIS, p < 0.001, Fig. 5A), confirming that the observation made with purified cells also holds true with unfractionated cells. Both B cells and T cells were able to respond to Con A and LPS under these conditions, indicating that the global B and T cell populations themselves and the APC function required to drive the Con A responses (27, 28, 29) are present (Fig. 5B).


Figure 5
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  		FIGURE 5. Spleen cells from AMIS mice do not respond to SRBCs in vitro. Mice were treated as described in Fig. 2. After 5 days, spleen cells were isolated from mice and cultured with SRBCs (A) or Con A and LPS (B). Cells were analyzed for proliferation by [3H]thymidine incorporation. The x-axis indicates the treatment groups; the y-axis indicates [3H]thymidine radioactivity measured in cpm. Data are expressed as mean ± SEM; n = 40 (A), n = 3 (B) mice/group. ***, p < 0.001 SRBC vs AMIS (ANOVA+Bonferroni).

 
To determine the lymphocyte subset from AMIS mice that proliferates in response to SRBCs in vitro, we labeled spleen cells with CFSE and analyzed the proliferation of CD4+ T cells, CD8+ T cells, and B220+ B cells in the presence and absence of SRBCs. The SRBC-induced proliferation of these cells was assessed by the diminished fluorescence of CFSE upon cell division (Fig. 6A) (30). The proliferative response of both CD4+ T cells and B cells from AMIS mice was significantly reduced (SRBC vs AMIS, p < 0.05, Fig. 6, B and D). The reduction in proliferation of CD8+ T cells (Fig. 6, A and C) was not statistically significant (p = 0.20). As expected, T cells and B cells from naive mice failed to respond to SRBCs in vitro (Fig. 6, A–D).


Figure 6
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  		FIGURE 6. The analysis of T and B cell proliferation by flow cytometry. Mice were treated as described in Fig. 2. CFSE-labeled spleen cells stimulated with SRBCs in vitro were stained with PE-conjugated rat anti-mouse CD4, PerCP-conjugated rat anti-mouse CD45R/B220, and PE-conjugated rat anti-mouse CD8{alpha}, and analyzed by flow cytometry. A, Dot plots showing the analysis of cells derived from naive, SRBC, or AMIS-treated mice and incubated with SRBCs in vitro. Markers were set based on the dot plot obtained from naive spleen cells incubated with medium. A decrease in CFSE fluorescence indicates the cell proliferation (i.e., compare cells in the top left vs top right quadrant). Dot plot for B220+ cells has been additionally gated to exclude all CD4+ and CD8+ cells. Representative data is shown. Bar graphs indicate the percentage of proliferating CD4+ (B), CD8+ (C), or B220+ (D) cells. The x-axis indicates the treatment groups; the y-axis indicates the percentage of dividing CD4+, CD8+, and B220+ cells. Data represent the mean ± SEM; n = 4 mice/data point from four independent experiments. *, p < 0.05 SRBC vs AMIS (ANOVA+Bonferroni).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The Ab response against RBCs as well as other particulate Ags can be effectively prevented by coadministration of Ag-specific IgG (23, 31, 32). This phenomenon has also been observed with vaccines (5). It is well known that immunization with IgG-opsonized cellular Ags effectively down-regulates the Ab response (23). However, there is an inadequate knowledge about B cell physiology under these conditions. This study represents the first look at the effect of IgG-opsonized RBCs on B cell priming at these early stages.

The ability of B cells from AMIS mice to present SRBCs to SRBC-primed T cells was significantly reduced. Because the APC function of B cells depends on successful priming (24, 25), B cell priming is probably either absent, down-regulated, or otherwise changed under AMIS conditions. We and others have observed that immunization at early time points after AMIS induction did not lead to suppression, but rather generated a secondary-like Ab response (19, 33, 34). This secondary-like response, a priori, excludes B cell anergy, B cell clonal deletion, or B cell clonal silencing as likely mechanisms for the observed lack of Ag-specific B cell APC function in vitro.

In contrast to the AMIS effect on B cells, AMIS had no significant effect on T cell priming, as previously reported by Kappler et al. (18) as well as ourselves (17). Interestingly, while it is not known whether CD8+ T cells have a role in the immune response against RBCs, we observed that both CD4+ and CD8+ T cells proliferated in response to SRBCs in vitro. It has recently been shown that IgG can inhibit Ab responses against T cell independent type 2 Ags, such as NP-Ficoll, further suggesting that disruption of T cells, per se, is not the mechanism of the AMIS effect under these conditions (35).

Although the AMIS effect was not previously detected at the T cell level (17, 18), we observed decreased Ag-specific proliferative responses of both CD4+ T cells and B220+ B cells in bulk splenic cell cultures from AMIS mice under conditions in which B cells are the preferred APC. We also did not detect a suppressive effect of AMIS B cells on the T cell response to SRBCs stimulated by other innate APCs in vitro. It appears therefore that the decreased proliferative response of Ag-primed CD4+ T cells is probably due to a lack of primed B cells from mice immunized under AMIS conditions. This lack of "properly" primed B cells has important implications for the developing Ab response, because this response can be critically dependent on the ability of B cells to recruit T cell help.

The lack of primed B cells from AMIS mice had a less pronounced effect on CD8+ T cell proliferation; this may occur if other APCs present SRBCs Ags to CD8+ T cells. Alternatively, CD4+ T cells may be indirectly promoting the responses of CD8+ T cells to SRBCs (36), and this effect may be reduced due to the B cell effect on CD4+ T cells.

In contrast to soluble Ags, with or without adjuvants, the B cell response to particulate Ags, such as RBCs, may involve a different method of Ag contact and Ag acquisition. Common to both responses is BCR-induced signaling. However, in the case of particulate Ags, BCR-induced signaling is followed by B cell spreading onto the particulate Ag surface (37, 38, 39, 40, 41). BCR-mediated Ag internalization is thought to promote Ag processing and assembly of MHC-peptide complexes (38, 39). It has also been shown that membrane Ags can be directly acquired following receptor-ligand interaction, possibly leading to the incorporation of membrane patches onto or into the B cells (42, 43). Ag processing and presentation to T cells should then occur. In the case of soluble Ags, accessory cells, such as dendritic cells (44, 45) or macrophages (46), can array Ags on their cell surface, facilitating B cell priming; this may not be necessary with particulate Ags (40, 41).

Under AMIS conditions, reduced B cell priming may be caused by disruption of any of these processes. It may be speculated that IgG that is bound to the surface of the particulate Ag may limit the number of Ags recognized by the BCR and prevent subsequent Ag acquisition by B cells. In support of this, several studies have shown that IgG can sometimes prevent the B cell presentation of specific epitopes to T cells (47, 48, 49, 50, 51, 52, 53, 54).

IgG-opsonized RBCs may also cross-link BCR with the inhibitory Fc{gamma}RIIB on the B cell surface and limit B cell spreading on the Ag surface. However, AMIS can occur independently of Fc{gamma}RIIB (19). Furthermore, reduced B cell priming was also observed in Fc{gamma}RIIB-deficient mice (data not shown), excluding the likelihood that Fc{gamma}RIIB-mediated B cell inhibition is the mechanism of reduced B cell priming.

The AMIS effect is thought to underlie the anti-D effect in hemolytic disease of the newborn prophylaxis (1). Worldwide efforts to replace polyclonal anti-D with monoclonal anti-D Abs have met with limited success; whereas some mAbs prevented the Ab response to the D Ag, others increased the incidence of D-Ag responders (55). Most assays used to evaluate and select mAbs have been based on the ability of these Abs to mediate RBC clearance (55). This study shows that successful AMIS Ab can prevent B cell priming; additional tests that could thus be used to help facilitate monoclonal anti-D design could include detection of primed B cells following monoclonal anti-D administration or testing of the ability of mAbs to prevent the early B cell response to Ags in vitro.

In conclusion, the data demonstrate that B cell priming is probably either absent, down-regulated, or otherwise altered under AMIS conditions. Decreased B cell priming may reflect impaired early B cell activation, including impaired B cell Ag presentation to T cells which is necessary for the recruitment of T cell help allowing a humoral immune response to occur.


   Acknowledgments
 
We thank Dr. J. W. Semple for a critical review of the manuscript, Drs. G. Denomme and S. Song, and S. Ma, N. Wragg, S. Pang, and L. Zhang for assistance and discussion, and the St. Michael’s Research Vivarium staff.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Canadian Blood Services (to A.H.L.) and a Graduate Student Fellowship Award from the Canadian Blood Services (to D.B.). Back

2 Address correspondence and reprint requests to Dr. Alan H. Lazarus, Transfusion Medicine Research, St. Michael’s Hospital, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8. E-mail address: lazarusa{at}smh.toronto.on.ca Back

3 Abbreviations used in this paper: AMIS, Ab-mediated immune suppression; ASC, Ab-secreting cell. Back

Received for publication March 6, 2008. Accepted for publication May 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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