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The Journal of Immunology, 2007, 178: 2328-2335.
Copyright © 2007 by The American Association of Immunologists, Inc.

Requirement for Phosphoinositide 3-Kinase p110{delta} Signaling in B Cell Antigen Receptor-Mediated Antigen Presentation1

Monther M. Al-Alwan*,{dagger}, Klaus Okkenhaug{ddagger}, Bart Vanhaesebroeck§,¶, Joel S. Hayflick|| and Aaron J. Marshall2,*

* Department of Immunology, University of Manitoba, Winnipeg, Canada; {dagger} Tumor Immunology Unit, Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; {ddagger} Laboratory of Lymphocyte Signalling and Development, Molecular Immunology Programme, The Babraham Institute, Cambridge, United Kingdom; § Ludwig Institute for Cancer Research, London, United Kingdom; Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom; and || ICOS, Bothell, WA 98021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The BCR serves to both signal cellular activation and enhance uptake and presentation of Ags by B cells; however, the intracellular signaling mechanisms linking the BCR to Ag presentation functions have been controversial. PI3Ks are critical signaling enzymes controlling many cellular processes, with the p110{delta} isoform playing a critical role in BCR signaling. In this study, we used pharmacological and genetic approaches to evaluate the role of p110{delta} signaling in Ag presentation by primary B lymphocytes. It was found that activation of allogeneic T cells is significantly reduced when B cells are pretreated with global PI3K inhibitors, but was intact when p110{delta} signaling was specifically inactivated. In contrast, inactivation of p110{delta} significantly impaired the ability of B cells to activate T cells in a BCR-mediated Ag uptake and presentation model. Prestimulation of p110{delta}-inactivated B cells with anti-CD40 or LPS could not rescue their BCR-mediated Ag presentation ability to normal levels. p110{delta} signaling was required for efficient presentation of either anti-Ig or protein Ag via a lysozyme-specific BCR. p110{delta}-inactivated B cells were able to internalize Ag normally, and no defects in association of Ag with lysosome-associated membrane protein 1+ late endosomes were observed; however, these cells were less effective in forming polarized conjugates with Ag-specific T cells. Our data demonstrate a role for p110{delta} signaling in B cell Ag presentation function, implicating 3-phosphoinositides and their targets in the latter stages of this process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The humoral immune response relies on the Ag-specific activation of B cells. This process is initiated by triggering the BCRs, which dictate the specificity of response and regulate B cell survival, activation, and differentiation. Cross-linking of the BCRs by Ag triggers a cascade of signaling events initiated by ITAM phosphorylation at the Ig{alpha} and Igbeta subunits of the BCR by Src family kinases (1, 2). BCR-Ag complexes are internalized into early endosomes and subsequently traffic into the late endocytic compartments, where they are degraded into peptides, loaded onto MHC class II, and displayed on the surface of B cells (3). The antigenic peptide-MHC class II complex is then presented to T cells, providing an essential step for B-T cell cognate interaction. Upon this cognate interaction, other factors such as receptors for cytokines and costimulatory molecules provide further bidirectional signaling enhancing the activation of the B cell and T cell (4, 5, 6).

Signaling pathways activated by BCR ligation play roles in BCR internalization, trafficking, and Ag presentation. Activation of Src family kinases, particularly Lyn, can regulate BCR internalization in some systems (7, 8, 9), perhaps via phosphorylation of clathrin H chain (10) and/or the adaptor protein Bam32 (11). Subsequent sorting of internalized BCR-Ag complexes into late endosomes and development of mature MHC class II-enriched compartment (MIIC)3 (12, 13, 14) serve to quantitatively enhance generation of peptide-MHC complexes (15), and may promote expression of peptide-MHC complexes in a functional conformation capable of activating Ag-specific T cells and acting as a signal transduction unit for the B cell (16). Proper BCR-Ag trafficking and formation of the MIIC require receptor-proximal signaling via Ig{alpha}beta, Syk, and B cell linker (17, 18); however, the downstream signaling pathways regulating BCR-Ag trafficking are not well characterized.

One key component of the BCR signaling cascade is activation of PI3Ks, which generate membrane-associated 3-phosphoinositide second messengers (19, 20). Receptor-mediated lymphocyte proliferation and survival are attenuated by pharmacological PI3K inhibitors or genetic inactivation of PI3K components, strongly supporting the role of PI3K signaling in regulating lymphocyte activation (reviewed in Refs. 21, 22, 23). A role for PI3Ks in B cell Ag presentation function was suggested from studies using pharmacological inhibition of PI3K activity in B cell lines (24, 25). However, a major limitation of PI3K inhibitors wortmannin (WM) and LY294002 is the global effect of these drugs on all PI3K classes and isoforms, as well as known off-target effects on other signaling molecules (26, 27, 28, 29). PI3K enzymes comprise several structurally and functionally distinct classes, with class 1A being responsible for most of the signaling downstream of tyrosine kinase-coupled receptors such as BCR-mediated signaling (30). Class 1A PI3Ks are heterodimers composed of a catalytic subunit (p110{alpha}, p110beta, or p110{delta}) and a regulatory subunit (p85{alpha}, p85beta, or p55{gamma}) (reviewed in Ref. 30). The expression of the p110{delta} isoform is restricted to immune cells and has been shown to play critical roles in B cell activation (31, 32, 33). In this study, we have investigated whether p110{delta} plays a role in Ag presentation function of primary B cells using both genetic inactivation of p110{delta} and a p110{delta}-specific inhibitor IC87114 (IC). Our data provide clear evidence for a role of this signaling pathway in BCR-mediated Ag presentation, implicating 3-phosphoinositides and their targets in this important process.


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

The p110{delta}D910A/D910A mice have been described previously (33) and were backcrossed on a C57BL/6 background for nine generations. For BCR-mediated Ag presentation experiments, the p110{delta}D910A/D910A mice were crossed to C57BL6 MHC congenic mice that express I-Ad haplotype (purchased from The Jackson Laboratory). The resulting p110{delta}D910A/WT I-Ad/b progeny were then intercrossed, and p110{delta}D910A/D910A I-Ad/d progeny were identified by PCR genotyping of the p110{delta} locus and FACS staining to identify MHC haplotypes. The age- and sex-matched wild-type (WT) mice were purchased from Charles River Laboratories. All animals were housed at the Central Animal Care Facility (University of Manitoba) in compliance with the guidelines established by the Canadian Council on Animal Care and were given standard rodent chow and water ad libitum.

Cells

B cells were purified by negative selection using the CD43 MicroBeads and MACS columns, using the manufacturer’s procedure (Miltenyi Biotec). This method generated >99% B cell purity, as assessed by FACS analysis using CD19 staining. T cells were purified by negative selection using T cell enrichment columns (R&D Systems). T cell purity was routinely >94%, as assessed by FACS analysis using CD3 staining. The 2R.50 T cell clone was a gift from J. Drake (Albany Medical College, Albany, NY). The 2R.50 T cells express MHC class II (I-Ad)-restricted TCR specific for rabbit IgG and were maintained and used as previously described (34, 35). The LK35.2 B cell transfectant (HyHEL10), which expresses a hen egg lysozyme (HEL)-specific IgM BCR, and 2G7 T cells, which express a MHC class II (I-Ek)-restricted TCR specific for HEL1–18, were gifts from F. Batista (Medical Research Laboratory of Molecular Biology, Cambridge, U.K.) and were maintained and used as previously described (36).

Abs and reagents

PE-labeled anti-CD86, FITC-labeled anti-CD19, and FITC-labeled anti-CD3 were from Cedarlane Laboratories. Rabbit anti-mouse IgM, Cy5 rabbit anti-mouse IgM, and the alkaline phosphatase-conjugated streptavidin were from Jackson ImmunoResearch Laboratories. The CD43 MicroBeads were from Miltenyi Biotec. The purified rat anti-mouse IL-2, biotin rat anti-mouse IL-2, FITC-labeled anti-MHC class II (clone 39-10-8; I-Ad specific), FITC-labeled anti-mouse lysosome-associated membrane protein 1 (LAMP1), and anti-H2M were from BD Pharmingen. Alexa488-labeled phalloidin was from Molecular Probes. The rIL-2 standard was from BioLegend. The p110{delta}-selective inhibitor IC (37, 38) was dissolved in DMSO. WM and LY294002 were purchased from Calbiochem and dissolved in DMSO. HEL was purchased from Sigma-Aldrich.

Mixed leukocyte reaction

B cells were isolated from WT or p110{delta} mutant C57BL6 mice. For some experiments, cells were pretreated for 30 min with various doses of the PI3K inhibitors or 0.5% DMSO (control), followed by extensive washing. The B cells were then treated with 25 µg/ml mitomycin C (Sigma-Aldrich) for 30 min at 37°C. Treated B cells were mixed at different ratios with allogeneic T cells isolated from BALB/c spleens (T cell number kept constant at 2 x 105 per well). Cultures were maintained in 200 µl of complete RPMI 1640 medium (10% FCS, 50 µM 2-ME plus antibiotics) in U-bottom 96-well plates (Nunc) for 3 days. The cells were pulsed with 1 µCi/ml [3H]thymidine (Valeant Pharmaceuticals) in the last 18 h of incubation. T cell proliferation was assessed by harvesting the cells on filtermats (Wallac) using a cell harvester (Tomtec) and measuring the [3H]thymidine uptake in a liquid scintillation counter (Wallac).

BCR-mediated Ag presentation

B cells (105) purified from C57BL/6 I-Ad or p110{delta} mutant I-Ad congenic spleens were cocultured in flat-bottom 96-well plates (Corning Glass) with Ag (rabbit anti-mouse IgM Ab) and 2R.50 T cells (3 x 104/well). Congenic B cells were confirmed to express I-Ad by staining with anti-I-Ad-specific Ab (BD Pharmingen) and failed to activate T cells from BALB/c background (I-Ad), indicating the lack of C57BL/6 I-Ab haplotype on these cells. For inhibitor experiments, B cells were pretreated for 30 min with various doses of the PI3K inhibitors or DMSO (control), followed by extensive washing before mixing with 2R.50 cells and Ag. The coculture and control supernatants were collected after 38–42 h, and levels of IL-2 were assessed by ELISA as a measure of T cell activation. For IL-2 detection, ELISA plates (Corning Glass) were coated with purified rat anti-mouse IL-2 Ab (1 µg/ml) overnight at 4°C, blocked for 2 h at 37°C, and washed, and the coculture supernatants were added along with IL-2 standards. Following overnight incubation at 4°C, the plates were washed, incubated with 1 µg/ml biotin rat anti-mouse IL-2 for 3 h at 37°C, washed, and incubated with alkaline phosphatase-conjugated streptavidin (1:2500) for 45 min at 37°C. Following washing, the phosphatase substrate (Sigma-Aldrich) was added and the absorbance was read. The detection limit for IL-2 was 2 pg/ml. The indicated B:T cell ratio and time of supernatant collection were predetermined to be the optimal conditions for IL-2 production. Detectable levels of IL-2 were generated only in cocultures containing B cells, T cells, and rabbit Ig Ag; no production was observed with omission of Ag, B cells, or T cells, or when rabbit anti-IgM was substituted with goat anti-IgM.

BCR internalization

B cells were incubated with biotin rabbit anti-mouse IgM for 10 min on ice, then either fixed immediately or warmed to 37°C for the indicated time. The cells were then diluted with ice-cold PBS, pelleted, and fixed with 2% paraformaldehyde for 10 at room temperature. The cells were washed and incubated with PE-streptavidin for 15 min to stain biotinylated Ab remaining on the cell surface. Cells were washed and fluorescence was analyzed on a FACSCalibur (BD Immunocytometry Systems).

Confocal microscopy

WT or p110{delta} mutant B cells were incubated with labeled Ag (Cy5 rabbit anti-mouse IgM Ab) and placed on ice for 10 min to allow binding. The cells were then warmed to 37°C for various time points, followed by fixation with 2% paraformaldehyde. The cells were then centrifuged onto glass slides and permeabilized with 0.1% saponin for 10 min at room temperature. The slides were washed with PBS and blocked with 5% goat serum for 30 min before adding the FITC anti-mouse LAMP1 Ab, FITC anti-MHC class II, or Alexa488 phalloidin for 1 h at room temperature. The rat anti-mouse H2M was incubated for overnight at 4°C, washed, and then incubated with Cy3 donkey anti-rat secondary Ab. Slides were washed and mounted in Prolong Gold antifade reagent (Molecular Probes). The colocalization of the Ag and various proteins was then visualized with a x100 objective on an inverted confocal microscope (Ultraview LCI; PerkinElmer Bioscience). Correlation coefficients used to determine the degree of colocalization were calculated using Ultraview image analysis software. For B:T conjugate analyses, purified B cells were incubated with Cy5 rabbit anti-mouse IgM (red) at 37°C for 1 h, mixed with 2R.50 T cells for another hour, fixed with 2% paraformaldehyde, cytospun onto slides, permeabilized, and stained with Alexa488-phalloidin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The p110{delta} signaling is required for BCR-mediated Ag presentation by primary B cells

To determine whether PI3K signaling is required for Ag presentation function in primary B cells, we used a model of BCR-mediated Ag presentation that requires BCR-mediated Ag uptake, Ag processing, and peptide-MHC class II loading on the cell surface. This system uses the 2R.50 T cell clone specific for processed rabbit anti-mouse Ab in the context of I-Ad on B cell surface (34, 35). In this system, T cells produce IL-2 only in the presence of both rabbit anti-mouse IgM Ab and B cells (Fig. 1A). Consistent with previous findings (35), no IL-2 secretion was observed by T cells when mixed with the B cells in the presence of goat anti-mouse IgM Ab (data not shown). Pretreatment of B cells with the PI3K inhibitor WM before their coculture with Ag and T cells abrogates the IL-2 release by the T cells (Fig. 1B). Similar results were observed using LY294002 inhibitor (data not shown). Because the PI3K catalytic subunit p110{delta} contributes a significant proportion of PI3K signaling in B cells (31, 32, 33), we examined the role of this isoform, using the p110{delta}-selective inhibitor IC (37, 38) and B cells from mice carrying an inactivating mutation of p110{delta} (33). Pretreatment of normal B cells with IC partially inhibited their ability to induce IL-2 production in a dose-dependent fashion (Fig. 1B). B cells from p110{delta}-inactivated mice showed a significant reduction in BCR-mediated Ag presentation (Fig. 1C). Together these findings demonstrate that PI3K signaling is critical for BCR-mediated Ag presentation function, and p110{delta} contributes a significant proportion of PI3K signaling in this process.


Figure 1
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  		FIGURE 1. The p110{delta} signaling is required for BCR-mediated Ag presentation by primary B cells. A, Various combinations of purified mouse B cells (B), 2R.50 T cells (T), and 10 µg/ml rabbit anti-IgM Ag (Ag) were cocultured, and the level of IL-2 produced was assessed by ELISA as a measure of T cell activation. Note that IL-2 is only produced in cocultures containing B cells, T cells, and Ag. On the far right, B cells were pretreated with 100 nM WM before coculture. B, B cells were pretreated for 30 min with various doses of WM or IC dissolved in DMSO, or 0.5% DMSO control, and washed thoroughly before coculture with 2R.50 and 10 µg/ml Ag. Inhibitor concentrations used were 2.5, 5, and 10 µM (IC) or 6.25, 25, and 100 nM (WM). ND, Not detectable. C, WT or p110{delta}-inactivated (p110{delta}-D910A) B cells were cocultured with different doses of Ag and a constant number of 2R50 T cells, and the level of IL-2 in supernatants was measured. All results are representative of at least five independent experiments.

 
Effect of preactivation on Ag presentation function of WT and p110{delta}-deficient B cells

Because B cell activation by CD40 ligation or LPS stimulation is known to up-regulate costimulatory molecules and enhance Ag presentation functions, we tested whether preactivation with anti-CD40 or LPS can restore the defect in BCR-mediated Ag presentation by p110{delta}-inactivated B cells. B cells from WT or p110{delta}-inactivated mice were stimulated with LPS or anti-CD40 overnight before mixing with Ag and T cells. This short activation enhanced both WT and p110{delta}-inactivated B cell Ag presentation (compare Fig. 1C with 2A); however, the relative impairment in Ag presentation function seen in p110{delta} mutant cells is maintained after stimulation (Fig. 2A). The basal surface expression of MHC class II and CD86 was comparable on WT and p110{delta}-inactivated B cells and was up-regulated to a similar extent after anti-CD40 or LPS prestimulation (Fig. 2B). WT preactivated B cells also showed dose-dependent inhibition of Ag presentation activity after treatment with p110{delta}-selective inhibitor, despite similar levels of MHC class II and CD86 expression (Fig. 2C). These results indicate that the requirement for p110{delta} signaling in BCR-mediated Ag presentation is not due to inadequate MHC or costimulator expression and cannot be overcome by strong activation of Ag presentation functions.


Figure 2
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  		FIGURE 2. BCR-mediated Ag presentation by p110{delta}-inactivated B cells is not restored to normal levels by prestimulation with anti-CD40 or LPS. A, WT or p110{delta} B cells were stimulated with LPS (1 µg/ml) or anti-CD40 (2.5 µg/ml) for 18 h, washed, and mixed with Ag and 2R.50 T cells. The level of IL-2 in supernatants was measured. B, Freshly purified or prestimulated (18 h) B cells were stained for MHC class II or CD86. The levels of expression were assessed by FACS, and mean fluorescence intensity (MFI) is shown on each histogram. Results are representative of three independent experiments. C, WT B cells were activated with LPS (1 µg/ml) for 18 h, washed, and pretreated with DMSO (control) or various doses of IC. The cells were extensively washed before mixing with various doses of Ag (rabbit anti-mouse IgM) and the 2R.50 cells. The level of IL-2 in supernatants and B cell expression of MHC II or CD86 were measured, as above. Results are representative of two independent experiments.

 
Requirement for p110{delta} signaling in BCR-mediated presentation of Ab vs protein ligands

To determine whether p110{delta} signaling is required for efficient presentation of monovalent protein Ags in addition to polyvalent Ab ligands, we used an HEL-specific cell line model (36). HyHEL10 B cell transfectants were incubated with varying concentrations of HEL Ag or rabbit anti-IgM, together with corresponding Ag-specific T cell lines (2G7 or 2R.50, respectively), and the resulting IL-2 production was measured (Fig. 3A). IL-2 was only generated when B cells, T cells, and Ag are incubated together. The 2G7 cells generally produce more IL-2 than the 2R.50 cells, most likely reflecting inherent characteristics of the T cell lines, rather than differences in presentation of the two types of BCR ligands, because 2G7 cells also produce more IL-2 in response to Con A stimulation (Fig. 3A). When the HyHEL10 cells were pretreated with the p110{delta}-selective inhibitor IC, presentation of both anti-Ig and HEL was inhibited in a dose-dependent manner (Fig. 3, B and C). Similar inhibition was seen using trinitrophenyl-specific A20-HL cells (39) together with trinitrophenyl-OVA Ag and 42-6A T cells (data not shown). Together these results indicate that the requirement for p110{delta} signaling in BCR-mediated presentation applies both to Ab and protein ligands.


Figure 3
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  		FIGURE 3. The p110{delta} signaling is required for BCR-mediated presentation of both anti-Ig and protein Ag. A, Various combinations of HyHEL10 B cells (B), 2R.50 T cells (T), and 10 µg/ml rabbit anti-IgM Ag (Ag) or 2G7 (T) and 100 nM HEL were cocultured, and the level of IL-2 produced was assessed by ELISA as a measure of T cell activation. Note that IL-2 is only produced in cocultures containing B cells, T cells, and Ag, and 2G7 produces substantially more IL-2 compared with 2R.50 when stimulated with Con A. B and C, HyHEL10 B cells were pretreated for 30 min with various doses of IC dissolved in DMSO, or 0.5% DMSO control, and washed thoroughly before coculture with 2R.50 and various doses of rabbit anti-IgM Ag (B) or with 2G7 and various doses of HEL (C). Results are representative of four independent experiments.

 
PI3K signaling, but not the p110{delta} isoform, is important for primary B cell Ag presentation to allogeneic T cells

To determine whether PI3K signaling may be required for B cells to present preformed Ag to T cells, we assessed the ability of B cells to stimulate allogeneic T cells. Purified splenic B cells were pretreated with WM or IC and mixed with allogeneic T cells and proliferation, and IL-2 production was assessed. WM-pretreated B cells were significantly less effective in activating allogeneic T cells when compared with untreated control (Fig. 4A). Similar results were observed using LY294002 (data not shown). In contrast, pretreatment with the p110{delta}-selective inhibitor IC had no effect on the ability of B cells to stimulate proliferation of allogeneic T cells (Fig. 4A). Similarly, B cells from p110{delta} mutant mice were comparable to WT B cells in stimulating allogeneic T cell proliferation (Fig. 4B). Consistent with the T cell proliferation results, pretreatment of B cells with WM significantly inhibited IL-2 release by T cells, whereas no effect was seen when B cells were pretreated with IC (Fig. 4C). The level of IL-2 induced by p110{delta} mutant B cells was also comparable to that induced by WT B cells. Altogether, these results show that whereas PI3K signaling as a whole is important for enhancing the ability of B cells to present preformed Ag, the contribution of p110{delta} signaling in this stage of Ag presentation is dispensable.


Figure 4
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  		FIGURE 4. PI3K signaling is required for B cell APC activity in an MLR, but the p110{delta} isoform is dispensable. A, B cells were pretreated with DMSO (control), WM (100 nM), or IC (10 µM) before coculture with allogeneic T cells at a 1:1 ratio. T cell proliferation was measured by [3H]thymidine uptake, and the results are expressed as mean cpm ± SD. B, B cells from WT or p110{delta} mice were mixed with allogeneic T cells at various ratios, and [3H]thymidine incorporation was measured as in A. C, IL-2 production in MLR cultures using untreated WT or p110{delta} B cells or WT B cells pretreated with the indicated inhibitors. Results are representative of at least five independent experiments.

 
Ag-BCR complexes are internalized and targeted to late endosomes in p110{delta}-inactivated B cells

Because impaired Ag presentation function of p110{delta}-inactivated B cells was only observed in the BCR-mediated Ag uptake and presentation system, we assessed whether the observed inhibition is due to defective Ag-BCR internalization or intracellular trafficking. The degree and the kinetics of BCR internalization triggered by rabbit anti-IgM Ab were similar in p110{delta}-inactivated B cells and WT B cells (Fig. 5A). Similar results were seen in WT B cells pretreated with the p110{delta}-selective inhibitor (data not shown). Confocal imaging of fluorescently labeled Ag did not reveal any visible differences in the pattern of Ag internalization by WT vs p110{delta}-inactivated B cells (Fig. 5B). These results indicate that the defect in BCR-mediated Ag presentation is not occurring at the level of initial Ag uptake.


Figure 5
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  		FIGURE 5. The p110{delta}-inactivated B cells have normal BCR internalization. A, WT and p110{delta} B cells were incubated with biotin rabbit anti-mouse IgM, and the degree of BCR internalization over time was determined by FACS. Data are expressed as mean MFI ± SD of each time point relative to MFI at time 0 and are representative of three independent experiments. B, WT and p110{delta} B cells were incubated with Cy5 rabbit anti-mouse IgM Ab for 10 min to allow binding. At the end of each time point, internalization was stopped by fixation and cells were centrifuged onto slides. BCR internalization was examined by confocal microscopy and representative images are shown.

 
We investigated whether impaired Ag presentation by p110{delta}-inactivated B cells is due to failure in targeting Ag to late endosomes/lysosomes. WT B cells showed a time-dependent colocalization of labeled Ag with LAMP1 (Fig. 6, A and B), a resident lysosomal membrane protein (40). The p110{delta} mutant B cells showed similar levels of Ag-LAMP1 colocalization (Fig. 6C). In addition, p110{delta} mutant B cells showed similar levels of Ag colocalization with H2M (Fig. 6D), a resident of the lysosome/late endosome (MIIC) compartments that catalyze loading of antigenic peptide onto the MHC class II (41, 42, 43, 44). These results suggest that p110{delta} mutant B cells are not defective in Ag-BCR internalization or targeting to the MIIC.


Figure 6
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  		FIGURE 6. The p110{delta}-inactivated B cells target BCR-Ag complexes to late endosomes. A, Representative images showing the colocalization between BCR and LAMP1 at the different time points. WT B cells were incubated with Cy5 rabbit anti-mouse IgM (red) at 37°C for various time points, fixed, permeabilized, and stained with FITC-labeled anti-LAMP1 (green). B, Correlation coefficient values show the degree of colocalization between BCR and LAMP1 at the different time points, representing the mean and SD of at least 50 cells per group. C, Top, Representative images demonstrate colocalization of BCR (red) and LAMP1 (green) after 60 min of Ag-BCR internalization. Bottom, Graph showing mean correlation coefficients of BCR and LAMP1 for at least 50 cells per group. D, Top, Representative images demonstrate colocalization of BCR (green) and H2M (red) after 60 min of Ag-BCR internalization. Bottom, Graph showing mean correlation coefficients of BCR and H2M for at least 50 cells per group.

 
The p110{delta}-inactivated B cells are less effective in forming polarized conjugates with Ag-specific T cells

To examine the ability of p110{delta} mutant B cells to form cognate interactions with T cells after Ag incubation, we fixed B:T cell conjugates formed after 2 h of coculture and examined them by confocal microscopy. Staining WT B cell conjugates with phalloidin revealed a marked polarization of F-actin toward the T:B interface (Fig. 7A). Although there were no obvious differences in the number of B:T conjugates formed by WT or p110{delta} mutant B cells, we found significantly less F-actin polarization toward the T:B interface in conjugates formed by p110{delta}-inactivated B cells compared with the WT control (Fig. 7, A and B). Consistent with this result, pretreatment of WT B cells with p110{delta}-specific inhibitor (Fig. 7C) or WM (data not shown) before Ag incubation led to a dose-dependent reduction in formation of polarized conjugates. These results imply a role for p110{delta} signaling in facilitating late stages of BCR-mediated Ag presentation leading to formation of stable polarized B:T conjugates, which is known to be a key step for productive T cell activation.


Figure 7
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  		FIGURE 7. The p110{delta}-inactivated B cells show impaired formation of polarized conjugates. A, WT or p110{delta} B cells were incubated with Cy5 rabbit anti-mouse IgM (red) at 37°C for 60 min, mixed with 2R.50 T cells for another 60 min, fixed, and stained for F-actin using Alexa488-labeled phalloidin (green). Shown is a representative image demonstrating the degree of F-actin polarization in B cell at the contact area with T cells. B, Graph showing percentage of F-actin polarization in at least 50 conjugates analyzed per group. C, WT B cells were pretreated with IC before their mixing with Cy5 rabbit anti-mouse IgM and 2R.50 T cells, as in A. At least 50 conjugates per condition were scored for F-actin polarization. **, p = 0.0016; ***, p < 0.0001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study provides the first evidence that the p110{delta} isoform of PI3K is an important regulator of BCR-mediated Ag presentation. We observed a requirement for p110{delta} signaling in multiple systems, including genetically deficient primary cells and pharmacological inhibition of primary B cells and cell lines. The requirement for p110{delta} signaling applies both to anti-Ig and protein Ags, suggesting that it is important for fundamental processes independent of ligand type. Although our data indicate that the p110{delta} isoform alone is contributing a functionally significant signaling activity, we cannot rule out the involvement of other PI3K isoforms in this process. Indeed, treatment with global PI3K inhibitors led to a more profound inhibition of BCR-mediated Ag presentation, suggesting compensatory effects of p110{alpha} and p110beta, which are expressed at normal levels in the p110{delta}-inactivated B cells (33) and in WT B cells pretreated with p110{delta}-selective inhibitor. We cannot rule out the possibility that the more dramatic effects of global PI3K inhibitors are due to additional off-target effects of the inhibitors; however, this seems unlikely given that similar effects were observed with two structurally unrelated inhibitors.

Our use of B cells from p110{delta}-inactivated mice avoids the caveats inherent in using inhibitors; however, this model has different caveats that need to be considered, associated with B cell developmental defects such as the lack of marginal zone B cells (33). Although marginal zone B cells are a numerically small B cell subset, we cannot rule out the possibility that they may influence the results under some conditions, for example, after LPS stimulation, in which these cells respond more robustly than conventional B cells (45). However, the combination of genetic and pharmaceutical inactivation data together provides consistent and strong evidence that p110{delta} signaling plays an important role in BCR-mediated Ag presentation.

Interestingly, WM and LY inhibitors both significantly affected the ability of B cells to activate T cells in an MLR, whereas p110{delta}-specific inactivation had no effect. It should be noted that primary mouse T cells also require p110{delta} signaling to respond optimally to both peptide Ag and alloantigen (33) (M. Al-Alwan, unpublished observation). In the present study, B cells were extensively washed following inhibitor pretreatment, minimizing the possibility of T cell exposure to the inhibitors. Although low-level leakage of inhibitors from the treated B cells cannot be ruled out entirely, this seems unlikely, given that IC-pretreated B cells can trigger normal allo-T cell responses, whereas WM treatment (which is known to irreversibly bind to and inactivate PI3Ks (46, 47) and is thus unlikely to leak) does inhibit. This result thus suggests a potential function of PI3Ks in B cell formation of a functional immune synapse with allogeneic T cells, requiring isoforms other than p110{delta}. Alternatively, a lower threshold for total PI3K signaling may be required for presentation function in the MLR vs BCR-mediated presentation, the latter of which may be more sensitive to partial reductions in 3-phosphoinositide production. Given the lethality of p110{alpha} or p110beta deletion (48, 49), future studies using other isoform-specific inhibitors and/or cell-specific gene disruption using cre-loxP system will be needed to study the function of these isoforms in Ag presentation.

It is well established that BCR-mediated Ag uptake reduces the Ag concentrations required for presentation, most likely due to enhanced targeting of BCR-bound Ag to specialized endocytic compartments (50). We find that p110{delta} signaling is not required for BCR internalization, consistent with findings that global PI3K inhibitors do not affect kinetics of Ag uptake (24, 25) (data not shown). Although the role of PI3K in intracellular trafficking of several receptors is well established, previous studies examining the effect of global PI3K inhibitors on B cell lines have given conflicting results regarding the potential role of this pathway in BCR trafficking. One study found that treatment with WM blocks trafficking of BCR complexes into late endosomes (24), whereas another study reported normal trafficking, but failure to form mature MIIC (25). Another study found that BCR signaling induces coalescence of LAMP1+ late endosomes into a multivesicular MIIC-like compartment, but this response is not blocked by WM (12). Although the interpretation of these results must be tempered by the knowledge of the off-target effects of WM (particularly regarding its effect on phosphatidylinositol 4-kinases), our results are consistent with the latter findings, in that we did not find evidence for abnormal trafficking of BCR-Ag complexes to late endosomes in p110{delta}-inactivated primary B cells. This suggests a role for p110{delta} at subsequent steps of BCR-mediated Ag presentation.

Generation of the MIIC involves recruitment of H2M and other molecules into the late endosomes/MIIC to facilitate Ag processing and loading (51). In our system, we did not detect impaired H2M recruitment to BCR-Ag containing late endosomes in p110{delta}-inactivated B cells, arguing against an overt failure in generation of the MIIC. Thus, we hypothesize that p110{delta} signaling is required for efficient maturation and trafficking of peptide-MHC complexes to generate functional Ag on the cell surface that can be recognized by the T cells. Because p110{delta} signaling is not required for B cells to activate alloreactive T cells, it seems unlikely that it plays a role in conjugate and synapse formation when peptide-MHC II complexes are abundant on the cell surface. However, we cannot rule out the possibility that p110{delta} signaling may also influence the efficiency of synapse formation under conditions in which peptide-MHC complexes are limiting, as this is likely the case for BCR-mediated Ag presentation.

T cells actively rearrange their cytoskeleton toward the contact point with the APCs (reviewed in Ref. 52), a key step for productive T cell activation. We previously found that dendritic cells also actively polarize their cytoskeleton toward the interface with naive CD4+ T cells and found evidence that DC F-actin polarization is required for T cell activation (53). In the present system, B cells form polarized conjugates with predominant F-actin accumulation at the cell-cell interface that appears to be mainly on the B cell side. However, we cannot rule out the possibility of additional F-actin accumulation on the T cell side of the contact site in this model: indeed, a minority of conjugates do show visible F-actin polarization in the 2R.50 T cells (M. Al-Alwan, unpublished observation). This F-actin polarization was significantly impaired in p110{delta}-inactivated B cells, most likely due to reduced levels of specific antigenic peptide-MHC II complexes on the cell surface, which are required to drive bidirectional polarization. An alternative interpretation of this result is that p110{delta} signaling is required for the bidirectional signaling processes mediating polarization; however, this seems unlikely because p110{delta} inactivation did not affect the ability of B cells to activate allogeneic T cells. We have also observed that in polarized B:T conjugates, labeled endocytosed Ag often appears to reorient to face the T cells (Fig. 6A and data not shown), suggesting that the polarization of B cell Ag-processing compartments may facilitate directional trafficking of loaded peptide-MHC complexes to the B cell surface facing the T cells, enhancing Ag presentation. Such directional trafficking has been reported recently in dendritic cells (54, 55), and raises the intriguing possibility that PI3K signaling may be required for targeted release of peptide-MHC II complexes generated via BCR-mediated uptake.

Collectively, our findings provide the first evidence that the PI3K p110{delta} isoform regulates BCR-mediated Ag presentation in primary B cells. Our results suggest that this signaling pathway is required at a late stage of Ag presentation after initial BCR trafficking to the MHC II loading compartment, but before immune synapse formation. These results should prompt further investigations of the roles of 3-phosphoinositides and their targets in Ag presentation.


   Acknowledgments
 
We thank Dr. James Drake for providing 2R.50 cells, Dr. Facundo Batista for providing HeyHEL10 transfectants and 2G7 T cells, Dr. Terutaka Kakiuchi for providing A20-HL and 42-6A T cells, and Dr. Michael Gold for critical review of the manuscript. We also thank Sen Hou for assistance with mouse genotyping, Baher Nashed for advice on IL-2 assays, and members of the Marshall laboratory for helpful discussions.


   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 an operating grant from the Canadian Institutes of Health Research (to A.J.M.) and infrastructure support from the Canadian Foundation for Innovation. M.M.A.-A. was supported by a postdoctoral fellowship from the King Faisal Specialist Hospital and Research Centre and University of Manitoba Faculty Fund. A.J.M. was supported by a Canadian Institutes for Health Research New Investigator Award. Back

2 Address correspondence and reprint requests to Dr. Aaron J. Marshall, Department of Immunology, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada R3E 0W3. E-mail address: marshall{at}ms.umanitoba.ca Back

3 Abbreviations used in this paper: MIIC, MHC class II-enriched compartment; HEL, hen egg lysozyme; LAMP, lysosome-associated membrane protein; MFI, mean fluorescence intensity; WM, wortmannin; WT, wild type; IC, IC87114. Back

Received for publication June 21, 2006. Accepted for publication December 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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