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/Ig
Complexes Generate Signals for B Cell Development Independent of Selective Plasma Membrane Compartmentalization1
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Abstract |
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/Ig
-dependent BCR signaling. Because a fraction of the BCR and an even larger fraction of the preBCR associates with lipid rafts in the apparent absence of ligand stimulation, it has been proposed that raft-associated receptor complexes mediate the ligand-independent basal signaling events observed in resting B lineage cells. However, there is no direct evidence that localization of Ig/Ig-containing complexes to detergent-resistant membrane compartments is absolutely required for the signaling events that drive B cell development. To address these issues we have designed surrogate preBCR/Ig/Ig complexes that are incapable of ligand-induced aggregation and that are preferentially targeted to either raft or nonraft compartments. An analysis of their ability to promote the preBCR-dependent proB
preB cell transition of murine B cell progenitors revealed that expression of these surrogate receptor complexes at levels that approximate that of the conventional preBCR can drive B cell development in a manner independent of both aggregation and lipid raft localization. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Ig. The arrest of B cell development at the proB cell stage demonstrates the importance of Ig/Ig-containing signaling complexes in promoting B cell development. However, early in B cell development, the Ig/Ig-containing complexes that are expressed (proBCR and preBCR) do not contain the conventional Ag binding surface Ig subunit, indicating that signals promoting proBpreB cell developmental progression are Ag independent, at least in terms of binding of polymorphic Ag.
The Ig/Ig-dependent signal transduction events responsible for promoting early B cell development remain enigmatic, but currently at least four models exist (reviewed in Ref.1). The first suggests that surrogate L chain-dependent homotypic interactions promote signal transduction through preBCR complexes via self-aggregation (2). The second model suggests that nonpolymorphic ligands, such as galectin-1 or an as yet unidentified stromal cell surface protein, would serve to aggregate the preBCR, thereby promoting signal transduction (3, 4, 5). The third model suggests that preferential localization of the preBCR to detergent-resistant plasma membrane compartments, known as lipid rafts, provides an environment rich in signal transduction components that allows for constitutive, ligand-independent preBCR signal transduction (6). Finally, the fourth model suggests that a homeostatic equilibrium established by a combination of positive and negative coreceptors results in the stochastic activation of tyrosine kinases and phosphatases associated with the Ig/Ig-containing signalosome, resulting in a constitutive or basal level of signal transduction through these complexes (7, 8, 9). Although each of these models is provocative, they are not mutually exclusive, and caveats that challenge their relevance to the physiologic preBCR-dependent signaling requirements driving early B cell development remain (reviewed in Ref.1).
To better understand the molecular mechanisms allowing the generation of a ligand-independent signal, we have synthesized a signaling-competent surrogate receptor formed by the cytoplasmic domains of Ig and Ig. This surrogate receptor is targeted to the plasma membrane by a fatty acid modification and therefore lacks extracellular and transmembrane domains (7, 8). Under conditions where we can clearly distinguish the structure and distribution of normal resting pre-BCR complexes from complexes that have been forced to undergo anti-BCR-induced aggregation, we have shown that the surrogate receptor closely resembles the resting preBCR (8, 9). Importantly, the nonaggregated surrogate receptor was able to trigger preBCR-dependent developmental functions, such as positive selection at the proB to preB checkpoint, proliferation, and allelic exclusion of the Ig H chain and L chain recombination, suggesting that these developmental processes are not dependent on a demonstrable aggregation of the receptor (8).
To better understand the mechanisms that regulate the Ig/Ig-dependent signaling events that promote early B cell development, variants of the Ig/Ig surrogate receptor complex were designed that preferentially localized to either the raft or nonraft compartments of the plasma membrane. An assessment of the Ig/Ig-dependent proBpreB cell transition driven by these surrogate receptor complexes indicated that the proBpreB cell transition is not dependent on receptor localization to the detergent-resistant membrane compartment.
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Materials and Methods |
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The construction of MAHB and its ITAM mutant variant has been described previously (7). To target MAHB inside and outside the lipid raft compartment, the Lck-derived myristoylation and palmitoylation sites were modified. The palmitoylated residues were modified first (Cys3Ser, Cys5Ala) by PCR using MAHB as template and oligonucleotides Myr-pp-S2 (5'-GCCGGAATTCCACCATGGGCAGTGTCGCCAGCTCAAACC-3') and antisense Ig (5'-CCCAAGCTTCATTCCTGGCCTGGATGCTCTCCTAC-3'). The resulting PPmut was then modified by the addition of sequences that encode six lysine residues immediately 3' of the modified Lck targeting sequence (MAHBnonraft). For the MAHBraft variant, the sequence encoding the six lysine residues was introduced into the region immediately 3' of the unmodified Lck targeting sequence of MAHB (Fig. 1A). Each variant was cloned into the retroviral vector MIGR1.
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Long term proB cell cultures from IgH3H9/+ and RAG2–/– (Taconic Laboratories) mice were generated and maintained in the presence of IL-7 as previously described (8, 10). By day 5 of culture, >99% of the cells were proB cells (B220+BP1+CD25–CD43+CD22–, Hardy’s fraction C/C') (11, 12). RAG2–/– proB cell cultures were spin-infected with the retroviral vectors, and GFP+ cells were sorted to obtain pure cultures. For analysis of developmental progression, cells were depleted of IL-7 for 2 days, then subjected to FACS analysis using Abs against the pan B cell marker B220 and the differentiation marker CD43. All Abs used in this study were purchased from BD Pharmingen. TO-PRO-3 iodide dye (Molecular Probes) was used to exclude dead cells.
Retroviral infection of bone marrow (BM)3 and adoptive transfer
Mice were housed and treated according to institutional animal care and use committee regulations at University of Pennsylvania. Infection of cells using MIGR1 retrovirus-containing supernatants was accomplished exactly as previously described (7). In addition, the details of the adoptive transfer of transduced BM from µMT mice into syngeneic hosts has been previously described (13). For the experiments in the current study, recipient mice were killed 4–6 wk post-transfer, and splenocytes were stained with Abs against the following surface proteins: B220, MHC class II, and CD23.
Electron microscopy
The proB cell lines were fixed with 4% paraformaldehyde plus 0.2% glutaraldehyde in 0.1 M sodium cacodylate buffer. Thin sections of the various samples were incubated with Abs anti-hemagglutinin (anti-HA; 12CA5; Roche) or anti-Igµ (B76) and then with 10 nm of gold particle-conjugated secondary Ab. Samples were analyzed using a JEOL JEM 1010 transmission electron microscope, and images were captured using a Hamamatsu CCD camera and analyzed with AMT 12-HR imaging software. For quantification of relative levels of receptor expression, the number of Immunogold complexes present in 400 µm of plasma membrane was counted (20 micrographs).
Sucrose gradients and immunoblotting
NIH-3T3 (1 x 107) cells expressing the chimeric proteins were lysed in 1% Triton X-100 lysis buffer precooled to 4°C and then subjected to sucrose gradient separation by a procedure similar to that described by Cheng et al. (14, 15). Gradient fractions were loaded, and proteins were separated through a 12% SDS-PAGE polyacrylamide gel, followed by transfer to nitrocellulose membranes. Western blot analysis was performed using mouse anti-HA (16B12; COVANCE) as primary Ab and peroxidase-conjugated horse anti-mouse IgG (Vector Laboratories) as secondary Ab. Membranes were stripped and reprobed with cholera toxin B-HRP conjugate (Calbiochem).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/Ig-containing variant complexes at the plasma membrane
We have previously documented that when expressed in preBCR-deficient B cell progenitors, a surrogate preBCR complex lacking ecto- and transmembrane sequences is able to generate the Ig/Ig ITAM-dependent basal signals necessary for developmental progression (7). This surrogate preBCR, named MAHB, is a fusion protein comprised of the cytoplasmic domains of Ig and Ig and the membrane targeting sequence of the murine Lck protein (aa 1–10). Like the resting preBCR and BCR, MAHB distributes across both the detergent-resistant and -soluble compartments at the plasma membrane (7). To assess whether the Ig/Ig-dependent proBpreB transition requires Ig/Ig complex localization to lipid rafts, we engineered MAHB variants that would preferentially localize to either the detergent-resistant or -soluble plasma membrane compartments (Fig. 1A).
The Lck membrane-targeting sequence present in MAHB contains a glycine residue (position 2) and two cysteine residues (positions 3 and 5) that are substrates for myristoylation and palmitoylation, respectively (16, 17). Elimination of the palmitoylation sites by cysteineserine substitutions in MAHB resulted in the generation of a protein that did not localize to the plasma membrane (not shown). Similarly, mutations in the palmitoylation sites of Lck resulted in cytoplasmic localization (16). In the latter studies, however, it was noted that insertion of a polylysine sequence C terminal to the myristoylation site generated a polybasic region that localized myristoylated Lck to the plasma membrane (16). Moreover, in proteins lacking a palmitoylation site, addition of a polybasic region resulted in localization to the detergent-soluble, nonraft compartment. Based upon this strategy, two MAHB variants were constructed (Fig. 1A). In the first, a six-amino acid, lysine-based polybasic region was appended to a mutated Lck targeting sequence in which the myristolyation and palmitoylation sites were replaced by serines (MAHBnonraft); in the second variant, the six-amino acid, lysine-based polybasic region was appended to a wild-type Lck-targeting sequence to accomplish preferential localization to lipid rafts (MAHBraft). MAHB and its modified variants were individually cloned into the MIGR1 retrovirus vector (13) and used to infect primary proB cells (Hardy’s fraction C') obtained by culturing BM progenitor cells from either RAG2–/– or IgH3H9/+ mice in the presence of IL-7 (10, 12). Fluorescent monitoring of GFP expressed from an internal ribosome entry site in the MIGR1 vector allowed for the purification of transduced populations and an assessment of relative expression levels.
We first assessed the relative expression levels of the fusion proteins by an immunoblot analysis of transduced proB cells, using an Ab to the HA epitope tag present in MAHB and each variant. This analysis revealed that although the parental MAHB and the raft-associated variant are expressed at comparable levels, the total expression level of the nonraft-associated variant was reproducibly 3- to 4-fold lower (Fig. 1B). We next assessed steady state levels of the both MAHBraft and MAHBnonraft variants at the plasma membrane using transmission electron microscopy. Cells were sorted for equal levels of GFP expression, and the number of gold complexes in micrographs of 20 individual cells (
400 µm of plasma membrane) were counted to estimate the relative number of surrogate receptors per linear micron of plasma membrane. From this analysis, we estimate that there are one and 2.4 complexes/µm plasma membrane for MAHBnonraft and raft variants, respectively (Fig. 1C). We conclude that although not identical, the steady state level of surface expression of MAHBraft and MAHBnonraft is not markedly different. As a negative control, proB cells derived from RAG2–/– mice were stained with a mixture of anti-IgM and anti-HA Abs. The absence of staining in these cells serves as a specificity control and confirms the absence of conventional preBCR that could have potentially contributed to signal transduction in RAG2–/– proB cell lines expressing MAHB variants.
A comparison of relative expression levels of the surrogate receptors with conventional preBCR expressed on primary proB cells (derived from B cell progenitors isolated from H chain transgenic IgH3H9/+ mice), revealed that they were quite similar (Fig. 1C). Although we acknowledge that different primary Abs (12CA5 anti-HA and B76 anti-IgM Abs) were used for this experiment, similar conclusions were reached in our previous studies (9) in which MAHB and preBCR expression levels were directly compared in primary IL-7-dependent lines using an Ab to the cytoplasmic domain of Ig. In these studies the level of expression of MAHB and preBCR were comparable and equivalent to the levels of the variants reported in this study. Therefore, although we cannot definitively state that expression levels of the MAHB model and variants are identical with those of preBCR, their expression levels are very similar.
As noted above, we have previously shown that MAHB is able to generate the Ig/Ig ITAM-dependent basal signals necessary for developmental progression when expressed in preBCR-deficient B cell progenitors (7). Although the absence of Ig and Ig ectodomains in these constructs precludes ligand-induced aggregation, it remained possible that signal transduction through these surrogate receptor complexes was facilitated by aggregation as a consequence of overexpression. However, our results indicate that both MAHBraft and MAHBnonraft variants are present at the plasma membrane in levels comparable to those of the conventional preBCR. Furthermore, we have previously demonstrated that the cell surface distribution of the MAHB variants is similar to that of the normal preBCR and is demonstrably different from that of preBCR aggregated by anti-BCR (8), suggesting that signal transduction through MAHB in not mediated by extensive oligomerization.
Differential plasma membrane localization of surrogate preBCR/Ig/Ig-containing variant complexes
To assess the relative ability of each MAHB variant to preferentially compartmentalize into the detergent-soluble and -resistant membrane fractions, transduced NIH-3T3 cells were sorted for GFP expression. Sorted GFP+ cells were solubilized in 1% Triton X-100 at 4°C, then subjected to sucrose gradient centrifugation. The detergent-resistant lipid rafts were enriched in the buoyant fractions (fractions 1–3), whereas the detergent-soluble compartments (nonrafts) were enriched in fractions 7–11. The detergent-resistant compartment was verified by selective enrichment of the sphingolipid GM1. In some experiments (not shown), these compartments were verified by localization of transferrin receptor (nonrafts) and Src kinase Lyn (predominately rafts). Fig. 2 confirms the dual distribution of MAHB inside and outside the lipid raft compartment. In contrast, the MAHBnonraft variant (Fig. 2, middle panel) localized exclusively to the nonraft containing fractions 9–11. The majority of the MAHBraft protein was found in the detergent-resistant buoyant fractions (fraction 2), but we were able to detect a small amount in the detergent-soluble fractions (7, 8, 9, 10, 11) as well. Importantly, we never detected the nonraft variant in the buoyant fractions, even when higher amounts of proteins were loaded. The preferential localization of the MAHB variants in raft or nonraft fractions provided us with a tool to assess whether the Ig/Ig-dependent proBpreB transition required the Ig/Ig-containing complexes to preferentially signal from the detergent-resistant plasma membrane compartment.
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/Ig ITAM-dependent signal required for the proBpreB transition is independent of lipid raft localization.
To assess whether the Ig/Ig ITAM-dependent signal required for the proBpreB transition depended upon localization of the signaling complex to lipid rafts, MAHB variants were transduced into primary proB cells obtained from long term IL-7-dependent cultures of B cell progenitors from RAG2–/– mice. IL-7 allows for the enrichment of primary proB cells, and the phenotypic characterization and criteria for assignment of these B cells to the late pro/pre B stage have been documented (8, 11).
We first established that selective targeting of the MAHBraft and MAHBnonraft variants to the detergent-resistant buoyant (raft) and -soluble nonbuoyant (nonraft) fractions observed in NIH-3T3 cells was also the case for IL-7-dependent primary proB cells. For this analysis, freshly sorted, GFP-expressing cells from cultures expressing each variant were lysed in 1% Triton X-100 at 4°C. Lysates were then fractionated through sucrose gradients to separate raft- and nonraft-associated proteins. The results of SDS-PAGE separation and Western blot analysis of proteins in the raft (fractions 2 and 3) and nonraft (fractions 10 and 11) fractions are shown in Fig. 3A. As in the NIH-3T3 cells depicted in Fig. 2, the MAHBnonraft variant appears to associate exclusively with the dense, detergent-soluble fractions, whereas the MAHBraft variant is detected predominantly, but not exclusively, in the detergent-resistant, buoyant fractions.
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preB transition. For this analysis, loss of CD43 expression after IL-7 withdrawal was used as an indicator of developmental progression (11). Expression of the parental MAHB construct triggered the proBpreB transition, as evidenced by the generation of a CD43– population in the MAHB-transduced proB cells, but not in cultures of proB cells transduced with either control MIGR1 or a variant of parental MAHB in which all four of the ITAM-associated tyrosines were replaced by phenylalanine (ITAMmut; boxed regions in Fig. 3B); this finding is consistent with those of our previous studies (7, 9). GFP– loss variants that arose in these stable IL-7-dependent cultures (to the left of the boxed regions) did not undergo transition to the CD43– phenotype, indicating that developmental progression is Ig/Ig dependent. To determine whether the proBpreB transition depended upon localization of the Ig/Ig-containing signaling complex to lipid rafts, MAHBraft- and MAHBnonraft-positive proB cells were also depleted of IL-7 to promote transition to the preB stage. The generation of CD43– cells in cells transduced with MAHB, MAHBraft, or MAHBnonraft variants suggested that all surrogate receptors were capable of mediating the proBpreB transition. For comparison, the absolute number of preB cells present in each culture at 48 h after IL-7 removal is presented in the table in Fig. 3B. Together, these results indicate that Ig/Ig-derived signals initiated outside of lipid rafts are competent in promoting the proBpreB transition. A comparison of the frequency of preB cells in the cultures of B cells expressing the raft and nonraft variants (Fig. 3B) suggests that raft-derived signals are somewhat more efficient in promoting this transition (32% MAHBraft-positive vs 21% MAHBnonraft-positive preB cells). However, we believe that this difference is due to the relatively higher expression level of the MAHBraft variant (Fig. 1, B and C). We normalized GFP levels based upon the relatively lower expression of MAHBnonraft compared with MAHBraft (2.4-fold), as determined by Western blotting. These normalized GFP gates (Fig. 4A) were then used to re-evaluate the frequency of CD43– cells present in the cultures described in Fig. 3B. The CD43– preB cell frequency to GFP level relationship was graphed, and each variant was compared using both unmanipulated (Fig. 4B) and normalized (Fig. 4C) GFP levels.
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/Ig signals for proBpreB transition.
Ig/Ig signaling initiated in the detergent-soluble plasma membrane compartment is sufficient to confer developmental progression in vivo
We have demonstrated that both the MAHBraft and MAHBnonraft surrogate preBCR complexes are capable of promoting the proBpreB transition in vitro. However, it remained possible that signaling through these complexes would not be efficient enough to drive B cell development beyond this transition point in vivo under physiological conditions, where positive selection, proliferation, and survival also play important roles in generating and maintaining the differentiating populations. Therefore, we next evaluated whether localization of Ig/Ig-containing complexes to the detergent-resistant lipid raft compartment was necessary to promote B cell development in vivo using adoptive transfer experiments.
B lineage cells in µMT mice are developmentally arrested at the proB stage due to a deletion of the transmembrane exon of the µ H chain that renders them unable to express a surface preBCR (18). To determine whether the surrogate preBCR/Ig/Ig-containing complex targeted to either the raft or nonraft compartments was capable of promoting further B cell development, MAHB variants were individually expressed in µMT BM progenitor cells and then adoptively transferred into lethally irradiated syngeneic hosts. Donor progenitors expressing MAHB or MAHB with nonfunctional ITAMs (ITAMmut) were included for comparison as positive and negative controls, respectively. Recipients were killed 4–6 wk after transfer, and BM and splenocytes were analyzed for the presence of B220+ B cells. Development was monitored by the expression of CD22 and MHC class II, which mark progression to the pre B and transitional immature-2 (T2) stages, respectively.
Fig. 5 shows that CD22+ B cell populations were present in the BM of lethally irradiated mice reconstituted with µMT BM progenitors expressing MAHB, MAHBraft, or MAHBnonraft. Notably, few if any B220+ cells were detected in the ITAMmut group, confirming the critical role of the Ig/Ig ITAM motif for developmental progression. These data demonstrate that the signaling complexes responsible for driving the continued developmental progression of B cells are not exclusively limited to those associated with the lipid raft compartment. Instead, signal transduction initiated in the detergent-soluble nonraft fraction of the plasma was also able to drive continued development beyond the proB to preB checkpoint, in this case at least to the preB and immature stages. Furthermore, the efficiency at which development was driven by signals originating in either compartment was not markedly different, although in this experiment MAHBraft appeared to be somewhat better at driving cells into the CD22+ gate (20 vs 15% for MAHBnonraft).
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preB checkpoint, the signals necessary for transition at least through to the T2 stage can be generated with equivalent efficiency inside or outside the lipid raft compartment. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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preB cell transition; it did not attempt to directly compare the efficiency of signal transduction through this surrogate receptor with that of a conventional preBCR.
The primary goal of this study was to evaluate the requirement for detergent-insoluble membrane domains (lipid rafts) in generating signals for transition through the pro-Bpre-B developmental checkpoint. The importance of detergent-insoluble membrane domains in the stabilization and persistence of Ag-induced signaling through the BCR has been well documented (6, 14, 15, 21). However, the importance of lipid rafts in either signal initiation or ligand-independent basal signaling remains largely unexplored. A significant fraction of the preBCR (40%) is constitutively localized to lipid rafts in resting preB cells (6), highlighting their potential importance in preBCR signaling. Based upon these previous studies, it was proposed that constitutive association of preBCR in lipid rafts may provide a means for the ligand-independent signaling that promotes the survival and expansion of preB cells that have successfully undergone Ig H chain rearrangement. However, the physiological relevance of raft-associated preBCR complexes was not directly assessed in these studies nor was it determined if the raft-associated preBCR complexes were present at the plasma membrane. The data presented in this study clearly indicate that preBCR basal signaling events can be initiated in either the lipid raft or nonraft membrane compartments, thereby severing the link between an absolute requirement for localization of the receptor to lipid rafts and the initiation of signal transduction pathways promoting preBCR/BCR-dependent developmental processes. In agreement with these findings, a preT variant that does not localize to lipid rafts is still capable of promoting thymocyte development (22).
Although our results do not support an absolute requirement for the preBCR to initiate signals from lipid rafts, it is still possible that compartmentalization into the liquid-ordered and disordered regions of the plasma membrane affects qualitative and/or quantitative aspects of the signals generated. Indeed, we have postulated that signals initiated outside of lipid rafts result in constitutive (basal) transient signals, whereas those resulting from receptor aggregation and consequent lipid raft localization will lead to more persistent signals (9). The potential biological impact of such partitioning would be significant, because it would allow the same Ig/Ig receptor complex to promote positive selection during early B cell development and survival of peripheral B cells (basal signals) and also promote effector cell differentiation after induction by Ag encounter (activating signals).
Based on our results, we favor a model in which ligand-independent basal or tonic signaling is sufficient for the initiation of signal transduction events by the resting preBCR. In this model, stochastic interactions between protein tyrosine kinases and phosphatases associated with the preBCR result in a basal level of signaling. The observed ability of the tyrosine phosphatase inhibitor pervanadate to shift the kinase/phosphatase equilibrium and promote a sustained and detectable BCR-dependent signal in otherwise unstimulated B cells (23) supports the idea that basal signaling is a constitutive event in B lymphocytes. Consistently, gain-of-function analysis revealed that constitutive activation of the tyrosine kinase Blk can mimic at least some aspects of preBCR signaling in preBCR-deficient proB cells (24). Furthermore, the negative coreceptor CD22 is expressed at low levels on preB cells, and this expression may contribute to the low basal signaling threshold for the preBCR (25). Although evidence suggests that tonic signaling through both the preBCR and the BCR exists (7, 8, 9), it is unlikely that under physiologic conditions, tonic signaling will be the sole mediator of signal transduction through these complexes. The challenge that remains is to determine how ligand-independent tonic signaling and ligand- and/or raft-dependent signaling events are integrated to promote both preBCR- and BCR-dependent developmental and homeostatic processes.
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Acknowledgments |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants AI32592, AI43620, and CA093615 (to J.G.M.), a Cancer Research Institute award (to E.M.F.-P.), and a National Cancer Institute training grant award (to G.B.). 
2 Address correspondence and reprint requests to Dr. John G. Monroe, University of Pennsylvania School of Medicine, Room 311 BRBII/III, 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: monroej{at}mail.med.upenn.edu
3 Abbreviations used in this paper: BM, bone marrow; HA, hemagglutinin.
Received for publication August 5, 2004. Accepted for publication November 5, 2004.
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References |
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5 mediates cell-autonomous pre-B cell receptor signaling. Nat. Immunol. 4:849.[Medline]
5 and stroma cell-associated heparan sulfate. J. Immunol. 171:2338./Ig signals trigger the coordinated initiation of pre-B cell antigen receptor-dependent processes. J. Immunol. 173:1000. in T lymphocyte development. Nat. Immunol. 3:483.[Medline]
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