IgG immune complexes (ICs) promote autoimmunity through binding fragment crystallizable (Fc) γ-receptors (FcγRs). Of these, the highly prevalent FcγRIIa (CD32a) histidine (H)-131 variant (CD32aH) is strongly linked to human autoimmune diseases through unclear mechanisms. We show that, relative to the CD32a arginine (R)-131 (CD32aR) variant, CD32aH more avidly bound human (h) IgG1 IC and formed a ternary complex with the neonatal Fc receptor (FcRn) under acidic conditions. In primary human and mouse cells, both CD32a variants required FcRn to induce innate and adaptive immune responses to hIgG1 ICs, which were augmented in the setting of CD32aH. Conversely, FcRn induced responses to IgG IC independently of classical FcγR, but optimal responses required FcRn and FcγR. Finally, FcRn blockade decreased inflammation in a rheumatoid arthritis model without reducing circulating autoantibody levels, providing support for FcRn’s direct role in IgG IC-associated inflammation. Thus, CD32a and FcRn coregulate IgG IC-mediated immunity in a manner favoring the CD32aH variant, providing a novel mechanism for its disease association.
Immune responses to IgG immune complexes (ICs) result from interactions between the fragment crystallizable (Fc) regions of IgG antibodies and diverse classical and atypical γ-receptors (FcγRs) primarily on hematopoietic cells (Pincetic et al., 2014; Bournazos et al., 2017). Many FcγRs are characterized by nonsynonymous single-nucleotide polymorphisms (SNPs), some of which affect the binding of FcγR variants to specific IgG subclasses (Bruhns et al., 2009; Shashidharamurthy et al., 2009). Of these, CD32a is particularly important, as it is widely expressed on hematopoietic cells and is characterized by the highly prevalent R131H SNP (rs1801274; Bournazos et al., 2017; Lehrnbecher et al., 1999). The H131 (CD32aH) is a high-affinity variant that is associated with several human autoimmune diseases, such as inflammatory bowel disease and rheumatoid arthritis (Shashidharamurthy et al., 2009; Zhang et al., 2016; Li et al., 2014), in contrast to its lower-affinity R131 counterpart (CD32aR), which is linked to an increased risk of infectious disease complications (Endeman et al., 2009; Bredius et al., 1994; Li et al., 2014). Critically, the residue 131 affected by this CD32a SNP is located on the CD32a-IgG Fc binding interface, where the R131H substitution alters CD32a binding to different isoforms of human IgGs (hIgGs) and the ICs they form (Shashidharamurthy et al., 2009; Bruhns et al., 2009). Specifically, one study found an ∼1.5- to 2-fold greater binding of ICs formed from hIgG1 and hIgG3 to CD32aH relative to CD32aR (Shashidharamurthy et al., 2009). In addition, CD32aR notably exhibits little binding to hIgG2 ICs (Shashidharamurthy et al., 2009; Parren et al., 1992; Salmon et al., 1992; Sanders et al., 1995). However, little is known about the functional consequences of these differences in CD32a variant binding or the precise intracellular mechanisms underlying their associations with increased risk of specific human diseases.
While CD32a variants disparately bind IgG ICs on the cell surface and direct their internalization into acidic intracellular compartments (Pincetic et al., 2014; Bournazos et al., 2017), their function in acidic conditions has not been examined. This is important, because the H131 residue (pKa ∼6.0) of CD32aH is prone to protonation in the pH 4.5–6.5 range observed in IgG IC-containing intracellular compartments (Baker et al., 2011; Haynes et al., 2016). Furthermore, intracellular endosomal compartments also contain FcRn, which binds monomeric IgGs and IgG ICs under mildly acidic conditions (pH <6.5) and at a site on IgG Fc that is distinct from FcγRs (Kim et al., 1999; Martin et al., 2001; Wines et al., 2000). FcRn is a nonpolymorphic atypical FcγR that is expressed in parenchymal and hematopoietic cells of mammals throughout life as a heterodimer of a MHC class I (MHCI)–related α-chain noncovalently associated with β-2-microglobulin (Pyzik et al., 2019; Simister and Mostov, 1989; Martin and Björkman, 1999). The pH-dependent binding of FcRn to IgG Fc occurs through salt bridge formation between several acidic residues of FcRn and critical histidine (H) residues on IgG Fc (Kim et al., 1999; Martin et al., 2001), whose pKa also renders them prone to protonation in the pH range observed in endosomes (pH <6.5), but not at neutral pH (Baker et al., 2011; Haynes et al., 2016). Functionally, FcRn is primarily recognized as an IgG salvage receptor, protecting IgG and IgG ICs from degradation and extending their circulating half-lives via pH-dependent binding in acidic endosomes and release under neutral pH cell surface conditions (Qiao et al., 2008; Blumberg et al., 2019; Roopenian et al., 2003; Borvak et al., 1998; Prabhat et al., 2007; Dickinson et al., 1999). Moreover, FcRn has also been shown to regulate a variety of functions in hematopoietic cells previously considered to be primarily determined by the classical FcγRs, such as phagocytosis (Vidarsson et al., 2006), innate cytokine production (Baker et al., 2013), antigen presentation (Qiao et al., 2008), and cross-presentation (Baker et al., 2011). However, the mechanisms whereby hematopoietic cells integrate CD32a- and FcRn-mediated processes in responding to IgG ICs is unclear. Because FcRn and CD32a both interact with IgG ICs under acidic conditions and regulate a variety of cellular responses to IgG ICs, we hypothesized that FcRn and CD32a functions are intimately coordinated in a CD32a allele-specific manner. We therefore investigated the mode of FcRn and CD32a interactions and the role of FcRn in CD32a-mediated immune processes. We assessed the effects of these interactions on innate and adaptive immune responses to IgG ICs in vitro in murine and human cells in the context of the CD32a variants and in vivo using the IC-mediated K/BxN model of rheumatoid arthritis.
We first assessed whether acidic conditions affected IgG IC binding to CD32a variants using an ELISA. As the FcRn-bearing compartments containing IgG ICs attain pH ∼5.6 (Baker et al., 2011), CD32aR and CD32aH variants were exposed at pH 5.6 to increasing concentrations of model hIgG1 ICs (Fig. 1 a), which were prepared from 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP)–conjugated OVA (OVANIP) and a monoclonal anti-NIP hIgG1 antibody (hIgG1WT). Under these conditions, CD32aH demonstrated significantly increased avidity for hIgG1WT IC relative to CD32aR (Fig. 1 b).
IgG contains two potential FcRn binding sites on each IgG at critical amino acids (I253, H310, and H435; IHH) contained within the CH2:CH3 domain interface of IgG Fc (Kim et al., 1999; Martin et al., 2001). These FcRn binding sites on IgG Fc are distinct from those associated with CD32a binding (Wines et al., 2000). Superimposition of FcRn:hIgG1 Fc and CD32aR:hIgG1 Fc crystal structures further suggested that a single hIgG1 Fc could accommodate simultaneous CD32a and FcRn engagement and formation of a ternary complex without steric clashing, as the FcRn and FcγR binding sites were separated by >39–53 Å (Fig. 1 c; Oganesyan et al., 2014; Ramsland et al., 2011). Alternatively, taking into consideration the tethering of the receptors to the cell membrane, it is also conceivable that distinct IgG Fc contained within ICs could also serve as a platform for ternary complex formation. We therefore next tested for simultaneous IgG IC binding by the CD32a variants and FcRn under acidic conditions by ELISA (Fig. 1 d). Consistent with formation of a ternary complex, hFcRn bound to hIgG1WT ICs captured by both CD32a variants, but in a manner that favored the CD32aH variant (Fig. 1 e). This ternary complex formation was abrogated if the applied ICs were composed of mutant hIgG1s that were specifically unable to bind FcRn (hIgG1IHH) or classical FcγRs (hIgG1N297A), including the CD32a variants (Table S1 and Fig. S1 a; Medesan et al., 1997; Kim et al., 1999; Tao and Morrison, 1989). We next used a proximity ligation assay (PLA) to investigate CD32a–IgG IC–FcRn ternary complex formation in an intracellular environment. To do so, mouse RAW 264.7 macrophage cells expressing endogenous mouse FcRn (mFcRn), which binds hIgG1 at acidic pH with comparable affinity to human FcRn (hFcRn; Neuber et al., 2014), were transfected with either the CD32aR or CD32aH variant (Fig. S1, b and c). PLA probes targeting CD32a and mFcRn yielded a fluorescence signal with both CD32a variants but only when both hIgG1 ICs and CD32a were present (Fig. 1 f and Fig. S1 d). We next used coimmunoprecipitation to biochemically identify the CD32a–IgG IC–FcRn ternary complex in the intracellular milieu. For this, we used human embryonic kidney cells (HEK 293) cells transfected with either CD32a variant and GFP-conjugated hFcRn (HEK 293hFcRn-GFP), which does not impact its hIgG1 binding or transcytosis functions, as a tractable tag for immunoprecipitation (Fig. S1 e; Christianson et al., 2012). Upon treatment of these cells with hIgG1WT ICs, but not hIgG1IHH ICs, more CD32aH relative to CD32aR was coimmunoprecipitated with hFcRn (Fig. 1 g and Fig. S1 f). Together, these data confirm ternary complex formation both under acidic conditions and in the intracellular environment when IgG IC can engage both CD32a and FcRn.
We next sought to determine whether formation of the CD32a–IgG IC–FcRn ternary complex was necessary for cellular responses to hIgG1 ICs. We first assessed innate immune responses in primary cells obtained from C57BL/6 mice transgenic (Tg) for either the CD32aR or CD32aH variant (hereafter “CD32aTg”; Fig. S2 a). To isolate any observed functional effects in these primary cells to CD32a and FcRn, these CD32aTg mice also lacked all classical FcγRs but maintained mFcRn expression (Fcgr2b−/−/Fcer1g−/−/Fcgrt+/+; Table S2; Fig. S2, b and c). Therefore, we isolated CD11c+MHCII+ dendritic cells (DCs) from spleens of CD32aR-Tg or CD32aH-Tg mice and observed increased secretion of TNF-α, IL-12/23p40, and IL-6 upon stimulation with hIgG1WT ICs relative to that observed with hIgG1IHH ICs and hIgG1N297A ICs, which are specifically disabled in FcRn or CD32a binding, respectively (Fig. 2, a–c; Medesan et al., 1997; Tao and Morrison, 1989; Kim et al., 1999). These data indicate that innate cytokine production in response to IgG ICs in APCs is both FcRn and CD32a dependent, consistent with the function of a CD32a–IgG IC–FcRn ternary complex.
As FcRn and classical FcγR are required for efficient antigen presentation and cross-presentation (Bonnerot et al., 1998; Qiao et al., 2008; Regnault et al., 1999; Baker et al., 2011), we examined the role of the CD32a–IgG IC–FcRn tripartite complex in these pathways. To do so, we used the antigen presentation machinery of the RAW 264.7 cells (MHC haplotype H-2d) described above. CD32aR or CD32aH variant- or control plasmid-transfected RAW 264.7 cells were loaded with hIgG1WT ICs at neutral pH and compared for their ability to stimulate co-cultured CD4+ T cells from DO11.10 mice Tg for a H2-Ad–restricted, OVA-specific T cell receptor (Qiao et al., 2008). Despite higher expression of CD32aR (Fig. S1 b), the CD32aH variant induced significantly more IL-2 production by CD4+ T cells over a range of hIgG1WT IC concentrations (Fig. 2 d), which was significantly decreased in the context of hIgG1IHH ICs or hIgG1N297A ICs (Fig. 2 e). We next examined cross-presentation in cells endogenously expressing hFcRn but lacking any classical FcγRs. For this, we turned to a previously described HEK 293T cell line stably expressing the murine MHCI molecule H2-Kb (HEK 293TH2-Kb; Faure et al., 2009; Giodini et al., 2009), which we transfected with either CD32a variant (Fig. S2, d–f). CD32aH-expressing HEK 293TH2-Kb cells treated with hIgG1WT ICs, but not hIgG1IHH ICs, stimulated greater IL-2 (Fig. 2 f) or IFN-γ (Fig. S2 g) production by primary H2-Kb–restricted, OVA-specific CD8+ OT-I T cells compared with CD32aR-expressing or vector-transfected HEK 293TH2-Kb cells (Hogquist et al., 1994; Faure et al., 2009). Similarly, treatment of primary CD11c+MHCII+ DCs from CD32aR-Tg and CD32aH-Tg mice (Fig. S2, a–c) with hIgG1WT ICs stimulated IFN-γ secretion by CD8+ OT-I T cells, which was lost if the hIgG1 ICs were composed of hIgG1IHH or hIgG1N297A (Fig. 2 g). Thus, antigen presentation and cross-presentation depend on both CD32a and FcRn, with the CD32aH variant inducing greater FcRn-dependent adaptive immune responses relative to the CD32aR variant.
We next sought to more precisely delineate the role of FcRn relative to FcγRs in eliciting these adaptive immune responses. DCs and monocytes uniquely express high levels of FcRn on the cell surface (Fig. S2 b; Blumberg et al., 2019; Zhu et al., 2001), which allowed us to examine whether FcRn could mediate cross-presentation independently of FcγR under conditions that enable cell surface FcRn–IgG interactions (e.g., acidic pH; Kim et al., 1999; Medesan et al., 1997; Dickinson et al., 1999). To do so, we incubated hIgG1WT ICs with DCs derived from C57BL/6 mice lacking all classical FcγRs but maintaining mFcRn expression (Fcgr2b−/−Fcer1g−/−Fcgrt+/+, hereafter "FcγRKO"; Table S2 and Fig. S2, a–c). When FcγRKO DCs were loaded with hIgG1WT at pH 5.6, but not at neutral pH or with hIgG1IHH ICs, co-cultured CD8+ OT-I T cells were induced to secrete IFN-γ (Fig. 2 h), though at lower levels than when CD32a is present (Fig. 2 g). Next, we performed cross-presentation experiments with hIgG1 ICs composed of the well-characterized hIgG1MST/HN mutant that exhibits augmented FcRn binding at both neutral (equilibrium dissociation constant [KD] = 7.4 nM at pH 7.2) and acidic (KD = 1.2 nM at pH 6) pH relative to hIgG1WT and therefore can bind FcRn on the cell surface (Table S1; Vaccaro et al., 2005; Grevys et al., 2015). When FcγRKO DCs were loaded with hIgG1MST/HN ICs at pH 7.4, we observed robust IFNγ production by co-cultured CD8+ OT-I T cells (Fig. 2 i). Pretreatment with a monoclonal mouse h/mFcRn-blocking monoclonal antibody, DVN24 (Christianson et al., 2012), significantly diminished cross-presentation of hIgG1MST/HN IC-delivered antigen to CD8+ OT-I T cells (Fig. 2 i). Thus, IgG IC-driven innate and adaptive immune responses require FcRn and can occur in the absence of classical FcγRs, but maximal responses result when both FcRn and FcγRs are engaged.
Previous studies have evaluated IgG IC interactions with CD32a variants (Shashidharamurthy et al., 2009; Zhu et al., 2001), yet no studies have directly assayed the contribution of FcRn to CD32a variant–associated immune responses in human cells. We therefore extended our investigations to leukocytes from healthy human volunteers homozygous for CD32aR (FCGR2AG/G) or CD32aH (FCGR2AA/A). Whole-blood samples from these donors were stimulated in vitro with hIgG1WT ICs, and the production of proinflammatory cytokines (TNF-α, IL-12/23p40, and IL-6) was assessed (Blumberg et al., 2019). Innate cytokine secretion from these cells incubated with soluble OVANIP or monomeric hIgG was negligible (data not shown). While TNF-α, IL-12/23p40, and IL-6 levels trended higher in CD32aH than CD32aR cells upon treatment with hIgG1WT ICs (Fig. 3, a–c), both the CD32aR and CD32aH cells exhibited significantly decreased cytokine production in response to the hIgG1IHH ICs and hIgG1N297A ICs, indicative of FcRn and classical FcγR dependence, respectively. Furthermore, we compared the differences in the FcRn-dependent production of TNF-α, IL-12/23p40, and IL-6 (Fig. 3, d–f) upon treatment with these hIgG1 mutants relative to hIgG1WT ICs. Notably, we observed that the reduction of IL-6 as a consequence of disabling hIgG1 interactions with FcRn (hIgG1IHH ICs) or FcγR (hIgG1N297A ICs) was significantly more prominent in cells from CD32aH individuals in vitro, although absolute IL-6 production levels were not different between CD32aH- and CD32aR-expressing cells (Fig. 3, c and f). A similar trend was noted for IL-12/23p40 (Fig. 3, b and e). This suggests not only that are human leukocyte innate immune responses to hIgG1 ICs dependent upon FcRn and classical FcγRs but also that individuals who are homozygous for CD32aH may be more affected by a loss of FcRn function compared with CD32aR-homozygous individuals.
Given the heterogenous nature of FcRn- and FcγR-expressing cells in the peripheral blood, we next focused our attention on peripheral blood monocytes that express CD32a and FcRn and secrete innate cytokines when stimulated by IgG ICs (Pincetic et al., 2014; Bournazos et al., 2017; Blumberg et al., 2019; Zhu et al., 2001). We isolated CD14+ monocytes (Fig. S3, a and b) from the blood of the same cohort of healthy volunteers described above and assessed their responses to hIgG1 ICs. Strikingly, CD14+ monocytes from the CD32aH donors produced significantly more TNF-α and IL-12/23p40 in response to hIgG1WT ICs compared with CD14+ monocytes from CD32aR donors (Fig. 3, g and h). The IC-dependent IL-6 responses by CD14+ monocytes could not be assessed due to the high nonspecific responses observed during treatment with soluble OVANIP only (Fig. S3 c). Furthermore, in response to the hIgG1IHH and hIgG1N297A ICs, monocytes displayed significant reductions in TNF-α and IL-12/23p40 levels compared with the IgG1WT ICs irrespective of CD32aR or CD32aH status (Fig. 3, g and h). Still, the reductions in TNF-α and, to a lesser extent, IL-12/23p40 production from hIgG1IHH and hIgG1N297A mutant ICs were most prominent in cells from CD32aH-homozygous donors (Fig. 3, i and j). As observed with whole-blood samples, these effects were observed in the absence of overall differences in the responses of CD32aR and CD32aH donors to hIgG1N297A ICs (Fig. 3, g–j). Together, these data confirm that the CD32aH variant induces augmented inflammatory innate immune responses to hIgG1 ICs in human leukocytes compared with the CD32aR variant. Further, these results suggest that immune cells expressing CD32aH exhibit greater dependence on FcRn interactions, relative to CD32aR-expressing cells, which is consistent with increased formation of a ternary complex by the CD32aH variant.
We finally sought to determine whether FcRn can regulate IgG IC-mediated inflammation in an FcγR-dependent disease model. To do so, we examined the K/BxN serum transfer model of rheumatoid arthritis (Matsumoto et al., 1999; Monach et al., 2008). As the K/BxN model is primarily mediated by murine IgG1 autoantibodies which avidly bind CD32aR, but not CD32aH (Matsumoto et al., 1999; Monach et al., 2008; Mancardi et al., 2011; Clark et al., 1991; Tate et al., 1992), we focused on the CD32aR-Tg mouse model (Table S2). We first verified that mIgG1 ICs can form a ternary complex with mFcRn and CD32aR at acidic pH (Fig. S4, a and b). To investigate the importance of FcRn in this ternary complex in vivo, we adoptively transferred bone marrow from CD32aR-Tg mice lacking other classical FcγRs (Fig. S2, a–c) into irradiated WT C57BL/6 mice, followed by K/BxN serum injection in the presence of isotype IgG2a control treatment or FcRn blockade with the DVN24 antibody by the schedule described in Fig. 4 a. As FcRn blockade has been shown to decrease circulating IgG levels in mice and humans (Blumberg et al., 2019), we designed an anti-FcRn DVN24 antibody treatment regimen that did not affect circulating autoantibody levels (Fig. 4 b) in order to more precisely investigate FcRn’s role in regulating CD32a-mediated cellular immune responses to IgG ICs in vivo. Despite the lack of associated decreases in autoantibody levels, DVN24 antibody treatment significantly decreased inflammatory disease burden as assessed by clinical inflammatory scoring (Fig. 4, c and d), histopathologic arthritic inflammation (Fig. 4, e and f) and animal mobility (Fig. 4 g). These data demonstrate that inflammation derived from CD32aR-expressing bone marrow cells is dependent upon FcRn.
In summary, we show that CD32a and FcRn directly cooperate through formation of a ternary complex on an IgG Fc scaffold under acidic conditions, as occurs in intracellular organelles (Baker et al., 2011). This ternary complex formation could impact a wide range of FcγR-mediated processes, including IgG IC induction of thrombosis, which has been previously recognized as a FcγRIIa-mediated process that is regulated by and dependent upon FcRn (Cines et al., 2020). Furthermore, we demonstrate that FcRn can independently execute adaptive cellular immune responses to IgG ICs when FcRn can engage IgG on the cell surface, but that the presence of an FcγR coreceptor for IgG ICs significantly augments these responses. Our studies thus predict that enhanced FcRn cooperation with classical FcγR will amplify their responses to IgG ICs. In line with this, passive immunization in nonhuman primates using anti-HIV IgG antibodies engineered to possess stronger FcRn binding demonstrated increased passive immune protection from HIV that depends on CD8+ T cells (Nishimura et al., 2017; Gaudinski et al., 2018). Thus, while engineered IgG with enhanced FcRn binding exhibit increased half-life, which probably improves their effectiveness (Pyzik et al., 2019), these engineered IgG antibodies will likely also induce greater FcRn-dependent cellular immune responses than would be expected of otherwise identical IgG antibodies with “normal” affinity for FcRn. Similarly, augmentation of IgG IC interactions with FcRn enhances antitumor CD8+ T cell responses (Baker et al., 2013).
Our studies also highlight the importance of acidic environments in shaping FcRn- and classical FcγR-dependent immune responses to IgG ICs. For FcγRs specifically, this is consistent with a study which found that hIgG1 IC binding to nongenotyped human neutrophils increases as extracellular pH declines (López et al., 1999). Our data are also consistent with published crystal structures of hIgG1 Fc complexed with CD32aH and CD32aR, respectively, which show distinct homodimers under neutral and acidic conditions that are predicted to affect both interactions with IgG Fc and conceivably formation of a ternary complex with FcRn (Ramsland et al., 2011), as illustrated here. Such pH-dependent synergy between CD32a and FcRn may also evolve from events at the cell surface, given our demonstration that surface FcRn can mediate cross-presentation of hIgG1WT ICs independent of classical FcγR when APCs encounter ICs under acidic conditions. This suggests that engagement of FcRn on the cell surface in a ternary complex with classical FcγRs could also amplify cellular immune responses to IgG ICs. Therefore, both intracellular and extracellular acidic environments, such as found in endosomes (Baker et al., 2011) or disease-associated tissue acidosis (e.g., neoplasia and inflammation; Riemann et al., 2016; Lindner and Raghavan, 2009), respectively, may affect FcRn-dependent immune responses that are regulated by classical FcγRs.
Further, we have found that FcRn-dependent innate and adaptive immune responses to IgG ICs are more actively promoted in the setting of CD32aH relative to CD32aR. This occurred through the increased propensity of CD32aH to associate with hIgG1 ICs and foster recruitment of FcRn to form a ternary complex under acidic conditions. Thus, our data also highlight the importance of allele-specific differences in CD32a’s handling of hIgG1 ICs and its functional interactions with FcRn, thereby delineating a potential mechanism underlying CD32aH contributions to human autoimmune disease (Carcao et al., 2003; Zhang et al., 2016; Li et al., 2014; Lehrnbecher et al., 1999; Dijstelbloem et al., 2000).
The functional demonstration of CD32a–hIgG1 IC–FcRn ternary complex formation may further implicate FcRn in classical FcγR-mediated immune responses more broadly. As observed with CD32a, FcRn may similarly cooperate with other classical FcγRs such as the polymorphic CD16a and CD16b, which are associated with human disease and expressed together with FcRn in polymorphonuclear leukocytes (Pincetic et al., 2014; Wu et al., 1997; Morgan et al., 2000; Lee et al., 2015; Li et al., 2014). This suggests that the monomorphic FcRn acts as a pH-dependent coreceptor of the highly polymorphic classical FcγR system and in an allele-specific manner as shown here for CD32a. These studies have implications for current clinical trials of anti-FcRn and anti-CD32a therapies (Chen et al., 2019; Kiessling et al., 2017; Ulrichts et al., 2018; Ling et al., 2019; Nixon et al., 2015; Blumberg et al., 2019), as they suggest that the therapeutic efficacy of these agents may derive in part from their effects on FcRn and classical FcγR interactions associated with cellular immunity independent of circulating IgG levels.
Materials and methods
All experiments involving human volunteers were performed consistent with ethical guidelines and with the approval of the Clinical Research Ethics Board at the University of British Columbia (H13-03524 and H14-00622). All volunteer subjects provided written, informed consent for blood collection for DNA isolation and genotyping, immune cell isolation, and functional characterization, as described previously (Kozicky et al., 2018).
Animals and cell lines
Animal experiments were approved by the institutional care and use committees of Harvard Medical School and Brigham and Women’s Hospital (Boston, MA). Mice (Table S2) were housed in specific pathogen–free facilities. WT C57BL/6, C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I mice), and C.Cg-Tg(DO11.10)10Dlo/J mice were from The Jackson Laboratory. Fcgrt−/−, Fcgr2b−/−/Fcer1g−/− (FcγRKO), and CD32aR-Tg (FCGR2AR-Tg/Fcgr2b−/−/Fcer1g−/−) mice have been previously described (Roopenian et al., 2003; McKenzie et al., 1999; Smith et al., 2012), and the latter two strains were generously provided by Dr. Jeffery Ravetch (The Rockefeller University, New York, NY), who also provided the CD32aH-Tg mice, the generation of which is described below.
HEK 293T cells stably expressing the mouse MHC I molecule H2-Kb (HEK 293TH2-Kb) were kindly provided by Dr. Peter Cresswell (Yale University, Hartford, CT; Giodini et al., 2009). RAW 264.7 cell lines were from American Type Culture Collection (#TIB-71). The GM-CSF–secreting B16-F10 melanoma cell line was previously described and was a kind gift from Dr. Glenn Dranoff (Dana Farber Cancer Institute; currently at Novartis, Boston, MA; Mach et al., 2000). Primary APCs and T cells were grown in RPMI 1640 medium (Corning; #10-040) with 10% fetal bovine serum (Life Technologies; #26400044), 1% sodium pyruvate (Lonza; #13-115E), 1% antibiotics (penicillin-streptomycin; Thermo Fisher Scientific; #15070063), 1% nonessential amino acids (Thermo Fisher Scientific; #11140076), and 8.6 µM β-mercaptoethanol (Sigma-Aldrich; #M6250; hereafter “cRPMI”) at 37°C, 5% CO2. Human CD14+ monocytes were maintained in RPMI with 10% fetal bovine serum. B16-F10, HEK 293GFP-hFcRn, and HEK 293TH2-Kb, and all derived cell lines were grown in complete DMEM (Corning; #10-017) in an environment and with additives as for cRPMI plus Hepes 1% (Corning; #25-060) but without β-mercaptoethanol (hereafter “cDMEM”). Details of cloning and associated primers, as well as methods of transfection and transduction of CD32a variants into these cell types, are outlined below. All unique reagents and mouse strains generated for or described in this article will be readily available upon request, with applicable restrictions as outlined in associated material transfer agreements.
Proteins and reagents
NIP hapten-conjugated OVA (OVANIP) was from Biosearch Technologies (N-5041-10), with 11 NIP per OVA. The mAbs DVN24 and ADM31 were produced as described previously (Christianson et al., 2012). Isotype IgG2a control was from BioXCell (clone c1.18.4, #BE0085). Staining for flow cytometry was done using the following antibodies against mouse tissues: CD64 (clone x54-5/7.2; BioLegend; #139307), CD16 (clone 93; BioLegend; #101302), CD16.2 (clone 9E9; BioLegend; #149517), and 2Kb (clone AF6-88.5; BioLegend; #116511). Antibodies for staining hFcγR for flow cytometry were specific for CD64 (clone 10.1; BioLegend; #305005), CD32a (clone FUN-2; BioLegend; #303208), CD16 (clone 3G8; BioLegend; #302010) and other cell markers, including CD15 (clone SSEA-1; BioLegend; #301924), CD66b (clone G10F5; BioLegend; #305112), and CD14 (clone M5E2; BioLegend; #301842). Staining for m/hFcRn or hFcRn was performed using mAbs DVN24 or ADM31, respectively, conjugated to a fluorophore by custom conjugation service (BioLegend). Flow cytometry isotype controls included mIgG2a,κ (clone MOPC-173; BioLegend #400240), mIgG2b,κ (clone 27–35; BioLegend; #402204), mIgG1,κ (clone MOPC-21; BioLegend; #400150). All cell acquisition for flow cytometry experiments were performed on MACSQuant (Miltenyi Biotec) or CytoFLEX flow cytometers (Beckman Coulter) and data were analyzed using FlowJo software (TreeStar), gating on whole cells→single cells→live cells.
Generation of CD32aH-Tg mice
CD32aH-Tg mice were generated by recombineering in EL350 cells (gift from Neal Copeland, National Cancer Institute, Frederick, MD) as described previously (Lee et al., 2001; Kotzamanis and Huxley, 2004). Homology arms were amplified by PCR, (including 16 kb upstream of Exon 1 and 6 kb downstream of Exon 7 of the FCGR2AH gene), subcloned into a pBeloBAC vector (gift from Clare Huxley, Imperial College, London, UK; Kotzamanis and Huxley, 2004) and electroporated into EL350 cells (CTD-2514J12 positive; Invitrogen; #96012) capturing ∼37 kb genomic DNA containing the FCGR2A locus, as described previously (Lee et al., 2001). The presence of rs1801274_His allele of FCGR2A was confirmed by DNA sequencing. The resulting captured construct was linearized using Not1 restriction enzyme (New England Biolabs; #R0189S) and microinjected into the pronuclei of fertilized oocytes from C57BL/6 mice. Tg FCGR2AH+/− founders were mated with C57BL/6 mice and maintained on this background. The CD32aH-Tg strain of mice expressing the FCGR2AH transgene as the only FcγR was created by crossing FCGR2AH mice with FcγRKO mice, producing FCGR2AH/Fcgr2b−/−/Fcer1g−/−, or CD32aH-Tg mice.
Generation of CD32a variant–expressing cell lines
Full-length FCGR2AH cDNA was purchased from Origene (#SC112914). The CD32aR variant was generated via site-directed mutagenesis using the QuickChange II site-directed mutagenesis kit (Agilent Genomics; 200523) and the following overlapping primer pairs: forward primer, 5′-ATCCCAGAAATTCTCCCGTTTGGATCCCACCTTCT-3′; reverse primer, 5′-AGAAGGTGGGATCCAAACGGGAGAATTTCTGGGAT-3′. The cDNAs encoding for CD32aR and CD32aH were subcloned into a pcDNA3.1 vector and sequences verified by DNA sequencing. HEK 293TH2-Kb and RAW 264.7 cells were transfected with pcDNA3.1-CD32aR, pcDNA3.1-CD32aH or empty pcDNA3.1 vector using the Lipofectamine 2000 reagent (Life Technologies) or by electroporation using the Amaxa Cell line nucleofector Kit V (Lonza; #VACA-1003), respectively, and maintained under constant selection with 0.2 mg/ml hygromycin B (Hygromycin B Gold; Invivogen; #ant-hg-1). CD32aR- or CD32aH-transfected HEK 293TH2-Kb and RAW 264.7 were processed by FACS (BD FACSAria II SORP). Transient transfection with pcDNA3.1 vectors was performed by separately mixing Opti-MEM (Thermo Fisher Scientific; #31985070) with DNA (40 µg/ml) and polyethylenimine (PEI; 65 µg/ml) MAX (Polysciences; #24765-1), adding DNA-Opti-MEM to the PEI-Opti-MEM for 30 min at room temperature before incubation with cells, and addition of growth medium, which was changed after 24 h and maintained for 72 h before transfection analysis and experimentation.
Production of recombinant human and mouse FcRn, and anti-NIP IgG mutants
His-tagged soluble mouse and human forms of FcRn proteins were produced using an insect cell–based system, as described previously (Firan et al., 2001; Popov et al., 1996), and site-specifically biotinylated FcRn variants were purchased from Immunitrack (#ITF07 or ITF02). Anti-NIP IgG mutants, as summarized in Table S1, were mouse/human chimeric mAbs and detailed previously (Grevys et al., 2015). hIgG1WT and derived mutants were produced by transient transfection of a vector containing the heavy chain gene from hIgG1 cotransfected with a vector encoding the mouse λ light chain, both with specificity for NIP, or from a stably transfected J558L cell line, as previously described (Grevys et al., 2015; Qiao et al., 2008). The IgG antibodies were purified either by affinity chromatography with anti-mouse λ L chain CaptureSelect resin (Thermo Fisher Scientific; #194323005), anti-hIgG-CH1 CaptureSelect column (Thermo Fisher Scientific; #494320001), or a column coupled with NIP followed by size exclusion chromatography using Superdex 200 10/300 column (GE Healthcare; #17517501).
ELISA was used to assess IgG IC binding to CD32a variants. Site-specific biotinylated monomeric human CD32aR and CD32aH (Sino Biological; #10374-H27H1-B-50 and 10374-H27H-B-50) were captured overnight on neutravidin-coated ELISA plates, blocked for 1 h at room temperature with 5% skim milk in PBS, washed with a pH 5.6 buffer (164 mM KH2PO4, 13 mM Na2HPO4 7H2OM NaCl, and 0.05% Tween [PBS-T], pH 5.6), and assayed at pH 5.6. Serial dilutions of ICs containing the anti-NIP IgGs (1.67–214.3 nM) in complex with OVANIP (Biosearch Technologies; #N-5041-100) at a ratio of 2:1 were added to the wells, incubated for 1 h, and washed at pH 5.6. The amount of IgG bound by CD32a variants was measured at pH 5.6 using peroxidase-conjugated goat anti-IgG Fc F(ab′)2 (Jackson ImmunoResearch; #109-036-008) diluted 1:1,000.
To assess IgG IC bridging of CD32a and FcRn, CD32aH and CD32aR were captured (4 µg/ml) on ELISA plates and incubated with titrated amounts of IgG ICs (1.7–214 nM) as described above. For hIgG ICs, biotinylated CD32a variants were captured on neutravidin-coated ELISA plates as above. For mIgG1 ICs, unconjugated CD32a variants (Sino Biological; #10374-H08H1-100 and 10374-H08H-100) were coated directly on ELISA plates overnight. Plates were then washed and blocked as above. His-tagged hFcRn or biotinylated mFcRn (10 µg/ml; Immunitrack; #ITF07) was added and incubated for 1 h at pH 5.6 with 2 µg/ml alkaline phosphatase (ALP)–conjugated anti-hFcRn nanobody for hIgG1 IC (Nb218-H4, which binds hFcRn at acidic pH and does not interfere with IgG binding; Andersen et al., 2013) or Streptavidin-HRP for mIgG1 ICs. After washing with PBS-T, pH 5.6, bound receptor was detected by adding 100 µl p-nitrophenyl phosphate (Thermo Fisher Scientific; #37620), or 3,3′,5,5′-tetramethylbenzidine substrate (Sera Care; #5120) for ALP and HRP, respectively. Protein concentrations were measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). ELISA plates were analyzed using a VERSAmax microplate reader (Molecular Devices).
Superimposition of the hFcRn-hIgG1 Fc crystal structure (PDB accession number 4N0U; Oganesyan et al., 2014) and hIgG1 Fc-CD32aR complex (PDB accession number 3RY6; Ramsland et al., 2011) with root mean square deviation of 1.378 Å was performed using PyMol (DeLano Scientific), yielding an hFcRn-hIgGFc-CD32aR ternary complex structural model.
IgG IC formation
For OVANIP antigen presentation and cross-presentation studies, innate immune stimulation, and ELISA experiments, anti-NIP IgG ICs were pre-formed in buffer or serum-free RPMI (for primary APCs) or DMEM (all others) at 37°C for 1 h, mixing every 15 min, and using 100 µg/ml of recombinant anti-NIP hIgG1 mutants and 0.5–20 µg/ml OVANIP, as specified.
RAW 264.7 cells were seeded onto sterile glass coverslips (12 mm, #1.0) coated with 0.1 mM poly-L-lysine, at 5 × 105 cells/ml and incubated overnight at 37°C, 5% CO2. ICs were formed in serum-free DMEM by complexing 0.1 mg/ml hIgG1 (hIgG1κ; Sigma-Aldrich; #15154) with 0.05 mg/ml Dylight 594–conjugated goat F(ab′)2 anti-human F(ab′)2 IgG (Jackson ImmunoResearch; #109-516-006) for 60 min at 37°C. Cells were washed with serum-free DMEM, treated with IC-containing DMEM for 15 min at 37°C and 5% CO2, and then washed with ice-cold PBS and fixed with 3% paraformaldehyde. Permeabilization and blocking was performed with PBS containing 0.1% saponin wt/vol, 1% BSA, and 5% goat serum for 1 h at room temperature. Primary antibody incubation occurred at 4°C overnight using antibodies specific for the cytoplasmic tails of CD32a (5 µg/ml mouse mAb clone 11B6; EMD Millipore; #mabf841) and rat/mFcRn (8 µg/ml rabbit polyclonal antibody made in-house). Goat anti-mouse and goat anti-rabbit secondary antibody PLA probes conjugated to complementary oligonucleotides (Sigma-Aldrich; #DUO92011 and #DUO92013, respectively) were then applied. Ligation of PLA probes in adequate proximity to form a closed, circular DNA template containing repeating sequences was performed followed by DNA amplification. After amplification, FarRed-labeled detection oligo probes (Sigma-Aldrich; #DUO92013) complementary to the repeated sequences were applied per instructions supplied by the manufacturer, except that the PLA probe incubation step was shortened from 60 to 30 min. Coverslips were mounted in antifade mounting medium containing DAPI (Invitrogen; #P36935). Images were acquired at 63× magnification under glycerin immersion using an inverted DMi6000 microscope (Leica) equipped with a CSU-X1 Yokogawa spinning disk, ZYLA SL150 sCMOS camera (Andor), with image analysis and overlay performed with ImageJ version 2.0.0-rc-68/1.52h, with Fiji plugins (https://imagej.net/Fiji/Downloads).
As FcγRs, including CD32a, and FcRn inherently contain binding sites for hIgG1 Fc and bind avidly to IgG from multiple species, we sought a system that would enable selective, Fc-free immunoprecipitation of the ternary complex. We therefore turned to previously published HEK 293hFcRn-GFP cells expressing full-length hFcRn with N-terminal GFP tag (Christianson et al., 2012), which could be immunoprecipitated using magnetic beads coated with anti-GFP high-affinity nanobodies, which lack a Fc region (Rothbauer et al., 2008) and therefore avoid CD32a binding to the immunoprecipitating agent per se. Accordingly, HEK 293hFcRn-GFP cells were transiently transfected using PEI-MAX transfection reagent and pcDNA3.1 vectors expressing either CD32aR of CD32aH (Genscript; #OHu27189D; CD32aR-containing vector was custom prepared by GenScript, introducing a A500G base substitution using OHu27189D as template). 3 d after transfection, CD32a transfection was assessed by flow cytometry and cells were treated for 15 min with hIgGWT or hIgGIHH ICs formed as described above for PLA but using an unconjugated anti–hIgG-Fc F(ab′)2 (Jackson ImmunoResearch; #109-006-006) and anti-NIP hIgG1WT or hIgG1IHH previously conjugated to a disulfide-containing, UV-activated, 13.5-Å, N-hydroxysuccinimide diazirine cross-linker (Thermo Fisher Scientific; #26169) per the manufacturer’s instructions. After IgG IC treatment, cells were washed with ice-cold PBS, exposed to direct UV light (380 nm) on ice for 30 min, and then lysed with ice-cold pH 8.0 CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate) lysis buffer containing 0.5% (wt/vol) CHAPS, 5% (vol/vol) glycerol, 150 mM NaCl, 2 mM CaCl2, 25 mM Tris-HCl, pH 8.0, and HALT Protease and Phosphatase Inhibitor (Thermo Fisher Scientific; #78442). Insoluble material was removed by centrifugation, protein content was quantified by bicinchoninic acid assay (Thermo Fisher Scientific; kit #23227), and immunoprecipitation of hFcRnGFP from lysate containing 0.5 mg protein was performed using GFP-Trap Magnetic Agarose (ChromoTek; #grma-20) or empty control agarose (data not shown; ChromoTek; #bmab-20) per the manufacturer’s protocol, washing with pH 7.4 wash buffer (0.01 M Tris-HCl, 0.15 M NaCl, and 0.5 mM EDTA). Anti-GFP magnetic agarose-bound proteins were eluted with Laemmli buffer containing β-mercaptoethanol at 95°C for 10 min. The eluate was resolved on 4–20% Tris-Glycine SDS-PAGE gel (Thermo Fisher Scientific; #XP04205BOX), followed by wet transfer to a nitrocellulose membrane. After membrane blocking with Odyssey Blocking Buffer (TBS; LI-COR; #927-50100) for 1 h at room temperature, immunoblotting was performed for hFcRn (Sigma-Aldrich; #HPA012122) and CD32a (clone 11B6) at 1:4,000 and 1:1,000 dilutions, respectively, in Odyssey Blocking Buffer 0.1% Tween 20 overnight at 4°C. Multiplex detection of hFcRn and CD32a was by a IRDye 800CW- or IRDye 680RD-conjugated antibodies (LI-COR; #926-32213 and #926-68072, respectively). Near-infrared–based imaging and densitometry quantification was performed using a LI-COR Odyssey Fc Imaging System running Image Studio version 5.6 (LI-COR).
Innate and adaptive APC stimulation with IgG ICs
APCs (RAW 264.7 cells, HEK 293TH2-Kb cells, or primary DCs; 5 × 104 cells/well) stably expressing CD32aR, CD32aH, or pcDNA3.1 control vector were incubated in 96-well plates in serum-free RMPI containing IgG ICs prepared as above at indicated concentrations for 3 h at 37°C. After washing, cells were co-cultured with 105 OVA-restricted T cells in cRPMI per well using CD8+ T cells for cross-presentation and CD4+ T cells for presentation experiments. Specifically, CD8+ OT-I T cells from mice Tg for a T cell receptor recognizing OVA257–264 peptide in the context of MHCI H-2b (expressed by CD11c+MHCII+ DCs and HEK 293TH2-Kb) or CD4+ T from DO11.10 mice Tg for a T cell receptor recognizing OVA323–339 peptide in the context of the MHCII H-2d (expressed by RAW 264.7) were purified from spleens and peripheral LNs by magnetically activated cell sorting using CD8α+ T cell Isolation kit (Miltenyi Biotec; #130-104-075) or CD4+ T cell Isolation kit (Miltenyi Biotec; #130-104-454), respectively. The cells were co-cultured with IC-stimulated APCs in cRPMI and supernatant collected at 24 or 48 h. The levels of IL-2 and/or IFN-γ in the co-culture supernatant were quantified by ELISA according to the manufacturer’s instructions (BD Biosciences; #555148 or 555138, respectively). For primary cell studies, CD11c+MHCII+ DCs were purified rom spleen by magnetically activated cell sorting in two steps, first using negative selection (CD19 MicroBeads; Miltenyi; #130-052-201) followed by positive selection (CD11c MicroBeads UltraPure; Miltenyi; #130-108-338), from CD32aH-Tg, CD32aR-Tg, or FcγRKO mice that had been inoculated subcutaneously with 5 × 106 GM-CSF–secreting B16-F10 melanoma cells 2 wk before spleen harvest, as described previously (Baker et al., 2013; Mach et al., 2000). For FcRn inhibition experiments, DCs were pretreated for 30 min with the indicated concentrations of the isotype IgG2a control or DVN24 before IC exposure.
For innate immune response assessment, murine splenic CD11c+MHCII+ DCs plated at 5 × 104 cells per well in 96-well plates were treated (or untreated) for 24 h with 100 µg/ml anti-NIP hIgG1 mutants in monomeric or IC form with or without OVANIP (1 µg/ml), formed as above. Mouse TNF-α, IL-12/23p40, and IL-6 levels in clarified supernatant were quantified by ELISA kits according to the manufacturer’s instructions (BD Biosciences; #555165, 558534, or 555240, respectively).
For studies of primary human APC, DNA was isolated from blood collected from volunteer human blood donors and genotyped for FCGR2A SNP rs1801274 by a Taqman assay (Thermo Fisher Scientific; #C_9077561_20) according to the manufacturer’s instructions as described (Kozicky et al., 2018). Blood for functional studies was collected in heparinized vials and either plated immediately or separated by density gradient using Mono-Poly medium (MP Biomedicals; #1698049). CD14+ cells were isolated from the upper Mono-Poly fraction by magnetic separation using EasySep Human CD14 Positive Selection Kit II (StemCell; #17858), according to the manufacturer’s instructions. Both whole-blood and CD14+ monocytes were stimulated as described above for murine cells (Blumberg et al., 2019), and TNF-α, IL-12/23p40, and IL-6 were quantified in clarified supernatant using ELISA kits according to the manufacturer’s instructions (BD Biosciences; #555171, 555212, or 555220, respectively).
K/BxN rheumatoid arthritis model
Rheumatoid arthritis was induced and assessed as described previously (Monach et al., 2008) in bone marrow chimeric mice prepared by adoptive transfer of bone marrow from sex-matched CD32aR-Tg donor mice to irradiated WT C57BL/6 recipient mice. mAb isotype IgG2a control or DVN24 (0.2 mg in 0.2 ml) was administered i.p. daily beginning on day −1 through day 5 from K/BxN sera injection (Fig. 4 a). Of note, the DVN24 dose employed in these experiments reflects its ∼17-fold lower affinity for mFcRn versus hFcRn (Sand et al., 2014). Therefore, these experiments required a proportionately higher DVN24 dose relative to that expected to be efficacious in the setting of hFcRn. After 1 wk (day 7), mobility was assessed by the cylinder test or the number side touches per mouse per minute in a 1-liter glass beaker (Brooks and Dunnett, 2009). Histopathological scoring of rear ankle joint inflammation and bone erosion on day 12 was by a blinded pathologist (J.N. Glickman) as described previously (Pettit et al., 2001). Escherichia coli expressing recombinant glucose-6-phosphate isomerase (GPI) were kindly provided by Dr. Christopher Benoist (Harvard Medical School, Boston, MA), and GPI production, purification, and quantification of circulating anti-GPI IgG by ELISA was performed as previously described (Monach et al., 2008).
Prism for Mac OS X version 8.2.1 (GraphPad Software) was used for statistical analysis. Analysis of ELISA binding curves was by nonlinear regression using variable slope (agonist) versus response equation for least squares fitting, with EC50 confidence intervals calculated using the likelihood ratio asymmetric method, goodness of fit assessed by R2, and extra sum-of-squares F test to detect differences between the EC50 values associated with the resulting best-fit curves. Nonnormal data were processed by loge transformation and resulting data evaluated for a normal distribution by inspection of homoscedasticity and QQ plots. Comparisons of two unpaired groups was made by a two-tailed Student’s t test for normal data or Mann–Whitney test for nonnormal data. For three or more groups with two parameters, two-way ANOVA was used as appropriate for data with a normal distribution, or a matched Friedman test was employed for nonnormally distributed data. Analysis of responses to multiple stimuli of a single cell type or of cells from a single donor were analyzed using paired tests. Post-hoc analysis to correct for multiple comparisons and detect differences between groups was by the two-stage linear step-up procedure of Benjamin, Krieger, and Yekutieli with a false discovery rate (FDR) < 0.05. A two-sided probability (P) of α error < 0.05 defined significance.
Online supplemental material
Fig. S1 (related to Fig. 1) shows control ELISA and PLA experiments, as well as flow cytometry assessments of transfection efficiency. Fig. S2 (related to Fig. 2) shows FcRn and CD32a transgene expression measured by flow cytometry and cellular activation data supportive of data in Fig. 2. Fig. S3 (related to Fig. 3) shows flow cytometric assessments of primary human cell preparation purity. Fig. S4 (related to Fig. 4) shows ELISA of ternary complex formation with CD32a, mFcRn, and mIgG1. Table S1 shows mutations defining anti-NIP hIgG1 mutants, with a summary of qualitative, relative pH-dependent binding strengths to FcRn and low-affinity FcγRs. Table S2 shows a summary of Tg mouse strains utilized in these studies.
The authors thank Victoria M. Thiele, Garrett Hauck, Thomas Hanley, Arianna Degruttola, Samantha Torquato, Mario Sablon, Victoria G. Aveson, and Anh Do for excellent technical assistance and Drs. Nitesh Shashikanth and Jerrold Turner for expert assistance with and access to confocal microscopy equipment. The authors also thank Jennifer Cusick for managerial assistance and Dr. Amanjot Riar for technical and managerial assistance. Dr. Jeffrey Ravetch (The Rockefeller University, New York, NY) generated and very generously shared the CD32aR-Tg and CD32aH-Tg mice. H2-Kb–expressing HEK 293T cells were a generous gift of Dr. Peter Cresswell (Yale University, Harford, CT). E. coli expressing recombinant GPI were kindly provided by Dr. Christopher Benoist (Harvard Medical School, Boston, MA).
This work was funded by Deutsche Forschungsgemeinschaft (RA 2040/1-1; T. Rath), the Canadian Institutes of Health Research (K. Baker, M. Pyzik, and L.K. Kozicky) and Canadian Institutes of Health Research/Canadian Blood Services project grant (CIHR2016-LS) to L.M. Sly, the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition Foundation Fellow to Faculty Transition Award NF-17-100 (J.J. Hubbard), the Crohn’s and Colitis Foundation Research Fellowship Award 511426 (J.J. Hubbard), the National Institutes of Health grants DK53056, DK053162, DK088199, DK044319 (R.S. Blumberg), and 1F32AI131511 (J.J. Hubbard), and the Harvard Digestive Diseases Center National Institutes of Health grant P30DK034854 (R.S. Blumberg). J.J. Hubbard was supported by the National Institutes of Health T32 grant DK007477 through the Division of Gastroenterology and Nutrition at Boston Children’s Hospital. I. Sandlie and J.T. Andersen were supported in part by the Research Council of Norway through its Centre of Excellence funding scheme (project 179573), the Research Council of Norway (grants 230526, 179573, and 287927), and South-Eastern Norway Regional Health Authority (grant 40018). K.M.K. Sand was supported by the University of Oslo, The U.S.A.-Norway Fulbright Foundation for Educational Exchange, and University of Oslo Life Science.
Author contributions: Conceptualization: J.J. Hubbard, M. Pyzik, T. Rath, L.K. Kozicky, K.M.K. Sand, J.T. Andersen, L.M. Sly, D.C. Roopenian, K. Baker, R.S. Blumberg. Methodology: J.J. Hubbard, M. Pyzik, T. Rath, L.K. Kozicky, K.M.K. Sand, A.K. Gandhi, A. Grevys, S. Foss, S.C. Menzies, J.T. Andersen, J.N. Glickman, I. Sandlie, L.M. Sly, K. Baker, and R.S. Blumberg. Validation: J.J. Hubbard, M. Pyzik, T. Rath, K.M.K. Sand, L.K. Kozicky, S.C. Menzies, J.T. Andersen, K. Baker, and R.S. Blumberg. Formal analysis: J.J. Hubbard, M. Pyzik, T. Rath, L.K. Kozicky, K.M.K. Sand, J.T. Andersen, and J.N. Glickman. Investigation: J.J. Hubbard, M. Pyzik, L.K. Kozicky, K.M.K. Sand, T. Rath, J.T. Andersen, K. Baker, S. Foss, and A. Grevys. Resources: J.T. Andersen, L.M. Sly, I. Sandlie, D.C. Roopenian, and R.S. Blumberg. Writing (original draft): J.J. Hubbard, M. Pyzik, L.K. Kozicky, K.M.K. Sand, L.M. Sly, K. Baker, and R.S. Blumberg. Writing (review and editing): J.J. Hubbard, M. Pyzik, T. Rath, L.K. Kozicky, K.M.K. Sand, J.T. Andersen, A.K. Gandhi, A. Grevys, E. Fiebiger, D.C. Roopenian, I. Sandlie, L.M. Sly, K. Baker, R.S. Blumberg. Visualization: J.J. Hubbard, M. Pyzik, L.K. Kozicky, K.S.M., T. Rath, J.T. Andersen, A.K. Gandhi, A. Grevys, I. Sandlie, K. Baker, and R.S. Blumberg. Supervision, L.M. Sly, I. Sandlie, K. Baker, and R.S. Blumberg. Project administration, K. Baker and R.S. Blumberg. Funding acquisition, R.S. Blumberg (primarily) but also L.M. Sly, J.J. Hubbard, M. Pyzik, T. Rath, K.M.K. Sand, J.T. Andersen, L.K. Kozicky, and K. Baker.
Disclosures: J.J. Hubbard reported a patent to US2019/017880 pending. M. Pyzik reported a patent to PCT/US2019/017880 pending, "Brigham and Women's Hospital." T. Rath reported a patent to 61/984,652 licensed and a patent to 61/909,229 licensed. A.K. Gandhi reported a patent for therapeutic FcRn-based bispecific monoclonal antibodies pending (PCT/US2019/107880). D.C. Roopenian reported a patent to US20190135915A1 licensed, "Alexion Pharmaceuticals"; and served as consultant with equity interests in Syntimmune Inc., a company developing therapeutic agents to target FcRn. Syntimmune is now a wholly owned subsidiary of Alexion Pharmaceuticals, Inc., following its acquisition by Alexion in November 2018. I. Sandlie has served as a consultant with equity interests in Syntimmune Inc., a company developing therapeutic agents to target FcRn. Syntimmune is now a wholly owned subsidiary of Alexion Pharmaceuticals, Inc., following its acquisition by Alexion in November 2018. K. Baker reported a patent to 61/984,652 issued and a patent to 61/909,229 issued. R.S. Blumberg served as consultant with equity interests in Syntimmune Inc., a company developing therapeutic agents to target FcRn. Syntimmune is now a wholly owned subsidiary of Alexion Pharmaceuticals, Inc., following its acquisition by Alexion in November 2018. In addition, R.S. Blumberg has a pending patent PCT/US2019/017880 to “BWH" and two patents licensed to "Alexion" (US2017/002073 and US2017/0045528). No other disclosures were reported.
J.J. Hubbard and M. Pyzik contributed equally to this paper.
K. Baker and R.S. Blumberg contributed equally to this paper.
T. Rath’s present address is Department of Medicine, Division of Gastroenterology, Erlangen University Hospital, Friedrich Alexander University Erlangen-Nürnberg, Erlangen, Germany.
K. Baker’s present address is Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.