A Double-Edged Kinase Lyn: A Positive and Negative Regulator for Antigen Receptor–mediated Signals

B cells from young lyn^−/− mice are hyperresponsive to anti-IgM–induced proliferation, suggesting involvement of Lyn in negative regulation of B cell antigen receptor (BCR)-mediated signaling. Here we show that tyrosine phosphorylation of FcγRIIB and CD22 coreceptors, which are important for feedback suppression of BCR-induced signaling, was severely impaired in lyn^−/− B cells upon their coligation with the BCR. Hypophosphorylation on tyrosine residues of these molecules resulted in failure of recruiting the tyrosine phosphatase SHP-1 and inositol phosphatase SHIP, SH2-containing potent inhibitors of BCR-induced B cell activation, to the coreceptors. Consequently, lyn^−/− B cells exhibited defects in suppressing BCR-induced Ca²⁺ influx and proliferation. Thus, Lyn is critically important in tyrosine phosphorylation of the coreceptors, which is required for feedback suppression of B cell activation.     

Differential Effects of B Cell Receptor and B Cell Receptor–FcγRIIB1 Engagement on Docking of Csk to GTPase-activating Protein (GAP)-associated p62

The stimulatory and inhibitory pathways initiated by engagement of stimulatory receptors such as the B cell receptor for antigen (BCR) and inhibitory receptors such as Fcγ receptors of the IIB1 type (FcγRIIB1) intersect in ways that are poorly understood at the molecular level. Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcγRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins. Stimulation of a B lymphoma cell line, A20, with intact anti-IgG antibody induced a direct, SH2-mediated association between Csk and a 62-kD phosphotyrosine-containing protein that was identified as RasGTPase-activating protein–associated p62 (GAP-A.p62). In contrast, stimulation of A20 cells with anti-IgG F(ab′)₂ resulted in little increase in the association of Csk with GAP-A.p62. The effect of FcγRIIB1 engagement on this association was abolished by blockade of FcγRIIB1 with the monoclonal antibody 2.4G2. Furthermore, the increased association between Csk and GAP-A.p62 seen upon stimulation with intact anti-Ig was abrogated in the FcγRIIB1-deficient cell line IIA1.6 and recovered when FcγRIIB1 expression was restored by transfection. The differential effects of BCR and BCR-FcγRIIB1–mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor–mediated signals in B cells.     

T Cell Development in Mice Lacking All T Cell Receptor ζ Family Members (ζ, η, and FcεRIγ)

The ζ family includes ζ, η, and FcεRIγ (Fcγ). Dimers of the ζ family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking ζ/η chains, T cell development is impaired, yet low numbers of CD4⁺ and CD8⁺ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a ζ family dimer can promote T cell maturation, or that in the absence of ζ/η, Fcγ serves as a subunit in TCR complexes. To elucidate the role of ζ family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only ζ/η or Fcγ. The data reveal that surface complexes that are expressed in the absence of ζ family dimers are capable of transducing signals required for α/β–T cell development. Strikingly, T cells generated in both ζ/η^−/− and ζ/η^−/−–Fcγ^−/− mice exhibit a memory phenotype and elaborate interferon γ. Finally, examination of different T cell populations reveals that ζ/η and Fcγ have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of ζ family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.     

Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies

Fc γ receptor (FcγR) coengagement can facilitate antibody-mediated receptor activation in target cells. In particular, agonistic antibodies that target tumor necrosis factor receptor (TNFR) family members have shown dependence on expression of the inhibitory FcγR, FcγRIIB. It remains unclear if engagement of FcγRIIB also extends to the activities of antibodies targeting immunoregulatory TNFRs expressed by T cells. We have explored the requirement for activating and inhibitory FcγRs for the antitumor effects of antibodies targeting the TNFR glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18; CD357) expressed on activated and regulatory T cells (T reg cells). We found that although FcγRIIB was dispensable for the in vivo efficacy of anti-GITR antibodies, in contrast, activating FcγRs were essential. Surprisingly, the dependence on activating FcγRs extended to an antibody targeting the non-TNFR receptor CTLA-4 (CD152) that acts as a negative regulator of T cell immunity. We define a common mechanism that correlated with tumor efficacy, whereby antibodies that coengaged activating FcγRs expressed by tumor-associated leukocytes facilitated the selective elimination of intratumoral T cell populations, particularly T reg cells. These findings may have broad implications for antibody engineering efforts aimed at enhancing the therapeutic activity of immunomodulatory antibodies.Activating Fc γ receptors (FcγRs) stimulate immune cell effector mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP), which combine to facilitate antibody-mediated tumor cell killing (Nimmerjahn and Ravetch, 2008; Hogarth and Pietersz, 2012). The importance of FcγR-mediated immune effector cell function has been demonstrated in preclinical efficacy studies for antibodies targeting a range of tumor cell–expressed receptors, including trastuzumab (HER2) and rituximab (CD20; Clynes et al., 2000; Nimmerjahn and Ravetch, 2012). The inhibitory FcγR, FcγRIIB, functions to modulate activating FcγR-mediated effector mechanisms in immune cells that coexpress both FcγR classes, such as macrophages and dendritic cells. FcγRIIB has recently been implicated in augmenting antibody-mediated receptor forward signaling through a mechanism of cross-linking in target cells expressing the TNF receptor (TNFR) family members TNFRSF10, TNFRSF10B (DR4 and DR5, respectively), and TNFRSF5 (CD40; Wilson et al., 2011; Li and Ravetch, 2012). It remains unclear what contribution FcγR biology has in the modality of antibody therapeutics that target other cell surface receptors. In particular, the emerging clinical benefit of agonistic antibodies targeting the T cell–APC interface raises the possibility that FcγR coengagement may contribute to their in vivo mechanism of action (Mellman et al., 2011).Preclinical studies in mice using agonistic antibodies targeted to glucocorticoid-induced TNFR-related protein (GITR)—a costimulatory TNFR expressed by regulatory and activated T cells—have shown compelling antitumor activity in syngeneic mouse tumor models (Turk et al., 2004; Ko et al., 2005). In vitro, stimulation of GITR with agonist antibodies can induce forward signaling into T cells, which promotes proliferation and cytokine production (Kanamaru et al., 2004; Ronchetti et al., 2007). In vivo, several mechanisms have been proposed to contribute to the antitumor activity of antibodies targeting GITR; however, the current paradigm stipulates that agonist properties of these antibodies promotes cytotoxic effector T cell generation, while dampening the immunosuppressive effects by FoxP3⁺ CD4⁺ T reg cells (Ronchetti et al., 2012; Schaer et al., 2012). The recent findings that antibodies targeted to TNFR family members require FcγRIIB interaction for their in vivo activities led us to explore a common mechanism for antibodies targeting TNFRs expressed by T cells, using GITR to test this paradigm.     

A Critical Role for Syk in Signal Transduction and Phagocytosis Mediated by Fcγ Receptors on Macrophages

Receptors on macrophages for the Fc region of IgG (FcγR) mediate a number of responses important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcγR cross-linking. Macrophages derived from Syk-deficient (Syk⁻) mice were defective in phagocytosis of particles bound by FcγRs, as well as in many FcγR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast, Syk⁻ macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected. Syk⁻ macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcγRs (actin cups), but failed to proceed to internalization. Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcγR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcγR-mediated phagocytosis. Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcγR engagement, accompanied by a delay in FcγR-mediated phagocytosis. These observations demonstrate that Syk is critical for FcγR-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcγR's analogous to models of signaling by the B and T cell antigen receptors.     

Functional Separation of Pseudopod Extension and Particle Internalization during Fcγ Receptor–mediated Phagocytosis

Receptors for the Fc portion of immunoglobulin (Ig)G (FcγR) mediate phagocytosis of IgG-opsonized particles by a process that can be divided into four major steps: receptor–ligand binding, pseudopod extension, internalization, and lysosomal fusion. We have expressed single classes of FcγR in COS fibroblasts to examine the structural determinants necessary to complete the four steps of phagocytosis. Using phase contrast, fluorescence, confocal, and electron microscopy we have demonstrated that FcγR-expressing COS cells can phagocytose in a manner similar to that of professional phagocytes. We have further analyzed the capacity of the three classes of FcγR to phagocytose, placing special emphasis on the FcγRIA–γ chain complex, which allowed us to examine independently the roles of the ligand-binding unit (FcγRIA) and the signaling unit (γ chain). We found that receptor complexes containing a conserved tyrosine activation motif (ITAM), as found in the cytoplasmic domain of FcγRIIA and in the γ chain associated with FcγRIA and FcγRIIIA, readily internalized target particles. In contrast, FcγRIA alone, having no ITAM, was unable to internalize target particles efficiently, but did mediate pseudopod extension. Cotransfection of γ chain with FcγRIA restored the ability of the receptor to internalize target particles. A mutant FcγRIA in which the cytoplasmic domain had been deleted was also capable of mediating pseudopod extension, showing that neither the γ chain nor the cytoplasmic domain of FcγRIA were required for this step. Cytochalasin D, an inhibitor of actin polymerization, blocked particle internalization by all FcγR, but did not block pseudopod extension. Staining the FcγRIA COS cells for F-actin and for tyrosine phosphoproteins, we found that actin did not polymerize during FcγRIA-mediated pseudopod extension, nor were tyrosine kinases activated. Our data suggest that pseudopod extension and internalization are functionally distinct steps mediated through different pathways.Fc receptor for IgG (FcγR)-mediated phagocytosis is the process whereby IgG-coated particulate pathogens are internalized and moved to lysosomes for degradation. The mechanism requires fine coordination of the action of a variety of molecules including cell membrane receptors, signaling molecules, and cytoskeleton components. The receptors initiate the process by a zipper mechanism whereby sequential interaction of particle-bound IgG with phagocyte FcγR stimulates the spreading of pseudopods around the particle resulting in its eventual engulfment (1). Complete circumferential coating of the target particle by ligand is required, and FcγR additional to those involved in the initial binding of the particle are recruited during the process (2).Several characteristics of the intracellular mechanism of FcγR-mediated phagocytosis have been described. Engulfment of attached particles requires actin polymerization as demonstrated by the blockade of phagocytosis by inhibitors of actin polymerization such as cytochalasin D. Along with F-actin, tyrosine phosphoproteins accumulate beneath attached IgG-coated particles before FcγR-mediated internalization. Both F-actin accumulation and particle internalization can be blocked by tyrosine kinase inhibitors, indicating that the signal pathway requires tyrosine kinases (3). Indeed, several nonreceptor tyrosine kinases of the src and syk family have been coisolated with FcγR, and their activities have been shown to increase after FcγR clustering (4–6). Other signaling molecules such as protein kinase C and phosphoinositide-3 kinase have also been implicated in FcγR-mediated phagocytosis (7, 8).FcγR comprise a family of integral membrane glycoproteins with three standard components, i.e., an extracellular ligand-binding portion consisting of two or three Ig-like domains, a short hydrophobic transmembrane region, and a cytoplasmic tail. In humans, three classes (I, II, and III) of these receptors have been characterized, differing in fine structure and affinity for IgG, and within these classes a total of eight subclasses (A, B, C) exist, each encoded by a separate gene (9). Of the eight FcγR in humans, three have convincingly been shown to mediate phagocytosis (FcγRIA, FcγRIIA, and FcγRIIIA; reference 10). Two of these three associate noncovalently with a common subunit, γ chain. A common amino acid sequence is present in the γ chain associating with FcγRIA and FcγRIIIA and in the cytoplasmic tail of FcγRIIA. This conserved motif, designated immunoreceptor tyrosine activation motif (ITAM)¹, links receptor clustering to the activation of tyrosine kinases (11– 13). Upon FcγR clustering, the tyrosines of the ITAM are phosphorylated (6, 14, 15) and, in turn, serve as docking sites for signaling proteins containing src homology 2 domains (16, 17).Analyzing the phagocytic mechanism of a single FcγR class has been difficult because monocytes and macrophages generally express multiple classes of FcγR. Therefore, structure–function studies of individual receptor classes have required expression of single FcγR classes into FcγR negative cells such as COS cell fibroblasts. This approach has allowed analysis of the phagocytic capacities of each receptor, the identification of receptor-associated molecules required for function (12, 18–20), and of receptor structural sequences necessary for phagocytosis (21–24).However, we have questioned two aspects of this model; first, whether phagocytosis by COS cells mimics the process as it occurs in a professional phagocyte, and second whether phase contrast and fluorescence microscopy alone are adequate indicators of bona fide phagocytosis. Therefore, before embarking intently upon the study of the COS cell model of phagocytosis, we critically scrutinized in these cells the four major steps of FcγR-mediated phagocytosis: receptor–ligand binding, pseudopod extension, internalization, and fusion of the phagosome with lysosomes, with four microscopic methods: phase contrast, fluorescence, confocal, and electron microscopy. Here we show that COS cells indeed complete these four steps in a manner morphologically similar to professional phagocytes, and we validate that phase contrast and fluorescence microscopy are reasonably accurate methods for estimating phagocytosis.We then focused on the signaling complex of FcγRIA with the γ chain to examine independently the effects of the ligand binding unit, FcγRIA, and the signaling unit, γ chain, in driving pseudopod extension and internalization. We found that although efficient particle internalization by the receptor complex requires the γ chain, pseudopod extension is mediated in the absence of γ chain and by a tailless FcγRIA. Moreover, pseudopod extension mediated by FcγRIA is not blocked by cytochalasin D, and F-actin does not polymerize nor do phosphotyrosine residues accumulate adjacent to pseudopod extensions. These data suggest that these two phases of phagocytosis, pseudopod extension and internalization, are functionally distinct steps mediated by different mechanisms.     

Induction of high-affinity IgE receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia

Respiratory viral infections are associated with an increased risk of asthma, but how acute Th1 antiviral immune responses lead to chronic inflammatory Th2 disease remains undefined. We define a novel pathway that links transient viral infection to chronic lung disease with dendritic cell (DC) expression of the high-affinity IgE receptor (FcεRIα). In a mouse model of virus-induced chronic lung disease, in which Sendai virus triggered a switch to persistent mucous cell metaplasia and airway hyperreactivity after clearance of replicating virus, we found that FceRIa^−/− mice no longer developed mucous cell metaplasia. Viral infection induced IgE-independent, type I IFN receptor–dependent expression of FcεRIα on mouse lung DCs. Cross-linking DC FcεRIα resulted in the production of the T cell chemoattractant CCL28. FceRIa^−/− mice had decreased CCL28 and recruitment of IL-13–producing CD4⁺ T cells to the lung after viral infection. Transfer of wild-type DCs to FceRIa^−/− mice restored these events, whereas blockade of CCL28 inhibited mucous cell metaplasia. Therefore, lung DC expression of FcεRIα is part of the antiviral response that recruits CD4⁺ T cells and drives mucous cell metaplasia, thus linking antiviral responses to allergic/asthmatic Th2 responses.     

The Motheaten Mutation Rescues B Cell Signaling and Development in CD45-deficient Mice

The cytosolic SHP-1 and transmembrane CD45 protein tyrosine phosphatases (PTP) play critical roles in regulating signal transduction via the B cell antigen receptor (BCR). These PTPs differ, however, in their effects on BCR function. For example, BCR-mediated mitogenesis is essentially ablated in mice lacking CD45 (CD45⁻), but is enhanced in SHP-1–deficient motheaten (me) and viable motheaten (me^v) mice. To determine whether these PTPs act independently or coordinately in modulating the physiologic outcome of BCR engagement, we assessed B cell development and signaling in CD45-deficient me^v (CD45⁻/SHP-1⁻) mice. Here we report that the CD45⁻/SHP-1⁻ cells undergo appropriate induction of protein kinase activity, mitogen-activated protein kinase activation, and proliferative responses after BCR aggregation. However, BCR-elicited increases in the tyrosine phosphorylation of several SHP-1–associated phosphoproteins, including CD19, were substantially enhanced in CD45⁻/SHP-1⁻, compared to wild-type and CD45⁻ cells. In addition, we observed that the patterns of cell surface expression of μ, δ, and CD5, which distinguish the PTP-deficient from normal mice, are largely restored to normal levels in the double mutant animals. These findings indicate a critical role for the balance of SHP-1 and CD45 activities in determining the outcome of BCR stimulation and suggest that these PTPs act in a coordinate fashion to couple antigen receptor engagement to B cell activation and maturation.     

Fcγ receptors enable anticancer action of proapoptotic and immune-modulatory antibodies

Antibodies have important roles in controlling cellular immunity through interaction with activating or inhibitory Fcγ receptors (FcγRs). FcγR engagement can facilitate receptor cross-linking on target cells, or induce retrograde FcγR signals to stimulate or suppress antibody-dependent, cell-mediated depletion of antigen-bearing target cells. Recent studies uncover unexpectedly important roles for FcγRs in the anticancer action of antibodies designed to trigger tumor cell apoptosis or enhance antitumor immunity. Here, we outline a conceptual framework for understanding these findings and discuss their mechanistic and translational implications.The adaptive immune system is capable of recognizing an essentially limitless number of antigens via the combinatorial assembly of gene segments that encode antibody variable domains. This diversity has been exploited successfully in a growing number of therapeutic antibodies that bind to a wide range of clinically validated targets. Antibodies that recognize soluble antigens, such as the cytokines tumor necrosis factor (TNF), vascular endothelial growth factor, or interleukin-6, act as antagonists by blocking the interaction of a target ligand with its cognate receptor. In some cases, such antibodies may also augment clearance of the target antigen.In contrast, antibodies that bind to cell surface antigens, often transmembrane receptors such as HER2, EGFR, or DR5, may act as antagonists or agonists, respectively, to block or stimulate the action of the cognate target. Alternatively, antibodies may bind a cell surface target that lacks signaling function, such as the CD20 antigen, and act as an anchor for FcγR-based recruitment of immune-effector cells to kill the antigen-expressing target by antibody-dependent, cell-mediated cytotoxicity (ADCC). Therefore, antibodies that recognize cell surface receptors can be categorized by their function of either mediating target cell killing or modulating target receptor signal transduction. However, two new studies in this issue demonstrate that these activities are not mutually exclusive and that antibodies harboring both properties may be advantageous for cancer immunotherapy. Due to shared expression of cell surface antigens, such as CTLA-4 or glucocorticoid-induced TNFR-related protein (GITR) on protumorigenic regulatory T (T reg) cells and antitumorigenic effector T (T eff) cells, antibodies that target such receptors are capable of inducing antitumor immunity both by depleting T reg cells and by stimulating T eff cells. However, antibodies that conform to this dual mechanism of action have the risk of depleting T eff cells, which are the final mediators of tumor cell killing. Therefore, understanding the principles that govern antibody–FcγR interactions is crucial for designing effective antibody-based immunotherapies.

Antibody–FcγR interactions

FcγRs fall into two functional classes: activating and inhibitory (Nimmerjahn and Ravetch, 2006). The FcγR family comprises three activating (mouse FcγRI, FcγRIII, and FcγRIV; human FcγRI, FcγRIIA, and FcγRIIIA) and one inhibitory (FcγRIIB) receptor. Activating FcγRs associate with a common signaling chain (FcRγ), containing an immunoreceptor tyrosine-based activation motif (ITAM) that recruits Syk family kinases to stimulate effector function. In contrast, FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that recruits specific phosphatases to oppose signaling by activating FcγRs. Innate-immune cells, such as macrophages, monocytes, dendritic cells, mast cells, and granulocytes, express both activating and inhibitory FcγRIIB (Amigorena et al., 1992; Nimmerjahn and Ravetch, 2008). IgG subtypes differ in FcγR affinity: human IgG1 and IgG3 have higher affinity for activating than inhibitory FcγR, as do mouse IgG2a and IgG2b (Dijstelbloem et al., 2001; Nimmerjahn and Ravetch, 2005, 2006).Antagonist antibodies may bind to a soluble ligand or a cell surface receptor to prevent signaling. Target inhibition per se typically does not require accessory FcγR-bearing cells; therefore, antagonist antibodies often act independently of FcγRs, and accordingly, IgG subtype. However, if the target is engaged at the cell surface and is sufficiently abundant, effector cells may be recruited via Fc–FcγR interactions to deplete the antigen-displaying cell, an outcome that can be desirable or undesirable depending on the context. Target cell depletion can be manipulated by selecting IgG subtypes that favor binding to activating or inhibitory FcγRs. Unwanted target cell depletion can be minimized by incorporating Fc mutations that decrease FcγR affinity (Presta et al., 2002; Carter, 2006; Lazar et al., 2006; Satoh et al., 2006; Jefferis, 2009). For example, asparagine 297, the site for N-linked glycosylation required for FcγR binding in the constant region, can be replaced by alanine. Further mutations to enhance or decrease specific FcγR interactions have also been reported. Alternatively, some antibody variants can be produced in Escherichia coli rather than mammalian cells to prevent Fc glycosylation. Fc effectorless antibodies have been demonstrated to be equally as potent at blocking ligand–receptor interactions as their wild-type counterparts.Recent work has revealed unexpectedly that agonist antibodies designed to stimulate the tumor necrosis factor receptor superfamily (TNFRSF) members DR4, DR5, or CD40 depend on FcγR interaction for robust agonist activity (Li and Ravetch, 2011; Wilson et al., 2011; Smith et al., 2012). As TNFRSF members usually require ligand-induced super-clustering for signal transmission, bivalent IgG molecules are unable to induce their efficient stimulation. In vitro activity can be enhanced by artificial cross-linking of the primary antibody, with secondary anti-Fc antibodies, or—perhaps more importantly—by providing contact with FcγR-bearing cells. Pretreating FcγR-expressing cells with actin polymerization inhibitors blocks this enhancement, suggesting that FcγR clustering is important for antibody-mediated stimulation of the target receptor (Wilson et al., 2011). Studies with mice deficient in specific FcγR subsets demonstrate that expression of the inhibitory FcγRIIB is sufficient—if not superior—for enabling in vivo efficacy of agonist antibodies targeting CD40, or the death receptors DR4 and DR5 (Li and Ravetch, 2011; Wilson et al., 2011). Reliance on FcγRIIB circumvents the potential for FcγR-mediated target cell depletion and is therefore advantageous for inducing signal transduction on target cells, such as CD40 signaling in dendritic cells. CD40 engagement on dendritic cells enhances their antigen presentation capabilities, thereby increasing T cell responses. Therefore, cross-linking CD40 via FcγRIIB on DCs enhances their T cell priming function, while minimizing depletion. Importantly, anti-CD40–induced adjuvant activity was unabated in mice deficient in activating FcγRs, yet was abrogated in FcγRIIB-deficient mice (Li and Ravetch, 2011). However, FcγR-dependent depletion of DR4- or DR5-expressing tumor cells could be useful to induce antitumor activity. These findings suggest that agonist antibodies targeting either proapoptotic or co-stimulatory TNFRSF members can rely on FcγRs as a dynamic cross-linking scaffold—a function that, at least in mice, may be supported more effectively by FcγRIIB.

Agonist GITR antibodies require activating FcγRs for antitumor efficacy

In this issue, Bulliard et al. report an apparent exception to the latter paradigm: agonist antibodies targeting the TNFRSF family member GITR require activating FcγR effector function to promote tumor regression in mice. GITR expression is induced on T eff cells upon T cell receptor (TCR) stimulation, and GITR cross-linking by GITR ligand or agonist antibodies co-stimulates TCR signaling (McHugh et al., 2002; Shimizu et al., 2002; Tone et al., 2003; Ronchetti et al., 2004; Stephens et al., 2004). In transplantable tumor models, anti-GITR treatment is hypothesized to induce tumor regression through T eff cell co-stimulation (Turk et al., 2004; Ko et al., 2005). Distinct from antibodies that target other TNFR superfamily members, anti-GITR activity was unaltered in Fcgr2b^−/− mice. However, anti-GITR treatment was ineffective in knockout mice lacking the ITAM-containing FcγR chain required for signaling by all activating FcγRs. Hence, an alternative (or additional) mechanism of action—distinct from FcγRIIB-mediated GITR cross-linking to promote co-stimulation—may be critical for antitumor efficacy. Given that GITR-expressing CD8⁺ T cells and CD4⁺ T eff cells are needed for tumor cell killing, this mechanism may involve FcγR-dependent depletion of GITR-expressing T reg cells.T reg cells frequently express T cell activation markers induced by TCR signaling (Gavin et al., 2002; McHugh et al., 2002). In addition to their well-characterized role in maintaining peripheral tolerance to self-antigens, T reg cells have been demonstrated to suppress tumor immunity (Nishikawa and Sakaguchi, 2010; Josefowicz et al., 2012). T reg cells are highly enriched in tumors, both in mouse models and in various human cancers. Furthermore, in cancer patients, abundance of T reg cells within tumors is associated with poor prognosis, suggesting that these cells play an important role in suppressing antitumor immunity. Therefore, strategies to deplete intratumoral T reg cells may enhance the generation of tumor-directed T eff cell responses.GITR is weakly expressed on naive T eff cells, but is present on resting T reg cells and up-regulated on activated T eff cells. Bulliard et al. (2013) observed a significant reduction in tumor-associated, but not peripheral, T reg cells upon anti-GITR treatment of tumor-bearing mice. Hence, anti-GITR antibodies may enhance antitumor immunity by depleting GITR-positive T reg cells, in addition to co-stimulating antigen-experienced T eff cells (Coe et al., 2010). However, the shared expression of GITR on T reg and T eff cells makes both populations potentially susceptible to antibody-dependent, FcγR-mediated depletion. Indeed, diminished effector CD4⁺ and CD8⁺ T cell numbers were reported to accompany T reg cell depletion in tumor tissues. Therefore, elimination of intratumoral T reg cells, coupled with T eff cell co-stimulation and minimal T eff cell depletion, might provide an integrated mechanism of action for anti-GITR antibodies. The relative contribution of each of these mechanistic components remains to be clarified.

CTLA-4 blocking antibodies mediate T reg cell depletion

CTLA-4 is an inhibitory receptor that is induced on antigen-experienced T eff cells as a negative feedback regulator (Walunas et al., 1994; Krummel and Allison, 1995). Whereas CTLA-4 expression is induced on activated T eff cells, CTLA-4 is constitutively expressed by T reg cells, which require CTLA-4 function to aggregate preferentially around dendritic cells and inhibit their antigen-presenting activity (Onishi et al., 2008). As such, the multiorgan autoimmunity detected in Ctla4 germline knockout mice is phenocopied in T reg cell–specific Ctla4 conditional knockout mice (Tivol et al., 1995; Waterhouse et al., 1995; Chambers et al., 1997; Wing et al., 2008). Although anti–CTLA-4 therapy is one of the first clinically validated examples of effective cancer immunotherapy, its mechanism of action is poorly understood (Hodi et al., 2010). It is hypothesized that antagonist anti–CTLA-4 antibodies enhance tumor immunity by relieving inhibitory CTLA-4 signals on antigen-experienced T eff cells, as well as curtailing the suppressive function of T reg cells. Following the paradigm of antagonist antibodies, CTLA-4–blocking antibodies would be expected to exert their efficacy in the absence of Fc effector function.However, studies from Bulliard et al. (2013) and Simpson et al. in this issue provide compelling evidence that the activity of anti–CTLA-4 antibodies requires activating FcγR engagement to deplete T reg cells. Similar to anti-GITR therapy, anti–CTLA-4–mediated tumor regression was abrogated in FcγR-deficient mice, suggesting that retrograde signaling by activating FcγRs and consequent T reg cell depletion is important for antitumor activity. Accordingly, intratumoral T reg cell numbers were dramatically reduced in response to anti–CTLA-4 treatment. Importantly, this reduction was not associated with dedifferentiation or impaired generation of T reg cells, thereby supporting cellular depletion as the most plausible mechanism of efficacy.Because activated T eff cells express CTLA-4, they are also susceptible to antibody-mediated depletion. In this regard, Bulliard et al. (2013) demonstrated that both T reg and T eff cell numbers were reduced in response to anti–CTLA-4 treatment. In contrast, Simpson et al. (2013) observed that in an adoptive T cell transfer model, intratumoral T eff cells were not depleted by anti–CTLA-4 treatment, but rather increased in number. Differences in tumor model, antibody isotype or clone, or vaccine co-administration could potentially account for these discrepant results. Nevertheless, T reg cells may be more sensitive to antibody-mediated depletion because of their increased expression of CTLA-4 within tumor tissues. Consistent with this notion, tumor-infiltrating T reg cells expressed nearly fourfold higher CTLA-4 levels than did tumor-associated T eff cells in the Colon26 carcinoma model. Additionally, in the B16-BL6 melanoma model, CTLA-4 surface expression was slightly elevated in tumor-associated versus peripheral T reg cells or intratumoral T eff cells. It remains to be investigated whether this variation in receptor expression is detected in human intratumoral T cells and leads to differential depletion in cancer patients.

Conclusions

Important roles have emerged for FcγRs in mediating the antitumor efficacy of a new generation of therapeutic antibodies aiming to attack cancer directly, by triggering tumor cell apoptosis, or indirectly, by enhancing antitumor immunity (Fig. 1). First, FcγRs on tumor-infiltrating innate immune cells provide a cross-linking scaffold to enhance antibody-mediated activation of proapoptotic TNFRSF members such as DR4 and DR5 on cancer cells. Similarly, FcγR-mediated cross-linking supports activation of T eff cells via co-stimulatory TNFRSF members such as GITR. In some instances, this cross-linking function can be performed redundantly by activating or inhibitory FcγRs; however, mouse studies suggest that the inhibitory FcγRIIB may provide a more effective molecular scaffold, for reasons that have yet to be defined. Fc mutations that enhance affinity for FcγRs may serve to improve the efficacy of agonist antibodies targeting various TNFRSF members. In addition to cross-linking cell surface receptors to support forward signaling in target cells, FcγRs can mediate antibody-driven reverse signaling to activate FcγR-bearing cells and promote target cell depletion. For cancer immunotherapy, antibodies that fulfill both functions may be beneficial to deplete immunosuppressive T reg cells and stimulate T eff cells. The high prevalence of T reg cells in tumor tissues poses a significant barrier to generating a productive antitumor T cell response. Expression of antigens such as GITR or CTLA-4 on T reg cells affords the opportunity for enhanced antibody-based depletion of such immune-suppressive cells from the tumor microenvironment, thereby augmenting antitumor immunity. A potential caveat is that desirable T eff cells also might express the same antigens and hence be subject to similar depletion. Thus, maximal therapeutic efficacy likely necessitates a delicate balance between depleting T reg cells while sparing T eff cells. Defining the FcγR interactions responsible for these different outcomes should help optimize the effectiveness of antibodies to biologically relevant T cell antigens, including GITR, CTLA-4, and beyond.Open in a separate windowFigure 1.How Fcγ receptors enable anticancer efficacy of proapoptotic and immune-modulatory antibodies. Inhibitory (Inh.) or activating (Act.) FcγRs can provide a dynamic scaffold for cross-linking agonist antibodies targeting proapoptotic receptors such as DR4 or DR5 on the surface of cancer cells, thereby promoting direct tumor cell apoptosis (A). Similarly, FcγRs can support agonist antibody-mediated cross-linking of immune cell co-stimulatory receptors, e.g., CD40 on antigen-presenting cells (B), or GITR on T eff cells (C), to augment antitumor immunity. T eff cell responses can be enhanced further, through direct antibody-based antagonism of the inhibitory molecule CTLA-4 (D). Finally, activating FcγRs can promote ADCC-based depletion of T reg cells upon antibody binding to GITR or CTLA-4, thus alleviating T reg suppression and strengthening antitumor immunity (E).     

Positive Selection of Extrathymically Developed T Cells by Self-antigens

Most T cells develop through the thymus, where they undergo positive and negative selection. Some peripheral T cells are known to develop in the absence of thymus, but there is insufficient information about their selection. To analyze the selection of extrathymically developed T cells, we reconstituted thymectomized male or female recipient mice with bone marrow cells of mice transgenic for male H-Y antigen–specific T cell receptor (TCR). It was revealed that the T cells bearing self-antigen–specific TCR were not deleted in thymectomized male recipients. More importantly, the absence of H-Y antigen–specific T cells in thymectomized female recipients suggests positive selection of extrathymically developed T cells by the self-antigen. The extrathymically developed T cells in male mice expressed interleukin (IL)-2 receptor β chain (IL-2Rβ) and intermediate levels of CD3 (CD3^int) but were natural killer cell (NK)1.1⁻. They rapidly produced interferon γ but not IL-4 after TCR cross-linking. Furthermore, a similar pattern of cytokine production was observed in CD3^intIL-2Rβ⁺NK1.1⁻ cells in normal mice which have been shown to develop extrathymically. These results suggest that extrathymically developed CD3^intIL-2Rβ⁺NK1.1⁻ cells in normal mice are also positively selected by self-antigens.     

Type II and III Receptors for Immunoglobulin G (IgG) Control the Presentation of Different T Cell Epitopes from Single IgG-complexed Antigens

T cell receptors on CD4⁺ lymphocytes recognize antigen-derived peptides presented by major histocompatibility complex (MHC) class II molecules. A very limited set of peptides among those that may potentially bind MHC class II is actually presented to T lymphocytes. We here examine the role of two receptors mediating antigen internalization by antigen presenting cells, type IIb2 and type III receptors for IgG (FcγRIIb2 and FcγRIII, respectively), in the selection of peptides for presentation to T lymphocytes. B lymphoma cells expressing recombinant FcγRIIb2 or FcγRIII were used to assess the presentation of several epitopes from two different antigens. 4 out of the 11 epitopes tested were efficiently presented after antigen internalization through FcγRIIb2 and FcγRIII. In contrast, the 7 other epitopes were efficiently presented only when antigens were internalized through FcγRIII, but not through FcγRIIb2. The capacity to present these latter epitopes was transferred to a tail-less FcγRIIb2 by addition of the FcγRIII-associated γ chain cytoplasmic tail. Mutation of a single leucine residue at position 35 of the γ chain cytoplasmic tail resulted in the selective loss of presentation of these epitopes. Therefore, the nature of the receptor that mediates internalization determines the selection of epitopes presented to T lymphocytes within single protein antigens.Antigen receptors on CD4⁺ helper T lymphocytes recognize short peptides presented by class II molecules of MHC (1, 2). Antigenic peptides are generated by proteolytic degradation in the endocytic pathway, where they associate with MHC class II molecules. Only a very limited set of peptides among all the potential peptides is actually loaded onto MHC class II molecules in APCs (3). The mechanisms underlying the selection of peptides for MHC class II–restricted antigen presentation are yet unclear. However, we know that two independent complex processes of intracellular transport towards endosomes are crucial for MHC class II–restricted antigen presentation: the traffic of MHC class II molecules and the delivery of antigens (4, 5).MHC class II intracellular transport has been analyzed in detail. Newly synthesized MHC class II molecules reach endosomes, either directly from the trans-golgi network or after a short appearance at the plasma membrane, in association to the invariant (Ii)¹ chain (5). Ii is then degraded and the class II–associated Ii chain peptide (CLIP) is replaced by an antigenic peptide under the control of HLA-DM (6). It has recently become clear that an alternative, Ii chain–independent pathway for MHC class II transport to endosomes also exists. Indeed, MHC class II molecules may reach the endocytic pathway from the cell surface by endocytosis (7), due to internalization signals present in the cytosolic domain of the MHC class II β chain (8). Newly synthesized and recycling MHC class II molecules may present different peptides (9). Accordingly, we have previously shown that different antigen receptors may also selectively target antigens for presentation by either of these MHC class II presentation pathways (10).In contrast, very little is known about the endocytic transport of antigen receptors. Physiologically, antigens are delivered to the endocytic pathway by different families of receptors, which strongly increase the efficiency of MHC class II–restricted antigen presentation (11). In B lymphocytes, surface Ig mediates both cell activation and the uptake of specific antigens (12), while the expression of a particular endocytosis-deficient receptor for the Fc portion of IgG (FcγRIIb1) prevents efficient presentation of irrelevant IgG-complexed antigens (13). Interestingly, the epitope specificity of surface Ig positively and negatively influences the presentation of various T cell epitopes (14, 15).In monocytes and dendritic cells, receptors for IgG (FcγRs), in addition to mannose receptors, mediate antigen internalization and strongly increase the efficiency of presentation to specific T cells (16). Two different FcγRs, type IIb2 and type III (FcγRIIb2 and FcγRIII) are expressed in dendritic cells. FcγRIIb2 is a monomeric receptor that, like FcγRIIb1, mediates the inhibition of cell activation when cocrosslinked to surface Ig (13). FcγIII is an heterotrimer consisting of an α chain and a dimer of γ chains, which couple the receptor to cytoplasmic effectors of signal transduction (17).To individually analyze the function of these two receptors, we expressed them by cDNA transfection into an FcγR-negative B cell lymphoma cell line (13, 18, 19). We have previously shown that the amino acid sequence, called immunoreceptor tyrosine kinase activation motif (ITAM), in the FcγIII-associated γ chain responsible for cell activation is also involved in receptor internalization (18, 19). In addition, mutation of either of the two tyrosine residues in the ITAM of the γ chain inhibits both cell activation and ligand internalization (19). Thus, in contrast to FcγRIIb2, which contains no ITAM, is not tyrosine phosphorylated, and does not induce cell activation, FcγRIII associates with and activates cytosolic tyrosine kinases after engagement by its ligand (18). In addition to this functional diversity of the two receptors, their expression is also selectively regulated, since TNF-α and IFN-γ increase the expression of FcγRIII and inhibit that of FcγRIIb2 in monocytes (20).The diversity in the signals required of FcγRIIb2 and FcγRIII internalization, as well as the differential regulation of their expression in APCs, suggest that the two receptors may have different antigen-presenting functions. We here examine the ability of FcγRIIb2 and FcγRIII to induce the presentation of various T cell epitopes from two different antigens, CI λ repressor and hen egg lysozyme (HEL). Internalization of antigen–antibody complexes through FcγRIII induced the efficient presentation of all the T cell epitopes tested, whereas FcγRIIb2 only induced the presentation of a few. Point mutation of leucine 35 to alanine (L35A) in the cytoplasmic tail of FcγRIII γ chain blocked signal transduction without affecting the internalization of immune complexes. This mutation also blocked the presentation of the epitopes that were only presented after internalization by FcγRIII. In contrast, the presentation of all the epitopes that were efficiently generated after internalization by FcγRIIb2 was not affected. Thus, the nature of the receptor that mediates antigen internalization influences the selection of the epitopes presented to T lymphocytes.     

Early Function of Pax5 (BSAP) before the Pre-B Cell Receptor Stage of B Lymphopoiesis

The formation of the pre-B cell receptor (BCR) corresponds to an important checkpoint in B cell development that selects pro-B (pre-BI) cells expressing a functionally rearranged immunoglobulin μ (Igμ) heavy chain protein to undergo the transition to the pre-B (pre-BII) cell stage. The pre-BCR contains, in addition to Igμ, the surrogate light chains λ5 and VpreB and the signal transducing proteins Igα and Igβ. The absence of one of these pre-BCR components is known to arrest B cell development at the pre-BI cell stage. Disruption of the Pax5 gene, which codes for the B cell–specific activator protein (BSAP), also blocks adult B lymphopoiesis at the pre-BI cell stage. Moreover, expression of the mb-1 (Igα) gene and V_H-to-D_HJ_H recombination at the IgH locus are reduced in Pax5-deficient B lymphocytes ∼10- and ∼50-fold, respectively. Here we demonstrate that complementation of these deficiencies in pre-BCR components by expression of functionally rearranged Igμ and chimeric Igμ-Igβ transgenes fails to advance B cell development to the pre-BII cell stage in Pax5 (−/−) mice in contrast to RAG2 (−/−) mice. Furthermore, the pre-BCR is stably expressed on cultured pre-BI cells from Igμ transgenic, Pax5-deficient bone marrow, but is unable to elicit its normal signaling responses. In addition, the early developmental block is unlikely to be caused by the absence of a survival signal, as it could not be rescued by expression of a bcl2 transgene in Pax5-deficient pre-BI cells. Together, these data demonstrate that the absence of Pax5 arrests adult B lymphopoiesis at an early developmental stage that is unresponsive to pre-BCR signaling.     

Syk Tyrosine Kinase and B Cell Antigen Receptor (BCR) Immunoglobulin-α Subunit Determine BCR-mediated Major Histocompatibility Complex Class II–restricted Antigen Presentation

Stimulation of CD4⁺ helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-α/Ig-β heterodimers which, second, target antigens to MHC class II–containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-α–associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-α cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide– MHC class II complexes through antigen targeting by BCR subunits.     

Regulatory CD4+ T Cells Expressing Endogenous T Cell Receptor Chains Protect Myelin Basic Protein–specific Transgenic Mice from Spontaneous Autoimmune Encephalomyelitis

The development of T cell–mediated autoimmune diseases hinges on the balance between effector and regulatory mechanisms. Using two transgenic mouse lines expressing identical myelin basic protein (MBP)–specific T cell receptor (TCR) genes, we have previously shown that mice bearing exclusively MBP-specific T cells (designated T/R⁻) spontaneously develop experimental autoimmune encephalomyelitis (EAE), whereas mice bearing MBP-specific T cells as well as other lymphocytes (designated T/R⁺) did not. Here we demonstrate that T/R⁻ mice can be protected from EAE by the early transfer of total splenocytes or purified CD4⁺ T cells from normal donors. Moreover, whereas T/R⁺ mice crossed with B cell–deficient, γ/δ T cell–deficient, or major histocompatibility complex class I–deficient mice did not develop EAE spontaneously, T/R⁺ mice crossed with TCR-α and -β knockout mice developed EAE with the same incidence and severity as T/R⁻ mice. In addition, MBP-specific transgenic mice that lack only endogenous TCR-α chains developed EAE with high incidence but reduced severity. Surprisingly, two-thirds of MBP-specific transgenic mice lacking only endogenous TCR-β chains also developed EAE, suggesting that in T/R⁺ mice, cells with high protective activity escape TCR-β chain allelic exclusion. Our study identifies CD4⁺ T cells bearing endogenous α and β TCR chains as the lymphocytes that prevent spontaneous EAE in T/R⁺ mice.     

Suppressive Role of B Cells in Chronic Colitis of T Cell Receptor α Mutant Mice

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-α^−/− mice was explored by creating double mutant mice (TCR-α^−/− × immunoglobulin (Ig)μ^−/−), which lack B cells. TCR-α^−/− × Igμ^−/− mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-α^−/− mice. Colitis was induced in recombination-activating gene-1 (RAG-1^−/−) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-α^−/− × Igμ^−/− mice. When purified B cells from TCR-α^−/− mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1^−/− mice. Administration of the purified Ig from TCR-α^−/− mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-α^−/− × Igμ^−/− mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-α^−/− × Igμ^−/− mice as compared to Igμ^−/− mice and TCR-α^−/− mice. Administration of the purified Ig from TCR-α^−/− mice into TCR-α^−/− × Igμ^−/− mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.     

SLP-65: A New Signaling Component in B Lymphocytes which Requires Expression of the Antigen Receptor for Phosphorylation

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-α/Ig-β heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H₂O₂. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H₂O₂-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.     

Rho is Required for the Initiation of Calcium Signaling and Phagocytosis by Fcγ Receptors in Macrophages

Phagocytosis of bacteria by macrophages and neutrophils is an essential component of host defense against infection. The mechanism whereby the interaction of opsonized particles with Fcγ receptors triggers the engulfment of opsonized particles remains incompletely understood, although activation of tyrosine kinases has been recognized as an early step. Recent studies in other systems have demonstrated that tyrosine kinases can in turn signal the activation of small GTPases of the ras superfamily. We therefore investigated the possible role of Rho in Fc receptor–mediated phagocytosis. To this end we microinjected J774 macrophages with C3 exotoxin from Clostridium botulinum, which ADP-ribosylates and inactivates Rho. C3 exotoxin induced the retraction of filopodia, the disappearance of focal complexes, and a global decrease in the F-actin content of J774 cells. In addition, these cells exhibited increased spreading and the formation of vacuolar structures. Importantly, inactivation of Rho resulted in the complete abrogation of phagocytosis. Inhibition of Fcγ receptor–mediated phagocytosis by C3 exotoxin was confirmed in COS cells, which become phagocytic upon transfection of the FcγRIIA receptor. Rho was found to be essential for the accumulation of phosphotyrosine and of F-actin around phagocytic cups and for Fcγ receptor–mediated Ca²⁺ signaling. The clustering of receptors in response to opsonin, an essential step in Fcγ-induced signaling, was the earliest event shown to be inhibited by C3 exotoxin. The effect of the toxin was specific, since clustering and internalization of transferrin receptors were unaffected by microinjection of C3. These data identify a role for small GTPases in Fcγ receptor–mediated phagocytosis by leukocytes.     

Lymphotoxin-α (LTα) Supports Development of Splenic Follicular Structure That Is Required for IgG Responses

LTα-deficient (LTα^−/−) mice show altered splenic microarchitecture. This includes loss of normal B cell–T cell compartmentalization, of follicular dendritic cell (FDC) clusters, and of ability to form germinal centers (GC). LTα^−/− mice immunized with sheep red blood cells (SRBC) produced high levels of antigen-specific IgM but no IgG in either primary or secondary responses, demonstrating failure of Ig class switching. This inability to switch to IgG could have been due to the altered splenic microarchitecture in these mice. Alternatively, it could have been due directly to a requirement for LTα expression by lymphocytes cooperating in the antibody response. To investigate this, we performed reciprocal spleen cell transfers. When irradiated LTα^−/− mice were reconstituted with wild-type splenocytes and immunized immediately with SRBC, splenic microarchitecture remained disturbed and there was no IgG response. In contrast, when irradiated wild-type animals received splenocytes from LTα^−/− mice, follicle structure and a strong IgG response were retained. These data indicate that LTα-deficient B cells and T cells have no intrinsic defect in ability to generate an IgG response. Rather, the altered microenvironment characteristic of LTα^−/− mice appears to result in impaired ability to switch to a productive IgG response. To investigate whether prolonged expression of LTα could alter the structure and function of spleen follicles, reciprocal bone marrow (BM) transplantation was performed. Six weeks after reconstitution of LTα^−/− mice with wild-type BM, spleen follicle structure was partially restored, with return of FDC clusters and GC. B cell/T cell compartmentalization remained abnormal and white pulp zones were small. This was accompanied by restoration of IgG response to SRBC. Reconstitution of wild-type mice with LTα^−/− BM resulted in loss of FDC clusters and GC, and loss of the IgG response, although compartmentalized B cell and T cell zones were largely retained. Thus, defective IgG production is not absolutely associated with abnormal B cell and T cell compartmentalization. Rather, expression of LTα supports the maturation of spleen follicle structure, including the development and maintenance of FDC clusters, which supports Ig class switching and an effective IgG response.Lymphotoxin-α (LTα)¹ shares structural features with the related cytokine, TNFα. Both LTα and TNFα exist in solution as homotrimeric proteins. In these forms, they share biological activities by virtue of their similar binding to the two defined TNF receptors, TNFR-I, and TNFR-II. Signaling via these two receptors modulates a wide variety of immune and inflammatory responses (1, 2). LTα also exists in a heteromeric form with the type II membrane protein LTβ, which in its most prevalent form on the membrane has the stoichiometry LTα₁LTβ₂. The LTα₁LTβ₂ heteromer has no measurable affinity for TNFR-I or TNFR-II, but does interact with high affinity with the TNFR-related protein (also designated the LTβ receptor, or LTβR) (3–5).LTα^−/− mice are born with defective development of LN and Peyer''s patches (PP) (6). In LTα^−/− mice, spleen structure is also disturbed, with small white pulp follicles that fail to segregate T cell and B cell zones, and fail to generate clusters of FDC or GC (6–8). Proper spleen microarchitecture, including the presence of primary and secondary lymphoid follicles that contain FDC, is thought to be required for all features of a mature T cell–dependent B cell response, including Ig class switching, affinity maturation, and development of antibody secreting cells (9–11). Consequently, it was striking that LTα^−/− mice were able to generate high affinity anti-NP IgG antibody after immunization with high doses of the T cell–dependent antigen 4-hydroxy3-nitrophenyl-ovalbumin (NP-OVA) adsorbed to alum (8). In contrast, there was impaired production of high affinity anti-NP IgG when LTα^−/− mice were immunized with low doses of NP-OVA absorbed to alum. Banks et al. (12) using an independently derived LTα^−/− mouse strain, have shown impaired IgG responses in LTα^−/− mice after subcutaneous immunization with KLH absorbed to alum or immunization with viral antigens. Further studies examining mice deficient in both TNFα and LTα demonstrated variable IgG responses, with deficient IgG responses after intraperitoneal immunization with SRBC but retained responses against vesicular stomatitis virus after an infectious challenge (13). Recently prepared TNFα^−/− mice (14) are reported also to have an impaired IgG anti-SRBC response but a strong response to DNP-KLH adsorbed to alum. Thus, the role of LTα in supporting an effective IgG response to SRBC remains incompletely defined.LTα^−/− mice manifest a complex phenotype that includes an absence of LTα expression and also established abnormalities of lymphoid tissue development and structure (6–8). The present study was undertaken to investigate the requirements for LTα expression in lymphoid cells and for intact lymphoid tissue structure to support production of an IgG responses to the T cell–dependent antigen SRBC. We report that LTα^−/− mice responded with high levels of IgM but very low levels of IgG after immunization with SRBC in the absence of adjuvant. Experiments in which suspensions of mature spleen cells or of  T cell–depleted bone marrow were transferred to wild-type or LTα^−/− mice demonstrated that certain elements of spleen follicle structure are plastic and are determined by the presence of LTα-expressing cells. These experiments also demonstrated that LTα^−/− B cells and T cells in a structurally intact lymphoid tissue environment are competent to perform Ig isotype switching. In contrast, disturbed lymphoid tissue structure caused by absence of LTα and manifested by absence of clusters of FDC is associated with an inability to form an effective IgG response. Thus, LTα produced by bone marrow (BM)–derived cells establishes a permissive environment for an effective IgG response.