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Blood, 1 September 2008, Vol. 112, No. 5, pp. 1557-1569.
CD4 T cells: fates, functions, and faults1 Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
In 1986, Mosmann and Coffman identified 2 subsets of activated CD4 T cells, Th1 and Th2 cells, which differed from each other in their pattern of cytokine production and their functions. Our understanding of the importance of the distinct differentiated forms of CD4 T cells and of the mechanisms through which they achieve their differentiated state has greatly expanded over the past 2 decades. Today at least 4 distinct CD4 T-cell subsets have been shown to exist, Th1, Th2, Th17, and iTreg cells. Here we summarize much of what is known about the 4 subsets, including the history of their discovery, their unique cytokine products and related functions, their distinctive expression of cell surface receptors and their characteristic transcription factors, the regulation of their fate determination, and the consequences of their abnormal activation.
CD4 T cells play a central role in immune protection. They do so through their capacity to help B cells make antibodies, to induce macrophages to develop enhanced microbicidal activity, to recruit neutrophils, eosinophils, and basophils to sites of infection and inflammation, and, through their production of cytokines and chemokines, to orchestrate the full panoply of immune responses. Beginning with the groundbreaking work of Mossman and Coffman in 19861 showing that long-term CD4 T-cell lines could be subdivided into 2 groups, those that made IFN  as their signature cytokine and those that produced IL-4, it has been realized that CD4 T cells are not a unitary set of cells but represent a series of distinct cell populations with different functions While some of these CD4 T-cell populations are actually distinct lineages of cells already distinguished from one another when they emerge from the thymus, such as "natural" regulatory T (nTreg) cells2,3 and natural killer T cells (NKT cells),4 several represent alternative patterns of differentiation of naive CD4 T cells. It is to the description of these cells, their functions, their patterns of differentiation, the sets of genes they express, and the consequences of abnormalities in them that this review is devoted. Naive conventional CD4 T cells have open to them 4 (and possibly more) distinct fates that are determined by the pattern of signals they receive during their initial interaction with antigen. These 4 populations are Th1, Th2, Th17, and induced regulatory T (iTreg) cells. Mossman and Coffman recognized the Th1 and Th2 phenotypes among the set of long-term T-cell lines that they studied and the early history of this field was devoted to understanding these 2 cell populations, with Th1 cells being regarded as critical for immunity to intracellular microorganisms and Th2 cells for immunity to many extracellular pathogens, including helminths.5,6 Abnormal activation of Th1 cells was seen as the critical event in most organ-specific autoimmune diseases while Th2 cells were responsible for allergic inflammatory diseases and asthma. Th17 cells have been recognized much more recently but there is now a growing body of work indicating not only that these cells exist but that they play a critical function in protection against microbial challenges, particularly extracellular bacteria and fungi.7 Further, some of the autoimmune responses formally attributed to Th1 cells, such as experimental autoimmune encephalomyelitis (EAE), collagen induced arthritis (CIA), and some forms of inflammatory bowel disease (IBD), have now been shown to be mediated, at least in part, by Th17 cells. iTreg cells are also now well established as an inducible cell population that phenotypically resembles nTreg cells, although distinguishing the function of iTreg cells from that of nTreg cells and, particularly, the relative importance of the 2 Treg populations in humans and experimental animals has been difficult. In this review, we will deal with the function of Treg cells as a group except where we explicitly speak of iTreg cells. There are also other regulatory CD4 T cells including Th3 and TR1 cells. Th3 cells are transforming growth factor β (TGF-β)–producing cells induced by oral tolerance.8 Most of them are likely inducible regulatory T cells that express Foxp3.9 Whether or not there are TGFβ-producing Foxp3– CD4 T cells is unclear. TR1 cells are IL-10 producing cells.10 Because all the CD4 T-cell sets including Th1, Th2, Th17 as well as Treg cells are capable of producing IL-10 under certain circumstances,11–13 TR1 cells may not be a distinct lineage but rather may represent a certain state of each existing lineage. Finally, there may well be other sets of conventional CD4 T cells and even among the more conventional sets, important differences exist, such as the detailed pattern of cytokines that they produce. Figure 1 summarizes much of what we know about the major sets of CD4 T cells, including their unique products, the characteristic transcription factors and cytokines critical for their fate determination and some of their functions. Each of these topics will be discussed in some depth in the subsequent sections of this review.
Initially, immunologists believed that there were fundamentally 2 types of immune responses that require the action of CD4 T cells. One was antibody-mediated and the other cell-mediated. However, there was very little progress in this area until the early 1980s, when T-cell cloning technology was developed, many cytokines were discovered and cloned, and assays for them became available. Tim Mosmman and Bob Coffman recognized that mature CD4 T cells could be subdivided into 2 distinct populations with different sets of products and that this would endow them with unique functions.1 Kim Bottomly was also working on this subject; she and her colleagues subdivided CD4 T-cell lines based on functional criteria, distinguishing inflammatory and helper CD4 T cells, with the latter being IL-4 producers.14 The translation of the differences observed in long-term CD4 T-cell lines to the behavior of normal CD4 T cells, first in vitro and then in vivo, constitutes the beginning of the Th field as a biologic subject. The earliest description of in vitro differentiation was reported in 1990 by our group and that of Susan Swain, demonstrating first that naive CD4 T cells failed to make IL-4 (or most other effector cytokines) and that these cells could be induced to develop into vigorous IL-4 producers if they were stimulated both with T-cell receptor ligands and IL-4, itself.15,16 Within 2 to 3 days after the initiation of culture, the stimulated cells acquire the capacity to produce IL-4. It was subsequently shown that this in vitro differentiation requires a signaling pathway that includes the IL-4 receptor, the signal transducer and activator of transcription (Stat) 6 and the DNA-binding factor GATA-3.17,18 As we will discuss later, this is far from the whole story, but "it gets us off to the races." We note in passing that in our original 1990 paper, we found that IL-2 was also necessary for cells to acquire IL-4–producing capacity, although that was largely overlooked and didn't come back for serious analysis for more than a decade.19
Three years later, Ken Murphy, Anne O'Garra, and their colleagues showed that naive CD4 T cells could acquire the capacity to produce IFN
At first, it appeared that there was a fundamental dichotomy between the logic of differentiation process for Th1 and Th2 cells, with a CD4 T-cell endogenous product, IL-4, playing a major positive feedback role in Th2 differentiation and an exogenous product, IL-12, probably mainly from dendritic cells, playing the major inductive role for Th1 cells. However, with time and attention, the logic of the differentiation processes appears to be much closer than initially appreciated. Neutralizing IFN
Immunologists attributed many autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and their experimental models, to the action of Th1 cells. However, they were puzzled by the paradoxical finding that neutralizing or knocking out IL-12 and IFN
In 2006, Stockinger, Weaver, Kuchroo, and their colleagues each showed that Th17 cells could be induced in vitro from naive mouse CD4 T cells by stimulation through their T-cell receptor (TCR) in the presence of IL-6 and TGF-β.26–28 ROR IL-21 produced by Th17 cells, induced in the course of Th17 differentiation,35–37 fulfills the role of the powerful positive feedback stimulant, reinforcing the Th17 induction process and showing that Th17 development has the logic similar to that of Th1 and Th2 cells. The Treg "revolution" has been one of the defining themes of modern immunology but reaching an understanding of how these cells differentiate has been complex. In 1995, Sakaguchi and his colleagues discovered that regulatory T cells express CD25.38 Transfer of CD4 T cells that had been depleted of the CD25+ population into congenitally athymic mice induced autoimmune diseases while transfer of intact populations of CD4 T cells did not. In 2001, the autoimmune Scurfy mice and a human immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) patient were found to have mutations in Foxp3.39–41 In 2003, Foxp3 was reported as the master transcriptional regulator for nTreg cells.42,43 Weiner and colleagues had reported in 1994 that oral tolerance regimens induced TGF-β–producing CD4 T regulatory cells.8 This cell population was designated Th3 cells. In 2003, Chen et al reported that TGF-β can convert Foxp3– naive CD4 T cells into Foxp3+ CD4 T cells, that is iTreg cells.44 It is now clear that activated naive CD4 T cells stimulated by TGF-β in the absence of proinflammatory cytokines develop into iTreg cells. The positive feedback factor here is TGF-β itself, although there is still much uncertainty as to the relative biologic importance of nTreg and iTreg cells, particularly in humans. Converting the Th paradigm from in vitro to in vivo situations initially met with much resistance but with time it became clear that memory and memory/effector T cells from normal priming events do display polarization in their cytokine-producing capacity, in their functions and in the range of cell surface molecules they express. Indeed, the recent description of the selective deficit in development of Th17 cells in patients with hyper-IgE syndrome (HIES or Job syndrome) strikingly validates this concept.45 HIES patients have a genetically determined inability to signal through Stat3, due to dominant negative mutations in the SH2 domain or the DNA-binding domain of this molecule.45–47 In humans and mice, the 3 major inducers and/or sustainers of Th17 differentiation, IL-6, IL-21 and IL-23, each use Stat3 for signal transduction. Indeed, the principal difficulties HIES patients face, recurrent staphylococcal and fungal infections, are precisely those observed in mice that cannot develop Th17 cells, strikingly validating the importance of the CD4 T-cell differentiation concept and indicating that lessons are learned, although not always perfectly, by studying experimental animals.
Th cells play critical roles in orchestrating the adaptive immune responses. They exert such functions mainly through secreting cytokines and chemokines that activate and/or recruit target cells.
Th1 cells mediate immune responses against intracellular pathogens.5,6 In humans, they play a particularly important role in resistance to mycobacterial infections. Th1 cells are also responsible for the induction of some autoimmune diseases. Their principal cytokine products are IFN
Th2 cells mediate host defense against extracellular parasites including helminths.5,6 They are important in the induction and persistence of asthma and other allergic diseases. Th2 cells produce IL-4, IL-5, IL-9, IL-10, IL-13, IL-25, and amphiregulin. IL-4 is the positive feedback cytokine for Th2 cell differentiation15,16 and is the major mediator of IgE class switching in B cells.53 IgE binds to Fc IL-5 plays a critical role in recruiting eosinophils.54 In addition to its effect on mast cells and lymphocytes, IL-9 induces mucin production in epithelial cells during allergic reactions.55 IL-10, produced by Th2 cells, suppresses Th1 cell proliferation.56 IL-10 can also suppress dendritic cell function.57 IL-13 is the effector cytokine in the expulsion of helminths and in the induction of airway hypersensitivity.58,59 Amphiregulin is a member of the epidermal growth factor (EGF) family. It induces epithelial cell proliferation. In the absence of amphiregulin, the expulsion of the nematode Trichuris muris is delayed.60 Amphiregulin may also be important for the induction of airway hypersensitivity.
IL-25 (also known as IL-17E) is also a Th2 cytokine.61,62 IL-25, signaling through IL-17RB, enhances the production of IL-4, IL-5, and IL-13 by a unique c-kit+Fc
Th17 cells mediate immune responses against extracellular bacteria and fungi.7 They are responsible for, or participate in, the induction of many organ-specific autoimmune diseases. Th17 cells produce IL-17a, IL-17f, IL-21, and IL-22. IL-17a was originally cloned as CTLA-8 and is homologous to a Herpesvirus saimiri gene. It was renamed IL-17 when its receptor was cloned.64 IL-17a and IL-17f are genetically linked and presumably under the control of the same locus control region (LCR). Thus, IL-17a and IL-17f are often coexpressed at the single cell level although there are also IL-17a- and IL-17f-single producing cells, suggesting the regulation of IL-17a and IL-17f expression in Th17 cells mirrors that of IL-4 and IL-13 in Th2 cells (see below). IL-17a and IL-17f both use the IL-17RA chain for their signaling, implying that they have similar functions, although IL-17a binds to IL-17RA with much higher affinity.65 IL-17a can induce many inflammatory cytokines, IL-6 as well as chemokines such as IL-8 (also known as CXCL8), and thus has an important role in inducing inflammatory responses.64 Both IL-17a and IL-17f recruit and activate neutrophils during immune responses against extracellular bacteria and fungi. IL-21 made by Th17 cells is a stimulatory factor for Th17 differentiation and serves as the positive feedback amplifier,35–37 as does IFN Treg cells play a critical role in maintaining self-tolerance as well as in regulating immune responses.2 Increasing Treg numbers and/or enhancing their suppressive function may be beneficial for treating autoimmune diseases and for preventing allograft rejection. Indeed, Treg cells stimulated in vitro with alloantigen prevent both acute and chronic allograft rejection in mice.72 On the other hand, depletion of Treg cells and/or inhibition of their function could enhance immunity against tumors and chronic infectious agents. Treg cells exert their suppressive functions through several mechanisms, some of which require cell-cell contact.3 The molecular basis of suppression in some cases is through their production of cytokines, including TGF-β, IL-10, and IL-35. TGF-β produced by Treg cells may also result in the induction of iTreg cells from naive CD4 T cells. Although TGF-β is not absolutely required for suppression in some settings, particularly in vitro, it is very important in mediating suppression in several circumstances in vivo.73,74 IL-10 production is critical for Treg-mediated prevention and cure of inflammatory bowel disease.75,76 Specific deletion of IL-10 in Treg cells by Foxp3-Cre results in the development of spontaneous colitis and enhanced lung inflammation.77 IL-10 also plays an important role in limiting the severity of EAE at later stages. During Leishmania infection, Treg IL-10 production in the lesion maintains a homeostasis between the host and the pathogen, allowing a low level of pathogen persistence and a consequent continued stimulation of protective immunity.78 IL-35, which consists of EBI3, a chain shared with IL-27, and IL-12 p35, is produced by Treg cells and contributes to suppressive activity.79 CD4 T cells other than Th2 and Treg cells can also produce IL-10. IL-10 production by Th1 or Th17 cells may play an important role in limiting their own effector function.11–13 IL-10, IL-27, and TGFβ can induce IL-10 production.10,13,80 Interestingly, Foxp3-deleted "Treg cells," judged by expression of GFP encoded by a Foxp3null locus, produce high levels of IL-10, suggesting that IL-10 production in Treg cells is independent of Foxp3.81 The originally described TR1 cells (IL-10–producing regulatory T cells) may include many different types of cells that are capable of producing IL-10. Thus, IL-10 production by all CD4 T cells serves as a negative regulatory mechanism for limiting the immune responses to prevent host tissue damage.
Th1 cells
IL-12Rβ2 expression is induced by TCR activation and then maintained by IL-12 as well as by IFN Th2 cells
IL-4R Th17 cells
Th17 cells express high levels of IL-23R.27,31,37 In addition, Th17 cells express substantial amounts of IL-1R1 and of IL-18R Treg cells
The majority of the nTreg cells express CD25.2 Although all activated T cells express CD25, Treg cells express the highest levels of CD25 and do so constitutively, whereas expression by conventional CD4 T cells is transient and lower. The high level of expression of CD25, IL-2R
Transcription factors including members of the nuclear factor of activated T cell (NFAT), NF-  B, and activator protein-1 (AP-1) families are critically involved in cytokine production upon TCR and/or cytokine stimulation. Presumably, those factors are also important during the process of T helper differentiation. However, they are not the factors directly determining T helper lineage fates and are usually expressed in all lineages. Below, we will focus on the transcription factors that either are specifically expressed, or function differently, in each of the lineages. Transcription factors for Th1 differentiation
T-bet,21 the Th1 master regulator, is up-regulated during Th1 differentiation. Stat1, the major transducer of IFN
However, T-bet–/– Th1 cells still produce some IFN
Stat4, an IL-12 signal transducer, is important for amplifying Th1 responses.103,104 In addition, Stat4 can directly induce IFN
Runx3,105,106 a transcriptional repressor important for silencing CD4 during CD8 T-cell development, is also up-regulated in Th1 cells. Overexpression of Runx3 in Th2 cells induces IFN
Hlx, a transcription factor induced by T-bet, interacts with T-bet and enhances T-bet-mediated IFN Transcription factors for Th2 differentiation Stat6, activated by IL-4, is the major signal transducer in IL-4–mediated Th2 differentiation.108–110 Stat6-deficient cells fail to develop IL-4–producing capacity in vitro; in vivo, Th2 responses independent of Stat6 activation can be obtained.111–113 In vitro, Stat6 activation is necessary and sufficient for inducing high expression levels of the Th2 master regulator gene, GATA-3.114,115 Overexpression of GATA-3 in Th1 cells induces IL-4 production116 and in the absence of GATA-3, Th2 differentiation is totally abolished in vitro and in vivo.117,118 Even in fully differentiated Th2 cells, deleting GATA-3 completely blocks the subsequent production of IL-5 and IL-13,117 although it has only a modest effect on IL-4 production, consistent with the presence of GATA-3-binding sites in the promoters of IL-5 and IL-13 but not in the IL-4 promoter. There are 2 Stat5 family members, Stat5a and Stat5b.119 They are important for cytokine-driven cell proliferation and cell survival. IL-2 potently stimulates Stat5 activation. Th2 cell differentiation requires strong Stat5 signaling.19,120 Thus, Stat5a single knockout cells have profound defects in Th2 cell differentiation both in vitro and in vivo despite the presence and activation of Stat5b. Stat5 has been shown to directly bind to DNase I hypersensitive sites (HSII and HSIII) in the second intron of the Il4 locus.120 c-Maf, which is selectively up-regulated in Th2 cells, also enhances IL-4 production but does not play a role in the production of other Th2 cytokines.121 IRF-4 expression is required for Th2 cell differentiation.122,123 IRF-4–deficient cells produce much less IL-4, but this defect can be rescued by overexpression of GATA-3, suggesting that IRF-4 up-regulates GATA-3.122 Gfi-1 is an immediate early IL-4–inducible gene.124 TCR activation also transiently induces Gfi-1 expression. Gfi-1 selects GATA-3hi cells for growth by modulating both the upstream and the downstream IL-2 signaling events.124,125 Transcription factors for Th17 differentiation
ROR
Another related nuclear receptor, ROR Stat3, the major signal transducer for IL-6, IL-21 and IL-23, is indispensable for IL-17 production and deletion of Stat3 results in the loss of IL-17 producing cells.127–129 Stat3 is also responsible for the induction of IL-23R.
Interferon regulatory factor–4 (IRF4) has been recently reported to be critical for Th17 cell differentiation.130 IRF4–/– T cells fail to produce any IL-17. EAE cannot be induced in IRF4–/– mice. IRF4 appears to play a role in ROR Transcription factors for Treg differentiation As noted above, most patients with IPEX and Scurfy mice have FOXP3/Foxp3 mutations, which result in loss of functional Treg cells. Overexpression of Foxp3 in conventional T cells converts them to a Treg phenotype and endows them with anergy and suppressive activity.42 TGF-β induces Foxp3 expression.44 Continuous expression of Foxp3 is critical for maintaining the suppressive activity of Treg cells.131 Diminishing the degree of Foxp3 expression may convert Treg cells to Th2 like cells, implying a close relationship of the Th2 and Treg lineages.132 Stat5 activation by IL-2, important for Th2 differentiation, is also required for Treg development.133 Stat5 may contribute to Foxp3 induction through binding to its promoter.134,135
Th1 cell differentiation
In the initiation of Th1 responses, antigen-presenting cells (APCs), particularly activated dendritic cells, stimulate naive CD4 T cells possessing cognate T-cell receptors. APCs that produce large amounts of IL-12 as a result of their activation136 (eg through either a combination of TLR3, TLR4, TLR7, TLR8, TLR9, and TLR11 stimulation or a single TLR activation in the presence of type I IFNs, IFN
At later stages of Th1 differentiation, IL-18R  is also up-regulated. IL-18R up-regulation requires IL-12/Stat4 signaling and is further increased by IFN. IL-12 and IL-18 jointly induce IFN production by Th1 cells in the absence of TCR stimulation. Such antigen-independent cytokine production is probably important for amplifying Th1 responses by recruiting other preexisting Th1 cells. Th2 differentiation Both IL-4 and IL-2 are required for Th2 differentiation (Figure 3) in vitro.15,19 IL-4 can be provided exogenously, in which case IL-4–mediated Stat6 activation induces GATA-3 expression. If exogenous IL-4 is not provided, naive CD4 T cells can produce limited amounts of IL-4, as a result of TCR-mediated Gata3 transcription and IL-2 mediated Stat5 activation.138 Such endogenous IL-4 production only occurs when cells receive low strength signals. The endogenous IL-4 then acts like exogenous IL-4 to up-regulate GATA-3 expression. GATA-3 has been reported to induce it own expression,139 probably when it has reached a threshold level. The IL-4/Stat6 pathway also induces expression of Gfi-1, a transcriptional repressor, which plays an important role in selecting GATA-3high cells to grow, providing a selective component in the Th2 development pathway124,125 (Figure 2). GATA-3 binds to regions of the Il4/Il13 loci including DNaseI hypersensitive site Va and CNS-1 sites (see "Epigenetic changes in Th differentiation"); however, GATA-3 alone is not sufficient to induce IL-4 production. IL-2–mediated activation of Stat5 plays a critical role in inducing/maintaining accessibility at the second intron HSII and HSIII DNase I hypersensitive sites of the Il4 locus.120 Indeed, Stat5 is bound to these 2 sites in Th2 but not Th1 cells. The collaboration of Stat5 and GATA-3 accounts for full Th2 differentiation in vitro.140
Accumulating in vivo studies indicate that IL-4 is not essential for Th2 differentiation in some settings, particularly for primary Th2 responses to Nippostrongylus brasiliensis and Schistosoma mansoni infection.111–113 The absence of IL-4 abolishes IgE switching in B cells in these infections, but Th2 cell differentiation is retained, at least partially. On the other hand, in vivo Th2 responses are completely dependent on GATA-3,117 suggesting that there is an IL-4–independent pathway for GATA-3 induction in vivo. It has been suggested that IL-4 can be induced by Notch signaling.141 However, Notch's role in IL-4–independent in vivo Th2 responses is still debatable. IL-4–independent Th2 responses in vivo may reflect hyperactivation of Stat5 by cytokines like IL-2, IL-7 or TSLP, because only limited amounts of GATA-3 are needed for Th2 differentiation when Stat5 is overexpressed.120 In fact, GATA-3 expression levels in in vivo–primed Th2 cells are substantially lower than those of in vitro–primed Th2 cells. Th17 differentiation
TGFβ is critical for Th17 cell differentiation.26–28,32–34 TGFβ1-deficient mice are devoid of Th17 cells. More importantly, T cell– specific deletion of TGFβ1 blocks differentiation of Th17 cells during EAE induction and such mice are resistant to EAE.74 IL-6 is produced by the cells of the innate immune system that have been activated through TLR signaling. In the presence of IL-6, TGFβ induces Th17 differentiation,26–28 production of IL-21 and expression of IL-23R and ROR Treg cell differentiation TGFβ also plays a major role in iTreg differentiation44 and is important for nTreg development.142 Deleting TGFβ from Treg cells results in diminished suppressive function and poor survival in vivo.74,143 In the absence of proinflammatory cytokines, TGFβ induces iTreg differentiation from naive mouse CD4 T cells.26 TGFβ activates Smad3 while TCR stimulation induces NFAT activation. Smad3 and NFAT collaborate in remodeling the Foxp3 enhancer region and promote Foxp3 expression.144 IL-2–mediated Stat5 activation is also required for the induction of Foxp3 expression.133,135,145 Both TGFβ and IL-2 are required for the survival and function of Treg cells even after they have differentiated.
As described, Th differentiation involves positive feedback by cytokines. The differentiation process also actively involves cross-inhibition of other lineage fates. Mutual suppression between IFN and IL-4 signaling was the take-off point for studies of cross-regulation.5,6 TGFβ was also found to suppress both Th1 and Th2 differentiation,146 and both IL-4 and IFN inhibit Th17 differentiation.24,25
The cross-regulation of Th cell differentiation by cytokines may be partly explained by interaction of master genes. T-bet suppresses GATA-3 function by direct binding of the factors.147 Although it has not been studied carefully, such interactions may also be important for IL-4–mediated suppression of Th1 development. TGFβ induces ROR Another level of cross-regulation is through transcriptional regulation of critical factors. GATA-3 has been reported to down-regulate Stat4.149 Strong Stat5 activation inhibits T-bet expression.120 On the other hand, T-bet can suppress GATA-3 expression.84
Finally, cross-regulation occurs at levels of cytokine transcription. Foxp3 suppresses IL-2 through its binding to NFAT150 as well as to Runx1.151 Runx3 inhibits IL-4 production through binding to the HSIV region of the Il4 locus.105 GATA-3 deficiency results in spontaneous IFN
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Abstract
Introduction
RI on basophils and mast cells and, when interacting with a multivalent ligand, cross-links Fc
