the structure and origin of rheumatoid factors

heumatoid arthritis (RA) is acommon crippling diseasecharacterized by destructivejoint inflammation and the

production of rheumatoid factor (RF) auto-antibodies1. RFs recognize epitopes in the Fcregion of IgG (Refs 2, 3) and, while their re-lationship to the pathogenesis of RA remainscontroversial, there is evidence to suggestthat they play an important role in a com-plex disease process4–6. IgM and IgG RFs areproduced abundantly by plasma cells in in-flamed synovial tissues, and form complexeswith the autologous IgG that is present inthe synovia of patients with RA. These complexes can cause inflammation by acti-vating complement or, in the case of self-associating IgG complexes, through cytokinerelease following ligation of Fcg receptors on macrophages7. RF titresin sera have been shown to correlate both with the severity of thearthritis and progressive joint destruction4,8, as well as with extra-articular vasculitis9.

However, RFs are not unique to RA, sometimes occurring in nor-mal elderly individuals, healthy immunized individuals and pa-tients with other autoimmune diseases or chronic infections5. Fur-thermore, anti-IgG antibodies, often indistinguishable from RFs, area common feature of secondary antibody responses and might havea physiological role in assisting the clearance of circulating immunecomplexes and improving the effectiveness of IgG against infectiousagents. The pathogenic RFs associated with joint disease may differfrom physiological (or ‘natural’) RFs in isotype, specificity, frequencyof mutation, site of production or ability to activate complement10,11.

Sequencing of monoclonal RFs from EBV-transformed lines andhybridomas from RA patients has shown that diverse VH and VL

genes are expressed, with considerable heterogeneity in D and J segments12,13. Thus immunoglobulin (Ig) gene usage is not restrictedin RFs and resembles that demonstrated in antibody responses to exogenous protein antigens. Somatic mutation is also well docu-mented in disease-related RFs and has been taken to indicate an antigen-driven response. Although its occurrence per se does notprove selection, mutation has occasionally been associated with increased affinity for Fc (Ref. 14), as well as with changes in grossand fine specificity11. However, there are several examples where the V genes of RFs are unmutated or only slightly mutated, sug-gesting that low-affinity IgG binding is encoded in the germline

repertoire12,15; these may represent the physiological RF population.

Hypotheses for the origin of RFautoantibodiesTriggering by antibody–antigen complexes(IgG as antigen)Several ideas have been proposed to accountfor the production of antibodies with RF ac-tivity. RF-producing B cells can receive T-cellhelp as a result of binding and processing ofimmune complexes16–19. The RF B-cell mem-brane Ig (mIg) receptor is specific for autolo-gous IgG Fc, whereas the foreign antigen is thesource of peptide recognized by T helper (Th)cells. This accounts for the occurrence of anti-IgG antibodies during active immunization

and is consistent with RF production during infections20. Activationof B cells through specific recognition of Fc, together with T-cell help,would be expected to produce monoreactive RFs that were of relativelyhigh affinity, heterogeneous in V-gene usage and isotype, somaticallymutated and idiotypically diverse. These features are typical of RFsfrom patients with RA (Refs 10, 11, 21, 22) and autoimmune mice23.

Crossreaction with foreign antigen or autoantigenRFs are often expressed during viral or bacterial infections24, well-documented examples including subacute bacterial endocarditis25

and Epstein–Barr virus (EBV) infection26. This could be the result ofclassical crossreactivity between microbial epitopes and IgG Fc. An-other possibility is that RFs are crossreactive with other autoantigens,and some RFs react with nuclear antigens27.

Anti-idiotypic mimicry of Fc-binding proteinsThe striking similarity between RFs and bacterial IgG-binding pro-teins in their binding specificity for human IgG subclasses3 has led tothe idea that RFs are anti-idiotypes reactive with antibodies againstStaphylococcus aureus protein A or similar microbial proteins. SuchRFs would be internal images of the bacterial proteins, mimickingthem functionally by binding to Fc (Refs 28, 29). A similar suggestionhas been made that Fc receptors (FcRs) expressed on cells infected byherpesviruses induce anti-FcR antibodies, and that anti-idiotypesagainst the latter are FcR mimics with RF activity30,31.

V I E W P O I N TI M M U N O L O G Y TO D AY

0167-5699/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-5699(00)01589-9

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The structure and origin of rheumatoid factors

Brian Sutton, Adam Corper, Vincent Bonagura and Michael Taussig

The recently determined X-ray

crystal structure of a human

rheumatoid factor Fab bound to IgG

Fc provides the basis of a new

hypothesis for the origin of these

autoantibodies in rheumatoid

arthritis. The observation that Fc is

bound outside the conventional

antigen combining site suggests a

novel form of crossreactivity with

simultaneous binding of another

antigen, potentiated by somatic

mutation. This article discusses the

implications for the induction of

these autoantibodies.

R

Polyclonal B-cell activationRF-producing B cells can be activated by the mitogenic effects of in-fectious agents (e.g. bacterial lipopolysaccharides, EBV) or through‘bystander effects’ during specific responses32. Stimulation of normalhuman B cells by EBV in vitro releases low-affinity IgM RFs from B-1 (CD51) cells33. Antibodies stimulated in this way are usuallypolyreactive with restricted V-gene usage, little somatic mutationand idiotypically crossreactive. These are typical properties of thephysiological RFs found in normal individuals.

RF production as a response to alteration in Fc glycosylationA correlation between RA disease activity and the composition ofthe N-linked carbohydrate in IgG Fc, in particular a deficiency ingalactose content, has been observed34,35. Such a structural change inFc might produce new epitopes that could elicit autoreactivity, andsome RFs are indeed sensitive to the state of Fc galactosylation36,37.Altered glycosylation could therefore enhance complex formationand complement activation, although the latter might also resultfrom the attachment of mannose-binding protein to immune complexes containing agalactosyl IgG (Ref. 38).

X-ray crystal structure of an RF–IgG complexThe recently determined structure of an RF bound to its autoantigenIgG Fc (Ref. 39) has revealed the precise nature of the autorecogni-tion event. The molecular details of the complex define both the epitope on IgG Fc and the RF combining site residues involved in theinteraction. The monoclonal RF IgM used in this study, RF-AN, wasderived from a peripheral blood B cell of an RA patient40, and theIgG Fc was from an IgG4 myeloma, Rea, with partially agalactosylcarbohydrate composition41. The properties of RF-AN are summa-rized in Box 1. Although it has a low affinity for IgG, its derivationfrom an RA patient, and the important role of somatic mutation inautorecognition (as described below), indicate its selection as part ofan autoimmune process.

The structure of the complex (Fig. 1) shows two RF-AN Fab frag-ments bound symmetrically on either side of the IgG4 Fc, demon-

strating the accessibility of the two identicalepitopes, one in each H chain. Both the Cg2and Cg3 domains contribute to the epitope,which includes residues lining the cleft be-tween them (for a detailed description of thestructure, see Ref. 39).

The most striking feature of the complexis the unusual use of the combining site,which contrasts strongly with the classicalmode of interaction familiar from structuresof high-affinity IgG antibodies against exoge-nous antigens43,44. Instead of employing thecentral region of the combining site aroundthe axis of the VH-VL b-barrel, and engagingresidues from five or six complementarity-determining region (CDR) loops, RF-ANrecognizes the Fc epitope via residues on oneside of the VH-VL dimer, at the edge of theconventional site (Fig. 2a). This is achievedthrough only eight contacts from four CDRloops, without involving L1 or L3 (Fig. 2b).Moreover, the RF-AN residues involvedpoint away from the central axis. The conse-quence of this topology is that the conven-tional binding area remains free and poten-tially accessible even in the complex (Fig. 2a).This raises the possibility that RF-AN


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Box 1. Properties of monoclonal humanrheumatoid factor RF-AN

• Derived from PBLs of an RA patient by EBV transformation40

• IgM/l; light chain is from the VlIIIa subgroup (gene V2-14);heavy chain is from the VH3 family (DP-31)42; somatically mu-tated with seven substitutions in VH and one in VL

(see Fig. 4)• Recognizes an epitope on human IgG subclasses 1, 2 and 4,

and G3m (st) allotype, but not IgG3 of Caucasian allotype(new Ga-related specificity)

• IgG Fc binding blocked by Staphylococcus aureus protein A• Affinity of Fab: Ka ,105 M21; avidity of IgM: Ka 5 2 3 106 M21

Abbreviations: EBV, Epstein–Barr virus; H, heavy chain; L, light chain; PBL, peripheral blood lymphocyte; RA, rheumatoid arthritis; V, variable.

Fig. 1. Crystal structure of a rheumatoid factor (RF)–IgG Fc complex39. Two Fabs of IgM RF-AN(heavy chain, dark blue; light chain, light blue) bind symmetrically, one to each heavy chain of IgG4Fc (red). The ribbons represent the polypeptide backbone. The epitope recognized on IgG Fc involvesboth the heavy chain Cg2 and Cg3 domains; the N-linked carbohydrate chains within Fc are not seenin the crystal structure, implying that they are disordered or mobile.

recognizes another, as yet unidentified, antigen at the conventionalsite, which is the primary specificity of the antibody and responsiblefor its induction. Thus, the structure may represent a novel form ofcrossreaction in which an antibody to an exogenous antigen has a si-multaneous weak affinity for an endogenous antigen, in this case IgGFc, at a different site. This site, on the side of VH–VL, is shown in Fig. 3.

There are other instances in which interactions with antibody Vdomains occur outside the conventional antigen-binding site. Idiotope/anti-idiotope recognition is one such example, although todate, the Id–anti-Id complexes studied crystallographically have revealed conventional use of all CDRs in both partners45. Other ex-amples are the so-called B-cell superantigens such as staphylococcalprotein A, which binds to VH3 domains; however, protein A bindsonly to VH framework residues (Ref. 46; M. Graille et al., unpub-lished), whereas the RF-AN binding site comprises CDRs (Fig. 3).

In the case of RF-AN, the amino acid sequence (Fig. 4) provides aclue to how the unconventional use of the binding site may havearisen, and indicates that crossreactivity for IgG Fc is not only gen-erated through the germline but also somatically (Table 1). Thus,critical contact residues in CDRs L2 and H3 are derived through so-matic processes (highlighted in Fig. 2c). The most striking example isVL residue L56, which is a point mutation from germline serine toproline. This position at the C-terminal end of CDR-L2 is rarely in-volved in recognition of exogenous antigen, and its presence as theonly somatic mutation in the entire VL segment strongly suggests se-lection by IgG Fc. The other contact region generated somatically isin CDR-H3, where three out of the four contact residues are coded by N-region nucleotides, added during the process of V–D rearrangement


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Fig. 2. The recognition of autoantigen IgG Fc involves an unconventionaluse of the RF combining site. The complex is viewed looking into the classi-cal combining site, along the axis of the b-barrel formed between the VH

(dark blue) and VL (light blue) domains of RF-AN. IgG Fc domains Cg2(upper) and Cg3 (lower) are shown in red. (a) Space-filling representationof the RF-AN–IgG Fc complex with the CDRs colour-coded as follows: L1,green; L2, purple; L3, pink; H1, white; H2, yellow; H3, orange. All areclearly accessible even in the complex. (b) The eight CDR contact residuesdefined in the crystal structure of the complex, together with the CDR backbones (L1, L2, L3; H1, H2, H3), are shown in white. (c) Germline gene segments and somatic origin of contact residues. The V-segment CDRs areshown in white, J segments in orange and the D segment in purple. Arrowsindicate residues arising in the H3 N-region (Arg H96 to Tyr H98) and thesomatic mutation Pro L56. Other somatic mutations that do not contributeto IgG Fc contact are shown but not labelled (in H2 and L3). Abbreviations:CDR, complementarity-determining region; D, diversity; H, heavy; J, joining;L, light; RF, rheumatoid factor; V, variable.

Fig. 3. View of the site with which RF-AN recognizes IgG Fc. Contactresidues are highlighted in red and labelled; the rest of the CDRs are inwhite, with the remainder of the VH and VL domains in dark and light blue,respectively. This view is perpendicular to that of Fig. 2, and shows that thesite lies on the side of the VH–VL domains. For abbreviations, see Fig. 2 legend.

(Fig. 4; Table 1). The germline contribution to the interaction comprises the remaining four of the eight CDR contacts, two in VH,one in D and one in VL (Fig. 4; Table 1; Fig. 2b), together with a main-chain framework contact at residue 1 in the VH (Fig. 3).

The structure of the complex addressestwo of the hypotheses discussed above forthe origin of RFs, namely, molecular (anti-idiotypic) mimicry of bacterial Ig-bindingproteins and the role of Fc oligosaccharides.Regarding the former, although the epitoperecognized by RF-AN substantially overlapsthe binding sites for both protein A andstreptococcal protein G, there is only partialidentity: only seven and nine out of the 15contact residues in the epitope recognizedby RF-AN are also contacts for proteins Aand G, respectively47. Furthermore, forthose Fc residues that are seen by both RF-AN and the Ig-binding proteins, there islittle correspondence at the atomic level between the interactions that are made39. Finally, the residues used by RF-AN and thebacterial proteins in making these contactsare quite different. Therefore, the structureslend little support to the concept of an idio-typic network as the driving force in RF production.

The structure of the complex does notsupport a role for carbohydrate in recogni-tion of IgG Fc by RF-AN. Indeed, the carbo-hydrate is not visible in the complex, imply-ing flexibility. Even if the carbohydrate wasmodelled according to the conformation ob-served in other Fc structures, it would notimpinge on the epitope, and there is no evi-

dence that the presence or absence of galactose would influencebinding in the case of this particular RF.

Implications for the origin of RFsA model for the origin of pathogenic RFs should take into accountthe above structural observations, incorporating the binding of twoRF Fabs to one Fc, the unusual topology of the combining site inter-action and the occurrence of somatic mutation in autoantigen con-tact residues. Three possible models for the events at the B-cell sur-face leading to RF production are shown in Fig. 5. In all three, B-cellactivation is specific (rather than polyclonal) and T-cell dependent,requiring presentation of peptides derived from an antigen otherthan Fc, as well as co-ligation of the complement receptor CR2 (CD21)–CD19 complex at the B-cell surface.

The bivalent, symmetrical nature of Fab binding to Fc suggests thatreceptor crosslinking by Fc could be part of the B-cell activation event.This enables RF B cells to trap and internalize immune complexes sothat foreign antigen can be presented to Th cells (Fig. 5a). The abilityof RF B cells to act as antigen-presenting cells in this way is experi-mentally well established in vitro17–19 and can account for the generationof RFs during secondary antibody responses16. In this model, speci-ficity for the exogenous antigen resides entirely in the trapped IgG.


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Fig. 4. Genetic segments of the RF-AN V regions and their contributions to contact with IgG Fc. Thecontact residues are shown in red type, and dashes indicate identity between RF-AN and the germlinesequences.

VH segment H1 RF-AN EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYAMHWVRQSPGKGLEW DP-31 ----------------------AA---------------A-------

H2 RF-AN VSGISWNTGTIIYADSVKGRFIISRDNAKNSLYLQMNSLRVEDTALYYCAKDP-31 -------S-S-G----------------------------A---------- N regions, D-JH segments

RF-AN: TRSYVVAAEY YFHYWGQGILVTD2-15 NNNN------ JH4b --D-----T---

VL segment L1 RF-AN YVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYV2-14 -----------------------------------------------

L2 RF-AN DDSDRPPGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHV2-14 ------S-----------------------------------------

JL segment

RF-AN AVFGGGTKLTJλ2 V--------- Immunology Today

Table 1. RF contact residues in the RF-AN–IgG Fccomplex and their genetic origin

Segment Contact residue Genetic origin

VH (DP-31) Asp H31 GermlineTrp H52a Germline

D (D2-15) Arg H96 N-regionSer H97 N-regionTyr H98 N-regionVal H99 Germline

JH ( JH4b) –a –

VL (Vl2-14) Asp L50 GermlinePro L56 Somatic mutation

JL ( Jl2) – –

aNo contact residues contributed.

Alternatively, recognition of the exogenous antigen could residewith the B-cell receptor (mIgM, Fig. 5b). In this case, RF activity is aresult of classical crossreactivity, and both antigen and Fc competefor the same binding site. The trapped IgG can contribute to B-cellactivation by exogenous antigen through crosslinking of mIg (Fig.5b); IgG may also restimulate antigen-activated B cells, providingthe basis for selection of mutations that enhance affinity for IgG Fcsuch as observed in the RF-AN structure. If the bound IgG also hasspecificity for the antigen, crosslinking of mIgM via IgG–antigenbridges could occur (not shown).

A key feature of the RF-AN complex is that the unusual mode ofFc binding could allow the RF to bind exogenous antigen and IgGsimultaneously. Binding of both IgG and antigen may enhance thestability of the complex through direct mutual interaction of the anti-gen and IgG Fc or by an indirect conformational effect. Stabilizationof the interaction of the B-cell receptor with antigen and Fc in aternary immune complex at the cell surface (Fig. 5c) could be par-ticularly significant where either the antigen is in low concentration,or the affinity for antigen or IgG Fc is too low to reach the thresholdfor activation by either ligand alone. RFs are commonly of low affin-ity for IgG Fc. As in the other models, the bivalency of Fc providesreceptor crosslinking, while the enhanced binding affinity will leadto selection of the RF-producing B cell by exogenous antigen andIgG, and hence to high-level RF production consistent with a patho-genic role. Although all three models are compatible with the struc-ture, the ternary interaction is the one that most completely explainsthe features of the RF-AN complex.

Figures 5b and 5c depict RF activity as a crossreaction of an anti-body-combining site directed primarily against a foreign antigen

but, whereas in Fig. 5b both antigen and IgG Fc are recognized at thesame site, in Fig. 5c the two sites are adjacent and potentially dis-tinct. Somatic mutation would be expected to have a different out-come in these two cases. If the binding sites overlap, mutations thatimprove antigen binding are likely to be deleterious (or at least neu-tral) for Fc binding, and vice versa. By contrast, where the antigenand Fc binding sites are spatially separated, somatic mutations canindependently enhance either interaction. Thus, for example, theCDR–L2 contact mutation involved in Fc binding lies well awayfrom the conventional binding area (Figs 2b, 3).

The concept of a ternary interaction is also applicable to com-plexes in solution. While complexes form between RF and IgG alone,they are generally of low affinity (Ka < 105–107 M21). The possibilityof increased stabilization of immune complexes through the ad-ditional presence of antigen would enhance subsequent events suchas complement activation, contributing to RF-induced pathogenesis.

Concluding remarksExtrapolating from the crystal structure of RF-AN bound to IgG, and the model shown in Fig. 5c, it can be proposed that: (1) some RF antibodies bind antigen and IgG Fc simultaneously using theconventional combining site and an adjacent site, respectively; (2)such RFs arise as a consequence of antibody responses to infectiousor environmental antigens (or other autoantigens); (3) they resultfrom V-gene somatic mutation that does not directly affect ‘classical’antigen binding; (4) the RF B-cell receptor binds both antigen andIgG Fc in a ternary complex, leading to high level RF production;and (5) the RFs localize in synovia as complement-activating ternary


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Fig. 5. Three possible models for the primary recognition event by the B-cell antigen receptor leading to rheumatoid factor (RF) production. (a) Trappingof an immune complex through recognition of IgG Fc; antigen specificity resides only with the trapped IgG. (b) Crossreactivity of the RF B-cell antigenreceptor (mIgM) for both antigen and IgG Fc. (c) Trapping of antigen and IgG in a ternary complex in which antigen and IgG Fc are bound simultaneouslyby the same RF Fab, as suggested by the crystal structure of the RF-AN–IgG Fc complex. Antigens are denoted by Ag-X, -Y and -Z. Note that peptidepresentation to T cells, co-ligation of the CD21 (CR2)–CD19 complex at the B-cell surface, and further downstream events are not shown.

Immunology Today

(a) (b) (c)

lgG lgG lgGAg-X

Ag-YAg-Z

mlgM mlgM mlgM

complexes, in which the affinity of RF for IgG is enhanced by thepresence of the second antigen. Clearly this hypothesis, althoughbased upon structure, derives from only one RF–autoantigen com-plex, and RF-AN does not bear all the hallmarks of a pathogenic RF.To ascertain the generality of these findings, structure determina-tions are currently being undertaken of RFs whose properties of highaffinity and pan-reactivity place them clearly in the group associatedwith a pathogenic role10,11.

These studies have been supported the Arthritis Research Campaign (UK).

We thank E. Stura for helpful discussions, I. Tomlinson for identification of the

germline gene segments, and R. Beavil and K. Kirwan for production of the

figures.

Brian Sutton ([email protected]) and Adam Corper are at the RandallCentre, King’s College London, Guy’s Campus, London, UK SE1 1UL;Vincent Bonagura is at the Dept of Pediatrics, Schneider Children’s Hospital of Long Island Jewish Medical Center, New Hyde Park, NY 11040,USA; Michael Taussig is at the Laboratory of Molecular Recognition, TheBabraham Institute, Babraham, Cambridge, UK CB2 4AT.

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he discovery of functionallydistinct types of CD41 T helper(Th) cells has provided a basisto explain the development

of polarized versions of the immune re-sponse1,2. Th1 cells induce phagocyte-dependent immune responses, exemplifiedby the delayed-type hypersensitivity reac-tion (DTH). Phagocytes play a critical role in fighting microbial and viral infections, butcan also cause tissue damage if dysregu-lated. Th2 cells promote IgE production andeosinophil function, both of which play arole in the pathogenesis of allergic reactions.Th2 cells can also exert an anti-inflammatoryaction by negatively regulating Th1-cell-mediated immunity. Thisfunctional heterogeneity in the adaptive immune system has beenproposed to explain a plethora of pathophysiologic phenomena3.The maintenance of functionally polarized immune responses re-quires different subsets of lymphocytes to localize to distinct sites ofinflammation. Here, we review recent studies indicating that differ-ential localization of Th-cell subsets results from multilayered

regulation of the recruitment process, andwe propose a model to integrate these find-ings with the recognized immunological roleof Th1 and Th2 cells.

General mechanisms of leukocyterecruitmentThe ability of leukocytes to traffic coordi-nately throughout the body is critical if theimmune system is to patrol tissues andmount specific responses aimed at eradicat-ing pathogens with minimal tissue damage.Leukocyte extravasation is a multistepprocess mediated by the interplay of adhe-

sion molecules and chemoattractants, and involves the sequentialrolling, firm adhesion and diapedesis of circulating leukocytes4,5.The expression of selectins on endothelial cells mediates rolling ofleukocytes via transient low affinity interactions with selectin li-gands. This initial rolling step allows the leukocyte to sample theendothelial surface for the presence of chemoattractants that signalto quickly upregulate integrin-mediated adhesion.

36 Soltys, A.J. et al. (1994) The binding of synovial tissue-derived humanmonoclonal immunoglobulin M rheumatoid factor toimmunoglobulin G preparations of differing galactose content. Scand.J. Immunol. 40, 135–143

37 Newkirk, M. (1996) in Abnormalities of IgG Glycosylation andImmunological Disorders (Isenberg, D.A. and Rademacher, T.W., eds),pp. 119–130, J. Wiley and Sons

38 Malhotra, R. et al. (1995) Glycosylation changes of IgG associated withrheumatoid arthritis can activate complement via the mannose-binding protein. Nat. Med. 1, 237–243

39 Corper, A.L. et al. (1997) Structure of human IgM rheumatoid factorFab bound to its autoantigen IgG Fc reveals a novel topology ofantibody–antigen interaction. Nat. Struct. Biol. 4, 374–381

40 Steinitz, M. et al. (1980) Continuous production of monoclonalrheumatoid factor by EBV-transformed lymphocytes. Nature 287, 443–445

41 Jefferis, R. et al. (1990) A comparative study of the N-linkedoligosaccharide structures of human IgG subclass proteins. Biochem. J.268, 529–537

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Localization of Th-cell subsets ininflammation: differential thresholds for

extravasation of Th1 and Th2 cellsDaniele D’Ambrosio, Andrea Iellem, Lucia Colantonio, Barbara Clissi,

Ruggero Pardi and Francesco Sinigaglia

The recruitment of T helper 1 (Th1)

and Th2 cells into peripheral tissues

is essential for inflammation and the

host response to infections. Here,

Daniele D’Ambrosio and colleagues

review the traffic signals that enable

the distinct positioning of Th1 and

Th2 cells and argue that differential

thresholds for extravasation

constitute a critical aspect of their

selective localization to

inflamed tissues.

T

the structure and origin of rheumatoid factors

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