A Ligand for CD5 Is CD5 Marion H

A Ligand for CD5 Is CD5 Marion H. Brown and Erica Lacey J Immunol 2010; 185:6068-6074; Prepublished online 15 This information is current as October 2010; of September 27, 2021. doi: 10.4049/jimmunol.0903823 http://www.jimmunol.org/content/185/10/6068 Downloaded from References This article cites 41 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/185/10/6068.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Ligand for CD5 Is CD5

Marion H. Brown and Erica Lacey

Recognition by scavenger receptor cysteine-rich domains on membrane proteins regulates innate and adaptive immune responses. Two receptors expressed primarily on T cells, CD5 and CD6, are linked genetically and are structurally similar, both containing three scavenger receptor cysteine-rich domains in their extracellular regions. A specific cell surface interaction for CD5 has been difficult to define at the molecular level because of the susceptibility of CD5 protein to denaturation. By using soluble CD5 purified at neutral pH to preserve biological activity, we show that CD5 mediates species-specific homophilic interactions. CD5 domain 1 only is involved in the interaction. CD5 mAbs that have functional effects in humans, rats, and mice block homophilic binding. Ag-specific responses by mouse T cells in vitro were increased when engagement of human CD5 domain 1 was inhibited by mutation or by IgG or Fab fragment from a CD5 mAb. This showed that homophilic binding results in productive engagement. Enhancement of polyclonal immune responses of rat lymph node cells by a Fab fragment from a CD5 mAb shown to block homophilic interactions provided evidence that the extracellular region of CD5 regulates inhibition in normal cells. These biochemical and in vitro func- Downloaded from tional assays provide evidence that the extracellular region of CD5 regulates immunity through species-specific homophilic interactions. The Journal of Immunology, 2010, 185: 6068–6074.

D5 and CD6 are two unusual T cell surface receptors in sponse is to deﬁne when and how the extracellular region is

that their extracellular regions are each composed of three engaged and how this influences the signaling pathway regu- http://www.jimmunol.org/ C scavenger receptor cysteine-rich (SRCR) domains instead latedbyCD5. of the more common Ig superfamily (IgSF) domains (1). SRCR The heterophilic interaction between the membrane-proximal domains are a primitive recognition domain stretching back to in- SRCR domain of CD6 and its IgSF ligand, CD166 (1), is not vertebrates and have been retained right through evolution in vari- a general paradigm for the type of domain recognized by SRCR ous tissues and the mammalian innate and adaptive immune sys- domains. Other types of recognition by SRCR domains include tems (2). the interaction between another immune system receptor, CD163, Expression of CD5 (3–5), like CD6 (6), has an inhibitory effect and hemoglobin-haptoglobin complexes (13), and the recognition on the cell that is not necessarily dependent on the extracellular of pathogens (2, 14). The interaction between CD6 and CD166 (1, region (3). High levels of CD5 are associated with resistance 12) is the only example that is understood in terms of a well- by guest on September 27, 2021 to activation (7–9). However, there is evidence of positive effects defined molecular interaction at the cell surface, of regulation of of CD5, like CD6 (6), that depend on extracellular engagement adaptive immune responses by a SRCR domain (6). Engagement of CD5 and by logical extrapolation that there is an endoge- of CD6 by CD166 in an Ag-specific immune response results in nous ligand. A soluble CD5-Ig fusion protein produced in vivo phosphorylation of CD6; direct recruitment of the adaptor protein, was inhibitory in a mouse experimental allergic encephalomy- SLP-76, by the C-terminal tyrosine motif; and a net positive out- elitis (EAE) model (10). The effect was species specific, mouse come (6). but not human (h)CD5-Ig being inhibitory (10). Positive effects Cell binding to immune cells has been observed with sol- of CD5 were revealed in vivo in the mouse EAE model in which uble forms of the extracellular region of CD5 (15–18) and with a cytoplasmic mutation in CD5 decreased the severity of dis- CD5 purified from cells (19). Consistent with it having a ligand- ease (11). An overall role for costimulation by CD5 was shown binding function, cell binding was affected by domain 1 alone by testing the effect of an immune challenge in the absence of (17). In none of these experiments was it possible to determine CD5. Mice lacking CD5 were resistant to EAE (10). CD5, like whether binding was mediated by biologically active material. CD6, is required for an optimal immune response (6, 12). The CD5 mAbs did not block binding (17) consistently (19, 20). key to understanding the function of CD5 in the immune re- The cell surface binding partner for CD5 purified from cells was identified as CD72 (19). An interaction between CD72 and CD5 has not been reproduced. Soluble recombinant CD5 did Sir William Dunn School of Pathology, Oxford, United Kingdom not bind cell surface CD72 (15–17), and there was no evidence Received for publication December 1, 2009. Accepted for publication September 11, for productive engagement of cell surface-expressed CD5 and 2010. CD72 (16). Attempts to define the molecular identities of al- This work was supported by Grant G0400808 from the Medical Research Council, ternative cell surface binding partners for recombinant soluble United Kingdom. CD5 have not been definitive (15–1ot8) probably due to its un- Address correspondence and reprint requests to Dr. Marion H. Brown, Sir William usual lability, which may be related to a high degree of con- Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, United Kingdom. E-mail address: [email protected] formational flexibility, as shown by nuclear magnetic resonance Abbreviations used in this paper: CHO, Chinese hamster ovary; EAE, experimental (NMR) spectroscopy of CD5 domain 1 (21). We show that pre- allergic encephalomyelitis; h, human; IgSF, Ig superfamily; m, mouse; NMR, nuclear servation of three-dimensional structure is key to molecular magnetic resonance; r, rat; RU, response unit; SPR, surface plasmon resonance; characterization of the ligand-binding properties of CD5, and SRCR, scavenger receptor cysteine rich. the extensive functional data can be interpreted by homophilic Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 interactions of CD5. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903823 The Journal of Immunology 6069

Materials and Methods for screening their effect on homophilic binding by hCD5-His or hrCD5- Recombinant protein production His at 25˚C. Chimericproteins of the extracellular regionof human(h; Genbankaccession Functional assays X04391) and mutants L26K, M124Q, V88D/V97K, E72K, L96P, and K69D 5 (21); rat (r; Genbank accession X78985: bp59A → T, bp258 T → C); Ag-specific IL-2 production of the mouse 2B4 hybridoma cells (10 ) and mouse (m; Genbank accession D10728) CD5 were made. XbaI sites in response to moth cytochrome c peptide presented by CHO cells K K 4 5 were introduced upstream of the initiation ATG (tctAGAAGGCCA), and expressing I-E (CHO I-E ;53 10 –10 ) was carried out as described SalI sites at the end of the extracellular region (join: gtg gca tcg tcg acc → previously in round-bottomed wells of 96-well plates (26). IL-2 production VAS ST). A BamHI site (bp 2035) in hCD5 was removed. CD5, hCD6, and after 18–24 h was measured by ELISA. mAbs and Fab fragments were control CD4 leader constructs in frame with rCD4 domains 3 and 4 and used at a final concentration of 5 mg/ml. Stimulation of lymph node cells a C-terminal peptide that can be biotinylated enzymatically (rCD4d3+3bio) from female Lewis rats was carried out as described (24). Following 5 in the pEFBOS-XB vector were expressed in 293T cells, supernatants bio- red cell lysis, lymph node cells were plated out at 2.5 3 10 /well in round- tinylated as described and tested for binding to a streptavidin-coated CM5 bottomed wells of 96-well plates and preincubated for 30 min at 0˚C BIAcore chip (see below) (22). As the biotin tag reduced expression levels, with Ab or Fab fragments at a final concentration, 5 mg/ml. Con A (5 mg/ the original rCD4d3+4 tag was used to construct hCD5CD4 and rCD5CD4 ml) and TCR mAb (R73) (5 mg/ml) were added and incubated along pEE14 vectors for production of stable lines (23). hCD5-His and rCD5-His with the cells for 3 d at 37˚C. Eighteen hours prior to harvesting, 0.5 ml 3 pEE14 vectors were constructed by shuttling fragments via pEFBOS-XB [methyl- H]thymidine (185 MBq/5 ml; Amersham, GE Healthcare UK, containing a SalI site and His tag (STHHHHHH). CD4hCD72 chimera Little Chalfont, Buckinghamshire, U.K.) was added per well and in- was made as a type II chimera with the C-terminal region of hCD72 con- corporation into cells was determined by scintillation counting. Student taining the C-type lectin domain fused to the C terminus of the leader and t test was carried out on samples in triplicate, and significance was de- domains 3 and 4 of rCD4. The amino acid sequence at the join was termined by p , 0.05. KGLNQQTNRV (CD4 in italics). Downloaded from Stable lines were produced by transfection using Fugene into Chinese hamster ovary (CHO) K1 cells and clones identified by either inhibition ELISA or binding to mAb at 25˚C immobilized via anti-mouse Fc on a CM5 BIAcore chip. Supernatant from typically one or two cell factories (Nunc, Naperville, IL) per selected clone was either purified by affinity chromatography on OX68 (rCD4d3+4 mAb)-Sepharose (Pharmacia, Pea- pack, NJ) with elution in 0.1 M Gly-HCl (pH 2.5) or nickel coupled to fast http://www.jimmunol.org/ flow Sepharose with elution in 250 mM imidazole (pH 8). Eluted material was immediately dialyzed into HEPES-buffered saline or PBS. Eluate was then concentrated with a Viviflow 200 (10,000 molecular mass cutoff; Viviscience, Hannover, Germany) and subjected to gel filtration using Superdex 200 or 75 and fast flow liquid chromatography (Pharmacia). Monomeric and dimeric fractions were collected and analyzed by SDS- PAGE (nuPAGE; Invitrogen, Carlsbad, CA) under nonreducing and reducing conditions. Yields of purified protein were in the order ∼15 mg/l for His-tagged proteins and CD4 chimaeras. Theoretical molecular masses and molar extinction coefficients are as follows: hCD5CD4, 59,932 kDa, 21 21 21 21 75,390 M cm ; rCD5CD4, 60,137 kDa, 64,020 M cm ; hCD5-His, by guest on September 27, 2021 40,445 kDa, 55,760 M21 cm21; rCD5-His, 40,650 kDa, 43,670 M21 cm21; mCD5-His, 39,762 kDa, 43,670 M21 cm21. mAbs mAbs used as tissue culture supernatant or purified IgG were specific for the following: hCD5 domain 1, LEU1 (mIgG2a; American Type Culture Collection, Manassas, VA) and UCHT2 (mIgG1; gift of D. Cantrell, Dundee, U.K.); hCD5 domain 3, CD5-48 (mIgG1; produced against soluble purified hCD5CD4 by G. Roncador and J. Cordell, Oxford, U.K.); rCD5 domain 1, OX19 (mIgG1) (24); mCD5 domain 1, 53.7.131 (rIgG2a; American Type Culture Collection), rTCR mAb, R73 (24), hCD72, BU40; hCD6, T12 (American Type Culture Collection); rCD4 domains 3 and 4, OX68 (23); and control OX21 (IgG1). Recognition by CD5-48 mAb was mapped using hCD5CD4, hCD5 domain 1 CD4, rCD5CD4, and de- finitively with CD5 domain 3 made in Escherichia coli by A. Garcia- Garza, Mill Hill, London, U.K. (21). Fab fragments were prepared by papain digestion and gel filtration. FIGURE 1. Species-specific homophilic binding of CD5. A and B, hCD5-His, rCD5-His, mCD5-His, and hrCD5-His migrated as monomers BIAcore analyses in SDS-PAGE analysis under nonreducing (nr; A, B) or reducing (r; B and data not shown) consistent with noncovalent dimer formation in gel fil- For analysis of homophilic interactions, CM5 chips were coated with C ∼ m streptavidin at 25˚C, and then the temperature was raised to 37˚C for im- tration (data not shown). , Monomeric hCD5-His ( 0.3 M) and rCD5- mobilization of biotinylated CD4d3+4 chimeric proteins (5 ml/min) at His (data not shown) bound immobilized CD5 domain 1 (LEU1) and 3 equivalent levels, in the range of 1500–3000 response units (RUs) and (CD5-48) mAbs. Over time, slow binding of presumably aggregated ma- subsequent injection (5 ml) of increasing concentrations of soluble protein terial to the rabbit anti-mouse Fc was observed. D, BIAcore sensor- (20 ml/min) to measure equilibrium binding (12, 22, 25). The rack con- gram traces of dimeric hCD5-His (2 mM) over hCD5CD4 (1474 RUs), taining proteins to be injected was cooled to 5–10˚C to minimize aggre- rCD5CD4 (1941 RUs), mCD5CD4 (2790 RUs), and control IgSF protein gation and denaturation during the time scale of the experiment. Specific rCD4d3+4 (1582 RUs) immobilized on streptavidin-coated flow cells. The equilibrium binding was determined by subtracting responses in a con- horizontal line indicates injection period. E and F, Equilibrium-binding trol flow cell. mAbs were injected as tissue culture supernatant or purified data at 37˚C from injections of increasing concentrations of monomeric (∼1–10 mg/ml) at 5 ml/min over chimeric proteins or polyclonal rabbit anti-mIgG (Pharmacia). CD5-His (∼1 mM) was injected (5 ml/min) over hCD5-His or hCD5CD4 over hCD5CD4 (1434 RUs), hCD6CD4 (1931 immobilized mAb. For analysis of mAb-blocking flow cells, double RUs), mCD5CD4 (1477 RUs), and control IgSF protein hTREM1CD4 volumes of CD5-His were prepared and injected before and after saturation (1439 RUs) immobilized on streptavidin-coated flow cells. At the end of of immobilized CD5 with mAb. CD5 domain 1 mutants were immobilized the experiment, species-specific CD5 mAb binding was injected over all on the chip from tissue culture supernatant via the rCD4d3+4 mAb, OX68, flow cells to confirm the identity of immobilized proteins. 6070 A LIGAND FOR CD5 IS CD5

Results on rate indicates this is probably due to traces of aggregated Species-specific homophilic interactions of CD5 material. We tested soluble CD5-His for self association by SPR and, to Similarities in the composition of the extracellular regions of mimic physiological conditions, all experiments were carried out at CD5 and CD6 suggested ligand-binding properties in common. 37˚C. Biotinylated CD5CD4 fusion proteins that had not been However, during screens using surface plasmon resonance (SPR) subjected to denaturing purification conditions were immobilized for possible interactions of CD5 with cell surface proteins, in- on a streptavidin-coated chip and serial dilutions of hCD5-His cluding CD166 and the related protein named BCAM or Lutheran, injected (Fig. 1D). Specific homophilic interactions of hCD5-His there was no binding (data not shown). Instead, we noticed a weak were detected (Fig. 1D). The interaction was weak, as indicated by self association of the purified recombinant hCD5CD4, a fusion a rapid dissociation whether the dimeric or monomeric fraction proteincomposedoftheextracellularregionofhCD5and was used (Fig. 1D and data not shown). Plotting the equilibrium- domains 3 and 4 of rCD4. We examined the potential for CD5 for binding data for binding a series of CD5 concentrations revealed self association and, to eliminate the risk of denaturation due to concentration-dependent species-specific binding of hCD5-His to purification at pH 2.5 using affinity chromatography, we produced hCD5CD4 (Fig. 1E). His-tagged versions of hCD5 and rCD5 that were purified at neu- In the complementary experiment, rCD5-His bound specifically tral pH. On gel filtration, hCD5-His and rCD5-His separated to immobilized rCD5CD4 and mCD5CD4 equally well (see Fig. as two peaks (data not shown), with elution volumes consis- 2B and data not shown) and vice versa mCD5-His to rCD5CD4 tent with asymmetric proteins forming dimers. The majority of and mCD5CD4 (see Fig. 2D,2F). Thus, CD5 binds CD5 repro- material was monomeric. Both peaks migrated as monomers ducibly in humans, mice, and rats. hCD5 reacts only with hCD5, Downloaded from on SDS-PAGE under nonreducing and reducing conditions con- whereas mCD5 and rCD5 can cross-react. sistent with a noncovalent association (Fig. 1A,1B). Species- specific saturable binding by purified hCD5-His to immobilized hCD5 domain 1 (LEU1 and UCHT2) and domain 3 mAbs was CD5 is susceptible to denaturation observed, indicating that the material was antigenically active The behavior of soluble hCD5CD4 purified at pH 2.5 was analyzed (Fig. 1C and data not shown). rCD5-His bound the rCD5 mAb, in the same way as hCD5-His in the same experiment using the OX19 (data not shown). The hCD5-His gave some binding to same chip (Fig. 1E,1F). Soluble hCD5CD4 purified at pH 2.5 http://www.jimmunol.org/ the high levels of immobilized rabbit IgG (Fig. 1C). Such a slow gave binding to both hCD5 and mCD5, but also the control by guest on September 27, 2021

FIGURE 2. Homophilic interactions between CD5 domain 1. A–D, Chimeric proteins containing rCD4 domain 3 and 4 (E, circles) hrCD5CD4, rhCD5CD4, hCD5CD4, and rCD5CD4 were immobilized (∼2000– 3000 RUs) on streptavidin-coated flow cells. Increasing concentrations of hCD5-His (A), rCD5-His (B), hrCD5-His (C), or mCD5-His (D) were injected over all flow cells at 37˚C. Equilibrium-binding data, relative to responses over rCD5CD4 (A, C)orhCD5CD4(B, D), are plotted. The identity and equivalent levels of immobilized proteins were con- firmed with mAb specific for hCD5 domain 1 (LEU1), rCD5 domain 1 (OX19), and hCD5 domain 3 (data not shown). In C and D, binding before (closed symbols) and after (open symbols) saturation of immobilized hCD5CD4 and hrCD5CD4 or rCD5CD4 and rCD5CD4 with LEU1 or OX19, respectively, is shown. E, Recombinant proteins are shown schematically. SRCR domains (squares) of human, rat, and mouse CD5 are depicted, domain 1uppermost.F, mCD5CD4 was saturated with 53.7 or control rCD4 mAb, OX68 mAb, and mCD5-His reinjected. The Journal of Immunology 6071 protein, hCD6CD4, and showed none of the discrimination seen with hCD5-His. Domain 1 is necessary for homophilic interactions of CD5 The N-terminal domain of CD5 is topologically suited to mediate ligand binding (2). To determine whether species-specific homophilic binding depends on the N-terminal membrane distal domain of CD5, we constructed chimeric CD5CD4 proteins containing human domain 1 and rat domains 2 and 3 (hrCD5CD4; see Fig. 2E) and the complementary protein containing rat domain 1 and human domains 2 and 3 (rhCD5CD4) (Fig. 2). Soluble hCD5-His or rCD5-His was injected over equivalent levels of CD5CD4 or the chimeras (Fig. 2). Species-specific binding was calculated by subtracting responses in the rCD5CD4 flow cell for hCD5-His and vice versa for rCD5-His. hCD5-His bound equally well to hCD5CD4 and hrCD5CD4, showing that hCD5 domain 1 was necessary for species-specific homophilic binding (Fig. 2A). rCD5- His bound in a species-specific manner to rCD5CD4, but poorly to rhCD5CD4 (Fig. 2B). Nevertheless, the weak binding indicates Downloaded from that rCD5 domain 1 mediates species-specific homophilic binding. The identity and levels of immobilization of all four proteins were checked using mAbs specific for hCD5 domains 1 (LEU1) and 3 (CD5-48) and rCD5 domain 1 (OX19) (data not shown). rCD5 mAb, OX19, was shown to recognize domain 1 using a chimeric

CD4 protein containing rat domain 1 and mouse domains 2 and 3 http://www.jimmunol.org/ (data not shown). Homophilic interactions between CD5 domain 1 FIGURE 3. The V88D/V97K CD5 domain 1 mutant does not bind homophilically. A and B, hCD5CD4, L26KCD4, M124QCD4, V88D/ Binding between hCD5-His and hrCD5CD4 shows that CD5 do- V97KCD4, and rCD5CD4 (∼3000 RUs) were immobilized on streptavi- main 1 is necessary for homophilic interactions. There are several din-coated flow cells. Increasing concentrations of hCD5-His were injected possible topologies of homophilic interactions of CD5 that comply over all flow cells at 37˚C (closed symbols). B, hCD5-His was injected with this result. Theoretically, any one of the domains in hCD5-His over hCD5CD4 saturated with UCHT2 mAb, hCD5CD4, and V88D/ could bind domain 1 of the immobilized material. To ask whether V97KCD4. V88D/V97KCD4 was then saturated with UCHT2 mAb and only CD5 domain 1 is involved in the species-specific homophilic hCD5-His reinjected (open symbols). Binding to hCD5CD4 saturated with by guest on September 27, 2021 interactions, we produced soluble hrCD5-His (Fig. 1B) and tested UCHT2 was the same in the two sets of injections. Equilibrium-binding it for binding to hrCD5CD4. hrCD5-His bound equally well to data, relative to responses over rCD5CD4, are plotted. hCD5CD4 and hrCD5CD4, but not to rCD5CD4 or rhCD5CD4 (Fig. 2C). These data show that hCD5 domain 1 binding to itself (53.7) mAbs (data not shown) showed that soluble CD5-His did is the dominant interaction in the species-specific homophilic in- not compete for immobilized mAb. teractions. In the complementary experiment with the same chip (Fig. 2D), mCD5-His bound the chimeras containing rCD5 do- The CD5 domain 1 V88D/V97K mutant does not bind main 1, confirming the identity and activity of the chimeras on the homophilically chip and the cross-reactivity between mCD5 and rCD5 homo- A clear separation between the epitopes of LEU1 and UCHT2 was philic interactions. The validity of CD5 homophilic binding is em- demonstrated by a single mutation, L26K, in hCD5CD4, which phasized by it being demonstrated in three species and with two specifically abolished recognition by UCHT2 mapping its epitope types of recombinant proteins, His-tagged and CD4d3+4 fusion to the N terminus of CD5 domain (21). The L26K hCD5CD4 proteins. mutant, which is not recognized by UCHT2, bound CD5-His as well as wild-type CD5 (Fig. 3A) consistent with the N terminus of CD5 domain 1 mAbs inhibit homophilic interactions of CD5 CD5 domain 1 not being essential for homophilic interactions. An understanding of the potential of CD5 domain 1 mAbs to block The mutant gave a clearer result than testing for blocking with ligand binding by CD5 aids in mapping the ligand binding site on UCHT2, which appeared to cross-link immobilized and soluble CD5 domain 1 and is important for interpretation of their effects in CD5 whether it be the wild-type or a nonbinding mutant (Fig. 3B). functional assays. Two hCD5 domain 1 mAbs, LEU1 and UCHT2, Mutation of a residue, M124Q, in the region defined as critical for recognize epitopes that do not overlap (21). We analyzed the effect the interaction between CD6 and CD166 did not have a drastic of saturating immobilized hCD5CD4 with LEU1 on homophilic effect on homophilic binding (Fig. 3A). We analyzed a panel of binding by hCD5-His and hrCD5-His. LEU1 specifically blocked hCD5 domain 1 mutants for their effect on homophilic binding binding of hCD5-His to hCD5CD4 (data not shown and Fig. 2C). (Table I). Two mutants showed reduced binding, E72K and V88D/ Blocking of homophilic binding by UCHT2 was not observed (see V97K (Fig. 3B, Table I). Expression and LEU1 mAb binding of Fig. 3B). E72K were reduced relative to wild-type CD5, which may indicate We tested the rCD5 domain 1 mAb (OX19) for its effect on CD5 structural instability. V88D/V97K identified as nonbinding is the ligand binding. OX19 blocked rCD5 homophilic binding (Fig. 2D). mutant from which the NMR structure (21) was derived and thus Similarly, saturation of mCD5CD4 with a mAb (53.7) specific for maintains structural integrity. In the screen of the panel of domain 1 (27) inhibited mCD5 homophilic binding (Fig. 2F). mutants, immobilized recombinant hCD72 was used as a negative Cross species blocking by the rCD5 (OX19) (Fig. 2D) and mCD5 control and its identity checked with the CD72 mAb, BU40. 6072 A LIGAND FOR CD5 IS CD5

Table I. Identification of residues critical for homophilic binding of pressed as monitored by CD5 domain 1 and 3 mAbs (Fig. 4A). Con- hCD5 domain 1 sistently, CD3 levels were higher on cells expressing mutant CD5 (Fig. 4A). Endogenous mCD5 was downregulated on cells ex- Protein hCD5 Binding LEU1 Binding pressing hCD5, whereas transfected mCD2 remained unchanged CD4hCD72A 22(Fig. 4A). The cells transfected with mCD2 were used as the pa- E72K 2 75% wild type rental line to boost levels of Ag-specific IL-2 in the stimulation L96P + + assays. Cells were stimulated with peptide presented by CHO V88D/V97K 2 80–100% wild type cells expressing the MHC class II, I-EK (CHO I-EK cells), or CHO K69D + + K M124Q + + I-E cells expressing mCD48 (28). A similar pattern of responses L26K + + was observed in response to both types of APC with higher IL-2 Soluble hCD5-His or hrCD5-His was passed over CD4 chimeras immobilized production for the ones expressing mCD48 (data not shown). directly from tissue culture supernatant onto a BIAcore chip coated with the Ag-specific IL-2 production by cells expressing mutant CD5 was CD4d3+4 mAb, OX68 at 25˚C, and binding at equilibrium measured. increased relative to wild-type CD5 (Fig. 4B). To ensure this was related to engagement of hCD5 domain 1 and not just the higher CD3 levels on the mutant cells, the effect of a soluble blocking Homophilic interactions of CD5 regulate an immune response CD5 mAb on responses by cells expressing wild-type hCD5 We developed a well-controlled in vitro assay in which we could was analyzed. The hCD5 mAb that blocked species-specific ho- detect functional effects of engagement of CD5 domain 1. Wild- mophilic interactions enhanced Ag-specific IL-2 production (Fig. type and mutant (V88D/V97K) CD5 were expressed in 2B4 4C). Confirmation that this was due to blocking was obtained by Downloaded from mouse T hybridoma cells. The hCD5 variants were highly ex- showing a Fab fragment also enhanced IL-2 production (Fig. 4C). http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Homophilic interactions of CD5 regulate an immune response. A, Mouse 2B4 T hybridoma cells were analyzed by flow cytometry for expression of transfected mCD2, mCD3, mCD5, and transduced hCD5 with hCD5 domain 1 (LEU1 and UCHT2) and domain 3 (hCD5d3) mAbs. B and C, Ag-specific IL-2 production by 2B4 hybridoma cells in response to CHO I-EK (B) or CHO I-EK expressing mCD48 (C) and different concentrations (B)or1 mM moth cytochrome c peptide (C) was enhanced by expression of V88D/V97K compared with wild-type hCD5 (B) or blocking (C) with a hCD5 domain 1 (LEU1) or Fab (LEU1) and not with a control mAb (OX21) or Fab (T12) (C). The difference between LEU1 and control is significant for both IgG and Fab (p , 0.001). D, Proliferation of polyclonally activated rat lymph node cells was enhanced by Fab fragments of rat CD5 mAb (OX19) and not control Fab (OX21) (pp , 0.01). The data shown are representative of three or more independent experiments. The Journal of Immunology 6073

Functional effects of hCD5 domain 1 in mouse cells are consistent 1. A ligand-binding function of CD6 domain 1 is also suggested with species-specific homophilic interactions, resulting in produc- by the existence of a form of CD6 that lacks the membrane- tive engagement. proximal CD166 binding domain on activated T cells (35). Dissection in the hybridoma model revealed the potential of the Homophilic binding by CD5 domain 1 has the potential to extracellular region of CD5 to regulate inhibitory effects of CD5. mediate interactions between cells, in trans or on the same cell, in To assess its physiological relevance, we examined the potential of cis. The lack of involvement or of a subsidiary role for domains 2 the extracellular region of CD5 to regulate inhibition of immune and 3 in CD5 homophilic interactions favors trans interactions as responses by a normal polyclonal cell population. We analyzed the might occur between T cells or T and B cells (16). Alternatively, mode of action of the rCD5 mAb, OX19, which has been shown to the absence of CD5 on dendritic cells at the initiation of an im- have costimulatory effects on primary cells (24). The rCD5 mAb, mune response favors cis interactions. The two topologies for CD5 OX19, blocks homophilic interactions (Fig. 2C). Comparison of homophilic binding are not mutually exclusive. There is good the effects of freshly gel-filtrated Fab fragments with Fab92 or IgG precedent for competition between cis and trans interactions in the [data not shown (24)] showed that all three reagents enhanced Siglec family (36) and interactions between Ly49 and MHC class polyclonal proliferation of rat lymph node cells (Fig. 4D). CD5 I (37). The highly conserved flexible stalk between CD5 domains was not modulated off the surface by OX19 IgG or Fab in vitro 1 and 2 is likely to be important in regulating potential trans (data not shown). The data are consistent with homophilic inter- versus cis CD5 interactions, as has been recently shown for Ly49 actions of CD5 regulating activation of normal cells. (37). The exclusive role of CD5 domain 1 in species-specific homophilic interactions differs from the involvement of all three

SRCR domains of CD5 in an interaction with fungal wall com- Downloaded from Discussion ponents (38). The involvement of domains 2 and 3 of CD5 in other Molecular characterization of homophilic binding by CD5 has interactions (39–41) is not excluded by the homophilic binding resolved two major sources of confusion over immune regulation data. by this receptor. First, speciﬁc and nonspeciﬁc binding by the Correlation of activation and inhibition by CD5 with a re- extracellular region of CD5 were distinguished. CD5 interacted quirement for extracellular engagement is not absolute. Dissection

with itself in a species-specific manner. Second, an understanding of engagement of CD5 in the cell line model shows the potential of http://www.jimmunol.org/ that the extracellular region of CD5 can engage in homophilic the extracellular region of CD5 to regulate its inhibitory effects. binding provides a molecular mechanism for functional effects of Caution must be exercised in dismissing the extracellular region in perturbing engagement of the extracellular region of CD5. regulating inhibition by CD5 based on experiments with geneti- The biochemical and functional data indicated that homophilic cally manipulated mice (3). To demonstrate a role for the extra- CD5 interactions were strong enough to mediate productive inter- cellular region of CD5, it is important to distinguish between actions to regulate immune responses. The micromolar concen- engagement of the extracellular and intracellular region. Simply trations of soluble protein that yielded measurable binding to im- observing differences between CD5+ and CD52 cells does not mobilized material were in the range of those observed for other make this distinction. An inhibitory role for the cytoplasmic re- weak, but physiological relevant interactions (12, 25). The kinetics gion of CD5 was shown in mice expressing CD5 lacking an ex- by guest on September 27, 2021 of CD5 binding revealed rapid on and off rates consistent with tracellular region (3). To test whether interactions of the extra- transient interactions typical for cell surface proteins (12, 22). cellular region contribute to regulation by CD5, we used a CD5 Low stoichiometry precluded good mathematical analysis, as we mAb that blocks CD5 homophilic interactions. In retrospective have done for other homophilic interactions (12, 25). Low stoi- analysis of the literature with the knowledge that a particular CD5 chiometry of binding measured by SPR could be due to the pres- mAb blocks homophilic binding, rigorous analysis of the mode ence of inactive protein or a consequence of self association by the of action is required before functional effects of the mAb, for soluble material competing with immobilized. example, OX19 (24), can be interpreted. To ensure the mode of ac- The presence of a region of particular flexibility in the NMR tion of OX19 was blocking, we used a Fab fragment. Enhance- solution structure of hCD5 domain 1 (21) provides a molecular ment of proliferation by rat lymph node cells by Fab fragments basis for the difficulty in identifying specific ligand binding. from OX19 provides good evidence that the extracellular region of Interactions of CD5 across species (15, 16, 19) or on cells that do CD5 is important for controlling activation in normal immune re- not express CD5 (17) (A. Johnstone, unpublished observations) sponses. A molecular understanding of the effect of ligand binding may be explained by the capacity of denatured CD5 in CD5 by the extracellular region of CD5 allows the relative contribu- preparations to give nonspecific binding. Promiscuous binding by tions of extracellular and intracellular engagement of CD5 to be denatured CD5 may reflect a general propensity of SRCR do- more clearly delineated and provides a molecular basis for inter- mains, including CD5, to multerimize (14, 29, 30). preting functional data. Homophilic binding by CD5 domain 1 may provide more of Homophilic binding by CD5 provides a molecular basis for a general paradigm for recognition by SRCR domains than the species-specific inhibition of EAE in mouse by soluble CD5-Ig interaction between an IgSF domain of CD166 and the membrane- (10). The mCD5 mAb (53.7) that blocks homophilic interactions proximal SRCR domain of CD6. Domain 1 of CD6 does not has also been reported to have functional effects, inhibiting B cell participate in the interaction with CD166 (1, 12), but by analogy activation (16), which, depending on the mode of action of the with CD5, its membrane distal position indicates a ligand-binding mAb in these experiments, would be evidence for ligand- function. There are data to indicate a significant role for CD6 dependent positive effects of CD5 in T-B cell collaboration. The domain 1 in regulation by CD6 in immune responses. Inhibitory difference between the overall effect of CD5 engagement being effects of two CD6 mAbs characterized as specific for different activating or inhibitory may depend on the number and activation epitopes on the membrane distal N-terminal domain have been status of responding cells in a polyclonal population. The evidence described (31–34). As they were effective in soluble form, it is is that the extracellular region of CD5 is important for regulating likely they are blocking an interaction. Importantly, the T1 mAb both activation and inhibition by CD5 and the molecular mecha- has shown therapeutic immunosuppressive effects in patients (33, nism involves species-specific homophilic interactions of CD5 34). This could involve a homophilic interaction of CD6 domain domain 1. 6074 A LIGAND FOR CD5 IS CD5

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