(OX40) Proliferation in the Absence of CD134 Robust B Cell Immunity But

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Robust B Cell Immunity but Impaired T Cell Proliferation in the Absence of CD134 (OX40) Susanne D. Pippig, Claudia Peña-Rossi, James Long, Wayne R. Godfrey, Deborah J. Fowell, Steven L. Reiner, Marian L. This information is current as Birkeland, Richard M. Locksley, A. Neil Barclay and Nigel of September 26, 2021. Killeen J Immunol 1999; 163:6520-6529; ; http://www.jimmunol.org/content/163/12/6520 Downloaded from

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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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Robust B Cell Immunity but Impaired T Cell Proliferation in the Absence of CD134 (OX40)1

Susanne D. Pippig,2,3* Claudia Pen˜a-Rossi,2,4* James Long,* Wayne R. Godfrey,* Deborah J. Fowell,† Steven L. Reiner,‡ Marian L. Birkeland,§ Richard M. Locksley,*†¶ A. Neil Barclay,§ and Nigel Killeen5*

CD134 (OX40) is a member of the TNF receptor family that is expressed on activated T lymphocytes. T cells from mice that lack expression of CD134 made strong responses to a range of challenges, but they showed impaired proliferation in response to direct stimulation through the TCR with monoclonal anti-CD3⑀ Ab. CD134-deﬁcient mice controlled infection with Leishmania major, Nippostrongylus brasiliensis, and Theiler’s murine encephalomyelitis virus, and they made overtly normal Ab responses to a variety of antigens. Thus, CD134 is not essential for many T cell responses in vivo, nor is it required for the provision of help to B cells. Nonetheless, a subtle role in the regulation of T cell reactivity is suggested by the effect of CD134 deﬁciency on in vitro T Downloaded from cell responses. The Journal of Immunology, 1999, 163: 6520–6529.

embers of the TNF receptor superfamily have promi- apoptotic or proliferative/differentiative outcomes through the re- nent roles in the regulation of immune responses and spective activation of caspases and transcriptional regulators such the formation of secondary lymphoid tissue (1–7). Li- as NF-␬B and AP-1 (7, 12). Mutant mice that lack expression of

M http://www.jimmunol.org/ gands for these receptors also constitute a superfamily whose TNF receptors, their ligands, or TRAFs show a range of defects in members share structural similarity to TNF and in many cases are lymphoid homeostasis and immune responses (3, 5, 6, 13–16). likely to form cell-bound or secreted trimers (8, 9). Lymphocyte The CD134 Ag was ﬁrst identiﬁed as a 50-kDa glycoprotein activation induces the expression of several TNF receptor-related target for the MRC OX40 Ab that was selectively expressed on proteins or their ligands including CD40L, CD95L, 4-1BB, CD30, activated rat CD4ϩ T cells (17). Mouse and human CD134 show and CD134 (10). This up-regulation of expression allows for li- a similar pattern of expression to the rat form, except they are also gand-dependent signals to be relayed from the receptors to the found on activated CD8ϩ T cells (18, 19). The ligand for CD134 nucleus through signaling molecules that include the TNF recep- (CD134L) is a type II trimeric transmembrane protein whose tor-associated factors (TRAFs)6 (4, 11, 12). These signals induce

mRNA is markedly induced in human T cell leukemia virus 1-in- by guest on September 26, 2021 fected cells through the action of the viral tax transactivator (19, 20).

Trimeric CD134L binds to cell surface CD134 with high affinity (Kd Ϫ Ϫ † ¶ ϭ ϭ ϫ 5 1 Departments of *Microbiology and Immunology and Medicine, and Howard 0.2–0.4 nM) and a slow off-rate (koff 4 10 s ) (8). ‡ Hughes Medical Institute, University of California, San Francisco, CA 94143; De- In several experimental settings, CD134 acts as a costimulator partment of Medicine, Committee on Immunology and the Gwenn Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637; and for T cells. For instance, anti-CD134 mAb augments the response §Medical Research Council Cellular Immunology Unit, Sir William Dunn School of of rat T cells activated with allogenic MHC (17) or anti-CD3 (21). Pathology, University of Oxford, Oxford, United Kingdom Similarly, mouse and human T cells make more robust prolifera- Received for publication July 20, 1999. Accepted for publication October 7, 1999. tive and cytokine responses when activated in the presence of The costs of publication of this article were defrayed in part by the payment of page transfected cells expressing CD134L (19, 20, 22). This costimu- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. latory activity appears to be synergistic with costimulation through 1 This work was supported by a grant-in-aid (to N.K.) from the American Heart CD28 and is particularly potent in prolonging the responses of Association (96-221) and by funds from the Lucille P. Markey Foundation through differentiated effector T cells (23). Interestingly, a similar type of the University of California, San Francisco, Program in Biological Sciences and the Howard Hughes Medical Institute University of California, San Francisco, Research costimulatory activity has also been described for CD137 (4-1BB), Resources Program. S.D.P., C.P-R., D.J.F., and M.L.B. were supported by postdoc- which is a related member of the TNF receptor family that is also toral fellowships from the Deutsche Forschungsgemeinschaft, the Human Frontiers in induced on activated T lymphocytes (24–26). Costimulatory sig- Science Program, the Juvenile Diabetes Foundation, and the Cancer Research Insti- tute (NY), respectively. W.R.G. was the recipient of a U.S. Public Health Service nals delivered through CD134 have been implicated in selectively Career Development Award. R.M.L. is an Investigator in the Howard Hughes Med- promoting the differentiation of Th2 cells (27–29), but other stud- ical Institute. A.N.B. was supported by the Medical Research Council (U.K.). ies suggest they may also regulate Th1 development (23, 30, 31). 2 S.D.P. and C.P-R. contributed equally to this work. The cytoplasmic tail of CD134 can associate with TRAF2, 3 Current address: Systemix Inc., Palo Alto, CA. TRAF3, and TRAF5 and may well mediate its costimulatory effect 4 Current address: Ares-Serono International S.A., 14, Chemin des Aulx, 1228 Plan- through these interactions (12, 32, 33). Finally, recent data indicate les Ouates, Switzerland. that like anti-CD137 (34), anti-CD134 signaling can block activa- 5 Address correspondence and reprint requests to Dr. Nigel Killeen, Department of Microbiology and Immunology, University of California, San Francisco, CA 94143- tion-induced T cell death in mice treated with superantigens (35). 0414. E-mail address: [email protected]. The physiological significance of costimulatory signaling 6 Abbreviations used in this paper: TRAF, TNF receptor-associated factor; TNP- through CD134 has not yet been resolved. CD134 is expressed on KLH, trinitrophenyl-keyhole limpet hemocyanin; ELISPOT, enzyme-linked immu- inflammatory T cells in vivo, as in the CNS of rodents with ex- nospot; NP, nitrophenyl; CFSE, carboxyfluorescein diacetate succinimidyl diester; LN, lymph node; TMEV, Theiler’s murine encephalomyelitis virus; SEB, staphylo- perimental allergic encephalomyelitis (36), in the joints of humans coccal enterotoxin B; ES, embryonic stem. with rheumatoid arthritis (37), in the peripheral blood of rats with

acute graft-vs-host disease (38), or on tumor-infiltrating lympho- targeted region was used on a BglII digest of the genomic DNA to identify cytes (39). CD134 is also expressed on T cells in the intestines of targeted clones. The structure of the mutant allele was subsequently con- Ј mice with inflammatory bowel disease wherein blockade of the firmed using a 3 probe on genomic DNA that had been digested with ApaI or BamHI. Approximately 1% of neor colonies were mutant at the cd134 CD134-CD134L interaction can lessen the severity of the disease locus. Chimeric mice were produced by injection of mutant ES cells into (40). Cumulatively, these expression and functional data raise the 3.5-day-old blastocysts according to standard procedures (48). These chi- possibility that CD134 signaling may help to prolong Ag-specific meras were then bred to C57BL/6 females, and germline transmission of proliferative responses or otherwise influence the persistence, dif- the cd134 mutation was confirmed by Southern blot analysis of tail DNA. CD134ϩ/Ϫ mice were interbred to produce the mice that were used in the ferentiation, or reactivation of effector/memory populations. experiments described below. The mRNA for CD134L is expressed in activated T cells, B cells, and also in the mouse brain and kidney (19). In humans, Flow cytometric analysis CD134L mRNA is found in the heart, skeletal muscle, pancreas, All flow cytometry was performed using a Becton Dickinson FACScan testes, and ovary (19). Human dendritic cells express CD134L, and flow cytometer (Becton Dickinson, Mountain View, CA) with CellQuest its ligation on these cells induces them to differentiate and secrete software Becton Dickinson). Fluorescent mAbs were purchased from PharMingen (San Diego, CA) and Caltag (South San Francisco, CA). The cytokines (41). CD134L is also expressed on various types of en- OX86 (18) and OX89 (D. Shipton, A. Al-Shamkhani, M. Puklavek, and dothelium, including that from umbilical cord, foreskin, and the A. N. Barclay, unpublished data) mAbs were used as supernatant or puri- aorta (42, 43). Thus, the expression of CD134L would allow for a fied Ig from the respective hybridomas. OX86 and OX89 are specific for role as a costimulatory ligand for T cells, but it also raises the mouse CD134 and CD134L, respectively. Labeling and analysis of cells possibility that CD134 may regulate the extravasation of activated with 5- (and 6-)carboxyfluorescein diacetate succinimidyl (CFSE; diester Molecular Probes, Eugene, OR) was performed as described previously T cells under certain circumstances. (49, 50). Downloaded from A possible function for CD134 in the regulation of T cell-dependent B cell responses has been proposed based on the finding In vitro T cell assays that a rabbit anti-mouse CD134 antiserum can inhibit Ag-specific Cell preparation and culture were performed in RPMI 1640 supplemented B cell responses in mice (44). Treated mice developed germinal with 10% FCS, 2 mM L-glutamine, 50 ␮M 2-ME, 100 ␮M nonessential amino acids, 50 U/ml penicillin, and 50 ␮g/ml streptomycin. Lymph node centers in response to trinitrophenyl-keyhole limpet hemocyanin Ϫ Ϫ (LN) cells were isolated from CD134 / and control (littermate) mice ϩ (TNP-KLH) immunization, but they were impaired in their secre- using a 40 ␮M nylon cell strainer (Becton Dickinson). To purify CD4 T http://www.jimmunol.org/ tion of TNP-specific IgG Abs and they also lacked TNP-reactive B cells, LN cells were incubated at 20 ϫ 106/ml in M5/114 or BP107 su- cell foci in the spleen. Nevertheless, these mice made adequate pernatant (anti-I-A mAbs) and 0.5 ␮g/ml purified 2.43 (anti-CD8 mAb). secondary responses, suggesting that the CD134 signal might reg- After two washes in medium, the LN cells were treated at 37°C with a 1/10 dilution of guinea pig complement (Accurate Chemicals, Westbury, NY) at ulate differentiation into Ig-secreting plasma cells but not memory 20 ϫ 106/ml for 30 min. After two additional washes in medium, the cells B cells. The induction of CD134L expression on activated B cells were rocked at room temperature for 30 min with sheep anti-mouse Ig and and the provision of a proliferative signal to B cells through it (45) sheep anti-rat Ig-coated magnetic beads according to the manufacturer’s would be consistent with this hypothesis. Costimulation through instructions (Dynal, Oslo, Norway). Bead-coated cells were then removed CD134 has also been implicated in the up-regulation of the che- from the culture using a magnet. The purity of the final preparation was determined by flow cytometry and was typically Ͼ90%. For anti-CD3 by guest on September 26, 2021 mokine receptor BLR-1 on T cells (29), which could be important proliferation assays, 5 ϫ 104 purified CD4ϩ LN T cells were incubated in for recruiting Ag-specific T cells into germinal centers and thereby microtiter wells with 5 ϫ 105 irradiated splenocytes (2000 rad) from influencing Ab responses. However, a unique role for CD134 in C57BL/6 ϫ 129/Sv mice in the presence of anti-CD3⑀ mAb (purified 145- this context has not yet been established. 2C11) for 1, 2, 3, 4, or 5 days. The plates were then incubated with 1 ␮Ci/well [3H]thymidine for 18–24 h. For mixed lymphocyte responses, To study the function of CD134 in more detail, we have gen- 0.5, 1, 2, or 4 ϫ 105 purified CD4ϩ LN T cells were incubated with 5 ϫ erated CD134-deficient mice by gene targeting in embryonic stem 105 allogenic stimulator cells from BALB/c (H-2d) or CBA (H-2k) mice in (ES) cells. The phenotype of these mice suggests that CD134 is not a volume of 200 ␮l in flat-bottom 96-well plates for 1, 2, 3, 4, or 5 days. a primary regulator of many T cell responses in vivo, nor is it To confirm the lack of expression of CD134 in the CD134-deficient Ϫ/Ϫ essential for B cell differentiation and Ab secretion. Nonetheless, mice, LN cells from CD134 and control mice were stimulated with PMA (1 ng/ml) and ionomycin (500 ng/ml) for 3 days. in vitro assays of T cell activation suggest that signals from CD134 may help to promote T cell proliferation under certain circumstances. Immunizations, ELISAs, and ELISPOT assays Mice were immunized i.p with T-dependent and T-independent Ags, in- Materials and Methods cluding 10 ␮g TNP-KLH, 10 ␮g TNP-Ficoll, and 5 ␮g nitrophenyl (NP)- Generation of CD134-deficient mice OVA (all given in alum). For ELISAs, plates were precoated with 0.5 ␮g/ml TNP-KLH, TNP-Ficoll, or NP (13)-BSA (Solid Phase Sciences, San The isolation of 129/Sv genomic phage ␭ clones that contained the cd134 Rafael, CA) in PBS. The plates were blocked with 10% FCS in PBS con- gene has been described previously (46). These phage clones were further taining 0.1% (v/v) Tween 20 and then incubated overnight at room tem- characterized by subcloning, restriction enzyme, and Southern blot analy- perature with 2-fold serial dilutions of sera from CD134Ϫ/Ϫ or control mice ses. A targeting construct was generated by inserting a 3.5-kb KpnI-SacI in PBS containing 2% FCS. TNP plates were washed and then incubated

fragment that contained exons 5, 6, and 7 into the pL2neo-2 vector (mod- with biotinylated goat anti-murine IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, or iﬁed from pL2neo, kind gift from Dr. Hua Gu, National Institutes of Health, IgE (PharMingen). This was followed by an additional wash before the ␮ Bethesda, MD). The pL2neo vector contains a neomycin resistance cassette addition of 1 g/ml HRP coupled to streptavidin (Jackson ImmunoRe- (neor) under the control of the MC1 (HSV-thymidine kinase) promoter. search, West Grove, PA). After 30 min at room temperature, the plates Neor and the CD134 genomic sequence were then reexcised using Hind III were washed and then 2,2Ј-azino-bis-(3-ethylbenzthiazoline-6-sulfonic

and inserted into pBluescript that contained a 4-kb KpnI-HindIII upstream acid) in a citric acid/phosphate buffer and 0.003% H2O2 was added as a fragment of the cd134 gene. substrate. Detection was performed using a Molecular Dynamics plate A total of 2 ϫ 107 RF8 ES cells (47) were transfected with 20 ␮gofthe reader (Molecular Dynamics, Sunnyvale, CA) set at ␭1–␭2, with ␭1 ϭ 405 linearized targeting vector by electroporation using a Bio-Rad Gene Pulser nm and ␭2 ϭ 490 nm. Results for the TNP-KLH assays were quantitated (Bio-Rad, Richmond, CA) set at 500 ␮F and 250 V. The cells were plated by comparison to standard anti-TNP mAbs (kindly provided by Dr. Robert on primary embryonic feeder cells, and G418 selection (200 ␮g/ml) was Coffman, DNAX, Palo Alto, CA). NP ELISAs were similar except alkaline applied 36 h after electroporation. Ten days later, individual G418-resistant phosphatase-coupled anti-mouse Ig Ab (PharMingen) and Sigma 104 sub- clones were picked to 96-well plates containing feeder cells. The clones strate (Sigma, St. Louis, MO) were used. were expanded and then split in half so that a portion of each could be To measure Abs produced against Theiler’s murine encephalomyelitis frozen in 96-well plates, and the remaining portion could be expanded for virus (TMEV), serial dilutions of sera were incubated for 45 min at 37°C DNA extraction and Southern blot analysis. A probe on the 5Ј side of the in commercially available ELISA plates coated with TMEV or control Ags 6522 PHENOTYPE OF CD134-DEFICIENT MICE

FIGURE 1. Inactivation of the murine cd134 locus by gene targeting in ES cells. A, Design of the cd134-targeting vector, structure of the cd134 locus before and after targeting, and the expected sizes of the mutant and wild-type bands on Southern blots. B,

Southern blot analysis: ES cell DNA cut with Downloaded from BglII and hybridized with probe A (left) and mouse tail DNA cut with ApaI and hybridized with probe B (right). Mouse and ES cell genotypes are indicated beneath the autora- diographic image. http://www.jimmunol.org/ by guest on September 26, 2021

(Charles River Breeding Laboratories, Wilmington, MA). The plates were (Amersham, Arlington Heights, IL) and hybridized with a virus-specific or washed and then incubated with alkaline phosphatase coupled anti-IgG Ab GAPDH 32P-labeled probe. Positive controls for the detection of viral RNA (Jackson ImmunoResearch) for 45 min at 37°C. Sigma 104 was used as a included brain and spinal cord RNA from a C57BL/6 mouse 7 days after substrate as above. TMEV inoculation and viral cDNA. IgE responses were determined as described previously (51). Briefly, Mice were inoculated in the hind footpads with 4 ϫ 105 metacyclic ELISA plates were coated with an anti-IgE Ab (B.1E.3) in PBS overnight promastigotes of Leishmania major. The progression of the disease was at 4°C and then blocked with 10% FCS in PBS/Tween 20. After several monitored weekly using a metric caliper to measure the hind footpad le- washes in PBS/Tween 20, serial dilutions of the sera were added and the sions. Parasite burden was determined as described previously (54). plates were incubated at room temperature. The plates were washed again Infective third stage larvae of Nippostrongylus brasiliensis were iso- and incubated with a secondary biotinylated anti-IgE Ab (EM-95). After lated from the feces of experimentally infected rats as described (51). The another round of washes, the plates were incubated with alkaline phospha- larvae were washed extensively with saline, counted, and injected s.c. at tase coupled to streptavidin (The Jackson Laboratory, Bar Harbor, ME). the base of the tail into CD134-deficient and control mice using 500 Sigma 104 substrate was added after a final wash. worms/mouse in 0.2 ml of PBS. After 10 days, the number of adult worms For enzyme-linked immunospot (ELISPOT) assays, ELISA plates were in the intestines was determined by direct visualization. The lungs of in- coated with anti-IL-4 Ab (11B11) in PBS overnight at 4°C. The plates were fected mice were completely blanched with ice-cold PBS, excised, minced then blocked with 10% FCS in PBS before incubation at 37°C overnight into fine fragments, and dispersed into a single-cell suspension in PBS with cells (2-fold serial dilutions starting at 1 ϫ 105). They were then using a syringe plunger. Cells were passed through a nylon strainer and washed again immediately before separate incubations with a secondary adjusted to 107 cells/ml in culture medium for use in cytokine ELISPOT anti-Il-4 Ab (BUD6), followed by streptavidin-alkaline phosphatase. After assays. a final round of washes, the plates were treated for 30 min in the dark with For delayed-type hypersensitivity responses, mice were sensitized with 1 mg/ml 5-bromo-4-chloro-3-indolylphosphate p-toluidine (Sigma) in 0.1 100 ␮l of 100 mg/ml oxazolone (4-ethoxymethylene-2-phenyloxazolone; M alkaline buffer/0.6% agarose. IL-4-secreting cells were detected as blue Sigma) in acetone/olive oil (4:1) applied evenly on a shaved hind flank (55, spots on the bottom of the wells. 56). Five days later, 5 ␮l of the same oxazolone solution was applied to each side of the right ear. Acetone/olive oil without oxazolone was applied Infections and delayed-type hypersensitivity assays to the left ear in a similar fashion. The thickness of the central portion of each ear was then measured at various times using a Dial thickness gauge. Mice were inoculated with 104 PFU intracranially or 106 PFU i.p. of the DA strain of TMEV as described (52, 53). Sera were collected on day 7 Adoptive transfer experiments with TCR transgenic T cells after i.p. inoculation or on day 90 after intracranial inoculation. Total RNA was prepared from PBS-perfused brain and spinal cord using Tri-Reagent These experiments were performed essentially as described (57). In brief, (Molecular Research Center, Cincinnati, OH). Five-fold serial dilutions cells from the LN of female H-2dd DO11.10 TCR transgenic mice starting with 10 ␮g RNA were filtered onto Hybond C-extra membranes (CD134Ϫ/Ϫ or controls, on a B6/129/B10.D2 background) were injected The Journal of Immunology 6523

i.v. into the tail veins of B10.D2 female mice. Donor populations were normalized by FACS so that each recipient received the same quantity of transgenic CD4ϩ T cells (6–7 ϫ 107). In some experiments, the donor populations were labeled with CFSE as above. After 24 h, the recipient mice were then immunized s.c. with 300 ␮g of the chicken OVA peptide (residues 323–329) in CFA. FACS analysis with the KJ-126 Ab was then used to monitor the expansion of DO11.10 transgenic T cells in the brachial and axillary LN at several time points after immunization. Activation-induced cell death A total of 2 ϫ 106 LN cells/ml were stimulated with 5 ␮g/ml Con A for 48 h. The cells were then washed extensively before incubation for 48 h with high (100 U) or low doses (3 U) of IL-2 to predispose for apoptosis as described previously (58). Cells were then stimulated with plate-bound anti-CD3⑀ Ab to induce apoptosis. Viable cells were distinguished from dead cells by size (forward light scatter) and propidium iodide incorporation. CD134-deﬁcient and control mice were injected i.p. with 100 ␮gof staphylococcal enterotoxin B (SEB; Sigma). FACS analysis was used to determine the proportions of LN T cells expressing V␤8.1 or 8.2 and V␤6 on day 0, 1, 2, 3, 4, and 7.

Results Downloaded from Generation of CD134-deﬁcient mice by gene targeting The gene encoding CD134 (46) was inactivated in murine ES cells according to the strategy depicted in Fig. 1A. Four exons of the gene encoding the ﬁrst 143 residues of the CD134 protein were

deleted and replaced by a neomycin resistance cassette. The ex- http://www.jimmunol.org/ pected structure of the resultant mutant allele was verified by FIGURE 2. Cell surface phenotype of CD134Ϫ/Ϫ lymphocytes. A, FACS Southern blot using probes from the 5Ј and 3Ј side of the targeted analysis of CD134 expression on LN T cells and thymocytes from wild-type region (Fig. 1B) and also using neomycin and CD134 cDNA and mutant mice after activation with PMA and ionomycin and anti-CD3⑀ probes (data not shown). CD134-mutant mice were generated from mAb. Dot plots on the left show indirect staining with anti-CD134 mAb, the targeted ES cells by standard blastocyst injection procedures whereas histograms on the right show staining with human IgG1 (control, (48, 59). shaded histogram) or human IgG1 fusion proteins containing the extracellular Activated T cells from homozygous mutant animals lacked ex- domains of CD134 (solid line) or CD134L (dashed line). PE-conjugated anti- pression of CD134 as detected by flow cytometry with the OX86 human IgG was used to detect the fusion proteins. Labels next to individual mAb (18) or with a soluble CD134L-Fc fusion protein (Fig. 2A). by guest on September 26, 2021 histograms refer to the expression of CD134 (as detected by CD134L-Ig staining), CD134L (as detected by CD134 staining), or background (as detected by No other receptor for CD134L could be detected on T cells in the staining with control human IgG1). B, FACS analysis of Con A-activated T absence of CD134 using soluble CD134L-Fc. The expression of Ϫ/Ϫ cells from wild-type and CD134Ϫ/Ϫ mice stained with anti-CD134 or anti- CD134L was significantly increased on activated CD134 T CD134L mAbs (OX86 and OX89, respectively). Control cell populations in- cells and thymocytes relative to wild-type cells as detected with clude nonactivated cells and cells stained with an isotype-matched mAb. either soluble CD134 or the OX89 mAb (Fig. 2, A and B). It re- OX86 and OX89 were detected with a biotinylated anti-rat IgG1 mAb fol- mains to be established whether this increase in CD134L expres- lowed by streptavidin-tricolor. The cells were costained with conjugated anti- sion has a transcriptional or posttranslational origin and whether it CD4 and anti-CD8 mAbs, and the histograms show relative fluorescence on has any impact on the phenotype of the mutant mice. cells gated for CD4 expression. Similar histograms were produced by gating The absence of CD134 had no detectable effect on the viability on CD8ϩ T cells. C, Lymphocyte populations in CD134-deficient mice. Lym- or fertility of mice, and there were no obvious indications that the phocytes from the thymus, spleen, LN, and bone marrow from wild-type and mutant mice were stained with the indicated Abs and analyzed by flow mice were unhealthy. Primary and secondary lymphoid tissues in cytometry. the mice were normal with respect to cellularity, the expression of

Table I. Lymphocyte populations in CD134-deﬁcient mice

% of Total Cells (%)a, c, d Total Cellsa,b Source CD134 (ϫ10Ϫ7) CD4ϩ CD8ϩ CD4ϩ/CD8ϩ CD4Ϫ/CD8Ϫ CD3ϩ B220ϩ/IgMϩ B220ϩ/IgMϪ

Thymus Ϫ/Ϫ 19 Ϯ 711Ϯ 35Ϯ 380Ϯ 10 5 Ϯ 4 ϩ/ϩ 16 Ϯ 812Ϯ 35Ϯ 478Ϯ 10 5 Ϯ 3 LNd, e Ϫ/Ϫ 7 Ϯ 166Ϯ 331Ϯ 167Ϯ 328Ϯ 3 ϩ/ϩ 7 Ϯ 161Ϯ 433Ϯ 463Ϯ 728Ϯ 3 Spleend Ϫ/Ϫ 10 Ϯ 464Ϯ 527Ϯ 447Ϯ 11 38 Ϯ 7 ϩ/ϩ 12 Ϯ 564Ϯ 726Ϯ 355Ϯ 331Ϯ 3 Bone marrow Ϫ/Ϫ 35Ϯ 11 3f 12 Ϯ 528Ϯ 4 ϩ/ϩ 4 Ϯ 14Ϯ 11 2f 12 Ϯ 429Ϯ 4

a All mice were between 6 and 17 wk of age. b n Ն 6. c n Ն 3; a blank space indicates that no determination was made. d Values for CD4ϩ and CD8ϩ LN and spleen cells are expressed as percentages of CD3ϩ cells. e Cervical, brachial, axillary, superﬁcial inguinal, and mesenteric. f n ϭ 2. 6524 PHENOTYPE OF CD134-DEFICIENT MICE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. In vitro responses by T cells from CD134-deficient mice. A, Purified CD4ϩ T cells were activated by the addition of various doses of anti-CD3⑀ mAb in the presence of irradiated spleen cells. [3H]Thymidine incorporation was used as an index of DNA synthesis and proliferation. The graphs show the results of a representative experiment assayed on day 3. A total of 50 U/ml of recombinant mouse IL-2 or 2 ␮g/ml of anti-CD28 mAb was added to some cultures. In each case, the results are representative of three or more independent experiments. B, Mixed lymphocyte response of CD134Ϫ/Ϫ T cells. Purified H-2b CD4ϩ T cells were stimulated with irradiated CBA (H-2k) spleen cells for 1–5 days before performing a [3H]thymidine uptake assay. The graph shows the result on day 3. CD134Ϫ/Ϫ T cells responded in an equivalent fashion to wild-type cells throughout the time course. This result is representative of more than four independent experiments. C, Relative representation of transgenic T cells in the draining LN of adoptive recipients of DO11.10 T cells. Mice were immunized at day 0 (24 h after adoptive transfer) with the OVA peptide emulsified in CFA as described in Materials and Methods. Each point represents a determination made on an individual recipient mouse (four donor populations and a nonimmunized control were analyzed at each time point). The experiment shown is representative of three others. In two of the experiments, populations of donor cells were labeled with CFSE so that the replicative history of the expanded DO11.10 T cells could be analyzed. Histograms of CFSE fluorescence at day 3 for CD4ϩ transgenic T cells from the draining LN of the indicated mice are shown in D. E, T cells from wild-type and CD134Ϫ/Ϫ mice were activated in vitro in the presence of 5 ␮g/ml Con A for 2 days, followed by culture for 2 days in 100 U/ml of recombinant murine IL-2 and activation for 2 days with anti-CD3⑀ mAb. The cells were then labeled with propidium iodide or stained with annexin V before analysis by flow cytometry. The result is representative of three independent experiments. The Journal of Immunology 6525 Downloaded from FIGURE 4. In vivo assays of T cell function in CD134Ϫ/Ϫ mice. A, Footpad measurements of mice infected with L. major. B, IL-4-secreting cells from the lungs of N. brasiliensis-infected mice. lymphocyte differentiation markers, and the presence of germinal Decreased proliferation was readily apparent when cells were ac- centers (Fig. 2C; Table I and data not shown). Thus, at first in- tivated with soluble but not plate-bound anti-CD3 mAb (data not http://www.jimmunol.org/ spection, the absence of CD134 had no detectable impact on lym- shown). CFSE-labeling experiments showed that proliferating phocyte development and homeostasis. CD134Ϫ/Ϫ cells progressed through successive cell divisions at a rate that was largely indistinguishable from control cells (data not T cell responses in the absence of CD134 shown and Fig. 3D). Furthermore, while proliferating, the cells Several previous reports have implicated CD134 in costimulation up-regulated the expression of activation Ags in a normal fashion. during T cell responses (17, 19–23, 35, 40). Therefore, we per- There was no apparent increase in the frequency of apoptotic cells formed a series of experiments to test for abnormalities in prolif- identified by staining with annexin V (data not shown), suggesting erative responses made by CD134-deficient T cells. As shown in that the defect in proliferation was not caused by enhanced apo- Ϫ/Ϫ ϩ Fig. 3A, CD134 CD4 T cells proliferated weakly when stim- ptosis. Curiously, other in vitro and in vivo assays of T cell pro- by guest on September 26, 2021 ulated with anti-CD3⑀ mAb in vitro, with the peak response made liferation, such as mixed lymphocyte responses, peptide-specific by CD134Ϫ/Ϫ T cells typically five times lower than that of responses of transgenic T cells, or stimulation with SEB, did not CD134-expressing control cells. This defect in T cell proliferation reveal the same sort of defect (Fig. 3, B–D, and data not shown). could not be corrected either by the addition of recombinant IL-2 We also found that CD134Ϫ/Ϫ T cells underwent activation-in- to the cultures or by treatment with anti-CD28 mAb (Fig. 3A). duced cell death in vitro and in vivo with apparently normal kinetics in response to anti-CD3 or SEB stimulation, respectively (Fig. 3E and data not shown). H-2d CD134Ϫ/Ϫ mice expressing the DO11.10 transgenic TCR (60) were generated so that peptide-specific T cell proliferation could be examined directly in adoptive transfer experiments (56). CD134-expressing or -deficient TCR transgenic LN cells were transferred to groups of nonirradiated mice that were then subsequently injected with OVA peptide in CFA. Mutant transgenic T cells expanded and declined in numbers in the draining LN of recipient mice in a fashion that was indistinguishable from control T cells (Fig. 3C). Furthermore, CFSE-labeling experiments showed that the mutant T cells also progressed through the cell cycle in vivo with apparently normal kinetics (Fig. 3D). As additional in vivo tests of the responses of CD134Ϫ/Ϫ T cells, we challenged the mutant animals with three kinds of infectious organisms. L. major and N. brasiliensis were used to evaluate the capacity of the mice to mount Th1- and Th2-dependent responses, respectively (61, 62), whereas TMEV was employed as a neuroinflammatory agent whose clearance from the CNS depends primarily on the action of cytotoxic T cells (63). As shown in Fig. 4 and described in more detail below, in all three cases, CD134Ϫ/Ϫ mice made equivalent responses to those of control littermates. FIGURE 5. Delayed-type hypersensitivity responses by CD134Ϫ/Ϫ Leishmania major is effectively controlled by Th1 responses in mice. Ear swelling after s.c. application of oxazolone to the ears of wild- mice of the C57BL/6 or 129 background. By contrast, BALB/c type or CD134Ϫ/Ϫ mice. mice make an inappropriate Th2 response and fail to clear the 6526 PHENOTYPE OF CD134-DEFICIENT MICE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Ab responses by CD134Ϫ/Ϫ mice. A, Anti-TNP-KLH-specific Ab titers in the sera of wild-type or CD134Ϫ/Ϫ mice immunized with TNP-KLH. Mice were immunized with TNP-KLH as described in Materials and Methods on day 0. The mice were then reimmunized in the same way on day 20. B, Anti-NP-specific Ab response by wild-type or CD134Ϫ/Ϫ mice 15 days after immunization with NP-chicken ␥ globulin. C, IgE production by the indicated mice after infection with L. major and N. brasiliensis. D, Anti-TMEV Ab response by the indicated mice 7 days after infection with TMEV. E, T cell independent anti-TNP Ab response by wild-type and CD134Ϫ/Ϫ mice 10 days after immunization with TNP-Ficoll. The Journal of Immunology 6527 parasite (54, 64). The absence of CD134 in mice of a mixed 129 ϫ ciency, the key observation is the reduced in vitro proliferative C57BL/6 background had no impact on their capacity to control response to anti-CD3 stimulation. This proliferative defect stands the replication of the parasite, as shown by footpad swelling assays in marked contrast, however, to the multiple robust T and B cell (Fig. 4A) and by a direct determination of the parasite burden in the responses that we have observed in vivo. Thus, our preliminary spleen and footpads (data not shown). Thus, CD134 is not essential analysis of these mutant mice indicates that CD134 does not me- for T cells to adopt the Th1 fate and mediate effective resistance to diate a prominent role in the various model immune responses we Leishmania. have examined. The clearance of N. brasiliensis from the intestines of infected Although we have uncovered a defect in T cell proliferation in mice is dependent on Th2 cells and is therefore a useful in vivo vitro, the underlying mechanism and physiological significance of assay for the function of these cells (62). Nine days after infection, this defect remain to be established. Annexin V staining experi- both CD134Ϫ/Ϫ and wild-type animals had cleared the parasite ments indicate that anti-CD3-activated CD134Ϫ/Ϫ T cells are not from their intestinal tract and showed evidence of a robust IL-4- abnormally prone to apoptosis (data not shown). CFSE-labeling dependent Th2 response in the form of IL-4-secreting cells in their experiments also show that they divide at a similar rate to that of lungs (Fig. 4B) and elevated levels of IgE in their sera (Fig. 6C). wild-type T cells in vivo (Fig. 3D) or in vitro (data not shown). Thus, by this assay, the absence of CD134 did not impair Th2 Moreover, while dividing, the cells change their pattern of expres- capability in vivo. sion of differentiation markers in an apparently normal fashion TMEV is a neurotropic agent that causes an autoimmune de- (data not shown). Additional kinetic experiments also indicated myelinating condition in susceptible mouse strains (63). Mice of that the requirement for CD134 function was evident at early time the C57BL/6 or 129 backgrounds are not susceptible to chronic points after stimulation, consistent with the very rapid and there- Downloaded from disease because of their expression of the H-2Db molecule, which fore atypical (23) up-regulation of CD134 under these in vitro allows for an effective cytotoxic T cell response to take place. conditions (data not shown). Thus, TMEV is effectively eliminated from the CNS of H-2b mice, The in vitro assay employing soluble anti-CD3 mAb generated whereas it persists in susceptible mice that lack H-Db. The signif- a dependency on CD134 that was not apparent when T cells were icance of cytotoxic T cells in eradicating TMEV is underscored by activated under other conditions (e.g., allo- or peptide-specific re- Ϫ/Ϫ

␤ http://www.jimmunol.org/ the persistence of the virus in the CNS of 2-microglobulin sponses; Fig. 3, B and C). At this point, it is unclear why there was mice that lack MHC class I expression (65). CD134Ϫ/Ϫ mice a requirement for CD134 under one set of circumstances, but not cleared TMEV from their brains and spinal cords, indicating that the other. It is possible, however, that the anti-CD3 assay was they were capable of mounting effective cytotoxic T cell responses deficient in supplying forms of costimulation that were more abun- in vivo (data not shown). dant in the other assays. Costimulation that might substitute for As a final specific test of the functional properties of CD134- CD134 signaling appeared to be distinct from that provided by deficient T cells, we examined the capacity of CD134Ϫ/Ϫ mice to CD28 because anti-CD28 mAb could not restore the proliferative mount a delayed-type hypersensitivity response after topical ap- response. Understanding the basis of the proliferative defect is plication of oxazolone (55, 56). As shown in Fig. 5, CD134Ϫ/Ϫ likely to provide important insight into the physiological condi- mice showed normal ear swelling after s.c. injection of oxazolone. tions that establish a need for the CD134 molecule. Such condi- by guest on September 26, 2021 This result indicated that the absence of CD134 did not obviously tions will also probably be revealed through the use of more sen- impair the capacity of T cells to initiate an inflammatory response. sitive in vivo assays than the ones employed here. The data presented here are consistent with previous work sug- B cell responses in the absence of CD134 gesting that CD134 provides a costimulatory function that can pro- Antagonizing the CD134 ligand/receptor with a rabbit anti-mouse mote T cell proliferation (19, 20, 22). Whereas this costimulatory CD134 antiserum was previously reported to interfere with B cell function is apparently not essential for T cell responses to several differentiation and Ab secretion (44). Therefore, we measured se- model challenges, it may act to sustain the duration of T cell im- rum Ab titers after immunization with model protein Ags and after munity in vivo and perhaps enhance the formation or persistence infection with the three agents mentioned above. Immunization of memory T cells. These possibilities are currently under analysis. with TNP-KLH (Fig. 6A) or NP-OVA (Fig. 6B) resulted in robust In vivo, CD134 expression is a characteristic of T cells involved and diverse Ag-specific responses that were no different from those in chronic inflammatory syndromes such as experimental allergic made by control animals. The mice also made enhanced responses encephalomyelitis (31), rheumatoid arthritis (37) and inflammatory after reimmunization (Fig. 6A), indicating that immunological bowel disease (40). It seems feasible therefore that costimulatory memory was not impaired by the absence of CD134. CD134Ϫ/Ϫ signals from CD134 may help to sustain T cell reactivity under mice made strong responses to Theiler’s virus (Fig. 6D) and they these types of chronic inflammatory circumstances. Indeed, recent showed elevated IgE levels that were equivalent to those of con- studies employing soluble CD134 in vivo lend experimental sup- trol-infected mice after inoculation with L. major and N. brasil- port to this notion (30, 40). It remains unclear, however, whether iensis (Fig. 6C). These last observations and the general finding the impact of CD134 signaling in such cases is most significant for that isotype diversity was unaffected (Fig. 6A) indicate that Th2 the T cell while in the LN draining an inflamed site, or, alterna- responses persist despite the absence of CD134. Finally, tively, within the inflamed tissues themselves. In this regard, CD30 CD134Ϫ/Ϫ mice made normal responses to the T cell-independent (also a member of the TNF receptor family) has recently been Ag TNP-Ficoll (Fig. 6E). Cumulatively, the results indicate that B implicated in the regulation of T lymphocyte proliferation in situ in cell responses are not obligatorily dependent on the function of the the pancreatic islets (66). Although the reported effect of CD30 in CD134 receptor/ligand system. this case is to suppress T lymphocyte responses, it is possible that CD134 may have the opposite effect and instead drive the localized Discussion expansion of Ag-reactive T cells. A chronic inflammatory response may therefore depend on the sustained inhibition and potentiation In this paper, we describe the generation and analysis of mutant of signals through CD30 and CD134, respectively. mice that lack expression of the TNF-receptor family member The finding that B cell responses are apparently unaffected by CD134. By way of substantial consequences of the CD134 defi- the loss of CD134 is superficially at variance with predictions from 6528 PHENOTYPE OF CD134-DEFICIENT MICE a study employing a rabbit antiserum specific for mouse CD134 6. Chaplin, D. D., and Y. Fu. 1998. Cytokine regulation of secondary lymphoid (44). In this study, the blockade of the CD134 receptor-ligand organ development. Curr. Opin. Immunol. 10:289. 7. Gravestein, L. A., and J. Borst. 1998. Tumor necrosis factor receptor family interaction interfered with the secretion of IgG, a result that was members in the immune system. Semin. Immunol. 10:423. suggestive of an important role for CD134 in T:B cell collabora- 8. Al-Shamkhani, A., S. Mallett, M. H. Brown, W. James, and A. N. Barclay. 1997. tion and B cell differentiation in vivo. Interestingly, the blockade Affinity and kinetics of the interaction between soluble trimeric OX40 ligand, a member of the tumor necrosis factor superfamily, and its receptor OX40 on did not impair recall responses to the priming Ag, indicating that activated T cells. J. Biol. Chem. 272:5275. the differentiation of Ag-primed B cells into memory cells was 9. Armitage, R. J. 1994. Tumor necrosis factor receptor superfamily members and unaffected. Why the absence of CD134 would have a different their ligands. Curr. Opin. Immunol. 6:407. 10. Gruss, H. J., and S. K. Dower. 1995. Tumor necrosis factor ligand superfamily: impact on B cell responses than a blocking Ab is not immediately involvement in the pathology of malignant lymphomas. Blood 85:3378. clear, but it is possible that the anti-OX40 antiserum had unantic- 11. Cao, Z., J. Xiong, M. Takeuchi, T. Kurama, and D. V. Goeddel. 1996. TRAF6 is ipated or indirect effects on the survival of T cells or on their a signal transducer for interleukin-1. Nature 383:443. 12. Arch, R. H., R. W. Gedrich, and C. B. Thompson. 1998. Tumor necrosis factor capacity to interact with B cells. Given that CD134 is expressed receptor-associated factors (TRAFs): a family of adapter proteins that regulates only on activated peripheral T cells, it seems unlikely that its ab- life and death. Genes Dev. 12:2821. sence would have a significant effect on the composition of the 13. Yeh, W. C., A. Shahinian, D. Speiser, J. Kraunus, F. Billia, A. Wakeham, Ϫ/Ϫ J. L. de la Pompa, D. Ferrick, B. Hum, N. Iscove, et al. 1997. Early lethality, naive T cell population. Thus, the capacity of CD134 T cells to functional NF-␬B activation, and increased sensitivity to TNF-induced cell death provide adequate help to B cells is probably not due to the selec- in TRAF2-deficient mice. Immunity 7:715. tion of an atypical precursor population that is abnormally CD134 14. Lomaga, M. A., W. C. Yeh, I. Sarosi, G. S. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, et al. 1999. TRAF6 deficiency independent in its function. Nonetheless, it is possible that the loss results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. of CD134 may have a subtle effect on the capacity of T cells to Genes Dev. 13:1015. Downloaded from promote certain B cell responses, and this effect may not be ob- 15. Matsumoto, M., S. Mariathasan, M. H. Nahm, F. Baranyay, J. J. Peschon, and D. D. Chaplin. 1996. Role of lymphotoxin and the type I TNF receptor in the vious in the settings that we have examined, perhaps because they formation of germinal centers. Science 271:1289. involve powerful antigenic stimulation. We have observed germi- 16. Koni, P. A., R. Sacca, P. Lawton, J. L. Browning, N. H. Ruddle, and nal centers in the spleens of unimmunized mice and in the LN of R. A. Flavell. 1997. Distinct roles in lymphoid organogenesis for lymphotoxins ␣ and beta revealed in lymphotoxin ␤-deficient mice. Immunity 6:491. immunized mice, but further work is required to determine 17. Paterson, D. J., W. A. Jefferies, J. R. Green, M. R. Brandon, P. Corthesy, whether these germinal centers are different from those present in M. Puklavec, and A. F. Williams. 1987. Antigens of activated rat T lymphocytes http://www.jimmunol.org/ normal mice, either in their rate of formation, their constituents, or including a molecule of 50,000 Mr detected only on CD4 positive T blasts. Mol. Immunol. 24:1281. their function. Other assays of T:B cell interaction, such as sensi- 18. al-Shamkhani, A., M. L. Birkeland, M. Puklavec, M. H. Brown, W. James, and tive adoptive transfer systems, may be useful in determining A. N. Barclay. 1996. OX40 is differentially expressed on activated rat and mouse whether the absence of CD134 has an effect on B cell function T cells and is the sole receptor for the OX40 ligand. Eur. J. Immunol. 26:1695. 19. Baum, P. R., R. B. Gayle, 3rd, F. Ramsdell, S. Srinivasan, R. A. Sorensen, beyond that which might be due solely to diminished T cell ex- M. L. Watson, M. F. Seldin, E. Baker, G. R. Sutherland, K. N. Clifford, et al. pansion (67, 68). 1994. Molecular characterization of murine and human OX40/OX40 ligand sys- The identity of cell surface molecules that might perform similar tems: identification of a human OX40 ligand as the HTLV-1-regulated protein gp34. EMBO J. 13:3992. functions to CD134 and thereby ameliorate the impact of its ab- 20. Godfrey, W. R., F. F. Fagnoni, M. A. Harara, D. Buck, and E. G. Engleman. sence is an issue of significance in terms of defining the physio- 1994. Identification of a human OX-40 ligand, a costimulator of CD4ϩ T cells by guest on September 26, 2021 logical function of CD134. Candidates in this respect would in- with homology to tumor necrosis factor. J. Exp. Med. 180:757. 21. Kaleeba, J. A., H. Offner, A. A. Vandenbark, A. Lublinski, and A. D. Weinberg. clude the CD137 molecule as well as other members of the TNF 1998. The OX-40 receptor provides a potent co-stimulatory signal capable of receptor family. CD137 is attractive because of its similar pattern inducing encephalitogenicity in myelin-specific CD4ϩ T cells. Int. Immunol. of expression and because it can activate NF-␬B through TRAF2, 10:453. 22. Akiba, H., M. Atsuta, H. Yagita, and K. Okumura. 1998. Identification of rat as can CD134 (24, 33, 69). Thus, it will be of interest to examine OX40 ligand by molecular cloning. 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