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Contributions of CD27 and relatives to the specific immune response

Hendriks, J.A.

Publication date 2004

Link to publication

Citation for published version (APA): Hendriks, J. A. (2004). Contributions of CD27 and relatives to the specific immune response.

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Download date:24 Sep 2021 Chapter 5

CD27 is acquired by primed B cells at the centroblast stage and promotes germinal center formation.

Yanling Xiao, Jenny Hendriks, Petra Langerak, Heinz Jacobs and Jannie Borst

The Journal of Immunology 2004 172: 7432-7441

67 68 CD27 Is Acquired by Primed B Cells at the Centroblast Stage and Promotes Germinal Center Formation1

Yanling Xiao, Jenny Hendriks, Petra Langerak, Heinz Jacobs, and Jannie Borst2

Studies on human B cells have featured CD27 as a marker and mediator of the B cell response. We have studied CD27 expression and function on B cells in the mouse. We find that B cells acquire CD27 at the centroblast stage and lose it progressively upon further differentiation. It is not a marker for somatically mutated B cells and is present at very low frequency on memory B ceils. Enrichment of CD27 among centroblasts and the presence of its ligand CD70 on occasional T and B cells in or near germinal centers (GCs) suggested a role for CD27/CD70 interactions in clonal B cell expansion. Accordingly, GC formation in response to influenza virus infection was delayed in CD27 knockout mice. CD27 deficiency did not affect somatic hypermutation or serum levels of virus-specific IgM, IgG, and IgA attained in primary and recall responses. Adoptive transfer of T and B cells into CD27/CD28-'- mice revealed that CD27 promotes GC formation and consequent IgG production by two distinct mechanisms. Stimulation of CD27 on B cells by C 1)28" Th cells accelerates GC formation, most likely by promoting centroblast expansion. In addition, CD27 on T cells can partially substitute for CD28 in supporting GC formation. The Journal of Immunology, 2004,172: 7432-7441.

xpression of TNFR family member CD27 is restricted to CD27" B cells as memory B cells. In particular, CD27 was re­ naive and activated CD4 : and CD8+ T cells and subsets cently found at low levels on all GC B cells from human tonsils, E of B and NK cells. Its membrane-bound ligand CD70 is and dramatically up-regulated upon their in vitro differentiation confined to activated lymphocytes and mature dendritic cells (1- into plasma cells (17). In this study, we have analyzed in detail the 4). In vitro studies have established that CD27 promotes expansion expression of CD27 on B cells in the mouse and find it in line with of newly activated T cells (1,5, 6). In CD27~~'~ mice, generation a contribution of CD27 to centroblast expansion. + + and maintenance of CD4 and CD8 effector T cells in response In vitro studies on human B cells indicate that CD27 can pro­ to antigenic challenge is impaired (7). We have recently resolved mote IgM, IgG, IgA, and IgE secretion (10, 16, 18, 19). Whether that CD27 rescues activated T cells from death, and in this way, CD27/CD70 interactions enhance Ig production by delivering dif­ increments the yield of live T cells upon their clonal expansion at ferentiation signals to B cells or by sustaining expansion of dif­ the site of priming. In addition, CD27 exerts a prosurvival effect on + ferentiating cells is unclear. Jacquot et al. (20) found that CD27 did CD4^ and CD8 T cells at tissue sites (8). not drive expansion of activated B cells, but promoted the gener­ In humans, CD27 is induced by B cell receptor triggering and ation of a plasma cell phenotype and IgG secretion. However, maintained long-term (9, 10). CD27+ B cells are predominantly 3 other data argue that CD27 does promote expansion of activated B found in germinal centers (GCs) and marginal zones (11, 12). It cells (19, 21). All in vitro studies rely on deliberate stimulation of is considered a marker for memory B cells (13) based on the fol­ CD27 with CD70 transfectants. Whether CD27 signals are invoked lowing observations: 1) among IgM+D+ B cells in blood and mar­ f in vivo will depend on availability of CD70. In both humans and ginal zones, CD27' ' cells contain somatic Ig gene mutations (12, mice, CD70 is induced by Ag receptor triggering in T and B cells 14); 2) Ig class switching is more frequent among CD27+ than (2, 3, 22). In humans, CD70 has been detected on B cells, which CD27" B ceils (9); 3) upon activation, CD27+ B cells secrete Ig may represent recent GC immigrants (23). CD70 expression is more efficiently than CD27 B cells (15); and 4) cord blood B very transient and plasma membrane levels are often extremely cells lack CD27. while the percentage of CD27+ B cells in blood low, making it difficult to detect (3). In this study, we have used increases with age (16). Although these findings are in line with CD27 being a hallmark of primed B cells, they do not classify all confocal laser microscopy to define at what point during the GC reaction CD27/CD70 interactions might play a part. Comparing wild-type and CD27 /_ mice, we have determined how CD27 contributes to the B cell response to influenza virus. An Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Neth­ erlands effect of CD27 deletion was expected, because anti-viral T cell Received for publication January 5, 2004. Accepted for publication March 26, 2004. responses in draining lymph nodes and the lung are severely im­ _/ The costs of publication of this article were defrayed in part by the payment of page paired in CD27 mice (7). Apart from determining its impact on charges. This article must therefore be hereby marked advertisement in accordance Ig production, we have studied the role of CD27 in GC formation. with 18 U.S.C. Section 1734 solely to indicate this fact. Present data suggest that CD27 promotes B cell expansion and/or 1 This work was supported by The Netherlands Organization for Scientific Research differentiation into plasma cells. Alternatively or in addition, CD27 and by the Dutch Cancer Society. may promote the B cell response indirectly by facilitating Th cell 2 Address correspondence and reprint requests to Dr. Jannie Borst. Division of Im­ munology. The Netherlands Cancer Institute. Plesmanlaan 121, 1066 CX Amsterdam. expansion. The costimulatory receptor CD28 and its ligands play The Netherlands. E-mail address: [email protected] a key role in GC formation. Mice lacking CD28 function are 1 Abbreviations used in this paper: GC. germinal center; DLN, draining lymph nodes; greatly defective in Th-dependent IgG responses to hapten-protein FDC. follicular dendritic cells; FSC. forward scatter; HPE, high performance EL1SA conjugates, and lack obvious GCs (24-27). To map a contribution buffer; OX40L, OX40 ligand; PNA. peanut agglutinin; SA. streptavidin; SHM, so­ matic hypermutation; TR, Texas Red. of CD27 to the GC reaction relative to that of CD28, we generated

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mice genetically deficient for both CD27 and CD28, and per­ with sera, serially diluted in high performance ELISA buffer tHPE: San- formed adoptive transfer experiments with T and B cells from quin. Amsterdam. The Netherlands) with 1% BSA. followed by biotinyl­ CD28~/_ or CD27_/~ mice. Collective experiments show that ated goat anti-mouse IgM, -GI. -G2a, -G2b. -G3. or -A mAb (Southern Biotechnology Associates), diluted 1:2,500 in HPE with 1% BSA. and CD27 can support GC formation and Ig production via a major SA-conjugated HRP (Sigma-Aldrich, St Louis. MO), diluted 1:10.000 in CD28-dependent route that proceeds via CD27 on B cells, and via HPE with \% BSA. Substrate 3.3'.5.5' tetramelhylbenzidin (Merck, Darm­ a minor CD28-independent route that requires CD27 on T cells. stadt, Germany) was added at 0.1 mg/ml (100 u-iAvell), together with 0.06% hydrogen peroxide in 0.1 M sodium acetate (pH 5.5). The reaction was stopped with 2 M H,SO„, and OD4M, was read by a Watlac 1420 Materials and Methods VICTOR2 multilabel counter (PerkinElmer Life Sciences, Boston. MA). Mice Endpoint titers were expressed as reciprocal log, of the last dilution, which gave an OD4St) of sO. 1 OD unit above the ODJ5„ of the negative control Wild-type, CD27 '" (7), and CD28~' mice (24) were on a C57BL/6 (pooled serum from nonimmunized mice). background and used for experiments at 6-10 wk of age. in accordance with national and institutional guidelines. Offspring were genotyped by Immunohistology PCR and phenotype was confirmed by flow cytometry. Spleens were embedded in OCT compound (Tissue-Tek; Miles. Torrance, Flow cytometry CA), frozen in liquid nitrogen, and stored at -80"C. Cryostat sections (6 Spleen and lymph nodes were forced through a nylon mesh in 1MDM with fi.ni) were mounted onto glass slides coated with 3-aminopropyltriethoxy- silane (Sigma-Aldrich). Slides were air dried for 1 h. fixed in ice-cold 8% FCS. Erythrocytes were lysed in 0.14 M NH4C1. 0.017 M Tris-HCl (pH 7.2). Cells were incubated with Fc Block (2.4G2; BD PharMingen, San acetone, dried, and stored at -20°C. Sections were rehydrated in PBS for 30 min and immersed in PBS and 5% BSA for 30 min at room temperature. Diego, CA), washed in staining buffer (PBS, 0.5% BSA. 0.01% sodium 3 azide), labeled as indicated, and analyzed using a FACSCalibur and They were incubated in the dark overnight at 4 C in PBS and 1% BSA with CellQuest software (BD Biosciences. Mountain View, CA). Abs used were mAb, which was directly conjugated to allophycocyanin, F1TC. or PE, or anti-mouse IgM, -IgD, pooled IgG (-1, -2a. -2b) mAb (Southern Biotech­ biotinylated and detected by Texas Red (TR)-conjugated SA (Molecular nology Associates. Birmingham. AL); anti-CD3e mAb 145.2CH, anti- Probes Europe, Leiden. The Netherlands). Abs used were anti-CD45R/ CD45R/B220 mAb RA3-6B2, anti-CD19 mAb 1D3. anti-CD27 mAb B220 mAb RA3-6B2. anti-GL7 mAb GL7. anti-CD3 mAb 500A2. anti- LG.3A10 (6). and anti-CD28 mAb 37.51 (from BD PharMingen). Dr. A. CD4 mAb RM4-5, anti-CD8 53-6.7 (1:200; BD PharMingen), and biotin­ Rolink (University of Basel, Basel, Switzerland) kindly provided 493 mAb ylated anti-foilicular dendritic cell (FDC) mAb FDC-M2 (30) (20 u.g/ml; (28). Biotinylated peanut agglutinin (PNA) was from Vector Laboratories AMS Biotechnology. Oxon. U.K.). Slides were mounted in Vectashield (Burlingame. CA). (Vector Laboratories) and observed under a Leica TCS NT confocal laser- scanning microscope (Leica Microsystems. Wet/Jar. Germany). For quan­ Selection for IgM expression titative analysis of GC formation, spleen sections were stained with anti- B220-allophycocyanin and anti-GL7-FlTC. For each data point, whole IgM' cells were selected by magnetic cell sorting. Spleen cells were in­ spleen cross-sections were analyzed. The total number of B cell follicles in cubated for 30 min on ice with 400 u.1 of anti-mouse IgM-microbeads each section was counted. B220/GL7 double-positive clusters within B (Miltenyi Biotec. Bergisch Gladbach. Germany) per 10' cells. After wash­ follicles were defined as GCs (31). GC formation was expressed as the ratio ing, cells were resuspended in IMDM with 4% FCS, followed by positive of GCs per total number of B cell follicles. selection using the autoMACSen (Miltenyi Biotec). The IgM-positive frac­ tion was stained with biotinylated anti-IgD mAb followed by slrepiavidin Preparation of purified T and B cells and total splenocytes (SA)-allophycocyanin, anti-lgM-PE. and anti-CD27-FITC. Alternatively, it was stained with biotinylated 493 mAb followed by SA-allophycocyanin. Splenocytes were passed over nylon wool to remove adherent cells (Poly- anti-IgD-PE, and anti-CD27-FITC. The IgM-negative fraction was stained sciences, Warrington, PA). For T cell purification, nonadherent splenocytes with biotinylated anti-pooled IgG, followed by SA-allophycocyanin. anti- were incubated on ice for 30 min with anti-MHC class II mAb CD19-PE, and anti-CD27-FITC. The IgM-positive fraction contained M5/114.15.2 (BD PharMingen), followed by 30 min incubation on ice with >98% B cells, and the IgM fraction contained —3-4% B cells. 100 u.1 of goat anti-mouse Jg-coated magnetic beads, and 20 u.1 of sheep anti-rat Ig-coated magnetic beads (Advanced Magnetics, Cambridge, MA) 7 Analysis of somatic hypermutation (SHM) per 10 cells. Beads were removed by magnetic sorting. For B cell puri­ fication, nonadherent splenocytes were incubated for 30 min on ice with rat Wild-type and CD27""'' mice were immunized i.p. with 50 fig chicken 7 anti-mouse mAb GK1.5 to CD4 and Lyt.2 to CD8. followed by 30 min globulin conjugated to (4-hydroxy-3-nitrophenyl) acetyl in alum. B cells incubation on ice with 100 u.1 of goat anti-rat Ig-coated magnetic beads per were enriched from spleen by means of MACS using anti- 107 cells. Purity of the resulting cell populations was checked with anti- CD 19-microbeads (Miltenyi Biotec). For analysis of SHM in CD27~' CD3e and anti-B220 mAbs. Only preparations that contained >98% T or mice, the MACS-soited population was stained with biotinylated PNA. B cells were used. followed by SA-allophycocyanin and PE-conjugated anti-VAl/2 mAb LS1.36 (29). PNAh'eh VA1/24 lymphocytes were soiled in the presence of Virus infection and adoptive transfer propidium iodide on a FACStarlus (BD Biosciences). For analysis of SHM in CD27 + and CD27 GC B cells of wild-type mice, the MACS-sorted Influenza vims strain A/NT/60/68 was obtained from the Department of population was stained with biotinvlated PNA. followed by SA-PerCP, Virology at Erasmus University (Rotterdam, The Netherlands) and stored ami-CD 19-Cy5. anti-CD27-FITC. and anti-VAI/2-PE. VA1/2* cells, all in aliquots at -80°C. Mice were anesthetized and infected intranasally expressing CD 19, were analyzed for PNA1"*"'' and PNAlow phenotype. with 25 hemagglutinin units of virus in 50 u.1 of HBSS. or with 100 U for Within the PNA1"8" gate, CD27' and CD27 cells were individually rechallenge. For adoptive transfer, total nonadherent spleen cells (50 x sorted. Single live cells were sorted into 96-well PCR plates, snap frozen. 10") or purified T or B cells (20 X 10") were resuspended in 200 u.1 of PBS and stored at - 20°C (29). Rearranged VA112 genes were amplified by PCR and injected into the tail vein of recipient mice. and 310 nt were sequenced using Bigdye Terminators Ready Reaction Mix (Applied Biosystetns, Foster City. CA), in combination with the nested Results VA1/2 primer (29). Sequences were analyzed using DNAstar software CD27 is transiently expressed during the GC reaction, primarily (Madison, WI). at the centroblasi stage ELISA A detailed analysis of CD27 expression on B cells in the mouse had not previously been performed. To determine CD27 expres­ Blood from the tail vein was collected in heparin-treated Microvette (Sarstedt Aktiengesellschaft. Niimbrecht, Germany) and centrifuged to ob­ sion on B cells of naive and memory phenotype, we purified IgM* tain sera, which were stored at -20°C. Density gradient-purified influenza B cells from spleens of mice, which had been infected with influ­ virus was incubated with 1% Triton X-100 at room temperature for I h and enza virus 6 mo earlier. In this population, 0.15% of IgM1 D f cells coated at 4°C overnight onto polystyrene 96-well flat-bottom microtiter and 5.65% of IgM+D" cells expressed CD27 (Fig. 1A). Because plates (Nunc, Roskilde, Denmark) at 2 /xg/well in 0.1 M sodium carbonate buffer (pH 9.6) with 0.01% Triton X-100. Subsequent incubations were the lgM*D cell population in the spleen contains both immature performed at room temperature with washing in between. Nonspecific transitional B cells and memory B cells (32), we also used 493 binding was blocked with PBS and \% BSA for 1 h. Wells were incubated mAb, which detects transitional B cells (28). Within the IgM+D

70 CD2 7 promotes germinal center formation

tgMpes^ fraction *tgM'gating IgM negative fraction + CD19" gating

10*

10*

10» 565* QTO1 . j^! O 10' a^ m »3% 4 Ï0w&' 101 103 IET* icr ic' io' IO3 io IgO —* IgG

^ positive traction

120 180 255 DLN

V V

FIGURE 1. CD27 expression is in line with a contribution to centrobiast expansion. A, Pooled splenocytes of two mice infected with influenza virus 6 mo earlier were separated into IgM* and lgM~ subsets by MACS, triple stained with the indicated Abs. and analyzed within the indicated gates for fluorescence intensity. Numbers give the percentage of positive cells in the quadrants. The experiment is representative of three experiments. B, One million pooled splenocytes or DLN cells of four mice obtained at day 8 after infection were stained with PNA, anti-CD19. and anti-CD27 mAb, and analyzed. Within CD194PNAhl|?h gates, FSC discriminates centroblasts and centrocytes. Percentages of positive cells per quadrant are indicated. C, At day 8 after virus infection, spleens were isolated, processed for confocal laser immunohistology, and stained with fluorescent Abs. CI and C2 show the same triple-stained section. In CI, GC cells are detected with anti-GL7-FfTC. In C2, B cells are detected with anti-B220-allophycocyanin. and arrows indicate some CD70* B cells. In C3, another section is shown, where T cells are detected with anti-CD3-FITC. CD70 was detected by anti-CD70-biotin and TR-SA.

population, the minor 493 + subset lacked CD27, while 5.6% of the essentially lacking on transitional and naive B cells and present on 493" IgM ^D" population of memory phenotype B cells expressed a few percent of lgM+ and IgG+ memory phenotype B cells. CD27 (Fig. M, red gate). Within the IgM fraction, 2.30% of To examine CD27 expression on GC B cells, we stained spleen IgG+ cells expressed CD27 (Fig. 1A). We conclude that CD27 is and lung-draining lymph nodes (DLN) at day 8 after influenza

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virus infection. CD19 was used as B cell marker and PNA as GC marker (33). Cells were examined for forward scatter (FSC) profile to discriminate between centrocytes and centroblasts (Fig. IB, red rectangles). CD27 was found on only 0.2% of PNAl°* non-GC B cells and -13% of PNAhigh GC B cells in both spleen and DLN, indicating that it is acquired during the GC reaction. About half of the CD27 * GC B cells were centroblasts and the other half cen­ trocytes. CD27 was found at 3- to 4-fold higher frequency among centroblasts than centrocytes (Fig. IB), suggesting that CD27 is most abundant during the expansion phase of primed B cells. lil To gain further insight into the GC B cell stage when CD27 is Day alter rt «awn acquired, we examined somatic mutations in Ig genes of CD27 + and CD27~ GC B cells of wild-type mice. At day 10 after immu­ nization with haptenated protein Ag, B cells were purified from pooled spleens of two mice and sorted individually on the basis of a PNAhishVAl/2+CD27+ or -CD27" phenotype. Single-cell PCR with VAI/2-specific primers allowed determination of the fre­ quency of somatic Ig gene mutations in each subset. For the CD27+ subset, 29 cells were analyzed, of which 4 contained mu­ tations (14%). For the CD27 subset, 42 cells were analyzed, of which 12 contained mutations (29%). The actual mutation fre­ quency, which is the percentage of nucleotides mutated among total nucleotides sequenced from mutated cells, was very similar for CD27+ and CD27 cells, namely 0.67% and 0.70%. These data argue that CD27 expression is not concordant with the pres­ ence of somatic mutations. 'I 14 31 a 58 S3 » Tt To examine CD70 expression, we used confocal immunohistol- •*» Day afiwfiinwryinleöi ogy, because CD70 is difficult to detect at the plasma membrane FIGURE 2. In CD27'"'"' mice, GC formation is delayed but serum Ig (3). GL7 was used as a marker for GC B cells, because it performs levels are normal. A, At the indicated days after infection, spleens were well on fixed tissue (34). Staining for CD70 and GL7 revealed isolated, processed for confocal laser immunohistology, and stained with multiple CD70-positive cells in GCs, or in close proximity of GCs, fluorescent Abs to B220 as B cell marker and GL7 as marker for GC cells. in spleens of influenza virus-infected mice (Fig. 1C). Staining of The percentage of follicles containing GCs was determined as described in the same section with B220 defined a significant proportion of Materials and Methods, using two spleens per time point. Error bar indi­ CD70 ' cells as B cells. CD70 was also found on T cells, which cates SD. B, At the indicated days after infection, serum titers of vims- were located in the B cell follicle near or in GCs, typically in specific Ig isotypcs were determined by ELISA. At day 56, mice were clusters (Fig. lO. reinfected (arrows). Each data point represents a measurement on pooled sera from four mice. Results are representative of two independent Our data indicate that, in mice, CD27 is essentially lacking on experiments. transitional and naive B cells and acquired during the GC reaction at the centroblast stage. CD27 is not a marker for somatically mutated B cells in the mouse and is absent from most memory B cells. Expression of CD27 and CD70 is in line with a role for this acteristic of SHM in the wild-type situation (35). We conclude that receptor/ligand pair during the B cell expansion phase in GCs. CD27 is not required for the somatic diversification of Ig genes. To examine the impact of CD27 deletion on Ig production, mice CD27 accelerates GC formation, but does not affect SHM or ig were infected with influenza virus and bled from the tail vein 0, 7, production 14, 21, 28, and 56 days later. Influenza virus-specific IgM, -Gl, Initial examination of GC formation at day 8 after influenza virus -G2a, -G2b, -G3, and -A isotypes increased in response to infec­ infection failed to reveal a defect in CD27 /_ mice. However, tion, with a transient increase in IgM preceding the others (Fig. when GC formation was followed kinetically, an impact of CD27 2B). No major differences in responsiveness were seen between _/ was found (Fig. 2A). At day 4 after infection, 32% of B cell fol­ wild-type and CD27 ~ mice. Virus-specific IgG and IgA levels licles in wild-type mice showed GCs, while this was about 2-fold were as high as the plateau level at day 56 after primary challenge less in CD27_/~ mice. At day 6, the frequency of GCs was still (Fig. 2B), and remained so for at least 6 mo in both wild-type and significantly lower in CD27_/~ mice, but at day 8 it had reached CD27~'~ mice (data not shown). Rechallenge at day 56 modestly the same level as in wild-type mice. Apparently, CD27 determines increased some Ig isotypes, but responsiveness in wild-type and the kinetics of GC formation, but is not absolutely required for this CD27 /" mjce was similar (Fig. 2fl). In conclusion, CD27 dele­ response. tion does not have a detectable impact on Ig class switching, gen­ To analyze whether CD27 affected SHM, wild-type and eration, and maintenance of virus-specific IgM, IgG, and IgA lev­ CD27 ' ~ mice were immunized with haptenated protein Ag, and els in primary and recall responses in this physiological model. single-cell PCR was performed on VAl/2+ GC B cells isolated 7 days later. Twenty-nine VA1/2 PCR products derived from pooled CD27 supports GC formation and IgG production in absence of cells of two CD27""'"" mice were sequenced, of which 34% carried CD28 mutations. The actual mutation frequency was 0.60%. These fig­ To determine whether CD27 contributes to GC formation down­ ures are in the wild-type range (29). The VA1/2 mutations in stream of CD28, we examined responses to influenza virus in / CD27 ""'~ cells showed an A-T bias, preferred transitions over CD28""/"' and CD27/CD28""'"" mice. In the spleen of these mice, transversions, and had a preference for RGYW motifs, as is char- T and B cell areas were normal in size and appropriately separated

72 CD27 promotes germinal center formation

(data not shown). To analyze GC formation, sections of spleen, sufficient to produce significant IgG levels. In the additional absence isolated at day 8 after virus infection, were double stained with of CD27, GC formation in spleens is undetectable and IgG production fluorescent Abs to B220 and GL7. B ceil follicles of wild-type and drops accordingly. The very low residual IgG production in CD27/ CD27 ' mice contained GCs of similar size and frequency. In CD28 '~ mice may be attributed to B cell maturation in the small spleens of CD28""'"" mice, GCs were still detectable, but these GCs that still form at low frequency in DLN of these mice (data not were small and low in frequency (Fig. 3A). Interestingly, in mice shown). lacking both CD27 and CD28, GC formation in the spleen was completely abrogated (Fig. 3,4). Apparently, CD27 can support GC A CD28-independent CD27-driven pathway to deliver T eel! formation in the absence of CD28, but these GCs are small and help to B cells occur in low frequency as compared widi wild-type. Our data indicate that CD27 can partially substitute for CD28 to At day 14 after influenza virus infection, when plateau levels are drive GC formation. Subsequent experiments were designed to de­ reached in IgG production (see Fig. 2B), IgGl, -G2a, and -G2b termine through what cell-cell interactions this was achieved. We levels were 8- to 10-fold reduced in CD28_/~ mice, as compared used CD27/CD28"''" mice as recipients and investigated to what with wild-type and IgG3 levels reduced —4-fold (Fig. 3B). How­ extent GC formation in the spleen could be restored by adoptive ever, significant IgG responses were still detectable. Interestingly, transfer of T cells that were deficient for CD28, but proficient for on the CD28_/~ background, CD27 deficiency further reduced levels CD27. As a positive control. CD27/CD28-'- mice were reconsti­ of all vims-specific IgG isotypes — 10-fold (Fig. 3B). Thus, CD27 can tuted with wild-type total nonadherent splenocytes, i.e., T and B support the generation of IgG-secreting plasma cells in the absence of cells. This permitted GC formation at a frequency approximating CD28. Apparently, the modest GC formation in CD28~/_ mice is that in wild-type mice (Fig. 4A). Transfer of CD27+/"!'CD28"/"

Wild-type CD27* CD28* CD27/CD28*

+ * f ^«/-//V * * * •••// lgG2b lgG3

* +* +f+ff -»> Serum dilution FIGURE 3- CD27 supports GC formation and IgG production in the absence of CD28. A. Spleens taken at day 8 after infection with influenza virus were stained with anti-B220-allophycocyanin and anti-GL7-FITC to detect GCs. Sections were examined by confocal microscopy. The percentage of follicles containing GCs was determined as indicated in Fig. 2A. B, Virus-specific serum Ig was measured as indicated in Fig. IB. Data points represent OD430 values in ELISA of pooled sera from three mice per group. IgGl, -G2a. -G2b, and -G3 levels were measured at day 14 after infection when plateau levels were reached (see Fig. 20). The experiment was repeated with very similar results.

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Recipient CD27/CD28*

J J + wild-type splenocytes + CD27*'* CD28 splenocytes + CD27*'* CD28 - T cells + wild-type B cells

%GC: 58.3% 5.2% 5.7% 0.0%

a CD28 ^fc~0 CD27 ^—0 CD27 CD70 A ro A CD70 A C027 ^Vï ^ CD70 © If70 ® &

CD27/CD28-'- Wild-type + CD27*'+ CD28'- splenocytes

FIGURE 4. GC formation in absence of CD28 is mediated by CD27+/' T cells. CD27/CD28'' mice were reconstituted by i.v. adoptive transfer of total nonadherent splenocytes (T and B cells) or purified T or B cells of the indicated phenotypes. Mice were infected at day 2 after transfer and examined 8 days after infection. A. Spieen sections were stained with anti-B220-allophycocyanin and anti-GL7-FFTC, and examined by confocal microscopy to determine the percentage and size of GCs. The experiment is representative of two experiments. The cartoons indicate the theoretical possibilities of CD27, CD28, and CD70 expression on T and B cells in the adoptive transfer setting, based on these genotypes of the cells present. B cells from donor (Bd) and recipient (B,) are indicated separately. B, Detection of FDC and CD4 ' T cells in GCs of CD27/CD28'"'" mice, which had received CD27"" CD28 ' splenocytes at day 8 after infection. Sections of adoptive transferred and wild-type control mice were stained with FDC-M2 TR (30) and anti-GL7-FlTC. or with anti-CD4-allophycocyanin and anti-GL7-FITC. as indicated.

74 CD27 promotes germinal center formation

sptenocytes allowed the formation of small GCs at low frequency, cells only. In the animals that received both CD27 ' CD28+/+ T exactly as seen in CD28_/~ mice (see Fig. 3A). Purified CD27+/~ cells and CD27+/+ B cells. GCs were of normal size, but some­ CD28~/_ T cells had the same effect, indicating that GC formation what lower in frequency than in the reconstituted wild-type situ­ in absence of CD28 requires CD27 on T cells, but not on B cells ation at day 8 after infection. However, at day 14 the frequency (Fig. 4A). In CD27/CD28~'~ mice that received CD27+/^CD28_/" was normal (Fig. 6, B and C). In mice that received purified splenocytes, GL7+ B ceils clustered next to the FDC network, as in CD27_/~CD28+/+ T cells only, a more significant deficit was wild-type mice. In addition. CD4+ T cells were present in these areas observed. At day 8 after infection, GCs were small and infrequent (Fig. AB). These findings indicate that CD27 on T cells can promote (Fig. 6B). Throughout the next 2 wk, GCs increased in size and the formation of small but normally organized GCs in the absence frequency to finally reach the wild-type situation (Fig. 6C). We of CD28 (36, 37). conclude that in this adoptive transfer setting, GC formation is Wt Production of anti-viral IgM, -Gl, -G2a, -G2b, and -G3 was slightly delayed when CD28 Th cells act in the absence of determined at days 7, 14. and 21 after infection in the reconstituted CD27 on T cells. In absence of CD27 on B cells, GC formation is mice, and compared with that in CD27/CD28_/~ mice or mice dramatically delayed, but not defective. In a wild-type adoptive reconstituted with wild-type splenocytes (control). Titers of all IgG transfer setting, GC formation is complete at day 8 after infection isotypes increased to plateau levels in this period, while IgM pro­ (Fig. 6CX while this situation is only reached at day 20 in absence duction peaked at days 7-14 (Fig. 5). Purified CD27+/+CD28~'~ of CD27 on B cells. We conclude that GC formation as driven by : T cells restored IgG3 production almost to control levels, while it CD28 T cells is accelerated in particular by CD27 on B cells, increased titers of the other IgG isotypes 3- to 6-fold, to a level that while CD27 on T cells also makes a small contribution. was —3-fold lower than control. Apparently, the modest GC for­ Virus-specific serum IgG production in CD27/CD28_/~ mice mation in these reconstituted mice allows for significant IgG pro­ reconstituted with CD27 _/"CD28+/+ T cells and CD27+/+ B duction. We conclude that there exists a CD28-independent path­ cells was as efficient as the response in wild-type mice (Fig. 7). way to deliver T cell help for GC formation and IgG production, This emphasizes that CD27 on T cells makes a nondetectable con­ which requires CD27 on T cells but not on B cells. tribution to the B cell response when CD28 is present. However, this experiment clearly revealed a contribution of CD27 on B cells CD27 on B cells accelerates CD28-dependent GC formation to CD28-dependent Ab production. Although the TgM response and facilitates IgG production was normal in reconstituted mice that lacked CD27 on B cells, the Subsequent experiments were designed to determine the contribu­ IgG responses were delayed. IgGl production had reached wild- tion of CD27 to CD28-dependent GC formation. Bringing back type plateau levels at day 21, but the other isotypes were still at a CD27_/~CD28~!_/+ splenocytes into CD28~'~ mice allowed for­ significantly lower level at this time point (Fig. 7). This is in line mation of GCs with wild-type size and frequency within 8 days with GC formation just reaching its optimum at day 20 after in­ after infection (Fig. 6A). This was expected, because both CD27 fection (Fig. 6C). In conclusion, CD28-dependent IgG production and CD28 were present in the reconstituted situation. Reconstitu- does not require CD27 on T cells, but is facilitated by CD27 on B tionofCD27/CD28w" mice with CD27~/~CD28+/+ splenocytes cells. created a situation where CD28 was present but CD27 was absent. In this case, only small GCs were observed at day 8 after infection, Discussion which occurred in ~5-fold lower frequency than in the reconsti­ We have found that in the mouse, B cells acquire CD27 during the tuted wild-type situation (Fig. 6A). GC reaction and express it there at the highest frequency. CD27 Next, we examined whether it was CD27 on T ceils or CD27 on was detected on —13% of GC B cells in spleen and DLN at the B cells that supported CD28-dependent GC formation. CD27/ peak of the primary response to influenza virus. In our hands, CD28W" mice received purified CD27"' CD28+/+ T cells and anti-CD27-biotin conjugates followed by SA-PE or SA-allophy- wild-type (CD27^/+) B cells, or purified CD27~/_CD28+/+ T cocyanin gave similar percentages of CD27 expression on B cells

C027/CD28' CD27/CD28' + wicMype spfanocytes CD211C07& +CD27"*CD28+Tcel!

FIGURE 5. Ig production in absence of CD28 is me­ diated by CD27 H+ T cells. CD27/CD28 ' mice were reconstituted with puritied T cells from CD28~'~ mice or total nonadherent spleen cells (T and B cells) from wild-type mice, as indicated. Mice were infected at day 2 after adoptive transfer and serum Ig titers were de­ termined at the indicated days after infection. Each data point represents a measurement on pooled sera from three mice.

75 Chapter 5

A Recipient: C028* Recipient: CD27/CD28J

J + CD27 - CD28*" splenocytes + CD27-'- CD28*" splenocytes

% GC: 54.5% 11.2%

CD2« •~0cO27

4 CO70 W CD7 m A C027QV ° ticoro ®(S° (S>

FIGURE ft. GC formation in presence of CD28 is Recipient: CD27/CD28J accelerated by CD27 on B cells. A, CD28 ' orCD27/ CD28~' mice were reconstituted with total nonadher­ + CD27JCD28*"T cells ent spleen cells (T and B cells) from CD27 ' CD28''' mice, and GC formation was determined at day 8 after & wild-type B cells + CD27* CD28" T ceMs only infection. B. CD27/CD28-'" mice were reconstituted with CD27 ' CD28'" T cells and wild-type (CD27 4' *) B cells or CD27 ' CD28 +/* T cells only. GC formation at day 8 is shown. Cartoons represent what is indicated in Fig. 4. C. Quantitative representa­ tion of GC incidence at days 8, 14. and 20 after infec­ tion in CD27/CD28 ' mice lh;il were reconstituted as indicated. Means ± SD are derived from three mice per time point.

• CD27/CD28' + wild-type T& B celts • CD27/CD28» • CD27' CD28"' T & wild-type B c • CD27/CD28' • C027' CD28" T cells only

day after Infection

76 CD 2 7 promotes germinal center formation

!<*• Wüd-type CD27/28^ + CD27+ CD28*" T & wfctype B c CD27/2*' +CD27* CIKM^T4CD27' B ce ki FIGURE 7. Ig production in the presence of CD28 is facilitated by CD27 on B cells. CD27/ CD28 ' mice were reconstituted with purified CD27~'~CD28+" T cells, plus purified wild- type or CD27~'~ B cells. Mice were infected with influenza virus at day 2 after adoptive transfer, and serum Ig titers were determined at the indicated days after infection. Responses in 14 21 wild-type mice were analyzed for comparison. Each data point represents a measurement on lgG3 pooled sera from three mice.

Day after i

as use of directly FITC-labeled anti-CD27 mAb. So, within the Th cells. We have recently established that on activated T cells, limits of detection, the frequency of CD27 expression on GC B CD27 promotes survival and not division (8). Therefore, we pos­ cells in the mouse appears lower than in humans, where all ton­ tulate that CD27 also promotes survival of activated B cells, and sillar GC B cells reportedly express CD27 (17). We found CD27 thus facilitates their expansion during the GC reaction. In humans, on —25% of centroblasts and only on —8% of centrocytes, sug­ CD27 is expected to fulfil the same role. Additionally, in humans, gesting that it is gradually lost from B cells after their expansion CD27 may promote the survival of previously primed B cells out­ phase. Within GCs, SHM was less frequent among CD27-positive side GCs when CD70 is available. cells than among CD27-negative cells, in line with its predominant We have also revealed a CD28-independent pathway where expression during the expansion phase. Among memory B cells, CD27+ T cells provide help to B cells to form small GCs and only a few percent express CD27. Consistently, CD27" B cells do produce IgG. This may involve interaction between CD27 on Th not accumulate in the mouse upon aging. However, the frequency cells and CD70 on B cells. Alternatively, CD27/CD70 interactions of CD27' B cells transiently increases upon secondary antigenic between T cells may support expansion of the Th cell population challenge (data not shown). Our collective data indicate that in the in the absence of CD28. A third scenario is that CD27 on T cells mouse, CD27 is a marker for recent B cell activation and that contributes to the B cell response like OX40, in that it would CD27/CD70 interactions come into play during the B cell expan­ interact with CD70 on CD4'3 accessory cells and stimulate T sion phase in GCs. The mutation status of CD27+' and CD27~ B cells to migrate into B cell follicles (38). Although a contribution cells within GCs is in line with CD27 acquisition preceding or of OX40 to the B cell response has been identified by Ab inter­ coinciding with the onset of SHM. In humans, CD27 is also in­ vention studies (39, 40), GC formation and IgG production in re­ duced upon B cell priming, but it appears to be expressed at a sponse to viruses and haptens are normal in OX40-deficient mice higher frequency among GC B cells and maintained long-term on (41, 42), while only a minor defect was found in OX40 ligand a much higher proportion of post-GC B cells than in the mouse. (OX40L)-deficient mice (43). We have found that lg production in Whether CD27 is engaged will depend on presence of CD70, response to influenza virus is normal in OX40L_/ mice, as well which both in humans and mice is tightly controlled by antigenic as in mice lacking both OX40L and CD27 (Y.X., unpublished stimulation and of a transient nature (1, 3). CD70 is difficult to observations). Possibly, the GC B cell response is finely tuned by document on live cells, but by confocal microscopy, we could partially overlapping costimulatory signals by TNFR family mem­ detect occasional CD70+ B cells in GC and CD70+ T cells at the bers and their ligands. The dominant effect of CD28 on GC for­ border of or inside GCs. This suggests that CD27/CD70 interac­ mation may be explained by its role in the up-regulation of mul­ tions come into play during Th cell-B cell communication just tiple receptors and ligands of this type. before or during B cell expansion in GCs. Studies on human cells We have found that CD27 deficiency reduces the survival of show unambiguously that stimulation of CD27 on activated B cells virus-specific CD8+ T cells in DLN and lung (8). How it impacts promotes the generation of plasma cells (1). We have indeed re­ on the CD4 * T cell response to influenza virus cannot be followed vealed such a pathway in vivo, but in the mouse it is not required in this model with the aid of MHC tetramers. Therefore, we have for production or maintenance of serum IgG and IgA levels in much less knowledge about it. CD27 deficiency does reduce the primary or recall responses to influenza virus (this study) or hap- accumulation of CD4+ T cells in the lung (7). However, GCs are -/ tenated protein Ags (our unpublished results). In CD27 mice, formed in DLN of CD27 ' mice (data not shown). Ongoing work the only observable B cell phenotype is a delay in GC formation, is focused on the CD4 T cell response to OVA, which we can indicative of less effective expansion of activated B cells in the follow both with MHC tetramers and with TCR transgenic cells. absence of CD27/CD70 interactions. Our adoptive transfer exper­ These studies should illuminate exactly how CD27 regulates CD4 iments reveal the same phenotype, with a larger window for read T cell function. out. They add that efficient GC B cell expansion requires CD27 on The detailed division of labor between different TNFR family B cells, while CD27 on T cells is virtually dispensable for it. Most members and their selective and transient expression on certain likely, CD27 on B cells is triggered by CD70 on activated CD28 + cell types during an ongoing immune response will allow for

77 Chapter 5

highly specific intervention in disease states. Because constitutive 21. Agematsu. K.. H Nagumo. Y. Oguchi. T. Nakazawa, K. Fukushima, K. Yasui, ligation of these receptors can be highly detrimental due to effects S. Ito. T. Kobata, C. Morimoto. and A. Komiyama. 1998. Generation of plasma cells from peripheral blood memory B cells: synergistic effect of interleukin-10 on lymphocyte homeostasis (e.g.. 44), such interventions should be and CD27/CD70 interaction. Blood 91:173. transient and properly localized. 22. Lens, S. M. A.. R. de Jong, B. Hooibrink, G. Koopman, S. T. Pals. M.H.J, van Oers. and R. AW. van Lier. 1996. Phenotype and function of human B cells expressing CD70 (CD27 ligand). Eur. J. Immunol. 26:2964. Acknowledgments 23. Lens, S. M. A., R. M. J. Keehnen, M. H. J. van Oers. R. A. W. van Lier, We thank G. Rimmelzwaan, G. Dingjan. R. Hendriks, and J. Laman for S. T. Pals, and G. Koopman. 1996. Identification of a novel subpopulation of assistance and advice, and R. A. W. van Lier, F. Scheeren, and K. Schepers germinal center B cells characterized by expression of IgD and CD70. Eur. J. /m- munol. 26:1007. for advice and critica! reading of the manuscript. We also thank the per­ 24. Shahinian. A.. K. Pfeffer. K. P. Lee, T. M. Kundig, K Kishihara. A. Wakeham, sonnel of the histology, confocal microscopy, flow cytometry, and exper­ K. Kawai, P. S. Ohashi. C. B. Thompson, and T. W. Mak, 1993. Differential T imental animal facilities of the Netherlands Cancer Institute for excellent cell costimuiatory requirements in CD28-deficient mice. Science 261:609. technical assistance. 25. Ferguson, S. E., S. Han, G. Kelsoe, and C. B. Thompson. 1996. CD28 is required for germinal center formation. J. Immunol. 156:4576. 26. Boriello, F.. M. P. Sethna, S. D. Boyd. A. N. Schweitzer, E. A. Tivol, D. Jacoby, References T. B. Strom, E. M. Simpson, G. J. Freeman, and A. H. Sharpe. B7-1 and B7-2 i Lt;ns, S. M, K. Tesselaar, M. H. J. van Oers. and R. A. W. van Lier. 1998. have overlapping, critical roles in immunoglobulin class switching and germinal Control of lymphocyte function through CD27-CD70 interactions. Semin. Immu­ center formation. Immunity 6:303. nol. 10:491. 27. Lane, P., C. Burdel, S. Hubele, D. Scheidegger. U. Mueller, F. McConneli, and 2. Oshima. H., H. Nakano, C. Nohara, T. Kobata, A. Nakajima. N. A, Jenkins. M. Kosco-Vilbois. 1994. B cell function in mice transgenic for mCTLA4-H7l: D. J. Gilbert. N. G. Copeiand, T. Muto, H. Yagita, and K. Okumura. 1998. lack of germinal centers correlated with poor affinity maturation and class switch­ Characterization of murine CD7Ü by molecular cloning and mAb. Int. Immunol ing despite normal priming of CD4f T cells. J. Exp. Med. 179:819. I'0:517. 28. Rolink. A. G., J. Andersson, andF. Melchers. 1998. Characterization of immature 3. Tesselaar. K., Y. Xiao, R. Arens, G. M. W. van Schijndel, D. H. Schiiurhuis, B cells by a novel monoclonal antibody, by turnover and by mitoaen reactivity. R. Mebius. J. Borst, and R. A. W. van Lier. 2003. Expression of the murine CD27 Eur. J. Immunol. 28:3738. ligand CD70 in vitro and in vivo. J. Immunol. 170:33. 29. Jacobs. H., Y. Fukita. G. T. J van der Horst, J, de Boer, G. Weeda, J. Essers, 4. Futagawa, T., H. Akiba, T. Kodama. K. Takeda, Y. Hosoda, H. Yagita, and N. de Wind. B. P. Engelward, L. Samson, S. Verbeek, et al. 1998. Hypermutation K. Okumura. 2002. Expression and function of 4-IBB and 4-IBB iigand on of immunoglobulin genes in memory B cells of DNA repair-deficient mice murine dendritic cells. Int. Immunol. 14:275. J. Exp. Med. 187:1735. 5. Hint/en, R Q.. S. M. Lens, K. Lammers, H. Kuiper. M. P. Beckmann, and 30. Taylor, P. R„ M. C. Pickering. M. H. Kosco-Vilbois, M. J. Walporl, M. Botto, R. A. W. van Lier. 1995. Engagement of CD27 with its ligand CD70 provides a S. Gordon, and L. Martinez-Pom ares. 2002. The follicular dendritic cell restricted second signal for T cell activation. J. Immunol. 154:2612. epitope. FDC-M2, is complement C4: localization of immune complexes in 6. Gravestein. L. A., J. D. Nieland, A. M. Kruisbeek, and J. Borst. 1995. Novel mouse tissues. Eur. J. Immunol. 32:1888. monoclonal antibodies reveal potent costimulatory activity of murine CD27. Int. 31. Pasparakis, M.. L. Alexopolou. V. Episkopou, and G. Kollias. 1996. Immune and Immunol. 7:551. inflammatory- responses in TNFa-deficient mice: a critical requirement for TNF« 7. Hendriks, J., L A, Gravestein. K. Tesselaar, R. A. W. van Lier, in the formation of primary B cell follicles, follicar dendritic cell networks and T. N. M. Schumacher, and J. Borst. 2000. CD27 is required for generation and germinal centers, and in the maturation of the humoral immune response J. Exp long-term maintenance of T cell immunity. Nut. Immunol. 1:433. Med. 184:1397. 8. Hendriks. J., Y. Xiao, and J. Borst. 2003. CD27 promotes activated Tcell sur­ 32. Loder. F.. B. Mutschler. R. J. Ray. C. J. Paige, P. Sideras, R. Torres, vival and complements CD28 in generation and establishment of the effector T M, C. Lamers, and R. Carsetti. 1999. B cell development in the spleen lakes place cell pool. J. Exp. Med. 198:1369. in discrete steps and is determined by the quality of B cell receptor-derived 9. Maurer, D., W. Holler. O. Majdic, G. F. Fischer, and W. Knapp. 1990. CD27 signals. J. Exp. Med. 190:75. expression by a distinct subpopulation of human B lymphocvles. Eur. J. Immu­ 33. Butcher. E, C, R. V. Rouse, R. L. Coflman, C. N. Nottenburg. R. R. Hardy, and nol. 20:2679. I. L. Weissman. 1982. Surface phenotype of Peyer's patch germinal center ceils: 10. Agematsu, K.. T. Kobata, F.-C. Yang, T. Nakazawa, K. Fukushima. M. Kitahara, implications for the role of germinal centers in B cell differentiation. J. Immunol T. Mori, K. Sugita, C. Morimoto, and A. Komiyama. 1995. 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Gaspal, H. E. Wiggestt, F. M. McConneil, 13. Agematsu, K., S. Hokibara, H. Nagumo, and A. Komiyama. 2000. CD27: a A. Guibranson-Judge, C. Raykundalia. L. S. K. Walker, M. D. Goodall, and memory B-cell marker. Immunol. Todav 21:204. P. J. L. Lane. 2003. CD4'CD3" accessory cells costimulaie primed CD4 T cells 14. Klein, U., K. Rajewsky, and R. Kuppers. 1998. Human immunoglobulin through OX40 and CD30 at sites where T cells collaborate with B cells. Immunity (lg)M + IgD' peripheral blood B cells expressing the CD27 cell surface antigen 18:643. carry somatically mutated variable region genes: CD27 as a general marker for 39. Walker. L. S. K„ A. Gulbranson-Judge. S. Flynn, T. Broeker, C. Raykundakia, somatically mutated (memory) B cells. J. Exp. Med. 188:1679. M, Goodall, R. Forster. M. Lipp, and P. Lane. 1999. Compromised OX40 func­ 15. Maurer, D., G. F. Fischer, I. Fae, O. Majdic, K. Stuhlmeier. N. von Jeney, tion in CD28-deficient mice is linked with failure to develop CXC chemokine W. Holter, and W. Knapp. 1992. IgM and IgG but not cytokine secretion is receptor 5-positive CD4 cells and germinal centers. J. Exp. Med. 190:1115. restricted to the CD27 ' B lymphocyte subset. J. Immunol. 148:3700. 40. Stueber, E., and W. Strober. 1996. The T cell-B ceil interaction via OX40-OX40L 16. Agematsu, K . H. Nagumo, F.-C. Yang, T. Nakazawa. K. Fukushima, S. Ito. is necessary for the T eel I-dependent humoral immune response. J. Exp Med K. Sugita, T. Mori. T. Kobata. C. Morimoto, and A. Komiyama. 1997. B cell 183:979. + subpopulations separated by CD27 and crucial collaboration of CD27 B cells 41. Kopf, M.. C. Ruedl, N. Schmitz, A. Gallimorc, K. Lefrang, B. Ecabert, and helper T cells in immunoglobulin production. Eur. J. Immunol. 27:2073. B. Odermatt, and M. F. Bachmann. 1999. OX40-deficient mice are defective in 17. Jung, J.. J. Choe. L. Li, and Y. S. Choi. 2000. Regulation of CD27 expression in Th cell proliferation but are competent in generating B cell and CTL responses the course of germinal center B cell differentiation: the pivotal role of IL-10. Eur. after virus infection. Immunity 11:699. J. Immunol. 30:2437. 42. Pippig, S. D., C. Pena-Rossi, J. Long, W. R. Godfrey. D. J. Foweli, S. L. Reiner. 18. Kobata. T., S. Jacquot, S. Kozlowski. K. Agematsu, S. F. Schlossman. and M, L. Birkeland. R, M. Locksley. A. N. Barclay, and N. Killeen. 1999. Robust B C. Morimoto. 1995. CD27-CD70 interactions regulate B-cell activation bv T cell immunity but impaired Tcell proliferation in the absence of CD 134 (OX 40). cells. Proc. Natl. Acad. Sci. USA 92:11249. J. Immunol. 163:6520. 19. Nagumo, H.. K. Agematsu. K. Shinozaki. S. Hokibara. S. ho, M. Takamoto. 43. Murata. K., N. Ishii. H. Takano, S. Miura. L. C. Ndhlovu, M. Nose, T. Noda, and T. Nikaido, K. Yasui, Y. Uehara, A. Yachie, and A. Komiyama. I99S. CD27/ K. Sugamura. 2000. Impairment of antigen-presenting cell function in mice lack­ CD70 interaction augments IgE secretion by promoting the differentiation of ing expression of OX40 ligand. J. Exp. Med. 191:365. memory B celis into plasma cells. / Immunol. 161:6496. 44. Tesselaar, K„ R. Arens, G. M. W, van Schijndel, P. A. Baars. M. A. van der Vaik. 20. Jacquot, S., T. Kobata, S. Iwata. C. Morimoto, and S. F. Schlossman. 1997. J. Borst, M. H. J. van Oers. and R. A. W. van Lier. 2003. Lethal T cell immu­ CDI54/CD40 and CD70/CD27 interactions have different and sequential func­ nodeficiency induced by chronic costimulation via CD27/CD70 interactions. Nat. tions in T cell-dependent B cell responses. J. Immunol. 159:2652. Immunol. 4:49.

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