Germinal Center B Cells Constitute a Predominant Physiological Source of IL-4: Implication for Th2 Development in Vivo

Germinal Center B Cells Constitute a Predominant Physiological Source of IL-4: Implication for Th2 Development In Vivo

This information is current as Bengt Johansson-Lindbom and Carl A. K. Borrebaeck of September 25, 2021. J Immunol 2002; 168:3165-3172; ; doi: 10.4049/jimmunol.168.7.3165 http://www.jimmunol.org/content/168/7/3165 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Germinal Center B Cells Constitute a Predominant Physiological Source of IL-4: Implication for Th2 Development In Vivo1

Bengt Johansson-Lindbom and Carl A. K. Borrebaeck2

Protective immunity depends upon the capability of the immune system to properly adapt the response to the nature of an infectious agent. CD4؉ Th cells are implicated in this orchestration by secreting a polarized pattern of cytokines. Although Th2 development in animal models and in human cells in vitro to a large extent depends on IL-4, the nature of the cells that provide the initial IL-4 in vivo is still elusive. In this report, we describe the anatomical localization as well as the identity of IL-4-producing cells in human tonsil, a representative secondary lymphoid organ. We demonstrate that IL-4 production is a normal and intrinsic feature of germinal center (GC) B cells. We also show that expression of IL-4 is highly conﬁned to the GCs, in which the B cells constitute the prevalent cellular source. Furthermore, immunoﬂuorescence analysis of colon mucosa reveals a strikingly similar pattern of IL-4-expressing cells compared with tonsils, demonstrating that IL-4 production from GC B cells is not a unique feature Downloaded from of the upper respiratory tract. Our results show that GCs provide the most appropriate microenvironment for IL-4-dependent Th2 polarization in vivo and imply a critical role for GC B cells in this differentiation process. The Journal of Immunology, 2002, 168: 3165Ð3172.

cell-dependent B cell responses culminate in the germi- The initial signals that trigger the Th polarization process may 3

nal center (GC) reactions in which Ag-specific B cell be derived from the infectious agent itself and may be transferred http://www.jimmunol.org/ T clones expand heavily, mutate the variable regions of to the lymphocyte compartment by innate cells of the immune their Ig genes, and undergo cytokine-dependent isotype switching. system. For example, LPS triggers IL-12 secretion from dendritic Late phase events include Ag-dependent selection of B cells ex- cells (DCs) and thereby initiates Th1 polarization (10, 11). How- pressing Ig with improved affinity and the development of memory ever, DCs do not necessarily produce IL-12 upon activation, but B cells. Thus, GCs represent anatomical sites that furnish two fun- rather can secrete other cytokines, including IL-4 and IL-13, af- damental features of B cell immunity: 1) a quantitatively and qual- fecting Th2 development and B cell differentiation (12, 13). The itatively improved response, and 2) immunological memory (1, 2). concept of Th cell polarization therefore may be extended to other CD4ϩ Th cells are required for normal GC development, provid- cells of the immune system, where several subsets of leukocytes in ing both cytokines and cell-associated molecules such as CD40 a joint effort undergo linked polarized differentiation in response to by guest on September 25, 2021 ligand (3, 4). However, the precise nature of the Th cell-derived a defined Ag. Such physiological polarization, comprising both B signals required for the initiation of GCs is not fully clarified (5). and T cells, was recently shown to occur at the level of cytokines By differential expression of cytokines and chemokine recep- being produced in mice immunized with typical Th1- or Th2-in- tors, Th1 and Th2 cells differ in their ability to activate distinct ducing Ags, respectively (14). cells and functions of the immune system in responses to patho- Activation and differentiation of naive Th cells take place in gens and Ags in general (6, 7). Th1 cells produce IFN-␥ and secondary lymphoid organs (2, 6). The effects of cytokines are TNF-␤, thereby activating CD8ϩ T cell and macrophage func- predominant among parameters known to have bearing on the po- tions, supporting switching toward complement-fixing isotypes, larization process, and Th1 development mainly depends on IL-12 and are hence key regulatory cells of delayed-type hypersensitivity (and in human also type 1 IFNs), whereas IL-4 most efficiently reactions. Th2 cells secrete a pattern of cytokines, including IL-4, drives Th2 differentiation (7, 15Ð17). In this report, we show that IL-5, IL-6, and IL-13, support a B cell response distinguished by production of IL-4 in human tonsils is highly restricted to GCs and the production of non-complement-fixing Abs, and also drive IL- that the predominant cellular source is the GC B cells themselves. 5-dependent eosinophil mobilization and function (7). Further- IL-4-producing B cells are also present within GCs of colon mu- more, Th2 cells and the cytokines they produce are key regulatory cosa, demonstrating that the phenomenon is not restricted to the components in development of atopic allergy, since dispropor- tonsils. These findings indicate that IL-4-dependent Th2 differen- tional IgE production is driven by IL-4 and IL-13 (7Ð9). tiation in vivo relies on a strong B cell participation and the development of GCs. They also provide a possible explanation, at the molecular level, for previous findings demonstrating the necessity Department of Immunotechnology, Lund University, Lund, Sweden to recruit B cells into Ag presentation in vivo for the Th2 polar- Received for publication July 2, 2001. Accepted for publication January 4, 2002. ization to occur (18). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 This work was supported by grants from the Vårdal Foundation and the European Commission (QLK3-2000-00270). Histology 2 Address correspondence and reprint requests to Dr. Carl A. K. Borrebaeck, Depart- Crystat sections (8 ␮m) of human tonsil and colon mucosa were fixed in ment of Immunotechnology, Lund University, P.O. Box 7031, Lund S-220 07, Swe- 4% paraformaldehyde, quenched for endogenous peroxidase activity with den. E-mail address: [email protected] 0.3% H2O2, blocked with 5% goat serum, and incubated with primary 3 Abbreviations used in this paper: GC, germinal center; DC, dendritic cell; Cy5, mouse mAbs to CD3 (UCHT1, IgG1; DAKO, Glostrup, Denmark), IgD indodicarbocyanine; IDC, interdigitating DC. (IgD26, IgG1; DAKO), CD20 (2H7, IgG2b; BD PharMingen, San Diego,

CA), or, in the presence of 0.2% saponin, to Ki67 (Ki-67, IgG1; Boehringer TUNEL analysis, whereas anti-IL-4 incubation was performed after. Mannheim, Mannheim, Germany). Binding of primary mouse mAbs was Sorting was performed with a FACSVantage SE (BD Biosciences). T cell- revealed by Alexa 568-conjugated goat anti-mouse IgG (Molecular Probes, depleted tonsil cells were stained with PECy5-conjugated mouse anti- Eugene, OR). Remaining anti-mouse reactivity on sections was blocked CD19 (DAKO) and PE-conjugated mouse anti-CD38 (BD PharMingen) with mouse serum, and endogenous biotin was blocked with a biotin block- and sorted into CD19ϩCD38Ϫ non-GC B cells and CD19ϩCD38ϩ GC B ing kit (DAKO) supplemented with 0.2% saponin. From this point, all cells (Ͼ99% purity). GC B cells were further fractionated by incubating the incubations and washing steps were performed in the presence of 0.2% cells with FITC-labeled annexin V (R&D Systems) in Ca2ϩ-containing saponin. Sections were incubated overnight with preformed complexes of buffer and sorting them into cells exhibiting no binding of annexin V and mouse anti-IL-4 (8D4-8, IgG1; BD PharMingen) and biotin-conjugated cells being stained by annexin V. Sorted cells were restained and analyzed Fab of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West on FACScan. This procedure did not result in two well-separated popula- Grove, PA) with unbound Fab blocked by mouse serum before applying tions but rather yielded one fraction lacking annexin V reactivity and one complexes on tissue samples. As a negative control for IL-4 detection, being enriched for cells exhibiting annexin V binding (Fig. 5b). Sorted aliquots of this complex preparation were further incubated with rIL-4 (20 cells were immediately lyzed in TRIzol (Life Technologies) and subjected ␮g/ml; R&D Systems, Minneapolis, MN) before being applied on sections. to RT-PCR as described above. Subsequently, anti-IL-4-dependent tissue deposition of biotin was increased by using a tyramide signal amplification biotin system (NEN Life Cell cultures for determination of IL-4 secretion Science, Boston, MA) and visualized by Alexa 488-conjugated streptavidin GC B cells were purified by negative selection as previously described (19, (Molecular Probes). For triple labeling, a polyclonal rabbit Ab preparation 20). Briefly, T cell-depleted tonsil lymphocytes were layered on 60% iso- ⑀ to a cytoplasmic epitope of CD3 (DAKO) was used in combination with tonic Percoll (Pharmacia Biotech) and, after centrifugation at 750 ϫ g for previous steps and revealed by indodicarbocyanine (Cy5)-conjugated goat 20 min, the buoyant fraction was further depleted using pan-mouse IgG anti-rabbit IgG (Jackson ImmunoResearch Laboratories). For imaging, a magnetic beads (Dynal Biotech) coated with mAbs to CD39 (AC2, IgG1; laser-scanning confocal device (model MRC-1024; Bio-Rad, Hercules, kindly provided by J. Gordon, Birmingham, U.K.) and IgD (TA 4.1, IgG3; Downloaded from CA) equipped with a 15 mW krypton/argon laser, and attached to an BD Biosciences). This procedure efficiently enriched for GC B cells (95 Ϯ Eclipse E800 microscope (Nikon, Melville, NY) was used. 0.7% CD20ϩ;84Ϯ 5.2% CD20ϩCD38ϩ, and 1 Ϯ 0.7% CD3ϩ cells (mean value Ϯ SD)). CD4ϩCD45ROϩ tonsil T cells were purified by use of a RT-PCR analysis CD4 Positive Isolation kit (Dynal Biotech) followed by depletion of ϩ Tonsils were minced and lymphocytes were enriched using density gradi- CD45RA cells with pan-mouse IgG magnetic beads (Dynal Biotech) coated with a mAb to CD45RA (4KB5, IgG1; DAKO). The resulting cell ent centrifugation on Ficoll-Isopaque (Pharmacia Biotech, Uppsala, Swe- ϩ ϩ den). Purification of B cells was done by rosetting T cells (and other CD2ϩ population contained Ͼ96% CD4 CD45RO T cells. Purified cells were 6 cells, including monocytes and DCs) with neuraminidase-treated sheep cultured at 5 ϫ 10 /ml in 1 ml of complete medium only or with the http://www.jimmunol.org/ RBCs, rendering Ͼ98% B cells and Ͻ0.2% T cells as judged by FACS addition of 10 ng/ml PMA and 1 ␮g/ml ionomycin (both from Sigma- analysis of CD20 vs CD3 expression. B cell depletion was performed with Aldrich, St. Louis, MO). GC B cells were also stimulated with 2 ␮g/ml Pan Mouse IgG dynabeads (Dynal Biotech, Oslo, Norway) coated with anti-CD40 mAb (S2C6, IgG1; kindly provided by S. Pauli, Stockholm mouse anti-CD20 (BD PharMingen), yielding Ͼ85% T cells and Ͻ4% B University, Stockholm, Sweden). To block consumption of the IL-4 being cells. The fractionated cells were lyzed in TRIzol (Life Technologies, Pais- released from cultured cells, all cultures contained 1 ␮g/ml of a blocking ley, U.K.) either directly after isolation or after3hofculture in complete anti-IL-4R mAb (25463.1, IgG2a; R&D Systems). After4hofculture, medium consisting of RPMI 1640 supplemented with 10% FCS, 2 mM supernatants were removed and stored at Ϫ20¡C until further use. Corre- L-glutamine, nonessential amino acids, and 50 ␮g/ml gentamicin (all from sponding cells were thoroughly recovered, washed in PBS, and then lysed Life Technologies). Total RNA was extracted with chloroform, precipi- in 0.5 ml of lysis buffer for 30 min at 4¡C with gentle agitation. Lysis buffer contained PBS with 1% Triton X-100 (v/v), 1% BSA (w/v), 2 mM EDTA,

tated with isopropanol, washed in 75% (v/v) ethanol, and dissolved in by guest on September 25, 2021 ␮ diethylpyrocarbonate-treated water. The mRNA was converted into cDNA 5 g/ml aprotinin, 0.2 mM PMSF (Sigma-Aldrich), and 0.02% NaN3 (w/ from total RNA using Superscript II Rnase HϪ reverse transcriptase primed v). Lysates were centrifuged at 14,000 ϫ g at 4¡C for 15 min and supernatants were stored at Ϫ20¡C until use. by oligo(dT)12Ð18 (Life Technologies). Target cDNA was amplified by PCR using AmpliTaq DNA polymerase (PerkinElmer/Cetus, Norwalk, ECL-based detection of IL-4 CT) and a cDNA template corresponding to 150 and 15 ng of total RNA for IL-4 and GAPDH amplification, respectively. Primer sequences were as The amounts of IL-4 in culture supernatants and cell lysates were deter- follows: IL-4 forward, 5Ј-GCGATATCACCTTACAGGAG-3Ј; IL-4 reverse, mined with a double-Ab method, using an ORIGEN Analyzer (IGEN, 5Ј-CGAACACTTTGAATATTTCTCTCTCAT-3Ј; GAPDH forward, 5Ј-AT Gaithersburg, MD), which is based on an ECL detection technique (21). GGGGAAGGTGAAGGTCGGAGTCAACGGA-3Ј; and GAPDH reverse, For the ORIGEN analysis, sheep anti-mouse IgG-coated magnetic beads 5Ј-AGGGGGCAGAGATGATGACCCTTTTGGCTC-3Ј. Cycling conditions (Dynabeads M-280, final dilution of 1/80; Dynal Biotech) were mixed with were 94¡C for 1 min, 55¡C for 1 min, and 72¡C for 2 min. PCR products were anti-IL-4 mAb (8D4-8, IgG1; BD PharMingen) at 50 ng/ml, biotinylated visualized by agarose gel electrophoresis with ethidium bromide. goat anti-IL-4 (polyclonal; R&D Systems) at 250 ng/ml, and 300 ng/ml of streptavidin, labeled with ruthenium (II) Tris-bipyridine chelate (TAG; FACS analysis and sorting IGEN) according to the manufacturer’s recommendations. Dilution buffer Tissue-freed tonsil cells were cultured in complete medium for 3 h with 10 used for these reagents was PBS supplemented with 1% Triton X-100 ␮ (v/v), 1% BSA (w/v), and 5% goat serum (v/v). After 30 min of incubation, g/ml brefeldin A added during the last 2 h. Cells were stained with Abs ␮ ␮ to cell surface Ags, fixed in 2% paraformaldehyde, permeabilized with 100 l of this reaction mixture was added to 125 l of each cell lysate or PBS containing 0.2% saponin and 2% FCS, and stained with mouse mAbs precleared culture supernatant (see below) in a 96-well polypropylene mi- against intracellular proteins, including PE-labeled anti-IL-4 (8D4-8, IgG1; croplate (Costar, Cambridge, MA) and further incubated for 2 h with con- BD PharMingen) in the presence of saponin. PECy5-labeled mouse anti- tinuous agitation at room temperature. Magnetic bead-associated lumines- CD3 (UCHT1, IgG1; DAKO) was used in combination with the following cence was thereafter determined with the ORIGEN analyzer. ECL signals FITC-conjugated mouse mAbs: anti-CD20 (B-Ly1, IgG1), anti-CD19 derived from cell culture supernatants and cell lysates were compared with (HD37, IgG1), anti-CD21 (1F8, IgG1), anti-CD79␤ (SN8, IgG1), anti- ECL signals obtained with a serial dilution of a human IL-4 standard (En- Bcl-2 (124, IgG1) (all from DAKO); anti-CD40 (5C3, IgG1) and anti- dogen, Woburn, MA) calibrated against World Health Organization IL-4 CD38 (HIT2, IgG1) (BD PharMingen); anti-HLA-DR (L243, IgG2a; BD reference standard (lot no. 88/656). For quantification of IL-4 in culture Biosciences, San Jose, CA); anti-Ki67 (Ki-67, IgG1; Boehringer Mann- supernatants and cell lysates, the IL-4 standard was diluted in complete medium and lysis buffer, respectively. The useful linear range spanned heim); and rat anti-CD77 (38.13, IgM; Immunotech, Marseille, France) Ͼ revealed by FITC-labeled mouse anti-rat IgM (G53-238, IgG1; BD Phar- from 3 to 2000 pg/ml. Because it was determined in initial experiments Mingen). A mix of FITC-labeled anti-␬ and anti-␭ (polyclonal rabbit that mAbs added to cultures to some degree interfered with the detection of F(abЈ) ; DAKO) was used for detection of L chain. Cells were analyzed on IL-4, probably by competing with the anti-IL-4 mAb for binding to the 2 sheep anti-mouse IgG-coated magnetic beads, supernatants were pre- a FACScan (BD Biosciences). Apoptosis was analyzed based on DNA ␮ fragmentation measured by TUNEL (Boehringer Mannheim) according to cleared with 20 l of the beads before being analyzed. This procedure the manufacturer’s instructions. In some experiments, cells were concom- completely prevented such interference. itantly analyzed for expression of CD10 or IL-4 using PE-labeled mouse anti-CD10 (SS2/36, IgG1; DAKO) and anti-IL-4. Staining with PECy5- Results conjugated anti-CD3 was done to electronically exclude T cells from col- To identify the nature of the cell(s) that could provide IL-4 in Th2 lected events. Cell surface staining was done previous to fixation and development in human tonsil, cryostat sections were analyzed by The Journal of Immunology 3167 immunofluorescence, using enzyme-catalyzed amplification of the approach, was confirmed by using a polyclonal anti-IL-4 prepara- IL-4-dependent signal. First, the anti-IL-4 mAb did not detect IL-4 tion instead, which indeed bound to the receptor-associated cyto- bound to cognate receptors, as ruled out by the inability to detect kine (data not shown). Second, the specificity of the IL-4 detection rIL-4 loaded onto IL-4R-bearing THP-1 cells (a human monocyte- was confirmed by the complete absence of fluorescence when the derived cell line), using flow cytometry. The stringency of this mAb was preincubated with rIL-4 (Fig. 1, a and b). Double label- Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. In situ IL-4 expression in human tonsil and lymphoid areas of colon mucosa is confined to GCs where B cells constitute the predominant cellular source. a, Anti-CD3 (red) and anti-IL-4 (green) immunofluorescent double labeling of human tonsil reveal a B cell follicle-restricted IL-4 expression. b, Preincubation of the anti-IL-4 mAb with rIL-4 completely abrogates IL-4-dependent fluorescence in an adjacent section, demonstrating the specific staining of IL-4-expressing cells. c, Higher magnification of B cell follicles reveals a few IL-4-expressing CD3ϩ T cells (arrows), whereas the vast majority of IL-4ϩ cells are non-T cells. d, Simultaneous anti-IgD (red) and anti-IL-4 (green) labeling establish that IL-4 is exclusively produced within the GCs of tonsil. eÐg, Triple labeling with anti-CD3 (blue), anti-IL-4 (green), and anti-CD20 (red) demonstrates overlapping immunoreactivity of anti-CD20 and anti-IL-4 in GCs of tonsil (e) and, at higher magnification (f), the CD20ϩ B cell identity of IL-4-producing non-T cells (arrows), of which several are in close proximity to T cells (arrowheads). Identical expression pattern is visualized in GCs of colon mucosa (g). hÐi, In some, often small-sized GCs, triple labeling with anti-CD3 (blue), anti-IL-4 (green), and anti-Ki67 (red) identifies a preferential localization of CD3ϪIL-4ϩ cells in the dark zone defined by proliferating centroblasts exhibiting nuclear staining of Ki67 (h), whereas, at higher magnification (i), IL-4 is visualized in both Ki67ϩ cells (arrows) and cells lacking anti-Ki67 reactivity (arrowhead). DZ, dark zone; LZ, light zone. Tonsil stainings are representative of five different donors. Tissue samples of colon from two donors were analyzed and yielded similar results. 3168 GC-RESTRICTED B CELL PRODUCTION OF IL-4

but the vast majority of IL-4ϩ cells did not express the T cell marker (Fig. 1c). Double labeling with anti-IgD and anti-IL-4 revealed a GC-restricted expression of the cytokine with no anti-IL-4 reactivity visualized in the mantle zone (Fig. 1d) or in primary follicles (data not shown). Three-color immunofluorescence using anti-CD20, anti-CD3, and anti-IL-4 identified CD20ϩ GC B cells as the major cellular source of IL-4 (Fig. 1, eÐg). In some of the GCs being examined, IL-4-expressing B cells were predominantly localized in the dark zone, defined by costaining of the proliferation-associated nuclear Ag Ki67 (Fig. 1h). However, IL-4 was visualized in both Ki67-positive and -negative cells (Fig. 1i), and most of the large GCs generally did not display such a clear bound- ary regarding IL-4 production. Finally, B cells expressing IL-4 were also identified in GCs in lymphoid areas of colon mucosa (Fig. 1g). Thus, at least in mucosa-associated lymphoid tissues, IL-4-expressing GC B cells appear to be a more general FIGURE 2. B cells contain lower levels of IL-4 transcripts compared with T cells. The mRNA from total tissue-freed tonsil cells (top), T cell- phenomenon. To confirm IL-4 expression in B cells at the level of mRNA enriched fraction (middle), or purified B cells (bottom) were prepared di- Downloaded from rectly after isolation (0 h) and after3hofculture and were subjected to transcripts, freshly isolated tonsil cells were processed for RT-PCR semiquantitative RT-PCR analysis using 32, 36, and 40 cycles of cDNA analysis as a bulk population, as a B cell-depleted fraction (Ͼ85% amplification with IL-4-specific primers. cDNA corresponding to equal T cells), or as purified B cells (Ͼ98% CD20ϩ cells, Ͻ0.2% CD3ϩ amounts of total RNA was used in each reaction and quantities were back- cells). Semiquantitative RT-PCR performed with RNA derived checked by amplification of GAPDH target cDNA at 32 cycles. Flow cy- from these cell preparations demonstrated IL-4 transcripts in B tometric analysis of CD20 vs CD3 expression for corresponding cell prep- cells, although at a lower level compared with T cells (Fig. 2). This arations is shown to the left. http://www.jimmunol.org/ was also true for cells cultured for 3 h, an in vitro procedure later used for IL-4 protein measurement at the single cell level (see ing with mAbs to CD3 and IL-4 revealed a highly anatomically below), and is in agreement with the relatively low level of secre- ordered expression pattern with almost all IL-4 localized to the tion of IL-4 from murine B cells, which can be induced by polar- follicles (Fig. 1a). Virtually no IL-4ϩ cells could be seen in the T ized Th2 cells (14). cell areas or in the reticulated epithelium of the crypts, a tonsil area Next, we applied flow cytometry to characterize IL-4-expressing in some aspects resembling the marginal zone in spleen (2). Ton- B cells at the single cell level. To this end, crude cell preparations sillar GC T cells previously have been reported to express IL-4 from tonsils were cultured for1htoallow the cells to recover after ϩ ϩ (22) and CD3 IL-4 cells were readily identified within follicles, the isolation procedure and for an additional2hinthepresence of by guest on September 25, 2021

FIGURE 3. IL-4 expression in tonsil cells in vitro is more frequent among B cells than T cells and is restricted to B cells exhibiting a GC B cell phenotype. Tissue-freed cells from tonsil were cultured for3hincomplete medium with brefeldin A (10 ␮g/ml) added during the last 2 h and were subsequently processed for three-color FACS analysis of intracellular IL-4 content in relation to marker protein expression. a, Majority of IL-4-producing cells do not express the T cell marker CD3. b, Preincubation of the anti-IL-4 mAb with rIL-4 (20 ␮g/ml) abrogates IL-4 staining. c, Phenotypic analysis of IL-4ϩCD3Ϫ cell with FITC-conjugated mAbs to indicated proteins. CD3ϩ cells have been excluded from data by electronic gating. The Journal of Immunology 3169 brefeldin A, imposing intracellular retention and accumulation of period of secretion, cells were cultured at a relatively high density otherwise secreted proteins. This period of time in culture did not of 5 ϫ 106 cells/ml. Low amounts of the cytokine could indeed be dramatically alter the levels of IL-4 transcripts in B and T cells, detected in supernatants from such short-term cultures, and an in- respectively (Fig. 2). In agreement with in situ observations, IL-4 crease in concentration was observed if an antagonizing mAb to production was more frequent among CD3Ϫ cells than CD3ϩ T the IL-4R was included at the beginning of cultivation (Fig. 6a). cells (20 Ϯ 7.2% vs 3.1 Ϯ 1.1%; mean value Ϯ SD, n ϭ 7) (Fig. Next, we compared purified GC B cells and the tonsillar 3, a and b). Three-color FACS analysis allowed CD3Ϫ cells to be CD4ϩCD45ROϩ T cells (memory Th cells) for their ability to electronically gated and further analyzed for marker expression in release IL-4. Fig. 6b summarizes the levels being released by GC relation to intracellular IL-4 content (Fig. 3c). CD3Ϫ IL-4ϩ cells B cells from three separate donors and by memory Th cells from displayed a characteristic GC B cell phenotype (1, 23) expressing two of these donors, respectively. Whereas GC B cells and mem- CD20, CD38, CD10 (data not shown), CD40, and HLA-DR and ory Th cells spontaneously secreted similar amounts, polyclonal staining negative for the intracellular anti-apoptotic protein Bcl-2, activation with PMA and ionomycin did not have any significant which is typically absent in GC B cells. In addition, the presence effect on the IL-4 production from any of the cell types during this of CD77 on most of the cells and a low level of surface Igs (i.e., short period of stimulation. Importantly, although released levels CD79␤, L chain (Fig. 3c), and IgD (data not shown)) indicate a cen- of the cytokine were measured for cells cultured for 4 h only, even troblast origin of the majority of IL-4-expressing GC B cells (23). this short period of time rendered a large fraction of the GC B cells The phenotype of the IL-4-producing cells that is presented in apoptotic, whereas the T cells remained viable (Fig. 6c). As shown Fig. 3, however, does reveal a slight deviation from the GC B cell in Fig. 6c, despite the fact that PMA and ionomycin and, to some phenotype, because the densities of several of the analyzed mark- degree, anti-CD40 stimulation could prevent the apoptotic cell Downloaded from ers are lower than corresponding levels on the IL-4-negative B death of cultured B cells, the increase in viability was not accom- cells. For example, when freshly isolated, GC B cells express the panied with a sustained production of IL-4 (see Fig. 6b). These highest levels of CD20 among the tonsil B cells, yet the IL-4ϩ reagents also failed to increase the concentration of IL-4 in super- cells shown in Fig. 3c display only a moderate density of this natants from GC B cell cultured for 16 h, in which the levels of particular protein. The explanation for this is that GC B cells IL-4 actually remained unchanged compared with supernatants quickly undergo apoptosis in culture and, because of exocytosis of taken after 4 h (data not shown). Furthermore, cell lysates from http://www.jimmunol.org/ apoptotic bodies, lose many of the membrane-associated proteins cultured GC B cell did not contain more IL-4 than lysates derived (24). The apoptotic progression of cultured IL-4-producing B cells was also confirmed by analysis of DNA fragmentation, using the TUNEL method (25) (Fig. 4). Freshly isolated cells, however, did not exhibit any detectable sign of nuclear fragmentation, and IL- 4-positive cells in situ also were not preferentially TUNEL positive (Fig. 5, a and b). We also sorted GC B cells by flow cytometry for their ability to bind annexin V (Fig. 5, c and d) because this property identifies apoptotic cells at an early stage of the apoptotic by guest on September 25, 2021 process (26). No major difference in mRNA content for IL-4 could be detected in the two fractions of GC B cells, which were divided on the basis of their annexin V binding capacity (Fig. 5e). Different amounts of IL-4 transcripts instead could be visualized if GC B cells were compared with the sorted fraction of non-GC B cells. Based on these experiments, we conclude that GC B cells with an IL-4-producing phenotype are not preferentially apoptotic in vivo or directly after isolation, but upon culture they initiate the apoptotic process, a feature which is indeed an important hallmark for GC B cells (20). GC B cells producing IL-4 thus rapidly die in culture. In an attempt to detect secreted IL-4, we therefore measured the amounts being spontaneously produced from purified GC B cells during a short period of only4hinculture. To compensate for this brief

FIGURE 5. Induction of apoptosis is not a prerequisite for IL-4 expression in GC B cells. a and b, Simultaneous in situ detection of IL-4 (red) and apotosis by the TUNEL method (green) demonstrate the presence of IL-4 expression in both apoptotic (arrows) and nonapoptotic (arrowheads) cells within a GC of tonsil. The separate image of TUNEL-mediated fluorescence is shown (a) to facilitate interpretation of the merged image (b). c, Isolated and T cell-depleted tonsil cells were incubated with anti-CD19 PECy5 and anti-CD38 PE mAbs and sorted by FACS into CD19ϩCD38ϩ GC B cells and CD19ϩCD38Ϫ non-GC B cells. d, GC B cells were stained with annexin V-FITC and subjected to further fractionation by FACS into FIGURE 4. IL-4 synthesis and apoptotic progression in GC B cells are an annexin VϪ fraction (black histogram) and into a fraction being en- concurrent events in vitro. Tissue-freed cells from tonsil were cultured for riched for annexin V binding cells (red histogram). e, mRNA was prepared 3 h in complete medium with brefeldin A (10 ␮g/ml) added during the last from the sorted cell populations in c and d and was analyzed by RT-PCR 2 h and were analyzed by FACS for apoptosis by TUNEL (FITC) con- with specific primers for IL-4. cDNA corresponding to equal amounts of comitantly to anti-CD3 PECy5 and anti-CD10 PE or anti-IL-4 PE staining, total RNA were used in each PCR, and quantities were back-checked by as indicated. Only CD3-negative events are shown. amplification of GAPDH target cDNA. 3170 GC-RESTRICTED B CELL PRODUCTION OF IL-4 Downloaded from http://www.jimmunol.org/

FIGURE 6. Purified GC B cells and CD4/CD45RO Th cells spontaneously produce and release similar amounts of IL-4 in short-term cultures. a, Freshly purified GC B cells (5 ϫ 106 cells/ml) were cultured in triplicate for 4 h with or without a neutralizing (blocking) mAb to the IL-4R, and supernatants were thereafter analyzed for IL-4. Bars represent mean values Ϯ SD for triplicate cultures. bÐd, Freshly purified GC B cells and CD4/CD45RO memory Th cells (both at 5 ϫ 106 cells/ml) were cultured in the presence of anti-IL-4R mAb, with or without PMA and ionomycin or anti-CD40 mAb, as indicated. by guest on September 25, 2021 Supernatants and cells were separately harvested after 4 h. IL-4 concentrations of supernatants were analyzed, and results obtained with cells from three separate donors are shown (b). Apoptotic progression of cultured GC B cells (upper panel) vs memory Th cells (lower panel) were measured by the TUNEL method (c). Cell lysates were prepared from cultured cells and analyzed for the presence of IL-4. Mean value Ϯ SD for triplicate cultures is shown (d). e, The relative proportions of the CD3ϩCD4ϩCD45ROϩ Th cells and the CD20ϩCD38ϩ GC B cells, respectively, among total tonsil leukocytes obtained from a typical tonsil specimen.

from the memory T cells, irrespective of under which conditions lineate mechanisms that drive Th2 development in vivo, identifi- cells were cultured (Fig. 6d). The significance of this finding is that cation of cells capable of providing an initial burst of IL-4 still B cells spontaneously release IL-4 and hence that concentrations in remains an important issue. This led us to investigate the cellular culture supernatants most likely reflect actual synthesis of the cy- sources of IL-4 in the human tonsil, and in this report we describe tokine. Although GC B cells and memory T cells accordingly pro- the identification of IL-4 production in GC B cells as a novel duce similar amounts of IL-4 in these cultures, it is important to cellular source of IL-4 in vivo. emphasize that secretion of IL-4 was measured using an equal Irrespective of which method is used for detection, the B cells number of B cells and T cells, respectively. In vivo, the tonsil consistently display a comparatively high production of IL-4 pro- generally contains more GC B cells than memory Th cells (Fig. tein, in terms of both fraction of cells producing the cytokine and 6e). In addition, the high incidence of cell death among cultured total quantity being produced. In contrast, the B cells clearly con- GC B cells clearly makes it difficult to compare levels of IL-4 tain less mRNA for IL-4 compared with the T cells. This imbal- being released in vitro to the in vivo situation. Although PMA/ ance suggests separate mechanisms for regulation of IL-4 synthe- ionomycin or the anti-CD40 mAb increased the viability of cul- sis in B and T cells, respectively. Inhibition of IL-4 production at tured cells, these entities may not be representative for the entire the posttranscriptional level, for example, is accomplished in mu- panel of survival and growth factors delivered, for example, by rine T cells by neuropeptides present in lymphoid organs (29) follicular DCs and activated T cells in vivo (3, 4, 27), which per- Our results support the interpretation that IL-4 is produced in B haps also can sustain the production of IL-4. cells only during their Ag-dependent differentiation within GCs because 1) in situ, IL-4 can be visualized virtually only within Discussion GCs, 2) ex vivo, IL-4 transcripts are enriched in the GC fraction of Although it appears that Th2 differentiation may proceed in an sorted B cells, 3) in vitro, IL-4-producing non-T cells homoge- IL-4-independent manner (28), the efficiency and magnitude of neously display an appropriate GC B cell phenotype, and 4) the polarization are severely reduced in IL-4 knockout mice or in mice intrinsic nature of GC B cells to rapidly undergo apoptosis when rendered deficient in IL-4R signaling (15, 16). Therefore, to de- being cultured (20) applies to the majority of IL-4-producing B The Journal of Immunology 3171 cells. Furthermore, CD40 ligation, most likely accounting for a ␥-dependent Ig isotypes, such as IgG2a, in the mouse. These re- decisive role in the final differentiation stage of centrocytes in vivo sponses are predominated by Th1 cells, and the development of (30, 31), appears to reduce the synthesis of IL-4 in cultured GC B GCs is probably driven by T cells being strongly influenced by, cells because the CD40-mediated increase in viability is not ac- e.g., IL-12 during their initial priming within the T cell zone (7, companied by an increase in IL-4 production. This may lend ev- 37). Furthermore, using an adoptive transfer system, it was re- idence to the idea that Th cells abort IL-4 production in GC B cells cently demonstrated by Smith et al. (38) that Ag-specific Th1 and when these differentiate into memory cells. Clearly, production of Th2 cells are equally capable of supporting B cell clonal expansion IL-4 is most frequent among the CD77ϩ centroblasts as judged by and Ab production in vivo. FACS analysis, indicating that light zone-associated events indeed The T cells residing within GCs of human tonsil have recently may be responsible for turning off the synthesis of IL-4 in their regained attention (39). This separate population of memory Th progeny. However, it is difficult to assess the precise function of cells, which appears to be specialized in providing help for the GC CD40 signaling in this context because of the property of the B cells, differs from other memory Th cells by its expression of CD40 pathway to both interfere with the apoptotic progression and CD57 as originally demonstrated by Poppema et al. (40) and re- simultaneously induce further B cell differentiation beyond the GC cently confirmed by Kim et al. (39). Even though they strongly state. That IL-4 is produced in GC B cells as a consequence of support Ig secretion from cultured GC B cells (39), they are, in apoptotic events, however, is unlikely because most IL-4-produc- contrast to their CD57ϪCD45ROϩ counterparts, poor in eliciting ing cells do not exhibit nuclear fragmentation in situ, nor are IL-4 responses from peripheral B cells (41). Accordingly, counteracting transcripts enriched in the annexin V-positive fraction of ex vivo- apoptotic cell death may be involved as an important mechanism analyzed GC B cells. for the helper activity of the GC T cells (20). In addition, in con- Downloaded from Because priming of naive Th cells appears to necessarily take trast to the extrafollicular subset of CD45ROϩ Th cells, the GC Th place on interdigitating DCs (IDCs) in the T cell zone (2), we cells produce large amounts of IL-10 (39), a cytokine that in the expected IL-4 to be produced in this area. Murine DCs have now presence of follicular DCs comprehensively promotes terminal dif- been identified as a potential source of IL-4 (12), and secretion of ferentiation of GC B cells into plasma cells (4, 42). IL-4 together IL-4 from IDCs would indeed provide a route for Th2 develop- with CD40 ligand instead favors the continuous differentiation ment in vivo. Detectable IL-4 production, however, was highly from centroblats via centrocytes to memory B cells (3, 4, 43). http://www.jimmunol.org/ restricted to the GCs in all donors analyzed by in situ immunoflu- Therefore, the comparable levels of IL-4 being released ex vivo by orescence (Fig. 1). Very few IL-4-producing cells were observed the GC B cells themselves and by the tonsillar memory Th cells in T cell areas, and this limited number of cells apparently did not suggest that memory development may constitute more of a de- include IDCs (defined as CD40ϩ and/or CD83ϩ cells intervening fault pathway for GC B cells in vivo, whereas plasma cell differ- within the dense T cell accumulations; data not shown), although entiation also requires IL-10 provided by the T cells. we cannot exclude that expression of IL-4 below the detection In this study, we focused on the predominant source of IL-4 in limit takes place in cells within the T cell zone. However, taking tonsils and, therefore, have not in greater detail studied the cyto- this into consideration, it is clear that GCs account for the absolute kine profile of the different subsets of T cells. Similar to B cells, predominant production of IL-4 in the tonsils, and a similar dis- IL-4-producing T cells appear to be mostly contained within GCs, by guest on September 25, 2021 tribution of IL-4-producing cells was also observed at inductive as we demonstrated in situ using immunofluorescence (Fig. 1). An sites of colon lamina propria. Therefore, we propose that GCs enrichment of IL-4-producing T cells within GCs is also in line provide the most favorable microenvironment for Th2 develop- with the results published by Butch et al. (22) and by Toellner et ment in these mucosa-associated lymphoid tissues. Considering al. (44), but was not clearly evident from the work by Kim et al. the prominent role IL-4 seems to play in mucosa-associated im- (39). However, in the latter report, the investigators used PMA/ munity compared with systemic immunity (32), it would be inter- ionomycin stimulation to induce secretion of IL-4 from T cells. esting to also investigate IL-4 production in the human spleen and Extended in vitro polyclonal activation may induce IL-4 expres- lymph nodes. sion in memory Th cells, which in vivo are relatively quiescent. Except for T and B cells, additional subsets of leukocytes are Furthermore, we feel that, in the chronically infected tonsil, it may indeed capable of producing IL-4. Eosinophiles and basophiles can not be appropriate to consider the cytokine profile of the GC T cell produce IL-4 and, under certain circumstances, perhaps support a subset as a criteria for how the cytokine microenvironment of the peripheral outgrowth of Th2 cells (9). An essential role for these follicles may influence the T cell polarization process. First, ex- cells in Th2 polarization, however, is unlikely, mainly because of trafollicular priming of T cells, preceding the migration toward an inappropriate anatomical localization. Rather than being present follicles (45, 46), may have influenced the acquisition of a certain in secondary lymphoid organs during early inductive events of cytokine-secreting phenotype of T cells within GCs. Second, al- immunity, they are recruited into inflamed target tissues, partially though the CD57ϩ GC T cells appear to be a resident population, by the action of preexisting Th2 cells (7). On the contrary, physical other Th cells may enter the B cell follicles and then migrate to- interaction between Ag-specific T and B cells take place within the ward chemokines produced in inflamed tissues (47Ð49). After an developing GC only a few days after primary immunization (33) initial expansion, a decrease in the number of T cells within the and, at this time point, a mutual agonistic outcome of such cellular growing GCs has indeed been reported to occur (45). Nevertheless, collaboration is evident by a shift from extra- to intrafollicular T production of high levels of IL-10 from the CD57ϩ GC T cells is cell proliferation (34). Although it has generally been believed that not in conflict with our suggestion of a GC B cell-mediated Th2 GC formation depends on Th2 cells, the initial T and B cell en- polarization (7). counter takes place within a time period that probably is not suf- In conclusion, our findings and supporting results provided by ficiently long to allow development of fully committed Th2 cells Harris et al. (14) of IL-4-producing B cells indeed being capable of (35, 36). 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