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Plasma cell differentiation in T-independent type 2 immune responses is independent of CD11chigh dendritic cells Katrin Hebel1, Klaus Griewank*1, Ayako Inamine1, Hyun-Dong Chang1, Brigitte Mller-Hilke2, Simon Fillatreau1, Rudolf A. Manz1, Andreas Radbruch**,§1 and Steffen Jung§3 1 2 3
German Arthritis Research Center Berlin, Berlin, Germany University of Rostock, Rostock, Germany Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel
Dendritic cells (DC) play an important role as antigen-presenting cells in T cell stimulation. Interestingly, a number of recent studies also imply DC as critical accessory cells in B cell activation, isotype switching and plasma blast maintenance. Here we use the conditional in vivo ablation of CD11chigh DC to investigate the role of these cells in Tindependent type 2 immune responses. We show that CD11chigh DC are dispensable for the initiation and maintenance of a primary immune response against the Tindependent type 2 antigen (4-hydroxy-3-nirophenyl)acetyl-Ficoll. Our results suggest that support for plasma cell formation in T cell-independent immune responses can be provided by non-DC such as stromal cells, or is independent of external signals. Interestingly, we found plasma blasts to express CD11c and to be diphtheria toxinsensitive in CD11c-diphtheria toxin receptor-transgenic mice, providing a unique tool for future analysis of in vivo aspects of plasma cell biology.
Received 7/6/06 Revised 14/8/06 Accepted 7/9/06 [DOI 10.1002/eji.200636356]
Key words: Dendritic cells (4-Hydroxy-3-nitrophenyl)acetyl-Ficoll Plasma cells T-independent type 2
Supporting information for this article is available at http://www.wiley-vch.de/contents/jc_2040/2006/36356_s.pdf
Introduction Dendritic cells (DC) are specialized mononuclear phagocytes that are believed to have co-evolved with adaptive immunity. Accordingly, DC are unrivaled in their potential to act as antigen-presenting cells (APC) in
§
Both senior authors contributed equally to the work
Correspondence: Steffen Jung, PhD, Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel Fax: +972-8-934-4141 e-mail:
[email protected] Abbreviations: AFC: antibody-forming cell AP: alkaline phosphatase B6: C57BL/6 DTR: diphtheria toxin receptor DTx: diphtheria toxin MZ: marginal zone NP: (4-hydroxy-3nitrophenyl)acetyl PC: plasma cell TI: T cell-independent f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the stimulation of naive T cells [1, 2]. Furthermore, DC also contribute critically to various mechanisms that ensure T cell tolerance [3]. The role of DC in B cell physiology remains less well understood. B cell responses can been subdivided according to the class of the eliciting antigen into T cell-dependent responses that require T cell help, and responses to T cell-independent (TI) antigens that stimulate antibody production in the apparent absence
* Current address: Department of Pathology, University of Chicago, IL 60637, USA ** Additional correspondence: Andreas Radbruch, German Arthritis Research Center Berlin, Schumannstrasse 20/21, 10117 Berlin, Germany Fax: +49-30-28460-603 e-mail:
[email protected] www.eji-journal.eu
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of T cell help [4, 5]. TI responses themselves can be of polyclonal nature, such as the responses stimulated by LPS (TI-1 antigens), or specific for repeated haptens of complex immunogens, as in the case of repetitive viral epitopes (TI-2 antigens). Anti-viral TI-2 responses have been shown to contribute critically to the generation of neutralizing antibodies against viral glycoproteins [6]. However, it remains a matter of debate, whether mere B cell receptor cross-linking is indeed sufficient to trigger the antigenspecific immune reaction or whether in vivo responses require additional signals provided by accessory cells [7, 8]. TI-2 responses involve marginal zone (MZ) or B1 B lymphocytes [9, 10] and take place in extrafollicular foci, where exponential growth leads to plasma cell (PC) production [11]. During the response, B cells move through various microenvironments and developmental stages before they finally end up as PC in the red pulp [12, 13]. TI-2 responses occur independent of T cellB cell contact and CD40-CD40L ligation [14, 15]. DC have been implied in antigen delivery and presentation to B cells. Thus, not all antigens that are ingested by DC are processed into peptides, but a fraction can be regurgitated in its native form [16, 17]. Furthermore, more recently, Bergtold et al. [18] proposed that mature DC retain antigen in its native state via the FccRIIB receptor and that efficient TI-2 responses require immune complexes presented by DC. Moreover, circulating CD11clow DC were shown to capture blood-borne TI-2 antigens and to provide critical survival signals to antigen-specific MZ B cells, promoting their differentiation into IgM-secreting plasma blasts [19]. TI-2 responses result in a characteristic directed Ig class switch and secretion of IgG3. Interestingly, in an in vitro model this c3 switch of activated B cells was supported by DC and has been proposed to depend on BAFF production by the latter [20]. Finally, a number of studies indicate that DC might play a role in the process of B cell differentiation into antibody-forming cells (AFC). Vinuesa et al. have shown that clustered CD11chigh CD8– DC [21] and IgM plasma blasts co-localize after TI and T cell-dependent immunization in the spleen and lymph nodes [11]. The authors reasoned that this intimate B-DC contact could be of functional importance for plasma blast maintenance, since an excess of plasma blasts over DC resulted in apoptosis of surplus plasma blasts. The study of TI immune responses enables to examine the contribution of DC in B cell activation and PC generation, without consideration of the DC impact on the generation of T cell help. Here we investigate the role of CD11chigh DC in the in vivo response of B cells to the TI-2 antigen (4-hydroxy-3nirophenyl)acetyl (NP)-Ficoll using a conditional CD11chigh DC ablation strategy [2]. By depleting f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Cellular immune response
CD11chigh DC in the early or late phase of the primary TI-2 response, we tested whether CD11chigh DC act as APC or provide differentiation/survival signals for the expanding splenic plasma blasts. We show that CD11chigh DC are dispensable for a primary TI-2 response, as well as for the switch to IgG3.
Results and discussion NP-specific antibody-forming cells co-localize with CD11chigh DC To investigate the contribution of CD11chigh DC in the process of plasma blast formation and maintenance during the primary TI-2 immune response, we chose to analyze mice that were challenged with NP-Ficoll in the presence or absence of CD11chigh DC. Analysis of the immunized BALB/c wild-type (WT) mice by ELISA and ELISPOT indicated that the peak number of NP6-specific IgM- and IgG (IgG3)-secreting AFC was reached 5 days after the NP-Ficoll injection ([11] and data not shown). To test for co-localization of plasma blasts and CD11chigh DC, spleens were recovered from C57BL/6 (B6) mice 3, 4, 5, and 6 days following injection with NP-Ficoll and analyzed by immuno-fluorescence microscopy. On the third day following immunization, Igk+ plasma blasts that specifically arise in response to the NP challenge [23, 24] were found in close contact with the CD11chigh DC. Clusters began to dissociate in the junction zones on the fifth day in accordance with published data (Fig. 1A, B) [22]. We hence chose to ablate the CD11chigh DC during this time window. Ablation of CD11chigh DC from the junction zones 24 h after diphtheria toxin (DTx) treatment of the CD11cDTx receptor (DTR)-Tg mice was confirmed by immunohistochemistry (Fig. 1C, D).
Plasma cells express the CD11c integrin aX subunit and are DTx-sensitive in CD11c-DTR-Tg mice Expression of the DTR gene in CD11c-DTR-Tg mice is driven by a 5.5-kb DNA fragment of the Itgax (CD11c) gene [25]. Murine CD11c expression (and potential activity of 5.5-kb promoter/enhancer element) is found in all DC (except for CD11clow–neg immature Langerhans' cells), but has also been reported for certain macrophages [26–28], activated T cells [29] and NK cells [30]. Not all of these cells are sensitive to ablation in CD11cDTR-Tg mice (line 57) (Jung et al., unpublished observations). CD11c expression has not been reported for murine plasma blasts so far.
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Figure 1. k+, NP-specific splenic plasma blasts co-localize with CD11chigh DC. Immunohistochemical analysis of frozen spleen sections of NP-Ficoll-immunized B6 mice on day 3 (A) and day 5 (B) and from CD11c-DTR-Tg mice without (C) or 12 h after DTx treatment (D). Sections were stained for CD11c (red) and Igk (green) (A, B) or for B220 (green), CD4 (red) and CD11c (blue) (C, D). Representative images from three mice each are shown. Original magnification 200 (A, B), 100 (C, D).
However, several reports suggest that activated human B cells can up-regulate expression of the aX integrin subunit [31, 32]. We therefore immunized WT and CD11c-DTR-Tg mice with NP-keyhole limpet hemocyanin/alum and analyzed the splenic CD138 (Syn-1+) cells on day 6 for expression of the DTR/GFP transgene. We found that a considerable fraction of in vivo generated CD138+ cells, i.e. plasma blasts [33], expressed the CD11c promoter-driven DTR/GFP transgene (Fig. 2A). Importantly, when we sorted the cells and performed ELISPOT assays we found that the GFP+ CD138+ cell fraction was significantly enriched in antibody-secreting cells. This suggests that CD11c promoter activity can serve as marker for AFC among CD138+ cells (Fig. 2B). Also the analysis of in vitro generated CD11c-DTR-Tg CD138+ plasma blast cells revealed a subpopulation of GFP-expressing cells, which again exclusively represented the AFC fraction (Fig. 2C). We persistently failed to establish a surface staining for CD11c for plasma blasts (data not shown). To determine whether CD11c promoter-driven GFP expression of CD138+ plasma blasts corresponded with transcriptional activity of the endogenous CD11c locus, we performed an reverse transcription PCR assay on sorted CD138+ GFP+, CD138+ GFP–, and CD138– GFP– cell fractions from CD11c-DTR-Tg mice. CD11c mRNA was significantly enriched in the CD138+ GFP+ cell fraction, as compared to the GFP– B cell fractions f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(Fig. 2D). Taken together these results suggest that murine plasma blasts express the aX integrin subunit CD11c, and that CD11c promoter activity is a marker for AFC among in vivo and in vitro generated CD138+ plasma blasts. Finally, we tested the DTx sensitivity of CD11c-DTRTg plasma blasts using a chimeric system. B cell-deficient JH–/– recipient mice [34] were lethally irradiated and reconstituted with a 1:1 mixture of bone marrow (BM) cells of CD11c-DTR (Ighb) and BM cells of B6.Cg (Igha) mice (Fig. 2E). The B cell compartment of the resulting chimeras was composed of 75% B cells expressing surface IgMb and 25% B cells expressing IgMa (data not shown). Also their humoral response to NP-Ficoll challenge was dominated by serum IgM of the b haplotype. Importantly, the DTx treatment completely abrogated the response of the CD11c-DTR-Tg B cells (Ighb), while concentrations of specific IgMa serum antibodies derived from WT plasma blasts were increased (Fig. 2E). Taken together, in vitro and in vivo generated murine plasma blasts express the CD11c integrin aX subunit. As a result, DTx treatment of CD11c-DTR-Tg mice results in direct in vivo ablation of CD11c-DTR-Tg plasma blasts. This conditional ablation system thus might provide a unique tool to analyze in vivo aspects of PC biology.
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Cellular immune response
Figure 2. Plasma blasts of CD11c-DTR-Tg mice express the CD11c and the DTR/GFP transgene. (A) Ex vivo CD138+ spleen cells of WT B6 and CD11c-DTR/GFP-Tg mice 6 days after immunization with NP-keyhole limpet hemocyanin in alum; n=4 mice. (B) Ex vivo fractions were sorted and tested for Ig secretion. (C) A 3-day LPS-stimulated B cell culture gated on CD138+ cells; fractions were sorted and tested for Ig secretion. (D) Expression levels of CD11c mRNA in the sorted subpopulations. The light cycler real-time PCR run was performed in duplicates and HPRT served as a reference gene. (E) BM chimeras that contained B cells from CD11c-DTR-Tg mice and B6.Cg WT mice were generated by reconstituting B cell-deficient JH–/– mice with 50% BM of CD11c-DTR-Tg (Ighb) and 50% BM of B6.Cg (Igha) mice; n=4–5 mice, *p=0.016. One representative experiment out of three is shown.
The differentiation of B cells to plasma cells is independent of CD11chigh DC To study the role of CD11chigh DC in plasma blast formation despite expression of CD11c by the latter, we developed a chimeric system composed of DTR-Tg DC and WT plasma blasts. Lethally irradiated WT B6 mice were reconstituted with 95% BM derived from CD11cDTR-Tg B cell-deficient JH–/– mice supplemented with 5% WT B6 BM (Fig. 3A). The analysis of the DC and f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
B cell compartments of the resulting chimeras is shown in Fig. 3B. As expected, the WT BM reconstituted the entire B cell compartment of the chimeras. In contrast, the splenic CD11chigh DC pool was essentially derived of JH–/– BM, as indicated by almost exclusive presence of GFP+ DC in the CD11c-DTR JH–/– BM recipients (Fig. 3B). To trigger the TI-2 response, the chimeras were immunized by intraperitoneal injection of 200 lg/ mouse NP30-Ficoll.
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It has been suggested that DC might be important for the maintenance of plasma blasts in the extrafollicular response [22]. We therefore decided to ablate CD11chigh DC in the late phase of the primary immune response against NP-Ficoll. To this end we treated the chimeras with DTx on day 3, 4, and 5 after immunization (20 ng/ g body weight/day) (Fig. 4). DTx-injection of the [WT/ DTR>WT] chimeras resulted in efficient and persistent ablation of CD11chigh DC, whereas B cell numbers were not affected (Fig. 3B). Analysis of the challenged chimeras revealed that the depletion of CD11chigh DC during the late phase of the anti-NP response neither
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resulted in a decrease in NP-specific IgM+ plasma blasts nor in reduced NP-specific IgM and IgG3 antibody titers, when compared to non-depleted chimeras (Fig. 4D–F). We therefore conclude that CD11chigh DC cells are dispensable for the maintenance of plasma blasts and their expression of IgG3 during the late time course of TI-2 immune responses. The above experiment does not rule out that DC play a role in the B cell priming process, either by capture [19] or by presentation of antigen [18, 35]. Furthermore, Litinskiy et al. [20, 36] proposed that DC provide via BAFF/APRIL secretion a critical trigger for the plasma blast switch to c3. To determine whether CD11chigh DC are needed for the initial B cell activation and the switch to IgG3 in the TI-2 immune responses, we ablated DC prior to immunization and in the early days of the response (day –1, 1, 3, 5). NP6-specific IgM AFC could be readily detected by ELISPOT analysis of the [WT/WT>WT] chimeras (Fig. 4A). Importantly, plasma blast generation was essentially unaffected by depletion of the splenic CD11chigh DC in [WT/DTR>WT] chimeras, as indicated by the AFC frequencies and IgM and IgG3 serum titers. This result establishes that CD11chigh DC are dispensable for display of antigen or co-stimulation of B cells in the priming process with NPFicoll and for the induction of switch to IgG3. It has been suggested that MZ macrophages play a critical role in the capture and presentation of carbohydrate antigens to MZ B cells [37] and may also be involved in the retention of lymphocytes [38]. However, mixed BM chimeras are known to have few MZ and metallophilic macrophages [39]. Furthermore, both of these cell types are depleted by DTx treatment of CD11c-DTR-Tg mice ([28] and Supplementary Fig. 1). In summary our results suggest that B cells that are triggered by TI-2 antigens have an intrinsic, cellautonomous capacity for differentiation into plasma blasts. Alternatively, support for PC formation in TI responses might be provided by non-hematopoietic stromal cells. Concluding remarks
Figure 3. Analysis of mixed [CD11c-DTR-Tg/WT] BM chimeras (A). (B) Evaluation of the reconstitution of the hematopoietic compartment in BM chimeras by flow cytometry. JH–/– DC of WT phenotype (row 1); JH–/– CD11c-DTR/GFP-Tg CD11chigh DC expressing GFP (row 2); loss of CD11chigh DC after DTx treatment (rows 3–4). (C) Reconstitution of the entire B cell compartment. Each flow cytometry profile shows about 5105 gated events. f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DC have been suggested to play a critical role in B cell activation and the formation of antibody-secreting PC. This includes their presentation of intact antigen or Ig complexes, their promotion of class switch recombination, and provision of crucial survival factors. Here we used an in vivo DC depletion model to specifically address the role of "classical" CD11chigh DC in the TI immune response to the TI-2 antigen NP-Ficoll. We show that CD11chigh DC are dispensable both in the early and late phase of the primary TI-2 response, and thus neither act as crucial APC nor provide essential differentiation/ survival signals for the expanding splenic plasma blasts. www.eji-journal.eu
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Rather our results suggest that support for PC formation in these responses stems from hematopoietic cells other than "classical" CD11chigh DC, such as blood-borne CD11clow DC and macrophages [19], or non-hematopoietic stromal cells. Alternatively, TI-2 antigen-triggered B cells might have an intrinsic, cell-autonomous
Cellular immune response
capacity for plasma blast differentiation. Interestingly, we found in vitro and in vivo generated murine plasma blasts to express the CD11c integrin aX subunit and to be DTx-sensitive in CD11c-DTR-Tg mice. This conditional ablation system thus might provide a unique tool to analyze in vivo aspects of PC biology.
Figure 4. CD11chigh DC are dispensable for the induction and maintenance of Ag-specific AFC. Triangles: BM chimeras that contained WT DC and WT B cells; squares: BM chimeras that contained CD11c-DTR-Tg DC and WT B cells; black symbols: BM chimeras were immunized with NP-Ficoll. Mice received DTx on day 3, 4, and 5 (A–C, late depletion) or on day –1, 1, 3, and 5 (D–F, early depletion). On day 6, leukocytes recovered from the spleen were counted and a serial dilution was placed in ELISPOT wells coated with NP6-haptenated BSA to detect high-affinity NP-specific IgM (D, A). Relative NP6-specific IgM (E, B) and IgG3 (F, C) antibody titers were measured by ELISA. The titers were relative to the same high-responder serum. Each symbol represents an individual mouse; n=5. Cells were recovered from four to five mice and treated independently. One out of two experiments is shown (left and right panel). f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Materials and methods Mice CD11c-DTR-Tg mice [B6.FVB-Tg (Itgax-DTR/GFP)57Lan/J] carry a DTR transgene under the murine CD11c promoter [2]; CD11c-DTR-Tg mice, B6.Cg-IghaThy1aGpi1a/J mice (kindly provided by H.-W. Mittrcker), and JH–/– mice [34] were all on B6 background. Mice were bred and maintained under SPF conditions at the "Bundesinstitut fr Risikobewertung" (BfR, Berlin, Germany) and experiments were performed according to institutional guidelines and German Federal laws on animal protection. For BM chimera generation recipient mice received 950 cGy of c-irradiation via a 137Cesium source. DTx treatment of [DTR>WT] BM chimeras does not result in any adverse side effects [40, 41]. Immunization and DTx treatment Mice were immunized with NP30-Ficoll (Biosearchtech, Novato, CA) in PBS. To systemically deplete CD11chigh DC, mice were injected with 4 and 20 ng/g DTx (Sigma) in PBS. Flow cytometry For surface staining we used anti-CD138-PE (BD Pharmingen), anti-B220-biotin followed by streptavidin-PerCP (BD), and CD11c-Cy5 (German Arthritis Research Center, DRFZ). Dead cells were stained with PI or DAPI (BD). Data were collected on a FACS Calibur cytometer or a LSRII cytometer (BD). Data analysis was performed with CellQuest (BD) software. ELISPOT and ELISA The ELISPOT was performed as described earlier [42]. Plates were coated with 5 lg/mL NP6-BSA (Biosearchtech). Relative NP-specific IgM and IgG3 titers were detected by ELISA. A pooled strong-responder serum served as reference. For detection we applied anti-IgM-alkaline phosphatase (AP), anti-IgG3-AP (Southern Biotech, AL) anti-IgMa-biotin, and anti-IgMb-biotin (BD Pharmingen) followed by streptavidin-AP (Roche). Immunohistochemistry
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Gladbach, Germany), followed by selection on the AutoMACS (Miltenyi). B220+ cells were cultured in RPMI 1640 mediun supplemented with 10 lg/mL LPS and incubated with 100 ng/ mL DTx. Three days thereafter, cultured cells were harvested, washed, and labeled for FACS. Real-time reverse transcription PCR To extract whole RNA from the FACS-sorted cells we applied the RNeasy kit (Qiagen). To prepare first-strand complementary DNA (cDNA) from the isolated RNA, TaqMan reverse transcription components (Applied Biosystems, Darmstadt, Germany) were applied. Expression of the aX integrin chain was analyzed using the primer pair up: 50 -CAGCCATGACCAGTTTACCAA-30 and down: 50 -CACTGTCCACA0 CAGCTTCTCC-3 . Quantification of the cDNA samples was performed with FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany). The cycling parameters were as follows: 95 C for 9 min, denaturation at 95 C for 10 s, annealing at 64 C for 10 s, and extension at 72 C for 10 s. A total of 45 cycles were performed for the detection. Statistical analysis Statistical significance was evaluated by the Mann-Whitney nonparametric (two-tailed) test, applying the Prism software (GraphPad, San Diego, CA). The differences were considered to be statistically significant, if p values were less than 0.05.
Acknowledgements: This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 421 and the Israel Science Foundation (ISF). S.J. is the incumbent of the Pauline Recanati Career Development Chair and a Scholar of the Benoziyo Center for Molecular Medicine.
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