IL-7 Is Required for the Development of the Intrinsic Function of Marginal Zone B Cells and the Marginal Zone Microenvironment

Leen Willems, Shengqiao Li, Omer Rutgeerts, Caroline Lenaerts, Mark Waer and An D. Billiau
J Immunol October 1, 2011, 187 (7) 3587-3594; DOI: https://doi.org/10.4049/jimmunol.1004012

Abstract

The characteristic microarchitecture of the marginal zone (MZ), formed by locally interacting MZ-specific B cells, macrophages, and endothelial cells, is critical for productive marginal zone B cell (MZB cell) Ab responses. Reportedly, IL-7–deficient mice, although severely lymphopenic, retain small numbers of CD21highCD23low B cells consistent with MZB cell phenotype, suggesting that IL-7 signaling is not exclusively required for MZB cell lymphopoiesis. In this study, we investigated the function of IL-7−/− MZB cells and the IL-7−/− microenvironment using a model of hamster heart xenograft rejection, which depends exclusively on MZB cell-mediated production of T cell-independent IgM xenoantibodies (IgMXAb). C57BL/6-IL-7−/− mice accepted xenografts indefinitely and failed to produce IgMXAb, even after transfer of additional IL-7−/− or wild-type C57BL/6 MZB cells. Transfer of wild-type but not IL-7−/− B cells enabled SCID mice to produce IgMXAb. When transferred to SCID mice, wild-type but not IL-7−/− B cells formed B cell follicles with clearly defined IgM+, MOMA-1+, and MAdCAM-1+ MZ structures. Conversely, adoptively transferred GFP+ C57BL/6 B cells homed to the MZ area in a SCID but not an IL-7−/− environment. Naive IL-7−/− mice showed absent or aberrant splenic B cell structures. We provide evidence that IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against T cell-independent type II Ags, for their homing potential, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.

The marginal zone (MZ) is a highly specialized anatomic structure of the spleen characterized by high blood flow and populated by B cells with an activated phenotype, the so-called marginal zone B (MZB) cells. The critical role of the MZ is to initiate fast and intense Ab responses to blood-borne pathogens, and MZB cells are typically capable of mediating rapid T cell-independent (TI) Ab response to bacterial Ags. TI MZB cells are thus a critical part of the immune defense against various pathogens (1). MZB cells express high levels of the complement receptors CD21 and CD35 and the nonclassical MHC molecule CD1d (2). In addition to MZB cells, the MZ harbors MOMA-1+ marginal metallophilic macrophages at the inner side and ER-TR9+ MZ macrophages at the outer side (3). Blood-borne lymphocytes enter the white pulp through fenestrated MAdCAM-1–expressing endothelial cells (4) that separate the MZ from the marginal sinus. MZB cells are highly motile cells, known to shuttle continuously between the MZ and the follicle (5), which is important for the efficient capture of complement-bound Ag from the blood and transport to splenic follicular dendritic cells, which present the Ag to Th cells and form a transition between innate and adaptive immunity (6).

MZB cell responses are typically studied using in vivo challenge with T cell-independent type II (TI-II) Ags, such as TNP-Ficoll or pneumococcal capsular polysaccharides (PPS) (7, 8) We recently showed in mice that concordant xenogeneic Ags, responsible for acute vascular rejection of hamster heart xenografts in mice, also behave as TI-II Ags (9). Indeed, many xenoantigens, such as Galα1,3Galβ1,4GlcNAc (αGal), as well as non-αGal Ags have a polysaccharide structure (10), and it is well known that B cells can reject xenografts independently of T cell help (11). Ohdan et al. (12) have shown in mice that anti-αGal xenoantibodies are produced by B-1b cells in the spleen. In contrast, we have shown in T cell-deficient mice that acute rejection of concordant hamster heart xenografts through rapidly induced IgM xenoantibodies (IgMXAb) against non-αGal xenoantigens is exclusively mediated by MZB cells (9), as adoptive transfer of MZB cells but not B-1b cells enabled SCID mice to mount an IgM xenoantibody response upon immunization with hamster Ags (9). This is in contrast to the Ab response to TNP-Ficoll and PPS, which are mediated by both B-1b and MZB cells (13–16).

In mice, IL-7 is considered an indispensable cytokine not only for T but also for B lymphocytes (17). Indeed, it has been demonstrated that in an IL-7–deficient environment, B cell potential is lost at the prepro-B stage (18, 19). However, others have reported that although the production of B cells in the marrow of adult IL-7−/− mice is almost completely abolished, a small but persistent peripheral pool of large activated B cells is preserved, with a phenotype closely resembling that of MZB cells (IgMhighIgDlowCD21highCD23low) (20). Absolute numbers of these IL-7−/− MZB cells are 4- to 5-fold lower than those of wild-type counterparts (8, 20), but the literature shows that these IL-7−/− splenic B cells respond with proliferation to the TI type I Ag LPS at doses as low as 0.25 μg/ml (20) and are capable of depositing IgM–immune complexes on follicular dendritic cells during a primary T cell-dependent humoral immune response (21), well-known characteristics of MZB cells (22). However, direct evidence of MZB cell-specific Ab responses or of the existence of a typical anatomical MZ microenvironment in IL-7−/− mice is not available (20).

Recently, we observed that IL-7−/− mice, despite the reported presence of a peripheral population of MZB cells, fail to reject a concordant xenogeneic hamster heart graft and fail to produce a TI-II IgM xenoantibody response. In this study, we used this model to explore the role of IL-7 signaling for the function of MZB cells and the MZ microstructure. We demonstrate that contrary to what the literature suggests, IL-7 signaling is critically required not only for the development of the intrinsic MZB cell function of rapidly induced IgM production against TI-II Ags but also for the functional and structural development of the highly specialized MZ microarchitecture.

Materials and Methods

Animals

C57BL/6 and BALB/c euthymic mice and AU/Hö Han Rj inbred golden hamsters were purchased from Centre d’Elevage R. Janvier (Le Genet-St-Isle, France) and C57BL/6 TCR-β−/− mice from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 IL-7−/− mice were kindly provided by P. Vieria (Institut Pasteur, Paris, France), CB17 SCID mice by D. Schols (Rega Institute, University of Leuven), and C57BL/6 GFPTg mice by C. Verfaillie (Stem Cell Institute Leuven, University of Leuven). TCR-β−/−, IL-7−/−, GFPTg mice and hamsters were maintained at the University of Leuven. All experiments were approved by the Ethics Committee on Research Animal Care of the University of Leuven.

Hamster heart transplantation and RBC immunization

Heterotopic xenoheart transplantation was performed as previously described (23). Briefly, hamster hearts (2- to 3-wk-old donors) were transplanted in the necks of recipient mice anesthetized by Nembutal (65 mg/kg i.p.; CEVA, Brussels, Belgium). The ascending aorta and pulmonary artery of the donor heart were anastomosed end-to-side to the common carotid artery, respectively the external jugular vein. Grafts were checked daily by inspection and palpation; cessation of beating indicated xenograft rejection, which was confirmed by histological examination. IgMXAb responses were induced in vivo by hamster red blood cell immunization (HRBCi), as described previously (9), using i.v. injection of 200 μl hamster whole blood.

Flow cytometry

Cell phenotype studies were performed using flow cytometry (23). Abs used were FITC-, PE-, or PerCP-conjugated anti-mouse CD19, CD1d, H-2Kb, B220, and CD3 (BD Biosciences Pharmingen, Erembodegem, Belgium) and anti-mouse CD21 and CD23 (eBioscience, Hatfield, U.K.). Serum levels of mouse anti-hamster IgMXAb were determined as described (23) using recipient serum as primary Ab and hamster RBCs as target cells. Briefly, 5 μl serum was incubated with 2 μl hamster RBCs for 20 min and then with FITC-conjugated goat anti-mouse IgM Ab (eBioscience) for 25 min. Cells were resuspended in PBS and analyzed using a FACSCanto (BD Biosciences).

B cell isolation

CD19+ B cells were isolated from spleens using MACS. In brief, after Percoll gradient isolation, spleen cells were incubated with anti-mouse CD19 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 20 min, and magnetic separation was performed using the LS column (Miltenyi Biotec). The purity of the selected cell population was >95% in TCR-β−/− and >85% in IL-7−/− mice; the lower purity in IL-7−/− mice is probably due to the low frequency of CD19+ cells in IL-7−/− spleen (15.4 ± 2.1%, n = 6) relative to TCR-β−/− mice (79.2 ± 1.5%, n = 4). Although remaining T cells in IL-7−/− are hyporesponsive (see Results), we verified at each isolation procedure that CD19+ populations did not contain T cells (data not shown).

Light microscopy and immunofluorescence studies

For routine microscopic examination, spleens or grafted hearts were formalin-fixed and stained with H&E. For immunofluorescence, tissues were snap-frozen in liquid nitrogen and kept at −80°C. Cryostat sections of 5–7 μm were air-dried overnight at room temperature and fixed in acetone for 10 min. Next, endogenous peroxidase activity was blocked by adding 0.3% H2O2, and nonspecific staining was blocked by incubation with 10% normal goat or donkey serum. Sections were then incubated overnight with FITC-conjugated anti-mouse IgM (eBioscience) at 4°C. For MOMA-1, MAdCAM-1, and GFP stainings, sections were incubated overnight at 4°C with purified rat anti-mouse MOMA-1 (AbD Serotec, Dusseldorf, Germany), purified rat anti-mouse MAdCAM-1 (BD Pharmingen), or purified anti-GFP rabbit IgG (Invitrogen, Molecular Probes, Merelbeke, Belgium) and then stained with Alexa Fluor 555 goat anti-rat or anti-rabbit secondary Ab (Invitrogen). The sections were mounted in a polyvinyl alcohol-based aqueous medium and analyzed with a Zeiss Axioplan 2 microscope (Zeiss, Göttingen, Germany). Images were acquired with a QICAM camera. Original magnifications were ×10/×20. All slides were analyzed and all pictures taken using the same camera settings.

MLR

MACS-purified T cells were stimulated with allo-lymphocytes or xeno-lymphocytes. Briefly, after Percoll gradient isolation, responder T cells were collected by negative selection using a Pan-T cell kit (BD Biosciences, Erembodegem, Belgium) and an LD column. Stimulator cells were obtained from allogeneic or xenogeneic splenocytes after Percoll gradient isolation and 2,000 cGy irradiation. Responders (0.5 × 106 cells/ml) were incubated with stimulators (1:1) for 5 d: during the last 18 h of culture, 1 μCi (0.037 MBq) of [3H]thymidine was added. Cells were harvested with an automated harvester. Stimulation index was determined as follows: [cpm(experiment) – cpm(spontaneous release)]/[cpm(maximum release) – cpm(spontaneous release)].

Treatment with FTY-720

Mice were injected i.p. with 3 mg/kg FTY-720 or with vehicle on days −1, 1, 3, and 5 and were challenged with a hamster heart or hamster blood on day 0.

Statistical analysis

The Mann–Whitney U test was used to estimate the level of significance between groups of data. A p value <0.05 was considered evidence for a statistically significant difference.

Results

IL-7−/− mice reject neither xenografts nor allografts

We transplanted xenogeneic hamster hearts in C57BL/6 IL-7−/− mice and compared graft survival with that in C57BL/6 wild-type (WT) and TCR-β−/− recipients. In IL-7−/− recipients, xenografts survived >3 mo (n = 5), whereas WT (n = 5) and TCR-β−/− (n = 6) mice showed acute rejection by day 6 (Fig. 1), along with high serum titers of IgMXAb (Fig. 2). IL-7−/− mice did not show a rise in serum IgMXAb levels after xenoheart transplantation (n = 4) or HRBCi (n = 3) (Fig. 2), a procedure previously shown to elicit an IgMXAb response in TCR-β−/− mice (9).

FIGURE 1.
FIGURE 1.

Survival of xenogeneic heart grafts in IL-7−/− mice. Xenogeneic hamster hearts were transplanted into IL-7−/− C57BL/6 mice (n = 5), T cell-deficient TCR-β−/− C57BL/6 mice (n = 5), WT C57BL/6 mice (n = 5), or IL-7−/− recipients after transfer of 90 × 106 IL-7−/− splenocytes (Δ n = 3), 90 × 106 TCR-β−/− splenocytes (n = 4), or 40 × 106 WT splenocytes (n = 4). Percent survival is shown per group. ▪, recipients were sacrificed with beating graft 2 mo posttransplantation.

FIGURE 2.
FIGURE 2.

Serum IgMXAb levels after hamster heart transplantation or HRBCi. Serum IgMXAb levels were determined using flow cytometry on serum samples collected on day 6 after hamster heart transplantation (HHTX) in WT mice (n = 4), T-deficient TCR-β−/− mice (n = 5), or IL-7−/− mice (n = 4) or after HRBCi in IL-7−/− mice (n = 3). Levels of IgMXAb are expressed as median fluorescence intensity (MFI). Bars represent mean ± SE. *p < 0.05 (Mann–Whitney U test).

Reportedly, IL-7−/− mice have a small residual T cell population (17). To determine the functionality of these IL-7−/− T cells, mice were given an allogeneic BALB/c heart graft. Allografts survived >3 mo in IL-7−/− recipients (n = 4), similar to TCR-β−/− mice (n = 3), whereas they were acutely rejected in WT recipients (n = 5, graft survival 7 d, 7 d, 7 d, 8 d, 10 d) (data not shown). In MLR, IL-7−/− T cells failed to generate a proliferative response against BALB/c and hamster stimulator cells (SI = 1.02, respectively SI = 1.31) in contrast to a clear MLR response generated by WT T cells (SI = 5.48, respectively SI = 2.98).

We conclude that IL-7−/− mice fail to generate an MZB cell-mediated TI-II IgMXAb response and are thus unable to reject a concordant hamster xenograft. They are equally incapable of mediating T cell-dependent rejection of allografts, confirming the T cell-deficient setting in these mice.

The failure of IL-7−/− mice to reject xenografts is not due to a quantitative reduction in MZB cells

We next specifically investigated MZB cell function in IL-7−/− mice. First, we confirmed that the cellular composition of IL-7−/− spleens was in accordance with that in the literature (20). Absolute cell numbers of lymphocytes in IL-7−/− spleens were 4- to 5-fold lower than those in TCR-β−/− mice [IL-7−/−, 12.8 ± 1.9 × 106 (n = 6); TCR-β−/−, 46.3 ± 3.5 × 106 (n = 4)]; IL-7−/− spleens contained 15.4 ± 2.1% (n = 6) CD19+ cells, relative to 79.2 ± 1.5% (n = 4) in TCR-β−/− mice, and IL-7−/− CD19+ B cells contained 28.3 ± 3.5% (n = 6) IgMhighCD21highCD23low cells (corresponding with the MZB cell phenotype), whereas in TCR-β−/− mice, this subpopulation amounted to 5.4 ± 1% (n = 4) only.

Despite the relative increase in MZB cells, the absolute number of MZB cells in spleens of IL-7−/− mice was still substantially lower than that in TCR-β−/− mice [IL-7−/−, 0.5 ± 0.1 × 106 (n = 6); TCR-β−/−, 1.8 ± 0.5 × 106 (n = 4), data not shown]. This is also in accordance with the literature (8). We have previously shown that adoptive transfer of only 2 × 106 purified MZB cells is sufficient to enable SCID mice to produce an IgMXAb response (9); however, we documented that adoptive transfer of 90 × 106 additional IL-7−/− splenocytes, containing ∼3 × 106 MZB cells, could not restore the capacity of IL-7−/− mice to reject a xenograft (n = 3, Fig. 1) or to produce IgMXAb (data not shown). This indicated that a quantitative defect of MZB cells cannot explain the lack of a xenoantibody response and xenorejection in IL-7−/− mice. Next, we found that after adoptive transfer of 90 × 106 TCR-β−/− splenocytes, containing ∼3 × 106 MZB cells, IL-7−/− mice still accepted xenografts indefinitely (n = 4, Fig. 1) and still failed to show an IgMXAb response (data not shown). In contrast, adoptive transfer of whole splenocytes from WT mice resulted in acute rejection of xenografts (n = 4) (Fig. 1). These observations indicate that the IL-7–deficient environment of the IL-7−/− spleen does not support the TI-II IgMXAb response of normally developed WT MZB cells. The rejection seen after transfer of whole splenocytes from WT mice can be assumed to depend on the response of xeno-reactive WT T cells and T-dependent B cells.

The microarchitecture of the IL-7−/− splenic MZ is not intact

Next, we explored the hypothesis that the microstructure of the MZ in IL-7−/− mice is defective and therefore unable to support an MZB Ab response. We performed immunofluorescence studies on spleens of naive IL-7−/− mice, and TCR-β−/− mice as controls, to detect the presence of the typical MZ microarchitecture.

A representative example of one of four TCR-β−/− and two of six IL-7−/− mouse spleens is shown in Fig. 3. Whereas in TCR-β−/− mice we documented a well-developed MZ B-cell area consisting of a mantle of IgMhigh cells (Fig. 3), corresponding with CD21+ cells (data not shown), this characteristic structure could not be documented in IL-7−/− spleens (Fig. 3). In IL-7−/− mice, the presence of IgM+ cells and the MZ structural anatomy was variable, with three patterns that could be distinguished (Fig. 3): 1) scarcely detectable IgM+ cell clusters are seen, forming small circular structures with a poorly defined MZ area; 2) normally sized follicle-like structures are detected, but IgM staining is weak, and despite the high proportion of CD21highCD23low MZB cells in IL-7−/− IgM+ B cells, the MZ is poorly differentiated from the follicular area; 3) in selected mice, IgM+ clusters could not be detected at all (data not shown).

FIGURE 3.
FIGURE 3.

The microarchitecture of the MZ in C57BL/6 TCR-β−/− and IL-7−/− mice. Spleens from TCR-β−/− and IL-7−/− mice were stained for B cells (IgM, green), marginal metallophilic macrophages (MOMA-1, red), and endothelial cells of the marginal sinus (MAdCAM-1, red). Sections were analyzed with a Zeiss Axioplan 2 microscope and images acquired with a QICAM camera. Stainings are shown from one representative animal of three C57BL/6 TCR-β−/− mice and for two of five IL-7−/− mice showing variation of defects in splenic microanatomy (see Results). Original magnification ×10.

Next, whereas staining for MOMA-1 and MAdCAM-1 in TCR-β−/− mice showed a brightly staining and continuous circular lining in the MZ area, in IL-7−/− spleens these structures were not intact and followed the pattern of IgM staining (Fig. 3), respectively: 1) scarce MOMA-1+ and MAdCAM-1+ small circular structures, with hazy and interrupted lining; 2) normally sized but weakly staining MOMA-1+ and MAdCAM-1+ circular structures, with hazy and interrupted lining; 3) no detectable MOMA-1+ and MAdCAM-1+ cells (data not shown).

These data demonstrate that in IL-7−/− mice, the microanatomy of the MZ, in particular the IgMhigh B cell area but also the adjacent macrophage and sinus-lining cell zones, is not intact.

A productive TI-II Ab response requires the localization of MZB cells in the MZ

We next confirmed that correct localization of MZB cells in the MZ area is a prerequisite for the initiation of a productive TI-II IgMXAb response in this specific model. We pretreated TCR-β−/− mice given HRBCi with FTY-720, an immunosuppressive agent that modulates lymphocyte trafficking and causes displacement of MZB cells from the MZ to the follicular area (24, 25). Immunofluorescence staining confirmed that, as early as 4 h after the start of FTY-720 treatment, IgM+ cells had completely disappeared from the MZ. This effect lasted for at least 72 h (Fig. 4). In control immunized TCR-β−/− mice, at 8 h, the MZ was less densely populated, consistent with the migration of MZB cells to the follicular area during an active Ab response (5), and by 72 h the MZ was again clearly detectable (Fig. 4). Next, we documented that xenografts in FTY-720–treated mice showed long-term survival (>3 mo, n = 4), whereas control TCR-β−/− mice rejected within 6 d (day 5, day 5, day 6, day 6, day 6, n = 5) (data not shown). Serum IgMXAb levels on day 6 were significantly higher in control than in FTY-720–treated mice (control mean MFI 37.7 ± 9.3 SE versus FTY-720–treated mean MFI 11.9 ± 4.2 SE; p < 0.05) (data not shown).

FIGURE 4.
FIGURE 4.

FTY-720 treatment prevents rejection of xenogeneic heart grafts and dislocates B cells from the MZ. A, Spleens from FTY-720–treated and naive TCR-β−/− mice, at different time points after HRBCi, were stained for B cells (IgM, green) and endothelial cells of the marginal sinus (MAdCAM-1, red). Sections were analyzed with a Zeiss Axioplan 2 microscope and images acquired with a QICAM camera. Stainings are shown for one representative animal in each group. Original magnification ×10. B, Xenogeneic hamster hearts were transplanted in naive C57BL/6 TCR-β−/− mice (n = 5) and C57BL/6 TCR-β−/− mice treated with FTY-720 (n = 4). Percent survival is shown per group. C, Serum IgMXAb levels were determined using flow cytometry on serum samples collected from naive C57BL/6 TCR-β−/− mice; samples collected on day 5 after hamster heart transplantation (HHTX) in C57BL/6 TCR-β−/− mice (n = 5) and on day 5 after HHTX in C57BL/6 TCR-β−/− mice treated with FTY-720 (n = 5). Levels of IgMXAb are expressed as MFI. Bars represent mean ± SE. *p < 0.05 (Mann–Whitney U test).

IL-7−/− MZB cells are not functional in an IL-7–sufficient environment

Next, we investigated the intrinsic function of IL-7−/− “marginal zone” B cells when transferred in an IL-7–sufficient milieu. We performed adoptive transfer studies of MACS-isolated CD19+ B cells of IL-7−/− or TCR-β−/− mice into B- and T-deficient CB17/SCID mice: 10 × 106, 20 × 106, or 50 × 106 IL-7−/− or TCR-β−/− CD19+ B cells were transferred on day 0, followed by HRBCi on day 7 (9). On day 14, serum IgMXAb levels were quantified. As shown in Fig. 5, neither SCID mice that received 10 × 106 IL-7−/− B cells nor those that received 20 × 106 IL-7−/− B cells produce significant levels of IgMXAb. In contrast, transfer of TCR-β−/− B cells did lead to a productive IgMXAb response; moreover, IgMXAb levels were higher after transfer of 20 × 106 TCR-β−/− B cells than after 10 × 106 TCR-β−/− B cells, but a further increase in cell dose to 50 × 106 B cells did not further enhance the Ab response.

FIGURE 5.
FIGURE 5.

IgMXAb response in CB17/SCID mice given IL-7−/− or IL-7–sufficient B cell transfer and HRBCi. Serum IgMXAb levels were determined using flow cytometry in serum samples collected from SCID mice 14 d after transfer of 10 × 106 (n = 5) or 20 × 106 (n = 2) IL-7−/− B cells or 10 × 106 (n = 6), 20 × 106 (n = 4), or 50 × 106 (n = 2) TCR-β−/− B cells. Mice were immunized using hamster RBCs 7 d after cell transfer. Levels of IgMXAb are expressed as MFI. Bars represent mean ± SE. As controls, serum IgMXAb levels are shown for C57BL/6 TCR-β−/− mice (n = 8) and CB17/SCID mice (n = 2), as well as for naive SCID mice (n = 3). *p < 0.05 (Mann–Whitney U test).

Because a productive Ab response would require B cells to migrate to the MZ, we also investigated the microanatomy of the MZ after adoptive transfer using immunofluorescence. In naive SCID spleens, expectedly, IgM+ cells were not detected (poorly cellular SCID spleen sections showed amorphous background staining only; Fig 6); in addition, MAdCAM-1+ and MOMA-1+ cells were scarcely detectable and not organized in circular structures as seen in TCR-β−/− mice (Fig. 6). This is in accord with published reports showing that the development and homeostasis of the MZ, including the characteristic presence of macrophages, is critically dependent on the presence of B cells (26) and CD19+ cells (27). After TCR-β−/− B cell transfer to SCID mice, we documented numerous follicle-like structures with IgM+ cells forming an MZ area, adjacent to clearly distinguishable MAdCAM-1+ and MOMA-1+ circular linings. In contrast, after IL-7−/− B cell transfer, very few and small IgM+ cell clusters were documented, which did not show organization in a follicle or MZ. In these spleens, MAdCAM-1+ and MOMA-1+ cells were seen with low frequency and did not form definable structures (Fig. 6).

FIGURE 6.
FIGURE 6.

Microarchitecture of the MZ in CB17/SCID mice after adoptive transfer of IL-7−/− or IL-7–sufficient B cells. Spleens from SCID mice were stained for B cells (IgM, green), marginal metallophilic macrophages (MOMA-1, red), and endothelial cells of the marginal sinus (MAdCAM-1, red) after transfer of 10 × 106 (n = 6) or 20 × 106 (n = 4) TCR-β−/− B cells or 10 × 106 (n = 5) or 20 × 106 (n = 2) IL-7−/− B cells. Sections were analyzed with a Zeiss Axioplan 2 microscope and images acquired with a QICAM camera. Stainings are shown for one representative naive SCID mouse (left), one SCID mouse transferred with 10 × 106 TCR-β−/− B cells (middle), and one SCID mouse transferred with 10 × 106 IL-7−/− B cells (right). Original magnification ×10.

These data indicate that IL-7−/− B cells, in contrast to TCR-β−/− B cells, are incapable of forming a structural MZ in an IL-7–sufficient milieu and are not functional Ab-producing cells.

The IL-7−/− microenvironment does not support migration and homing of WT MZB cells

Finally, we explored whether the IL-7−/− splenic environment supports the appropriate homing of WT MZB cells to the MZ area, as could be seen when WT B cells were transferred to SCID mice (Fig. 6). We transferred 50 × 106 MACS-purified C57BL/6 GFPTg CD19+ B cells to IL-7−/− mice or SCID mice, and used immunofluorescence to detect the formation of MZ structures by transferred GFP-expressing B cells 7 d later. Similar to the results obtained in Fig. 6, in SCID mice we observed formation of IgM+GFP+ B cell follicular structures with a clearly detectable MZ (Fig. 7). In IL-7−/− mice, however, IgM staining showed vague follicle-like structures, but no clearly distinguishable MZ; GFP stain was either negative (data not shown) or showed weak positive staining in the follicular area only, with only few double-staining IgM+GFP+ B cells in this area (Fig. 7). Using a double staining for MAdCAM-1 and GFP, we confirmed that transferred GFP+ B cells in SCID mice home to the outer side of the MAdCAM-1 lining, in contrast to transferred GFP+ B cells in IL-7−/− mice, which could only be seen in the follicular area, at the inner side of the MAdCAM-1 lining (Fig 7).

FIGURE 7.
FIGURE 7.

Microarchitecture of the MZ in SCID and IL-7−/− mice after adoptive transfer of GFP-expressing IL-7–sufficient B cells. On day 7 after adoptive transfer of 50 × 106 GFP+ C57BL/6 B cells, spleens from SCID (n = 3) and IL-7−/− (n = 5) mice were stained for B cells (IgM, green), GFP (red or green to identify donor-derived B cells), and endothelial cells of the marginal sinus (MAdCAM-1, red). Sections were analyzed with a Zeiss Axioplan 2 microscope and images acquired with a QICAM camera. Stainings are shown for one representative SCID mouse (left) and IL-7−/− mouse (right). Original magnification ×20.

This indicates that the IL-7−/− microenvironment does not support adequate migration and homing of normally developed WT MZB cells to the IL-7−/− MZ area.

Discussion

In the MZ, the presence of MZB cells, marginal metallophilic and MZ macrophages, and MAdCAM-1+ sinus-lining cells in a characteristic microarchitecture, as well as the local interaction between these cells and the dynamics of intrasplenic MZB cell migration during Ab responses, are critical for the highly specialized function of the MZ (3, 5, 26–28). In this study, we provide evidence that, contrary to what is suggested by the literature on IL-7−/− mice (19, 20), IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against TI-II Ags, for the homing potential of MZB cells, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.

This study was inspired by our observation that IL-7−/− mice do not reject concordant xenogeneic hamster heart grafts, whereas prior studies by others had suggested that IL-7–deficient mice have a relative preservation of the MZB cell compartment (19, 20), and that B cells of IL-7–deficient mice exhibit some typical functions of MZB cells (20, 21). We previously established that the xenoantibody response accompanying hamster heart transplantation and RBC immunization in T cell-deficient mice is a TI-II Ab response exclusively mediated by MZB cells (9). In the current study, we show that the inability of IL-7−/− mice to xenoreject is due to a qualitative defect in the intrinsic function of the residual MZB cells and in the intrinsic function of the MZ microenvironment, or a combination of both. This was evident from the following observations. First, absolute numbers of MZB cells in IL-7−/− mice are 4- to 5-fold lower than those in WT mice, but although we previously demonstrated that transfer of 2 × 106 MZB cells only is sufficient to establish an IgMXAb response in SCID mice (9), in IL-7−/− mice, a quantitative correction of MZB cell numbers did not reestablish rejection capacity. Second, IL-7−/− B cells transferred into SCID mice failed to generate an IgMXAb response after hamster Ag challenge, in contrast to B cells from IL-7–sufficient TCR-β−/− mice. Third, transfer of IL-7–sufficient B cells to IL-7−/− mice also failed to restore the capacity to produce an IgMXAb response or rejection, whereas in SCID mice this readily led to a productive IgMXAb response.

We next showed that one of the mechanisms explaining the qualitative defects of IL-7−/− MZB cells and of the IL-7−/− MZ microenvironment relates to defective homing of MZB cells to the MZ area. First, using immunofluorescence studies on SCID mice given IL-7−/− or IL-7–sufficient B cells, we documented that the defect of IL-7−/− to restore the IgMXAb response is associated with the inability of MZB cells to locate efficiently in the MZ area. Accordingly, although IL-7–sufficient B cells formed an MZ area along with MOMA-1+ and MAdCAM-1+ cell linings, these structures were not induced by IL-7−/− B cells. Using flow cytometric analysis, we ascertained, however, that donor-derived B cells had migrated to the spleens of the transferred mice (data not shown). Second, using adoptive transfer experiments with IL-7–sufficient GFP-expressing B cells, we documented that whereas IL-7–sufficient B cells consistently migrate to the follicular and MZ area in an IL-7–sufficient environment of SCID mice, they fail to do so when transferred to IL-7−/− mice; instead, the B cells could not be localized or scarcely located in the follicular area. Consistent with these findings, we documented that in naive IL-7−/− mice, the presence of typical IgM+, MOMA-1+, and MAdCAM-1+ structures is variable, and, if present, they showed an aberrant microarchitecture. In the past, a study in IL-7R−/− mice has documented the presence of a MOMA-1+ MZ macrophages structure, but a distinct marginal or follicular area formed by IgM+ cells was not seen (29), and MAdCAM-1+ structures were not reported on.

In a similarly designed study to the current one, Shriner et al. (8) recently described that IL-7−/− and IL-7R−/− mice show strongly reduced or completely absent Ab responses to respectively PPS Ags and TNP-Ficoll. According to adoptive transfer studies in Rag−/− mice (13–16), it is the B-1b cells that produce the majority of anti-PPS3 and TNP-Ficoll Abs, and Shriner et al. equally showed that in the peritoneal cavity of TNP-Ficoll–challenged euthymic mice, the B-1b population contains the majority of TNP-specific B cells. In the current study, we specifically examined the role of IL-7 signaling in a TI-II Ab response of which we previously showed in a T cell-deficient in vivo model that it depends exclusively on MZB cells, and not on B-1 cells (20). In accordance with Shriner et al. (8) and others (13–16), we had found in this experimental system that the TNP-Ficoll IgM Ab response is only in part mediated by MZB cells (9). Ohdan et al. (12) reported that TI-II natural Ab response to the xenoantigen αGal is dependent on splenic B-1a cells. Along this line, others have concluded that the different nature of various TI-II Ags used in experimental studies can explain the differential involvement of TI B-cell types and/or other components such as macrophages and C3 and complement receptors in the respective Ab (7, 30). Although Shriner et al. (8) attributed the effects of IL-7 deficiency on PPS3 and TNP-Ficoll specific Ab responses predominantly to an effect on B-1b cells, our findings indicate that a qualitative defect in intrinsic MZB cell function and MZ microarchitecture contribute to the defective Ab levels seen in the TNP-Ficoll system as well. Indeed, others have also shown that MZB cells contribute significantly to the Ab response to these Ags (7).

In contrast to most studies on TI-II Ab responses, in our previous and current study we used a T cell-deficient setting, which excludes any influence of T cell reactivity on the TI-II Ab response. This may further account for differences seen in the Ab responses profiles or B cell populations involved between our system in this study and those of others (8, 24). Indeed, as reviewed by Vos et al. (1), the modulation by conventional T cells of the response to TI-II Ags, although mechanistically unresolved, has been well established. In the current study, the T cell deficiency of TCR-β−/− mice was checked regularly by documenting their failure to reject allografts (data not shown), and along this line, we documented that IL-7−/− mice do not reject allografts and that residual IL-7−/− T cells fail to respond in vitro to allogeneic and xenogeneic targets. This illustrates profound T cell deficiency in IL-7−/− mice and confirms the T cell-deficient setting of the IgMXAb response studied in this setting.

Whereas we found that IL-7−/− B cells do not exhibit the characteristic ability of MZB cells to produce a rapidly induced IgM response to TI-II Ags, others have reported that such B cells do show other functions typical of MZB cells, in particular, a proliferative response to a low dose of the TI type I Ag LPS (20), and the ability to deposit IgM–immune complexes on follicular dendritic cells during a primary T cell-dependent humoral immune response (21). Although a difference in the IL-7−/− phenotype may theoretically account for functional differences in IL-7−/− mice across studies, we postulate that IL-7 deficiency may variably affect the different characteristic functions of MZB cells, in particular, responses to a T cell-independent type I or type II Ag or to a T cell-dependent Ag.

The processes of MZB cell homing to the MZ area and of intrasplenic shuttling during Ab responses, which are critical for the functional integrity of the MZB cell compartment, have recently been described (5, 24). With respect to the TI-II IgMXAb response studied in the current work, we confirmed that correct localization of MZB cells in the MZ is critically required for an in vivo TI-II xenoantibody response and xenorejection: displacement of MZB cells from the MZ by FTY-720 treatment, previously also demonstrated by others (24, 25), prevented xenograft rejection and the associated IgMXAb response. Similar to the long-term xenograft survival observed in the FTY-720 study, we have shown in previous studies that a temporary but specific depletion of MZB cells in C57BL/6 nude mice by 2 Gy total body irradiation also sufficed to induce long-term xenograft survival (9). In fact, in these 2 Gy-treated mice, we found that long after the transplant, low levels of circulating IgMXAb could be detected and that long-term surviving xenografts showed low immunoreactivity for IgM in the absence of signs of vascular rejection (9). This indicates that in this xenograft model—once acute vascular rejection is prevented and MZB cells return—IgMXab production may recover to some extent, but that grafts still survive owing to a mechanism of graft accommodation. Of note, Vora et al. (24) previously reported that FTY-720 treatment did not influence the Ab response against TNP-Ficoll, and the reasons for the difference with the effect in our model may be twofold. First, as mentioned, variability in the nature of the TI-II Ag determines the TI B-cell subsets involved in the Ab response, and specifically, our previous data in the same T cell-deficient model showed that TNP-Ficoll only partially exhibits the characteristics of the anti-hamster IgMXAb response by MZB cells (9). Second, the TNP-Ficoll study by Vora et al. (24) was performed in euthymic mice, and as mentioned, it is known that T cells can modulate TI-II Ab responses.

Possible mechanisms of qualitative changes in B cells as a result of IL-7 deficiency, as suggested by Shriner et al. (8), are an underdeveloped BCR repertoire diversity and a lack of appropriate signals for proliferation and/or differentiation. First, our observation that adoptive transfer of WT MZB cells in IL-7−/− mice still failed to restore Ab production indicates that a disturbed BCR repertoire is not the only causal mechanism. Second, IL-7 deficiency is likely to result in a defect of proliferation: although all studies reporting on IL-7−/− mice have demonstrated a relative preservation of the MZ compartment, absolute B cell counts within these compartments are still significantly lower than those of naive controls. However, we verified that the lack of the IgMXAb response is not merely due to a reduction in MZB cells. Finally, our data provide evidence for the hypothesis that IL-7 deprivation during B cell lymphopoiesis qualitatively affects the development of both MZB cells and the MZ microenvironment; specifically, it affects the intrinsic capacity of MZB cells to home to the MZ and affects the microenvironment of the MZ itself in such a way that WT MZB cells fail to migrate to and lodge in this area. Possibly these two phenomena are interrelated. It is known that both the development and the homeostasis of the MZ anatomy are critically dependent on the presence of B cells. Nolte et al. (26) demonstrated in CD70-transgenic mice that progressive loss of B cells is associated with the loss of the specific anatomic structure of the MZ, and as a consequence, these mice are unable to take up TI-II Ags. You et al. (27) subsequently showed using CD19−/− mice that expression of CD19 on B cells in this process is critical. The failure of IL-7−/− MZB cells to locate in the MZ may therefore secondarily affect the further organization of the MZ environment. However, the observation that WT B cells do not efficiently home to an IL-7−/− MZ area, whereas they do so in an IL-7–sufficient B cell-deficient SCID environment (thereby instructing the development of typical MOMA-1+ and MAdCAM-1+ macrophage structures), indicates that the defect in the MZ microarchitecture, in terms of attracting and lodging MZB cells, is due at least in part to an intrinsic effect of IL-7 deficiency on non-B cell compartments of the MZ, and not to the prior prolonged absence of B cells in the MZ as occurs in the SCID environment. Indeed, macrophages also are indispensable for the integrity and proper function of the MZ, as it has been shown in mice lacking the macrophage scavenger receptors MARCO and SR-A that the MZM layer is not intact and that the MAdCAM-1 lining and IgM are discontinuous. Moreover, these mice have an impaired response to pneumococcal capsular polysaccharides (28). To our knowledge, the role of IL-7 in MZ macrophages, sinus-lining cells, and stromal cells has to date not been studied.

We thus provide evidence that the reported small population of phenotypic “MZB cell-like” B cells that persists in IL-7−/− mice does not correspond with a B cell population with the typical MZB cell function of rapidly induced IgM formation against TI-II Ags. It has been postulated that residual MZB cell lymphopoiesis in IL-7−/− mice is dependent on FltL3, a potent cofactor for the growth of primitive B cell progenitors (31, 32). We propose that the MZB cell phenotype of these B cells arises as a phenomenon secondary to lymphopenia. It has been suggested that in a lymphopenic milieu, B cells preferentially acquire and maintain an activated phenotype, which is similar to that of MZB cells (33). Whereas in spleens of WT mice, the ratio of follicular to MZB cells is 6 to 1, in IL-7−/− mice, this ratio is 1 to 2, and our data argue that this lymphopenia-induced MZB cell phenotype occurs also in IL-7−/− mice. In fact, we observed this phenomenon also in TCR-β−/− cell-transferred SCID mice: whereas in naive TCR-β−/− mice, most of the B cells have a follicular B cell phenotype, after transfer in a lymphopenic milieu, these cells changed phenotype to that of MZB cells (data not shown).

In conclusion, IL-7 signaling is critically required for the development of the intrinsic MZB cell ability to produce a rapidly induced IgM response to TI-II Ags. In addition, IL-7 is required for the functional and structural development of the highly specialized MZ microarchitecture. Specifically, IL-7 deficiency gives rise to “MZB-like” B cells that fail to locate in the MZ area and results in an MZ microenvironment that fails to attract and lodge MZB cells.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • Abbreviations used in this article:

    αGal
    Galα1,3Galβ1,4GlcNAc
    HRBCi
    hamster red blood cell immunization
    IgMXAb
    IgM xenoantibodies
    MFI
    median fluorescence intensity
    MZ
    marginal zone
    MZB
    marginal zone B
    PPS
    pneumococcal capsular polysaccharides
    TI
    T cell-independent
    TI-II
    T cell-independent type II
    WT
    wild-type.

  • Received December 8, 2010.
  • Accepted July 25, 2011.

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