Architectural Defects in the Spleens of Nkx2-3-Deficient Mice Are Intrinsic and Associated with Defects in Both B Cell Maturation and T Cell-Dependent Immune Responses

David Tarlinton, Amanda Light, Donald Metcalf, Richard P. Harvey and Lorraine Robb
J Immunol April 15, 2003, 170 (8) 4002-4010; DOI: https://doi.org/10.4049/jimmunol.170.8.4002

Abstract

Mice lacking the homeodomain transcription factor Nkx2-3 are either asplenic or develop a spleen of significantly reduced size with poorly organized white pulp. In this report, we analyze the effect of this mutation on B lymphocyte development and differentiation. Follicular dendritic cells in spleen, but not lymph node, of Nkx2-3−/− mice fail to express a developmental Ag (follicular dendritic cell-M2) and show an abnormal association with B cells, despite essentially normal expression of several chemokine genes. Bone marrow reconstitution studies show the splenic disorganization and absence of marginal zone B cells to be of stromal rather than hemopoietic origin. Furthermore, Nkx2-3−/− mice show an excess of conventional B cells in mesenteric lymph node and peritoneal cavity, whereas transitional B cells are rare in spleen but overrepresented in bone marrow. Finally, immunization of Nkx2-3−/− mice with a T cell-dependent Ag elicits clusters of germinal center B cells, although these fail to develop to the same extent as in controls and there is no evidence of affinity maturation in serum Ab. Similarly, Ab-forming cells fail to aggregate into foci early in the response. Collectively, these data indicate a substantial role for Nkx2-3 in the correct association of lymphocytes and splenic stromal elements that is independent of chemokine expression.

The spleen has three principal functions: it acts as a filter to remove senescent and damaged erythrocytes, it is the point where newly generated lymphocytes enter the peripheral lymphocyte pool, and it is a site of immune reactivity, especially against blood-borne particulate Ags. These roles are facilitated by the organization of the spleen into red and white pulp areas (reviewed in Ref.1). The red pulp is rich in erythrocytes and monocytes and is the site where arterial blood flow in the spleen enters the venous compartment. After entry, the splenic artery undergoes successive branching until its smallest branches, called central arterioles, leave the trabeculae, enter the red pulp, and terminate as capillaries in or near venous sinuses, thus completing a complex set of open and closed circulatory routes (reviewed in Ref.2). Before termination in the red pulp, the central arterioles are sheathed in lymphocytes forming the periarteriolar lymphoid sheath (PALS).3 Collectively, the PALS constitute the spleen’s white pulp. The red and white pulp are separated from each other by the marginal sinus, a lining of cells that envelope the white pulp and may have a role in the selective migration of leukocytes into the white pulp by means of adhesion molecules such as mucosal addressin cell adhesion molecule-1 (3) and L1 (4). Surrounding the marginal sinus is a ring of B lymphocytes and monocytes forming the marginal zone (MZ). The MZ is the site for the termination of one branch of the central arteriole (2, 5), and it forms a potential entry point of immature B lymphocytes and particulate Ags into the white pulp (1). Thus, the structure of the spleen underlies aspects of both B lymphocyte maturation and immune reactivity.

The white pulp of the spleen comprises distinct B and T lymphocyte areas (6). T cells are enriched in the inner PALS, whereas B cells reside primarily in follicles in the outer PALS, a segregation that is mediated by chemokines secreted by stromal and/or dendritic cells unique to the respective lymphocyte areas (7). Recirculating B lymphocytes are attracted into follicles by CXC chemokine ligand (CXCL)13 secreted by follicular stromal cells and acting through the receptor CXCR5. Naive T lymphocytes are most strongly attracted by CC chemokine ligands (CCL)21 and 19, both operating through the receptor CCR7 and produced by T-zone stromal cells and interdigitating dendritic cells, respectively. The development of stromal cells to the point where they secrete chemokines is poorly understood, but the involvement of the lymphotoxin and TNFR-ligand pathways has been well documented using both Ab treatment and gene ablation studies (7, 8, 9). Analysis of genetically modified mice also identified the NF-κB family of transcription factors as having critical roles in transducing the developmental signals within, for example, the stromal cells of the spleen necessary for secretion of CXCL13 (10). Thus, while the genetic pathways involved in establishing the chemokine gradients within secondary lymphoid organs are being defined, other developmental pathways remain unexplored.

The immune function of the spleen is dependent on white pulp organization. Ags that localize in the spleen result in the formation of germinal centers (GCs) within B cell follicles of the white pulp (11). These GCs form in close association with follicular dendritic cells (FDCs) due to the ability of these cells to trap and retain immune complexes on their surface by virtue of receptors for both the Fc component of Ig and complement components (12). GCs function as sites for the efficient selection of B cell variants with improved affinity for Ag, resulting in the eventual generation of persistent, high-affinity Ab-forming cells (AFCs) and memory B cells (13, 14). Preceding AFC generation in the GC, Ab production is restricted to foci of AFCs that develop in the outer PALS during the first week of the response (15). The AFCs of the foci show little evidence of Ig V gene somatic mutation or affinity maturation and undergo apoptosis within 2 wk of immunization (16). The localization of GCs is also dependent on the microarchitecture of the spleen. For example, when the white pulp is disorganized, GCs may form ectopically (17, 18, 19). Similarly, positioning and migration of AFCs requires coordinate expression of various chemokines during the course of the immune response (20, 21). Therefore, appropriate development and organization of the follicular stromal cells is important for determining the efficiency of the processes driving affinity maturation in the immune response.

Recently we and others described the phenotype of a mouse strain lacking the homeodomain transcription factor Nkx2-3 (22, 23). Approximately one-third of these mice are asplenic, and the remainder have spleens that are on average 10-fold smaller by weight compared with littermate controls and that, histologically, lack MZ and have poorly demarcated B and T cell areas. Furthermore, the cells lining the marginal sinus do not express mucosal addressin cell adhesion molecule-1, apparently due to a direct effect of Nkx2-3 on the promoter of this gene (23, 24). These knockout mice also show abnormalities in gut development and are blocked in the formation of Peyer’s patches. In this report, we examine the basis and immunological consequences of the malformed splenic white pulp in Nkx2-3−/− mice.

Materials and Methods

Mice

Mice deficient in Nkx2-3 were generated as described (23). These mice were on a mixed C57BL/6.129Sv background, having been backcrossed two generations to C57BL/6 before being brother-sister mated to generate homozygous knockouts. The heterozygous mice generated during these matings were used as controls and to generate subsequent knockout mice.

Flow cytometry and Abs

Tissue preparation, Ab staining, and flow cytometry were performed as described (13). Abs used in this study were anti-CD45R-PE and anti-CD45R-FITC (B220; clone RA3-6B2), anti-IgM-PE (goat anti-mouse; Southern Biotechnology Associates, Birmingham, AL), anti-CD21-biotin (clone 7G6), anti-CD23-FITC (clone B3B4), anti-CD5-FITC (Ly1; clone 53-7.3), FDC (clone FDC-M2) (25), anti-IgD-biotin (clone 11-26c), anti-Thy1-PE (clone T24.3.2.1), and anti-CD22-biotin (clone Cy34.1). Biotinylated Abs were revealed with Streptavidin-PE or Streptavidin-Tricolor (Caltag Laboratories, Burlingame, CA) in three-color staining. Dead cells were excluded on the basis of propidium iodide uptake.

Immunohistology

Tissue isolation, freezing, and preparation of sections were as described (26). Abs used in immunochemistry were as follows: anti-IgM-HRP (goat anti-mouse; Southern Biotechnology Associates), anti-IgD-biotin (clone 11-26c), anti-CD45R (B220; clone RA3-6B2), anti-CD3-biotin (clone KT3), and anti-CD35-biotin (clone 8C12; BD Biosciences, Mountain View, CA). Rat Abs were revealed with HRP-conjugated mouse anti-rat Igκ (BD PharMingen, San Diego, CA) and biotinylated Abs with avidin-alkaline phosphatase. Color detection used peroxidase and alkaline phosphatase substrate kits (Vector Laboratories, Burlingame, CA). Immunofluorescence used anti-CD45R-FITC and anti-FDC (clone FDC-M2) together with Texas Red-conjugated donkey anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Stained sections were photographed using a Leitz DMR microscope (Leika, Wetzlar, Germany) for immunochemical stains and a Zeiss Axioplan 2 (Zeiss, Oberkochen, Germany) for two-color immunofluorescence. Three-color stainings used FITC-conjugated peanut agglutinin (PNA) (Vector Laboratories), biotinylated anti-CD45R revealed with Cy5-avidin (Caltag Laboratories), and FDC-M2 revealed with Texas Red-donkey anti-rat Ig. Cross-reactivity was blocked by incubation with normal rat serum where necessary. Images were generated on a Leica TCS 4D scanning confocal laser microscope (Leica, Deerfield, IL) and then compiled using Photoshop software (Adobe Systems, Mountain View, CA).

RNA isolation and Northern blot analysis

On two occasions, spleens from four controls and seven knockouts were collected and snap-frozen in liquid nitrogen. Total RNA was prepared from each batch using Trizol (Life Technologies, Rockville, MD), electrophoresed under denaturing conditions, transferred to nylon membrane (Bio-Rad, Hercules, CA), hybridized with labeled probes, and subsequently washed as described (27). Probes used were representative cloned cDNAs generated by RT-PCR and verified by nucleotide sequencing. The primers and regions amplified for CXCL13, CCL19, CCL21, and CXCL12 were as described (28). GAPDH was used as a loading control. Thus, each band on the Northern blot is an independent sample of its respective population. Quantitation of band intensities used scans of nonsaturating exposures in conjunction with a computing densitometer (Molecular Dynamics, Sunnyvale, CA). The values presented are the average of the bands from the independent samples of each genotype after division by the intensity of the average GAPDH signal of that sample on that blot.

Bone marrow reconstitutions

Recipient mice were irradiated twice at 5.5 Gy, 4 h apart, and reconstituted 1 day later with 10 × 106 T cell-depleted donor bone marrow cells injected i.v. Recipients were fed antibiotic-containing water for 2 wk after irradiation to minimize the risk of infection. Mice were analyzed 8 wk after reconstitution.

Immunization

For T cell-dependent immune responses, mice were immunized once with 100 μg of the hapten 4(hydroxy-3-nitrophenyl)acetyl (NP) coupled to the carrier protein keyhole limpet hemocyanin (KLH) precipitated on alum and injected i.p. as described (13). Immunized mice were bled weekly, and the level and relative affinity of NP-specific IgG1 was determined by ELISA using differentially haptenated plate coats as described (13). T cell-independent responses were induced by immunizing mice with a single i.p. injection of 10 μg of DNP-dextran in PBS and assaying elicited serum IgM by cross-reactive binding to trinitrophenol coupled to BSA. OD was measured at 405 nM with a reference wavelength of 490 nM. The OD from the 1/10,000 dilution is plotted for all time points, this being in the linear region of the titration.

Peripheral blood examination

Retro-orbital blood was analyzed using a hematology system analyzer (Advia 120; Bayer Diagnostics, Tarrytown, NY). Peripheral blood smears and bone marrow cytospins were stained with May Grunwald Giesma for differential counts. Differences were assessed for significance using the Student t test.

Progenitor cell analysis

Bone marrow progenitor cells were assayed using semisolid agar and cultures as previously described (29). Semisolid 1-ml agar cultures containing 2.5 × 104 bone marrow cells or 5.0 × 104 spleen cells in 0.3% agar in DMEM supplemented with 20% newborn calf serum (selected batch) were plated in triplicate and stimulated by purified recombinant growth factors. For progenitor cell assays, individual growth factors were added to replicate cultures at the following final concentrations: mouse IL-3 (mIL-3; Peprotech, Rocky Hill, NJ) at 100 ng/ml, mGM-CSF (Peprotech) at 10 ng/ml, mM-CSF (Cetus Corporation, Emeryville, CA) at 10 ng/ml, and human G-CSF (a gift from Amrad Pharmaceuticals, Melbourne, Australia) at 10 ng/ml. Preprogenitor cells were assayed in bone marrow cultures stimulated with mouse stem cell factor (produced by expression in Pichia pastoris) at 100 ng/ml or with Flk ligand (produced by expression in Chinese hamster ovary cells) at 500 ng/ml and LIF (Chemicon, Melbourne, Australia) at 10 ng/ml (30). After 7 days of incubation at 37°C in a fully humidified atmosphere of 10% CO2 in air, colonies were enumerated by examination of cultures at ×35 magnification. Cultures were fixed with 2.5% glutaraldehyde in normal saline and stained using acetylcholinesterase, luxol fast blue, and hematoxylin, and the colonies were classified at ×100 or ×400 magnification. Megakaryocyte colonies were defined as those containing three or more megakaryocyte cells.

CFU-spleen (CFU-S) assay

CFU-S assays were performed using 75,000 nucleated bone marrow cells from control and mutant mice. Cells were washed in serum-free DMEM and injected i.v. via the retro-orbital sinus into four control recipients. Several hours before transplantation, the recipient mice were irradiated as described above. Transplanted mice were maintained on oral antibiotic. Spleens were removed after 12 days and were fixed in Carnoy’s solution (60% ethanol, 30% chloroform, 10% acetic acid), and the numbers of macroscopic colonies were enumerated.

Results

Splenic disorganization of Nkx2-3−/− mice extends to FDCs

We first determined the organization of the splenic white pulp in the Nkx2-3−/− mice used in the current experiments. Spleens from adult Nkx2-3−/− mice were examined by immunohistology to define the distribution of B and T lymphocytes and the presence of MZ B cells. As previously reported (23, 24), B and T cells segregate poorly in knockout spleens relative to controls, and MZ lymphocytes fail to develop (Fig. 1⇓, A–D). The T and B cell localization in Nkx2-3−/− mice is similar to that of mice with defective FDC development (9). To determine whether this was also the case in Nkx2-3 knockout spleens, we examined by histology the distribution of cells staining with either CD35 or, separately, the Ag recognized by the mAb FDC-M2 (25). B cell follicles, revealed with anti-B220, were defined clearly in control spleens and were associated with CD35+ FDCs, showing an underlying reticular pattern (Fig. 1⇓, E and F). Nkx2-3-deficient spleens, however, showed an unusual association of B cells with the CD35+ staining cells. Rather than being clustered around the CD35+ FDCs, B cells in the Nkx2-3−/− spleen were dispersed in a manner that appeared to be independent of localization of the FDCs. Staining with FDC-M2 also revealed differences between control and knockout spleens (Fig. 1⇓, G and H). Although the Ag recognized by this Ab has recently been defined as complement component C4 (31), staining in the spleen is primarily restricted to FDCs (25). In control spleens, FDC-M2-positive cells appeared as clusters dispersed semiregularly throughout the spleen that coincided with B cell follicles (data not shown). In the spleens of knockout mice, however, FDC-M2 staining was not clustered but rather had a reticular pattern. Collectively, these results reveal presumptive FDCs in the spleens of Nkx2-3−/− mice and show their association with B cells to be abnormal. Although the unusual FDC-M2 staining may indicate a developmental abnormality in the splenic FDCs of Nkx2-3−/− mice, the normal pattern in the mesenteric lymph nodes of mutant mice reveals it to be organ specific (Fig. 1⇓, I and J). This is also in keeping with the essentially normal B and T cell distribution in the lymph nodes of Nkx2-3 mutant mice (24).

FIGURE 1.
FIGURE 1.

Splenic white pulp is disorganized in the absence of Nkx2-3. Spleen cryosections were stained with anti-B220 (red) and anti-CD3 (blue) (A and B), anti-IgM (red) and anti-IgD (blue) (C and D), anti-B220 (green) and anti-CD35 (red) (E and F), or FDC-M2 (red) (G and H). Cryosections of mesenteric lymph node were also stained with FDC-M2 (red) (I and J). MZs are indicated by arrows (C). Stainings shown are representative of at least five mice of each genotype.

Expression of chemokine genes in Nkx2-3−/− spleen

The abnormal segregation of B cells and T cells in the spleens of Nkx2-3−/− mice could result from defective maturation of stromal cells and thus could be associated with a failure to secrete lymphocyte-attracting chemokines such as CXCL13 (7). We examined levels of mRNA encoding several chemokines important for lymphocyte organization in the spleen to determine whether the splenic stromal cells in the Nkx2-3−/− mice had matured to the point of transcribing these genes at detectable levels (Fig. 2⇓). Total RNA extracted from the spleens of knockout and control mice was assessed by Northern blot for mRNA encoding the chemokines CXCL13, CCL19, CCL21, and CXCL12. All chemokine mRNAs were readily detectable, although the levels of some differed in the knockouts relative to controls. Using expression of the housekeeping gene GAPDH as a reference, the level of CXCL13 was approximately equal in control and knockout spleens (0.07 and 0.09, respectively), whereas those of CXCL12 (+/−, 0.3; −/−, 0.7), CCL19 (+/−, 0.7; −/−, 1.4), and CCL21 (+/−, 0.4; −/−, 1.1) were all approximately twofold higher in Nkx2-3−/− samples. Although the reason for and consequences of these differences in mRNA levels are currently unresolved, the abnormal distribution of lymphocytes in the spleens of Nkx2-3−/− mice cannot be attributed to the failure of follicular stromal cells to transcribe genes encoding the lymphocyte-attracting chemokines.

FIGURE 2.
FIGURE 2.

Chemokine genes are expressed in the spleens of Nkx2-3−/− mice. Total RNA, extracted from pooled spleens of each genotype, was separated by electrophoresis, transferred to a nylon membrane, and probed with labeled cDNAs corresponding to the indicated chemokines. Membranes were stripped and rehybridized with GAPDH to assess RNA loading. Each lane is an independently prepared RNA sample from a unique tissue pool.

Hematological and immune parameters in intact Nkx2-3−/− mice

Given the obvious abnormalities in splenic morphology in the Nkx2-3−/− mice and the importance of the spleen in immunological and hematological homeostasis, we examined parameters associated with these processes. We measured serum Ig levels in mice that had not been experimentally immunized and found that they did not differ significantly from controls (data not shown). In comparison with their littermates, Nkx2-3−/− mice were anemic but had a markedly raised white cell count (control: 8.0 ± 3.3, n = 20; Nkx2-3−/−: 30.7 ± 18.6, n = 13). Differential counts revealed that in Nkx2-3−/− mice, circulating lymphocytes, neutrophils, eosinophils, and monocytes were all significantly increased in comparison with Nkx2-3+/+ and Nkx2-3+/− mice (Fig. 3⇓). Absolute numbers and differential counts of nucleated bone marrow cells did not differ between wild-type and mutant mice. To investigate further the blood leukocytosis in Nkx2-3−/− mice, bone marrow and preprogenitor cells and progenitor cells were assayed in semisolid agar cultures stimulated as described in Materials and Methods. The number of bone marrow-derived colony-forming cells (CFCs) did not differ in Nkx2-3−/− mice (e.g., IL-3-stimulated myeloid CFCs, 62.5 ± 2 per 25,000 cells, n = 2) compared with littermate controls (57.5 ± 1, n = 2). Differential colony counts did not reveal significant differences in the percent of colony types in the two groups (data not shown). In keeping with the reduction in spleen size in the Nkx2-3−/− mice, total nucleated cells were reduced 10-fold, and there was also a reduction in the frequency of CFCs in the spleens of Nkx2-3−/− mice (e.g., IL-3-stimulated myeloid CFCs, 0.8 ± 1, per 50,000 cells, n = 2) compared with littermate controls (8.0 ± 4, n = 2). CFU-S day 12 are a population of stem cells with capabilities of self-renewal and generation of multilineage precursors (32). The number of CFU-S per 75,000 nucleated bone marrow cells did not differ between control and mutant mice (wild-type: 7.8 ± 3, n = 8; Nkx2-3−/−: 8 ± 2, n = 8).

FIGURE 3.
FIGURE 3.

Differential blood cell counts. Peripheral blood from 20 control and 13 Nkx2-3−/− mice was examined. The number of all cell types was significantly elevated in knockouts compared with littermate controls (∗∗, p < 0.001), although the relative ratios remained normal.

Examining the frequency of B and T lymphocytes in several lymphoid organs revealed a number of significant differences between knockout and control mice (Fig. 4⇓). The bone marrow of Nkx2-3−/− mice contained significantly more IgM+ B cells than did the bone marrow of controls. Partitioning IgM+ bone marrow B cells into immature (B220lowIgMlow), transitional (B220lowIgMhigh), and recirculating (B220highIgMlow) subpopulations (33) revealed significant increases in the latter two compartments. In blood, Nkx2-3−/− mice again displayed an increased proportion of B cells although the difference failed to reach significance. The mesenteric lymph nodes of Nkx2-3−/− mice were found to contain a significantly increased proportion of B lymphocytes compared with controls, perhaps as a consequence of the failed Peyer’s patch development in these mice. The representation of conventional (B2) B lymphocytes in peritoneal lavage (PerC) was increased significantly in mutant mice compared with controls. B1 cells were infrequent in both strains and the observed differences were not significant (data not shown). Analysis of the lymphocyte populations in spleen confirmed the virtual absence of B cells with an MZ phenotype (24) and revealed a fivefold reduction in the percentage of cells with a transitional B cell phenotype (34). B cells with a follicular phenotype (CD23+) were present at an essentially normal frequency in the spleens of Nkx2-3−/− mice, despite an average overall 20% reduction in B cell representation in knockout spleens (Fig. 4⇓).

FIGURE 4.
FIGURE 4.

B cell distribution is altered in Nkx2-3−/− mice. Two-color flow cytometric analysis of bone marrow (A), mesenteric lymph node (B), and peritoneal cavity (PerC) (C) of control and knockout mice. Abs used are indicated. D, Three-color analysis of B cell subsets in spleen. Splenocytes were partitioned according to CD23 expression and the distribution of CD21 and IgM assessed on the positive and negative subsets. Transitional 1 (T1) and MZ B cells are boxed with the percentages of gated cells given. Percentages are the average of three mice of each genotype. FACS data are depicted as probability plots with logarithmic scales. E, B cell subpopulation frequencies from control (filled symbols) and knockout (open symbols) mice. Each point is a single mouse. B cell frequencies in PerC are a percentage of recovered lymphocytes as defined by light scatter. Significant differences are indicated (∗, p < 0.01; ∗∗, p < 0.001).

Abnormal structures found in the spleens of Nkx2-3−/− mice are a property of the stromal elements and are not corrected by bone marrow reconstitution

Analysis of intact Nkx2-3−/− mice had revealed a significant alteration in the distribution of B cells, T cells, and FDC-M2+ staining in the spleen as well as abnormalities in B lymphocyte populations in bone marrow, mesenteric lymph nodes, and peritoneum. Given that Nkx2-3 is not expressed in hemopoietic cells (23), these defects are most likely due to abnormalities outside the hemopoietic compartment in the knockout mice. To determine whether this was the case, we performed reciprocal bone marrow reconstitutions between mice homozygous and heterozygous for the Nkx2-3 mutation (extensive analysis revealed no differences between Nkx2-3+/− and Nkx2-3+/+; data not shown). Mice of both genotypes were lethally irradiated and then reconstituted with bone marrow from donors of either the identical or alternate genotype. Immunological parameters found to be abnormal in the intact knockout mice were examined in the recipients 8 wk after reconstitution.

Examination of the lymphoid populations in the bone marrow, spleen, mesenteric lymph nodes, and PerC revealed that the differences observed between intact Nkx2-3−/− and Nkx2-3+/− mice were not a property of the Nkx2-3−/− bone marrow-derived cells (Fig. 5⇓). Thus, reconstituted Nkx2-3−/− mice contained a substantially increased IgMhighB220+ cell population in their bone marrow irrespective of the donor’s genotype. Conversely, the B cell populations in the bone marrow of Nkx2-3+/− hosts did not differ with different donors, except for the pre-B cell compartment that was expanded in recipients of Nkx2-3−/− bone marrow for reasons that remain unclear. Similarly, mesenteric lymph nodes of reconstituted Nkx2-3−/− mice showed increased B cell representation irrespective of the donor, whereas the spleens of the same mice showed reduced B cell representation. There were no differences apparent in the lymphoid representation of peritoneal lavage of the reconstituted mice. Therefore, it remains unclear whether the differences observed in PerC of the intact mice (Fig. 4⇑) represent a long-term effect or whether irradiation has had an equalizing effect on all recipients.

FIGURE 5.
FIGURE 5.

B cell developmental defects in Nkx2-3−/− mice are host dependent. Flow cytometry was used to assess the frequency of B cell subsets in lymphoid tissues of bone marrow-reconstituted mice. A, Representative example of bone marrow is shown using the Abs indicated. FACS data are depicted as probability plots with logarithmic scales. B, The result of analyzing three mice of each donor-recipient pairing is shown. Only two data points are present for the spleens of Nkx2-3−/− recipients because one mouse in each group was asplenic. Architecture of the spleens of bone marrow-engrafted recipients was examined by staining cryosections with anti-B220 (red) and anti-CD3 (blue) (C–F), anti-IgM (red) and anti-IgD (blue) (G–J), and FDC-M2 (red) and anti-B220 (green) (K–N). G and H, MZ indicated by arrows.

We examined by histology the recipients’ spleens to determine whether bone marrow transplantation had rectified problems in the architecture of intact Nkx2-3−/− spleens. Staining frozen sections of spleen from reconstituted mice with anti-B220 and anti-CD3 revealed poor resolution of B and T cell areas in Nkx2-3−/− recipients irrespective of the bone marrow donor, whereas both knockout and wild type-derived lymphocytes were appropriately segregated in the spleens of Nkx2-3+/− recipients (Fig. 5⇑, C–F). Staining for IgM and IgD identified follicular and MZ structures in the spleens of Nkx2-3+/− recipients irrespective of the bone marrow origin, whereas Nkx2-3−/− recipients lacked histologically defined MZ B cell populations (Fig. 5⇑, G–J) irrespective of bone marrow donor’s genotype. Consistent with the poor organization of the splenic white pulp in Nkx2-3−/− recipients, the distribution of FDC-M2 staining remained abnormal irrespective of the genotype of the bone marrow donor (Fig. 5⇑, K–N). Thus, bone marrow transplantation confirms the stromal origin of the microarchitectural defects in the spleens of Nkx2-3−/− mice.

Abnormal T cell-dependent immune responses in Nkx2-3−/− mice

Despite the irregularities apparent in the immune systems of intact Nkx2-3−/− mice, serum Ig levels were approximately normal (data not shown). However, because these titers represent an accumulation of Ig over a lengthy period and because they are regulated at many levels, we wished to determine Ig production in response to defined stimuli. Nkx2-3−/− mice were immunized with either a T cell-dependent (TD) or a T cell-independent (TI) Ag, and specific Ab production was measured. The IgM response of knockout mice to DNP-dextran, a TI Ag delivered i.p., was essentially normal over the time period of the experiment in that the increase in specific Ab titer during this period paralleled that of controls, albeit with a slight delay apparent at day 9 (Fig. 6⇓A).

FIGURE 6.
FIGURE 6.

Nkx2-3−/− mice show an abnormal response to TD but not TI Ags. A, Induction of Ag-specific serum IgM in knockout and control mice at the indicated time points after immunization with DNP-dextran. OD of 405 nM for the 1/10,000 dilution of serum is plotted for each sample at each time point. Filled and open symbols are from control and knockout mice, respectively. B, Serum titers taken at the indicated times after primary immunization with NP-KLH. Titers of total and high-affinity Ag-specific IgG1 are indicated in arbitrary units. Analysis of serum from pre-bleeds revealed no NP-binding IgG1 in all mice. Filled and open symbols are from control and knockout mice, respectively. C–J, Cryosections of spleens from immunized mutant and control mice, taken at day 7 (C–F) or day 14 (G–J) of the response, were stained for IgG1 AFCs with a polyclonal anti-IgG1 serum (C and D), GCs using anti-B220 (blue), PNA (green), and FDC-M2 (red) (E and F), and PNA (blue) and B220 (red) (G–J) with GCs indicated by arrows. Sections from at least three mice of each genotype were examined with identical results.

Immunization of Nkx2-3−/− and Nkx2-3+/− mice with the TD Ag NP-KLH precipitated in alum and injected i.p. induced a higher serum Ab response from controls than from knockout mice at all time points measured (Fig. 6⇑B). Indeed, serum titers of Ag-specific IgG1 increased throughout the period of assessment in controls, whereas knockouts showed either no increase or a slight decrease on the day 7 titers. This was apparent in both total and high-affinity Ab. To investigate this difference, we examined spleen histology at different times after immunization (Fig. 6⇑, C and D). Foci of IgG1 AFCs were clearly visible at the outer PALS in the spleens of control mice immunized 7 days previously. In Nkx2-3−/− mice, however, IgG1 AFCs were not clustered into foci but rather were dispersed. Despite the poor organization of IgG1 AFCs in the knockout spleens, however, the cells clearly existed and therefore were not the cause of the diminished serum response.

The failure of Nkx2-3−/− mice to show affinity maturation of serum Ab during the course of the response (Fig. 6⇑B) suggested a defect in the processes underlying the generation of high-affinity variant B cells. Production of high-affinity Ab in response to TD Ags depends in large part on AFCs derived from the GC reaction and residing in the bone marrow (13). To determine whether GC formation was normal in knockout mice, spleens were examined by histology at day 14 of the primary response. Sections were stained with Abs specific for B220 and FDC-M2 and with the lectin PNA to identify GC B cells (Fig. 6⇑, E and F). As expected, controls showed aggregates of PNA-binding B cells located within B cell areas and clustered above concentrations of FDC-M2 reactive processes. In the knockout spleens, PNA-binding B cell aggregates were rare and did not correspond with concentrations of FDC-M2 staining, nor did they all occur within B cell areas. The extent of the GC deficiency in knockout spleens was examined by staining spleen sections from additional immunized mice with PNA and B220 (Fig. 6⇑, G–J). In control spleens, large clusters of PNA+ cells were seen within the majority of B cell follicles, whereas in Nkx2-3−/− spleens, clusters of PNA+ B cells were rare and small. Thus, GC formation in the spleens of Nkx2-3−/− mice is abnormal and may explain the diminished production of high-affinity Ab.

Discussion

Analysis of Nkx2-3-deficient mice has revealed a critical role for this homeobox transcription factor in spleen development and organization (23, 24). In its absence, B and T cells fail to segregate appropriately in the white pulp, and an MZ does not develop. In this report, we have addressed the cellular basis of this disorganization and the consequences it has on B lymphocyte development and differentiation. We find the poor resolution of B and T cells and the absence of the MZ to depend on Nkx2-3-deficient stromal elements in the spleen and not on bone marrow-derived cells. Interestingly, the disorganization of the splenic white pulp is not due to a failure of FDCs to develop, nor to a failure to express the genes encoding the chemokines responsible for the distribution of lymphocytes in the periphery (7). We noted also abnormalities in B cell development in the bone marrow, spleen, mesenteric lymph nodes, and PerC of Nkx2-3−/− mice, some of which may be secondary to the defects in the spleen. Finally, we observed defects in TD immune responses in that immunized Nkx2-3−/− mice showed a diminished Ab response with no apparent affinity maturation. Histological analysis of the spleens of immunized mice revealed poorly developed GCs and inappropriately positioned AFCs in knockout compared with control spleens. Collectively, our data identify a role for Nkx2-3 in establishing the correct environment for normal B cell development and differentiation.

The lymphoid disorganization in the spleens of Nkx2-3−/− mice is reminiscent of mice lacking components of the lymphotoxin and TNF pathways (35). In these mice, FDCs fail to develop from splenic stromal cells, correlating with reduced expression of the chemokine CXCL13 (28) and a consequent inability of B lymphocytes to move out of the T cell areas. Data presented in this report show that, in contrast with these cases, Nkx2-3−/− mice possess FDCs, as defined by expression of CD35 (Fig. 1⇑), and express the chemokine genes required to organize B and T lymphocytes (Fig. 2⇑). Thus, the defects induced by the absence of Nkx2-3 do not appear to relate to the failure of a cell type to develop, but rather to the positioning of cells within the organ. This is apparent in the reduced colocalization of FDCs and B cells (Fig. 1⇑), the scattered distribution of IgG1+ AFCs induced by immunization, and the failure of GCs to develop fully (Fig. 6⇑). These data suggest a role for Nkx2-3 in the patterning of the spleen, a function that would be consistent with the morphogenetic properties of the Nkx2 family of transcription factors in other tissues such as the heart, brain, and lungs (36, 37, 38) and with the extensive expression of Nkx2-3 in the spleen during organogenesis (23).

In this report, we also extend our previous observation of the presence of FDCs in the spleens of Nkx2-3−/− mice (23) to show that the development of these cells is abnormal. Whereas histology with Abs specific for FDC-M1 (23) and CD35 (Fig. 1⇑) reveals essentially normal FDCs in spleen, staining with the mAb FDC-M2 gave a reticular rather than dendritic pattern (Fig. 1⇑). FDC-M2 has been reported to stain CR1/2− cells in normal spleen (31) with a reticular pattern similar to that reported here for the Nkx2-3−/− spleens (Fig. 1⇑). This distribution was interpreted as revealing sites of immune complex capture distinct from FDCs (31). That we find FDCs in the Nkx2-3−/− spleens in the absence of complement-containing immune complexes suggests that the defect in GC development may be mechanical rather than cellular. That is, structural defects in the Nkx2-3−/− spleen may prevent or impede the colocalization of Ag and/or cells necessary to initiate and maintain a TD immune response, possibilities that are currently under investigation. It is important to keep in mind that this deficiency is restricted to spleen, because FDCs in mesenteric lymph nodes of control and knockout mice are indistinguishable (Fig. 1⇑). In the bone marrow transfer studies reported here, we noted the failure of the FDC-M2 epitope to appear in Nkx2-3−/− mice reconstituted with either control or knockout marrow (Fig. 4⇑). This suggests that the immunological defect represented by the absence of FDC-M2 staining is intrinsic to the organ and is not dependent on bone marrow-derived cells.

B cell maturation in the absence of Nkx2-3

There are three major B cell populations identifiable in the spleen: the transitional B cell population, the MZ population, and the follicular B cell population. The spleens of Nkx2-3−/− mice lack an identifiable MZ and, not surprisingly, we find few B cells with an MZ phenotype, confirming previous results (24). The reduced frequency of transitional B cells in the spleens, however, was somewhat unexpected. B cell maturation occurs in defined stages and locations. Immature or transitional B cells emigrate from the bone marrow (33) and are thought to enter the spleen in the MZ, where branches of the central arterioles terminate (1, 3). From here, a fraction of transitional B cells migrate to the follicles and assume the phenotype, lifespan, and recirculating properties of follicular B cells. Maturation from transitional to follicular B cell involves up to three stages (34, 39) and may be selective for particular B cell receptor specificities (40). The sparsity of transitional B cells but essentially normal proportion of B cells with a follicular phenotype in Nkx2-3−/− spleens indicates either that the transition from immature to mature B cell occurs rapidly in the knockouts or, alternatively, that it does not have to occur in the spleen. Finding an elevated frequency of B cells with a transitional phenotype in the bone marrow of knockout mice raises the possibility that at least part of the maturation process could occur therein. The expanded IgMhighB220low population in the bone marrow of knockout mice is not a consequence of a B cell leukocytosis in the blood of these mice because the CD22high population is not grossly overrepresented in the bone marrow of knockouts (data not shown), and this is a reasonable marker of the recirculating B cell population (41). Interestingly, we did not observe B cells with a transitional phenotype in the lymph nodes of the knockout mice (data not shown), indicating that the bone marrow and spleen may be the only permissive environments for such cells.

The expanded B cell population in the mesenteric lymph nodes and PerC of the Nkx2-3−/− mice may be a reflection of the gut abnormalities induced by this mutation. The knockout mice fail to develop Peyer’s patches and have a shortened small intestine with malabsorption. This may place a greater reliance on the immunological capabilities of the mesenteric nodes and may expand the population of peritoneal B1 cells. Bone marrow reconstitution experiments support this hypothesis by revealing a requirement for an Nkx2-3−/− host for the B cell expansion to occur in mesenteric nodes. The B cells in the lymph nodes of both intact and reconstituted mice have the appearance of being resting and naive. They are small, nondividing, and express IgM and IgD, all indicating that they are not actively engaged in an immune response. So, if the B cell expansion in these sites is secondary to the abnormalities in gut development, it is an accumulation of resting cells rather than cells actively engaged in immune responses.

Immune responsiveness of Nkx2-3−/− mice

Splenic organization is central to the efficient functioning of the immune response. In instances where B and T cells fail to segregate appropriately, immunization fails to induce appropriate GC formation and, consequently, has an impact on affinity maturation (17), although this is not always the case (19). Immunization of Nkx2-3−/− mice with a TD Ag via i.p. injection, a route that drains primarily to the spleen, induced substantially less Ab production than did controls. Histological analysis indicated that both the extrafollicular and intrafollicular pathways of B cell differentiation were affected. Early in the response, IgG1 AFCs were detectable in the spleens of knockout mice, but they were not clustered into foci in the outer PALS (Fig. 6⇑), although CXCL12 mRNA was clearly present (Fig. 2⇑). Similarly, PNAhigh clusters of B cells develop in the spleens of Nkx2-3−/− mice, but they are small and are not associated with FDC-M2+ cells, as is the case in controls (Fig. 6⇑). This difference may explain the failure of knockout mice to produce AFCs secreting high-affinity Ab. Alternatively, the abnormal architecture of the spleen may preclude the appropriate localization and/or migration of such cells and therefore may prevent their accumulation to any significant degree, despite the expression of the chemokines thought to be necessary for this process. Despite the absence of a histological MZ or B cells with an MZ phenotype, Nkx2-3−/− mice responded to TI immunization, a result that apparently contradicts the proposal that such responses are dependent on this structure (42). The presence or absence of this structure, however, is not always predictive of TI responsiveness as exemplified by Bruton’s tyrosine kinase-deficient and Rac2-deficient mice, which possess and lack MZ B cells, respectively, yet do not and do respond to TI-2 Ags (43, 44, 45). Furthermore, the route of immunization (i.p. vs i.v.) and the activity of B1 B cells may also confuse the interpretation (46). A final complication in the assessment of the immunological activity of the MZ is the fact that many mutations affecting the formation of the MZ also affect B cell responsiveness to external signals. This is the case, for example, for CD22 (47), Lsc (48), Pyk (42), Lyn (49), and Aiolos (43). Thus, the responsiveness of Nkx2-3−/− mice to TI-2 immunization may have several explanations that will require additional investigation to identify and clarify.

Concluding remarks

Organization of secondary lymphoid organs is critical to their efficient functioning in the immune system. To date, studies of the mechanism underlying lymphocyte organization have focused primarily on chemokines and their receptors in positioning lymphocytes both before and during immune responses. In this report, we present data suggesting that elements in addition to chemokines yet derived from the stroma and regulated by Nkx2-3 are critical for the correct organization of lymphocytes in the spleen. Further investigation of these factors may allow the requirements for normal stromal cell positioning and development in the spleen, an area that is currently poorly understood.

Footnotes

  • ↵1 This work was supported by the National Health and Medical Research Council of Australia. L.R. is a fellow of the Sylvia and Charles Viertel Foundation and D.T. is a fellow of the National Health and Medical Research Council of Australia.

  • ↵2 Address correspondence and reprint requests to Dr. David Tarlinton, Immunology Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Victoria, Australia. E-mail address: tarlinton{at}wehi.edu.au

  • ↵3 Abbreviations used in this paper: PALS, periarteriolar lymphoid sheath; MZ, marginal zone; CXCL, CXC chemokine ligand; CCL, CC chemokine ligand; GC, germinal center; FDC, follicular dendritic cell; AFC, Ab-forming cell; PNA, peanut agglutinin; NP, 4(hydroxy-3-nitrophenyl)acetyl; KLH, keyhole limpet hemocyanin; m, mouse; CFU-S, CFU-spleen; CFC, colony-forming cell; PerC, peritoneal cavity; TD, T cell dependent; TI, T cell independent.

  • Received April 8, 2002.
  • Accepted February 6, 2003.

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