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The Journal of Immunology, 1998, 161: 49-59.
Copyright © 1998 by The American Association of Immunologists

Requirements for B7-CD28 Costimulation in Mucosal IgA Responses: Paradoxes Observed in CTLA4-H{gamma}1 Transgenic Mice1

Eva Gärdby*, Peter Lane{dagger} and Nils Y. Lycke2,*

* Department of Medical Microbiology and Immunology, University of Goteborg, Goteborg, Sweden; and {dagger} Department of Immunology, University of Birmingham Medical School, Birmingham, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The block in the CD80/CD86-CD28/CTLA-4 pathway in CTLA4-H{gamma}1 transgenic (Tg) mice results in strongly impaired systemic IgG immunity and failure to develop germinal center reactions. By contrast, here we report that mucosal immunity and IgA B cell differentiation are not affected by this block. We found abundant germinal centers and evidence of IgA switch differentiation in Peyer’s patches, normal total IgA levels, and normal numbers of IgA-labeling cells in the gut mucosa. The distribution of B-1 and B-2 cells and the relative contribution of B-1 cells to the total IgA B cells were similar in Tg and wild-type mice. Despite this, oral immunizations with keyhole limpet hemocyanin plus cholera toxin adjuvant failed to stimulate Ag-specific mucosal IgA responses in CTLA4-H{gamma}1 Tg mice. This was not due to a lack of adjuvant activity of cholera toxin in Tg mice, nor was this secondary to an inability to take up Ag from the gut lumen. Rather, CD4+ T cells stimulated by oral immunization in Tg mice appeared to be inappropriately primed, as evidenced by a significantly reduced level of CD40 ligand and CD44 expression and an increased expression of CD95 compared to those in wild-type mice. This study reveals a paradox in the regulation of mucosal IgA responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ahallmark of mucosal immunity is the predominance of IgA Ab formation. In fact, approximate calculations have indicated that a majority of B cells that undergo isotype-switch differentiation will become IgA-producing cells at various mucosal surfaces (1). One of the most active sites for IgA production is the intestinal lamina propria (LP)3 and the gut-associated lymphoid tissues (GALT) (1). Despite much recent progress in the field of mucosal immunology we still lack fundamental information on factors that govern intestinal immune responses and IgA B cell differentiation, in particular.

Our current understanding of IgA B cell differentiation ascribes a central role to the CD4+ T cell as a provider of cytokines as well as cell-cell membrane interactions (2). Using IL-4 knockout mice we recently demonstrated that Th2 activity is important for induction of intestinal immune responses following oral immunizations (3). The Th2 cytokines associated with IgA B cell differentiation are IL-5 and IL-6, which act on already IgA-switched cells, while TGF-ß, probably IL-10, and perhaps IL-4 affect switching from IgM to IgA (2). The dependency on CD4+ T cells also heavily relies on membrane interactions, and the CD40L has been found to be a key factor in controlling IgA B cell differentiation. Concomitant stimulation of B cells with cytokines and CD40L may both initiate IgA switching in naive IgM+/IgD+B cells as well as promote terminal differentiation of IgA+ B cells (4, 5, 6, 7, 8, 9, 10) It is, perhaps, this latter effect that is the most striking example of a strong influence of cognate interaction with CD4+ T cells for mucosal IgA production (8).

Cross-linking of the CD40 molecule on the B cell not only promotes isotype-switch differentiation and cell proliferation, but counteracts apoptosis and is required for memory development in activated B cells (11, 12, 13). Another critical function of the CD40L interaction is the induction of costimulatory molecules, CD80, CD86 and others, on the B cells (14, 15, 16). Thereby, CD40 engagement of the B cell may also affect CD28-dependent as well as CD28-independent signaling in the T cells (16, 17, 18). Such a bidirectional effect of CD40-CD40L interaction on T and B cell development (19, 20, 21) is supported by the recent observations that CD40L expression may be especially critical for Th2 differentiation of CD4+ T cells via the CD86-CD28 costimulatory pathway (22, 23, 24, 25, 26). Whether these costimulatory molecules play a regulatory role in mucosal IgA responses has hitherto not been investigated.

Isotype-switch differentiation and clonal expansion of Ag-activated B cells occur in the germinal centers (GC) within primary follicles of lymphoid tissues (27, 28). The GC reaction itself is under strict CD4+ T cell control, and with few exceptions, only T cell-dependent (TD) Ag can stimulate significant GC development (27, 28). Both CD40L and CD28 interactions by activated CD4+ T cells have been found to control the GC reaction, and mice deficient in these signaling systems lack GC in spleen and peripheral lymph nodes (29, 30, 31, 32). GC formation and intestinal IgA B cell switch differentiation are evident in the Peyer’s patches (PP) and follicles of the GALT (33). These IgA-committed GC cells are believed to be the main progeny of the intestinal IgA response (34). Additional LP IgA precursor cells may be contributed by B-1 cells enriched in the peritoneal cavity, but the extent to which this occurs is poorly understood (35).

The development of a normal PP morphology is dependent on local exposure to antigens, since gnotobiotic or neonatal mice have few IgA-producing LP B cells and poorly developed PP (36). In conventionally reared animals and even without specific immunization, PP, in contrast to spleen and lymph nodes, exhibit GC reactions, demonstrating the continuous presence of foreign Ag at this site (37). In the PP, the GC contains 75 to 80% IgA+ B cells, whereas in peripheral lymph nodes, very few IgA+ B cells are seen (37). Therefore, it is thought that the GC in PP differ intrinsically from those of spleen and lymph nodes in their content of particular kinds of APC, stromal cells, or regulatory CD4+ T cells (2, 34). Many of the mutant mice recently generated via gene targeting have provided new insights into factors required for GC formation in spleen and peripheral lymph nodes (38). While this knowledge applies to systemic IgG responses, our own experience using IL-4 and CD4-targeted mice have provided conflicting information with regard to IgA B cell differentiation, indicating that gut mucosal immune responses may be differently regulated compared with systemic responses (3, 39).

The present study was undertaken to investigate to what extent the CD80/86-CD28/CTLA4 pathway of interaction is critical for mucosal immunity and IgA B cell differentiation. To this end we employed the mCTLA4-H{gamma}1 transgenic (Tg) mice that carry the mCTLA4-H{gamma}1 transgene under the Ig heavy chain promoter (40, 41). The mice stably express the protein, which binds to CD80 and CD86 and blocks the CD80/86-CD28/CTLA4 pathway (42). Recently, Tg mice were demonstrated to have poor responsiveness to TD Ag and impaired IgG class switching and to lack GC reactions in spleen and peripheral lymph nodes following systemic immunizations (41). Preliminary observations made by these researchers indicated that these mice might be differently responsive at mucosal surfaces and that IgA B cell differentiation was differently regulated compared with systemic IgG responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male mCTLA4-H{gamma}1 Tg mice were bred to C57BL/6 female mice under pathogen-free conditions using ventilated microisolator cages and sterile workbenches at the Department of Medical Microbiology and Immunology, University of Goteborg (Goteborg, Sweden). The progeny was tested at 4 to 6 wk of age for the presence of the transgenic product, mCTLA4-H{gamma}1; the plasmid is encoding murine CTLA4 and the human IgG1 constant domains (H{gamma}1) (40). Its transcription is driven from the Ig heavy chain promoter, and the protein is secreted as a soluble dimer. A routine ELISA was used to identify the Tg mice by detection of H{gamma}1 in serum. The mice stably expressed 10 to 30 µg/ml of mCTLA4-H{gamma}1 in serum. All experiments were conducted with sex-matched 8- to 14-wk-old mice. C57BL/6 littermates were used as wild-type (wt) control mice.

Immunizations

Mice were given three oral immunizations with KLH (Sigma, St. Louis, MO) at 2.0 mg/dose in the presence or the absence of 10 µg/dose of cholera toxin (CT; List Biological Laboratories, Campbell, CA) as previously described (43). Systemic immunizations in the presence or the absence of 1 µg of CT adjuvant were given twice by i.p. injections of KLH at 100 µg/dose or of DNP-KLH at 200 µg/dose or once by a single injection with TNP-LPS at 10 µg/dose. Six to eight days following the final immunization mice were sacrificed, and the immune responses were analyzed.

Preparation of lymphoid cells

Spleen, mesenteric lymph node (MLN), and PP lymphoid cells were prepared by teasing the tissue through a nylon screen. RBC were lysed by osmotic shock, and single-cell suspensions were prepared and washed twice in Ca2+- and Mg2+-free HBSS (Life Technologies, Paisley, Scotland) containing 10% FCS (Life Technologies). Peritoneal cavity (PerC) cells were obtained by flushing the peritoneum with fresh chilled HBSS (Life Technologies) supplemented with 5% FCS. Intestinal lamina propria lymphocytes were prepared as previously described (44).

Adoptive transfer experiment

C57BL/6 nu/nu mice were injected i.v. with 2.4 x 106 purified MLN CD4+ T cells from sex-matched mCTLA4-H{gamma}1 Tg or wt mice that had been previously primed twice by oral immunizations with KLH plus CT adjuvant. Highly enriched CD4+ T cells were prepared from single-cell suspensions of MLN lymphocytes by depleting B cells and CD8+ T cells using anti-B220 (mouse Pan B) and anti-CD8 (mouse CD8, Lyt2) Dynabeads (Dynal, Oslo, Norway). The beads were adjusted to a final density of five beads per cell diluted in PBS followed by magnetic adherence, according to the manufacturer’s instructions. The enriched CD4+ T cell preparation was 90 to 95% pure as determined by FACS analysis after labeling with the appropriate Abs (see below; Becton Dickinson, Mountain View, CA). Two days after the adoptive transfer, the recipient nu/nu mice were immunized i.p. with 100 µg of DNP-KLH in RIBI-adjuvant system (RAS) (TriChem ApS, Copenhagen, Denmark), and 14 days later they were boosted with 100 µg of DNP-KLH in PBS. Serum was collected 9 days after the priming as well as after the booster immunization and analyzed for DNP-specific serum IgG Abs by ELISA.

FACS analysis

Freshly isolated PP, LP, MLN, and PerC lymphocytes were washed twice in cold 0.1% BSA in PBS (BSA/PBS) buffer, and the density was adjusted to 5 x 106 cells/ml. To each tube containing 100 µl of cell suspension we added 1 µl of the 24G2 FcR-blocking Ab (PharMingen, San Diego, CA), and the tube was incubated for 5 to 15 min at 4°C. Phenotypic analysis was then performed by single or double labeling using FITC- or PE-conjugated anti-mouse Abs specific for CD5, IgM, IgA, Mac-1, B220, CD3, CD4, CD8, {alpha}ßTCR, CD95, CD44, CD40, CD40L (all Abs from PharMingen), or biotin-conjugated goat anti-mouse IgA or IgM (Southern Biotechnology, Birmingham, Al) used together with streptavidin-phycoerythrin (Dako, Glostrup, Denmark). For detection of germinal center B cells we used FITC-labeled peanut (Arachis hypogaea) hemagglutinin (PNA-FITC; Sigma). The Abs were diluted to a final concentration of 5 to 10 µg/ml in BSA/PBS, and the cells were incubated for 30 min on ice. After thorough washing in BSA/PBS, phenotypic analysis was performed using a FACScan analyzer (Becton Dickinson, Mountain View, CA). Gates were set on live cells, excluding dead cells and debris by forward and side scatter light emission. For determinations of frequencies of B-1 and B-2 cells and extended analysis of CD4+ T cells, 10,000 events were registered using a live gate, and the fluorescence profiles were analyzed by quadrant and histogram representations. All experiments were performed at least three to five times.

ELISPOT assay

Spleen and LP lymphocytes were analyzed for specific Ab production at the single-cell level using the ELISPOT assay as previously described in detail (45). Briefly, anti-KLH, anti-DNP, or anti-CT spot-forming cells (SFC) were determined using petri dishes (Nunc, Roskilde, Denmark; KLH- and CT-specific SFC) or nitrocellulose HA plates (Triton-free mixed cellulosa, 0.45 µm pore size) (Millipore, Boston, MA; DNP-specific SFC) that were coated with 100 µg/ml KLH, 200 µg/ml DNP-OVA, or 3 nmol/ml ganglioside GM1 followed by 3 µg/ml of CT at 4°C overnight. To duplicate petri dishes we added 400 µl/well of cells at 106 cells/ml in complete Iscove’s medium containing 10% FCS in duplicate dishes. To triplicate nitrocellulose HA wells we added 100 µl/well of cells at 4 x 106 cells/ml, and twofold serial dilutions in corresponding subwells were performed. The cells were allowed to incubate for 2.5 h (petri dishes) or 4 h (HA plates) at 37°C. The SFC activity was visualized using HRP-conjugated rabbit anti-mouse Ig (spleen SFC; Dako) or goat anti-mouse IgA (lamina propria lymphocyte SFC; Cappel, Organon Teknika, West Chester, PA) followed by HRP-conjugated anti-goat Ig (Dako). The SFC reaction was developed by adding the HRP substrate, paraphenylenediamine (0.5 mg/ml) and 0.01% H2O2 in 1% agar in PBS, which was applied as a thin film. For nitrocellulose HA plates we added the substrate (10 mg/ml 3-amino-9-ethylcarbazole (AEC) in citrate buffer and 0.01% H2O2) for 5 to 7 min before rinsing the plates in water.

Serum and gut lavage ELISA

The mice were killed, and gut lavage and serum were collected as described in detail previously (39, 46) and stored at -70 and -20°C, respectively, until analyzed. Specific or total IgA concentrations were determined by ELISA. Polystyrene microtiter plates (Nunc) were coated with unlabeled rabbit anti-mouse IgA Abs (PharMingen) for assaying total IgA or with KLH or DNP-OVA at 100 µg/ml or GM1 ganglioside (0.5 nmol/ml) (Sigma) followed by CT (0.5 µg/ml; List) for detection of Ag-specific responses. Sera or lavage at 1/100 dilution were added, and serial threefold dilutions were performed in corresponding subwells. The plates were incubated at 4°C overnight. Total Ig- and isotype-specific Abs were visualized with HRP-conjugated anti-mouse Ig (Dako) at a 1/200 dilution or with alkaline phosphatase-conjugated isotype-specific rabbit anti-mouse Abs (Southern Biotechnology) at a 1/500 dilution. Ag-specific HRP-labeled Abs bound to the plate were visualized using ortho-paraphenylenediamine (1 mg/ml)-0.04% H2O2 substrate in citrate buffer, and the reaction was read at 450 nm using a Titer-Tek Multiscan MS spectrophotometer (Labsystems, Stockholm, Sweden). Alkaline phosphatase-labeled Abs were visualized using phosphatase substrate tablets. Nitrophenyl substrate (Sigma) at 1 mg/ml in ethanolamine buffer (pH 9.6) was added to the wells, and the reaction was read at 405 nm in the Multiscan MS spectrophotometer. Ab titers were defined as the interpolated OD reading giving rise to an absorbance of 0.4 above background, which consistently gave OD readings on the linear part of the curve. Titers were given as the log10 mean ± SD. Total IgA in micrograms per ml was calculated from a standard curve generated by serial dilutions of purified hybridoma IgA protein (PharMingen) of known concentration. The isotype-specific antisera were highly specific and did not cross-react with purified proteins of the other isotypes.

Immunohistochemistry

Frozen sections (5 µm) from unimmunized or KLH- and CT adjuvant-primed C57BL/6 or CTLA4-H{gamma}1 Tg mice were prepared on microslides using a cryostat (model 1720, Leitz, Wetzlar, Germany) and were frozen at -70°C. The slides were fixed in 50% acetone for 30 s followed by 100% acetone for 5 min at 4°C. After washings in PBS, the slides were treated with 5% horse serum in PBS for 15 min in a humid chamber. For detection of GC reactions and IgA+ cells in the GALT, fixed sections were double labeled with FITC-conjugated PNA (Sigma) and Texas Red-conjugated anti-mouse IgA (Southern Biotechnology Associates), both at a 1/100 dilution. The slides were mounted with fluorescence mounting medium (Dako). For detection of the CTLA4-H{gamma}1 protein in tissue, sections were incubated with 0.3% H2O2 for 5 min followed by rinsing in PBS containing 5% horse serum. Thereafter, biotin-labeled anti-human IgG1 was added for 30 min followed by peroxidase-conjugated avidin-biotin complexes (Dako) for an additional hour at room temperature. The CTLA-4 protein was visualized by addition of the peroxidase AEC substrate (Sigma; 10 mg AEC in 6 ml DMSO mixed with 50 ml Na-acetate and 4 N H2O2). After 15 min the tissue was washed, counterstained with HTX (HistoLab Products, Goteborg, Sweden), and mounted in Aquamount (BDH Laboratory Supplies, Poole, U.K.). Sections were evaluated and photographed using DAS Mikroskop, LEICA DMLD (Leica Microscope System, Welzar, Germany).

Analysis of T cell functions

Ag-specific and polyclonal T cell activation was performed on primed or naive cells as indicated. Cells from MLN or PP were cultured at 1 to 2 x 106/ml in complete Iscove’s medium with 10% FCS in 96-well flat-bottom plates (Nunc) in the presence or the absence of anti-CD3 or anti-CD3 + CD28 (PharMingen) at 5 µg/ml or endotoxin-free KLH (Calbiochem) at 100 µg/ml. Cell proliferation was analyzed in triplicate wells on day 3 of culture. During the final 6 h of culture 1 µCi of [3H]thymidine (5 µCi/mmol; Amersham International, Aylesbury, U.K.) was added to the cultures as previously described (47). Values obtained as were the mean counts per minute ± SD of triplicate wells. In parallel cultures we determined cytokine production from activated T cells after stimulation with recall Ag or after polyclonal activation as described above. The concentrations of IFN-{gamma}, IL-4, and IL-5 in the supernatants were determined using ELISA as described in detail previously (47). The concentrations of cytokines in the supernatants were expressed as nanograms per ml, as calculated from the plotted standard curves of the recombinant cytokines. The detection levels for the respective assays were: IFN-{gamma}, 0.2 ng/ml; IL-5, 0.2 ng/ml; and IL-4, 40 pg/ml.

TUNEL technique

An apoptosis detection kit (Boehringer Mannheim, Mannheim, Germany) was used according to the manufacturer’s instructions.

Statistical analysis

We used Student’s t test with independent samples for analysis of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Normal total IgA levels in serum and gut lavage in CTLA4-H{gamma}1 transgenic mice

Whereas earlier studies in CTLA4-H{gamma}1 Tg mice have clearly shown that a block of the costimulatory pathway between CD80/86 and CD28/CTLA4 dramatically inhibited systemic IgG responses and abolished GC formation, there is no information available about mucosal immune responses and IgA B cell differentiation (40, 41). Since IgA B cell responses and isotype-switch differentiation are thought to be critically dependent on CD4+ T cell help, one would expect to find low levels of IgA in Tg mice (2). On the contrary, we found that both serum and gut lavage contained normal levels of total IgA compared with those in wt mice (Fig. 1Go, A and B). Moreover, IgA labeling of frozen sections of gut mucosa revealed normal levels of IgA plasma cells in the lamina propria of naive CTLA4-H{gamma}1 Tg mice (Fig. 1GoC). In PP, the inductive sites for intestinal IgA responses, we noted large numbers of GC to which membrane IgA+ B cells colocalized, suggesting isotype-switch differentiation in situ from IgM to IgA (Fig. 2Go). Thus, despite impaired systemic responses and lack of GC in peripheral lymph nodes and spleen (41), IgA B cell differentiation and GC formation in mucosal lymphoid tissues appeared unaffected in CTLA4-H{gamma}1 Tg mice.



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  		FIGURE 1. Normal levels of total IgA in CTLA4-H{gamma}1 Tg mice. The total IgA concentration in serum (A) or gut lavage (B) was determined in CTLA4-H{gamma}1Tg (open symbols) or wild-type (closed symbols) mice. In serum the level of IgA was compared with that of total IgG1 or IgG2a subclasses (A). The values in A and B represent mean log10 titers ± SD of six to eight mice in each group taken from one representative experiment of at least three that produced similar results. In C we have illustrated the distribution of IgA-containing cells in the small intestinal bowel of naive CTLA4-H{gamma}1 Tg mice. This was similar to that found in wild-type control mice. Ten visual fields of small intestine were counted for presence of IgA-containing cells per animal, and at least three mice were analyzed per group.

 


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  		FIGURE 2. IgA+ and PNA+ germinal center B cell areas in PP of CTLA4-H{gamma}1 Tg mice. Light level micrographs demonstrating the presence of a GC (upper panels) in PP from a naive CTLA4-H{gamma}1 Tg (right side) and a wild-type (left side) mouse (upper panels). Frozen sections of PP were stained with PNA-FITC (brightly green) and were double labeled with Texas Red-labeled anti-IgA Abs (brightly red), showing colocalization of IgA+ B cells (middle panels) to the PNA+ GC area. The presence of the mCTLA4-Hg1 protein in PP sections from Tg mice, but not in PP from wt control mice, was demonstrated by labeling a consecutive section with biotin anti-human IgG1 followed by HRP-conjugated avidin (lower panels). The brown-red staining depicts B cells producing and follicular dendritic cells binding the mCTLA4-H{gamma}1 protein. These results are representative of four analyses performed on separate occasions with two to four mice at each time. Sections of peripheral lymph nodes or spleen from these mice did not show labeling with PNA.

 
To exclude the simple explanation that the CTLA4-H{gamma}1 protein was not expressed in PP, as opposed to peripheral lymph nodes, we incubated tissue sections with peroxidase-conjugated anti-human IgG1. Immunohistochemical analysis demonstrated strong and comparable expression of the CTLA4-H{gamma}1 protein in PP (Fig. 2Go) as well as in peripheral lymph nodes of CTLA4-H{gamma}1 Tg mice. No labeling was observed in wt mice (Fig. 2Go).

Distribution of B-1 and B-2 cells in the gut immune system of CTLA4-H{gamma}1 transgenic mice

Two populations of IgA B cells in the LP have been identified: those migrating from the inductive sites, primarily the PP, and those coming from the PerC (34, 35). Whereas the PP provides conventional B-2 cells, the PerC contributes largely B-1 cells. In mice, the relative proportion of the latter population to the total number of gut LP IgA cells has varied in different studies from low to almost 50% in chimeric or transgenic models (48, 49, 50, 51). Phenotypic analysis by FACS of B-1 and B-2 cells in the LP revealed similar patterns in CTLA4-H{gamma}1 Tg and wt mice (Fig. 3Go). Both strains hosted comparable frequencies of LP IgM+-bright cells, and the fraction of IgM+ cells expressing CD5 (B-1a cells) or Mac1 (B-1b cells) was low in both strains. Likewise, low and comparable frequencies of LP IgA+ cells expressing CD5 or Mac1 were found in Tg and wt mice. Extended analysis using live gates set at 10,000 IgM+ or IgA+ LP B cells demonstrated similar levels of Mac1+ or CD5+ expression in both strains.



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  		FIGURE 3. Low distribution of B-1 cells in the gut LP in CTLA4-H{gamma}1 Tg mice. A FACS analysis of the distribution of B-1 cells in the gut LP of CTLA4-H{gamma}1 Tg and wt mice was performed. Isolated lymphocytes in single-cell suspensions from three mice were double labeled with PE-conjugated anti-IgM or anti-IgA Abs in combination with FITC-conjugated anti-CD5 or anti-Mac1. The IgM+ CD5+ frequency was higher in wt than in Tg mice, and the mean values were significantly different (p < 0.05). No difference in the distribution of B-1b cells (Mac1+; upper right quadrants) between Tg and wt mice was found. The dot blots depict the results of one representative analyses of at least three that produced similar results.

 
As illustrated in Table IGo, the distribution of B-1 cells in other compartments of the GALT gave a comparable pattern in Tg and wt mice. In both strains and only in the PerC B cell population did we observe a predominance of B-1 cells, while the PP, similar to the LP, contained a majority of B-2 cells and only few B-1 cells (Table IGo). Therefore, the distribution of B-1 and B-2 cells in the GALT and the contribution of B-1 cells to the total production of IgA in LP appeared to be low and similar in both Tg and wt mice. In conclusion, this analysis suggested that most of the IgA in the LP was produced by B-2 cells, probably derived from precursor cells in the PP or other inductive sites.


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  		Table I. Distribution of B-1 and B-2 cells in the GALT of CTLA4-H{gamma}1 transgenic mice1

 
Paradoxically impaired mucosal IgA responses to oral immunizations in CTLA4-H{gamma}1 transgenic mice

As reported above, contrary to systemic IgG responses, local mucosal immunity and IgA B cell differentiation appeared unaffected by the block in the CD80/86-CD28/CTLA4 pathway. To further test the validity of this observation, we performed oral immunizations with the soluble protein KLH, given together with CT adjuvant. However, we found no or poor LP IgA SFC responses in Tg mice to either CT or KLH as opposed to the strong responses observed in wt control mice (Fig. 4Go). Also, splenic SFC responses following oral immunizations were undetectable or low (not shown). Interestingly, the splenic SFC activity against CT was undetectable, while that against KLH was weak but, nevertheless, clearly detectable in the Tg mice. The poor responsiveness to oral immunizations was also reflected in the anti-KLH-specific serum response, which was at least 10-fold weaker in Tg compared with wt mice (not shown). Reduced titers were observed for all IgG isotypes as well as for IgA, suggesting that the impaired responsiveness did not selectively affect Th1 or Th2 CD4+ T cell functions. In agreement with the lack of a splenic anti-CT SFC response, no serum response to CT could be detected.



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  		FIGURE 4. Impaired intestinal IgA responses in CTLA4-H{gamma}1 Tg mice following oral immunization. Mice were given three oral doses with KLH plus CT adjuvant, and their gut LP CT-specific (A) and KLH-specific (B) IgA SFC responses were assessed 8 days after the final dose. The mucosal IgA responses in the CTLA4-H{gamma}1 Tg (open bars) and wild-type (closed bars) mice were compared. Specific SFC per 107 isolated lymphocytes are expressed as the mean ± SEM of three experiments. The differences in SFC activity between Tg and wt mice were statistically significant: A, p < 0.02; and B, p < 0.01.

 
CT exhibits potent adjuvant effects on systemic immune responses in CTLA4-H{gamma}1 transgenic mice

To investigate whether the impaired mucosal responsiveness to oral immunizations was secondary to a defect in the adjuvant capacity of CT in the Tg mice, we challenged animals with KLH systematically, i.e., i.p., in the presence or the absence of CT adjuvant. We found that serum responses to KLH alone were strongly reduced in CTLA4-H{gamma}1 Tg mice, in agreement with previous reports (41). In particular, IgG2a and IgG2b, but also IgG1, serum responses were reduced in the CTLA4-H{gamma}1 Tg mice (Fig. 5GoA). In contrast, in the presence of CT adjuvant, significant increases (p < 0.05) in serum responses were recorded, clearly demonstrating a functional adjuvant effect of CT in CTLA4-H{gamma}1 Tg mice (Fig. 5GoB). The Tg mice exhibited roughly equivalent enhancement (fold increase) in specific serum IgG-subclass titers as the wt mice, although the total response in each isotype was significantly lower than that in the wt mice (Fig. 5GoB). In fact, Tg mice immunized with KLH and CT adjuvant exhibited IgG subclass titers close to those observed in wt mice immunized with KLH alone. The response to CT per se in Tg mice was weak or undetectable in all isotypes analyzed (Fig. 5GoA), while the anti-CT response in wt mice was strong and >10-fold greater than that to KLH (Fig. 5GoA). Thus, CT exerted its adjuvant functions despite the lack of an immune response to itself, suggesting that the adjuvant effect of CT may not be directly linked to its immunogenic properties.



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  		FIGURE 5. CT exerts potent systemic adjuvant function in CTLA4-H{gamma}1 Tg mice. A, Distribution of anti-KLH Ab titers in different isotypes in individual mice after i.p. priming and booster immunizations of CTLA4-H{gamma}1 transgenic (open symbols) and wild-type (closed symbols) mice with KLH alone (diamonds). In this panel we have also included mean anti-CT titers (circles) in the various isotypes following i.p. immunizations with KLH plus CT. B, Illustrates the fold increase in various isotypes of anti-KLH Abs as a consequence of the addition of CT adjuvant to the i.p. immunization protocol in CTLA4-H{gamma}1 transgenic (open symbols) and wild-type (closed symbols) mice. The results are representative of two experiments and are expressed in log10 titers as the mean ± SD.

 
An extended analysis of the adjuvant effect of CT in CTLA4-H{gamma}1 Tg mice using hapten-specific Abs as markers of immune responsiveness to systemic immunizations with TNP-LPS (T cell-independent system (TI Ag)) or DNP-KLH (T cell-dependent system (TD Ag)) revealed that CT adjuvant can enhance both types of responses. However, while T-independent IgM responses were augmented by three- to fourfold in both strains, the T-dependent IgG1 response to DNP-KLH appeared more strongly enhanced in wt than in Tg mice (Fig. 6Go). This would be in agreement with our previous findings that CT may enhance humoral immunity by direct effects on B cell differentiation as well as by indirect effects via augmented priming of CD4+ T cell help (47, 52). Moreover, the experiment demonstrated that CTLA4-H{gamma}1 Tg mice are impaired in their responses to TD Ag, whereas their responses to TI Ag are normal (41).



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  		FIGURE 6. CT adjuvant enhances humoral immune responses to both TI and TD Ag. The immune responses to TI (TNP-LPS) and TD (DNP-KLH) Ag after a priming and a booster i.p. immunization in the presence or the absence of CT adjuvant were assessed. The DNP-specific IgM or IgG1 SFC per 107 isolated spleen cells was determined by the ELISPOT technique using DNP-OVA-coated petri dishes. CT significantly increased the immune responses to both TI and TD Ag (p < 0,01). Of note, the increase in TD responses due to CT adjuvant was significantly stronger in wt (closed bars) than in Tg (open bars) mice (p < 0.01). These data represent the mean ± SD of two experiments with five or six mice in each group.

 
Oral immunizations in CTLA4-H{gamma}1Tg mice appear to inappropriately prime CD4+ T cells in organized gut lymphoid tissues

A phenotypic analysis of T cells in the GALT demonstrated that the frequencies of CD3+, {alpha}ßTCR+, CD4+, and CD8+ cells in MLN were unaltered compared with those in wt mice, while CD4+ cells were less frequent in PP of CTLA4-H{gamma}1 Tg mice (not shown). Similar to the results of previous studies, polyclonal activation with anti-CD3 and anti-CD28 Ab ex vivo caused comparable proliferative and cytokine production (IFN-{gamma}) in PP and MLN T cells from CTLA4-H{gamma}1 Tg and wt mice (not shown) (40). Also, the response to recall Ag in vitro by MLN T cells from orally KLH- plus CT adjuvant-immunized CTLA4-H{gamma}1 Tg mice was comparable with or stronger than that observed in wt mice, clearly indicating that the Tg mice were able to take up Ag from the gut and stimulate T cells in the GALT. MLN T cell proliferation was augmented threefold above that in unimmunized controls, while IFN-{gamma} and IL-5 concentrations in supernatants, undetectable in supernatants from unimmunized mice, were 3 and 0.4 ng/ml, respectively.

However, despite the seemingly normal functional data on CD4+ T cells in PP and MLN of CTLA4-H{gamma}1 Tg mice, a phenotypic analysis after a single oral immunization revealed striking differences. The frequency of CD4+ T cells expressing CD40L was consistently lower in Tg mice, in particular in the PP (Fig. 7GoA). In addition, and most evident in PP, we observed significantly lower expression of the CD44 marker on CD4+ T cells in GALT in CTLA4-H{gamma}1 Tg compared with that in the wt mice (Fig. 7GoB). By contrast, CD95 expression (Fas/APO-1) was dramatically higher on CD4+ T cells from CTLA4-H{gamma}1 Tg mice than on that from wt mice. In the latter case not only the expression but also the frequency of MLN CD4+T cells expressing CD95 was higher, with 91 ± 4% in CTLA4-H{gamma}1Tg as opposed to 61 ± 14% in wt mice (Fig. 7GoC). We also observed increased apoptosis (as determined by TUNEL technique) in PP, with roughly twice as many CD4+ T cells undergoing cell death in CTLA4-H{gamma}1 Tg mice (18%) as in the wt control mice (9%) following oral immunization.



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  		FIGURE 7. Oral immunizations in CTLA4-H{gamma}1 Tg mice inappropriately primes CD4+ T cells in the GALT. The effect of a single oral immunization with KLH plus CT adjuvant appeared to inappropriately prime CD4+T cells in the GALT in CTLA4-H{gamma}1 Tg mice. Cells were isolated from PP and MLN and double labeled with PE-conjugated anti-CD4 together with FITC-conjugated anti-CD40L, CD44, or CD95. Phenotypic analysis was performed on 10,000 sorted cells with live gates set on CD4+ lymphocytes using a FACScan. We found that the surface expression of the activation markers was lower for CD40L in CD4+ T cells in PP in CTLA4-H{gamma}1 Tg mice (right) than that in wt mice (left; A). Moreover, the level of CD44-high expressing CD4+ T cells in PP was significantly (p < 0.01) lower in CTLA4-H{gamma}1 Tg (dark profile) than in wt mice (striped profile) mice (B). By contrast, the CD4+ MLN T cells labeled strongly with anti-CD95, and both an increased frequency and higher levels of surface CD95 (Fas/Apo1) expression (p < 0.01) were found in CTLA4-H{gamma}1 Tg mice (dark profile) compared with those in wt mice (striped profile; C). Isotype-matched FITC-conjugated Abs directed against rat IgG (white profile) were used in all analyses as a specificity control. This is one representative experiment of five. In D, we illustrate the ability of highly enriched MLN CD4+ T cells from orally KLH- plus CT adjuvant-primed CTLA4-H{gamma}1 Tg (open symbols) or wt (closed symbols) mice to support anti-DNP-specific Ab production in recipient nu/nu syngeneic mice following adoptive transfer. The values represent the serum anti-DNP IgG log10 titer ± SD of five mice per group after i.p. immunizations with ONP-KLH, p < 0.02, when comparing the DNP-specific IgG responses in nu/nu mice receiving CD4+ T cells from wt transgeric mice. Unimmunized control mice had undetectable levels of anti-DNP IgG Abs.

 
These results argued that oral immunization inappropriately primed Ag-specific CD4+ T cells in the GALT of CTLA4-H{gamma}1 Tg mice. To examine the ability of these Ag-specific CTLA4-H{gamma}1 Tg MLN CD4+ T cells to act as helper cells for Ab responses, an adoptive transfer experiment was performed. Highly enriched MLN CD4+ T cells from orally KLH- plus CT adjuvant-immunized Tg or wt mice were injected into syngeneic nu/nu recipient mice. Following a priming and a booster immunization with DNP-KLH, the serum anti-DNP Ab titers were determined. We found that KLH-primed MLN CD4+ T cells from CTLA4-H{gamma}1 Tg mice were significantly weaker helper cells than T cells derived from wt mice (Fig. 7GoD). This result provided additional evidence that oral immunization in CTLA4-H{gamma}1 Tg mice appears to inappropriately prime CD4+ T cells in the GALT. Thus, the poor Ab responses to oral immunization in CTLA4-H{gamma}1 Tg mice may partly be explained by weak helper activity in GALT CD4+ T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge this is the first study to investigate the regulatory role of costimulation along the B7-CD28 axis for mucosal IgA responses. The findings presented here suggest that mucosal IgA and systemic IgG responses are differently regulated. Confirming the initial observation by Lane et al., we found that the GC reaction in PP was not inhibited by a block in the CD80/86-CD28/CTLA4 pathway, which was clearly the case for GC formation in systemic lymphoid tissues (41), and, contrary to the strongly impaired IgG Ab formation and subclass switching, we observed that the total IgA Ab production was normal in the mCTLA4-H{gamma}1 Tg mice. Since both IgG and IgA B cell differentiation are dependent on CD4+ T cell help, our results suggest that the microenvironment in the GALT and spleen differ intrinsically in the composition of regulatory elements required for B cell expansion and isotype switching (2, 34, 38, 53).

One such determining factor, which we previously found to play a pivotal role in the induction of mucosal immune responses is IL-4, which is essential for Th2 development (3). Whereas IL-4-/- mice failed to develop GC in the PP and were impaired in their mucosal IgA response to oral immunizations, systemic IgG responses and subclass switching (apart from IgG1 and IgE under direct IL-4 control) following i.v. immunizations were not impaired in these mice (3). The results indicated that IL-4 and Th2 activity are critical factors for induction of mucosal immunity, while for induction of systemic immune responses IL-4 appeared to be dispensable. Several recent studies also support the idea that IL-4 and Th2 activity are fundamental components in the regulation of mucosal immune responses (23, 25, 54, 55).

Having stated that mucosal IgA and systemic IgG Ab responses appear to be differently regulated, however, we have to explain the somewhat paradoxical finding that oral immunization using KLH and CT adjuvant, despite a seemingly intact IgA differentiation pathway, failed to elicit specific intestinal IgA responses. This was particularly evident as we examined the response to CT, which despite being a potent TD Ag (56), did not stimulate anti-CT IgG or IgA responses in mCTLA4-H{gamma}1 Tg mice. A simple explanation for this paradox would be to assume that the B cells, which are responsible for the total IgA production at the gut mucosal level, are different from the progeny giving rise to the specific anti-CT or anti-KLH LP IgA responses. It is well documented that a population of CD5+ B-1 cells, enriched in the PerC, can contribute to the total intestinal IgA production (35). These B-1 cells would be the perfect candidate for such an IgA-producing population with a different requirement for regulatory elements compared with the B-2 cells in spleen and peripheral lymph nodes. Several investigators have reported that intestinal IgA production to a large extent may rely on these self-replenishing B-1 cells, although their repertoire of Ag recognition is thought to be restricted mostly to autoantigens and bacterial TI Ag (48, 49, 50). However, our detailed phenotypic analysis of the presence of B-1 and B-2 cells in the various compartments of the GALT and PerC failed to demonstrate a difference between mCTLA4-H{gamma}1 Tg and wt mice. In particular, we found no increase in B-1 cells in LP or in the PP that would account for the poor responsiveness to KLH or CT despite the normal total IgA production in the gut. It is, therefore, most likely that the total IgA production in gut LP in mCTLA4-H{gamma}1 Tg mice stems from the activation of conventional B-2 cells in PP or at other inductive sites in the GALT. From the point of view of responsiveness to KLH or CT, we must conclude that the specific intestinal IgA responses, but not IgA B cell differentiation in general, required an intact B7-CD28 signaling system. Thus, in this regard mucosal IgA and systemic IgG responses to specific soluble TD Ag appear to share similar regulatory requirements.

The fact that B-2 cells in the PP, but not in spleen or peripheral lymph nodes, can be activated to form GC reactions in mCTLA4-H{gamma}1 Tg mice is indirect evidence of that the microenvironment in PP and that in systemic lymphoid tissues differ significantly. This could be secondary to the selection or load of Ag in PP as opposed to that which accesses the systemic lymphoid tissues. Perhaps the continuous uptake of Ag, in particular bacterial products, from the gut lumen may stimulate the presence of factors, such as cytokines, that promote GC formation and IgA switching in B-2 cells in PP (33). It is difficult to understand why such a process would not allow for CT, reported to selectively bind and be taken up by the M cells overlaying the PP (57), to stimulate significant GC formation in the PP of mCTLA4-H{gamma}1 Tg mice (E. Gärdby, unpublished observation). Rather, we have to assume that the quality of the Ag at the inductive sites in GALT differ from that in systemic lymphoid tissues. Normally the GC reaction is indicative of a response to a TD Ag in the mCTLA4-H{gamma}1 Tg mice. Therefore, it may be that the GC in PP represent responses to TI Ag. Indeed, a few bacterial products, such as dextran, have been found to be capable of stimulating GC reactions in spleen following i.v. injection (58). Although such GC reactions have not been associated with strong isotype switching, it is possible that the GC reaction and the IgA B cell-switch differentiation observed in PP in mCTLA4-H{gamma}1 Tg mice is caused by TI rather than TD Ag. It is likely, though, that there is some T cell involvement in these GC reactions, because nu/nu mice fail to show GC reactions and are poor producers of IgA (34, 59). Such a limited requirement for T cell help would also be sufficient to explain the paradoxical findings of significant GC formations observed in PP of TCR {alpha}-/- or CD4-/- mice (39, 60). Although previously not reported, it is possible that {gamma}{delta}TCR+ cells in PP may be involved in GC formation. However, such cells are infrequent in PP in wt mice. Taken together, the presence of GC to which IgA+ B cells colocalize in PP does not in itself constitute an ability to respond to soluble TD Ag given orally together with CT adjuvant, but may reflect responses to, e.g., TI Ag produced by the bacterial flora. Studies are underway to dissect the Ag recognition pattern of the IgA Abs in mCTLA4-H{gamma}1 Tg mice to establish the relative contribution of TI Ag in driving this response.

In agreement with previous work using the mCTLA4-H{gamma}1 Tg mouse model, we found that naive T cells from GALT were normally responsive to anti-CD3- and CD28-mediated costimulation. Also, Ag-specific T cell priming appeared to be unaltered because the proliferative and cytokine responses of MLN T cells to recall Ag in vitro were comparable in both quality and magnitude to those recorded for wt mice. This observation is important because it clearly documents that Ag was, indeed, taken up and able to prime an immune response in the GALT of mCTLA4-H{gamma}1 Tg mice in the presence of CT adjuvant. However, our phenotypic analysis of CD4+ T cells in GALT argued that the Th cells in mCTLA4-H{gamma}1 Tg mice were inappropriately primed by oral immunization and that because of the block in the CD80/86-CD28/CTLA4 pathway, they failed to generate significant numbers of activated Th cells expressing sufficient levels of CD40L and CD44. The adoptive transfer experiment with KLH-primed MLN CD4+ T cells clearly demonstrated that such cells from mCTLA4-H{gamma}1 mice were significantly weaker helper cells for Ab responses than MLN CD4+ T cells from immunized wt mice. It is feasible to think that following oral immunization, an impaired CD40L expression in the GALT CD4+ T cells would dramatically inhibit a productive T-B cell interaction, affecting GC formation, isotype switching, as well as B cell survival (11). It would also have negative implications for Th2 differentiation in CD4+ T cells, and together with the lack of CD28 signaling in the mCTLA4-H{gamma}1 Tg mice, it would affect CD4+ T cell function and survival (61, 62, 63). In support of this scenario, we found an increase in the frequency and above all the expression of CD95 (Fas) on CD4+ T cells in the GALT of immunized mCTLA4-H{gamma}1 Tg mice. A high expression of CD95 is known to constitute a high vulnerability to Fas ligand-induced apoptosis (62, 64, 65). This was also observed for the PP, where the frequency of CD4+ T cells undergoing apoptosis was twice as high in mCTLA4-H{gamma}1 Tg mice as in wt mice. Collectively, these findings argue that oral immunizations in mCTLA4-H{gamma}1 Tg mice inappropriately prime CD4+ T cells and, thereby, fail to generate functionally adequate B cell help.

CT is the best-characterized mucosal adjuvant we know of today (56). Here we demonstrate for the first time in vivo that the adjuvant mechanism affects both TI and TD Ag responses. Whereas IgM responses to TI Ag were augmented 3- to 4-fold, the increase in TD Ag titers was substantially stronger, with IgG1 titers alone increased by at least 10-fold in wt mice. In addition, and at variance with earlier theories, we provide strong evidence to suggest that the adjuvant effect of CT may be entirely separate from its immunogenic properties (56, 66). This idea is based on the fact that CT strongly augmented Ab responses to the unrelated Ag in the absence of significant anti-CT Ab production following i.v. immunizations in mCTLA4-H{gamma}1 Tg mice. A reciprocal finding was made in IL-4-/- mice, which failed to display any mucosal adjuvant effect of CT, while CT stimulated a significant mucosal anti-CT IgA response (3). These results support our earlier observations that CT has direct immunomodulating effects on B cell isotype switching and differentiation and that it greatly promotes priming of Ag-specific CD4+ T cells (47, 52). We have proposed that the latter effect is secondary to an enhanced costimulation provided by CT-exposed APCs in general and B cells in particular (67). The mechanism for this, we believe, is linked to the strong increase in CD86 (and to some extent CD80) expression on CT-exposed APC (67). However, since CT was also effective as an adjuvant in mCTLA4-H{gamma}1 Tg mice, we have to consider that the costimulation induced by CT can be either completely independent of or at least only partly dependent on CD28 signaling in CD4+ T cells. Ongoing studies using CD4+ T cells from CD28-/- mice will be instrumental in allowing us to discriminate between these two possibilities. Nevertheless, by inference we have to predict that other costimulatory molecules, other than CD80 and CD86, on the APC are also involved in the adjuvant mechanism of CT (16, 17, 18). In this context it is of interest that we recently observed that ICAM-1 (CD54) can be up-regulated after CT treatment of B cells in vitro (N. Lycke, unpublished observation).

In an attempt to reconcile the paradoxical findings in the present and previous studies we put forward the following hypothesis. The microenvironment in PP and GALT differ intrinsically from that in spleen and systemic secondary lymphoid tissues, in that B cells, rather than dendritic cells and macrophages, play a critical role in the induction of immune responses to soluble TD Ag. Thereby, B cells, which fail to produce IL-12 (68) and readily express CD86 upon Ig receptor interaction with Ag, promote Th2 rather than Th1 CD4+ T cell differentiation. IL-4 strongly up-regulates CD86 expression on B cells, augmenting their costimulatory capacity (69), and recent studies have indicated that CD86-CD28 signaling is required when B cells are the primary APC (70). This is why IL-4 deficiency negatively affects mucosal immunity more than systemic immune responses. The failure of CT to act as a mucosal adjuvant in mCTLA4-H{gamma}1 Tg mice may be explained by the lack of CD28-independent costimulation in B cells, whereas, by contrast, CD28-independent costimulation may readily be induced in macrophages and dendritic cells, securing the systemic adjuvant effect of CT (71). Additional studies focusing on the role of B cells as APC for mucosal immune responses are much needed.


   Footnotes
 
1 This work was supported by the World Health Organization Global Programme for Vaccines and Immunizations-Transdisease-Vaccinology Program, the Swedish Medical Research Council, the National Institutes of Health (Grant 1R01AI40701-01), the Swedish Cancer Foundation, the Martin Bergvalls Foundation, and the Nanna Svartz Foundation. Back

2 Address correspondence and reprint requests to Dr. Nils Lycke, Department of Medical Microbiology and Immunology, University of Goteborg, S-413 46 Goteborg, Sweden. E-mail address: Back

3 Abbreviations used in this paper: LP, lamina propria; GALT, gut-associated lymphoid tissues; CD40L, CD40 ligand; GC, germinal centers; TD, T cell dependent; PP, Peyer’s patches; Tg, transgenic; wt, wild type; KLH, keyhole limpet hemocyanin; CT, cholera toxin; MLN, mesenteric lymph node; PerC, peritoneal cavity; PE, phycoerythrin; PNA, peanut hemagglutinin; ELISPOT, enzyme-linked immunospot; SFC, spot-forming cells; HRP, horseradish peroxidase; TI, T cell independent; AEC, 3-amino-9-ethylcarbazole. Back

Received for publication November 20, 1997. Accepted for publication February 26, 1998.


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