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The Journal of Immunology, 1998, 160: 778-787.
Copyright © 1998 by The American Association of Immunologists

Presence of Activated Antigen-Binding B Cells During Immunization Enhances Relative Levels of IFN-{gamma} in T Cell Responses1

Chandrashekhar Pasare*, Vivian Morafo{dagger}, Maureen Entringer{dagger}, Pratima Bansal*, Anna George*, Vineeta Bal*, Satyajit Rath2,* and Jeannine M. Durdik2,{dagger}

* National Institute of Immunology, New Delhi, India; and {dagger} Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To examine the influence of Ag presentation by B cells on immune responses, we have used mice transgenic for an Ig heavy chain from a monoclonal anti-azobenzenearsonate (Ars) Ab to deliver Ag to B cells during immunization. A large proportion of transgene-expressing B cells in these mice binds Ars, while transgenic serum Ig shows poor Ars binding. Transgenic B cells present Ars proteins better than their nonhaptenated counterparts. This is associated with an increase in the proliferative responses of transgenic T cells to Ars protein immunization. Although B cell numbers in the transgenic mice are lower, many B cells in them show an activated phenotype, as identified by altered surface levels of peanut agglutinin reactivity, CD23, CD24, CD44, CD62L, and CD86. Even against nonhaptenated immunogens, transgenic responses show significant enhancement in the relative proportions of the Th1 cytokine IFN-{gamma} over the Th2 cytokines IL-4 and IL-10. Haptenated immunogens further enhance the predilection of transgenic mice to produce relatively more IFN-{gamma}. Consistent with this, there is an increase in IgG2a/IgG1 ratios in serum Abs in response to haptenated immunogens in transgenic mice. Adoptive transfer of primed hapten-specific secondary B cells into nontransgenic mice also induces an increase in relative levels of IFN-{gamma} in response to haptenated immunogens. Thus, presentation of immunogen in vivo by activated Ag-binding B cells contributes to enhanced immunogenicity and a Th1 cytokine bias.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The capacity of B cells to act as APCs is well known (1, 2, 3, 4, 5), and their ability to process and present Ag taken up through their surface Ig receptors on MHC class II to CD4 T cells to attract cognate T cell help for B cell differentiation is fundamental to the generation of T-dependent Ab responses (6). However, the role of B cells as APCs in modulating CD4 T cell responsiveness is less well established, both in regulating T cell tolerance vs responsiveness and in altering the cytokine profiles of the triggered T cell response between the Th1 (IFN-{gamma} and TNF-ß) and the Th2 (IL-4, IL-5, and IL-10) groups (7). Two major variables are relevant in this context.

First, CD4 T cell activation requires two signals, one being the specific peptide-MHC class II ligand cognately engaged by the TCR, and the second being the range of noncognate costimulatory signals provided by the APC to the T cell during cognate recognition. The quantity and quality of costimulatory signals provided play a significant role in determining the outcome of T cell stimulation (8), both in terms of response vs tolerance (9) as well as in terms of biases in favor of either Th1 or Th2 cytokine responses (10). All costimulatory signals are not constitutively expressed by APCs; some are, in fact, triggered on APCs by T cell contact (11), and costimulatory molecule profiles of B cells are quite different from those of professional T cell-priming APC populations such as dendritic cells (6).

The second variable relevant to a consideration of the role of B cells as APCs in T cell priming is the state of activation of B cells. B cell activation is likely to modify the cognate signal to T cells by increasing the level of MHC class II expressed on them (12) and is also likely to alter their costimulatory molecule profile (13). Both of these consequences of B cell activation will have profound implications for their function as priming APCs for T cells.

Naive, resting B cells are deficient in costimulatory functions (14), and appear to induce tolerance in naive T cells rather than activate them (15, 16). The role of activated B cells has been far more controversial, with reports that even costimulation-sufficient B cell blasts induce tolerance in T cells (16). On the other hand, there are also reports that the recruitment of B cells as APCs during T cell priming in vivo confers a predisposition toward Th2 cytokines in the resultant T cell response (17).

Using various modalities to deplete B cells in vivo, B cells have been shown to be either crucial for CD4 T cell priming in vivo (18, 19) or for priming against protein but not peptide Ags (20), or not to be so (21). They have been shown to be essential for triggering memory CD8 T cell responses (22) or not to be needed for the maintenance of memory (23).

Rather than depleting B cells in vivo, in the present report we have used transgenic mice expressing an IgM heavy chain from an anti-p-azobenzenearsonate (anti-Ars)3 mAb or secondary B cells in a nontransgenic adoptive transfer model to make Ag delivery to B cells possible early during immunization so as to examine the quantitative and qualitative modulation of T cell immunogenicity of protein Ags under the influence of B cell APCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and immunization

Three transgenic founder lines, R16.7µ39, R16.7µ5, or R16.7µ33, and their normal littermates were used in these studies. The transgenic mice were 6- to 8-wk-old C57BL/6 mice transgenic for an Ig heavy chain from a hybridoma specific for the hapten Ars (24). Mice were bred either at the Small Animal Facility, National Institute of Immunology (New Delhi, India), or at the University of Arkansas Biomass Center (Fayetteville, AR). For studies involving adoptive transfers, normal C57BL/6 mice bred and maintained at the same facilities were used. Transgenic and littermate nontransgenic mice were immunized with 100 to 1000 µg nonhaptenated or Ars proteins in CFA or PBS as appropriate. Where necessary, mice were irradiated (400 rad) before receiving adoptive i.p. transfers of splenocytes (5 x 107 cells/mouse) from immunized donors.

Reagents

Chicken OVA, conalbumin (CA), BSA (Sigma Chemical Co., St. Louis, MO), and keyhole limpet hemocyanin (KLH; Pierce Chemical Co., Rockford, IL) were the Ags used. Coupling of the hapten Ars to these proteins was performed using standard protocols (25). The anti-idiotypic mAb specific for the transgenic heavy chain, AD8, was a gift from Dr. Peter Hornbeck, University of Maryland (Baltimore, MD). Ars-BSA and the monoclonal anti-I-Ab Ab Y-3P (26) were biotinylated using biotin-succinylamide ester (Sigma Chemical Co.). Ars-BSA was also fluoresceinated using FITC (Sigma Chemical co.). Peanut agglutinin (PNA)-fluorescein, PNA-biotin, mouse anti-rat Ig-phycoerythrin (PE; Accurate Chemical Corp., Westbury, NY), goat anti-mouse IgM-fluorescein (Southern Biotechnology, Birmingham, AL), rat anti-mouse IgG2a-biotin, rat anti-mouse IgG1-biotin, and rat anti-mouse Ig{kappa}-biotin (Zymed, San Francisco, CA), avidin-fluorescein and avidin-horseradish peroxidase (HRP; Vector Laboratories, Burlingame, CA), streptavidin-PE (Caltag, San Francisco, CA), anti-IgMa-fluorescein, anti-IgMa-biotin, anti-IgMb-PE, anti-CD86-fluorescein, anti-CD23-biotin, anti-CD24-biotin, anti-CD44-PE, and anti-CD62L-PE (PharMingen, San Diego, CA) were the other reagents used. The rat anti-mouse Thy-1 mAb Y-19 (gift from Dr. C. A. Janeway, Yale Medical School, New Haven, CT) was used for T cell depletion protocols as culture supernatant.

T cell activation assays

T cells were purified, where appropriate, from spleens of C57BL/6 mice immunized with OVA in CFA i.p. 10 days after immunization over nylon wool (27). In some experiments, further panning on anti-Ig-coated plates was performed to ensure complete APC depletion. Efficient depletion of APCs was confirmed by the failure of these cells to respond significantly to OVA without any further addition of APCs. For B cell enrichment of spleen cell populations, T cells were depleted by anti-Thy-1 followed by rabbit complement (Low-Tox-M, Accurate), and non-B cell APCs were depleted by plastic adherence.

For Ag presentation assays in vitro, OVA-immune T cells (1 x 106 cells/ml) were incubated with B cell-enriched splenocytes (1 x 106 cells/ml) from either normal or transgenic mice and graded doses of OVA or Ars-OVA. For analysis of immunogenicity, normal or transgenic mice immunized with nonhaptenated or Ars proteins were killed 7 to 14 days after immunization, and single-cell suspensions from spleens were cultured at 1.5 x 106 cells/ml with graded doses of Ags in triplicate cultures. All T cell activation assays were performed in 200 µl of Click’s medium (Irvine, Santa Ana, CA) containing 10% FCS (HyClone, Logan, UT), antibiotics, and 0.05 mM 2-ME in 96-well flat-bottom plates (Falcon-Becton & Dickinson, Lincoln Park, NJ). Proliferative responses were measured by pulsing the plates with 0.5 to 1.0 µCi/well of [3H]thymidine (Amersham, Aylesbury, U.K.) on day 4 for 12 to 16 h. Plates were harvested onto glass-fiber filters for scintillation spectroscopy (Betaplate, Wallac, Finland). Replicate wells were harvested at 60 h of incubation for cytokine estimation by enzyme-linked immunoassays.

Cytokine analyses

Enzyme-linked immunoassays were performed on culture supernatants using appropriate purified and biotinylated Ab pairs for IFN-{gamma} (Genzyme, Cambridge, MA) and IL-10 (PharMingen) according to the manufacturers’ protocols. Purified monoclonal anti-mouse IFN-{gamma} or IL-10 Abs were adsorbed for capture on polystyrene microtiter plates (Nunc, Roskilde, Denmark). The culture supernatants were followed by either biotinylated polyclonal goat anti-mouse IFN-{gamma} or biotinylated monoclonal rat anti-mouse IL-10. Streptavidin-HRP followed by hydrogen peroxide and tetramethylbenzidine (Sigma Chemical Co.) were used for detection. Titration curves of rIFN-{gamma} (Genzyme) and IL-10 (PharMingen) were used as standards for calculating cytokine concentrations in the culture supernatants tested. The limits of detection for both cytokines were routinely in the range of 15 to 30 pg/ml.

Flow cytometry

For flow cytometry, cells (1 x 105 to 1 x 106/well) were incubated with the primary staining reagents in 50 µl of staining buffer (PBS containing 0.1% NaN3 and 1% FCS) for 45 min on ice. After washing in staining buffer, similar incubations were used for secondary reagents as needed. Stained cells were fixed in 0.5% paraformaldehyde and stored at 4°C until analyzed. Samples were analyzed on an EPICS 752 flow cytometer (Coulter Electronics, Hialeah, FL) or a Bryte flow cytometer (Bio-Rad, Hemel Hampstead, U.K.), and data analysis was conducted using WinMDI shareware.

Ab assays

Microtiter plates (Dynatech, Chantilly, VA) were coated with Ag (10 µg/ml). Specific Abs in sera were detected with biotinylated mouse IgG isotype-specific Abs and avidin-HRP followed by 2,2'-azinobis-(3-ethylbenzthiazoline-sulfonic acid (Kirkegaard and Perry, Gaithersburg, MD) and hydrogen peroxide. Color was read at 405 nm on a microplate reader (Biotek Winooski, VT). IgG2a/IgG1 indexes were expressed as the ratio of absorbances obtained for each isotype at a serum dilution of 1/300 (previously determined to lie in the titrating range).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
R16.7µ transgene-expressing B cells bind to Ars proteins

For these studies we have used C57BL/6 mice transgenic (R16.7µ) for the Ig heavy chain from a mAb, R16.7, which is specific for the hapten Ars (24). We have previously shown that transgene-expressing B cells from these mice can undergo both isotype switching events across chromosomes and extensive somatic mutation to make high affinity anti-Ars Abs upon immunization with Ars proteins (24, 28). Only those B cells with the right {kappa} light chain participate in switching and somatic mutation (24). In agreement with this finding, nonimmunized R16.7µ mice have a significant amount of the transgenic heavy chain in their serum (30–40 µg/ml), but less than one-fifth of this shows any binding to Ars (29), since most of the transgenic Ig in the serum of these mice represents the transgenic heavy chain in association with endogenous noncanonical {kappa}- and {lambda}-chains. The transgenic serum Ig thus lacks sufficient affinity to bind Ars well, and therefore is unlikely to clear haptenated proteins efficiently in vivo.

However, when we look at whether B cells from transgenic R16.7µ mice can bind Ars, a significant proportion of transgene-expressing B cells, some 60 to 70%, bind to Ars-BSA (Fig. 1Go). As reported previously, the numbers of B cells in R16.7µ mice are lower than those in their nontransgenic littermates (30). Using either an anti-allotype Ab against IgMa (an allotype present on transgenic, but not on endogenous, IgH protein in R16.7µ mice) or a transgene IgH-specific anti-idiotypic mAb, AD8 (31), it can be seen that about half of all B cells express transgenic heavy chains in three independent founder strains of R16.7µ mice, and some of these, in fact, express both transgenic and endogenous proteins as inferred previously (29) (Fig. 1Go). Unlike in serum (29), a high proportion of these transgene-expressing B cells bind to Ars-BSA in all three strains (Fig. 1Go). The data in Figure 1Go represent three to five independent experiments. Thus, while only a small fraction of the serum transgenic Ig in these mice binds to Ars, most transgene-expressing B cells can recognize it, possibly due to the multivalency of the cell surface. This makes it possible to deliver haptenated proteins to the high frequency of transgene-expressing B cells found in vivo without the intervention of efficient clearance by serum Abs.



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  		FIGURE 1. R16.7µ transgenic B cells show binding to Ars-BSA. Nontransgenic (A, D, G, J,L, and N) or transgenic (R16.7µ39 (B, E, and H), R16.7µ33 (C, F, and I), R16.7µ5 (K, M, and O)) spleen cells were stained for the markers shown in two-color flow cytometric analyses. Nontransgenic cells (which have the IgMb endogenous allotype) do not show any staining for the transgenic allotype IgMa (D and J), nor do they bind to Ars-BSA (A, G, and N) or show staining for the transgenic Id (L). All three strains of transgenic mice have a high proportion of cells bearing IgMa (B, C,E, F, and K), and a large subset of these cells binds Ars-BSA (B, C, and O) even when they also express the endogenous allotype, IgMb (H and I). The transgenic Id is also well expressed on them (M).

 
Transgenic B cells present Ars proteins better than nonhaptenated proteins to T cells

The next issue is whether the binding of Ars to transgene-expressing B cells has any consequences in terms of Ag presentation. When T cells purified from OVA-immune C57BL/6 mice are presented either OVA or Ars-OVA by irradiated adherence-depleted spleen cells from C57BL/6 mice, the antigenic dose-response curves for the haptenated and nonhaptenated Ags are overlapping (Fig. 2GoA). However, when nonadherent splenic APCs from either R16.7µ5 or R16.7µ39 mice are used, Ars-OVA is presented much better than OVA (Fig. 2Go, B and C). Similarly, even if total splenic cells from OVA-immune normal or R16.7µ39 mice are stimulated with either OVA or Ars-OVA, the dose-response curves of the resultant T cell responses overlap for the two Ags in normal mice, but Ars-OVA is still presented better than OVA by transgenic cells (data not shown). Figure 2Go represents three independent experiments.



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  		FIGURE 2. R16.7µ transgenic B cells present Ars-OVA better than OVA to OVA-primed T cells. Purified OVA-immune T cells from C57BL/6 mice were stimulated with titrated doses of OVA (hollow symbols) or Ars-OVA (filled symbols) either without additional APCs (A, broken lines) or with B cell APCs from nontransgenic (A, continuous lines), R16.7µ39 transgenic (B, circles), or R16.7µ5 transgenic (C, squares) mice. The horizontal line in A represents the background counts per minute in the absence of Ag. Proliferation is presented as the mean ± SE counts per minute of [3H]thymidine incorporation in triplicate cultures.

 
Targeting of immunogen to B cells in vivo enhances T cell proliferative immune responses

The above data indicate that Ars-binding B cells take up Ars-OVA preferentially and present it better than OVA. The next question is whether there are any consequences of this preferential presentation of Ars proteins by transgenic B cells in vivo, since immunization with Ars protein would lead to more B cell-mediated Ag presentation during T cell priming in these transgenic mice. Normal or R16.7µ39 mice were immunized with either OVA or Ars-OVA in CFA i.p., and T cell proliferation responses were assayed on day 11 of immunization using titrating doses of OVA for all four groups. Figure 3Go shows one such representative experiment of three, in which the spleen cells from Ars-OVA-immune transgenic mice show a significantly higher response compared with those of the other three groups, suggesting that targeting Ags to B cells enhances the immunogenicity of the Ag in these transgenic mice.



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  		FIGURE 3. Transgenic mice mount a better immune response to Ars-OVA than to OVA. Nontransgenic (triangles) or R16.7µ39 (circles) mice were injected i.p. with 100 µg of OVA (hollow symbols) or Ars-OVA (filled symbols) in CFA. Eleven days later, splenocytes from all four groups were stimulated with graded concentrations of OVA. The resultant T cell proliferative response is shown as the mean ± SE counts per minute of [3H]thymidine incorporation in triplicate cultures.

 
Activated B cells are numerous in R16.7µ transgenic mice

The enhancement of quantitative immunogenicity is surprising, since naive B cells have been reported to tolerize naive T cells (15, 16). Given reports that activated B cells, on the other hand, can indeed prime naive T cells (32, 33, 34), perhaps in part by virtue of being able to express the costimulatory CD80/CD86 molecules (13), the surface phenotype of B cells in transgenic mice has been examined with regard to their activation status. Although transgenic mice have fewer B cells than normal mice, they have a relatively higher proportion of activated B cells that show increased levels of PNA reactivity, CD24, CD44, and even CD86 and lower levels of CD23 and CD62L (Fig. 4Go). The levels of MHC class II and CD80 do not appear greatly changed (data not shown). These data are representative of findings in multiple mice. Thus, it is possible that the higher numbers of activated and costimulation-competent B cells in transgenic mice are contributing to the increase in immunogenicity of haptenated immunogen in these mice.



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  		FIGURE 4. B cells in transgenic mice show an activated phenotype. Nontransgenic (A, C, E, G, and I) or R16.7µ5 transgenic (B, D, F, H, and J) mouse spleen cells were stained for the various markers shown in a two-color flow cytometric analysis. Staining for CD86 (thick lines) or negative controls (thin lines) is shown as single-color histograms of gated IgM-bearing spleen cells in K (nontransgenic mice) and L (R16.7µ5 transgenic mice).

 
Th1/Th2 cytokine profiles in transgenic mice

Given the altered phenotype of B cells in transgenic mice in terms of numbers and activation, the next issue examined was the effect of these alterations on the cytokine output preferences of their T cell responses. Mice immunized with CA, KLH, or OVA in PBS or CFA were killed 1 to 2 wk after immunization, and spleen cells were stimulated with various doses of Ag in vitro for inducing cytokine production. A representative experiment with CA-PBS-immune mice is shown in Figure 5GoA, where it can be seen that the amounts of IFN-{gamma} produced in transgenic cultures appear to be more than those produced in nontransgenic ones, while the converse is true for IL-10. However, for ease of comparison, the data are presented as IFN-{gamma}/IL-10 ratios, and results from immunizations with various antigenic proteins in either PBS or CFA are shown. Surprisingly, even such immunization with nonhaptenated Ags consistently induces cytokine profiles in which the relative levels of IFN-{gamma} in relation to IL-10 are higher in transgenic mice (Fig. 5GoB).



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  		FIGURE 5. Th1 and Th2 cytokine profiles differ between R16.7µ transgenic and normal mice. A shows the results of IFN-{gamma} and IL-10 assays performed using culture supernatants from spleen cell cultures from normal (N) or R16.7µ39 transgenic (T) mice immunized with CA in PBS and activated in vitro with graded doses of CA. B shows the IFN-{gamma}/IL-10 ratios (shown as the mean ± SE of values at various Ag doses) obtained in similar experiments using various immunization Ags and protocols in either normal (stippled bars) or R16.7µ39 transgenic (hatched bars) mice. In the case of the OVA-PBS* immunization, the transgenic mice used were of the R16.7µ5 strain.

 
To rule out the possibility that transgenic B cells could nonspecifically induce higher levels of IFN-{gamma} during restimulation from any primed T cell population (or from non-T cells), APC-depleted T cells from CA-CFA-immunized nontransgenic mice were incubated with titrating doses of CA and adherence-depleted spleen cells from either nontransgenic or R16.7µ39 as APCs. Figure 6Go shows that there is antigenic dose-dependent increase in the production of both IFN-{gamma} and IL-10 from the nontransgenic T cells, and that the relative quantities of these cytokines are not different regardless of whether nontransgenic or transgenic APCs are used. Thus, it is clear that the enhanced production of IFN-{gamma} shown in Figure 5Go is due to differences in T cell priming in vivo rather than due to restimulation-related events. The basis of such a shift in cytokine commitment was the focus of the next experiments.



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  		FIGURE 6. Transgenic APCs do not alter the cytokine profile of immune T cells during restimulation in vitro. CA-immune T cells purified from nontransgenic mice were stimulated with CA in the presence of either no additional APCs (-) or adherence-depleted splenic cells from nontransgenic (N) or R16.7µ39 (39T) mice. The levels of IFN-{gamma} (A) and IL-10 (B) produced are shown.

 
Th1/Th2 cytokine ratios increase upon Ag delivery to B cells

The enhanced Th1 bias even for nonhaptenated immunogens in transgenic mice could be due to two distinct reasons. One, there are fewer B cells in transgenic mice (Figs. 1Go and 4Go) (30), the Ab levels generated in transgenic mice against nonhaptenated Ags are lower than those in normal mice (data not shown), and therefore Ag-negative B cells would be rarer contributors to Ag presentation during T cell priming in vivo. B cells have been reported to be responsible for a shift of the T cell cytokine profile to the Th2 end of the spectrum (17). Therefore, in their relative paucity, it may be expected that the other APC lineages, macrophages and dendritic cells, which have been implicated in inducing Th1 responses (35), would shift the balance toward Th1 cytokines. An alternative possibility, however, is that the higher numbers of activated B cells in these transgenic mice allow better costimulation-sufficient Ag presentation to T cells in vivo, and that this increase in Ag presentation would alter the cytokine balance in favor of Th1 responses. We have tested this by delivering Ag to Ars-binding B cells in the transgenic mice. If the former possibility were correct, the relative prominence of IFN-{gamma} would decrease, while the latter possibility would predict that this Th1 bias should be enhanced.

Again, cytokine profiles of splenic cells from normal and transgenic mice immunized with either nonhaptenated or haptenated OVA, CA, or KLH with or without CFA were examined upon restimulation with the immunizing Ag in vitro. Figure 7Go, A and B, show the cytokine profiles resulting from KLH or Ars-KLH immunization in PBS for nontransgenic or R16.7µ39 transgenic mice. While the levels of IL-10 are not altered significantly in either group by immunogen haptenation (Fig. 7GoB), such haptenated immunogen increases IFN-{gamma} production by immune T cells from transgenic, but not nontransgenic, mice (Fig. 7GoA). The use of a different protocol of immunization does not affect this relative shift toward Th1 cytokines, as shown in Figure 7Go, C and D, which show cytokine profiles resulting from OVA or Ars-OVA immunization in CFA for nontransgenic or R16.7µ39 transgenic mice. Once again, the levels of IFN-{gamma} are enhanced by the use of haptenated immunogen only for transgenic and not for nontransgenic mice (Fig. 7GoC), while the levels of IL-4 decrease concurrently (Fig. 7GoD).



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  		FIGURE 7. Th1/Th2 cytokine ratios increase upon Ag delivery to B cells in R16.7µ transgenic mice. The IFN-{gamma} (A) and IL-10 (B) levels generated upon restimulation with KLH in vitro after immunization with either KLH-PBS or Ars-KLH-PBS of nontransgenic (N) or R16.7µ39 transgenic (39T) mice show a shift toward IFN-{gamma} in Ars-KLH-immune transgenic mice. The IFN-{gamma} (C) and IL-4 (D) levels generated upon restimulation with Ars-OVA in vitro after immunization with either OVA-CFA or Ars-OVA-CFA of nontransgenic (N) or R16.7µ39 transgenic (39T) mice also show similar results. E shows a summary of data from multiple experiments, in which normal (stippled bars) or R16.7µ39 (or R16.7µ5 for the OVA-PBS* immunization) transgenic mice (hatched bars) were immunized with the nonhaptenated or the Ars form of the Ags shown. Their spleen cells were stimulated 1 to 2 wk later with either Ag, and the IFN-{gamma}/IL-10 ratios were calculated from the culture supernatants. The data are shown as the IFN-{gamma}/IL-10 index obtained for Ars-Ag-immune cells to that obtained for nonhaptenated Ag-immune cells. Since haptenation should make little difference in normal mice to the cytokine profile, this ratio should be close to 1 in normal mice, as can be seen (stippled bars).

 
For ease of presentation, data from multiple such experiments are shown as the ratio of the Th1/Th2 balance (which is the IFN-{gamma}/IL-10 ratio) generated by haptenated vs nonhaptenated immunogens in normal and transgenic mice (Fig. 7GoE). In these experiments, this value remains close to 1, as expected in normal mice, indicating that similar IFN-{gamma}/IL-10 ratios are generated by haptenated and nonhaptenated immunogens in them. On the other hand, transgenic mice show a significant and consistent increase in multiple experiments in the relative Th1 component upon immunization with Ars proteins. Thus, delivering Ags to activated B cells during priming in the transgenic mice enhances the Th1 bias of cytokine responses in them.

IgG2a/IgG1 ratios in serum Abs generated in normal and transgenic mice by haptenated vs nonhaptenated Ags

Since there is less than perfect allelic exclusion in these transgenic mice (Fig. 1Go) (29), it is possible to look at Ab production to antigenic epitopes other than Ars. The relative prominence of the various IgG isotypes produced would be expected to reflect the cytokine balance of the T cell help they receive. Mice were immunized with two doses of 100 µg of KLH or Ars-KLH in CFA 1 wk apart, and sera were collected for anti-KLH IgG2a and IgG1 Ab assays at 1, 2, 3, and 4 wk after the second dose. The IgG2a/IgG1 ratios of the anti-KLH Abs in the sera are shown in Figure 8Go. While KLH and Ars-KLH in normal mice generate comparable ratios, Ars-KLH immunization of transgenic mice shows a significant shift in favor of IgG2a, an IFN-{gamma}-dependent isotype, over IgG1, an IL-4-dependent isotype.



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  		FIGURE 8. Ars immunogen generates a higher IgG2a/IgG1 ratio in transgenic mice than that in normal mice. IgG2a/IgG1 ratios were calculated as described in sera collected 1, 2, 3, and 4 wk after immunization from normal (N) or R16.7µ5 transgenic mice (T) immunized with nonhaptenated KLH (U) or Ars-KLH (H) at 100 µg in CFA in two weekly doses.

 
Targeting Ags to secondary B cells in vivo in nontransgenic mice leads to a shift in favor of IFN-{gamma}

Thus far, all data shown here have used transgenic mice. It is possible that these findings do not necessarily apply to normal situations. To determine whether B cell-mediated Ag presentation in vivo in nontransgenic mice also induces a bias in the T cell response in favor of IFN-{gamma}, protein immunogens were delivered to secondary B cells in adoptive transfer experiments. Splenic cells from mice immunized 10 days earlier with 100 µg of KLH or Ars-KLH were transferred to normal, lightly irradiated recipients. The recipients were then immunized with CA or Ars-CA and killed 2 wk later, and their splenic cells were stimulated with graded doses of CA or Ars-CA for cytokine assays. T cells from mice receiving KLH-primed cells show no differences in either IFN-{gamma} or IL-10 levels whether they are immunized with CA or Ars-CA (Fig. 9Go, A and B). However, in mice receiving Ars-KLH-primed cells, Ars-CA as an immunogen induces more IFN-{gamma} and less IL-10 than CA immunization does (Fig. 9GoB). An independent experiment is shown as IFN-{gamma}/IL-10 ratios in Figure 9GoC, demonstrating that whether CA or Ars-CA is used for restimulation in vitro makes no difference to the cytokine shift toward IFN-{gamma} induced by priming with haptenated Ag in the presence of hapten-primed cells. Thus, the presence of hapten-specific secondary B cells during priming with haptenated protein can lead to a bias in the resultant T cell response in favor of IFN-{gamma}. These data have been reproduced in independent experiments with another Ag combination, namely, Ars-OVA or OVA followed by Ars-BSA or BSA (data not shown).



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  		FIGURE 9. Adoptive transfer of hapten-primed spleen cells into naive mice enhances the Th1 cytokine response to haptenated Ag in the recipients. Spleen cells from mice primed with either Ars-KLH or KLH (as indicated) were transferred to irradiated recipients who were immunized 24 h later with either Ars-CA or CA. Spleen cells were harvested 1 wk later for stimulation in vitro with Ag. A shows the IFN-{gamma} and B shows the IL-10 levels generated from these variously immunized T cells; the legends show the immunogens used as immunogen for donor/immunogen for recipient. C shows the IFN-{gamma}/IL-10 ratios (mean ± SE of values at various Ag doses) calculated from such culture supernatants in an independent experiment after restimulation by either CA (stippled bars) or Ars-CA (hatched bars).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The efficiency of specific B cells in presenting Ag to T cells has been well documented. We have used two systems to examine the nature of the T cell response in animals in which Ag presentation is augmented by B cells. Our observation that B cells of mice transgenic for the Ig heavy chain alone from an Ars-specific hybridoma can bind to Ars-coupled proteins (Fig. 1Go) has led us to an investigation of the biologic consequences of this binding. Since the serum transgene-containing Ig from these mice is far less capable of binding to Ars (29), Ars binding by their B cells is likely to be of lesser affinity than the binding observed by normal Ag-specific "secondary" B cells for their Ag. Despite this, transgenic B cells present Ars-Ags better to Ag-specific T cells in vitro than nontransgenic B cells do (Fig. 2Go), and this advantage in presentation can be observed even in the presence of other APC types, such as macrophages and dendritic cells, in whole spleen cell populations (data not shown). Since a substantial proportion of B cells in the transgenic mice bind to Ars-Ags and present them, immunization with an Ars-Ag would involve a far larger population of B cell APCs presenting the Ag during T cell priming in vivo than would normally be the case.

B cell APCs have been argued to be tolerogenic in various situations (15, 16, 36), and it is therefore something of a surprise to find that Ars-OVA is actually quantitatively more immunogenic than OVA in transgenic, but not in normal, mice (Fig. 3Go). One relevant difference may be that B cell tolerogenicity has been most frequently seen in situations where the Ag is being delivered almost exclusively to B cells, whereas in our experimental system, presentation would involve all categories of APCs, especially since the B cell recognition of Ars is likely to have low affinity. It is thus possible that these B cell APCs, when added to, for example, dendritic cell APCs during in vivo priming, increase the total amount of Ag presentation available to the responding T cells, inducing a greater response.

However, for this argument to apply, the increased Ag presentation made available by B cells must be accompanied by sufficient costimulatory signals. By most accounts, B cells acquire costimulatory signaling capabilities upon activation (13, 37, 38), and therefore, if the transgenic B cells are to function as effective APCs in vivo, they would be expected to be activated. Such is, indeed, the case. Higher levels of MHC class II, PNA reactivity, CD24, and CD44 and lower levels of CD23 and CD62L are features characteristic of activated B cells (39, 40, 41, 42, 43, 44), and many of these characteristics are present in a very large proportion of B cells in transgenic spleens (Fig. 4Go). As expected, there is an increase in the proportion of B cells expressing CD86 in transgenic mice (Fig. 4Go), although no B cell-specific expression of CD80 is induced (data not shown).

Since the presence of Ag-binding B cells appears to play an immunomodulatory role in these transgenic mice, it was appropriate to investigate their effect on another decision that responding T cells make, namely, whether to make the Th1 or the Th2 group of cytokines (7). This is especially relevant because there are conflicting data about the role of B cell APCs in such Th1/Th2 commitment. Some immunization systems suggest that B cell-mediated Ag presentation during T cell priming may generate more IFN-{gamma} (45, 46), while a study in a transgenic TCR-bearing mouse strain suggests that their presence may lead to greater production of IL-4 (17) in keeping with earlier speculations based on data with T cell clones (47). We find, in the first place, that even nonhaptenated Ags induce a greater IFN-{gamma}/IL-10 ratio in the transgenic mice (Fig. 5Go), and that this is related to events during priming in vivo rather than restimulation in vitro (Fig. 6Go). In transgenic mice, the use of a B cell-binding immunogen enhances the already existing bias of these mice to generate an IFN-{gamma}-dominated response (Fig. 7Go). This shift in cytokine balance upon use of an Ars-Ag is not only seen in vitro, it shows its presence in vivo as an increase in the IgG2a/IgG1 ratio of the Abs generated (Fig. 8Go), suggesting a dominance of the IFN-{gamma}-dependent IgG2a isotype (48). Interestingly, this finding is in agreement with the production of IgG2a in the in vivo experiments reported in the TCR-transgenic system, from which in vitro data suggest that B cells may help the Th2 pathway choice (17). Together, these data are incompatible with the possibility that a paucity of B cell-mediated Ag presentation leads to a bias in favor of IFN-{gamma} in the transgenic mice, since that would have meant a shift back toward Th2 cytokines once B cells were recruited by Ars-mediated delivery. It thus appears more likely that activated B cells induce a shift in favor of IFN-{gamma}, and that delivering Ag to B cells simply accelerates this trend further.

It was necessary to determine whether these data from a transgenic mouse system in which the B cells show an unusual phenotype (30) are also applicable to normal situations. The present data suggest that involvement of activated B cells as APCs during T cell priming in vivo biases the immune response toward a Th1 cytokine phenotype. This means that prior exposure to an Ag that expands the pool of activated B cells would result in a shift toward Th1 cytokines in the future in normal animals. To test this, we have immunized normal mice with either nonhaptenated or Ars protein, then transferred spleen cells from them to irradiated recipients and immunized these with a second unrelated protein, either nonhaptenated or arsanylated. Ars-primed spleen cell recipients respond to Ars protein immunization with more IFN-{gamma} and less IL-10 than they do to nonhaptenated protein immunization (Fig. 9Go), supporting the data we have shown above for the transgenic mouse system. Serum anti-Ars Abs are minimal in recipients of Ars-Ag-immune spleen cells (data not shown), suggesting that the low levels of Ars binding by serum Ig in transgenic mice are unlikely to be a relevant feature in the Th1/Th2 decisions made.

Thus, our data suggest that activated B cells, when included as APCs in a maturing immune response, tend to increase the amount of T cell priming and to shift the cytokine balance of the response toward the Th1 category. Other data from our laboratory suggest that immunogen delivery to macrophage-specific scavenger receptors (49) also tilts the Th1/Th2 balance toward Th1.4 Thus, it appears that regardless of the APC lineage involved, increasing levels of MHC-peptide ligand generated on the APCs may be able to generate a Th1 bias in the immune response, provided that sufficient costimulation is available on the APCs concerned.


   Acknowledgments
 
We thank Drs. R. K. Anand and R. K. Juyal for advice, and Inderjit Singh and Leonard Dunn for work on breeding and maintenance of transgenic mice. The help of Mr. T. A. Nagarjuna with flow cytometry is gratefully acknowledged.


   Footnotes
 
1 This work was supported by a grant (to J.M.D.) from the National Institutes of Health (R01GM48691) and a grant (to S.R.) from the Department of Biotechnology, Government of India. The National Institute of Immunology is supported by the Department of Biotechnology, Government of India. Back

2 Address correspondence and reprint requests to Dr. Satyajit Rath, National Institute of Immunology, New Delhi, India 110 067; or to Dr. J. M. Durdik, Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701. Back

3 Abbreviations used in this paper: Ars, p-azobenzenearsonate; CA, chicken conalbumin; KLH, keyhole limpet hemocyanin; PNA, peanut agglutinin; PE, phycoerythrin; HRP, horseradish peroxidase. Back

4 Singh, N., S. Bhatia, R. Abraham, S. K. Basu, A. George, V. Bal, and S. Rath. Modulation of T cell cytokine profiles and peptide-MHC complex availability in vivo by delivery to scavenger receptors via antigen maleylation. Submitted for publication. Back

Received for publication June 25, 1997. Accepted for publication October 7, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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