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The Journal of Immunology, 2003, 171: 1758-1767.
Copyright © 2003 by The American Association of Immunologists

Incomplete Activation of CD4 T Cells by Antigen-Presenting Transitional Immature B Cells: Implications for Peripheral B and T Cell Responsiveness1

James B. Chung*, Andrew D. Wells{dagger}, Scott Adler{dagger}, Anand Jacob{ddagger}, Laurence A. Turka{dagger} and John G. Monroe2,{ddagger}

Divisions of * Rheumatology and {dagger} Nephrology, Department of Medicine, and {ddagger} Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
B cells leave the bone marrow as transitional B cells. Transitional B cells represent a target of negative selection and peripheral tolerance, both of which are abrogated in vitro by mediators of T cell help. In vitro, transitional and mature B cells differ in their responses to B cell receptor ligation. Whereas mature B cells up-regulate the T cell costimulatory molecule CD86 (B7.2) and are activated, transitional B cells do not and undergo apoptosis. The ability of transitional B cells to process and present Ag to CD4 T cells and to elicit protective signals in the absence of CD86 up-regulation was investigated. We report that transitional B cells can process and present Ag as peptide:MHC class II complexes. However, their ability to activate T cells and elicit help signals from CD4-expressing Th cells was compromised compared with mature B cells, unless exogenous T cell costimulation was provided. A stringent requirement for CD28 costimulation was not evident in interactions between transitional B cells and preactivated CD4-expressing T cells, indicating that T cells involved in vivo in an ongoing immune response might rescue Ag-specific transitional B cells from negative selection. These data suggest that during an immune response, immature B cells may be able to sustain the responses of preactivated CD4+ T cells, while being unable to initiate activation of naive T cells. Furthermore, the ability of preactivated, but not naive T cells to provide survival signals to B cell receptor-engaged transitional immature B cells argues that these B cells may be directed toward activation rather than negative selection when encountering Ag in the context of a pre-existing immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mature B cells may act as APCs to activate CD4 T cells. They drive T cell expansion (1, 2, 3) and direct T cell differentiation (4). T cells, in turn, provide help to B cells, allowing them to participate in T cell-dependent Ab responses (5). Although small resting B cells appear to be ineffective APC for naive CD4 T cells (6, 7) and may even be tolerogenic (8), B cells activated through the Ag receptor or through CD40 up-regulate costimulatory molecules, such as B7, and become effective APC for T cells (6, 7, 9, 10, 11). In fact, the specificity of the Ag receptor allows the B cell to capture and concentrate even minute amounts of soluble Ags for presentation to T cells in a highly efficient manner (12, 13).

Although Ag encounter together with cognate interactions with CD4 T cells (14) leads to activation, proliferation, and further differentiation into Ab-producing cells of mature B cells, the fate of transitional immature B cells in these processes has not been studied. Past in vivo and in vitro studies support the idea that immature and transitional immature B cells undergo negative selection in response to Ag encounter (15, 16, 17). These developmental differences are felt to play a significant role in maintaining B cell tolerance to peripheral self Ags. T cell help, however, has been shown to rescue both immature and mature self-reactive B cells from deletion in hen egg lysozyme (HEL)3 and anti-HEL double-transgenic mice (18, 19). Furthermore, purified transitional immature B cells that are triggered to undergo apoptosis through their Ag receptors are capable of being rescued from apoptosis by T cell help signals, such as IL-4 or anti-CD40 (20, 21). Transitional immature B cells, although not found in lymph nodes, circulate through the white pulp of the spleen and are found within the B cell follicle and in the outer periarterial lymphatic sheath (21, 22). They are, thus, optimally positioned to interact with circulating T cells.

Numerous investigators have linked the role of intact B cell Ag receptor (BCR)-mediated signaling pathways with the proper targeting of Ag to late endosomal compartments and the degradation and loading of resultant peptides onto the MHC class II molecules (23, 24, 25, 26, 27, 28, 29, 30, 31). Given the differences in intracellular signal transduction pathways in the transitional immature compared with mature B cells after BCR ligation (32, 33, 34), the contribution of the surface expression of peptide:MHC class II complexes to the interaction between transitional B cells and CD4 T cells is an important issue that has not yet been addressed experimentally. The inability of transitional B cells to up-regulate the costimulatory molecule CD86 (B7.2) in response to Ag stimulus (21) adds additional complexity to understanding the results of Ag presentation by transitional B cells. Previous studies of the Ag-presenting function of developing B cells showed that bone marrow immature B cells and neonatal B cells after 21–28 days are able to induce IL-2 production in a T cell hybridoma specific for pigeon cytochrome c (35). Although instructive, these studies did not specifically examine the circulating peripheral immature B cells, which have the potential to interact with primary resting and activated T cells in restricted lymphoid compartments, and these studies lacked the detail necessary to dissect differences in the APC function of transitional B cells compared with fully mature resting B cells. The use of a T cell hybridoma also made the results of T cell responses difficult to interpret.

We sought to study the capability of transitional immature B cells to process and present Ag and the response of cognate CD4 T cells to this interaction. We also designed these studies so that we could ascertain whether productive cognate interactions between transitional immature B and T cells could affect changes on B cell negative selection. The effect of naive vs activated T cells and bystander vs cognate interactions was compared.


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

Female BALB/c mice were bred in the animal facility of the University of Pennsylvania and used between the ages of 8 and 12 wk for all experiments. Transitional B cells were harvested and purified from the spleens of mice 13 or 14 days after irradiation with 500 rad (36). These autoreconstituted B cells are >98% heat stable Aghigh. Female adult CBA/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility of the University of Pennsylvania. DO11.10 transgenic mice expressing transgenic TCR specific for an OVA323–339 in the context of I-Ad have been previously described and were also bred in the animal facility of the University of Pennsylvania School of Medicine (37).

Abs and reagents

Goat anti-mouse IgG F(ab')2 were used to stimulate B cells (Jackson ImmunoResearch Laboratories, West Grove, PA). C4H3 hybridoma-producing rat IgG2b mAb specific for the HEL46–61 peptide in the context of I-Ak was provided by R. Germain (National Institutes of Health, Bethesda, MD) (38). CTLA4-Ig was provided by R. Peach (Bristol-Myers Squibb, Princeton, NJ). The 37.51 hybridoma producing an anti-CD28 mAb was provided by J. Allison (University of California, Berkeley, CA). The following Abs used for FACS analysis were purchased from BD PharMingen (Franklin Lakes, NJ): anti-CD86 (GL-1), anti-heat stable Ag (M1/69), anti-CD23 (B3B4), AA4.1, anti-CD69 (H1.2F3), anti-CD4 (L3T4), anti-CD45RB (16A), low endotoxin/no azide anti-LFA-1 (M17/4), and anti-B220 (RA3-6B2). CFSE and TOPRO-3 iodide were purchased from Molecular Probes (Eugene, OR). Chicken OVA and HEL were purchased from Sigma-Aldrich (St. Louis, MO). The OVA peptide OVA323–339 was synthesized by the Protein Chemistry Laboratory of the University of Pennsylvania. The peptide HEL46–61 was purchased from PeptidoGenic (Livermore, CA).

Conjugation of goat anti-mouse IgG F(ab')2 with OVA

One milligram (10 nM) of goat anti-mouse IgG F(ab')2 was added to 40-fold molar excess sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (Pierce, Rockford, IL) in 1 ml PBS and incubated for 1 h at room temperature. A Slide-A-Lyzer small-volume dialysis cassette (Pierce) was used to dialyze the mixture overnight at 4°C in PBS. The next day, OVA (5-fold molar excess protein compared with the F(ab')2) was dissolved in 250 ml of a reaction buffer (50 mM triethanolamine, pH 8, 0.15 M NaCl, 1 mM EDTA) and added to an equal volume of 2-iminothiolane (5-fold molar excess compared with the OVA) dissolved in the reaction buffer. The mixture was incubated for 1 h at room temperature, then a D-Salt Polyacrylamide Plastic Desalting Column (10 ml) (Pierce) was used to collect 250 ml fractions. Spectrometer readings were taken at OD280 to pool 1–1.5 ml of the protein peak. The F(ab')2:sulfo-SMCC and thiolated OVA were mixed and incubated overnight at 4°C.

B cell purification

Transitional B cells were harvested and purified from the spleens of mice 13 or 14 days after irradiation with 500 rad, as described previously (20, 36). Spleens from nonirradiated or postirradiated mice were dissected, and cells were dispersed with the frosted surface of glass slides. The cell suspension was collected and treated with anti-Thy-1.2, rabbit complement, and DNase I for 1 h. The small population of immature B cells was depleted from nonirradiated adult spleens with the addition of AA4.1 Abs, a recently isolated mAb specific for immature B cells (39), followed by MAR18.5 during the complement lysis step. MAR18.5 is an anti-rat-{kappa} Ab that enhances cytolysis by noncomplement fixing Abs. After RBC lysis in Gey’s solution, the cells were centrifuged over a 50/75% Percoll gradient (Pharmacia Biotech, Uppsala, Sweden). Cells separating at the 50/75% interface were collected for use in the experiments. Typical preparations were 90–95% pure for B cells.

CD4 T cell purification

Single cell suspensions were prepared from the spleen and lymph nodes of DO11.10 TCR transgenic mice. The cells were centrifuged over a Lympholyte-M density cell separation medium (Cedarlane Laboratories, Hornby, Ontario, Canada) to separate out the RBCs and dead cells. The cells in the interface were resuspended in PBS, 2 mM EDTA, and 0.5% BSA; labeled with anti-CD4 magnetic beads (L3T4); and passed through a magnetic column (Miltenyi Biotec, Auburn, CA). The retained positively selected CD4+ T cells were used for the coculture experiments.

B cell apoptosis assay

A total of 2 x 105 purified B cells was cultured in 200 ml of assay medium in sterile snap-top tubes with the indicated reagents, then cocultured with 4 x 104 CD4+ DO11.10 T cells. After 18 h of culture in a 37°C CO2 incubator, cells were harvested by one wash with PBS, 2% FCS, and 0.02% azide. The cells were stained with anti-CD23 and anti-B220 and then fixed with 500 ml 0.0625% paraformaldehyde for 1 h at 4°C. Cells were washed, resuspended in 1 ml of 0.2% Tween 20, and then incubated in 37°C CO2 incubator for 15 min. After washing once, 500 ml of propidium iodide (PI) (Sigma-Aldrich) staining buffer (PBS, 10 mg/ml PI, 50 mg/ml RNase, 0.02% azide) was added to each tube. The cells were incubated overnight in the dark at room temperature, then analyzed on a BD Biosciences (Franklin Lakes, NJ) FACSCalibur using CellQuest software. The percentage of apoptotic cells was represented as the percentage of B cells with subdiploid DNA content.

CFSE labeling of CD4 T cells

A total of 2–3 x 107 purified CD4 T cells was resuspended in 5 ml of PBS (room temperature). An equal volume of PBS/2 µM CFSE was added, and the suspension was gently rocked back and forth for 3 min. The reaction was quenched with one-fifth volume cold FCS, washed, and resuspended in the culture medium.

B and T coculture experiments

A total of 2 x 105 purified mature or transitional B cells was cultured in B cell assay medium (2 x 106 cells/ml) containing RPMI 1640, supplemented with 10% heat-inactivated FCS (defined; HyClone, Logan, UT), 2 mM L-glutamine, nonessential amino acids, penicillin/streptomycin, and 5 x 10-5 M 2-ME at 37°C in 6% CO2 in round-bottom 96-well plates alone or with a combination of noted reagents. After 4 h of incubation, 100 µl containing 4 x 104 purified DO11.10 CD4 T cells resuspended in assay medium was added to the B cell culture for the additional time, as noted.

Calculation of responder frequency

After 3 days of B/T cell coculture, CFSE-staining patterns of live CD4 T cells were analyzed by gating on those CD4+ cells that exclude the vital dye TOPRO-3. Making use of the mathematical relationship that a single T cell dividing n times will generate 2n daughter cells, the frequency of precursors that respond by proliferating (responder frequency) can be extrapolated from the number of daughters under each division peak (40).

Measurement of IL-2 and IFN-{gamma}

After centrifuging the samples, supernatants (150 ml) from the cocultures were collected and stored at -80°C until ready for the assay. Seventy-five milliliters of each sample were thawed for the experiment. First, anti-IL-2 capture Abs (JES6-1A12) (BD PharMingen, Franklin Lakes, NJ) were bound onto sulfonated latex beads (Interfacial Dynamics, Portland, OR) by incubating 5 µl of Ab to 107 beads in 1 ml of PBS for 1 h in a 37°C water bath. One hundred thousand coated beads were then added to each supernatant sample and further incubated at 37°C for 1 h. The samples were then placed into FACS tubes, washed once with PBS/0.5% BSA, then stained with anti-IL-2-PE Ab (BD PharMingen). Serial dilutions of rIL-2 were used as a standard for calculating concentrations. For measurements of IFN-{gamma} levels, tissue culture supernatants were collected at 72°C and assayed for IFN-{gamma} by standard ELISA using anti-IFN-{gamma} capture Ab (BD PharMingen) and anti-IFN-{gamma} detection Ab (BD PharMingen). The biotinylated secondary Ab was detected by streptavidin HRP and SureBlue detection kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transitional immature B cells present Ag to T cells less efficiently than mature B cells, but this deficit can be overcome with costimulation

To evaluate the Ag-specific proliferative response of CD4 T cells to mature or transitional B cells, CFSE-labeled DO11.10 CD4 T cells were cocultured for 3 days with mature or transitional immature B cells purified from wild-type BALB/c mice. Before the addition of CD4 T cells, the B cells were pulsed with OVA323–339 peptide, and activated through the Ag receptor by anti-mouse Ig F(ab')2 (anti-BCR). By pulsing with preprocessed peptide, the BCR-mediated signaling component could be separated from the Ag processing and presenting components of B cell function. In labeling the DO11.10 CD4 T cells with CFSE before coculture with the B cells, the proliferation history of individual CD4-expressing T cells could be ascertained.

When the antigenic peptide was added alone at a limiting dose of 0.005 µg/ml, neither mature (left panel) nor transitional immature (right panel) B cells induced T cells to proliferate (Fig. 1). By comparison, when anti-BCR was added with the peptide Ag in the coculture, T cells cocultured with mature B cells proliferated, whereas those cocultured with transitional immature B cells did not. The addition of CTLA4-Ig to the coculture decreased the T cell response to Ag-pulsed mature B cells, and anti-CD28 increased the proliferative response of T cells cocultured with transitional B cells to levels similar to those found in mature B/T cell cocultures. At higher doses of peptide Ag (0.1 µg/ml), differences in the proliferative response to mature and transitional B cells were decreased. Costimulatory blockade with CTLA4-Ig failed to decrease the T cell proliferative response (data not shown), indicating a reduced dependence on CD28 costimulation at these higher peptide concentrations. This finding shows that for proliferation, a sufficiently strong stimulatory signal through the TCR obviates the need for costimulation. Moreover, under these conditions in which the requirement for costimulation is relaxed, the difference in the T cell activation response that is mediated by mature vs transitional immature B cells is minimized.



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  		FIGURE 1. Transitional B cells present peptide Ag less efficiently than mature B cells. Resting mature or transitional immature B cells were cocultured with CFSE-labeled DO.11.10 CD4 T cells in the presence or absence of 5 ng/ml of OVA323–339 and the indicated reagents for 72 h. CFSE labeling results in a homogeneous distribution of the dye within and at the surface of the cell. The dye segregates equally between daughter cells upon cell division, resulting in sequential halving of cellular fluorescence intensity with each successive generation. Cell division of populations of cells can be visualized as distinct peaks of various intensity by flow cytometry. The resulting CFSE fluorescence histograms gated on CD4-positive, TOPRO-3-negative lymphocytes are shown.

 
These effects indicate that at lower doses of Ag, CD28 costimulation is necessary for productive T cell activation. The lack of T cell responsiveness in the transitional immature B cell cocultures is a consequence of ineffective costimulation rather than ineffective primary signals generated through TCR-peptide:MHC class II interactions (signal 1). These conclusions will be explored more directly in studies that follow.

Transitional immature B cells are capable of processing and presenting Ag as peptide:MHC class II complexes on the surface

The above studies used pulsed peptide rather than Ag that had been endocytosed by BCR-dependent processes, processed, and then presented onto the cell surface. To directly measure the ability of transitional B cells to present processed antigenic peptide in the context of MHC class II molecules, the C4H3 mAb (38), which recognizes the HEL46–61 peptide in the context of I-Ak, was used. For these experiments, B cells were purified from spleens of nonirradiated and autoreconstituting adult CBA/J mice (H-2k) (see Materials and Methods). AA4.1 is a marker that allows transitional immature and mature B cells to be distinguished (21, 36). In this case, transitional immature B cells are AA4.1high, whereas mature B cells are AA4.1low. AA4.1 staining of the purified B cells confirmed that the spleens of day 14 sublethally irradiated CBA/J mice consisted almost entirely of transitional immature B cells, as determined by the high AA4.1 expression by B220+ cells compared with levels expressed by splenic B cells from nonirradiated mice (Fig. 2A). Purified splenic mature or transitional B cells from CBA/J mice were pulsed with HEL46–61 peptide or the whole HEL protein for 2.5 h and then stained with the C4H3 mAb (Fig. 2B). There was significant background C4H3 staining without specific Ag, a characteristic of this Ab that has been previously described (38). Nevertheless, there was a clear dose-related increase in the level of C4H3 staining after pulsing with the HEL46–61 peptide, as well as with the whole HEL protein in both the mature and transitional B cells. Pulsing with equimolar amounts of the MHC class II-incompatible OVA323–339 and whole OVA protein did not result in increased staining, confirming the Ag specificity of the C4H3 mAb (data not shown). Immature and mature B cells differ with respect to their expression level of MHC class II complexes: mature B cells express more than immature B cells. Comparative analysis of the FACS histograms from this experiment shows that for each dose of the whole protein used in the pulse, both mature and transitional B cells displayed comparable levels of peptide:MHC class II complexes on the surface (Fig. 2C).



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  		FIGURE 2. Transitional immature B cells are capable of processing protein Ag and presenting in the context of class II complexes. A, Purified B cells from spleens of CBA/J mice sublethally irradiated 14 days previously are transitional immature, as determined by AA4.1 expression (dotted line) relative to mature B cells (solid line). B, Mature or transitional immature B cells were pulsed for 2.5 h with medium, HEL46–61 peptide, or whole HEL at the indicated doses, then washed and stained with the C4H3 mAb that detects HEL46–61:I-Ak complexes. There is a dose-related increase in the level of C4H3 staining after pulsing with the HEL peptide and with whole HEL in both mature and transitional B cells. C, Overlay histograms comparing C4H3 staining on mature and transitional immature B cells pulsed with equimolar amounts of whole Ag demonstrate comparable levels of peptide:MHC class II complexes on the cell surface.

 
These results represent the first direct demonstration of the efficiency of Ag processing and presentation by transitional B cells. Although this experiment shows Ag processing in the setting of fluid-phase uptake and not by BCR-mediated endocytosis, it demonstrates that the processing and presentation mechanisms are indeed intact.

Transitional immature B cells display a failure to up-regulate CD86 in coculture with CD4 T cells

Unlike mature B cells, purified transitional B cells do not up-regulate CD86 in response to anti-BCR stimulation (21). Given the importance of costimulation in T cell responses to B cell Ag presentation, the level of CD86 on transitional B cells in coculture with intact cognate CD4+ T cells was evaluated. Having demonstrated the ability of transitional B cells to efficiently process and present Ag onto the surface as MHC class II:peptide complexes, whole Ag was used for the cocultures in addition to pulsing with the peptide component. The heterogeneity of the BCR repertoire in the primary B cells purified from a wild-type mouse prevented us from using a conventional protein Ag to test BCR-mediated uptake. The inability to use Ig transgenic mice to isolate transitional immature B cells of sufficient purity prevented us from using these models for this study. Instead, anti-mouse Ig F(ab')2 were covalently conjugated to OVA using the cross-linking agent sulfo-SMCC (see Materials and Methods). At appropriate concentration, conjugated Ag Fab2:OVA) would allow us to direct internalization and processing by mature or transitional immature B cells toward BCR-dependent processes, regardless of the specificity of their BCR.

The transitional immature B cells failed to up-regulate CD86 after coculture with CD4 T cells in the presence of both OVA323–339peptide and Fab2:OVA, although mature B cells very effectively demonstrated this response (Fig. 3A). The low level of CD86 expression on transitional B cells did not, however, greatly affect the interaction with and early responses by CD4+ T cells. Although less than mature B cells (Fig. 3B), transitional B cells activated a significant percentage of T cells, as seen by the induction of surface CD69 on interacting CD4 T cells. These latter results confirm that both transitional immature as well as mature B cells deliver signal 1 (mediated via peptide:MHC class II:TCR interaction) to the T cell.



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  		FIGURE 3. Differential up-regulation of CD86 on mature compared with transitional immature B cells after coculture with DO11.10 CD4 T cells. A, CD86 (B7.2) expression on B cells after coculture with T cells in the presence of conjugated Ag. Depicted is CD86 expression on gated viable B220+ B cells. B, Mature or transitional immature B cells were pulsed with varying doses of conjugate or peptide Ags and then CD4 T cells were added in coculture for an additional 14 h. CD69 expression is reported as the percentage of CD4 T cells that have up-regulated CD69 on the surface. These results demonstrate that both B cell populations are able to provide signal 1 to resting CD4 T cells.

 
BCR-mediated endocytosis and processing of Ag by transitional immature B cells lead to less efficient presentation to Ag-specific T cells, which can be overcome with costimulation

Ag-dependent proliferative and IL-2 responses by DO11.10 CD4 T cells cocultured with mature or transitional B cells were evaluated. The use of CFSE labeling allows for the division history of individual cells to be tracked and for proliferation of subpopulations to be quantified. Given that a single T cell dividing n times will generate 2n daughter cells, the frequency of precursors can be extrapolated from the number of daughters under each division peak (40). The responder frequency represents the frequency of starting T cells that respond to the stimulating Ag by proliferating, and it serves as a quantitative index of proliferation for comparative analysis.

The results using the conjugated Fab2:OVA Ag confirm what was seen by coculture experiments using peptide Ags. At lower doses (0.05–1 µg/ml), T cells cocultured with mature B cells proliferated significantly more when compared with those cocultured with transitional B cells (Fig. 4A). This response difference was eliminated by the addition of anti-CD28 Abs to the transitional B:T cell cocultures. In the absence of Ag, CD4 T cell proliferative responses were negligible in both the mature and transitional immature B cell-containing cocultures.



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  		FIGURE 4. Transitional B cells are less effective than mature B cells in inducing proliferation and IL-2 production by cocultured CD4 T cells. A, CFSE-labeled CD4+ DO11.10 T cells were cocultured with mature or transitional immature B cells in the presence of Fab2:OVA or OVA alone. The doses indicated for Fab2:OVA correspond to the OVA component of the conjugate, and thereby allow direct comparison between conjugate- and OVA-containing cocultures. TOPRO-3 was used to identify and eliminate dead cells in the cultures. In all cases, there was no significant difference noted in the frequency of dead cells detected by this method when comparing any of the culture conditions out to 72 h of culture. Responder frequency was used as an index of the number of precursors that were recruited into a proliferative response (see Materials and Methods). The data represent the mean of triplicates ± SD. B, A parallel set of cocultures to allow comparison of T cell responses to B cells presenting Fab2:OVA (A) compared with OVA (B). C, For IL-2 measurements, the supernatant was collected after 20 h of coculture. Then an ELISA in solution using flow cytometry was used to measure the level of IL-2 (see Materials and Methods). D, IL-2 levels are plotted against the responder frequency for all Ags and doses shown in C. Best fit curves drawn through the cell type interacting with the T cells demonstrate discoordinate IL-2 production and proliferative responses of T cells presented Ag by transitional immature B cells.

 
A concern in coculture studies is that processing and presentation of Ag may be occurring through contaminating non-B cell APC. To address whether or not this contamination was a concern at the Ag doses relevant to our results, we compared the results depicted in Fig. 4A using Fab2:OVA with responses obtained using unconjugated OVA. Using OVA, uptake, processing, and presentation can only occur through fluid-phase uptake in all but OVA-reactive B cells. The presence of such B cells would be expected to be extremely rare, if present at all, in these polyclonal B cell isolates. If the results from using Fab2:OVA were not the consequence of direct presentation by BCR-expressing cells, then the dose-response relationship using unconjugated OVA should be similar. The efficiency of presenting conjugated Ag through the BCR compared with using fluid-phase uptake of unconjugated whole Ag was apparent in the greater than 3 log difference in Ag dose required to reach similar responder frequency (Fig. 4B). This finding is in keeping with previous data showing a 1000-fold difference in efficiency of BCR-mediated Ag endocytosis and presentation compared with fluid-phase uptake (13). These results also show that non-B cell APCs contaminating the B cell preparation do not significantly contribute to the T cell responses in these assays.

As with the proliferative responses, IL-2 production was also greatly reduced when transitional immature B cells were used as APC, relative to mature B cells (Fig. 4C). In fact, the differences at all doses were even more pronounced than those observed using proliferation as a readout. However, as with proliferation, this difference in IL-2 was ameliorated by providing costimulation with the addition of anti-CD28. Finally, we also observed the same difference in proliferation and IL-2 levels between transitional and mature B cells when higher concentrations of nonconjugated OVA were used. This observation most likely indicates a requirement for costimulation following cognate interactions involving transitional immature B cells presenting peptide derived from protein Ag following fluid-phase rather than BCR-mediated uptake.

Plotting IL-2 levels of the coculture supernatants under all conditions against the responder frequency of the T cells clearly demonstrates the disproportionate decrease in the level of IL-2 in cocultures with immature B cells (Fig. 4D). Therefore, there appears to be a dissociation between proliferation and IL-2 levels seen with CD4 T cells cocultured with transitional B cells that is made up with anti-CD28 treatment. The significance of this low level of IL-2 is unclear. It most likely represents underproduction rather than overconsumption, given that there is no corresponding increase in proliferation compared with T cells cocultured with mature B cells.

The levels of IL-2 were measured 20 h after the initiation of the cocultures. As noted, the decreased levels in cultures in which Ag was presented by transitional immature B cells, compared with mature B cells, correlate with the decreased T cell proliferation in the transitional B cell cultures. These results clearly establish an impairment in late T cell responses involving proliferation when Ag was presented by transitional immature B cells. Interestingly, as with the earlier CD69 responses shown in Fig. 3, not all measures of T cell responsiveness are impaired when transitional and mature B cells are compared. Shown in Fig. 5, IFN-{gamma} levels were comparable between cocultures of DO11.10 CD4 T cells and transitional or mature B cells containing various doses of Fab2:OVA. These results further indicate selectivity in the impairment of functional T cell responses following Ag presentation by transitional immature B cells.



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  		FIGURE 5. Transitional immature and mature B cells elicit comparable IFN-{gamma} responses from DO11.10 CD4 T cells. IFN-{gamma} levels were measured by ELISA on supernatants from cultures containing DO11.10 CD4 T cells and transitional immature or mature B cells with various doses of Fab2:OVA. The details of the cultures were identical with those described for IL-2 measurements, except that supernatants were harvested at 72 h after initiation of cocultures.

 
Transitional immature B cells poorly elicit protective signals from CD4+ Th cells unless CD28 costimulation is provided

Previous studies have shown that T cell help signals in the form of IL-4 and activating anti-CD40 mAb can rescue transitional immature B cells from undergoing Ag-induced apoptosis (20). Although instructive, soluble factors do not replicate the actual interactions that occur between two intact cells. Specifically, these previous studies did not address whether transitional immature B cells can elicit these protective signals from Ag-reactive T cells. To test this signaling, purified transitional immature B cells were cocultured with DO11.10 CD4 T cells in the presence of anti-BCR alone or with the OVA323–339 peptide. The amount of peptide used to pulse mature and transitional immature B cells was chosen based upon the dose shown for mature B cells to stimulate maximal T cell proliferation (Fig. 1). The amount (0.1 µg/ml) was tested in these experiments for its ability to deliver equivalent signal 1 responses by DO11.10 CD4 T cells. As seen in Fig. 6A, equivalent frequencies of CD69+ CD4+ cells are observed 14 h following coculture with either peptide-pulsed transitional immature or mature B cells. Furthermore, the frequency of CD69+ T cells after 14 h was not significantly different in response to either peptide-pulsed B cell population at 1 µg/ml OVA peptide, indicating that the ability to initiate signaling in T cells was maximal and equivalent for both B cell populations at the lower dose.



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  		FIGURE 6. Transitional immature B cells can be rescued from Ag-induced apoptosis in the presence of Ag-specific CD4 T cells. A, The activation of DO11.10 CD4 T cells measured by the percentage of CD69+ cells after 14 h of coculture with transitional immature B cells in the presence of medium or with the addition of anti-CD28. The data represent the mean of triplicates ± SD. B, Transitional immature B cells were cocultured with DO11.10 CD4 T cells with (solid line, squares) or without (solid line, X) anti-BCR (10 µg/ml) and OVA323–339 peptide, as indicated. The cells were stained with B220. After fixation and permeabilization, the cells were stained with PI. Percentage of apoptotic cells refers to the percentage of B220+ cells with subdiploid DNA content. Some cocultures that were stimulated with anti-BCR were also provided with anti-CD28 Ab (dotted line, diamonds).

 
Fig. 6B depicts the ability of DO11.10 T cells to protect transitional B cells from BCR-induced apoptosis. Maximal anti-BCR-induced apoptotic responses were observed in those cultures containing T cells and transitional immature B cells without peptide loading (0 µg/ml). This response was only minimally decreased by addition of anti-CD28 to the coculture (69 vs 66%, respectively). A statistically significant, but small T cell-dependent modulation of the BCR-induced apoptotic responses was observed for those cultures receiving transitional immature B cells loaded with 0.01 and 0.1 µg/ml peptide. However, this suboptimal level of protection conferred by the T cells at these lower Ag doses was significantly increased in cocultures that received anti-CD28. This increase represents a 50% reduction (after subtracting background levels) in the frequency of apoptotic B cells present. In fact, at all Ag doses, anti-CD28 markedly enhanced the antiapoptotic effects of DO11.10 CD4 T cells. In the case of the 1 µg/ml peptide dose, the T cells provided with exogeneous costimulation (anti-CD28) completely abrogated the BCR-induced apoptotic response, returning it to baseline levels.

These results confirm and extend our previous published results (21); they show that T cell help signals can modulate BCR-dependent negative selection. However, they further establish that the ability of T cells receiving optimal TCR signal is compromised, unless exogeneous costimulation is provided.

T cell-mediated protection from BCR-induced apoptosis is less costimulation dependent with cognate interactions involving activated T cells

In vivo, Ag-reactive transitional immature B cells theoretically could have access not only to compartments containing naive T cells, but also to activated T cells. The latter might occur as a consequence of an ongoing immune response present at the time that the newly emerging transitional immature B cells encounter Ag. One could argue teleologically that it might be advantageous to preferentially rescue those Ag-reactive developing B cells that have recognized and processed an Ag that the T cell compartment has already identified as dangerous to the host. This argument predicts that protection might be more easily elicited from preactivated rather than naive T cells. This prediction was tested in the following set of experiments.

As a source of preactivated T cells, DO11.10 splenocytes were pulsed with OVA323–339 peptide and cultured for 4 days, after which time CD4 T cells were purified (Fig. 7A). Naive, resting CD4 T cells were isolated by sorting CD45RBhigh CD4+ cells from DO11.10 splenocytes (see Fig. 7B). This sorting protocol was indicated because there exists a small, but significant amount of endogenous TCR {alpha}-chain expression in DO11.10 TCR Tg mice, resulting in the presence of CD4 T cells with dual TCR specificities. These dual expressers have the possibility of being triggered by endogenous Ag and differentiating into a memory phenotype. Eliminating the CD45RB- population eliminates this potential caveat.



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  		FIGURE 7. Preactivated T cells confer increased antiapoptotic protection of BCR-stimulated transitional immature B cells. A, Preactivated T cells were isolated by magnetic column positive selection after a 4-day culture of RBC-depleted DO11.10 splenocytes with high doses of OVA323–339 peptide (5 mg/ml). B, Naive CD4 T cells were sorted by staining leukocytes from the spleen and lymph nodes of a DO11.10 mouse and sorting for CD4-positive cells with a high expression of CD45RB. Approximately 70% of CD4 T cells are CD45RBhigh before the sort. After the sort, 98% of the total cells are CD45high naive CD4 T cells. C, Apoptosis protection of transitional immature stimulated with anti-BCR (10 µg/ml) by resting, naive, and preactivated or without (none) T cells with varying doses of peptide. Sorted naive T cells and CD4 T cells purified as in previous results (resting) did not show a difference in the degree of B cell apoptosis protection. In anti-BCR-stimulated transitional B cells cocultured with preactivated T cells, there is protection from apoptosis even in the absence of Ag, unlike that seen with anti-CD28. D, Apoptosis protection of transitional immature and mature splenic B cells by resting, naive, and preactivated or without (none) T cells with Fab2:OVA conjugate (1 µg/ml) as Ag (filled bars) or no Ag (open bars). As in C, preactivated, but not naive T cells block the anti-BCR apoptotic response of the transitional immature B cells. The mature B cells do not exhibit an induced apoptotic response either in the presence or absence of CD4 T cells in the culture.

 
Resting or preactivated T cells were added in coculture with transitional B cells treated with anti-BCR and pulsed with varying amounts of OVA323–339 peptide (Fig. 7C). At doses shown previously to trigger optimal primary signals through the TCR (1.0 and 0.1 µg/ml of OVA peptide; Fig. 6), the levels of apoptotic B cells were reduced to background levels (i.e., no anti-BCR stimulation), even though anti-BCR was present throughout the culture period. Notably, even in the absence of Ag, preactivated CD4 T cells provided extra protection, suggesting that there is an Ag-nonspecific bystander effect that may augment the Ag-specific protective effects.

A small, but significant amount of endogenous TCR {alpha}-chain expression in DO11.10 TCR Tg mice resulted in the presence of CD4 T cells with dual TCR specificities. Although the resting CD4 T cells used for these studies were purified from nonimmunized TCR transgenic mice and were mostly naive, T cells exposed to Ag through the endogenous {alpha}-chain may convert the cells to a memory phenotype. To confirm that the results seen with resting CD4 T cells are indeed what is observed with a truly naive population of CD4 T cells, RBC-depleted leukocytes from the spleen and lymph nodes of DO11.10 mice were sorted by flow cytometry by CD4 and CD45RB status (Fig. 7B). The naive CD45RBhigh CD4+ T cells were added to anti-BCR-treated transitional B cells, and the degree of apoptosis protection was compared with nonsorted resting CD4 T cells. The results showed that there was essentially no difference at all doses studied (Fig. 7C). Finally, similar results were obtained when the Fab2:OVA conjugate was used to allow Ag uptake through BCR-mediated processes. In this case, responses were compared also to mature B cells (Fig. 7D). The latter do not exhibit an anti-BCR-induced apoptotic response regardless of the source of CD4 T cells in the coculture.

Together, the results comparing the effects of resting and activated T cells in the previous two studies demonstrate that while naive CD4+ Th cells can productively interact and respond to Ag-presenting transitional immature B cells, the ability to elicit antiapoptotic signals is severely compromised relative to cocultures in which CD28-mediated cosimulation is exogenously provided. The observation that exogeneous costimulatory signals mediated such a profound effect suggests that cognate interactions between B7.2 low transitional immature B cells and naive T cells are unlikely to block BCR-induced negative selection of these B cells. In contrast, preactivated T cells are very efficient in mediating this protection and do so even without exogeneous CD28-mediated costimulatory signals. Such effects, which one might predict as a component of an ongoing T cell response, are more efficient when the B cells present the appropriate peptide, but they also exist to a limited degree in the absence of peptide.

LFA-1-dependent cognate interactions between CD4 T cells and transitional immature B cells are required for optimal protection from BCR-induced apoptosis

In addition to CD28/B7, the LFA-1:ICAM-1 molecules have been shown to be an important costimulatory receptor/ligand couple for CD4 T cell interactions with APCs (41, 42, 43, 44). Wulfing et al. (45) have recently demonstrated that the engagement of these molecules leads to their accumulation at the T cell-APC interface, which is not a passive, but rather an active, TCR-independent cytoskeletal mechanism that amplifies any TCR-mediated signals (45). When anti-LFA-1 was added to the transitional B cell/CD4 T cell coculture, the protective effect of the CD4 T cells was decreased with a consequent increase in transitional B cell apoptosis (Fig. 8A). This decrease suggests that the CD4 T cells exert their protective effects at least partly through cell contact with transitional B cells. The addition of anti-LFA-1 Ab presumably disrupts the stability of the immunological synapse and prevents the activation of CD4 T cells. Indeed, there is dramatically reduced percentage of CD4 T cells that are positive for CD69 expression when anti-LFA-1 is added to the coculture (Fig. 8B).



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  		FIGURE 8. LFA-1 blockade disrupts T cell-mediated protection of transitional immature B cells from BCR-induced apoptosis. A, Anti-BCR-treated transitional B cells were cocultured with DO11.10 CD4 T cells in the presence of varying doses of OVA323–339 peptide. The addition of anti-LFA-1 led to an increase in apoptosis at all doses studied. B, Anti-BCR-treated transitional B cells were cocultured for 14 h with DO11.10 CD4 T cells in the presence of varying doses of OVA323–339 peptide. There is a dramatically reduced percentage of CD4 T cells positive for CD69 expression when anti-LFA-1 is added to the coculture. Mean of triplicates is shown ± SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The migration of transitional B cells to splenic compartments enriched in Ag and CD4 T cells provides an ideal setting for the selection processes to operate. Although intrinsic processes lead developing B cells to undergo negative selection upon encountering Ag, the proximity of CD4 T cells allows for the possibility of rescue through cognate interactions or bystander effects. We have shown that transitional immature B cells are capable of processing and presenting Ag onto the surface as MHC class II:peptide complexes. However, because transitional immature B cells are unable to up-regulate CD86 upon Ag stimulation, the consequence of this presentation has implications for the fates of both the Ag-presenting transitional immature B cells and the interacting CD4 T cells.

It was found that CD4 T cells cocultured with transitional immature B cells proliferated less well and had a dramatic decrease in IL-2 levels relative to those cocultured with mature B cells. These deficits were made up when exogenous stimulation was provided by anti-CD28. These results are not unexpected, as disruption of the CD28/B7 interaction previously has been shown to reduce the frequency of proliferating T cells (40, 46). The low IL-2 levels can be attributed to the findings of others that CD28 increases transcription of IL-2 and stabilizes its mRNA (47, 48, 49). It may also reflect differences in the efficiency of IL-2 consumption, although this possibility was not directly tested.

The lack of CD86:CD28 costimulation by the transitional immature B cells might predict that cognate interactions with naive T cells may result in anergy. Jenkins and Schwartz (50) made the important initial observation with T cell clones that in the absence of costimulatory signals, T cells fail to proliferate and become refractory to further activation. Despite multiple attempts, we were unable to consistently demonstrate Ag-induced anergy of DO11.10 CD4 T cells using transitional immature B cells as the APC. This lack of a clearly inducible anergic phenotype was observed for both proliferation and IL-2 secretion as readouts in response to restimulation by peptide-pulsed splenocytes. There may be several reasons for a failure to induce T cell unresponsiveness in this model. Other investigators have reported difficulty in inducting anergy in primary T cells in vitro (51, 52, 53, 54). Initially defined in Th1 cell lines, anergy may perhaps be a mechanism limited to preactivated T cells rather than a process to tolerize primary T cells. Alternatively, the signals that lead to anergy may involve not only TCR engagement in the absence of costimulation, as is supplied in our model, but in addition they may involve other interactions and signals not provided in our experimental design.

In vivo, the interaction between transitional immature B cells and CD4 T cells has the potential for shaping the repertoire of the long-lived B cell compartment. The presented studies have demonstrated that CD4 T cells can rescue Ag-activated transitional B cells from apoptosis, and that this process can be modulated by costimulation through CD28 as well as through processes dependent upon LFA-1. Providing exogenous costimulation through CD28 led to increased protection from Ag-induced apoptosis, suggesting a possible mechanism through which self-reactive transitional B cells in the periphery can escape deletion. Preactivated CD4 T cells, as well, were shown to have an enhanced ability to rescue transitional B cells, and to a limited extent, they did so even in the absence of specific Ag. This finding suggests that strong bystander effects, such as those that may occur in situations of chronic unregulated T cell activation or inflammation, have the potential to play a role in the selection process in the spleen. Although self-reactive B cells may escape deletion nonspecifically, it may be outweighed by the benefits of recruiting newly emerging B cells in mounting a response against a legitimate foreign Ag. Transient production of autoantibodies is a well-described phenomenon associated with acute infections. However, as expected, protection from BCR-induced negative selection was observed to be most efficient under conditions in which cognate interactions between the B and T cells were maintained. The initiation and stabilization of the immunological synapse has been described as a major mechanism of costimulation provided by CD28/B7 and ICAM-1:LFA-1 receptor/ligand pairs (45). The presented studies have shown that both these pathways are important modulators of B cell negative selection. Another potential mechanism for LFA-1 to confer protection for transitional B cells is through its interaction with ICAM-2, which is an activator of the protein kinase B pathway, leading to inhibition of apoptosis (55).

In addition to its role in T cell activation and the induction of tolerance, CD28 has been shown to regulate Th1/Th2 differentiation (56, 57, 58), maintain the homeostasis of the CD25+CD4+ immunoregulatory T cells (59, 60), and affect cell migration and inflammation through the regulation of key chemokines (61, 62). The exact nature of these downstream changes may prove critical for understanding the development of the T cell immune repertoire and the development of autoimmunity. The restricted anatomic compartment in which transitional B cells interact with CD4 T cells provides a rich environment for both contact and soluble mediators to play a role in affecting T cell fates and modulating B cell negative selection. Continued dissection of the array of factors involved in this complex interaction will shed light on the process of immune repertoire formation of B and T cell compartments.


   Acknowledgments
 
We acknowledge the editorial assistance of Justina Stadanlick during the final preparation of this manuscript.


   Footnotes
 
1 This work was partially supported by National Institutes of Health Grants AI32592 and AI43620 to J.G.M. and AI43620 to L.A.T. J.B.C. was supported by the Arthritis Foundation’s Physician Scientist Development Award, and by National Institutes of Health Grant AI49323. Back

2 Address correspondence and reprint requests to Dr. John G. Monroe, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 311 BRB II/III 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: monroej{at}mail.med.upenn.edu Back

3 Abbreviations used in this paper: HEL, hen egg lysozyme; BCR, B cell receptor; PI, propidium iodide; sulfo-SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. Back

Received for publication November 22, 2002. Accepted for publication June 4, 2003.


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