Role of B cells as antigen-presenting cells in vivo revisited: antigen-specific B cells are essential for T cell expansion in lymph nodes and for systemic T cell responses to low antigen concentrations

Amariliz Rivera, Chiann-Chyi Chen, Naomi Ron, Joseph P. Dougherty and Yacov Ron

Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA

Correspondence to: Y. Ron


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Studies in B cell-deficient mice generated by continuous injection of anti-µ antibodies (µSM) showed that T cell priming in lymph nodes was dependent on antigen presentation by B cells. This concept has recently become controversial since a wide range, from complete deficiency to near normal T cell responses, was reported in studies carried out with B cell-deficient mice generated by gene disruption (µMT). In this study we show that in the absence of B cells, T cell responses are greatly reduced in all the available µMT mouse strains although responses in µMT of the C57BL/6 background (which were used for most studies with µMT) were much more variable and could reach up to 42% of control. In contrast, T cell responses in µMT -> F1 bone marrow chimeras which have the same phenotype as µMT were totally impaired, suggesting a principle difference between mice developing without B cells (µMT mice) and µSM which are made B cell deficient only after birth. Normal T cell priming was completely restored by reconstitution of µMT and µMT -> F1 mice with syngeneic B cells. Interestingly, only B cell populations containing antigen-specific B cells were capable of reconstituting T cell responses. Monoclonal B cells taken from Ig transgenic mice could not reconstitute responses to an irrelevant antigen. We also found that B cells were also required for systemic T cell priming when antigen concentrations were limiting but were not required for priming (for T cell help) when mice were immunized with a high antigen dose.

Keywords: antigen presentation, B cell-deficient mice, clonal expansion, T-B cell collaboration, T cell activation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD4+ T cells are activated by the recognition of peptide in the context of self-MHC class II molecules expressed by professional antigen-presenting cells (APC) such as macrophages, dendritic cells (DC) and B cells. For complete T cell activation, APC also have to express a variety of co-stimulatory molecules, mainly CD40, CD80 and CD86 (1). Although all professional APC can present antigen, it is unclear whether individual subsets of APC have unique properties in certain immune responses or environments. The first in vivo evidence that B cells are essential APC in lymph nodes (LN) came from studies using mice that lacked peripheral B cells due to the administration of anti-µ antibodies from birth [µ-suppressed mice (µSM)] (26). µSM mice showed a severe impairment in CD4+ T cell priming when antigen was injected locally in complete Freund's adjuvant (CFA). These mice also had increased susceptibility to viral infections (7,8), reduced delayed-type hypersensitivity (DTH) responses (4,9), and were resistant to autoimmune diseases like experimental autoimmune encephalomyelitis (EAE) (1013) and diabetes (µSM NOD mice) (14), strongly suggesting that in the absence of B cells, CD4+ T cells could not be efficiently primed. All the CD4+ T cell defects observed in µSM could be reversed upon adoptive transfer of B cells prior to antigenic challenge, which pointed to a direct role for B cells as APC and argued against a deleterious effect caused by the prolonged exposure to high levels of anti-µ antibodies (5,6,15).

The role of B cells as important APC in vivo became controversial after B cell-deficient mice were generated by gene disruption of the transmembrane domain (µMT) or the JH segment of the Ig heavy chain (JHD) (16,17). Although both µSM and µMT mice lack mature peripheral B cells, studies of T cell priming in µMT mice gave conflicting results ranging from near normal to severely impaired responses. T cell proliferative responses to foreign proteins like keyhole limpet hemocyanin (KLH), human {gamma}-globulin or purified protein derivative (PPD) were reported to be similar to normal controls, although at least in the work of Epstein et al. control responses were unusually low making it difficult to evaluate differences in responses between control and µMT mice (18,19). In studies employing JHD B cell-deficient mice, Liu et al. reported a severe impairment of both T cell proliferation and T cell help to proteins like KLH and ovalbumin (20). Normal priming of CD4+ T cells could be restored by adoptive transfer of mature B cells before priming (20). In contrast, Macaulay et al. reported that CD4+ T cell proliferative responses to KLH were normal in JHD mice, but that the cytokine profile was skewed such that the primed CD4+ cells were unable to provide B cell help for antibody production (21).

T cell responses to small peptide antigens (which do not require processing) in B cell-deficient mice were also variable. While Vella et al. reported reduced CD4+ T cell responses to small peptide antigens irrespective of whether the T cells were derived from LN or spleen (22), Constant et al. reported normal T cell proliferative responses to peptide antigens in µMT mice, but greatly reduced responses to large protein antigens (23,24). Differences in responses to whole proteins versus responses to small peptides were also reported in the induction of EAE. µMT mice have been found to be consistently susceptible to EAE induction with peptides [myelin oligodendrocyte glycoprotein (MOG) 35–55 and Ac-myelin basic protein 1–11] in two different mouse strains with similar disease severity and onset as normal controls (2528). In contrast, Lyons et al. reported that although µMT mice were susceptible to EAE induction with the MOG35–55 encephalitogenic peptide, the mice were resistant to EAE induction with the whole MOG protein (27).

In spontaneous autoimmune diseases, an absolute requirement for B cells as APC has been reported for the development of diabetes in NOD mice and autoimmune responses in MRL/lpr/lpr mice. µMT.NOD mice do not exhibit the characteristic T cell infiltration in the pancreas and do not develop insulin-dependent diabetes (2932). Furthermore, a direct role for B cells as APC in the development of disease was clearly demonstrated in NOD mice that did not express I-Ag7 molecules exclusively on B cells (NOD BCIID). Although NOD BCIID mice had normal class II expression in all APC other than B cells, these mice were resistant to autoimmune diabetes (33). Similarly, when the JHD mutation was backcrossed to the MRL/lpr/lpr mouse, the animals did not develop spontaneous autoimmune nephritis and vasculitis with complete absence of the typical T cell infiltrates (3437). The fact that in order for autoimmunity to develop in the MRL/lpr/lpr mice, B cells were required as APC and not as antibody-producing cells, was elegantly demonstrated utilizing transgenic mice that expressed cell-surface IgM but not secreted IgM (Tg mIgM). Tg mIgM on the MRL/lpr/lpr background had no serum antibodies and still developed nephritis. Moreover, these mice had T cell infiltrates, similar numbers of memory cells as controls and greater disease-related mortality, phenotypes that were not observed in JHD-MRL/lpr/lpr mice (35).

In order to clarify the requirement for B cells as APC for T cell activation in vivo, we analyzed a variety of CD4+ T cell responses in µMT mice of various genetic backgrounds and in µMT -> F1 chimeras. We also investigated whether antigen-specific B cells are essential for antigen presentation.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
C57BL/6 µMT (µMT.B6) male and female mice were initially purchased from the Jackson Laboratory (Bar Harbor, ME) and were maintained in a germ-free environment. Breeding pairs of µMT mice backcrossed to the BALB/c genetic background (µMT.BALB/c) were a kind gift from Dr Walter Gerhard. SJL µMT (µMT.SJL) mice were a generous gift of Drs Bonnie N. Dittel and Charles A. Janeway. C57BL/6, BALB/c and SJL male and female normal control mice were all obtained from the Jackson Laboratory. CB6F1 (BALB/cxC57BL/6) mice were either obtained from the Jackson Laboratory or bred in our facilities. C57BL/6 mice transgenic for the Ig heavy and {kappa} chain complex (IgHelMD4) specific for hen egg lysozyme (HEL) were purchased as breeding pairs from the Jackson Laboratory or were obtained from the breeding colony of Dr James Kenny. Sex- and age-matched animals were used in all experiments.

Media
For most procedures cells were prepared and manipulated in PBS (Cellgrow, Herdon, VA). For in vitro assays cells were grown in T cell media consisting of RPMI 1640 (Cellgrow) supplemented with 10% FCS (HyClone, Pittsburgh, PA), 5x10-5M 2-mercaptoethanol (Sigma, St Louis, MO) and 50 mg/ml of gentamycin (Gibco/BRL, Grand Island, NY).

Antigens
Highly purified KLH and foul {gamma}-globulin (FGG) were obtained from Calbiochem (La Jolla, CA). BSA was obtained from Sigma. Sheep red blood cells (SRBC) were purchased from Colorado Serum (Denver, CO).

mAb
Hybridomas J1j (rat anti-Thy-1.2), J11d (rat anti mouse HSA), GK1.5 (rat anti-mouse CD4), 3.168 (rat anti-mouse CD8), 120.1.2 (anti mouse I-Ab), RA3.3 (rat anti mouse B220), 34-4-21S (anti mouse H-2Dd) and Y3P (anti-class I, all haplotypes except d, absorbed on BALB/c spleens to eliminate any cross-reactivity) were originally obtained from the ATCC (Manassas, VA). Antibody purification and labeling with FITC or biotin were performed in our laboratory. Monoclonal phycoerythrin (PE)-labeled anti-mouse CD19 antibodies were purchased from PharMingen (San Diego, CA). Polyclonal goat anti-mouse IgG and PE-conjugated Streptavidin were obtained from Jackson ImmunoResearch (West Grove, PA). mAb specific for IgM of the a allotype were provided by Dr James Kenny.

T cell proliferation assay
µMT and control mice were injected with 50 µl/footpad of antigen emulsified in CFA containing 1 mg/ml of antigen. Eight days later, draining LN were removed and single-cell suspensions were prepared by using the frosted ends of two glass slides. Total LN cells were plated in 96-well plates at a concentration of 4x105 cells/well (or at 2x105 cells/well in half-area 96-well plates) in T cell media. Cells were cultured for 90–96 h together with increasing doses of the relevant antigen and with 0.5–1 µCi/well of [3H]thymidine during the last 8–12 h. Cells were then harvested and processed for liquid scintillation measurements of radioactivity. Unless otherwise stated, LN preparations tested for T cell proliferation were not supplemented with exogenous APC. In several experiments, B cells were depleted from the normal control LN cells prior to in vitro culture with magnetic beads coated with goat-anti mouse IgG (H & L) (BioMag, Framingham, MA) according to the manufacturer's instructions. In experiments where only CD4+ T cells were used, B cells were removed in the same manner, and CD8+ T cells were depleted by antibody and complement treatment. Cell depletions were verified by FACS analysis.

Bone marrow chimeras
Recipient mice (2–3 months old) were lethally irradiated using a 137Cs source, (110 rad/min; Atomic Energy of Canada). C57BL/6 mice were irradiated with 900 rad and CB6F1 mice with 1050 rad at least 2 h before bone marrow transfer. Bone marrow from donor µMT or control mice were obtained from femur and tibia, and depleted of T cells by antibody plus complement using a mixture of J1j, GK1.5 and 3.168 mAb and guinea pig/rabbit complement mixture (Colorado Serum). All recipient mice were injected with 2–8x106 bone marrow cells and allowed to reconstitute for 2 months. All chimeras were kept on drinking water containing 100,000 U/l of Polymyxin B and 25 mg/l of neomycin sulfate (Pharma-Tek, Hungtington, NY) for 4–6 weeks before returning to regular water

FACS analysis
The absence of peripheral B cells in µMT mice and bone marrow chimeras was determined by staining 1x106 LN or spleen cells with anti-CD19 antibodies or anti-IgG antibodies. The samples were processed in an Epics profile analyzer (Coulter, Hialeah, FL). The chimeric status of bone marrow reconstituted mice was analyzed by two-color staining with J1j and 34-4-21S or Y3P for typing of T cells, and with CD19 or RA3.3 plus 34-4-21S or Y3P for typing of B cells. Samples from all mice used in the experiments were FACS analyzed.

Preparation of B cells
For adoptive transfers, B cells were obtained from the spleens of normal or Ig transgenic (IgHelMD4) mice. Spleen cells were depleted of T cells by treatment with J1j and complement for 45 min at 37°C. Cells were washed 4 times and doses of 50x106–1x107 cells in PBS were injected i.v. into recipient mice 1 week before antigen challenge. Reconstitution of B cells in the recipients was confirmed by FACS analysis.

DTH
Mice were sensitized on day 0 by i.v. injection of 200 µl of 0.01% SRBC in PBS solution. At day 4, sensitized and control animals were challenged in the right hind footpad with 50 µl of a 20% SRBC suspension. At 24 and 48 h later footpad swelling was measured with a micrometer. The magnitude of the response was determined by subtracting measurements of uninjected left footpads from experimental right ones.

Th assay
Mice that were to be used as a source of B cells for measuring Th function were injected i.p. with 0.5 ml of a 10% SRBC solution 2–6 months before. B cells were prepared by depleting the spleens of T cells with J1j plus complement lysis. As a source of primed T cells the spleens of mice that had been injected i.v. with 0.5 ml of 10% SRBC 8 days before were used after depletion of B cells with J11d and complement treatment. To measure Th function in vivo, 5x106 primed B cells and 5x105 primed T cells were transferred together with 100 µl of 5% SRBC to lethally irradiated syngeneic recipient mice. The spleens of recipient mice were removed 6 days later and were assayed for direct (IgM, non-enhanced) and indirect (IgG, enhanced) plaque-forming cells as described (2).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Variable responses to protein antigens in µMT.B6 mice but not in µMT of other genetic backgrounds
The source of the discrepancy in the results obtained in µSM and µMT B cell-deficient mouse models is unclear. A close scrutiny of the results obtained in µMT mice reveals that most studies used µMT on the C57BL/6 (B6) background (µMT.B6). Since the reported results in this strain were variable and inconsistent, a large number of µMT.B6 mice were immunized to three different protein antigens in order to better assess the responses in this strain. Figure 1Go depicts the result of eight independent experiments where a total of 18 µMT.B6 and 16 B6 control mice were examined. The results demonstrate that secondary T cell responses to KLH in µMT mice were consistently reduced in all experiments; however, the relative response of µMT mice as compared to normal controls varied from as low as 4% to as high as 42%. Similar results were also obtained for other antigens like FGG (Fig. 2AGo) and BSA (data not shown). The reasons for the variable results obtained in µMT of the B6 background are not clear, and we could not establish any correlation between level of responses and the age, sex or the general health condition of the tested animals. In this context, it is perhaps worth mentioning that C57BL/6 is generally a poor responder strain to most antigens (38,39) and it is also known to be skewed toward a Th1 phenotype (40), factors that might play a role in the observed fluctuation.



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Fig. 1. Variable T cell proliferative responses in µMT.B6 mice. Normal B6 and µMT.B6 mice were primed with KLH in the hind footpads. Eight to 10 days later T cell recall responses were measured as described in Methods. The results presented are the summary of eight independent experiments. Each bar represent one mouse: black bars represent µMT.B6 mice and gray bars represent B6 control mice. The magnitude of µMT.B6 responses relative to control mice for each antigen concentration in each experiment was calculated by averaging the c.p.m. values obtained per µMT mouse (after subtraction of the media control) over the average response obtained for B6 micex100. The percent response is shown above each group. The results are expressed as the mean of triplicate cultures ± SD.

 


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Fig. 2. Removal of B cells prior to a T cell proliferation assay has no effect on the intensity of the response measured in normal control mice. Mice were primed with KLH or FGG in CFA in the hind footpads and T cell responses were measured as described before. For this experiment, untreated samples were plated with various antigen concentrations. To rule out an influence of B cells in the proliferation observed in the normal control mice, the same samples were depleted of mature B cells by magnetic bead cell sorting as described in Methods. The percentages of CD4+ T cells and B cells (CD45R+ cells) in untreated and treated samples were analyzed by FACS; B cells were completely depleted in treated samples. (A) Responses to FGG in µMT mice (black bars) and control animals in the presence (gray bars) or absence of B cells (white bars). Each bar represents one mouse. (B) Responses to KLH in µMT mice (black bars) and control animals in the presence (gray bars) or absence of B cells (white bars). (C) To examine T cell responses when only purified CD4+ T cells are cultured, we depleted mature B cells from the normal B6 samples by magnetic bead cell sorting and CD8+ T cells by antibody and complement lysis as described in Methods. The depletion of unwanted cell populations was confirmed by FACS. Each bar represents one mouse: unmanipulated µMT (black bars), unmanipulated controls (gray bars), µMT CD4+ T cells only (hatched bars) and normal control CD4+ T cells only (white bars).

 
Since in these experiments total LN cells were used to measure T cell proliferation in vitro, it could be argued that the presence of B cells in the normal control cultures contributes to the measured proliferation in these samples. Although, based on our experience, this is unlikely, we performed experiments where T cell proliferation was measured in the presence or absence of B cells. Primed LN T cells from normal control mice were either plated unmanipulated or depleted of B cells prior to culture and their proliferative response to the relevant antigen was measured. In all experiments, the presence or absence of B cells during culture had no effect on T cell proliferation by normal control mice (Fig. 2A and BGo). The same was true for cultures depleted of both B cells and CD8+ cells (Fig. 2CGo). In fact, when purified CD4+ T cells were used, control B6 responses were higher (Fig. 2CGo). This is expected since the percent of T cells is obviously higher in the responding cultures. This result is consistent with previous reports (4144) and, therefore, in most experiments B cells were not depleted from controls in order to keep manipulation of the samples to a minimum.

In contrast to the results reported for µMT.B6 mice, in most of the experiments utilizing the µSM mouse model, in which T cell priming was dramatically reduced, various types of F1 strain combinations were used. To assess the possible contribution of genetic background to immune responses in µMT mice, T cell responses were compared in SJL, BALB/c and (C57BL/6xBALB/c)F1 [(CB6)F1] mice. The results shown in Fig. 3Go indicate that unlike µMT.B6, responses in these strains were much more consistent and the amplitude of the response in all mice was never >20% of the normal controls and in most experiments responses were <10%. These results seem to suggest that the variability of responses observed in µMT.B6 is particular to that strain.



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Fig. 3. Reduced T cell proliferative responses in µMT mice of various genetic backgrounds. Responses to KLH in µMT and normal control mice were measured by in vitro T cell proliferation as described in Methods. Each bar represents one mouse: black bars represent µMT mice and open bars represent normal control mice. Data are representative of results from three independent experiments. (A) µMT.SJL and normal control SJL mice. (B) µMT.BALB/c and normal BALB/c mice. (C) µMT.(CB6)F1 and normal control (CB6)F1 mice. The results are expressed as the mean of triplicate cultures ± SD.

 
Profound decrease in T cell responses in µMT -> F1 bone marrow chimeras
Another potentially important difference between the µMT and the µSM B cell-deficient models is that µSM mice are made B cell deficient only after birth while µMT mice are B cell deficient throughout their development. It was recently shown that LN development and tissue organization is dependent on the presence of B cells or B cell-specific chemokines during organogenesis of the LN (4548). It is therefore possible that the structure and cellularity of the LN in µMT animals is different than in normal and µSM mice, which might be another contributing factor to the observed difference between these two B cell-deficient mouse models. To test this, µMT -> F1 and µMT -> B6 irradiation bone marrow chimeras were made. T cell-depleted µMT.B6 bone marrow was used to reconstitute lethally irradiated B6 or CB6F1 mice. These chimeras are totally B cell deficient (as the bone marrow donor µMT mice); however, their LN have developed in the presence of B cells and therefore should have a normal structure. Two months after transfer, chimeras were challenged with KLH and secondary T cell responses were analyzed as before. All µMT chimeras were practically unresponsive to antigen, whereas control chimeric mice reconstituted with normal B6-derived bone marrow had normal T cell responses (Fig. 4A and BGo). The absence of B cells in the µMT -> B6 and µMT -> F1 chimeras was confirmed in each individual mouse by FACS analysis using B cell-specific markers. Moreover, T cell responses in these chimeras were consistently lower (<4% of controls) than in any of the µMT mice used for the experiments described earlier.



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Fig. 4. Severely impaired T cell responses in µMT.B6 -> B6 and µMT.B6 -> (CB6)F1 chimeras. T cell-depleted bone marrow from µMT.B6 or control B6 mice was used to reconstitute lethally irradiated recipient B6 or (CB6)F1 mice as described in Methods. Two months after bone marrow transfer T cell responses to KLH (and in some experiments, to PPD) were measured as described. The absence or presence of B cells was analyzed for each mouse by FACS analysis; representative staining profiles are depicted. Each bar represent one mouse. Data are representative of results from three independent experiments. (A) T cell responses to KLH in µMT.B6 -> B6 (black bars) and control B6 -> B6 (gray bars) chimeras. (B) T cell responses to KLH and PPD in µMT.B6 -> (CB6)F1 (black bars) and control B6 -> (CB6)F1 (gray bars) chimeras.

 
T cell proliferation in µMT and µMT -> F1 bone marrow chimeras can be restored by the adoptive transfer of B cells prior to antigen challenge
To ascertain that the deficiency in T cell proliferation observed in these B cell-deficient mice was due solely to the absence of B cells, normal B6-derived B cells were adoptively transferred into both µMT.B6 and µMT -> F1 chimeras 1–2 weeks before antigen/CFA injection and T cell proliferation to the relevant antigen was assayed as before. In all mice reconstituted with B cells, T cell proliferation was restored to normal levels, whereas T cell responses in untreated B cell-deficient mice remained very low, especially in unreconstituted µMT -> F1 chimeras (Fig. 5A and BGo). The presence of adoptively transferred B cells in the LN and spleen was confirmed in all reconstituted animals by FACS analysis. These results suggest that the deficiency in T cell proliferation observed in µMT mice is caused exclusively by the absence of B cells. In fact, studies by Linton et al. found that DC derived from µMT were as competent APC as DC from normal mice in an adoptive transfer system (49). Furthermore, analysis by many laboratories have failed to detect any intrinsic deficiency in macrophages or T cell functions in B cell-deficient animals (18,20,25), suggesting that the phenotype observed in these mice is solely due to the absence of B cells.



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Fig. 5. T cell responses in µMT.B6 and µMT.B6 -> (CB6)F1 mice are restored by adoptive transfer of B cells prior to antigen priming. Mature B cells (50x106) purified from normal syngeneic spleen or LN were adoptively transferred into each recipient by i.v. injection in the lateral tail vein as described in Methods. Between 1 and 2 weeks after adoptive transfer mice were challenged with KLH in CFA and recall T cell proliferation was measured as described. The extent of B cell reconstitution was analyzed for each mouse by specific antibody staining as indicated. Representative staining profiles for one mouse of each group is shown. Each bar represents one mouse. Data are representative of results from three independent experiments. (A) Gray bars represent normal B6 mice, hatched bars represent µMT.B6 mice reconstituted with purified B cells and black bars lines represent unreconstituted µMT.B6. (B) Gray bars represent normal B6 -> (CB6)F1 mice, hatched bars represent µMT.B6 -> (CB6)F1 mice reconstituted with purified B cells and black bars lines represent unreconstituted µMT.B6 -> (CB6)F1 mice.

 
B cells bearing a BCR specific for HEL do not restore T cell proliferation to KLH
The data presented so far indicates that B cells play an important role in the clonal expansion of T cells during local responses in LN, which is consistent with previous work by us and others (2–6,49). However, it remains unclear whether all B cells, irrespective of their specificity, can act as APC in vivo. Although several B cell tumors were shown to be able to present a variety of antigens to T cells in vitro, various studies have suggested that antigen-specific B cells are more potent APC due to their ability to internalize antigen through their BCR (15,5059). To evaluate the relative contribution of antigen non-specific B cells to T cell clonal expansion in vivo, µMT -> F1 chimeras were reconstituted with either a monoclonal or a polyclonal population of B cells prior to antigen priming. Monoclonal B cells were isolated from IgHELMD4 (MD4) (Ig transgenic mice bearing a BCR specific for HEL) and polyclonal B cells were prepared from normal B6 mice. Since T cell proliferation in µMT -> F1 chimeric mice is negligible, they were used to measure the contribution of adoptively transferred B cells to T cell priming. As shown in Fig. 6Go, HEL-specific B cells did not restore any degree of T cell proliferation to KLH, while B6-derived B cells completely restored the response. The level of B cell reconstitution in all mice was monitored by FACS using isotype-specific and CD19-specific antibodies (Fig. 6Go). Similar levels of B cells were detected in both groups of reconstituted mice. Since B6 animals are extremely low responders to HEL (60,61 and unpublished observations) we were unable to measure the contribution of the transgenic MD4 B cells to priming to their cognate antigen in this system. However, studies by other groups have shown MD4 B cells to be normal in all their functions and to be competent APC in presentation of HEL to responsive mice of the H-2k background (58,59,62,63). It is therefore unlikely that the observed absence of priming to KLH by MD4 B cells in vivo is due to an inherent defect in antigen presentation. These results suggest that in vivo, antigen-specific B cells are the main APC responsible for the clonal expansion of antigen-specific T cells in the LN.



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Fig. 6. BCR transgenic B cells specific for HEL can not reconstitute T cell responses to KLH in µMT.B6 -> (CB6)F1 B cell-deficient mice. µMT.B6 -> (CB6)F1 mice were reconstituted with either polyclonal spleen B cells isolated from C57BL/6 mice or monoclonal BCR transgenic B cells specific for HEL derived from the spleens of MD4 mice. One week after adoptive transfer mice were challenged with KLH/CFA, unreconstituted µMT -> (CB6)F1 mice (lacking any B cells) as well as B6 -> (CB6)F1 chimeric mice were also primed. Recall T cell responses were measured as described in Methods. The level of B cell reconstitution in each mouse was determined by antibody staining and FACS analysis, transgenic B cells are of the a allotype while B6 B cells are of the b allotype. Cells were stained with either anti-CD19 antibodies or with anti-IgMa antibodies. Representative staining profiles for one mouse of each group are shown. Data are representative of results from five independent experiments. Each bar represents one mouse. Black bars are µMT -> (CB6)F1, hatched bars are µMT -> (CB6)F1 mice that received polyclonal B cells, open bars are µMT -> (CB6)F1 mice that received monoclonal B cells (the response was extremely low so that these bars are barely visible) and gray bars are control B6 -> (CB6)F1.

 
Systemic priming in µMT is only affected in responses to low antigen concentrations
It was previously shown that systemic T cell priming (i.v. or i.p. immunization) as assessed by T cell help was unaffected in µSM which was attributed to the fact that the spleen, the primary site for immune responses initiated by systemic priming, contains ample non-B cell APC such as DC and macrophages. We re-examined this in µMT mice of the B6 and BALB/c backgrounds using the same antigen (SRBC) but in two different experimental systems which differ significantly in the priming antigen dose. The first one was a DTH reaction which is a recall response where the initial priming is performed with a very low dose of antigen (0.2 ml of 0.01% SRBC) injected i.v. followed by a local s.c. antigenic challenge with high antigen dose (0.05 ml of 20% SRBC) 4 days later. Representative results of DTH in µMT mice are shown in Fig. 7Go. At 24 h after challenge, µMT.B6 mice barely showed any footpad swelling, but by 48 h the swelling had increased and the magnitude of the DTH response was ~50% of the response measured in normal B6 controls (Fig. 7AGo). On the other hand, µMT.BALB/c mice primed and challenged in the same fashion showed greatly reduced DTH responses to SRBC both at 24 and 48 h post-challenge (Fig. 7BGo).



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Fig. 7. Reduced DTH responses to SRBC in µMT mice of B6 and BALB/c genetic background. µMT and control mice were primed i.v. with SRBC and 4 days later all mice were challenged in the right footpad as described in Methods. Footpad swelling was measured with a micrometer. The extent of the DTH response was determined by subtracting the values obtained for uninjected left footpads from the values obtained for injected right ones. Data are representative of results from two independent experiments. Each symbol represent one mouse. Horizontal lines represent the mean value of the response per group as determined with the graph PRISM program. (A) DTH responses for µMT.B6 mice and B6 control mice. Black squares are µMT.B6 mice, open circles are primed B6 controls and black circles are challenged B6 mice that had not been primed. Responses determined at 24 and 48 h post-challenge are shown. (B) DTH responses to SRBC in µMT mice of the BALB/c background as compared to normal BALB/c mice. Black diamonds are µMT.BALB/c mice and gray triangles are BALB/c control mice.

 
The second system used for measuring systemic responses was an in vivo Th cell assay in which the ability of primed T cells to provide antigen-specific help for antibody production is assessed in an adoptive transfer model. T cells primed to a high dose of SRBC (0.2 ml of 20%) in µMT and normal control mice were adoptively transferred together with SRBC-primed B cells into lethally irradiated recipients. Six days later the number of antibody-producing B cells in the spleen was quantified using a direct and indirect plaque assay. As shown in Table 1Go, T cells primed systemically in either µMT.B6 and µMT.BALB/c were equally capable of providing B cell help as T cells primed in normal control mice. No significant differences between µMT.B6 and µMT.BALB/c responses were observed in this assay.


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Table 1. Normal T cell priming in the spleen to high antigen dose (SRBC) in µMT and normal control micea
 
The results show that after systemic priming with the same antigen, DTH responses are dependent on B cells, whereas Th priming is not. The contrasting results in these two systems are most likely due to the different antigen doses used for the initial priming and possibly to differences in the effector T cell function assayed. The dependency on B cells when low antigen dose is used support the notion that efficient B cell antigen presentation is restricted to antigen-specific B cells.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It was first reported >15 years ago that T cell clonal expansion was impaired in B cell-deficient mice produced by continuous injections of anti-µ antibodies from birth (µSM) (2,46). The requirement for B cells as APC for T cell priming in the environment of the LN has become a source of controversy since the introduction of the µMT B cell-deficient mouse in which T cell priming was reported to be less affected or not affected at all (1820,2224). The source of the discrepancy in the results obtained in these two similar mouse models is unclear.

The results presented here suggest that the reason for the discrepancies in the literature between the two B cell-deficient mouse models, µSM and µMT, are at least 2-fold. The first is that C57BL/6 mice were used in most of the reported experiments utilizing the µMT model. We find that unlike the results obtained in BALB/c, SJL and (BALB/cxC57BL/6)F1 responses in the C57BL/6 strain were variable, and ranged from 4 to 42% of controls. In fact, a close scrutiny of the results obtained in µMT mice by other laboratories reveals that in most cases, T cell responses were reduced but not as dramatically as was reported for µSM.

The second is the fact that µSM mice are made B cell deficient only after birth. The absence of B cells during organogenesis was shown to be crucial for the proper development of LN in mice (49). This is supported by our finding that µMT -> F1 and µMT -> B6 bone marrow chimeras, which have the same phenotype as `regular' µMT mice but had their LN develop in the presence of B cells, are invariably unresponsive. We also show that T cell priming in these mice could be fully restored by reconstituting the mice with B cells prior to immunization, which indicates that the inefficient T cell priming in these mice was only due to the lack of B cells in LN. The ability to reconstitute T cell priming by adoptive transfer of B cells allowed us to test whether antigen presentation in vivo can be mediated by all B cells or only by antigen-specific ones. The results strongly suggest that antigen-specific B cells account for most of the antigen presentation to T cells in the environment of the LN. Monoclonal B cells from Ig transgenic animals could not restore responses to an irrelevant antigen. The ability of antigen-specific B cells to present antigen in vivo was shown by many laboratories (15,5059). Mechanistically, it has been demonstrated that when B cells uptake antigen through their BCR, B7 co-stimulatory molecules are up-regulated and B cells become very potent APC (54,6466). This mechanism also explains why anti-peptide responses were reported to be less dependent on B cells (23,24,27). Peptide antigens do not require antigen processing and can bind directly to surface MHC class II molecules and therefore are much more efficient antigens, which makes them less dependent on antigen-specific B cells as APC (67,68). Similarly, one would predict that low concentrations of a protein antigen even when delivered systemically would be dependent upon antigen-specific B cells for efficient presentation (50,58). This was indeed the case in the DTH experiments described above. It could also be argued that in autoimmune diseases directed against tissue-specific antigens, those antigens are present in limited concentrations and therefore their presentation would be much more dependent on antigen-specific B cells (54,69). Indeed, the development of spontaneous autoimmune diseases like diabetes in NOD mice and nephritis and vasculitis in MRL-lpr//lpr mice were the most B cell-dependent T cell-mediated responses reported in µMT mice (2932,3437). In both of these animal models the dependency on B cells was definitively shown to be restricted to their antigen-presenting function (33,35).


    Acknowledgments
 
This work was supported in part by National Institute of Health grants 5 R01 NS38272 (Y. R.) and RO1-CA50777 (J. D.), a National Multiple Sclerosis Society grant 2626A2/1 (Y. R.), a Milstein Foundation Grant (Y. R and J. D.), an Individual National Service award, GM19331 (A. R.), and a National Research Service award 2 T32 AI 07403-07 (C.-C. C.)


    Abbreviations
 
APC antigen-presenting cell
B6 C57BL/6J mice
CFA complete Freund's adjuvant
DC dendritic cells
DTH delayed-type hypersensitivity
EAE experimental autoimmune encephalomyelitis
FGG foul {gamma}-globulin
HEL hen egg lysozyme
JHD JH segment of the Ig heavy chain
KLH keyhole limpet hemocyanin
LN lymph node
MD4 Ig transgenic B cells specific for HEL
MOG myelin oligodendrocyte glycoprotein
PPD purified protein derivative
SRBC sheep red blood cells
µMT µ-knockout mice
µSM µ-suppressed mice

    Notes
 
Transmitting editor: D. R. Green

Received 6 July 2001, accepted 14 September 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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