Address correspondence to Lawrence J. Wysocki, Integrated Dept. of Immunology, K902a, National Jewish Medical and Research Center and University of Colorado School of Medicine, 1400 Jackson St., Denver, CO 80206. Phone: (303) 398-1385; Fax: (303) 270-2182; email: wysockiL{at}njc.org
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Abstract |
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Key Words: lymphocytes SLE antinuclear antibody self-tolerance glomerulonephritis
Abbreviations used in this paper: BCR, B cell receptor; CFSE, carboxyfluorescein diacetate succinimidyl ester; FR1, framework-1; GVH, graft-versus-host; H&E, hematoxylin and eosin; RT, room temperature; Tg, transgenic.
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Introduction |
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Various experimental models have revealed that failure to develop or maintain tolerance to Ig-derived peptides results in either B cell death, or survival of B cells that no longer present antigenic T cell epitopes (e.g., through isotype switching; references 37). However, in models where tolerance is not attained, T cells specific for self-antigens that are ubiquitously expressed by B cells can induce B cell activation and autoantibody production (811). Given that B cells can self-present clonally unique B cell receptor (BCR)derived peptides (1, 1216), we and others have proposed that such peptides might provide an unregulated avenue of help for B cells in spontaneous systemic autoimmunity. Several studies in spontaneously lupus-prone mice have provided support for this idea (1723).
To investigate the consequences of a T cell encounter with a BCR-derived peptide, we generated a complementary pair of transgenic (Tg) mice. Our Tg mice express the
chain of mAb 36-71, in which a class IIrestricted T cell epitope is located within V
framework-1 (FR1). This epitope was originally created by two somatic mutations in codons 7 and 8 (1, 2). CA30 Tg mice express corresponding
ß TCR transgenes encoding a receptor specific for the V
36-71 epitope. We found that central tolerance was tight in double Tg mice. However, adoptive transfer of mature CA30 T cells into
Tg recipients resulted in multiclonal B cell activation, antinuclear antibody production, and lupus nephritis. This was associated with a persistence of CA30 T cells that, paradoxically, appeared to be refractive in proliferation assays. Although our results underscore the importance of maintaining specific T cell tolerance to Ig V regions, they suggest that nondeletional forms of tolerance may be insufficient to protect against the development of antinuclear Ab.
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Materials and Methods |
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sMHC Tetramer Construction.
Using an overlapping primer strategy, an oligonucleotide encoding the V 36-71 FR1 peptide (DIQMTQIPSSLSA) was linked to the 5' end of the gene encoding the ß chain of I-Ak. To this end, the vector pBACp10ph (provided by J. Kappler, National Jewish Medical and Research Center, Denver, CO; reference 29), containing an I-Ak construct, was used in two rounds of PCR as a template for primers 11 and 12, and 11 and 13. Primers 12 and 13 contained DNA encoding the V
36-71 peptide sequence. The resulting DNA consisting of the 5' end of the I-Ak ß construct connected to DNA encoding the V
36-71 peptide was cloned into pBACp10ph (29) using the EcoRI and SpeI sites, and the integrity of the construct was verified by sequencing.
To increase protein stability, a leucine zipper was introduced to the carboxy terminal ends of the and ß chains. A plasmid encoding the zipper templates was provided by J. Bill (University of Colorado School of Medicine, Denver, CO). The basic zipper chain was linked to an I-Ak
chain, and the acidic zipper chain was linked to the I-Ak ß chain. The acidic zipper template was amplified with primers 14 and 15 and cloned into the I-Ak ß gene using the NheI and SphI restriction sites. The basic zipper template was amplified with primers 17 and 18, introduced into the I-Ak
gene via amplification with primers 16 and 17 and cloned into the pBACp10ph vector (29) using XhoI and KpnI sites. This strategy destroyed the thrombin cleavage sites. Sequence verification revealed zipper sequences that were otherwise identical to those published previously (30).
Soluble I-Ak-peptide tetramers were produced as described previously (29, 31, 32). Soluble I-Ak protein was detected with mAb 10.3.6 (33) and biotinylated mAb 11.5.2 (33) and affinity purified with sepharose conjugate of antiI-Ak mAb 10.3.6. Bound protein was washed in sodium bicarbonate (50 mM, pH 8) and eluted with sodium carbonate (50 mM, pH 11). Staining with the tetrameric reagent was performed at 37°C for 3 h before the addition of secondary antibodies for FACS® analysis.
Cell Purification.
Single cell suspensions were obtained from thymi, LNs, or spleens as described previously (1). Thymocytes and LN cells were used without further manipulation unless otherwise indicated. Erythrocytes were removed from splenocyte preparations by lysis with ammonium chloride. In some cases, splenocyte APCs were fractionated on the basis of their specific gravity with Percoll as described previously (1). For most proliferation assays and intracellular cytokine detection, T cells were isolated by negative selection following the StemSepTM magnetic separation protocol (StemCell Technologies Inc.). Typically, this protocol yielded T cells that were 8595% pure as judged by FACS® analysis.
Adoptive Transfers, Immunizations, and Injections.
CA30 LN cells were isolated as aforementioned, washed once in PBS, and injected into a lateral tail vein of A/J or Tg recipient mice (not irradiated). Some recipient mice were immunized with 30 µg of V
36-71 peptide emulsified in CFA and injected either intraperitoneally or subcutaneously. Spleens or draining LNs were harvested for proliferation and cytokine assays.
Flow Cytometry.
Conjugated mAbs were purchased from BD Biosciences, and cells were stained using standard staining procedures. Biotinylated antibodies were visualized with streptavidin-PE (Biosource International) in most cases. Cells were analyzed on FACsCaliburTM or FACscanTM flow cytometers using CELL QuestTM software (Becton Dickinson).
Proliferation Assays.
Various numbers of purified T cells were cultured with a fixed number (2 x 105) of lightly irradiated (1,100 rad) splenocytes as a source of APCs from an unmanipulated mouse. Stimulator peptide was added at the indicated concentrations, and the culture was incubated for 45 d at 37°C in 5% CO2. [3H]Thymidine (1 µCi/well) was added during the final 20 h of culture, and incorporation was detected with a Wallac Microbeta, using Beta-Scint scintillation fluid (both obtained from Wallac).
Analysis of Serum Antibody.
Chromatin-specific serum IgG was detected on plates coated with either 10 µg/ml of mouse chromatin or 10 µg/ml BSA. Total IgG was detected on plates coated with 5 µg/ml Fc-specific goat antimouse IgG (Sigma-Aldrich). All assay plates (Corning) were coated overnight at 4°C and incubated with blocking buffer (2% BSA, 1% gelatin in PBS containing 0.05% Tween 20) for 12 h at 37°C or room temperature (RT). Mouse serum was incubated at the indicated dilutions for 1 h at 37°C or RT. IgG antibodies were detected with biotinylated goat antimouse IgG (Southern Biotechnology Associates, Inc.) followed by europium-labeled streptavidin (Wallac). Europium fluorescence at 615 nm was measured as described previously (1). Chromatin-specific Ig isotypes were detected with horseradish peroxidasecoupled goat antimouse isotype-specific antibodies (Southern Biotechnology Associates, Inc.) followed by colorimetric detection of 2,2'-azino-bis(3ethylbenz-thiazoline-6-sulfonic acid) at 405 nm (Victor 2; Wallac).
Analysis of Antinuclear Antibodies.
Sera from individual mice were incubated for 30 min in a humidified chamber on slides containing NOVA-Lite HEP-2 cells (Inova Diagnostics). Antinuclear antibodies were detected with a FITC-labeled F(ab)2 fragment of goat antimouse IgG (Fc specific; Sigma-Aldrich).
Analysis of Kidney Pathology.
For tests of Ig deposition, kidneys were quick frozen in Tissue-Tek OCT (Sakura Finetek Europe B.V.) and stored at 80°C. Serial sections of 68 µm were transferred to positively charged microscope slides (ProbeonPlus; Fisher Scientific), fixed in acetone, and stored at 80°C. Sections were thawed at RT and blocked with 5% FCS in PBS for 10 min at RT. Subsequently, the sections were incubated with FITC-coupled goat antimouse isotype antibodies for 30 min at RT. Sections were mounted using Gel/Mount (Biomeda). Images were taken on a Marianas workstation using Slidebook 4.0.6.4 (both obtained from Intelligent Imaging Innovations, Inc.). Hematoxylin and eosin (H&E) staining was performed on kidneys fixed in 1% formalin by the Histology Core Laboratory at the National Jewish Medical and Research Center.
Online Supplemental Material.
Table S1 lists the primers used in the project. Table S2 gives weights and numbers of cells in LNs and spleens of Tg recipients on day 10. Table S3 illustrates activation markers on B cells at day 10 in
Tg mice that received different numbers of CA30 cells. Table S4 shows the Ig isotypes of antichromatin antibodies. Fig. S1 illustrates normal expression levels and allelic exclusion of the CA30 transgene-encoded TCR. Fig. S2 shows intracellular IL-2 in stimulated CA30 cells. Fig. S3 illustrates poor allelic exclusion in peripheral CD4 cells of double Tg mice. Fig. S4 shows the total IgG and antichromatin titers in sera of recipients of 107 CA30 cells. Kidney pathology in recipients of CA30 cells is compared at days 28 and 130 in Fig. S5. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20031234/DC1.
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Results |
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Specificity and Function of CA30 Tg T Cells.
To assess the biological activity of CA30 T cells, we performed a series of in vitro and in vivo tests. Initially, LN cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated with various concentrations of V 36-71 peptide. 2 d later, the cultures were analyzed by FACS® for tetramer staining and CFSE fluorescence. At high peptide concentrations, virtually all of the tetramer+ cells had undergone at least one round, and most cells had undergone two rounds of division (Fig. 1 C). Furthermore, nearly all of these cells produced IL-2 as measured by intracellular cytokine staining (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20031234/DC1).
CA30 T cells were also directly stimulated by Tg splenocytes. As predicted from our previous studies (1) and shown in Fig. 1 D, CA30 Tg T cells proliferated vigorously to low density splenocytes (
< 1.079) from
Tg mice. Similarly, CA30 T cells were strongly stimulated by splenocytes containing anergic Ars/A1 Tg B cells, which express the mAb 36-71
chain in conjunction with a µ
Tg (24). In contrast, proliferation induced by high density resting splenocytes was weak in agreement with our previous findings (1). For in vivo tests of biological activity, 2 x 106 CA30 Tg LN cells were transferred into nonTg recipients. Immediately after transfer, the recipient mice were immunized subcutaneously with the V
36-71 peptide emulsified in CFA. T cells recovered from peripheral LNs of recipient mice at day 10 responded strongly to the V
36-71 peptide in vitro, both by proliferation and intracellular IL-2 production (Fig. 1 E and not depicted).
Thymic Deletion of CA30 T Cells in Mice Expressing V 36-71.
In a test for central tolerance, CA30 Tg mice were bred to Tg mice expressing the chain of mAb 36-71. Fig. 2 A shows representative FACS® analyses of thymic and LN cells from double Tg mice and age-matched controls. Compiled data from several mice are shown in Fig. 2 (B and C). In addition to the percent decrease in CD4 cells shown in Fig. 2 (AC), a total reduction of 3040-fold in CD4+ cell numbers was seen in thymi and LNs of double Tg mice (not depicted). This large reduction in cell numbers is consistent with central thymic deletion at the CD4+/CD8+ double-positive stage.
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Central Tolerance Induced by Maternally Derived V 36-71.
To test whether APCs that process and present exogenous antibody could mediate thymocyte deletion, we analyzed offspring of Tg mothers that had inherited the TCR Tg but not the
transgene. These mice should only be exposed to maternally derived
Ig transmitted either transplacentally or through milk. In very young mice (23.5 wk of age), we observed profound deletion of Tg T cells in both the thymus and the periphery (Fig. 2, F and G). However, the CD4 T cell populations began recovering shortly after weaning, and by 7 wk of age, the percentages of CD4, Vß8, and tetramer-staining populations were nearly normal (Fig. 2 G and not depicted). Moreover, V
2-expressing T cells were more abundant in young than adult CA30 Tg offspring of
Tg mothers (unpublished data). These results demonstrate that V
-specific T cells are subject to deletion by maternally derived antibody, and that the repertoire recovers with time presumably due to antibody clearance.
T Cell Activation in Tg Adoptive Recipients of CA30 Cells.
Although our results implicated central tolerance as a mechanism to regulate Ig V regionspecific CD4 T cells, they did not address possible redundant regulation by peripheral tolerance mechanisms. To test for peripheral tolerance, we transferred 107 CA30 LN cells into Tg recipients or control A/J recipients. By 3 d after transfer, the Tg T cells had expanded dramatically in
Tg recipients, as assessed by tetramer staining and loss of CFSE fluorescence in cells that were labeled before transfer. At day 10, tetramer-binding T cells uniformly expressed elevated levels of CD44 and still comprised a large percentage of splenic and LN T cells (Fig. 3 A). Furthermore,
Tg recipients of CA30 LN cells that were killed 10 d after transfer displayed enlarged LNs and spleens, suggesting massive proliferation (Fig. 3 B, left, and Table S2, available at http://www.jem.org/cgi/content/full/jem.20031234/DC1). Even 28 d after transfer, the spleens of
Tg mice receiving 107 CA30 cells were still dramatically enlarged, and tetramer-binding T cells comprised
30% of the splenic CD4 T cell population (Fig. 3 C). Although splenic architecture was generally intact, the white pulp was significantly expanded, particularly follicles and germinal centers. Similar but less dramatic results were obtained upon transfer of fewer CA30 cells into
Tg recipients (Fig. 3 B, right). Transfer of 106 CA30 cells induced less severe lymphadenopathy and splenomegaly at day 10, in which 711% of LN and splenic CD4+ cells were tetramer+. 105 CA30 T cells induced lymphadenopathy without splenomegaly, in which tetramer staining cells comprised
3% of the peripheral CD4+ population.
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B Cell Activation in Tg Adoptive Recipients of CA30 Cells.
We also investigated the effect of transferred CA30 T cells on B cells. As shown in Fig. 4 A, Tg B cells in recipients of 107 CA30 cells expressed increased levels of activation markers. On day 3 after transfer, CD86 levels were increased on most B cells in LNs and spleen, whereas high levels of CD69 were expressed on most B cells in LNs and
35% of B cells in the spleen. By day 10, CD86 expression remained high on a substantial fraction of splenic B cells, whereas CD69 was elevated on
50% of splenic B cells. In contrast, B cells of A/J mice that received CA30 cells had staining profiles identical to those of control uninjected
Tg mice (unpublished data). Qualitatively similar results were seen in
Tg mice that received fewer CA30 cells. However, in these recipients, frequencies of B cells expressing CD86 and CD69 were reduced relative to those of mice that received 107 CA30 cells (Table S3, available at http://www.jem.org/cgi/content/full/jem.20031234/DC1).
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Development of Antichromatin Ab in Tg Adoptive Recipients of CA30 Cells.
Significant titers of antichromatin IgG antibodies were observed in sera of all Tg recipients tested at days 14 and 28 after adoptive transfer (n = 9; Fig. 5, A and B, and Fig. S4 B). The presence of antinuclear antibodies was verified by staining of HEP-2 cells (Fig. 5, C and F). However, only a small fraction of total serum IgG bound chromatin at these early time points. On day 14, the concentration of antichromatin IgG in
Tg recipients was
12 µg/ml, whereas total IgG concentration was several milligrams per milliliter. Moreover, from days 14 to 28, chromatin-specific IgG increased only proportionally to the total increase in IgG. These data indicate that the transferred T CA30 cells initially activated not only chromatin-specific B cells but also B cells with other antigenic specificities.
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Persistent CA30 T Cells in Most Tg Recipients.
We speculated that failed tolerance in CA30 T cells might account for a prolonged production of autoantibodies in most of the recipients. To test this idea, we first assayed for tetramer-binding cells in blood samples from the four Tg recipients of the initial experiment. Tetramer+ cells were only observed in the two recipients (nos. 3 and 4) that contained high titers of antichromatin Ab at day 76 (unpublished data). In a more rigorous test for Tg T cells, we immunized the two mice with lower autoantibody levels (nos. 1 and 2) and stained splenocytes 3 d later (day 126) for tetramer-binding cells. Despite immunization, the frequencies of tetramer+ cells were not significantly greater than those seen in control A/J recipients (Fig. 6 A). In contrast, clearly defined tetramer+ populations were evident in the recipients with high antichromatin titers.
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Evidence of Inflammation and Kidney Involvement.
To test for antibody deposition in glomeruli, we stained frozen kidney sections from Tg recipients of CA30 cells with a panel of antiisotype antibodies. Little or no staining was observed in kidneys of mice killed at day 10 after transfer. However, by day 28, some recipients of higher numbers of CA30 cells displayed infrequent segmental staining in a few glomeruli (3/10 mice). However, no evidence of disease was observed upon H&E staining. At day 130, in contrast, kidneys of all five
Tg recipients of 107 CA30 cells demonstrated global deposition of antibody in virtually all glomeruli (Fig. 7 and Fig. S5, available at http://www.jem.org/cgi/content/full/jem.20031234/DC1). Moreover, H&E staining revealed enlarged glomeruli in four out of five mice, with cellular infiltrates in two of these four animals (unpublished data). In all five mice, antibodies of multiple classes, including IgM, IgG1, IgG2a, and IgG3 were deposited in glomeruli, with IgG1 and IgG2a predominating. Of note, these are the same two classes that were found to predominate in kidney deposits within spontaneously autoimmune (NZB x NZW)F1 mice (36). Because these were the same five mice aforementioned that contained persistent antichromatin Ab in sera, we were able to define their isotypes as well. In the case of sera, the predominant serum antichromatin IgG was IgG2a, followed by IgG3 and IgG2b (Table S4, available at http://www.jem.org/cgi/content/full/jem.20031234/DC1). The IgG2a and IgG3 subclasses are associated with the inflammatory cytokine, IFN-
, which is known to induce class switch recombination to
2a and
3 constant region genes. IgG2a is also the predominant serum isotype of antinuclear Ab in two widely studied murine models of spontaneous lupus: the (NZB x SWR)F1 mouse and the MRL lpr/lpr mouse (37). Collectively, these data reveal manifestations of chronic lupuslike disease in recipients of T cells that react to a BCR-derived peptide.
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Discussion |
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Evidence for central CD4+ T cell tolerance to Ig sequences has been reported by several groups (3842). However, two Tg systems previously developed to address this question yielded contrasting results. Bogen et al. (40) found that T cells specific for a V region peptide from the light chain of MOPC-315 underwent thymic deletion in mice that either expressed the cognate Ig or that were injected with soluble antibody at a high concentration. These results are consistent with others suggesting that wild-type IgG2ab-specific T cells attain tolerance in the thymus (41). In contrast, Granucci et al. (7) found that IgG2ab-specific T cells from a TCR Tg mouse failed to undergo negative selection in mice endogenously expressing IgG2ab. In agreement with the MOPC-315 result, we observed a near complete deletion of the Tg T cells. Although indirect evidence has been published suggesting that maternally derived Ig can induce central tolerance (3, 4244), our results provide the first direct evidence of thymocyte deletion resulting from interactions with maternally derived Ig. Nevertheless, although it is clear from our data that high levels of serum Ig can induce central tolerance in Ig V regionreactive CD4+ T cells, it is still unclear whether this tolerance can be induced centrally by physiologically low levels of IgM bearing a particular V region.
The consequences of a cognate T cellB cell interaction mediated by a BCR-derived V region peptide are largely unknown. Two different outcomes might be predicted from clues in the literature. Some evidence suggests that B cells might be suppressed (35, 45). This outcome is predicted from studies using Ig allotype-specific T cells or clones that induce allotype suppression. Results from other models suggest instead that an Igpeptide-directed interaction between a CD4+ T cell and a B cell will result in B cell activation. Singh et al. (22) demonstrated that immunization of BALB/c mice with a V region peptide derived from a lupus-associated Ab, resulted in transient autoantibody production. The striking implication of this result is that chromatin-specific B cells received T cell help directed to BCR V regionderived peptides that were self-presented by B cells in the context of class II MHC. The temporary nature of autoantibody production observed by Singh et al. is similar to what was seen in two of our Tg recipients of CA30 T cells. Our results are also reminiscent of those from several models in which T cell help was directed to peptides derived from natural or Tg-encoded membrane antigens expressed by B cells (811). The early hypergammaglobulinemia, splenomegaly, and lymphadenopathy seen in our transfer model are particularly reminiscent of CD4+ T cellinduced GVH disease with lupuslike manifestations (10, 11). Although it is clear that initial B cell activation is multiclonal, more interesting is the time-dependent specific increase in antichromatin IgG, which is accompanied by a significant reduction of total serum IgG. This indicates that chromatin-reactive B cells are selectively stimulated with time after CA30 cell transfer. A common feature of spontaneous murine lupus is a progressive, oligoclonal antichromatin response (17, 46, 47).
Although the outcomes of our transfer model and the previously described models are similar in some respects, our model is distinct in that recipient mice contained high levels of serum Ig bearing the cognate T cell epitope. In this sense, it is striking that CA30 Tg T cells were not immediately rendered tolerant due to high levels of 36-71 chain in recipient mice. There is considerable evidence that IgG can be tolerogenic for CD4+ T cells, and our recipient mice contained milligram per milliliter quantities of serum IgG
(4853). However, studies from one group have provided evidence that IgM can be immunogenic (5355). Thus, it is possible that APCs presenting V
36-71 peptide derived from IgM stimulated the transferred CA30 T cells at early time points before tolerance could be induced by APCs presenting V
36-71 peptide derived from IgG. The subsequent rise in total IgG levels could explain an observed delay in the acquisition of T cell tolerance; CA30 Tg T cells recovered 10 d after transfer proliferated strongly in vitro in response to the V
36-71 peptide, whereas T cells recovered 28 d after transfer were much less responsive to antigenic stimulation.
Although all Tg recipients of CA30 cells consistently developed early manifestations of GVH-like disease, there was considerable variation in the duration and level of the specific antichromatin IgG response. Seven out of nine mice had high serum Ab titers at day 76, and in six of these mice, the titers were maintained or further increased to varying levels at the final, 4-mo time point. The two recipients with only low titers of antichromatin IgG at day 76 had no clearly detectable CA30 cells at the end point of the experiment. In all others, defined populations were evident upon tetramer staining. At face value, these observations suggest that a persistent CA30 population is required for long-term production of antichromatin Ab. Nevertheless, the recovered CA30 cells responded only poorly to the V
36-71 peptide in proliferation assays.
In a well-defined model of peripheral tolerance, transfer of Tg CD4+ T cells into a host ubiquitously expressing cognate antigen led to a state of "adaptive tolerance" that was manifested by poor proliferative and cytokine responses to antigen (56). An interesting feature of adaptive tolerance is its apparent reversibility, which can occur if T cells are removed from the antigenic environment that provides constant stimulation (57, 58). In our transfer model, it is intriguing that an antichromatin IgG response persisted in the face of antigen-refractive CA30 T cells. This apparent discrepancy could be explained by the adaptive tolerance idea if chromatin-specific B cells are more effective stimulators of T cells than are other APCs. CA30 T cells that are refractive to the V 36-71 peptide presented by professional APCs may be able to respond to the same peptide that is self-presented by chromatin-specific B cells. This might occur if the B cells present a higher concentration of the V
peptide due to BCR internalization after chromatin engagement. Alternatively, chromatin-activated B cells may provide enhanced or unique costimulatory signals for T helper cells. CpG motifs in chromatin apparently enhance B cell activation by triggering Toll-like receptor 9 (59, 60). It is conceivable that this pathway might enhance T cell costimulation as well. Further studies will be required to address this hypothesis.
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Acknowledgments |
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This work was supported by National Institutes of Health grants (nos. AI33613, AI48108, and AI22295) and a predoctoral training grant from the Department of Defense Breast Cancer Research Program (award no. DAMD17-00-1-0359).
Submitted: 23 July 2003
Accepted: 19 May 2004
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References |
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