Department of Microbiology and Immunology, and the Kimmel Cancer Institute, BLSB 708 ,233 South 10th Street, Jefferson Medical College, Philadelphia, PA 19107, USA
Correspondence to: T. Manser
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: antigens, autoantibodies, CD95, clonal deletion, germinal centers, immunohistochemistry
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There is a growing body of evidence suggesting that T cells play an active role in these peripheral B cell tolerance pathways (1014). For example, the use of Ig transgenic systems has shown that certain autoreactive B cells, in the presence of cognate self-antigens, are eliminated after migrating to the TB interface of splenic follicles, thereby implicating T cells as a regulatory factor, possibly through FasFas ligand-mediated programmed cell death (1519). Moreover, it has recently been observed that B cells expressing autoreactive, transgene-encoded BCR display distinct phenotypes and behaviors in T cell-deficient mice, as compared to when they are present in a T cell sufficient environment (20,21).
In this report we show that autospecific B cells are present in T cell-deficient TCRß/ mice and that these mice mount a vigorous serum autoantibody response and develop symptoms of autoimmune disease shortly after they are injected with mature, syngeneic splenic T cells. However, this autoimmune response is transient, suggesting that, on the one hand, reconstitution of the T cell compartment allows activation of quiescent autoreactive B cells and, on the other, that reconstituting T cells ultimately participate in the restoration of peripheral B cell tolerance mechanisms. This T cell reconstitution system provides a means to evaluate the role of various T cell subsets and T cell regulatory factors in peripheral B cell tolerance, and has advantages over transgenic systems since it examines the activity of natural autoreactive B cells.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell-surface staining
Cell-surface staining was performed as described (28). Briefly, cells were preincubated in PBS (50 mM phosphate, 0.15 M NaCl, pH 7.2) staining buffer (0.5% BSA) and then incubated on ice in an optimal concentration of fluorochrome-labeled or biotinylated antibody for 30 min and then washed in PBS staining buffer. Biotinylated antibodies were detected by incubating the cells with Red670 coupled to streptavidin. Cells were fixed in PBS containing 1% paraformaldehyde and analyzed on a Coulter Epics Elite using WinMDI software (Scripps Research Institute, La Jolla, CA). Forward and side scatter plots were used to gate on live lymphocytes in all analyses.
Antibodies
Purified mAb FITCanti-CD3 clone 145-2C11, FITCanti-CD4 clone H129.19, allophycocyaninanti-CD8 clone 53-6.7, biotinanti-CD35 (CR1) clone 8C12, phycoerythrin (PE)anti-CD23 (IgE Fc Receptor) clone B3B4, FITCanti-CD24 (heat stable antigen) clone M1/69, PEanti-CD28 clone 37.51, FITCanti-CD40 clone 3/23, biotinanti-CD43 clone S7, PEanti-CD44 (Pgp1) clone IM7, PE or FITCanti-CD45R B220 clone RA3-6B2, biotinanti-CD69 (very early activation antigen) clone H1.2F3, FITCanti-CD86 (B7-2) clone GL1, FITCanti-CD90.1 (Thy1.1) clone HIS51, biotinanti-CD90.2 (Thy1.2) clone 53-2.1, biotinanti-CD95 (Fas) clone Jo2, biotinanti-CD138 (syndecan-1) clone 281-2, PEanti-CD154 (CD40 ligand) clone MR1, anti-Fas ligand clone MFL3, biotinanti-NK1.1 (NKR-P1C) clone PK136 and PEanti-DX5 (Pan NK cells) clone DX5 were all purchased from PharMingen (San Diego, CA). Biotinrat anti-mouse IgD was purchased from Southern Biotechnology Associates (Birmingham, AL) and FITCdonkey anti-mouse IgM from Jackson ImmunoResearch (West Grove, PA). StreptavidinRed670 was purchased from Gibco/BRL (Grand Island, NY). PL9-6 was the kind gift of Marc Monestier (30).
Immunohistochemistry
Immunohistochemical studies were performed as described previously (31,32). Briefly, spleens were quick frozen in OCT (Elkhart, Indianapolis, IN) and 6 µm sections were prepared. Sections were fixed in acetone and air dried. The slides were incubated in 0.3% H2O2 to destroy endogenous peroxidase activity. The slides were blocked with TBS + 5% BSA. The sections were incubated with horseradish peroxidase (HRP)- and biotin-labeled antibodies followed with streptavidinalkaline phosphatase (Southern Biotechnology Associates). HRP-labeled antibodies were revealed with 3-amino-9-ethyl-carbazole and alkaline phosphatase-labeled antibodies were revealed with Fast BB Blue Base (Sigma, St Louis MO).
B cells were identified using a rat anti-B220 (6B2) ascites and mouse anti-rat IgGalkaline phosphatase (Jackson ImmunoResearch) or peroxidase-labeled donkey anti-mouse IgM (Jackson ImmunoResearch). T cells were identified using anti-CD3biotin and streptavidinalkaline phosphatase (Dako, Glostrup, Denmark). Germinal centers were identified using peanut agglutinin (PNA) coupled to horseradish peroxidase (Sigma).
Serology
Mice were bled at various intervals pre- and post-transfer via the retro-orbital sinus and the levels of anti-chromatin, anti-single-stranded DNA and anti-double-stranded DNA, and various antibody isotypes were determined by previously described ELISA assays (7,31,33). Chromatin was prepared as described elsewhere (34).
Anti-nuclear antibody (ANA) assays
ANA staining activity was analyzed using human epitheloid HEp-2 cells on prepared slides (Antibodies Inc., Davis, CA). HEp-2 cells were stained with a 1:50 dilution of sera from time points corresponding to the peak of autoantibody production as analyzed by ELISA. The slides were washed with PBS and the presence of mouse antibodies was revealed using a goat anti-mouse IgGFITC (Southern Biotechnology Associates).
Immune complex deposition
Kidneys from mice at days 0 and 28 post-T cell transfer were frozen in OCT and 6 µm sections were prepared. Sections were stained with a FITC-labeled anti-mouse mAb, clone EM-34.1 (Sigma), to reveal immune complex (IC) deposition.
Mitogen stimulation of B cells in vitro
Spleen cells were depleted of T cells via treatment with anti-Thy1 (mAb J1j) and complement, and small, resting B cells purified on Percoll gradients. Cells were plated at 106 cells/ml in RPMI media containing 10% FCS, 50 µg/ml lipopolysaccharide (LPS; Difco, Detroit, MI) and 5 µg/ml dextran sulfate (Amersham Pharmacia, Piscataway, NJ). After 5 days, supernatants were harvested and assayed for levels of antibodies with various specificities by ELISA.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
T cell reconstitution kinetics in the spleen were further examined by flow cytometry. T cells could be easily detected by day 7 post-transfer (Fig. 1B) when ~1x107 T cells were found per spleen (Fig. 1C
). This is about one-third the amount characteristic of C57BL/6 mice. This number remained relatively constant throughout the time course. Previous studies of the reconstitution of SCID mice with mature T cells have reported similar levels of splenic reconstitution (35,36). Control experiments in which T cells from Thy1a congenic donors were transferred to TCRß
/ mice showed that all of the T cells in the recipient mice were of donor origin. Moreover, both CD4+ and CD8+ and
ß+ and
+ subsets were found in the spleens of reconstituted mice in normal ratios (data not shown).
T cell reconstitution of TCRß/ mice results in the appearance of GC
Staining of spleen sections with PNA showed that GC appeared in follicular areas as early as day 14 post-T cell transfer. By day 28 large GC were observed in most follicles (Fig. 1A, lower right panel, red staining PNA, blue staining B220). By day 35 the size of the GC had diminished, but even at day 56 small GC were still visible in most follicles. Double staining of sections with PNA and anti-CD3 revealed numerous T cells in these GC. During the height of the GC response, abundant tingible body macrophages containing TUNEL-positive cell nuclei could be seen in all GC, suggesting that GC cells were undergoing extensive apoptosis at this stage (data not shown).
Reconstitution of the T cell compartment of TCRß/ mice coincides with increases in spleen weight and B cell numbers
An increase in spleen size was observed as early as three days post-T cell transfer. The weight of day 7 post-transfer spleens was approximately twice that seen in pre-transfer mice. Pre-transfer spleens weighed an average of 130 mg, while day 7 spleens increased to an average of 220 mg. Spleen weights were observed to decrease at later time points. At day 35 the weight of spleens decreased to 125 mg and by day 49 they began to approximate that seen in C57BL/6 mice which weighed an average of 114 mg.
Concomitant with the increase in spleen size was an increase in various splenocyte subsets (Fig. 1C). Control C57BL/6 mice had ~5x107 splenic B cells and 3x107 splenic T cells. Pre-transfer TCRß
/ mice had increased numbers of splenic B cells compared to C57BL/6 mice with ~9x107 B cells (Fig. 1C
). Following transfer there was an average of 1.6x108 B cells in the spleens of transfer mice at day 7. These numbers decreased over time to ~5x107 cells by days 3549. Flow cytometry also revealed a transient increase in a population of non-B/non-T cells during reconstitution (data not shown). Preliminary phenotypic analysis of this subpopulation revealed that it included NK cells.
During T cell reconstitution TCRß/ mice produce a vigorous but transient serum antibody response to self-antigens
As expected, TCRß/ pre-transfer mice had lower total serum Ig concentrations, especially of switched isotypes G1, G2a and G2b, compared to C57BL/6 mice. However, IgM and IgG3, isotypes associated with T-independent responses, were elevated. After T cell reconstitution, mice displayed an increase in total serum antibody. Individual isotypic classes of the antibodies varied in their degree of increase. The most pronounced elevation was seen in IgG2a and there was no increase in IgG3 (data not shown).
During the course of T cell reconstitution of TCRß/ mice we observed temporary symptoms consistent with systemic disease, including reduced skin elasticity, tufted fur and lethargy. These symptoms peaked 23 weeks after injection of T cells and subsequently disappeared. Mice followed up to 1 year after T cell transfer showed no recurrence of disease symptoms. For this reason, sera obtained from TCRß
/ mice at various times after T cell transfer were assayed for levels of autoantibodies with specificities associated with systemic autoimmune disease. Figure 2
(A) shows that
light chain-bearing serum antibodies capable of binding single-stranded DNA, double-stranded DNA and chromatin appeared at high levels shortly after T cell transfer. At 23 weeks post-transfer the titers of these autoantibodies approached 50% of those characteristic of aged autoimmune MRL lpr/lpr mice. However, this autoantibody response was transient. An increase in autoantibody titers was seen as early as day 7 and reached a peak in the day 1421 time frame, and then declined to levels characteristic of normal mice. Heavy chain isotype analysis of serum anti-chromatin (Fig. 2B
) and anti-DNA antibodies (data not shown) revealed high levels of IgG2a, particularly 28 days after T cell transfer, consistent with the overall increase in total serum IgG2a observed after T cell injection. Interestingly, there was substantial mouse to mouse variation seen in the kinetics of expression of autoantibodies with particular specificities.
|
|
|
To investigate whether activation of autoantibody producing B cells in the TCRß/ reconstituted mice required direct T cell co-stimulation, congenic C57BL/6 CD40 ligand-deficient mice were used as a source of T cells for transfer. Figure 5
illustrates that transfer of splenic T cells from these mice into TCRß
/ mice resulted in long-term partial T cell reconstitution equivalent to when C57BL/6 T cells were used (Fig. 5B
) and modest increases in total serum antibody at day 28 post-T cell transfer (IgM and IgG, Fig. 5A
). However, post-transfer sera obtained from these mice at this time lacked detectable levels of anti-chromatin (Fig. 5A
) and anti-double-stranded DNA antibodies (data not shown). The mice were also bled a multiple time points before and after day 28, up to the time when they were sacrificed to evaluate T cell reconstitution. At no time were anti-chromatin or anti-DNA antibodies levels above background controls detected (data not shown). This indicated that the autoantibody response in TCRß
/ mice receiving normal T cells is not a T-independent response stimulated by antigens expressed by T cells and that conventional T cellB cell co-stimulatory interactions are required for autoantibody production. However, since T cell reconstitution by CD40 ligand-deficient T cells was only evaluated at a late time point, we cannot exclude the formal possibility that the kinetics of reconstitution differed in this situation as compared to when CD40 ligand-sufficient T cells were used and that this difference influenced the magnitude of the autoimmune response in the recipients.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We considered that a by-product of T cell reconstitution was a transient and perhaps non-specific polyclonal activation of the B cell compartment. Our data strongly argue against this possibility. Transfer of syngeneic CD40 ligand/ T cells into TCRß/ mice did not result in a serum autoantibody response, demonstrating that conventional B cell co-stimulation by T cells is required for this response. The unique ANA staining patterns obtained from the sera of individual C57BL/6 T
TCR
ß/ reconstituting mice and large mouse to mouse variation in expression levels of various autospecificities are indicative of a restricted number of stochastically selected B cell clones giving rise to the majority of autoantibody in each mouse. Most convincingly, flow cytometric analysis of the B cell compartment in C57BL/6 T
TCRß
/ reconstituting mice revealed that only a subpopulation of B cells in this compartment transiently expressed cell-surface markers characteristic of activation.
Nonetheless, transfer of mature T cells may have other, more global effects on the B cell population of TCRß/ mice. This was suggested by our observation that sIgD levels were low on the majority of splenic B cells in these mice, but returned to normal shortly after injection of T cells. A role for T cells in the primary development of B cells has been previously suggested (21,40,41). For example, Wortis and colleagues have shown that mice bearing both the nude mutation and the mutation in Btk characteristic of CBA/N mice lack both mature B cells and T cells, whereas CBA/N mice display an absence of only certain mature B cell subpopulations. The influence of T cells on the development of B cells in this system appears to be manifested during pre-B cell development. While further experiments on the influence of mature T cells on primary B cell development in TCRß
/ mice is clearly warranted, we think it unlikely that such an effect could alone account for the transient autoimmune response we have described, for reasons detailed above.
Autoantibody production has not been detected in previous experiments in which mature splenic T cells were transferred into T cell-sufficient mice (42,43). However, temporary autoantibody responses to single-stranded DNA and cardiolipin have been observed after repeated immunization of non-autoimmune mice with apoptotic thymocytes (42). Indeed, there is mounting evidence that perturbation of apoptotic pathways or of the removal of apoptotic cells is a contributory factor to the development of systemic autoimmune disease (44,45). It is possible that transfer of T cells to TCRß/ mice results in increased levels of apoptotic cells in the secondary lymphoid organs. This might result from death of a major fraction of the transferred cells or from perturbation of homeostatic regulatory pathways leading to death of large numbers of host cells. Further studies will be required to stringently test these ideas. However, we did not observe a reduction in T cell numbers following transfer, and B and NK cell populations were observed to expand. Moreover, immunization with apoptotic cells does not give rise to antibodies characteristic of frank systemic autoimmune disease (e.g. ANA+, anti-double-stranded DNA) (42), whereas we observed high titers of such antibodies in C57BL/6 T
TCRß
/ mice.
The autoantibody response in C57BL/6 T TCR
ß/ mice appears to be derived from a pre-existing subpopulation of autoreactive cells in TCR
ß/ mice that is absent in T cell-sufficient mice. Transfer of TCRß
/ B cells into syngeneic, B cell-deficient µMT mice resulted in increased and sustained serum levels of anti-DNA and anti-chromatin autoantibodies as compared to B cells from control mice. The level of autoantibody production in the TCRß
/ B
µMT mice was lower than in the C57BL/6 T
TCRß
/ mice, but this could have resulted from a poor reconstitution of the B cell compartment in the non-irradiated hosts, as previously observed in experiments using SCID mice (35,36). In addition, polyclonal activation of purified TCRß
/ B cells in vitro resulted in secretion of 2- to 3-fold higher levels of anti-chromatin and anti-DNA antibodies, as compared to B cells from T cell-sufficient mice.
Peripheral B cell tolerance is effected at several stages of differentiation (46,47). The first stage seems to target a population of `transitional' B cells that are recent emigrants from the adult bone marrow. Studies exploiting mice expressing transgene-encoded, autoreactive BCR have shown that engagement of autoantigen by recent B cell bone marrow emigrants inhibits their developmental maturation and results in a state of `anergy'. In several transgenic systems, this state of `anergy' is associated with partial activation, alterations in homing and a reduced life span (1012,48). In these systems, autoreactive B cells accumulate in the T cell rich PALS of the spleen, close to the follicular boundary. Studies by Goodnow and colleagues have also suggested that in this microenvironment, the death of anergic B cells is facilitated by interaction with CD4 T cells via the FasFas ligand pathway (17).
Given the results of these previous studies, and the data presented here, we suggest that the deficiency in T cells in TCRß/ mice leads to perturbations in this first step of peripheral B cell tolerance induction. In such mice, T cells and the T cell-rich microenvironments that prevent autoreactive transitional/anergic B cells from entering the follicle are absent. Once such B cells have gained access to the follicle in TCRß
/ mice, we suggest they often undergo further maturation and their life span is increased, despite the autoreactivity of their BCR. Upon introduction of T cells into TCRß
/ recipients, those B cells whose BCR are being cross-linked by multivalent antigens would be receptive to T cell help while other B cells would not. These B cells would then mount a vigorous GC and antibody response. These conclusions are consistent with our findings that TCRß
/ mice have elevated levels of peripheral B cells, many of which express reduced levels of sIgD, and that a subset of B cells in these mice can produce autoantibodies after stimulation with LPS in vitro or after transfer to B cell-deficient mice.
However, since the T cells transferred in our experiments are self-tolerant, how could they provide cognate help to follicular autoreactive B cells? Although a general, yet transient breakdown of T cell tolerance during reconstitution cannot be ruled out, we suspect that this is due to the presence of T cells, many of which are previously primed and have specificities for commonly encountered environmental antigens. In TCRß/ mice, all B cells, including the pre-existing autoreactive subset, would be processing and presenting such antigens at low levels, irrespective of the specificity of their BCR (49,50). In addition, a large of fraction B cells in normal mice have been shown to express `multireactive' BCR, capable of binding both self and foreign antigens (5156). The subset of the latter type of B cells specific for environmental antigens would be particularly receptive to the help provided by incoming T cells specific for these same antigens, due to their ability to efficiently endocytose and shunt this antigen into the MHC class II antigen processing and presentation pathway via the BCR.
Recent studies have shown that subsequent to transfer of mature T cells into syngeneic, irradiated, T depleted or congenitally T cell-deficient mice donor T cell proliferation is induced. This proliferation does not take place in hosts with intact T cell compartments and seems to require the recognition of the same MHCpeptide ligands by the donor cells that positively selected their precursors in the thymus (57,58). In the experiments described here, such homeostatic and self-ligand-induced proliferation might have promoted both the reconstitution of the T cell compartment, as well as the activation of T cells specific for environmental antigens, that could then provide help for `multireactive' B cells. Further studies will be required to investigate these ideas.
Given the scenario for the development of the autoimmune response in C57BL/6 T cell TCR
ß/ mice discussed above, why would this response be transient? The establishment of a functional T cell compartment would be expected to reduce the further entry of autoreactive `transitional/anergic' B cells into the follicular microenvironment. The production and maintenance of serum levels of antibodies to environmental antigens would result in the clearance of these antigens, inhibiting the further recruitment of naive Th cells into this response. These processes would inhibit the continued induction of a primary immune responses to autoantigens, as well as the differentiation of autoreactive pre-plasma cells that would require high levels of T cell help for further differentiation (59,60).
It seems unlikely that the activity of previously primed autoreactive and `multireactive' B cells participating in the GC reaction would be substantially influenced by reduction in circulating environmental antigen levels, however. In fact, the stimulatory form of antigen in the GC is most probably IC on the surface of follicular dendritic cells (FDC) (6163). However, during this phase of antigen-driven differentiation B cells appear to become highly susceptible to activation-induced cell death, as they express elevated levels of Fas and low levels of Bcl-2 and Bcl-xL (64,65).
Current data suggest that GC B cells undergoing V region somatic hypermutation are subjected to two forms of selection that are strongly influenced by the affinity and specificity of their BCR (7,61,66,67). In the first step, B cells that efficiently interact with FDC via binding of their BCR to the IC on the FDC surface receive survival signals and also endocytose these IC. In the second step, the antigen obtained from these IC is processed and presented by the GC B cell to CD4 T cells, resulting in the receipt of secondary survival and differentiation signals. B cells that do not receive these T cell-derived signals are thought to be eliminated by the FasFas ligand pathway. Thus, the proposed role of T cells in this peripheral tolerance pathway is 2-fold: they are required for the efficient priming of B cells and the induction of the GC reaction and they play a direct role in the deletion of Fashi, (Bcl-2, Bcl-xL)low GC B cells that express autoreactive BCR.
We suggest that deletion of autoreactive and `multireactive' B cells driven to participate in the GC reaction by environmental antigen-specific CD4 T cells in C57BL/6 T TCR
ß/ mice takes place via this pathway. The observation that diminution in serum antibodies levels in such mice coincides with a vigorous GC reaction and the disappearance of a subpopulation of activated, Fashi B cells supports this idea. However, further evaluation of this hypothesis will require determining whether the Fashi B cells are a GC subpopulation expressing autoreactive or `multireactive' BCR. We have previously proposed that in normal mice, a GC peripheral tolerance pathway results in the deletion of `multireactive' B cells that escape the `transitional/anergic' pathway of peripheral tolerance induction or that acquire autoreactivity via somatic hypermutation of their BCR (7).
In total, our data support the conclusion that regulation of peripheral autoreactive B cells takes place via dynamic and multifaceted processes in which T cells play a pivotal role. This is in keeping with previous data indicating that the development of autoantibody-mediated systemic disease is nearly always dependent on, or associated with, defects in the T cell compartment (68,69). The use of completely T cell-deficient TCRß/ mice as recipients for various syngeneic T cell subsets and T cells with defined genetic defects in regulatory factors should allow further elucidation of the nature of the T cellB cell interactions that culminate in a state of self-tolerance in the humoral immune system.
![]() |
Acknowledgments |
---|
![]() |
Abbreviations |
---|
ANA anti-nuclear antibody |
FDC follicular dendritic cell |
GC germinal center |
HRP horseradish peroxidase |
IC immune complex |
LPS lipopolysaccharide |
PALS periarteriolar lymphoid sheath |
PE phycoerythrin |
PNA peanut agglutinin |
SCID severe combined immune deficiency |
![]() |
Notes |
---|
Received 24 March 2000, accepted 10 July 2000.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|