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Departments of
* Internal Medicine and
Pediatrics, University of Cincinnati College of Medicine and Veterans Affairs Medical Center, Cincinnati, OH 45220;
Division of Rheumatology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095;
La Jolla Institute of Allergy and Immunology, San Diego, CA 92121; and
¶ University of Tennessee, Memphis, TN 38163
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain or peptides derived from its VH region
induced anti-VH Th cells, IgG anti-dsDNA Ab, and
proteinuria. The breakdown of B cell tolerance in nonautoimmune mice,
however, was short-lived: anti-DNA Ab and nephritis subsided
despite subsequent immunizations. The recovery from autoimmunity
temporally correlated with the appearance of T cells that inhibited
anti-DNA Ab production. Such inhibitory T cells secreted TGF
;
the inhibition of anti-DNA Ab production by these cells was partly
abolished by anti-TGF Ab. Even without immunization,
nonautoimmune mice possess T cells that can inhibit autoantibody
production. Thus, inhibitory T cells in nonautoimmune mice may normally
inhibit T-dependent activation of autoreactive B cells and/or reverse
such activation following stimulation by Th cells. The induction of
such inhibitory T cells may play a role in protecting nonautoimmune
mice from developing chronic autoimmunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To understand the mechanisms that prevent autoreactive B cells from producing Ab, several laboratories have generated transgenic mice in which most B cells express Ig of a single specificity. These studies have identified several mechanisms, such as clonal deletion and anergy, that protect mice from breaking self-tolerance in B cells (8, 9, 10, 11, 12, 13). Not many studies, however, have addressed the protective mechanisms that come into play once self-tolerance has been broken in normal mice.
Here, we ask: 1) Can B cells in nonautoimmune mice be activated to secrete anti-DNA Ab by exposure to autoreactive Th cells? 2) Will such a breakdown in the state of inactivation in B cells cause persistent anti-DNA Ab production and disease in these mice? 3) Can these mice develop protective mechanisms that contain or reverse the loss of self-tolerance in B cells?
To address these questions, we used a nontransgenic model in which MHC class II-restricted Th cells help B cells produce IgG anti-DNA Ab (4); the (NZB x NZW)F1 (BWF1) strain of lupus-prone mice; and two nonautoimmune mice that have MHC class II molecules identical to those in BWF1 mice. Using this system, we show that nonautoimmune mice, upon persistent exposure to autoreactive Th cells, develop inhibitory T cells that can inhibit autoantibody production and renal disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NZB/B1nj, NZW/Lacj, BALB/c, and B10.PL mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and intercrossed to generate BWF1 (NZB x NZW) F1, CWF1 (BALB/c x NZW) F1, and BPF1 (NZB x B10.Pl) F1 mice in the Cincinnati Veterans Affairs Medical Center or University of California, Los Angeles Vivarium. Female mice were used in all experiments. Experiments were performed in accordance with the institutional animal research committee guidelines.
Peptides
15-mer overlapping peptides representing the entire
VHD region sequence of an anti-dsDNA mAb,
A6.1, were synthesized using a modified pin synthesis or a macrocrown
method (14). Each 15-mer peptide overlapped its neighbor
by 10 residues. Several 11- to 15-mer T cell epitopes and control
peptides (Table I
) were synthesized in large quantities at the Chiron
Laboratories (Clayton, Australia), using F-moc chemistry. The synthetic
peptides were analyzed for purity by HPLC and by mass spectrometry.
Each peptide chromatographed essentially as a sharp single peak. All
purified peptides had the expected molecular mass.
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Young CWF1 or BWF1 mice were immunized i.p. with 20 µg of A6.1 (purified from ascites fluid as IgG), emulsified in CFA, and boosted with 10–20 µg of A6.1 in IFA, as described in the figure legends.
Purification of cells
Spleen cells were pooled from 3 to 12 mice and purified as mononuclear cells on Ficoll-Hypaque. Enrichment of CD4+, CD8+, and B cells was performed with the Vario MACS magnetic purification system using microbead-coated Ab (Miltenyi Biotec, Auburn, CA). Purity of the cell populations as determined by FACS varied from 92 to 99%.
Determination of T cell help for anti-DNA Ab synthesis by ELISPOT and ELISA
Sonicated, nitrocellulose-filtered calf thymus dsDNA (100 µg/ml; Sigma-Aldrich, St. Louis, MO) was coated onto 96-well microtiter plates (catalogue no. 3590; Costar, Cambridge, MA) and incubated overnight at 4°C. After washing with PBS, DNA-coated plates were blocked for 1 h with 10% FCS in PBS at room temperature. Splenic T and B cells in complete medium (DMEM with 10% FBS) and varying concentrations of peptides were added to each well. After 8–12 h of incubation at 37°C, cells were poured off and plates were washed eight times. Plates were then incubated overnight at 4°C with alkaline phosphatase-conjugated antimouse IgG or IgA, diluted 1/500 in 10% FCS in PBS. Plates were washed again with Tris/Tween 20, and incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate with agarose gel at a final concentration of 0.6%. Anti-dsDNA Ab-forming cells (AFC)3 were enumerated as blue spots using an inverted microscope (Leitz Canada, Midland, Ontario, Canada) by two of us (F.M.E. and R.R.S.) in a blinded fashion and recorded as number of AFC per 106 B cells. The specificity of anti-dsDNA ELISPOT was confirmed by competitive inhibition studies; the anti-dsDNA ELISPOT was inhibited by DNA but not with cardiolipin, keyhole limpet hemocyanin, or hen egg lysozyme (HEL) (15, 16, 17). The anti-dsDNA ELISPOT was inhibited by both dsDNA and ssDNA but it required two times more ssDNA than dsDNA for 50% inhibition (15, 17). This is consistent with observations that some populations of anti-DNA Abs share dsDNA and ssDNA binding specificities.
The results of ELISPOT assays for anti-dsDNA Ab were further confirmed in simultaneous ELISA. Briefly, spleen cells were cultured with or without peptides for 5 days; supernatants were tested for IgG anti-dsDNA Ab, as described previously (3, 4). Briefly, 96-well plates (Costar) were coated with dsDNA (100 µg/ml; Sigma-Aldrich) overnight at 4°C. After blocking with 10% FCS, samples were added. Bound Ab was detected with alkaline phosphatase-conjugated goat anti-mouse IgG (Fisher, Pittsburgh, PA). Reactions were developed with p-nitrophenyl phosphate (Sigma-Aldrich) and A405 was measured with an ELISA reader. The results of ELISA and ELISPOT assays correlated well with each other (15).
Establishing T cell lines
Splenic single-cell suspensions from the CWF1 or BWF1 mice immunized with A6H31–45 were fractionated into CD4+ or CD8+ T cells. The fractionated CD4+ and CD8+ T cells were cultured and periodically stimulated with syngeneic APCs (irradiated T cell-depleted spleen cells), A6H31–45 and Con A supernatant, as described previously (4).
B cell hybridomas
Four B cell hybridomas, including a dsDNA-specific (A6.1) and three non-DNA binding (1GE6, 375-57, and 375-100), were established from BWF1 mice by fusion of their spleen cells with a nonsecreting murine myeloma cell, as described previously (3, 18).
Cytokine assays by ELISA
Resting T cell lines were cultured with APCs and peptide;
supernatants were tested for cytokines by ELISA, as previously
described (19). TGF was assayed by sandwich ELISA using
a TGF1-specific kit (G1230; Promega, Madison, WI) that is designed
to measure biologically active TGF1. To assay for total TGF1, the
samples were acid treated and neutralized according to the
manufacturer’s instructions (www.promega.com). In brief, multiwell
ELISA plates (Nunc MaxiSorp; Nunc, Naperville, IL) were coated with 100
µl of an anti-TGF1 mAb in carbonate buffer (pH 9.6) overnight
at 4°C. Plates were washed three times with Tris-HCl (pH 7.6)
containing 0.05% Tween 20, followed by blocking for 35 min at 37°C.
Samples and standard were added, and plates were incubated for 90 min
at room temperature. Plates were washed five times before the addition
of 100 µl per well of a second polyclonal anti-TGF1 Ab. After
2 h at room temperature, plates were washed and 100 µl of a
tertiary HRP-conjugated species-specific Ab was added. The plates were
incubated for 2 more hours, then washed six times, and 100 µl of
peroxidase substrate/tetramethylbenzidine solution was added.
Fifteen minutes later, the reaction was stopped with 1 M phosphoric
acid. A405 was measured with a
Labsystem ELISA reader.
Assessment of clinical disease
Proteinuria and blood urea nitrogen were tested using Albustix and Azostix (range, 0–4+), respectively, and creatinine was estimated by reagents from Stanbio Laboratories (San Antonio, TX), as described previously (4, 20).
Statistical analysis
Student’s t test was performed to compare the test
and control groups. A Shapiro-Wilk test of normality was performed to
determine whether the distribution of the effect of
CWF1-derived T cell lines on anti-DNA Ab
production (see Fig. 4C) matches a normal distribution.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The BWF1 mice, which are the
F1 intercross of NZB (H-2d)
and NZW (H-2z) mice, develop an
autoantibody-mediated disease that shares many features with human
lupus (2, 4). The autoantibody production in these mice is
mostly dependent on MHC class II-restricted
TCR
+ cells (2, 3, 4). To
determine whether F1 mouse strains that express
MHC class II molecules (H-2dxz) identical to
those of BWF1 mice develop a lupus-like disease,
we crossed BALB/c (H-2d) with NZW mice to
generate CWF1 mice. Since NZW class II molecules
have an identical sequence to the H-2u haplotype
(21), we crossed NZB with B10.Pl (H-2u =
z) to generate another F1 strain,
BPF1, that expresses H-2dxu =
dxz. Twenty BWF1, 20
CWF1, and 32 BPF1 female
mice were monitored for serum IgG anti-DNA Ab and proteinuria up to
1 year of age. All BWF1 mice had anti-DNA Ab
by 15 wk of age, proteinuria by 28 wk, and died by 42 wk. In contrast,
all CWF1 and BPF1 mice
appeared to be healthy during the study period; <10% had anti-DNA
Ab, but none had proteinuria. A similar observation in
BPF1 mice has been reported by others
(21).
Autoreactive B and Th cells are inactive or tolerant in nonautoimmune mice
To confirm the absence of IgG anti-DNA Ab-secreting B cells in
naive nonautoimmune mice, we cultured spleen cells from
CWF1, BPF1, or
BWF1 mice for 8–12 h in microtiter plates coated
with DNA and estimated the numbers of IgG anti-DNA AFC by ELISPOT.
Less than 10 AFC per 106 B cells were detected in
CWF1 or BPF1 mice, while
BWF1 cultures had 180 AFC (Fig. 1, T plus B). Similar results were
obtained when spleen cells from these mice were cultured for 5 days and
supernatants were tested for anti-DNA Ab in an ELISA.
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), and IgG anti-DNA Ab production
was estimated by ELISPOT (Fig. 1). In the presence of T cell
stimulatory peptides, the anti-DNA AFC significantly increased in
BWF1, but not in CWF1 or
BPF1 mice. Thus, in contrast to
BWF1 mice, MHC class II-identical
CWF1 and BPF1 mice do not
exhibit spontaneous activation of DNA-specific B and
anti-VH Th cells. These cells may either be
absent, ignorant, or tolerant in these mice. Th cells disrupt B cell self-tolerance (or inactive state) in nonautoimmune mice
In four separate experiments, CWF1 or
BPF1 B cells were cultured with
BWF1 T cells and vice versa. Strikingly,
CWF1 and BPF1 B cells were
induced to produce IgG anti-DNA Ab in the presence of
BWF1 T cells (Fig. 2A, left panel). In contrast,
T cells from the nonautoimmune mice did not promote anti-DNA Ab
production by BWF1 B cells (Fig. 2A, right
panel). This indicates that DNA-specific B cells exist in
nonautoimmune mice and can be activated to secrete IgG anti-DNA Ab
by autoimmune Th cells in vitro.
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-chain or VH epitopes (listed in Table I) in adjuvant induced VH peptide-specific T cell
proliferative or Ab responses in CWF1 mice (data
not shown), suggesting that anti-VH Th cell
repertoire exists in nonautoimmune mice. To determine whether these in
vivo-primed anti-VH Th cells can promote
anti-DNA Ab production, 20-wk-old CWF1 mice
(n = 12) were immunized with A6.1 in CFA and boosted
twice at 2-wk intervals with A6.1/IFA. Splenic T cells from these mice
were cultured with syngeneic B cells without or with 23 overlapping
15-mer peptides representing the entire VH
sequence of the A6.1 (Fig. 2B). Results show that three
peptides (peptides 7–9) increased anti-DNA AFC 4- to 5-fold above
the background. Importantly, all immunized CWF1
mice developed high levels of serum anti-DNA Ab (Fig. 2C) and most developed renal involvement. Three of 20 mice
had a severe nephritis (proteinuria, 300 mg/dl with increased blood
urea nitrogen (80 mg/dl) and creatinine (1.8 mg/dl)); 14 developed a
mild disease (proteinuria, 100 mg/dl with normal blood urea nitrogen
(<20 mg/dl) and creatinine (<0.6 mg/dl); and 3 remained normal.
Injection of a control, nonautoreactive mAb, MOPC-21, did not induce
IgG anti-DNA Ab or proteinuria (Ref. 3 and data
not shown). This suggests that potentially autoreactive B cells in
nonautoimmune mice can be activated to secrete pathogenic IgG
anti-DNA Ab by appropriate Th cells.
To exclude the possibility that serum anti-DNA titers in
A6.1-immunized mice (Fig. 2C) were simply coming from the
injected IgG2a mAb, we showed that the IgG anti-DNA Abs of all IgG
isotypes were detected in the immunized CWF1 mice
(data not shown).
Breakdown of self-tolerance in B cells in nonautoimmune mice is transitory in nature
The induction of serum IgG anti-DNA Ab and mild proteinuria
upon immunizations with A6.1 prompted us to test whether persistent
anti-DNA response and severe nephritis would be induced following
chronic immunization of CWF1 mice. Serum
anti-DNA Ab levels did increase between immunizations two and four,
and 75% (8 of 12) of the mice developed proteinuria (100–300 mg/dl)
with normal to mildly elevated plasma creatinine (mean ± SD,
0.9 ± 0.3). Thereafter, serum anti-DNA levels decreased (Fig. 3A). All immunized mice slowly
recovered from disease by the sixth immunization, at which time all had
30 mg/dl proteinuria and blood urea nitrogen and creatinine in the
normal range. Subsequent immunizations did not induce a vigorous
anti-DNA Ab response (Fig. 3A) or proteinuria. This
suggests that, although CWF1 mice are susceptible
to breakdown of B cell tolerance, this loss of tolerance is
self-limited.
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2-fold of the background.
Thereafter, none of the peptides elicited increases in anti-DNA
production (Fig. 3B). Activation of inhibitory T cells is involved in inhibiting autoantibody response
Multiple mechanisms may contribute to the decrease in anti-DNA Ab production after repeated immunizations: 1) induction of inhibitory or regulatory T cells or cytokines; 2) induction of anergy or deletion in anti-VH Th cells; 3) induction of regulatory anti-idiotypic Ab; and 4) development of Ab against A6.1 and formation of A6.1-anti-A6.1 immune complexes that could alter Ag processing and presentation. Although we have not excluded the relative contribution of each of these possibilities, we demonstrate several lines of evidence supporting the activation of inhibitory T cells that down-regulate anti-DNA Ab production.
First, we demonstrate that the freshly isolated T cells from the
"recovered" CWF1 mice decrease anti-DNA
Ab production by BWF1 spleen cells in a
peptide-specific manner (Fig. 4A). This decrease in AFC was
not due to a changed proportion of T plus B cells or crowding, since
addition of the same numbers of BWF1 T cells or
CWF1-derived, HEL106–116-reactive T cells to
BWF1 T plus B cell cultures did not affect
anti-DNA AFC numbers (Fig. 4A and data not shown).
Second, we asked whether a similar immunization regimen would activate
inhibitory T cells in lupus mice (Fig. 4B). In two
independent experiments, 8–10 short-term T cell lines were established
from pooled spleen cells of 5 each of CWF1 or
BWF1 mice that were immunized with A6H31–45
(stimulatory peptide, no. 7 in Fig. 2B) five times at 2-wk
intervals. Three sets of independent, resting short-term T cell lines
(in varying numbers from 102 to
105) were cocultured with
105–106 B cells from 26-
to 32-wk-old unimmunized BWF1 mice. Results show
that while CWF1-derived T cell lines decreased
anti-DNA production by BWF1 B cells in a dose
(T cell number)-dependent manner, BWF1-derived T
cell lines increased anti-DNA Ab production (Fig. 4B).
This suggests that the development of inhibitory T cells may be
inadequate or defective in BWF1 mice.
Third, 42 long-term lines or clones were established by subcloning
CWF1-derived short-term T cell lines and tested
for the ability to influence anti-DNA Ab production by
BWF1 B cells (Fig. 4C). Although only
2 (5%) T cell lines increased anti-DNA AFC numbers by 2-fold, 13
(31%) T cell lines decreased the AFC numbers to <50% of the
background. The remaining T cell lines had no or <2-fold effect on
anti-DNA Ab production (Fig. 4C). This distribution of
the effect of T cell lines on anti-DNA Ab production did not match
a normal distribution (p = 0.003, Shapiro-Wilk
test). Thus, both inhibitory T and Th cells coexist in
CWF1 mice.
Although most (16 of 20 tested (80%)) inhibitory cell T lines suppressed anti-DNA AFC only in the presence of A6H31–45, some (20%) inhibitory T cell lines were activated in the presence of control peptides (HYHEL31–45 or A6H110–121) or even without any added peptide. To test the specificity of inhibition of anti-DNA Ab production by inhibitory T cells, an inhibitory T cell line (CWF1 no. 3) or purified T cells from CWF1 mice were cultured for 5 days with three BWF1-derived B cell hybridomas—A6.1 that expresses anti-DNA Ig that contains the A6H31–45 sequence and 375–57 and 375–100 that express non-DNA binding Ig that do not contain the A6H31–45 sequence. Results show that inhibitory T cells decreased total Ig production by A6.1 by 40–60%, but not by 375–57 and 375–100.
Inhibition of autoantibody production by some inhibitory T cells is
partly dependent on TGF
TGF is an immunoregulatory cytokine that can inhibit
autoantibody production (31, 32). To determine whether
inhibitory T cells selectively secrete this cytokine, we cultured APCs
with peptide and eight T cell lines each from
CWF1 and BWF1 mice and
measured TGF in culture supernatants (Fig. 5A). Collectively,
CWF1-derived T cell lines secreted higher levels
of TGF than BWF1-derived T cell lines
(p < 0.05, Student’s t test).
Essentially similar results were obtained with another set of T cell
lines. Interestingly, all CD8+ inhibitory T cell
lines tested secreted 2- to10-fold higher amounts of TGF when
compared with all CD4+ T cell lines from
CWF1 or BWF1 mice.
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secreted by
CWF1-derived T cells contributes to the
inhibition of autoantibody production. First, we show that splenic T
cells from the hyperimmunized CWF1 mice when
cocultured with BWF1 T plus B cells decreased the
IgG anti-DNA AFC number. The addition of an anti-TGF mAb
(1.D.11.16; Celltrix, Santa Clara, CA), but not an isotype-matched
control Ig, to these cultures abolished the inhibition of anti-DNA
Ab (Fig. 5B). The TGF effect on the inhibition of IgG
anti-DNA Ab production was not associated with the isotype skewing
of anti-DNA Ab toward IgA (data not shown). Naive CWF1 mice also have T cells that inhibit anti-DNA Ab production by BWF1 spleen cells
So far, we have shown that primed T cells from
CWF1 mice suppress anti-DNA Ab production. To
determine whether such inhibitory T cells exist in naive mice, we
cultured BWF1 spleen cells for 5 days and tested
supernatant for anti-DNA Ab by ELISA. To these cultures, addition
of T cells from 10-wk-old, naive CWF1 mice
significantly decreased anti-DNA Ab; this inhibition by naive T
cells was also partially abrogated in the presence of an
anti-TGF mAb (Fig. 6).
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Nonautoimmune CWF1 mice have more active TGF than
lupus-prone BWF1 mice
We hypothesized that the tendency to develop TGF-secreting
inhibitory T cells is due to the intrinsic ability of nonautoimmune
mice to make more active TGF in vivo. To test this, we determined
serum levels of active and total TGF. Serum-active TGF1 was
significantly more in young CWF1 mice than in
BWF1 mice (Fig. 7).
Total TGF1 levels, however, were similar in the two strains; thus,
the mean ratio of active:total TGF was significantly higher in
CWF1 (0.11 ± 0.016) than in
BWF1 (0.05 ± 0.009) mice
(p = 0.02, n = 6 each).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Addition of anti-TGF Ab to the cultures partly abrogated
the ability of regulatory T cells to inhibit anti-DNA Ab
production.
In nonautoimmune mice bearing transgenes that encode anti-DNA Ig,
DNA-specific B cells accumulate at the T-B interface of the splenic
follicle, whereas in lupus-prone MRL/lpr mice the
anti-DNA B cells are present in the follicle, thus allowing T-B
cell interaction (10, 11, 12). Anti-DNA B cells from
nonautoimmune transgenic mice proliferate in response to CD40 ligand
and IL-4 (13), suggesting that B cell self-tolerance is
due primarily to lack of availability of T cell help. Indeed, that is
the case in our nontransgenic system: B cells from nonautoimmune mice
could readily make IgG anti-DNA Ab when exposed to autoreactive Th
cells from lupus mice or syngeneic Th cells that were activated
following immunization with an anti-DNA mAb (Fig. 2). Such B cell
activation induced a strong anti-DNA Ab response in vivo (Fig. 2C) and caused renal disease. Thus, potentially pathogenic
autoreactive B cells that may have escaped clonal deletion exist in
nonautoimmune mice.
Although anti-DNA Ab production by CWF1 mice
was readily induced by immunizations that activated Th cells (Fig. 2B), this effect was short-lived, as anti-DNA Ab
decreased despite further immunizations (Fig. 3). It is possible to
explain this decrease in anti-DNA Ab response in nonautoimmune mice
by the emergence of T cells that decreased anti-DNA Ab in these
mice. Remarkably, adoptive transfer of such inhibitory T cells into
BWF1 mice that are genetically prone to develop
lupus resulted in decreased serum anti-DNA Ab levels, delayed onset
of nephritis, and improved survival (our unpublished data).
Although the mechanisms by which inhibitory T cells arise and inhibit
autoimmunity are not well defined, our studies offer some interesting
clues. Most Th and inhibitory T cells appear to recognize the same
peptides (Figs. 2B and 4C). Analogous findings
have been reported in other models (33, 34, 35). For instance,
the same self-MHC-reactive T cells helped as well as suppressed B cell
Ig production under different conditions (33). The
different conditions, in our case, could be created by differences in
the in vivo TGF levels between nonautoimmune
CWF1 and lupus-prone BWF1
mice. Naive, young CWF1 mice that exhibit a
dominant inhibitory T cell response had more active TGF than
BWF1 mice that develop a mostly Th response
(Figs. 4B and 7). Such a cytokine milieu might contribute to
the development of inhibitory T cells in vivo. An exposure of
CD8+ T cells to TGF can condition these cells
to become suppressors of Ab production (36). TGF
selectively activates CD8+ T cells to proliferate
and augments maturation of naive to memory T cells (37).
The regulatory role of TGF (33, 36, 38) was confirmed
in our experiments in which anti-TGF Ab abrogated, at least in
part, the T cell-mediated inhibition of anti-DNA Ab production
(Fig. 5B).
Although, TGF-mediated suppression appears to be a major mechanism
in our model (Fig. 5), it does not represent the sole mechanism of
regulation of autoantibody production. Although the inhibition of
anti-DNA Ab production by CD8+ inhibitory T
cell lines was markedly inhibited by an anti-TGF mAb (Fig. 5B), there was no or minimal effect of anti-TGF mAb
on the inhibition of autoantibody production by
CD4+ inhibitory T cell lines (data not
shown).
Most Th and inhibitory T cells recognized a peptide, A6H31–45 that
contains at least two epitopes: one epitope binds an MHC class II
molecule, I-Ed, and activates
CD4+ Th cells that promote anti-DNA Ab
production in BWF1 mice (4), and the
other epitope binds an MHC class I molecule, Kd,
and activates CD8+ T cells that inhibit
anti-DNA Ab
production.4 The
15-mer peptide, A6H31–45, activated Th cells in both
BWF1 (Fig. 4B) and
CWF1 mice (Figs. 2B, 3B,
and 4C), but activated inhibitory T cells only in
CWF1 mice (Figs. 4, A–C, and
5B). A shorter version of this peptide, a 12-mer A6H34–45
that was used in initial experiments (Figs. 1 and 2A), also
contains the same helper and inhibitory epitopes. It remains to be
determined whether a differential processing of the
VH molecule into the helper vs inhibitory
epitopes are responsible for the predominant helper response in
BWF1 mice vs prominent inhibitory response in
CWF1 mice, respectively, shown in Fig. 4B.
Numerous examples illustrate that inhibitory T cells recognize
molecules involved in Ag recognition (33, 39, 40, 41).
TCR-reactive T cells arise during recovery from experimental autoimmune
encephalomyelitis and suppress it (39);
MHC-reactive T cells protect rats from autoantibody-mediated disease
(40); and Ig-reactive T cells confer resistance to
induction of autoimmune keratitis (41). In our
experiments, Ig peptide-reactive inhibitory T cells protect mice from
developing persistent autoantibody-mediated disease (Fig. 3A
and data not shown).
Finally, the development of inhibitory T cells did not appear to be an
artifact of multiple immunizations, as even without immunization
splenic T cells from CWF1 mice inhibit
autoantibody production, albeit to a much smaller extent than T cells
from immunized CWF1 mice (Fig. 6). Thus, it
appears that the normal immune repertoire is composed of potentially
autoreactive Th as well as inhibitory T cells (Fig. 4C)
(35, 42, 43) and inhibitory T cells expand whenever there
is an impending danger of pathologic autoimmunity.
In conclusion, inhibitory T cells may normally prevent activation of autoreactive B cells and/or reverse their activation following stimulation by Th cells. We suggest that the ability to develop inhibitory T cells may be one of many checkpoints that protect nonautoimmune mice from developing clinical autoimmunity. Such inhibitory T cells may be responsible for spontaneous regression or waxing and waning course of autoimmune diseases, while defective regulation may contribute to sustained autoimmunity and its consequences such as systemic lupus erythematosus.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ram Raj Singh, Department of Internal Medicine, Division of Immunology, Autoimmunity and Tolerance Laboratory, University of Cincinnati College of Medicine, ML 0563, 231 Albert Sabin Way, Cincinnati, OH 45267-0563. E-mail address: singhrm{at}email.uc.edu
3 Abbreviations used in this paper: AFC, Ab-forming cell; HEL, hen egg lysozyme.
4 G. C. Fan and R. R. Singh. Vaccination with minigenes encoding VH-derived MHC class I-binding epitopes activates cytotoxic T cells that ablate autoantibody-producing B cells and inhibit lupus. Submitted for publication.
Received for publication February 4, 2002. Accepted for publication April 30, 2002.
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  R. P. Singh, A. La Cava, and B. H. Hahn pConsensus Peptide Induces Tolerogenic CD8+ T Cells in Lupus-Prone (NZB x NZW)F1 Mice by Differentially Regulating Foxp3 and PD1 Molecules J. Immunol., February 15, 2008; 180(4): 2069 - 2080. [Abstract] [Full Text] [PDF]
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V. Saxena, D. W. Lienesch, M. Zhou, R. Bommireddy, M. Azhar, T. Doetschman, and R. R. Singh Dual Roles of Immunoregulatory Cytokine TGF-{beta} in the Pathogenesis of Autoimmunity-Mediated Organ Damage J. Immunol., February 1, 2008; 180(3): 1903 - 1912. [Abstract] [Full Text] [PDF]
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H.-K. Kang, M. Liu, and S. K. Datta Low-Dose Peptide Tolerance Therapy of Lupus Generates Plasmacytoid Dendritic Cells That Cause Expansion of Autoantigen-Specific Regulatory T Cells and Contraction of Inflammatory Th17 Cells J. Immunol., June 15, 2007; 178(12): 7849 - 7858. [Abstract] [Full Text] [PDF]
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U. S. Deshmukh, H. Bagavant, D. Sim, V. Pidiyar, and S. M. Fu A SmD Peptide Induces Better Antibody Responses to Other Proteins within the Small Nuclear Ribonucleoprotein Complex than to SmD Protein via Intermolecular Epitope Spreading J. Immunol., February 15, 2007; 178(4): 2565 - 2571. [Abstract] [Full Text] [PDF]
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B. H. Hahn, R. P. Singh, A. La Cava, and F. M. Ebling Tolerogenic Treatment of Lupus Mice with Consensus Peptide Induces Foxp3-Expressing, Apoptosis-Resistant, TGF{beta}-Secreting CD8+ T Cell Suppressors J. Immunol., December 1, 2005; 175(11): 7728 - 7737. [Abstract] [Full Text] [PDF]
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W. Guo, D. Smith, A. Guth, K. Aviszus, and L. J. Wysocki T Cell Tolerance to Germline-Encoded Antibody Sequences in a Lupus-Prone Mouse J. Immunol., August 15, 2005; 175(4): 2184 - 2190. [Abstract] [Full Text] [PDF]
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 E N Voynova, A I Tchorbanov, T A Todorov, and T L Vassilev Breaking of tolerance to native DNA in nonautoimmune mice by immunization with natural protein/DNA complexes Lupus, July 1, 2005; 14(7): 543 - 550. [Abstract] [PDF]
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H.-K. Kang, M. A. Michaels, B. R. Berner, and S. K. Datta Very Low-Dose Tolerance with Nucleosomal Peptides Controls Lupus and Induces Potent Regulatory T Cell Subsets J. Immunol., March 15, 2005; 174(6): 3247 - 3255. [Abstract] [Full Text] [PDF]
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) or from
unimmunized BWF1 mice (control; 
). Results are expressed
as the mean triplicate IgG anti-DNA AFC per 106 B
cells. The addition of CWF1 T cells to BWF1
spleen cell cultures decreased anti-DNA AFC in a peptide-specific
manner (*, p < 0.01). Similar results were
obtained using fractionated CD4+ or CD8+ T
cells and with another set of T cell determinant (A6H41–55) and
control (A6H93–107) peptides. Data are the pool of three independent
experiments. B, Short-term T cell lines from
CWF1 mice (•) decrease anti-DNA Ab production by
BWF1 B cells in a dose (T cell number)-dependent manner,
while T cell lines from similarly immunized BWF1 mice (
)
promote anti-DNA Ab production. Results are expressed as the mean
triplicate IgG anti-DNA AFC per 106 B cells from one of
two similar experiments; deviations from the mean were <10%. Similar
results were obtained using another two sets of short-term T cell lines
from CWF1 and BWF1 mice. C,
Although most long-term T cell lines and clones from CWF1
mice inhibit anti-DNA Ab production, some promote autoantibody
production. The resting T cell lines or clones that were established by
subcloning short-term lines (shown in B) were cocultured
with A6H31–45 and BWF1 splenic T plus B cells. Results are
expressed as the mean IgG anti-DNA AFC per 106 B cells.
The first bar on the left (BW B plus T cells without any
added peptide) represents the background AFC number. The horizontal
dashed line crossing this figure denotes one-half of the background AFC
number. We arbitrarily used this cut off to label the T cell lines as
inhibitory (
, labeled
arbitrarily as helper T cell lines). The remaining T cell lines that
included 15 CD4+ and 12 CD8+ T cell lines had
no or <2-fold effect on AFC (
). Data are representative of two
separate experiments.
, p < 0.001), but the AFC numbers decreased
below the background level when T cells from eight times immunized
CWF1 mice (n = 5) were added to these
cultures (
(an isotype-matched control rat IgG1)).
