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Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or TCR
but not Ig L chains (1).
The N nucleotides are added by the enzyme terminal deoxynucleotidyl
transferase (TdT)4 in
an untemplated fashion, thus adding much diversity to the
complementarity-determining region 3 (CDR3) regions (2, 3). The Ab and TCR repertoire of fetal and newborn mice is very
different from that of adult mice due to the delayed onset of
expression of TdT until the first week after birth (1, 2, 3, 4).
The absence of TdT not only eliminates N region diversity, but also
limits the diversity of junctions created, because Ig junctions created
in the absence of TdT are often formed at the site of short sequence
homologies, which are very common between the 3' end of DH
and 5' end of JH segments (4, 5, 6). This
"homology-directed recombination" also restricts the diversity at
the VH-DH junction to a more variable extent,
thus making the fetal/neonatal Ig repertoire both much smaller than,
and importantly, also different from, the adult repertoire
(5). The coding ends of TCR V, D, and J genes do not have
many short sequence homologies, thus the diversity of TCR junctions in
the neonate is merely restricted by the lack of N regions
(7). The reason why the immune system evolved to have an early smaller repertoire, and later, a more diverse repertoire is not known. We and others have suggested that the early repertoire may be restricted so that certain specificities, such as the anti-PC Abs, which are critical for clearing Streptococcus pneumonia infection, can be reproducibly made (5, 8). These Abs lack N regions, and have characteristic VDJ junctions that are created by homology-directed recombination. Mice have been generated in which the TdT gene has been disrupted, and these TdT-/- mice are healthy and respond to most complex Ags (2, 3, 9). The T cell repertoire of these TdT-deficient mice has been shown to be more promiscuous with regard to peptide recognition (10). Also, the early N-region-lacking T cells appear to undergo positive selection in the thymus more readily (11). In contrast, the B cell repertoire from these TdT-/- mice has been shown to be less polyreactive than that of the N region-containing repertoire in a wild-type mouse (12).
To test the role of the TdT+ and TdT- repertoires in autoimmunity, lupus-prone mice were made that were TdT deficient by crossing TdT-/- mice to New Zealand Black (NZB) and New Zealand White (NZW) mice (13). The resulting TdT-deficient (NZB x NZW)F1 mice showed increased lifespan and decreased incidence of glomerulonephritis (GN), suggesting a dramatic effect of repertoire on this autoimmune disease. However, surprisingly, the level of anti-dsDNA was not altered. Anti-dsDNA Abs have been shown to be enriched in arginines, which are likely to aid in binding to the anionic Ag, DNA (14). Within CDR3, arginines are most often encoded by N region addition, whereas some arginines are also encoded by using D regions in alternative reading frames, or the use of inverted D regions or D-D joinings (14). Therefore, one might have predicted that the absence of N regions might reduce the level or affinity of anti-dsDNA Abs. Therefore, it was very surprising that these (NZB x NZW)F1 TdT-/- mice produced similar levels of anti-dsDNA to their wild-type counterparts. Because TdT is expressed only in developing T and B cells, and its function is to add N region diversity, the decrease in pathology in these mice is likely to be due to a repertoire change in either B or T cells, or both. Thus, these studies either suggested that the limitation in the T or B cell repertoire due to the absence of N region nucleotides was for a different specificity, or that the fine specificity or affinity for dsDNA might be very different in the two groups of mice.
In this study we bred TdT-deficient mice to MRL/lpr, Fas-deficient (MRL-Faslpr) mice to determine whether the decrease in autoimmune disease due to TdT deficiency was generalizable to another lupus mouse model. Also, we were interested in examining the anti-dsDNA Abs, as well as to broaden the spectrum of analyzed autoantibodies. We found, as in the TdT-deficient (NZB x NZW)F1 mice, that TdT deficiency led to an increase in lifespan. However, in contrast to the TdT-deficient (NZB x NZW)F1 mice, we observed significantly lower levels of anti-dsDNA. In addition, we observed a decrease in levels of anti-chromatin and IgM-rheumatoid factor (RF) Abs and in the generalized hypergammaglobulinemia characteristic of MRL-Faslpr mice. Also, there was a reduction in the characteristic CD4-CD8- (double negative, DN) B220+ T cell population and in lymphoid hyperplasia, and a decrease in the incidence of skin lesions. Thus, essentially all aspects of the lupus disease were lessened in these mice due to the absence of TdT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MRL-Faslpr mice were obtained from
the breeding colony at The Scripps Research Institute.
TdT-/- mice, backcrossed approximately three
generations to C57BL/6, were given to us by Drs. D. Mathis and C.
Benoist (both from Harvard University, Cambridge, MA) via Dr. M. Bevan
(University of Washington, Seattle, WA), and have been
maintained as a TdT-/- stock in our breeding
colony. Breeding and maintenance were performed under specific
pathogen-free conditions. TdT-deficient
MRL-Faslpr background mice were generated
by backcrossing the TdT-deficient mice to the
MRL-Faslpr strain and screening for
lpr and TdT on tail DNA using previously
described PCR conditions (10, 15). Of the initial 105 mice
screened, 47 were Faslpr homozygous.
Forty-two of these were TdT+/+ and five were
TdT-/-. Because TdT and
Fas are both on chromosome 19, 
16.5 cM apart (Mouse
Genome Database); The Jackson Laboratory, Bar Harbor, ME), the
five TdT-/- mice had each undergone a crossover
event. Fas-/-TdT-/-
mice were then backcrossed to MRL-Faslpr.
The data shown here were derived from intercross mice derived from the
third through the fifth backcross generations (N3–N5), and the
survival data also included 13 N2 intercross mice. For each assay
performed in this study, similar numbers of littermate
TdT-/- and TdT+/+ mice
were analyzed.
Pathology
Autopsies were performed at 6 mo. Pathologic examinations were conducted as previously described (16). Tissue sections were fixed in Bouin’s solution and stained with periodic acid-Schiff reagent and hematoxylin. GN was graded blindly using a 0–4 scale (16). Skin lesions were assessed by visual inspection.
Flow cytometry
Fluorescent dye-conjugated Abs reactive with B220, CD3, CD4,
CD8, CD19, 
TCR, V8.3, V8.1 + V8.2, and CD44 were
purchased from BD PharMingen (San Diego, CA). Standard procedures were
used to isolate and stain spleen and lymph node (LN) cells.
5-Bromo-2'-deoxyuridine (BrdU) labeling was performed as described
(17). Briefly, mice received drinking water containing 0.8
mg/ml BrdU (Sigma, St. Louis, MO) prepared fresh every other day for 9
days. Mice were then sacrificed, and the splenocytes were surface
stained for CD3, CD4, CD8, and CD44 expression. The cells were then
fixed, permeabilized, and intracellularly stained with FITC-conjugated
anti-BrdU (BD Biosciences, Mountain View, CA). Data were acquired
on a FACSort and analyzed with CellQuest software (BD Biosciences).
Propidium iodide was used to exclude dead cells.
Serology
Serum levels of polyclonal IgG, anti-dsDNA, anti-chromatin, and IgM-RF were determined by ELISA. Wells were coated with either Fc-specific F(ab')2 of goat anti-mouse IgG (3 µg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA), mouse chromatin (3.5 µg/ml, a gift from Dr. R. Rubin, The Scripps Research Institute, La Jolla, CA). or, after overnight precoating with poly-L-lysine, dsDNA (3.5 µg/ml; Sigma). Bound serum total IgG and IgG subclasses were measured using alkaline phosphatase-conjugated goat anti-mouse IgG and subclass-specific Abs (Caltag Laboratories, Burlingame, CA). Serum IgM-RF levels were determined by adding diluted sera to wells coated with purified IgG subclasses (3 µg/ml; all obtained from Caltag Laboratories, except IgG2a, which was purchased from BD PharMingen) and then developing with alkaline phosphatase-conjugated goat anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL). Ab concentrations were determined by comparison to standard Ig-calibrated mouse sera (The Binding Site Limited, Birmingham, U.K.).
Immunohistology
Detecting Abs included FITC-conjugated anti-mouse IgG (Vector Laboratories, Burlingame, CA), biotinylated anti-mouse CD3 (BD PharMingen), and biotinylated F4/80 (Caltag Laboratories). Standard immunohistologic staining procedures were used. Briefly, OCT-embedded, snap-frozen kidneys were sectioned, air-dried, and acetone-fixed. Nonspecific background was reduced by an avidin/biotin blocking kit (Vector Laboratories) and incubated with 10% goat or rabbit serum in PBS. Sections stained with FITC-conjugated Abs were directly examined, whereas sections stained with biotinylated Abs were incubated with streptavidin-HRP, developed with aminoethyl carbazole (Vector Laboratories), and counterstained with Mayer’s hematoxylin. Sections were graded blindly on a 1–4 scale.
CDR3 analyses
DNA from spleens of 4.5-mo-old N5 mice was prepared as
previously described (18). VHJ558
rearrangements were amplified using the VH primer AF326
(5'-GGGGCTTAAGCTTAGCTGTCCAGCAAGGCT) with the previously described
JH primers (JH1 + 4, JH2, and
JH3) (19). Amplified products from three
independent PCRs for each group of mice were cloned and sequenced as
previously described (19). RNA was prepared with TRIzol
from sorted B220+ DN T cells from LNs of 5-mo-old
N5 mice, and from CD4+ spleen cells from 4-mo-old
N5 mice. Rearranged V8 transcripts were amplified with a V8 and a
C primer set as previously described (20). Amplified
products from three to four independent PCRs were cloned and sequenced
from each group of mice.
Statistics
Survival was analyzed by Kaplan-Meier statistic using censored events with significance determined by log-rank (Mantel-Cox) test. The unpaired t test, Mann-Whitney U test, or Fisher exact test were used to compare groups as indicated. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TdT+/+ and TdT-/-
MRL-Faslpr mice were produced by
intercrossing
TdT+Fas+/TdT-Faslpr
heterozygous backcross mice. The first group of intercross mice,
consisting of 13 N2 and 25 N3 mice, were analyzed for survival up until
6 mo, when the survivors were sacrificed. Despite the limited
backcrossing, the median survival of TdT wild-type mice (5.6 mo) was
similar to that of our MRL-Faslpr colony
(5.5 mo) (Fig. 1
). In contrast,
TdT-deficient mice had increased survival with 82.3% (4 of 5 N2 and 10
of 12 N3 mice) alive at 6 mo compared with only 47.6% (4 of 8 N2 and 6
of 13 N3 mice) of TdT+/+ mice
(p = 0.04) (Fig. 1). An additional 10 mice were
followed until 7.5 mo of age. At that time, 5 of 5 of the
TdT-/- mice were still alive, whereas only 2 of
5 of the TdT+/+ mice were alive. Thus, TdT
deficiency greatly increases the lifespan of
MRL-Faslpr mice.
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One striking macroscopic finding was a marked reduction in the
incidence of skin lesions in the TdT-deficient
MRL-Faslpr mice. Of the N3 mice, only 1 of
9 TdT-/-
MRL-Faslpr mice had skin lesions vs 4/5 of
TdT+/+ MRL-Faslpr
mice at 6 mo of age, when the mice were sacrificed for histologic
examination. An additional 19 N5 intercross mice that were 6 mo old
were also examined for skin lesions, and 60% of the
TdT+/+ mice but only 22% of the
TdT-deficient mice displayed skin lesions (Table I).
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and Fig. 2). Both mesangial and capillary wall
deposits were observed in both groups, although the amount of staining
was greatly reduced in the TdT-/- animals.
Despite the reduction in IgG deposits, no decrease in GN scores was
detected in the TdT-/- group (Table I). It
should be recalled that half of the TdT+/+ mice,
but few of the TdT-/- mice, had already died
before this 6-mo time point, and these mice presumably all had severe
GN. Thus, although TdT-/- mice have reduced
early mortality, a significant number develop some pathologic evidence
of glomerular injury by 6 mo of age.
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The point mutation in the Fas gene results in massive
lymphoaccumulation, and the presence of large numbers of T cells with
the unusual phenotype of
B220+CD4-CD8-
TCR+ (DN B220+ T
cells). MRL-Faslpr mice also develop
increases in memory/effector phenotype (CD44high)
T cells. The TdT-deficient MRL-Faslpr mice
displayed a 2-fold reduction in the percentage of
B220+ DN T cells in cervical LN and spleen as
compared with TdT wild-type littermates (p
< 0.002) (Fig. 3). As might be expected
with the decrease in B220+ DN cells, we also
observed a decrease in the lymphadenopathy, which is a hallmark of
MRL-Faslpr mice in
TdT-/- mice (Table II). B cells, CD8+,
and CD4+ cells were compensatorily increased in
proportion in spleen and LNs of TdT-deficient
MRL-Faslpr mice by 1.5- to 2.5-fold
(p = 0.009 and 0.017 for B cells;
p = 0.04 and 0.008 for CD8+
cells, p = 0.008 and 0.003 for
CD4+ cells). Activated
CD4+CD44high and
CD8+CD44high T cells were
not significantly reduced in either organ, but DN
CD44high T cells in both organs were modestly but
significantly reduced in the TdT-deficient animals as compared with
their TdT+/+ littermates
(p = 0.011 and 0.03).
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As observed in wild-type MRL-Faslpr
mice, the N3 generation TdT-expressing
MRL-Faslpr mice exhibited marked
hypergammaglobulinemia and elevated anti-chromatin and
anti-dsDNA responses, and produced significant amounts of IgM-RF
(21). In contrast, TdT-/-
MRL-Faslpr mice displayed a 57% reduction
in total IgG (143 ± 37 vs 62 ± 21 mg/ml) and an even
greater reduction of anti-chromatin Abs (151 ± 28 vs 52
± 24 ng/ml), anti-dsDNA Abs (68 ± 18 vs 24 ± 9 ng/ml),
and IgM-RF (1005 ± 241 vs 290 ± 77 ng/ml), compared with
their TdT wild-type littermates (Fig. 5).
In the case of the polyclonal IgG, the only significant reduction was
in the IgG2a subclass (p = 0.04), whereas for
the anti-dsDNA autoantibodies, a significant reduction was observed
in both IgG2a and IgG2b subclasses (p = 0.02),
and a reduction in IgG1 was also seen (p =
0.055). IgM-RF Abs reactive with all IgG subclasses were significantly
decreased (p values ranging from 0.05 to 0.0003).
Although the reduction in total levels of these specific autoantibodies
exceeded the reduction in total polyclonal IgG, most of the
autoantibodies and polyclonal Ig are of the IgG2a subclass, and all
IgG2a values were reduced 3-fold. Thus, the decrease in total IgG2a
could account for the decrease in IgG2a autoantibody titers, thus
leaving it unclear whether there was a specific decrease in
autoantibody levels in addition to the generalized decrease in
hypergammaglobulinemia.
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To analyze CDR3 diversity in VDJ rearrangements, we amplified
VHJ558 rearrangements from splenic DNA of 4.5-mo-old
littermate MRL-Faslpr
TdT-/- or TdT+/+ mice. As
expected, the CDR3 regions of Ig from the
TdT-/- mice were 1.1 aa shorter than those of
the TdT+/+ mice (Table III). Importantly, only one of the
38 IgH rearrangements from the MRL-Faslpr
TdT-/- mice had any arginines in CDR3, whereas
10/21 of the CDR3 regions from the
MRL-Faslpr TdT+/+
mice contained arginines, most of which were encoded by N regions.
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DN B220+ T cells have been shown to be
enriched in V8.3-expresssing TCR (22, 23, 24). Because the
DN B220+ T cells are reduced in the
TdT-/- MRL-Faslpr
mice, we stained LN cells of 5-mo-old littermate mice with mAbs that
react with V8.1 + V8.2, or that react with
V8.3+. Cells were also stained with
anti-CD3, anti-CD4, and anti-CD8, and were analyzed by
FACS. We observed that DN B220+ T cells are
similarly enriched in V8.3+ cells in the
TdT-deficient mice as well as the TdT+/+
MRL-Faslpr mice (data not shown).
This enrichment in V8.3+ T cells was also
observed in sequences derived from RNA from DN
B220+ T cells from these same LN preparations.
TCR sequences were amplified with a V8 primer that equally amplifies
all three V8 genes, and both the TdT+/+ and
TdT-/- mice showed 55% representation of
V8.3 sequences among the total V8 sequences (Table IV). As previously observed (22, 23), a wide variety of J sequences were used. The CDR3
regions are an average of 0.8 aa shorter in length in the
TdT-/- mice (Table IV). Thus, despite a 30%
reduction in the number of DN T cells in cervical LNs, the DN
B220+ T cells that are present display the same
enrichment for V8.3 expression. Because they contain shorter CDR3
regions and use a variety of J regions, this suggests that the CDR3
region of this unique subpopulation of cells is not as critical as the
CDR1 or CDR2 regions of V8.3 for recognition of their yet
undescribed Ag(s), and thus may suggest an indirect rather than a
direct effect of TdT deficiency on the reduced percentage of this
subpopulation in the TdT-/- mice.
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rearrangements
RNA from splenic CD4+ T cells was amplified with V8
and C primers, and rearranged TCR were sequenced. The average
CDR3 length is 1.4 aa shorter in the TdT-/-
mice (Table V). Most of these
sequences contained the acidic amino acids glutamic acid or
aspartic acid, which have been associated with the CDR3 regions of T
cells that can provide help for an anti-dsDNA response
(25). However, the majority of these acidic amino acids
are encoded by the V8 or the J region, and there is only a slight
increase in the average number of acidic residues in CDR3 from the
TdT+/+ mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thus, given that the (NZB x NZW)F1 TdT-/- mice had a much milder course of disease, it was surprising that they produced similar levels of anti-dsDNA to their wild-type counterparts. This suggested that some specificity other than anti-dsDNA autoantibodies was being affected by the lack of N regions. Therefore, we investigated the effect of TdT deficiency on several aspects of autoimmune disease in another lupus-prone strain of mice, MRL-Faslpr, to determine whether we would obtain similar disease amelioration yet a lack of decrease in anti-dsDNA titers. We show here that in MRL-Faslpr mice also, the onset of autoimmunity is delayed, and we observed a significant increase in longevity. However, in contrast to the (NZB x NZW)F1 mice, we observed a significant decrease in circulating anti-dsDNA. Also, we observed a significant decrease in IgG deposits in the kidney of the MRL-Faslpr TdT-/- mice. The decrease in circulating anti-dsDNA levels is likely to be responsible for the decrease in IgG deposits in the kidney. In addition, we also observed a significant reduction in the generalized hypergammaglobulinemia characteristic of MRL-Faslpr mice, and also a decrease in RF levels. However, we did observe some renal damage at the 6-mo time point that we studied, so the disease was likely delayed, not eliminated. The incidence of skin lesions, which are characteristic of human lupus and are also present in MRL-Faslpr mice (21), was also reduced in the absence of TdT. Thus, the effect of TdT deficiency in both murine models was to slow down the course of disease, but the manner in which it affected various disease manifestations in both strains of mice is different.
Because TdT is expressed only in developing T and B cells, and its function is to add N region diversity to V-D-J junctions in CDR3, the decrease in pathology in the MRL-Faslpr TdT-/- mice must be due to a repertoire change in either B or T cells, or both. An obvious candidate is the anti-dsDNA B cell precursors. Not all anti-dsDNA Abs contain arginines, so this is not an absolute requirement, but arginines are far more prevalent in these Abs than in most other Abs (14, 31). Arginines in CDR1 and CDR2 arise predominantly by somatic hypermutation and thus would not be affected by the absence of TdT. However, the arginines in CDR3 can also arise by N region addition or the use of alternative reading frames for the D regions (14). For Abs, the majority of DH regions are used in reading frame 1 in the absence of N regions, due to homology-directed recombination (4). However, the addition of N regions precludes homology-directed recombination, and thus the other two reading frames are observed at higher frequencies than in the fetal repertoire. The amino acids encoded by the three different reading frames are very different, with reading frame 1 not encoding any arginines (except for DSP2.10), whereas the other reading frames do. Thus, TdT has a direct influence on the amino acid composition of CDR3, both in the N segment itself, and for the reading frame of the D segment.
These mechanisms, which act during the process of V(D)J recombination, could potentially increase the affinity of the primary repertoire of anti-dsDNA, and thus provide a precursor pool with receptors enriched in arginines, and upon which somatic hypermutation could exert additional affinity and subsequent selection. An important issue is what determines the ability of a naive B cell to have some minimal affinity for dsDNA, sufficient to be subsequently triggered by Ag, and undergo somatic hypermutation and become a high-affinity anti-dsDNA B cell. Twenty percent of all anti-dsDNA from MRL-Faslpr or (NZB x NZW)F1 mice are encoded by one VHJ558 gene, 3H9 (32). Almost all pathogenic high-affinity anti-dsDNA Abs are somatically mutated, as is the VH region in the 3H9 hybridoma. When a more germline 3H9 Ab was created by reversion of the arginines generated by somatic mutation, a low level of DNA binding was still present (33). However, this residual binding was abolished by the elimination of the arginine at the V-D junction, which was N region encoded (33). Thus, we propose that the B cell precursors using this VH gene, and most likely other VH genes also, will benefit by the presence of N region-produced arginines in CDR3 to have a reasonable chance to have enough affinity for DNA to be initially stimulated. However, when stimulated, somatic hypermutation plays a major role in generating high-affinity pathogenic anti-dsDNA Abs.
However, the decrease in dsDNA B cell precursors is clearly unable to
explain all of the observations that we have made, because
MRL-Faslpr TdT-/-
mice also have decreased generalized hypergammaglobulinemia, decreased
RF titers, decreased lymphoid hyperplasia, decreased skin disease, and
decreased numbers of DN B220+ T cells. The
reduction in anti-chromatin titers may reflect the decrease in
anti-dsDNA titers, because the target Ag in chromatin is DNA in
combination with histones. However, the other parameters of disease
that are ameliorated are unlikely to be due to any potential decrease
in anti-dsDNA precursors. RF are present in some lupus patients,
and Faslpr-homozygous mice are one of the
few mouse models that recapitulate this feature of human systemic lupus
erythematosus. However, not much is known about the Ig structural
requirements of RF, and there is no known association between N regions
in H chain CDR3 and RF Abs. RF from patients with rheumatoid arthritis
have 
-chains that have longer than normal CDR3 regions due to N
region addition (28, 29). However, because mice, in
contrast to humans, have very few N region nucleotides in their light
chain junctions (34), the lack of TdT cannot affect L
chain CDR3 in the MRL-Faslpr
TdT-/- mice. One study showed that the H chain
CDR3 of an RF Ab was critical for RF binding by exchanging CDR3s
(35). However, in contrast to the light chain
junctions, the IgH-CDR3 associated with RF was the shorter of the two
CDR3s.
The effects of the TdT deficiency cover both a general reduction in hypergammaglobulinemia, and a decrease in the levels of autoantibodies characteristic of this disease. It may be that the decrease in levels of these autoantibodies is due to a reduction in the polyreactive, rather than monoreactive, Abs, as observed in the B6 TdT-deficient mice (12). Because the IgG2a anti-DNA Abs are decreased by approximately the same extent as the total IgG2a, it is not clear whether the anti-dsDNA levels are specifically reduced. In this regard, it may be that the absence of N regions in T cells that provide help for B cells causes a generalized decrease in the production of all serum Abs.
The autoantibody profile of the MRL-Faslpr
TdT-/- mice, although unlike that of the
(NZB x NZW)F1
TdT-/- mice, was more similar to that of
TdT-deficient B6-Faslpr mice
(36). In that study, which was published while we were
analyzing our mice, B6-Faslpr mice were
made TdT deficient by backcrossing to TdT-/-
mice, and the mice showed reduced levels of anti-ssDNA as compared
with their TdT+/+ counterparts. However,
B6-Faslpr mice do not make significant
levels of anti-dsDNA Abs, which are the only pathogenic
anti-DNA Abs, nor do they develop kidney or skin disease; MRL
background genes are necessary for pathology. Splenic B cells from the
B6-Faslpr TdT+/+
mice displayed a higher incidence of arginines in the IgH CDR3 regions
as compared with the B6-Faslpr
TdT-/- mice, as has been shown previously for
TdT+ vs TdT- repertoires
of normal mice. We found an even more pronounced difference in the
frequency of arginines in the MRL-Faslpr
TdT+/+ vs TdT-/- mice
(Table IV). Similar to our finding that
MRL-Faslpr TdT-/-
mice also had reduced levels of anti-chromatin Abs and IgM-RF
compared with their wild-type littermates, the
B6-Faslpr TdT-/-
mice were shown to have reduced titers of anti-histone components
and of IgM-RF and IgG-RF. However, a significant difference is that the
B6-Faslpr mice did not show any decrease
in their more modest hypergammaglobulinemia in the absence of TdT,
whereas we observed an over 2-fold reduction in the massive
hypergammaglobulinemia in the MRL-Faslpr
TdT-/- mice (36). Thus,
autoantibody production and RF titers are decreased in both the
autoimmune and nonautoimmune TdT-deficient mouse strains carrying the
defective Faslpr allele, but are not
decreased in the (NZB x NZW)F1 autoimmune
strain of mice.
In addition to effects on the primary B cell repertoire, T cells could
also be affected by the absence of TdT, and may be responsible for some
of the effects that were observed in these
MRL-Faslpr mice. T cell help is required
for the somatic hypermutation and affinity maturation of high-affinity
IgG anti-dsDNA Abs, and the production of mutated high-affinity Abs
may be an initiating event in the pathogenesis of lupus. The
specificity of the T cells that provide help for the anti-dsDNA B
cells in vivo is not certain, but there is evidence that these T cells
may be specific for basic DNA binding proteins such as histones. One
study isolated T cells from (SWR x NZB)F1
mice that helped the production of anti-dsDNA Abs in vitro
(25). Another group studied T cells specific for a
DNA-binding basic peptide Fus1 (37). The T cells from
both studies showed V skewing and overrepresentation of
V8. TCR V8 genes have a germline-encoded aspartic acid at
position 99 that is often retained in CDR3, and V8 are
overrepresented in these clones. The CDR3 regions all had at least one
acidic amino acid, some of which were generated by N nucleotides. Thus,
these two studies would suggest that the T cells that may be required
for providing T cell help in the production of pathogenic
anti-dsDNA Abs in vivo might be decreased in relative frequency in
N region-lacking populations. A different type of candidate Ag for the
T cells required for lupus induction is peptides from VH
genes used in anti-dsDNA autoantibodies. Because B cells present
peptides from their own Ig molecules, T cells specific for Ig peptides
should be able to help B cells using the VH gene in its
BCR. Many peptides from the CDR3 region of anti-dsDNA Abs have been
shown to elicit proliferative responses from autoimmune T cells, and
treatment of mice with these peptides can delay or accelerate the onset
of disease (38, 39). These peptides are enriched in
charged amino acids (40). The structural characteristics
of such TCR is unknown, but may be presumed to have complementary
charged amino acids. Thus, these T cells also may be decreased in
MRL-Faslpr TdT-deficient mice.
We amplified V8 sequences from CD4+ T cells
and observed that both TdT-deficient and TdT-containing
CD4+ T cells had a high frequency of aspartic
acids or glutamic acids in CDR3 (Table V). The majority of acidic
residues in all cases were derived from the V or J sequences,
although some of the acidic residues in the
TdT+/+ mice were N region encoded, and we did
note a slight decrease in the frequency of acidic residues in CDR3 of
CD4+ T cells derived from TdT-deficient
MRL-Faslpr mice.
These MRL-Faslpr TdT-deficient mice also
showed a decrease in the incidence of skin lesions. Many cell types
have been implicated in the production of skin lesions. Some studies
have shown that IgG3 cryoglobulins from
MRL-Faslpr mice, but not IgG3 Abs lacking
this property, are capable of inducing skin lesions in mice
(41). T cells are also clearly involved in the development
of skin lesions. MRL-Faslpr mice that lack
T cells displayed delayed onset of skin lesions, whereas mice
deficient in both TCR and CD40 ligand (CD40L) developed a
similar slow course of pathology as mice deficient only in T
cells, except that they did not develop skin disease at all
(42). In contrast, CD40L deficiency alone led to less
severe renal disease and less autoantibody production, but those mice
had equivalent skin pathology to the wild-type
MRL-Faslpr. Thus, T cells may be
involved in the development of skin disease by a CD40L-independent
pathway, and CD40L interactions must also influence the development of
skin disease in a non- T cell pathway. How the lack of N regions
affects these cells is not known.
MRL-Faslpr mice that lack CD4 T cells show greatly reduced autoantibodies and reduced incidence of GN (43). Because we observed a decrease in autoantibodies as well as a decrease in generalized hypergammaglobulinemia, we suggest that the lack of N regions is also affecting the CD4 T cells, which are required for the production of these autoantibodies, as well as for the somatic hypermutation required to generate high affinity, presumably pathogenic, anti-dsDNA Abs. Experiments are currently in progress to determine whether it is the T cell or B cell compartment, or both, which is critically affected by the absence of N regions, and which results in the increased lifespan and other aspects of disease amelioration in these TdT-deficient lupus-prone mice.
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Acknowledgments |
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Footnotes |
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2 This is manuscript 14132-IMM from The Scripps Research Institute.
3 Address correspondence and reprint requests to Dr. Ann J. Feeney, Department of Immunology IMM-22, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: feeney{at}scripps.edu
4 Abbreviations used in this paper: TdT, terminal deoxynucleotidyl transferase; GN, glomerulonephritis; RF, rheumatoid factor; DN, CD4-CD8- double negative; BrdU, 5-bromo-2'-deoxyuridine; CDR3, complementarity-determining region 3; MRL-Faslpr, MRL/lpr, Fas-deficient; CD40L, CD40 ligand; LN, lymph node.
Received for publication May 17, 2001. Accepted for publication July 11, 2001.
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