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Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Strasbourg, France
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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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The presence of pathogenic autoantibody-inducing Th cells specific for chromatin subparticles or histones has also been demonstrated in human lupus (12, 13, 14). In these studies, histone H4 was again found to contain epitope(s) recognized by pathogenic autoantibody-inducing Th cells. Lu et al. (14) characterized several autoepitopes in the four core histones, and remarkably some of them overlap major autoepitopes identified in SNF1 mice. Two additional Th epitopes were identified in residues 34–48 of H2A and 49–63 of H4. Among histone subtypes, H4 is the most highly conserved through evolution, but is also the site of many posttranslational modifications (8). The observation that, within the nucleosome structure, H4 seems to play an important role at the T cell level in human and murine lupus is an intriguing feature. To further address this question, we studied the ability of H4 as well as that of overlapping peptides spanning the whole sequence of H4, to elicit Th and B cell responses in normal, non-SLE-prone mice. We also tested in vitro the capacity of H4 and H4 peptides to stimulate Th cells primed in vivo with purified nucleosomes and examined their cytokine profiles. The overall results indicate that in the normal repertoire of BALB/c H-2d mice, autoreactive Th cells specific for histones are not deleted and that in terms of specificity, the reactivity of these Th cells seems to be relatively restricted and resembles that of Th clones generated from SNF1 lupus mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histone peptides (calf thymus sequence) were synthesized using classical Fmoc (N-[9-fluorenyl] methoxycarbonyl) solid-phase chemistry (15). Each peptide was purified by reversed-phase HPLC using a Perkin-Elmer (Roissy, France) preparative HPLC system on an aquapore ODS 20-µm column (10 x 100 mm). The elution was achieved by a linear gradient of aqueous 0.1% trifluoroacetic acid (TFA; solvent A) and 0.08% TFA in 80% acetonitrile-20% water (solvent B) at a flow rate of 6 ml/min with UV detection at 220 nm. The homogeneity of each peptide was checked by analytical HPLC on a nucleosil C18, 5-µm column (4.6 x 150 mm), using a linear gradient of 0.1% TFA in water and acetonitrile containing 0.08% TFA. The identity of purified peptides was assessed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry using a protein TOF apparatus (Bruker Spectrospin, Bremen, Germany).
Histone H4 and (H3-H4)2 tetramer were prepared
from calf thymus and purified, as described previously
(16). There is no change in the primary structure of calf
and mouse core histones. Some experiments were also performed with
commercial histone preparations purchased from Roche (Mannheim,
Germany) and Sigma (St. Louis, MO). The homogeneity of each histone
fraction was checked by 18% SDS-PAGE. Nucleosomes were prepared from
calf thymus, as described previously (17), and purified on
a 5–29% (w/v) sucrose gradient. The nucleosome preparations were
characterized by 1.5% agarose gel electrophoresis, and the content in
histones was checked by 18% SDS-PAGE. An example of preparation is
shown in Fig. 1
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Female BALB/c and NZB/NZW F1 (B/W) mice were purchased from Janvier (Le Genest St. Isle, France) and Harlan (Gannat, France), respectively. For T cell experiments using BALB/c mice, two 8–10-wk-old mice/Ag/experiment were injected s.c. in hind footpads and at the base of the tail with 100 µg of peptide or histone diluted in H2O and mixed (v/v) with CFA. Alternatively, BALB/c mice were immunized with 10 µg (expressed in terms of histone content) of mono- or tetranucleosome in sucrose in the presence of CFA (v/v). To study the B cell response to H4 and nucleosome, BALB/c mice (two to three mice/Ag/experiment) were immunized, as described above, or s.c. in the flanks. Booster injections were performed in IFA on a fortnightly basis, alternatively with bleedings. A prebleeding of each mouse was performed and used as control in each assay. B/W mice, 8–10 wk old at the beginning of the experiments, as well as nonimmunized control BALB/c mice of the same age, were bled regularly during 40 wk.
Lymphocyte proliferation assay and measurement of cytokine secretion
In the case of BALB/c mice immunized with the different Ags, the
proliferation assay was essentially as described previously
(18). Ten days after immunization of mice, inguinal,
popliteal, and periaortic lymph nodes were removed and washed in RPMI
1640-Glutamax I (Life Technologies, Cergy-Pontoise, France) containing
10% FCS (DAP, Vogelgrun, France), 10 µg/ml gentamicin, 10 mM HEPES,
and 5 x 10-5 M 2-ME.
Lymph node cells (LNC) were resuspended at a concentration of 5 x
106 cells/ml in the same culture medium, and 100
µl of this suspension was added to microtiter wells (96-well
flat-bottom culture plates; Costar, Cambridge, MA) containing 100 µl
of medium with different concentrations of peptides or protein. Each
concentration was tested in triplicate, and tests were repeated at
least three times in independent experiments. The cells were cultured
at 37°C in 5.5% CO2. After 24 h, 50-µl
supernatant was taken off to test the production of IL-2 using CTL-L
cell (18). For the detection of other cytokines (IFN-
,
IL-4, IL-6, and IL-10), culture supernatants (50 µl) were collected
after 24–48 h and tested in ELISA, as described below. After 54
h, the cultures were pulsed during 18 h with tritiated thymidine
(methyl [3H]thymidine, 2% ethanol, 6.7
Ci/mmol, 1 µCi/well; ICN, Orsay, France). The cells were subsequently
harvested on filter with an automatic cell-harvesting device (Packard,
Meriden, CT), and DNA-incorporated radioactivity was measured using a
Matrix 9600 direct beta counter (Packard). The results are expressed as
the arithmetic mean of thymidine uptake expressed as cpm. Proliferative
responses were considered to be significantly positive when the
[3H]thymidine uptake was equal to or above
twice the uptake by LNC cultured in medium alone without Ag. The SD of
triplicate cultures was always below 20% of the mean. Control tests
were performed by adding Con A (100 µl/well; 0.2 and 2.5 µg/ml;
Sigma) to cells during the time (72 h) of the culture.
Some experiments were performed with purified CD4+ Th cells. These cells were purified using magnetic beads coated with anti-CD4 mAb (Dynabeads; Dynal, Oslo, Norway). Briefly, 40 x 106 LNC were incubated with 8 x 107 magnetic beads for 45 min at 4°C. After four washings and recovery of the negative population, beads were treated with a Detach bead Ab (Dynal) for 45 min at room temperature (RT), and the CD4+ population was harvested. The purity of the two populations was assessed by immunofluorescence analysis on a FACScalibur apparatus (Becton Dickinson, Mountain View, CA) using a PE-labeled anti-CD4 mAb (PharMingen, San Diego, CA). In the proliferation assay, 2.5 x 105 purified CD4+ T cells were cultured with 5 x 104 mitomycin C-treated autologous spleen cells as APCs in the presence of peptide, Con A, or medium alone. As an additional control, treated APCs were cultured alone in the presence of the peptide. IL-2 secretion was evaluated after 24 h, and cell proliferation was measured after 72 h, as described above.
ELISA for cytokine detection
IFN-, IL-4, IL-6, and IL-10 secretion was evaluated by
sandwich ELISA using commercial Abs from PharMingen and polyvinyl
plates (Falcon, Oxnard, CA; reference 3912). Standard curves performed
with known concentrations of recombinant cytokines (PharMingen) were
used for the test calibration. All steps were performed according to
manufacturer’s recommendations. In these conditions, the minimal
levels of detectable cytokine were 3 U/ml IFN-, 0.5 U/ml IL-4, 0.3
U/ml IL-6, and 5 U/ml IL-10.
ELISA and Western immunoblotting for Ab detection
For the test of mouse sera, polyvinyl microtiter plates (Falcon)
were coated overnight at 37°C with 1 µM histone peptide or 800
ng/ml purified histone diluted in 0.05 M carbonate buffer, pH 9.6. In
each assay, sera were also tested in a noncoated well incubated with
coating buffer alone as a control. Abs raised in rabbits against each
of histone peptides tested in this study (17) were used to
check that plates were satisfactorily coated with each peptide. The
subsequent steps of the test were performed, as described previously
(17), using mouse sera at different dilutions in PBS
containing 0.05% Tween and 0.5% BSA (PBS-T-BSA) and either
HRP-conjugated goat anti-mouse IgG diluted 1/15,000 or
HRP-conjugated goat anti-rabbit IgG diluted 1/40,000. The cutoff
points of each assay were determined using the sera from twelve
8-wk-old female BALB/c mice. Mouse sera were considered positive when
the OD values were higher than the mean OD value + 2 SD, i.e., OD
values 
0.1 in the case of all H4 peptides except 1–29, 28–42,
30–47, and 42–59 (calculated cutoff = 0.2), and 0.2 in the
case of histones except H3 (calculated cutoff = 0.1).
The binding of Abs to H4 was also tested by Western immunoblotting using calf thymus H4 (200 ng/lane) subjected to an 18% SDS-PAGE for 1 h at 200 V and then electrophoretically transferred to 0.2-µm nitrocellulose for 20 min at 15 V and 400 mA. The blotted strips were saturated for 1 h at RT in 0.5 M Tris buffer saline, pH 8, containing 0.05% Tween-20 (TBS-T) and 5% nonfat milk, and then incubated for 2 h at RT with mouse or rabbit antisera diluted 1/500 in TBS-T containing 5% milk. After washing, the membranes were incubated with HRP-conjugated goat anti-mouse IgG Abs (diluted 1/10,000 in TBS-T) or goat anti-rabbit IgG Abs (diluted 1/5,000). Enhanced chemiluminescent (ECL) reagents (Amersham Pharmacia Biotech, Buckinghamshire, U.K.; reference RPN 2109) were used to reveal positive reactions.
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Results |
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A panel of nine 15- to 29-mer overlapping H4 peptides spanning the
entire sequence of H4 was used to map T epitopes of H4. Their
composition is shown in Table I. In a
first set of experiments, we either immunized female BALB/c mice (8–10
wk old) with the nine H4 peptides (two mice/peptide) in CFA or injected
CFA without peptide as control. After 10 days, we removed the draining
lymph nodes and tested each suspension with the original priming
peptide for its ability to induce cell proliferation ex vivo. No
proliferation and IL-2 secretion were observed when any of the H4
peptides was added to cultures containing T lymphocytes removed from
control mice injected with CFA alone. However, proliferative responses
with IL-2 secretion in varying magnitudes were reproducibly found with
several peptides when they were added to cultures containing T
lymphocytes removed from immunized mice (Fig. 2, A and B). The
strongest responses were systematically measured with the C-terminal
peptides 72–89 and 85–102, while relatively weak or no reactivity was
found with the N-terminal peptides 1–29, 18–34, and 42–59 (Figs. 2 and 3A).
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). No
proliferation and IL-2 secretion were observed when peptide 81–92,
which overlaps the two positive peptides 72–89 and 85–99, was added
to cultures containing T lymphocytes removed from mice immunized with
the same homologous peptide. However, three peptides were recognized by
T lymphocytes from BALB/c mice primed with the homologous peptides,
namely peptides 85–99, 88–99, and 88–101 (Fig. 3A).
When instead of homologous peptides, the whole histone H4 was added to
cultures containing T lymphocytes removed from mice injected with the
13 peptides, very weak proliferative response and IL-2 production were
observed. The highest levels of IL-2 secretion were reproducibly
observed in the case of LNC primed in vivo after immunization with H4
peptides 30–47 and 88–99 (Fig. 3B).
To further study the T cell response to H4, BALB/c mice were immunized
with purified histone H4, and we tested the ability of LNC to
proliferate ex vivo in the presence of the same protein or H4 peptides.
No proliferation and IL-2 secretion were observed with any of the 13 H4
peptides. However, H4-primed LNC responded well to H4; this response
was specific and histone dose dependent (Fig. 2, C and
D).
The H4 peptide 88–99 is recognized by nucleosome-primed T cells from BALB/c mice and corresponds to a dominant Th epitope
Although we found that ex vivo H4 peptides failed to
stimulate T cell response primed in vivo with H4, we then examined
whether these peptides may induce the proliferation of
nucleosome-primed T cells. We first determined whether in nonautoimmune
BALB/c mice, nucleosomes could elicit an efficient Th cell response.
BALB/c mice were primed s.c. with purified calf thymus mono- and
tetranucleosomes in CFA. After 10 days, draining lymph nodes were
removed and LNC were cultured in the presence of nucleosome and
purified histones. As above, each suspension was tested for its ability
to proliferate and produce IL-2 ex vivo. No proliferation and IL-2
secretion were observed when purified mono- or tetranucleosomes were
added to cultures. However, a significant proliferative response with
IL-2 secretion was reproducibly observed with H4 as well as with the
(H3-H4)2 tetramer (Fig. 2, E and
F). No or nonsignificant response was found with purified
H2A, which presents at its N terminus a sequence of eight residues that
are identical to those located in the N terminus of H4. These data thus
show that H4 and the (H3-H4) tetramer, but not nucleosomes and isolated
H2A, can be efficiently processed and presented by APCs from
H-2d BALB/c mice and are recognized in this
context by T cells primed in vivo with purified nucleosomes.
We next tested the 13 partially overlapping H4 peptides with
nucleosome-primed T cells. It appeared that of 13 H4 peptides, peptide
88–99, and at a lower extent peptide 28–42, induced a significant and
peptide dose-dependent IL-2 secretion (Fig. 4). Although cell proliferation was
systematically weak with SI about 1.5, IL-2 production was repetitively
found in independent experiments, indicating that with regard to
nucleosome, the Th cell epitope in peptide 88–99 is dominant.
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The immune response against peptide 88–99 of H4, which
constitutes a dominant Th cell epitope of nucleosome in normal BALB/c
mice, was studied in more detail. We first confirmed that
CD4+ cells were effectively engaged in this
response. LNC from mice primed with peptide 88–99 were separated using
CD4-conjugated magnetic beads, and the purity of the
CD4+ and CD4- populations
was assessed by immunofluorescence analysis. The purified cell
population obtained by positive selection contained 92.9%
CD4+ cells, whereas the negative population
contained 1.7% CD4+ cells. Purified cells were
cultured with mitomycin C-treated APCs from autologous spleen in the
presence of peptide 88–99. CD4+ cells showed a
significant proliferation with IL-2 secretion (Fig. 5, A and B), whereas
no response was observed with either CD4- cells
or mitomycin-treated APCs incubated with peptide without
CD4+/- T cells.
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, C–F). Culture supernatants were collected after 24 and
48 h, and cytokine secretion was evaluated by ELISA. In addition
to IL-2 secretion evaluated using the CTL-L cell line (results
described above), a significant and peptide dose-dependent IFN-
secretion was induced in response to peptide 88–99 added ex vivo to
the LNC cultures (Fig. 5C). A basal production of IL-10 was
found (Fig. 5F). However, no Ag dose-dependent IL-10, IL-4,
or IL-6 production was observed, suggesting that the Th cell response
to H4 peptide 88–99 is Th-1 dependent. Abs to peptide 88–99 react with H4, but this peptide does not contain a dominant B cell epitope recognized by anti-H4 Abs
To determine whether the H4 peptides that contain Th cell epitopes also contain B cell epitopes, BALB/c mice (three mice/peptide) were repetitively injected with six nonconjugated peptides covering the N- and C-terminal regions, namely peptides 1–29, 72–89, 85–99, 85–102, 88–99, and 88–101. Antisera were tested in ELISA for their reactivity with homologous or heterologous H4 peptides. No reactivity was observed in the sera from mice injected with CFA alone. Four s.c. injections in the presence of Freund’s adjuvant were necessary to raise detectable anti-peptide IgG Abs. Sera from mice immunized with peptides 72–89, 85–102, and 88–99 gave a strong homologous anti-peptide response, whereas a relatively weak reaction was detected in the sera from mice injected with peptide 85–99 and no reactivity was found in the sera from mice that received peptides 1–29 and 88–101. No phenomenon of spreading could be observed between nonrelated H4 peptides, for example between N- and C-terminal peptides. As far as the peptide 88–99 was concerned, the same results were obtained when mice were injected s.c. in the flanks or in footpads and the tail.
Peptide antisera were then tested in ELISA for their reactivity with histone H4. Antisera from mice immunized against unconjugated peptides 72–89, 85–99, and 85–102 (3/3 mice) and 88–99 (1/3 mice) strongly reacted with H4. The other sera were negative with H4. Similar results were found with histone H4 tested in Western immunoblotting. Antisera from mice immunized with peptides 72–89 (3/3 mice), 85–99 (1/3 mice), 85–102 (2/3 mice), and 88–99 (1/3 mice) possessed Abs reacting with blotted H4. Taken together, these results indicate that several sequences in the C-terminal region of H4 covered by peptides 72–89, 85–99, 85–102, and 88–99 are located at the surface of H4. The surface accessibility of the region 85–102 of H4 at the surface of the free nucleosome in solution was previously demonstrated (9).
The panel of peptides was also used to map B cell epitopes on H4 free
or associated to nucleosome. No reactivity with H4 and H4 peptides was
detected in ELISA with sera from three BALB/c mice that received five
injections of nucleosome or H4-RNA complex in the presence of CFA/IFA
or CFA alone. Antisera from two of three mice injected with non-RNA
complexed H4 reacted relatively weakly (OD value 
0.4) with H4 in
ELISA. The serum from one of these mice cross-reacted strongly with
histones H2A, H2B, and H3; the second positive serum recognized H4 only
and not the other core histones. These three sera were tested with the
panel of 13 H4 peptides. Three peptides, namely peptides 1–29, 28–42,
and 30–47, all located in the N terminus half of H4, were found
positive with the serum, which also showed the initial highest
reactivity with H4 (Fig. 6A).
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We tested the sera from fourteen 36–38-wk-old
H-2d/z B/W lupus mice. All sera except one showed
a strong IgG reactivity (0.8<OD<3) with dsDNA in ELISA. Most of them
(12/14) also reacted with H2A, H2B, and H3, and 6 reacted with H4 in
ELISA. In Western immunoblotting, 10/14 sera reacted with histone H4.
The 14 sera were tested in ELISA with the panel of H4 peptides, and it
was found that 7 reacted with peptide 1–29, 6 with peptide 28–42, 2
with peptide 30–47, and one with peptides 18–34, 42–59, and 88–101
(results shown in individual mice in Fig. 6B). No reaction
was found with the other H4 peptides including peptide 88–99, and no
reactivity with any of these peptides or histones was detected in the
sera from four BALB/c mice of the same age collected in parallel (Fig. 6B). Thus, although this study has no statistical value, it
is interesting to note that the reactivity of B/W mice resembles that
of the H4-positive BALB/c mouse immunized against H4 (Fig. 6A) with apparently a dominant specificity for epitopes
located in the N terminus of H4 and not in the C terminus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (10). Certain of
these T cell epitopes, namely those located in the N terminus of H2B
and H4, overlap domains that are also often targeted by lupus Abs from
mice and humans (7, 8, 17, 19). It is well established that healthy humans and mice have CD4+ T cell specific for self components (20, 21). The relationship between the pool of those potentially autoimmune, inactive CD4+ cells and the activated pathogenic CD4+ cells involved in autoimmune diseases is not clear. It is not known whether they share similar properties or whether autoimmune T cells have unique characteristics, for example in terms of their fine specificity. In this study, we showed that immunization of normal BALB/c mice with several H4 peptides emulsified in Freund’s adjuvant induced a strong Th cell proliferative response that could be recalled by the homologous peptides. These peptides spanned residues 28–42 (and the overlapping peptide 30–47), 66–83, 72–89, and 85–102. Within the C-terminal region 85–102 of H4, a minimal T cell epitope containing residues 88–99 was characterized. Th cells primed in vivo by at least two of these peptides (30–47 and 88–99) responded ex vivo to the whole histone H4. When we explored the immunogenicity of H4, we found that immunization of BALB/c mice with autologous histone H4 generated a T cell response that could be recalled by H4, but not by any of the partially overlapping H4 peptides tested. Intact nucleosomes failed to prime T cells that could be activated ex vivo by the same nucleosome. It has been similarly observed that nonautoimmune strains of mice failed to mount a T cell response to immunization with autologous purified small nuclear ribonucleoproteins (22, 23). However, CD4+ T cells generated after immunization of mice against nucleosomes proliferated and secreted IL-2 ex vivo in the presence of autologous H4, (H3-H4)2 tetramer, and particularly one of the 13 overlapping H4 peptides tested, namely peptide 88–99. According to the criteria of Sercarz et al. (24), H4 peptide 88–99 represents a cryptic epitope with regard to H4 and an immunodominant epitope presented by nucleosome, which forms a supramolecular complex. We might call it a supradominant epitope. This result is important if we consider the apparent key role of the nucleosome in lupus. It is known that the initial form in which an Ag is delivered can influence the APC type involved in T cell priming (25). It is possible that using different cell surface receptors, H4 and H4 bound to nucleosomes are not delivered to the same APC subsets and therefore are not internalized, processed, and presented with the same efficacy. It is also likely that even though the same APCs are engaged, important differences exist regarding the processing of individual histones and histones bound to nucleosomes. Regions of histones in contact with DNA within the nucleosome are most probably protected from autoantigen processing and should be presented preferentially to Th cells.
The most prominent of our findings was the discovery in normal mice of a supradominant epitope (H488–99) recognized by nucleosome-specific T cells. At this stage, we do not know whether these T cells can help B cells from autoimmune mice. On the other hand, of course, we realize that our failure to detect epitopes recognized by H4-specific Th cells or additional epitopes recognized by nucleosome-specific T cells might be the result of the insensitivity of our method. Other studies with normal mice have identified Th cell epitopes of proteins that are often targeted by lupus Abs (23, 26, 27). For example, Reynolds et al. studied the T cell response to murine and human La in normal mice (26). No autologous T epitope could be identified when mice were immunized with murine La and the T cells recalled with peptides corresponding to the murine sequence. However, multiple xenogeneic T epitopes (containing one or a few amino acid exchanges between human and mouse) were identified in murine La after immunizing mice with the human recombinant protein. Deshmukh et al. (27) recently identified several Ro60 Th epitopes in non-SLE-prone mice. Three peptides spanning residues 121–140, 281–300, and 311–330 were able to recall the proliferative response in SJL/J mice after immunization of mice with the mouse rRo60 protein. It is not known whether Ro particle-specific T cells react with any of these peptides.
Peptides 28–42 and 88–99 recognized by nucleosome-specific T cells
partially overlap the promiscuous epitopes 16–39 and 71–94
characterized in SNF1
H-2d/q mice and lupus patients (6, 14) (Fig. 7). In addition to these
two peptides, the sequences that are the most strongly recognized by T
cells generated against H4 peptides contain residues 30–47, 66–83,
and 72–89. It is noticeable that these Th epitopes also overlap
partially most of the H4 Th epitopes recognized by nucleosome-specific
T cells from lupus patients (14) (Fig. 7). From these
observations, we can conclude first that in the normal repertoire of
BALB/c H-2d mice, autoreactive Th cells specific
for histones are not deleted, and second, that in terms of specificity,
the reactivity of these Th cells is relatively restricted and resembles
that of Th clones generated from SNF1 lupus mice
previously described by Datta and collaborators.
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The observation that certain histone peptides can be promiscuously presented and recognized in the context of diverse MHC alleles and are recognized by autoimmune T cells from lupus mice and patients is of considerable importance for developing therapeutic strategies in humans despite their HLA diversities (29). To this regard, it is remarkable that after immunization with nucleosomes or histone peptides in Freund’s adjuvant that induces the costimulatory APC functions that are necessary for T cell priming and mimics a transient inflammation state, the specificity of Th cells from normal mice resembles that of Th clones generated from SNF1 lupus mice. This might indicate that in lupus, the multiple functional defects among the cells of the immune system and their signaling (30, 31, 32, 33) do not directly affect the nature of nucleosomal peptides that are presented by MHC molecules and recognized by the TCRs. It is obviously also possible that still unknown discrete neo-determinants for T cells are created and contribute to the breakdown of peripheral tolerance to nucleosomal proteins. Such possible determinants may be located in any of the histones and, as suggested in several studies (27, 29, 34, 35), a deleterious T cell activation and a diversified T and B cell response could occur via an intramolecular spreading phenomenon.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. S. Muller, Institut de Biologie Moléculaire et Cellulaire, UPR 9021 CNRS, 15 rue René Descartes, 67000 Strasbourg, France.
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; LNC, lymph node cell; RT, room temperature; SI, stimulation index; SNF1, (SWR x NZB)F1; TFA, trifluoroacetic acid.
Received for publication February 2, 2000. Accepted for publication April 24, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain. J. Exp. Med. 187:367.This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or 48 h (
), and cytokine
secretion was measured by ELISA. The results are expressed as OD units
measured at 450 nm (left scale) and U/ml evaluated using
known concentrations of each recombinant cytokine (right
scale). The only clear dose-response production in the presence
of increasing amounts of peptide 88–99 was found in the case of
IFN-
) in the presence of CFA/IFA. B, Reactivity of sera
from 14 individual 36- to 38-wk-old B/W lupus mice (