close

Вход

Забыли?

вход по аккаунту

?

Identification and characterization of SmD183119-reactive T cells that provide T cell help for pathogenic antidouble-stranded DNA antibodies.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 48, No. 2, February 2003, pp 475–485
DOI 10.1002/art.10762
© 2003, American College of Rheumatology
Identification and Characterization of
SmD1
-Reactive T Cells That Provide T Cell Help for
Pathogenic Anti–Double-Stranded DNA Antibodies
83–119
Gabriela Riemekasten,1 Dirk Langnickel,2 Fanny M. Ebling,3 George Karpouzas,3
Jatinderpal Kalsi,3 Gunda Herberth,2 Betty P. Tsao,3 Peter Henklein,2 Sven Langer,2
Gerd-R. Burmester,2 Andreas Radbruch,4 Falk Hiepe,5 and Bevra H. Hahn3
Objective. The C-terminal peptide of amino acids
83–119 of the SmD1 protein is a target of the autoimmune response in human and murine lupus. This
study was undertaken to test the hypothesis that
SmD183–119-reactive T cells play a crucial role in the
generation of pathogenic anti–double-stranded DNA
(anti-dsDNA) antibodies.
Methods. Splenic or lymph node T cells derived
from unmanipulated as well as SmD183–119-immunized
NZB/NZW mice were analyzed in vitro by enzyme-linked
immunospot (ELISpot) assay to determine T cell help
for anti-dsDNA generation induced by the SmD183–119
peptide. Cytokines expressed by these T cells were
measured by ELISpot assay, enzyme-linked immunosorbent assay, and flow cytometry. SmD183–119- and
ovalbumin-specific T cell lines were generated and
characterized.
Results. The SmD183–119 peptide, but not the
control peptides, significantly increased the in vitro
generation of anti-dsDNA antibodies in cultures from
unmanipulated NZB/NZW mice. Interferon-␥ (IFN␥),
interleukin-2 (IL-2), IL-4, transforming growth factor
␤, and IL-10 production increased in response to the
peptide in young mice; only IFN␥ and IL-2 were
increased in older, diseased mice. Activation of
SmD183–119-reactive T cells by immunization of NZB/
NZW mice resulted in elevated anti-dsDNA synthesis
and, later, increased antibodies to SmD183–119. Most
cells in SmD183–119-specific CD4ⴙ T cell lines helping
both antibodies had increased intracellular expression
of IFN␥, and most expressed both IFN␥ and IL-4.
Conclusion. The SmD183–119 peptide plays an important role in generating T cell help for autoantibodies,
including anti-dsDNA, and activates different subsets of
T cells as defined by distinct cytokine expression. This
peptide is an interesting target structure for the modulation of autoreactive T cells, and its characterization
may contribute to our understanding of the role of
autoantigen-reactive T cells in the pathogenesis of SLE.
Supported by grants from University Research Foundation of
Humboldt University, the DFG (Ri 1056/2-1, SFB 421, C4 and C6),
Merck Sharp & Dohme (Arthritis/Arthrose 2000 grant), the NIH
(R37-AI-46776 and P60-AR-36834), and the Arthritis Foundation
Southern California Chapter, and by gifts from the Paxson Family, the
Mitchell Family, and the Dorough Foundation.
1
Gabriela Riemekasten, MD: University of California, Los
Angeles and Charité University Hospital, Berlin, Germany; 2Dirk
Langnickel, Dipl Biotech, Gunda Herberth, Dipl Biol, Peter Henklein,
PhD, Sven Langer, Dipl Biol, Gerd-R. Burmester, MD: Charité
University Hospital, Humboldt University, Berlin, Germany; 3Fanny
M. Ebling, PhD, George Karpouzas, MD, Jatinderpal Kalsi, PhD,
Betty P. Tsao, PhD, Bevra H. Hahn, MD: University of California, Los
Angeles; 4Andreas Radbruch, PhD: Deutsches Rheumaforschungszentrum, Berlin, Germany; 5Falk Hiepe, MD: Charité University
Hospital and Deutsches Rheumaforschungszentrum, Berlin, Germany.
Address correspondence and reprint requests to Gabriela
Riemekasten, MD, Department of Rheumatology and Clinical Immunology, Charité University Hospital, Schumannstrasse 20/21, D-10117
Berlin, Germany. E-mail: gabriela.riemekasten@charite.de.
Submitted for publication May 29, 2002; accepted in revised
form October 9, 2002.
High-affinity antibodies against the Sm proteins
as well as against double-stranded DNA (dsDNA) are a
hallmark of the immune response in systemic lupus
erythematosus (SLE) (1,2). Despite the importance of
these antibodies as diagnostic and prognostic markers,
the target structures and the mechanisms of autoantibody production are not well understood. The Sm
antigens are part of the spliceosomal complex that plays
an essential role in the generation of messenger RNA
and DNA (for review, see ref. 3). It comprises at least 9
different polypeptides, designated B, B⬘, N, D1, D2, D3,
E, F, and G (4). Anti-Sm antibodies are predominantly
directed against the SmD1 protein (5). Anti-dsDNA as
475
476
RIEMEKASTEN ET AL
well as anti-Sm antibody responses are polyclonal,
antigen-driven, somatically mutated, and high-titered
(6), suggesting that both responses are driven by autoreactive T helper cells. Since uncomplexed mammalian
DNA is poorly immunogenic (7,8), and T cells are
unlikely to recognize dsDNA in the context of the major
histocompatibility complex (MHC) (9), models have
emerged in which peptides of anti-dsDNA antibodies
themselves (10), anti-dsDNA antibody binding peptides
(11), or nucleosomal peptides (12) provide the T cell
epitopes that drive anti-dsDNA production. As shown
for other autoimmune diseases, the frequency of autoreactive T cells is low (13). Until now, data characterizing autoantigen-specific T cells were obtained by analyses of T cell clones or of T cells after immunization with
the autoantigens (9,14).
Recently, we identified the C-terminal peptide of
amino acids (aa) 83–119 of the SmD1 protein as a major
target for the autoimmune response in SLE (15,16).
Approximately 70% of patients with SLE have antibodies to SmD183–119, compared with ⬍7% of disease
controls and healthy individuals (17). These antibodies
are closely associated with disease activity and with
concentrations of anti-dsDNA in individual patients,
suggesting a functional link between dsDNA and the
SmD183–119 peptide. In the NZB/NZW mouse, a murine
model of SLE, acceleration of lupus and increased
concentration of pathogenic anti-dsDNA antibodies after immunization with the SmD183–119 peptide indicate a
role of this peptide in the generation of anti-dsDNA
antibodies (17). We have hypothesized that the highly
positively charged SmD1 peptide could be a further
candidate for the activation of T cell help for dsDNAspecific B cells and could therefore be responsible for
the association between anti-dsDNA and anti-Sm antibodies (17). The present study examined this hypothesis.
MATERIALS AND METHODS
Mice. (NZB ⫻ NZW)F1 mice were obtained from
Jackson Laboratories (Bar Harbor, ME) or from Harlan
Winkelmann (Borchen, Germany). Strains were bred either in
the University of California, Los Angeles Rheumatology Vivarium or in the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin in Berlin-Marienfelde,
Germany. Only female mice were used in these experiments.
Antigens. The SmD1 83–119 peptide (VEPKVKSKKREAVAGRGRGRGRGRGRGRGRGRGGPRR), as
well as a randomized peptide with amino acids identical to the
SmD1 83–119 peptide (CREKGRVGRGRPAVGRRGVGRPGRRGSRARGEGKGRK), used as control antigen,
were synthesized according to a protocol described previously
(17). For T cell epitope mapping, 7 overlapping 15-mer
peptides, peptides aa 83–97, aa 85–99, aa 87–101, aa 90–104, aa
93–107, aa 97–111, and aa 105–119, were synthesized in the
same way. Other control peptides were histone peptide 3
(91–105; QSSAVMALQEASEAY), Ro 480 peptide (CAIALREYRKKMDI), a peptide based on complementaritydetermining region 1 of a murine anti-dsDNA monoclonal
antibody that bears the common idiotype (16/6Id [18]), and
pCONS (FIEWNKLRFRQGLEW), an artificial peptide
based on an algorithm describing T cell stimulatory sequences
from the VH regions of murine IgG antibodies to DNA (19).
In vivo activation of SmD183–119-reactive T cells by
immunization of NZB/NZW mice. For stimulation of
SmD183–119-reactive T cells and for T cell epitope mapping,
NZB/NZW mice at different ages were immunized subcutaneously (SC) in the hind limbs with either the SmD183–119
peptide or the randomized peptide, each emulsified with
Freund’s complete adjuvant (CFA) (1:1). After 9–10 days, cells
from draining popliteal and inguinal lymph nodes and spleens
were harvested. Six additional NZB/NZW mice (6 weeks old)
were repeatedly immunized SC (in the neck and finally in
the hind limbs) with 100 ␮g keyhole limpet hemocyanin–
SmD183–119 peptide and subsequently boosted with 50 ␮g
SmD183–119 emulsified in incomplete Freund’s adjuvant (IFA)
once per week (5 times during a period of 9 weeks). Nine days
after the last immunization, at age 14 weeks, the mice in
this group were killed and serum, splenic, and lymph node T
cells were harvested. For intracellular cytokine determination,
12-week-old NZB/NZW mice were immunized SC in the neck
with 100 ␮g of the SmD183–119 peptide in CFA, followed 1
week later by 2 weekly injections of 50 ␮g of the SmD183–119
peptide emulsified with IFA. As control, mice were immunized
with the randomized peptide of SmD183–119. Spleen cells were
harvested 14 days after the last immunization (when the age of
the mice was ⬃16 weeks).
Enzyme-linked immunosorbent assay (ELISA). An
anti-dsDNA ELISA was used with calf thymus as the source of
dsDNA; it was performed as described previously (20). AntiSmD183–119 peptide antibodies were detected by ELISA using
polystyrene-coated SmD183–119 peptide as described (17). Supernatants or sera were analyzed in triplicate in 1:4 and 1:100
dilutions, respectively.
Purification of T and B cells. Single cell suspensions
derived from spleens and lymph nodes were enriched for
Thy-1,2⫹ and B220⫹ cells with the Vario MACS magnetic
purification system (Miltenyi, Auburn, CA), using appropriate
microbead-coated antibodies. Purity of the isolated cell populations as determined by fluorescence-activated cell sorter
(FACS) varied from 92% to 99%. For enzyme-linked immunospot (ELISpot) assay, B and T cells were cultured at a ratio
of 1:10 in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% fetal calf serum (FCS) plus 20% concanavalin
A supernatant with or without peptides at optimal concentrations, in a 12-well culture plate (Gibco BRL Life Technologies,
Paisley, UK) at 37°C. After 5 days, cells were separated by
centrifugation and cultured in fresh DMEM with 10% FCS
and used for ELISpot assay. Supernatants were harvested for
detection of antibodies.
Determination of T cell help for anti-dsDNA and
anti-SmD183–119 secretion by ELISpot assay. B cells (105) and
T cells (106) cultured in complete medium (DMEM with 10%
FCS) with various concentrations of peptides were added to
ROLE OF SmD183–119-REACTIVE T CELLS IN ANTI-dsDNA GENERATION
each well of a 96-well plate coated with dsDNA or the
SmD183–119 peptide, respectively, and blocked for 2 hours with
sterile 3% bovine serum albumin (Sigma, St. Louis, MO) in
phosphate buffered saline at room temperature. After 8–10
hours of incubation at 37°C, cells were poured off and plates
were washed 12–16 times. Spots were developed as described
previously (21). Antibody-forming cells (AFCs) were enumerated as blue spots using an inverted microscope (Leitz, Wetzlar, Germany) and recorded as the number of IgG-producing
AFCs/105 B cells. Results of ELISpot assays were further
confirmed in simultaneous ELISAs of the supernatants derived from the cultures.
Cytokine ELISAs. T cells (2 ⫻ 105) and B cells (2 ⫻
4
10 ) were cultured in serum-free AIM V medium (Gibco BRL
Life Technologies) with or without peptides. Interleukin-2
(IL-2), interferon-␥ (IFN␥), and IL-4 (all from PharMingen,
San Diego, CA) were measured in individual supernatants
using commercial ELISA kits, according to the manufacturer’s
instructions. Each sample of supernatants was assayed in
triplicate in 2 or 3 separate experiments, at a dilution of 1:4. A
standard curve was constructed for each experiment, using
recombinant cytokines (PharMingen).
Cytokine ELISpot assays. Ninety-six–well plates
(Costar, Cambridge, MA) were coated with purified anticytokine antibodies (anti-IFN␥, anti–IL-4, anti–IL-10, anti–
transforming growth factor ␤ [anti-TGF␤]) (100 ␮l/well; all
from PharMingen) overnight according to the instructions of
the manufacturer and blocked as described above. T cells (2 ⫻
105) derived from spleens or lymph nodes of treated or
untreated mice, as well as B cells (2 ⫻ 104) from diseased
NZB/NZW mice exhibiting proteinuria of ⬎100 mg/dl were
suspended in AIM V medium and cocultured with or without
peptides (5 ␮g/well). After 72 hours in 5% CO2 at 37°C, cells
were removed and the reaction was visualized by addition of
the individual biotinylated anticytokine antibody and subsequent alkaline phosphatase–conjugated streptavidin. Spots
representing cytokine-expressing cells were detected as was
done for AFCs. The expression index was calculated as the
quotient between numbers of spots in T and B cell cultures
with peptide and spots in T and B cell cultures without peptide.
Intracellular cytokine detection by FACS. Cells were
fixed, permeabilized, and stained for intracellular cytokines
and surface markers as described previously (22). Monoclonal
antibodies (all from PharMingen) were used for intracellular
and surface staining. Cells were divided into 2 groups for
further staining. The first group of cells, which had been
marked with biotinylated CD69, were stained using fluorescein
isothiocyanate (FITC)–conjugated anti-IFN␥, phycoerythrin
(PE)–conjugated anti-CD4, and Cy5-stained anti-CD3. The
second subset were stained with FITC-conjugated anti–tumor
necrosis factor ␣ (anti-TNF␣), PE-conjugated anti–IL-4, anti–
IL-2 antigen-presenting cells (APCs), and biotinylated antiCD4 according to the instructions of the manufacturer (Becton
Dickinson). Completely unstained controls and stained isotype
controls were also used. Samples were analyzed by 4-color
analysis on a FACSCalibur flow cytometer (Becton Dickinson), using FCS-Express software, version 1.0.
Generation of T cell lines. T cells derived from splenic
cells of unmanipulated 8–10-week-old female NZB/NZW mice
were cultured with irradiated (10 minutes, 30 Gy) CD90depleted splenocytes (APCs) from 5-month-old untreated fe-
477
Figure 1. In vitro T cell help for anti–double-stranded DNA (antidsDNA) antibodies by the SmD183–119 peptide in untreated NZB/
NZW mice. Addition of SmD183–119 to a culture of splenic T cells from
10–15-week-old nonimmunized prenephritic NZB/NZW mice plus B
cells from older nonimmunized mice resulted in an increase in
anti-dsDNA antibody–forming cells (AFCs), compared with the spots
obtained from T and B cells cultured without the peptide. Cultures of
SmD183–119 with B cells alone, with the addition of the randomized
peptide (rand. pept.) to B cells alone, and with T ⫹ B cells did not
influence the generation of anti-dsDNA AFCs. Values are the mean ⫾
SEM from 3 independently performed experiments (n ⫽ 8 in each). P
values are versus all cultures without SmD183–119.
male NZB/NZW mice at a ratio of 1:4 in appropriate culture
wells at 37°C/5% CO2 in the presence of 5 ␮g/ml SmD183–119.
As a control, ovalbumin (OVA) was used for repeated in vitro
restimulation of T cells derived from mice previously immunized with OVA (50 ␮g, emulsified in CFA). As shown in our
previous work (17), OVA immunization has no influence on
anti-dsDNA response and survival. CD90 depletion was performed by MACS technology according to the instructions of
the manufacturer (Miltenyi).
Every 14–17 days, cells were restimulated with irradiated CD90-depleted APCs as well as with 5 ␮g/ml SmD183–119
peptide or OVA. Three days after restimulation, cells were
stimulated with 50 units/ml of IL-2; this was repeated every
3–4 days, followed by a resting period of 4–5 days. Characterization of T cells was performed after 6 weeks of culture or
later. T cells were analyzed by flow cytometry after staining
with carboxyfluorescein diacetate succinimidyl ester (Molecular Probes, Eugene, OR) for 5 days with or without stimulation
either with the corresponding antigen or with anti-CD3 or
SmD183–119. Simultaneously, T cell lines were functionally
analyzed in vitro for help for anti-dsDNA and anti-SmD183–119
antibodies, by ELISpot assay.
Statistical analysis. Comparisons between groups
were performed by Student’s t-test and by nonparametric
478
RIEMEKASTEN ET AL
Figure 2. In vitro activation of SmD183–119-reactive T cells by 5 ␮g/ml SmD183–119 peptide in immunized 7–12-week-old NZB/NZW mice, and the
influence on in vitro help for autoantibody generation. A, Increased T cell help for anti-dsDNA AFCs after immunization with the SmD183–119
peptide, as detected by enzyme-linked immunospot (ELISpot) assay. T cells were obtained from draining lymph nodes 9 days after subcutaneous
immunization. B cells were from unmanipulated 35-week-old mice with proteinuria. B, Detection of anti-SmD183–119 AFCs, by ELISpot assay, in
15-week-old NZB/NZW mice (n ⫽ 5) after repeated immunization with SmD183–119 (5 times during 9 weeks). T cells were obtained from draining
lymph nodes after paw immunization. Values are the mean ⫾ SEM from 3 independently performed experiments (n ⫽ 8 in each). P values are versus
cultures of T and B cells without any peptide. See Figure 1 for other definitions.
Mann-Whitney test. Values are presented as the mean ⫾ SEM.
P values less than 0.05 were considered significant.
RESULTS
The SmD183–119 peptide activates in vitro T cells
derived from untreated NZB/NZW mice to help in the
generation of anti-dsDNA–producing plasma cells. The
addition of the SmD183–119 peptide to a culture of
splenic T cells harvested from nonimmunized prenephritic NZB/NZW mice plus B cells from older nonimmunized NZB/NZW mice resulted in an increase in
anti-dsDNA AFCs, compared with the spots obtained
from T and B cells cultured without the peptide (P ⬍
0.001 for cultures with 5 ␮g or 20 ␮g of the SmD183–119
peptide) (Figure 1). Cultures of SmD183–119 peptide with
B cells alone, with the addition of the randomized
peptide to B cells alone, and with T ⫹ B cells did not
influence the generation of anti-dsDNA AFCs. AntiSmD183–119 antibodies were not found in the supernatants of T ⫹ B cells cocultured for 5 days, while
anti-dsDNA antibodies were readily detectable (data
not shown). Extended cultivation of T and B cells with
the SmD183–119 peptide over 9 days resulted in detectable anti-dsDNA as well as anti-SmD183–119 in the
supernatant (data not shown).
Comparative potencies of various self peptides to
induce T cell help for anti-dsDNA. The capability of
different peptides to induce T cell help for anti-dsDNA
antibodies in T cells derived from unmanipulated mice
was studied. In 3 independently performed experiments,
the effect of the SmD183–119 peptide on anti-dsDNA
generation was slightly higher than that induced by the
pCONS peptide, but in all of these experiments, the T
cell help was clearly increased by both peptides (data not
shown). The histone peptide 3 (91–105; QSSAVMALQEASEAY) was also compared in 3 independently performed experiments with the SmD183–119 peptide, showing in all experiments a lower capability to
induce anti-dsDNA antibodies in vitro. As a negative
peptide, the Ro 480 peptide (CAIALREYRKKMDI),
showing no T cell help for the generation of anti-dsDNA
antibodies, was used (data not shown). In all of these
experiments, 8-week-old mice were used as T cell donors; the results might vary with donors at older ages.
ROLE OF SmD183–119-REACTIVE T CELLS IN ANTI-dsDNA GENERATION
In vivo stimulation of SmD183–119-reactive T cells
and their influence on autoantibodies. A single in vivo
immunization of 7-week-old mice with the SmD183–119
peptide and subsequent in vitro restimulation with
SmD183–119 enhanced the generation of anti-dsDNA–
secreting plasma cells. T cells derived from draining
lymph nodes that were stimulated in vitro with the
SmD183–119 peptide enhanced the number of antidsDNA AFCs compared with spontaneous T cell help
(⬃5-fold; P ⬍ 0.001) (Figure 2A). In contrast, antidsDNA–specific AFCs were not significantly increased
by immunization and subsequent in vitro restimulation
of T cells with the randomized peptide as compared with
cultures of B and T cells without any peptide.
T cell help for anti-dsDNA AFCs was also increased by immunization of 27-week-old NZB/NZW
mice with SmD183–119. However, anti-dsDNA AFC
numbers increased only 1.7-fold (data not shown), which
was not as great an increase as that observed with young
NZB/NZW mice.
After single immunization, no anti-SmD183–119
antibodies were detectable in the supernatants of lymph
nodes or in spleen cell cultures. Repeated immunization
(up to 5 times) of NZB/NZW mice with the SmD183–119
peptide emulsified in CFA or IFA resulted in significant
T cell help for anti-SmD183–119 AFCs (P ⫽ 0.03 with 5
␮g) by T cells from draining lymph nodes, as detected by
ELISpot (Figure 2B) and confirmed by ELISA (data not
shown), as well as help for anti-dsDNA antibody generation. The sera from these repeatedly immunized mice
contained high levels of anti-SmD183–119 and antidsDNA antibodies compared with sera from untreated
NZB/NZW mice (data not shown).
In an analysis of the fine specificity of the
SmD183–119 peptide in lymph node T cells from SC
SmD183–119–immunized NZB/NZW mice, using overlapping 15-mer peptides spanning the SmD183–119 peptide, the largest response was found for the N-terminus
of the peptide. This was observed for both splenic T cells
(data not shown) and lymph node T cells (Figure 3).
Peptide aa 87–101 induced the highest response.
Cytokine profile of SmD183–119-reactive T cells
derived from untreated NZB/NZW mice. To analyze the
cytokine expression pattern of SmD183–119-reactive T
cells, splenic Thy-1,2⫹ T cells derived from untreated
NZB/NZW mice of different ages were reactivated for
72 hours with SmD183–119 ex vivo in the presence of B
cells from older nonimmunized NZB/NZW mice and
analyzed by cytokine ELISpot. T cells from 7-week-old
mice without proteinuria and from 27-week-old mice
with proteinuria (⬎100 mg/dl) were compared (Table 1).
479
Figure 3. T cell fine epitope mapping by 7 overlapping 15-mer
peptides spanning the whole SmD183–119 peptide, after subcutaneous
immunization of NZB/NZW mice with SmD183–119. T cells were
obtained from lymph nodes 9 days after immunization. The dominant
T cell epitope of the SmD183–119 peptide was found in the N-terminus.
P values are versus T and B cells without any peptide. aa ⫽ amino acid
(see Figure 1 for other definitions).
In young prenephritic mice, SmD183–119 resulted in
expression (above background levels) of various cytokines, including IFN␥, IL-2, IL-4, and IL-10. In response
to the SmD183–119 peptide, TGF␤ expression was detectable only in young prenephritic NZB/NZW mice; however, the number of spots was few (1/105 B cells). In
27-week-old mice with clinical nephritis, baseline production of all cytokines was higher than in young mice,
and addition of SmD183–119 resulted in increases of
IFN␥ and IL-2 only. The numbers of cells expressing
IL-4 decreased, and IL-10 secretion did not change
(Table 1). The randomized peptide control did not
induce significantly increased levels of IFN␥ or IL-4
above background. B cells alone or stimulated with
SmD183–119 did not contain any ELISpot-forming cells.
Activation of SmD183–119 peptide–reactive T cells
by in vivo immunization with the SmD183–119 peptide
and its influence on cytokine expression. Splenic T cells
from 7-week-old and 27-week-old NZB/NZW mice that
had been immunized with the SmD183–119 peptide were
analyzed for cytokine expression 9 days after immuniza-
480
RIEMEKASTEN ET AL
Table 1. Number of cytokine-specific spots, as detected by enzyme-linked immunospot assay, in cells from young and
nephretic unmanipulated and from young and nephritic SmD183–119-immunized female NZB/NZW mice, with and without in
vitro stimulation with the SmD183–119 peptide*
Cytokine-specific spots/2 ⫻ 105 cells/ml
7-week-old mice
T⫹B
Unmanipulated
IFN␥
IL-4
IL-10
IL-2
Immunized
IFN␥
IL-4
IL-10
IL-2
6.3
68
8.8
52
1
14
21
17
T ⫹ B ⫹ SmD1
27-week-old nephritic mice
83–119
T⫹B
T ⫹ B ⫹ SmD183–119
26 (4.1 [P ⬍ 0.001])
104 (1.5 [P ⬍ 0.004])
22.3 (2.5 [P ⫽ 0.0016])
75 (1.4 [P ⫽ 0.02])
19
85
22
75
34 (1.7 [P ⫽ 0.01])
68 (0.8 [P ⫽ 0.02])
24 (1.1)
110 (1.5 [P ⫽ 0.04])
15 (15 [P ⬍ 0.001])
67 (4.8 [P ⬍ 0.001])
27 (1.3)
31 (1.8 [P ⬍ 0.01])
2
42
66
53
25 (12.5 [P ⬍ 0.001])
74 (1.8 [P ⫽ 0.02])
66 (1)
150 (2.8 [P ⬍ 0.02])
* Values are the median number of spots, from triplicate specimens pooled from 2 mice in 1 representative experiment (3
experiments [total of 6 mice; 2 in each experiment] were performed, and all had similar results). Values in parentheses are the
expression index (EI), estimated as the number of cytokine-expressing cells after peptide stimulation divided by the number of
cytokine-expressing cells without peptide stimulation. When the EI was significant, the P value is indicated. IFN␥ ⫽
interferon-␥; IL-4 ⫽ interleukin-4.
tion (Table 1). Immunization with the SmD183–119 peptide influenced the number of T cells expressing a given
cytokine after restimulation with SmD183–119 peptide,
but it also affected the secretion of T cells without any in
vitro peptide restimulation (T ⫹ B cells alone). The
absolute number of T cells expressing a distinct cytokine
does not reflect the SmD183–119-specific stimulation
after immunization. Therefore, cytokine expression after
restimulation with SmD183–119 was compared with basal
secretion of B and T cells without SmD183–119 and
described by an index of cytokine expression.
In 7-week-old female NZB/NZW mice, the expression index of IFN␥ was strongly increased in immunized mice compared with untreated mice (P ⬍ 0.001).
Immunization with the SmD183–119 peptide markedly
increased secretion of IL-4 (P ⬍ 0.001), with a 5-fold
increased expression above background. IL-2 expression
was not significantly increased by immunization with
SmD183–119 compared with nonimmunized mice. TGF␤
expression was lower in immunized mice compared with
nonimmunized controls (P ⫽ 0.03). IL-10 expression was
slightly, but not significantly, lower in SmD183–119immunized mice than in untreated animals. Modulation
of T cells by immunization in 27-week-old nephritic
NZB/NZW mice confirmed the results obtained by
immunization of young NZB/NZW mice. The results of
the ELISpots are summarized in Table 1.
Analysis of supernatants of B and T cell cultures
from repeatedly immunized mice cocultured with
SmD183–119 confirmed the enhancement of IFN␥, and to
a lesser degree of IL-2 expression, detected by ELISpot
assay ex vivo. Both CD4⫹ and CD8⫹ T cells produced
IFN␥ (data not shown), whereas IL-4 was not detectable
in culture supernatants.
Intracellular cytokine expression of SmD183–119reactive T cells after immunization with SmD183–119, as
detected by flow cytometry. Two weeks after repeated
immunizations with the SmD1 83–119 peptide,
CD4⫹,CD3⫹ T cells were analyzed cytometrically and
the results compared with those found in age-matched
(16-week-old) mice without treatment. In unmanipulated mice, there was no increase in IFN␥ expression in
T cells after stimulation with the SmD183–119 peptide,
compared with unstimulated T cells (Figures 4a and b).
In contrast, in SmD183–119-immunized mice, the frequency of IFN␥-expressing T cells was increased to
0.08% after stimulation with the SmD183–119 peptide,
while in unstimulated T cells and in T cells stimulated
with the randomized peptide, no IFN␥-expressing T cells
could be detected. (Figures 4c–f).
CD69 expression did not serve as an appropriate
marker to reflect autoantigen-mediated stimulation. As
suggested previously (23), CD69 expression is disturbed
in lupus, and the permanent in vivo activation by autoantigens probably prevents further up-regulation.
In 16-week-old SmD183–119-treated mice, the frequency of IL-2–expressing cells among SmD183–119restimulated T cells increased to 0.08%, but no increase
was observed in unstimulated T cells or in T cells that
had been restimulated with the randomized peptide
ROLE OF SmD183–119-REACTIVE T CELLS IN ANTI-dsDNA GENERATION
481
with the SmD183–119 peptide in vitro to generate T cell
lines. T cell help for anti-dsDNA and anti-SmD183–119
synthesis was detected in vitro by ELISpot. After stimulation with the SmD183–119 peptide, SmD183–119specific lines cocultured with B cells increased help
for both anti-dsDNA and anti-SmD183–119. In contrast,
an OVA-specific T cell line, when stimulated with
SmD183–119 (Figure 5), but not with OVA (data not
shown), provided slightly increased help for anti-dsDNA
antibody–secreting cells, but still, no anti-SmD183–119
response could be detected by ELISpot.
In the SmD183–119-specific T cell line derived
from unmanipulated mice, 92% of the SmD183–119reactive T cells were CD4⫹, whereas a minority (4%)
were CD8⫹. The remaining 4% of the cells were double
negative. A high proportion of CD4⫹ T cells in the
SmD183–119-specific T cell line that helped anti-dsDNA
synthesis expressed intracellular IFN␥, and the majority
of these IFN␥⫹ T cells also expressed cytoplasmic IL-4
after stimulation with anti-CD3 (Figure 6). Only a
minority of T cells (⬍4%) expressed IL-4 without IFN␥.
IL-2 expression was detectable in ⬃50% of the T cells
and IL-10 in 10–15% (Figure 6). Similar results were
Figure 4. Intracellular cytokine expression, as measured by flow cytometry, in CD4⫹,CD3⫹ splenic T cells from 16-week-old unmanipulated NZB/NZW mice (a and b) and from age-matched NZB/NZW
mice that were subcutaneously immunized 3 times with the SmD183–119
peptide (c–f). T cells were stained with no stimulation (a and c) or after
stimulation with SmD183–119 (b and d), the randomized (rand.) peptide
of SmD183–119 (e), or phorbol myristate acetate (PMA)/ionomycin (f).
After immunization of mice with the SmD183–119 peptide, interferon-␥
(IFN␥) expression was stimulated by in vitro culture with the SmD183–119
peptide. In contrast, immunization with the randomized peptide and in
vitro restimulation with this peptide did not result in IFN␥ expression.
IFN␥ expression was not increased in unmanipulated mice after 3
hours of restimulation with SmD183–119. PerCP ⫽ peridin chlorophyll
protein; FITC ⫽ fluorescein isothiocyanate.
(data not shown). Restimulation of T cells from immunized mice with the SmD183–119 peptide also resulted in
an increased frequency of cells coexpressing TNF␣ and
IL-2 (0.05%; data not shown). In general, the frequencies of cytokine-expressing cells were low. IL-4 expression was not detectable in immunized or untreated mice
after 3 hours of restimulation.
Characterization of SmD183–119-reactive T cell
lines. T cells from unmanipulated 8-week-old NZB/
NZW mice were harvested and repeatedly restimulated
Figure 5. In vitro T cell help for anti-dsDNA and anti-SmD183–119
antibodies by SmD183–119-specific T cell lines. T cell lines were
obtained from 8–10-week-old unmanipulated female NZB/NZW mice
and restimulated in vitro with the SmD183–119 peptide and irradiated
antigen-presenting cells several times for up to 8 weeks. As a control,
ovalbumin (OVA)–specific T cell lines were obtained from OVAimmunized NZB/NZW mice. SmD183–119-specific as well as OVAspecific T cell lines were stimulated with SmD183–119 peptide. In the
presence of SmD183–119, SmD183–119-specific T cells gave help for
anti-dsDNA as well as anti-SmD183–119 synthesis. In contrast, OVAspecific cell lines gave only slightly increased help for anti-dsDNA
synthesis, but not for anti-SmD183–119 antibodies. Restimulation of
OVA-specific T cell lines with OVA did not result in significant help
for anti-dsDNA or anti-SmD183–119 antibodies. See Figure 1 for other
definitions.
482
RIEMEKASTEN ET AL
Figure 6. Cytokine expression in SmD183–119-specific T cell lines, as
detected by flow cytometry. SmD183–119-specific T cells from lines that
provide help for both anti–double-stranded DNA (anti-dsDNA) and
anti-SmD183–119 antibodies were analyzed. As a control, ovalbumin
(OVA)–specific T cell lines were measured for intracellular cytokine
expression. After stimulation with anti-CD3, interferon-␥ (IFNg) was
the cytokine dominantly expressed in SmD183–119-specific T cells. The
majority of these T cells coexpressed IFNg and interleukin-4 (IL-4). In
contrast, OVA-specific T cell lines revealed a completely different
cytokine pattern, with only a few IFNg/IL-4 double-positive T cells.
SmD183–119-specific T cells also expressed other cytokines, such as IL-2
and IL-10. FL ⫽ fluorescence; PE ⫽ phycoerythrin; APC ⫽ antigenpresenting cells.
obtained after stimulation with SmD183–119, with a high
frequency of IFN␥⫹,IL-4⫹ T cells (data not shown). In
contrast, OVA-specific T cell lines exhibited a completely different cytokine pattern, with only a few IFN␥/
IL-4 double-positive T cells (Figure 6). Another
SmD183–119-specific T cell line expressing only IFN␥ and
no IL-4 did not provide T cell help for autoantibody
generation in vitro (data not shown).
DISCUSSION
In previous work, we hypothesized that
SmD183–119-reactive T cells play a crucial role in the
generation of pathogenic anti-dsDNA antibodies via T
cells (17). In the present study, we characterized
SmD183–119-reactive T cells and demonstrated that they
indeed promote the development of anti-dsDNA antibodies. Involvement of the protein SmD1 in the generation of anti-dsDNA antibodies presents a novel concept
of interaction between the immune responses toward
small nuclear RNP and dsDNA via T cell recognition
and could explain the close relationship between antiSm and anti-dsDNA antibodies. To our knowledge, the
present investigation is the first in which in vitro stimulation of freshly isolated T cells with a defined autoantigen has been used to characterize direct cytokine
expression by autoantigen-specific T cells in unmanipulated mice with SLE.
We previously found that SmD183–119 strongly
influences the generation of anti-dsDNA antibodies in
immunized NZB/NZW mice (17). We have now demonstrated that in cultures of B cells derived from diseased NZB/NZW mice, with SmD183–119 in the absence
of T cells, plasma cells specific for either dsDNA or
SmD183–119 were not generated. This rules out the
polyclonal activation of dsDNA-specific B cells by
SmD183–119. In contrast, SmD183–119-reactive T cells
restimulated with SmD183–119 did increase the frequency
of dsDNA-specific plasma cells ⬃3-fold, suggesting that
SmD183–119-reactive T cells provide T cell help for
anti-dsDNA–specific plasma cells. Our objective was to
determine the mechanism by which SmD183–119-reactive
T cells are activated.
In vivo experiments and in vitro cultures demonstrated that the effect of SmD183–119 on the anti-dsDNA
response is stronger than that on the anti-SmD183–119
response. Therefore, the dsDNA-specific B cells themselves could play a decisive role in the activation of
SmD183–119-reactive T cells. Such B cells could initially
be activated in a T cell–independent manner (e.g., by
crosslinkage of surface antigen receptors) or independent of antigen receptors (e.g., by activation of Toll-like
receptor 9, which has been suggested to play a role in
lupus pathogenesis [24,25]). A precondition for these
models is that the highly positively charged SmD183–119
or SmD1 molecules bind to DNA and do not critically
impair the binding of DNA to anti-dsDNA antibodies on
the B cell surface. Several groups (26,27) have hypothesized that such SmD1–dsDNA complexes are targets
for both anti-dsDNA and anti-Sm responses. Our previous studies demonstrated that the addition of dsDNA to
SmD1 increased the frequency of SmD1-reactive T cells
in human SLE (17). In the experimental setting described here for the in vitro cultures, dsDNA for the
ROLE OF SmD183–119-REACTIVE T CELLS IN ANTI-dsDNA GENERATION
483
Figure 7. Possible mechanism for the generation of anti-dsDNA and anti-SmD183–119 antibodies. After
activation of dsDNA-specific B cells in a T cell–independent manner, these cells become potent
antigen-presenting cells for dsDNA-associated proteins such as SmD1, accompanied by up-regulation of
major histocompatibility complex and costimulatory molecules such as interferon-␥ (IFN␥) or IL-4. As a
result, SmD183–119 could be presented to SmD183–119-reactive T cells, and T cells would be activated and
in turn provide help for cells secreting high-affinity anti-dsDNA as well as anti-SmD183–119 antibodies.
TLR9 ⫽ Toll-like receptor 9; TcR ⫽ T cell receptor; CD40L ⫽ CD40 ligand (see Figure 6 for other
definitions).
initial activation of B cells could have been supplied by
dead cells, or preactivation of B cells could be a result of
culture.
Once activated, the dsDNA-specific B cell blasts
could become potent antigen-presenting cells, thereby
up-regulating MHC and costimulatory molecules. SmD1
as a DNA-associated protein could be presented either
directly or indirectly to SmD183–119-reactive T cells. The
activated T helper cells could in turn help the activated
B cells to perform hypermutation, class switching, and
differentiation into plasma cells, resulting in the generation of secreted, high-affinity anti-dsDNA antibodies, as
observed in the present study. The activated SmD183–119reactive T cells could then further activate SmD183–119specific B cells. This would depend on T cell help since
SmD183–119 is not likely to activate B cells directly via
repetitive epitopes.
In the case of the mice that received repeated
immunizations, the SmD183–119-reactive T cells had
been activated in vivo and could directly mediate the
rapid production of anti-SmD183–119 by B cells. In this
model, the same SmD183–119-reactive T cells provide the
driving force for both anti-dsDNA and anti-SmD183–119
antibody production, as was previously shown for
histone-reactive T cells that drive antihistone, antinucleosome, anti-dsDNA, and anti–single-stranded DNA
responses (12). Figure 7 illustrates a proposed model for
the activation of dsDNA- and SmD183–119-specific B
cells by SmD183–119-reactive T cells. However, further
studies are needed to determine whether naive T cells
can also be activated by such B cell blasts, or only by T
cells that have been preactivated by dendritic cells in
vivo. Moreover, the mechanisms by which autoimmunization against the peptide SmD183–119 specifically occurs
in human and murine lupus is still unclear.
Studies to characterize SmD183–119-reactive T
cells, especially after immunization, have shown a dominance of cells expressing the Th1 cytokine IFN␥, as can
readily be demonstrated by ELISpot, flow cytometry,
and cytokine ELISA of supernatants from short-term
cultures (72 hours) and T cell lines. T cell–derived IFN␥
is believed to play a key role in the pathogenesis of lupus
484
since IFN␥ hyperproduction is a consistent finding in
murine (28,29) and human (14) lupus. Treatment of
NZB/NZW mice with IFN␥ has been shown to accelerate the manifestations of lupus, whereas treatment with
anti-IFN␥ antibodies or soluble IFN␥ receptors early in
life delayed disease progression (30,31). Both antidsDNA and anti-SmD183–119 antibodies contain the
IgG2a subtype (results not shown), further suggesting an
important role of IFN␥, which activates synthesis of this
subtype (32).
Data on the role of IL-4 in murine lupus are
somewhat contradictory. Ectopic expression of IL-4
prevented the development of lethal lupus-like glomerulonephritis in a (NZW ⫻ C57BL/6.Yaa)F1 murine
lupus model (33). Conflicting evidence provided by
Nakajima and colleagues suggests that systemic blocking
of IL-4 by monoclonal antibodies can prevent the onset
of lupus nephritis in NZB/NZW mice (34). In the
present study, the ex vivo addition of IL-4 and anti-IFN␥
to cultures of T and B cells derived from unmanipulated
prenephritic NZB/NZW mice resulted in an increase in
the SmD183–119-related anti-dsDNA antibody response.
These findings demonstrate the relevance of IL-4 (formerly described as B cell–stimulating factor 1 [35]), at
least in a period of disease development, for the generation of plasma blasts that secrete dsDNA-specific antibodies.
Furthermore, T helper cell cultures in SmD183–119specific lines derived from young prenephritic NZB/
NZW mice were rich in cells exhibiting cytoplasmic
expression of both IFN␥ and IL-4. Th cells that produce
both Th1 and Th2 cytokines have been described and
classified as Th0 (36,37). It is still unclear whether they
represent a developmental stage occurring during Th1/
Th2 differentiation, a stable differentiated population,
or a mixture of various subpopulations (38). However,
the majority of histone-specific T cell clones derived
from SLE patients display such an unrestricted cytokine
secretion pattern, suggesting a possible role of this Th0
subset in the pathogenesis of lupus (14). Such T cells
would also explain the activation of both humoral and
inflammatory processes during SLE.
In conclusion, our findings suggest a new mechanism for functional linkage of anti-Sm and anti-dsDNA
responses. T helper cells from lupus-prone NZB/NZW
mice can be activated by the peptide SmD183–119, an
autoantigen unrelated to nucleosomes. It promotes antiDNA production and probably serves as a link between
the anti-dsDNA and antispliceosomal response. The
role of SmD183–119-reactive T cells in the generation of
pathogenic anti-dsDNA antibodies suggests that these
RIEMEKASTEN ET AL
cells may be useful targets for autoantigen-specific treatment strategies.
ACKNOWLEDGMENTS
We are grateful to Claudia Klein for technical support,
and to Rudi Manz and Thomas Kamradt for advice and
support.
REFERENCES
1. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol
1989;44:93–151.
2. Hahn BH. Antibodies to DNA. N Engl J Med 1998;338:1359–65.
3. Mattaj IW, Tollervey D, Seraphin B. Small nuclear RNAs in
messenger RNA and ribosomal RNA processing. FASEB J 1993;
7:47–53.
4. Lehmeier T, Foulaki K, Lührmann R. Evidence for three distinct
D proteins, which direct differentially with anti-Sm autoantibodies,
in the core of the major snRNPs U1, U2, U4/6, and U5. Nucleic
Acids Res 1990;18:6475–84.
5. Hoch SO, Eisenberg RA, Sharp GC. Diverse antibody recognition
patterns of the multiple SmD antigen polypeptides. Clin Immunol
1999;92:203–8.
6. Maddison PJ, Reichlin M. Quantitation of precipitating antibodies
to certain soluble nuclear antigens in SLE: their contribution to
hypergammaglobulinemia. Arthritis Rheum 1977;20:819–24.
7. Madaio MP, Datta SK. Journal club: major peptide autoepitopes
for nucleosome-specific T cells of human SLE. Am J Kidney Dis
2000;35:992–6.
8. Pisetsky DS. Immune responses to DNA in normal and aberrant
immunity. Immunol Res 2000;22:119–26.
9. Khalil M, Inaba K, Steinman R, Ravetsch J, Diamond B. T cell
studies in a peptide-induced model of systemic lupus erythematosus. J Immunol 2001;166:1667–74.
10. Ebling FM, Tsao BP, Singh RR, Sercarz E, Hahn BH. A peptide
derived from an autoantibody can stimulate T cells in the (NZB ⫻
NZW)F1 mouse model of systemic lupus erythematosus. Arthritis
Rheum 1993;36:355–64.
11. Puttermann C, Diamond, B. Immunization with a peptide surrogate for double stranded DNA (dsDNA) induces autoantibody
production and renal immunoglobulin deposition. J Exp Med
1998;188:29–38.
12. Datta SK, Kaliyaperumal A. Nucleosome-driven autoimmune
response in lupus: pathogenic T helper cell epitopes and costimulatory signals. Ann N Y Acad Sci 1997;815:155–70.
13. Kuwana M, Feghali CA, Medsger TA Jr, Wright TM. Autoreactive
T cells to topoisomerase I in monozygotic twins discordant for
systemic sclerosis. Arthritis Rheum 2001;44:1654–9.
14. Voll RE, Roth EA, Girkontaite I, Fehr H, Herrmann M, Lorenz
H-M, et al. Histone-specific Th0 and Th1 clones derived from
systemic lupus erythematosus patients induce double-stranded
DNA antibody production. Arthritis Rheum 1997;40:2162–71.
15. Riemekasten G, Marell J, Trebeljahr G, Klein R, Hausdorf G,
Häupl T, et al. A novel epitope on the C-terminus of SmD1 is
recognized by the majority of sera from patients with systemic
lupus erythematosus. J Clin Invest 1998;102:754–63.
16. Riemekasten G, Weiss C, Schneider S, Thiel A, Bruns A, Schumann F, et al. T cell reactivity against the SmD183–119 C-terminal
peptide in patients with systemic lupus erythematosus. Ann
Rheum Dis 2002;61:779–85.
17. Riemekasten G, Kawald A, Wei␤ C, Meine A, Marell J, Klein R,
et al. Strong acceleration of murine lupus by injection of the
SmD183–119 peptide. Arthritis Rheum 2001;44:2435–45.
ROLE OF SmD183–119-REACTIVE T CELLS IN ANTI-dsDNA GENERATION
18. Waisman A, Ruiz PJ, Israeli E, Eilat E, Konen-Waisman S, Zinger
H, et al. Modulation of murine systemic lupus erythematosus with
peptides based on complementarity determining regions of a
pathogenic anti-DNA monoclonal antibody. Proc Natl Acad Sci
U S A 1997;94:4620–5.
19. Hahn BH, Singh RR, Wong WK, Tsao BP, Bulpitt K, Ebling FM.
Treatment with a consensus peptide based on amino acid sequences in autoantibodies prevents T cell activation by autoantigens and delays disease onset in murine lupus. Arthritis Rheum
2001;44:432–41.
20. Panosian-Sahakian N, Klotz J, Ebling F, Kronenberg M, Hahn
BH. Diversity of Ig V gene segments found in anti-DNA autoantibodies from a single (NZB ⫻ NZW)F1 mouse. J Immunol
1989;142:4500–6.
21. Singh RR, Hahn BH, Tsao BP, Ebling FM. Evidence for multiple
mechanisms of polyclonal T cell activation in murine lupus. J Clin
Invest 1998;102:1841–9.
22. Assenmacher M, Schmitz J, Radbruch A. Flow cytometric determination of cytokines in activated murine T helper lymphocytes:
expression of interleukin-10 in interferon- and in interleukin-4expressing cells. Eur J Immunol 1994;24:1097–101.
23. Crispin JC, Martinez A, de Pablo P, Velasquillo C, Alcocer-Varela
J. Participation of the CD69 antigen in the T-cell activation
process of patients with systemic lupus erythematosus. Scand
J Immunol 1998;48:196–200.
24. Takeshita F, Leifer CA, Gursel I, Ishii KJ, Takeshita S, Gursel M,
et al. Cutting edge: role of toll-like receptor 9 in CpG DNAinduced activation of human cells. J Immunol 2001;167:3555–8.
25. Krieg AM. CpG DNA: a pathogenic factor in systemic lupus
erythematosus? J Clin Immunol 1995;15:284–92.
26. Reyes PA, Tan EM. DNA-binding properties of Sm nuclear
antigen. J Exp Med 1977;145:749–54.
27. Dempsey LA, Hanakahi LA, Meizels N. A specific isoform of
hnRNP D interacts with DNA in the LR1 heterodimer: canonical
RNA binding motifs in a sequence-specific duplex DNA binding
protein. J Biol Chem 1998;273:29224–9.
28. Murray LJ, Lee R, Martens C. In vivo cytokine gene expression in
T cell subsets of autoimmune MRL/Mp-lpr/lpr mouse. Eur J Immunol 1990;20:163–170.
485
29. Prud’homme GJ, Kono DH, Theofilopoulos AN. Quantitative
polymerase chain reaction analysis reveals marked overexpression
of interleukin-1, interleukin-10, and interferon-gamma mRNA in
the lymph nodes of lupus-prone mice. Mol Immunol 1995;32:
495–503.
30. Jacob CO, van der Meide PH, McDevitt HO. In vivo treatment of
(NZB ⫻ NZW)F1 lupus-like nephritis with monoclonal antibody
to ␥ interferon. J Exp Med 1987;166:798–803.
31. Ozmen L, Roman D, Fountoulakis M, Schmidt G, Ryffel B,
Garotta G. Experimental therapy of systemic lupus erythematosus:
treatment of NZB/W mice with mouse soluble interferon-gamma
receptor inhibits the onset of glomerulonephritis. Eur J Immunol
1995;25:6–12.
32. Snapper CM, Paul WE. Interferon-gamma and B cell stimulatory
factor-1 reciprocally regulate Ig isotype production. Science 1987;
236:944–7.
33. Santiago ML, Fossati L, Jacquet C, Muller W, Izui S, Reininger L.
Interleukin-4 protects against a genetically linked lupus-like autoimmune syndrome. J Exp Med 1997;185:65–70.
34. Nakajima A, Hirose S, Yagita H, Okumura K. Roles of IL-4 and
IL-12 in the development of lupus in NZB/W F1 mice. J Immunol
1997;158:1466–72.
35. Rabin EM, Ohara J, Paul WE. B-cell stimulatory factor 1 activates
resting B cells. Proc Natl Acad Sci U S A 1985;82:2935–9.
36. Assenmacher M, Löhning M, Scheffold A, Richter A, Miltenyi S,
Schmitz J, et al. Commitment of individual Th-1 like lymphocytes
to expression of IFN␥ versus IL-4 and IL-10: selective induction of
IL-10 by sequential stimulation of naı̈ve Th cells with IL-12 and
IL-4. J Immunol 1998;161:2825–32.
37. Firestein GS, Roeder WD, Laxer JA, Townsend KS, Weaver ST,
Hom JT, et al. A new murine CD4⫹ T cell subset with an
unrestricted cytokine profile. J Immunol 1989;143:518–25.
38. Löhning M, Grogan JL, Coyle AJ, Yazdanbakhsh M, Meisel C,
Gutierrez-Ramos JC, et al. T1/ST2 expression is enhanced on
CD4⫹ T cells from schistosome egg-induced granulomas: analysis
of Th cell cytokine coexpression ex vivo. J Immunol 1999;162:
3882–9.
Документ
Категория
Без категории
Просмотров
1
Размер файла
480 Кб
Теги
antibodies, stranded, smd183119, pathogens, dna, provider, characterization, identification, reactive, antidouble, help, cells
1/--страниц
Пожаловаться на содержимое документа