Mechanism of Action of Transmembrane Activator and
Calcium Modulator Ligand Interactor-Ig in Murine Systemic
Meera Ramanujam,*†Xiaobo Wang,*†Weiqing Huang,*†Lena Schiffer,*†Christine Grimaldi,†
Alla Akkerman,*†Betty Diamond,*†Michael P. Madaio,‡and Anne Davidson2*†
B cell-activating factor belonging to the TNF family (BAFF) blockade prevents the onset of disease in systemic lupus erythem-
atosus (SLE)-prone NZB/NZW F1mice. To determine the mechanism of this effect, we administered a short course of TACI-Ig
with and without six doses of CTLA4-Ig to 18- to 20-wk-old NZB/NZW F1mice and evaluated the effect on B and T cell subsets
and on anti-dsDNA Ab-producing B cells. Even a brief exposure to TACI-Ig had a beneficial effect on murine SLE; CTLA4-Ig
potentiated this effect. The combination of TACI-Ig and CTLA4-Ig resulted in a temporary decrease in serum IgG levels. However,
after cessation of treatment, high titers of IgG anti-dsDNA Abs appeared in the serum and IgG Abs deposited in the kidneys.
Despite the appearance of pathogenic autoantibodies, the onset of proteinuria was markedly delayed; this was associated with
prolonged depletion of B cells past the T1 stage, a decrease in the size of the spleen and lymph nodes, and a decrease in the absolute
number of activated and memory CD4?T cells. TACI-Ig treatment normalized serum levels of IgM that are markedly elevated
in NZB/W F1mice; this appeared to be due to a prolonged effect on the ability of the splenic microenvironment to support
short-lived IgM plasma cells. Finally, a short course of combination TACI-Ig and CTLA4-Ig prolonged life and even reversed
proteinuria in aged NZB/W F1mice, suggesting that BAFF blockade may be an effective therapeutic strategy for active SLE. The
Journal of Immunology, 2004, 173: 3524–3534.
molog that activates apoptosis, NF-?B, and JNK; and zTNF4), is an
important survival factor for B cells. BAFF is produced predomi-
nantly by myeloid cells and binds to three distinct receptors on the B
cell surface, transmembrane activator and calcium modulator ligand
interactor (TACI), B cell maturation Ag (BCMA), and BAFF-R (1,
2). Engagement of BAFF-R by BAFF is an absolute requirement for
B cell survival and maturation in secondary lymphoid organs. BAFF
also mediates class switching to IgG (3) and promotes the survival of
plasmablasts (2, 4, 5). A proliferation-inducing ligand (APRIL), an-
other TNF family member, is highly homologous to BAFF, but binds
to only two of the three BAFF receptors, TACI and BCMA (6, 7).
Recombinant APRIL induces B cell proliferation and class switching
(3), and APRIL transgenic mice have increased levels of serum IgM
and increased IgM responses to T-independent Ags (8).
cell-activating factor belonging to the TNF family
(BAFF3; also known as B lymphocyte stimulator; TNF-
and ApoL-related leukocyte-expressed ligand 1; TNF ho-
BAFF-deficient mice have small spleens with few B cells, and
they mount an attenuated humoral immune response to T-depen-
dent and T-independent Ags (9, 10). Conversely, BAFF transgenic
mice develop B cell hyperplasia and an autoimmune lupus-like
disease (2, 11, 12). Overexpression of BAFF has been observed in
spontaneous murine systemic lupus erythematosus (SLE) models
and in humans with autoimmune diseases (13, 14). These findings
have suggested that antagonism of BAFF may be a useful thera-
peutic approach for autoimmune disease.
Antagonism of BAFF can be achieved by administration of a
soluble form of any of the three BAFF receptors. Both TACI-Ig
and BAFF-R-Ig have been reported to delay the onset of SLE in
the murine NZB/W F1model (2, 15). We have generated an ad-
enovirus expressing a fully murine TACI-Ig fusion protein to study
the mechanism for this effect and to determine whether antagonism
of BAFF would be synergistic with an agent that blocks T cell
costimulation. We show that administration of a single dose of
adenovirus expressing TACI-Ig to prenephritic NZB/W F1mice
together with six doses of CTLA4-Ig results in profound depletion
of splenic B cells and a delay in disease onset. The long delay in
disease onset after a short course of TACI-Ig, with or without
CTLA4-Ig, is associated with a decrease in the absolute number of
activated lymphocytes of all lineages. TACI-Ig treatment also re-
sults in a long term survival disadvantage for IgM-producing
plasma cells in the spleen, but does not appear to have a sustained
effect on the survival of IgG-producing plasma cells. Finally, a
short course of TACI-Ig and CTLA4-Ig can reverse nephritis and
prolong survival even in older mice, suggesting that B cell deple-
tion may be an effective therapeutic strategy for active SLE.
Materials and Methods
Generation of an adenovirus expressing fully murine TACI-Ig
The extracellular domain of murine TACI (aa 2–129) was cloned from
NZB/W F1spleen cDNA by RT-PCR and fused to a protrypsin signal
Departments of *Medicine and†Microbiology and Immunology, Albert Einstein Col-
lege of Medicine, Bronx, NY 10461; and‡Department of Medicine, University of
Pennsylvania, Philadelphia, PA 19104
Received for publication February 4, 2004. Accepted for publication June 29, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the New York Systemic Lupus Erythematosus Foundation
(to M.R. and L.S.) and the National Institute of Allergy and Infectious Diseases (to A.D.).
2Address correspondence and reprint requests to Dr. Anne Davidson, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, U505, Bronx, NY 10461. E-mail
3Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the
TNF family; TACI, transmembrane activator and calcium modulator ligand interac-
tor; BCMA, B cell maturation Ag; APRIL, A proliferation-inducing ligand; Ad-L,
LacZ adenovirus; Ad-L/C, LacZ adenovirus and CTLA4-Ig; Ad-T, TACI-Ig adeno-
virus; Ad-T/C, TACI-Ig adenovirus and CTLA4-Ig; AID, activation-induced cytidine
deaminase; MZ, marginal zone; Ox, oxazolone; SLE, systemic lupus erythematosus;
T1, transitional type 1; T2, transitional type 2.
The Journal of Immunology
Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00
sequence and to the CH2-CH3 domains of murine IgG2a. The construct
was expressed in the Adeno-X adenovirus according to manufacturer’s
instructions (BD Clontech, Palo Alto, CA). This is a nonreplicating virus
based on Ad-5 with tropism for mouse liver (16). The expression of
TACI-Ig dimer was confirmed by Western blot of serum from a SCID
mouse injected 4 days previously with 1 ? 109PFU of plaque-purified
virus and by TACI-specific ELISA.
Treatment of NZB/W F1mice
NZB/NZW F1females were purchased from The Jackson Laboratory (Bar
Harbor, ME) and maintained in a conventional animal housing facility.
Mice were tested at 18 wk for IgG anti-dsDNA Abs and were distributed
into matched treatment groups depending on the Ab titer. Mice were
treated at the age of 18–20 wk with TACI-Ig adenovirus (Ad-TACI-Ig) or
?-galactosidase-expressing adenovirus (Ad-LacZ) 1 ? 109PFU (10 mice
each). Treatment was also combined with CTLA4-Ig, six doses of 100 ?g
administered over a 2-wk period (Ad-TACI-Ig/CTLA4-Ig, 15 mice; Ad-
LacZ/CTLA4-Ig, 10 mice). Comparisons were performed with 10 mice
that received CTLA4-Ig alone and 20 untreated controls.
Fisher Scientific, Pittsburgh, PA) every 2 wk. Mice were followed until death.
Separate groups of Ad-TACI-Ig/CTLA4-Ig- and Ad-LacZ/CTLA4-Ig-treated
mice were killed at 26 wk for ELISPOT and flow cytometry experiments and
at 36 wk for renal histology. Finally, a separate group of 10 mice was treated
with Ad-TACI-Ig/CTLA4-Ig at the age of 30 wk. We elected to use the com-
bination treatment for remission induction studies, because it appeared to be
more efficacious at preventing the onset of proteinuria than Ad-TACI-Ig alone.
These mice were compared with five mice treated at 30 wk with Ad-LacZ/
CTLA4-Ig and 15 untreated controls.
Anti-dsDNA ELISA was performed as previously described (17). For mea-
surement of affinity for dsDNA, sera with maximal IgG anti-dsDNA titers
were identified for each mouse, and inhibition assays were performed as
previously described (18) by preincubating sera at the predetermined con-
centration that resulted in 80% maximal binding to the plate with increas-
ing amounts of dsDNA from 2–200 ?g/ml.
Oxazolone (Ox) immunization of NZB/W F1mice
To evaluate the effect of TACI-Ig on the humoral immune response to a T
cell-dependent Ag, groups of five NZB/W F1mice were immunized with
750 ?g of Ox by skin paint 8 wk after treatment with Ad-TACI-
Ig/CTLA4-Ig or Ad-LacZ/CTLA4-Ig. Mice were bled at weekly intervals,
and anti-Ox Abs were measured by ELISA as previously described (19).
Mice were challenged with 50 ?g of Ox by skin paint 4–6 wk later, and
spleens were harvested for ELISPOT analysis as previously described (20)
3 days after boosting. To determine whether TACI-Ig interfered with es-
tablished memory B cells in treated mice, 10 NZB/W F1mice, aged 14 wk,
were immunized with 750 ?g of Ox. Four weeks later, Ad-TACI-
Ig/CTLA4-Ig or Ad-LacZ/CTLA4-Ig was administered as described above
to groups of five mice. Once serum TACI-Ig levels became undetectable (6
wk after virus administration), mice were challenged with 50 ?g of Ox by
skin paint. The mice were bled 7 days later for measurement of anti-Ox
Abs, and bone marrow was harvested 14 days after challenge for analysis
of anti-Ox responses by ELISPOT as described above.
Anti-dsDNA ELISPOT assay
Spleens were harvested at 35–37 wk of age by survival splenectomy. At
this time control mice were beginning to develop proteinuria, but treated
mice remained well. ELISPOT assays for total Ig-secreting cells and for
anti-dsDNA-secreting B cells were performed as previously described (17).
Generation of hybridomas
Hybridomas were generated from spleen cells by standard techniques using
NSO cells as the fusion partner. Hybridomas were screened for anti-Ox or
anti-dsDNA activity, and positive hybridomas were isotyped as previously
Analysis of class switching and somatic mutation
Evidence of active class switching was sought by semiquantitative RT-
PCR analysis of IgG2b I-C transcripts from spleens of five Ad-TACI-Ig/
CTLA4-Ig-treated and five age-matched control mice as previously de-
scribed (17). PCR of these transcripts yielded a single band. The amount of
activation-induced cytidine deaminase (AID) was measured by semiquan-
titative RT-PCR using specific primers (5?-CCTCCTGCTCACTGGACTTC
To determine whether somatic mutation was occurring in autoreactive B
cells, we analyzed the autoreactive VHBW-16 gene, a marker gene for the
anti-dsDNA response (21). IgG cDNA libraries were constructed by RT-
PCR from the spleens of seven Ad-TACI-Ig/CTLA4-Ig treated mice, four
Ad-TACI-Ig treated mice, and eight age-matched controls and were
screened for VHBW-16 as we have previously described in detail (17).
Sequences were compared with the germline VHBW-16 sequence using
BLAST search (www.ncbi.nlm.nih.gov/blast/).
Flow cytometry and immunofluorescence analysis of spleens
Spleen and peripheral blood cells were analyzed for B and T cell markers
using Abs to CD4 (Caltag Laboratories, Burlingame, CA), CD8 (Caltag
Laboratories), and CD19. T cell subsets were identified using FITC-anti-
CD4, PE-anti-CD69, CyChrome-anti-CD44, and PE-anti-CD62L. B cell
subsets were identified using biotin-anti-CD23, FITC-anti-CD21, PE- or
CD5, PE-anti-B220, PE-anti-CD43, and PE-anti-CD69. Streptavidin-
PerCP was used as a second stain for biotinylated Abs. Spleen and periph-
eral blood dendritic cells were identified using PE-anti-CD11b and FITC-
anti-CD11c. Except where indicated, all Abs were purchased from BD
Pharmingen (San Diego, CA).
For immunofluorescence staining, 5-?m cryosections were stained with
FITC-anti-mouse IgM, PE-anti-mouse IgD, FITC-anti-CD11c, FITC-anti-
CD4, FITC-peanut agglutinin, and PE-anti-mouse CD45R/B220 (BD
Pharmingen) in 10% normal goat serum-FCS/PBS for 1 h at room tempera-
ture. Images were captured using a digital CCD-camera system (Diagnostic
Instruments, Sterling Heights, MI) connected to a microscope (Nikon,
Immunohistochemical analysis of kidneys
Examination and scoring of the kidneys were performed as previously
Proteinuria and survival data shown in Fig. 2, A and B, and Fig. 10A were
analyzed using Kaplan-Meier curves and log-rank test. The comparisons
shown in Figs. 2C, 3, 4, 6, 7, 10B, 11, and 12G and in Tables I and II were
performed using Wilcoxon’s rank-sum test. The comparisons shown in Fig.
10C were performed using paired t test. The comparisons in Table III were
performed using ?2analysis or Fisher’s exact test. Only significant p values
All animal experiments were approved by the institutional animal review
board of Albert Einstein College of Medicine
Clinical effects of TACI-Ig in NZB/W F1mice
TACI-Ig was only detected in the serum of NZB/W F1mice for
7–10 days after Ad-TACI-Ig injection. For this reason and because
Ad-TACI-Ig treatment alone had only a modest effect on the de-
velopment of proteinuria, in some mice the adenovirus was ad-
ministered together with six doses of CTLA4-Ig. This resulted in
the expression of TACI-Ig protein for 4–5 wk (Fig. 1). No IgG1 or
NZB/W F1mice injected on day 0 with 109PFU of Ad-TACI-Ig alone (left
panel) or together with six doses of 100 ?g of CTLA4-Ig (right panel).
Mean ? SD serum TACI-Ig levels over time in five
3525 The Journal of Immunology
IgG2b Abs to TACI-Ig were detected 6 wk after virus administra-
tion (not shown).
The life span of NZB/W F1mice treated with either Ad-
TACI-Ig alone or both Ad-TACI-Ig and six doses of CTLA4-Ig
(Ad-TACI-Ig/CTLA4-Ig) was significantly prolonged; however,
Ad-TACI-Ig-treated mice developed proteinuria significantly ear-
lier than those treated with Ad-TACI-Ig/CTLA4-Ig (Fig. 2C).
NZB/W F1mice have a polyclonal increase in serum levels of
IgM that continues to increase with age. Both Ad-TACI-Ig and
Ad-TACI-Ig/CTLA4-Ig treatment caused a drop in serum IgM lev-
els that was sustained for up to 6 mo after treatment (Fig. 3A).
Serum IgG levels decreased for a short time after Ad-TACI-Ig/
CTLA4-Ig, but were unaffected by treatment with Ad-TACI-Ig or
Ad-LacZ/CTLA4-Ig (Fig. 3B). Serum IgA levels followed a sim-
ilar pattern as IgG (Fig. 3C). The drop in serum levels of IgG after
Ad-TACI-Ig/CTLA4-Ig was due predominantly to a decrease in
levels of IgG1 and, to a lesser extent, of IgG2a (Fig. 3C).
IgM anti-DNA Abs were undetectable in the serum of ?70% of
the Ad-TACI-Ig-treated mice up to the time that proteinuria de-
veloped, even in mice that had high serum titers of IgG anti-DNA
Abs (Fig. 4A). The appearance in serum of IgG anti-DNA Abs was
delayed in mice that received Ad-TACI-Ig/CTLA4-Ig compared
with untreated controls (p ? 0.001), but not compared with mice
that received Ad-LacZ and CTLA4-Ig (Ad-LacZ/CTLA4-Ig; p ?
0.09), suggesting that this delay was partly due to CTLA4-Ig (Fig.
4B). The affinity of the Abs for dsDNA, measured by inhibition
assay, was the same in treated and control mice (Fig. 4C).
Analysis of B cell phenotype
At 26 and 36 wk of age (8 and 18 wk after treatment), the spleens
of TACI-Ig-treated mice were significantly smaller and contained
fewer cells than the spleens of control 16-, 26-, and 36-wk-old
NZB/W F1mice (Tables I and II). Thirty-six-week-old control
NZB/W F1mice had more B cells in the spleen than 16-wk-old
controls due to an increase in follicular B cells and CD19?/IgM?/
IgD?class-switched B cells (Table II). The spleens of both 26-
and 36-wk-old, Ad-TACI-Ig/CTLA4-Ig-treated mice consistently
had a lower percentage and absolute number of CD19-positive B
cells than the spleens of age-matched, Ad-LacZ/CTLA4-Ig-treated
mice and untreated controls. A decrease in the percentage of B
cells was also observed at 26 wk in the lymph nodes (not shown)
and peripheral blood of Ad-TACI-Ig/CTLA4-Ig treated mice com-
pared with controls (25.2 ? 4.7 vs 45.4 ? 4.3%; p ? 0.001).
To determine whether certain B cell subsets were preferentially
depleted in the treated mice, CD19-gated cells were examined for
markers to distinguish T1 (CD23?/CD21?/IgMhigh), T2 (CD23?/
CD21high/IgMhigh), marginal zone (MZ; CD23?/CD21high), and
B1 (B220low/CD5?) subsets. Because down-regulation of CD21
and overall survival (B) in the six groups of NZB/W F1mice described in
Materials and Methods. Mice treated with Ad-TACI-Ig had prolonged sur-
vival (Ad-TACI-Ig/CTLA4-Ig vs Ad-LacZ/CTLA4-Ig, p ? 0.003; Ad-
TACI-Ig vs Ad-LacZ, p ? 0.004), but only mice treated with Ad-TACI-Ig
and CTLA4-Ig had a significantly prolonged proteinuria-free survival (Ad-
TACI-Ig/CTLA4-Ig vs Ad-LacZ/CTLA4-Ig, p ? 0.0001; Ad-TACI-Ig vs
Ad-LacZ, p ? NS; Ad-TACI-Ig/CTLA4-Ig vs Ad-TACI-Ig, p ? 0.002).
Mice treated with CTLA4-Ig alone were similar to Ad-LacZ/CTLA4-Ig
mice and were not further studied. C, Median age of onset of IgG anti-
dsDNA Abs, proteinuria ?300 mg/dl, and death for each group.
A and B, Kaplan-Meier plots of proteinuria-free survival (A)
in both Ad-TACI-Ig/CTLA4-Ig- and Ad-TACI-Ig-treated mice and stayed at this level for ?24 wk. Serum IgG levels decreased as long as TACI-Ig was
detectable in the serum in Ad-TACI-Ig/CTLA4-Ig-treated mice. Levels of IgG1, IgG2a, and IgA 4 wk after treatment are shown as a percentage of the
prebleed level (C). All were significantly decreased (IgG1, p ? 0.001; IgG2a, p ? 0.03; IgA, p ? 0.01).
Mean ? SD serum IgM (A) and IgG (B) in 15 treated and 10 untreated control NZB/W F1mice. Serum IgM levels decreased to normal
3526 TACI-Ig IN MURINE SLE
and CD23 has been reported in BAFF-deficient mice (23), imma-
ture (IgMhigh/IgDlow) and mature (IgMint/IgDhigh) B cells were
also distinguished using staining with anti-IgM and anti-IgD with
similar results (Fig. 5). Twenty-six-week-old, Ad-TACI-Ig/
CTLA4-Ig-treated mice had the same number of B1 and T1 cells
as controls, but fewer T2, MZ, and follicular B cells, consistent
with a TACI-Ig-induced block in B cell maturation. This abnor-
mality persisted in mice examined at 36 wk of age. The number of
peritoneal B1 cells was unaffected by Ad-TACI-Ig/CTLA4-Ig
treatment (not shown). Splenic CD19?cells were also examined
for class-switched IgM?/IgD?cells and for the CD69 activation
marker. Although the frequency of these cells was unchanged in
Ad-TACI-Ig/CTLA4-Ig treated mice (Table I), their total number
was diminished compared with that in age-matched controls (Ta-
ble II). Of the four Ad-TACI-Ig spleens examined, two resembled
Ad-TACI-Ig/CTLA4-Ig mice, and two resembled age-matched
control mice, reflecting a similar, but more short-lived, treatment
effect in the absence of CTLA4-Ig.
Analysis of B cell function
To enumerate the frequency of Ig-secreting cells, ELISPOT assay
was performed on equal numbers of spleen cells from treated and
control mice. At both 26 and 36 wk of age, the frequency of total
Ig-secreting cells was 2- to 3-fold less in Ad-TACI-Ig/CTLA4-Ig-
treated mice than in Ad-LacZ/CTLA4-Ig-treated controls (Fig. 6,
A and B), paralleling the decrease in frequency of B cells in the
spleens of Ad-TACI-Ig/CTLA4-Ig-treated mice. The total number
of Ig-secreting cells per spleen was ?2- to 3-fold less in the 26-
wk-old, Ad-TACI-Ig/CTLA4-Ig-treated mice than in controls, but
due to the spleen enlargement in older untreated mice, it was 10-
to 20-fold less in 36-wk-old, Ad-TACI-Ig/CTLA4-Ig-treated mice
than in age-matched controls (Fig. 6C). Ad/TACI-Ig-treated mice
displayed a mixed picture, and the mean frequency of Ig-secreting
cells was between those in young and age-matched controls.
To determine whether Ad-TACI-Ig- and Ad-TACI-Ig/CTLA4-
Ig-treated mice could mount a humoral immune response to a for-
eign Ag during the period of delay in disease onset, mice were
immunized with Ox at 30 wk of age. IgM anti-Ox Abs were un-
detectable in the serum of both Ad-TACI-Ig- and Ad-TACI-Ig/
CTLA4-Ig-treated mice 7 days after primary immunization. The
IgG serum response to Ox was diminished compared with that in
NZB/W F1controls only in the Ad-TACI-Ig/CTLA4-Ig-treated
mice, but was higher than that in immunized BALB/c mice (Fig.
7A). The frequency of IgM and IgG anti-Ox-secreting splenic B
cells, as evaluated by ELISPOT, was decreased by 2- to 3-fold in
Ad-TACI-Ig/CTLA4-Ig-treated mice compared with controls, but
not in Ad-TACI-Ig-treated mice, reflecting the decrease in the total
number of Ig-secreting cells in Ad-TACI-Ig/CTLA4-Ig mice (Fig.
6). Few hybridomas were recovered from the Ad-TACI-Ig/
CTLA4-Ig-treated mice, but hybridomas were readily recovered
from the spleens of two Ad-TACI-Ig-treated mice that had low
serum IgM anti-Ox Ab levels; the frequency of both IgM and IgG
anti-Ox hybridomas in these mice was the same as that in controls
(percent Ox-positive hybridomas, 8.8% with an IgG:IgM ratio of
measurable titers of IgM anti-DNA Abs. However IgG anti-DNA Abs could be detected even in the absence of IgM anti-DNA Abs (A). Median levels of
IgM anti-DNA Abs were lower at all ages in mice treated with TACI-Ig than in controls (B). Inhibition assays showed that the affinity of anti-dsDNA Abs
was equivalent in treated and control mice (C).
The effect of TACI-Ig treatment on anti-dsDNA Abs. At 42 wk of age, only four of 15 Ad-TACI-Ig/CTLA4-Ig-treated mice had developed
Table I. Frequency of spleen cell subsetsa
% of Cells
16 wk NZB/W
(n ? 5)
26 wk NZB/W
(n ? 5)
Ig, 26 wk
(n ? 5)
Ig, 26 wk
(n ? 5)
36 wk NZB/W
(n ? 8)
Ig, 36 wk
(n ? 7)
Ig, 36 wk
(n ? 4)
CD19 (mean ? SD)
38.2 ? 2.1
3.7 ? 0.4
65.1 ? 1.3
8.2 ? 1.8
4.6 ? 0.9
11.8 ? 2.5
5.5 ? 1.0
25.3 ? 2.0
7.4 ? 2.3
28.9 ? 10.1
42.6 ? 13.5
8.8 ? 1.6
43.7 ? 12.6
3.5 ? 1.1
61.2 ? 5.3
5.9 ? 3.2
4.4 ? 2.8
13.8 ? 6.1
30.0 ? 9.0
17.1 ? 4.2
48.1 ? 13.7
29.5 ? 13.6
9.6 ? 6.7
46.8 ? 3.3
3.7 ? 1.3
65.5 ? 5.1
4.2 ? 1.7
5.1 ? 1.9
14.5 ? 4.9
3.8 ? 1.4
26.3 ? 4.8
16.1 ? 9.2
9.0 ? 4.3
24.2 ? 7.8b
4.2 ? 1.4
53.5 ? 4.2c
15.1 ? 10.4d
3.7 ? 1.6
7.2 ? 2.0c
12.0 ? 2.9d
42.5 ? 5.3e
11.0 ? 4.1
18.3 ? 3.2e
40.1 ? 5.9
16.5 ? 2.7
69.3 ? 4.9
5.6 ? 1.1
3.5 ? 1.1
4.3 ? 2.0
13.2 ? 3.5
29.8 ? 6.0
30.7 ? 2.9
83.7 ? 3.6
5.7 ? 1.6
5.2 ? 1.0
22.2 ? 3.7b
18.6 ? 5.2
52.0 ? 1.3c
16.1 ? 2.7c
3.1 ? 0.8
4.1 ? 1.8
12.0 ? 1.8
33.3 ? 10.6
16.2 ? 1.4d
60.0 ? 9.0e
15.0 ? 3.3e
20.5 ? 3.3e
36.7 ? 11.4
24 ? 4.8
61.6 ? 5.8
7.8 ? 3.5
14.6 ? 5.6
25.9 ? 5.4
36.8 ? 12.2
aThe p values are compared with age-matched controls. Only significant p values are shown.
bp ? 0.002.
cp ? 0.05.
dp ? 0.01.
ep ? 0.02.
3527 The Journal of Immunology
2.03 in Ad-TACI-Ig-treated mice vs 5.7% with an IgG:IgM ratio
of 2.2 in controls).
To determine the effect of treatment on somatic mutation, anal-
ysis of the VH-Ox-1 gene derived from IgG cDNA libraries from
spleen was performed in Ad-TACI-Ig/CTLA4-Ig-treated mice. Ex-
amination of the VHregion showed a 28% decrease in the fre-
quency of somatic mutations (35 mutations in 17 sequences com-
pared with 65 in 23 control sequences), but no difference in the R:S
ratio in the CDR regions (14:5 vs 22:7; Table III). Furthermore, the
affinity of anti-Ox Abs of the IgG1 and IgG2a isotypes, measured
using different coupling frequencies of Ox-BSA to coat the plates,
was no different in Ad-TACI-Ig/CTLA4-Ig- or Ad-TACI-Ig-
treated mice than in untreated NZB/W F1controls (not shown).
This suggests that functional germinal centers were formed. The
difference in IgG titers between Ad-TACI-Ig/CTLA4-Ig and con-
trol mice appears to reflect a decrease in the total number of B cells
in the treated mice.
The results presented above indicate that despite the small
spleen size and the decrease in B cell frequency observed in Ad-
TACI-Ig/CTLA4-Ig-treated mice at 36 wk of age, their B cells
were still able to respond to T cell signals, undergo class switching
and somatic mutation, and generate IgG-producing plasma cells.
To confirm this result, we performed semiquantitative RT-PCR on
spleen cDNA for IgG2b class switch transcripts and AID. We
found that IgG2b class switch transcripts and AID expression cor-
related with each other and were diminished by ?50% in the
TACI-Ig-treated mice (ratio of IgG2b:actin, 0.27 ? 0.18 vs 0.63 ?
0.11; p ? 0.005), consistent with the 50% decrease in B cell
Immunohistochemical studies of spleens from 36-wk-old, Ad-
TACI-Ig/CTLA4-Ig-treated NZB/W F1mice revealed that lym-
phoid organization was intact, but follicles were smaller and con-
tained fewer B cells and more CD4?T cells than those in controls
(Fig. 8, A–D). However germinal centers were still present within
the follicles (Fig. 8, E and F). Cells staining intensely for cyto-
plasmic IgM were abundant in the splenic red pulp of control mice,
but were diminished in both number and staining intensity in the
spleens of Ad-TACI-Ig/CTLA4-Ig-treated mice (Fig. 8, G–J).
It is still not known whether flares of SLE occur as a result of
recruitment of naive B cells to the autoreactive response or
whether autoreactive memory cells are reactivated. Because of the
marked depletion of B cells in the spleen, we determined whether
6 and 7). Twenty-six-week-old controls are shown in A, panels 1–4, and B, panel 6. Thirty-six-week-old controls are shown in A, panels 5–7. Ad-TACI-
Ig/CTLA4-Ig-treated mice are shown in B, panels 1–4 (26 wk) and panels 5 and 7 (36 wk). Panel 1, Loss of B cells and relative increase in T cells in
treated mice. Panel 2, Only the CD23?population is shown. Note the decrease in marginal zone B cells (upper box) and increase in T1 cells (lower box)
in treated mice. Panel 3, The oval shows marginal zone B cells; panel 4, the square shows B1 B cells. Panel 5, Immature (IgMhigh/IgDlow), mature
(IgM?/IgDhigh), and class-switched (IgM?/IgD?) B cells are shown. Note the increase in immature B cells in treated mice. Panel 6, Loss of naive
CD44low/CD62Lhighand increase in memory CD44high/CD62LlowCD4 cells are shown in 36-wk-old compared with 26-wk-old control NZB/W. Panel 7,
The square shows activated T cells.
Representative FACS plots of B and T cell subsets. Cells were gated on CD19 (panels 3–5), both CD19 and CD23 (panel 2) or CD4 (panels
Table II. Absolute number of spleen cellsa
No. of Cells/Spleen
(n ? 5)
26 wk, NZB/W
(n ? 5)
Ig, 26 wk
(n ? 5)
Ig, 26 wk
(n ? 5)
36 wk, NZB/W
(n ? 8)
Ig, 36 wk
(n ? 7)
Ig, 36 wk
(n ? 4)
Total cell number ? 107(mean ? SD)
CD19 ? 107
CD19/CD69 ? 106
Follicular ? 107
T1 ? 106
T2 ? 106
MZ ? 106
B1 ? 106
CD4 ? 107
CD4/CD69 ? 107
CD4/CD44?/CD62L?(memory) ? 107
CD4/CD44?/CD62L?(naive) ? 107
CD8 ? 107
9.0 ? 1.0
2.8 ? 0.1
1.9 ? 3.0
2.2 ? 0.1
2.8 ? 0.8
1.6 ? 0.2
4.0 ? 0.7
1.9 ? 0.4
2.3 ? 0.3
1.4 ? 0.2
0.6 ? 0.2
0.9 ? 0.3
0.8 ? 0.1
13.0 ? 1.1
5.7 ? 1.8
2.0 ? 1.0
3.6 ? 0.1
3.4 ? 2.3
2.3 ? 1.1
5.1 ? 5.7
3.9 ? 1.1
6.3 ? 1.4
1.5 ? 0.7
1.0 ? 0.7
1.1 ? 0.9
12.8 ? 2.8
5.8 ? 1.5
2.2 ? 1.3
3.9 ? 1.0
2.3 ? 0.8
3.1 ? 1.5
8.3 ? 2.3
2.2 ? 0.9
3.2 ? 0.4
5.3 ? 3.6
1.0 ? 0.3
6.1 ? 3.8b
1.7 ? 1.6d
0.6 ? 0.5c
0.9 ? 0.9c
2.1 ? 1.4
0.7 ? 0.8d
1.3 ? 1.6c
2.2 ? 2.4
2.5 ? 1.2
2.7 ? 1.7
1.1 ? 0.6
18.0 ? 8.0
5.1 ? 2.9
7.3 ? 3.2
3.9 ? 2.3
3.2 ? 1.9
2.1 ? 1.6
2.5 ? 2.1
6.7 ? 2.5
5.0 ? 1.8
15.5 ? 7.0
4.2 ? 1.7
0.3 ? 0.2
0.8 ? 0.1
4.5 ? 0.9c
1.0 ? 0.1e
1.8 ? 6.0b
0.5 ? 0.1c
1.3 ? 1.0d
0.4 ? 0.2c
0.3 ? 0.2c
1.1 ? 0.1d
1.6 ? 0.5e
2.6 ? 1.0e
1.0 ? 0.3b
0.2 ? 0.1
0.9 ? 0.1
5.8 ? 4.2b
2.5 ? 2.2
3.6 ? 2.6
1.5 ? 1.6
1.6 ? 1.5
4.1 ? 2.2
1.4 ? 0.8d
3.6 ? 2.6d
aThe p values are compared with age-matched controls. Only significant p values are shown.
bp ? 0.05.
cp ? 0.002.
dp ? 0.02
ep ? 0.01.
3528TACI-Ig IN MURINE SLE
a short course of TACI-Ig treatment would result in decreased
survival of memory B cells. No difference in the secondary IgG
anti-Ox response was detected in mice that had been immunized
with Ox 4 wk before Ad-TACI-Ig/CTLA4-Ig treatment and
boosted 6 wk post-treatment by either serologic assay (Fig. 7B) or
ELISPOT (controls, 24.3 ? 15.6; treated, 22.6 ? 9.9/105bone
marrow cells vs ?4 in naive controls), indicating that memory B
cells survived Ad-TACI-Ig/CTLA4-Ig treatment and could be re-
activated upon Ag boosting after TACI-Ig and CTLA4-Ig were no
longer present in the serum.
Effect of TACI-Ig treatment on autoreactive B cells
The above data indicate that despite their low numbers, some B
cells in the Ad-TACI-Ig/CTLA4-Ig-treated mice were activated,
could undergo class switching, and could acquire autoreactivity.
ELISPOT analysis revealed that the decrease in the frequency of
anti-dsDNA-secreting B cells of the IgM and IgG isotypes in Ad-
TACI-Ig/CTLA4-Ig-treated mice paralleled the decrease in total
Ig-secreting cells, indicating that there was no specific deletion of
autoreactive B cells (Fig. 6).
To determine whether somatic mutation of an autoreactive V
gene could occur in the small spleen, we analyzed the autoreactive
VHBW16 H chain gene. This gene is overexpressed among anti-
DNA Abs in NZB/W F1mice and is regulated in normal mice (21).
The amount of IgG VHBW16 transcript was decreased in Ad-
TACI-Ig/CTLA4-Ig-treated mice, reflecting the decreased number
of mature B cells. Thirty-seven different IgG VH-BW-16 se-
quences were obtained from the Ad-TACI-Ig/CTLA4-Ig-treated
mice, and their mutation frequency and pattern were compared
with those of untreated mice (44 sequences) or mice treated with
Ad-LacZ/CTLA4-Ig (14 sequences). The overall mutation fre-
quency of VHBW-16 was decreased by 43% in the Ad-TACI-Ig/
CTLA4-Ig-treated mice compared with untreated controls (p ?
0.06), but the frequency of replacement mutations in the CDR
sponse to Ox in NZB/W F1mice (p ? 0.02; A). Only the IgG2a response
to Ox in Ad-TACI-Ig/CTLA4-Ig-treated mice was significantly different
from controls (p ? 0.02). In contrast, if the mice were primed before
administration of TACI-Ig and were boosted after TACI-Ig was no longer
present in the serum, the memory response to Ox was intact (B). Naive
mice in this experiment received the boost immunization only. Five mice
were examined per group, and values are expressed as the mean ? SD. A
titration assay is shown in C.
TACI-Ig had a prolonged effect on the primary IgM re-
(A) and IgG (B) isotypes from spleens of control and treated NZB/W F1
mice (four or five per group). The frequency is per 103cells for total Ig and
per 105cells for anti-dsDNA Ig and anti-Ox Ig. Ad-LacZ/CTLA4-Ig-
treated mice were indistinguishable from untreated controls (not shown).
Ad-TACI-Ig/CTLA4-Ig-treated mice had fewer IgM and IgM anti-dsDNA-
and anti-Ox-secreting cells than age-matched controls at both times stud-
ied. Ad-TACI-Ig/CTLA4-Ig-treated mice had fewer IgG and IgG anti-
dsDNA- and anti-Ox-secreting cells than controls at 36 wk. Only signifi-
cant p values are shown. C, Shown are absolute numbers of spots per
spleen for the 36-wk mice.
Mean ? SD frequency of Ab-secreting B cells of the IgM
3529 The Journal of Immunology
regions was not decreased, and the pattern of mutations, including
the accumulation of positively charged residues in CDR2, was
similar in all three groups of mice analyzed (Table III and data not
shown), indicating little effect of treatment on selection of B cells
Finally, to determine whether pathogenic Abs were present in
Ad-TACI-Ig/CTLA4-Ig-treated mice, kidneys from three mice
killed at the age of 36 wk were examined and compared with
kidneys from three Ad-LacZ/CTLA4-Ig-treated controls. Glomer-
ular damage was present in all three Ad-LacZ/CTLA4-Ig-treated
mice (glomerular score, 2–3?), but none was evident in Ad-TACI-
Ig/CTLA4-Ig-treated mice (glomerular score, 0–1?). Deposition
of IgG was present in the glomeruli of all three Ad-TACI-Ig/
CTLA4-Ig-treated mice as well as in the controls, indicating that
the autoantibodies generated in Ad-TACI-Ig/CTLA4-Ig-treated
mice had pathogenic potential (Fig. 9).
Phenotypic analysis of spleen T cells and dendritic cells
Changes in the phenotype of T cells induced by TACI-Ig were less
dramatic than those observed with B cells and were consistent with
the state of B cell deficiency in the treated mice. Thirty-six-week-
old control NZB/W F1mice had a 2-fold increase in the number of
splenic CD4?T cells compared with 16-wk-old NZB/W F1con-
trols, but a 7- to 10-fold increase in the number of activated and
memory CD4 T cells. Ad-TACI-Ig/CTLA4-Ig-treated mice had a
20–30% decrease in the number of CD4 T cells compared with
pretreatment controls. The frequency of activated and memory
CD4 T cells in 36-wk-old, Ad-TACI-Ig/CTLA4-Ig-treated mice
was intermediate between young and age-matched untreated con-
trols. Because of the small spleen size, however, the absolute num-
bers of activated CD4?T cells were significantly decreased in
Ad-TACI-Ig/CTLA4-Ig- and Ad-TACI-Ig-treated mice compared
(C–E, G, H, and K) stained with anti-B220 (red; A–F, K, and L), anti-CD4 (green; A–D), peanut agglutinin-FITC (green; E and F), anti-IgM (green; G–J),
anti-IgD (red; G–J), and anti-CD11c (green; K and L). B cell follicles in treated mice contain fewer B cells, more CD4 T cells (A–D), and a similar number
of CD11c?cells as control mice (K and L). Germinal centers are present in both treated mice and controls (E and F). IgM-containing plasma cells are
abundant in the red pulp of control mice, but are smaller and less frequent in treated mice (G–I, representative of five mice).
Immunohistochemistry of spleens from Ad-TACI-Ig/CTLA4-Ig-treated mice (A, B, F, I, J, and L) and age-matched control NZB/W F1mice
Table III. Analysis of mutations of the VHBW-16 and VH-Ox-1 genesa
Total Mutations/SequenceR/S Ratio
7 23 13
5 6 10
66 20 79 44 209
226 25 17
549 42 19 124
aFR, Framework; R, replacement; S, silent.
3530TACI-Ig IN MURINE SLE
with age-matched controls (Tables I and II). These data indicate a
significant inhibitory effect of TACI-Ig treatment on CD4 T cell
activation and proliferation.
No difference in the percentage or distribution of CD11c-
positive cells was observed between treated and control mice in
either spleen (Fig. 8, K and L) or peripheral blood, but again,
because of the small spleen size, the absolute number of these
cells in the spleen was reduced in TACI-Ig-treated mice (data
Induction of remission by TACI-Ig and CTLA4-Ig
To determine whether Ad-TACI-Ig/CTLA4-Ig therapy would be
as effective in treating established disease in NZB/W F1mice as it
was in delaying disease onset, we studied its effects at 30 wk when
high titer IgG anti-dsDNA Abs were present and ?50% of the mice
had developed proteinuria. The combination of TACI-Ig and
CTLA4-Ig was found to reverse or delay the onset of proteinuria and
prolong the life span in these mice (Fig. 10A). The two treated mice
that died shortly after treatment had established ?300 mg/dl protein-
uria at the time of treatment and expressed low levels of TACI-Ig in
the serum after treatment, probably because of renal excretion of
TACI-Ig. Even at this late age, Ad-TACI-Ig/CTLA4-Ig treatment re-
sulted in a prompt decrease in serum levels of IgM and IgG and a
decrease in IgM and IgG anti-DNA Abs (Fig. 10B). Similar to our
observations with Ad-TACI-Ig/CTLA4-Ig treatment in younger mice,
the decrease in IgG anti-DNA Abs was temporary, lasting only as
long as TACI-Ig was expressed in the serum, whereas the decrease in
IgM anti-DNA Abs was sustained for up to 3 mo (Fig. 10C).
Analysis of the spleens of surviving mice, performed at 40–44
wk of age, 10–14 wk after treatment, revealed that they were
smaller (11.7 ? 8.1 vs 21.2 ? 6.7 ? 107cells; p ? 0.09) and
contained fewer B cells than control, 40- to 42-wk-old spleens
(32.8 ? 5.3 vs 48.8 ? 3.1%; p ? 0.02), but had no change in the
absolute number of CD4?and CD8?T cells. The only other sta-
tistically significant difference from controls was an increase in the
frequency of naive CD4?T cells (23.7 ? 14.0 vs 8.3 ? 4.0%; p ?
0.02) and a decrease in the frequency of memory CD4?T cells
(60.1 ? 9.7 vs 75.9 ? 3.0%; p ? 0.01), suggesting that the tran-
sition from the naive to the memory phenotype is still occurring at
30 wk and can be delayed either directly or as a result of B cell
depletion, by TACI-Ig. Consistent with the flow cytometry data,
H&E-stained sections of kidneys from two mice treated with Ad-TACI-
Ig/CTLA4-Ig (A and C) and two mice treated with Ad-LacZ/CTLA4-Ig (B
and D) show no glomerular damage or cellular infiltrates in the treated
mice. Immunofluorescence staining shows IgG deposits in the glomeruli of
both treated (E) and control (F) mice.
TACI-Ig treatment delays renal damage in NZB/W F1mice.
Ad-LacZ/CTLA4-Ig or remained untreated. Ad-TACI-Ig/CTLA4-Ig treatment reversed proteinuria in three of the five proteinuric mice (left panel; p ?
0.01) and prolonged survival (right panel; p ? 0.04). B, Ad-TACI-Ig/CTLA4-Ig treatment at 30 wk induced a decrease in serum levels of total IgM (left)
and IgG (right) that was maximal 1–3 wk after treatment. Serum IgG levels rebounded by wk 5, but serum IgM levels remained lower than pretreatment
levels for ?9 wk (significant p values for IgG: wk 1, p ? 0.01; significant p values for IgM: wk 1, p ? 0.01; wk 5 and 7, p ? 0.03; wk 3 and 9, p ? 0.05).
C, IgG anti-dsDNA levels rebounded promptly after TACI-Ig was no longer present in the serum, but IgM anti-dsDNA levels remained lower than
pretreatment levels for ?11 wk (significant p values for IgG: wk 1, p ? 0.015; wk 3, p ? 0.002; significant p values for IgM: wk 1 and 5, p ? 0.003;
wk 3, p ? 0.001; wk 7 and 9, p ? 0.01; wk 11, p ? 0.05). Values are expressed as the mean ? SD of 10 control and eight treated mice.
A, Ten previously untreated NZB/W F1mice were treated with Ad-TACI-Ig/CTLA4-Ig at 30 wk of age. Twenty controls were treated with
3531The Journal of Immunology
the frequency of IgM- and IgG-secreting B cells as measured by
ELISPOT was 2-fold lower in spleens from treated mice compared
with controls. In contrast, there was no difference in the frequency
of IgG-secreting cells or IgG anti-DNA-secreting cells in the bone
marrow of treated and control mice (Fig. 11).
Immunofluorescence studies of the kidneys of treated mice
10–14 wk after treatment revealed IgG deposits in the glomeruli of
treated mice. However, compared with controls, the Ad-TACI-Ig/
CTLA4-Ig-treated mice had less glomerular damage and less in-
terstitial lymphocytic infiltrate (Fig. 12).
The studies presented in this report have confirmed that a short
course of the murine BAFF and APRIL inhibitor TACI-Ig, given
as a single dose of adenovirus, has a profound effect on disease
onset in NZB/W F1mice. We also showed that the addition of a
2-wk course of CTLA4-Ig prolonged the duration of TACI-Ig ex-
pression and the nephritis-free survival of the mice; this combina-
tion also had beneficial effects in older mice with established
Our studies addressed the question of how TACI-Ig modulates
the immune system to induce these clinical outcomes. As has been
observed in nonautoimmune mice (10), TACI-Ig induced a pro-
found depletion of MZ, T2, and mature B2 B cells that persisted
for many months after treatment, with sparing of T1 and B1 B
cells. The B cells that remained in the spleen after TACI-Ig treat-
ment, however, were able to form germinal centers and respond to
T cell help. Once TACI-Ig was no longer present in the serum,
mice could mount a class-switched and high affinity IgG response
to the hapten Ox, and IgG anti-dsDNA Abs were produced after
only a short delay. These Abs had a similar affinity for DNA as
Abs from control mice, and they were able to deposit in the kid-
neys. Furthermore, the frequency of somatic mutation of both the
VH-Ox-1 gene and the autoreactive VHBW-16 gene, a marker
gene for anti-DNA Ab-producing B cells, was only modestly de-
creased, and replacement to silent ratios of mutation in the CDR
regions were comparable with those found in controls. Finally, in
Ad-TACI-Ig/CTLA4-Ig-treated mice there was no selection
against mutations that resulted in arginine substitutions in VHBW-
16, a feature associated with anti-dsDNA specificity. This suggests
(as has recently been reported by others for nonautoimmune mice
(24)) that germinal centers were functional in the small spleens and
lymph nodes of TACI-Ig-treated mice. Our findings further show
that the selection, migration, and survival of high affinity IgG-
producing plasma cells in bone marrow could be supported.
In contrast to the temporary effect of TACI-Ig treatment on se-
rum levels of IgG, TACI-Ig with or without CTLA4-Ig induced a
profound decrease in serum levels of IgM and IgM anti-DNA Abs
that was sustained for many months after cessation of therapy.
TACI-Ig-treated mice were also unable to mount a serum IgM
response to a T-dependent Ag, Ox, despite the presence of anti-Ox
IgM-secreting B cells in the spleen that could be detected by ELIS-
POT and rescued as hybridomas. This result might be directly
related to the loss of MZ B cells and/or B2 cells. However, ad-
ministration of the selective BAFF antagonist BAFF-R-Ig had an
even more profound effect on the number of MZ and B2 B cells,
but had no effect on serum levels of IgM or the IgM response to Ox
(M. Ramanujam and A. Davidson, manuscript in preparation).
Serum IgM levels reflect both the frequency of IgM-secreting
cells and the amount of Ig secreted per cell. Our data suggest that
the low serum IgM level in treated mice is due to the absence of
a few B cells that produce large amounts of Ig. TACI-Ig has pre-
viously been shown to prevent the IgM response to a T-indepen-
dent Ag in nonautoimmune mice (5) due to decreased survival of
plasmablasts. Immunohistochemical staining of the spleens from
treated mice showed that IgM-producing plasma cells were present
in the splenic red pulp of untreated NZB/W F1mice, and that both
the frequency and the size of these plasma cells in the red pulp of
the spleen were significantly decreased in treated mice compared
isotypes from spleens and bone marrows of control and treated NZB/W F1
mice (seven or eight mice per group) after late treatment with Ad-TACI-
Ig/CTLA4-Ig, shown as the mean ? SD. The frequency is per 103cells for
total Ig and per 105cells for anti-dsDNA Ig. There are significantly fewer
total Ig-secreting cells (p ? 0.01 for IgM and IgG) and anti-
dsDNA-secreting cells (p ? 0.03 for IgM and IgG) in the spleen, but not
in the bone marrow, of treated mice.
Frequency of Ab-secreting B cells of the IgM and IgG
histology in NZB/W F1mice. All mice still alive at 44 wk were killed for
analysis. Control mice, shown in A, C, and E, had evidence of proliferative
glomerulonephritis, tubular casts (A), and vasculitis (C). Ad-TACI-Ig/CTLA4-
Ig-treated mice are shown in B, D, and F. These mice had much less glomer-
ular damage (B) and interstitial disease (D), but deposition of IgG in the glo-
meruli (F) was no different from that in controls (E). Mean glomerular and
interstitial disease/vasculitis score are shown in G for the six surviving control
mice and for six Ad-TACI-Ig/CTLA4-Ig-treated mice that remained free of
proteinuria. A score of 5 indicates a mouse that died within 15 wk of treatment
with ?300 mg/dl proteinuria. The p values for treated vs control mice were
calculated for the whole group, with scores of 3 attributed to dead mice: glo-
merular score, p ? 0.01; tubular score, p ? 0.002.
Effect of Ad-TACI-Ig/CTLA4-Ig treatment at 30 wk on renal
3532TACI-Ig IN MURINE SLE
with controls. We therefore believe that TACI-Ig induces an en-
vironment in which these extrafollicular plasma cells, elicited by
either T-dependent or T-independent Ags, do not survive. The re-
duction of serum IgM levels by TACI-Ig, but not BAFF-R-Ig, is
consistent with the finding that TACI-deficient mice have low se-
rum IgM levels (25), whereas IgM levels are normal in BAFF-R-
deficient mice (26). These data suggest that the survival of short-
lived IgM plasma cells in the spleen is dependent on the interaction
of BAFF and APRIL with TACI. However, the recent identifica-
tion of BCMA as an important BAFF receptor on human and mu-
rine bone marrow plasma cells (27–29) suggests that this point
needs further investigation.
It is unlikely that the decrease in serum levels of IgM autoan-
tibodies was responsible for the long delay in onset of renal dis-
ease, because mice treated with BAFF-R-Ig also had prolonged
delay in disease onset (15) (M. Ramanujam and A. Davidson,
manuscript in preparation). The beneficial effect of TACI-Ig could
not be explained by a decrease in the titer or affinity of IgG auto-
antibodies or their ability to deposit in the kidney. It has previously
been reported that T cells are important mediators of renal damage
in mice with SLE even in the absence of circulating Ig (30). As
NZB/W F1mice age, they have increased numbers of CD4?cells,
increased expression of T cell activation markers, and loss of the
naive T cell marker CD62L (31). We have shown that kidneys of
diseased NZB/W F1mice contain large aggregates of lymphoid
cells, including B cells, T cells, and dendritic cells, resulting in
local secretion of inflammatory mediators and cytokines (22). We
therefore asked whether there was an alteration in T cell activation
in treated mice. In TACI-Ig-treated mice, the proliferation of
CD4?T cells that accompanies aging and disease onset did not
occur, and the frequency of activated and memory T cells was
intermediate between those in young and aged controls. Of note,
despite previous reports of a costimulatory function for TACI on T
cells (32, 33), we were unable to demonstrate any effect of
TACI-Ig on delayed-type hypersensitivity in BALB/c mice (not
shown). Finally, although the frequency of dendritic cells in spleen
and peripheral blood was not changed, their absolute numbers
were diminished as a result of the small spleen size.
Our findings suggest that the profound effect of TACI-Ig on
disease onset in NZB/W F1mice is secondary to B cell depletion.
B cells produce soluble mediators involved in the organization of
lymphoid tissues, and their absence results in shrinkage of the
spleen and lymph nodes (34, 35). B cells also function as APCs
that can participate in cycles of autoantigen presentation to auto-
reactive T cells (36). Because of the small spleen size and the
decreased number of activated B cells in TACI-Ig-treated mice
(36), the total number of activated T cells was decreased 4- to
5-fold. In addition, the small spleen size resulted in a decrease in
the total number of dendritic cells. Dendritic cells that migrate to
the kidneys of NZB/W F1mice secrete chemokines that attract
inflammatory cells, including B and T cells, into the kidney (22).
The suppression of T cell activation and the decrease in the total
number of dendritic cells probably contributed to the delay in onset
of proteinuria despite the deposition of Abs in renal glomeruli.
A striking observation in this study was the ability of Ad-TACI-
Ig/CTLA4-Ig to induce improvement of established disease in
NZB/W F1mice with prolongation of life span and in some cases
even reversal of proteinuria. As in the younger NZB/W F1mice,
the major effect of Ad-TACI-Ig/CTLA4-Ig was a decrease in the
frequency of B cells accompanied by a sustained decrease in IgM
and a temporary decrease in IgG anti-DNA Abs. Thus, even in
older autoimmune mice, B cell survival and maintenance of high
circulating autoantibody levels are BAFF and APRIL dependent.
The requirement for CTLA4-Ig to mediate the observed effect on
IgG anti-DNA Abs needs to be examined further, but it is possible
that both BAFF/APRIL and T cell cytokines contribute to the sur-
vival of plasmablasts (28).
The findings reported in this study with TACI-Ig treatment of
NZB/W F1mice are in contrast to previous studies using other
costimulatory blocking reagents. In preventive studies neither
CTLA4-Ig, anti-CD40L, nor the combination of the two agents
induced either a change in serum levels of Ig or a decrease in titers
of IgM anti-DNA Abs, even in young mice (17, 20, 31). In con-
trast, these treatments inhibited class switching and somatic mu-
tation and appeared to alter selection of autoreactive V region
genes, consistent with their effect on delivery of T cell help to B
cells (37). In remission induction studies, a 2-wk course of com-
bination anti-CD40L and CTLA4-Ig did not induce remission in
nephritic NZB/W F1mice (22). The combination of CTLA4-Ig and
cyclophosphamide induced remission in nephritic mice, but it did
not alter serum Ig or autoantibody levels (22). Anti-CD40L treat-
ment of 26-wk-old mice did result in a decrease in the titer of IgG
anti-DNA Abs (31), but this took much longer to occur than the 1
wk effect with TACI-Ig treatment, consistent with an effect of anti-
CD40L on an earlier autoreactive precursor rather than on the ef-
fector B cell (31). Similar findings were reported in human patients
treated with anti-CD40L Ab (38). Taken together, our findings are
consistent with the hypothesis that a large amount of the abnor-
mally high circulating autoantibody levels, even in older NZB/W
F1mice, derives from plasma cells that are at least in part depen-
dent for survival on signals from BAFF and/or APRIL. TACI-Ig
does not, however, appear to affect memory B cells, and the rapid
rebound of IgG anti-dsDNA to pre-treatment levels once TACI-Ig
has cleared from the serum might reflect the generation of new
plasma cells from the memory compartment, which appears func-
tionally resistant to BAFF blockade.
As large numbers of early plasmablasts are found in the periph-
eral blood of patients with active SLE (39), blockade of BAFF and
APRIL may be an effective means of rapidly depleting these cells
during an acute flare. Once TACI-Ig is no longer present in the
serum, treated mice are capable of mounting an effective response
to a T cell-dependent Ag, and established memory cells can be
readily reactivated. However, the profound and prolonged effect of
TACI-Ig on spleen size, B cell number, and serum levels of IgM
raises concerns about its immunosuppressive potential. In view of
these considerations, additional experiments are needed to deter-
mine the optimal means of BAFF blockade and to define potential
toxicities so that this strategy for treatment can be safely used in
humans with autoimmune diseases.
We thank Dr. H. Keiser for critical reading of the manuscript.
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