The influence of effector T cells and Fas ligand on lupus-associated B cells.
ABSTRACT Circulating autoantibodies against dsDNA and chromatin are a characteristic of systemic lupus erythematosus in humans and many mouse models of this disease. B cells expressing these autoantibodies are normally regulated in nonautoimmune-prone mice but are induced to secrete Abs following T cell help. Likewise, anti-chromatin autoantibody production is T cell-dependent in Fas/Fas ligand (FasL)-deficient (lpr/lpr or gld/gld) mice. In this study, we demonstrate that Th2 cells promote anti-chromatin B cell survival and autoantibody production in vivo. FasL influences the ability of Th2 cells to help B cells, as Th2-gld/gld cells support higher titers of anti-chromatin Abs than their FasL-sufficient counterparts and promote anti-chromatin B cell participation in germinal centers. Th1 cells induce anti-chromatin B cell germinal centers regardless of FasL status; however, their ability to stimulate anti-chromatin Ab production positively correlates with their level of IFN-gamma production. This distinction is lost if FasL-deficient T cells are used: Th1-gld/gld cells promote significant titers of anti-chromatin Abs regardless of IFN-gamma production levels. Thus, FasL from effector T cells plays an important role in determining the fate of anti-chromatin B cells.
- SourceAvailable from: onlinelibrary.wiley.com[Show abstract] [Hide abstract]
ABSTRACT: The maintenance of B-cell anergy is essential to prevent the production of autoantibodies and autoimmunity. However, B-cell extrinsic mechanisms that regulate B-cell anergy remain poorly understood. We previously demonstrated that regulatory T (Treg) cells are necessary for the maintenance of B-cell anergy. We now show that in Treg-cell-deficient mice, helper T cells are necessary and sufficient for loss of B-cell tolerance/anergy. In addition, we show that the absence of Treg cells is associated with an increase in the proportion of CD4(+) cells that express GL7 and correlated with an increase in germinal center follicular helper T (GC-T(FH) ) cells. These GC-T(FH) cells, but not those from Treg-cell-sufficient hosts, were sufficient to drive antibody production by anergic B cells. We propose that a function of Treg cells is to prevent the expansion of T(FH) cells, especially GC-T(FH) cells, which support autoantibody production.European Journal of Immunology 07/2012; 42(10):2597-607. · 4.52 Impact Factor
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ABSTRACT: Autoimmunity results from loss of mechanisms controlling self-reactivity. Autoimmune disorders play an increasingly important role and CD40-CD40 ligand (CD40L) interaction on immunocompentent cells is able to break established immunotolerance. To study the effects of the calcineurin-inhibitor FK506 on CD40L-induced systemic autoimmunity, CD40L transgenic (tg) mice, which spontaneously develop a mixed connective tissue-like disease, were treated with FK506 after onset of overt autoimmunity. Interestingly, FK506-treated CD40L tg mice showed significantly reduced autoimmune dermatitis scores and markedly decreased numbers of lesional infiltrating leukocytes. This finding was associated with diminished lymphadenopathy induced by FK506 treatment. Furthermore, FK506 suppressed the development of cytotoxic/autoreactive CD8(+) T cells as evidenced by the reduced expression of T cell activation markers in treated CD40L tg mice. Importantly, FK506 induced a significant reduction in autoantibody titers in the serum of CD40L tg animals. As CD40L tg mice develop nephritis leading to loss of renal function proteinuria was determined after FK506 injections. Notably, FK506 treatment re-established renal function as indicated by significantly reduced uric protein concentrations of treated CD40L tg mice. Together, these findings show the beneficial therapeutic effects of FK506 for the treatment of CD40L-induced autoimmunity. Additionally, these results demonstrate that FK506 is able to suppress ongoing severe autoimmune responses.Journal of Investigative Dermatology 06/2006; 126(6):1307-15. · 6.37 Impact Factor
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ABSTRACT: The absence of regulatory T cells (Tregs) results in significant immune dysregulation that includes autoimmunity. The mechanism(s) by which Tregs suppress autoimmunity remains unclear. We have shown that B cell anergy, a major mechanism of B cell tolerance, is broken in the absence of Tregs. In this study, we identify a unique subpopulation of CD4(+) Th cells that are highly supportive of Ab production and promote loss of B cell anergy. Notably, this novel T cell subset was shown to express the germinal center Ag GL7 and message for the B cell survival factor BAFF, yet failed to express markers of the follicular Th cell lineage. We propose that the absence of Tregs results in the expansion of a unique nonfollicular Th subset of helper CD4(+) T cells that plays a pathogenic role in autoantibody production.The Journal of Immunology 04/2012; 188(11):5223-6. · 5.36 Impact Factor
The Influence of Effector T Cells and Fas Ligand on
Lupus-Associated B Cells1
Michele L. Fields, Simone A. Nish, Brian D. Hondowicz, Michele H. Metzgar, Gina N. Wharton,
Andrew J. Caton, and Jan Erikson2
Circulating autoantibodies against dsDNA and chromatin are a characteristic of systemic lupus erythematosus in humans and
many mouse models of this disease. B cells expressing these autoantibodies are normally regulated in nonautoimmune-prone mice
but are induced to secrete Abs following T cell help. Likewise, anti-chromatin autoantibody production is T cell-dependent in
Fas/Fas ligand (FasL)-deficient (lpr/lpr or gld/gld) mice. In this study, we demonstrate that Th2 cells promote anti-chromatin B cell
survival and autoantibody production in vivo. FasL influences the ability of Th2 cells to help B cells, as Th2-gld/gld cells support
higher titers of anti-chromatin Abs than their FasL-sufficient counterparts and promote anti-chromatin B cell participation in
germinal centers. Th1 cells induce anti-chromatin B cell germinal centers regardless of FasL status; however, their ability to
stimulate anti-chromatin Ab production positively correlates with their level of IFN-? production. This distinction is lost if
FasL-deficient T cells are used: Th1-gld/gld cells promote significant titers of anti-chromatin Abs regardless of IFN-? production
levels. Thus, FasL from effector T cells plays an important role in determining the fate of anti-chromatin B cells. The Journal
of Immunology, 2005, 175: 104–111.
tosus (1). Using the VH3H9 Ig transgenic (Tg)3model, a popula-
tion of anti-chromatin B cells (VH3H9/V?1) has been character-
ized in both healthy and autoimmune mice (2–7). In healthy mice,
these anti-chromatin B cells persist in the periphery but their Abs
are undetected (2, 5). They have a decreased life span, are pre-
dominantly developmentally arrested and activated, and localize to
the edges of the B cell follicles near the T cell areas in the spleen
(5, 8). We hypothesize that this phenotype is a consequence of
chronic exposure to Ag, in the absence of T cell help (9–13).
To dissect the responses of anti-chromatin B cells to CD4?Th
cells, we established an in vivo model of cognate interaction be-
tween these two cell types. Mice engineered to express the neoself
Ag hemagglutinin (HA) on MHC class II-bearing cells (including
B cells) were mated to VH3H9 Ig Tg mice (14, 15). Anti-HA
CD4?T cells from TCR Tg mice and HA-expressing anti-
chromatin B cells were then transferred together into a third-party
recipient mouse and their fates tracked (16, 17). Using this strat-
egy, we have demonstrated that anti-chromatin B cells from
healthy mice respond to CD4?Th cells in vivo by producing au-
toantibodies (16). These findings contrast with those obtained from
the hen-egg lysozyme model of B cell tolerance (18, 19), where
ntibodies that bind dsDNA and chromatin are a serologic
hallmark of systemic lupus erythematosus and are found
in several murine models of systemic lupus erythema-
autoreactive anti-hen-egg lysozyme B cells were shown to be re-
sistant to CD4?T cell help (20–23). Importantly, Fas/ Fas ligand
(FasL) interactions mediated this resistance (20, 21, 24).
Both Th1 and Th2 cells can help nonautoreactive B cells to
produce Abs (25–27), although Th2 cells appear more efficient in
this regard (28–31). Th1 cells reportedly express higher levels of
FasL than Th2 cells (32–35), which could modulate T-B interac-
tions by either limiting the availability of Th cells (32, 33, 35)
and/or by direct killing of the autoreactive B cells (20, 21). Fur-
thermore, Th1 cells may be more susceptible to Fas-mediated
death even in cases where they express comparable levels of
FasL (32, 33, 35).
Once activated, B cells can follow a number of differentiation
pathways–toward short-lived Ab-forming cells (AFCs), memory B
cells, or long-lived AFCs. Although much has been learned about
the transcription factors that govern the fate decisions of B cells
(36), less is known about the precise cues that determine these
decisions. Given the critical role that CD4?T cells play in auto-
antibody production, and the seemingly contradictory data on au-
toreactive B cell responses to T cell help (20–23), we have inves-
tigated the impact of Th1 and Th2 cells, with and without FasL, on
anti-chromatin B cells. We report here the differential capabilities
of T effector cells to influence anti-chromatin B cells in vivo, and
suggest that FasL may play a role in controlling the differentiation
pathways and magnitude of the autoreactive B cell response.
Materials and Methods
TS1 BALB/c (FasL-sufficient or -deficient) and VH3H9 Tg/HACII/Ig??/?
mice were bred in specific-pathogen-free conditions at The Wistar Institute
under the approval and supervision of the Institutional Animal Care and
Use Committee (IACUC), and genotyped as described (2, 6, 16, 17, 37).
Similarly, site-directed VH3H9 Tg BALB/c mice in which the VH3H9 Tg
was targeted to the JHlocus (Refs. 38–40; generously provided by Dr. M.
Weigert (University of Chicago, Chicago, IL) were bred to produce site-
directed VH3H9 Tg/HACII/Ig??/?mice. CB17 mice were purchased from
Charles River Laboratories. Only young (6–12 wk old) TS1 BALB/c-gld/
gld mice were used. All other mice were used at 6–16 wk, and both gen-
ders were used.
The Wistar Institute, Room 276, 3601 Spruce Street, Philadelphia, PA 19104
Received for publication January 19, 2005. Accepted for publication April 12, 2005.
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.
1Funding has been provided by the National Institutes of Health (AI32137, AR47913,
and 2T32AI007518) and the Commonwealth Universal Research Enhancement Pro-
gram, PA Department of Health.
2Address correspondence and reprint requests to Dr. Jan Erikson, The Wistar Insti-
tute, Room 276, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address:
3Abbreviations used in this paper: Tg, transgenic; HA, hemaglutinin; FasL, Fas li-
gand; AFC, Ab-forming cell; AP, alkaline phosphatase; PNA, peanut agglutinin; GC,
germinal center; EFF, extrafollicular foci.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
Th1 and Th2 cell cultures
TS1 BALB/c or TS1 BALB/c-gld/gld lymph nodes were depleted (?90%)
of CD8?cells using anti-CD8 Dynalbeads (Dynaltech). A total of 0.5 ?
106CD8-depleted lymphocytes were then cultured for Th1/Th2 deviation
as previously described (41). rIL-12 was a generous gift from Dr. G.
Trinchieri (National Institutes of Health, Bethesda, MD) or was purchased
from R&D Biosystems or PeproTech. Cells received fresh media contain-
ing IL-2 at days 3 and 5, and were rested in the absence of IL-2 at day 7.
At day 9, cells were harvested and an aliquot cultured with PMA and
ionomycin (Sigma-Aldrich) in the presence of brefeldin A (Cytofix/Cyto-
perm kit; BD Pharmingen) for another 4–6 h. Cells were stained for CD4
expression, fixed, permeabilized, and stained for intracellular cytokines,
using anti-IFN-?-FITC, anti-IL-4-PE, and/or anti-IL-10-PE (BD Pharmin-
gen) (42). The cells that were not restimulated with PMA/ionomycin were
purified by centrifugation with Lympholyte M (Cedarlane Laboratories)
T cell/influenza virus injections
A total of 5–10 ? 106Th1- or Th2-deviated CD4?cells from in vitro
cultures were suspended in sterile PBS with 1000 hemagglutinating units
(43) of purified PR8 influenza virus (15) and injected i.v.
Anti-chromatin B cell injections
Splenocytes from VH3H9 Tg/HACII/Ig??/?or site-directed VH3H9 Tg/HA-
CII/Ig??/?mice were depleted of RBC and an aliquot was stained by flow
cytometry to determine the frequency of anti-chromatin B cells (B220?
Ig?1?). CB17 recipient mice (allotype Igb) were injected with spleen prepa-
rations containing 4–10 ? 106anti-chromatin B cells (allotype Iga). Con-
trol mice received B cells without previous injection of Th cells or virus.
Blockade of CD40-CD154 interactions
Mice were i.p. injected with 250 ?g of purified anti-CD154 mAb (MR1) (a
kind gift of Dr. R. Noelle, Dartmouth Medical School, Lebanon, NH) on
the same day as B cell transfer, and 3 days afterward.
Chromatin (a generous gift of Dr. M. Monestier, Temple University, Phil-
adelphia, PA) was diluted to 2 ?g/ml and plated overnight as described
(44). ELISAs were done as described (45) with the following modifica-
tions: the block used was 1% BSA/PBS/azide, developing Abs were anti-
Ig?1, anti-IgMa, anti-IgG1, or anti-IgG2aa(all biotinylated, BD Pharmin-
Biotechnology Associates). Plates were developed for 14–18 h with Im-
munoPure PNPP (Pierce) as the substrate. OD values were recorded and
background values were subtracted out (background was defined as the OD
value generated by a hybridoma supernatant of irrelevant specificity, typ-
ically ?0.07). All developing Abs were allotype-marked (Iga) except for
IgG1, (due to poor sensitivity of the anti-IgG1aAb in ELISAs). Although
the IgG1 reagent was not allotype-marked, sera from uninjected CB17
mice showed no significant staining above background for IgG1 anti-
chromatin Abs. Points derived from the linear range of the ELISAs were
used for generating graphs. For studies examining whether long-lived
plasma cells are generated when T cell help is provided, mice were serially
bled and their sera tested by ELISA. When anti-chromatin OD values re-
turned to baseline levels for a mouse, they were no longer bled or tested.
A total of 0.5–1 ? 106splenocytes were surface stained (46) using the
following Abs: anti-B220-FITC (RA3-6B2), anti-CD4-PE (GK1.5), anti-
IgMa-PE or -bio (DS-1), anti-Ig?1-bio (R11-153), IgDa-bio (217–170), IgG1a-
bio (10.9), IgG2aa-bio (8.3) (BD Pharmingen), and 6.5-biotin (grown as su-
pernatant and biotinylated). Biotinylated peanut agglutinin (PNA; Vector
Laboratories) was also used to mark germinal center (GC) B cells.
Determination of cell recovery
The frequency of IgMa?Ig?1?B cells or CD4?6.5?T cells in the spleen
was determined by flow cytometry and multiplied by the total number of
live splenocytes to determine the absolute number of cells. The percent
recovery of transferred B or T cells was determined by dividing the abso-
lute number of cells recovered by the number of cells injected.
Spleens were frozen, sectioned, and stained (5) using the following Abs:
anti-CD4-bio (GK1.5), anti-CD22-FITC or -bio (Cy34.1), anti-IgMa-FITC
(DS-1), anti-IgG1a-bio (10.9), anti-IgG2aa-bio (8.3) (BD Pharmingen),
and/or PNA-bio (Vector Laboratories). Secondary reagents were anti-
FITC-AP, anti-FITC-HRP, streptavidin-AP, or streptavidin-HRP (South-
ern Biotechnology Associates). Developed slides were read by multiple (at
least four) investigators without prior knowledge of the experimental
Statistical significance was determined via the unpaired, two sample Stu-
dent’s t test provided by Microsoft Excel software unless otherwise noted.
Significance was ascribed when p ? 0.05.
The VH3H9 H chain paired with the V?1 L chain generates an Ab
that binds dsDNA and chromatin (47, 48). Therefore, the VH3H9
Tg can be used to directly track autoreactive B cells in vivo by stain-
ing for endogenous V?1. To test the impact of cognate T cell help on
anti-chromatin B cells in vivo, HA-bearing anti-chromatin B cells
were injected into third-party recipient CB17 mice that had received
distinct subsets of anti-HA CD4?T cells (Fig. 1; Ref. 16).
In vitro polarization of anti-HA CD4?T cells from FasL-
sufficient and -deficient mice
Before injection, anti-HA CD4?T cells from FasL-sufficient or
-deficient mice were polarized into either the Th1 or Th2 subset
(Fig. 2). Th2 cells from FasL-sufficient and -deficient mice pro-
injected with influenza virus and anti-HA CD4?T cells (from wild-type or
FasL-deficient mice) that were cultured in vitro under Th1 or Th2 condi-
tions. The following day, anti-chromatin B cells (?-chromatin-B); IgMa
allotype from either VH3H9/HACII/Ig??/?or site-directed VH3H9 Tg/
HACII/Ig??/?BALB/c mouse spleens) were injected. Mice were analyzed
on day 8.
Experimental protocol. CB17 mice (allotype IgMb) were
cells. Th1 or Th2 cells were generated in vitro from
either TS1 Tg BALB/c or TS1 Tg BALB/c-gld/gld cells
and tested for production of IFN-? and either IL-4 (top)
or IL-10 (bottom). The range for cells producing IFN-?
from Th1 cultures was 2–70%. Thus, Th1 cells are
grouped into high or low IFN-?-producing categories.
Graphs are representative of n ? 4 for each cell type.
Cytokine production by in vitro-deviated
105The Journal of Immunology
duced IL-4 (20–40% of cells) and IL-10 (3–10% of cells) (Fig. 2).
In addition, a small population of Th2-gld/gld cells produced
IFN-? (Fig. 2, 2.5% of cells vs 0.53% of cells from FasL-sufficient
mice, p ? 0.01).
Th1-deviated cells derived from wild-type or gld/gld mice pro-
duced little or no IL-4 or IL-10, and varied widely according to the
amount of IFN-? they made (Fig. 2). One group of Th1 cells had
40–70% of the cells producing IFN-? without detectable IL-4 or
IL-10, and such cells are termed IFN-?highTh1 cells. In contrast,
the second group, which also did not make IL-4 or IL-10, yielded
fewer IFN-?-producing cells (2–35%), and less IFN-? per cell, as
visualized by lower intracellular IFN-? fluorescent intensity (Fig.
2). This second group is termed IFN-?lowTh1 cells. This variation
in IFN-? production proved to be significant in terms of the ability
of the T cells to help anti-chromatin B cells (see below) and ap-
peared dependent upon the source of rIL-12 in cultures. To directly
examine the effects of limiting IL-12 on deviated Th cells, cultures
were set up in the absence of exogenous rIL-12, but in the presence
of anti-IL-4 Ab to prevent acquisition of a Th2-like phenotype
(cells resulting from such cultures are termed IFN-?low?cells). A
low frequency of CD4?T cells (2–8%) from these cultures pro-
duced IFN-?, with low levels of IFN-? per cell, and ?1% pro-
duced IL-4 or IL-10. Both the IFN-?highand IFN-?lowintracellular
cytokine levels are within the range of those reported by others for
Th1-deviated cultures (41, 42, 49, 50).
Anti-chromatin Ab production is induced by Th2 cells and IFN-
?highTh1 cells in a CD40-dependent manner
Th2 cells promoted anti-chromatin autoantibodies from the trans-
ferred B cells (Fig. 3). Autoantibody production from mice given
Th1 cells was variable and correlated (p ? 0.01) with the level of
IFN-? produced by the Th1 cells before their injection. IFN-?high
Th1 cells induced levels of anti-chromatin autoantibodies similar
to those seen in Th2 recipients, whereas levels in recipients of
IFN-?lowand IFN-?low?Th1 cells were not above the baseline
(Fig. 3). The observation that IFN-? levels affect the ability of Th1
cells to help B cells may resolve a controversy regarding the ability
of Th1 cells to serve as helpers (25–31), as laboratories using
distinct in vitro deviation protocols may generate differing fre-
quencies of IFN-?-producing Th1 cells.
CD40-CD154 interactions play a vital role in Th cell activity,
including autoimmune settings (51–54). Likewise, the blocking
anti-CD154 mAb (MR1) abrogated anti-chromatin Ab production
stimulated by either Th2 or IFN-?highTh1 cells (Fig. 3).
FasL influences the quality of T cell help for anti-chromatin
Injection of Th2-deviated gld/gld cells enhanced the production of
anti-chromatin Abs in recipient mice compared with the titers ob-
served with Th2 cells derived from wild-type mice (Fig. 3). There
was no difference in anti-chromatin Ab titers between IFN-?high
Th1 cells and IFN-?highTh1-gld/gld cells (Fig. 3, p ? 0.34),
whereas IFN-?lowTh1-gld/gld T cells promoted higher titers of
anti-chromatin Abs than their IFN-?lowFasL-sufficient counter-
parts (Fig. 3).
autoantibody production. Sera were assayed by ELISA for IgMaanti-
chromatin Abs (serum dilution 1/100). Bar graphs show mean OD value for
each experimental condition, ?SEM; ?, significant difference (p ? 0.05).
Th1 recipients are divided according to whether they received IFN-?highor
IFN-?lowTh1 cells. Also shown are results from mice that received T cells
from either FasL-sufficient or -deficient mice, cultured with anti-IL-4 and
no exogenous rIL-12 (marked as IFN-?low?). All graphs with an average
above 1.0 are significantly different from the condition with B cells alone.
Ø, B cells alone. Sample sizes: B cells alone, n ? 4; Th2, n ? 10; Th2 ?
MR1, n ? 3; IFN-?highTh1, n ? 7; IFN-?highTh1 ? MR1, n ? 3; IFN-
?lowTh1, n ? 7; IFN-?low?Th1, n ? 6; IFN-?highTh1-gld/gld, n ? 3;
IFN-?lowTh1-gld/gld, n ? 3; IFN-?low?Th1-gld/gld, n ? 3.
Th2 cells and IFN-?highTh1 cells induce anti-chromatin
isotype-switched anti-chromatin Abs. Sera were assayed
by ELISA for anti-chromatin Abs for (A) total Ig?1, (B)
IgMa, (C) IgG1, and (D) IgG2aa. OD values were re-
corded at the following serum dilutions for the ELISAs:
1/400 for Ig?1, 1/800 for IgMa, and 1/100 for both IgG1
and IgG2aa. Bar graphs show mean values ? SEM; ?,
significant difference (p ? 0.05) between experimental
groups. ?, Significant difference compared with mice
given B cells alone. Ø, B cells alone. Sample sizes: B
cells alone, n ? 10; Th2, n ? 7; IFN-?highTh1, n ? 4;
Th2-gld/gld, n ? 6; IFN-?highTh1-gld/gld, n ? 6.
Th2 cells and IFN-?highTh1 cells induce
106IMPACT OF EFFECTOR T CELLS AND FasL ON LUPUS B CELLS
Anti-chromatin B cells undergo isotype switching in response to
Th2- or Th1-type help
The site-directed VH3H9 Tg was used to monitor anti-chromatin
isotype switching. CB17 mice that received Th2 cells or IFN-?high
Th1 cells and site-directed Tg anti-chromatin B cells had detect-
able titers of Ig?1 anti-chromatin Abs (Fig. 4A). These Abs in-
cluded not only IgMaAbs (Fig. 4B) but also isotype-switched Abs
(Fig. 4, C and D). IFN-?highTh1 cell help resulted in higher titers
of IgG2aaautoantibodies than mice given either Th2 cells or B
cells alone. Th2 recipients produced high levels of IgG1 anti-
chromatin Abs and low, but also significant (relative to recipients
of B cells alone), titers of IgG2aaanti-chromatin Abs. Like what
was observed using the non-site-directed VH3H9 Tg donors, IFN-
?lowTh1 cells did not induce anti-chromatin Abs.
Th2-gld/gld cells induced higher titers of Ig?1 anti-chromatin
Abs from site-directed VH3H9 Tg B cells than their FasL-sufficient
counterparts (Fig. 4). This increase included higher titers of the
IgMaand IgG2aaisotypes, but similar levels of the IgG1 isotype.
The increased level of IFN-? production by gld/gld-derived Th2
cells (Fig. 2) may account for the higher IgG2a isotype anti-
chromatin Abs (25). In contrast, the absence of functional FasL on
IFN-?highTh1 cells did not have an effect on the levels of Ig?1
anti-chromatin Abs produced or their isotype distribution (Fig. 4,
p ? 0.05 for Ig?1, IgMa, or IgG2aaELISAs).
Anti-chromatin B cell recovery correlates with autoantibody
In the absence of exogenous Th cells, very few anti-chromatin B
cells remained 7 days after transfer (Fig. 5), consistent with their
rapid in vivo turnover rate (8). Both Th2 and IFN-?highTh1 cells
supported anti-chromatin B cell recovery and this was dependent
upon CD40-CD154 interactions (Fig. 5). FasL-deficient Th2 and
IFN-?highTh1 cells promoted similar recoveries compared with
their wild-type counterparts. In contrast, IFN-?lowTh1 cells de-
rived from gld/gld mice promoted anti-chromatin B cell survival
well above that from FasL-sufficient IFN-?lowTh1 cells (Fig. 5).
Anti-chromatin B cells form AFC clusters and GCs in response
to T cell help
Immunohistochemistry was used to further examine the differen-
tiation status of the anti-chromatin B cells (Fig. 6). Clusters of
extrafollicular anti-chromatin B cells were observed in the spleens
of mice given Th2 cells or IFN-?highTh1 cells (Fig. 6A). These
cells exhibited substantially higher Ig staining levels than follicular
B cells, and expressed CD138 (data not shown), a marker of AFCs.
Consistent qualitative differences in the AFCs are apparent, with
AFCs in Th2-recipient mice being more tightly clustered in ex-
trafollicular foci (EFF), than those in mice given IFN-?highTh1
cells (Fig. 6A). Anti-chromatin AFCs were not observed in mice
that received IFN-?lowTh1 cells, consistent with the absence of
anti-chromatin Abs (Fig. 6B).
Few GCs are visible in CB17 mice given only anti-chromatin B
cells, whereas mice also injected with Th cells and virus developed
GCs in at least 50% of the B cell follicles (Fig. 6, A and B) and this
was blocked by MR1 (data not shown). Although GCs were ob-
served in Th2 recipients, they do not include the transferred Iga
cells and are likely a consequence of endogenous CB17 B cells
(Igb) responding to virus. In contrast, recipients of IFN-?highTh1
cells have many instances of IgMacells in B cell follicles, often
localizing in GCs (Fig. 6A, arrows). Strikingly, the few transferred
anti-chromatin B cells that persist in IFN-?lowTh1 recipients are
primarily associated with GCs (Fig. 6B). Flow cytometry was used
to quantitate the higher frequency of GCs in Th1 recipients (IFN-
?highand IFN-?low) compared with Th2 recipients (Fig. 7).
When FasL-deficient Th2 cells were administered, tight clusters
of anti-chromatin AFCs were observed, similar to their FasL-suf-
ficient counterparts (Fig. 6A). Anti-chromatin B cells from Th2-
gld/gld recipients, however, were also present in GCs with a higher
frequency compared with mice given FasL-sufficient Th2 cells
(Fig. 6A, arrows, and Fig. 7). Recipients of IFN-?highTh1-gld/gld
T cells had anti-chromatin GCs as did their wild-type counterparts,
but their EFF were even more diffuse. Most strikingly, IFN-?low
B cells given T cell help. A, Flow cytometry was used to
determine the percent recovery of transferred anti-
chromatin B cells (Ig?1?IgMa?cells). Shown are rep-
resentative graphs. B, Bar graphs show mean values ?
SEM; ?, significant difference (p ? 0.05) between ex-
perimental groups. ?, Significant difference compared
with mice given B cells alone. Ø, B cells alone. Sample
sizes: B cells alone, n ? 12; Th2, n ? 16; Th2 ? MR1,
n ? 3; Th2-gld/gld, n ? 8; IFN-?highTh1, n ? 10;
IFN-?highTh1 ? MR1, n ? 3; IFN-?lowTh1, n ? 10;
IFN-?highTh1-gld/gld, n ? 4; IFN-?lowTh1-gld/gld,
n ? 6.
Recovery of transferred anti-chromatin
107The Journal of Immunology
Th1-gld/gld cells induced not only anti-chromatin GCs but also
AFCs (data summarized in Table I).
With site-directed VH3H9 Tg mice used as a source of anti-
chromatin B cells, in conjunction with T cell help, isotype-
switched AFCs were detected. As was observed in the serum, re-
cipients of Th2 cells had anti-chromatin EFF mainly comprised of
IgMaand IgG1acells, with rare IgG2aacells, whereas recipients of
IFN-?highTh1 cells have EFF containing both IgMaand IgG2aa
but not IgG1acells (Fig. 6C). Minimal Igacells were visualized in
recipients of site-directed VH3H9 Tg B cells in the absence of
exogenous Th cells (data not shown).
Transferred Th cells can be tracked via clonotypic staining
To test the fate of the transferred Th1 or Th2 cells, splenocytes of
recipient mice were stained with the 6.5 clonotypic Ab (Fig. 8). A
distinct population of CD4?6.5?cells could be detected in spleens
of mice given Th cells, but not in the absence of exogenous Th
cells (Fig. 8A). CD40-CD154 blockade significantly inhibited re-
covery of both Th2 and IFN-?highTh1 cells (Fig. 8B). Of the
CD4?Th cells, IFN-?highTh1 cells displayed the highest level of
cell recovery; notably, recovery of these cells was decreased when
they were derived from FasL-deficient mice (Fig. 8B). This sug-
gests that, under some conditions, FasL may provide a positive
signal, as was suggested for CD4?cells in vitro (55).
Anti-chromatin Abs do not persist
To determine whether long-lived anti-chromatin plasma cells are
generated when T cell help is provided, CB17 mice were injected
with IFN-?highTh1 or Th2 cells and anti-chromatin B cells from
either VH3H9 Tg or site-directed VH3H9 Tg donors, and then se-
rially bled for up to 10 wk. In all mice given Th cells, anti-
chromatin Ab titers peaked at days 8–11 and then declined such
that by day 36, levels were similar to those in uninjected mice and
mice given B cells without exogenous T cells (Table II).
The abilities of Th1 and Th2 cells, with and without FasL, to elicit
autoantibody production, promote autoreactive B cell survival, and
trigger participation in the GC reaction in vivo are summarized in
Table I. These studies were done using anti-chromatin B cells from
both VH3H9 Tg and site-directed VH3H9 Tg mice because of dif-
ferences documented in signaling between IgM/IgD and IgG re-
ceptors (56, 57) as well as disparities seen between randomly in-
tegrated transgenes and those that have been targeted to the JH
locus (58). The only distinction we detected between the site-di-
rected and the non-site-directed transgenes was that the former
generated isotype-switched Abs in response to T cell help.
Although polarized CD4?T cell populations are typically de-
scribed by the production of Th1 vs Th2 cytokines, it has been
cells given T cell help. Mice received deviated Th cells,
virus, and anti-chromatin B cells from VH3H9/HACII/
??/?mice (A and B), or site-directed VH3H9 Tg/HACII/
??/?mice (C), and were sacrificed 7 days after B cell
transfer. Serial spleen sections were stained with Abs to
CD22 and either IgMa, IgG1a, IgG2aa, or PNA, as in-
dicated. Pictures are representative of n ? 5 for each
Splenic localization of anti-chromatin B
108 IMPACT OF EFFECTOR T CELLS AND FasL ON LUPUS B CELLS
documented that within these populations a wide range of cyto-
kines can be produced (29, 59). In this study, this variability was
most pronounced for the Th1 polarized cells and proved to be
significant in terms of B cell fate. Although Th2 cells consistently
induced high titers of anti-chromatin Abs in a CD40-dependent
manner, the ability of Th1-deviated cells to help anti-chromatin B
cells correlated with T cell IFN-? production. IFN-?highTh1 cells
induced anti-chromatin AFCs and GCs, but IFN-?lowTh1 cells
promoted only GCs, and a much lower degree of anti-chromatin B
cell recovery. Unlike the tightly clustered anti-chromatin AFCs ob-
served in Th2 recipients, the AFCs found under IFN-?highTh1 con-
mice (4, 60–62). This phenotype is even more pronounced in recip-
ients of Th1-gld/gld cells, which is consistent with the possibility that
the T cells driving the autoimmune response in Fas/FasL-mutant mice
are of the Th1 type (61–65). The significance of the distinct local-
ization sites for AFCs has not been determined, but in one model,
these extrafollicular cells were somatically mutated (60).
FasL plays an important role in influencing the outcome of T
cell help for anti-chromatin B cells. Relative to their FasL?coun-
terparts, Th2-gld/gld cells induced higher titers of IgM and IgG2a
isotype anti-chromatin Abs, and more frequent anti-chromatin B
cell GCs. Strikingly, the inability of IFN-?lowTh1 cells to induce
anti-chromatin Ab production appears dependent on FasL, as only
FasL-deficient IFN-?lowTh1 cells supported significant anti-
chromatin B cell survival and high titers of anti-chromatin Abs.
FasL may alter anti-chromatin B cell fate indirectly by limiting
T cell help (32, 33, 35) or by direct B cell killing (20). T cell loss
due to FasL expression, however, was not observed in our study.
IL-4, which has been shown to protect B cells from Fas-mediated
death (66), may be responsible for the similar recovery of anti-
chromatin B cells in recipients of Th2 FasL-sufficient and -defi-
cient cells (see Fig. 5). The finding that IFN-?high, but not IFN-
?low, Th1 cell help results in anti-chromatin Ab production and B
cell survival, prompts us to consider that, like IL-4 (66), IFN-?
with 6.5 (clonotype) and anti-CD4 to determine recovery of transferred Th
cells in recipient mice. Plots are representative of n ? 10 mice. B, The
absolute number of CD4?clonotype?Th cells was determined and the
percent recovery of transferred cells was plotted. Bar graphs depict mean
values ? SEM; ?, significant difference (p ? 0.05). Sample sizes: Th2, n ?
14; Th2 ? MR1, n ? 3; Th2-gld/gld, n ? 9; IFN-?highTh1, n ? 10; Th1
? MR1, n ? 3; IFN-?lowTh1, n ? 8; IFN-?highTh1-gld/gld, n ? 4;
IFN-?lowTh1-gld/gld, n ? 6.
Th cell recoveries in the spleen. A, Spleen cells were stained
Table I. Fate of transferred anti-chromatin B cells and Th cells
ConditionTh cellsAnti-Chromatin B Cells
Anti-chromatin B cells
No exogenous T cells
aData were obtained from analysis at day 8 (7 days after B cell transfer). With
regard to cell recovery and serum autoantibody levels, “?”, “?”, and “??” indicate
statistically significant differences (p ? 0.05).
bN/A, not applicable.
cSerum autoantibody titers from Th2-gld/gld recipients were significantly higher
than their FasL-sufficient counterparts.
Table II. T cell-induced anti-chromatin Abs do not persista
Anti-chromatin Abs (IgMa)
B cells alone (n ? 4)
IFN-?highTh1 (n ? 5)
IFN-?highTh1-gld/gld (n ? 6)
Th2 (n ? 4)
Th2-gld/gld (n ? 5)
Site-directed VH3H9 Tg donors-anti-chromatin Abs (Ig?1)
B cells alone (n ? 4)
IFN-?highTh1 (n ? 2)
IFN-?highTh1-gld/gld (n ? 4)
Th2 (n ? 4)
Th2-gld/gld (n ? 3)
aCB17 mice received Th cells, virus, and anti-chromatin B cells from VH3H9
Tg/HACII/??/?or site-directed VH3H9 Tg/HACII/??/?mice, and were bled at in-
tervals thereafter. IgMaor Ig?1 anti-chromatin Abs were detected via ELISA. Days
denote number of days after T cell injection. The “?” indicates significance above
mean titer of sera from uninjected CB17 mice. NT, not tested.
was used to determine frequency of IgMa?cells that were PNA?in mice
given T cell help. Bar graphs show mean values and circles represent
values from individual mice. ?, Significant difference (p ? 0.05). Sample
sizes: Th2, n ? 10; Th2-gld/gld, n ? 7; IFN-?highTh1, n ? 10; IFN-?low
Th1, n ? 4; IFN-?highTh1-gld/gld, n ? 4; IFN-?lowTh1-gld/gld, n ? 4.
GC phenotype of anti-chromatin B cells. Flow cytometry
109The Journal of Immunology
may impart resistance to Fas-mediated death. Consistent with this
hypothesis, one report showed a synergistic effect on B cells if
IFN-? was combined with stimulatory CpG oligonucleotides (67),
but the direct effect of IFN-? on B cells remains controversial
(68–73). An alternate hypothesis is that the IFN-?lowTh1 cells
have diverged to a distinct differentiation pathway, such that they
provide unique helper functions compared with the IFN-?highTh1
cells. Because we have previously documented that undeviated Th
cells can promote autoantibody production (16), the failure of the
IFN-?lowTh1 cells to induce autoantibodies is not likely to be a
consequence of them being less differentiated.
It has been postulated that Fas-mediated killing is involved in B
cell negative selection within GCs (74–77). Our experiments pro-
vide no evidence that B cell death via FasL borne on Th cells
curtails anti-chromatin GCs: Th1 cells (both IFN-?highand IFN-
?low) induce anti-chromatin GCs with or without FasL, and al-
though Th2-gld/gld but not FasL-sufficient Th2 cells induce anti-
chromatin GCs, a difference in B cell survival is not observed.
Rather, we found a correlation between IFN-? production by Th
cells and B cell GC differentiation. In all cases where some degree
of IFN-? is produced (including Th2-gld/gld conditions), anti-
chromatin B cells are found in GCs. Notably, older Fas/FasL-de-
ficient mice have a predominance of IFN-??T cells (78), and
IFN-? is critical for autoantibody production and autoimmune dis-
ease in MRL-lpr/lpr mice (79, 80). Studies to examine the direct
effect of IFN-? on anti-chromatin B cell proliferation, activation,
and sensitivity to Fas-mediated death are under way.
Although we have documented that anti-chromatin B cells can
form GCs, there is no evidence that long-lived AFCs are generated
by the addition of T cell help (Table II). Other reports have de-
scribed anti-DNA B cells in GCs in the absence of detectable se-
rum Ab production (81, 82), suggesting a fail-safe mechanism that
guards against memory formation and long-term autoantibody pro-
duction. The observation documented here that only short-lived
AFCs are generated in healthy mice by provision of T cell help
suggests a means of curtailing the liability of autoreactive B cells.
This conclusion is tempered, however, as another study, again us-
ing a transfer model but this time with nonautoreactive B cells,
also failed to induce long-lived AFCs (26). This raises the possi-
bility that their generation may have particular requirements that
are not met under the transfer conditions used. Studies are under-
way to determine whether autoantibodies that arise naturally in
autoimmune settings are derived from long-lived or short-lived
AFCs. In the New Zealand Black/White and New Zealand M2410
models, the presence of long-lived AFCs has been documented,
and interestingly, they have been shown to reside at unique sites
(83–85). Clearly, more studies are warranted to better understand
what governs B cell fate decisions in healthy vs autoimmune set-
tings, the outcome of which may have important implications for
B cell depletion strategies currently used to treat human autoim-
mune diseases (86, 87).
We thank A. Acosta and J. Faust of The Wistar Institute Flow Cytometry
Facility; S. Alexander and A. Paga ´n for excellent technical assistance; and
R. Noelle, M. Weigert, G. Trinchieri, and M. Monestier for reagents.
The authors have no financial conflict of interest.
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111 The Journal of Immunology