of December 21, 2015.
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Both CD4 T Cell- and B Cell-Intrinsic
Chronic Graft-versus-Host Disease through
IL-21 Promotes Lupus-like Disease in
Ching Chen and Violeta Rus
Vinh Nguyen, Irina Luzina, Horea Rus, Cosmin Tegla,
2012; 189:1081-1093; Prepublished online 20
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The Journal of Immunology
IL-21 Promotes Lupus-like Disease in Chronic
Graft-versus-Host Disease through Both CD4 T Cell- and
B Cell-Intrinsic Mechanisms
Vinh Nguyen,* Irina Luzina,*,†Horea Rus,†,‡Cosmin Tegla,†,‡Ching Chen,xand
T cell-driven B cell hyperactivity plays an essential role in driving autoimmune disease development in systemic lupus eryth-
ematosus. IL-21 is a member of the type I cytokine family with pleiotropic activities. It regulates B cell differentiation and
function, promotes T follicular helper (TFH) cell and Th17 cell differentiation, and downregulates the induction of T regulatory
cells. Although IL-21 has been implicated in systemic lupus erythematosus, the relative importance of IL-21R signaling in CD4+
T cells versus B cells is not clear. To address this question, we took advantage of two induced models of lupus-like chronic graft-
versus-host disease by using wild-type or IL-21R2/2mice as donors in the parent-into-F1 model and as hosts in the Bm12→B6
model. We show that IL-21R expression on donor CD4+T cells is essential for sustaining TFHcell number and subsequent help for
B cells, resulting in autoantibody production and more severe lupus-like renal disease, but it does not alter the balance of Th17
cells and regulatory T cells. In contrast, IL-21R signaling on B cells is critical for the induction and maintenance of germinal
centers, plasma cell differentiation, autoantibody production, and the development of renal disease. These results demonstrate
that IL-21 promotes autoimmunity in chronic graft-versus-host disease through both CD4+T cell- and B cell-intrinsic mechanisms
and suggest that IL-21 blockade may attenuate B cell hyperactivity, as well as the aberrant TFHcell pathway that contributes to
lupus pathogenesis.The Journal of Immunology, 2012, 189: 1081–1093.
ronment (1). IL-21R is expressed on a variety of immune cells,
including B, T, NK, and dendritic cells (DCs), whereas IL-21
production is restricted to activated CD4+T cells, T follicular
helper (TFH) cells, Th17 cells, and NKT cells (1, 2). IL-21 promotes
the expansion of NK cells and augments their antitumor activity,
enhances CD8+T cell maturation into cytotoxic T lymphocytes,
and promotes the differentiation and expansion of TFHcells (1, 3–
5). In addition, within the T cell lineage, IL-21 regulates the re-
ciprocal differentiation of Th17 cells and regulatory T cells (Tregs)
by promoting Th17 cell expansion and inhibiting the generation and
function of induced Tregs (iTregs) (6–9). Within the B cell lineage,
IL-21 regulates B cell proliferation and survival; Ig production
and class switching, particularly to IgG1; germinal center (GC)
nterleukin-21 is a member of the type I cytokine family, with
pleiotropic effects on the immune system depending on the
cellular context, nature of costimulation, and cytokine envi-
formation; plasma cell (PC) differentiation; and memory B cell
responses (10–13). IL-21 can also induce B cell apoptosis when
B cells are activated with LPS, CpG, anti-IgM, and IL-4 (14).
Recent evidence suggests that IL-21 may play an important role
in autoimmune diseases, including systemic lupus erythematosus
(SLE), rheumatoid arthritis, and Sjo ¨gren’s syndrome (15–18). In
humans, an association of IL-21 and IL-21R polymorphisms with
SLE, along with elevated levels of IL-21 in serum and in CD4+
T cells, was reported (16–20). Studies in murine models of lupus
indicated increased production of IL-21 in MRL-Faslpr, BXSB-
Yaa mice and in the knockout (KO) mouse sanroque (21–23).
Furthermore, IL-21 blockade was beneficial in MRL-Faslprmice,
whereas it had a biphasic effect in BXSB-Yaa mice, negatively
influencing survival early on and positively influencing survival
at later stages of disease (22, 23). In addition, IL-21R–deficient
BXSB-Yaa mice showed none of the autoimmune abnormalities
characteristic of IL-21R–competent BXSB-Yaa mice (24).
Thewide range of costimulatory and inhibitory signals delivered
by IL-21 on T and B cells suggests a complex role for IL-21 in
21/IL-21R interaction in promoting SLE through CD4+T cell-
dependent mechanisms that may affect TFHcells, Th17 cells, or
Tregs or through B cell-intrinsic mechanisms has not yet been
determined. In the absence of conditional KO mice, it has not been
technically possible to investigate this issue in autoimmune-prone
lupus models in vivo. Therefore, to address this question, we took
advantage of the induced lupus-like model of chronic graft-versus-
host disease (cGVHD) that allowed us to independently manipu-
late Tand B cell responses and dissect the requirements of the IL-
21/IL-21R interaction for the initiation and progression of the
disease. To this end, IL-21R–sufficient and -deficient mice on the
B6 background were used as donors in the parent-into-F1 (P→F1)
model or as hosts in the Bm12→B6 model of cGVHD. In addition,
because the exact timing of disease onset is known, these models
*Division of Rheumatology and Clinical Immunology, Department of Medicine,
University of Maryland School of Medicine, Baltimore, MD 21201;†Research Ser-
vice, Veteran Affairs Medical Center, Baltimore, MD 21201;‡Department of Neu-
rology, University of Maryland School of Medicine, Baltimore, MD 21201; and
xDepartment of Pathology, University of Maryland School of Medicine, Baltimore,
Received for publication January 27, 2012. Accepted for publication May 13, 2012.
This work was supported by National Institutes of Health Grant AR053704 (to V.R.),
a Veterans Affairs Merit Review Grant (to V.R., H.R., and I.L.), and a grant from the
Mid-Atlantic Chapter of the Arthritis Foundation (to V.R.).
Address correspondence and reprint requests to Dr. Violeta Rus, University of Mary-
land School of Medicine, MSTF Building, Room 8-34, 10 S. Pine Street, Baltimore,
MD 21201. E-mail address: email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this article: B6, C57BL/6; Bm12, B6.C-H2bm12/KhEg;
cGVHD, chronic graft-versus-host disease; DC, dendritic cell; GC, germinal center;
GN, glomerulonephritis; iTreg, induced regulatory T cell; KO, knockout; nTreg,
natural regulatory T cell; P→F1, parent-into-F1; PC, plasma cell; PNA, peanut ag-
glutinin; SLE, systemic lupus erythematosus; TFH, T follicular helper; Treg, regula-
tory T cell; WT, wild-type.
by guest on December 21, 2015
allowed us to perform a kinetic analysis of Tand B cell activation,
differentiation, and effector functions (25). Our results indicate
that lack of the IL-21/IL-21R interaction on either B cells or Ag-
specific CD4+T cells independently impairs the development of
autoimmune manifestations of cGVHD and results in an attenu-
ated disease phenotype.
Materials and Methods
Six- to eight-week-old male B6D2DF1, B6.C-H-2bm12KhEg (Bm12), and
C57BL/6J (B6) mice were purchased from The Jackson Laboratory (Bar
Harbor, ME). Breeding pairs of IL-21R2/2mice on the B6 background
were provided by Dr. Michael Grusby (Harvard School of Public Health,
Boston, MA) (26). C57BL/6-Tg(UBC-GFP)30Scha mice, C57BL/6J-
transgenic mice that express GFP, were provided by Dr. David Trisler
(University of Maryland School of Medicine). IL-21R2/2mice used in this
study were housed and bred at the University of Maryland Animal Care
Facility. All procedures were approved by the University of Maryland
School of Medicine Office of Animal Welfare Assurance.
Induction of cGVHD
Single-cell suspensions of splenocytes were prepared in RPMI 1640, fil-
tered through sterile nylon mesh, washed, and diluted to a concentration of
108viable (trypan blue-excluding) cells/ml. P→F1 cGVHD was induced
with CD8+T cell-depleted splenocytes containing 10–15 3 106CD4+
donor cells from either B6 wild-type (WT) or B6 IL-21R2/2donors
injected i.v. into B6D2DF1mice, as described (27). Flow cytometry was
used prior to injection to confirm that equal numbers of CD4+T cells were
injected into recipient F1 mice. Donor CD8+T cells were depleted using
Dynabeads Mouse CD8 (Lyt 2) (Invitrogen, San Diego, CA). Flow cyto-
metric analysis demonstrated ,1% contaminating CD8+T cells. Controls
consisted of uninjected age- and sex-matched F1 mice. Bm12→B6
cGVHD was induced in B6 WT or B6 IL-21R2/2recipients by i.p. in-
jection of 1 3 108Bm12 donor splenocytes. Recipient and donor mice
were age- and sex-matched within each independent experiment. In all
experiments, we used male mice to avoid artifacts due to the sex-based
difference in IL-21 gene expression (28).
Cell isolation and in vitro generation and measurement of
Spleen cells from control or P→F1 cGVHD mice were pooled (three mice/
pool), and CD4+T cells were negatively selected using MACS beads
(Miltenyi Biotec). Donor and host cells from cGVHD mice were further
purified using biotinylated anti–H2-KdAbs and anti-biotin MACS beads.
Purified donor CD4+T cells were cultured for 5 d with plate-bound anti-
CD3 (5 mg/ml), anti-CD28 (1 mg/ml), and IL-23 (10 ng/ml) and then
restimulated with PMA/ionomycin for 4 h. Supernatants were tested in
duplicates for IL-17A expression by ELISA. B cells were purified from
control or Bm12→B6 cGVHD mice using MACS beads.
Abs and flow cytometry
Spleen cells were first incubated with anti-murine FcgRII/III mAb (2.4G2)
for 10 min and then stained with saturating concentrations of Alexa
Fluor 488-conjugated, allophycocyanin-conjugated, biotin-conjugated,
PE-conjugated, FITC-conjugated, PE/Cy5-conjugated, or PE/Cy7-conjugated
mAb against CD4, CD8, B220, H-2Kb, H-2KdI-Ab, I-Ad, CD80, CD86,
CD69, CD44, FAS, ICOS, PD-1, GL-7, CXCR5, annexin, FOXP3, Helios,
and Ki-67. Abs were purchased from BD Biosciences (San Jose, CA),
BioLegend (San Diego, CA), eBioscience (San Diego, CA), and Sigma-
Aldrich (Saint Louis, MO). Biotinylated primary mAbs were detected using
streptavidin-allophycocyanin (BioLegend), streptavidin-FITC, streptavidin-
PE, or streptavidin–PE–Cy5 (BD Biosciences). Cells were fixed in 1%
paraformaldehyde before flow cytometric analysis. Intracellular staining for
FOXP3, Helios, and Ki-67 was performed using the FOXP3 Buffer Staining
Set from eBioscience, according to the manufacturer’s protocol. Annexin V
staining was performed with the FITC Annexin VApoptosis Detection Kit I
(BD Pharmingen), according to the manufacturer’s protocol, and analyzed
by flow cytometry within 1 h of staining. Multicolor flow cytometric
analyses were performed using a FACScan, Accuri C6, and LSR II flow
cytometer (BD Biosciences). Lymphocytes were gated by forward and side
scatter, and fluorescence data were collected for a minimum of 10,000
gated cells. Studies of donor T cells in the P→F1 model were performed
using a lymphocyte gate that was positive for CD4 and negative for MHC
class I of the uninjected parent (H-2Kd2).
Immunohistochemistry staining of splenic sections
Sections of formalin-fixed spleen (5 mm) were deparaffinized, rehydrated,
and then stained, as previously described (29). The following Abs, with
their specificities and conjugations, were used: biotinylated peanut ag-
glutinin (PNA) from Sigma-Aldrich, unlabeled rat anti-human/mouse
CD45R(B220) (clone RA3-6B2) from eBioscience, unlabeled rabbit
anti-GFP (Invitrogen), streptavidin HRP (Vector Laboratories), and alka-
line phosphatase-conjugated goat anti-rabbit and alkaline phosphatase-
conjugated goat anti-rat Ab (both from Jackson ImmunoResearch). Sub-
strates used were AEC and Vector Blue Alkaline Phosphatase Kit (Vector
Preparation of CFSE-labeled donor cells
CFSE (Molecular Probes, Eugene, OR) labeling of donor splenocytes and
analysis of donor cell proliferation by flow cytometry were performed as
previously described (27). Cells were adjusted to 5 3 107/ml in PBS/0.1%
BSA and then incubated in the dark for 10 min at 37˚C with CFSE (10 mM
stock solution diluted in DMSO to a final concentration of 5 mM). Staining
was quenched with five volumes of ice-cold RPMI 1640/10% FBS, and
cells were washed three times in PBS before injection into F1 mice.
Proliferating CFSE+donor CD4+T cells were distinguished by multipa-
rameter flow cytometry.
Ninety-six–well cellulose membrane plates (MAIPS4510; Millipore) were
prewet with 35% ethanol for 5 min and then coated overnight with 100 mg/
ml prefiltered herring sperm DNA (Promega) in PBS at 4˚C. Two-fold
serial dilutions of spleen cells or bone marrow cells were plated in du-
plicate overnight on DNA-coated plates, starting at 0.5 3 106/well, in 3%
BSA/DMEM. After washing, the plates were incubated with anti-mouse
IgG alkaline phosphatase (Sigma-Aldrich) at 1:1000 in PBS/3% BSA for 1
h at 37˚C. Plates were then developed with bromochloroindolyphosphate
(KPL, Gaithersburg, MD). Spots were counted using an automated reader
(CTL- Europe GmbH Reader System, software version 4).
ELISA for anti-dsDNA, anti-ssDNA, total IgG, and IgG1 Abs
were coated with heat-denatured calf thymus DNA (Sigma-Aldrich), fol-
The plates were then incubated with alkaline phosphatase-conjugated anti-
mouse IgG and anti-mouse IgG1 (Sigma-Aldrich), respectively, and OD
was quantitated at 405 nm. For measurement of IgG and IgG1 anti-dsDNA
Ab, plates were coated with 100 mg/ml prefiltered herring sperm DNA
(Promega). Following blocking with 3% BSA/PBS, sera were added to the
plate at dilutions starting at 1/50. The respective secondary Abs were al-
kaline phosphatase-conjugated anti-mouse IgG and anti-mouse IgG1
(Sigma-Aldrich). For each experiment, murine MRL-Faslprsera were used
as a standard, and the results were converted to arbitrary units.
For total IgG and IgG1, goat anti-mouse IgG or IgG1 (Southern Biotech,
Birmingham AL) was coated at 5 mg/ml onto plates. Following blocking
with 3% BSA/PBS, sera were added to the plate at dilutions starting at 1/
50,000 and 1/25,000, respectively. Mouse IgG and IgG1 (Rockland, Gil-
bertsville, PA), respectively, were used as the standards. The plates were
incubated with alkaline phosphatase-conjugated anti-mouse IgG (Sigma-
Aldrich) and anti-mouse IgG1 (Jackson ImmunoResearch), respectively.
Total RNA isolation, quantitation, and reverse transcription were per-
formed, as described (27). 18S rRNA was used as an internal control. All
primers and probes (IL-21, IL-4, IL-10, IL-6, bcl-6, RORgt, IL-17A,
Prdm1, Aicda, and 18S) were purchased from SABiosciences (Frederick,
MD). RT-PCR was carried out on an Applied Biosystems Step1 Plus PCR
machine (Foster City, CA).
Kidney histopathology and immunofluorescence
Formalin-fixed kidney sections (4 mm) were stained with H&E. The sec-
tions were examined in a blinded fashion (by C.C. and H.R.) for glo-
merular, tubular, and interstitial pathology. Disease was scored on
a semiquantitative scale using the published criteria, with modifications
(30). The severity of glomerulonephritis (GN) was graded on a 0–3 scale:
0 = normal; 1 = mild to moderate increase in cellularity with mesangial
proliferation; 2 = moderate increase in cellularity with endocapillary and
mesangial proliferation, increased matrix, and/or karyorrhexis; and 3 =
1082 IL-21R DEFICIENCY IN THE CHRONIC GVHD MODEL OF LUPUS
by guest on December 21, 2015
marked increase in cellularity with endocapillary proliferation, crescent
formation, and/or necrosis, and/or sclerosis. Scores from 20 glomeruli
were averaged to obtain a mean score for each kidney section. Deposits of
IgG in the glomeruli were detected by incubating acetone fixed, 5-mm-
thick cryostat sections of kidney in 20% normal goat serum for 30 min,
followed by a 1-h incubation with FITC-conjugated goat anti-mouse IgG
(1/500; Southern Biotech). Fluorescence in glomerular capillary walls and
in the mesangium was subjectively scored blindly on a scale of 0–3 (0 =
none, 1 = weak, 2 = moderate, 3 = strong); 10 glomeruli/section were
Normally distributed data were analyzed by the unpaired t test, and non-
parametric data were analyzed by the Mann–Whitney U test using Prism
4.0 (GraphPad) software.
Upregulation of IL-21 production and TFHcell differentiation
of donor CD4+T cells
We first evaluated whether IL-21 is upregulated in the P→F1 and
Bm12→B6 models of cGVHD. To this end, we measured IL-21
mRNA expression by RT-PCR in splenocytes of cGVHD mice
at 2 wk after disease induction, when the autoimmune features
are already present. Compared with normal control mice, IL-21
mRNA production increased by 30- and 28-fold in P→F1
cGVHD and Bm12→B6 cGVHD mice, respectively (Fig. 1A, 1B).
In both models of cGVHD, donor CD4+T cells, activated by host
MHC class II, expand and provide cognate help to host B cells.
Because activated CD4+T cells are known to produce IL-21, we
evaluated whether donor CD4+T cells are the major source of IL-
21. We addressed this question in the P→F1 model, in which
donor CD4+T cells (H-2Kd2) can be distinguished by flow
cytometry and separated from H-2Kd+host cells. As seen in Fig.
1C, IL-21 mRNA transcripts were significantly higher in purified
donor CD4+T cells than in host CD4+T cells.
Recent studies highlighted a role for IL-21 as a growth factor
for TFHcells and as a regulator of the reciprocal differentiation of
Th17 cells and Tregs (6, 9, 31). To assess the effect of IL-21R
signaling on these subsets, we evaluated whether TFHcells, Th17
cells, and Tregs of donor origin would be readily detectable in
cGVHD mice. To this end, we first assessed whether donor CD4+
T cells differentiated into TFHcells, with upregulation of the TFH
cell transcription factor Bcl-6 and localization in the GCs. TFH
cells detected by flow cytometry as ICOShiCXCR5hicells among
CD4+H2-Kd2donor cells expanded from 0.45 6 02% at baseline
in naive donor CD4+T cells prior to transfer to 14 6 2% of donor
were induced, as described in Materials and Methods. Splenocytes were analyzed after 14 d for IL-21 mRNA expression by RT-PCR. Results were
normalized to 18S RNA. (C) Two weeks after P→F1 cGVHD induction, donor and host CD4+T cells were isolated from pooled mice, as described in
Materials and Methods. RT-PCR for IL-21 was performed. (D) Mean percentage of donor TFHcells detected by flow cytometry as ICOShiCXCR5hiCD4+
H2-Kd2T cells in P→F1 cGVHD mice at 2 wk after induction. (E) Naive donor CD4+T cells prior to transfer and engrafted donor CD4+T cells from
cGVHD mice were isolated, as described in Materials and Methods, and RT-PCR for Bcl-6 was performed. (F) P→F1 cGVHD was induced with GFP-
expressing WT donor cells. Immunohistochemistry staining of spleen section for PNA+GCs (brown) and donor GFP+cells (blue) was performed at 10 d
after disease induction (original magnification 3200). (G) mRNA expression for IL-17A and RORgt was assessed ex vivo from donor CD4+T cells purified
on day 14 after cGVHD induction. Purified, naive CD4+T cells prior to transfer were used as controls. (H) Purified donor CD4+T cells were stimulated
with anti-CD3, anti-CD28, and IL-23 for 5 d and then restimulated with PMA/ionomycin. Supernatants were tested for IL-17 expression by ELISA. (I)
Mean percentage of donor Tregs identified by flow cytometry as FOXP3+cells among uninjected, naive donor CD4 cells and engrafted donor CD4+cells at
day 14. Helios staining was used to distinguish FOXP3+Helios+nTregs and FOXP3+Helios2iTregs. Data are representative of two independent experi-
ments (n = 5 mice/group). *p , 0.05, ***p , 0.001.
Donor CD4+T cells produce IL-21 and display TFHcell, Th17 cell, and Treg phenotypes. P→F1 cGVHD (A) and Bm12→B6 cGVHD (B)
The Journal of Immunology1083
by guest on December 21, 2015
CD4+T cells engrafted in cGVHD mice (Fig. 1D). Furthermore,
by RT-PCR, we detected a significant upregulation of Bcl-6
mRNA in purified donor CD4+T cells from cGVHD mice com-
pared with naive, uninjected donor CD4+T cell controls (Fig. 1E).
Consistent with their differentiation into TFHcells, in mice with
cGVHD induced with GFP-expressing cells, donor GFP+cells
detected by immunohistochemistry were preferentially localized
in GCs, as well as in the B cell area and at the T–B border (Fig.
1F). Similarly, mRNA transcripts for IL-17A and RORgt, the
transcription factor required for Th17 cell differentiation, are
upregulated in donor CD4+T cells purified from cGVHD mice
compared with naive, uninjected CD4+T cells (Fig. 1G). Fur-
thermore, purified donor CD4+T cells from cGVHD mice, cul-
tured in vitro with anti-CD3/anti-CD28 Ab and IL-23, a cytokine
known to expand differentiated Th17 cells, produced significant
levels of IL-17 in supernatants (Fig. 1H). We could not detect IL-
17 production from naive, uninjected CD4+T cells under the same
conditions (Fig. 1H). The percentage of CD4+FOXP3+Tregs was
similar in the uninjected, naive donor CD4+T cells and in donor
CD4+T cells from cGVHD mice at 2 wk after disease induction.
Furthermore, the proportions of natural Tregs (nTregs) and iTregs
determined by intracellular Helios staining were comparable in
naive, uninjected and engrafted donor CD4+T cells. These results
indicate that, in cGVHD, donor CD4+T cells upregulate IL-21
mRNA expression and differentiate into TFHcells and Th17 cells
while maintaining the same proportion of natural and induced
IL-21R deficiency on donor CD4+T cells attenuates B cell
parameters and kidney disease in P→F1 cGVHD
After activation, donor CD4+T cells provide MHC class II-
restricted cognate help to host B cells, resulting in chronic
B cell hyperactivity and expansion. Consequently, IgG anti-
ssDNA Ab levels are elevated early (day 14) after donor cell
transfer, and renal disease occurs after 2 mo (25, 32). To assess the
contribution of the IL-21/IL-21R interaction on donor CD4+
T cells to the disease phenotype, we compared early and late
disease parameters of P→F1 cGVHD induced with donor cells
from IL-21R2/2mice, which abrogates the IL-21/IL-21R inter-
action on donor cells but not on host B cells, versus cGVHD in-
duced with donor cells from WT mice.
At 7 and 14 d after cGVHD induction with WT or IL-21R2/2
CD8-depleted donor cells, we determined the number of B cells,
B cell MHC class II expression, the proportions of GL-7+Fas+
B220+GC B cells, and the levels of anti-ssDNA autoantibody.
After 7 d, no significant differences were observed in any of these
parameters between the two groups. By comparison, after 14 d,
mice injected with IL-21R2/2donor cells had a significantly lower
number of B cells (Fig. 2A) and a trend toward decreased MHC
class II expression, although this did not reach significance (data
not shown). The proportion of GL-7+Fas+B220+GC B cells was
also significantly lower in the spleens of cGVHD mice injected
with IL-21R2/2donor cells (Fig. 2B, 2C). Consistent with the flow
cytometry data, GCs were smaller by immunohistochemistry (Fig.
2E–G). Furthermore, IgG anti-ssDNA Ab levels were significantly
lower in mice injected with IL-21R2/2donor cells. These data
suggest that the IL-21/IL-21R interaction on donor CD4+T cells is
dispensable for the initiation of the autoimmune B cell response
but is required for the optimal expansion of host B cells and GC
B cells and for maximal autoantibody production.
donor cells developed an attenuated lupus-like renal disease
compared with mice with cGVHD induced with WT donor cells;
they also displayed much milder GN (Fig. 3A–C, 3H), signifi-
cantly lower proteinuria (Fig. 3G), and decreased deposition of
IgG (Fig. 3D–F).
IL-21R–deficient donor T cells exhibit diminished TFHcell
expansion and persistence
The diminished host B cell and GC expansion and decreased anti-
ssDNA Ab production at 2 wk after disease induction in the ab-
sence of the IL-21/IL-21R interaction on donor CD4+T cells
suggest a defect in sustained CD4+Th function. We evaluated
whether this effect is due to altered donor TFHcell differentiation
and/or expansion. In mice receiving WT CD8-depleted donor
cells, CXCR5+PD-1+donor TFHcells increased from 1.4 6 0.5%
in naive donor CD4+T cells at the time of transfer to a peak of
18 6 3% at day 7 and then decreased slightly to 14 6 1.3% by
day 14. In mice with cGVHD induced with IL-21R2/2donor cells,
the proportion of TFHcells was lower at both days 7 and 14 but
reached statistical significance only on day 14 (Fig. 4A, Supple-
mental Fig. 1B). The decrease in IL-21R2/2TFHcell frequency
was mainly due to decreased proliferation, because the proportion
of dividing Ki-67+donor TFHcells was significantly lower at 2 wk
of disease (Fig. 4B, Supplemental Fig. 1D). The frequency of
apoptotic donor TFHcells was slightly higher in mice injected with
IL-21R2/2donor cells at 2 wk of disease, but the difference was
not statistically significant (Fig. 4C). Next, we assessed whether
the decreased expansion of donor TFHcells is specific for this
T cell subset or whether it also affects non-TFH donor CD4+
T cells. As seen in Fig. 4D, the percentage of engrafted donor
CD4+T cells is significantly lower on both days 7 and 14 in the
absence of IL-21R. Similar to donor TFHcells, in WT donor cells,
the frequency of proliferating CD4+T cells had increased 4.2-fold
from baseline at day 7 and 3-fold at day 14. By comparison, in IL-
21R2/2donor cells, the percentage of proliferating donor CD4+
T cells was lower, but not statistically significant, on day 7; it
decreased further to significantly lower levels by day 14 (Fig. 4E,
Supplemental Fig. 1C). Similar to donor TFHcells, the apoptotic
rate tended to be higher in IL-21R2/2–injected mice, although the
difference was not statistically significant (Fig. 4F). These results
suggest that IL-21R is important for the sustained proliferation
of TFHcells, as well as non-TFHcell donor CD4+T cells, from
days 7 to 14. Although we were unable to demonstrate differences
in apoptotic rates, altered survival of the TFHcells, as well as non-
TFHcell donor CD4+T cells, cannot be completely ruled out.
IL-21 and IL-4, known for their ability to promote GC devel-
opment, are the main cytokines secreted by TFHcells (33, 34).
We determined whether IL-21 and IL-4 mRNA expression is
altered in donor CD4+T cells from mice injected with IL-21R–
deficient donor cells. As seen in Fig. 5, both IL-21 and IL-4
mRNA transcripts are decreased in IL-21R2/2donor CD4+
T cells, suggesting that the expression of these cytokines parallels
the decrease in donor CD4+T cells and TFHcells.
Donor Tregs and Th17 cells are not altered in the absence of
To assess whether the lack of IL-21R signaling on donor CD4+
T cells alters the proportion of donor Tregs and donor Th17 cells,
we initially compared the percentage of FOXP3+CD4+donor cells
in cGVHD mice induced with IL-21R–sufficient and -deficient
cells. At baseline, there was no difference in the frequency of
CD4+FOXP3+Tregs in the injected donor cells of IL-21R+/+and
IL-21R2/2origin (data not shown). At 2 wk after disease induc-
tion, the frequency of donor CD4+FOXP3+Tregs did not differ
between IL-21R+/+and IL-21R2/2donor CD4+T cells (Fig. 6A,
6B). Furthermore, we did not observe a significant difference
between the proportions of FOXP3+Helios+nTregs and FOXP3+
1084IL-21R DEFICIENCY IN THE CHRONIC GVHD MODEL OF LUPUS
by guest on December 21, 2015
Helios2iTregs. Similarly, we observed no difference in either IL-
17 mRNA expression or IL-17 secretion of in vitro-stimulated
donor CD4+T cells purified from mice with cGVHD induced
with IL-21R+/+or IL-21R2/2donor cells (Fig. 6C, 6D).
Decreased B cell activation, autoantibody production, and
kidney disease severity in Bm12→B6 cGVHD induced in
To assess the contribution of IL-21R signaling on host B cells to
the disease phenotype independent of IL-21R signaling on donor
CD4+T cells, we compared early and late disease parameters in
Bm12→B6 cGVHD induced in IL-21R–deficient recipients,
which abrogates the IL-21/IL-21R interaction on host cells but not
on donor cells, versus cGVHD induced in WT hosts.
B cell activation that characterizes cGVHD is a complex mul-
tistep process involving a nonstringent step, in which activation of
donor T cells induces polyclonal activation and proliferation of all
B cells, and a stringent, cognate interaction, resulting in the ac-
tivation of autoreactive B cells, such as anti-dsDNA B cells, and
autoantibody production (35). We first assessed whether parame-
ters of B cell activation were altered in the absence of IL-21R
signaling on host B cells. MHC class II and CD69 were upregu-
lated to a similar extent on B cells from IL-21R–sufficient and
-deficient cGVHD recipients at 7 d after disease induction.
However, at 2 wk of disease, MHC class II expression and CD69
were further upregulated on B cells from IL-21R+/+hosts but not
IL-21R2/2hosts (Fig. 7). These data suggest that, in the absence
of IL-21R signaling, B cells fail to reach optimal levels of acti-
vation. However, we cannot exclude the possibility that the lack of
IL-21R on activated B cells impairs their survival.
Serum levels of total IgG Abs increased significantly in IL-21–
sufficient hosts as early as 2 wk, peaked around week 7, and then
gradually decreased by week 18 to levels reached at weeks 2 and
4. IL-21R–deficient hosts displayed significantly lower levels of
total IgG at all time points before week 18, when they reached
a plateau at levels similar to those detected in IL-21R–sufficient
hosts (Fig. 8A). IgG anti-dsDNA autoantibody levels were only
transiently detected at low titer at 2 and 4 wk in IL-21R2/2hosts
compared with IL-21R+/+hosts and were significantly lower at all
time points (Fig. 8B). Because IL-21 induces isotype switching to
IgG1, we assessed the levels of total and anti-dsDNA IgG1 Abs.
Both total IgG1 and anti-dsDNA IgG1 Abs were detected only in
IL-21R–sufficient hosts and displayed a kinetic similar to IgG Ab
levels (Fig. 8C, 8D). IL-21R2/2hosts had total and anti-dsDNA
Ab IgG1 levels similar to control groups.
anti-dsDNA autoantibodies suggested that these mice may develop
less severe renal disease. Indeed, proteinuria was significantly
decreased in IL-21R2/2cGVHD mice compared with IL-21R+/+
cGVHD mice at 10 and 13 wk after disease induction (Fig. 9H).
Furthermore, typical histological features of autoimmune GN,
such as mesangial and capillary cell proliferation and crescent
Methods. Serum and spleens were collected at the specified time points. (A) Absolute number of host B cells. (B) Frequency of GL-7+Fas+GC B cells. (C)
Flow cytometric contour plots of GL-7+Fas+GC B cells (B cell gate). (D) Anti-ssDNA Ab levels were determined by ELISA. (E–G) Spleen sections
obtained at 2 wk were stained for PNA-R+GC B cells (brown) and B220 (blue) (original magnification 3100). Data are representative of two independent
experiments (n = 5 mice/group). *p , 0.05, **p , 0.01.
IL-21R deficiency on donor CD4+T cells attenuates B cell parameters of cGVHD. P→F1 cGVHD was induced as described in Materials and
The Journal of Immunology1085
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formation, were observed in the kidneys of WT but not IL-21R2/2
cGVHD mice (Fig. 9A–C). The latter had glomerular histological
scores similar to the control groups (Fig. 9G). In addition, WT, but
not IL-21R2/2, recipients of Bm12 cells displayed glomerular IgG
deposition at 13 wk after disease induction (Fig. 9D–F). There was
no difference between WT and KO recipient groups in the degree
of perivascular lymphoid infiltration and tubulointerstitial dam-
age (data not shown).
Decreased GCs and PC differentiation in IL-21R2/2cGVHD
In models of protein immunization, the role of the IL-21/IL-21R
interaction in the initiation and/or maintenance of GCs has been
controversial (5, 13, 36, 37). We assessed the importance of IL-
21R signaling on B cells for GC formation and maintenance in
Bm12→B6 cGVHD at 1 wk after disease induction, when GCs are
initially formed, and at 3 wk, when GCs have already peaked. By
flow cytometry, the percentages of GC B cells decreased by 40%
at day 7 and by 60% at day 21 after disease induction in IL-21R–
deficient hosts (Fig. 10G). By immunohistochemistry, splenic GCs
in IL-21R2/2hosts were smaller, ill-formed, disrupted, and
without apparent polarization compared with those in IL-21R+/+
hosts, both at 7 and 21 d after disease induction (Fig. 10A–F).
These differences were maintained at day 28 (data not shown).
These data suggest that, in cGVHD, the absence of IL-21R sig-
T cells ameliorates lupus-like nephritis in P→F1
cGVHD. P→F1 cGVHD was induced as described in
Materials and Methods. (A–C) H&E-stained kidney
sections of normal F1 control mice, cGVHD mice
induced with WT, or IL-21R2/2donor cells at 13 wk
after disease induction. Enlarged glomerulus with
crescent formation, glomerular sclerosis, and intersti-
tial infiltrate is noted in (B) (original magnification
3400). (D–F) Immunofluorescent staining of IgG
deposits (original magnification 3200). (G) Protein-
uria. (H) GN pathologic scores. Data are representa-
tive of two independent experiments (n = 5 mice/
group). *p , 0.05.
IL-21R deficiency on donor CD4+
described in Materials and Methods. Recipient mice were sacrificed at the times indicated. Mean percentage of donor TFHcells (A), Ki-67+donor TFHcells
(B), annexin V+donor TFHcells (C), donor CD4+T cells (D), Ki-67+donor CD4+T cells (E), and annexin V+donor CD4+T cells (F). Data are repre-
sentative of two independent experiments (n = 5 mice/group). *p , 0.05, **p , 0.01.
IL-21R–deficient donor CD4 and TFHcells exhibit decreased expansion and proliferation in P→F1 cGVHD. P→F1 cGVHD was induced as
1086IL-21R DEFICIENCY IN THE CHRONIC GVHD MODEL OF LUPUS
by guest on December 21, 2015
naling on B cells reduces the magnitude of the initial GC response,
as well as its maintenance.
Consistent with the decrease in GC B cell numbers, we observed
a significant decrease in the proportion of proliferating GC B cells
in the absence of IL-21R signaling. The percentage of Ki-67+Fas+
GL-7+GC B cells decreased by 1.5-fold at 7 d and by 2.8-fold at
21 d after disease induction compared with controls (Fig. 10H).
Thus, although the absence of IL-21R signaling decreases GC
B cell proliferation at both early and late time points, the mag-
nitude of this reduction is greater at later time points, suggesting
that IL-21R signaling has a predominant role in the maintenance
of GCs. Among GC B cells, centroblasts are highly proliferative
cells that express AID (encoded by Aicda). We detected signifi-
cantly decreased Aicda mRNA expression in purified B cells from
IL-21R2/2hosts (Fig. 10I).
The decreased level of anti-dsDNA Abs and the attenuated GC
response in IL-21R2/2hosts suggest the possibility of decreased
PC differentiation in the GCs. We used ELISPOT to examine the
number of anti-dsDNA Ab-secreting PCs in the bone marrow at
28 d after disease induction, when PCs have already migrated out
of the spleen. Anti-dsDNA–secreting PCs were significantly lower
in the bone marrow of IL-21R2/2hosts for both IgG (Fig. 11A,
11B) and IgG1 (Fig. 11C, 11D) Abs. Consistent with the decrease
in PC formation, mRNA expression of Prdm1, the gene encoding
BLIMP-1, the master regulator of PC differentiation, was signif-
icantly decreased in purified B cells from IL-21R2/2cGVHD
mice (Fig. 11E).
Normal donor CD4+T cell priming and TFHcell formation in
The diminished GC response and autoantibody levels in the IL-
21R2/2recipients raise the question of the quality of the T cell
help in these mice. Therefore, we assessed whether the decreased
GC and autoantibody response in IL-21R2/2cGVHD mice could
be due to a failure of T cell priming or to a suboptimal donor TFH
cell response. T cell priming was assessed using Bm12 donor cells
labeled with CFSE prior to cGVHD induction, followed by de-
tection of activation markers and proliferation rate of CFSE+
donor cells on day 3 after cGVHD induction. CD4+CFSE+donor
cells exhibited similar expression of the activation markers CD44
and CD69 whether they were injected into WTor IL-21R2/2hosts
(Fig. 12A). Similar results were obtained on day 5 (data not
shown). Interestingly, as previously reported for CD8+T cells, the
proliferative response of CFSE-labeled donor CD4+T cells was
greater in IL-21R2/2hosts than in IL-21R+/+hosts (Fig. 12B,
12C), possibly as a result of the increased concentration of IL-21
on donor cells in IL-21R2/2host mice (38). These data indicate
that donor CD4+T cells injected into IL-21R2/2mice were ac-
tivated and expanded to the same, if not greater, extent as were
those transferred to the WT mice.
donor CD4+T cells. Two weeks after P→F1 cGVHD induction, donor
CD4+T cells, purified as described in Materials and Methods, were ana-
lyzed for IL-21 (A) and IL-4 (B) mRNA expression by RT-PCR. Results
were normalized to 18S RNA. Results represent mean 6 SEM (n = 6 mice/
group). Data are representative of two independent experiments. *p ,
Decreased IL-21 and IL-4 mRNA expression in IL-21R2/2
are not altered in mice with cGVHD in-
duced with IL-21R2/2donor cells. (A)
Two weeks after P→F1 cGVHD induction,
spleens from recipient F1 mice were ex-
amined by flow cytometry for the fre-
quency of FOXP3+donor Tregs. Helios
staining was used to further identify
FOXP3+Helios+nTregs and FOXP3+He-
lios2iTregs. (B) Flow cytometric contour
plots of FOXP3+CD4+(donor cell gate).
(C) mRNA expression for IL-17A was
assessed ex vivo on day 14 from naive,
uninjected CD4+T cells and donor CD4+
T cells purified from cGVHD mice. (D)
Purified donor CD4+T cells from cGVHD
mice at day 14 were stimulated for 5 d
with plate-bound anti-CD3 (5 mg/ml),
anti-CD28 (1 mg/ml), and IL-23 (10 ng/
ml) and were restimulated with PMA/
ionomycin for 4 h. Supernatants were
tested for IL-17 expression by ELISA.
Data are representative of two independent
experiments (n = 5–6 mice/group).
Donor Tregs and Th17 cells
The Journal of Immunology1087
by guest on December 21, 2015
Cognate interaction between primed T cells and B cells at the
T–B border provide the required signals for TFHcell differentia-
tion (5, 34, 39–41). We assessed whether the lack of IL-21R signal-
ing on B cells impairs the differentiation of TFHcells. To this end,
CFSE-labeled donor CD4+T cells were assessed on day 7 after
disease induction for the percentage of PD-1+CXCR5+TFHcells.
The proportion of CFSE+donor TFHcells was similar in both
IL-21R–sufficient and -deficient cGVHD mice (Fig. 12D). Thus,
these data demonstrate that, at early time points, donor CD4+
T cells from IL-21R2/2host mice were activated, proliferated,
and developed into TFHcells to the same extent as did the donor
cells from WT mice. Therefore, the attenuated humoral response
is not a consequence of a failure of donor T cell priming or TFH
In this study, we combined the P→F1 and Bm12→B6 models of
lupus-like cGVHD to investigate the importance of IL-21R sig-
naling on CD4+T cells, independently of IL-21R signaling on
B cells, in the initiation and progression of the disease. Previous
studies addressing this issue in bone marrow chimeras and adoptive-
transfer systems in recipients immunized with protein Ags reported
conflicting results. Although some studies reported that the effect of
IL-21 on the immune response was exclusively CD4+T cell in-
trinsic, others showed a B cell-intrinsic mechanism (5, 13, 36, 37).
In contrast, our data demonstrate that, in cGVHD, IL-21 promotes
disease parameters, such as GC formation, PC differentiation, au-
toantibody production, and GN, through both CD4 cell-dependent
and B cell-intrinsic mechanisms. Specifically, cGVHD induced
with either IL-21R–deficient donor CD4+T cells in the P→F1
model or with IL-21R–sufficient donor cells in IL-21R–deficient
hosts (hence, IL-21R2/2B cells) in the Bm12→B6 model displayed
an attenuated lupus-like phenotype with respect to GC formation,
autoantibody production, PC differentiation, and renal disease.
CD4+T cell help provided by TFHcells is the primary limiting
factor for GC formation and subsequent GC B cell responses.
host B cells is attenuated in the absence
of IL-21R signaling in Bm12→B6
cGVHD. Bm12→B6 cGVHD was in-
duced in IL-21R+/+and IL-21R2/2re-
cipient B6 mice as described in
Materials and Methods. At 7 and 14 d
after disease induction, spleens were
examined by flow cytometry. (A) Mean
fluorescence intensity (MFI) of MHC
class II (left panel) and CD69 (right
panel) on B cells. (B) Flow cytometric
graphs of MHC class II and CD69 on B
cells from B6→ B6 mice (dotted line),
Bm12→WT B6 mice (thick line), and
Data are representative of two inde-
pendent experiments for day 7 and three
independent experiments for day 14
(n = 5 mice/group). *p , 0.05.
Activation phenotype of
mice (thin line).
impairs total and anti-dsDNA IgG and IgG1 Ab
production. Sera from Bm12→B6 cGVHD mice
were assayed by ELISA for the presence of total IgG
and IgG1 (A, C) and anti-dsDNA IgG and IgG1 (B,
D). Data are representative of two independent
experiments (n = 6–8 mice/group). *p , 0.05, **p ,
0.01, ***p , 0.001, IL-21R–sufficient versus IL-
21R–deficient cGVHD groups.
IL-21R deficiency on host B cells
1088 IL-21R DEFICIENCY IN THE CHRONIC GVHD MODEL OF LUPUS
by guest on December 21, 2015
Conflicting data have been reported on the role of IL-21 in the
differentiation or persistence of TFHcells. In the context of protein
immunizations or viral infections, IL-21 promoted TFHcell dif-
ferentiation and function in a number of studies, whereas, in
others, IL-21 had a modest impact or no role (4, 5, 13, 36, 37, 42).
In addition, in the autoimmune sanroque mice characterized by
excessive TFHcell numbers, loss of IL-21 did not correct the in-
creased TFHcell population (43). Our kinetic analysis of the TFH
cell response in the P→F1 cGVHD model demonstrates that lack
of the IL-21/IL-21R interaction on donor CD4+T cells resulted in
a modest decrease in the expansion of donor-derived TFHcells at
earlier time points and a more pronounced decline at later time
points. These results indicate that, in our model, IL-21 contributes
primarily to the persistence of TFHcells and, to a lesser extent, to
their differentiation and initial expansion. Furthermore, our data
suggest that IL-21 promotes the persistence of TFHcells primarily
by sustaining their proliferation and, to a lesser extent, their sur-
vival. The decrease in GCs and GC B cells, as well as the levels of
anti-ssDNA autoantibodies, detected after the first week of disease
in cGVHD mice that received IL-21R2/2donor cells suggests that
the decreased number of TFHcells available to provide B cell help
was sufficient to attenuate the response of autoreactive IL-21R+/+
host B cells. The decreased B cell help was likely mediated by
decreased production of IL-21 and IL-4 by TFHcells (33). In
addition to fully differentiated TFHcells, the observed decrease in
IL-21 mRNA expression in IL-21R2/2donor CD4+T cells could
have resulted from fewer activated donor CD4+T cells and Th17
cells. However, in the P→F1 model, we did not observe any
changes in IL-17 mRNA expression and protein secretion in the
absence of IL-21R signaling; additionally, in BXSB-Yaa mice, IL-
21 was not a product of Th17 cells (24). In addition, although we
observed a decreased number of donor CD4+T cells in the second
week of disease due to a faster contraction and decrease in pro-
liferation, it was reported that IL-21 from non-TFHcells is less
likely to contribute to GC reaction and B cell responses (44).
Although controversial, it was suggested that IL-21 has the
ability to regulate the reciprocal differentiation of Treg and Th17
cells by promoting Th17 cell differentiation and expansion and
by downregulating the differentiation of iTregs and/or their sup-
pressive capacity (6, 8, 9, 31, 45). Increased Th17 cells and a de-
creased number and/or functionally deficient Tregs are well-
known abnormalities in murine and human lupus (46–49). An
important question we sought to address is whether the attenuation
of autoimmune parameters in the absence of IL-21R signaling on
donor cells could be due to the correction of the balance between
pathogenic Th17 and Tregs. Our observation that upregulation of
IL-17 mRNA expression and protein secretion are not altered in
the absence of IL-21R signaling exclude the possibility that the
amelioration of B cell parameters in P→F1 cGVHD is due to
decreased IL-17 production. Our results parallel those obtained in
BXSB-Yaa lupus-like mice, in models of bone marrow transplant,
in experimental allergic encephalitis induced in IL-6–sufficient
mice, as well as in several models of organ-related autoimmune
diseases (25, 50–52). As previously reported, it is possible that the
contribution of IL-21 to Th17 differentiation/expansion is less
important in an autoimmune setting in which IL-6 production is
abundant (53). In addition, the observation that the proportion of
natural or induced donor Tregs was not altered when IL-21R
signaling was disrupted suggests that the attenuated phenotype
in this model is not due to expanded Tregs. However, that does
not exclude the possibility that IL-21 blockade may restore the
function of Tregs or reverse the resistance of responder T cells to
Materials and Methods. (A–C) H&E-stained kidney sections of B6→B6 control mice, cGVHD mice induced in WT, or IL-21R2/2hosts at 16 wk after
disease induction. Enlarged glomerulus with crescent formation, glomerular sclerosis, and interstitial infiltrate is noted in (B). Original magnification 3200.
(D–F) Immunofluorescent staining of IgG deposits (original magnification 3200). (G) GN pathologic scores. (H) Proteinuria scores. Data are representative
of one experiment (n = 8 mice/group). *p , 0.05, ***p , 0.001.
IL-21R deficiency on host B cells ameliorates lupus-like nephritis in Bm12→B6 cGVHD. Bm12→B6 cGVHD was induced as described in
The Journal of Immunology1089
by guest on December 21, 2015
Treg-mediated suppression (6, 9). Further studies will address this
In the Bm12→B6 model of cGVHD, disruption of IL-21R
signaling on host B cells resulted in a significant impairment of
a number of B cell parameters, such as B cell activation, GC
formation, GC B cell proliferation, PC formation, and autoanti-
body production. Lupus-like GN was also attenuated. Contrasting
data were reported in different models of protein immunization
with respect to the kinetics of GC formation and Ab response in
the absence of IL-21R signaling. In the setting of SRBC immu-
nization, IL-21R signaling was critical for both GC initiation and
long-term maintenance, whereas following immunization with
keyhole limpet hemocyanin, GC formation was comparable to
control mice at day 14 but decreased significantly at day 28 due to
an accelerated resolution and increased memory B cell generation
(36, 37). After immunization with NP-chicken g globulin, early
GC formation and Ab production were dependent on IL-21R but
not GC maintenance or long-term Ab production. Our results
showing decreased GC formation and GC B cell numbers at
7 d after disease induction, which was even more striking at 21 d,
indicate that in cGVHD, IL-21R signaling is important both for
reaching the optimal initial GC response and autoantibody pro-
duction and for their maintenance. Consistent with the attenuated
GC response, we observed a decreased number of anti-dsDNA
Ab-secreting cells in the bone marrow of IL-21R2/2hosts and
decreased expression of BLIMP-1 (encoded by Prdm1) in spleen
B cells, indicating that the differentiation of high-affinity
autoantibody-secreting PCs from the follicular GC is impaired
in the absence of IL-21R signaling. Although we did not examine
the effect of IL-21R signaling on the extrafollicular response in
B cells decreases GCs in Bm12→B6
cGVHD. Bm12→B6 cGVHD was induced
as described in Materials and Methods.
Recipient mice were sacrificed at the
times indicated. Spleen sections from
Bm12→B6 (A–C) and Bm12→IL-21R2/2
(D–F) cGVHD mice were stained for B220
(blue) and PNA (brown) (original mag-
nification 3200). (G) Flow cytometry
graphic analysis and contour plots of
GL-7+Fas+GC cells gated on B220+lym-
phocytes. (H) Quantification of proliferating
Ki-67+GC B cells and representative flow
cytometry profiles. (I) RT-PCR for Aicda
in purified B cells. mRNA levels were
normalized to 18S and reported as fold
increase over normal control. Data are
representative of two independent experi-
ments (n = 5 mice/group). *p , 0.05.
IL-21R deficiency on host
1090 IL-21R DEFICIENCY IN THE CHRONIC GVHD MODEL OF LUPUS
by guest on December 21, 2015
our model, the contribution of the IL-21/IL-21R interaction on
the extrafollicular pathway was reported in MRL-Faslprmice, in
which the extrafollicular response is the dominant pathway of
autoantibody production (54).
The priming of T cells by DCs in the T cell zone, followed by T–
B cell interaction at the T-B border resulting in TFHcell differ-
entiation are the initial steps in the multistage, multifactorial
process of GC formation (5, 34, 39–41). In view of reports
showing an inhibitory effect of IL-21 on DC maturation and ac-
tivation, we considered the possibility that the attenuation of
cGVHD parameters in IL-21R2/2hosts is not due to a direct
effect on B cells but to an indirect effect on the ability of DCs to
prime donor T cells. Our data contradict this idea, because donor
T cell priming was not impaired and was even enhanced in IL-
affects PC formation after Bm12→B6 cGVHD
induction. Bm12→B6 cGVHD was induced as
described in Materials and Methods. Bone marrow
cells were collected at 28 d after immunization.
The frequency of IgG (A, B) and IgG1 (C, D) anti-
dsDNA Ab-secreting cells (ASC) was assessed
by ELISPOT. The results are presented as anti-
dsDNA ASC/million splenocytes. (E) Prdm1
mRNA expression was determined by RT-PCR in
purified B cells at 14 d after disease induction.
mRNA levels were normalized to 18S and reported
as fold increase over normal control. Data are
representative of two independent experiments
(n = 5 mice/group). *p , 0.05.
Lack of IL-21R on host B cells
host B cells does not alter the priming
and TFHcell differentiation of donor
CD4+T cells in Bm12→B6 cGVHD.
Bm12→B6 cGVHD was induced us-
ing CFSE-labeled donor cells. (A)
Mean percentage of CD44hi
CD69hidonor cells detected as CFSE+
CD4+T cells at 3 d after disease in-
duction. (B) Proliferation of donor
CD4+T cells detected by generational
analysis of CFSE+cells. (C) Percent-
age of proliferating donor cells. (D)
Percentage of PD-1+CXCR5+donor
TFHcells at 7 d after disease induction.
Data are representative of two inde-
pendent experiments (n = 5 mice/
group). **p , 0.01.
Lack of IL-21R on
The Journal of Immunology1091
by guest on December 21, 2015
21R2/2hosts. In addition, the expression of maturation markers
MHC class II, CD80, and CD86 on CD11c+DCs was similar in
IL-21R–deficient and -sufficient mice (data not shown). Further-
more, although a number of activation markers were decreased on
host B cells, the number of TFHcells did not differ in IL-21R–
sufficient and -deficient hosts, suggesting that, at least at the early
time points examined, IL-21R deficiency on B cells did not impair
In conclusion, our data suggest that the genetic inactivation of
IL-21R attenuates the T cell-dependent B cell hyperactivity that
contributes to lupus pathogenesis by impairing both the aberrant
TFHcell pathway and the hyperactive B cell response. Further
studies to evaluate whether in vivo blockade of the IL-21 pathway
achieves similar effects are needed.
We thank Dr. Michael Grusby for initially providing the IL-21R–deficient
mice, Dr. David Tisler for providing the C57BL/6J-transgenic mice that
expressed GFP, and Dr. Wendy Davidson (National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD) for help-
The authors have no financial conflicts of interest.
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