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To Support Regional Th17 Responses in
Autoreactive Th1 Cells Activate Monocytes
Aitken, Victoria Garcia, Alissa Basehoar and Andrew J.
Donald M. Simons, Soyoung Oh, Elizabeth Kropf, Malinda
2013; 190:3134-3141; Prepublished online 18
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The Journal of Immunology
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The Journal of Immunology
Autoreactive Th1 Cells Activate Monocytes To Support
Regional Th17 Responses in Inflammatory Arthritis
Donald M. Simons,1Soyoung Oh, Elizabeth Kropf, Malinda Aitken, Victoria Garcia,
Alissa Basehoar, and Andrew J. Caton
We have examined mechanisms underlying the formation of pathologic Th17 cells using a transgenic mouse model in which auto-
reactive CD4+T cells recognize influenza virus hemagglutinin (HA) as a ubiquitously expressed self-Ag and induce inflammatory
arthritis. The lymph nodes ofarthritic mice contain elevated numbers ofinflammatory monocytes (iMO) with an enhancedcapacity
to promote CD4+Th17 cell differentiation, and a regional inflammatory response develops in the paw-draining lymph nodes by an
IL-17–dependentmechanism.The activationofthese Th17-trophic iMOprecedes arthritisdevelopmentandoccurs inthecontext of
an autoreactive CD4+Th1 cell response. Adoptive transfer of HA-specific CD4+T cells into nonarthritic mice expressing HA as a
self-Agsimilarly ledtotheformationofTh1cellsandofiMOthatcouldsupport Th17cellformation,and,notably,theaccumulation
of these iMO in the lymph nodes was blocked byIFN-g neutralization. These studies show that autoreactive CD4+Th1 cells directed
to a systemically distributed self-Ag can promote the development of a regional Th17 cell inflammatory response by driving the
recruitment of Th17-trophic iMO to the lymph nodes.The Journal of Immunology, 2013, 190: 3134–3141.
are also important participants in a variety of autoimmune dis-
The processes that drive Th17 cell formation are not fully under-
stood, particularly in the setting of autoimmune disease, where
Th17 cell induction is pathological and presumably reflects a dys-
regulation of processes that are normally protective to the host.
Both in vitro and in vivo studies have shown that cytokines such
as TGFb1, IL-6, and IL-23 can play important roles in promoting
the differentiation of naive CD4+T cells into Th17 cells (3–5).
More recently, an important role for gut-residing bacteria in Th17
cell formation in vivo has become apparent, such that colonization
of mice with particular commensal microbes (such as segmented
filamentous bacterium) can profoundly influence the magnitude of
Th17 cell responses that can be induced (6). Notably, these effects
on Th17 cell formation were found to influence the development
of autoimmune arthritis in K/BxN mice, indicating that commensal
bacteria can play a role in promoting the formation of pathologic
autoreactive Th17 cells in vivo (7). However, it is clear that addi-
helper 17 cellsareasubsetofeffectorCD4+Tcellsthatcan
play an important role in maintaining effective antimi-
crobial immunity at mucosal surfaces such as the gut, but
tional factors must contribute to autoimmune disease develop-
ment, in addition to those received from commensal bacteria,
because most strains of mice carrying these bacteria do not develop
autoimmunity. In particular, these findings do not explain why
many autoimmune diseases show strong genetic linkages with
MHC class II (MHC II) alleles, which imply an important role for
CD4+T cell recognition of self-peptides in the disease process
(8). Indeed, mechanisms by which CD4+T cell recognition of
self-peptides might participate in and/or promote the formation
of pathologic Th17 cells in autoimmune settings remain poorly
We have examined this question in a mouse model of inflam-
matory arthritis, in which autoreactive CD4+T cells responding to
a self-peptide expressed by APCs induce arthritis by an IL-17–
dependent mechanism (9). We show that autoimmune disease is
initiated by a systemic autoreactive CD4+Th1 response, which
drives the formation of Th17-trophic inflammatory monocytes
(iMO) that mobilize to the lymph nodes (LN). These studies
provide a basis by which autoreactive CD4+T cells responding to
a systemically distributed Ag can promote a regional IL-17–me-
diated inflammatory response.
Materials and Methods
TS1, HACII, and TS1xHACII mice were previously described (10, 11).
HACII.Ca2/2mice were generated by crossing HACII mice with TCR.
Ca2/2mice that were bred to homozygosity for the H-2dhaplotype and
backcrossed onto the BALB/c background for at least seven generations
(12). TS1.CD45.1+/2congenic mice were generated by breeding TS1 mice
with CD45.1+/+mice on the BALB/c background that were purchased from
The Jackson Laboratory. Mice were housed under specific pathogen-free
conditions at The Wistar Institute Animal Facility. All experiments were
performed according to protocols approved by The Wistar Institutional
Animal Care and Use Committee.
Assessment of arthritis and anti–IL-17 treatment
Mice were assessed weekly for the number of arthritic limbs beginning at
4 wk of age and continuing for at least 12 wk. For anti–IL-17 treatment,
TS1xHACII mice received three i.p. injections per wk of 150 mg IL-17A–
neutralizing Ab (M210; provided by Amgen) or of an isotype control Ab
(2A3; BioXCell) beginning at 4 wk of age.
The Wistar Institute, Philadelphia, PA 19104
1Current address: Abbvie Bioresearch Center, Worcester, MA.
Received for publication November 20, 2012. Accepted for publication January 17,
This work was supported by grants from the National Institutes of Health (AI24541),
the National Cancer Institute (P30 CA10815), Sibley Memorial Hospital, and the
Commonwealth of Pennsylvania (to A.J.C.). D.M.S. was supported by National
Cancer Institute Grant T32 CA09171.
Address correspondence and reprint requests to Dr. Andrew J. Caton, The Wistar
Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: caton@wistar.
The online version of this article contains supplemental material.
Abbreviations used in this article: cDC, conventional dendritic cell; HA, hemagglutinin;
iMO, inflammatory monocyte; LN, lymph node; MHC II, MHC class II; PDCA,
plasmacytoid dendritic cell Ag; pdLN, paw draining lymph node; PMN, polymor-
phonuclear cell; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus.
by guest on October 18, 2015
Abs and flow cytometry
Single-cell suspensions from spleens or lymph node were stained with the
Live/Dead Fixable Aqua Dead Cell stain kit from Invitrogen (except when
sorting) and then with fluorochrome-labeled Abs purchased from eBio-
science or BD Biosciences unless stated otherwise. The following clones
were used in all experiments. For analysis and sorting of APCs: F4/80
(BM8), plasmacytoid dendritic cell Ag-1 (PDCA-1) (eBio927), CD19
(1D3), CD11b (M1/70), CD11c (N418), Ly6G (1A8), Ly6C (HK1.4),
MHC II (M5/114.15.2), B220 (RA3-6B2), CD115 (AF598), CD49b
(DX5), and CCR2 (475301; R&D Systems); and for analysis and sorting
of T cells: CD4 (RM4-5), CD25 (PC61.6), IL-17 (eBio17B7), IFN-g
(XMG1.2), CD45.1 (A20), and CD8 (53-6.7). Biotinylated 6.5 was pre-
pared in-house and detected with APC (BD Biosciences) or Qdot655-
conjugated (Invitrogen) streptavidin.
For flow cytometric analysis and sorting of APC subsets, spleens and
LN were minced and digested with 400 U/ml collagenase D (Roche) at
37˚C prior to use. Spleen and/or LN cells were sorted with a MoFlow
(DakoCytomation) or FACSAria (BD Biosciences) cell sorter into the
following subsets: inflammatory monocytes (B2202CD11c2Ly6G2
CD11b+Ly6Chigh), conventional dendritic cells (cDC) (B2202Ly6G2
Ly6C2CD11b+/2CD11chigh), and B cells (CD11b2CD11c2Ly6C2B220high).
For coculture experiments, hemagglutinin (HA)-specific CD4+CD2526.5+
cells were sorted from the pooled spleens and LN of TS1.CD45.1+/2or
In vitro cocultures
FACS-purified APCs and HA-specific T cells were combined at T cell/APC
ratios ranging from ∼1:2 (LN APCs) to 1:10 (spleen APCs) and cultured at
∼1.5 3 106total cells/ml in cel-culture media supplemented with 50 ng/ml
IL-23 (R&D Systems). After 7 d, PMA, ionomycin, and brefeldin A were
added to the cultures, and IL-17 and IFN-g production were assessed 5 h
later by intracellular cytokine staining and flow cytometry.
Serum cytokine analysis
Serum was collected from whole blood, and inflammatory cytokine ex-
pression was determined by multiplex ELISA using the Luminex platform
(Millipore) at the University of Pennsylvania Human Immunology Core.
CD4+T cells were purified from the spleens and LN of TS1.CD452/2
mice by MACS depletion of unwanted cells with Abs against B220, I-Ad,
CD11b, F4/80, and CD8. A total of 106cells/mouse were adoptively
transferred into HACII.Ca2/2recipients by retro-orbital injection, and
recipient mice were sacrificed 7 d later for analysis. In some experiments,
recipients were injected i.p. with 0.5 mg anti–IFN-g (XMG1.2; BioXCell)
or 0.5 mg isotype control Ab (HRPN; BioXCell) on days 0 and 3. For
analysis of in vivo proliferation, CD4+T cells purified as described above
were labeled with 5 mM CFSE (Sigma-Aldrich) prior to adoptive transfer
into BALB/c or HACII recipients, which were sacrificed 3 d later for
All statistical analyses were performed using GraphPad Prism software
package (GraphPad). Unpaired, two-tailed t tests were used for data analysis
and the generation of p values.
Arthritis in TS1xHACII mice is accompanied by a regional
Th17 response to a systemic self-Ag
TS1 mice express a transgenic I-Ed–restricted TCR that recognizes
the S1 determinant of influenza PR8 HA and can be detected with
the clonotypic mAb 6.5 (10). HACII mice express HA as a neo–
self-Ag under the control of a MHC II promoter, and we previ-
ously reported that the majority of TS1xHACII mice (generated
by mating TS1 mice with HACII mice) spontaneously develop
TS1 and arthritic TS1xHACII mice. Middle and bottom panels show front and rear paws. Graph (right panel) shows arthritis incidence in a cohort of
TS1xHACII mice. (B) CFSE dilution by CD4+T cells from TS1 mice 3 d posttransfer into HACII or BALB/c recipients. (C) Histograms showing 6.5
staining by CD4+T cells in pdLN and spleens of TS1 and arthritic TS1xHACII mice. (D) Numbers of 6.5+CD4+T cells in pdLN and spleens of TS1 and
arthritic TS1xHACII mice. (E) Ex vivo production of IFN-g and IL-17 by 6.5+CD4+T cells from pdLN and spleens of TS1 and arthritic TS1xHACII mice.
(F) Arthritis incidence in 15-wk-old TS1xHACII mice given weekly injections of anti–IL-17 beginning at 4 wk of age. All data are means 6 SEM of $5
independent determinations. *p , 0.05, **p , 0.01.
Arthritis in TS1xHACII mice is accompanied by a regional Th17 response to a systemic self-Ag. (A) Photographs of age- and sex-matched
The Journal of Immunology3135
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inflammatory arthritis as they reach adulthood (Fig. 1A) (11). Ar-
thritis depends on coexpression of the TS1 and HACII transgenes
(because neither TS1 nor HACII mice develops the disease) and is
driven by CD4+T cells without a requirement for B cells, because
TS1xHACII mice that congenitally lack B cells also develop ar-
thritis (11). It is noteworthy that TS1xHACII mice develop arthritis
as a prominent autoimmune manifestation, because the targeting of
HA to MHC II+cells results in its expression as a self-Ag by APCs
throughout the body. Indeed, CD4+T cells from TS1 mice underwent
extensive division in both the spleens and LN following transfer
into HACII mice (Fig. 1B), and HA-specific 6.5+CD4+T cells are
extensively deleted in TS1xHACII mice (Fig. 1C, 1D), indicating
that HA is a systemically expressed self-Ag in TS1xHACII mice.
To examine how autoreactive 6.5+CD4+T cells that evade de-
letion can promote arthritis development, we first analyzed their
representation and phenotype in the spleens and paw draining LN
(pdLN) of arthritic TS1xHACII mice. There were increased fre-
quencies of both IFN-g– and IL-17–producing 6.5+CD4+T cells
(Th1 and Th17 cells, respectively) relative to TS1 mice (Fig. 1E).
Notably, however, the percentage of 6.5+CD4+T cells that were
Th17 cells was significantly higher in the pdLN of arthritic
TS1xHACII mice than in the spleens, whereas the percentages of
6.5+CD4+T cells that were Th1 cells at these sites did not differ.
Moreover, administration of an anti–IL-17A mAb led to a signif-
icant decrease in the numbers of arthritic paws that developed in
TS1xHACII mice, consistent with other studies demonstrating
a role for Th17 cells in inflammatory arthritis (Fig. 1F) (7, 9, 13).
Together, these data show that TS1xHACII mice spontaneously
develop an IL-17–dependent inflammatory arthritis that is ac-
companied by a regional increase in the frequency of HA-specific
CD4+Th17 cells in the pdLN.
Th17-trophic inflammatory monocytes accumulate in arthritic
The HA self-Ag that is recognized by 6.5+CD4+T cells in
TS1xHACII mice is expressed by MHC II–expressing cells (in-
cluding APCs), and we wanted to determine whether a particular
APC type might be playing a prominent role in promoting the
formation of autoreactive Th17 cells. We addressed this by first
comparing the frequencies of various APC subsets in the pdLN of
arthritic TS1xHACII mice with HACII mice. There were 6–8-fold
increases in the numbers of CD19+B cells and CD192PDCA2
CD11chighcDC in the pdLN of arthritic TS1xHACII mice relative
to HACII mice. However, there was a much more dramatic in-
crease in the number of CD192PDCA-12CD11c2CD11b+
Ly6GlowLy6ChighiMO (Fig. 2A); these cells were almost unde-
tectable in the pdLN of HACII mice and increased in frequency at
least 30-fold in the pdLN of arthritic TS1xHACII mice. The iMO
also expressed the markers CD115, F4/80, and CCR2 that are
typical of monocytes and lacked expression of the markers B220
and CD49b used to identify Ly6C+plasmacytoid DCs and NK
cells, respectively (Supplemental Fig. 1) (14–16). CD192PDCA-12
CD11c2CD11b+Ly6GhighLy6Cint.polymorphonuclear cells (PMN)
were also present in elevated numbers in arthritic mice, which is
consistent with the ability of IL-17 to enhance the formation of
these cells (17).
The iMO in the pdLN of arthritic TS1xHACII mice also
expressed MHC II (along with endogenously synthesized HA-
derived peptides synthesized under control of the MHC II pro-
moter), indicating that they were capable of acting as APCs for
6.5+CD4+T cells. We therefore examined their ability to promote
the formation of IL-17–secreting 6.5+CD4+T cells in vitro relative
to other APC populations from the pdLN of arthritic TS1xHACII.
pdLN. The percent of total live cells falling within each gate is indicated. MHC II expression by iMO and the total numbers of each APC are shown in the
accompanying panels. (B) Percentage of 6.5+CD4+T cells that produced IL-17 or IFN-g following 7 d of coculture with APC subsets purified from the LN
of arthritic TS1xHACII mice. (C) As in (A), except for spleen. (D) As in (B), except coculture was with APC subsets purified from the spleens of HACII
mice or arthritic TS1xHACII mice. All data are means 6 SEM of $5 independent determinations. *p , 0.05, **p , 0.01, ***p , 0.005. pDC, Plas-
Th17-trophic inflammatory monocytes accumulate in arthritic TS1xHACII mice. (A) Representative flow data showing APC subsets in the
3136MONOCYTES PROMOTE Th17 RESPONSES IN ARTHRITIS
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Notably, iMO purified from the pdLN of arthritic TS1xHACII
mice induced a significantly higher proportion of 6.5+CD4+cells
from TS1 mice to become IL-17 secretors than were induced by
either B cells or cDC that had also been isolated from the pdLN
(Fig. 2B). Inflammatory monocyte and cDC numbers were also
elevated in the spleens of arthritic TS1xHACII mice, and, al-
though lower than was the case for iMO in the pdLN, the splenic
iMO from arthritic TS1xHACII mice expressed higher levels of
MHC II than iMO from the spleens of HACII mice (Fig. 2C).
Splenic iMO from arthritic TS1xHACII mice again induced the
formation of IL-17–secreting 6.5+CD4+T cells more efficiently
than was the case for cDC isolated from arthritic TS1xHACII mice,
and they were also more efficient at inducing the formation of IL-
17–secreting 6.5+CD4+T cells than were either iMO or cDC ob-
tained from the spleens of control HACII mice (Fig. 2D). Thus, the
accumulation of IL-17–secreting 6.5+CD4+T cells in the pdLN of
arthritic TS1xHACII mice is associated with a sizable increase in
the representation of an iMO population that is a more potent in-
ducer of Th17 cells than is the case for other APC populations in
the pdLN. Together, these data strongly suggest that these Th17-
trophic iMO are important in promoting the regional Th17 cell
response that develops in the pdLN of arthritic TS1xHACII mice.
spleens of TS1, prearthritic TS1xHACII, and arthritic TS1xHACII mice. (B) Percentage of 6.5+CD4+splenocytes that produced IL-17 or IFN-g. (C) Heat
map showing the concentrations of Th17 and Th1 cytokines in the serum of prearthritic and arthritic TS1xHACII mice. Average values from seven control
(CTL) mice are shown in the leftmost column. The concentration of each cytokine is indicated within the tiles. The p values refer to comparisons between
prearthritic and arthritic TS1xHACII mice. Number of APCs in the pdLN (D) and spleen (E) and percentages of cells in the blood (F) of 5-wk-old
TS1xHACII and TS1 mice. (G) MHC II expression by iMO in the spleens and blood of 5-wk-old TS1xHACII and TS1 mice. (H) Percentage of 6.5+CD4+
T cells from TS1 mice that produced IL-17 or IFN-g following 7 d of coculture with APC subsets purified from the spleens of 5-wk-old TS1xHACII or
HACII mice. All data are means 6 SEM of n $ 5 independent determinations. *p , 0.05, **p , 0.01, ***p , 0.005. N/A, Equivalent subset not present;
pDC, plasmacytoid DC.
A systemic CD4+Th1 cytokine response precedes arthritis in TS1xHACII mice. (A) Number of CD4+and 6.5+CD4+T cells in the pdLN and
The Journal of Immunology3137
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A systemic CD4+Th1 cytokine response precedes arthritis in
Arthritis arises spontaneously in adult TS1xHACII mice, and we
were interested to evaluate processes occurring in younger mice
that might play a predisposing role in arthritis development. The
pdLN of 4- to 5-wk-old prearthritic TS1xHACII mice exhibited
a marked hypocellularity, with total CD4+T cells and 6.5+CD4+
T cells substantially underrepresented relative to both TS1 and
arthritic TS1xHACII mice (Fig. 3A). The spleens of prearthritic
TS1xHACII mice contained CD4+and 6.5+CD4+cells in numbers
that were more comparable to the spleens of TS1 and arthritic
TS1xHACII mice. Notably, .50% of 6.5+CD4+splenocytes in
prearthritic TS1xHACII mice were IFN-g–secreting Th1 cells,
compared with 25 and 5% of 6.5+CD4+splenocytes in arthritic
TS1xHACII and TS1 mice, respectively (Fig. 3B). Moreover, serum
obtained from prearthritic TS1xHACII mice contained significantly
higher levels of Th1-associated cytokines and chemokines (IFN-g,
CXCL9, and CXCL10) than serum from arthritic TS1xHACII mice;
by contrast, the serum from arthritic TS1xHACII mice expressed
significantly higher levels of Th17-associated cytokines (IL-17, IL-6,
and IL-1b) than were found in serum from prearthritic TS1xHACII
mice (Fig. 3C).
When we examined the APC subsets present in prearthritic
TS1xHACII mice, we again found very low numbers of cells in
the pdLN (Fig. 3D). By contrast, the spleens of prearthritic
TS1xHACII mice contained similar numbers of APCs (including
iMO) as were found in age-matched TS1 mice, and there was
a significant increase in the frequency of circulating iMO in the
blood of prearthritic TS1xHACII mice relative to their TS1
counterparts (Fig. 3E, 3F). Notably, iMO in the spleens and blood
of prearthritic TS1xHACII mice expressed MHC II at levels that
were comparable to those found on iMO in the pdLN of arthritic
TS1xHACII mice (compare Figs. 3G and 2A, respectively). More-
over, splenic iMO from prearthritic TS1xHACII mice were signif-
icantly better at inducing 6.5+CD4+T cells from TS1 mice to
become Th17 cells than was the case for either cDC from prear-
thritic TS1xHACII mice or cDC or iMO obtained from the spleens of
age-matched HACII mice (Fig. 3H). Together, these data indicate
that the autoreactive CD4+T cell response in prearthritic TS1xHACII
mice differs from that observed in arthritic TS1xHACII mice in
that it is characterized by IFN-g production and occurs predom-
inantly in the spleen rather than the pdLN. Notably, the formation
of Th17-trophic iMO precedes the onset of an inflammatory re-
sponse in the pdLN and arthritis.
Autoreactive CD4+T cells promote activation of Th17-trophic
inflammatory monocytes via IFN-g production
To examine whether the autoreactive CD4+Th1 cells that arise in
prearthritic TS1xHACII mice can play a direct role in the for-
mation of Th17-trophic iMO, we used an adoptive transfer ap-
proach in which CD4+T cells from TS1 mice were introduced into
T cell–deficient mice that express the HACII self-Ag (HACII.
Ca2/2mice). By 7 d posttransfer, 80% of the donor CD4+T cells
in the spleens and LN of recipient mice were IFN-g–secreting
cells, with a smaller subset coproducing IL-17 or producing IL-17
alone (Fig. 4A). The serum of the recipient mice also contained
elevated concentrations of proinflammatory cytokines relative to
mice that had not received T cells, with IFN-g showing the
greatest increase (.100-fold), consistent with the high frequency
of IFN-g–secreting cells detected by intracellular cytokine stain-
HACII.Ca2/2mice (recipient mice). All data were collected at 7 d posttransfer. (A) Production of IFN-g and IL-17 by CD4+T cells from a TS1 mouse
analyzed directly ex vivo or at 7 d posttransfer. (B) Fold increase in the indicated cytokines in the serum of recipient versus unmanipulated mice. (C)
Representative flow data of iMO and PMN in the pdLN and spleens. The frequency of each subset as a percentage of total cells is indicated on each plot.
The total number of iMO in the spleens and pdLN of recipient mice is shown in the accompanying chart (right panel). (D) Histograms showing MHC II
expression by iMO in the spleens and pdLN of recipient mice. (E) Percentages of 6.5+CD4+T cells that produced IL-17 or IFN-g following 7 d of coculture
with APCs isolated from the spleens of unmanipulated HACII or recipient mice. All data are means 6 SEM of n $ 3 independent determinations. *p ,
0.05, **p , 0.01, ***p , 0.005.
Autoreactive T cells promote the activation of Th17-trophic inflammatory monocytes. CD4+T cells from TS1 mice were transferred into
3138MONOCYTES PROMOTE Th17 RESPONSES IN ARTHRITIS
by guest on October 18, 2015
ing (Fig. 4B). The introduction of HA-specific CD4+T cells also
led to increased numbers of MHC II+iMO in the pdLN and
spleens of recipient mice (Fig. 4C, 4D), and as had been observed
in intact TS1xHACII mice, these iMO were significantly more
capable of inducing Th17 cell formation than were cDC from
HACII.Ca2/2mice that had received CD4+T cells or iMO or
cDC from unmanipulated HACII mice (Fig. 4E).
To identify signals that the CD4+Th1 cells were providing to
induce the formation of Th17-trophic iMO, we first generated
mixed bone marrow chimeras in which 75% of marrow-derived
cells expressed the HACII transgene and were CD45.1+, and the
remaining 25% were CD45.12and lacked expression of HA.
When CD4+T cells from a TS1 mouse were transferred into these
chimeras, there was a significant increase in the number of iMO in
their pdLN compared with control HA-chimeric mice that had not
received T cells (Fig. 5A). However, the frequencies of HA+to
HA2iMO (based on CD45.1 expression) in the LNs of recipient
mice were no different from in chimeras that had been left un-
manipulated as controls and were similar to that of PMN, which
were uniformly MHC II2and would therefore not be expected to
interact with the transferred T cells in an Ag-specific manner (Fig.
5B). Moreover, MHC II was upregulated by iMO in the spleens of
recipient mice independent of HA expression (Fig. 5C).
We then repeated the approach of transferring CD4+T cells into
HACII.Ca2/2mice and in this case treated the recipients either
with an anti–IFN-g mAb or an isotype control Ab. The number of
iMO was significantly reduced in the pdLN of mice that received
the anti–IFN-g mAb (Fig. 5D). Moreover, there was little or no
expression of MHC II on the iMO that were present in either the
pdLN or the spleens of mice that were treated with anti–IFN-g
mAb, whereas MHC II expression by cDC in the spleens of re-
cipient mice was unaffected by anti–IFN-g treatment (Fig. 5E).
Together, these data indicate that IFN-g produced by CD4+Th1
cells responding to a self-Ag can play a direct role in the activa-
tion of Th17-trophic iMO and that CD4+T cell recognition of self-
Ag expressed by the iMO themselves is not required for this ac-
Inflammatory arthritis is a prominent disease manifestation in a
number of systemic autoimmune disorders including rheumatoid
arthritis (RA) and systemic lupus erythematosus (SLE). Because
MHC II alleles display by far the strongest genetic linkages with
diseases such as SLE and RA, it is likely that CD4+T cell rec-
ognition of self-peptides drives these disease processes, but how
autoreactive CD4+T cells can cause both systemic and joint-
targeted autoimmune manifestations remains poorly understood,
at least partly because the Ags that are recognized by autoreactive
CD4+T cells in human inflammatory arthritis are mostly unde-
fined (18, 19). In TS1xHACII mice, the target autoantigen rec-
mixed bone marrow from HACII.CD45.1+(75%) and BALB/c.CD45.12(25%) mice. Four weeks after reconstitution, CD4+T cells from a TS1 mouse were
adoptively transferred into the chimeras, and data were collected at 7 d posttransfer. (A) Representative flow data showing the accumulation of iMO in the
pdLN of chimeric mice at day 7 posttransfer. Gating for live CD192PDCA-12cells is shown. (B) The frequency of iMO and PMN that were CD45.1+in the
pdLN of chimeric mice at day 7 posttransfer. (C) Representative histograms showing MHC II expression by iMO and PMN in the spleens of chimeric mice
at day 7 posttransfer. Flow data are representative of three independent experiments with at least one mouse per condition per experiment. (D and E) As in
Fig. 4, except mice were treated at days 0 and 3 with an anti–IFN-g neutralizing mAb. (D) Number of iMO in the pdLN of recipient mice treated with anti–
IFN-g or an isotype control mAb. (E) Histograms showing MHC II expression by iMO and cDC in the spleens and pdLN of recipient mice treated with anti–
IFN-g or an isotype control mAb. All numerical data are means 6 SEM of at least three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.005.
IFN-g promotes in vivo activation of Th17-trophic inflammatory monocytes. (A–C) Lethally irradiated BALB/c mice were reconstituted with
The Journal of Immunology3139
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ognized by autoreactive CD4+T cells (i.e., HA) is expressed se-
lectively by APCs, which is notable in light of findings indicating
that PBLs from RA patients exhibit an autologous MLR sugges-
tive of an autoreactive CD4+T cell response to APCs (and APC-
derived peptides) (19, 20). Moreover, in both SLE and RA, tissues
other than the joints can also be affected by inflammatory pro-
cesses. Indeed, these autoimmune diseases are typically classified
as systemic because they involve multiple tissues and organ sys-
tems, and it is noteworthy in this regard that TS1xHACII mice
develop inflammatory processes affecting the lungs and exhibit
additional evidence of systemic immune activation resembling the
processes that can occur in patients who develop autoimmune
inflammatory arthritis (11). The studies in this paper describe an
autoimmune process in which an initial systemic Th1-dominated
autoreactive CD4+T cell response against an APC-derived peptide
leads to the activation of iMO that are biased toward inducing
Th17 cell responses. Because iMO have an intrinsic capacity to
mobilize from sites such as the blood and the spleen to the LNs
and tissues, their activation by autoreactive CD4+T cells provides
a basis by which an autoimmune response that is systemic in
nature can exhibit regional manifestations.
The reason that inflammatory arthritis is a prominent manifes-
tation of the autoimmune response in TS1xHACII mice most
likely reflects the sensitivity of the joints to the activities of IL-17
(21). We have shown that blockading IL-17A inhibits arthritis
development in TS1xHACII mice, and IL-17 has been shown to
promote joint pathology in other mouse models of inflammatory
arthritis. In SKG mice (which harbor a mutation in the ZAP-70
gene that causes increased self-reactivity and autoreactive Th17
cell formation), synoviocytes produce CCL20, which attracts
CCR6-bearing Th17 cells to the joints (22). In the K/BxN arthritis
model, IL-17–producing T cells can augment autoantibody-
induced arthritis and were enriched in inflamed joints of ar-
thritic mice (23). Increased frequencies of IL-17–secreting CD4+
T cells were similarly found to promote arthritis development in
another mouse model, even when these T cells lacked specificity
for joint Ags (24). Our data provide evidence that the mobiliza-
tion of iMO by autoreactive CD4+T cells can also contribute to
the development of a regional autoimmune response in inflam-
matory arthritis and resembles studies showing that the inflamed
synovium and synovial fluid of patients with active RA contain
activated monocytes that specifically promote Th17 responses
(25). Increased iMO activity may also promote arthritis develop-
ment through activation of CD4+Th17 cells that react with joint-
derived Ags because recent studies in this system suggested that
the CD4+Th17 cells that cause joint inflammation include cells
using nonclonotypic TCRs, suggesting the a process of epitope-
spreading may contribute to the development of arthritis in
TS1xHACII mice (9).
As is also the case in RA, TS1xHACII mice develop inflam-
matory arthritis as adults, and we have shown in this study that the
autoimmune environment is distinct in young prearthritic and older
arthritic TS1xHACII mice. Thus, the accumulation of autoreactive
Th17 cells in the pdLN of arthritic TS1xHACII mice was ac-
companied by a shift in serum cytokines toward elevated ex-
pression of Th17-related cytokines and away from the elevated
levels of Th1-related cytokines found in younger mice. It was also
notable that the LNs of prearthritic mice exhibited a marked
hypocellularity relative to age-matched TS1 controls. This hypo-
cellularity in the LNs, but not in the spleens, could be an indication
that the inflammatory environment of young TS1xHACII mice
interferes with the formation and/or activity of lymphoid tissue
inducer cells, which play a crucial role in LN formation (26).
Additional studies will be necessary to determine why LN for-
mation is impaired in young TS1xHACII mice, but at this stage,
it is notable that impaired LN formation in young TS1xHACII
mice may contribute to the delay in arthritis development until
TS1xHACII mice become adults.
The biphasic cytokine response in TS1xHACII mice (Th1-
dominated in prearthritic mice, Th17-dominated in arthritic mice)
is also likely a reflection of an autoimmune environment in which
various cytokines exert counterregulatory effects. For example,
IFN-g can be a negative regulator of IL-17 production and may
tend to inhibit IL-17 production at early stages of the autoimmune
response (27, 28). The decreased levels of IFN-g in older mice
may in turn be a consequence of increased IL-6 production (which
was elevated in the serum of arthritic TS1xHACII mice), because
IL-6 has been found to inhibit IFN-g production and promote
Th17 cell formation (4, 29). However, it is also possible that the
IFN-g–induced activation of iMO contributes to this effect, be-
cause monocytes secrete a number of inflammatory cytokines that
support Th17 responses (including IL-6), and monocyte produc-
tion of IL-6 is enhanced by IFN-g (30, 31). Similarly, activated
monocytes and other myeloid cells can produce IL-23, which is
likely to play an important role in supporting Th17 cell formation,
and IL-23 can itself directly and indirectly induce myeloid cell
activation, as indicated by studies in which elevated levels of IL-
23 were found to lead to arthritis development (32–34). In these
respects then, the activation of iMO by autoreactive CD4+Th1
cells during the initial phases of the autoimmune response may be
viewed as both a consequence of and a contributor to an evolving
The authors have no financial conflicts of interest.
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