American Journal of Transplantation 2012; 12: 835–845
Wiley Periodicals Inc.
C
Copyright 2012 The American Society of Transplantation
and the American Society of Transplant Surgeons
doi: 10.1111/j.1600-6143.2011.03971.x
Th17 Cells Induce a Distinct Graft Rejection Response
That Does Not Require IL-17A
E. I. Agorogiannis, F. S. Regateiro, D. Howie,
H. Waldmann and S. P. Cobbold∗
University of Oxford, Sir William Dunn School of
Pathology, South Parks Road, Oxford OX1 3RE, UK
*Corresponding author: Stephen P. Cobbold,
[email protected]
recombination activating gene; SD, standard deviation.
Received 5 September 2011, revised 17 November 2011
and accepted for publication 12 December 2011
Introduction
IL-17A-producing helper T (Th17) cells have been implicated in the pathogenesis of autoimmune disease, inflammatory bowel disease and graft rejection, however
the mechanisms by which they cause tissue damage
remain ill-defined. We examined what damage Th17
cell lines could inflict on allogeneic skin grafts in the
absence of other adaptive lymphocytes. CD4+ Th17 cell
lines were generated from two TCR transgenic mouse
strains, A1(M).RAG1−/− and Marilyn, each monospecific for the male antigen Dby. After prolonged in vitro
culture in polarizing conditions, Th17 lines produced
high levels of IL-17A with inherently variable levels of
interferon gamma (IFNc ) and these cells were able to
maintain IL-17A expression following adoptive transfer into lymphopenic mice. When transferred into lymphopenic recipients of male skin grafts, Th17 lines
elicited a damaging reaction within the graft associated with pathological findings of epidermal hyperplasia and neutrophil infiltration. Th17 cells could be
found in the grafted skins and spleens of recipients and
maintained their polarized phenotype both in vivo and
after ex vivo restimulation. Antibody-mediated neutralization of IL-17A or IFNc did not interfere with Th17induced pathology, nor did it prevent neutrophil infiltration. In conclusion, tissue damage by Th17 cells
does not require IL-17A.
Key words: graft rejection, mouse skin allograft, T-cell
cytokine, T helper cells,
Abbreviations: APC, antigen-presenting cell; BMDCs,
bone marrow-derived dendritic cells; ChIP, chromatin
immunoprecipitation; cDNA, complementary DNA;
FBS, fetal bovine serum; FACS, fluorescence-activated
cell sorting; CSF3, granulocyte colony stimulating factor; CSF2, granulocyte-macrophage colony stimulating
factor; GVHD, graft-versus-host disease; rm, recombinant murine; H&E, hematoxylin and eosin; IHC, immunohistochemical; IFNc , interferon gamma; IMDM,
Iscove’s modified Dulbecco’s medium; MPO, myeloperoxidase; nM, nanomolar; PMA phorbol myristate acetate; PBS, phosphate-buffered saline; (RT-)PCR, (reverse transcriptase) polymerase chain reaction; RAG,
Th17 cells can orchestrate a wide range of proinflammatory
activities in both normal host defense and autoimmune
disease pathogenesis (1,2). A contribution to graft rejection
has been proposed (3), but despite early claims for a role
of IL-17A (4–10), convincing experimental evidence for the
involvement of Th17 cells in this process has only recently
emerged (11–13). Further work has implicated Th17 cells
in dysfunction and acute rejection of lung transplants (14–
16), in chronic kidney rejection (17) and also graft-versushost disease (GVHD) (18). Nonetheless, the redundancy
of effector mechanisms able to induce transplant damage
(19) has not allowed for clear evidence of a specific role of
Th17 cells in this setting. Even though antagonism of IL-17A
activity could interfere with forms of transplant rejection
(5–7), Th17 cells are not essential to the process, as Th1
or Th2 cell lines were able to elicit graft rejection in the
absence of other lymphocytes (20).
Th17 cells can mediate graft rejection of MHC-IImismatched cardiac allografts in T-bet-deficient mice (13).
IL-17A produced by CD4+ T cells and neutrophils were
shown to be important in this case. For rejection of fully
MHC-mismatched heart transplants by T-bet-deficient mice
the main source of IL-17A was CD8+ T cells (21,22). Previous findings had revealed increased neutrophil infiltration within grafts in IFNc (23) or IL-4 deficient recipients
(24), and depletion of neutrophils was associated with prolonged transplant survival. The involvement of IL-17A was
not assessed. There may, however, be a necessary role
for Th17 cells in rejection of male skin grafts by female
C57BL/6 hosts that absolutely depended on IL-17A and
neutrophil infiltration (12). Th17 cells were detected early
in both skin grafts and draining lymph nodes, their abundance decreasing over time paralleled by the emergence
of IFNc -producing CD8+ T cells (12).
As previous studies did not exclude the presence of other
lymphocyte populations, the exact contribution of Th17
cells to transplant rejection remained unclear. We needed
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Agorogiannis et al.
to know whether Th17 cells could induce graft rejection
in their own right or whether they merely form part of an
inflammatory cascade involving other types of T cells. Here
we describe the effector properties of long-term polarized,
stable Th17 cells in single minor histocompatibility antigen
(H-Y)-mismatched transplantation, in the absence of other
T- or B-cell populations.
Materials and Methods
Mice
C57BL/6, CBA/Ca, Marilyn (25) bred to a C57BL/6 recombination activating gene 1 deficient (RAG1−/− ) background, A1(M).RAG1−/− (20),
C57BL/6.RAG1−/− and CBA/Ca.RAG1−/− mice were bred and maintained in
the specific pathogen-free animal facility of the Sir William Dunn School of
Pathology (Oxford, UK), and used at the age of 5–25 weeks. All experiments
were performed in accordance with the Animals (Scientific Procedures) Act
1986.
Generation of long-term CD4 + T-cell lines
For the generation of long-term Th1 and Th17 lines, A1(M).RAG1−/− or
Marilyn naive CD4+ T cells, purified using the CD4+ T cell isolation kit
(Miltenyi Biotec, CD4+ cell purity >80%), were stimulated weekly in the
presence of female syngeneic mitomycin C-treated T-cell-depleted splenocytes and cognate peptide (100 nM) under the following conditions: for
Th1 cells 5 ng/mL recombinant murine (rm)IL-12, 10 ng/mL rmIL-2 and
10 lg/mL anti-IL-4 (clone 11B11) (26) and for Th17 cells 2 ng/mL recombinant human TGFb1, 50 ng/mL rmIL-6, 50 ng/mL rmIL-21, 10 lg/mL antiIFNc (clone XMG1.2) (27) and 10 lg/mL anti-IL-4. For the preparation of Tcell-depleted splenocytes, mice were injected with 2 mg of each YTA3.1.2
and YTS191.1.2 (anti-CD4), and YTS156.7.7 and YTS169.4.2.1 (anti-CD8)
antibodies (28,29) on days −5 and −3, and spleens were recovered on day
0. Recombinant cytokines were purchased from R&D Systems and PeproTech EC. Peptide sequences recognized by T cells from A1(M).RAG1−/−
and Marilyn mice were REEALHQFRSGRKPI (Dby479–493 ) in the context of
H2Ek and NAGFNSNRANSSRSS (Dby608–622 ) in the context of H2Ab respectively (30). At the end of each activation round, live cells were separated
on a histopaque (Sigma-Aldrich, St. Louis, MO, USA) gradient and cultured
again with fresh splenocytes, antigen, cytokines and neutralizing antibodies. Th1 cells were cultured in RPMI 1640 with 10% fetal bovine serum
(FBS), whereas for Th17 cells IMDM with 10% FBS was used.
at the indicated time points and concentrations. Isotype controls were anticanine antibody YCATE55 (rat IgG1) (33) and murinized IgG104.1 (isotype
control for anti-IL-17A antibody, UCB Celltech, Brussels, Belgium).
Imaging of skin grafts
Mice were anesthetized using isoflurane (IsoFlo, Abbott Animal Health,
Abbott Park, IL, USA) and black and white photos of skin grafts were taken
using a Maestro in vivo imaging system (CRi) weekly starting on day 7 after
transplantation. The skin graft surface area was calculated using Adobe
Photoshop CS2 software (version 9.0.2). Data are expressed as relative
graft surface area normalized to the baseline area measured on day 7 after
grafting. Color photos of skin grafts were taken with an Olympus SP-500
UZ digital camera.
Histological analysis of skin grafts
Skin grafts were recovered and fixed in a solution of 4% wt/vol formaldehyde in PBS. After 24 h, skin grafts were embedded in paraffin and microscopic sections were stained with hematoxylin and eosin (H&E) at the
histology facility of the Sir William Dunn School of Pathology. Immunohistochemical (IHC) analysis was performed with polyclonal rabbit antihuman
myeloperoxidase (Dako, Glostrup, Denmark, A0398) and antihuman CD3
(clone SP7, rabbit IgG, Lab Vision). Photos of histological slides were taken
with a Nikon CoolScope digital microscope (Nikon, Tokyo, Japan).
Fluorescence-activated cell sorting (FACS) analysis
Live cells separated on a histopaque gradient or total splenocytes recovered from mice injected with CD4+ T cells, were restimulated for 4–5 h with
50 ng/mL phorbol myristate acetate (PMA) and 500 ng/mL ionomycin, for
the last 2–3 h in the presence of 10 lg/mL brefeldin A (all from SigmaAldrich). Restimulated cells were stained for membrane markers and subsequently fixed with formaldehyde and permeabilized with the saponin solution (Sigma-Aldrich) in PBS before staining with anticytokine antibodies.
Antibodies used were anti-CD3e (145–2C11), anti-CD4 (RM4–5), anti-Vb6
(RR4–7), anti-Vb8 (F23.1), anti-IL-17A (TC11–18H10.1), anti-IFNG (XMG1.2)
(all from BD Biosciences, Franklin Lakes, NJ, USA) and anti-FoxP3 (FJK16s) (eBioscience, San Diego, CA, USA). Cells were analyzed on a 4-colour
FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using
FlowJo software version 7.6.1 (Tree Star, Ashland, OR, USA). In all FACS
plots, numbers in gates represent percentages of cells.
Analysis of gene expression
Measurement of cell proliferation using tritiated ( 3 H) thymidine
To assess T-cell proliferation, CD4+ T cells were cultured in 96-well round
bottom plates at 5×104 per well with 1×104 bone marrow-derived dendritic
cells (BMDCs) or 25×104 T-cell-depleted splenocytes in the presence of
the indicated conditions. Generation of BMDCs has been described before
(31). Cells were incubated for a total of 72 h, for the last 18–24 h in the
presence of 0.5 lCi 3 H-thymidine (Amersham, Buckinghamshire, UK) per
well of culture. Incorporation of 3 H-thymidine was assessed using a flat-bed
scintillation counter (Perkin-Elmer, Waltham, MA, USA). Data are expressed
as means and standard deviations (SD) of triplicate cultures.
In vivo T-cell transfers and skin transplantation
AutoMACS-isolated naive CD4+ T cells or live-cultured CD4+ T cells separated on a histopaque gradient were resuspended at appropriate concentrations in phosphate-buffered saline (PBS) and injected in the lateral tail vein.
Control mice were injected with PBS. When necessary, skin transplantation
was performed 1 day after injection, as previously described (28,29). Skin
grafts were considered rejected when no viable tissue was visible.
In vivo antibody treatment
Neutralizing antibodies against IFNc (clones XMG1.2 and R46-A2, both rat
IgG1) (27,32) and IL-17A (ab13, UCB Celltech) were injected intraperitoneally
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DNase-treated total RNA was purified using the SV total RNA isolation system (Promega) from cell lysates or from skin graft samples homogenized
using a FastPrep-24 system (MP Biomedicals, Irvine, CA, USA). The SuperScript III first-strand synthesis system for reverse transcriptase polymerase
chain reaction (RT-PCR) with random hexamers (Invitrogen, Carlsbad, CA,
USA) was used to synthesize complementary DNA (cDNA) from 1 lg
of total RNA. TaqMan gene expression assays (Applied Biosystems, Foster City, CA, USA) for Hprt1 (Mm01545399_m1), Cd3g
(Mm00438095_m1), Il17c (Mm00439619_m1), Ifng (Mm00801778_m1),
Mpo (Mm00447886_m1), Rorc (Mm03682796_m1) and Tbx21
(Mm00450960_m1) were used for real-time RT-PCR analysis on the
Applied Biosystems 7500 Fast Real-Time PCR System with SDS software
v1.3.1. Using the 2−Ct method, expression of a target gene was
normalized to an endogenous control gene, and data were presented
relative to the same sequence in a calibrator sample (34).
Chromatin immunoprecipitation (ChIP) analysis
The “fast ChIP” method (35) was used for the immunoprecipitation of
histone 3 trimethylated at lysine 4 (H3K4me3) or 27 (H3K27me3) with
anti-H3K4me3 (17–614, Millipore, Billerica, MA, USA) and anti-H3K27me3
(17–622, Millipore) antibodies, respectively. Real-time PCR was performed
American Journal of Transplantation 2012; 12: 835–845
Th17-Mediated Graft Rejection
using SYBR green master mix (Applied Biosystems) on the Applied Biosystems 7500 fast real-time PCR system with SDS software v1.3.1. The density of an immunoprecipitated factor (target) at a locus was expressed
relative to 10% input DNA according to the equation “relative density
= 2Ct(input)−Ct(target) ”, where Ct(input) and Ct(target) indicate the threshold PCR cycles for amplification of input DNA and immunoprecipitated
factor-associated DNA respectively. The following primers were used for
real-time PCR analysis; RORc t promoter: 5 -CATTCCTAGGCCCGCTGAA3 , 5 -GGGACACACCCTGCCAAAG-3 (36); T-bet promoter: 5 -TGGGCATACAGGAGGCAGCA-3 , 5 -TCGCTTTTGGTGAGGACTGAAG-3 (37).
Statistical analysis
Where indicated, statistical analysis was performed using GraphPad Prism
(version 5.01) for Windows (GraphPad Software, San Diego, CA, USA) with
unpaired two-tailed Student’s t-test. p-Values were classified as not significant (ns; p > 0.05), significant (∗ 0.01<p < 0.05), very significant (∗∗ 0.001
< p < 0.01) or extremely significant (∗∗∗ p < 0.001).
Results
Prolonged culture under polarizing conditions confers
greater stability of IL-17A expression
Previous studies have highlighted the propensity of Th17
cells, generated in short-term cultures, to lose IL-17A expression and up-regulate IFNc following transfer in vivo
(38–43). Although such phenotypic flexibility might be a
physiological property, it does not allow a clear interpretation of adoptive transfer experiments. We hypothesized
that repetitive stimulation in the presence of polarizing conditions would enable the generation of Th17 cells with a
more stable phenotype. As shown in Figure 1A and B, longterm culture of either A1(M).RAG1−/− or Marilyn CD4+ T
cells in the presence of TGFb, IL-6, IL-21 plus neutralizing anti-IFNc and anti-IL-4 antibodies, led to a persistently
high production of IL-17A with variable levels of IFNc coexpression. Importantly, even after 160 days of culture,
cells still maintained specific antigen-dependent proliferation. When transferred into female RAG1−/− hosts, lacking
any endogenous B or T cells, these long-term Th17 lines
were able to maintain expression of IL-17A in the majority
of cells and did not generate any detectable IL-17A− IFNc +
cells (Figure 1C), suggesting they could be used for in vivo
transfer studies.
Th17 cells induce a slow skin graft rejection
In order to test the direct influence of Th17 cells on
graft survival, female C57BL/6.RAG1−/− hosts were infused with Marilyn “long-term” Th17 cells, and subsequently grafted with male C57BL/6.RAG1−/− skin. As positive controls we monitored graft rejection caused by naive
Marilyn CD4+ T cells or “long-term” Th1 cells. Flow cytometry analysis validated the Th1 and Th17 cells as
properly polarized (Figure 2A). As shown in Figure 2B,
both Th1 and Th17 cells were able to enter skin grafts,
as transplants recovered on day 7 from Th1- or Th17injected hosts had increased levels of CD3c mRNA, compared to PBS-injected mice. Nevertheless, grafts on Th1injected hosts had higher levels of CD3c transcripts,
American Journal of Transplantation 2012; 12: 835–845
Figure 1: Prolonged culture of Th17 cells under polarizing conditions confers greater stability of IL-17A expression. (A–B)
Summary FACS analysis for the expression of IL-17A and IFNc
from n = 3 A1(M).RAG1−/− Th17 lines growing for 77 days in
the presence of TGFb, anti-IFNc and anti-IL-4, with either IL-6
or IL-21, or both and n = 4 Marilyn Th17 lines growing for 135
days in the presence of TGFb, IL-6, anti-IFNc and anti-IL-4, with
or without IL-21 and/or IL-23. IL-23 was used at 20 ng/mL. Numbers next to gated populations indicate percentages presented
as means ± SD. Also shown is antigen-dependent proliferation
by A1(M).RAG1−/− (A) and Marilyn (B) Th17 cells growing in the
presence of polarizing conditions for 160 and 57 days, respectively. For 3 H-thymidine incorporation study, cells were restimulated for 3 days with female syngeneic splenocytes or BMDCs
under different conditions. Peptides were used at a concentration
of 100 nM, unless otherwise indicated. (C) A1(M).RAG1−/− Th17
cells growing in the presence of polarizing conditions for 162 days
were transferred into female CBA/Ca.RAG1−/− mice. Mice were
sacrificed on days 10 or 21, and FACS analysis was performed on
splenocytes restimulated with PMA and ionomycin. Numbers next
to gated populations indicate percentages presented as means ±
SD of n = 3 mice/group. FACS plots are gated on live CD4+ cells (A
and B) or live CD3+ CD4+ splenocytes (C). Data are representative
of at least two experiments.
suggesting a more efficient migration and/or increased
proliferation of Th1 cells (Figure 2B). The total numbers
of Vb6+ cells detected in spleens of graft recipients on
day 22 after transplantation were also higher when Th1
cells were injected (Figure S1), suggesting that Th17
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Agorogiannis et al.
Figure 2: Th17 cells enter skin grafts
and induce distinct transplant damage compared to Th1 or naive CD4+
T cells. A total of 0.1×106 Marilyn naive
CD4+ T cells or 10×106 long-term cultured Th1 or Th17 cells were transferred
into female C57BL/6.RAG1−/− hosts on
day −1. Other mice were injected only
with PBS. Mice were grafted with male
or female C57BL/6.RAG1−/− tail skin on
day 0. (A) Phenotype of Marilyn longterm Th1 and Th17 cells before transfer. (B) Real-time RT-PCR analysis for the
expression of CD3c , IL-17A, IFNe and
MPO in male skin grafts recovered on day
7. Data are normalized to expression of
HPRT1 and presented as means and SD of
n = 3 mice/group, except for Th17injected mice (n = 5). Grafts on PBSinjected mice were used as calibrator. Statistical analysis was performed using unpaired two-tailed Student’s t-test. (C) Representative photographs (not to scale) of
male skin grafts on mice injected with
naive CD4+ T cells or PBS only, and female or male skin grafts on Th17-injected
mice (day 14). White-dashed rectangles
are indicative of the position of the graft
only, and do not represent the measurements described in (D). (D) Survival curves
are shown for male skin grafts on PBS-,
CD4+ - or Th1-injected mice, whereas the
relative surface area is presented at the
indicated time points as mean ± SD for
male grafts on PBS-injected hosts, and
male or female grafts on Th17-injected
mice. Statistical analysis was performed
using unpaired two-tailed Student’s t-test
in order to compare male grafts on PBSand Th17-injected mice.
cells might also have a reduced ability to homeostatically
expand in lymphopenic recipients compared to Th1
cells.
A1(M).RAG1−/− Th17 cells into female CBA/Ca.RAG1−/−
hosts and transplantation with male CBA/Ca.RAG1−/− skin
(Figure S2A).
As expected, intragraft levels for IL-17A and IFNc mRNA
were higher after transfer of Th17 and Th1 cells respectively (Figure 2B). Myeloperoxidase (MPO) expression at
this time point, a measure of neutrophil infiltration, was
not increased by the transfer of either Th1 or Th17 cells
above the background levels induced by nonspecific inflammatory changes associated with skin grafting (Figure 2B).
Similar findings were observed following the transfer of
Both Th1 and naive CD4+ T cells induced acute rejection
of male skin grafts (Figure 2C and D), manifesting as massive epithelial necrosis and scab formation within 2 weeks
after grafting, followed by contraction and replacement by
scar tissue. Male grafts on PBS-injected hosts remained
healthy, similar to female grafts on Th17-injected mice
(Figure 2C and D). Nevertheless, Marilyn Th17 cells did not
induce a classic acute rejection, but caused a number of
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American Journal of Transplantation 2012; 12: 835–845
Th17-Mediated Graft Rejection
rapid pathological changes in male skin grafts (Figure 2C),
characterized by hair loss and focal erythema that soon
resolved, and succeeded by a gradual loss of graft volume
that could be followed over time (Figure 2D). A similar
pattern of rejection was induced by A1(M).RAG1−/− Th17
cells (Figures S2B and C). Forty-eight days after transplantation, residual tissue could still be observed in skin grafts
on hosts that received Th17 cells (not shown), so we could
not define a specific time of rejection, but the absence of
any normal skin texture (e.g. hair or dermal ridges) was
proof for the potential of transferred Th17 cells to induce
antigen-specific tissue damage in the absence of other
lymphocyte populations.
Th17 cells promote unique pathological changes and
neutrophil infiltration
The differential rejection phenotypes induced by Th1 and
Th17 cells were further investigated by histological analysis. As shown in Figure 3A, PBS-injected hosts retained
healthy grafts, whereas both Th1 and Th17 cells were
able to induce distinct pathological patterns explaining the
macroscopic changes. As early as day 7 after transplantation, Th1 cells had elicited dermal hemorrhage, signaling
imminent scab formation, and epithelial damage had led to
epidermal thinning (Figure 3A). On the contrary, skin grafts
on Th17-injected hosts showed epidermal hyperplasia, loss
of skin appendages (hair follicles and sebaceous glands)
and extensive dermal inflammation (Figure 3A), without
any obvious signs of inflammatory involvement of the vasculature. Such histological findings were reproducible in
the A1(M).RAG1−/− transplantation system (Figure S2D).
In order to further characterize the Th17-mediated inflammatory reaction, IHC staining for CD3 and MPO was performed in grafts from mice injected with Th17 cells. Figure
3B shows that on day 48 after transplantation dense MPO+
cell infiltrates were present in these grafts, suggesting neutrophils as mediators of Th17 effector function, whereas
the presence of CD3+ cells verified efficient homing of
Th17 cells into grafted tissue. As no difference was observed in MPO mRNA levels between grafts from PBSand Th17-injected mice on day 7 (Figures 2B and S2A), it
is likely that neutrophil infiltration remains active at later
stages during Th17-mediated rejection.
Transferred Th17 cells maintain memory of IL-17A
expression in vivo
We subsequently examined the phenotype of transferred
cells in spleens of grafted mice. Th1 cells maintained their
phenotype, and naive CD4+ T cells differentiated into Th1
cells expressing high levels of IFNc , but no IL-17A (Figure
4A), IL-22 or IL-4 (data not shown). Most Marilyn-derived
Th17 cells maintained IL-17A expression. There was, however, an increase in the percentage of IL-17A− IFNc + cells
that represented only a minor fraction prior to transfer (Figure 4A). This contrasted with A1(M).RAG1−/− Th17 cells
that maintained IL-17A expression without up-regulation of
IFNc (Figure S3A). The phenotype of transferred MarilynAmerican Journal of Transplantation 2012; 12: 835–845
Figure 3: Th17 cells induce pathological changes distinguishable from Th1-mediated rejection and drive intragraft
neutrophil infiltration. (A) Female C57BL/6.RAG1−/− mice, injected with Marilyn Th1 or Th17 cells, were grafted with male
C57BL/6.RAG1−/− skin on day 0, as described in Figure 2. Other
mice were injected with PBS only. H&E staining was performed on
grafts from mice killed on day 7. Different examples of skin grafts
are shown. In grafts from PBS-injected hosts, there is a normal
appearance of epidermis and dermis, with retention of hair follicles and sebaceous glands. Th1 cells-induced epithelial damage
with thinning of the epidermis and red blood cell extravasation,
indicative of dermal hemorrhage. Th17 cells-promoted epidermal
hyperplasia and dermal inflammatory cell infiltration, associated
with loss of skin appendages. (B) Female C57BL/6.RAG1−/− mice
were injected with PBS or Marilyn Th17 cells on day −1, and
grafted with male C57BL/6.RAG1−/− skin on day 0, as described
in Figure 2. IHC staining for CD3 and MPO (in brown) was performed on grafts from mice killed on day 48. Note the abundance
of CD3+ and MPO+ cells in grafts from Th17-injected hosts relative to PBS-injected mice.
derived T cells was also examined after in vitro restimulation in the presence of Dby608–622 peptide. After 4 days of
culture, the vast majority of CD4+ and Th1 cells expressed
IFNc (Figure 4B). The expression of both IL-17A and IFNc
by Th17 cells increased, and IL-17A+ IFNc + cells now represented the most abundant population (Figure 4B). A small
percentage of Th17 cells were seen to up-regulate FoxP3
in vivo, although they lacked any FoxP3 expression before
transfer (Figure S4). It is unclear whether these FoxP3+
cells can interfere with Th17-mediated rejection.
As Th17 lines maintained memory of IL-17A expression in vivo, unlike short-term cultured cells in previous
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Agorogiannis et al.
Figure 4: Maintenance of permissive histone modifications in the Rorc locus
allows transferred Th17 cells to retain
IL-17A expression, while up-regulating
IFNc , in recipients of male skin grafts. (A)
Female mice injected with Marilyn CD4+
(n = 4), Th1 (n = 3), or Th17 (n = 7) cells,
and grafted with male skin as described
in Figure 2, were killed on day 48 after
transplantation, and FACS analysis was performed on splenocytes restimulated with
PMA and ionomycin. Expression of IL-17A
and IFNc by Vb6+ cells is shown. (B) Total splenocytes from (A) were stimulated
at a density of 2.5×105 cells per well in
96-well plates with 100 nM Dby608–622 in
IMDM. After 4 days, cells were restimulated with PMA and inomycin and analyzed
by flow cytometry for expression of IL-17A
and IFNc . All FACS plots are gated on live
Vb6+ cells. Numbers next to gated populations indicate percentages presented as
means ± SD. (C) ChIP analysis of longterm cultured Marilyn Th17 cells stimulated
for 7 days in the presence (Th17) or absence (Th17medium ) of polarizing conditions.
As control, long-term cultured Marilyn Th1
cells were analyzed. Real-time PCR was
performed using DNA from samples immunoprecipitated with antibodies against
H3K4me3 and H3K27me3. Data are normalized to 10% input DNA and presented
as ratios of H3K4me3 to H3K27me3 density at the promoters of RORc t (Rorc+5.0)
and T-bet (Tbx21) genes. Means and SD of
n = 3 technical replicates from one experiment (representative of two) are shown.
studies (38,40), we tested transferred cells for histone 3
modifications in the promoters of RORc t (Rorc+5.0) and
T-bet (Tbx21), two gene loci regulating fidelity to the Th17
phenotype. RORc t drives the differentiation program of
Th17 cells and induces IL-17A expression (44), whereas
T-bet has been implicated in the phenotypic instability of
Th17 cells (38,40,45). ChIP analysis was performed on Marilyn long-term Th17 lines stimulated for 7 days in vitro in
the presence or absence of polarizing cytokines. As shown
in Figure 4C, withdrawal of polarizing conditions was associated with an increase in the H3K4me3/H3K27me3 ratio
for Tbx21, but despite this the permissive modifications
in the RORc t promoter were maintained. In contrast, Th1
cells cultured for the same duration showed predominance
of repressive marks in the RORc t promoter and a predominance of H3K4me3 in Tbx21 as previously described (46).
Similar findings were observed for A1(M).RAG1−/− Th17
cells activated under various conditions (Figure S3B), and
importantly histone 3 modifications in the promoters of
RORc t and Tbx21 mirrored gene expression (Figure S3C).
These results indicate that activation of polarized Th17 cells
in the absence of Th17-inducing cytokines, as may have oc840
curred within skin grafts, should facilitate stable expression
of IL-17A while allowing up-regulation of IFNc .
Th17-induced rejection proceeds independently of
IL-17A and IFNc
We sought to determine whether neutralization of IL-17A
or IFNc could have an effect on Th17-induced rejection. IL17A is a potent inducer of neutrophil mobilization (1, 2) and
Figure 3B clearly indicated participation of neutrophils in
the response to the graft. Additionally, because long-term
Th17 lines produced IFNc as an inherent feature (Figures
1A and B), the significance of this cytokine for Th17 effector function needed clarification. Exposure to IFNc can
up-regulate MHC molecules by nonimmune cells (47,48),
and could thus enhance antigen presentation and the propagation of Th17-associated tissue damage. A combination
of two antibodies, XMG1.2 (27) and R46-A2 (49), known to
neutralize in vivo IFNc activity, and confirmed by ourselves
to block an IFNc -specific ELISA in vitro (data not shown),
was used.
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Th17-Mediated Graft Rejection
Figure 5: Th17-mediated pathology
proceeds independently of IL-17A
or IFNc neutralization. A total of
10×106 long-term cultured Marilyn
Th17 cells were transferred into female C57BL/6.RAG1−/− hosts on day
−1. Mice were grafted with male
C57BL/6.RAG1−/− tail skin on day 0
and treated either with IgG (n = 8) or
anti-IL-17A antibody (n = 10) (3 doses
× 0.75 mg, weekly, starting on the day
of grafting), or with IgG1 (n = 4) or
anti-IFNc antibodies (n = 8) (8 doses
× 1 mg of each R46-A2 and XMG1.2,
every other day, starting on the day
of grafting). (A) Relative surface area
on day 21 is presented as mean and
SD for male grafts on antibody-treated
recipients as described above or female grafts (n = 4). Statistical analysis was performed using unpaired twotailed Student’s t-test. (B) H&E and IHC
stainings for MPO (in brown) were performed on grafts from IgG- or anti-IL17A-treated mice killed on day 22. Note
that the abundance of MPO+ cells remains unchanged despite IL-17A neutralization. The arrows indicate the border between host and grafted skin. (C)
Grafted mice were killed on day 22 after transplantation, and FACS analysis
was performed on splenocytes restimulated with PMA and ionomycin. Expression of IL-17A and IFNc by Vb6+
cells is shown. Statistical analysis was
performed using unpaired two-tailed
Student’s t-test in order to compare relevant groups for the same gated population (∗∗ 0.001<p < 0.01, highlighted
in red). (D) Expression of IL-22 by Vb6+
cells is shown like in (C). Statistical
analysis was performed using unpaired
two-tailed Student’s t-test.
Neither IL-17A nor IFNc neutralization interfered with
Th17-mediated graft contraction on day 21, which was
a consistent finding in both the Marilyn (Figure 5A) and
A1(M).RAG1−/− (Figures S2B and C) systems. Despite an
apparent increase in graft size in the control IgG- compared
to anti-IL-17A-treated Marilyn groups (Figure 5A), histological analysis confirmed the absence of any biological difference between them (Figure 5B and Figure S2D). Neutrophil infiltration, as judged by IHC staining for MPO, was
not influenced by IL-17A neutralization (Figure 5B), suggesting compensatory activity by other Th17-derived molecules
such as IL-17F (50).
Blockade of IL-17A activity did not alter the expression of
IL-17A or IFNc by Th17 cells (Figure 5C and Figure S3A),
American Journal of Transplantation 2012; 12: 835–845
whereas IFNc neutralization reduced the frequency of IL17A− IFNc + cells amongst transferred Marilyn Th17 cells
(Figure 5C). Antigen exposure was responsible for driving IFNc up-regulation by Th17 cells, as Vb6+ splenocytes
recovered from female graft recipients did not show increased expression of IFNc (Figure 5C). Finally, IL-22 expression by transferred cells remained unchanged despite
antibody treatments (Figure 5D).
Discussion
Despite previous reports implicating Th17 cells in transplant rejection (51,52), the exact dimensions of such involvement remain unclear. To our knowledge, our study is
841
Agorogiannis et al.
the first to investigate the individual contribution of Th17
cells to graft rejection in the absence of other adaptive
lymphocytes.
It is still uncertain whether Th17 cells can arise endogenously in response to grafted tissues in wild-type recipients. The implication of IL-17A and Th17 cells in transplant rejection by T-bet-deficient mice (13,21,22), unable to
mount efficient Th1 responses, may suggest a redundant
role for Th17 cells in wild-type hosts. Moreover, transferred
naive Marilyn CD4+ T cells did not seem to differentiate into
Th17 cells (Figure 4A), unless regulatory T cells were also
present (12). Even if Th17 cells have little or no involvement
in graft rejection in unmanipulated animals, it is possible
they might participate in patients receiving immunosuppression that inhibits other effector populations, or where
graft antigens are recognized by preexisting memory Th17
cells acquired through heterologous immunity (53).
Th17 lines with sustainable IL-17A expression were generated after long-term culture under polarizing conditions.
Cells polarized either with IL-23 or without IL-21 displayed
comparable phenotypes, and variable levels of IFNc expression remained an inherent property of these Th17 lines
even after neutralization of both IL-12 and IL-23 (with antiIL-12p40 antibody) (data not shown). Marilyn Th17 cells
were found to generally produce higher levels of IFNc
compared to A1(M).RAG1−/− both in vitro and after in vivo
transfer. The dose of antigen has been shown to influence the level of IFNc expression of Th17 cells (43) and
the higher surface expression of the TCR by Marilyn compared to A1(M).RAG1−/− T cells (54) may have a similar
consequence.
Importantly, epigenetic stability in the Rorc locus following
withdrawal of polarizing conditions indicated that long-term
lines can maintain their core Th17 differentiation status and
expression of IL-17A, despite enrichment for permissive
modifications in the T-bet promoter and subsequent IFNc
production. Overall, while these data might be explained
by plasticity in the Th17 lineage (38–43), an alternative view
would be that production of IFNc is an inherent effector
property of Th17 cells elicited following recognition of antigen. In any case, as tissue damage was not ameliorated
following IFNc neutralization and as the graft pathology
induced by Marilyn and A1(M).RAG1−/− Th17 cells was indistinguishable, it is unlikely that IFNc expression by Th17
cells plays an essential part in this model of graft rejection.
Injected Th17 cells were able to enter skin grafts and induce rapid pathological changes evident as early as day 7
after grafting. Epidermal hyperplasia (acanthosis), loss of
skin appendages and dermal inflammation with neutrophil
infiltration were consistent, suggesting parallels with psoriasis models (55,56), although Munro’s microabscesses
were absent. A murine model of Th17-induced GVHD was
also associated with epidermal hyperplasia (57), and longterm Th17 lines grown with additional IL-23 +/− IL-1b, in842
duced identical pathology (unpublished). These changes
were distinct from Th1-induced acute rejection that was
characterized by epidermal thinning and dermal hemorrhage.
Th17-induced pathology was not prevented following IL17A or IFNc neutralization, suggesting redundancy of effector mechanisms. For example, IL-17F is also known to
promote neutrophil recruitment (50), and IL-17A-deficient
mice show increased neutrophil counts associated with
increased IL-17F and granulocyte colony stimulating factor (CSF3) plasma concentrations (58). It is also possible
that neither IL-17A nor IL-17F are essential to the rejection process mediated by Th17 cells (59), with granulocytemacrophage colony stimulating factor (CSF2) (60,61) and
IL-22 (62) as other candidates to be excluded. Indeed, IL22 has been recognized as an important player in tissuespecific inflammation (63, 64), and IL-22 (65), but not IL-17A
(66), is involved in IL-23-induced acanthosis, and in a mouse
model of psoriasis IL-22 neutralization prevented disease
manifestations (63).
In summary, our data indicate that Th17 cells can induce
graft damage in their own right, as is the case for Th1
and Th2 cells (20), but with a distinct pathology due to different underlying effector mechanisms (67–69). Our study
confirms IL-17A-independent pathways to Th17-induced inflammation (59–61) and suggests new experimental opportunities to resolve mechanisms by which these cells
mediate tissue damage.
Acknowledgments
We are indebted to Mr. Richard Stillion for preparation of histological slides,
Dr. Teresa Marafioti for IHC staining of histological sections, Dr. Diane Marshall (UCB Celltech) for the kind gift of anti-IL-17A and control IgG antibodies
and members of the Pathology Support Building Staff for excellent care of
experimental mice. This work was supported by a MRC grant and an Edward
Penley Abraham studentship (to E.I.A.).
Disclosure
The authors of this publication have no conflicts of interest to disclose as described by the American Journal of
Transplantation.
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Supporting Information
Additional supporting information may be found in the online version of this article.
This section contains four supplementary figures (S1–S4).
Figure S1: Reduced expansion of Th17 cells in female
lymphopenic recipients of male skin grafts compared
to Th1 and naive CD4+ T cells. Female mice injected with
Marilyn CD4+ (n = 4), Th1 (n = 3) or Th17 (n = 7) cells,
and grafted with male skin as described in Figure 2, were
killed on day 48 after transplantation, and FACS analysis
was performed on splenocytes. Total numbers of Vb6+
cells within recovered spleens are shown.
Figure S2: A1(M).RAG1−/− Th17 cells enter skin grafts
and induce transplant pathology independently of IL17A, similar to Marilyn Th17 cells. A total of 10×106
long-term cultured A1(M).RAG1−/− Th17 cells were transferred into female CBA/Ca.RAG1−/− hosts on day −1.
Other mice were injected only with PBS. Mice were
grafted with male CBA/Ca.RAG1−/− tail skin on day 0. Th17injected mice were left untreated (n = 3), or injected with
IgG (n = 5) or anti-IL-17A antibody (n = 5) (3 doses × 0.75
mg, weekly, starting on the day of grafting). (A) Real-time
RT-PCR analysis for the expression of CD3Y, IL-17A, IFNc
and MPO in male skin grafts recovered on day 7. Data
are normalized to expression of HPRT1 and presented as
means and SD of n = 4 (PBS) or 3 (Th17) mice/group.
Grafts on PBS-injected mice were used as calibrator.
American Journal of Transplantation 2012; 12: 835–845
Th17-Mediated Graft Rejection
Statistical analysis was performed using unpaired twotailed Student’s t-test. (B) Representative photographs (not
to scale) of male skin grafts on day 21. White-dashed rectangles are indicative of the position of the graft only, and
do not represent the measurements described in (C). (C)
Relative surface area is presented at the indicated time
points as mean ± SD for male grafts on PBS- or Th17injected hosts. Statistical analysis was performed using
unpaired two-tailed Student’s t-test in order to compare
male grafts on IgG- and anti-IL-17A-injected mice. (D) H&E
staining was performed on grafts from mice killed on day
22. Normal appearance of epidermis and dermis in grafts
on PBS-injected hosts contrasts with epidermal hyperplasia, dermal inflammatory cell infiltration and loss of skin
appendages observed in grafts from recipients of Th17
cells, regardless of IL-17A neutralization. The arrow indicates the border between host and grafted skin, whereas
the arrowheads show fibrin aggregates interspersed with
neutrophils.
Figure S3: A1(M).RAG1−/− Th17 cells maintain IL-17A
expression without upregulation of IFNc , in recipients
of male skin grafts. (A) Female CBA/Ca.RAG1−/− mice
injected with A1(M).RAG1−/− Th17 cells were grafted with
male skin and either left untreated (n = 3) or treated with
IgG (n = 5) or anti-IL-17A antibody (n = 5), as described in
Figure S2. Mice were killed on day 22 after transplantation,
and FACS analysis was performed on splenocytes restimulated with PMA and ionomycin. Expression of IL-17A and
IFNc by Vb8+ cells is shown. (B) ChIP analysis of longterm cultured A1(M).RAG1−/− Th17 cells stimulated for 7
American Journal of Transplantation 2012; 12: 835–845
days in the presence of different conditions. Real-time PCR
was performed using DNA from samples immunoprecipitated with antibodies against H3K4me3 and H3K27me3.
Data are normalized to 10% input DNA and presented as
ratios of H3K4me3 to H3K27me3 density at the promoters
of the RORc t (Rorc+5.0) and T-bet (Tbx21) genes. Means
and SD of n = 3 technical replicates from one experiment
(representative of two) are shown. (C) Cells from (B) were
analyzed for expression of Rorc and Tbx21 mRNA. Data
are normalized to expression of HPRT1 and presented as
means and SD of n = 3 biological replicates for each different condition, except for cells activated in the presence
of Th17-polarizing conditions (n = 4). CBA/Ca splenocytes
were used as calibrator.
Figure S4: Th17 cells upregulate FoxP3 expression in
female lymphopenic recipients of male skin grafts. (A)
Expression of FoxP3 by Marilyn and A1(M).RAG1−/− Th17
cells before in vivo transfer. (B) Female mice injected with
Marilyn CD4+ (n = 4), Th1 (n = 3) or Th17 (n = 7) cells,
and grafted with male skin as described in Figure 2, were
killed on day 48 after transplantation, and FACS analysis
was performed on splenocytes. Expression of FoxP3 by
CD4+ cells is shown.
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should be directed to the corresponding author for the
article.
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