A Lymphotoxin/Type I IFN Axis Programs
CD8 + T Cells To Infiltrate a Self-Tissue and
Propagate Immunopathology
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of June 18, 2017.
Dennis Ng, Blandine Maître, Derek Cummings, Albert Lin,
Lesley A. Ward, Ramtin Rahbar, Karen L. Mossman,
Pamela S. Ohashi and Jennifer L. Gommerman
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2015; 195:4650-4659; Prepublished online 12
October 2015;
doi: 10.4049/jimmunol.1501053
http://www.jimmunol.org/content/195/10/4650
The Journal of Immunology
A Lymphotoxin /Type I IFN Axis Programs CD8+ T Cells To
Infiltrate a Self-Tissue and Propagate Immunopathology
Dennis Ng,* Blandine Maı̂tre,* Derek Cummings,† Albert Lin,‡ Lesley A. Ward,*
Ramtin Rahbar,‡ Karen L. Mossman,† Pamela S. Ohashi,‡ and Jennifer L. Gommerman*
T
ype I IFNs (IFN-I) are pleiotropic cytokines comprised of
a single IFN-b, numerous IFN-a, and other IFN subtypes,
including IFN-ε, -k, -v, and -d, that facilitate various
immune responses. Despite this diversity in IFN members, all
IFN-I bind exclusively to the IFN-a receptor expressed ubiquitously on all cell types (1). One of the confounding issues concerning IFN-I is the multifaceted effects of this cytokine that exert
discrepant biological outcomes, such as cell proliferation versus
cytotoxicity and immune activation versus immune suppression (2,
3). Various studies have demonstrated the importance of IFN-I in
the generation of CTLs (4–6), whereas others have shown the
suppressive effects of IFN-I on CD8+ T cell–mediated immune
responses (7–10). How IFN-I can exert both proliferative and cytotoxic effects remains unclear, although factors such as the dose,
timing, persistence, and location of IFN-I production may be relevant factors. Given that an IFN-I gene signature is associated with
numerous chronic autoimmune diseases (11–13), understanding the
*Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8,
Canada; †Department of Pathology and Molecular Medicine, McMaster University,
Hamilton, Ontario L8S 4L8, Canada; and ‡Campbell Family Cancer Research Institute, University Health Network, Toronto, Ontario M5G 2M9, Canada
ORCID: 0000-0002-3264-7330 (B.M.).
Received for publication May 6, 2015. Accepted for publication September 18, 2015.
This work was supported by an Ontario Graduate Student Scholarship (to D.N.) and
Canadian Institutes of Health Research Operating Grant MOP 67157 (to J.L.G.).
Address correspondence and reprint requests to Dr. Jennifer L. Gommerman, Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, ON
M5S 1A8, Canada. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BMDC, bone marrow–derived DC; DC, dendritic cell;
IRF3, IFN regulatory factor 3; LCMV-GP, lymphocytic choriomeningitis virus glycoprotein; LT, lymphotoxin; LTbR, LT-b receptor; PRR, pattern recognition receptor;
RANK, NF-kB; RANKL, NF-kB ligand; RIP-GP, rat insulin promoter-glycoprotein;
TNFRSF, TNFR superfamily; TNFSF, TNF superfamily member; WT, wild-type.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501053
impact of this cytokine on T cell responses is important for rationalizing immunomodulatory therapies.
Dendritic cells (DC) survey the body and rely on pattern recognition receptors (PRRs) such as TLRs for the detection of invading microbes. As efficient APCs, DCs activate the adaptive
immune response by providing several signals for optimizing T cell
activation. These include 1) Ag-specific peptide–MHC complexes,
2) costimulatory molecules such as CD80 and CD86, and 3)
cytokines such as IL-12 and IFN-I. In the case of bacterial or viral
infection, the presence of inflammatory cytokines triggered by
DC-intrinsic PRR activation generates highly immunogenic DC
that can directly prime CD8+ T cell responses. However, during
immune responses that elicit lower levels of inflammation, DCs
often require help from CD4+ T cells to boost DC activation and
their capacity to secrete cytokines (14). Previous studies have
shown that activated CD4+ T cells upregulate several TNF superfamily members (TNFSF), including CD40-L (CD154), receptor activator for NF-kB ligand (RANKL), and LTab during an
immune response. These TNFSF ligands signal cognate TNFRs
expressed on DC as a form of help to potentiate the T cell–priming
capacity of DC for the generation of CTLs in the context of helpdependent immune responses (15–18).
Dendritic cells (DCs) are major producers of IFN-I, and PRR
activation has been well characterized as the primary means of
triggering IFN-I production from DC. Signaling through TLR-3, -4,
-7, and -9 as well as the RIG-I–like receptors can induce rapid and
robust IFN-I production in response to infection (2). Interestingly,
recent studies have hinted that the TNFR superfamily (TNFRSF)
members, TNFR-1, TNFR-2, receptor activator for NF-kB (RANK),
and lymphotoxin (LT)-b receptor (LTbR), are also capable of inducing IFN-I production; however, in contrast to PRR activation,
TNFRSF members typically elicit a very modest, albeit sustained
level of IFN-I production (19–23).
Previously, we showed that expression of LTab on Ag-specific
CD4+ T cells provides a help signal through the LTbR on DC, and
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Type I IFNs (IFN-I) are cytokines that can mediate both immune suppression and activation. Dendritic cells (DC) are significant
producers of IFN-I, and depending on the context (nature of Ag, duration of exposure to Ag), DC-derived IFN-I can have varying
effects on CD8+ T cell responses. In this study, we report that in the context of a CD8+ T cell response to a self-Ag, DC-intrinsic
expression of IFN regulatory factor 3 is required to induce optimal proliferation and migration of autoreactive CD8+ T cells,
ultimately determining their ability to infiltrate a target tissue (pancreas), and the development of glucose intolerance in rat
insulin promoter–glycoprotein (RIP-GP) mice. Moreover, we show that signals through the lymphotoxin-b receptor (LTbR) in DC
are also required for the proliferation of autoreactive CD8+ T cells, the upregulation of VLA4/LFA1 on activated CD8+ T cells,
and their subsequent infiltration into the pancreas both in vitro and in vivo. Importantly, the defects in autoreactive CD8+ T cell
proliferation, accumulation of CD8+ T cells in the pancreas, and consequent glucose intolerance observed in the context of priming
by LTbR2/2 DC could be rescued by exogenous addition of IFN-I. Collectively, our data demonstrate that the LTbR/IFN-I axis is
essential for programming of CD8+ T cells to mediate immunopathology in a self-tissue. A further understanding of the IFN-I/
LTbR axis will provide valuable therapeutic insights for treatment of CD8+ T cell–mediated autoimmune diseases. The Journal
of Immunology, 2015, 195: 4650–4659.
The Journal of Immunology
Materials and Methods
Mice
C57BL/6 wild-type (WT) mice were purchased from Charles River Laboratories. LTbR2/2 mice (25) were a gift of R. Newberry (Washington
University School of Medicine, St. Louis, MO). Rat insulin promoter–glycoprotein (RIP-GP) (26), P14 (27), and SMARTA transgenic mice (28) were
gifts of P. Ohashi (Campbell Family Cancer Research Institute, University
Health Network). All animals were housed under specific pathogen-free
conditions, and all experiments were performed according to animal-use
protocols approved by the University of Toronto.
Bone marrow–derived DC culture
Femurs and tibia were dissected, and bone marrow cells were harvested by
flushing with PBS. The bone marrow–derived DC (BMDC) cultures were
prepared at a density of 2 3 106 cells/ml in RPMI 1640 medium (SigmaAldrich) supplemented with 10% FBS (Life Technologies inactivated for
60 min at 56˚C), 0.05 mM 2-ME, 100 U/ml penicillin, 100 ug/ml streptomycin, and 40 U/ml murine rGM-CSF (Peprotech). Fresh medium was
added every 3 d until day 6, and every 2 d thereafter. Nonadherent cells
were harvested on day 10 for analysis.
RIP-GP diabetes model
For induction of diabetes in RIP-GP mice, we activated BMDC with
10 ng/ml LPS (Sigma-Aldrich) for 18 h and pulsed with 1 mg/ml gp33–41
(KAVYNFATM), gp276–286 (SGVENPGGYCL), and gp61–80 (GLNGPDIYKGVYQFKSVEFD) for 10 h. Pulsed DCs were washed three times
with PBS and transferred i.v. at 2 3 106 cells per mouse. For IFN-a addback experiments, mice were injected with 10,000 U IFN-a (purchased
from PBL Assay Science catalogue 12100-1) i.v. on day 3 post-BMDC
transfer. Diabetes induction was monitored by blood glucose measurements on the day of treatment and every 3 d after transfer of BMDC. Blood
glucose levels were measured using AccuChek III glucometers and
Chemstrips (Roche).
Flow cytometry
Abs against CD4, CD8, CD11c, CD11a, CD49d, CD45.1, CD45.2, MHCII,
CD11b, IFN-g, TNF-a, as well as streptavidin-fluorochrome conjugates
were purchased from eBioscience. For analysis of lymphocytic
choriomeningitis virus glycoprotein (LCMV-GP)-specific CD8+ T cells,
monomers of H-2Kb:KAVYNFATM were purchased from the Baylor
College of Medicine, and tetramers were made by conjugating with PE or
allophycocyanin streptavidin purchased from Molecular Probes and Life
Technologies.
Pancreas histology
Pancreata were perfused with PBS, washed, and placed into 10% formalin
for 24 h. Paraffin embedding, sectioning, and H&E staining were performed
at the Toronto Centre for Phenogenomics (Toronto, Ontario, Canada). For
fluorescent immunohistochemistry staining, pancreata were embedded in
OCT compound (Tissue-Tek, Sakura Finetek). Tissue sections were
blocked with mouse serum and stained with CD3-allophycocyanin, CD4FITC, and CD8-PE (eBioscience). Stained sections were captured with
a QImaging Retiga Exi camera through a Leica DMRA2 microscope.
Statistical analysis
Data were analyzed based on two-tailed Student t test using GraphPad
Prism software. Responses were considered significant when a p value
,0.05 was obtained.
Results
DC-intrinsic LTbR signaling is required to induce pancreatic
inflammation and glucose intolerance
To study the effect of DC-intrinsic LTbR signaling and the priming
of CD8+ T cells in the context of an autoimmune disease, we
compared the immunogenic potential of WT and LTbR 2/2
BMDC in provoking an autoimmune response to a neo–self-Ag.
Specifically, we used RIP-GP mice that express the LCMV-GP
under the control of the rat insulin-inducible promoter such that
LCMV-GP is expressed by pancreatic islet b cells. Normally, the
peripheral LCMV-GP–reactive T cells are ignorant to the expression of LCMV-GP Ag on pancreatic islet b cells (26). This
tolerance can be breached through the adoptive transfer of LCMVGP peptide–loaded BMDC that have been activated with a TLR
agonist, thereby triggering the activation of LCMV-GP–specific
CD8+ T cells in the endogenous T cell repertoire. Activated
LCMV-GP–specific CD8+ T cells subsequently infiltrate the
pancreas and contribute to islet cell mass loss (29, 30).
We first determined whether diabetes induction by peptideloaded BMDC is dependent on CD4+ T cell help in RIP-GP
mice by monitoring diabetes incidence induced by BMDC that
were stimulated with LPS and loaded with or without the MHCII–restricted LCMV-GP peptide. Indeed, BMDC loaded with only
MHC-I–restricted peptides did not develop diabetes, suggesting
that CD4+ T cell help is critical for the proper activation of
LCMV-GP–specific CD8+ T cells in vivo, and that CD4+ T cell:
DC cross talk is important for instigating autoimmunity under
these conditions (Supplemental Fig. 1).
Because we had previously shown that the expression of LTab on
CD4+ T cells was involved in T cell:DC cross talk (15), we next
asked whether LTbR signaling in DC was required for diabetes
induction in RIP-GP mice. BMDC derived from WT or LTbR2/2
donor mice were loaded with LCMV-GP peptide and adoptively
transferred into RIP-GP mice to compare the immunogenic potential of WT versus LTbR2/2 DC in priming LCMV-GP–specific
CD8+ T cells for their capacity to mediate immune pathology in
the pancreas. Adoptively transferred WT and LTbR2/2 BMDC
were found to survive equally well in recipient mice (data not
shown). The majority of RIP-GP mice that received WT BMDC
exhibited a marked increase in blood glucose levels between days 6
and 10 post-DC transfer, and the mice remained diabetic for the
remainder of the experiment. In contrast, RIP-GP mice that received
LTbR2/2 BMDC were less susceptible to diabetes, with on average
,15% of mice developing glucose intolerance (Fig. 1A for three
representative experiments, and Table I for summary of all eight
experiments). Histological and immunofluorescence examination of
pancreata from RIP-GP mice that had received WT BMDC revealed
significant immune cell infiltration (primarily CD8+ T cells) into the
islets, whereas pancreata from RIP-GP mice that received LTbR2/2
BMDC displayed a lesser degree of immune cell infiltration into the
islets (Fig. 1B, 1C). Quantitative analysis by flow cytometry of
pancreas-resident cells showed a ∼5-fold difference in the frequency of CD8+ T cells within the pancreas on day 7 after transfer
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this LTbR-dependent T cell:DC cross talk was required to optimize CD8+ T cell expansion in vitro and in vivo in response to
protein Ag (20). Although the induction of IFN-I via PRR signaling has been well characterized (24), the relevance and
mechanism of TNFRSF-facilitated IFN-I, specifically in the context of an autoimmune attack on a self-tissue, are not well understood. In this study, we have examined the involvement of DCintrinsic LTbR signaling and IFN-I production in the context of
a CD8+ T cell–mediated disease model. In this study, we found
that LTbR-deficient DC were inefficient at promoting Ag-specific
CD8+ T cell expansion in response to a neo–self-Ag expressed in
the pancreas, resulting in significantly lower expression of adhesion molecules on Ag-specific CD8+ T cells that restrict their
access from the target organ. However, mice that received LTbRdeficient DC, but supplemented with exogenous IFN-a, successfully restored CD8+ T cell expansion and the infiltration of CD8+
T cells into the pancreas, resulting in the destruction of islet b cell
mass and glucose intolerance. Taken together, our study sheds new
light on the importance of a LTbR/IFN-I axis in shaping the early
CD8+ T cell response to a nonreplicating self-Ag, thus providing
a therapeutic rationale for inhibiting the LT pathway in the context
of autoimmune disease.
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LTbR SIGNALING IN DC MODULATES DISEASE PATHOLOGY
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FIGURE 1. DC-intrinsic LTbR signaling is required for the induction of diabetes. (A) Diabetes incidence in RIP-GP mice that received an adoptive transfer of
LCMV-GP peptide–loaded WT or LTbR2/2 BMDC. Data show blood glucose levels post-BMDC transfer and disease incidence. Data shown are the cumulative
incidence curve of three experiments (n = 23–27 mice per group) and are representative of eight independent experiments (see Table I for results from all experiments).
(B) Pancreata isolated from RIP-GP mice 6 d after transfer of WT or LTbR2/2 LCMV-GP peptide–loaded BMDC were stained with H&E to assess infiltration
of islets. Scale bar at the lower right corner represents 100 mm. (C) Immunofluorescent staining was performed on pancreata from the experiment in (B) using
fluorescent-conjugated Abs specific for CD8 and CD3. Data shown for (B) and (C) is representative of three or more independent experiments with n = 3–7 mice per
group. (D) The frequency of total CD8+ T cells found in the pancreas at 7 and 10 d after transfer was determined by FACS. Control refers to mice that did not receive
LCMV-GP–loaded BMDC—this was only relevant when examining total CD8+ T cells, as LCMV-GP–specific CD8+ T cells were below the limit of detection in mice
not receiving LCMV-GP–loaded BMDC. Data shown are representative of three or more independent experiments (n = 3–7 mice per group). **p , 0.01, *p , 0.05.
The Journal of Immunology
4653
Table I. Average incidence of glucose intolerance in RIP-GP mice receiving either WT or LTbR2/2 LCMV-GP peptide–loaded BMDC
Frequency of Glucose Intolerance with
WT BMDC Vaccination (%)
Frequency of Glucose Intolerance with
LTbR2/2 BMDC Vaccination (%)
89
89
67
100
67
100
100
78
86
12
29
17
43
0
14
0
10
16
Experiment 1
Experiment 2
Experiment 3
Experiment 4
Experiment 5
Experiment 6
Experiment 7
Experiment 8
Average incidence across experiments
Average incidence of glucose intolerance in eight independent experiments with RIP-GP mice receiving either WT or LTbR2/2 LCMV-GP peptide–loaded BMDC.
Proliferation and upregulation of VLA4/LFA-1 on CD8+ T cells
correlate with the induction of diabetes in RIP-GP mice
We previously showed that LTbR signaling in DC is essential for
CD8+ T cells to fully expand in response to a nonreplicating
protein Ag, OVA (20). To assess whether CD8+ T cell expansion
in response to an endogenous autoantigen is likewise dependent
on DC-intrinsic LTbR signaling, we examined the frequency of
LCMV-GP–specific CD8+ T cells in recipient mice post-BMDC
transfer (note that for mice that did not receive LCMV-GP peptide–
loaded DC, LCMV-GP–specific CD8+ T cells were not detectable
by tetramer analysis and therefore not shown). Interestingly, we
noted a ∼50% reduction in the frequency (Fig. 2A) and numbers
(Supplemental Fig. 2A) of CD8+ T cells specific for LCMV-GP
peptide in the blood, pancreatic lymph node, and the pancreas itself.
This observation was further confirmed by transferring gp33specific transgenic T cells derived from P14 transgenic mice
(Supplemental Fig. 2B). However, despite impaired CD8+ T cell
expansion in the mice that received LTbR2/2 LCMV-GP peptide–
loaded BMDC, we found that CD8+ T cells restimulated with
LCMV-GP peptides could still produce equivalent levels of the
cytokines IFN-g and TNF-a (Fig. 2B). These data indicate that
some priming of Ag-specific CD8+ T cells in response to LTbR2/2
LCMV-GP peptide–loaded BMDC may occur (thus eliminating the
possibility that LTbR2/2 LCMV-GP peptide–loaded BMDC simply
fail to persist in RIP-GP mice). However, this limited priming by
LTbR2/2 LCMV-GP peptide–loaded BMDC is not sufficient to
induce optimal expansion of Ag-specific CD8+ T cells in the blood,
or their accumulation in the pancreas.
Even though DC-intrinsic LTbR signaling contributes to reduced Ag-specific CD8+ T cell numbers in the periphery, the
paucity of CD8+ T cells in the pancreas of mice that received
LCMV-GP peptide–loaded LTbR2/2 BMDC (Fig. 1D) may not
necessarily be explained by impaired expansion alone. For effector T cells to successfully migrate into target organs, they must
upregulate the expression of key adhesion molecules such as
VLA-4 and LFA-1. Indeed, all of the infiltrating CD8+ T cells
located in the pancreas of RIP-GP mice expressed high levels of
both VLA-4 and LFA-1, and they were found to be actively
proliferating, as evidenced by the expression of Ki67 (Fig. 3A).
We therefore examined the expression of VLA-4 and LFA-1 on
LCMV-GP–specific CD8+ T cells in the periphery to determine
whether there was a defect in the upregulation of these markers on
CD8+ T cells in mice with poor pancreatic CD8+ T cell infiltration
and diabetes induction. Indeed, we found that the expression of
both VLA-4 and LFA-1 on LCMV-GP–specific CD8+ T cells was
significantly reduced in the mice that received LTbR2/2 BMDC,
particularly on day 7 post-BMDC transfer (Fig. 3B). Hence, a
reduction in VLA4 and LFA1 expression on peripheral CD8+
T cells in RIP-GP mice receiving LCMV-GP peptide–pulsed
LTbR2/2 BMDC correlates with a reduction in CD8+ T cells in
the pancreas.
Expression of IFN regulatory factor 3 by BMDC is required for
optimal CD8+ T cell activation/expansion and diabetes
induction in RIP-GP mice
Because LTbR signaling can facilitate IFN-I expression in DCs
(20) and IFN-I is particularly important for the programming of
CTLs (4, 5, 31, 32), we queried whether adoptive transfer of
LCMV-GP peptide–loaded BMDC that are incapable of producing IFN-I would have a similar effect in RIP-GP mice
compared with RIP-GP mice that received LCMV-GP peptide–
pulsed LTbR2/2 BMDC. Accordingly, we transferred BMDC
generated from IFN regulatory factor 3 (IRF3)–deficient mice
into the RIP-GP host to assess their capacity to induce disease.
IRF3 is a transcription factor that mediates IFN-I expression
downstream of TLR-3, TLR-4, and the RIG-I–like receptor in
response to infection. IRF32/2 DC cannot produce IFN-I in
response to LPS (33). When we transferred IRF32/2 BMDC into
RIP-GP mice, they also failed to develop diabetes (Fig. 4A),
concomitant with a reduction in the frequency and number of
LCMV-GP–specific CD8+ T cells in the blood (Fig. 4B) and
a significant reduction in total infiltrating CD8+ T cells into the
pancreas (Fig. 4C). Moreover, LCMV-GP–specific CD8+ T cells
primed by IRF32/2 BMDC in vivo expressed significantly lower
levels of VLA-4 and LFA-1 (Fig. 4B), reminiscent of the LTbRdeficient setting (Fig. 3B). Thus, adoptive transfer of IRF32/2
BMDC or LTbR2/2 BMDC to RIP-GP mice results in similar
outcomes with respect to CD8+ T cell phenotype and diabetes
susceptibility.
Exogenous administration of IFN-I restores CD8+ T cell
activation and immunopathology in RIP-GP mice that received
LTbR2/2 BMDC
Given the strong phenocopy observed between IRF32/2 versus
LTbR2/2 BMDC in the context of the RIP-GP model, we hypothesized that the production of IFN-I facilitated by DC-intrinsic
LTbR signaling was important for optimizing CD8+ T cell proliferation and their potential to invade and destroy pancreas tissue.
To test this hypothesis, we added rIFN-a to our established in vitro
and in vivo systems. For the in vitro experiments, we isolated
gp61-specific CD4+ T cells from transgenic SMARTA mice and
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comparing RIP-GP mice that received WT BMDC versus LTbRdeficient BMDC, and an even greater disparity was observed on day
10 (Fig. 1D). Therefore, the induction of CD8+ T cell infiltration
into the pancreas and subsequent development of diabetes by the
transfer of LCMV-GP peptide–pulsed BMDC into RIP-GP mice
requires CD4+ T cell help and BMDC-intrinsic LTbR signaling.
4654
LTbR SIGNALING IN DC MODULATES DISEASE PATHOLOGY
gp33-specific CD8+ T cells from P14 mice, and cocultured
these T cells with LPS-activated WT or LTbR2/2 BMDC
(Supplemental Fig. 3). When we titrated the amount of gp33
peptide in the culture (data not shown), we were able to generate
a help-dependent system whereby LCMV-GP–specific CD8+ T cells
failed to expand in the absence of CD4+ SMARTA T cells. Using
this in vitro system, we found that LCMV-GP–specific CD8+ T cells
cocultured with peptide-loaded LTbR2/2 BMDC expanded poorly
when compared with LCMV-GP–specific CD8+ T cells cocultured
with peptide-loaded WT BMDC (Supplemental Fig. 3A). Furthermore, CD8+ T cells cocultured with peptide-loaded LTbR2/2
BMDC also expressed significantly lower levels of VLA-4 and
LFA-1 (Supplemental Fig. 3A, 3B), reminiscent of our in vivo
observation (Fig. 3B).
Interestingly, we found that in the presence of exogenously
added rIFN-a, the proliferation and the expression of VLA4 and
LFA1 on LCMV-GP–specific CD8+ T cells primed by LTbR2/2
LPS-activated BMDC were restored (Supplemental Fig. 3A,
3B). Likewise, in a similar experimental setup, the add-back
of rIFN-a restored the expression of VLA4 on OVA-specific
CD8+ T cells to WT levels, confirming that the defect in
VLA4 expression on CD8+ T cells we observed in the presence
of LTbR2/2 DC was not restricted to the LCMV-GP system
(Supplemental Fig. 4A). However, in the in vitro culture, the
LTbR help can be overridden with increasing concentrations of
Ag, suggesting that LTbR-derived signals in DC provide a form
of help that shifts the set point of responsiveness for CD8+
T cells such that they require less Ag for their optimal activation
(Supplemental Fig. 4B).
Lastly, to test whether IFN-I facilitated by DC-intrinsic LTbR
signaling was relevant for inducing CD8+ T cell responses targeted at the pancreas, RIP-GP mice that were injected with
LTbR2/2 BMDC were subsequently treated with IFN-a. Consistent with our findings shown in Fig. 1, LCMV-GP peptide–
loaded LTbR2/2 BMDCs could not induce diabetes in the RIPGP mice. However, when RIP-GP mice were also provided with
rIFN-a, .80% RIP-GP mice that received LCMV-GP peptide–
loaded LTbR2/2 BMDCs became diabetic by day 9 after
transfer (Fig. 5A). Of the RIP-GP mice that became diabetic,
LCMV-GP–specific CD8+ T cells were found to expand normally and upregulated VLA-4 and LFA-1 to comparable levels
as that found on LCMV-GP–specific CD8+ T cells derived from
RIP-GP mice administered LCMV-GP–pulsed WT BMDC
(Fig. 5B). Moreover, CD8+ T cell infiltration into the pancreas
was partially restored in mice that received peptide-pulsed
LTbR2/2 BMDCs along with exogenously added IFN-a
(Fig. 5C). This implies that LTbR2/2 LCMV-GP peptide–
loaded BMDCs were capable of limited priming in RIP-GP
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FIGURE 2. DC-intrinsic LTbR
signaling is required for optimal
expansion of Ag-specific CD8+ T cells
in the periphery and their accumulation in the pancreas. RIP-GP mice
were given an adoptive transfer of
activated WT or LTbR2/2 BMDC
loaded with LCMV-GP peptides to
induce diabetes. (A) At 7 and 10 d
after transfer, the frequency of
LCMV-GP–specific CD8+ T cells
was measured in the blood, pancreatic LN, and pancreas. **p , 0.01,
*p , 0.05. (B) CD8+ T cells in the
blood harvested on day 7 were
restimulated with LCMV-GP peptides to assess cytokine production.
Frequency of CD8+ T cells that
produce cytokines in response to
peptides is depicted. Control samples were derived from mice that
did not receive LCMV-GP–loaded
BMDC. Data shown are representative of three or more independent
experiments (n = 3–7 mice per
group).
The Journal of Immunology
4655
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FIGURE 3. Pancreas-infiltrating LCMV-GP–specific CD8+ T cells express high levels of adhesion molecules VLA-4 and LFA-1. (A) The pancreata of
RIP-GP mice that had previously received LCMV-GP peptide–loaded WT BMDC were analyzed by FACS. Expression of VLA4, LFA1, and Ki67 was
assessed on CD8+ T cells isolated from pancreata (empty histograms) relative to the respective FMO controls (shaded histograms). (B) The expression of
VLA-4, LFA-1, and Ki67 on peripheral LCMV-GP–specific CD8+ T cells was compared in mice that received either WT or LTbR2/2 LCMV-GP peptide–
loaded BMDC on days 7 and 10 after transfer. Data are representative of three or more independent experiments (n = 3–7 mice per group). **p , 0.01,
*p , 0.05.
mice, but, without an appropriate amount of IFN-I, disease induction is suboptimal.
Collectively, these data indicate that IFN-I production by DCs
is necessary for CD8+ T cell expansion, expression of VLA-4/
LFA1 on CD8+ T cells, and diabetes induction in the absence of
DC-intrinsic LTbR signaling. These results highlight an impor-
tant role for the LTbR/IFN-I axis in promoting autoaggressive
CD8+ T cell responses.
Discussion
Type I IFNs can act on different immune cell types with varying
effects and functions. They play an important role in antiviral re-
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LTbR SIGNALING IN DC MODULATES DISEASE PATHOLOGY
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FIGURE 4. DC-intrinsic expression of IRF3 is required to prime CD8+ T cells and induce diabetes in RIP-GP mice. (A) Blood glucose level and diabetes
incidence of RIP-GP mice that received an adoptive transfer of activated WT or IRF32/2 BMDC were monitored. (B) The frequency and numbers of
LCMV-GP–specific CD8+ T cells and their expression of VLA-4, LFA-1, and Ki67 were quantified in the periphery of RIP-GP mice on days 7 and 10 after
transfer of WT versus IRF32/2 LCMV-GP peptide–loaded BMDC. *p , 0.05. (C) Pancreata from RIP-GP mice receiving WT or IRF32/2 LCMV-GP
peptide–loaded BMDC were compared using H&E staining to assess leukocyte infiltration of islets at day 10 after transfer and by FACS to determine the
frequency of CD8+ T cells within the pancreata on days 7 and 10 after transfer. Scale bar at the lower right corner represents 100 mm. Control refers to mice
that did not receive LCMV-GP–loaded BMDC. These data are pooled from two independent sets of experiments (n = 4–6 mice per group). ***p , 0.001,
**p , 0.01, *p , 0.05.
The Journal of Immunology
4657
sponses during infection and can promote immune cell recruitment
and activation. In contrast, IFN-I is also widely used as a therapeutic
treatment for some autoimmune diseases, ostensibly dampening
overzealous immune responses (34), and IFN-I has been shown
to have both antiproliferative and proapoptotic functions in
various systems (35, 36). Whether IFN-I are pro- or antiinflammatory, the relevant cells that produce IFN-I, and how
the effects of IFN-I are regulated in vivo, remain largely unclear.
One possible molecular explanation for differential effects of
IFN-I may be that varying doses/kinetics of IFN-I exposure result in differential expression of STAT transcription factors, ultimately governing the type of biological response incurred (24,
36). Our study provides new insights into how TNFSF and PRR
cooperate in provoking an autoimmune response directed at
a self-tissue in an IFN-I–dependent manner.
Immune responses that do not elicit strong inflammatory cytokines require CD4+ T cell help for optimal generation of CTLs
(37). Our study shows that DC-intrinsic LTbR signaling represents
an important help signal that regulates the induction of IFN-I. The
lack of this help signal, or alternatively the absence of a DCderived IFN-I response (as in IRF32/2 DC), results in poor expansion of Ag-specific CD8+ T cells and impaired upregulation of
VLA-4 and LFA-1. LFA-1 is a promigratory receptor that is
expressed on all leukocytes as well as activated T cells, and its
expression allows immune cells to bind ICAM family members
that are largely expressed on endothelial and target tissue cells
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FIGURE 5. Exogenous administration of IFN-I restores CD8+ T cell expansion and immunopathology in RIP-GP mice that received LTbR2/2 BMDC.
(A) The LCMV-GP peptide–loaded WT or LTbR2/2 BMDC were administered to RIP-GP mice and on day 3 after transfer, and mice receiving LTbR2/2
BMDC were further administered rIFN-a i.v. Glucose levels and disease incidence up to 15 d post-BMDC administration are depicted. (B) The frequency of
LCMV-GP–specific CD8+ T cell in the blood and their expression of VLA-4 and LFA-1 were compared by FACS at day 10 post-BMDC administration,
**p , 0.01, *p , 0.05, Student’s t test. (C) Pancreata extracted at day 10 postimmunization were subjected to flow cytometry to examine CD8+ T cell
infiltration. Control refers to mice that did not receive LCMV-GP–loaded BMDC. Data are representative of two independent sets of experiments (n = 9–10
mice per group).
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in response to Listeria monocytogenes (51). A recent study
showed that both IFN-I and LTbR signaling are important for the
control of hepatitis B virus in human hepatocytes; however, in this
case, LTbR ligation does not trigger IFN-I expression within hepatocytes (52). It is therefore possible that the facilitation of IFN-I
production in APCs (DC) by LTbR signaling is unique to APCs
and is particularly manifested in the context of CD8+ T cell
priming.
In summary, our current study characterizes LTbR signaling as
a crucial help signal that regulates IFN-I expression in DCs and
demonstrates the importance of this signal for CD8+ T cell programming and generation of tissue-infiltrating CD8+ T cells for the
induction of diabetes. Importantly, a recently published clinical trial
in psoriatic arthritis has shown that LTbR-Ig therapy has the capacity to reduce the IFN-I signature in those arthritic patients that
exhibit such a signature pretreatment, linking the LT pathway with
IFN-I production in humans in vivo (53). In conclusion, inhibitors
of the LT pathway, through their effect on IFN-I production, may
have therapeutic potential in CD8+ T cell–driven disease states.
Acknowledgments
We thank the entire Gommerman laboratory for help and suggestions. We
also thank Dionne White of the flow cytometry facility, Faculty of Medicine, University of Toronto. We thank Derek Cloutier for providing
SMARTA mice and technical assistance.
Disclosures
J.L.G. has a granted patent (Browning, J.L. and Gommerman, J.L. “Treatment of immunological renal disorders by lymphotoxin pathway inhibitors.” United States patent no. U.S.S.N. 50/422,588). This patent does not
have any bearing on the content of this manuscript. The other authors have
no financial conflicts of interest.
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