T cells from epicutaneously immunized mice are prone to T cell receptor revision

Yale University, New Haven, Connecticut, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2005; 102(8):2898-903. DOI: 10.1073/pnas.0409880102
Source: PubMed
ABSTRACT
Epicutaneous immunization of T cell receptor (TCR) transgenic (Tg) mice whose CD4(+) T cells are specific for the Ac1-11 fragment of myelin basic protein (MBP) with Ac1-11 elicits T cells with dominant suppressor/regulatory activity that confers protection against Ac1-11-induced experimental autoimmune encephalomyelitis. We now report that such disease-resistant MBP TCR Tg mice also harbor a sizeable fraction of peripheral CD4(+) T cells lacking surface expression of the Tg TCR beta chain and expressing diverse, endogenously rearranged TCR beta chains. Ex vivo incubation at physiological temperature caused the loss of neo-beta-chain expression and reversion to the MBP alphabeta TCR(+) phenotype. The presence of recombination activating gene 1 and 2 proteins in CD4(+) T cells with revised TCRs was consistent with effective V(D)J recombination activity. The emergence of these cells did not depend on the thymic compartment. We conclude that in mice epicutaneously immunized with an autoantigen, peripheral specific T cells are susceptible to multiple mechanisms of tolerance.

Full-text

Available from: Richard A Flavell
T cells from epicutaneously immunized mice are
prone to T cell receptor revision
Margaret S. Bynoe*, Christophe Viret
, Richard A. Flavell*, and Charles A. Janeway, Jr.
Section of Immunobiology, Howard Hughes Medical Institute and Yale University School of Medicine, New Haven, CT 06520
Contributed by Richard A. Flavell, January 6, 2005
Epicutaneous immunization of T cell receptor (TCR) transgenic (Tg)
mice whose CD4
T cells are specific for the Ac1-11 fragment of
myelin basic protein (MBP) with Ac1-11 elicits T cells with dominant
suppressorregulatory activity that confers protection against Ac1-
11-induced experimental autoimmune encephalomyelitis. We now
report that such disease-resistant MBP TCR Tg mice also harbor a
sizeable fraction of peripheral CD4
T cells lacking surface expres-
sion of the Tg TCR
chain and expressing diverse, endogenously
rearranged TCR
chains. Ex vivo incubation at physiological
temperature caused the loss of neo-
-chain expression and rever-
sion to the MBP
␣␤
TCR
phenotype. The presence of recombina-
tion activating gene 1 and 2 proteins in CD4
T cells with revised
TCRs was consistent with effective V(D)J recombination activity.
The emergence of these cells did not depend on the thymic
compartment. We conclude that in mice epicutaneously immunized
with an autoantigen, peripheral specific T cells are susceptible to
multiple mechanisms of tolerance.
CD4 T cells tolerance skin recombination
T
he direct application of purified protein antigen onto the
epidermis of mice, or epicutaneous immunization (ECi), is
capable of inducing a potent adaptive immune response biased
toward the so-called type 2 of CD4
T helper (Th) cell response,
that is, helper T cells with prominent secretion of antiinflam-
matory cytokines (1–3). Hypothesizing that ECi with purified
forms of autoantigens might then interfere with the development
of Th1 CD4
T cell-driven proinflammatory autoimmune dis-
orders, we previously applied the Ac1-11 fragment of myelin
basic protein (MBP) to the skin of mice transgenic (Tg) for the
rearranged
and
chain of the Ac1-11:I-A
u
complex-specific
MBP T cell receptor (TCR) (MBP
␣␤
TCR Tg mice) (4, 5).
Because of the principle of allelic exclusion (6–10) that operates
at the TCR chain loci, such engineered mice harbor a T cell
repertoire dominated by the MBP
␣␤
TCR, yet not strictly
monoclonal, because allelic exclusion is not absolute, particularly
at the TCR
-chain locus. TCR chains encoded by endogenously
rearranged TCR genes can thus be found on T cells from TCR
Tg mice. MBP TCR Tg mice, as well as other TCR Tgs (11), are
commonly used to study experimental autoimmune encephalo-
myelitis (EAE), a chronic proinflammatory autoimmune pathol-
ogy that targets the white matter of the CNS and serves as an
animal model for the human disease multiple sclerosis (12). We
have observed that MBP TCR Tg mice epicutaneously immu-
nized with pure Ac1-11 in the absence of any adjuvant are
resistant to EAE that can be normally induced upon standard
immunization with Ac1-11 plus adjuvant (13). Interestingly,
rather than involving immune deviation from the classical Th1
profile that prevails in mice with clinical signs of EAE, disease
protection was mediated by CD4
T cells that acquired regula-
torysuppressor capacity. Such suppressor T cells did not pro-
duce major antiinflammatory cytokines and could confer pro-
tection to unmanipulated hosts upon adoptive transfer,
indicating that suppression was dominant. Importantly, ECi with
other CNS antigens could protect non-TCR Tg mice from
relapsing-remitting forms of EAE, as well (13).
We report here that, in addition to induction of CD4
T cells
with suppressive activity, ECi of MBP TCR Tg mice with only
purified Ac1-11 endowed peripheral, specific CD4
T cells with
the capacity to replace the MBP TCR
chain by a diverse set of
endogenously produced
chains, leading to revision of the
surface-expressed TCR. The data point to a model where the
epicutaneous delivery of autoantigens makes peripheral T cells
susceptible to multiple mechanisms of tolerance to self-
determinants.
Materials and Methods
Mice. MBP TCR Tg mice (MBP TCR Tg
/
) express the
rearranged genes encoding the
(V
4
) and
(V
8.2
)
chains of an autoreactive, I-A
u
-restricted TCR specific for the
acetylated N-terminal peptide (Ac1-11) of MBP. MBP TCR Tg
(Thy1.1 or Thy1.2) mice were mated to TCR C
/
or to
rag2
/
mice to generate MBP TCR C
/
and MBP TCR
rag2
/
mice, respectively. Four-week-old to 1-month-old
adult thymectomized B10.PL(H2
u
) mice were purchased from
The Jackson Laboratory. All mice were bred and housed in the
pathogen-free mouse facility at the Section of Immunobiology
at Yale University.
Epicutaneous (Skin) Patch Immunization, EAE Induction, and Monitor-
ing of Clinical Signs of Disease.
The procedures are fully described
in ref. 13. Very briefly, the backs of mice were shaved, and, one
day later, Ac1-11, myelin oligodendroglial glycoprotein (MOG)
31–55, or ovalbumin (Grade V, Sigma) at 10
gml in 100
lof
PBS or PBS alone was loaded onto an occlusive patch (Duo-
DERM Extra Thin, Convatec, Princeton) and applied to the
shaved area. The patch was left in place for 1 week, and the
procedure was repeated for a second week. For EAE induction,
Ac1-11 (3
gml in PBS) was mixed with an equal volume of
complete Freund’s adjuvant, and 50
l was injected s.c. in each
flank of MBP TCR Tg mice that were previously skin-patched.
Pertussis toxin (0.2
g) (Biological Laboratories, Campbell, CA)
was given i.v. at the time of immunization and again 2 days later.
Mice were scored daily for clinical signs of disease, and a
numerical score was assigned to mice based on the severity of the
disease symptoms: 0, no disease; 1, weak tail; 2, weak tail and
partial hind limb paralysis; 3, total hind limb paralysis; 4, both
hind limb and forelimb paralysis; 5, death. Mice with a score of
4 were euthanized. All procedures involving mice were approved
by Yale University’s Institutional and Animal Care and Use
Committee.
Abbreviations: TCR, T cell receptor; ECi, epicutaneous immunization; MBP, myelin basic
protein; Tg, transgenic; EAE, experimental autoimmune encephalomyelitis; RAG, recom-
bination-activating gene; PE, phycoerythrin; MOG, myelin oligodendroglial glycoprotein.
*To whom correspondence may be addressed. E-mail: margaret.bynoe@yale.edu or richard.
flavell@yale.edu.
Present address: Commissariat a` l’Energie Atomique–Department Response et Dynamique
Cellulaires and Institut National de la Sante´ et de la Recherche Me´ dicale U548, 38000
Grenoble, France.
Deceased April 12, 2003.
© 2005 by The National Academy of Sciences of the USA
2898–2903
PNAS
February 22, 2005
vol. 102
no. 8 www.pnas.orgcgidoi10.1073pnas.0409880102
Page 1
Antibodies and FACS Sorting. Cells were stained with anti-CD4-
FITC conjugate or anti-CD4-phycoerythrin (PE) (Caltag, South
San Francisco, CA); anti-CD4-CyChrome; anti-V
8.12-FITC
PE; anti-TCRC
-PE; anti-CD69-PE; anti-CD44-PE; anti-V
2,
3, 4, 6, 7, 10, 13, 14-FITC; or anti-CD62L-FITCPE (BD
Biosciences). Clonotypic antibody 3H12 was a kind gift from
Juan Lafaille (Skirball Institute of Biomolecular Medicine, New
York). The clonotypic mAb 19G was produced in C.A.J.’s
laboratory. Cells were incubated with antibodies at 4°C for 30
min, washed, and sorted on a FACStar
plus
or FACSVantage
instrument (Becton Dickinson). Sorted cells were examined on
a FACSScan analyzer (Becton Dickinson). For occasional do-
norrecipient T cell discrimination, anti-Thy 1.1 (CD90)-biotin,
streptavidin-FITC, and anti-Thy1.2 (CD90.2)-PE antibodies
were used (BD Biosciences).
Cell Culture and Proliferation Assay. For phenotypical analysis ex
vivo, sorted CD4
V
8.2
T cells were incubated at 37°C at a
density of 5 10
6
cells per well in a 12-well tissue culture plate
in Click’s Eagle–Hanks’ amino acid (EHAA) medium (Life
Technologies, Grand Island, NY) supplemented with 5% FCS
without antigen or exogenous cytokines. Cells were removed at
various time points (4–72 h) for analysis by flow cytometry-
coupled immunofluorescence, in particular for detection of
components of the transgene-encoded MBP TCR. For T cell
proliferation assay, sorted CD4
V
8.2
or CD4
V
8.2
cells
from spleen and lymph nodes of Ac1-11 epicutaneously immu-
nized mice or CD4
V
8.2
cells from naı¨ve MBP TCR Tg mice
were plated at 5 10
4
cells per well in the presence of 2 10
5
irradiated syngeneic (B10.PL) spleen cells per well as antigen-
presenting cells in Click’s EHAA medium supplemented with
5% FCS. The Ac1-11 peptide was added at concentrations
ranging from 0 to 100
gml. After 48 h of incubation at 37°C,
cells were pulsed with 1
Ci (1 Ci 37 GBq) of [
3
H]thymidine
and harvested 18–24 h later. Counts were determined in a
-plate scintillation counter.
Adoptive Transfer of Sorted T Cells. CD4
V
8.2
T cells from
pooled spleen and lymph nodes from naı¨ve (as controls) or
Ac1-11 epicutaneously immunized mice were washed in sterile
PBS two to three times. Cells (1 10
7
) were resuspended in a
final volume of 200
l and administered i.v. to MBP TCR Tg
C
/
recipients. Five days later, EAE was induced by standard
procedure. In some experiments, naı¨ve CD4
V
8.2
T cells
were sorted from Thy1.2
MBP TCR Tg mice and transferred
into B10.PL (Thy1.1
) hosts.
Western Blotting. Cell lysates were obtained from sorted
CD4
V
8.2
or CD4
V
8.2
cells from Ac1-11 epicutaneously
immunized mice or controls. Aliquots (10
g) of each cell lysate
were run on SDSPAGE under reducing conditions and trans-
ferred to a transblot transfer medium nitrocellulose membrane
(Bio-Rad). Blocking was with 5% milk for2hatroom temper-
ature. Membranes were incubated for3hwithpolyclonal
antibodies against recombination-activating gene (RAG) 1 and
RAG2 proteins by using specific polyclonal antibodies (a gift
from D. Schatz, Yale University). The blots were washed three
times with PBS plus 0.1% Tween 20. Protein bands were
visualized by using secondary horseradish peroxidase-
conjugated antibodies and the ECL detection system (Amer-
sham Biosciences) according to the manufacturer’s instructions.
Results
Endogenous TCR
-Chain Expression in Peripheral CD4
T Cells from
MBP TCR Tg Mice. MBP TCR Tg mice that are epicutaneously
immunized with the cognate (Ac1-11) peptide are resistant to
EAE induction. MBP TCR Tg mice that lack surface expression
of the endogenously rearranged TCR
chains (MBP TCR Tg
C
/
mice) are well known for their spontaneous and rapid
development of EAE, and these mice could also be protected
from disease upon ECi. Such resistance relied on the induction
of CD4
T cells with dominant suppressiveregulatory activity
(13). Disease-resistant MBP TCR Tg or MBP TCR Tg C
/
mice will be referred to hereafter as Ac1-11 ECi mice. The
phenotypic analysis of peripheral blood, spleen, or lymph node
CD4
T cells from Ac1-11 ECi mice revealed that a sizeable
fraction of them stained negative for V
8.2, that is, for the V
domain used by the transgene-encoded TCR
chain (Fig. 1 A
and B). This fraction varied from mouse to mouse and could
comprise 50% of CD4
T cells, or more, over time. This
percentage was in sharp contrast with the low frequency (2–4%)
of CD4
V
8.2
T cells detected in unmanipulated MBP TCR
Tg mice and with the virtual absence of these cells in MBP TCR
Tg C
/
mice (Fig. 1 C and D). The accumulation of
CD4
V
8.2
T cells was detected exclusively in mice that were
epicutaneously immunized with Ac1-11 and immunized with
Ac1-11 plus adjuvant for disease induction. In addition, the
increase in frequency of CD4
V
8.2
T cells was never ob-
served in MBP TCR Tg mice that were epicutaneously immu-
nized with vehicle (PBS), an irrelevant antigen (ovalbumin), or
a CNS antigenic peptide other than Ac1-11 (such as MOG 35-55)
before Ac1-11 challenge (Fig. 1E), indicating that the detection
of a substantial fraction of CD4
V
8.2
T cells in EAE-resistant
MBP TCR Tg mice required both ECi and challenge with the
cognate antigenic peptide. The CD4
V
8.2
T cell fraction
remained readily detectable for at least 3 months after the
attempt to induce EAE. The simultaneous use of the anti-C
-
specific H57 and anti-V
8.2 F23.2 mAbs showed that spleen and
lymph node cells isolated from Ac1-11 ECi mice contained both
C
V
8.2
and C
V
8.2
cells (Fig. 1F). Again, C
V
8.2
cells were scarce and absent in unmanipulated MBP TCR Tg
mice and MBP TCR Tg C
/
mice, respectively (data not
shown). Collectively, the results document the presence of a
substantial population of peripheral T cells with surface expres-
sion of endogenous (H57
V
8.2
), rather than Tg (V
8.2
),
Fig. 1. Surface expression of endogenously rearranged TCR
chains in CD4
T cells from MBP TCR Tg mice epicutaneously immunized with Ac1-11 and
treated for EAE induction. (A and B) FACS analysis of peripheral blood, spleen,
or lymph node cells of disease-resistant Ac1-11 ECi mice after being stained
with mAbs to CD4 and to the Tg
chain (anti-V
8.2). (C) Unmanipulated MBP
TCR Tg mice produce 2–5% of CD4 T cells expressing the endogenous
chain
(CD4
V
8.2
). (D) Unmanipulated MBP TCR Tg C
/
mice produce virtually
no CD4
V
8.2
T cells. (E) MBP TCR Tg mice that were epicutaneously immu-
nized with a control CNS peptide or treated with PBS before disease induction
and survived did not display any increase in CD4
V
8.2
T cells. (F) Spleen and
lymph node CD4 T cells from Ac1-11 ECi mice were stained with mAbs specific
for the constant region of the TCR
chain (H57) or for the Tg
chain (F23.2).
Bynoe et al. PNAS
February 22, 2005
vol. 102
no. 8
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IMMUNOLOGY
Page 2
TCR
chain(s) in EAE-resistant, Ac1-11 ECi MBP TCR Tg
mice.
Cultured CD4
V
8.2
Peripheral T Cells Gain Surface Expression of the
MBP TCR.
The detection of a large fraction of CD4
T cells with
surface expression of endogenously rearranged TCR
chains
could reflect at least two phenomena. One possibility is that the
rare CD4
V
8.2
T cells present in untreated MBP TCR Tg
mice are induced to proliferate extensively, and their detection
follows the expansion of a minor, preexisting population. On the
other hand, MBP TCR
-chain expression may be altered in
CD4
T cells from mice that were epicutaneously immunized
and challenged with Ac1-11. The first possibility was rather
improbable for two reasons. First, CD4
V
8.2
T cells from
Ac1-11 ECi mice were not blasting, as assessed by examination
of their side scatterforward scatter flow cytometry profile (data
not shown). Second, the frequency of Ac1-11:I-A
u
complex
reactive T cells would have to be high among the few T cells
lacking expression of the Tg MBP TCR for such a massive
expansion to occur upon challenge. Most importantly, we ob-
served that sorted CD4
V
8.2
T cells from spleenlymph
nodes of Ac1-11 ECi mice acquired a V
8.2
surface phenotype
after overnight incubation at 37°C in complete media in the
absence of antigen or of any exogenous cytokines (Fig. 2 AC).
By 48 h of culture, most cells were V
8.2
and, remarkably, were
positive when stained with the MBP TCR-specific 3H12 clono-
typic mAb (Fig. 2 D and E), indicating that ex vivo incubation at
physiological temperature induced them to express the complete
MBP
␣␤
TCR on their surface. We also observed that, over time,
the CD4
V
8.2
population increased with concomitant de-
crease in the CD4
V
8.2
population (Fig. 2F). The data are
most compatible with the idea that MBP TCR
-chain expres-
sion is repressed in peripheral CD4
H57
V
8.2
T cells from
Ac1-11 ECi mice and, therefore, that these cells have undergone
TCR revision in vivo.
To determine whether the repertoire of endogenously rear-
ranged TCR
chains expressed by CD4
V
8.2
T cells was
diverse or rather restricted, we first performed flow cytometry-
coupled immunostaining using a panel of anti-V
-specific mAbs.
The results showed that multiple variable domains were used by
the
chain of endogenous origin in CD4
T cells with revised
TCR from Ac1-11 ECi mice. Fig. 2G shows, for instance, that the
V
3, V
4, V
6, V
10, and V
14 domains were all used by
substantial fractions of peripheral CD4
T cells from Ac1-11
ECi mice. Thus, in CD4
V
8.2
T cells, distinct TCR
chains
could be endogenously synthesized and used to form TCRs able
to traffic intracellularly and get expressed on the cell surface.
Expression of endogenous V
chains was further confirmed by
detection of transcripts (data not shown).
CD4
V
8.2
T Cells Express the RAG1 and RAG2 Proteins. Because the
TCR gene rearrangement process requires both rag1 and rag2
gene products for sequence-specific DNA recognition and DNA
cleavage and postcleavage functions (14, 15), we examined
whether we could detect RAG expression in CD4
T cells from
Ac1-11 ECi mice that had acquired expression of endogenously
rearranged TCR
chains. We carried out RAG1- and RAG2-
specific Western blot analysis of lysates from sorted CD4
T cells
isolated from Ac1-11 ECi mice and of thymic and splenic cells
from unmanipulated animals as controls. Although WT spleen
cells and thymocytes were respectively negative and positive for
RAG protein expression, peripheral CD4
V
8.2
T cells from
the same mice were found to express substantial amounts of
RAG1 and RAG2 proteins (Fig. 3A). CD4
V
8.2
T cells from
Ac1-11 ECi mice, however, showed either no expression or,
Fig. 2. CD4
V
8.2
T cells acquire surface expression of the MBP
␣␤
TCR ex
vivo.(A and B) Cells from Ac1-11 ECi mice with 50% or more CD4 T cells lacking
surface expression of the Tg
chain were sorted into CD4
V
8.2
(A)or
CD4
V
8.2
(B) populations. (C) Sorted CD4
V
8.2
T cells reexpressed the Tg
(V
8.2
) TCR
chain after 24 h of ex vivo incubation at physiological tem-
perature. (D and E) Sorted CD4
V
8.2
T cells gained a V
8.2
phenotype
after 48 h at 37°C in complete media (D) stained positive for the 3H12
clonotypic mAb (E), demonstrating that they express the MBP
␣␤
TCR. (F)In
Ac1-11 Eci mice, the increase in V
8.2
cell frequency among CD4
cells was
associated with a concomitant decrease in the V
8.2
frequency. (G)
CD4
V
8.2
cells used a variety of endogenously rearranged TCR
chains.
Fig. 3. CD4
V
8.2
cells from Ac1-11 ECi mice express RAG12 proteins,
display a suppressive potential in vivo, and harbor an ‘‘effectormemory’’
mouse T cell phenotype. ( A) RAG1 (120 kDa) and RAG2 (60 kDa) proteins are
expressed in CD4
V
8.2
cells as shown by Western blot analysis of sorted cells
from Ac1-11 ECi mice. (Seven of 16 mice expressed RAG1, 5 of 16 expressed
RAG2, and 5 of 16 expressed both RAG1 and RAG2.) Sorted CD4
V
8.2
cells
from spleen and lymph nodes of control MOG ECi mice (three mice per group)
or control naı¨ve MBP TCR Tg mice (three mice per group) expressed neither
RAG1 nor RAG2. An anti-
-actin mAb was used for control. Spleen and thymic
cells from naı¨ve mice were included as negative and positive controls, respec-
tively. (B) Both CD4
V
8.2
and CD4
V
8.2
cells display suppressive activity
in vivo.1 10
6
CD4
V
8.2
, CD4
V
8.2
,ornaı¨ve CD4
T cells from MBP TCR
Tg C
/
mice were adoptively transferred into naı¨ve MBP TCR Tg C
/
recipients, and disease was induced 3 days later. (C) Sorted CD4
V
8.2
cells
from spleen and lymph nodes of Ac1-11 ECi (open histograms) or unmanipu-
lated MBP TCR Tg (filled histograms) mice were stained with mAbs to CD44,
CD62L, and CD69 molecules.
2900
www.pnas.orgcgidoi10.1073pnas.0409880102 Bynoe et al.
Page 3
occasionally, trace amounts of RAG proteins (data not shown).
There were rare CD4
V
8.2
T cell samples with detectable
expression of RAG1 but not RAG2. RAG protein expression
was typically observed for CD4
V
8.2
T cell samples from
one-third to half of disease-resistant Ac1-11 ECi mice analyzed
at a given time point. In contrast, spleenlymph node cells from
MOG-ECi mice or unmanipulated MBP TCR Tg mice remained
negative for RAG12 protein expression (Fig. 3A). These ob-
servations demonstrate that expression of the RAG proteins,
which is turned off upon intrathymic positive selection and is
normally absent in mature peripheral T lymphocytes (16, 17),
was induced in peripheral CD4
T cells that surface-express
endogenously rearranged TCR
chains in Ac1-11 ECi mice.
Both CD4
V
8.2
and CD4
V
8.2
T Cells Confer Protection Against
EAE.
We then asked whether receptor revision was necessary for
disease resistance to occur in Ac1-11 ECi mice. Sorted
CD4
V
8.2
and CD4
V
8.2
T cells from Ac1-11 ECi mice
were adoptively transferred into young MBP TCR Tg C
/
hosts. Upon disease induction, MBP TCR Tg C
/
mice that
received naı¨ve MBP TCR Tg CD4
T cells developed rapid and
severe disease. In contrast, recipients of either CD4
V
8.2
or
CD4
V
8.2
epicutaneously immunized T cells showed signs of
protection, with a greater resistance in the case of the
CD4
V
8.2
T cell transfer (Fig. 3B). Thus, although
CD4
V
8.2
T cells seemed to have a slightly enhanced sup-
pression potential under the conditions used, CD4
V
8.2
T
cells were protective as well. That is, there was no strict depen-
dence on receptor revision for purified CD4 T cells from Ac1-11
mice to confer disease resistance upon adoptive transfer. To
further examine whether receptor revision is required for pro-
tective activity, we turned to a more stringent assay. We used
MBP TCR Tg rag2
/
mice that exhibit spontaneous EAE and
a fulminate disease course; all such mice develop severe clinical
signs by 5 weeks of age and succumb very rapidly (ref. 5;
unpublished data). We reasoned that if receptor revision was a
requirement for ECi treatment to confer protection, we would
repeatedly fail to observe any protection against EAE in epicu-
taneously immunized MBP TCR Tg rag2
/
mice whose T cells
are devoid of any V(D)J rearrangement potential. Conversely,
the detection of disease resistance in such mice would strongly
argue against a true requirement for receptor revision in order
for ECi-induced protection to occur. We observed that two of
eight Ac1-11 ECi MBP TCR Tg rag2
/
mice remained free of
clinical signs of disease over the time of observation (up to 3
months). In contrast, all mice died rapidly from EAE when
epicutaneously immunized with MOG 35-55 (Table 1). The data
support the notion that the ECi-induced protection against
disease does not depend on TCR revision.
CD4
V
8.2
Peripheral T Cells Harbor an Antigen-Experienced Mouse
T Cell Phenotype.
To gain insight into the nature of CD4
V
8.2
T cells from Ac1-11 ECi mice, we analyzed the expression of a
few cell surface markers. Unlike sorted CD4
V
8.2
T cells
from unmanipulated MBP TCR Tg mice, spleen and lymph node
CD4
V
8.2
T cells from Ac1-11 ECi mice displayed aug-
mented expression of CD44 and reduced expression levels of
L-selectin (CD62L) (Fig. 3C). CD4
V
8.2
T cells from Ac1-11
ECi mice also displayed modified CD44 and CD62L expression
(data not shown). This phenotype is reminiscent of that of
antigen-experienced mouse T lymphocytes (18) and is consistent
with the assumption that T cells expressing the CD4
V
8.2
phenotype have been involved in antigen recognition in vivo.
Occasionally, increased CD69 surface expression could also be
observed among CD4
V
8.2
T cells (Fig. 3C).
Peripheral T Cells with Revised TCR Do Not Require the Thymic
Microenvironment.
Because developing thymocytes appear sus-
ceptible of modifying their receptor by de novo V(D)J rear-
rangements in response to self-determinants (19–21), a process
often referred to as editing by reference to antigen-receptor
modifications during B-cell maturation in bone marrow (22–26),
we specifically asked whether the emergence of cells with revised
TCR depended on the thymic compartment. To examine this
issue, we sorted naı¨ve CD4
V
8.2
T cells from normal MBP
TCR Tg mice and transferred them into regular B10.PL hosts
that were thymectomized and sublethally irradiated (Fig. 4A).
FACS analysis showed that donor cells were detectable in the
peripheral blood at 24 h posttransfer (Fig. 4B). Six to ten days
later, the recipient mice were epicutaneously immunized with
Ac1-11 or a control peptide for 2 weeks and then immunized
Table 1. EAE incidence in Ac1-11 ECi and control MBP TCR Tg rag2
mice
Antigen for ECi n
No. of mice free
of clinical signs
of EAE
Day of disease
onset
No. of mice
recovered
Mean EAE
score
Ac1-11 8 2 15 06 3.6
MOG 35-55 7 0 14 075
MBP TCR Tg rag2
mice whose T cells are devoid of V(D)J rearrangement capacity were epicutaneously
immunized with Ac1-11 or MOG 35-55 (both 10
g), treated with Ac1-11 plus adjuvant for disease induction, and
monitored for clinical signs of EAE over several months.
Fig. 4. CD4
V
8.2
cells from Ac1-11 ECi mice do not depend on the thymic
compartment for their emergence in vivo and do not react to Ac1-11 stimu-
lation in vitro.(A and B) Peripheral blood of naı¨ve thymectomized B10.PL mice
that were sublethally irradiated before transfer (day 0) (A) or 24 h after they
were adoptively transferred with 1 10
7
sorted CD4
V
8.2
cells from
unmanipulated MBP TCR Tg mice (B). Six to 10 days later, recipient mice were
epicutaneously immunized with Ac1-11 or a control peptide for 2 weeks, and
disease was induced. (C and D) Peripheral blood cells obtained from Ac1-11 ECi
(C) or control (D) mice were stained with mAbs to CD4 and V
8.2. (E) Sorted
populations of CD4
V
8.2
or CD4
V
8.2
cells from Ac1-11 ECi mice or
CD4
V
8.2
cells (5 10
4
cells per well) from naı¨ve MBP TCR C
/
mice were
stimulated in vitro with varying concentrations of Ac1-11 and irradiated
antigen-presenting cells (2 10
5
cells per well).
Bynoe et al. PNAS
February 22, 2005
vol. 102
no. 8
2901
IMMUNOLOGY
Page 4
with Ac1-11 plus adjuvant. Five of 10 mice died (data not shown).
Two of the five disease-resistant mice clearly had a substantial
fraction of T cells that were CD4
V
8.2
at 6 weeks postchal-
lenge (Fig. 4C). Upon isolation by sorting and incubation at 37°C,
these cells switched again to a V
8.2
3H12
phenotype (data
not shown). In contrast, we repeatedly failed to detect
CD4
V
8.2
T cells in the few mice that could survive disease
induction in the MOG 35-55 ECi control group (Fig. 4D). Thus,
receptor revision can occur among peripheral MBP TCR Tg T
cells transferred into thymectomized, irradiated hosts. This
result indicates that the thymic microenvironment itself is not
required for the emergence of CD4
T cells with revised TCR
in Ac1-11 ECi mice.
CD4
V
8.2
T Cells from Ac1-11 ECi Mice Are Not Reactive to
Ac1-11:I-A
u
Stimulation. To determine whether CD4
V
8.2
T
cells are responsive to Ac1-11, we isolated CD4
V
8.2
and
CD4
V
8.2
T cell subsets from Ac1-11 ECi mice along with
naı¨ve CD4
T cells from unmanipulated MBP TCR Tg mice and
challenged them in vitro with serial doses of Ac1-11 in the
presence of syngeneic (B10.PL) antigen-presenting cells. Al-
though CD4
V
8.2
T cells gave a detectable proliferative
response, they were clearly hyporesponsive when compared with
the response of naı¨ve MBP TCR Tg T cells (Fig. 4E). This
hyporesponsiveness was consistent with our previous observa-
tion that purified CD4
T cells from Ac1-11 ECi mice, which are
able to transfer protection to naı¨ve hosts, were only marginally
responsive to specific stimulation in culture (13). CD4
V
8.2
T cells, however, did not divide in response to Ac1-11 stimulation
under the conditions used, although they could respond to
anti-CD3 stimulation (data not shown). The data indicate that
freshly isolated CD4
V
8.2
T cells from Ac1-11 ECi mice do
not proliferate in response to the Ac1-11:I-A
u
complex in vitro.
Discussion
MBP TCR Tg mice epicutaneously immunized with only the
cognate (Ac1-11) peptide before challenge for disease induction
(Ac1-11 plus adjuvant) are protected against full EAE. Such
resistance relies on the induction of CD4
suppressor T cells
capable of conferring protection to naı¨ve hosts upon adoptive
transfer (13). We document here the presence of a substantial
fraction of peripheral blood, spleen, or lymph node CD4
T cells
that lack expression of the Tg TCR
chain (CD4
V
8.2
T
cells) in 30–50% of Ac1-11 ECi MBP TCR Tg mice that are
resistant to EAE. The emergence of CD4
V
8.2
cells required
a double in vivo encounter with Ac1-11 because these cells were
not detected in MBP TCR Tg mice that were epicutaneously
immunized with Ac1-11 and not challenged for EAE induction.
In addition, MBP TCR Tg mice epicutaneously immunized with
MOG 35-55 (or ovalbumin) developed full EAE after Ac1-11-
mediated disease induction and were devoid of peripheral
CD4
V
8.2
T cells. The H57
phenotype of CD4
V
8.2
T
cells demonstrated that these cells surface-express non-Tg, that
is, endogenously rearranged, TCR
chain(s).
The presence of a sizeable fraction of peripheral CD4
V
8.2
T cells expressing a diverse set of non-Tg
chains in disease-
resistant MBP TCR Tg mice could be explained by at least two
mechanisms. One can imagine that CD4
V
8.2
T cells that are
detected at low frequency in unmanipulated MBP TCR Tg mice
are induced to proliferate extensively in vivo. Alternatively, it is
possible that under the conditions used, Tg CD4
T cells revise
their surface-expressed
␣␤
TCR. Evidence supporting the sec-
ond possibility is twofold. First, Western blot data demonstrated
that the lymphoid lineage specific components of the V(D)J
recombination machinery (RAG12), whose expression is nor-
mally turned off in mature peripheral T lymphocytes, were
induced in CD4
V
8.2
T cells from Ac1-11 ECi mice. Second,
freshly isolated CD4
V
8.2
T cells acquired a MBP
␣␤
TCR
phenotype (3H12
) ex vivo after few hours in culture without
further manipulation. Indeed, the possibility that CD4
V
8.2
T cells could have originated from the sustained expansion of
few such cells in response to ECichallenge of MBP TCR Tg
mice with Ac1-11 would imply that CD4
V
8.2
T cells react to
Ac1-11. One may then expect them to respond to Ac1-11
stimulation in vitro because they are not modifying their TCR
composition. Clearly, this was not the case, even in the presence
of exogenous IL-2. Hence, the corpus of data supports the
occurrence of RAG-mediated receptor revision in CD4
V
8.2
T cells from disease-resistant mice. Such a TCR revision phe-
nomenon (27) among peripheral T cells was perhaps best
characterized by studying the fate of Tg CD4
V
5
T cells
exposed in vivo to a mouse mammary tumor virus 8-encoded
peripheral superantigen (20, 28–31). In our system, maintenance
of TCR revision appeared to require factor(s) present in vivo,
because acquisition of the 3H12
phenotype occurred relatively
rapidly e x vivo. We speculate that the presence of Ac1-11 could
be a contributing factor, as sorted CD4
V
8.2
T cells incu-
bated with syngeneic antigen-presenting cells plus Ac1-11 in vitro
did not gain the 3H12
phenotype (data not shown). Although
RAG1 and RAG2 protein expression was often codetected in
sorted CD4
V
8.2
T cells, it happened that only RAG1 was
readily detectable. The reason for this difference is unclear, but,
interestingly, a similar phenomenon has been reported for
human mature CD4
CD3-low T cells that display RAG reex-
pression and initiate secondary V(D)J rearrangements (32).
Sorted CD4
V
8.2
T cells from Ac1-11 ECi mice, that is,
CD4
T cells bearing endogenously rearranged TCR
chains,
effectively mediate suppression of T cells that cause EAE in vivo.
It is reasonable to assume that among CD4
V
8.2
T cells with
revised TCR, there were many T cells with TCR synonymous to
those expressed by the non-TCR Tg CD4
T cells that protect
unmanipulated MBP TCR Tg mice from the spontaneous
occurrence of disease (33) and that also display a diverse TCR
repertoire (34). Thus, the protective potential of CD4
V
8.2
T cells with revised TCR observed in vivo in transfer experiments
is not unexpected. Indeed, the high incidence (66%) of spon-
taneous EAE among MBP TCR Tg mice lacking only endoge-
nous TCR
chains (MBP TCR Tg C
/
) proved that cells with
high suppressive potential in MBP TCR Tg mice are not subject
to tight
-chain allelic exclusion (33).
When we first observed TCR chain replacement in Ac1-11 ECi
mice that were resistant to EAE induction, we wondered whether
TCR revision was a requirement for protection. This scenario
appears unlikely, because in a given group of simultaneously
manipulated mice, at most one-third to one-half of EAE-
resistant mice displayed a substantial fraction of CD4
V
8.2
T
cells at the time of analysis. The reason for this frequency is
unclear, but it was not related to the sex or age of the mice (data
not shown). It is possible that TCR revision occurs in most mice,
but with a differential efficiency because of subtle variability in
efficiency of Ac1-11 diffusion from the skin patch andor during
s.c. immunization. More importantly, MBP TCR Tg rag2
/
mice that normally display a fulminate disease course with 100%
mortality by 5 weeks of age (5) could show long-term protection
from EAE when epicutaneously immunized with Ac1-11.
In summary, the ECi of MBP TCR Tg mice with the CNS
autoantigen Ac1-11 before immunization not only elicits T cells
with dominant suppressorregulatory activity that confer pro-
tection against Ac1-11-induced EAE, but also causes CD4
T
cells to repress surface expression of the autoreactive TCR
chain and to express a broad repertoire of endogenously rear-
ranged TCR
chains because of RAG reexpression. The
emergence of T cells with such revised TCRs was not dependent
on the thymic compartment. Thus, the epicutaneous adminis-
tration of pure autoantigens renders peripheral specific T cells
2902
www.pnas.orgcgidoi10.1073pnas.0409880102 Bynoe et al.
Page 5
susceptible to multiple mechanisms of tolerance in a nonmutu-
ally exclusive fashion.
This work is dedicated to the memory of Dr. Charles A. Janeway, Jr.
We thank E. Robinson for assistance with sequencing, L. Corbett
(Yale University) and Dr. D. Schatz for the RAG antibodies, Dr. J.
Lafaille (SIBM, New York) for the 3H12 antibody, Dr. A. Etgen and
Wook-Jin Chae for reading the manuscript, and F. Manzo for assis-
tance with manuscript preparation. We also thank L. Gorelik, E. Tran,
and A. Bothwell for discussion; Dr. O. Henegariu, P. Preston-
Hurlburt, and J. Shanabrough for technical assistance; and C.
Annicelli and J. Apicelli for help with mice. This work was supported
by the Howard Hughes Medical Institute, National Institutes of Health
(NIH) Minority Supplement, and NIH Grants Al14579 and
Po1A136529 to (C.A.J.). R.A.F. is an Investigator of the Howard
Hughes Medical Institute.
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  • Source
    • "Some combinations of TCRs and antigens, however, fail to induce TCR revision, so the generality of this process is not yet fully known [63,65]. Several intriguing hypotheses have been generated about how the ability to dynamically transition among different antigen-specific states may relate to the induction of tolerance, autoimmunity, or pathogenspecific immune responses [63,65,66,68,69]. Considering the history-dependent and heterogeneous nature of TCR revision, it is likely that single-cell technologies that combine TCR sequencing with tracking of progeny will help to address unresolved questions about the functional significance of TCR revision and its regulation. "
    [Show abstract] [Hide abstract] ABSTRACT: Activated T cells have classically been thought to progress unidirectionally through discrete phenotypic states and differentiate into static lineages. It is increasingly evident, however, that T cells exhibit much more complex and flexible dynamic behaviors than initially appreciated, and that these behaviors influence the efficacy of T cell responses to immunological challenges. In this review, we discuss how new technologies for monitoring the dynamics of T cells are enhancing the resolution of the fine phenotypic and functional heterogeneity within populations of T cells and revealing how individual T cells transition among a continuum of states. Such insights into the dynamic properties of T cells should improve immune monitoring and inform strategies for therapeutic interventions.
    Preview · Article · Nov 2012 · Trends in Immunology
  • Source
    • "Furthermore, the reinduction of RAG expression that initiates TCR revision may tap into a tried and true regulatory pathway that operates during T cell development, in which TCR mediated signaling extinguishes RAG expression, while the absence of this signaling prompts its maintenance [15] [16] [17]. As such, the modulation of sTCR expression levels represents a key control point for TCR revision, and sTCR downregulation is a hallmark of this process in humans [22] [24] [25] [26] and both TCR Tg [4] [8] [10] [33] and nonTg (reference [9] and Figure 3) mice. "
    [Show abstract] [Hide abstract] ABSTRACT: TCR revision is a tolerance mechanism by which self-reactive TCRs expressed by mature CD4(+) peripheral T cells are replaced by receptors encoded by genes generated by post-thymic DNA rearrangement. The downmodulation of surface TCR expression initiates TCR revision, and serves as a likely trigger for the induction of the recombinase machinery. We show here in a Vβ5 transgenic mouse model system that downregulation of the self-reactive transgene-encoded TCR is not maintained by transgene loss or diminished transcription or translation. The downregulation of surface TCR expression likely occurs in two stages, only one of which requires tolerogen expression.
    Preview · Article · Nov 2011 · Cellular Immunology
  • Source
    • "Previous studies that successfully identified targets of CD4 + responses in complex pathogens such as Mycobacterium and Salmonella [24,25,26] have focused on antigens targeted by B cell response. However, the success of this approach seemed to be at odds with classical studies of CD4 + responses that showed no linkage between antigens recognized by CD4 + and B cell responses, but rather a predominance of 'intermolecular help' [27,28,29,30]. This apparent contradiction may be explained by a recent study that found a strong correlation between CD4 + and antibody responses on the antigen level in vaccinia virus infected mice [31]. "
    [Show abstract] [Hide abstract] ABSTRACT: Coxiella burnetii is an obligate intracellular gram-negative bacterium that causes acute Q fever and chronic infections in humans. A killed, whole cell vaccine is efficacious, but vaccination can result in severe local or systemic adverse reactions. Although T cell responses are considered pivotal for vaccine derived protective immunity, the epitope targets of CD4(+) T cell responses in C. burnetii vaccination have not been elucidated. Since mapping CD4(+) epitopes in a genome with over 2,000 ORFs is resource intensive, we focused on 7 antigens that were known to be targeted by antibody responses. 117 candidate peptides were selected from these antigens based on bioinformatics predictions of binding to the murine MHC class II molecule H-2 IA(b). We screened these peptides for recognition by IFN-γ producing CD4(+) T cell in phase I C. burnetii whole cell vaccine (PI-WCV) vaccinated C57BL/6 mice and identified 8 distinct epitopes from four different proteins. The identified epitope targets account for 8% of the total vaccination induced IFN-γ producing CD4(+) T cells. Given that less than 0.4% of the antigens contained in C. burnetii were screened, this suggests that prioritizing antigens targeted by antibody responses is an efficient strategy to identify at least a subset of CD4(+) targets in large pathogens. Finally, we examined the nature of linkage between CD4(+) T cell and antibody responses in PI-WCV vaccinated mice. We found a surprisingly non-uniform pattern in the help provided by epitope specific CD4(+) T cells for antibody production, which can be specific for the epitope source antigen as well as non-specific. This suggests that a complete map of CD4(+) response targets in PI-WCV vaccinated mice will likely include antigens against which no antibody responses are made.
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