From the Institut Necker, Institut National de la Santé et de la Recherche Médicale 373, F-75730
Paris, Cedex 15, France
Continuous antigenic stimulation in vivo can result in the generation of so-called "anergic"
CD4+ or CD8+ T cells that fail to proliferate upon antigenic stimulation and fail to develop
cytolytic effector functions. Here we show that class II major histocompatibility complex-restricted
T cells specific for influenza hemagglutinin (HA) that become anergic in mice expressing HA
under control of the immunoglobulin
promoter exhibit an impaired effector function in
causing diabetes in vivo, as compared to their naive counterparts, when transferred into immunodeficient recipients expressing HA under the control of the insulin promoter. Furthermore,
HA-specific T cells anergized in vivo contain higher levels of interleukin (IL)-4 messenger
RNA (mRNA) than naive and recently activated T cells with the same specificity and more than a 100-fold higher levels of IL-10 mRNA. The higher expression of the IL-10 gene is also
evident at the protein level. These findings raise the interesting possibility that T cells rendered anergic in vivo have in fact become regulatory T cells that may influence neighboring immune
responses through the release of IL-10.
 |
Introduction |
Many T cells reactive to self peptides are deleted in the
thymus (1). This is also true for T cells reactive to
superantigens (2). Central deletion is not the only mechanism of immune tolerance, and additional mechanisms
must exist to ensure tolerance to antigens that are not encountered in the thymus in sufficient quantity. In vivo tolerance experiments conducted with mature T cells have shown that peripheral tolerance may result from deletion as
well as anergy induced by MHC-presented peptides (3)
as well as superantigens (7) in mature CD4+ (5) as well
as CD8+ T cells (3, 4). It has also been reported that some
antigens expressed in certain tissues may be ignored either
because T cells have no access to them or perhaps because
cells of such tissue cannot stimulate T cells (10, 11). It appears unlikely that ignorance is the reason for tolerance to a
large variety of antigens since, except for privileged sites
where antigens released from certain tissues cannot be presented by hemopoietic APCs, fragments of tissue-specific
proteins can be presented by neighboring APCs (12). In in
vitro experiments, it was reported that already activated T
cell clones could be rendered "anergic" by antigen presented on nonprofessional APCs that lack costimulatory
molecules (13). Anergic cells were reported to have a block
in p21ras activation, a decrease in the activities of MAP kinase pathways, a reduced induction of c-Fos and JunB proteins, and a failure to form and phosphorylate the activator
protein 1 required for IL-2 gene transcription (14).
More recently, negative regulation of TCR-mediated IL-2
gene transcription has been described as an additional mechanism of T cell anergy in vitro (19, 20). In all cases, anergy
induced in vitro could be overcome by the addition of exogenous IL-2 (21, 22). It remains to be seen whether anergy induced in vivo rests on similar mechanisms. Although some experiments showed a similar type of anergy of in
vivo tolerized CD8+ T cells (23), others found that the anergic state could not be overcome by the addition of IL-2
(3, 6, 24). Moreover, several experiments strongly indicate
that anergy in vivo can be preceded by an activation of
T cells (3, 6, 9, 25) and it was recently shown that blocking
the B7/CD28 interaction prevents in vivo priming, whereas
blocking the B7/CTLA4 interaction prevents in vivo tolerance induction (25). The latter observation suggests that
tolerance was induced by B7 expressing APCs. We have observed that in transgenic mice expressing both a class II-
restricted TCR-
/
specific for influenza hemagglutinin and the hemagglutinin under the control of the Ig-
promoter,
thymic deletion of hemmagglutinin (HA)1-specific T cells
takes place but some self-reactive cells also accumulate in
the periphery (6). These cells show signs of activation in
vivo, but are anergic in terms of proliferation when restimulated in vitro. In contrast to in vitro anergized T cell
clones, unresponsiveness could not be overcome by addition of exogenous IL-2. In this system, one can study T
cells that have been anergized in vivo by mechanisms that
are perhaps physiologically more relevant than those analyzed in vitro. Furthermore, the possibility of isolating the
TCR transgenic cells by means of a clonotypic antibody,
permits the analysis of the anergic cells from TCR-HA × IG-HA mice in greater detail. In the present study we have
characterized the anergic HA-specific cells in terms of their
effector function and cytokine production in vivo. We
show that the anergic cells, in contrast to their naive precursors, fail to induce rapid and fulminant diabetes when
injected into recipients that express HA under the control
of the insulin promoter (INS-HA). Nevertheless, these cells
do not appear to be entirely anergic, as they produce much
higher levels of IL-10 messenger RNA (mRNA) and protein than their naive counterparts, suggesting that they may
have a regulatory role.
 |
Materials and Methods |
Mice.
The TCR-HA transgenic mice expressing a TCR-
/
specific for peptides 111-119 from influenza HA presented by I-Ed
have been previously described (6, 26). Mice expressing HA
(IG-HA) have the HA transgene under control of the Ig-
promoter and enhancer elements and are on BALB/c background
(6). TCR-HA × IG-HA double transgenic mice were bred in our
animal facilities. TCR-HA mice on recombination activating
gene (RAG)-2
/
background have been described (25). INS-HA mice, already described (27), were backcrossed onto the
RAG
/
background and were bred onto the H-2d haplotype.
Transgene expression was determined by PCR. Animal care was
in accordance with institutional guidelines.
T Cell Proliferation Assays.
In vitro assays were performed in
complete IMDM as previously described (6). Responder cells (2 × 105/well) were isolated from the spleen of TCR-HA × IG-HA
or TCR-HA mice and cultured in the presence of 5 × 105 total
irradiated (2,200 rads) BALB/c splenocytes and different concentrations of the HA peptide. Responder cells were also stimulated with coated anti-CD3 at 10 µg/ml or with coated 6.5 mAb at
100 µg/ml in absence or presence of IL-2 (30 UI/ml). 3H incorporation was measured over the last 18 h of a 66-h culture.
Antibodies and Flow Cytometry.
The following mAbs were
used for staining: F23.1-biotinylated (specific for the TCR-
chain of HA-reactive TCR), 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS)-labeled 6.5 clonotypic mAb, and
SA-PE (Southern Biotechnology, Birmingham, AL). All stainings
were done in 96-well plates (5 × 105 cells/well) in 20 µl of mAb
in PBS plus 2% FCS plus 0.1% sodium azide for 20 min on ice.
Between first and second step reagents, cells were washed in PBS
plus 2% FCS plus 0.1% sodium azide, as was done after the last
step. Data were analyzed on a FACScan® (Beckton Dickinson,
Mountain View, CA), using Lysis II software.
For cell sorting experiments, splenocytes were depleted of surface Ig+ cells using Dynabeads (Dynal, Oslo, Norway), and cells
were subsequently stained with F23.1 and 6.5 mAbs. F23.1+6.5hi
cells were sorted with a FACS® Vantage.
Transfer Experiments.
Sorted F23.1+6.5hi spleen cells from
TCR-HA × IG-HA mice, TCR-HA mice, or total lymph node
and spleen cells from TCR-HA mice on RAG
/
background
were washed in serum-free IMDM and were injected into the
lateral tail vein of RAG
/
INS-HA+/+ mice. Recipients were
tested for urine glucose levels every day and diabetes was confirmed by blood glucose levels.
Histology.
Pancreata were quick frozen in O.C.T. compound,
and 5-µm sections were obtained and fixed in acetone for 10 min.
For immunohistochemistry, primary antibodies consisting of biotin-conjugated rat anti-mouse CD4 (clone GK1.5) and rat anti-
mouse CD8 (clone 53-67.2) were applied on sections for 45 min
at the appropriate dilution. After washing in PBS, slides were incubated with a biotinylated anti-rat
antibody (Immunotech, Marseille, France) for 30 min, washed again, and finally incubated
with streptavidin-peroxidase (Vector Laboratories, Inc., Burlingame, CA). Color reaction was revealed with 3-amino-9-ethyl-carbazole (AEC; Sigma Chemical Co., St. Louis, MO), and the slides
were counterstained with hematoxylin. For the insulin staining, a
guinea pig anti-porcine insulin antibody (Dako, Glostrup, Denmark) and a peroxidase-conjugated anti-guinea pig antibody (Dako)
were used as primary and secondary antibodies, respectively.
Competitive Reverse Transcriptase PCR for Cytokine mRNA Expression.
Total RNA was isolated from F23.1+6.5hi sorted splenocytes (2 × 105 cells) from TCR-HA single transgenic and
TCR-HA × IG-HA double transgenic mice using RNeasy Mini
Kit (Qiagen, Hilden, Germany). Reverse transcription was carried out using Superscript II reverse transcriptase (RT; GIBCO
BRL, Gaithersburg, MD). The cytokine polycompetitor plasmid
pQRS was used to quantitate amounts of transcripts for IL-4, IL-10,
IFN-
, and the constitutively expressed hypoxanthine guanine
phosphoribosyltransferase (HPRT), using the primers and PCR conditions as described previously (28). PCR products were analyzed
by electrophoresis on 2% agarose gels and visualized by ethidium
bromide staining under UV illumination, and then photographed
(665 film; Polaroid, St. Albans, Hertfordshire, UK). Image densitometric analysis was performed using National Institutes of Health
Image 1.61 software (SCR, Bethesda, MD) by integrating the volume in individual amplicons. After subtraction of background
values, the density ratio of the competitor band to the target
mRNA was determined and relative amounts of cytokine mRNA
were calculated based on the starting amount of competitor.
Enzyme-linked Immunospot Assay for IL-10.
The enzyme-linked
immunospot (ELISPOT) assay was performed as previously described (29). In brief, F23.1+6.5hi sorted splenocytes from TCR-HA
single transgenic and TCR-HA × IG-HA double transgenic mice
were transferred by threefold dilutions (starting at 104 cells/well)
in duplicate to 96-well microtiter plates (Millipore, Bedford,
MA) that had been coated with the capturing mAb to IL-10 (JES5-2A5; PharMingen, San Diego, CA). After 20 h, cells were removed and spot-forming cells visualized with biotinylated detecting mAb (SXC-1; Pharmingen) and avidin peroxidase in conjunction with 3-amino-9-ethylcarbazole (Sigma Chemical Co.)
substrate. Spots were counted under a dissecting microscope, and
the frequency of antigen-specific cells was determined in both
types of mice.
 |
Results and Discussion |
HA-specific T Cells from TCR-HA × IG-HA Mice Cannot
Respond to Further Antigenic Stimulation in Vitro.
As described
previously (6), mice that express a transgenic TCR specific
for peptides 111-119 from influenza HA (TCR-HA), as
well as influenza HA under the control of the Ig-
promoter (IG-HA) contained CD4+ T cells in periphery that
expressed high levels of the transgenic TCR (detected by
the idiotypic 6.5 antibody). The proportion of these cells,
which increased with the age of these mice, exhibited activation markers (like CD69), but did not proliferate when
stimulated with different concentrations of antigen in vitro
(Fig. 1). Such cells did not respond when stimulated with
the clonotypic antibody, even after addition of exogenous
IL-2. In the following, we have further characterized these
cells that appear to behave differently from in vitro anergized cells.

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Fig. 1.
Proliferation of splenocytes from TCR-HA or from
TCR-HA × IG-HA mice upon
antigenic stimulation in vitro. 2 × 105 spleen cells were stimulated with 5 × 105 irradiated
BALB/c splenocytes in the presence of the indicated amounts of
peptide or with coated 6.5 antibody at 100 µg/ml. IL-2 (30 UI/ml) was added to cells stimulated with the 6.5 antibody. Total cpm values were divided by the number of 6.5+ cells/well as determined by flow cytometry. Each bar represents mean values of triplicate
determinations for each mouse. Response of splenocytes from each
mouse to stimulation with immobilized CD3 antibody was similar (not
shown).
|
|
T cells from TCR-HA Single but Not from TCR-HA × IG-HA Double Transgenic Mice Cause Rapid and Fulminant Diabetes.
To determine whether the lack of proliferation in
vitro was reflected by a lack of effector function in vivo, it
was tested whether anergic 6.5hi cells could cause diabetes
after transfer into rearrangement-deficient RAG
/
mice expressing HA under the control of the rat insulin promoter (INS-HA). In this system, transfer of 104 or 105 6.5+ T cells
from TCR-HA single transgenic mice on the RAG
/
background or of 105 6.5hi cells sorted from TCR-HA single transgenic mice induced diabetes at days 13, 9, and 10, respectively, after transfer (Table 1). The injected mice died
within 12 to 16 d after transfer. In contrast, 105 6.5hi sorted
cells from TCR-HA × IG-HA double transgenic mice did
not induce diabetes with the same kinetics or the same severity, since recipients only became diabetic 16 d after transfer of cells (Table 1) and survived for >40 d after transfer.
The significant delay in the onset of diabetes in the RAG
/
INS-HA mice that received anergic cells was not due to
the fact that these cells could not repopulate the host or
could not home to the pancreas, as T cells were detected
both in the lymph nodes and in the pancreas 11 d after
transfer (Fig. 2). Histological examination of the pancreas
of recipients of naive versus anergic 6.5hi cells revealed
striking differences; in the former, infiltrating cells were detected in both the exocrine and endocrine (islets) tissue, the
islets were completely disrupted, and at the day of diabetes
onset, insulin-producing cells were no longer detectable. On the contrary, the distribution of anergic 6.5hi cells was
restricted to the islets and insulin-producing cells were still
detectable after diabetes onset (Fig. 3). Thus, the impaired
in vitro response of the anergic HA-specific cells was reflected by an impaired effector function in vivo that may be caused by a defect in in vivo proliferation and/or effector
function in the pancreas. The precise reason for the late development of diabetes is unclear. We cannot exclude the
possibility that among the 6.5hi cells from TCR-HA × IG-HA double transgenic mice, there are recent thymic emigrants
that have not yet been rendered anergic and that are causing the slow and mild diabetes observed in the recipients.

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Fig. 2.
Detection of transferred cells in the lymph nodes and pancreas of RAG / INS-HA recipients 11 d after transfer. (A) Splenocytes
from TCR-HA or from TCR-HA × IG-HA mice were stained with
F23.1 and 6.5 mAbs, and F23.1+6.5hi cells were gated as shown and were
sorted accordingly. The purity of the cell populations after sorting was 97 and 95%, respectively. RAG / × INS-HA recipients were injected with
105 6.5hi cells from TCR-HA or TCR-HA × IG-HA mice, and were
analyzed 11 d after transfer; the recipient of T cells from TCR-HA shown
was diabetic, whereas the recipient of cells from the TCR-HA × IG-HA donor was not. For B, two-color staining with the F23.1 and the 6.5 mAbs was performed on lymph node cell suspensions from each recipient.
For C, the pancreas of the same mice (the left and right sections correspond to the recipient of 6.5 cells from the TCR-HA or the TCR-HA × IG-HA mouse, respectively) was frozen and sections were stained with a
mixture of CD4 and CD8 antibodies and revealed with AEC as described
in Materials and Methods. The 6.5 mAb was not used because of its poor
performance on histology sections, but isolation and staining of infiltrating
lymphocytes in the pancreas of other transfered mice confirmed that they
were all 6.5 positive. Note the total disruption of the anatomical structure
of the pancreas having received the naive cells (left). Original magnification was 160 for both sections.
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Fig. 3.
Detection of insulin-producing cells in the pancreas of RAG / INS-HA recipients. Mice transferred with 6.5hi cells from a TCR-HA (left)
or from a TCR-HA × IG-HA mouse (right) were killed at the day of diabetes onset or 7 d after diabetes onset, respectively. Pancreata were embedded in
OCT and frozen; 5-µm sections were stained with insulin antibodies and revealed with AEC. Insulin (red staining) was clearly detected in recipients of
6.5hi anergic cells and not in recipients of 6.5hi naive cells (original magnification: 400).
|
|
Anergic T Cells Contain High Levels of IL-10 mRNA.
In a first attempt to analyze the lack of effector function of
the 6.5hi T cells from the double transgenic mice in some
greater detail, the mRNA level of various cytokines was
determined by competitive PCR. To this end, 6.5hi expressing cells from either TCR-HA single or TCR-HA × IG-HA double transgenic mice were sorted, and mRNA
levels were compared (Table 2). Although IFN-
mRNA
was not detectable in T cells from both mice, only cells
from the double transgenic mice contained low levels of
IL-4 mRNA. By far, the most significant difference was
observed with regard to IL-10; very high levels of this
cytokine mRNA were found in 6.5hi cells from TCR-HA × IG-HA double transgenic, but not from TCR-HA single transgenic mice (Fig. 4). These results were confirmed with sorted CD4+6.5+ cells from the single and double
transgenic mice. The lack of IL-10 transcripts in 6.5+ cells
from single TCR transgenic mice was not due to the lack of activation since 6.5+ cells transferred to INS-HA recipients did not produce IL-10 mRNA up to a point when the
recipient mice became diabetic (not shown).

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Fig. 4.
Competitive RT-PCR for IL-10 mRNA expression in 6.5hi sorted splenocytes
from TCR-HA and TCR-HA × IG-HA mice. IL-10 mRNA
transcripts were compared between mice after standardization
for the expression of the constitutively expressed HPRT gene
(not shown). Constant amounts
of cDNA samples were amplified
in the presence of serial fourfold dilutions of a multispecific internal plasmid control (pQRS). PCR products were separated on 2% agarose gels
and visualized by ethidium bromide staining under UV illumination, and
photographed. Image densitometric analysis was performed using National Institutes of Health Image 1.61 software. The concentrations of
competitor used are indicated on the top. In all cases, the upper band is due to amplification of the competitor construct and the lower band is
due to amplification of the cDNA. The point of equivalence between the
competitor and the cDNA indicates the relative concentration of mRNA
(15.6 fg/ml). Similar results were obtained in two separate experiments
for each group.
|
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High Levels of IL-10 mRNA Are Reflected in Protein Secretion.
To determine whether the increased levels of IL-10
mRNA reflected an increased secretion of the protein, we
performed ELISPOT assays on 6.5hi sorted cells from single
and double transgenic mice. Fig. 5 clearly confirms that the
anergic 6.5hi cells produced and secreted significantly higher
levels of IL-10 than T cells from single transgenic mice.

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Fig. 5.
ELISPOT for IL-10.
The frequency of 6.5hi sorted splenocytes from TCR-HA single
transgenic and TCR-HA × IG-HA double transgenic mice that
secreted IL-10 was determined.
(A) Results are expressed as mean ± standard error spot-forming
units per 1.1 × 103 cells of duplicate determinations. (B) Representative alkaline phosphatase-
based ELISPOT demonstrating
IL-10 secretion by 6.5hi sorted
splenocytes from TCR-HA × IG-HA sorted splenocytes. Similar results were obtained in two
separate experiments.
|
|
These data raise the interesting possibility that so called
anergic cells, as obtained in vivo through continuous stimulation with antigen, are in fact not really anergic but have
assumed the phenotype of regulatory cells. IL-10 has been
reported to downregulate costimulation by APCs (30),
dendritic cell-driven IFN-
production by T cells (31), and
T cell responses to antigen through inhibition of IL-2 production and IL-2R
chain expression (32, 33). Furthermore, adoptive transfer of autoreactive T cells genetically
designed to secrete IL-10 were able to delay the onset of
experimental autoimmune encephalomyelitis in mice (34).
Finally, a recent study by Groux et al. (35) showed that
OVA-specific T cell clones derived from in vitro cultures in the presence of IL-10 and chronic antigenic exposure,
were able to prevent T cell-mediated inflammatory bowel
disease when cotransferred with CD4+CD45RBhi cells into
SCID mice. These cells exerted their influence only upon
stimulation with antigen, i.e., when the recipients were fed
with OVA.
We show here that IL-10-producing T cells cannot only
result from unphysiological maneuvers like genetic alteration or growth in IL-10-containing media, but also develop as a result of chronic antigenic stimulation in vivo.
The IL-10 production by anergic T cells is in line with previous observations that repeated superantigen stimulation results in anergy as well as in IL-10 production (36) and with
other data showing IL-10 production in SCID patients transplanted with HLA-mismatched hemopoietic stem cells
(37). The exact source of IL-10 was not obvious from these
experiments and it was not clear whether the unresponsive
state of T cells in terms of proliferation and effector function correlated with IL-10 production. The results presented here directly show that T cells unable to proliferate
in response to antigen in vitro and with an impaired specific effector function in vivo, can nevertheless produce high
levels of IL-10 and therefore may regulate immune response
through the release of IL-10.
Address correspondence to Harald von Boehmer, Institut Necker, INSERM 373, 156, rue de Vaugirard,
F-75730 Paris, Cedex 15, France. Phone: 33-1-4061-5381; Fax: 33-1-40615590; E-mail: vonBoehm{at}infobiogen.fr
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