U.S. patent application number 10/536316 was filed with the patent office on 2006-06-15 for rapamycin and il-10 for the treatment of immune diseases.
Invention is credited to Manuela Battaglia, Maria Grazia Roncarolo.
Application Number | 20060127357 10/536316 |
Document ID | / |
Family ID | 32469339 |
Filed Date | 2006-06-15 |
United States Patent
Application |
20060127357 |
Kind Code |
A1 |
Roncarolo; Maria Grazia ; et
al. |
June 15, 2006 |
Rapamycin and il-10 for the treatment of immune diseases
Abstract
The invention discloses a combined preparation containing IL-10
and rapamucin able to induce immunosuppression and antigen-specific
immune tolerance, and the use thereof in the treatment of diseases
involving an excessive, dysfunctional or uncontrolled immune
response mediated by T cells.
Inventors: |
Roncarolo; Maria Grazia;
(Segrate, IT) ; Battaglia; Manuela; (Milano,
IT) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET
2ND FLOOR
ARLINGTON
VA
22202
US
|
Family ID: |
32469339 |
Appl. No.: |
10/536316 |
Filed: |
November 27, 2003 |
PCT Filed: |
November 27, 2003 |
PCT NO: |
PCT/EP03/13351 |
371 Date: |
January 6, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60429561 |
Nov 29, 2002 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
424/144.1; 514/291 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 38/1777 20130101; A61P 25/00 20180101; A61K 38/2066 20130101;
A61K 31/436 20130101; A61K 38/13 20130101; A61P 31/00 20180101;
C07K 2317/626 20130101; A61K 38/1777 20130101; C07K 2317/73
20130101; A61P 43/00 20180101; A61P 37/08 20180101; A61P 37/02
20180101; A61K 31/436 20130101; A61K 38/1793 20130101; A61K 38/13
20130101; A61P 3/10 20180101; C07K 16/289 20130101; A61K 38/2066
20130101; A61P 19/02 20180101; A61P 1/04 20180101; A61P 11/06
20180101; A61K 38/1793 20130101; C07K 16/244 20130101; A61P 11/00
20180101; A61P 17/00 20180101; A61P 37/06 20180101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/085.2 ;
514/291; 424/144.1 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 39/395 20060101 A61K039/395; A61K 31/4745 20060101
A61K031/4745 |
Claims
1. A combined pharmaceutical preparation containing IL-10 and
rapamycin, analogs, derivatives or conjugates thereof, for use in
the modulation of T cell-mediated immune responses.
2. A pharmaceutical preparation according to claim 1, for
simultaneous, separate or sequential use in the preventive or
therapeutic treatment of diseases involving an excessive,
dysfunctional or uncontrolled self-or non-self T cell mediated
immune responses.
3. A pharmaceutical preparation according to claim 2, for
simultaneous, separate or sequential use in the preventive or
therapeutic treatment of allogeneic organ rejection, allograft
.beta.-islets rejection, graft versus host disease, and type I
diabetes.
4. A pharmaceutical preparation according to claim 2, for
simultaneous, separate or sequential use in the preventive or
therapeutic treatment of autoimmune diseases selected from
rheumatoid arthritis, multiple sclerosis, systemic lupus
erithematosus; chronic inflammatory diseases; inflammatory bowel
diseases selected from Crohn's disease and ulcerative colitis;
chronic obstructive pulmonary diseases; allergic diseases selected
from asthma and atopic dermatitis, fibrotic diseases; and immune
reactions to gene therapy derived products.
5. A pharmaceutical preparation according to claim 1, further
comprising an active substance selected from immunosuppressive or
immunomodulating agents, antibodies and cytokines.
6. A pharmaceutical preparation according to claim 5, wherein said
active substance is selected from cyclosporine, FK506,
pimecrolimus, mycofenolate, antibodies against CD45, LFA-1
molecules or the IL-2 receptor.
7. A pharmaceutical preparation according to claim 6, containing
antibodies to the IL-2 receptor alpha, beta, and gamma chains.
8. A pharmaceutical composition containing IL-10 and rapamycin, or
analogs, derivatives or conjugates thereof, together with
pharmaceutically acceptable excipients.
9. A pharmaceutical composition according to claim 8, which is in
the form of solution, suspension, tablet or capsule.
10. The use of a combination of IL-10 and rapamycin, or analogs,
derivatives or conjugates thereof, for the preparation of an
immune-modulating agent.
11. The use according to claim 10, for the preparation of an
immune-suppressive agent.
12. The use according to claim 11, wherein the immune-suppressive
agent induces antigen-specific immune tolerance.
13. The use according to claim 11, wherein the immunosuppression
and antigen-specific immune tolerance is mediated by Tr1 cells
and/or CD4+CD25+ Tr cells.
14. The use according to claim 12, wherein the immunosuppression
and antigen-specific immune tolerance is mediated by Tr1 cells
and/or CD4+CD25+ Tr cells.
Description
[0001] The present invention regards methods and compositions for
inducing immunosuppression and/or antigen-specific immune tolerance
in subjects in need thereof. More precisely, the invention provides
a combined preparation of rapamycin and IL-10 for use in the
treatment of diseases involving an excessive, dysfunctional or
uncontrolled self- or non-self immune response mediated by T cells.
The invention is also directed to pharmaceutical compositions
containing IL-10 and rapamycin and to their use as modulators of
the immune response.
BACKGROUND OF THE INVENTION
[0002] Transplantation and immunosuppressive drugs. Transplantation
is the treatment of choice for most patients with end stage
kidney-failure, hearth or liver disease, autoimmune type 1 diabetes
and it is a developing possibility for patients with deficiencies
in small-bowel and lung function. Graft survival depends on a
number of factors but the most significant of these is the
administration of powerful immunosuppressive drugs. Transplantation
between genetically disparate individuals evokes a rapid and
potentially destructive alloreactive immune response that, if left
uncontrolled, can lead to complete destruction of the transplanted
organ. Administration of immunosuppressive drugs attenuates this
response and thus prevents acute graft rejection. However,
continued graft survival depends on life-long immunosuppression
because withdrawal of immunosuppression results in re-activation of
the rejection response, leading to rapid graft destruction.
[0003] Recently, among the immunosuppressive drugs, selective T
cell inhibitors have been developed including cyclosporine A (CsA),
FK506 and rapamycin. Both CsA and FK506 inhibit T cell activation
by blocking calcineurin function and thereby prevent the generation
of the potent nuclear factor of activated T cells (NFAT). This step
is essential for up-regulating the mRNA of several cytokines,
including IL-2. The major limitations of CsA and FK506 are their
various toxicities. Moreover, both CsA and FK506 prevent T cell
apoptosis (reviewed in Yu.et al. 2001).
[0004] On the contrary, rapamycin is a potent immunosuppressant
that inhibits T cell proliferation by binding a cytosolic protein
(FKBP-12) and blocking IL-2 signaling (Sehgal 1998). The complex
binds to and blocks the mammalian target of rapamycin (mTOR),
resulting in the inhibition of cytokines induced T-cell
proliferation. Importantly, in contrast to CsA and FK506, rapamycin
does not block TCR-mediated T cell activation (Blaha et al, 2003)
and IL-2 T cell priming for activation-induced cell death (AICD).
This latter is a form of T cell apoptosis which seems to play a
role in the induction of peripheral transplantation tolerance
(Wells et al. 1999). Unlike CsA, which has no effects on dendritic
cells (DC), rapamycin profoundly affects DC phenotype and function
(Hackstein et al. 2002). It markedly reduces their antigen uptake
capacity, thereby favoring the differentiation of DC with a
tolerogenic phenotype. This effect, present at a low,
physiologically relevant concentration of rapamycin (1 ng/ml) is
independent of DC maturation and has been demonstrated both in
vitro and in vivo (Hackstein et al. 2002).
[0005] Although the currently available immunosuppressive drugs are
very effective in short term, substantial problems indicate a
pressing need to develop alternative and more sophisticated ways of
preventing graft rejection. The main obstacle is the inability to
distinguish between beneficial immune responses against infectious
pathogens and destructive immune responses against the graft. Thus,
immunosuppressive therapies can lead to increased risk of
opportunistic infections. Several studies show that non specific
immunosuppression would lead to an increased incidence of cancer in
transplanted patients (Hojo et al. 1999). Therefore, the full
potential of transplantation will be fulfilled only when
alternatives to non specific immunosuppression are found. The major
aim of transplantation immunology is to develop protocols that
prevent immune responses towards the graft but leave the rest of
the immune system intact. This accomplishment will lead to
transplantation tolerance.
[0006] Autoimmunity. In autoimmune diseases, undesired immune
responses to self-antigens lead to destruction of peripheral
tissues. Treatments of autoimmune diseases are currently based on
downmodulation of inflammation and non-antigen (Ag) specific
immunosuppression. As for prevention of allograft rejection, this
strategy is frequently not effective in the long term with high
risk of relapse once the drug is withdrawn and hazards of excessive
immunosuppression, including infections and tumors. The alternative
approach is based on the induction of transient immunosuppression
and/or specific immune tolerance, aimed at "silencing" the
pathogenic response to self-Ag, while keeping host defense
mechanism intact.
[0007] The immune system has evolved two distinct mechanisms to
induce tolerance to self or non-harmful antigens. These are
referred to as central and peripheral T cell tolerance. Central
tolerance is realised during fetal development and the very early
natal period and is mediated by clonal deletion of self-reactive T
cells during thymic development. Peripheral mechanisms induce
tolerance in mature T cells and occur in the periphery during the
whole life. These mechanisms include functional inactivation of
antigen specific lymphocytes (named anergy) and activation of T
cell subsets with suppressive and regulatory capacities (T
regulatory cells reviewed in Battaglia et al. 2002).
[0008] Tolerance and T regulatory cells. Recently, there has been a
growing interest in the induction of T regulatory (Tr) cells as a
strategy to achieve graft specific tolerance. The majority of Tr
cells identified to date lie within the CD4.sup.+ population,
although other T cell subsets, such as CD8.sup.+,
CD8.sup.+CD28.sup.- and TCR.sup.+CD4.sup.-CD8.sup.- have also been
shown to contain cells with regulatory capacity. Within the
CD4.sup.+ population, various fractions with suppressive properties
have been identified. Our group has characterised a subset of Tr
cells, defined as type 1 regulatory T cells (Tr1), which have a
cytokine production profile distinct from that of Th1 and Th2
cells. Human and mouse Tr1 cells produce high levels of IL-10,
significant amounts of IL-5, TGF-.beta., and IFN-.gamma., but low
levels of IL-2 and no IL-4 (Groux et al. 1997). IL-10 is a crucial
cytokine for the differentiation and effector functions of Tr1
cells. Culture of CD4.sup.+ T cells in the presence of antigen and
IL-10 leads to generation of Tr1 cells that are able to suppress
antigen-specific T cell responses in vitro and the development of
autoimmune colitis in vivo (Groux et al. 1997). Tr1 cells can also
be generated in vivo. Trl cells have indeed been isolated from
peripheral blood of SCID-reconstituted patients, in whom high
levels of IL-10 were associated with successful allogeneic stem
cell transplantation (Bacchetta et al. 1994).
[0009] Tolerance and IL-10. IL-10 plays a key role in
immunoregulation (reviewed in Moore et al. 2001). It inhibits
proliferation and IL-2 production of T lymphocytes. IL-10 has
strong anti-inflammatory properties by inhibiting production of
pro-inflammatory cytokines such as TNF-.alpha., IL-1, IL-6 and
chemokines such as IL-8, MIP1.alpha., and MIP1.beta. by activated
monocytes/macrophages, neutrophilis, eosinophilis, and mast cells.
Moreover, IL-10 suppresses antigen-presenting capacities of antigen
presenting cells such as monocytes/macrophages/DC by downregulating
MHCII and co-stimulatory molecules. The ability of IL-10 to inhibit
induction and effector function of T cell-mediated and
anti-inflammatory immune responses led to numerous studies on IL-10
expression, function, and potential utility in bone marrow and
organ transplantation. In studies of vascularized heart allograft
in mice, IL-10 treatment of recipient animals prior to grafting
enhanced graft survival, whereas providing IL-10 at or after the
time of grafting had little beneficial effect or even enhanced
rejection (Li et al. 1999). Patients exhibiting elevated levels of
IL-10 production prior to BMT have lower incidence of GVHD and
improved survival (Baker et al. 1999). On the contrary, high IL-10
levels in post-BMT GVHD patients indicates a poor prognosis for
survival (Hempel 1997). However, Blazar and colleagues showed that
treatment of mice with small amounts of IL-10 (10.sup.-3, 10.sup.-4
of the amount that increased mortality) protects against
GVHD-associated lethality (Blazar et al. 1998).
[0010] Combination of immunosuppressive drugs with IL-10. The
majority of immunosuppressive drugs in current clinical uses act by
inhibiting T cell activation and thus prevents graft rejection.
However, this may be counter-productive, as under appropriate
circumstances, T cell activation may lead to the induction of
processes facilitating the development of graft-specific tolerance.
Therefore, the usage of immunosuppressive drugs might not be
optimal when the aim is tolerance induction. A clear demonstration
of this phenomenon comes from SCID patients in whom tolerance was
achieved after allogeneic hematopoietic stem cell transplantation
without any immunosuppressive therapy (Bacchetta et al. 1994). In
these patients the presence of donor derived Tr1 cells specific for
the host alloantigens correlated with stable mixed chimerism, high
levels of IL-10 production in vivo, and normal immune functions in
the absence of any immunosuppressive therapy. In contrast, in BMT
patients who received an immunosuppressive regimen to control
acute-GVHD, Tr1 cells could not be isolated from peripheral blood,
although donor derived T cells specific for host alloantigens were
detectable (Bacchetta et al. 1995).
[0011] Rapamycin represents a novel compound with interesting
immunomodulatory properties. For this reason we combined the in
vivo administration of rapamycin with IL-10 in order to prevent
allograft rejection or modulate type 1 diabetes and to allow the in
vivo development of Tr cells.
STATE OF THE ART
[0012] U.S. Pat. No. 6,277,635 relates to the use of IL-10 for
suppressing transplant rejection. This patent teaches methods of
treating and inhibiting tissue rejection, inhibiting GVHD and
antigen specific responses. It further describes T cells that
exhibit anergy for a particular antigen.
[0013] U.S. Pat. No. 6,428,985 describes mammalian, including
human, immunosuppressive compositions containing IL-10 polypeptides
with at least one mutation in the native sequence (Mut IL-10 ),
either alone or in combination with other agents, and various in
vitro and in vivo methods of using such compositions and
combinations thereof. Uses include immunosuppressive and
combination therapies for a number of diseases and disorders
related to inflammation, transplantation, fibrosis, scarring, and
tumor treatment. The effect of Mut IL-10 has been shown in animal
studies but not in human clinical settings.
[0014] U.S. Pat. No. 5,624,823 describes DNA encoding porcine IL-10
and a method for inducing tolerance in a recipient mammal, e.g. a
primate, receiving an allogeneic transplant. Rapamycin,
cyclosporine and FK506 are mentioned as "help reducing agent", i.e.
agents which reduce the cytokine release. Porcine IL-10 is used in
a context of thymus transplantation only.
[0015] U.S. Pat. No. 6,022,536 describes the combined use of IL-10
and cyclosporine as immunosuppression therapy for treating
autoimmune diseases and GVHD. Synergistic combination of low doses
of IL-10 and cyclosporine and a pharmaceutical carrier are
proposed.
[0016] U.S. Pat. No. 6,403,562 describes methods for treating
autoimmune-related diseases, such as multiple sclerosis, by
administering IL-10 together with TGF-.beta., to a person afflicted
with or predisposed to an autoimmune disease. These cytokines act
in a synergistic manner as suppressor factors to inhibit the
activation of self-reactive T cells that are involved in autoimmune
disease.
DESCRIPTION OF THE INVENTION
[0017] The invention provides a combined pharmaceutical preparation
containing IL-10 and rapamycin for use in the modulation of T-cell
mediated immune response, in particular for inducing
immunosuppression and antigen-specific immune tolerance in a
subject in need thereof. The induction of Tr1 and
CD4.sup.+CD25.sup.+ Tr cell-mediated antigen-specific immune
tolerance is useful for the treatment of pathological conditions
that involve an excessive, dysfunctional, unregulated or
uncontrolled self- or non-self T cell-mediated immune response.
[0018] In a preferred embodiment of the invention, IL-10 and
rapamycin are in the form of a combined preparation for
simultaneous, separate or sequential use in the preventive or
therapeutic treatment of allogeneic organ rejection, type 1
diabetes, autoimmune and chronic inflammatory diseases including
psoriasis, multiple sclerosis, inflammatory bowel disease, Crohn's
disease, rheumatoid arthritis, or other T-cell mediated diseases
such as GVHD, asthma, atopic dermatitis, chronic obstructive
pulmonary disease, and immune reactions to gene therapy derived
products. In addition, treatment of fibrotic diseases including
liver and lung fibrosis is envisaged.
[0019] Preferably, the combined preparation is used for the
preventive or therapeutic, treatment of solid allogeneic organ
rejection, particularly allograft .beta.-islets rejection, and
autoimmune diseases, especially type 1 diabetes.
[0020] The combined preparation may contain human or viral IL-10,
analogs, derivatives or conjugates thereof improving the
bioavailability or biological efficacy of the natural molecule,
such as polyethylene glycol (PEG) conjugated IL-10. IL-10
functional analogs include small molecules that mimic IL-10 effects
and monoclonal antibodies (mAbs) against the IL-10 receptor or
IL-10 fusion proteins, which trigger IL-10 signaling pathway.
[0021] The combined preparation may contain rapamycin analogs or
derivatives. Besides rapamycin and IL-10, derivatives.or analogs
thereof, the combined preparation may further contain
immunosuppressants or immunomodulating agents, monoclonal
antibodies or cytokines. Preferred biologically active substances
that may be used in combination with IL-10 and rapamycin include:
a) calcineurin inhibitors such as cyclosporine, FK506 (tacrolimus),
pimecrolimus, b) other immunosuppressant such as micofenolate, c)
antibodies against different isoforms of CD45, or adhesion
molecules such as LFA-1 and d) antibodies against the IL-2 receptor
alpha, beta and gamma chains. Suitable immunosuppressive agents
include those that act through the IL-2 signaling pathway (e.g.
JAK1 and JAK3 and STAT5 inhibitors). The combination of
rapamycin+antiTac (a humanized antibody to the IL-2 receptor a
chain)+IL-10 proved particularly effective in preventing allogeneic
rejection, especially in a murine model of allograft .beta.-islets
rejection, by inducing a state of tolerance instead of the
persistent immunosuppression generated by conventional therapeutic
protocols. Moreover, the combination of rapamycin+IL-10 proved to
be effective in treating autoimmune diabetes and inducing long term
immunomodulation in NOD mice. Tolerance is achieved as a result of
the rapamycin+IL-10 induced expansion and differentiation of type 1
T regulatory (Tr1) and CD4.sup.+CD25.sup.+ Tr cells, which mediate
antigen-specific tolerance through different mechanisms including
the production of suppressive cytokines (IL-10 and TGF-.beta.), and
inhibition of T cell activation.
[0022] Rapamycin+IL-10 combined preparations according to the
invention exert a long term protection, which can be maintained
after drug withdrawal despite recovery of T cell
immunocompetence.
[0023] In a further embodiment the invention provides
pharmaceutical compositions containing IL-10 and rapamycin and
optionally further active ingredients selected from
immunosuppressant or immunomodulating agents, monoclonal antibodies
and cytokines, together with pharmaceutically acceptable
excipients. Suitable pharmaceutical compositions are administered
by the oral, intravenous, parenteral, or subcutaneous route, and
are preferably in the form of solutions, suspensions, injectables,
tablets, or capsules. Effective amounts of rapamycin may range from
0.001 mg/Kg to 100 mg/Kg and effective amounts of IL-10 may range
from 0.001 .mu.g/Kg to 1000 .mu.g/Kg.
[0024] The invention is further illustrated by the following
examples and the enclosed figures.
DESCRIPTION OF THE FIGURES
[0025] FIG. 1 Mice treated with the IL-10 protocol and the Edmonton
protocol have comparable graft survival.
[0026] Balb/c mice that had been rendered diabetic by
streptozotocin injection were transplanted under the kidney capsule
with purified allogeneic C57BL/6 .beta.-islets. Mice were not
treated (control, n=13 mice), or treated with
rapamycin+antiTac+IL-10 (IL-10 protocol, n=16 mice) or
rapamycin+antiTac+FK506 (Edmonton protocol, n=4 mice) for 30 days.
Graft survival was monitored by glycemia levels. A graft was
considered rejected when glycemia was higher than 250 mg/dl.
[0027] Replacement of FK506 (Edmonton protocol) with IL-10 (IL-10
protocol) resulted in comparable graft survival: in mice treated
with the IL-10 protocol graft survival was 89% whereas 100%
survival was observed in mice treated with the Edmonton
protocol.
[0028] FIG 2 The absence of antiTac from the IL-10 protocol
slightly increases allogeneic .beta.-islets rejection.
[0029] Balb/c mice that had been rendered diabetic by
streptozotocin injection were transplanted under the kidney capsule
with purified allogeneic C57BL/6 .beta.-islets. Mice were not
treated (control, n=8 mice), or treated with rapamycin in
combination with IL-10 (rapa+IL10, n=8 mice) or IL10 only (n=4
mice). Graft survival was monitored by glycemia levels.
[0030] The absence of antiTac from the IL-10 protocol (see FIG. 1)
slightly affected graft survival. Rapamycin in combination with
IL-10 allowed graft survival in 78% of the animals. Treatment with
IL-10 alone was not efficient in preventing graft rejection.
[0031] These data suggest that the antiTac is not required to
prevent allograft rejection.
[0032] FIG. 3 T cells from mice treated with the IL-10 protocol
maintain an in vitro proliferative capacity.
[0033] T cells from control untransplanted mice (white bars) and
mice treated with the IL-10 protocol (gray bars), or the Edmonton
protocol (black bars) were isolated from the spleen and stimulated
in vitro polyclonally with antiCD3 and antiCD28 mAbs. Cells from
mice treated with the Edmonton protocol were strongly reduced in
their in vitro proliferative capacity while only a mild reduction
in proliferation was observed in T cells isolated from mice treated
with the IL-10 protocol.
[0034] These data suggest a profound state of immunosuppression in
T cells isolated from mice treated with the Edmonton protocol but
not from mice treated with the IL-10 protocol.
[0035] FIG. 4 T cells from mice treated with the IL-10 protocol
preserve an antigen-specific proliferative capacity.
[0036] Mice transplanted 280 days before and treated only for 30
days with the IL-10 protocol (gray bars), or the Edmonton protocol
(black bars), were immunized in vivo in the hind foot pad with
CFA+OVA. Draining lymph nodes were collected and re-stimulated in
vitro with OVA and self APC.
[0037] OVA-specific T cell proliferation was strongly reduced in
mice treated with the Edmonton protocol while OVA-response in mice
treated with the IL-10 protocol was comparable to that observed in
untransplanted immunized mice.
[0038] These data further demonstrate a general state of
immunosuppression in mice treated with the Edmonton protocol, but
not with the IL-10 protocol
[0039] FIG. 5 T cells isolated from mice treated with the IL-10
protocol produce IL-10.
[0040] CD4.sup.+ T cells isolated from the kidney of control
untransplanted mice or mice treated with the IL-10 protocol (gray
bars) and the Edmonton protocol (black bars) were stimulated in
vitro with antiCD3 and antiCD28 mAbs. Supernatants were collected
96 hours after stimulation and IL-10 production was evaluated by
ELISA.
[0041] T cells from mice treated with the IL-10 protocol produced
higher levels of IL-10 compared to mice treated with the Edmonton
protocol and control untransplanted mice
[0042] FIG. 6 A distinct population of IL-10 producing T cells can
be isolated from mice treated with the IL-10 protocol.
[0043] (A) T cells from mice treated with IL-10 protocol, Edmonton
protocol, and from control untransplanted mice were isolated and
stimulated polyclonally in vitro to induce cytokine production.
After 3 hours, the cells were labeled with a diabody consisting of
one mAb that binds an ubiquitous cell surface marker and the other
mAb able to catch IL-10. The labeled cells were then incubated for
an additional hour at 37.degree. C. in order to release cytokines
accumulated during polyclonal stimulation. IL-10 produced by the
labeled cells was captured by the diabody. Cells were further
labeled with an antiIL-10 mAb labeled with PE. Anti-PE microbeads
were used in order to magnetically separate IL-10.sup.+ enriched
(filled histogram) and IL10.sup.- (empty histogram) cells. A
distinct population of IL-10+ enriched T cells was isolated only
from transplanted mice treated with the IL-10 protocol.
[0044] (B). Intracytoplasmic staining was performed on this
distinct IL-10.sup.+ enriched T cell population and a significant
proportion of cells with a Tr1 cytokine profile (i.e.
IL-10.sup.+,IL-4.sup.-) was identified in mice treated with the
IL-10 protocol but not in mice treated with the Edmonton protocol
or control untransplanted mice
[0045] FIG. 7 IL-10 is required to induce IL-10 producing T cells
in vivo.
[0046] To understand the requirement for IL-10 administration to
induce IL-10.sup.+ Tr1 cells in vivo, transplanted mice were
treated with the IL-10 protocol (rapamycin+antiTac+IL-10 ) or
rapamycin+antiTac only. The production of IL-10 by CD4.sup.+
splenic T cells in the two groups of mice was then evaluated.
[0047] T cells from mice treated with the IL-10 protocol (gray
bars), or rapamycin+antiTac (black bars), or control untransplanted
(white bars) were isolated and stimulated in vitro polyclonally
with antiCD3 and antiCD28 mAbs.
[0048] Significant levels of IL-10 were produced only by cells
isolated from the IL-10 protocol treated mice.
[0049] These data suggest that IL-10 is required in order to induce
IL-10 producing cells in vivo.
[0050] FIG. 8 Preliminary results with rapamycin+IL-10 for the
treatment of type I diabetes in NOD mice.
[0051] NOD mice at 11 weeks of age are at a stage of pre-diabetes.
These mice have insuilitis and infiltrating autoimmune T cells in
the pancreas, however they still have enough normal .beta.-islets
left able to produce sufficient insulin to be normoglycemic.
[0052] Pre-diabetic mice were treated daily starting from 11 weeks
of age with either rapamycin, rapamycin+IL-10 or IL-10 alone. Six
weeks after treatment, 33% of control mice developed diabetes while
mice treated with rapamnycin and IL-10 are still all
normoglycemic.
[0053] These preliminary results suggest that rapamycin+IL-10 can
be used to block diabetes in its early stage and to prevent the
further spontaneous development of full blown autoimmune
diabetes.
[0054] FIG. 9 The IL-10 protocol inhibits diabetes induced in
NOD.SCID mice following transfer of diabetogenic T cells.
[0055] 5.times.10.sup.6 splenocytes from NOD diabetic mice were
transferred intravenously in NOD.SCID mice. The recipient mice were
either untreated or treated with the Edmonton protocol
(rapamycin+antiTac+FK506), IL-10 protocol
(rapamycin+antiTac+IL-10), or rapamycin+antiTac for 40 days after
transfer.
[0056] Fifty days post transfer all the control untreated mice were
diabetic.
[0057] All the mice treated with the Edmonton protocol and 75% of
the mice treated with rapamycin+antiTac became diabetic.
Interestingly, only 33% of the mice treated with the IL-10 protocol
became diabetic.
[0058] These preliminary data indicate that the IL-10 protocol
inhibits type I diabetes induced in NOD.SCID mice by transferring
autoimmune diabetogenic NOD T cells.
[0059] FIG. 10 Use of rapamycin+IL-10 for treatment of type I
diabetes.
[0060] A) Treatment of Diabetes in NOD Mice.
[0061] NOD mice were treated from 11 weeks to 31 weeks of age with
IL-10 (IL-10, n=7 mice), or rapamycin (RAPA, n=13 mice), or
rapamyciil+IL-10 (RAPA/IL-10 n=16 mice), or vehicle (CNTR, n=22
mice). Diabetes incidence, monitored by glycemia levels, was stable
at least up to 60 weeks of age. Administration of rapamycin alone
reduced diabetes incidence from 95% to 46%. IL-10 administration
had no significant effect on the development of diabetes. The
protective effect of rapamycin was significantly improved when
IL-10 was added to the treatment reducing diabetes incidence to
13%.
[0062] B) Ability of Splenocytes From Treated NOD Mice to Transfer
Diabetes in NOD.SCID Mice.
[0063] 5.times.10.sup.6 total splenocytes from untreated-diabetic
NOD mice (DIABETIC NOD n=8) or mice treated with rapamycin (RAPA
NOD, n=5) or rapamycin+IL-10 (RAPA/IL-10 NOD n=13) were transferred
in NOD.SCID mice and diabetes incidence was monitored by glycemia
levels.
[0064] Transfer of splenocytes from rapamycin-treated mice resulted
in a significant delay in onset of the disease, compared to mice
injected with splenocytes from diabetic NOD mice. Importantly,
splenocytes from mice treated with rapamycin+IL-10 even further
delayed diabetes transfer.
[0065] FIG. 11 Tr cells content in mice treated with
rapamycin.+-.IL-10.
[0066] Cytokine production by CD4.sup.+ T cells (left panels) and
percentages of CD4.sup.+CD25.sup.+ T cells (right panels) were
evaluated by CBA and FACs analysis respectively, in the spleen
(upper panel), pancreatic lymph nodes (PLN) (middle panel), and
islet infiltrating cells (IIC) (lower panel) of untreated-diabetic
NOD mice (gray bars), rapamycin-treated mice (white bars), or
rapamycin+IL-10-treated mice (black bars). A high proportion of
CD4.sup.+ Tr1 cells, as determined by their cytokine production
profile (i.e. IL-10.sup.++ IL-15.sup.+ TGF-.beta..sup.+), was
present only in spleens of tolerant mice treated with
rapamycin+IL-10 (black bars). The percentages of
CD4.sup.+CD25.sup.+ T cells were higher in the spleen, PLN, and IIC
of both mice treated with rapamycin alone or rapamycin+IL-10 (black
bars). Therefore, in rapamycin+IL-10 treated mice, Tr1 cells are
present in the spleen and CD4.sup.+CD25.sup.+ Tr cells are present
in the spleen, lymph nodes, and pancreas.
[0067] FIG. 12 Ability of Tr cells to suppress immune responses in
vitro.
[0068] In vitro suppressive activity of Tr cells on proliferation
of CD4.sup.+ naive NOD T cells labeled with CFSE and cultured in
the presence of antiCD3 mAb was tested. Either CD4.sup.+ IL-10
enriched splenic T cells (purity .about.40%) (left panel) or
CD4.sup.+CD25.sup.+ T cells (MACs purified, purity.gtoreq.75%)
(right panel) were used as suppressor cells added in equal number
to naive T cells. Naive T cells divided in the absence of any added
cells (gray bars) were used as control. Cell division in the
presence of CD4.sup.+ IL-10 enriched Tr1 cells or CD4.sup.+
CD25.sup.+ Tr cells isolated from untreated-diabetic NOD mice
(black bars), or rapamycin-treated mice (white bars), or
rapamycin+IL-10-treated mice (dotted bars) was evaluated and
percentages of suppression relative to control were determined
(numbers on top of each histogram). Tr1 cells from spleens of
rapamycin+IL-10 treated mice mildly suppress the proliferative
responses of CD4.sup.+ T cells obtained from NOD mice. Strong
suppression was observed with CD4.sup.+CD25.sup.+ T cells isolated
from PLN and IIC of both rapamycin and rapamycin+IL-10 treated
mice.
EXAMPLES
Example 1
[0069] 1. Allogeneic .beta.-islet transplantation. A model of fully
mismatched murine islet allotransplantation (C57BL/6 into Balb/C)
was used. Allogeneic pancreatic .beta.-islet transplantation is
becoming a valid alternative to insulin replacement therapy or to
pancreas transplantation for the cure of type 1 diabetes. In the
past years improved methods for the isolation and preservation of
human .beta.-cells and development of new immunosuppressive agents
have significantly improved the clinical outcome of these
transplants. Specifically, a new steroid-free immunosuppressive
regimen based on rapamycin+antiTac+FK506 (the Edmonton protocol)
has been recently shown to induce insulin independence in 80% of
the patients at 1 year after transplant (Shapiro et al. 2000).
These results largely exceed the ones obtained with all previous
immunosuppressive combination therapies. However, the demonstration
that this regimen may induce tolerance has not been produced.
Importantly, the mechanism of action of FK506 might prevent a state
of tolerance induction due to prevention of apoptosis and
inhibition of Tr cells development
[0070] In an effort to develop a tolerogenic protocol we designed a
regimen in which FK506 in the Edmonton protocol was replaced by
IL-10 (i.e. IL-10 protocol: rapamycin+antiTac+IL-10 ).
[0071] Balb/c mice that had been rendered diabetic by
streptozotocin injection were transplanted under the kidney capsule
with purified allogeneic C57BL/6 .beta.-islets. Graft survival was
similar (89% and 100% at 240 days post transplant) in mice treated
with the IL-10 protocol and the Edmonton protocol, respectively
(FIG. 1).
[0072] The in vivo usage of antiTac has been strongly supported in
the past years by the need to block activated T cells with high
IL2R.alpha. chain expression. However, it has been widely
demonstrated that a subset of Tr cells constitutively express the
IL2R.alpha. chain (i.e. CD4.sup.+CD25.sup.+ Tr cells) and that this
T cell population is able to suppress allograft rejection (Taylor
et al. 2002). Therefore, the usage of a mAb which blocks the
CD25.sup.+ T cell population might be counterindicated when in vivo
tolerance induction mediated by Tr cells is sought. For this
reason, removal of antiTac from the treatment protocol was
evaluated. We treated transplanted mice for 30 days with
rapamycin+IL-10 or IL-10 alone in order to determine whether it
would be sufficient to prevent islet rejection (FIG. 2). Long-term
graft survival was obtained in 30% of the mice treated with IL-10
alone and it increased to 78% in mice treated with rapamycin+IL-10.
This level of graft survival was only slightly lower than that of
mice that in addition were treated with antiTac (as shown in FIG.
1). These data indicate that the absence of antiTac mAb from the
IL-10 protocol allows allograft survival while CD4.sup.+CD25.sup.+
Tr cells are not affected and in vivo tolerance induction by these
cells could be preserved.
[0073] Since one of the desired outcomes is tolerance rather than
imnimunosuppression, we examined whether T cells from mice treated
with IL-10 and Edmonton protocols were responsive to polyclonal and
antigen-specific stimulation. First, T cells were isolated from
spleens of mice at day 240 after transplantation (210 days after
cessation of treatment) and stimulated with antiCD3 and antiCD28
mAbs (FIG. 3). Proliferation of T cells from mice treated with the
Edmonton protocol was strongly suppressed when compared to that of
T cells from control untransplanted mice. Suppression of T cell
proliferation was not as strong in mice treated with the IL-10
protocol (FIG. 3). Next, 280 days post transplant (250 days after
cessation of treatment) mice were immunised with CFA plus OVA in
the hind, foot pad and proliferative responses of T cells isolated
from the draining lymph nodes was measured (FIG. 4). T cells from
mice treated with the Edmonton protocol did not proliferate in
response to OVA, whereas T cells from mice treated with the IL-10
protocol had similar responses as untransplanted immunised control
mice (FIG. 4).
[0074] In order to determine whether the replacement of FK506 by
IL-10 potentially promoted Tr1 cell expansion, CD4.sup.+ T cells
infiltrating the site of islet transplantation were isolated from
mice 200 days after transplant and their cytokine production was
examined. CD4.sup.+ T cells isolated from mice treated with the
IL-10 protocol produced significantly higher amounts of IL-10 after
stimulation with antiCD3 and antiCD28 mAbs than mice treated with
the Edmonton protocol (FIG. 5). Purified spleen T cells were then
stimulated with antiCD3 and antiCD28 mAbs and the IL-10 secreting
cells were enriched using IL-10 capture beads (FIG. 6A). The IL-10
/IL-4 cytokine profile was examined by intracytoplasmic staining
(FIG. 6B). Interestingly, a distinct population of
IL10.sup.+IL4.sup.- cells (i.e. reflecting the cytokine profile of
Tr1 cells) was identified only in mice treated with the IL-10
protocol.
[0075] Infiltrating cells from mice treated with rapamycin+antiTac
in the absence of IL-10 did not produce IL-10, indicating that
increased in vitro IL-10 production was due to the in vivo
administration of IL-10 (FIG. 7).
[0076] Collectively, these data indicate that:
1. The combination of rapamycin+antiTac+FK506 (Edmonton protocol)
protects mice from allo-rejection but induces a state of
long-lasting chronic immunosuppression.
[0077] 2. The combination of rapamycin+antiTac+IL-10 (IL-10
protocol) provides long-term protection against allo-rejection.
This treatment results in expansion of a distinct population of T
cells with cytokine profiles consistent with Tr1 cells and
protection is maintained after drug withdrawal despite recovery of
T cell immunocompetence.
Example 2
[0078] 1. Preliminiary results in type I diabetes. The tolerogenic
effect of rapamycin+IL-10 was also evaluated in a setting of type I
diabetes.
[0079] We believe that prevention of .beta.-cell destruction, which
is associated with progression to type I diabetes and is found at
disease onset, can be prevented by:
1. Down-regulation of the general `bystander` inflammation within
the pancreas.
2. Blockade of the expansion of islet specific T effector
cells.
3. Induction and expansion of antigen-specific Tr cells.
[0080] In our proposed protocol, down-regulation of inflammation
should be achieved by IL-10, and blockade of T effector cell
expansion should be achieved by rapamycin. Neither IL-10 nor
rapamycin prevent T cell priming and therefore they should allow
induction of antigen-specific T regulatory cells, and as described
below, IL-10 should promote the induction and expansion of Tr1
cells. We investigated the effect of rapamycin alone or in
combination with IL-10 in treating autoimmunity in the NOD mouse a
model for type I diabetes. The NOD mouse develops overt disease at
15-30 weeks of age with destruction of the .beta.-cells of the
islets and elevations in blood glucose and shares many key features
with the human disease (Tisch et al. 1996, Delovitch et al. 1997).
Inhibition of type I diabetes was evaluated by treating NOD mice
daily starting at 11 weeks of age (i.e. pre-diabetic mice with
periinsultis) with rapamycin, rapamycin+IL-10, or IL-10 alone. Six
weeks after treatment, 33% of untreated control mice started
developing diabetes while mice treated with rapamycin+IL-10 are
still all normoglycemic (FIG. 8).
[0081] The efficacy of our protocol was also tested in a model of
adoptive transfer. NOD.SCID mice, which lack endogenous T and B
cells and therefore do not develop diabetes spontaneously, develop
diabetes in 15-20 days after transfer of 5.times.10.sup.6
splenocytes from a diabetic NOD mouse,. NOD.SCID recipient mice
were either untreated or treated for 40 days after transfer of
diabetic cells with rapamycin+antiTac+FK506 (Edmonton protocol),
rapamycin+antiTac+IL-10 (IL-10 protocol), and rapamycin+antiTac
(FIG. 9). Control mice started developing diabetes 15 days after
transfer. All the mice treated with the Edmonton protocol and 75%
of the mice treated with rapamycin+antiTac became diabetic within
33 days after transfer. Interestingly, only one mouse out of three
treated with the IL-10 protocol (33%) became diabetic 35 days after
transfer.
[0082] All together these preliminary data provide strong rationale
for the use of rapamycin+IL-10 to inhibit full development of type
I diabetes.
[0083] 2. Rapamycin +IL-10 therapy inhibits autoimmune diabetes and
induces long-term tolerance. Based on the promising preliminary
results obtained in the NOD mouse model (FIG. 8) we treated NOD
mice for 20 weeks with rapamycin.+-.IL-10 starting at 11 weeks of
age, a time point at which pancreatic-cell autoimmunity is clearly
established as judged by insulitis and auto-insulin antibodies.
Administration of rapamycin alone reduced the incidence of diabetes
from 95% to 46% (FIG. 10A). Previous observations indicated that
the effects of IL-10 therapy in NOD mice vary depending on route,
dose, and timing of administration (Roncarolo et at. 2003).
However, here we show that administration of IL-10 alone over the
same time period had no significant effect on the development of
diabetes. The protective effect of rapamycin was significantly
improved when IL-10 was added to the treatment, further reducing
the incidence of diabetes to 13% (FIG. 10A). Interestingly,
protection was maintained for an additional 30 weeks after the
treatment was stopped, demonstrating establishment of long-term
immunomodulation.
[0084] The mechanism by which rapamycin or rapamycin+IL-10 prevents
development of autoimmune diabetes was further investigated in
transfer experiments with cells from tolerant mice. Transfer of
splenocytes from untreated-diabetic NOD mice in immunodeficient
NOD.SCID mice rapidly induced diabetes, while transfer of
splenocytes from rapamycin-treated mice resulted in a significant
delay in onset of the disease. Interestingly, transfer of
splenocytes from mice treated with the combination of
rapamycin+IL-10 even further delayed diabetes transfer (FIG. 10B).
These data indicate that treatment with rapamycin down-regulates
the ability of splenic autoreactive T cells to transfer diabetes
and that this effect is strongly enhanced when IL-10 is added to
the treatment.
[0085] The mechanisms underlying long-term tolerance were analysed
in tolerant mice of 50 weeks of age or older. Although spleens from
untreated-diabetic NOD mice or rapamycin+IL-10 -treated mice
contained comparable cell numbers and the same proportion of
CD4.sup.+ and CD8.sup.+ T cells, their cytokine production profiles
were distinct. A high proportion of CD4.sup.+ Tr1 cells, as
determined by their cytokine production profile (i.e.
IL-10.sup.++IL-5.sup.+TGF-.beta..sup.+), was present in spleens of
tolerant mice treated with rapamycin+IL-10, but not in spleen of
mice treated with rapamycin alone or untreated-diabetic NOD mice.
(FIG. 11). However, the proportion of splenic CD4.sup.+ T cells
producing IL-4 was the same in both untreated and treated mice. In
addition, the percentages of splenic CD4.sup.+CD25.sup.+ T cells
were higher in both mice treated with rapamycin alone or
rapamycin+IL-10, as compared to untreated-diabetic NOD mice (FIG.
11). In contrast, no Tr1 cells could be detected in pancreatic
lymph nodes (PLN) and islet infiltrating cells (IIC) (FIG. 11),
indicating that Tr1 cells are not present at the site of
autoimmunity. On the other hand, CD4.sup.+CD25.sup.+ T cells were
observed in high numbers in PLN, and represented almost 100% of the
CD4.sup.+ T cells isolated from the IIC of mice treated with either
rapamycin alone or rapamycin+IL-10 but not in untreated-diabetic
mice (FIG. 11). These CD4.sup.+CD25.sup.+ T cells from IIC were
anergic and did not produce significant levels of cytokines with
the exception of TGF-.beta..
[0086] Next we determined whether the Tr1 cells present in spleens
of rapamycin+IL-10 treated mice and the CD4.sup.+CD25.sup.+ T cells
from spleens, PLN, and ITC of rapamycin and rapamycin+IL-10 treated
mice had suppressive activity in vitro. Tr1 cells from spleens of
rapamycin+IL-10 treated mice mildly suppressed the proliferative
responses of CD4.sup.+ T cells obtained from NOD mice (FIG. 12).
Suppression was also observed with CD4.sup.+CD25.sup.+ T cells
purified from spleens of both treated and untreated-diabetic NOD
mice (FIG. 12), which indicates that CD4.sup.+CD25.sup.+ Tr cells
are also present in spleens of diabetic NOD mice, but at much lower
frequencies (shown in FIG. 11). Interestingly, strong suppression
was observed with CD4.sup.+CD25.sup.+ T cells isolated from PLN and
IIC of both rapamycin or rapamycin+IL-10 treated mice. In contrast,
CD4.sup.+CD25.sup.+ T cells isolated from untreated-diabetic NOD
mice did not have any measurable suppressive activity (FIG. 12).
These data indicate that pancreatic tissue of diabetic NOD mostly
contain activated Teff cells rather than Tr cells, whereas PLN and
IIC of treated mice contain predominantly Tr cells among the
CD4.sup.+CD25.sup.+ subset.
[0087] Overall, these data show that the steady-state tolerance
observed following rapamycin+IL-10 treatment is associated with
accumulation of Tr1 cells in the spleen and of CD4.sup.+CD25.sup.+
Tr cells in the lymph nodes and pancreas.
Materials and Methods
[0088] Mice. Balb/c, C57BL/6, NOD/Lt, and NOD.SCID female mice were
purchased from Charles River Laboratories (Calco, Italy). All mice
were kept under specific pathogen free conditions. Glucose level in
the tail venous blood was quantified using Glucometer Elite system
(Bayer, Wuppertal, Germany). Diabetes was induced in Balb/c mice by
intravenous injection of streptozotocin (Sigma, St. Louis, Mo.) at
170 mg/kg. A diagnosis of diabetes was made after two sequential
glucose measurements higher than 250 mg/dl.
[0089] Islet transplant. Hand picked C57BL/6 pancreatic islets were
transplanted (300 islets/mouse), after overnight cultures at
37.degree. C., under the kidney capsule of Balb/c diabetic mice as
previously described (Davalli et al. 1996).
[0090] Treatment of transplanted mice. Treatment of transplanted
Balb/c mice began the day after transplant and lasted for 30 days.
Rapamycin (Rapamune, Wyeth-Ayerst Research, Pearl River, N.Y.) was
diluted in peanut oil (Sigma) and administered once daily at a dose
of 1 mg/kg by gavage. Human IL-10 (BD Biosciences, Mountain View,
Calif.) was diluted in PBS and administered twice a day at a dose
of 0.05 mg/Kg IP. FK506 (Prograf, Fujisawa, Milano) was diluted in
saline solution and administered once daily at a dose of 0.3 mg/kg
IP. AntiIL-2 R.alpha. chain mAb (antiTac) (clone 7D4, BD) was
diluted in saline solution and administered IP at time 0 and 4 days
post transplant to reach a final dose of 1 mg/mouse. Diabetes
incidence was monitored by blood glucose levels.
[0091] Diabetes inhibition study. Female NOD mice were treated from
age 11 weeks to 31 weeks of age with rapamycin, rapamycin+IL10, or
IL-10 alone at the same doses used in transplanted mice. Diabetes
incidence was monitored by blood glucose levels.
[0092] Diabetes transfer study. Splenocytes from diabetic NOD
female mice were collected and injected IV in NOD.SCID at a dose of
5.times.10.sup.6 per mouse. Recipient mice were untreated or
treated with rapamycin+antiTac+FK506, or rapamycin+antiTac+IL-10,
or rapamycin+antiTac for 40 days after transfer at the same doses
used in transplanted mice. Diabetes incidence was monitored by
blood glucose levels.
[0093] Adoptive cell transfer in NOD.SCID mice. Spleens from
control and treated NOD mice were collected after stopping the
treatment. Five millions total splenocytes were adoptively
transferred by IV injection into NOD.SCID mice. Diabetes
development was monitored by glucose levels.
[0094] In vivo immunisation. Ovalbumin (OVA) peptide 323-339
(Primm, Milano, Italy) emulsified in CFA (Difco, Detroit, Mich.)
was injected at a dose of 100 .mu.g/mouse once S.C. in the hind
footpads of transplanted Balb/c mice. Draining lymph nodes were
collected and used in the in vitro assays.
[0095] Cell sorting. The cells infiltrating the pancreas were
isolated as described (Gregori et al., 2003). The obtained cell
population was incubated with antiCD90 mAb-coated microbeads and
applied onto MiniMacs columns (Miltenyi Biotec, Bergisch Gladbach,
Germany) to obtain purified T cells. CD4.sup.+CD25.sup.+ T cells
were sorted with a Multisort kit (Miltenyi) (average
purity.gtoreq.75%). In some experiment CD4.sup.+CD25.sup.+ T cells
were sorted aseptically on a FACStar cell sorter (BD) (average
purity=99%). IL-10 producing cells were sorted with the murine
IL-10 secretion assay enrichment and detection kit (Miltenyi)
(average purity.gtoreq.40%).
[0096] Enrichment of IL-10 positive cells. IL-10 producing cells
were enriched by means of a commercially available kit (Miltenyi).
Purified T cells were cultured at a concentration of 10.sup.6/ml in
the presence of immobilised antiCD3 and soluble antiCD28. After 3
hours of culture, cells were harvested and labeled for 10 minutes
at 4.degree. C. with a diabody consisting in a mAb directed against
CD45 and another mAb capturing murine IL-10. The cells were then
diluted at a final concentration of 10.sup.5/ml and allowed to
secrete cytokines for 45 minutes at 37.degree. C. After the
cytokine capture period, cells were harvested, resuspended
10.sup.8/ml in PBS containing 0.5% BSA and 5 nM EDTA (buffer) and
stained for 10 min. at 4.degree. C. with PE-conjugated .alpha.Il10
mAb (BD). Cells were wvashed in buffer once, resuspended
10.sup.8/ml and stained with anti-PE microbeads for 10 min at
4.degree. C. IL-10 enriched cell population was isolated on
magnetic columns. Cell samples were analysed on a FACScalibur flow
cytometry (BD).
[0097] Cell cultures. For suppression experiments, naive CD4.sup.+
NOD T cells were stained with CFSE (Molecular Probes, Eugene.
Oreg.) as described elsewhere (Lyons et al. 1994) and cultured in
96 well plates (1.times.10.sup.5/well) in the presence of 10
.mu.g/ml antiCD3 mAb (BD). CD4.sup.+ T cells obtained from NOD mice
treated for 20 weeks with rapamycin, or rapamycin+IL-10 were added
in 1:1 ratio to the culture and percentage of divided naive cells
was evaluated and compared to percentage of divided cells in the
absence of any added cells. The divided cells were evaluated by
dividing the events contained in the proliferating population by
the total events CFSE.sup.+. For measurement of cytokines released
in the media, purified T cells (1.times.10.sup.5/well) were
cultured in 96 well plates stimulated with 10 .mu.g/ml immobilized
antiCD3 (BD) and 1 .mu.g/ml soluble antiCD28 (BD). Supernatants
were collected after 48 (for IL-5 detection), and 96 hours (for
IL-10 and TGF-.beta. detection) of culture.
[0098] Flow cytometry. Cells were stained with the indicated Abs
(all from BD), and were analyzed with a FACScan flow cytometer
equipped with CellQuest software (BD).
[0099] Cytokine measurement. Cytokines present in the collected
supernatants were quantified by sandwich ELISA or flow cytometry
based assay (CBA), using standard commercially available kits (BD).
The percentage of cells producing specific cytokines was measured
by intracellular staining. Purified T cells were stimulated for 6
hours with 10 .mu.g/ml immobilised antiCD3 and 1 .mu.g/ml soluble
antiCD28 (BD) at a concentration of 1.times.10.sup.6/ml. Brefeldin
A was added for the final 3 hours of culture. Intracellular
staining was performed as previously described (Trembleau et al.
2000).
REFERENCES
[0100] Bacchetta, R., M. Bigler, et al. (1994). "High levels of
interleukin 10 production in vivo are associated with tolerance in
SCID patients transplanted with HLA mismatched hematopoietic stem
cells. Growth and expansion of human T regulatory type 1 cells are
independent from TCR activation but require exogenous cytokines."
Journal of Experimental Medicine 179(2): 493-502. [0101] Bacchetta
R., Parkman R., et al. (1995) "Dysfunctional cytokine production by
host-reactive T-cell clones isolated from a chimeric severe
combined immunodeficiency patient transplanted with haploidentical
bone marrow." Blood 85: 1944-1953. [0102] Baker, K. S., M. G.
Roncarolo, et al. (1999). "High spontaneous IL-10 production in
unrelated bone marrow transplant recipients is associated with
fewer transplant-related complications and early deaths." Bone
Marrow Transplant 23(11): 1123-9. [0103] Battaglia, M., B. R.
Blazar, et al. (2002). "The puzzling world of murine T regulatory
cells." Microbes Infect 4(5): 559-66. [0104] Blaha, P. Bigenzahn,
S., et al. (2003). "The influence of immunosuppressive drugs on
tolerance induction through bone marrow transplantation with
costimulation blockade." Blood 101: 2886-2893. [0105] Blazar, B.
R., P. A. Taylor, et al. (1998). "Interleukin-10 dose-dependent
regulation of CD4+ and CD8+ T cell-mediated graft-versus-host
disease." Transplantation 66(9): 1220-9. [0106] Davalli, A. M., L.
Scaglia, et al. (1996). "Vulnerability of islets in the immediate
posttransplantation period. Dynamic changes in structure and
function." Diabetes 45(9): 1161-7. [0107] Delovitch, T. L., B.
Singh. (1997) "The nonobese diabetic mouse as a model of autoimmune
diabetes: immune dysregulation gets the NOD" Immunity 6 (7):
727-738. [0108] Gregori S., Giarratana N., et at. (2003) "Dynamics
of pathogenic and suppressor T cells in development of autoimmune
diabetes" J. Immunol 171: 4040-4047. [0109] Groux, H., A. O'Garra,
et al. (1997). "A CD4+ T-cell subset inhibits antigen-specific
T-cell responses and prevents colitis." Nature 389(6652): 737-42.
Hackstein, H., T. Taner, et al. (2002). "Rapamycin inhibits
macropinocytosis and mannose receptor-mediated endocytosis by bone
marrow-derived dendritic cells." Blood 100(3): 1084-7. [0110]
Hempel, L., D. Korholz, et al. (1997). "High interleukin-10 serum
levels are associated with fatal outcome in patients after bone
marrow transplantation." Bone Marrow Transplant 20(5): 365-8.
[0111] Hojo, M., T. Morimoto, et al. (1999). "Cyclosporine induces
cancer progression by a cell-autonomous mechanism." Nature
397(6719): 530-4. [0112] Li, W., F. Fu, et al. (1999). "Recipient
pretreatment with mammalian IL-10 prolongs mouse cardiac allograft
survival by inhibition of anti-donor T cell responses." Transplant
Proc 31(1-2): 115. [0113] Lyons, A. B. Parish, C. R. (1994)
"Determination of lymphocyte division by flow cytometry" J Immunol
Methods 171:131-137. [0114] Moore, K. W., R. de Waal Malefyt, et
al. (2001). "Interleukin-10 and the interleukin-10 receptor." Annu
Rev Immunol 19: 683-765. [0115] Roncarolo M. G., Battaglia M., et
al. (2003) "The role of interleukin 10 in the control of
autoimmunity" J. Autoimm 4: 269-272 [0116] Sehgal, S. N. (1998).
"Rapamune (RAPA, Rapamycin, sirolimnus): mechanism of action
immunosuppressive effect results from blockade of signal
transduction and inhibition of cell cycle progression." Clin
Biochem 31(5): 335-40. [0117] Shapiro, A. M., J. R. Lakey, et al.
(2000). "Islet transplantation in seven patients with type 1
diabetes mellitus using a glucocorticoid-free immunosuppressive
regimen." N Engl J Med 343(4): 230-8. [0118] Taylor, P. A., C. J.
Lees, et al. (2002). "The infusion of ex vivo activated and
expanded CD4(+)CD25(+) immune regulatory cells inhibits
graft-versus-host disease lethality." Blood 99(10): 3493-9. [0119]
Tisch, R. and H. McDevitt (1996). "Insulin-dependent
diabetes-mellitus." Cell 85(3): 291-7. [0120] Trembleau, S., G.
Penna, et al. (2000). "Early Th1 response in unprimed nonobese
diabetic mice to the tyrosine phosphatase-like
insulinoma-associated protein 2, an autoantigen in type 1
diabetes." J Immunol 165(12): 6748-55. [0121] Wells, A. D., X. C.
Li, et al. (1999). "Requirement for T-cell apoptosis in the
induction of peripheral transplantation tolerance." Nat Med 5(11):
1303-7. [0122] Yu, X., P. Carpenter, et al. (2001). "Advances in
transplantation tolerance." Lancet 357(9272): 1959-63.
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