difuhgdlkhjl

MODELLING COMPLEX BIOLOGICAL SYSTEMS IN THE CONTEXT OF GENOMICS
Evry, May 21-25, 2012

On the cellular and supracellular networks
controlling
regulatory T cells and autoimmunity

Jorge Carneiro
Instituto Gulbenkian de Ciência, Portugal
http://qobweb.igc.gulbenkian.pt

Quantitative Organism Biology

Cells of multicellular organism cooperate to ensure body
development and maintenance. They do this in a collective
distributed manner, without a global plan.

How cells collectively generate organism’s properties?

General principles of biological organisation

The design and control of artiﬁcial systems

http://www.embl-heidelberg.de/digitalembryo/

How cells get along without a GPS ?

How cells get along without a GPS ?

global information (system)

autoimmune disease and immunopathology,

2
2

2
2
B Foxp3 represents a more specific marker than
5RBhi alone +Foxp3/MIGR1 +MIGR1 None CD25-CD45RBhi alone cell-surface molecules (such as
currently used +Foxp3/MIGR1 +MIGR1 None
CD25, CD45RB, CTLA-4, and GITR), which

Multiple are unable to completely discriminate between
regulatory T cells and activated, effector, or
memory T cells.
scales
Colon
Mutations in the Foxp3 gene culminate in
the development of a fatal lymphoprolifera-
tive disorder associated with multiorgan pa-
thology both in mice and humans (12–20).
FOXP3 is predominantly expressed in human
CD25ϩCD4ϩ T cells as well (32). Further-
Stomach more, transduction of a mutant Foxp3 lacking
the forkhead domain, similar to the mutated
Foxp3 in scurfy mice (17), failed to confer
suppressive activity to CD25–CD4ϩ T cells
Fig. 4. Prevention(fig.IBD and autoimmune gastritis by Foxp3-transduced T cells. (A) C.B-17 scid
IBD and autoimmune gastritis by Foxp3-transduced T cells. (A) C.B-17 scid of S7). The present results therefore sug-
105 fresh CD25–CD45RBhighCD4ϩ cells either alone (n ϭ 6, where n is the 4 ϫ 10that mutations of thehighCD4ϩgene may alone (n ϭ 6, where n is the
mice received gest 5 fresh CD25–CD45RB Foxp3 cells either
derived of mice)cause squares)disorders through1.2develop- GFPϩ sorted cells derived from
pen squares) or together with 1.2 ϫ 106 GFPϩ sorted cells number from (open these or together with ϫ 106
D4ϩ cells infected with Foxp3/MIGR1 (n ϭ 7) (closed circles) or MIGR1 –(n ϭ 5) highmentalcells infected with Foxp3/MIGR1 (n ϭthe (closed circles) or MIGR1 (n ϭ 5)
CD25 CD45RB CD4ϩ or functional abnormality of 7)
weight is represented as the percentage of initial weight (mean Ϯ SD). Body weight is represented as the percentage of initial weight (mean Ϯ SD)
(open circles). CD25ϩCD4ϩ T population.
R
gniﬁcant difference, P Ͻ 0.01, Foxp3/MIGR1 versus other two groups indicate signiﬁcant difference, P of T cells by Foxp3 versus other two groups by
Astericks by Potentially, generation Ͻ 0.01, Foxp3/MIGR1
R
(B) Histopathology of the colon and stomach in each group and in an test. (B) Histopathology of the colon and stomach in each group and in an
Mann-Whitney
transduction of naıve T cells may provide a
¨
D mouse (None). (C) Colitis (left) and gastritis (right) were unreconstituted SCID mouse (None). (C) Colitis (left) and gastritis (right) were histologically
histologically
the group cotransferred with MIGR1-infected cells and one transferredTwo micepreviously unstudied therapeutic mode for cells and one transferred with
scored. with in the group cotransferred with MIGR1-infected
ϩ CD25 CD45RB treatment of autoimmunedebilitation before histological examination. Results
–
D4 cells alone died of debilitation before histological examination. Results high
CD4ϩ cells alone died of and inflammatory
are from a total of three independent experiments. shown in (A) to (C) are from a total of three independent experiments.
diseases and in transplantation tolerance.

1060
14 FEBRUARY 2003 VOL 299 SCIENCE www.sciencemag.org 14 FEBRUARY 2003 VOL 299 SCIENCE www.scien


2
2

2
2

memory T cells.
scales
Colon

Astericks by
R Cell population dynamics
Potentially, generation Ͻ 0.01, Foxp3/MIGR1
(ODE)
R
Mann-Whitney
¨
histologically
–

1060


2
2

2
2

memory T cells.
scales
Colon

Astericks by
R Cell population dynamics
Potentially, generation Ͻ 0.01, Foxp3/MIGR1
(ODE)
R
Mann-Whitney
¨
histologically
–

1060

Gene regulatory networks
(Logical network dynamics)

overview
Tolerance, autoimmunity, and regulatory T cells
Regulatory T cells 101

How regulatory T cells mediate tolerance ?
Modelling T cell population dynamics
Ordinary differential equations ··························· CRM

What makes a T cell be a regulatory T cell ?
Modelling gene regulatory networks and T cell differentiation
Logical network formalism ······················ Th cell plasticity

If regulatory T cells are plastic how can tolerance be robust ?
Multiscale modelling of cellular and supracellular networks
Agent-based stochastic simulations

What does the immune system do in the vertebrate organism?

Fighting infection

Assimilating
intestinal flora

Fighting infection

Assimilating
intestinal flora

Rejecting cancer cells

Fighting infection

Assimilating
intestinal flora

House keeping


Homeostasis
Fighting infection and Regulation

Assimilating
intestinal flora

House keeping


Homeostasis
Fighting infection and Regulation

Assimilating
intestinal flora

House keeping


http://www.sciencemuseum.org.uk/exhibitions/lifecycle/116.asp http://ww.grc.org/Graphics/ programs/2003/cells3.jpg
http://www.leukemia-web.org/images/cells.jpg

Failure of homeostasis and regulation
unleashes pathologic autoimmunity

Failure of homeostasis and regulation
unleashes pathologic autoimmunity
Rheumatoid arthritis

Type I diabetes

Multiple sclerosis

IPEX sindrome

Cortesy: Magda Carneiro-Sampaio, São Paulo

From: Jean-François Bach, New England J Med

“In general, the management of human systemic
autoimmune disease is empirical and unsatisfactory. For the
most part, broadly immunosuppressive drugs, such as
corticosteroids, are used in a wide variety of severe
autoimmune and inﬂammatory disorders (…)”

– Philipe Cohen In: Fundamental Immunology (Ed. W. Paul)

Clonal selection theory
Stem Cell

Jerne, 1953
Burnet, 1957 G.O.D.
Ag A

1 2 3 4 ... 111 112 ... 623 ... 1245 ... n

Ag B

1 2 3 4 ... 111 112 ... 623 ... 1245 ... n

111 111 1245 1245

111 111 111 111 1245 1245 1245 1245

CTL
CTL
CTL
CTL CTL CTL
TH CTL

Antigen TH
TH
TH
TH TH TH
TH

B
B
B B B
B
B
TH

CTL
CTL
CTL
CTL CTL CTL
TH CTL

Antigen TH
TH

TH
“Self” tolerance by deletion
TH
TH
TH
TH

B
B
B B B
B
B
TH

Reductionist molecular biology approach to Immunology

TOLERANCE = absence of clonal expansion = CLONAL DELETION

Therapy of autoimmune diseases should aim at
“deleting” autoreactive cells or clones

In the absence of clonal-speciﬁc therapies: kill them all !!!

Tolerance is mediated by regulatory T cells
Mason-Sakaguchi

WT

Mason-Sakaguchi

CD25- CD4+ T cells
E E E
T E T T
T T

(Effector T cells, TE, E)

WT

Mason-Sakaguchi
“empty” rag-/-

CD25- CD4+ T cells
E E E
T E T T
T T


WT

Mason-Sakaguchi
“empty” rag-/-
AID
CD25- CD4+ T cells
E E E
T E T T
T T


WT

Mason-Sakaguchi
“empty” rag-/-
AID
CD25- CD4+ T cells
E E E
T E T T
T T


WT
CD25+CD4+T cells
R
R
R R R
R R
R

(Regulatory T cells, TR, R)

Mason-Sakaguchi
“empty” rag-/-
AID
CD25- CD4+ T cells
E E E
T E T T
T T


WT
CD25+CD4+T cells

R R
R
R
R
Healthy
R R
R


Mason-Sakaguchi
“empty” rag-/-
AID
CD25- CD4+ T cells
E E E
T E T T
T T
AID
(Effector T cells, TE, E) or
Healthy
WT
CD25+CD4+T cells

R R
R
R
R
Healthy
R R
R



CTL
CTL
CTL CTL
CTL CTL
TH CTL

Self
Antigen TH
TH
TH
TH TH
TH
TH
TH

B
B B B
B
B
TH B

TOLERANCE = absence of clonal expansion = control by regulatory T cells

Therapy of autoimmune diseases should aim at
stimulating autoreactive regulatory T cells or clones

… BUT …

there are many open questions

CD25+CD4+T cells
R
R
R R R
R R
R


What makes a T cell
be a regulatory T cell ?

CD25+CD4+T cells
R
R
R R R
R R
R


What makes a T cell

CD25+CD4+T cells
R
R

How do they interact
R R R
R R
R

with other cells ? (Regulatory T cells, TR, R)

What makes a T cell

CD25+CD4+T cells
R
R

R R R
R R
R


How do they prevent
autoimmune diseases ?

What makes a T cell

CD25+CD4+T cells
R
R

R R R
R R
R

How do they allow
efﬁcient immune responses ?
How do they prevent

What makes a T cell

How many and how
CD25+CD4+T cells diverse are they ?
R
R

R R R
R R
R

How do they allow
How do they prevent

How does their repertoire
What makes a T cell compare to that of other T
be a regulatory T cell ? cells ?

How many and how
CD25+CD4+T cells diverse are they ?
R
R

R R R
R R
R

How do they allow
How do they prevent

Balance between regulatory and effector T cell subpopulations

Is it that simple ?

“Everything should be made as simple as possible,
But not simpler.”

Albert Einstein, XX century

http://www.medscape.com/content/2004/00/46/84/468446/468446_ﬁg.html

http://www.sewingmachines.us/sewing-machine-636.jpg


http://www.sewingmachines.us/sewing-machine-636.jpg

http://www.roselabiche.com/blog/index.php?2007/11

http://web.mit.edu/2.972/www/reports/sewing_machine/a-sewing_machine.gif

J. theor. Biol. (2000) 207, 231}254
doi:10.1006/jtbi.2000.2169, available online at http://www.idealibrary.com on

Modelling T-cell-Mediated Suppression Dependent on Interactions
in Multicellular Conjugates
KALET LEON*-?, ROLANDO PEREZ*, AGUSTIN LAGE*
D D AND JORGE CARNEIRO-

MODELLING T LYMPHOCYTE LINKED SUPPRESSION 249
*Centro de ImmunolognH a Molecular, P.O. Box 16040, Habana 11600, Cuba and
-Instituto Gulbenkian de Ciencia, Apartado 14, 2781-901, Oeiras, Portugal
ore than two cell types with the alternative mechanisms of linked sup-
mechanisms can be speci- (Received on 7 February 2000, Accepted in revised form onreported 2000)
pression. Overall, the modelling results 10 August
setting of the interaction here and the whole set of observations that we
the consequences of each discussed would strongly favour two candidate
Tolerance to peripheral body antigens involves multiple mechanisms, namely T-cell-mediated
able 1). The application of
suppression of potentially autoimmuneare Recent in vivo and in vitro evidence indicates that
mechanisms. These cells. the ones that are trans-
regulatory T cells suppress the response of e!ector T cells by a mechanism that requires the
tions other than the cur- lated by the "nal model: regulatory T cells inhibit
simultaneous conjugation of regulatory and e!ector T cells with the same antigen-presenting
minant tolerance can be (APC). Despite this strong requirement, it is not yet clearwhile theywhile both cells are
cell the proliferation of e!ector cells what happens are
conjugated. Several hypotheses are discussed in the literature. Suppression may result from
For example, it was competition of regulatory and e!ectoracells for activation resources on the APC;
simple nevertheless dependent on growth factor that the
the interaction between regulatory T cellsproduce; an inhibitory signal tocells inhibit the same conjugate; or
latter may deliver or regulatory e!ector T cells in the ex-
e!ector T cells may acquire the regulatory phenotype during their interaction with regulatory
Ls does not involve thecells. The present article tries to further our understanding of T-cell-mediated suppression,
T pansion of the population of e!ector cells because
they convertnumber of multicellular conjugates of T propose the "rst general
ns between the two cells to narrow-downthe formation of to the regulatoryWe cells and APCs. Using this
and
formalism describing
the
them candidate mechanisms. phenotype.
formalism we derive three particularor both may be operative in of T-cell-
resenting cell, as classi- Either mechanism models, representing alternative mechanisms
mediated suppression. For each model, we make phase plane and bifurcation analysis, and
son & O'Malley, 1987; vivo, maybe even dependent on with the history of
identify their pros and cons in terms of the relationshipthe life large body of experimental
hat the APC may act as observations on T-cell-mediated Modigliani et al.that accounting for the quantitative
the e!ector cells. suppression. We argue (1996b) actually
details of adoptive transfers of tolerance requires models with bistable regimes in which either
nnett et al., 1998; Ridge regulatory cells or e!ectors cells dominate the steady cellsFrom this thymic conclude
demonstrated that regulatory state. from analysis, we
r et al., 1998). The formal- cells actively inhibit mechanism of T-cell-mediated suppression the maintenance of the
that the most plausible
T
epithelium the growth of can both suppressrequires re- regulatory
chimeras e!ector T cells, and that the that


net growth = inﬂux + interaction-dependent — death
growth

dTi
= i + ↵i (T, A, m, k) · Ti · Ti
dt


net growth = inﬂux + interaction-dependent — death
growth

dTi
= i + ↵i (T, A, m, k) · Ti · Ti
dt

How to choose appropriate functional forms
for the interaction terms ?


A taxonomy of putative mechanisms of cell-to-cell interaction
R

T
IL-X
R

R

[Leon et al. J Theor Biol 2000; Carneiro et al. Immunol Rev 2007]


A taxonomy of putative mechanisms of cell-to-cell interaction
R

T
IL-X
R

R

CRM


Modelling T cell population dynamics: the crossregulation model

Interactive simulation

Quantitative assay for cell proliferation

+
CFSE

Nº Cells

Log FL1-H
Log Intensity of CFSE staining

[Leon et al. J.Theor.Biol. 2004]

Putting the CRM to the test

+ anti-CD3 antibody
APCs

Cell Cycle
R R R R E
R R R R
R R
R R

B6 Thy1.2 Analysis
CD25+CD4+Thy1.2+

3 day culture
E E E E
E E E E
E E E E E E
E E E E

B6 congenic Thy1.1
CD25-CD4+Thy1.1+

[Carneiro et al. Immuno. Rev. 2007]


+ anti-CD3 antibody
APCs Proportions Effector Cells Regulatory Cells
Thy1.1:Thy1.2
Thy1.1 Thy1.2
Cell Cycle
R R R E
R R R
R R
Day 0 Day 3
R R
R R

Analysis
CD25+CD4+Thy1.2+

? ?
3 day culture
E
E E
E E
E
E

E
E
+ + anti-CD3100:0 N.D.
E
E E
E E
E
antibody
E

E
E

ND
y1.1
CD25-CD4+Thy1.1+

80:20 47:53
Cell Cycle
R R E
R R
R
R

Analysis
.2+
+ anti-CD3 antibody
50:50 24:76
APCs 3 day culture
E E
E E
E E E

+
E

+ anti-CD30:100 N.D. Cycle
antibody
E
Cell
R R R R E
R R R R
R R
R R ND
Analysis
1.1+
CD25+CD4+Thy1.2+

3 day culture Log FL1-H / CFSE intensity
E E
E E E E E E
E E E E E E

Cell Cycle
R
E
R
E
E E E

R R
R
R
CD25-CD4+Thy1.1+
Analysis et al. Immuno. Rev. 2007]
[Carneiro
.2+


+ anti-CD3 antibody
Thy1.1:Thy1.2
Thy1.1 Thy1.2
Cell Cycle
R R R E
R R R
R R
Day 0 Day 3
R R
R R

Analysis
CD25+CD4+Thy1.2+

?
3 day culture
E
E E
E E
E
E

E
E
+ + anti-CD3100:0 N.D.
E
E E
E E
E
antibody
E

E
E

ND
y1.1
CD25-CD4+Thy1.1+

80:20 47:53
Cell Cycle
R R E
R R
R
R

Analysis
.2+
+ anti-CD3 antibody
50:50 24:76
APCs 3 day culture
E E
E E
E E E

+
E

antibody
E
Cell
R R R R E
R R R R
R R
R R ND
Analysis
1.1+
CD25+CD4+Thy1.2+

E E
E E E E E E
E E E E E E

Cell Cycle
R
E
R
E
E E E

R R
R
R
CD25-CD4+Thy1.1+
[Carneiro
.2+


+ anti-CD3 antibody
Thy1.1:Thy1.2
Thy1.1 Thy1.2
Cell Cycle
R R R E
R R R
R R
Day 0 Day 3
R R
R R

Analysis
CD25+CD4+Thy1.2+

3 day culture
E
E E
E E
E
E

E
E
+ + anti-CD3100:0 N.D.
E
E E
E E
E
antibody
E

E
E

ND
y1.1
CD25-CD4+Thy1.1+

80:20 47:53
Cell Cycle
R R E
R R
R
R

Analysis
.2+
+ anti-CD3 antibody
50:50 24:76
APCs 3 day culture
E E
E E
E E E

+
E

antibody
E
Cell
R R R R E
R R R R
R R
R R ND
Analysis
1.1+
CD25+CD4+Thy1.2+

E E
E E E E E E
E E E E E E

Cell Cycle
R
E
R
E
E E E

R R
R
R
CD25-CD4+Thy1.1+
[Carneiro
.2+


+ anti-CD3 antibody
Thy1.1:Thy1.2
Thy1.1 Thy1.2
Cell Cycle
R R R E
R R R
R R
Day 0 Day 3
R R
R R

Analysis
CD25+CD4+Thy1.2+

3 day culture
E
E E
E E
E
E

E
E
+ + anti-CD3100:0 N.D.
E
E E
E E
E
antibody
E

E
E

ND
y1.1
CD25-CD4+Thy1.1+

80:20 47:53
Cell Cycle
R R E
R R
R
R

Analysis
.2+
+ anti-CD3 antibody
50:50 24:76

? ?
APCs 3 day culture
E E
E E
E E E

+
E

+ anti-CD30:100 N.D.
antibody
E
Cell Cycle
R R R R E
R R R R
R R
R R ND
Analysis
1.1+
CD25+CD4+Thy1.2+

E E
E E E E E E
E E E E E E

Cell Cycle
R
E
R
E
E E E

R R
R
R
CD25-CD4+Thy1.1+
[Carneiro
.2+


+ anti-CD3 antibody
Thy1.1:Thy1.2
Thy1.1 Thy1.2
Cell Cycle
R R R E
R R R
R R
Day 0 Day 3
R R
R R

Analysis
CD25+CD4+Thy1.2+

+ anti-CD3 antibody
3 day culture
APCsE EE EE
E
E

E
E
+ + anti-CD3100:0 N.D.
antibody E
E E
E E
E
E

E
E

ND
y1.1
CD25-CD4+Thy1.1+
Cell Cycle
R R R E
R R R
R R

+ anti-CD3 antibody

? ?
R R
R R

Analysis
CD25+CD4+Thy1.2+
APCs 80:20 47:53
Cell Cycle
R R E 3 day culture
R R

+ + anti-CD3 antibody
R

? ?
+
E E
E E R E E E E

Analysis Cell Cycle
E E E E E E R E
E E RE R E
R
R
R
R R
R R
R R

.2+
CD25-CD4+Thy1.1+
Thy1.2 Analysis
CD25+CD4+Thy1.2+ 50:50 24:76
3 day culture 3 day culture

Cell Cycle
R R EE E E E
E
E E E
E E
R E ER E E E E E E
R E E
E E E E E
R E E

Analysis
0:100 N.D.
ongenic Thy1.1
ND
CD25-CD4+Thy1.1+
+
1.1+
E E
E E
E E E
E E

+


Proportions Effector Cells Regulatory Cells
Thy1.1:Thy1.2
Thy1.1 Thy1.2
Day 0 Day 3

100:0 N.D. ND

80:20 47:53

50:50 24:76

0:100 N.D. ND

Log FL1-H / CFSE intensity



R

T
IL-X
R

R

CRM



Regulatory T cell populations grow as a
function of the effector T cells they
suppress R
R

Leon et al. JTB (2000) R

E

E
E

01
. Imm unol 20
iol 2 000 , J
al. J Theor B
eon et Thesis
L alh o PhD
sis , Caram
hD The

the control of the insulin promoter. We had previously observed
Authorship note: Yenkel Grinberg-Bleyer and David Saadoun contributed equally to
this work. Eliane Piaggio and Benoît L. Salomon are co–senior authors.
that HA-specific Tregs (HA-Tregs) preferentially proliferated and
expanded at days 5–7 after transfer in draining pancreatic LNs
Conflict of interest: The authors have declared that no conflict of interest exists. (PLNs) of ins-HA homozygous mice (29). When we repeated the
Citation for this article: J Clin Invest. 2010;120(12):4558–4568. doi:10.1172/JCI42945. experiment in ins-HA hemizygous recipients, which express lower

The Journal of Clinical Investigation http://www.jci.org Volume 120 Number 12 December 2010

Related Commentary, page 4190

Regulatory T cell populations grow as a
function of the effector T cells they
suppress
Yenkel Grinberg-Bleyer,1,2,3 David Saadoun,1,2,3 Audrey Baeyens,1,2,3 Fabienne Billiard,1,2,3
Jérémie D. Goldstein,1,2,3 Sylvie Grégoire,1,2,3 Gaëlle H. Martin,1,2,3 Rima Elhage,1,2,3
Nicolas Derian,1,2,3 Wassila Carpentier,1,4 Gilles Marodon,1,2,3 David Klatzmann,1,2,3
R
R
Eliane Piaggio,1,2,3 and Benoît L. Salomon1,2,3
1Université Pierre et Marie Curie — Univ Paris 06, 2CNRS UMR 7211, 3INSERM U959, Paris, France.
4Plate-forme Post-Génomique P3S, Hôpital Pitié-Salpêtrière, Paris, France.

Leon et al. JTB (2000)
CD4+CD25+Foxp3+ Tregs play a major role in prevention of autoimmune diseases. The suppressive effect of
Tregs on effector T cells (Teffs), the cells that can mediate autoimmunity, has been extensively studied. How-
R

E
ever, the in vivo impact of Teff activation on Tregs during autoimmunity has not been explored. In this study,
we have shown that CD4 + Teff activation strongly boosts the expansion and suppressive activity of Tregs.

This helper function of CD4+ T cells, which we believe to be novel, was observed in the pancreas and draining
lymph nodes in mouse recipients of islet-specific Teffs and Tregs. Its physiological impact was assessed in
autoimmune diabetes. When islet-specific Teffs were transferred alone, they induced diabetes. Paradoxically,
when the same Teffs were cotransferred with islet-specific Tregs, they induced disease protection by boost-
E
ing Treg expansion and suppressive function. RNA microarray analyses suggested that TNF family members
were involved in the Teff-mediated Treg boost. In vivo experiments showed that this Treg boost was partially
E
dependent on TNF but not on IL-2. This feedback regulatory loop between Teffs and Tregs may be critical to
preventing or limiting the development of autoimmune diseases.

bers of mature DCs in inflamed tissues may favor the activation of
01
unol 20
The peripheral T cell repertoire of any individual contains autoreac- autoreactive Tregs (21–23), which would then turn on their sup-
tive T cells specific for a variety of self antigens (1). Their activation pressive activity and exert bystander suppression (24, 25).
Thus, during an autoimmune process, there is a local enrichment 00 , J
. Imm
could induce an autoimmune process, eventually leading to an auto-
Bi ofl 2 0
of both autoreactive Teffs and Tregs. Since heefficacy o Treg-
immune disease. Severe and prolonged inflammation in a tissue may
J T the orbetween acti-
mediated suppression depends onal. equilibrium
eon et the factor that could tip this Thesis
o PhD
lead to the activation of pathological autoreactive T cells by several
L Tregs (26), any
mechanisms (2, 3). At the site of inflammation or in draining LNs, vated Teffs and activated
balance to one side or the other could then determine thes, Ca
ramalh
tissue damage results in an enhanced presentation of autoantigens
hD Th esi outcome

difuhgdlkhjl

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to difuhgdlkhjl

Similar to difuhgdlkhjl (20)

Recently uploaded

Recently uploaded (20)

difuhgdlkhjl

Editor's Notes