Homeostasia e clonalidade

Homeostasia e clonalidade
http://qobweb.igc.gulbenkian.pt/4u/fmul
Homeostasia
Claude Bernard
(1813-1878)
“La constance du milieu intérieur est la condition d’une vie libre"
— Introduction to Experimental Medicine
“The constancy of the internal environment is the condition
that life should be free and independent.... So far from the
higher animal being indifferent to the external world, it is on
the contrary in a precise and informed relation with it, in
such a way that its equilibrium results from a continuous
and delicate compensation, established as by the most
sensitive of balances”
Walter Cannon
(1871-1945)
Homeostasis is the maintenance of equilibrium, or constant conditions, in a
biological system by means of feedback mechanisms that counteract
perturbations tending toward disequilibrium.
— The Wisdom of the Body (1932)
Qual é o paradigma do mecanismo de retroação / feedback negativo ?
Watt’s steam engine
http://visite.artsetmetiers.free.fr/watt.html
“flyball governor”
http://en.wikipedia.org
Homeostasia = feedback negativo
O número de linfócitos é tão constante que sugere um
mecanismo de regulação homeostático
1012 lymphocytes in humans
108 lymphocytes in mice
Clonalidade
Geração de diversidade por recombinação V(D)J
V-segments
rag-1
rag-2
J-segments
C-segment
V(D)J recombination
Transcription
mRNA processing
Translation
Light Chain Protein
Antigen receptor proteins
CDRs
CDRs
CDRs
TCR αβ
(or γδ)
Antibody (IgG)
Geração de diversidade por recombinação V(D)J
“NHEJ repair+TdT” resulta em junção imprecisa
Fluorescence
intensity
Hori et al. / Journal of Immunological Methods 268 (2002) 159–170
CDR3 length (aminoacid residues)
Seleção clonal: expansão e contração de clones
Stem Cell
G.O.D.
Ag A
1
2
3
4
...
111
112
...
623
...
1245
...
n
1
2
3
4
...
111
112
...
623
...
1245
...
n
Ag B
111
111
111
111
111
1245
111
1245
1245
1245
1245
1245
>1015
Figure 1. T-cell counts and percentages of naive T-cell
subsets. (A) Total, naive, and memory CD4" T-cell numbers in counts per microliter of blood. (B) Total, naive, and
memory CD8" T-cell numbers in counts per microliter of
blood. (C) Percentage of naive CD4" and naive CD8"
T cells. Œ represents values after thymectomy; ‚, samples
taken just before thymectomy; and E, healthy controls.
Lines connect longitudinal samples (n ! 7).
Humans
Effect of thymectomy on naive T-cell proliferation levels
Because IL-7 is known to be essential for the survival and
homeostatic proliferation of naive T cells, and because its availability has been shown to be inversely related to the size of the naive
T-cell population,29,30 we hypothesized that increased IL-7 levels in
the first years after thymectomy
the naive T-cell compartment in
the level of IL-7 in plasma from
2.5 years after thymectomy
(P ! .012) than in healthy age
Humans
Proliferação
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~1 % células / dia
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Como se mantêm o número e diversidade dos linfócitos ?
Como se mantêm o número de linfócitos ?
O que fariam para investigar
os mecanismos de manutenção do número de linfócitos ?
... linfócitos T ?
Proliferação
Produção
tímica
ro s T
e
m ito
ú
N fóc
in
l
de
Turnover: Produção + proliferação = morte
Morte
Proliferação
Produção
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l
de
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Morte
y: Berzins et al.
Proc. Natl. Acad. Sci. USA 96 (1999)
ted), and blood by previously established
usly
established
ow Cytometry. For flow cytometric anal-
(PE)-labeled anti-CD25, anti-CD69, and
ed anti-V! 3, 4, 6, 8.1!8.2, 10, 11, and 12;
44; FITC-labeled CD45.2 (Ly5.2); and
Thymicwere
grafting
PC)-labeled anti-CD8
purchased
Biotinylated anti-CD4 and streptavidinwere purchased from Caltag (South San
C Isomer 1 (for intrathymic injection) was
ton Dickinson. Annexin V-Biotin (Pharto detect cells undergoing apoptosis by
pid phosphatidylserine on the cell surface
sions were stained for analysis by
-bottom tubes (3 ! 106 cells per test) by
cribed techniques. Flow cytometry data
using a FACScalibur machine (Becton
yzed by using CELL QUEST software (Bec-
ytometric analanti-CD69, and
10, 11, and 12;
2 (Ly5.2); and
ere purchased
d streptavidinag
(South San
Thymocytes. Details of this technique
hose
described elsewhere
(23). Briefly,
cetized
injection)
was
and the chest was opened (or kidney
case of grafted mice)
to reveal the thymic
V-Biotin
(Pharinjected with approximately 10 #l of 350
BS), which typically resulted in random
gof theapoptosis
by
thymocyte population (70–80% for
The wound was closed with a surgical
the
cell until
surface
e was warmed
fully recovered from
killed by CO asphyxiation "24hr
rere
analysis
raft and
lymphoid organs by
taken for analBlood (2007) 109: 954-960
2
e washed between removal of each organ,
FIG. 1. (A) Thymic grafting causes a significant increase in T cell
pool size proportional to the number of grafted lobes. The T cell pool
size of mice grafted with two, six, or nine thymic lobes was significantly
higher than those of normal mice (two lobes, P $ 0.001; six or nine
lobes, P $ 0.0001; unpaired Student’s t test) and significantly different
from each other (P $ .0001; ANOVA test). (B) The increase in T cell
pool size caused by thymic grafting is consistent with separate regulation of thymic emigrants. Estimations of the extent of the increases
caused by thymic grafting based on the number of grafted lobes were
made by using a modified theory of peripheral T cell homeostasis. This
theory proposes that emigrant T cells survive outside the restrictions
of homeostatic regulation for up to 3 weeks postthymic export. The
resultant estimates closely matched the pool sizes of mice engrafted
with two, six, and nine thymic lobes.
the number of grafted thymic lobes and the resultant increase
Berzins
al. (1999)
Natl.were
Acad.
Sci. U.S.A.
, 9787
in T celletnumbers.
MiceProc.
typically
grafted
for 4–896
weeks
with no difference in pool size detected between mice in this
Proliferação
Produção
tímica
ro s T
e
m ito
ú
N fóc
in
l
de
Turnover: Produção + proliferação = morte
Morte
Proliferação
Produção
tímica
ro s T
e
m ito
ú
N fóc
in
l
de
Turnover: Produção + proliferação = morte
Morte
Proliferação
Produção
tímica
ro s T
e
m ito
ú
N fóc
in
l
de
Turnover: Produção + proliferação = morte
Morte
Homeostasia
Feedback dependente de densidade / número de células
Como demonstrar feedback ?
Quantificação da proliferação por diluição de CFSE
Nº Cells
+
CFSE
Log FL1-H
Log Intensity of CFSE staining
hat enter the lymphoid periphery daily (14).
eferential niche was not revealed when lower
were transferred. Previous thymus graft data
Es
haveata early
preferential
nicheafter
in the
lymphoid
ur data
time points
transfer
may
nfferences
the observation
that
excess
numbers
of
RTEs
in trafficking in addition to differe
(14,
15).
However,
now ishave
clearan
thatincreased
this time
ecause CD8 RTEsit may
thegut
kinetics
oththe
(22). for the disappearance of cells
omeostatic
survival
signals
(24), eliminating
the
that under
normal
homeostatic
conditions,
e an RTE niche. Our studies in which RTEs
ed preferentially into the lymphoid periphery.
nsferred MN T cells directly test the notion of an
o not appear to have a separate niche. It is
vide strong data against such a compartment.
mber
of RTEs
transferred
our experiments
conditions,
RTEs
competein well
with 6MN T
ch
a
niche.
Our
transfers
of
1–2
×
RTEs
lightly faster rate and accumulating to10a higher
∼10%
engraftment
(23)
that is well
below
the
s corroborated
by the
observation
that,
relative
at
lymphoid
(14).
Es enter
expressthe
high
levels of periphery
CD24 (2), adaily
molecule
erential
niche was
not revealed
when
lower
mal
homeostatic
proliferation
(25).
Because
were
transferred.
Previous diversity
thymus graft
data
source
of new repertoire
(1), their
sbe
have
a preferential
niche
especially
important
wheninTthe
cellslymphoid
are unthe
that excess
numbers
RTEs
the observation
lymphoid periphery.
Our
resultsofsuggest
(14, 15).circumstances,
However, it now
is clear
this time
nuating
RTEs
arethat
capable
of
halthe
kinetics
for the disappearance of cells
niche
effectively.
meostatic
survival
signals
(24), eliminating
the
RTE expansion
under
lymphopenic
conditions
RTEs
are niche.
somewhat
givenRTEs
that
an RTE
Our self-reactive,
studies in which
ls undergo
lymphopenia-induced
prosferred
MN Tfaster
cells directly
test the notion of
an
Furthermore,
RTEs
appear
to ahave
the greatest
ide strong data
against
such
compartment.
tage
in chronically
lymphopenic
whichT
onditions,
RTEs compete
well mice,
with MN
oad
gut flora
that to
RTEs
may
ghtlyoffaster
rate(28),
andsuggesting
accumulating
a higher
eactive to antigens
commensal
corroborated
by thefrom
observation
that,bacteria
relative-/nvironment
maylevels
allowof
RTEs
to (2),
overcome
their
s express high
CD24
a molecule
ompete
for IL-7. The
TCR repertoire
RTEs
al homeostatic
proliferation
(25). of
Because
f MN Tofcells
suggesting
that RTEs
may
source
new(5),
repertoire
diversity
(1), their
toreactive
potential
that generally
is masked
by Fig. 6. RTEs out-compete MN T cells in lymphopenic environments. CFSEbe
especially
important
when T cells
are unlabeled naïve CD4 or CD8 RTEs and MN T cells were coinjected into lymespond
less
robustly
upon
stimulation.
he lymphoid periphery. Our results suggest
gest that naïve CD4 T cells from mice ≥6 mo of phopenic recipients. (A) At the indicated times after transfer into irradiated
uating circumstances, RTEs are capable of recipients, the splenic RTE:MN ratio relative to input was calculated for CD4
“lymphoreplete”
linfopenia
induzida
linfopenia
crónica
(TCR βδ )
PNAS (2011) 108, 5366–5371
F
e
T
a
f
*
T
c
0
o
t
m
s
s
1
Proliferação
Produção
tímica
ro s T
e
m ito
ú
N fóc
in
l
de
Turnover: Produção + proliferação = morte
Morte
Proliferação
Produção
tímica
ro s T
e
m ito
ú
N fóc
in
l
de
Turnover: Produção + proliferação = morte
Morte
Proliferação
Produção
tímica
ro s T
e
m ito
ú
N fóc
in
l
de
Turnover: Produção + proliferação = morte
Morte
ells, RTEs express high levels of CD24 (2), a molecule
for optimal homeostatic proliferation (25). Because
the sole source of new repertoire diversity (1), their
on would be especially important when T cells are unnted in the lymphoid periphery. Our results suggest
ese extenuating circumstances, RTEs are capable of
peripheral niche effectively.
ferential RTE expansion under lymphopenic conditions
est that RTEs are somewhat self-reactive, given that
ve T cells undergo faster lymphopenia-induced pro26, 27). Furthermore, RTEs appear to have the greatest
e advantage in chronically lymphopenic mice, which
creased load of gut flora (28), suggesting that RTEs may
trongly reactive to antigens from commensal bacteria
hat this environment may allow RTEs to overcome their
bility to compete for IL-7. The TCR repertoire of RTEs
m that of MN T cells (5), suggesting that RTEs may
reater autoreactive potential that generally is masked by Fig. 6. RTEs out-compete MN T cells in lymphopenic environments. CFSElabeled
naïve
CD4after
or CD8
RTEs and
RTE
levels
seen in our data at early
time
points
transfer
mayMN T cells were coinjected into lymency to respond less robustly
upon
stimulation.
recipients.
(A) At the
to differences
trafficking
in addition
to indicated
differ- times after transfer into irradiated
data suggest that naïve CD4beT attributable
cells from mice
≥6 mo of inphopenic
recipients,
the
splenic
RTE:MN
ratio
relative to input was calculated for CD4
in survival,
because
RTEs may have an increased
ncreased survival relative ences
to naïve
CD4 T cells
fromCD8(Left)
home to of
thethe
gut (22). and CD8 (Right) RTE-MN competitions. Solid bars depict means from
mice, in part because of tendency
reduced to
expression
or four independent experiments analyzing 4–12 mice per time point.
Our
data suggest
underthree
normal
homeostatic conditions,
tic molecule Bcl-2-interacting
mediator
of cellthat
death
Open bars denote RTE:MN ratios from Fig. 1 A and B, analyzing RTE:MN
RTEs
not accepted
preferentially
into
the lymphoid periphery.
30). Our data suggest that
the are
decreased
survival
of transfers
into lymphoreplete recipients. (B) CFSE profiles from CD4 (Left) and
Furthermore,
do notthe
appear
haveRTEs
a separate
It determined
is
CD8to
(Right)
and MN Tniche.
cells were
1 wk after transfer. (Upper)
aused primarily by the young
age of RTEs,they
although
that
the number
of RTEs
transferredCFSE
in our
experiments
Representative
dilution
profiles; numbers (black for RTEs and gray for
(3–5 mo) of our MN T cellsunlikely
also may
contribute
to en6
such athan
niche. MN
OurT transfers
of 1–2
10 RTEs
cells) represent
the×
percentage
of cells falling into the undivided gate.
rvival. The youngest CD4would
RTEsoverwhelm
declined faster
(Lower) The
of undivided
cells. n = 4 per sample, combining
should result
in an
engraftment
(23)mean
thatproportions
is well below
the
s that had been out in the periphery
longer.
In ∼10%
addition,
from twoperiphery
independent
experiments.
(C) At the indicated time points
∼1 × 106 occurred
RTEs that
lymphoid
daily
(14).
ed that this decreased persistence
forenter
both thedata
−/−
after
transfer
into
TCR
βδ
recipients,
the
splenic RTE:MN ratio relative to
Furthermore,
a preferential
was not revealed when lower
CD8 RTEs, and in our preliminary
studies,
MN T cells niche
input was
calculated
for CD4
(Left)
and CD8 (Right) RTE-MN competitions.
of RTEs.
RTEs were transferred.
Previous
thymus
graft
data
ve reduced Bim expressionnumbers
relative to
Solid bars depict means from two independent experiments analyzing six or
suggested
that RTEs
have a preferential niche in the lymphoid
a from lymphoreplete conditions
suggest
that joining
seven mice per time point. Open bars denote the same ratio as in A. (D) CFSE
periphery,
on the task,
observation
that excess numbers of RTEs
of recirculating lymphocytes
may be based
a nontrivial
profiles from CD4 (Left) and CD8 (Right) RTEs and MN T cells were determined
take
up
to
3
wk
to
die
(14,
15).
However,
it now
is clear
this time
transfer.
CFSEthat
dilution
profiles are representative of three recipio the historical view that T cells are full-fledged mem- 1 wk after
is in lineupon
with thymic
the kinetics
forNumbers
the disappearance
cells
ents.
(black for RTEsof
and
gray for MN T cells) represent the perhe peripheral T-cell poolframe
immediately
failing
to receive
homeostatic
eliminating
the
centagesignals
of cells(24),
falling
into the undivided
gate. Error bars represent SEM.
seed the periphery properly,
RTEs
must exit
the thy- survival
*P < 0.05,
< 0.01,inand
***P RTEs
< 0.001, using a paired Student’s t test.
need to across
hypothesize
an RTE niche.
Our**P
studies
which
late in the blood, and extravasate
endothelium
compete
with
cotransferred
MN
T
cells
directly
test
the
notion
of
an
dary lymphoid organs (23). RTEs lost under normal
RTEwith
niche
and provide
strong
ic conditions may include cells
marginal
defects
in data against such a compartment.
perhaps
those that
In lymphopenic
conditions,
compete
well receive
with MNtheT strongest survival signals, inm or other sustaining properties
that affect their
ability RTEs
rate andcells
accumulating
toTCR
a higher
with useful
specificities, are incorporated into
e the lymphoid periphery. cells, dividing at a slightly faster cluding
level.
finding
corroborated
the observation
that,Under
relative
periphery.
lymphopenic conditions, in which
ogether, our results suggest
thatThis
RTEs
enteristhe
lym- thebylymphoid
to MN
T cells,
RTEs express
levels
CD24
(2), a molecule
RTEs
areofmost
needed,
these youngest T cells are able to fill the
iphery in a vulnerable state,
with
a tendency
to be high
necessary
for optimal
homeostatic
proliferation (25). Because
out of the peripheral T-cell
pool. Only
some RTEs,
void effectively.
RTEs are the sole source of new repertoire diversity (1), their
contribution would be especially important when T cells are unw.pnas.org/cgi/doi/10.1073/pnas.1015286108
Houston et al.
derrepresented in the lymphoid periphery. Our results suggest
that in these extenuating circumstances, RTEs are capable of
filling the peripheral niche effectively.
The preferential RTE expansion under lymphopenic conditions
may suggest that RTEs are somewhat self-reactive, given that
autoreactive T cells undergo faster lymphopenia-induced proliferation (26, 27). Furthermore, RTEs appear to have the greatest
competitive advantage in chronically lymphopenic mice, which
have an increased load of gut flora (28), suggesting that RTEs may
be more strongly reactive to antigens from commensal bacteria
(12) and that this environment may allow RTEs to overcome their
reduced ability to compete for IL-7. The TCR repertoire of RTEs
differs from that of MN T cells (5), suggesting that RTEs may
harbor a greater autoreactive potential that generally is masked by Fig. 6. RTEs out-compete MN T cells in lymphopenic environments. CFSElabeled naïve CD4 or CD8 RTEs and MN T cells were coinjected into lymtheir tendency to respond less robustly upon stimulation.
Recent data suggest that naïve CD4 T cells from mice ≥6 mo of phopenic recipients. (A) At the indicated times after transfer into irradiated
recipients, the splenic RTE:MN ratio relative to input was calculated for CD4
age have increased survival relative to naïve CD4 T cells from (Left) and CD8 (Right) RTE-MN competitions. Solid bars depict means from
younger mice, in part because of reduced expression of the three or four independent experiments analyzing 4–12 mice per time point.
proapoptotic molecule Bcl-2-interacting mediator of cell death Open bars denote RTE:MN ratios from Fig. 1 A and B, analyzing RTE:MN
(Bim) (29, 30). Our data suggest that the decreased survival of transfers into lymphoreplete recipients. (B) CFSE profiles from CD4 (Left) and
RTEs is caused primarily by the young age of RTEs, although the CD8 (Right) RTEs and MN T cells were determined 1 wk after transfer. (Upper)
older age (3–5 mo) of our MN T cells also may contribute to en- Representative CFSE dilution profiles; numbers (black for RTEs and gray for
hanced survival. The youngest CD4 RTEs declined faster than MN T cells) represent the percentage of cells falling into the undivided gate.
CD4 RTEs that had been out in the periphery longer. In addition, (Lower) The mean proportions of undivided cells. n = 4 per sample, combining
experiments. (C) At the indicated time points
we observed that this decreased persistence occurred for both data from two independent
after transfer into TCR βδ−/− recipients, the splenic RTE:MN ratio relative to
CD4 and CD8 RTEs, and in our preliminary studies, MN T cells input was calculated for CD4 (Left) and CD8 (Right) RTE-MN competitions.
did not have reduced Bim expression relative to RTEs.
Solid bars depict means from two independent experiments analyzing six or
The data from lymphoreplete conditions suggest that joining seven mice per time point. Open bars denote the same ratio as in A. (D) CFSE
the pool of recirculating lymphocytes may be a nontrivial task, profiles from CD4 (Left) and CD8 (Right) RTEs and MN T cells were determined
contrary to the historical view that T cells are full-fledged mem- 1 wk after transfer. CFSE dilution profiles are representative of three recipibers of the peripheral T-cell pool immediately upon thymic ents. Numbers (black for RTEs and gray for MN T cells) represent the peregress. To seed the periphery properly, RTEs must exit the thy- centage of cells falling into the undivided gate. Error bars represent SEM.
mus, circulate in the blood, and extravasate across endothelium *P < 0.05, **P < 0.01, and ***P < 0.001, using a paired Student’s t test.
into secondary lymphoid organs (23). RTEs lost under normal
Proliferation
T cell density
Fig. 3. RTEs express more CD5 and less GM1 than MN T cells. CD5 and GM1
expression levels were determined on GFP+ CD4 and CD8 single-positive (SP)
TCRβ+ thymocytes and naïve (CD44lo) GFPhi young RTEs, GFPlo older RTEs,
and GFP− MN T cells from RAG2p-GFP Tg mice. Bars represent mean MFI
from three or four mice per group. Error bars represent SEM. **P < 0.01 and
***P < 0.001, using a paired Student‘s t test.
T cells (6.0 ± 0.7 × 106 for CD4 and 5.5 ± 0.5 × 106 for CD8)
compared with euthymic mice (12.7 ± 0.9 × 106 for CD4 and 8.1 ±
0.5 × 106 for CD8). However, at all analysis time points, 70–90%
of transferred RTE and MN T cells were CD44lo/mid in both
thymectomized and euthymic mice, suggesting very little homeostatic proliferation.
To test whether a separate RTE niche could be revealed if
smaller numbers or only the youngest RTEs were transferred, we
sorted GFPhi young RTEs and transferred low numbers (2.5 ×
105) of each cell type. Even under these conditions, RTEs fared
worse than MN T cells in both euthymic and thymectomized
hosts (Fig. 5B).
Under Lymphopenic Conditions, RTEs Out-Compete MN T Cells. Upon
cotransfer of RTEs and MN T cells into mice made acutely
lymphopenic by sublethal irradiation, RTE numbers increased
slightly relative to MN T cells for both CD4 and CD8 T cells
(Fig. 6A). The difference in dynamics in this lymphopenic environment compared with normal homeostatic conditions (open
bars in Fig. 6A) is notable. A lower percentage of RTEs remained undivided at 1 wk posttransfer (Fig. 6B Lower), and
more CD4 RTEs had undergone two or more divisions (Fig. 6B
Upper Left). At 1 wk posttransfer, the bulk of each transferred
cell population had not fully diluted CFSE, suggesting these cells
were undergoing slower, lymphopenia-induced proliferation
WT
IL-7-/-
WT
IL-7-/-
Proliferation
O suplemento de IL-7 aumenta a proliferação de
populações de células policlonais em recipientes
“lymphoreplete”
Nature Immunology (2009) 10, 149 - 157
Persistencia de células T CD4 em animais com deficiência
condicional de MHC classe II
Um suplemento de ligandos MHC-peptido aumenta a
proliferação de populações de células em recipientes
“lymphoreplete”
(ex. animais “germ-free” tem menos linfócitos)
“[Peripheral T cell] homeostasis is maintained by
competition for two limiting factors, IL-7 and MHC-loaded
self peptides”
PNAS (2011) 108, 5366–5371
Naïve
CD4+
CD8+
CD4
CD8
TCR
TCR
α β
CD28
IL-7R
α β
CD28
IL-7R
Survival
Ag-specific
activation
Memory
Survival
Ag-specific
activation
CD4+
CD8+
CD4
CD8
TCR
TCR
α β
IL-7R
α β
IL-15R
IL-7R
Survival
Homeostatic
proliferation
Ag-specific
activation
IL-15R
Survival
Homeostatic
proliferation
Ag-specific
activation
Figure 1. Survival, homeostatic proliferation, and antigen-specific activation of T-cell subsets depend on different signals. For naı̈ve CD41 and
CD81 T cells, antigen (Ag)-specific activation (in blue) of these cells requires strong TCR triggering and co-stimulatory molecules such as CD28.
Conversely, survival (in bright green) of naı̈ve T cells relies on weak stimulation of TCR and IL-7 receptor (IL-7R). Ag-specific secondary stimulation
(in blue) of memory CD41 and CD81 T cells depends on a strong TCR stimulus without engagement of CD28. Survival (in bright green) of memory
CD41 and CD81 T cells is TCR-independent but requires IL-7 signals, while IL-15 might also contribute to memory CD81 T-cell survival. Conversely,
homeostatic proliferation (in red) of memory CD81 T cells depends mainly on IL-15, whereas memory CD41 T cells undergo homeostatic
Eur. J. Immunol. (2009) 39: 2088–2094
Homeostasia não é mais do que competição
por IL-7 e complexos MHC-péptido limitantes ?
“empty” rag-/-
CD25- CD4+ T cells
T
E E E
T
E T
T
T
(Effector T cells, TE, E)
WT
CD25+CD4+T cells
R
R
R
R
R
R
R
R
(Regulatory T cells, TR, R)
The organ distribution of the CD4! cells in all groups of mice
is shown in Table I. The majority of T cells were found in the
spleen, accounting for roughly half of the recovered CD4 T cells.
In the intestine, with the exception of recipients of the 25! RBlow
cumulation and the respective homeostatic equifraction, similar numbers of CD4 T cells were recovered in both
ected T cell populations, at the time of sacrifice
healthy and sick animals in all other groups (Table I). However, it
D4! cells" was scored in
the spleen;
axillary, in#
low
high
2. CD25 , but not CD25 CD45RB , CD4 T cells inhibit the
accumulation
of CD45RB
CD4
cellsobserved
transferreddifferences
into RAG-2° in
hosts.
cannot
be
formally
excluded
thatT the
cell
nteric
lymph
nodes;
as
well
as
blood
and
#
low
#
"
lowintestine
"
5
5
high
25 CD45RB (CD25 ) or CD25 CD45RB (CD25 ) CD4 numbers
T cells (3 !between
10 ) weredifferent
coinjectedCD4
with 3T!cell
10 subsets
CD45RBare CD4
cells
due Tnot
to into
difts described in the previous section. In this series
Annacker et al. J Immunol. (2001)166, 3008-3018
Downloaded from www.
sion of CD4 T cell subsets: CD25!
T cells reach homeostatic equilibrium at low
Downloaded from www.jimmunol.org on October 5, 2011
dence of wasting disease and T cell numbers at equilibrium in alymphoid recipients reconstituted with different CD4 T cell subsets.
T cells of the indicated phenotype (3 " 105) were transferred into syngenic RAG-2° hosts. A, FACS profiles of sorted CD4 populations
The time of sacrifice for each recipient (six to eight recipients per group), either 12–14 wk after transfer or when they lost at least 20%
ght. C, Body weight of the recipients at the time of sacrifice. Sick animals were defined by weight loss below their initial weight, which
diarrhea and a markedly enlarged colon. D, Sum of the number of CD4! cells in spleen; axillary, inguinal, and mesenteric lymph nodes;
ml of blood per animal); and intestine. The background of noninjected control RAG-2° mice was 1.3 " 105 CD4! cells (n % 4). The
m three or four independent experiments per group.
om www.jimmunol.org on October 5, 2011
REGULATORY T CELLS IN CD4 T CELL HOMEOSTASIS
Células reguladoras Foxp3+ enriquecidas no pool de
células CD25+ controlam a expansão e níveis das restantes
células CD4
Além da competição a “ecologia” linfocitária inclui um
feedback do tipo predador-presa
Carneiro et al. (2007) When three is not a crowd: A crossregulation model of the dynamics and repertoire selection of regulatory CD4 T cells.
Immunol. Reviews 216:48-68.
Como se controla a diversidade ?
Se a população se mantém por competição como se
mantêm a diversidade ?
“nichos” e diversidade
a Intraclonal competition
b Interclonal dominance through promiscuity
REVIEWS
c Interclonal dominance through fitness
APC
MHC
Peptide A
Peptide B
T cell
Cell
division
Cytokine
receptor
Apoptotic
cell
Figure 1 | Fighting for a niche. Models for the ability of naive T cells to compete with each other for ‘space’.a | In this
model the competition is strictly intraclonal between the two groups of T cells specific for different self-peptide–MHC
| Immunology
complexes and results in maintenance of these two pools independently. There are too manyNature
T cells Reviews
of one specificity
(green T cells) for the available niche (defined here by the availability of self-peptide–MHC complexes) and so a T cell
dies. By contrast, there are fewer T cells of a different specificity (yellow T cells) for the available niche, and hence
another naive T cell of this specificity can be accommodated from recent thymic emigrants (not shown) or by limited cell
division of the resident naive T cells. b | In this model, the promiscuity of the T cell receptor (TCR) of one population of
naive T cells allows them to compete not only for their ‘own’ self-peptide–MHC complexes but also for the
self-peptide–MHC complexes that support the T cells of a different specificity, effectively limiting the T cell space
available for the T cells with different specificity.c | In this model, a mechanism of interclonal competition is active, in
which each T cell interacts with its own unique set of self-peptide–MHC complexes but the consequences for this
interaction differ, allowing the T cells of one specificity to more effectively compete for a different limited survival factor
(shown here as a secreted factor, such as a cytokine).
self-peptide–MHC complexes are not equivalent for all
naive T cells, allowing ‘winner’ T cells to compete more
effectively for (non-self-peptide–MHC-dependent)
resources than ‘loser’ T cells (FIG. 1c).
Also, it is interesting to note that the competitive
hierarchy of TCR-transgenic T cells correlates, in some
experiments involving inducible loss of TCR expression,
several groups have shown that TCR signals are required
for long-term naive T cell survival36,37, and a combined
loss of LCK and FYN (but not either kinase on its own)
leads to the demise of naive T cells38,39. However, whether
the effect of losing TCR expression or TCR signalling is
Takada & Jameson Nature Rev Immunol (2009) 10: 823-832
“nichos” e diversidade
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Molecular Immunology.
2, 1-10
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C. M. Capitini et al.
|
Review: IL-7 and T-cell homeostasis
Terapia baseada em Il-7
lation that gradually show diminished expression of
CD44, and acquisition then loss of CD25, as they
progress through well defined DN1, DN2, DN3 and
DN4 subsets. The DN1 subset that has T lineage
potential does not express CD127 [32], however upregulation of CD127 is observed in DN2 to DN3
compartments followed by low to absent expression
in the DN4 subset [33, 34]. With the loss of CD44,
which occurs at the DN2-3 transition, cells undergo
rearrangement of the T cell receptor (TCR) b, c and d
genes. During this phase, IL-7R-mediated survival
signals and recombinase activity appear to be critical
[35]. Subsequently, DN cells acquire expression of
CD3 and CD4 ⁄ CD8, to become so-called double
positive thymocytes. Double positive thymocytes
comprise the majority of the thymocyte population,
but only a minority of these cells is finally selected to
become CD4+ or CD8+ single positive thymocytes
and ultimately emigrate from the thymus. CD127 is
not expressed by CD4+CD8+ double positive thymocytes, but is expressed again by mature single positive
cells [3] and is highly expressed on recent thymic
emigrants [36, 37].
r
o
g
s
o
n
u
m
m
i
n
i
p
ty
Fig. 2 Naı̈ve T-cell production is hampered through a normal ageing-associated involution of the thymus. Whilst children have the capacity to generate large numbers of naı̈ve T
cells by thymopoiesis, thymic-dependent T-cell production
in adults is limited, rendering lymphopenic adults dependent
upon homeostatic peripheral expansion for T-cell regeneration. A similar dependence of peripheral expansion to maintain T-cell numbers occurs with advancing age, and leads to
limited T-cell receptor repertoire diversity. IL-7 therapy may
selectively expand small numbers of recent thymic emigrants and naı̈ve T cells, which would lead to increased
diversity of the peripheral T-cell repertoire. IL-7 therapy
may also enhance thymic throughput in some settings, also
potentially increasing TCR repertoire diversity. Figure
adapted from the 2007 NIH Annual Report.
a
r
t
c
e
p
S
Thymic cellularity is reduced 20-fold in IL-7) ⁄ )
mice, and 0.01–10% of normal in CD127) ⁄ ) mice
[3]. Despite diminished thymocyte numbers in these
mice, all of the major thymic subsets are present in
a similar proportion to wild-type controls, and in
IL-7) ⁄ ) mice, thymopoiesis can be augmented by
IL-7 therapy. These results are due to a requirement
for IL-7 in thymocyte expansion during the early
phases of thymic development as well as IL-7’s contribution to expression of the Rag-1 gene, which is
required for TCR recombination. IL-7’s effects on
recombinase expression are nonredundant for development of cd T cells [8, 38] as this subset does not
develop in IL-7) ⁄ ) mice [14]. IL-7 signalling also
plays an essential role in preventing apoptosis in
developing thymocytes. In fact, thymocytes of
IL-7 in lymphocyte development
Capitini
et al J Internal Medicine (2009) 266, 141-153
T-cell progenitors emigrate from the bone marrow to
e
p
co
Como se quantifica a diversidade ?
Como contar quantos clonotipos distinctos existem numa
população de células ?
Análise do CDR3 por Imunoscope / Spectratyping
164
S. Hori et al. / Journal of Immunological Methods 268 (2002) 159–170
Fig. 2. Repeated measurements of CDR3 length distributions using two different numbers of spleen cells reveal increased relative variability of
peak area among the profiles at the smaller number of cells. Immunoscope analysis for Vh8.3 – Jh1.1 rearrangements was carried out on 12
independent samples of 5 " 104 (left panels) or 5 " 105 (right panels) spleen cells of a C57BL/6 mouse.
te hierarlonal size
ons with
where w
ρ. In this
xpression,
: (i) draw
ameter ρ,
ue x from
epeat this
n approxonal sizes
on of the
mate TCR
onotypes.
kely that
nalysis in
fits of the
s outliers
stimation
One first
discarded
ulation is
copies in
lonotype
discounts
ion size,
ma/n). By
outlying
ma/n), is
onotypes
data was
p://www.
AM (from
ommand
fit, as well
diversity is restricted to the imprecise joining of the Vα region
with either Jα segments. Further experimental details can be
found in the original reference (Correia-Neves et al., 2001).
A large collection of thymic and peripheral CDR3 TCRα
sequences is available from different mice (see supplementary
material of Correia-Neves et al., 2001). Data under analysis
will be of double positive CD4+ CD8+ thymocytes expressing
low levels of CD3 protein (DP CD3low) from mouse 17, and of
single positive (SP) thymocytes, either CD4+ or CD8+, and
lymph node (LN) T cells from mouse 57 (Table 1). Data from
other mice were not included in the analysis due to their even
smaller sample sizes. Experimentally, TCRα's of SP thymocytes
and of LN T cells were sequenced by single-cell RT-PCR, while
those of DP CD3low by RT-PCR and cloning. TCR uniqueness
(Pacholczyk et al., 2006).
To compute these different curves, we used Monte Carlo
resampling. We first obtain a random order by which TCR
sequences are successively added to the samples. For each
sampling step, we calculate the proportion of simulations in
which the added TCR sequence increased the sampled
diversity (Fig. 1A), the mean of sampled diversity (Fig. 1B),
and the mean of estimated diversity according to the
Homogeneous Poisson model (Fig. 1C).
As expected, the probability of obtaining a new TCR
variant is a decreasing function of the number of accumulated
sequences (Fig. 1A). However, it is far from zero at the current
Sequenciação massiva do CDR3
Existem dificuldade de natureza estatística
observation that mature T
(Bosco et al., 2009; Hale an
expanded in the periphery c
populations upon reentry in
in theory be assessed by stud
and LN T cell repertoires us
Sepúlveda (2009) while atte
tion between conventiona
compartments. However, th
scope of this paper.
To quantify the diversity
index as reported before (Ve
Table 1
Number of distinct CDR3 TCRα sequences with i copies in the samples, where
M is the total number of distinct sequences (clonotypes) in the samples, n is
the respective sample size, and Ds is the Simpson's diversity index. DP
CD3low data are from mouse 17, while remaining data from mouse 57. After
proper standardization of sample sizes, Simpson's diversity index was
estimated by the median of the diversity distribution resulting from 10,000
random samples of a subset without replacement.
Thymus
i
DP CD3low
SP CD4
Lymph nodes
+
SP CD8
+
LN CD4+
LN CD8+
1
2
3
4
5
6
7
8
10
11
16
20
21
28
52
79
17
6
5
1
1
1
33
6
2
2
0
0
0
0
1
0
1
0
0
1
16
3
3
5
3
1
1
1
0
1
34
8
2
1
0
0
0
1
1
0
0
1
17
8
1
2
1
0
0
0
0
0
0
0
1
0
1
M
n
Ds
110
169
0.99
46
113
0.91
34
98
0.96
48
98
0.94
31
122
0.79
Fig. 1. Assessment of the information contained in the samples assuming that TCR sequences were obtained by a se
obtaining a new TCR variant in 10,000 simulations (A), mean of sampled diversity (B), and mean of estimated diversit
model (C) as a function of the number of TCR sequences accumulated in the samples.
Sepulveda et al. J Immunol Methods (2010) 353, 125-137.
Homeostasia dos níveis séricos de anticorpos
Produção
Turnover:
e
o
ã
ç
a
tr ade
n
ce rsid al
n
Co dive utur
r )
t
s
e (Ig
Produção = catabolismo
Catabolismo
The notable difference in the turnover rates
of serum IgM and IgG
IgM has a half-life of about 5 days
IgG has a half-life of about 3 weeks
!2-microglobulin-containing neonatal intestinal transport receptor
Prolonged IgG half-life mediated by the FcN or Brambell
Fc-receptor
(Brambell receptor!Fc receptor!IgG survival!recycling!differential catabolism)
R. P. JUNGHANS*†
AND
C. L. A NDERSON‡
*Biotherapeutics Development Lab, Department of Medicine, Harvard Medical School, New England Deaconess Hospital, Boston, MA 02215; and ‡Departments
of Internal Medicine, Molecular Genetics, and Medical Biochemistry, The Ohio State University, Columbus, OH 43210
Communicated by Henry Metzger, National Institutes of Health, Bethesda, MD, April 23, 1996 (received for review March 5, 1996)
Immunology: More
Junghans
and
Anderson
ABSTRACT
than 30
years
ago, Brambell published
the hypothesis bearing his name [Brambell, F. W. R., Hemmings, W. A. & Morris, I. G. (1964) Nature (London) 203,
model 5500
dual channel
gamma
with
1352–1355]
that remains
as thecounter,
cornerstone
for corrections
thinking on
applied for
radioactive
crossover
and
decay.
IgG catabolism. To explain the long survival of IgG relative to
In Vivoother
Studies.
were
byoftail
vein for
pharplasmaMice
proteins
andinjected
its pattern
increased
fractional
catabolism
with
high was
concentrations
of indicated
IgG, Brambell
postumacokinetic
studies.
Blood
sampled at
times
and
IgG ‘‘protection
receptors’’
(FcRp) that would
processedlated
forspecific
protein-bound
counts
by trichloroacetic
acid
bind IgG in pinocytic vacuoles and redirect its transport to the
precipitation
as described (17). Rapidly catabolized proteins
circulation; when the FcRp was saturated, the excess unbound
require confirmation
the toprotein-bound
fractioncatabolism.
of radioIgG then wouldofpass
unrestricted lysosomal
activity to
distinguish
protein
from
radioactive
catabolites
in
Brambell subsequently postulated the neonatal gut transport
125
serum. Some
were
injected
i.p. saturable
with
I-labeled
receptoranimals
(FcRn) and
showed
its similar
character.
FcRn was recently cloned
but on
FcRp
has not been
human immunoglobulin
(Miles)
schedules
to identified.
maintain
Using
a
genetic
knockout
that
disrupts
the
FcRn
and
intesdifferent steady-state blood levels of IgG for the duration
of
tinal IgG transport, we show that this lesion also disrupts the
the experiment.
Mice
received
1–3
i.p.
doses
of
human
IgG
IgG protection receptor,
supporting the identity of these two
131I-labeled anti-Tac, and 0–8 i.p doses
before i.v.
injection
ofcatabolism
receptors.
IgG
was 10-fold faster and IgG levels
131I-labeled anti-Tac. Mice were
after thewere
i.v.correspondingly
injection of lower
in mutant than in wild-type mice,
131I-labeled
whereas
was the
same
groups,
demonstrating
the
injected i.v.
with IgA
a single
dose
ofbetween
anti-Tac,
six hours
specific
effects
on
the
IgG
system.
Disruption
of
the
FcRp
in
subsequent to the last prior i.p. dose of human IgG. Blood
the
mutant
mice
was
also
shown
to
abrogate
the
classical
125
levels of administered human IgG were determined from I
pattern of decreased IgG survival with higher IgG concentracounts and
specific
activity,
and
added
to the estimated
tion.IgG
Finally,
studies
in normal
mice
with monomeric
antigentotal murine
IgG.complexes showed differential catabolism in which
antibody
131I-soluble Tac
Monomeric
Complexes.
antigenAntigen-Antibody
dissociates in the endosome
and passes
to the lysosome,
whereas
the
associated
antibody
is
returned
circu[0.3 !g (10 pmol)] was mixed with 100 !g (1200 pmol to
binding
125
lation;
in
mutant
mice,
differential
catabolism
was
lost
and
site) of nonspecific (UPC) or specific (anti-Tac) I-antibody
the whole complex cleared at the same accelerated rate as
and injected
i.v. as above. The concentration of specific
albumin, showing the central role of the FcRp to the differantibody ential
binding
site ranged
from
1200
to 300
nM protein
in the
catabolism
mechanism.
Thus,
the same
receptor
plasma over
the
duration
of
the
experiment,
"1000!
the
that mediates the function of the FcRn transiently in the
is shown
to have
its antigen
functionally
dominant
expression
antibody neonate
Kd, thus
ensuring
that
binding
is essentially
the FcRp throughout
life,
resolving a longstanding
mystery
complete.asAntibody
survival is
unaffected
by antigen binding
of
the
identity
of
the
receptor
for
the
protection
of
IgG.
This
(17) and ‘‘antigen excess’’ is accordingly not represented
in
result also identifies an important new member of the class of
these tests.
Samples
werereceptors
collected
processed
forofproteinrecycling
surface
andand
enables
the design
protein
bound counts
as
above.
adaptations to exploit this mechanism to improve survivals of
Pharmacokinetic
Modeling.
other therapeutic
proteins Kinetic
in vivo. parameters were obtained by two-compartment modeling of the composite data of
Thirty-two
ago, Brambell
published
the hypothesis
(1)
each group
using years
PCNONLIN
4.2 (SCI,
Durham,
NC). The
Natl.
Acad. Sci.
USA(6–8).
93 (1996)
saturable nature (5)Proc.
which was
confirmed
by others
The
connection was made early and often between these two
systems in which the same mechanism or receptor system was
postulated (5, 6, 8), although it could not be demonstrated
directly. Common features include IgG saturation and transendosomal transport (1–8), acid-enhanced binding (5–9), a
shared site on the Fc for binding (10, 11), and widespread
expression of both the heavy and light chain of the cloned
FcRn in normal adult tissues (9, 12) that corresponds generally
to diverse sites of IgG catabolism (13, 14). In 30 years, the
FcRp has not been identified, and the problem has attracted
little further attention in the absence of genetic markers to
define its activity.
The intestinal receptor was cloned and characterized by
Simister and colleagues (15, 16). It is a heterodimer of a
membrane-integral class I-like heavy chain and a !2-microglobulin (!2m) light chain (15) in which both chains make
essential contacts with Fc (11). When Fc is mutated in the
domains contacting either FcR heavy or light chain (11), survival
and transport are both adversely affected (10). In mice with a light
chain deletion (!2m!/!), FcRn surface expression is lost and
neonatal pups are devoid of maternal IgG transport (16). The
same study noted that older !2m!/! mice had autologous IgG
levels 1!10th that of normal mice, which was proposed to reflect
decreased IgG synthesis. We considered that this could instead be
due to increased catabolism from parallel impairment of the IgG
protection mechanism. Using a genetic knockout that disrupts
the FcRn and intestinal IgG transport, we demonstrate that this
lesion similarly disrupts the IgG protection receptor activity of
these mice, providing genetic and functional links to support the
identity of these two molecules.
5513
FIG. 1. Abbreviated IgG survival in #2m$/$ mice. Animals were
MATERIALS
METHODS
131I-labeled
injected with
mixtures of AND
murine anti-Tac antibody (f,
Animals. Wild-type
!2m knockout
(!2125
m!/!
) mice were
wild-type
mice; !, and
mutant
mice) and
I-labeled
murine albumin
purchased
from
The
Jackson
Laboratory,
with
either awere
mixedprocessed for
(Inter-Cell) (data not shown), and blood samples
C57BL!6
" 129!Ola
or an
inbred
C57BL!6J
protein bound
counts.background
Five mice were
used
per group.
Error bars # "1
background.
Animals
were
raised
under
low
pathogen
SE, shown only on last points; fractional errors ofcondiother points are
tions
(3, 4)
yield low endogenous IgG levels.
similar
ortoless.
Proteins. Purified murine anti-Tac antibody was a gift of T.
A.mice.
Waldmann
(National
Institutes
Health).coadministered
Anti-Tac is an
§ When
normalized
to of
albumin
in these