Transcript Ontogeny of ex-Foxp3 T cells

Christina Ziegler
Feb 15th 2010
Mechanisms of tolerance induction
(1) Clonal deletion
- negative selection of thymocytes with high affinity TCR for MHC:selfantigen (central tolerance)
(2) Clonal anergy
- auto-reactive T cells encountering their Ag in absence of co-stimulatory
signal become non-responsive to Ag (peripheral tolerance)
(3) Clonal ignorance
- removal of auto-reactive T cells not encountering their Ag in periphery
(4) Anti-idiotypic antibody
- Ab against specific idiotypes of other Ab or TCR
(5) Regulatory T cells (suppressor cells)
- suppressive function via production of TGF-β and IL-10 or cell-cell contact
(6) Termination of tolerance
- By prolonged absence/exposure to tolerogen, damage of immune system or
immunization with cross-reactive Ag
Development of autoimmune diseases
AUTOIMMUNITY
Breakdown of mechanisms controlling central and/or peripheral
tolerance by
(1) Sequestration of antigen
- antigen develops late or is only expressed in particular organ
(2) Escape of autoreactive clones
- defective negative selection in thymus
(3) Lack/deficiency of regulatory T cells
(4) Cross-reactive antigens
- pathogens antigen may cross-react with self-antigens leading
to an autoimmune response like e.g. streptococcal nephritis
Characteristics of regulatory T cells
Natural Tregs (nTregs) developed in thymus with high affinity for selfantigen
- CD25+ Foxp3+ CTLA-4+ (5–10% of total CD4+ αβ T cells)
Adaptive Tregs (aTregs) develop from conventional T cells in periphery
and can be divided into
(a) Th3 cells (CD4+ CD25 - Foxp3-)
-activated by IL-10 which induced its secretion; acts autocrine
(b) Tr1 cells (CD4+ CD25 - Foxp3-)
- require IL-10 for maturation, then secrete TGF-β and IL-10
- like Th3 cells, Tr1 are abundant in intestine and likely induce
tolerance to food Ag
(c) CD8+ Tregs (CD8+ CD25 - Foxp3-)
- shown to suppress CD4+ cells in vitro
Stability of regulatory T cells
– STATUS QUO –
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•
•
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Tregs retain Foxp3 expression under homeostatic conditions after
adoptive transfer maybe via positive feedback loop
During inflammation, Tregs have lower Foxp3 expression
Possible that IL-6 acts in synergy with IL-1 to downregulate
Foxp3
CD4+CD25-Foxp3+ were shown to convert into Th cells
SUM: Peripheral Tregs can become unstable under certain
conditions.
Mouse model to analyse stability of Tregs
Adapted from
http://commons.wikimedia.org/wiki/File:CreLoxP_experiment.png
Development of Foxp3+ T cells in
Foxp3-GFP-Cre x R26-YFP mice
Majority of Foxp3+ cells developed from CD4SP thymocytes (a).
Most Foxp3+ transcription is initiated after maturation of CD4SP thymocytes in
the thymus (b ).
Conclusion: Foxp3+ Treg cells develop as ´escape´ mechanism during negative
selection process after exposure to self-Ag.
´Ex-Foxp3´ T cells show fading Foxp3
translation in periphery
15% - 20% of YFP cells lack Foxp3 and
GFP expression in thymus and peripheral
lymphoid organs, respectively (c).
Different peripheral lymphoid organs
showed similar proportions of CD4+ T
cells expressing Foxp3 at various
maturation stages (e).
Conclusion: Certain population of T cells
called ´ex-Foxp3´ had ceased translation
of Foxp3.
Methylation status of ex-Foxp3+ Treg
as indicator for their stability
Differentiation of Tconv, Tregs and
ex-Foxp3 Tregs using CD4 vs
Foxp3 or GFP vs YFP (A).
Methylation of CpG islands is the
principle control mechanism: 90%
of CpG motives in TSDR of
Foxp3 locus of naive CD4+ Foxp3Tconv cells are methylated (d).
Tregs were mostly de-methylated
(GFP+YFP+), while ex-Foxp3 Tregs
(GFP-YFP+) Tregs had random
methylation status (d).
Conclusion: Factors controlling
the expression of the Foxp3 led to
re-methylation of this locus at
certain stage in ex-Foxp3 Tregs .
´Ex-Foxp3´ T cells have a non-Treg cell
surface phenotype in the periphery
YFP+ ex-Foxp3 T cells were
CD25-GITRlowCD127high
and
thus differ considerably from
Foxp3+ Tregs (a).
Loss of ´signature´ Treg markers
FR4, CTLA-4 and CD103 on
ex-Foxp3 T cells in comparison
to Tconv and Foxp3 + Tregs (b).
(b) thick line: Tconv cells
thin line: Foxp3+ Tregs
filled:
ex-Foxp3 T cells
Conclusion: Ex-Foxp3 T cells
do no longer show Treg specific
phenotype
indicating
their
instability
in
homeostatic
conditions.
´Ex-Foxp3´ T cells show an effectormemory phenotype
Ex-Foxp3 T cells (GFP-YFP+) showed an
activated-memory T cell phenotype (CD62LlowhighCD44high) (a).
Stimulated YFP+ T cells secreted IFN-γ (b) and
IL-17 in GALT (c).
 Th1 or Th17?
Conclusion:
ExFoxp3 T cells show
an effector-memory
T cell phenotype
those
cytokine
profile depends on
the microenvironment.
Mouse model to study the Foxp3
expression during an autoimmune disease
NOD MOUSE
• the non-obese diabetic mouse is a model of autoimmune disease
•develops spontaneous autoimmune diabetes similar to T1D in humans incl.
- pancreas-specific autoantibodies
- autoreactive CD4+ and CD8+ T cells
•Inflamed pancreatic β islets have lower Treg to Teffector ratio
Theory: Lower Foxp3 expression in the autoimmune disease shifts balance
of Tregs to ex-Foxp3 cell phenotype.
Approach: Crossing of Foxp3-GFP-Cre mouse with R26-YFP-NOD mouse
Autoimmune enviroment favours
loss of Foxp3
Fig. legend
Panc: pancreas
PLN: pancreatic LN
ILN: inguinal LN
Pancreas contained sig. lower amount of Tregs (GFP+YFP+) but higher
percentage of ex-Foxp3 T cells (GFP-YFP+) (a).
These ex-Foxp3 T cells were CD25-CD127+ and secreted IFN-γ (b).
Conclusion: The autoimmune microenvironment altered the T cell
phenotype and promoted pathogenicity.
Appearance of ex-Foxp3 T cells was likely consequence of
antigen recognition in inflamed area.
Mouse model to study if auto-reactive T
cells favour development of ex-Foxp3 T cells
BDC2.5 TCR-tg mouse
TCR of CD4+ T cells in the BDC2.5 TCR-tg mouse are reactive to a natural
pancreatic islet β cell antigen
Theory: Auto-reactive T cells in pancreas changes the percentage of exFoxp3 cells and their pathogenic potential.
Approach: Crossing of Foxp3-GFP-Cre x R26-YFP mouse with BDC2.5
TCR-tg mouse.
Autoimmune environment favours
loss of Foxp3
Proportions of thymic CD4+Tconv and ex-Foxp3 T cells (GFP-YFP+) similar
between non-tg and BCD2.5 mice (d).
However, spleen and LN of BCD2.5 mice had more ex-Foxp3 cells (d and
e) similar to situation in pancreas of NOD mice.
Conclusion: Strong affinity to self-antigen especially during inflammation
promotes generation of ex-Foxp3 T cells.
Mouse model to study if auto-reactive T
cells favour development of ex-Foxp3 T cells
NOD Tcra-/- mouse
Lack αβ T cells and thus are completely protected from autoimmune diabetes.
NOD Rag2-/- mouse
Has immunodeficiency and combined cellular and humoral immune defects.
Theory: Tregs are unstable and potentially pathogenic in autoimmune conditions.
Approach: Adoptive transfer of Tregs from Foxp3-GFP-Cre x R26-YFP x
BDC2.5 TCR-tg mouse into
a) NOD Tcra-/- mouse and
b) NOD Rag2-/- mouse
Ex-Foxp3 cells can be generated
from nTregs or aTregs
Adoptively transferred nTregs from
BDC2.5 TCR-tg Foxp3-GFP-Cre x
R26-YFP mouse into the NOD Tcra-/a) had to 1/3 down-regulated Foxp3,
b) effector-memory phenotype (a).
After adoptive transfer of Foxp3cells into the NOD Rag2-/- mouse,
those expressing BDC2.5 TCR were
0.3% YFP+ in the pancreas.
Conclusion: Ex-Foxp3 cells can be
generated from instable nTregs or to a
lesser extend from abortive aTregs.
Auto-reactive ex-Foxp3 T cells turn into
effector cells and then induce T1D
Ex vivo expansion of ex-Foxp3, Tconv and Tregs
from BDC2.5 TCR-tg mice for 7-9 d.
20% of GFP+YFP+ and 2% of YFP+ lost
Foxp3 expression (b).
Adoptive transfer of three T cell subtypes into
the NOD Rag2-/- mouse
i) Tregs did not alter the blood glucose levels
ii) Tconv and ex-Foxp3 T cells induced
diabetes (c and d).
Conclusion: Auto-reactive Ex-Foxp3 T cells
turn into effector cells after self-antigen
recognition and induce T1D.
Ontogeny of ex-Foxp3 T cells
Unclear if ex-Foxp3 originate from
i) aborted Fopx3+ aTreg cells that had converted from Tconv or
ii) Tconv in the periphery or
iii) loss of Foxp3 expression in true CD4+Foxp3+ nTreg cells
Analysis of the CDR3 in various CD4+ T cell subsets from BDC2.5 TCRtg mice showed that
i) all subsets had productive VJ gene rearrangement
ii) Treg and Tconv cells had dinstinct TCR Vα2 repertoire as only 13% of
CDR3 sequence was present in Tconv
iii) Ex-Foxp3 cells shared 24% and 36 % sequence CDR3 similarity to Treg
and Tconv, respectively.
Conclusion: Ex-Foxp3 cells have substantial overlap of TCR repertoire
with Treg and Tconv and can probably originate from both T
cell subtypes.
Summary and conclusions
Substantial fraction of Tregs are unstable in the periphery as a significant
percentage
(a)
(b)
(c)
(d)
(e)
(f)
down-regulates Foxp3
loses their characteristic Treg phenotype
exhibits an activated-memory phenotype and
produces pathogenic cytokines
loses their suppressive function
triggers development of autoimmune disease
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•
´ex-Foxp3´ T cell levels were elevated in autoimmune conditions
cells share ontogeny with Foxp3+ Tregs and Tconv thus likely originate
from nTregs and aTregs
THEORY: Foxp3 instability can lead to the generation of pathogenic
effector-memory T cells that promote autoimmunity.
Thank you for
your attention!
Possible reasons for the development
of autoimmune diseases
• Foxp3 instability can lead to the generation of pathogenic effectormemory T cells that promoter autoimmunity
• Functional deficiency of IL-2 signalling in Treg cells in autoimmunity
may disturb the positive feedback loop that controls Foxp3 stability
• Dysfunctional microRNA or Dicer can affect Foxp3 stability
• Destabilized Foxp3 possibly involves epigenetic changes in the Foxp3
locus
• Early inflammatory cytokines induced by the innate immune system may
disable Tregs and enhance immunity by creating locally pathogenic
autoreactive T cell repertoire