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Christina Ziegler Feb 15 th 2010. Mechanisms of tolerance induction. Clonal deletion negative selection of thymocytes with high affinity TCR for MHC:self -antigen (central tolerance) (2) Clonal anergy

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Christina Ziegler Feb 15 th 2010

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Christina ziegler feb 15 th 2010

Christina Ziegler

Feb 15th 2010


Mechanisms of tolerance induction

Mechanisms of tolerance induction

  • Clonal deletion

  • negative selection of thymocytes with high affinity TCR for MHC:self-antigen (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

Development of autoimmune diseases

  • AUTOIMMUNITY 

  • Breakdown of mechanisms controlling central and/or peripheral tolerance by

  • 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

Characteristics of regulatory T cells

Natural Tregs(nTregs) developed in thymus with high affinity for self-antigen

- 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

Stability of regulatory T cells – STATUS QUO –

  • Tregsretain Foxp3 expression under homeostatic conditions after adoptive transfer maybe via positive feedback loop

  • During inflammation, Tregshave lower Foxp3 expression

  • Possible that IL-6 acts in synergy with IL-1 to downregulateFoxp3

  • CD4+CD25-Foxp3+ were shown to convert into Th cells

  • SUM: Peripheral Tregscan become unstable under certain conditions.


Mouse model to analyse stability of t regs

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

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

´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 t reg as indicator for their stability

Methylation status of ex-Foxp3+ Tregas 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+ Foxp3- Tconv 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 t reg cell surface phenotype in the periphery

´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).

Conclusion: Ex-Foxp3 T cells do no longer show Treg specific phenotype indicating their instability in homeostatic conditions.

(b) thick line: Tconv cells

thin line: Foxp3+ Tregs

filled: ex-Foxp3 T cells


Ex foxp3 t cells show an effector memory phenotype

´Ex-Foxp3´ T cells show an effector-memory phenotype

Ex-Foxp3 T cells (GFP-YFP+) showed an activated-memory T cell phenotype (CD62Llow-highCD44high) (a).

Stimulated YFP+ T cells secreted IFN-γ (b) and IL-17 in GALT (c).

 Th1 or Th17?

Conclusion: Ex-Foxp3 T cells show an effector-memory T cell phenotype those cytokine profile depends on the microenviron-ment.


Mouse model to study the foxp3 expression during an autoimmune disease

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

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

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 ex-Foxp3 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

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 cells1

Mouse model to study if auto-reactive T cells favour development of ex-Foxp3 T cells

NOD Tcra-/- mouse

Lack αβT 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 nt regs or at regs

Ex-Foxp3 cells can be generated from nTregs or aTregs

Adoptively transferred nTregsfrom 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-memoryphenotype(a).

After adoptive transfer of Foxp3- cells into the NOD Rag2-/- mouse, thoseexpressing BDC2.5 TCR were 0.3% YFP+ in thepancreas.

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

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

  • Tregs did not alter the blood glucose levels

  • Tconvand 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

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 TCR-tg mice showed that

i) all subsets had productive VJ gene rearrangement

ii) Treg and Tconvcells 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

Summary and conclusions

  • Substantial fraction of Tregs are unstable in the periphery as a significant percentage

  • 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

  • ´ex-Foxp3´ T cell levels were elevated in autoimmune conditions

  • cells share ontogeny with Foxp3+Tregs and Tconvthus 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

Thank you for your attention!


Possible reasons for the development of autoimmune diseases

Possible reasons for the development of autoimmune diseases

  • Foxp3 instability can lead to the generation of pathogenic effector-memory 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


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