Christina Ziegler Feb 15 th 2010

Mechanisms of tolerance induction (1)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 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 (nT regs ) developed in thymus with high affinity for self- antigen - CD25 + Foxp3 + CTLA-4 + (5–10% of total CD4 + αβ T cells) Adaptive T regs (aT regs ) 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 + T regs (CD8 + CD25 - Foxp3 - ) - shown to suppress CD4 + cells in vitro

Stability of regulatory T cells – STATUS QUO – T regs retain Foxp3 expression under homeostatic conditions after adoptive transfer maybe via positive feedback loop During inflammation, T regs 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 T regs can become unstable under certain conditions.

Mouse model to analyse stability of T regs Adapted from

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 + T reg 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 + T reg as indicator for their stability Differentiation of T conv, T regs and ex-Foxp3 T regs 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 - T conv cells are methylated (d). T regs were mostly de-methylated (GFP + YFP + ), while ex-Foxp3 T regs (GFP - YFP + ) T regs 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 T regs.

´Ex-Foxp3´ T cells have a non-T reg cell surface phenotype in the periphery YFP + ex-Foxp3 T cells were CD25 - GITR low CD127 high and thus differ considerably from Foxp3 + T regs (a). Loss of ´signature´ T reg markers FR4, CTLA-4 and CD103 on ex-Foxp3 T cells in comparison to T conv and Foxp3 + T regs (b). Conclusion: Ex-Foxp3 T cells do no longer show T reg specific phenotype indicating their instability in homeostatic conditions. (b) thick line: T conv cells thin line: Foxp3 + T regs filled: ex-Foxp3 T cells

´Ex-Foxp3´ T cells show an effector- memory phenotype Ex-Foxp3 T cells (GFP - YFP + ) showed an activated-memory T cell phenotype (CD62L low- high CD44 high ) (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 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 T reg to T effector ratio Theory: Lower Foxp3 expression in the autoimmune disease shifts balance of T regs to ex-Foxp3 cell phenotype. Approach: Crossing of Foxp3-GFP-Cre mouse with R26-YFP-NOD mouse

Autoimmune enviroment favours loss of Foxp3 Pancreas contained sig. lower amount of T regs (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. Fig. legend Panc: pancreas PLN: pancreatic LN ILN: inguinal LN

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 Proportions of thymic CD4 + T conv 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: T regs are unstable and potentially pathogenic in autoimmune conditions. Approach: Adoptive transfer of T regs 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 Adoptively transferred nT regs 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 Foxp3 - cells 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 nT regs or to a lesser extend from abortive aT regs.

Auto-reactive ex-Foxp3 T cells turn into effector cells and then induce T1D Ex vivo expansion of ex-Foxp3, T conv and T regs 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)T regs did not alter the blood glucose levels ii)T conv 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 + aT reg cells that had converted from T conv or ii) T conv in the periphery or iii) loss of Foxp3 expression in true CD4 + Foxp3 + nT reg 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) T reg and T conv cells had dinstinct TCR V α 2 repertoire as only 13% of CDR3 sequence was present in T conv iii) Ex-Foxp3 cells shared 24% and 36 % sequence CDR3 similarity to T reg and T conv, respectively. Conclusion: Ex-Foxp3 cells have substantial overlap of TCR repertoire with T reg and T conv and can probably originate from both T cell subtypes.

Summary and conclusions Substantial fraction of T regs are unstable in the periphery as a significant percentage (a)down-regulates Foxp3 (b)loses their characteristic T reg phenotype (c)exhibits an activated-memory phenotype and (d)produces pathogenic cytokines (e)loses their suppressive function (f)triggers development of autoimmune disease ´ex-Foxp3´ T cell levels were elevated in autoimmune conditions cells share ontogeny with Foxp3 + T regs and T conv thus likely originate from nT regs and aT regs THEORY: Foxp3 instability can lead to the generation of pathogenic effector-memory T cells that promote autoimmunity.

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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 T reg 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 T regs and enhance immunity by creating locally pathogenic autoreactive T cell repertoire