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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Feb 15th 2010
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
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.
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.
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 .
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 (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.
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.
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.
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
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,
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.
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.