Inducing and expanding regulatory T cell populations by foreign antigen Karsten Kretschmer NATURE IMMUNOLOGY 2005; 6:1219

Background The extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immunity. The contributions of thymically and extrathymically generated suppressor T cells to the peripheral pool of CD4+CD25+ suppressor T cells is not known. The ability to generate de novo in secondary lymphoid tissue represent an important tool in the induction of antigen-specific tolerance in the fully mature immune system. Also, this pathway of generating suppressor cells may be used by tumors to avoid tumor-specific immune responses.

CD4+CD25+ regulatory T cells expressing the lineage marker Foxp3 CD4+CD25+ regulatory T cells expressing the lineage marker Foxp3 The cellular and molecular mechanisms involved in the peripheral generation of CD4+CD25+ suppressor T cells are not known The cellular and molecular mechanisms involved in the peripheral generation of CD4+CD25+ suppressor T cells are not known recombination activating gene 2–deficient (Rag2–/–) recombination activating gene 2–deficient (Rag2–/–) Thy-1.2 BALB/c Rag2–/– TCR-HA mice and Thy-1.1 BALB/c congenic Rag2–/– TCR-HA mice express a TCR specific for H-2IEd HA Thy-1.2 BALB/c Rag2–/– TCR-HA mice and Thy-1.1 BALB/c congenic Rag2–/– TCR-HA mice express a TCR specific for H-2IEd HA Antigens can be targeted to DCs in vivo by means of the DEC-205 endocytosis receptor Antigens can be targeted to DCs in vivo by means of the DEC-205 endocytosis receptor

Objection As the exact cellular and molecular mechanisms involved in the peripheral generation of CD4+CD25+ suppressor T cells are not known, we investigated whether this process can be initiated through antigen presentation by dendritic cells (DCs). This seemed an important issue to study, as cross- presentation of tumor-specific antigens by major histocompatibility complex class II molecules on DCs could constitute an essential pathway by which tumor cells induce Foxp3-expressing suppressor T cells.

Question What conditions are suited for the induction of antigen-specific Foxp3+CD4+CD25+ suppressor T cells in the context of a fully mature immune system using anti-DEC–HA?

90% or more of the CD25+ cells were stained with the clonotypic 6.5 TCR antibody 1.Conversion of naive CD4+CD25– T cells into CD4+CD25+ T cells. single-dose injection of anti-DEC–HA resulted in little TCR internalization

Question Does CD4+CD25+ T cells express the Foxp3 which is the marker of T regulatory cells? Does the converted Foxp3+CD25+ T cells have regulatory function?

2. Foxp3 expression and antigen-specific suppression by CD4+CD25+ T cells

Question What conditions are favoring for suppressor cell generation? Antigen dose? DC activation? Cell division? Routes of antigen administration?

3. Efficient induction of CD4+CD25+ regulatory T cells requires low doses of anti-DEC–HA and lack of costimulation.

Injection of either a substantial amount of anti-DEC–HA or injection together of minute amounts of anti-DEC–HA plus anti-CD40 did not result in efficient and long-lasting conversion of CD4+CD25– naive T cells into Foxp3+CD4+CD25+ suppressor T cells

4. Inverse relationship of cell division and CD25 expression. converted suppressor T cell populations generated in subimmunogenic conditions could subsequently be expanded by delivering antigen in immunogenic conditions

Different routes of administration of anti-DEC–HA (intraperitoneal, intravenous and subcutaneous) were similarly efficient in CD25+ suppressor T cell induction (data not shown). Also, prolonged subcutaneous infusion of small amounts of anti-DEC–HA (10 ng/d for 14 d) by means of osmotic mini- pumps did not increase the absolute numbers of induced Foxp3+CD4+CD25+ suppressor T cells (data not shown). Furthermore, injection of less than 40 ng anti-DEC–HA resulted in a much reduced recovery of initially seeded T cells 14 d after injection (data not shown).

Background TGF-beta could help the conversion of in vitro–stimulated peripheral CD4+CD25– T cells into suppressor cells suggests that TGF-b signaling could be involved in the antigen-driven conversion of suppressor cells in vivo. Consistent with that idea, mice deficient in components of the TGF-beta–TGF-beta receptor system have reduced numbers of peripheral CD4+CD25+ T cells

Question The role of TGF-beta in antigen induced T regulator cells generation? The role of TGF-beta in antigen induced T regulator cells generation?

5.Impaired TGF-betaR signaling diminishes conversion of naive T cells into CD4+CD25+ regulatory T cells.

Conclusion TGF-beta receptor signaling–dependent inhibition of proliferation correlated with more efficient in vivo conversion of naive T cells into CD4+CD25+ regulatory T cells. Q : whether increased TGF-b receptor signaling could reduce proliferation and enhance anti-DEC–HA–mediated conversion in vivo?

6.Costimulation of naive CD4+CD25– T cells in vitro by TCR and TGF-beta receptor.

In vitro, TGF-b1 slightly inhibited anti-CD3- and anti-CD28-mediated T cell proliferation at 36 and 60 h of culture Foxp3 mRNA expression was induced in vitro only when cells were stimulated with TGF-b1 plus anti-CD3 and anti-CD28 cells stimulated with TGF- b1 plus anti-CD3 and anti- CD28 expressed Foxp3 protein 36 h after stimulation, and this increased to 23% at day 3. Foxp3 expression correlated with CD25 surface expression

7. In vivo conversion of naive CD4+CD25– T cells costimulated in vitro with TCR and TGF- beta.

Background IL-2 is a cytokine that controls proliferation of antigenically stimulated T cells. Q : whether IL-2 interfered with or enhanced the conversion of naive T cells into CD4+CD25+ suppressor T cells??

8.Efficient conversion of Il2–/–CD4+CD25– naive T cells into regulatory T cells.

lack of autocrine IL-2 reduced proliferation mediated by anti- DEC – HA converted Il-2 – / – CD T cells proliferated in antigen-draining lymph nodes after immunization with peptide in IFA

Conclusion Conversion was achieved by minute antigen doses with suboptimal dendritic cell activation. Conversion was achieved by minute antigen doses with suboptimal dendritic cell activation. The addition of transforming growth factor-b or the absence of interleukin 2 production, which reduces proliferation, enhanced the conversion rate. The addition of transforming growth factor-b or the absence of interleukin 2 production, which reduces proliferation, enhanced the conversion rate. regulatory T cell populations induced in subimmunogenic conditions could be expanded by delivery of antigen in immunogenic conditions. regulatory T cell populations induced in subimmunogenic conditions could be expanded by delivery of antigen in immunogenic conditions. The extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immunity. The extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immunity.