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

Inducing and expanding regulatory T cell Inducing and expanding regulatory T cell

populations by foreign antigenpopulations by foreign antigen

Karsten Kretschmer Karsten Kretschmer NATURE IMMUNOLOGY 2005; 6:1219NATURE IMMUNOLOGY 2005; 6:1219

BackgroundBackground The extrathymic generation and proliferation of regulator

y 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 marCD4+CD25+ regulatory T cells expressing the lineage marker Foxp3ker Foxp3

The cellular and molecular mechanisms involved in the perThe cellular and molecular mechanisms involved in the peripheral generation of CD4+CD25+ suppressor T cells are nipheral generation of CD4+CD25+ suppressor T cells are not knownot 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 BALThy-1.2 BALB/c Rag2–/– TCR-HA mice and Thy-1.1 BAL

B/c congenic Rag2–/– TCR-HA mice express a TCR specifiB/c congenic Rag2–/– TCR-HA mice express a TCR specific for H-2IEd HAc for H-2IEd HA

Antigens can be targeted to DCs in vivo by means of the DAntigens can be targeted to DCs in vivo by means of the DEC-205 endocytosis receptorEC-205 endocytosis receptor

ObjectionObjection As the exact cellular and molecular mechanisms i

nvolved 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.

QuestionQuestion

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 ofanti-DEC–HA resulted in little TCR internalization

QuestionQuestion 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-specificsuppression by CD4+CD25+ T cells

Lenovo User

Foxp3 protein expression in Thy-1.2+CD4+CD25– T cells from Rag2–/– TCR-transgenic donor mice was belowthe limit of detection

Lenovo User

In vitro, converted CD25+ cells suppressed naive 6.5+ cells as efficiently as CD25+cells from TCR-HA � pgk-HA mice that coexpressed the transgenic TCR forHA(107–119) and hemagglutinin

USER

At 14 d after transfer into Thy-1-mismatched hosts and administration of anti-DEC–HA, about 85%of Thy-1.2+CD4+ CD25+ T cells were Foxp3+ (Fig. 2a, bottom). Lessthan 5% of Thy-1.2+CD4+CD25– T cells stained positively for Foxp3(Fig. 2a, bottom)

USER

We adoptivelytransferred purified naive CD4+CD25– T cells (2 � 106) fromThy-1-matched donor Rag2–/–6.5+ mice (Thy-1.2+) into BALB/cThy-1.2+ mice and injected recipient mice with 40 ng anti-DEC–HAon the next day. Then, 3 weeks later, we gave the recipient mice smallnumbers of Rag2–/–6.5+Thy-1.1+ effector T cells (2 � 105), followed by

QuestionQuestionWhat 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 co

stimulation.

USER

strong activation of DCs and high doses ofantigen induced increased T cell proliferation but no stable conversioninto Foxp3+ suppressor cells.

Injection of either a substantial amount of anti-DEC–HA or injectiontogether of minute amounts of anti-DEC–HA plus anti-CD40 did notresult 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 immunogenicconditions

USER

anti-DEC–HA–inducedCD4+CD25+ suppressor T cells proliferated extensively in antigendraininglymph nodes of mice immunized with HA(107–119) in IFA3 weeks after administration of anti-DEC–HA

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).

BackgroundBackground

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

QuestionQuestion

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

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

Lenovo User

Analysis 14 d after antibodyinjection showed that the percentage of dnTGF-bRII+CD4+Thy-1.2+T cells with CD25 surface and Foxp3 expression was diminishedconsiderably

Lenovo User

Foxp3 expression was diminishedconsiderably (Fig. 5a, top and bottom) and was accompanied bymuch more extensive proliferation than that of wild-type CD4+6.5+T cells

ConclusionConclusion TGF-beta receptor signaling–dependent inhibit

ion 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.

BackgroundBackground IL-2 is a cytokine that controls proliferation of ant

igenically 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–/–CD4+6.5+ T cells proliferated in

antigen-draining lymph nodes after immunization

with peptide in IFA

USER

The reduced proliferation in response to antigen deliveredby anti-DEC did not seem to be due to an intrinsic defect inproliferation of converted cells,

ConclusionConclusion Conversion was achieved by minute antigen doses witConversion was achieved by minute antigen doses wit

h suboptimal dendritic cell activation. h suboptimal dendritic cell activation. The addition of transforming growth factor-b or the aThe addition of transforming growth factor-b or the a

bsence of interleukin 2 production, which reduces probsence of interleukin 2 production, which reduces proliferation, enhanced the conversion rate. liferation, enhanced the conversion rate.

regulatory T cell populations induced in subimmunogregulatory T cell populations induced in subimmunogenic conditions could be expanded by delivery of antienic conditions could be expanded by delivery of antigen in immunogenic conditions.gen in immunogenic conditions.

The extrathymic generation and proliferation of regulThe extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well atory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exas the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immploited clinically to prevent or reverse unwanted immunity.unity.

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