specific inhibitors of rejection with different phenotypes, suggesting separate pathways of...

doi:10.1182/blood-2008-05-156612Prepublished online September 30, 2008;

Boyd, Suzanne J. Hodgkinson and Bruce M HallNirupama D. Verma, Karren M. Plain, Masaru Nomura, Giang T. Tran, Catherine Robinson, Rochelle suggesting separate pathways of activation by Th1 and Th2 responsesalloantigen specific inhibitors of rejection with different phenotypes, CD4+CD25+T cells alloactivated ex vivo by IL-2 or IL-4 become potent

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1

CD4+CD25+T cells alloactivated ex vivo by IL-2 or IL-4 become potent alloantigen

specific inhibitors of rejection with different phenotypes, suggesting separate

pathways of activation by Th1 and Th2 responses.

Running Title: IL-2 and IL-4 induce Ag-specific CD4+CD25+Tregs

Nirupama D. Verma*, Karren M. Plain*,1, Masaru Nomura2, Giang T. Tran, Catherine

Robinson, Rochelle Boyd, Suzanne J. Hodgkinson, Bruce M. Hall.

Faculty of Medicine, The University of New South Wales, Suite 302E Biomedical

Building, Australian Technology Park, Eveleigh and Department of Medicine,

Liverpool Hospital, Liverpool, NSW, Australia.

* These authors contributed equally to this work.

1 Current Address; Farm Animal and Veterinary Public Health, Faculty of Veterinary

Science, University of Sydney (Camden), 425 Werombi Road Camden 2560.

2 Current Address; First Department of Surgery, Hokkaido University School of

Medicine N15, W7 Kita-Ku Sapporo Hokkaido, Japan

Key words: tolerance/suppression, rodents, transplantation, tolerance, cytokines

Corresponding Author;

Prof. Bruce Hall [email protected] Dept Medicine. University of New South Wales Level 3, Biomedical Building, Australian Technology Park Eveleigh, NSW 2015, Australia,

Blood First Edition Paper, prepublished online September 30, 2008; DOI 10.1182/blood-2008-05-156612

Copyright © 2008 American Society of Hematology




2

ABSTRACT

CD4+CD25+Foxp3+T cells are regulatory/suppressor cells (Treg) that include non-

antigen(Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naïve

CD4+CD25+Treg develop into specific Tregs is unknown. Here, we generated adaptive

Tregs by culture of naïve CD4+CD25+Foxp3+T cells with alloAg and either IL-2 or IL-

4. Within days, IL-2 enhanced IFN-γR and IL-5 mRNA and IL-4 induced a reciprocal

profile with de novo IL-5Rα and increased IFN-γ mRNA expression. Both IL-2 and IL-

4 alloactivated CD4+CD25+Tregs within 3-4 days of culture had enhanced capacity to

induce tolerance to specific donor but not to third party cardiac allografts. These hosts

became tolerant as allografts functioned >250 days, with a physiological ratio of <10%

CD4+CD25+Foxp3+T cells in the CD4+ population. CD4+CD25+T cells from tolerant

hosts given IL-2 cultured cells had increased IL-5 and IFN-γR mRNA. Those from

hosts given IL-4 cultured cells had enhanced IL-5Rα mRNA expression and IL-5

enhanced their proliferation to donor but not third party alloAg. Thus, IL-2 and IL-4

activated alloAg-specific Tregs with distinct phenotypes that were retained in vivo.

These findings suggested that Th1 and Th2 responses activate two pathways of adaptive

Ag-specific Tregs that mediate tolerance. We propose they be known as Ts1 and Ts2

cells.




3

Bone marrow and organ transplantation are curative for an increasing number of

diseases. The major barriers are graft versus host disease (GVHD) and rejection, which

currently are inhibited by toxic non-specific immunosuppression. Induction of donor

specific tolerance without the requirement for immunosuppressive drugs would be

highly desirable. Adoptive therapy with ex vivo induced antigen (Ag) specific T

regulatory cells (Tregs) has considerable potential.

CD4+CD25highFoxp3+T cells are potent regulators of transplant rejection1-5 and GVHD6-

8. Natural Tregs are produced as a separate lineage in the thymus that constitute 2-10%

of peripheral CD4+T cells9, inhibit in a non Ag-specific manner and protect normal

tissue from immune injury2,10. The CD4+CD25+T cells that mediate transplant

tolerance are Ag-specific1-4 but how they develop and differ to natural non-Ag-specific

CD4+CD25+Tregs is poorly understood9.

In fully allogeneic models, naïve CD4+CD25highT cells if given at a ratio of 1:1 with

naïve CD4+T cells can totally prevent rejection5 and GVHD7, but only partially block

GVHD at a ratio of 1:28. At a ratio of 1:10 naïve CD4+CD25highT cells do not block

rejection5 or GVHD7. Given the very low number of CD4+CD25+T cells in the T cell

population, it is impractical to prepare enough cells for therapeutic usage at a ratio of

1:1. Ex vivo polyclonal activation of CD4+CD25+T cells with IL-2 and anti-CD3 mAb6

or IL-2, anti-CD3 and anti-CD2811 results in 200-250 fold cell number expansion but no

enhanced Ag-specific regulatory capacity12. Culture of CD4+CD25+Tregs with alloAg

and IL-2 for a week or more also increases cell numbers but does not induce high

potency Ag-specific CD4+CD25+Tregs, in that a ratio of 2:113 or 5:114 with naïve T cells

are required to suppress skin graft rejection. Similarly in GVHD, a 7:10 ratio of IL-2




4

and alloAg cultured Tregs to naïve cells is less effective than fresh naïve CD4+CD25+T

cells, in that they delay but do not fully prevent GVHD8.

In contrast, CD4+CD25+T cells within CD4+T cells from hosts with alloAg-specific

tolerance to a graft can transfer alloAg-specific tolerance at an effective ratio of

<1:202,3. These tolerant CD4+T cells when cultured in vitro lose the capacity to transfer

tolerance unless stimulated by specific donor Ag in media supplemented with T cell

cytokines2,15,16. Which cytokines are most effective at maintaining specific Tregs is

unknown, but IL-2 alone is insufficient16. This suggests that Ag specific CD4+CD25+T

cells may become dependent on cytokines other than IL-2. In unpublished studies, we

have identified that IFN-γ and IL-5, but not other Th1 and Th2 cytokines, promoted

proliferation and survival of Ag-specific tolerance mediating Tregs from rats tolerant to

an allograft.

Th1 and Th2 responses promote different pathways of activation in other immune cells,

including activated CD8+T cells17, B cells18 and macrophages19. As both Th1 and Th2

responses activate CD4+CD25+T cells20, we examined whether Th1 and Th2 cytokines

promoted different pathways of activation of natural CD4+CD25+T cells. We found

that the only Th1 or Th2 cytokines that could markedly enhance proliferation of naïve

CD4+CD25+T cells to alloAg were IL-2 or IL-4. Short-term culture of natural

CD4+CD25+T cells with allo-Ag and IL-2 or IL-4 produced alloAg-specific Tregs that

at a ratio of 1:10 with naïve CD4+T cells inhibited rejection of specific donor but not

third party allografts. Within three days, IL-2 or IL-4 induced Ag-specific

CD4+CD25+Foxp3+Tregs had acquired different profiles of cytokine receptor and




5

cytokine expression, consistent with two separate pathways of activation of Tregs by

Th1 and Th2 responses.




6

MATERIALS AND METHODS

Animals. DA (Rt-1a), PVG (Rt-1c) and Lewis (Rt-1l) rats were bred at the Liverpool

Hospital Animal House or the Animal Resources Centre, Perth. The University of New

South Wales Animal Care and Ethics Committee approved all experiments.

Cytokines: IL-2, IFN-γ, IL-5, IL-10, IL-12p70, IL-13, IL-15 and IL-23 were produced

from transfected CHO-K cell lines, characterised and quantified using bioassays, as

described21-24. The cell line for IL-4 was a kind gift from N. Barclay, Sir William Dunn

School of Pathology, Oxford, UK. IL-2 was used at 100 units/ml and IL-4 at 125

units/ml, the dose that induced nearly maximal proliferation of CD4+CD25+T cells in

dilutional MLC assays. This concentration inhibited proliferation of CD4+CD25-T

cells, which peaked with lower concentrations of 5-25 units/ml (data not shown).

Lymphocyte preparation and culture. Cells were prepared from spleens and lymph

nodes of DA rats and immunofluorescence staining performed as described5,25. Anti-rat

mAb used were R7.2(TCR-αβ), G4.18(CD3), MRCOx35(CD4), MRCOx8(CD8),

MRCOx39(CD25), MRCOx33(CD45RA), CyTM5 AnnexinV(Pharmingen-BD, San

Diego, CA) and FITC anti-mouse/rat Foxp3(eBioscience, San Diego, CA).

T cell subsets were enriched by indirect panning to deplete CD8+T cells and B cells,

followed by CD25 enrichment using PE conjugated MRCOx39 and anti-PE magnetic

beads using the MACS system(Miltenyi, Bergisch Gadenbach, Germany) as

described5,26.




7

Mixed lymphocyte cultures (MLC) were performed as described5,26 and proliferation

measured by 3H-thymidine incorporation. APC were irradiated (25Gy) thymus cells,

media was supplemented with 20% normal rat serum and cytokines were in serum-free

media at 50-200 units/ml. Syngeneic control counts were <200 cpm, equivalent to

media alone.

Microcultures contained 1-2 x 105 responder and 2 x 104 stimulator cells/well with 4-6

replicates/group. Bulk cultures (25cm2 flasks) were harvested at 2, 4 or 6 days; the

CD4+CD25+T cells were re-separated post-culture by MACS separation before RNA

extraction for cytokine and cytokine receptor mRNA analysis.

To test suppressive capacity of alloactivated CD4+CD25+T cells, serial two fold

dilutions of cells starting at 105 cells per well, were admixed with 105 naïve

CD4+CD25T cells and in vitro proliferative response to priming and third party

stimulator cells was assessed at day 4.

RT-PCR. RNA extraction, cDNA synthesis and semi-quantitative PCR were

performed as described 27. Primers and conditions for GAPDH, IL-2, IFN-γ, IL-4, IL-5,

IL-10, IL-13, and TGF-β were as described27. Primers (forward; reverse) for rat

cytokine receptors were; IFN-γR:(5’-AGAAGCACCAGAGCAGGAAGAAC-3’;5’-

CACGAGAACAAAGCAGGAAAAC-3’), IL-4Rα:(5’-

GGTGAGTGTGCTGTTGTTGCTGA-3’;5’-AATGAGTCCCTGAATCCCTTGTG-3’),

IL-5Rα:(5'-CTTCTGCCACCTGTCAATTTTACC-3';5'-

AACAAGCCAGGTGCAACGAAGAGA-3'); and for rat transcription factors were;

Foxp3:(5’-GCTCCTGCTGCCTCGTAG-3’;5’-TTGTGGAAGAACTCTGGAAAGG-

3’), GATA3:(5’-ACTGTGGCGGCGAGATGGTA-3’; 5’-




8

ATGAACGGGGAGATGTGGCT-3’), T-bet:(5’-AACCAGTATCCTGTTCCCAGC-

3’; 5’-TGTCGCCACTGGAAGGATAG-3’).

Real time RT-PCR was performed using a Rotorgene(Corbett Research, Mortlake,

NSW, Australia) with SYBR Green I and HotMaster Taq polymerase(Eppendorf AG,

Hamburg, Germany) or SensiMix DNA kit(Quantace, London, UK). Gene copy

numbers were derived from a standard curve run in parallel and were normalized

against GAPDH expression.

Allograft adoptive transfer assay. DA recipient rats were given 7Gy whole body

irradiation and grafted with fully allogeneic PVG or Lewis heterotopic heart allograft.

Graft function was scored using a semi-quantitative scale: 4+ robust contraction with

normal auxiliary heart graft rate; 3+ minor slowing of heart rate and/or minor reduced

contraction; 2+ obvious slowing and swelling of the graft with significant impairment of

contraction on palpation; 1+ marked slowing, very poor palpable contraction, or very

reduced ECG amplitude; and 0 no palpable contraction, equivalent to no ECG activity,

as described5.

In this model, rejection is ablated unless hosts are restored with CD4+T cells2,5,28,29 with

5x106 naïve CD4+T cells reconstituting severe rejection in 10-20 days5. Some of these

allografts recover days or weeks later and function long-term5. As poor cardiac function

would not support life if the graft was orthotopic, our aim was to assay the ability of

activated Tregs to abolish severe rejection, defined as <2+ using the semi-quantitative

score.




9

In preliminary studies, naïve CD4+CD25+T cells co-administered at a ratio of 1:1 with

naïve CD4+T cells prevented severe rejection of all grafts but at a ratio of 1:10, 8 of 9

rats had severe rejection episodes5. In vitro alloactivation of naïve CD4+CD25+T cells

with IL-2, did not activate effector T cells and suppressed rejection at a ratio of 1:15.

Here, we examined if in vitro IL-2 or IL-4 alloactivated CD4+CD25+Treg had enhanced

capacity to suppress rejection at a ratio of 1:10 with naïve CD4+T cells. Thus, 5x105

CD4+CD25+T cells activated with either IL-2 or IL-4 and PVG alloAg were adoptively

transferred with 5x106 naïve CD4+T cells to irradiated DA hosts with either PVG or

Lewis cardiac allografts. All groups had 6-10 animals.

Adoptive hosts that accepted their graft for >250 days were examined for graft

histology, as described5, peripheral lymphocyte subset composition and cytokine and

cytokine receptor mRNA expression by peripheral CD4+CD25+T cells. The reactivity

of CD4+CD25+ and CD4+CD25-T cells from tolerant hosts to donor and third party Ag

was examined in MLC, as was the effect of IL-5 and IFN-γ on their proliferation. To

obtain sufficient CD4+CD25+T cells to undertake the studies, lymph nodes and spleens

of all long-term surviving hosts were combined.

Statistics. Significance was determined using ANOVA with a Bonferroni-Dunn post-

hoc test on StatView 5.0. Graft survival used the Kaplan-Meier method and differences

were determined using the Mann-Whitney log-rank test on Statview (Abacus Concepts,

Berkley, CA). Significance was p<0.05.




10

Results

Effect of Th1 and Th2 cytokines on naïve CD4+CD25+T cells’ response to alloAg in

MLC.

Either IL-2 or IL-4 enhanced proliferation of CD4+CD25+T cells to both self and

allogeneic APC (Figure 1A) with counts of 1500-5000 cpm and ongoing proliferation at

6 days. The proliferation to alloAg was always greater than to self in all seven

experiments. The original CD4+CD25+ enriched populations were ≥96% CD25+, >98%

CD4+, <1% CD8+ (Figure 1B), with >99% TCR-α/β+and CD3+, confirming they were T

cells. After 3 days in culture with alloAg and IL-2 or IL-4, 99% of cells expressed

CD4, CD25, TCR-αβ and CD3. Foxp3 expression post-culture with IL-2 (97%) and

IL-4 (78%) (Figure 1B lower panel) was similar to naïve CD4+CD25+T cells (88%)

indicating retention of their suppressor phenotype. In contrast, CD4+CD25+T cells

cultured with alloAg alone had very low proliferation (<500 cpm) peaking at 3-4 days

(Figure 1A) and only ~30% of cells were CD4+CD25+highFoxp3+. At 24 hours and 96

hours respectively 45% and 50% of CD4+CD25+T cells cultured with alloAg expressed

Annexin V, compared to 29% and 50% with IL-2, and 28% and 22% with IL-4,

indicating both cytokines reduced early apoptosis.

Other Th1 cytokines, including IFN-γ and IL-12p70, as well as Th2 cytokines, including

IL-5, IL-10 and IL-13, did not enhance proliferation (Figure 1A) in all three

experiments. In one experiment IL-15 and IL-23, did not induce proliferation of naïve

CD4+CD25+T cells (data not shown).




11

Th1 cytokine and cytokine receptor mRNA expression in naïve CD4+CD25+T cells

stimulated in MLC with IL-2 or IL-4.

There was no induction of IL-2 mRNA in any culture, consistent with Foxp3 inhibition

of IL-2 transcription in CD4+CD25+T cells and no induction of Th1 cells30.

IFN-γ mRNA expression was increased in CD4+CD25+T cells cultured with alloAg and

IL-4, but not IL-2 (Figure 1C), with the greatest differences (>20 fold) observed at day

2 in culture. IFN-γR mRNA expression was enhanced on CD4+CD25+T cells cultured

with IL-2, but not with IL-4 or nil cytokine (Figure 1C), with the greatest differences

observed at day 4-6. The data in Figure 1C was representative of five experiments.

The Th1 transcription factor T-bet was not induced in either IL-2 or IL-4 cultured

CD4+CD25+T cells.

Th2 cytokine and cytokine receptor mRNA expression in naïve CD4+CD25+T cells

stimulated in MLC with IL-2 or IL-4.

mRNA for IL-5Rα, the IL-5 binding component of the receptor, was induced in

CD4+CD25+T cells cultured with IL-4 for 4-6 days (Figure 1C; representative of four

experiments). IL-5Rα mRNA was not detected on freshly isolated CD4+CD25+T cells

nor was it induced by culture with IL-2 or nil cytokine and alloAg, or culture with self

stimulator cells and IL-2 or IL-4.

IL-5 mRNA expression tended to be higher after culture with IL-2 and alloAg at day 2-

4 (Figure 1C) and in one experiment was ten fold greater. In cultures with IL-4 and

alloAg, IL-5 mRNA expression was similar to that observed in fresh CD4+CD25+T

cells.




12

IL-4 mRNA expression in cells cultured with either IL-2 or IL-4 (Figure 1D) was

similar to fresh CD4+CD25+T cells(data not shown). IL-4Rα, the IL-4 binding

component of the receptor, was enhanced after culture with either IL-2 or IL-4 in

allogeneic MLC but not in cultures with self-APC or nil cytokine(data not shown). IL-

10 and TGF-β mRNA expression was similar in IL-2 and IL-4 cultured cells (Figure

1D). The Th2 transcription factor, GATA3, was modestly increased with IL-4, but not

with IL-2.

Comparison of the cytokine and cytokine receptor expression in CD4+CD25-T cells

cultured in MLC with either IL-2 or IL-4.

In some experiments CD4+CD25-T cells cultured, in parallel with CD4+CD25+T cells,

and had greater proliferation at 4 and 6 days26. Foxp3+ cells did not increase in

CD4+CD25-T cells cultures, with 3% before and 2-4% after culture with either IL-2 or

IL-4. AnnexinV expression was similar with no cytokine or IL-2 at 24 hours, 75% vs

73%, and at 96 hours, 21% vs 21%. There was reduced early apoptosis with IL-4 as

only 24% expressed AnnexinV at 24 hours and 20% at 96 hours.

There was no induction of IL-5Rα in any CD4+CD25-T cell cultures (data not shown).

In cultures with IL-2, there was also no enhanced expression of IL-5 mRNA in

CD4+CD25-T cells, unlike the CD4+CD25+T cells which had enhanced IL-5 mRNA

expression. There was increased IL-5 mRNA expression in CD4+CD25-T cells cultured

with IL-4, consistent with induction of a Th2 profile. IFN-γ mRNA was detected in

CD4+CD25-T cell cultured with either IL-2 or IL-4. CD4+CD25-T cells had no enhanced




13

expression of IFN-γR mRNA in MLC supplemented with either IL-2 or IL-4 compared

to freshly isolated cells.

This confirmed that the distinct changes in cytokine and cytokine receptor mRNA

expression in naïve CD4+CD25+T cells following alloactivation with IL-2 or IL-4, were

not due to induction of Th1 and Th2 phenotypes.

Capacity of IL-2 or IL-4 alloactivated CD4+CD25+T cells to suppress in vitro.

Limiting dilutions of CD4+CD25+T cells with a constant number of CD4+CD25-T cells

were assayed in MLC. Both IL-2 and IL-4 alloactivated CD4+CD25+T cell populations

had greater suppression of CD4+CD25-T cell proliferation to specific priming Ag (PVG)

than to third party Lewis stimulator cells, manifest at a ratio of 1:16 and 1:32

respectively (Figure 2B&D). At higher ratios there was non-Ag specific suppression

with similar effects against PVG or Lewis stimulators. Parallel experiments with naïve

CD4+CD25+T cells showed similar suppression against PVG and Lewis at all ratios

tested, with near complete suppression only at a ratio of 1:1 (Figure 2A&C). The

enhanced inhibition at lower ratios, by IL-2 and IL-4 alloactivated CD4+CD25+T cells,

suggested induction of alloAg-specific Tregs.

Examination of the capacity of IL-2 or IL-4 cultured CD4+CD25+T cells to suppress

allograft rejection and induce tolerance.

5x105 CD4+CD25+T cells cultured with PVG alloAg and either IL-2 or IL-4 were

transferred to irradiated hosts that had been grafted with either specific donor PVG or

third party Lewis cardiac grafts and restored with 5x106 naïve CD4+T cells (Figure 3A).




14

At this ratio of 1:10, the transferred CD4+CD25+T cells suppressed specific donor but

not third party allograft rejection (Figure 3B).

PVG grafts were accepted long term with IL-2 alloactivated Tregs (median survival

time (MST) >250 days, p=0.0066) and IL-4 alloactivated Tregs (MST 66->250 days,

p=0.035), whilst hosts treated with 5 x 106 naïve CD4+T cells alone had severe rejection

episode (rejection score <2+) between 10-20 days (n=12). All grafts that survived

>100 days (Figure 3B) continued to function normally for >250 days without any

immunosuppression. Fresh naïve CD4+CD25+T cells did not suppress early severe

PVG graft rejection episode at a ratio of 1:10 with naïve CD4+T cells, but do at a ratio

of 1:1, as described5 (Figure 3B).

Rejection of third party Lewis grafts by 5x106 naive CD4+T cells was not prevented by

co-transfer of either IL-2 alloactivated Tregs (MST 11 days) or with IL-4 alloactivated

Tregs (MST 13-24 days). Time to severe rejection was not significantly different to

rats restored with 5 x 106 naïve CD4+T cells alone (MST 13 days). This demonstrated

that IL-2 and IL-4 alloactivated CD4+CD25+T cells suppressed rejection in an Ag-

specific manner.

Examination of hosts with long surviving grafts after restoration with IL-2 or IL-4

alloactivated CD4+CD25+T cells.

Specific donor heart grafts in animals restored with either IL-2 or IL-4 alloactivated

CD4+CD25+T cells and 5x106 naïve CD4+T cells that functioned >250 days had normal

muscle morphology with only scattered areas of mononuclear cell infiltration (Figure




15

3C). These hosts had normal peripheral lymphocyte population, with only 5%

CD4+CD25+T cells. >80% of CD4+CD25+highT cells expressed Foxp3 indicating they

were mainly Tregs (Figure 4A).

We also analysed the phenotype and proliferation responses of CD4+T cell subsets from

hosts made tolerant to an allograft by transfer of IL-2 or IL-4 alloactivated

CD4+CD25+T cells. CD4+CD25+T cells from hosts restored with IL-2 activated Tregs

had greater expression of IL-5 and IFN-γR mRNA than CD4+CD25+T cells from naïve

rats or from tolerant hosts restored with IL-4 alloactivated Tregs (Figure 4B).

IL-5Rα mRNA was detected in CD4+CD25+T cells from hosts restored with IL-4

activated Tregs but was not found in CD4+CD25+T cells from naïve rats or tolerant

hosts restored with IL-2 activated Tregs. IFN-γ mRNA was increased in CD4+CD25+T

cells from hosts restored with IL-2 activated Tregs but not those given IL-4 activated

Tregs. IFN-γ was the only marker not consistent with the original phenotype. It may

be explained by IFN-γ mRNA induction in Th1 cells that express CD25. Thus, hosts

had CD4+CD25+T cells with a cytokine and cytokine receptor phenotypes similar to the

alloactivated Tregs they were restored with.

In vitro, CD4+CD25+T cells from hosts made tolerant with IL-4 activated Tregs had

significantly enhanced proliferation to specific donor APC when cultured with IL-5

(p<0.05), but not IFN-γ (Figure 4C). CD4+CD25+T cells from hosts restored with IL-2

activated cells, did not have enhanced proliferation to PVG alloAg in the presence of

IL-5 (1447+401 cpm) compared to cultures without cytokine (1511+593 cpm). This




16

demonstrated that the IL-5Rα expressed on the CD4+CD25+T cells from tolerant hosts

restored with IL-4 activated Tregs was functional and that IL-5 could promote

proliferation of these cells.

IFN-γ suppressed proliferation of all cultured cells, including CD4+CD25-T cells as well

as CD4+CD25+T cells. In other studies, we reported that IFN-γ induces iNOS in the

APC in MLC, which in turn produces nitric oxide that inhibits T cell proliferation5,26 .

CD4+CD25-T cells from hosts given IL-2 activated Tregs had similar responses to PVG

and Lewis (Figure 5A). The response to syngeneic cells was also high, but less than the

response to alloAg, as observed with naïve cells5,26. The response of the CD4+ and

CD4+CD25-T cell subset from hosts given IL-4 alloactivated Tregs was tested against

both specific donor PVG and third party Lewis in a limiting dilution MLC. At all

dilutions, the response to PVG was as great as that to Lewis for both CD4+(Figure 5B)

and CD4+CD25- T cells (Figure 5C). This demonstrated there was no clonal deletion of

alloreactive CD4+CD25-T cells in these hosts with long surviving allografts.




17

Discussion

This study using rat CD4+CD25+T cells found only IL-2 and IL-4, but not other Th1 or

Th2 cytokines, activated these cells as described in man31. Our findings suggested Th1

and Th2 cytokines induced separate pathways of activation of CD4+CD25+T cells, that

we have termed Ts1 and Ts2 (Figure 6). This is not surprising as Th1 and Th2 cells,

through the cytokines they produce, induce separate pathways of activation in other

immune associated cells. These include CD8+T cells that differentiate to Tc1 with IL-2

and IFN-γ or to Tc2 cells with IL-417, B cells that have different Ig isotype switching

with IFN-γ or IL-4 and IL-518, and macrophages that differentiate to M1 with IFN-γ and

to M2 with IL-4 and IL-1319.

In vivo, Tregs develop in parallel with Th1 and Th2 responses20. Th1 cells only

transiently produce IL-2 and later produce IFN-γ and TNF-β32. Th2 cells produce IL-4

early and later produce IL-5, IL-10 and IL-1332. Our findings with Ts1 and Ts2 cells

are consistent with IL-2 and IL-4 being critical cytokines for the early activation of

Tregs. It is possible that they later become dependent upon late Th1 and Th2 cytokines,

such as IFN-γ and IL-5. Naïve CD4+CD25+T cells cultured with IL-2 and alloAg

expressed IFN-γR and IL-4Rα but not IL-5Rα mRNA and maintain IL-5 but not IFN-γ

mRNA expression. In contrast, naïve CD4+CD25+T cells activated with IL-4 and

alloAg expressed IFN-γ, IL-5Rα and IL-4Rα but not IFN-γR mRNA. Neither IL-2 nor

IL-4 activated Tregs expressed IL-2 but both expressed IL-4, IL-10 and TGF-β mRNA.

These findings suggested there are two separate pathways of activation of Tregs; Ts1

cells induced by Th1 cytokine (IL-2) and Ts2 cells activated by Th2 cytokine (IL-4).




18

The phenotype of Ts1 and Ts2 cells was shown to be distinct from Th1 and Th2 cells;

particularly, the enhanced expression of IL-5Rα and IFN-γ mRNA by IL-4 alloactivated

Ts2 cells and enhanced expression of IL-5 mRNA by IL-2 alloactivated Ts1 cells. IL-

5Rα has not been previously identified on T cells but is expressed by B cells,

eosinophils and mast cells33.

Ts1 and Ts2 cells are different to Tr1 cells that are induced in culture with IL-10 or by

IL-10 producing DC, and function via release of IL-10 and TGF-β34. Tr1 cells do not

express CD25 but do also produce IL-534. The cells we activated also differ in their

cytokine profile from Th3 cells that mediate oral tolerance and produce TGF-β35.

CD4+CD25+T cells are a heterogeneous population and in our studies >75% expressed

Foxp3, the transcription factor that may define CD4+CD25+Tregs9,30. In our studies,

culture with either IL-2 or IL-4 and alloAg increased the proportion of Foxp3+ cells,

suggesting the initial Foxp3- cells are not the cells proliferating in MLC. If IL-2

promoted activation of Foxp3- cells, it would have induced mRNA for Th1 transcription

factor T-bet and for cytokines such as IL-2 and IFN-γ. Further, these cells would effect

rejection. The opposite occurred in this study, IL-2 alloactivated CD4+CD25+T cells

had enhanced Foxp3, and no T-bet. They also had reduced IFN-γ and no IL-2 mRNA

induction but enhanced expression of IL-5 mRNA, a Th2 cytokine. In addition, even

5x106 IL-2 alloactivated CD4+CD25+T cells do not effect rejection5. Similarly, if IL-4

promoted activation of CD4+CD25+Foxp3-T cells it would have enhanced Th2

transcription factor GATA3 and cytokine expression, but there was no increased

expression of GATA3, IL-4, IL-5, IL-10 or IL-13 mRNA in this study. Instead there




19

was enhanced expression of Foxp3 and IFN-γ mRNA, with de novo induction of IL-

5Rα mRNA, which is not expressed by Th2 cells.

In this study over 100 units/ml of IL-2 or IL-4 were required to induce maximum

proliferation CD4+CD25+T cells in MLC. This was 30 fold greater than required to

induce maximal proliferation of CD4+CD25-T cells in MLC. The doses of IL-2 and IL-

4 used to promote Tregs partially suppressed the CD4+CD25-T cell proliferation,

however. Both IL-2 and IL-4 alloactivated Tregs suppressed rejection of specific donor

allografts at the physiological ratio of 1:10 to CD4+CD25-T cells. A 2-4 fold enhanced

alloAg-specific suppression by Ts1 and Ts2 cells was observed in MLC, albeit there

was also enhanced non-Ag specific suppression.

We believe this is the first study to show such rapid and enhanced alloAg-specific

induction of activated CD4+CD25+T cells that can regulate an allograft response. The

Ts1 and Ts2 cells we studied, had ten-fold greater suppressive capacity than fresh naïve

CD4+CD25+T cells, which require a ratio of 1:1 to prevent severe rejection and induce

tolerance5. Naïve CD4+CD25+T cells given at a ratio of 1:10 with naïve CD4+T cells

could not prevent severe rejection episodes, although grafts recovered later5.

Previous studies found that culture of naïve CD4+CD25+T cells with IL-2 and alloAg

can produce CD4+CD25+T cells that suppress GVHD8 and graft rejection13,14,36, but

these used longer culture times. CD4+CD25+T cells cultured for 7 days suppressed

rejection of skin allografts by naïve CD25- cells only at a ratio of 5:114. A cell line of

CD4+CD25+T cells stimulated with IL-2 and autologous dendritic cells pulsed with an




20

allopeptide given at a ratio of 2:1 with CD4+CD25-T cells suppressed skin graft

rejection in thymectomized, T cell depleted mice13. Polyclonal activation of

CD4+CD25+T cells with IL-2 and anti-CD3/anti-CD28, before stimulating them with

alloAg together with IL-2 and TGF-β, produced Tregs that protected specific donor but

not third party cardiac allografts from rejection; but the ratio of Tregs to CD25-T cells

was not determined36. In our studies, within 3-4 days in culture, CD4+CD25+T cells

acquired enhanced donor specific suppressive capacity and inhibited rejection at a ratio

of 1:10, which was 20-50 fold lower ratio than reported for skin grafts13,14.

The ratio required with our allactivated Tregs is similar to the physiological ratio of

<1:10 of CD4+CD25+: CD4+CD25-T cells in the CD4+T cells that transfer tolerance

from animals with long surviving cardiac grafts2,29. These Ag-specific CD4+Tregs lose

their function in culture unless stimulated with both donor alloAg and T cell derived

cytokines15,16 but IL-2 alone is not sufficient to maintain their full suppressor function16.

In our unpublished studies, IFN-γ or IL-5 alone can maintain Ag-specific CD4+Tregs,

but IL-4 alone cannot. Therefore, we hypothesized that activation of Ag-specific

CD4+CD25+Tregs may initially be dependent upon IL-2 but they may later depend upon

other T cell cytokines. This led us to examine if culture with alloAg and IL-2 or IL-4

can induce expression of the receptors for IFN-γ and IL-5 on CD4+CD25+T cells.

Taken together, these findings suggest that the lack of enhanced suppressive potency

with longer cultures of CD4+CD25+T cells reported in other studies13,14 may be due to

lack of other cytokines.




21

The notion that IL-5Rα and IFN-γR expression is important in the function of alloAg-

specific CD4+CD25+Treg was supported by the continued expression of IFN-γR and IL-

5 mRNA by CD4+CD25+T cells from tolerant hosts restored with Ts1 cells over 250

days earlier. Also, by the expression of IL-5Rα mRNA in CD4+CD25+T cells in

tolerant hosts restored with Ts2 cells and the ability of IL-5 to enhance proliferation of

these cells in MLC to specific donor alloAg but not to third party or self-Ag. These

findings suggest that the expression of IL-5Rα is a marker of an Ag-specific

CD4+CD25+Treg.

There is now clear evidence that Th2 cells reactive to alloAg effect rejection, rather than

mediating tolerance37,38. We are not aware of any previous reports showing IL-4 can

promote CD4+CD25+Tregs that prevent allograft rejection. The ability of IL-4 to

activate Ag-specific CD4+CD25+Treg, may explain why IL-421 or a Th2 response may

under certain conditions promote transplant tolerance induction39,40. Thus, tolerance

induced by Th2 responses, may be due to IL-4 and other Th2 cytokines promoting

Tregs, not the Th2 cells themselves.

Tolerant hosts restored with either IL-2 or IL-4 alloactivated CD4+CD25+T cells had

normal proportion of CD4+CD25+Foxp3+T cells, which was <10% of CD4+T cells.

Thus, tolerance was not due to an abnormally high ratio of CD4+CD25+T cells to

CD4+CD25-T cells. The restoration of a physiological ratio probably occurred due to

normal homeostatic mechanism that have been described in irradiated41 and normal

hosts42.




22

The regeneration of CD4+CD25-T cells in animals with long surviving grafts was not

associated with clonal deletion, as CD4+T cells had similar alloreactivity to donor

alloAg and third party in MLC. Thus, transferred alloactivated CD4+CD25+T cells did

not deplete donor reactive cells with the potential to effect rejection. In this respect,

these animals were similar to transplant tolerant adult hosts, which have CD4+T cells

with the capacity to respond to donor alloAg in vitro43,44 and have Ag-specific adaptive

CD4+CD25+T cells that maintain tolerance1-4. These adult tolerant hosts also have a

ratio of CD4+CD25+ to CD4+CD25-T cells of <1:102,3,44.

We have shown that IL-5 can promote allograft survival and may be important in

tolerance induction, associated with reduction in IL-2 and IFN-γ mRNA expression22.

Other Tregs express IL-5 but its function in these cells has not been examined34. We

have found that IL-5 therapy promoted recovery from active experimental autoimmune

neuritis, and this was associated with induction of more IL-5Rα expressing cells, and

CD4+CD25+T cells whose proliferation to specific Ag was enhanced by IL-5 (Tran et.

al. manuscript in preparation). The findings in this study suggest that IL-5 therapy may

promote Ag-specific Ts2 cells.

The expression of IFN-γR on Ts1 cells and the effect of IFN-γ produced by Ts2 cells, is

harder to isolate, given the multiple and paradoxical effects of IFN-γ. Sawitzki et. al.45

found IFN-γ is an effector of Tregs in transplant tolerance, and our study suggested this

could be by Ts2 cells. Tr1 cells also produce IFN-γ34, however. IFN-γ plays a key

role in transplant tolerance induction by co-stimulation blockade46, in spontaneous

acceptance of liver allografts47 and transplant tolerance maintenance45 but is not always




23

essential for transplant tolerance induction48. IFN-γ inhibits Th2 cell development49

and induces apoptosis of activated T cells50. Release of IFN-γ by activated Th1 cells

can induce iNOS in macrophages, which in turn produce nitric oxide that limits

alloreactive T cell proliferation5,26. Our findings suggested that IFN-γ may promote

Ts1 cells.

As production of IL-2 and IL-4 is early and transient in the immune response32, we

propose that activated Tregs must acquire receptors for cytokines that are produced by

Th1 and Th2 cells late in the immune response. For Th1 responses, this could be IFN-γ,

and in Th2 responses, IL-5, consistent as with Ts1 and Ts2 respectively expressing IFN-

γR and IL-5Rα. (Figure 6). This requirement for other cytokines could explain why

longer cultures of CD4+CD25+T cells with IL-2 and alloAg do not increase alloAg-

specific Tregs13,14.

This hypothesis is consistent with our original description of Ag-specific

CD4+CD25+Treg in transplant tolerance, which die without T cell derived cytokines16.

IL-216 or IL-4 do not fully maintain tolerance transferring cells but either IL-5 or IFN-γ

can (manuscripts in preparation). These findings led us to examine IFN-γR and IL-5Rα

expression in this study and demonstrate that expression of these cytokine receptors is

functionally important for Ts1 and Ts2 cells in the maintenance of alloAg-specific

tolerance. The maintenance of the Tregs in adoptive hosts may depend upon the

production of cytokines produced by the co-administered CD4+T cells, which are

activated by alloAg and induced to express cytokines even in the presence of tolerance

transferring T cells44.




24

The rapid development of alloantigen specific Tregs in culture, may allow ex vivo

induction of these cells for use to prevent GVHD and allograft rejection. The

identification of two pathways of Treg activation with different cytokine and cytokine

receptor expression, suggests that these cells may have different suppressor

mechanisms.




25

Acknowledgements:

This work was funded by a generous donation from Bob and Jack Ingham of Ingham

Enterprises, Liverpool NSW Australia, Liverpool Hospital, and UNSW, especially Vice

Chancellor Professor Rory Hume as well as unencumbered grants from Bayer

Pharmaceuticals/ Schering, Australia. Grants were from the South Western Sydney

Area Health Foundation, Juvenile Diabetes Foundation of Australia and Multiple

Sclerosis Australia. We appreciate the assistance of Ms N. Carter, Dr J. Chen,

Associate Professor M. Killingworth and Mr M. Botros for technical assistance. We

thank the Radiation Oncology Unit and Blood Bank at Liverpool Hospital for use of

irradiators. Some cytokine clones were kindly provided by Neil Barclay, MRC

Cellular Immunology Unit, Oxford UK and X.Y. He, Liverpool Hospital, NSW,

Australia.

Contribution of Authors:

NV and KMP are equal first authors and have contributed equally to this work.

NV: designing and performing experiments, growing IL-2 and IL-4 activated T cells,

drafting and writing manuscript.

KMP: Planning and performing experiments, growing IL-2 and IL-4 activated T cells,

drafting and writing manuscript.

MN: Planning and performing heart graft experiments.

GT: Cytokine production and development of RT-PCR analysis.

CR: Development of in vitro assays.




26

RB: Planning and conduct of experiments with heart grafts.

SJH: Planning and interpretation of all aspects of project, drafting and writing

manuscript.

BH: Planning and interpretation of all aspects of project, drafting and writing

manuscript.

The authors declare no financial conflicts of interest.




27

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32

FIGURE LEGENDS

Figure 1. Proliferation and phenotype of IL-2 and IL-4 cultured naïve

CD4+CD25+T cells. (A) Representative MLC experiment showing proliferation of

naïve CD4+CD25+T cells cultured for 4 days with self (DA, ) or allogeneic (PVG, )

APC and different Th1 and Th2 cytokines. Proliferation induced by IL-2 and IL-4 was

greater to alloAg than to self. CTL: cultured with no cytokine added. Data presented as

mean ± standard deviation (n=4). * p<0.0001 compared to CTL. (B) Representative

FACS profiles showing CD4+CD25+T cells retained CD25 and Foxp3 expression post-

culture. Expression of surface CD4(Cy5), CD25(PE) and intracellular Foxp3(FITC)

analysed by three colour staining. Top panel; naïve unfractionated peripheral LN and

spleen and freshly isolated CD4+CD25+T cells pre-culture. Middle and lower panel;

CD4+CD25+T cells cultured with alloAg and either IL-2 (left) or IL-4 (right). (C)

Expression of cytokines and cytokine receptors by CD4+CD25+T cells: fresh ( ) or

cultured with allogeneic-APC (/PVG) with no cytokines (CTL, ), IL-2 ( ) or IL-4 ( )

for 2-6 days. Representative quantitative real time RT-PCR data expressed as ratio of

specific gene copy number per 103 copies of GAPDH. Culture with IL-2 and alloAg

increased expression of IFN-γR and IL-5 mRNA while IL-4 and alloAg induced IL-5Rα

and enhanced IFN-γ expression. (D) Composite semi-quantitative RT-PCR of mRNA

from CD4+CD25+T cells cultured for 2-4 days with allogeneic APC (/PVG) and no

cytokine ( ), IL-2 ( ) or IL-4 ( ) (n=4-5). Y-axis shows cDNA dilutions (neat to 1 in

100) after normalisation for GAPDH. PCR reactions were at 40 cycles, except for

GAPDH (30 cycles).




33

Figure 2. Ability of IL-2 or IL-4 and allo(PVG)-activated CD4+CD25+Tregs to

suppress in vitro proliferation of CD4+CD25-T cells in MLC. Response to specific

PVG and third party Lewis stimulators were compared. Effect of specific suppression

was evident at ratios of activated CD4+CD25+Tregs to CD4+CD25- T cells of 1:16 and

1:32 respectively. At ratios above this, there was non-Ag-specific suppression by both

IL-2 and IL-4 alloAg activated Tregs.

A and B: Proliferation of 1 x 105 naïve CD4+CD25-T cells to PVG and Lewis APC

cultured with naïve (A) and IL-2/PVG activated (B) CD4+CD25+Tregs in different ratio;

no T regulatory cells ( ), IL-2/PVG activated CD4+CD25+T regs and CD4+CD25-T

cells at ratios of 1: 16 ( ), 1: 4 ( ) and 1:1 ( ).

C and D: Proliferation of 1 x 105 naïve CD4+CD25-T cells to PVG and Lewis APC

cultured with naïve (C) and IL-4/PVG activated (D) CD4+CD25+Tregs in different ratio;

no CD4+CD25+ Tregs ( ), IL-4/PVG activated CD4+CD25+Tregs and CD4+CD25-T

cells at ratios of 1: 32 ( ), 1: 4 ( ) and 1:1 ( ).

Figure 3: Graft survival without a severe rejection episode and histology of heart

grafts from DA hosts that were restored with CD4+CD25+T cells cultured with

allogeneic APC and IL-2 or IL-4.

(A) Experimental plan to test the ability of IL-2 or IL-4, PVG alloactivated

CD4+CD25+T cells from DA rats to suppress severe rejection of heterotopic adult heart

grafts from PVG (specific) or Lewis (third party) donors. All irradiated DA hosts were

restored with 5x106 naïve CD4+T cells. CD4+CD25+T cells, either fresh or cultured for

3-4 days with allogeneic APC (PVG) and IL-2 or IL-4, were co-transferred with naïve

CD4+T cells at varying ratios. (B) Heart graft survival results showing the ability of




34

naïve CD4+CD25+T cells cultured with allogeneic APC and IL-2 (Ts1 cells) or IL-4

(Ts2 cells) to suppress allograft rejection. Irradiated hosts restored with 5x106 naïve

CD4+T cells (�) reject both PVG (n=12) and Lewis (n=9) grafts. CD4+CD25+T cells

cultured with IL-2 (▲) or IL-4 (∆) and PVG alloAg, mixed at a ratio of 1:10 with naïve

CD4+T cells (5x105 CD4+CD25+ with 5x106 CD4+ cells), suppressed donor specific

(PVG) (p=0.0066 for IL-2, n=6 and p=0.035 for IL-4 cultured, n=6) but not third party

allograft rejection (NSD, n=5 for IL-2 and n= 6 for IL-4 cultured). Naïve CD4+CD25+T

cells suppressed severe rejection of PVG allografts at a ratio of 1:1 with naïve CD4+T

cells (�) (p<0.0006, n=9) but not at a ratio of 1:10 (�) (NSD, n=9). Some control data

has previously been published5. Graft survival without a severe rejection episode is

only shown to 100 days, but all grafts functioning at that time were monitored to at least

250 days and none had a rejection episode. (C) Photomicrographs (200x) of hearts from

DA hosts given CD4+CD25+T cells cultured with PVG alloAg and IL-2 (i and ii) or IL-

4 (iii and iv). Comparison of histology of PVG long surviving donor heart (left panels)

to the recipient’s own heart (right panels) showed similar morphology with preserved

muscular structure except for scattered patches of infiltrating mononuclear cells in

donor hearts.

Figure 4: Phenotype and in vitro reactivity of CD4+CD25+T cells from hosts with

long surviving grafts given CD4+CD25+T cells that had been cultured with

allogeneic APC and IL-2 or IL-4.

(A) FACS profiles showing normal proportions of CD4+CD25+T cells in tolerant hosts

and Foxp3 expression by the majority of CD4+CD25+high T cells. Expression of surface

CD4(Cy5), CD25(PE) and intracellular Foxp3(FITC) was analysed by three color




35

staining. Isotype control mAb staining showed specificity of Foxp3 Ab. (B) Cytokine

and cytokine receptor expression profiles of fresh naïve CD4+CD25+T cells from

normal DA rats ( ) or CD4+CD25+T cells from DA hosts with long surviving PVG

allograft after being restored with IL-2 cultured Ts1 ( ) or IL-4 cultured Ts2 ( ) cells.

CD4+CD25+T cells from Ts1 restored hosts had higher IFN-γR and IL-5 but no IL-5Rα

mRNA expression. CD4+CD25+T cells from hosts that had been restored with Ts2 cells

had higher IL-5Rα and low IL-5 mRNA. Thus, tolerant hosts’ CD4+CD25+T cells had

a cytokine and cytokine receptor profile similar to the alloactivated CD4+CD25+T cell

population given to induce tolerance. (C) In vitro reactivity of CD4+CD25+T cells from

DA hosts with long surviving PVG allografts after being restored with IL-4/PVG

activated (Ts2) CD4+CD25+Tregs . CD4+CD25+T cells from these tolerant hosts were

cultured either without stimulator cells ( ) or against self (DA), specific donor (PVG)

or third party (Lewis) stimulator cells. Control cultures not supplemented with

cytokine/mAb ( ) were compared to those supplemented with IFN-γ ( ), IL-5 ( ) or a

combination of IL-5 and anti-IL-5 mAb ( ). These cells had higher proliferation to

specific donor alloAg in the presence of IL-5 that was blocked by anti-IL-5 mAb,

consistent with the IL-5Rα expression on tolerant CD4+CD25+T cells having a

functional role.

Figure 5: Proliferation in MLC of CD4+ and CD4+CD25- T cells from DA rats with

long surviving PVG allografts after restoration with IL-2 or IL-4 alloactivated

CD4+CD25+Tregs. A. The proliferative response of CD4+CD25-T cells from hosts

restored with IL-2/PVG alloactvated (Ts1) cells against PVG and Lewis stimulator cells

was not significantly different, but both were greater than response without allogeneic




36

stimulator cells (p<0.03 for PVG and p<0.0001 for Lewis). B and C. Proliferative

response of CD4+T cells (B) and CD4+CD25-T cells (C) from tolerant hosts restored

with IL-4/PVG alloactivated CD4+CD25+T cells (Ts2) to PVG (�) and Lewis (�)

stimulator cells in a limiting dilution assay. At all dilutions of responder cells the

proliferation to specific donor PVG was as great as to Lewis, indicating there was no

clonal deletion. For CD4+T cells the response to PVG at one dilution (*) was greater,

p<0.05.

Figure 6 . Proposed scheme of the initial activation of naïve CD4+CD25+T cells

with allogeneic APCs and either IL-2 or IL-4, showing two separate activation

pathways of CD4+CD25+Tregs. We propose these be known as Ts1 and Ts2 cells, as

they have distinct phenotypes, defined by the cytokines and cytokine receptors they

express. Ts1 express IFN-γR, less IFN-γ and more IL-5 than Ts2 cells and Ts2 express

IL-5Rα and have greater IFN-γ mRNA induction. We hypothesise that expression of

IFNGR or IL-5Rα occurs as the Ts1 and Ts2 cells respectively become dependent upon

IFN-γ and IL-5 for survival and function as alloAg specific Tregs.




Figure 1

A.

(Cop

ies/

GA

PD

H c

opie

s) x

103

C.

Neat

1/10

1/100

IL-4 IL-10 IL-13 TGF-β

cDN

A d

ilutio

n

D.

CP

M (

x 10

3 )

0

1

2

3

4

5

0

10

20

IFN-γR

0

10

20

30

048

1216 IL-5Rα

0

2

4

6 IFN-γ

IL-5

8

1

40 96

11

2

CD

4

CD25 CD25

UnfractionatedSpleen and LN

Freshly isolatednaive CD4+CD25+

B. Fresh naïve CD4+CD25+

Short-term cultured CD4+CD25+

99.4

0.20.2

0.297

00

3

97 78

IL-2/CD4+CD25+high

CD25

IL-2 cultured CD25+ IL-4 cultured CD25+

Foxp3

CD25

Foxp3

IL-4/CD4+CD25+high

CD

4C

D4

* *

IL-10

*

*

CTL IL-2 IL-12p70 IL-5 IL-13IL-4IL-12p35 IFN-γIL-12p40




vs PVG vs Lewis vs PVG vs Lewis

A. Naive

010203040

05

10152025

0

10

20

30

01020304050

B. IL-2 cultured

C. Naive D. IL-4 cultured

Figure 2C

PM

(x

103 ) *

**

*

* **

*




PVG (specific donor) heart graft

1008060402000

20

40

60

80

100

Sur

viva

l to

acut

e re

ject

ion

B.

A.PVG or Lewis rat

Adoptive host

Donorheart

CD4+ T cells Naïve DA

7 Gy

Fresh orIL-2/IL-4 CulturedCD4+CD25+T cells

Graft survival

Figure 3

i ii

iii iv

C. Donor heart Recipient’s heart

Days post-transplantation100806040200

0

20

40

60

80

100Lewis (3rd party) heart graft




Figure 4

(Cop

ies/

GA

PD

H c

opie

s) x

10 3

B.

C.

CC

PM

0

100

200

300

400

500

600

vs Self APC

vs Specific donor APC

vs Third party APC

ND

*

Cellsalone

5

10

15

20

0

10

20

30

40

50

60

IFN-γR IFN-γ IL-5 IL-5Rα

25

0

85%

CD4+CD25+

CD25

CD

4

5%

86%

CD

4

CD4+CD25+highA.

Unfractionated

CD25C

D4

3%

Foxp3

CD

4

Isotype control




Figure 5

0

4

8

12

16

2x105 1x105 0.5x105 2.5x104 1.25x104

Dilution

CP

M X

100

0

Dilution

02468

101214

2x105 1x105 0.5x105 2.5x104 1.25x104

CP

M X

100

0

*

0 5 10 15 20

CD25- alone

CD25-/Lew

CD25-/PVG

CCPM x 1000

*

*

A.

B.

C.




Figure 6

IL-4

CD4+

CD25+

IL-2

APC IL-5Rα

Ag non-specificCD4+CD25+

T cell

Ag-specificCD25+ Treg

Th2IL-5

IFN-γTh1

CD4+

CD25+

IFN-γR

CD4+

CD25+

↑ IL-5Rα↑

IFN-γ

↑ IL-5↓

IFN-γ

↑ IFN-γR

Ts1

Ts2




specific inhibitors of rejection with different phenotypes, suggesting separate pathways of...

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