on September 28, 2015
` on September 28, 2015
` on September 28, 2015
` on September 28, 2015
` on September 28, 2015
`
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`
`Downloaded from
`Downloaded from
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`
`R E P O R T S
`
`8. Materials and methods are available as supporting
`material on Science Online.
`9. D. R. Piperno, D. M. Pearsall, The Origins of Agriculture
`in the Lowland Neotropics (Academic Press, San Di-
`ego, CA, 1998).
`10. T. C. Andres, R. W. Robinson, in Cucurbitaceae 2002,
`D. N. Maynard, Ed. (ASHS Press, Alexandria, VA,
`2002), pp. 95–99.
`11. M. Nee, Econ. Bot. 44, 56 (1990).
`12. Despite extensive searches, no other wild Cucurbita
`has been found in Ecuador (10, 11). C. ecuadorensis is
`believed to have been at least semi-domesticated
`and used more commonly in the past than it is at the
`present time (10, 11). Many free-living populations,
`which are common in dry forest and along streams
`and road banks, have small (⬃8 to 10 cm long) and
`bitter fruits. Vines found today in house gardens,
`where they are grown or maintained and consumed
`by humans or more commonly fed to domestic ani-
`
`mals, have fruits with sizes of domesticated species
`(from 12 to 14 cm long and 12 to 18 cm in diameter)
`and other domesticated characteristics, such as non-
`bitter flesh, nonlignified rinds, and considerable vari-
`ability in color and pattern (10).
`13. S. R. Bozarth, Am. Antiq. 52, 607 (1987).
`14. D. R. Piperno, I. Holst, L. Wessel-Beaver, T. C. Andres,
`Proc. Natl. Acad. Sci. U.S.A. 99, 10923 (2002).
`15. Phytoliths are placed in fruit rinds in such a way that
`they straddle the interface between the hypodermis
`and the outermost stone (lignified) cells. Phytolith
`thickness is greatly influenced by how large the stone
`cells are. Domesticated fruits make longer and larger
`stone cells than do wild fruits, hence phytolith thick-
`ness is also a sensitive indicator of domestication
`(14).
`16. O. I. Sanjur, D. R. Piperno, T. C. Andres, L. Wessel-
`Beaver, Proc. Natl. Acad. Sci. U.S.A. 99, 535 (2002).
`17. D. W. Lathrap, D. Collier, H. Chandra, Ancient Ecua-
`
`dor: Culture, Clay, and Creativity, 3000-300 B.C.
`(Field Museum of Natural History, Chicago, IL, 1975).
`18. D. M. Pearsall, in Dumbarton Oaks Conference on the
`Ecuadorian Formative, J. S. Raymond, R. Burger, Eds.
`(Dumbarton Oaks, Washington, DC, 1996), pp. 213–
`257.
`19. J. R. Harlan, Science 174, 468 (1971).
`20. Supported by the Smithsonian Tropical Research In-
`stitute and the Museo Antropolo´gico, Banco Central
`del Ecuador (Guayaquil).
`
`Supporting Online Material
`www.sciencemag.org/cgi/content/full/299/5609/1054/
`DC1
`Materials and Methods
`Fig. S1
`Table S1
`
`11 November 2002; accepted 26 December 2002
`
`Control of Regulatory T Cell
`Development by the
`Transcription Factor Foxp3
`Shohei Hori,1 Takashi Nomura,2 Shimon Sakaguchi1,2*
`
`Regulatory T cells engage in the maintenance of immunological self-tolerance
`by actively suppressing self-reactive lymphocytes. Little is known, however,
`about the molecular mechanism of their development. Here we show that
`Foxp3, which encodes a transcription factor that is genetically defective in an
`autoimmune and inflammatory syndrome in humans and mice, is specifically
`expressed in naturally arising CD4⫹ regulatory T cells. Furthermore, retroviral
`gene transfer of Foxp3 converts naı¨ve T cells toward a regulatory T cell phe-
`notype similar to that of naturally occurring CD4⫹ regulatory T cells. Thus,
`Foxp3 is a key regulatory gene for the development of regulatory T cells.
`
`reduction or functional alteration in rodents
`leads to the spontaneous development of var-
`ious organ-specific autoimmune diseases in-
`cluding autoimmune thyroiditis, gastritis, and
`type 1 diabetes (6–9). TR cells also appear to
`maintain a balanced response to environmen-
`tal antigens, preventing inflammatory bowel
`disease (IBD) and allergy in rodents (10, 11).
`Several studies have provided findings
`that offer clues to the potential pathway by
`which TR cells develop. Similar multiorgan
`autoimmune diseases, allergy, and IBD de-
`velop in the X-linked recessive disease, IPEX
`(immune dysregulation, polyendocrinopathy,
`enteropathy, X-linked syndrome) or XLAAD
`(X-linked autoimmunity-allergic dysregula-
`tion syndrome) (12). A mouse mutant strain,
`
`To maintain immunological unresponsive-
`ness to self-constituents (i.e., self-tolerance),
`potentially hazardous self-reactive lympho-
`cytes are eliminated or inactivated during
`their development (1). Activation and expan-
`sion of self-reactive T lymphocytes that have
`escaped thymic clonal deletion is actively
`
`suppressed in the periphery by naturally oc-
`curring CD4⫹ regulatory T cells (TR), the
`majority of which constitutively express
`CD25 [interleukin (IL)-2 receptor ␣-chain]
`(2–6). CD25⫹CD4⫹ TR cells are at least in
`part produced by the thymus as a functionally
`mature T cell subpopulation (7, 8), and their
`
`1Laboratory of Immunopathology, Research Center
`for Allergy and Immunology, Institute for Physical and
`Chemical Research, Yokohama 230 – 0045,
`Japan.
`2Department of Experimental Pathology, Institute for
`Frontier Medical Sciences, Kyoto University, Kyoto
`606 – 8507, Japan.
`
`*To whom correspondence should be addressed. E-
`mail: shimon@frontier.kyoto-u.ac.jp
`
`Fig. 1. Expression of Foxp3 in a subpopulation of CD4⫹ T
`cells in the thymus and periphery. (A) BALB/c thymocytes
`were sorted into CD4–CD8– (DN), CD4⫹CD8⫹ (DP), CD4–
`8⫹ (CD8) or CD4⫹8– (CD4) cells (left). CD4⫹8– thymo-
`cytes were further separated into CD25⫹ or CD25– cells
`(right). cDNA from each population was subjected to non-
`saturating PCR using Foxp3- or HPRT (hypoxanthine-
`guanine phosphoribosyl-transferase)–specific primers (21).
`(B) Pooled lymph node and spleen cells from BALB/c mice
`were sorted into the indicated compartments, and nonsat-
`urating RT-PCR analyses were carried out. CD4⫹ cells were
`further separated into CD25⫹ or CD25– cells. (C) Quanti-
`fication of relative Foxp3 mRNA levels in indicated CD4⫹ T
`cell subsets. cDNA samples were subjected to real-time
`quantitative PCR analyses using primers and an internal
`fluorescent probe specific for Foxp3 or HPRT. The relative
`quantity of Foxp3 in each sample was normalized to the
`relative quantity of HPRT (21). (D) CD25–CD4⫹ (open
`symbols) or CD25⫹CD4⫹ cells (closed symbols) were ac-
`tivated for indicated hours with plate-bound CD3 mAb in
`the presence of IL-2 (circles) or CD28 mAb (squares) and
`assessed for the expression of Foxp3 by real-time quanti-
`tative RT-PCR. (A) to (D) each show one representative
`result of three independent experiments.
`
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`R E P O R T S
`
`Fig. 2. Retroviral transduction of Foxp3 into naı¨ve CD4⫹ T cells. (A) Foxp3 cDNA was inserted
`into the MIGR1 retrovirus vector (21). This vector simultaneously expresses two cDNAs, Foxp3
`and GFP, with the use of an internal ribosomal entry site (IRES). (B) CD3 mAb–stimulated
`proliferative response of freshly isolated CD25–CD4⫹ cells and GFP⫹ Foxp3/MIGR1- or
`MIGR1-infected CD25–CD4⫹ cells. [3H] thymidine incorporation was measured as an indicator
`of cell proliferation and expressed as the mean (⫾ SD) of triplicate cultures. cpm, counts per
`IFN-␥,
`minute. (C) IL-2,
`IL-4, or IL-10 concentration in the culture supernatant of CD3
`mAb–stimulated culture of GFP⫹ Foxp3/MIGR1- or MIGR1-infected CD25–CD4⫹ cells (mean ⫾
`SD). (D) Foxp3/MIGR1- or MIGR1-infected T cells derived from CD25–CD4⫹ cells were stained
`with phycoerythrin (PE)–labeled mAb for CD25, GITR, CD103, CD4, CD44, or an irrelevant
`antigen (control), along with detection of GFP. Intracellular CTLA-4 molecules were detected
`with PE-labeled CTLA-4 or isotype-matched control mAb (21). (B) to (D) each show one
`representative result of three independent experiments.
`
`scurfy, also succumbs to similar X-linked
`recessive autoimmune and inflammatory dis-
`eases as a result of uncontrolled activation
`and expansion of CD4⫹ T cells (13–16).
`Recent efforts to identify the genetic defect in
`IPEX/XLAAD patients or scurfy mice have
`revealed mutations
`in a common gene,
`Foxp3, which encodes a forkhead-winged–
`helix transcription factor designated Scurfin
`(17–20). Immunological similarities between
`the autoimmunity and inflammation pro-
`duced by manipulating CD25⫹CD4⫹ TR
`cells and those induced by genetic defects in
`Foxp3 prompted us to investigate the possible
`contribution of Foxp3 to the development or
`function of regulatory T cells.
`We first examined the expression of Foxp3
`mRNA in the thymus and the periphery of
`normal mice by reverse transcriptase–polymer-
`ase chain reaction (RT-PCR) (21). In the thy-
`mus, CD25⫹CD4⫹CD8– thymocytes, which
`constitute 5% of CD4⫹CD8– thymocytes (7),
`predominantly transcribed Foxp3, whereas
`CD4–CD8⫹ and other immature thymocyte
`populations did not (Fig. 1A). In the periphery,
`CD4⫹ T cells specifically transcribed the gene,
`whereas CD8⫹ T cells and CD19⫹ B cells did
`not (Fig. 1B) (17). Among CD4⫹ T cells, the
`CD25⫹ subset, which constitutes 5 to 10% of
`CD4⫹ T cells in normal naı¨ve mice, exhibited
`predominant transcription. Real-time quantita-
`tive PCR analyses revealed that the Foxp3
`mRNA level in CD25⫹CD4⫹ cells was 100-
`fold more abundant than in CD25–CD4⫹ cells
`(Fig. 1C). A low level of expression in CD25–
`CD4⫹ cells was confined to the CD45RBlow
`population, which has been previously reported
`to contain regulatory activity (22, 23). These
`results indicate that Foxp3 expression is pre-
`dominantly restricted to the CD25⫹CD4⫹ pop-
`ulation in both the thymus and periphery.
`Expression of Foxp3 is not a mere conse-
`quence of T cell activation, because in vitro
`stimulation of CD25–CD4⫹ cells for 3 days
`by monoclonal antibodies (mAbs) to CD3 in
`the presence of IL-2 or CD28 mAb failed to
`elicit Foxp3 expression (Fig. 1D). Neither
`effector Th1 nor effector Th2 cells prepared
`from naı¨ve T cells expressed Foxp3 (fig. S1).
`Furthermore, stimulation of CD25⫹CD4⫹
`cells with CD3 mAb and IL-2 did not alter
`their expression levels of Foxp3. Thus, Foxp3
`expression is stable in CD25⫹CD4⫹ TR cells
`irrespective of the mode or state of activation.
`We next determined whether forced ex-
`pression of Foxp3 in naı¨ve T cells could
`convert these cells toward a regulatory T cell
`phenotype. Bicistronic retroviral vectors ex-
`pressing Foxp3 and green fluorescent protein
`(GFP)
`(Foxp3/MIGR1)
`or GFP alone
`(MIGR1) were generated (Fig. 2A). Periph-
`eral CD25–CD4⫹ cells from normal naı¨ve
`mice were stimulated with CD3 mAb and
`IL-2 and infected with either retrovirus. One
`week after infection,
`the proliferative re-
`
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`
`with the level of GFP (Foxp3) expression in the
`infected population [Supporting Online Ma-
`terial (SOM) Text, fig. S3A]. These GFPhigh
`cells also suppressed IL-4 secretion by
`CD25–CD4⫹ responder T cells
`(fig.
`S3B). CD25⫹CD4⫹ cells, whether infected
`with Foxp3/MIGR1 or MIGR1,
`showed
`
`equally potent suppressive activity (Fig. 3A).
`To examine whether antigen-specific na-
`ı¨ve T cells could be converted to regulatory T
`cells by ectopic Foxp3 expression, we used
`recombination-activating gene (RAG)–defi-
`cient DO11.10 CD4⫹ T cells, which express
`ovalbumin (OVA) peptide–specific trans-
`
`R E P O R T S
`
`CD25+CD4+
`
`B
`
`150
`
`IL-2
`
`100
`
`50
`
`pg/ml
`
`CD25-CD4+
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`0.0
`
`+ MIG R1
`
`+C D25+
`
`0
`
`C D25- alone
`+ Foxp3/MIG R1
`
`anti-CD3
`
`0.0
`
`0.5
`
`1.0
`
`1.5
`
`2.0
`2.5
`0.0
`0.5
`No. of GFP+ cells (x10-4)
`
`1.0
`
`1.5
`
`2.0
`
`2.5
`
`1.5
`
`1.2
`
`0.9
`
`0.6
`
`0.3
`
`0.0
`
`OVA peptide
`
`1.5
`
`1.2
`
`0.9
`
`0.6
`
`0.3
`
`0.0
`
`D
`
`cpm (x10-5)
`
`DO.RAG-
`
`2.0
`0.0
`0.5
`1.0
`1.5
`No. of GFP+ cells (x10-4)
`
`0.0
`
`0.5
`
`1.0
`
`1.5
`
`2.0
`2.5
`0.0
`0.5
`1.0
`No. of GFP+ cells (x10-4)
`
`1.5
`
`2.0
`
`2.5
`
`1.0
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`0.0
`
`1.4
`
`1.0
`
`0.6
`
`0.2
`
`0.1
`
`0.0
`
`A
`
`cpm (x10-5)
`
`C
`
`cpm (x10-5)
`
`E
`
`1.5
`
`1.0
`
`0.5
`
`cpm (x10-5)
`
`0.75
`
`0.50
`
`0.25
`
`0.00
`
`PBS
`
`ααααIL-10R
`
`ααααTGF-ββββ
`
`ααααIL-10R
`+ααααTGF-ββββ
`
`0
`
`C D25- alone
`+Foxp3/MIG R1
`+Foxp3/MIG R1
`(transwell)
`
`Fig. 3. Suppressive activity of Foxp3-transduced naı¨ve CD4⫹ T cells. (A) Graded doses of GFP⫹ cells
`infected with either Foxp3/MIGR1 (closed circles) or MIGR1 (open circles) derived from BALB/c
`CD25–CD4⫹ (left) or CD25⫹CD4⫹ cells (right) were cultured with 2.5 ⫻ 104 freshly prepared
`CD25–CD4⫹ cells for 72 hours with CD3 mAb and 5.0 ⫻ 104 antigen-presenting cells (APCs).
`Proliferation of cells was assessed as in Fig. 2B. Freshly isolated CD25⫹CD4⫹ cells were also
`included in the assay (open squares). (B) Freshly prepared CD25–CD4⫹ cells alone or mixed with
`the same number of GFP⫹ Foxp3/MIGR1-(⫹Foxp3/MIGR1) or MIGR1-infected CD25–CD4⫹ cells
`(⫹MIGR1) or freshly isolated CD25⫹CD4⫹ cells (⫹CD25⫹) were stimulated with CD3 mAb, and
`IL-2 concentration in the culture supernatant was measured as in Fig. 2C. (C) GFP⫹ cells from
`DO11.10/RAG-2–/– CD4⫹ cells infected with Foxp3/MIGR1 (closed circles) or MIGR1 (open circles)
`were cultured with 2.0 ⫻ 104 freshly prepared DO11.10 CD4⫹ cells in the presence of OVA peptide
`and 4.0 ⫻ 104 APCs. (D) BALB/c CD25–CD4⫹ cells were transduced with Foxp3 (closed circles) or
`GFP alone (open circles) and sorted for GFP. Indicated numbers of GFP⫹ cells were mixed with
`2.5 ⫻ 104 DO11.10 TCR transgenic CD4⫹ cells and stimulated with either specific OVA peptide
`(left) or CD3 mAb (right). (E) GFP⫹ Foxp3/MIGR1-infected T cells derived from BALB/c CD25–CD4⫹
`cells and an equal number of freshly prepared CD25–CD4⫹ cells were separated or nonseparated
`by a semipermeable membrane and stimulated with CD3 mAb and APCs on each side (left). IL-10
`receptor (IL-10R) mAb, TGF-␤ mAb, or the mixture of these two was added to the culture of freshly
`prepared CD25–CD4⫹ cells either alone (white bars) or in the presence of GFP⫹ Foxp3/MIGR1-
`infected CD25–CD4⫹ cells (gray bars) or freshly prepared CD25⫹CD4⫹ cells (black bars) (right)
`(21). (A) to (E) each show one representative result of three independent experiments.
`
`sponses of GFP⫹ cells to T cell receptor
`(TCR) stimulation, cytokine production, and
`expression of cell surface molecules were
`examined. Retroviral transduction led to ex-
`pression of GFP in 30 to 60% of CD4⫹ cells.
`Upon TCR stimulation with CD3 mAb,
`GFP⫹ cells from Foxp3/MIGR1-infected cul-
`tures proliferated poorly in contrast to the
`vigorous proliferation of GFP⫹ cells from
`control MIGR1-infected cells or freshly pre-
`pared CD25–CD4⫹ cells (Fig. 2B). In addi-
`tion, Foxp3/MIGR1-infected GFP⫹ cells pro-
`duced very little but detectable IL-2, IFN-␥,
`IL-4, and IL-10 as compared to GFP⫹
`MIGR1-infected cells, which secreted large
`amounts of these cytokines (Fig. 2C). Al-
`though these Foxp3-expressing cells pro-
`duced higher amounts of cytokines than
`freshly isolated CD25⫹CD4⫹ cells (fig. S2),
`cytokine-producing cells were largely con-
`fined to cells expressing low levels of GFP
`(and hence Foxp3).
`Naturally arising CD25⫹CD4⫹ TR cells
`characteristically express CD25, cytotoxic T
`lymphocyte-associated antigen– 4 (CTLA-4),
`glucocorticoid-induced tumor necrosis factor
`receptor family–related gene (GITR), and
`CD103 (␣
`(6–10, 24–28). Al-
`E integrin)
`though the activation of CD25–CD4⫹ cells
`for retroviral infection led to expression of
`these molecules in both Foxp3/MIGR1- and
`MIGR1-infected cells, GFP⫹ cells in the
`former expressed CD25, GITR, and CTLA-4
`at higher levels than GFP– cells or GFP⫹
`MIGR1-infected cells (Fig. 2D, table S1).
`Among the GFP⫹ Foxp3-transduced cells,
`the higher the level of GFP (Foxp3) expres-
`sion, the higher the expression was of these
`molecules
`(Fig. 2D). Furthermore, only
`GFPhigh (Foxp3high) cells in the Foxp3/
`MIGR1-infected cells expressed CD103,
`with no expression on MIGR1-infected cells.
`Because Foxp3 transduction did not affect the
`expression levels of either CD4 or CD44, the
`Foxp3-induced high expression of these TR-
`associated molecules is most likely due to
`specific genetic instruction and not simply
`excessive cell activation by Foxp3. From
`these experiments, we conclude that trans-
`duction of Foxp3 could render naı¨ve T cells
`hyporesponsive to TCR stimulation, inhibit
`cytokine production, and up-regulate expres-
`sion of cell surface molecules closely associ-
`ated with the regulatory function of naturally
`occurring CD25⫹CD4⫹ TR cells (29, 30).
`We next assessed the potential of Foxp3-
`transduced T cells to show suppressive activity.
`Foxp3/MIGR1-infected CD25–CD4⫹ T cells
`could specifically reduce proliferation of fresh-
`ly prepared CD25–CD4⫹ responder T cells
`when stimulated with CD3 mAb (Fig. 3A).
`This suppression was associated with the inhi-
`bition of IL-2 production of the responder pop-
`ulation in a fashion similar to that of natural
`CD25⫹CD4⫹ TR cells (Fig. 3B) and correlated
`
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`
`of T cell regulation. For this, we used a model
`of IBD and autoimmune gastritis that can be
`induced
`in
`severe
`combined
`immuno-
`deficiency (SCID) mice by the transfer of
`CD25–CD45RBhighCD4⫹ T cells from normal
`BALB/c mice and prevented by cotransfer of
`CD25⫹CD4⫹ TR cells (10, 31) (Fig. 4). CD25–
`CD45RBhighCD4⫹ cells from BALB/c mice
`were infected with Foxp3/MIGR1 or MIGR1,
`and GFP⫹ cells were cotransferred with freshly
`prepared CD25–CD45RBhighCD4⫹ cells. The
`GFP⫹ Foxp3-transduced cells inhibited weight
`loss, diarrhea, and histological development of
`colitis and gastritis induced by the transfer of
`CD25–CD45RBhighCD4⫹ cells as effectively
`as naturally occurring CD25⫹CD4⫹ cells. By
`contrast,
`the cells transduced with GFP
`alone failed to prevent disease, and rather
`enhanced weight loss and the development
`of colitis. Thus, transduction of Foxp3 can
`render naı¨ve T cells capable of preventing
`autoimmune gastritis and IBD caused by
`dysregulated immune responses
`toward
`gastric self-antigens and commensal bacte-
`ria, respectively (SOM Text).
`The present study shows that Foxp3 is pre-
`dominantly expressed in the CD25⫹CD4⫹ TR
`population naturally arising in the thymus and
`periphery and that Foxp3 expression in naı¨ve T
`cells can convert these cells to a regulatory T
`cell phenotype functionally similar to naturally
`occurring CD25⫹CD4⫹ TR cells. This result
`suggests that Foxp3 may be a master regulatory
`gene for cell-lineage commitment or develop-
`mental differentiation of regulatory T cells in
`the thymus and the periphery. Our results also
`indicate that, in defining the naturally occurring
`CD4⫹ regulatory T cells engaged in preventing
`autoimmune disease and immunopathology,
`Foxp3 represents a more specific marker than
`currently used cell-surface molecules (such as
`CD25, CD45RB, CTLA-4, and GITR), which
`are unable to completely discriminate between
`regulatory T cells and activated, effector, or
`memory T cells.
`Mutations in the Foxp3 gene culminate in
`the development of a fatal lymphoprolifera-
`tive disorder associated with multiorgan pa-
`thology both in mice and humans (12–20).
`FOXP3 is predominantly expressed in human
`CD25⫹CD4⫹ T cells as well (32). Further-
`more, transduction of a mutant Foxp3 lacking
`the forkhead domain, similar to the mutated
`Foxp3 in scurfy mice (17), failed to confer
`suppressive activity to CD25–CD4⫹ T cells
`(fig. S7). The present results therefore sug-
`gest that mutations of the Foxp3 gene may
`cause
`these disorders
`through develop-
`mental or
`functional abnormality of
`the
`CD25⫹CD4⫹ TR population.
`Potentially, generation of TR cells by Foxp3
`transduction of naı¨ve T cells may provide a
`previously unstudied therapeutic mode for
`treatment of autoimmune and inflammatory
`diseases and in transplantation tolerance.
`
`R E P O R T S
`
`natural
`in
`deficient
`are
`genic TCRs,
`CD25⫹CD4⫹ TR cells (7), and scarcely ex-
`press Foxp3 (SOM Text, fig. S4). In the
`absence of specific antigen, almost all T cells
`from these animals remain in a naı¨ve state.
`Transgenic CD4⫹ T cells infected with
`Foxp3/MIGR1 suppressed the proliferation
`of freshly prepared noninfected transgenic T
`cells upon stimulation with specific OVA
`peptides, whereas those infected with MIGR1
`did not (Fig. 3C). These results collectively
`indicate that ectopic expression of Foxp3 was
`sufficient to convert otherwise nonregulatory
`naı¨ve T cells toward a regulatory T cell phe-
`notype capable of suppressing proliferation
`of other T cells, presumably through inhibi-
`tion of IL-2 production (SOM Text, fig. S5).
`Foxp3-transduced CD4⫹ T cells appeared
`to exert the suppressive activity in a similar
`manner
`to
`that
`of
`naturally
`occurring
`CD25⫹CD4⫹ TR cells (SOM Text, fig. S6).
`First, Foxp3-transduced cells derived from
`BALB/c CD25–CD4⫹ cells failed to suppress
`the proliferative response of DO11.10 TCR
`transgenic CD4⫹ T cells when stimulated with
`
`OVA peptide, whereas polyclonal stimulation
`with CD3 mAb induced suppression (Fig. 3D).
`This indicates that Foxp3-transduced T cells
`require stimulation through TCRs to exert sup-
`pression (29, 30). Second, Foxp3-infected T
`cells failed to suppress other T cells when sep-
`arated by a semipermeable membrane, in con-
`trast to effective suppression that was observed
`when cell-cell contact was allowed (Fig. 3E).
`These separated Foxp3-infected T cells even
`enhanced T cell responses across the mem-
`brane, presumably via cytokines they pro-
`duced (fig. S2). In addition, neutralization of
`transforming growth factor–␤ (TGF-␤) or
`blocking of IL-10 receptor, either alone or in
`combination, failed to abrogate suppression,
`as was the case with natural CD25⫹CD4⫹ TR
`cells. These results indicate that in vitro sup-
`pression by Foxp3-transduced T cells may
`not be mediated by humoral factors but re-
`quires cell contact (29, 30).
`Finally, we examined whether Foxp3/
`MIGR1-infected T cells could suppress in vivo
`the inflammation and the autoimmune disease
`that have been shown to develop in the absence
`
`3
`
`2
`
`1
`
`0
`
`gastritis score
`
`012345
`
`C
`
`colitis score
`
`*
`
`*
`
`*
`
`*
`
`*
`
`0
`
`1
`
`2
`3
`4
`5
`Weeks after transfer
`
`6
`
`7
`
`None
`
`25-45R Bhi alone
`+Foxp3/MIG R1
`+ MIG R1
`
`25-45R Bhi alone
`+Foxp3/MIG R1
`+ MIG R1
`
`None
`
`CD25-CD45RBhi alone
`
`+Foxp3/MIGR1
`
`+MIGR1
`
`None
`
`A
`
`120
`
`110
`
`100
`
`90
`
`80
`
`70
`
`60
`
`% of initial body weight
`
`B
`
`Colon
`
`Stomach
`
`Fig. 4. Prevention of IBD and autoimmune gastritis by Foxp3-transduced T cells. (A) C.B-17 scid
`mice received 4 ⫻ 105 fresh CD25–CD45RBhighCD4⫹ cells either alone (n ⫽ 6, where n is the
`number of mice) (open squares) or together with 1.2 ⫻ 106 GFP⫹ sorted cells derived from
`CD25–CD45RBhighCD4⫹ cells infected with Foxp3/MIGR1 (n ⫽ 7) (closed circles) or MIGR1 (n ⫽ 5)
`(open circles). Body weight is represented as the percentage of initial weight (mean ⫾ SD).
`Astericks indicate significant difference, P ⬍ 0.01, Foxp3/MIGR1 versus other two groups by
`Mann-Whitney test. (B) Histopathology of the colon and stomach in each group and in an
`unreconstituted SCID mouse (None). (C) Colitis (left) and gastritis (right) were histologically
`scored. Two mice in the group cotransferred with MIGR1-infected cells and one transferred with
`CD25–CD45RBhighCD4⫹ cells alone died of debilitation before histological examination. Results
`shown in (A) to (C) are from a total of three independent experiments.
`
`1060
`
`14 FEBRUARY 2003 VOL 299 SCIENCE www.sciencemag.org
`
`Page 4 of 6
`
`YEDA EXHIBIT NO. 2070
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`R E P O R T S
`
`31. E. Suri-Payer, H. Cantor, J. Autoimmun. 16, 115
`(2001).
`32. S. Hori, T. Nomura, S. Sakaguchi, unpublished data.
`33. We thank K. J. Wood, Z. Fehervari, and T. Takahashi
`for critically reading the manuscript; W. S. Pear and T.
`Kitamura for reagents; and T. Matsushita for histol-
`ogy. Supported by grants-in-aid from the Ministry of
`Education, Sports and Culture, the Ministry of Human
`Welfare, and the Organization for Pharmaceutical
`Safety and Research of Japan.
`
`Supporting Online Material
`www.sciencemag.org/cgi/content/full/1079490/DC1
`Materials and Methods
`SOM Text
`Figs. S1 to S7
`Table S1
`References
`
`17 October 2002; accepted 23 December 2002
`Published online 9 January 2003;
`10.1126/science.1079490
`Include this information when citing this paper.
`
`placed with a heterologous protein-protein
`interaction (Fig. 2A): the well-characterized
`heterodimerization interaction between PDZ
`domains from the mammalian proteins neu-
`ronal nitric oxide synthase (nNOS) and syn-
`trophin (13, 14). PDZ domains are interaction
`modules involved in assembly of diverse sig-
`naling complexes in higher eukaryotes (15,
`
`References and Notes
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`I. Caramalho, J.
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`Itoh, M.
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`11. M. A. Curotto de Lafaille et al., J. Exp. Med. 194, 1349
`(2001).
`12. R. S. Wildin, S. Smyk-Pearson, A. H. Filipovich, J. Med.
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`13. V. L. Godfrey, J. E. Wilkinson, L. B. Russell, Am. J.
`Pathol. 138, 1379 (1991).
`
`14. P. J. Blair et al., J. Immunol. 153, 3764 (1994).
`15. L. B. Clark et al., J. Immunol. 162, 2546 (1999).
`16. J. L. Zahorsky-Reeves, J. E. Wilkinson, Eur. J. Immunol.
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`17. M. E. Brunkow et al., Nature Genet. 27, 68 (2001).
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`20. C. L. Bennett et al., Nature Genet. 27, 20 (2001).
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`material on Science Online.
`22. L. A. Stephens, D. Mason, J. Immunol. 165, 3105 (2000).
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`Ishida, S.
`Sakaguchi, Nature Immunol. 3, 135 (2002).
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`
`Rewiring MAP Kinase Pathways
`Using Alternative Scaffold
`Assembly Mechanisms
`
`Sang-Hyun Park, Ali Zarrinpar, Wendell A. Lim*
`
`How scaffold proteins control information flow in signaling pathways is poorly
`understood: Do they simply tether components, or do they precisely orient and
`activate them? We found that the yeast mitogen-activated protein (MAP) kinase
`scaffold Ste5 is tolerant to major stereochemical perturbations; heterologous pro-
`tein interactions could functionally replace native kinase recruitment interactions,
`indicating that simple tethering is largely sufficient for scaffold-mediated sig-
`naling. Moreover, by engineering a scaffold that tethers a unique kinase set, we
`could create a synthetic MAP kinase pathway with non-natural input-output
`properties. These findings demonstrate that scaffolds are highly flexible orga-
`nizing factors that can facilitate pathway evolution and engineering.
`
`Scaffold proteins are known to play a critical
`role in a growing number of signaling path-
`ways, including several mitogen-activated pro-
`tein kinase (MAPK) cascades (1–4). In the
`budding yeast Saccharomyces cerevisiae, the
`scaffold proteins Ste5 and Pbs2 are essential for
`the mating and high–osmolarity response
`MAPK pathways, respectively (5–8). These
`scaffold proteins contain binding sites for each
`of the pathway kinases, as well as for upstream
`signaling input proteins (Fig. 1).
`Despite their importance, little is known
`about the mechanism by which scaffold pro-
`teins such as Ste5 contribute to efficient and
`specific signaling (9). One model is that scaf-
`fold proteins simply tether pathway compo-
`nents, increasing their likelihood of acting on
`one another. However, one might expect a
`simple tethering scaffold to enhance but not
`
`Department of Cellular and Molecular Pharmacology
`and Department of Biochemistry and Biophysics, Uni-
`versity of California, 513 Parnassus Avenue, San Fran-
`cisco, CA 94143, USA.
`
`*To whom correspondence should be addressed. E-
`mail: wlim@itsa.ucsf.edu
`
`be required for signaling. Thus, because Ste5
`is essential for signaling, and because of ev-
`idence supporting conformational changes in-
`duced by scaffold-kinase association (10, 11),
`an alternative model is that Ste5 plays a more
`complex catalytic role, precisely orienting
`and/or allosterically regulating pathway ki-
`nases (4). One way to distinguish between
`these models would be to probe pathway
`sensitivity to perturbations
`in assembly
`mechanisms. If pathway function depended
`on precise catalytic participation of the scaf-
`fold, then strict stereochemical requirements
`for kinase recruitment would be expected.
`We therefore tested whether non-native
`protein-protein interactions could be used to
`build a scaffolded assembly capable of me-
`diating proper mating pathway connectivity
`and function. We took advantage of several
`known mutations in Ste5 that selectively de-
`stroy recruitment of the MAPK kinase kinase
`(MAPKKK) Ste11 and the MAPK kinase
`(MAPKK) Ste7. These mutations, respective-
`ly termed Ste5* and Ste5**, each resulted in
`a nonfunctional mating pathway (12). Defec-
`tive recruitment interactions were then re-
`
`Fig. 1. Yeast mating and high-osmolarity MAPK
`pathways require scaffold proteins Ste5 and
`Pbs2. Both pathways require the shared MAP-
`KKK Ste11 but exhibit no cross-signaling under
`normal conditions. Ste5 has distinct docking
`sites for Ste11, the MAPKK Ste7, and the MAPK
`Fus3 (or the partially redundant MAPK Kss1,
`not shown for simplicity) (5–7). Input to Ste5
`occurs through a docking site for Ste4, the G␤
`subunit of the heterotrimeric guanine nucleo-
`tide–binding protein activated upon phero-
`mone binding to the ␣-factor receptor, Ste2.
`Pbs2 functions as both the scaffold and MAPKK
`of the osmolarity pathway:
`It has a MAPKK
`domain, and it has been shown to bind Ste11
`and the MAPK Hog1 ( precise binding sites have
`not been identified) (8). Pbs2 also binds the Src
`homology 3 (SH3) domain from the upstream
`osmosensor Sho1 through a proline-rich dock-
`ing site (residues 94 to 100), indicated by PxxP
`(26). A second branch of the osmoresponse
`pathway involving the two-component sensor
`protein Sln1 has been omitted for simplicity
`(27). This branch of the pathway does not
`require Sho1 or Ste11. All of the studies de-
`scribed here were performed with strains lack-
`ing this pathway branch (ssk2⌬ and ssk22⌬).
`
`www.sciencemag.org SCIENCE VOL 299 14 FEBRUARY 2003
`
`1061
`
`Page 5 of 6
`
`YEDA EXHIBIT NO. 2070
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`Control of Regulatory T Cell Development by the Transcription Factor
`Foxp3
` et al.
`Shohei Hori
` 299 Science
`
`, 1057 (2003);
`DOI: 10.1126/science.1079490
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