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Independently of NKT Cell-Related Signalsof Memory/Effector Functions in T Cells PLZF Induces the Spontaneous Acquisition

Maggie Eidson, Kim E. Nichols and Derek B. Sant'AngeloDamian Kovalovsky, Eric S. Alonzo, Olisambu U. Uche,

http://www.jimmunol.org/content/184/12/6746doi: 10.4049/jimmunol.1000776May 2010;

2010; 184:6746-6755; Prepublished online 21J Immunol 

Referenceshttp://www.jimmunol.org/content/184/12/6746.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

PLZF Induces the Spontaneous Acquisition of Memory/Effector Functions in T Cells Independently of NKTCell-Related Signals

Damian Kovalovsky,* Eric S. Alonzo,*,† Olisambu U. Uche,* Maggie Eidson,‡

Kim E. Nichols,x and Derek B. Sant’Angelo*,†,{

The broad complex, tramtrack, bric-a-brac–zinc finger (BTB-ZF) transcription factor promyelocytic leukemia zinc finger (PLZF) is

required for development of the characteristic innate/effector functions of NKT cells. In this study, we report the characterization

and functional analysis of transgenic mouse T cells with forced expression of PLZF. PLZF expression was sufficient to provide some

memory/effector functions to T cells without the need for Ag stimulation or proliferation. The acquisition of this phenotype did not

require the proliferation typically associated with T cell activation. Furthermore, PLZF transgenic cells maintained a diverse TCR

repertoire, indicating that there was no preferential expansion of specific clones. Functionally, PLZF transgenic CD4 and CD8

lymphocytes were similar to wild type memory cells, in that they had similar requirements for costimulation and exhibited a similar

pattern of cytokine secretion, with the notable exception that transgenic T cells produced significantly increased levels of IL-17.

Whereas transgene-mediated PLZF expression was not sufficient to rescue NKT cell development in Fyn- or signaling lymphocytic

activation-associated protein (SAP)-deficient mice, the acquisition of memory/effector functions induced by PLZF in conventional

T cells was independent of Fyn and SAP. These data show that PLZF is sufficient to promote T cell effector functions and that PLZF

acts independently of SAP- and Fyn-mediated signaling pathways. The Journal of Immunology, 2010, 184: 6746–6755.

Invariant NKT lymphocytes have unique characteristics thatplace them at the interface of the innate and adaptive immunesystems. One defining characteristic is the use of a highly

restricted TCR that is composed of an invariant Va14-Ja18 TCRa-chain that associates with Vb8, Vb7, and Vb2 chains in mice.This TCR recognizes glycolypids presented by the nonclassicalMHC-I molecule CD1d on cortical CD4+CD8+ double positive(DP) thymocytes, triggering the NKT cell differentiation pathway(1, 2). Homotypic interactions of Slamf1 and Slamf6 receptors (3)between cortical DP thymocytes and NKT cell precursors trigger

signals through the adaptor signaling lymphocytic activation(Slam)-associated protein (SAP) and the Src kinase Fyn, whichcontribute to the positive selection of NKT cells (4–8). Down-stream of these signaling pathways, activation of PKCu, Bcl-10and NF-kBp50 have been implicated in the differentiation andsurvival of NKT cells (9–11). After positive selection, NKT cellprecursors undergo a proliferative burst (12) in the thymus thatdepends on the activity of c-myc (13, 14), followed by the acqui-sition of effector/memory functions. In contrast to conventionalT cells, NKT cells that egress from the thymus can rapidly re-spond to stimulation without the need for Ag education in theperiphery. Their invariant TCRa subunit, memory phenotype,presence of NK markers, ability to cosecrete IFN-g andIL-4 among other cytokines, and preformed cytotoxic granzymeB+ granules are unique characteristics that distinguish NKT cellsfrom conventional T cells (15).We and others recently identified that promyelocytic leukemia

zinc finger (PLZF), a member of the bric-a-brac, tramtrack, broadcomplex-poxvirus zinc finger (BTB-ZF) family of transcriptionfactors, is highly expressed during the differentiation of NKT cellsand is essential for the acquisition of effector/memory innate-likefunctions (16, 17). Lack of PLZF expression severely affectedNKT cell development and function. PLZF-deficient NKT cellslose their characteristic memory phenotype, accumulated in lymphnodes instead of the liver, and their function was greatly impaired.In particular, PLZF-deficient NKT lymphocytes lose their uniqueability to cosecrete IL-4 and IFN-g upon ex vivo stimulation anddid not express NK cell-specific markers (16, 17). The failure toproduce cytokines was not a general defect in the cells; however,upon secondary activation, PLZF-deficient NKT cells producedcytokines comparable to wild type (WT) cells (16). Therefore,in the absence of PLZF, NKT cells do not acquire innate effectorfunctions, but instead exhibit behaviors similar to naive conven-tional T cells. The loss of PLZF did not in any measurable wayaffect the function of conventional T cells (16).

*Immunology Program, Sloan-Kettering Institute, †Louis V. Gerstner Jr. GraduateSchool of Biomedical Sciences, and ‡Department of Pediatrics, Memorial Sloan-Kettering Cancer Center; {Weill Graduate School of Medical Sciences of CornellUniversity, New York, NY, 10065; and xDivision of Oncology, Children’s Hospital ofPhiladelphia, Philadelphia, PA 19104

Received for publication March 9, 2010. Accepted for publication April 14, 2010.

This work was supported by National Institutes of Health, National Institute ofAllergy and Infectious Diseases Grant AI059739 and R21AI083769; The Mayand Samuel Rudin Family Foundation; and National Cancer Institute GrantP30-CA 08748, which provides partial support for the Memorial Sloan-KetteringCancer Center Monoclonal Antibody, Flow Cytometry, Glassware Washing corefacilities and the Research Animal Resource Center. D.K. is supported by the RuthL. Kirschstein National Research Service Award (NRSA) Institutional TrainingGrant T32-CA-09149-32. E.S.A. is supported by the Ruth L. Kirschstein NRSAF31CA130744. K.E.N is supported by National Heart, Lung, and Blood InstituteGrant R01HL089745-01.

Address correspondence and reprint requests to Derek B. Sant’Angelo, MemorialSloan-Kettering Cancer Center, 1275 York Avenue, Box 492, New York, NY10065. E-mail address: santangd@mskcc.org

Abbreviations used in this paper: BTB-ZF, broad complex, tramtrack, bric-a-brac–zinc finger; DN, double negative; DP, CD4+CD8+ double positive; Id3, inhibitor ofDNA binding 3; ITK, IL-2–inducible T cell kinase; KO, knockout; LIV, liver; LN,lymph node; M, memory; MFI, mean fluorescence intensity; MSKCC, MemorialSloan-Kettering Cancer Center; N, naive; ND, not detected; PLZF, promyelocyticleukemia zinc finger; SAP, signaling lymphocytic activation-associated protein;Slam, signaling lymphocytic activation; SPL, spleen; TG, transgenic; THY, thymus;WT, wild type.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

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High PLZF expression was also recently shown in a subset ofgd T cells (18–20). These Vg1.1Vd6.3 T cells exhibit innate T cell-like features similar to NKT cells (21) and these innate T cell char-acteristics are dependent on PLZF expression (18, 20). Interestingly,the frequency of these innate gd T cells was dramatically increasedin IL-2–inducible T cell kinase (ITK)-deficient mice (19). The in-creased frequency of the Vg1.1Vd6.3 T cells was subsequentlyshown to be a direct consequence of reduced TCR signaling thatwas controlled by inhibitor of DNA binding 3 (Id3) (20). Interest-ingly, these gd T cells were also shown to require the CD4 T celldeterminant, ThPOK, for normal development (20), suggesting func-tional interplay between these two BTB-ZF proteins.Ectopic expression of PLZF confers an effector/memory phe-

notype in CD4 T cells, associated with the acquisition of doubleIFN-g/IL-4 secretion (17). Similarly, transgenic PLZF expressionin CD8 lymphocytes leads to increased IFN-g secretion (22).Although these studies establish that PLZF expression is suf-ficient to confer an NKT cell-like memory phenotype to T cells,it is not clear whether this transformation corresponds to a lineagechange or to a lower threshold for peripheral activation and ac-quisition of memory as it occurs in WT lymphocytes. Therefore,PLZF transgenic T cells might be functionally equivalent to WTmemory T cells. Moreover, it is also not clear whether PLZFfunctions depend on the Slam/Fyn/Sap signals that are essentialfor the development of NKT cells and other innate T lymphocytes,but not conventional T cell lineages (3, 5).In this work, we characterized a transgenic mouse model in

which PLZF is expressed in all T cells. We observed that ectopicexpression of PLZF inT cells led to the acquisition of amemory-likephenotype. These memory-like T cells accumulated in the liverand acquired high CXCR3 expression. Notably, PLZF-expressingT cells did not induce the upregulation of NK markers, and onlya small proportion of CD4 lymphocytes became double IFN-g/IL-4 secretors, indicating that PLZF is not sufficient to transformall T cells into NKT cell or innate-like T cell functional equivalents.Acquisition of the memory/effector phenotype was spontaneousand not correlated with increased T cell proliferation in vivo orthe amplification of specific T cell clones. PLZF-transgenic T cellshad a cytokine secretion profile similar to WT memory cells withthe exception of a bias toward increased IL-17 secretion. PLZFtransgenic T cells were also more sensitive to TCR stimulation thanwere WT T cells. Finally, transgenic PLZF expression induceda memory-like (innate-like) phenotype in conventional T cells inthe absence Fyn or Sap expression. Therefore, PLZF functions in-dependently of NKT cell-related signaling pathways.

Materials and MethodsMice

The LCK-PLZF transgene was constructed by inserting the ∼2KB geneinto the LCK vector as described (23). Transgene DNA was injected intofertilized C57BL/6 eggs by the Memorial Sloan-Kettering Cancer Center(MSKCC) Mouse Genetics Core Facility. All animal work was done incompliance with the MSKCC Institutional Animal Care and Use Commit-tee guidelines. PLZF-deficient, Fyn-deficient, and Sap-deficient mice havebeen previously described (5, 7, 24).

Flow cytometry

Surface staining was performed in FACS buffer (PBS with 2% heatinactivated FBS) for 20 min on ice using the indicated surface Abs. Dataacquisition was performed on an LSRII cytometer (BD Biosciences, SanJose, CA), and exclusion of dead cells was performed by DAPI staining.Cell doublets were removed by monitoring the pulse width channel. Datawere analyzed using FlowJo software (TreeStar, Ashland, OR). Fixation andpermeabilization for intranuclear staining with PLZF and FoxP3 wasperformed using the FoxP3 eBioscience (San Diego, CA) kit. Intracellularstaining for the analysis of cytokine secretion was performed using the BDBiosciences intracellular kit (San Jose, CA).

Abs

CD1d:PBS57 tetramers, used for some experiments, were obtained from theNational Institutes of Health Tetramer Core Facility. The Armenian hamsteranti-human PLZF (Mags-21F7) mAb was generated by the MSKCC mAbCore Facility using standard approaches by immunizing animals with a com-bination of peptides covering the amino, carboxyl, and hinge regions of theprotein. Other Abs were obtained from BD Biosciences, eBioscience, or theMSKCC Ab Core Facility. The following Ab clones were used: anti-CD4(RM4-5), anti-CD8a (53.6.7), anti-TCRb (H57.597), anti-CD44 (IM7),anti-CD62L (MEL-14), anti-CD69 (H1.2F3), anti-CXCR3 (CXCR3), anti-FoxP3 (NRRF-30), anti-Ly6C (AL-21), anti-CD122 (TM-b1), anti-NKG2D(CX5), anti-NK1.1 (PK136), anti-CD94 (MFL3), anti-DX5 (DX5), anti-2B4(2B4), FITC conjugated anti-Vb3 (KJ25), Vb4 (KT4), Vb5.1/5.2 (MR9-4),Vb6 (RR4-7), Vb7 (TR310), Vb8.1/8.2 (F23.1), Vb9 (MR10-2), Vb10b(B21.5), Vb12 (MR11-1), Vb13 (MR12-3), Vb14 (MR14-2), Va2 (B20.1),Va3.2b,c (RR3-16), Va8 (B21.14 and Va11.1/11.2b,d (RR8-1). For theanalysis of intracellular cytokines: anti–IFN-g (XMG1.2), anti–IL-4 (11B11),anti-GMCSF (MP1-22E9), anti–IL-17 (eBio17B7), anti–IL-2 (JESd-544). Abswere used with different fluorochrome conjugations: FITC-, PE-, PERCP-Cy5.5-, PE–Cy7-, APC- and APC-Cy7.

Analysis of CDR3 lengths

Naive (CD44loCD62Lhi) and memory (CD44hiCD62Llo) CD4 and CD8lymphocytes were purified by cell sorting by the MSKCC FACS facility.mRNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA),and cDNA was obtained using the reverse transcriptase Superscript-III 1ststrand kit (Invitrogen). Amplification of CDR3 lengths from the variableVb5 and Vb3 regions was performed by PCR using the following primers:6-FAM-labeled-reverse Cb mCv (59-CTTGGGTGGAGTCACATTTCTC-39); forward mVb3 (59-CCTTGCAGCCTAGAAATTCAG-39); forwardmVb5 (59-ACAGTTTGATGACTATCACTCTG-39). PCR was performedusing 40 cycles with a 55˚C annealing temperature. The product of thePCR reaction was between 150 and 250 bp. Size resolution was deter-mined by the MSKCC sequencing facility.

Proliferation

Analysis of in vivo proliferating lymphocytes was performed using theFITC BrDU flow kit (BD Biosciences) according to the manufacturer’sinstructions. Mice were injected i.p. with a single dose of BrDU (1 mg);18 h later, mice were sacrificed, lymphocytes were stained for surfacemarkers, and the percentage of BrDU positive lymphocytes was identifiedby FACS.

Analysis of T cell proliferation in vitrowas performed usingmagneticallypurified Thy1.2+ labeled T cells using MACS columns (Miltenyi Biotec,Auburn, CA). After purification, cells were labeled with 5 mg/ml CFSE byincubation at 37˚C for 10 min in medium. Cells were subsequently washedand plated in round-bottom 96-well plates containing plate-bound anti-CD3 Abs at different concentrations. Stimulation was performed in thepresence or absence of soluble anti-CD28 (5 mg/ml) Abs. After 72 h in-cubation, cells were stained for surface markers to identify the differentlymphocyte populations and analyzed for CFSE dilution by FACS.

Cytokine secretion

CD4 and CD8 lymphocytes were purified by cell sorting and incubated withplate-bound anti-CD3 (5 mg/ml) in the presence or absence of soluble anti-CD28 (5mg/ml) for 48 h. Secreted cytokines were detected with cytokinebead arrays (BD Biosciences). IL-17 was detected using the eBioscienceIL-17 ELISA kit. For intracellular cytokine detection, total splenocyteswere stimulated with PMA (100 ng/ml) plus ionomycin (50 ng/ml) fora total of 6 h. One hour after stimulation, brefeldin-A was added to theculture. After incubation, cells were stained for surface markers, fixed,stained for intracellular cytokine contents, and analyzed by FACS as pre-viously described.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (La Jolla, CA) soft-ware. All samples were analyzed using unpaired, two-tailed Student t tests.

ResultsNormal NKT cell and T cell differentiation in Lck-PLZFtransgenic mice

To test the effects of ectopic PLZF expression on T cell function,we generated PLZF transgenic mice in which expression of PLZFwas restricted to T cells by the control of the proximal Lck

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promoter. Four original founders were produced that expressed thetransgene at different levels, as observed by intranuclear stainingand FACS analysis (data not shown). The two founders expressingthe highest levels of PLZF were used for these studies. Expressionof PLZF in transgenic mice was restricted to T cells (Fig. 1A).Overall T cell development appeared to be normal (Fig. 1B), andthe total number of TCR-positive lymphocytes in transgenic ani-mals was similar to that observed in WT mice (Fig. 1C). Therewas, however, a statistically significant skewing of the CD4/CD8ratio in the thymus in favor of CD8+ T cells. In contrast, the CD4/CD8 ratio was reversed with more CD4+ than CD8+ T cells in thespleen and lymph nodes (Fig. 1D). A similar skewing was seen inmice with ectopic expression of MHCII on thymocytes as a con-sequence of T cell-specific ectopic expression of CIITA (25). In-terestingly, a sizable percentage of the CD4 T cells in these CIITAtransgenic mice express PLZF (26). In the CIITA transgenic mice,the increase of CD8SP in these mice was shown to be due toincreased IL-4 signaling, presumably produced by the PLZF-

expressing T cells (25). The percentage of CD4 T cells was largelysimilar in tissues from WT and PLZF transgenic mice, with a sig-nificant difference found only in the spleen (Fig. 1E). The per-centage of PLZF transgenic CD8 T cells, however, was signifi-cantly increased in the thymus and reduced in the lymph nodesand spleen (Fig. 1F). Finally, FoxP3+ regulatory T cells werepresent at WT levels in the spleen (Fig. 1G).Interestingly, NKT cell percentages were not increased in the

thymus, lymph nodes, spleen, or liver of PLZF transgenicmice (Fig.2A). Furthermore, the capacity of NKT cells from PLZF transgenicmice to cosecrete IL-4 and IFN-g upon ex vivo stimulation withPMA/ionomycin was not altered (Fig. 2B). Finally, the frequency ofNKT cells in various tissues was not significantly altered in PLZFtransgenic mice (Fig. 2C). Therefore, ectopic expression of PLZFin addition to expression from the endogenous locus skews T celldevelopment, but does not appear to alter NKT cell developmentor cytokine production.

PLZF transgenic T cells have a memory phenotype but do notexpress NK markers

Consistent with previous reports (17, 22), transgenic PLZF ex-pression in both CD4 and CD8 T cells led to upregulation of CD44and downregulation of CD62L, a phenotype characteristic ofNKT cells, innate-like T cells, and memory T cells. TCRb+ T cellswith the activated phenotype were clearly more prominent in pe-ripheral tissues compared with the thymus. In particular, TCRb+

T cells in the liver were nearly all CD62Llo and CD44hi, consistentwith selective enrichment in nonlymphoid organs (Fig. 3A).CD27, a member of the TNFR family, is downregulated on acti-vated T cells (27). PLZF transgenic T cells, however, expressedCD27 similarly to WT T cells (Fig. 3B). KLRG1, a killer celllectin-like receptor, has been shown to be expressed on Ag-experienced T cells (28). Again, expression of this marker ontransgenic PLZF T cells was essentially identical to expressionon T cells from WT littermates (Fig. 3C). Interestingly, we foundthat IL-7R (CD127) expression was markedly increased on bothCD4+ and CD8+ PLZF transgenic T cells compared with T cellsfrom WT littermates (Fig. 3C–E). Upregulation of IL-7R occurs

FIGURE 1. A, FACS analysis showing intranuclear PLZF expression in

lymphocytes from WT and PLZF transgenic (TG) spleens. B, FACS

analysis for CD4 and CD8 expression from WT and PLZF TG mice in the

indicated tissues. C, Total TCRb+ cells in the thymus (THY), lymph nodes

(LNs), spleen (SPL), and liver (LIV) from WT (white) or PLZF TG (black)

mice. D, The ratio of CD4 to CD8 single positive thymocytes in the

thymus THY or CD4+ to CD8+ T cells in the LNs, SPL, or LIV in WT

(white) or PLZF TG (black) mice. E, Percentage of TCRbhi CD4SP or

TCRb+ CD4+ T cells or (F) TCRbhi CD8SP or CD8+ T cells in WT and

transgenic mice in the indicated tissues. Percentages exclude NKT cells. G,

Intranuclear FACS analysis for FoxP3 expression within TCRb+ spleen

T cells fromWTand PLZF TG mice. The p values for C–F were calculated

using two-tailed Student t tests. In all panels, the numbers within the

graphs represent the percentage of events within the gates. Data are rep-

resentative of four or more independent experiments.

FIGURE 2. A, FACS analysis showing NKT cells, identified with CD1d/

PBS57-tetramers and anti-TCRb, in THY, LNs, SPL, and LIV. B, FACS

analysis of intracellular IFN-g and IL-4 levels in NKT cells from WT and

PLZF TG mice either not stimulated or after ex vivo PMA and ionomycin

stimulation for 5 h. Numbers represent the percentage of events within the

gates. C, Percentage of NKT cells in the indicated tissues in WT (white) or

PLZF TG (black) mice. The p values were calculated using two-tailed

Student t tests. Data are representative of four or more experiments.

6748 PLZF FUNCTIONS INDEPENDENTLY OF Sap AND Fyn

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on long-lived memory T cells (29, 30). IL-7 is also important forthe expansion and survival of NKT cells (31, 32). Overall, theseresults demonstrated that ectopic expression of PLZF results inthe upregulation of some, but not all, markers associated withmemory/effector T cells.Other markers associated with a memory T cell phenotype were

differentially upregulated on CD8 or CD4 transgenic T cells. Ly6Clevels, for example, were elevated on CD4 SP thymocytes and bothCD4 and CD8 T cells in the spleen (Fig. 4A). A subset of CD8T cells in the spleen upregulated CD122 (IL-2R b-chain), which isnormally expressed on NK cells and some activated T cells.Nearly all PLZF transgenic CD8 T cells were also found to ex-press CXCR3. This chemokine receptor is thought to be involvedin directing T cells to sites of inflammation (33). Interestingly,

CXCR3 was also recently shown to retain NKT cells in the thy-mus (34). PLZF-expressing T cells did not show upregulation ofNK/NKT cell-associated markers, such as NKG2D, NK1.1, CD94,DX5, and 2B4. This finding suggests that ectopic expression ofPLZF was insufficient to convert conventional T cells into bonafide NKT cells (Fig. 4B).

PLZF-expressing memory T cells are not proliferating andhave a variable repertoire

Conventional T cells generally acquire a memory phenotype afterundergoing Ag-induced proliferation. We next tested whether theacquisition of thememory phenotype by transgenic PLZF expressioninvolved a similar process. For this purposewe analyzed the prolifer-ation of T cells in vivo by BrdU incorporation. Eighteen hours aftera single injection of BrdU, the thymus and spleen were isolated andincorporation ofBrdU inT cellswas analyzed by FACS.As a positivecontrol for the experiment,BrdU incorporation in highly proliferatingdouble negative thymocytes was analyzed. Our results showed thatthe acquisition of the memory phenotype in T cells was not relatedto increased proliferation in PLZF transgenic mice (Fig. 5A).Although PLZF transgenic T cells did not seem to be actively

proliferating in vivo, it was still possible that the memory phenotypewas the consequence of a selective expansion of certain T cell clones.This expansion would lead to a restricted variability of the T cellrepertoire. To evaluate this possibility, we analyzed the distributionsof the CDR3 lengths (spectratyping) of Vb3 and Vb5 TCRb sub-units from naive and memoryWTand PLZF transgenic CD4 T cells.Our analysis showed that the distribution of the CDR3 lengths inPLZF transgenic CD4 T cells followed a Gaussian curve as in WTnaive T cells, indicating a high variability of the TCR (Fig. 5B). Asimilar result was observed for CD8 lymphocytes (data not shown).Clonal expansionwas also examined by comparingTCRb andTCRausage on CD4+ T cells from the spleens of WTand PLZF transgenicmice. No significant differences were seen for CD4 T cells (Fig. 5C)or for CD8 T cells (data not shown).

FIGURE 3. A, FACS analysis of TCRb+ CD44 and CD62L levels on

gated CD4 or CD8 SP thymocytes or T cells from the indicated tissues

from WT and PLZF TG mice. The numbers in the graph represent the

frequency of events within the gates. B, FACS analysis of CD1d-tetramer

negative, gd TCR2, CD3+ spleen T cells from WT or PLZF TG mice

stained with Abs against CD62L and CD27 or (C) KRG1 and IL-7R.

Numbers indicate the percentage of cells in each region. D, Histogram

overlay of IL-7R levels on CD1d-tetramer negative, gd TCR2, CD3+

spleen T cells from WT (filled), and PLZF TG (solid black line) mice.

E, MFI for IL-7R expression from three independent experiments. The p

values were calculated using two-tailed Student t tests. MFI, mean fluo-

rescence intensity.

FIGURE 4. A, FACS analysis of CD4+ or CD8+ TCRb+ T cells from the

thymus and spleen of PLZF transgenic (solid black line) or WT mice (thin

dotted line). Cells were stained with Abs for Ly6C, CD122, and CXCR3.

B, FACS analysis for the expression of NKT cell-associated marker ex-

pression on TCRb+ spleen cells from PLZF transgenic mice (thick black

line), WT mice (dotted line) and WT NKT cells (shaded). Data are repre-

sentative of more than four independent experiments.

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Altered cytokine secretion pattern in PLZF transgenic T cells

To assess the functional effects of ectopic PLZF expression onT cells, we next evaluated the cytokine secretion patterns followingPMA/ionomycin stimulation. Our first observation was that theeffect of PLZF expression in CD4 T cells was different from that inCD8 T cells. For example, PLZF expression conferred the ability tosecrete IL-4 to CD4 but not to CD8 T cells (Fig. 6A). Furthermore,in contrast to WT NKT cells, only a small fraction of transgenicCD4 lymphocytes were able to produce both IL-4 and IFN-g.More than half of the CD8 T cells from PLZF transgenic mice

produced IFN-g secretion; however, the frequency of IFN-gproducing CD4 T cells did not increase. PLZF expression inCD4 and CD8 lymphocytes led to a reduction of IL-2 secretion,but no change in the percentage of IFN-g/IL-2 double producers.PLZF expression led to increased GM-CSF secretion in CD4 andCD8 lymphocytes and to an increased proportion of IL-17–secreting CD8 lymphocytes (Fig. 6A).Memory T cells are less dependent on costimulation for activation

than are naive T cells (35, 36). To analyze the dependency ofcostimulation for cytokine secretion in PLZF transgenic T cells, wesorted WT naive, WT memory, and PLZF transgenic CD4 and CD8lymphocytes and analyzed their cytokine secretion followingstimulation with plate-bound anti-CD3 Abs in the presence or ab-sence of soluble anti-CD28 Abs (Fig. 6B). This analysis allowedthe direct comparison of the functionality of PLZF transgenicT cells to WT naive and memory T cells. PLZF transgenic T cellswere functionally equivalent to WT memory cells, with respect tothe dependency for costimulation. Moreover, levels of IL-4, IL-2, GM-CSF, IFN-g, and IL-3 production were similar betweenthese two populations. The notable exception was IL-17, whichwas substantially increased in PLZF-expressing CD8 lymphocytesrelative to the other groups, suggesting a direct role for PLZF inthe acquisition of the IL-17–secreting phenotype (Fig. 6B).Finally, we explored the mechanistic basis for the increased

capacity of PLZF transgenic CD4 T cells to produce IL-4 upon pri-mary activation. NKT cells are able to rapidly produce IL-4, in partbecause of the constitutive transcription of the IL-4 gene. Indeed,IL-4 mRNA is easily detected in NKT cells, although IL-4 proteinis produced only after activation (37). The presence of preformedIL-4 mRNA is dependent on the expression of PLZF (16). To testfor the presence of IL-4 mRNA, we crossed the LCK-PLZF to the“4get” IL-4 GFP reporter mice (38). NKT cells in these mice havebeen shown to constitutively express GFP (39). Only a small in-crease of GFP-expressing CD4 T cells was found in 4get x PLZFtransgenic mice (Fig. 6C). Therefore, it is possible that PLZF-induced constitutive activity of the IL-4 gene may account forsome of the increased production of IL-4, but it certainly doesnot account for the entire increase.

PLZF transgenic T cells are more responsive to stimulation

Memory T cells are more responsive to TCR stimulation than arenaive cells (40, 41). To test how T cells were affected by trans-genic expression of PLZF, we analyzed the proliferation of T cellsafter in vitro stimulation with variable doses of plate-bound anti-CD3 (Fig. 7). T cells were magnetically purified according toThy1.2 expression, stained with CFSE, and stimulated with dif-ferent concentrations of plate-bound anti-CD3 Abs in the presenceor absence of soluble anti-CD28 for 72 h. Proliferation was ana-lyzed by dilution of CFSE staining. We observed that at the high-est anti-CD3 concentrations (2.5 and 0.5 mg/ml), proliferation wassimilar between WT and PLZF transgenic CD4 and CD8 T cells.In these conditions, costimulation with abs against CD28 slightlyincreased the proliferation of T cells. This effect was also simi-larly observed in WT and PLZF transgenic T cells (Fig. 7). Undersuboptimal conditions of stimulation (0.1 mg/ml); however, onlyPLZF transgenic CD4 and CD8 T cells were able to proliferate inthe presence of costimulation, suggesting a lower threshold foractivation.

PLZF transgenic expression does not rescue the NKT celldifferentiation defect in Fyn- and Sap-deficient mice

Differentiation of NKT cells depends on the homotypic interactionandactivationof theSLAMfamilyproteins,which signal throughFynand Sap (4–7). Although PLZF expression is necessary for efficient

FIGURE 5. A, BrdU was administered to WT or PLZF TG mice by i.p.

injection. Lymph nodes, spleens, and thymuses were harvested 18 h later.

Single-cell suspensions were stained with Abs against TCRb, CD4, and

CD8 and then fixed, treated with DNAse, and stained with Abs against

BrdU. CD42CD82 DN thymocytes are shown as a positive control for

BrdU incorporation. Numbers indicate the percentage of cells that incorpo-

rated BrdU. B, Spectratyping analysis of the TCRb CDR3 length in Vb3

and Vb5 TCRs from sorted CD62L+CD442 (WT naive) or CD62L2CD44+

(WT memory) CD4+ T cells from WT mice or total CD4+ T cells from

PLZF TG mice. These data are representative of two independent experi-

ments. C, Percentage of TCRb+ cells expressing TCRs with the indicated

TCRb or TCRa from spleens of WT (white) or PLZF TG (black) mice.

Approximately 30,000 CD4+ events were collected per experiment. Data

represent the average from two different mice. CD1d tetramer positive

NKT cells were excluded from the analysis. DN, double negative.

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differentiation of NKT cells, it is not clear whether PLZF actsdownstream of the Slam/Sap/Fyn pathway. We previously showedthat the fewNKT lymphocytes that differentiate inFyn-deficientmicehave PLZF expression levels equivalent toWTNKT cells (16). How-ever, these NKT cells may represent “escapees” that differentiatedthrough the Slam/Sappathway, but in amanner independent of Fyn. Itis still therefore possible that PLZF expression and function lie down-stream of these signaling cascades.

To test whether PLZF functions are dependent on Fyn or Sapactivity, we generated PLZF transgenic mice that are Fyn- or Sap-deficient by breeding, and we analyzed whether PLZF expressioncould rescue the defects in NKT cell differentiation. As a control,we introduced the LCK-PLZF transgene into PLZF-deficient miceby breeding. Transgenic expression of PLZF in PLZF knockout(KO) mice did not fully restore NKT cell development (Fig.8A). There was, however, a consistent increase in the frequency

FIGURE 6. A, Spleen cells from WT and PLZF TG

mice were activated with PMA and ionomycin for

a total of 5 h. Brefeldin A was added to the culture for

the last 4 h. Cells were harvested stained with Abs

against the TCR, CD4, and CD8, and then made per-

meable followed by staining with Abs against GM-

CSF, IL-4, and IFN-g or IL-2, IL-17, and IFN-g.

Numbers indicate the percentage of cells in each

quadrant. Data are representative of four independent

experiments. B, Cytokine secretion after in vitro

stimulation of sorted WT naive (N) CD62L+CD442,

WT memory (M) CD62L2CD44+ or PLZF TG CD4

and CD8 lymphocytes with plate-bound anti-CD3 in

the presence or absence of soluble anti-CD28. All data

points correspond to n $ 6. C, FACS analysis of CD4+

T cells from WT, NKT cells from “4get” IL-4 GFP

reporter mice (4get NKT), CD4+ T cells from 4get

mice (4get CD4), and CD4+ T cells from 4get/PLZF

transgenic mice (4get PLZF TG). Percentage of cells in

each quadrant is shown. Two independent experiments

gave similar results. M, memory; N, naive.

FIGURE 7. Analysis of CFSE dilution after ex vivo

stimulation of magnetically purified T cells from WTor

PLZF TG mice. Proliferation was analyzed at 72 h after

stimulation with different concentrations of plate-bound

anti-CD3 in the presence or absence of soluble anti-

CD28 Abs. Analysis is shown on electronically gated

CD4 and CD8 lymphocytes. Data are representative of

three independent experiments.

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of CD44hiCD69hi NKT cells in the thymus of the PLZF-deficient,PLZF transgenic mice compared with PLZF deficient mice with-out the transgene (Fig. 8A). The partial reconstitution was moreclearly seen in the liver (Fig. 8B). Whereas liver NKT cells in thePLZF-deficient, PLZF transgenic mice acquired a mature CD44hi

CD69hi phenotype and accumulated at a significantly higher fre-quency (Fig. 7C) compared with PLZF-deficient mice without thetransgene, they were not reconstituted to the levels seen in WT

mice. The partial rescue is likely related to the level of PLZFexpression from the transgene. PLZF expression from the trans-gene is essentially equivalent to WT levels in mature NKT cells inthe liver (Fig. 7D). Immature stage 1 WT NKT cells, however,express substantially higher levels than thymocytes from thePLZF-deficient, PLZF transgenic mice. Consistent with a require-ment for high levels of PLZF specifically during NKT cell de-velopment, mice carrying one WT and one gene-targeted allele ofPLZF mice also exhibit a partial reduction in the number ofNKT cells (data not shown).Fyn-deficient mice have a severe block in NKT cell development

(6, 7, 42). Some NKT cells, however, mature and accumulate in theliver (Fig. 8E). Introducing the PLZF transgene into Fyn-deficientmice, however, had no effect on the development of NKT cells (Fig.8E, 8F). The absence of SAP has an even more profound effect onthe development of NKT cells. Although a few immature NKT cellscan be detected in the thymus, there are none in the liver. Like inthe Fyn-deficient mice, the PLZF transgene had no effect on NKTcell development in the absence of SAP (Fig. 8G).

Memory/effector functions in PLZF transgenic T cells areinduced independently of Fyn and SAP

Transgenic expression of PLZF failed to rescue NKT cell differ-entiation in the Fyn- and SAP-deficient mice. Therefore, the activ-ity of PLZF in NKT cells cannot supersede the requirement forsignaling via these twomolecules.We next asked whether the spon-taneous acquisition of effector/memory phenotype and function asa consequence of ectopic expression of PLZF in conventionalT cells was also dependent on Fyn and/or SAP signaling pathwaysas previously shown for other innate like T cells, such as NKT cells(5–7) and Vg1.1Vd6.3 gd T cells (20). T cells from the spleensand livers of Fyn-deficient, PLZF-transgenic mice were predom-inately CD44hiCD62Llo compared with transgene-negative, Fyn-deficient littermates (Fig. 9A). T cells from SAP-deficient, PLZF-transgenic mice also exhibited an activated phenotype comparedwith PLZF transgene-negative littermates (Fig. 9B).Transgenic PLZF expression also led to alteration of the cytokine

secretion pattern in T cells from these strains. In particular, therewas a clear increase in the percentage of IFN-g expressing CD8T cells from the PLZF transgenic both in the absence of Fyn (Fig.9C) and in the absence of SAP (Fig. 9D). There also was a slightincrease in the percentage of IL-4 producing CD4 T cells in theabsence of these two signaling molecules (Fig. 9C, 9D). Notably,even the small increase in the frequency of IL-4 and IFN-g doublesecretors was still observed in both Fyn-deficient and SAP-deficient PLZF transgenic CD4 T cells. These data indicate that,in contrast to NKT cells, PLZF-mediated phenotypic changes ofconventional T cells are not dependent on Fyn or SAP.

DiscussionThe transcription factor PLZF is necessary for the development ofNKT cell effector functions and the characteristic activated phe-notype of these innate T cells (16, 17). Furthermore, ectopic ex-pression of PLZF in conventional T cells has been shown toinduce a memory-like phenotype similar to NKT cells (17, 22).Using a transgene to drive T cell-specific ectopic expression ofPLZF, we further explore how PLZF controls the development ofNKT cells and also the effects of PLZF expression on CD4 andCD8 T cell function.Consistent with previous reports using different PLZF transgenic

models (17, 22), we observed that transgenic expression of PLZFis sufficient to confer an effector/memory phenotype to T cells.We observed that T cells with this activated phenotype accumu-lated in the spleen and liver, but were almost undetectable in the

FIGURE 8. NKT cells in the (A) thymus and (B) liver from PLZF-

deficient mice (PLZF KO) or PLZF deficient mice carrying the Lck-PLZF

transgene (PLZF KO/PLZF TG) were identified with CD1d tetramer and

anti-TCRbAb. Number indicates the percentage of NKT cells in each tissue.

CD44 and CD69 expression on the NKT cells in each tissue is shown in the

bottom panels of (A) and (B). The numbers indicate the percentage of cells in

each quadrant. C, A statistically significant (p = 0.0029) increase in the

percentage of NKT cells was observed in PLZF-deficient mice carrying

the PLZF transgene. n = 3 Fyn-deficient, 3 SAP-deficient, 5 Fyn KO x PLZF

TG, 5 SAP KO x PLZF TG.D, PLZF levels for WT (dotted line) and PLZF-

deficient mice carrying the PLZF transgene (solid line) in stage 1 thymic

NKT cells (CD44lo) and liver NKT cells. E, FACS analysis showing the

percentage and phenotype of NKT cells in the livers of Fyn-deficient mice

and Fyn-deficient mice carrying the PLZF transgene. F, The percentage of

NKT cells in the livers of Fyn-deficient (TG2) and Fyn-deficient PLZF

transgenic (TG+) mice. n = 4. G, FACS analysis showing the percentage

and phenotype of NKT cells in the livers of SAP-deficient mice and SAP-

deficient mice carrying the PLZF TG. Numbers indicate the percentage in

each region. ND, not detected.

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thymus. We also found that a large percentage of CD8 T cellsupregulated CD122 (IL-2Rb), suggesting that these cells wereresponsive to IL-15, similar to NK (43) and NKT cells (32). In-terestingly, nearly all transgenic CD8 T cells expressed high levelsof CXCR3. This chemokine receptor was recently shown to benecessary for retaining NKT cells in the thymus (34), although itis generally associated with T cell migration to sites of inflamma-tion (33). The conventional T cell repertoire in PLZF- transgenicmice showed a similar degree of variability as compared with WTT cells, as measured by the distribution of the CDR3 lengths ofTCRb molecules. Furthermore, there was no obvious increase ofproliferation of PLZF transgenic T cells in vivo. Acquisition of theeffector memory phenotype and functions of PLZF transgenicT cells, therefore, appears to be spontaneous and not the conse-quence of amplification of certain clones or immune activation.Previous reports suggested that PLZF overexpression was suffi-

cient to confer innate functions to T cells (22), providing charac-teristics that are unique to NKT cells, such as the double productionof IFN-g and IL-4 cytokines (17). In this study, we performed a di-rect comparison betweenWTmemory T cells and PLZF transgenicT cells. These experiments showed that, overall, the function ofT cells overexpressing PLZF was similar to WT memory cells inregard to cytokine secretion and the requirements for costimulation.Interestingly, the effects of transgenic PLZF expression on cytokinesecretion were not uniform. For example, a large percentage ofPLZF transgenic CD8 became IFN-g secretors, yet thepercentage of IFN-g secreting CD4 T cells was not substantiallyincreased. Alternatively, PLZF transgenic CD4 T cells producedsubstantially more IL-4, but CD8 T cells did not. Furthermore,a small percentage of CD4 T cells became IFN-g/IL-4 double secre-tors. The effects of PLZF also varied within T cell lineages. For

example, only∼20% of CD8 T cells secreted GM-CSF and of these,approximately half also secrete IFN-g. Furthermore, transgenicPLZF expression resulted in ∼5% of CD8 T cells to secrete IL-17. Overall these results show that the specific effects of PLZFexpression are highly variable in different T cells, but it is sufficientto confer a memory phenotype to all T cells.Endogenous PLZF expression and transgenic PLZF expression

clearly differ both in the timing and the level of expression.Transgenic PLZF expression did not, however, result in a grosschange in the selection of T cells, NKT cells, regulatory T cells, orgdT cells (20) as their numbers and proportions were similar toWT mice. The failure to increase the frequency of NKT cells andVg1.1+Vd6.3+ T cells, which normally express PLZF, suggeststhat selection of these cells via the TCR is the predominant factorin controlling their overall frequency. Interestingly, transgene-driven expression of PLZF only partially restored NKT cell de-velopment in PLZF deficient mice. The failure to restore normalNKT cell development was likely due to lower levels of PLZFexpression from the transgene compared with the endogenousgene. This finding is consistent with the finding that haploinsuffi-ciency of PLZF results in a significant defect in NKT cell de-velopment (E.S. Alonzo and D.B. Sant’ Angelo, unpublishedobservations). Therefore, extremely high levels of PLZF expres-sion at the most immature stage of NKT cell development appearsto be necessary to establish the programming required for theinnate T cell lineage.The mechanisms involved in the acquisition of a memory

phenotype by PLZF expression in T cells without Ag stimulationare not known. Spontaneous acquisition of memory has beenidentified in WT CD8 lymphocytes, because memory cells can bedetected in germ-free mice. This phenotype might be related to ho-meostatic proliferation and maintenance of memory features (44).It is possible, therefore, that PLZF expression drives a similarprocess in NKT cells, however we did not detect increased pro-liferation of lymphocytes as a result of PLZF expression. Further-more, NKT cells are not dependent on PLZF to undergoproliferation, as has been shown in PLZF-deficient mice (16,17). Indeed, PLZF has been reported to inhibit cell cycle progres-sion (45, 46) and to cause cell quiescence (47) and suppression ofcell growth (48). Therefore, it is possible that the NKT cell pro-liferative burst is actually dependent on the rapid downregulationof PLZF that we reported (16).We and others have shown that residual NKT cells that appear in

Fyn- and SAP-deficient mice express PLZF (16, 17). This findingsuggests that PLZF expression is not sufficient to drive the fulldifferentiation of NKT cells. To more directly test this importantaspect of NKT cell development, we introduced a T cell-specificPLZF transgene into both Fyn- and SAP-deficient mice. Data fromthese mice clearly showed that expression of PLZF did not rescuethe development of NKT cells in the absence of Fyn- or SAP-mediated signaling.In contrast, in the absence of Fyn or SAP, PLZF transgenic

T cells still acquired an activated/memory phenotype as seen by theupregulation of CD44 and the loss of CD62L. Furthermore, thetransgenic cells retained the increased capacity to produce cyto-kines upon primary activation. Therefore, the effects of ectopicexpression of PLZF on conventional T cells was independent ofsignalling mediated by SAP and Fyn. These data indicate that thefunction of PLZF is independent of SAP and Fyn signals.Recent data have shown that loss of RORgt (49, 50) or the E

protein HEB (51) results in a near complete loss of NKT cells. Theloss of NKT cells in these mutant mice, however, was due toa failure to rearrange the invariant TCR, and development wasfully restored by introducing a Va14 TCR transgene (50, 51). E2A

FIGURE 9. CD44 versus CD62L expression of electronically gatedCD3+

T cells from spleens or livers from (A) Fyn-deficient or Fyn-deficient PLZF

TG mice and (B) SAP-deficient or SAP-deficient PLZF TG mice. C and D,

Intracellular staining for IFN-g versus IL-4 after 5 h stimulation with PMA

and ionomycin in electronically gated CD4 and CD8 T cells from the in-

dicated strains. The numbers in the graphs indicate the frequency of events

within the respective gates. Data are representative of four independent

experiments.

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is also dispensable for the development of NKT cells (51); this wassurprising, considering the role that Id3 and E2A appear to play inthe development of PLZF-expressing gd T cells (20, 52). HighTCR avidity for self ligands has long been proposed as a criticalfactor for the development of NKT cells (53). Consistent with thishypothesis was the finding that ITK-deficient and SLP76:Y145Fmutant mice, which have reduced TCR signaling capacities, havesignificant defects in NKT cell development (54, 55). Furthermore,PMA plus ionomycin stimulation of DP thymocytes followed by 5d in culture with OP9-dl1 cells was shown to induce PLZF in gdT cells (18). Both ITK-deficient and SLP76:Y145F mutant mice,however, actually have a massive increase of PLZF-expressing gdT cells (19, 20). It is therefore difficult to reconcile a strength ofTCR signaling model with the induction of PLZF. Combined withour new data showing that PLZF functions independently of Fynand SAP, it seems likely that the factors that control the cell-specific expression of PLZF may require the identification of novelsignaling pathways that are potentially unique to innate T cells.

AcknowledgmentsWe thank Tom Martillotti for excellent technical support, Dr. Peter Savage

for providing reagents and advice for TCR spectratyping analysis, Drs. Lisa

Denzin and AimeeM. Beaulieu for critical review of the manuscript, and the

National Institutes of Health Tetramer Facility at Emory University for pro-

viding CD1d tetramers.

DisclosuresThe authors have no financial conflicts of interest.

References1. Zhou, D., J. Mattner, C. Cantu, 3rd, N. Schrantz, N. Yin, Y. Gao, Y. Sagiv,

K. Hudspeth, Y. P. Wu, T. Yamashita, et al. 2004. Lysosomal glycosphingolipidrecognition by NKT cells. Science 306: 1786–1789.

2. Speak, A. O., V. Cerundolo, and F. M. Platt. 2008. CD1d presentation of gly-colipids. Immunol. Cell Biol. 86: 588–597.

3. Griewank, K., C. Borowski, S. Rietdijk, N. Wang, A. Julien, D. G. Wei,A. A. Mamchak, C. Terhorst, and A. Bendelac. 2007. Homotypic interactionsmediated by Slamf1 and Slamf6 receptors control NKT cell lineage de-velopment. Immunity 27: 751–762.

4. Pasquier, B., L. Yin, M. C. Fondaneche, F. Relouzat, C. Bloch-Queyrat,N. Lambert, A. Fischer, G. de Saint-Basile, and S. Latour. 2005. DefectiveNKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product. J. Exp. Med. 201: 695–701.

5. Nichols, K. E., J. Hom, S. Y. Gong, A. Ganguly, C. S. Ma, J. L. Cannons,S. G. Tangye, P. L. Schwartzberg, G. A. Koretzky, and P. L. Stein. 2005. Reg-ulation of NKT cell development by SAP, the protein defective in XLP. Nat.Med. 11: 340–345.

6. Eberl, G., B. Lowin-Kropf, and H. R. MacDonald. 1999. Cutting edge: NKT celldevelopment is selectively impaired in Fyn- deficient mice. J. Immunol. 163:4091–4094.

7. Gadue, P., N. Morton, and P. L. Stein. 1999. The Src family tyrosine kinase Fynregulates natural killer T cell development. J. Exp. Med. 190: 1189–1196.

8. MacDonald, H. R., and M. P. Mycko. 2007. Development and selection ofValpha l4i NKT cells. Curr. Top. Microbiol. Immunol. 314: 195–212.

9. Cannons, J. L., L. J. Yu, B. Hill, L. A. Mijares, D. Dombroski, K. E. Nichols,A. Antonellis, G. A. Koretzky, K. Gardner, and P. L. Schwartzberg. 2004. SAPregulates T(H)2 differentiation and PKC-theta-mediated activation of NF-kappaB1. Immunity 21: 693–706.

10. Stanic, A. K., J. S. Bezbradica, J. J. Park, L. Van Kaer, M. R. Boothby, andS. Joyce. 2004. Cutting edge: the ontogeny and function of Va14Ja18 natural Tlymphocytes require signal processing by protein kinase C theta and NF-kappaB. J. Immunol. 172: 4667–4671.

11. Stanic, A. K., J. S. Bezbradica, J. J. Park, N. Matsuki, A. L. Mora, L. Van Kaer,M. R. Boothby, and S. Joyce. 2004. NF-kappa B controls cell fate specification,survival, and molecular differentiation of immunoregulatory natural T lympho-cytes. J. Immunol. 172: 2265–2273.

12. Benlagha, K., T. Kyin, A. Beavis, L. Teyton, and A. Bendelac. 2002. A thymicprecursor to the NK T cell lineage. Science 296: 553–555.

13. Dose, M., B. P. Sleckman, J. Han, A. L. Bredemeyer, A. Bendelac, and F. Gounari.2009. Intrathymic proliferation wave essential for Valpha14+ natural killer T celldevelopment depends on c-Myc. Proc. Natl. Acad. Sci. USA 106: 8641–8646.

14. Mycko, M. P., I. Ferrero, A. Wilson, W. Jiang, T. Bianchi, A. Trumpp, andH. R. MacDonald. 2009. Selective requirement for c-Myc at an early stage of V(alpha)14i NKT cell development. J. Immunol. 182: 4641–4648.

15. Matsuda, J. L., T. Mallevaey, J. Scott-Browne, and L. Gapin. 2008. CD1d-restricted iNKT cells, the ‘Swiss-Army knife’ of the immune system. Curr. Opin.Immunol. 20: 358–368.

16. Kovalovsky, D., O. U. Uche, S. Eladad, R. M. Hobbs, W. Yi, E. Alonzo, K. Chua,M. Eidson, H. J. Kim, J. S. Im, et al. 2008. The BTB-zinc finger transcriptionalregulator PLZF controls the development of invariant natural killer T cell effec-tor functions. Nat. Immunol. 9: 1055–1064.

17. Savage, A. K., M. G. Constantinides, J. Han, D. Picard, E. Martin, B. Li,O. Lantz, and A. Bendelac. 2008. The transcription factor PLZF directs theeffector program of the NKT cell lineage. Immunity 29: 391–403.

18. Kreslavsky, T., A. K. Savage, R. Hobbs, F. Gounari, R. Bronson, P. Pereira,P. P. Pandolfi, A. Bendelac, and H. von Boehmer. 2009. TCR-inducible PLZFtranscription factor required for innate phenotype of a subset of gammadelta T cellswith restricted TCR diversity. Proc. Natl. Acad. Sci. USA 106: 12453–12458.

19. Felices, M., C. C. Yin, Y. Kosaka, J. Kang, and L. J. Berg. 2009. Tec kinase Itk ingammadeltaT cells is pivotal for controlling IgE production in vivo. Proc. Natl.Acad. Sci. USA 106: 8308–8313.

20. Alonzo, E. S., R. A. Gottschalk, J. Das, T. Egawa, R. M. Hobbs, P. P. Pandolfi,P. Pereira, K. E. Nichols, G. A. Koretzky, M. S. Jordan, and D. B. Sant’Angelo.2010. Development of promyelocytic zinc finger and ThPOK-expressing innategamma delta T cells is controlled by strength of TCR signaling and Id3. J.Immunol. 184: 1268–1279.

21. Gerber, D. J., V. Azuara, J. P. Levraud, S. Y. Huang, M. P. Lembezat, andP. Pereira. 1999. IL-4-producing gamma delta T cells that express a very re-stricted TCR repertoire are preferentially localized in liver and spleen. J. Immu-nol. 163: 3076–3082.

22. Raberger, J., A. Schebesta, S. Sakaguchi, N. Boucheron, K. E. Blomberg,A. Berglof, T. Kolbe, C. I. Smith, T. Rulicke, and W. Ellmeier. 2008. Thetranscriptional regulator PLZF induces the development of CD44 high memoryphenotype T cells. Proc. Natl. Acad. Sci. USA 105: 17919–17924.

23. Lewis, D. B., C. C. Yu, K. A. Forbush, J. Carpenter, T. A. Sato, A. Grossman,D. H. Liggitt, and R. M. Perlmutter. 1991. Interleukin 4 expressed in situ se-lectively alters thymocyte development. J. Exp. Med. 173: 89–100.

24. Barna, M., N. Hawe, L. Niswander, and P. P. Pandolfi. 2000. Plzf regulates limband axial skeletal patterning. Nat. Genet. 25: 166–172.

25. Li, W., M. G. Kim, T. S. Gourley, B. P. McCarthy, D. B. Sant’Angelo, andC. H. Chang. 2005. An alternate pathway for CD4 T cell development: thymocyte-expressedMHC class II selects a distinct T cell population. Immunity 23: 375–386.

26. Lee, Y. J., Y. K. Jeon, B. H. Kang, D. H. Chung, C. G. Park, H. Y. Shin,K. C. Jung, and S. H. Park. 2010. Generation of PLZF+ CD4+ T cells via MHCclass II-dependent thymocyte-thymocyte interaction is a physiological process inhumans. J. Exp. Med. 207: 237–246.

27. Watts, T. H. 2005. TNF/TNFR family members in costimulation of T cellresponses. Annu. Rev. Immunol. 23: 23–68.

28. Sarkar, S., V. Kalia, W. N. Haining, B. T. Konieczny, S. Subramaniam, andR. Ahmed. 2008. Functional and genomic profiling of effector CD8 T cellsubsets with distinct memory fates. J. Exp. Med. 205: 625–640.

29. Huster, K. M., V. Busch, M. Schiemann, K. Linkemann, K. M. Kerksiek,H. Wagner, and D. H. Busch. 2004. Selective expression of IL-7 receptor onmemory T cells identifies early CD40L-dependent generation of distinct CD8+memory T cell subsets. Proc. Natl. Acad. Sci. USA 101: 5610–5615.

30. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, and R. Ahmed.2003. Selective expression of the interleukin 7 receptor identifies effector CD8T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191–1198.

31. Vicari, A. P., A. Herbelin, M. C. Leite-de-Moraes, U. Von Freeden-Jeffry,R. Murray, and A. Zlotnik. 1996. NK1.1+ T cells from IL-7-deficient mice havea normal distribution and selection but exhibit impaired cytokine production. Int.Immunol. 8: 1759–1766.

32. Matsuda, J. L., L. Gapin, S. Sidobre, W. C. Kieper, J. T. Tan, R. Ceredig,C. D. Surh, and M. Kronenberg. 2002. Homeostasis of V alpha 14i NKT cells.Nat. Immunol. 3: 966–974.

33. Frigerio, S., T. Junt, B. Lu, C. Gerard, U. Zumsteg, G. A. Hollander, and L. Piali.2002. Beta cells are responsible for CXCR3-mediated T-cell infiltration in insu-litis. Nat. Med. 8: 1414–1420.

34. Drennan, M. B., A. S. Franki, P. Dewint, K. Van Beneden, S. Seeuws, S. A. vande Pavert, E. C. Reilly, G. Verbruggen, T. E. Lane, R. E. Mebius, et al. 2009.Cutting edge: the chemokine receptor CXCR3 retains invariant NK T cells in thethymus. J. Immunol. 183: 2213–2216.

35. Croft, M., L. M. Bradley, and S. L. Swain. 1994. Naive versus memory CD4 Tcell response to antigen. Memory cells are less dependent on accessory cellcostimulation and can respond to many antigen-presenting cell types includingresting B cells. J. Immunol. 152: 2675–2685.

36. Lovett-Racke, A. E., J. L. Trotter, J. Lauber, P. J. Perrin, C. H. June, andM. K. Racke. 1998. Decreased dependence of myelin basic protein-reactiveT cells on CD28-mediated costimulation in multiple sclerosis patients. A markerof activated/memory T cells. J. Clin. Invest. 101: 725–730.

37. Godfrey, D. I., and M. Kronenberg. 2004. Going both ways: immune regulationvia CD1d-dependent NKT cells. J. Clin. Invest. 114: 1379–1388.

38. Mohrs, M., K. Shinkai, K. Mohrs, and R. M. Locksley. 2001. Analysis of type 2immunity in vivo with a bicistronic IL-4 reporter. Immunity 15: 303–311.

39. Stetson, D. B., M. Mohrs, R. L. Reinhardt, J. L. Baron, Z. E. Wang, L. Gapin,M. Kronenberg, and R. M. Locksley. 2003. Constitutive cytokine mRNAs marknatural killer (NK) and NK T cells poised for rapid effector function. J. Exp.Med. 198: 1069–1076.

40. Kersh, E. N., S. M. Kaech, T. M. Onami, M. Moran, E. J. Wherry, M. C. Miceli,and R. Ahmed. 2003. TCR signal transduction in antigen-specific memory CD8T cells. J. Immunol. 170: 5455–5463.

41. Curtsinger, J. M., D. C. Lins, and M. F. Mescher. 1998. CD8+ memory T cells(CD44high, Ly-6C+) are more sensitive than naive cells to (CD44low, Ly-6C-) toTCR/CD8 signaling in response to antigen. J. Immunol. 160: 3236–3243.

6754 PLZF FUNCTIONS INDEPENDENTLY OF Sap AND Fyn

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.jimm

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ownloaded from

42. Dao, T., D. Guo, A. Ploss, A. Stolzer, C. Saylor, T. E. Boursalian, J. S. Im, andD. B. Sant’Angelo. 2004. Development of CD1d-restricted NKT cells in themouse thymus. Eur. J. Immunol. 34: 3542–3552.

43. Waldmann, T. A., and Y. Tagaya. 1999. The multifaceted regulation of in-terleukin-15 expression and the role of this cytokine in NK cell differentiationand host response to intracellular pathogens. Annu. Rev. Immunol. 17: 19–49.

44. Haluszczak, C., A. D. Akue, S. E. Hamilton, L. D. Johnson, L. Pujanauski,L. Teodorovic, S. C. Jameson, and R. M. Kedl. 2009. The antigen-specific CD8+T cell repertoire in unimmunized mice includes memory phenotype cells bearingmarkers of homeostatic expansion. J. Exp. Med. 206: 435–448.

45. Shiraishi, K., K. Yamasaki, D. Nanba, H. Inoue, Y. Hanakawa, Y. Shirakata,K. Hashimoto, and S. Higashiyama. 2007. Pre-B-cell leukemia transcriptionfactor 1 is a major target of promyelocytic leukemia zinc-finger-mediated mel-anoma cell growth suppression. Oncogene 26: 339–348.

46. Costoya, J. A., R. M. Hobbs, and P. P. Pandolfi. 2008. Cyclin-dependent kinaseantagonizes promyelocytic leukemia zinc-finger through phosphorylation. On-cogene 27: 3789–3796.

47. Filipponi, D., R. M. Hobbs, S. Ottolenghi, P. Rossi, E. A. Jannini, P. P. Pandolfi,and S. Dolci. 2007. Repression of kit expression by Plzf in germ cells. Mol. Cell.Biol. 27: 6770–6781.

48. Bernardo, M. V., E. Yelo, L. Gimeno, J. A. Campillo, and A. Parrado. 2007.Identification of apoptosis-related PLZF target genes. Biochem. Biophys. Res.Commun. 359: 317–322.

49. Egawa, T., G. Eberl, I. Taniuchi, K. Benlagha, F. Geissmann, L. Hennighausen,A. Bendelac, and D. R. Littman. 2005. Genetic evidence supporting selection ofthe Valpha14i NKT cell lineage from double-positive thymocyte precursors.Immunity 22: 705–716.

50. Bezbradica, J. S., T. Hill, A. K. Stanic, L. Van Kaer, and S. Joyce. 2005.Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+stage of thymic ontogeny. Proc. Natl. Acad. Sci. USA 102: 5114–5119.

51. D’Cruz, L. M., J. Knell, J. K. Fujimoto, and A. W. Goldrath. 2010. An essentialrole for the transcription factor HEB in thymocyte survival, Tcra rearrangementand the development of natural killer T cells. Nat. Immunol. 11: 240–249.

52. Ueda-Hayakawa, I., J. Mahlios, and Y. Zhuang. 2009. Id3 restricts thedevelopmental potential of gamma delta lineage during thymopoiesis. J.Immunol. 182: 5306–5316.

53. Capone, M., M. Troesch, G. Eberl, B. Hausmann, E. Palmer, andH. R. MacDonald. 2001. A critical role for the T cell receptor alpha-chainconnecting peptide domain in positive selection of CD1-independentNKT cells. Eur. J. Immunol. 31: 1867–1875.

54. Felices, M., and L. J. Berg. 2008. The Tec kinases Itk and Rlk regulate NKT cellmaturation, cytokine production, and survival. J. Immunol. 180: 3007–3018.

55. Jordan, M. S., J. E. Smith, J. C. Burns, J. E. Austin, K. E. Nichols,A. C. Aschenbrenner, and G. A. Koretzky. 2008. Complementation in trans ofaltered thymocyte development in mice expressing mutant forms of the adaptormolecule SLP76. Immunity 28: 359–369.

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