Unexpected Role for the B Cell-Specific Src Family Kinase B ... · PDF fileLymphoid Kinase in...

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of May 21, 2018. This information is current as T Cells δ γ Producing - Development of IL-17 Family Kinase B Lymphoid Kinase in the Unexpected Role for the B Cell-Specific Src Renee M. Laird, Karen Laky and Sandra M. Hayes http://www.jimmunol.org/content/185/11/6518 doi: 10.4049/jimmunol.1002766 October 2010; 2010; 185:6518-6527; Prepublished online 25 J Immunol Material Supplementary 6.DC1 http://www.jimmunol.org/content/suppl/2010/10/25/jimmunol.100276 References http://www.jimmunol.org/content/185/11/6518.full#ref-list-1 , 26 of which you can access for free at: cites 55 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. All rights reserved. 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on May 21, 2018 http://www.jimmunol.org/ Downloaded from by guest on May 21, 2018 http://www.jimmunol.org/ Downloaded from

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of May 21, 2018.This information is current as

T CellsδγProducing −Development of IL-17 Family Kinase B Lymphoid Kinase in the

Unexpected Role for the B Cell-Specific Src

Renee M. Laird, Karen Laky and Sandra M. Hayes

http://www.jimmunol.org/content/185/11/6518doi: 10.4049/jimmunol.1002766October 2010;

2010; 185:6518-6527; Prepublished online 25J Immunol 

MaterialSupplementary

6.DC1http://www.jimmunol.org/content/suppl/2010/10/25/jimmunol.100276

Referenceshttp://www.jimmunol.org/content/185/11/6518.full#ref-list-1

, 26 of which you can access for free at: cites 55 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. All rights reserved.1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Unexpected Role for the B Cell-Specific Src Family Kinase BLymphoid Kinase in the Development of IL-17–Producing gdT Cells

Renee M. Laird,* Karen Laky,† and Sandra M. Hayes*

The Ag receptors on ab and gd T cells differ not only in the nature of the ligands that they recognize but also in their signaling

potential. We hypothesized that the differences in ab- and gdTCR signal transduction were due to differences in the intracellular

signaling pathways coupled to these two TCRs. To investigate this, we used transcriptional profiling to identify genes encoding

signaling molecules that are differentially expressed in mature ab and gd T cell populations. Unexpectedly, we found that B

lymphoid kinase (Blk), a Src family kinase expressed primarily in B cells, is expressed in gd T cells but not in ab T cells. Analysis

of Blk-deficient mice revealed that Blk is required for the development of IL-17–producing gd T cells. Furthermore, Blk is

expressed in lymphoid precursors and, in this capacity, plays a role in regulating thymus cellularity during ontogeny. The

Journal of Immunology, 2010, 185: 6518–6527.

The conservation, in all jawed vertebrates, of two T celllineages suggests that ab and gd T cells have comple-mentary and nonredundant roles in immunity. There is

growing evidence to support this idea. First, ab and gd T cellshave different Ag specificities, with gd T cells recognizing native,unprocessed Ags and ab T cells recognizing peptides in associationwith MHC molecules (1–3). Second, ab and gd T cells localize todifferent peripheral tissues. Whereas most ab T cells circulatethrough secondary lymphoid tissues, most gd T cells reside in ep-ithelial tissues, such as skin, intestine, lung, tongue, and femalereproductive tract (1–3). Third, although ab and gd T cells shareeffector functions, epithelial resident gd T cells display specializedroles in immunity, as evidenced by their ability to mediate epithe-lial cell homeostasis and wound healing (4–6). Fourth, ab and gdT cells respond at different stages during the host immune response,with gd T cells often acquiring effector functions days before ab

T cells (7–10).The ability of gd T cells to manifest their unique functions and

their rapid effector response during an immune response may beexplained in part by the different signaling properties of the ab-and gdTCRs. In a direct comparison of ab- and gdTCR signaltransduction, in assays that measure calcium mobilization and ERKactivation, the gdTCR signaled with faster kinetics and greatermagnitude than the abTCR (11). Importantly, the enhanced sig-

naling proficiency of the gdTCR affected the kinetics of T cell ac-tivation, as evidenced by the ability of stimulated gd T cells toupregulate the expression of genes associated with T cell effectorfunction faster than stimulated ab T cells (12) and to undergo morerounds of proliferation than stimulated ab T cells (11).The molecular basis for the difference in ab- and gdTCR sig-

naling properties is currently unknown. One explanation for thisdifference is that the signaling pathways triggered by the gdTCRare distinct from those triggered by the abTCR. To test this, weused global gene expression profiling to identify signaling mole-cules that are differentially expressed between mature ab and gdT cells. Using this strategy, we discovered B lymphoid kinase(Blk), which encodes a B cell-specific member of the Src family ofprotein tyrosine kinases (SFKs) (13), to be preferentially expressedin gd but not ab T cells. Interestingly, protein expression studiesshowed that Blk is expressed in only a small subset of mature gd

T cells, indicating that Blk cannot be responsible for the enhancedsignaling ability of the gdTCR per se. To determine the biologicalsignificance of Blk expression in gd lineage cells, we analyzed gd

T cell development and function in Blk2/2 mice and found thatBlk is required for the development of IL-17–producing gd T cells.In addition, we discovered that Blk, which is expressed in variouslymphoid precursor populations, regulates thymus cellularity bycontrolling the number of early thymic progenitors (ETPs) and theproliferative capacity of immature thymocytes during ontogeny.

Materials and MethodsMice

C57BL/6J (B6), B6.SJL-Ptprca Pep3b/BoyJ (B6-CD45.1+), B6;129S7-Fyntm1Sor/J (Fyn2/2), B6.129S2-Lcktm1Mak/J (Lck2/2) B6.129P2-Tcrbtm1Mom/J (TCRb2/2), and B6.129S2-Tcratm1Mom/J (TCRa2/2) micewere all purchased from The Jackson Laboratory (Bar Harbor, ME). B6-Blktm1 (Blk2/2) mice (14) were provided by A. Tarakhovsky (The Rock-efeller University, New York, NY), B6-IL-23R-GFP knockin mice(IL-23R-GFP.KI) (15) were provided by M. Oukka (Seattle Children’sResearch Institute, Seattle, WA), and B6-Vg6/Vd1 gdTCR transgenic(gdTCR Tg; line 134) (16) mice were provided by P. Love (NationalInstitutes of Health, Bethesda, MD). All mice used in this study were bredand maintained in the Department of Laboratory Animal Resources at StateUniversity of New York (SUNY) Upstate Medical University (Syracuse,NY) in accordance with the specifications of the Association for Assess-ment and Accreditation of Laboratory Animal Care. Mouse protocols were

*Department of Microbiology and Immunology, State University of New York Up-state Medical University, Syracuse, NY 13210; and †Laboratory of Cellular andMolecular Immunology, National Institutes of Allergy and Infectious Disease, Na-tional Institutes of Health, Bethesda, MD 20892

Received for publication August 13, 2010. Accepted for publication September 24,2010.

This work was supported by the Hendricks Fund for Medical Research and NationalInstitutes of Health Grant AI081068.

Address correspondence and reprint requests to Dr. Sandra M. Hayes, Departmentof Microbiology and Immunology, State University of New York Upstate Med-ical University, 750 East Adams Street, Syracuse, NY 13210. E-mail address:[email protected]

The online version of this article contains supplemental material.

Abbreviations used in this paper: B6, C57BL/6J; Blk, B lymphoid kinase; BM, bonemarrow; CLP, common lymphoid progenitor; DN, double-negative; DP, double-positive; ETP, early thymic progenitor; i.c., intracellular; iIEL, intestinal intraepithe-lial lymphocyte; LN, lymph node; LSK, lineage-negative Sca-1+c-Kit+ cell; SFK, Srcfamily kinase; SUNY, State University of New York; Tg, transgenic.

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approved by the SUNY Upstate Medical University Committee on theHumane Use of Animals.

Abs and reagents

mAbs used for flow cytometric analysis and magnetic bead separationincluded anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-TCRgd (UC7-13D5), anti-TCRb (H57-597), anti-CD3 (145-2C11), anti-CD11b (M1/70), anti-CD19 (6D5), anti-CD25 (PC61), anti-CD44 (IM7), anti-NK1.1(PK136), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD117 (2B8), anti–Ly6-G/Ly6-C (RB6-8C5), anti–I-Ab (AF6-120.1), anti-CCR6 (29-2L17),and anti–TER-119 (TER-119), which were purchased from BioLegend(San Diego, CA), eBioscience (San Diego, CA), and BD Pharmingen (SanJose, CA). mAbs against Vg1 (2.11), Vg4 (UC3-10A6), and Vg5 (F536)were purified from their respective hybridoma supernatants using Im-munoPure (A/G) IgG Purification kit (Pierce, Rockford, IL) and thenbiotinylated using Pierce’s sulfosuccinimidyl-6-(biotinamido) hexanoate,according to manufacturer’s instructions. PE-streptavidin was purchasedfrom BioLegend. Abs used in intracellular flow cytometric assays wereanti-Blk (Cell Signaling Technology, Danvers, MA), anti-Fyn (FYN-59;BioLegend), anti-Lck (3A5; Millipore, Billerica, MA), Ki-67 (B56;BD Pharmingen), anti–IL-17A (TC11-18H10.1; BioLegend), anti–IFN-g(XMG1.2; BD Pharmingen), anti-mouse IgG2b (R12-3; BioLegend), anddonkey anti-rabbit IgG (Invitrogen, Carlsbad, CA).

Purification of lymphocyte subsets

CD4+ ab T cells, CD8+ ab T cells, CD42CD82(double-negative [DN])gd T cells, and DN gd thymocytes were purified by negative selectionusing the magnetic bead separation system (Miltenyi Biotec, Auburn, CA)as described previously (11, 12).

B cells were purified by positive selection using magnetic bead sepa-ration. Briefly, spleen cells from B6 mice were stained for 10 min withPE-conjugated anti-CD19 mAb, washed, and then incubated with anti-PEbeads (Miltenyi Biotec) for 15 min, with all steps at 4˚C. The purity of theresulting cell population was typically $95%.

Quantitative Western blot analysis

DN gd thymocytes purified from gdTCR Tg mice were lysed in a buffercontaining 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4,protease inhibitors (Roche Laboratories, Indianapolis, IN), and 1% Non-idet P-40 alternative (Calbiochem, Gibbstown, NJ). Serial dilutions ofcleared lysates from a known number of DN gd thymocytes were resolvedby SDS-PAGE in parallel with serial dilutions of known amounts ofrecombinant His-tagged Blk or Lck (Millipore), transferred to a polyvi-nylidene difluoride membrane, and immunoblotted with anti-Blk or LckAbs. Abs used for Western blot analysis were anti-Blk (Santa Cruz Bio-technology, Santa Cruz, CA), anti-Lck (Millipore), and anti–b-actin(Sigma-Aldrich, St. Louis, MO). The volume (defined as intensity3 mm2)of each band was determined using densitometric analysis. A standardcurve was generated by plotting the concentration of Blk or Lck standard(in nanograms) versus the volume of each corresponding band. The con-centration per cell was then estimated based on this standard curve.

Isolation of intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (iIELs) were isolated as describedpreviously (17).

Microarray analysis

The GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara,CA) was used to screen for genes encoding signaling molecules whose geneexpression was significantly different between DN gd T cells and CD8+ abT cells. Total RNA from both T cell populations was extracted and pro-cessed for analysis as described previously (12). The labeled cRNA washybridized to the GeneChip Mouse Genome 430 2.0 Array (containing45,000 probe sets that represent 39,000 transcripts and 34,000 mousegenes) at 45˚C for 16 h with constant rotation (60 rpm), washed, and thenstained on a Fluidics Station 400 (Affymetrix), according to the EukGE-WS2v4 protocol. GeneChips were scanned using the Agilent G2500AGene Array Scanner. After scanning, the Affymetrix GCOS MicroarrayAnalysis Suite version 5.0 software was used to determine the presence (P)or absence (A) of a transcript, the differential change in gene expression,and the magnitude of the change in gene expression. The complete micro-array data set can be found at the Gene Expression Omnibus repository(http://www.ncbi.nlm.nih.gov/projects/geo/) accession number GSE24281.

Flow cytometric analysis

Flow cytometric analysis for both surface and intracellular Ags was per-formed as described previously (18).

Real-time RT-PCR analysis

RNA was extracted from B cells and T cell subsets using the RNeasy kitfromQiagen (Valencia, CA). cDNAwas then synthesized using Invitrogen’sSuperScript First-Strand Synthesis System. Quantitative real-time RT-PCRanalysis was performed using a Bio-Rad iQ5 real-time PCR machine (Bio-Rad, Hercules, CA). All of the primer sets for the quantitative real-timeRT-PCR analysis, which include Gapdh, Blk, and Lyn, were purchasedfrom SABiosciences (Frederick, MD).

Mixed bone marrow chimeras

Bone marrow (BM) cells from 6- to 8-wk-old B6-CD45.2+ and Blk2/2mice(also CD45.2+) were mixed in a 1:1 ratio with BM cells from age-matchedB6-CD45.1+ mice. A total of 5 3 106 cells of this cell mixture were theninjected i.v. into the tail vein of lethally irradiated (1100 rad) 6-wk-old B6-CD45.1+ mice. Three weeks postinjection, thymocytes were harvested andstained with mAbs against various surface Ags as well as mAbs againstCD45.1 and CD45.2 to determine the degree of chimerism.

In vitro stimulation of gd T cells

gd T cells from gdTCR Tg Blk2/2 and gdTCR Tg Blk+/+ mice werestimulated as described previously (18).

ResultsBlk is expressed in gd T cells but not ab T cells

To identify signaling molecules that are differentially expressedbetween ab and gd T cells, we transcriptionally profiled matureDN gd T cells and CD8+ ab T cells. We reasoned that comparingthe gene expression profiles of two T cell populations that sharefunctional properties, such as the propensity to produce IFN-gfollowing TCR activation (19), would highlight any qualitative orquantitative differences in their gene expression. To this end, wepurified DN gd T cells from the lymph nodes (LNs) of Vg6/Vd1gdTCR Tg mice (hereafter referred to as gdTCR Tg) and CD8+ abT cells from the LNs of B6 mice, both by negative selection. TotalRNA from the two T cell populations was extracted and thenprocessed for microarray analysis. This approach identified manygenes that are expressed at higher levels in DN gd T cells than inCD8+ ab T cells. Table I lists genes that exhibited the greatestdifference in expression between DN gd T cells and CD8+ ab

Table I. List of genes preferentially expressed in ex vivo DN gd T cellscompared with CD8+ ab T cells

Gene Symbol Fold Changea

TCRg constant region Tcrg-C 9.8S100 calcium-binding protein A6 s100a6 6.8Ubiquitin-specific peptidase 18 Usp18 6.8S100 calcium-binding protein A4 s100a4 6.5Regulator of G protein signaling 1 Rgs1 6.2Tetraspanin 32 Tspan32 5.6Placental protein 11 related Pp11r 5.5IGF-binding protein 4 Igfbp4 5.4Caspase 1 Casp1 5.1Lymphocyte Ag 6 complex, locus A Ly6a 5.1Niban protein Niban 4.9B lymphoid kinase Blk 4.9Leukocyte-associated Ig-like receptor 1 Lair1 4.8Thyroid hormone receptor interactor 4 Trip4 4.8Integrin b3 Itgb3 4.7Deltex 1 homolog Dtx1 4.7SRY-box containing gene 13 Sox13 4.3Regulator of G protein signaling 2 Rgs2 3.9

aGene expression is shown as log2-fold change in expression in DN gd T cellsversus CD8+ ab T cells.

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T cells. Some of these genes, such as Rgs1, Rgs2, and Sox13, havebeen previously reported to be preferentially expressed in gd lin-eage cells (20, 21). Because our goal was to identify signalingmolecules that are differentially expressed in ab and gd T cells, weconsidered Blk to be the most interesting in this list of genes, be-cause it encodes a kinase known to be involved in Ag receptorsignal transduction (22–24). The differential expression of Blk wasconfirmed by performing real-time RT-PCR analysis on not onlypurified DN gd T cells and CD8+ ab T cells but also purified CD4+

ab T cells and B cells. Specifically, we observed a significantdifference in the relative expression of Blk between gd T cells andthe two ab T cell subsets but not between gd T cells and B cells(Fig. 1A). Expression of Lyn, another SFK typically found inB cells, was absent in the purified gd T cell population (Fig. 1A),indicating that the expression of Blk by gd T cells was not due toB cell contamination.Next, we developed an intracellular (i.c.) flow cytometric assay to

examine both the pattern and level of Blk expression in variouslymphocyte populations (Supplemental Fig. 1). Using this technique,we found, in both gdTCR Tg and B6 mice, that Blk is expressedin all gd thymocytes but only in a small percentage of maturegd T cells (Fig. 1B). Notably, the expression level of Blk in gd

thymocytes was lower than that in B cells, whereas its expressionlevel in mature gd T cells was comparable to that in B cells.Many of the genes that are preferentially expressed in gd

lineage cells require a thymic microenvironment containing aquorum of lymphotoxin-producing CD4+CD8+ (double-positive;DP) thymocytes for their proper expression (20, 25). To de-termine whether Blk expression by gd thymocytes is similarlyregulated, we assayed gd lineage cells from TCRb2/2 mice,which do not possess the appropriate DP thymocyte compartmentto support the expression of the gd-biased genes (25). As shown inFig. 1B, we found that Blk is indeed expressed in TCRb2/2 gdthymocytes and at levels comparable to those observed in B6mice. These findings indicated that Blk expression in gd lineagecells is not dependent on interactions with immature ab lineagecells.Unlike ab T cell development, which is completely blocked in

Lck2/2Fyn2/2 mice, gd T cell development is impaired but notabrogated (26). In fact, a small number of gdTCR+ cells are de-tected in secondary lymphoid tissues, small intestine, and epidermisof Lck2/2Fyn2/2 mice (26, 27). The differential requirements forSFKs in ab and gd T cell development may be explained by theexpression of another SFK, namely Blk, in gd lineage cells. To test

FIGURE 1. Blk is expressed in gd lineage cells. A, Quantitative real-time RT-PCR analysis of the relative transcript levels of Blk and Lyn in DN gd

T cells purified from gdTCR Tg mice and in CD8+ ab T cells, CD4+ ab T cells, and CD19+ B cells purified from B6 mice. Data were normalized to Gapdh

transcript levels and are presented as fold change over B cells (set to 1). Bars represent mean 6 SEM of at least three samples of each lymphocyte

population; **p # 0.01. B, Black histograms show representative staining of the i.c. levels of Blk in gated populations of thymic and peripheral gd lineage

cells from gdTCR Tg, B6, TCRb2/2, and Lck2/2Fyn2/2 mice. DP thymocytes (dark shaded histogram) and splenic B cells (light shaded histogram) are

shown as negative and positive controls, respectively. C, Black histograms show representative staining of the i.c. levels of Blk in gated populations of Vg1+

and Vg4+ gd T cells from the LNs of B6 mice. DP thymocytes (dark shaded histogram) are shown as a negative control. D, Black histograms show

representative staining of the i.c. levels of Blk in gated populations of Vg5+ and Vg52 thymocytes from B6 embryonic day 17 (E17) fetuses. DP thy-

mocytes (dark shaded histogram) are shown as a negative control. E, Black histograms show representative staining of the i.c. levels of Blk in gated

populations of DN and CD8aa+gdTCR+ iIELs from B6 mice. DP thymocytes (dark shaded histogram) are shown as a negative control. F, Serial dilutions

of cleared lysates from a known number of DN gd thymocytes were resolved by SDS-PAGE in parallel with serial dilutions of known amounts of

recombinant His-tagged Blk or Lck, transferred to a polyvinylidene difluoride membrane, and immunoblotted with anti-Blk, anti-Lck, or anti–b-actin Abs.

Data are representative of four independent experiments. G, Mean number of Blk and Lck molecules per DN gd thymocyte; #p # 0.001.

6520 ROLE OF Blk IN DEVELOPMENT OF IL-17–PRODUCING gd T CELLS

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this, we measured Blk expression levels in DN gdTCR+ thymocytesand LN cells from Lck2/2Fyn2/2 mice. Significantly, Blk expres-sion was detected not only in gd thymocytes but also in two-thirdsof the gd T cells present in the LNs of Lck2/2Fyn2/2 mice (Fig.1B). These data suggested that gd lineage cells that express Blkdevelop and survive in the absence of both Lck and Fyn.Because murine gd T cells can be divided into subsets based on

their Vg usage and their anatomical location (1–3), it was of interestto determine which gd T cell subsets express Blk. In the LNs of B6mice, only 14% of the gd T cells expressed Blk (Fig. 1B). Pheno-typic analysis of these Blk+ gd T cells, using Vg1- and Vg4-specificmAbs, demonstrated that they are restricted to the Vg4+ gd T cellsubset (Fig. 1C). In fact, we noted that 50% of the Blk+ gd T cells inB6 LNs were Vg4+ (data not shown). Next, we assayed the gdT cell populations that develop in the fetal thymus for Blk ex-pression. We found that, although all fetal gd thymocytes expressedBlk, the Vg5- subset, the vast majority of which represent Vg6+ gdT cells that home to lung, tongue, and female reproductive tract, con-tained a small population of Blkhi cells, whereas the Vg5+ subset,which represents the skin-homing dendritic epidermal T cells), didnot (1–3) (Fig. 1D). Last, we performed a phenotypic analysis ofthe gd T cells that reside in the small intestine (iIELs) and de-tected minimal Blk expression in either the DN or CD8aa+ gd

iIEL subset (Fig. 1E). Taken together, these data indicated that Blkexpression in mature gd T cells is restricted to Vg4+ and Vg6+ gdT cell subsets.If Blk were to contribute to the signaling proficiency of the

gdTCR, then we would expect the expression level of Blk in gdlineage cells to be comparable to that of the T cell-specific SFKLck. To test this, we measured the protein concentrations of Blkand Lck in purified gd thymocytes by quantitative Western blotanalysis using His-tagged Blk and Lck as protein standards (Fig.1F). We found that gd thymocytes expressed ∼4-fold more Blk(∼80,000 molecules/cell) than Lck (∼20,000 molecules/cell) (Fig.1G). Because flow cytometric analysis showed that the peripheralBlk+ gd T cell subset expresses 2.5-fold more Blk than gd

thymocytes (Fig. 1B), we estimate that these gd T cells express∼200,000 molecules of Blk/cell. This amount is even more strikinggiven that LN gd T cells express significantly less Lck and Fyn thangd thymocytes (18). These data indicated that Blk is the pre-dominant SFK expressed in gd thymocytes and in the mature Blk+

gd T cell subset.

Blk is expressed in thymic precursors

Given the relatively high expression levels of Blk in gd thymocytes,we sought to determine whether its expression is induced or up-regulated as a consequence of commitment to the gd lineage. Usingthe i.c. flow cytometric assay, we found that Blk is expressed inimmature DN thymocytes, which contain precursors with the po-tential to develop into ab or gd lineage cells (28) but at lower levelsthan those detected in gd thymocytes (Fig. 1B; data not shown).When the immature DN thymocyte population was subdivided intofour populations based on expression of CD44 and CD25 (29), wefound that Blk is highly expressed in DN1 and DN2 thymocytes,slightly downregulated in DN3 thymocytes, and virtually absent inDN4 thymocytes (Fig. 2A). Notably, this expression pattern con-trasted with those of Lck and Fyn, whose expression is maintainedat relatively high levels throughout the DN4 stage (Fig. 2A). Be-cause the divergence of ab and gd lineages has been shown tooccur at the DN2 stage and, to a lesser extent, the DN3 stage (30)and because DN4 thymocytes represent immature ab lineage cellstransitioning to the DP stage (31), these findings demonstrated thatBlk is expressed in thymic progenitors, and following ab/gd line-age choice, its expression is upregulated in gd lineage cells butdownregulated in ab lineage cells.

Blk deficiency affects the early stages of T cell development

The expression of Blk at the earliest DN1 stage prompted us toevaluate Blk expression in defined precursor populations, such asETPs, common lymphoid progenitors, and lineage-negative Sca-1+

c-Kit+ cells. Strikingly, all of these precursor populations expressedBlk and at relatively high levels (Fig. 2B–D). To determine the

FIGURE 2. Blk is expressed in thymic and BM progenitor cells. A, Black histograms show representative staining of the i.c. levels of Blk, Lck, and Fyn

in gated DN1 (Lin2CD252CD44+), DN2 (Lin2CD25+CD44+), DN3 (Lin2CD25+CD442), and DN4 (Lin2CD252CD442) thymocyte populations from

TCRa2/2 mice. Lin2 is defined as CD42CD82CD11b2TCRb2TCRgd2CD192NK1.12IAb2TER-1192Ly6-G/Ly6-C2. Dark shaded histograms represent

negative staining controls (DP thymocytes for i.c. Blk staining, Lck2/2 thymocytes for i.c. Lck staining, and Fyn2/2 thymocytes for i.c. Fyn staining). Light

shaded histograms represent positive staining controls (splenic B cells for i.c. Blk staining and mature CD4+ T cells for both i.c. Lck and Fyn staining). B,

Black histograms show representative staining of the i.c. levels of Blk in Lin2Sca-1+c-Kithi (lineage-negative Sca-1+c-Kit+ cell [LSK]) progenitors from B6

BM. DP thymocytes (dark shaded histogram) are shown as a negative control. C, Black histograms show representative staining of the i.c. levels of Blk in

Lin2Sca-1loc-Kitlo cells or common lymphoid progenitors (CLPs) from B6 BM. DP thymocytes (dark shaded histogram) are shown as a negative control.

D, Black histograms show representative staining of the i.c. levels of Blk in lin2CD252CD44+c-Kit+ thymocytes or ETPs from TCRa2/2 thymus. DP

thymocytes (dark shaded histogram) are shown as a negative control.

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significance of Blk expression in T cell progenitors, we analyzedT cell development in various aged Blk+/+ (B6) and Blk2/2 mice.During ontogeny, we found that the cellularity of the Blk2/2 thy-mus did not reach wild-type numbers until 4 wk of age, whereasthe cellularity of Blk2/2 LNs did not reach wild-type numbers until5 wk of age (Fig. 3A). Because the most significant difference inthymus cellularity was observed between 3-wk-old Blk+/+ andBlk2/2 mice, we chose this age to perform a more detailed analysisof T cell development.Notably, we observed significant decreases in both the percent-

age and number of DP thymocytes in 3-wk-old Blk2/2 micecompared with age-matched Blk+/+ mice (Fig. 3B; data not shown).This phenotype contrasts with the one observed at 6 wk of age,when T cell development has reached steady-state levels, in thatwe did not detect any differences in the percentage and numberof DN, DP, and SP thymocytes between Blk+/+ and Blk2/2 mice(data not shown). The reduction in the number of DP thymocytesin young Blk2/2 mice was not to due to a defect in the maturationof the DN subsets, because no appreciable difference was observedbetween the two genotypes in the relative frequency of each DNsubset (Fig. 3B). However, when we compared the proliferationstatus of the DN subsets in Blk+/+ and Blk2/2 thymi by measuringexpression of the Ki-67 proliferation marker, we found that allBlk2/2 DN subsets underwent significantly less proliferation thantheir wild-type counterparts (Fig. 3C). In addition, there was a sig-nificant reduction in the percentage and number of ETPs (c-Kithi

DN1 thymocytes) in Blk2/2 mice relative to Blk+/+ mice (Fig. 3D,3E). It is interesting to note that we detected a significant population

of cells in the Blk2/2 DN1 subset that were c-Kitlo (Fig. 3D). Thesurface phenotype of these cells is similar to that of DN1c cells,which have been shown to have T lineage potential but to exhibit alow proliferative capacity (32). Taken together, these data demon-strated that Blk controls the frequency of the different thymicprogenitors as well as the proliferative capacity of immature DNthymocytes.To determine whether the defects in the early stages of T cell

development that were observed in young Blk2/2 mice are due tothe loss of Blk in thymic precursors, we generated BM chimeras inwhich BM from Blk2/2 (CD45.2+) or Blk+/+ (CD45.2+) mice weremixed in a 1:1 ratio with B6-CD45.1+ BM and then injected intolethally irradiated B6-CD45.1+ hosts. Three weeks after trans-plantation, we found that, in comparison with Blk+/+-origin pre-cursors, Blk2/2-origin precursors did not compete as well againstB6-CD45.1-origin precursors in repopulating the thymus (Fig.4A). In addition, the CD4 versus CD8 staining profile of the Blk2/2

-origin thymocytes in the chimeras mirrored that of 3-wk-old in-tact Blk2/2 mice (Figs. 3B, 4C). Taken together, these findingsindicated that the T cell developmental defects observed in Blk2/2

mice are cell intrinsic.

Blk is not required for commitment to the gd T cell lineage

The differential expression of Blk between committed ab and gdlineage cells in the thymus suggested that it plays a role in gdT cell commitment and/or development. To investigate this, weassessed gd T cell development in various aged Blk+/+ and Blk2/2

mice. At 3 wk of age, we found that the percentages of DN

FIGURE 3. Effects of Blk deficiency on T cell development. A, Cellularity of the thymus and LNs of Blk+/+ (gray shaded diamond) and Blk2/2 (black

square) mice plotted as a function of age. Mean cell number 6 SEM from 3 to 10 mice are shown for each age. *p # 0.05; **p # 0.01; #p # 0.001. B,

Phenotypic analysis of thymocytes from 3-wk-old Blk+/+ and Blk2/2 mice. Top panel, Dot plots show representative CD4 versus CD8 staining profiles of

total thymocytes. Bottom panel, Dot plots show representative CD44 versus CD25 staining on gated Lin2 thymocytes, where Lin2 is defined as in Fig. 2A.

Numbers in quadrants represent percentage of cells in each quadrant. The mean thymus cell number6 SEM for each genotype are displayed above the two-

color plots. C, Effect of Blk deficiency at 3 wk of age on the proliferative capacity of the four DN subsets. Bars represent the mean percentage of Ki-67+

cells 6 SEM from at least three mice per genotype; #p # 0.001. E, Dot plots show representative CD44 versus CD117 (c-Kit) staining profiles on gated

Lin2CD252CD44+ (DN1) thymocytes from 3-wk-old Blk+/+ and Blk2/2 mice. Numbers represent percentages of ETPs. F, Comparison of the number of

ETPs in 3-wk-old Blk+/+ and Blk2/2 mice. The mean number of ETPs 6 SEM from three mice are shown; *p # 0.05.

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TCRgd+ cells in the thymus and periphery are comparable betweenBlk+/+ and Blk2/2 mice, but their absolute numbers are decreasedbecause of the reduction in thymus and LN cellularity (Fig. 5A,5C). By 6 wk of age, however, no differences were observed

between Blk+/+ and Blk2/2 mice in the percentage or number ofthymic and LN gd T cells (data not shown). Even though these datasuggested that Blk is not required for commitment to the gd lin-eage, we took another approach by mating a gdTCR transgene onto

FIGURE 4. Defects in T cell development in Blk2/2 mice are cell intrinsic. A, Degree of chimerism in the thymus of Blk+/+:CD45.1- and Blk2/2:CD45.1-

mixed BM chimeras. Bars represent the percentages of CD45.2+ thymocytes 6 SEM in Blk+/+:CD45.1 chimeras (n = 4) and Blk2/2:CD45.1 chimeras (n =

3); *p # 0.05. B, Phenotypic analysis of thymocytes from Blk+/+:CD45.1-mixed BM chimeras. Two-color plots show expression of CD4 versus CD8 on

total and on gated CD45.2+ and CD45.1+ thymocytes. Numbers in quadrants of the two-color plots represent the percentage of cells in each quadrant. C,

Phenotypic analysis of thymocytes from Blk2/2:CD45.1-mixed BM chimeras. Two-color plots show expression of CD4 versus CD8 on total and on gated

CD45.2+ and CD45.1+ thymocytes. Numbers in quadrants of the two-color plots represent the percentage of cells in each quadrant.

FIGURE 5. Effects of Blk deficiency on gd T cell development. A, Phenotypic analysis of gd lineage cells in Blk+/+ and Blk2/2 mice. Left panel, Dot

plots show representative Vg5 versus CD3 staining profiles on gated DN thymocytes from E17 Blk+/+ and Blk2/2 fetuses. Right panel, Dot plots show

representative TCRgd versus CD3 staining on total thymocytes and LN cells from 3-wk-old Blk+/+ and Blk2/2 mice. Numbers represent percentage of cells

in each gate. B, Quantification of Vg5+ and Vg52 fetal thymocytes in E17 Blk+/+ and Blk2/2 fetuses. Bars represent mean 6 SEM of at least eight thymic

lobes per genotype; #p # 0.001. C, Quantification of DN TCRgd+ cells in the thymus and LN of 3-wk-old Blk+/+ and Blk2/2 mice. Bars represent mean 6SEM of 9–10 mice per genotype; *p # 0.05; #p # 0.001. D, Percentage of Vg1+ and Vg4+ cells in gated DN TCRgd+ LN cells from 3-wk-old Blk+/+ and

Blk2/2 mice. Bars represent mean 6 SEM of five mice per genotype; #p # 0.001. E, Percentage of Vg1+ and Vg4+ cells in gated DN TCRgd+ LN cells

from 6-wk-old Blk+/+ and Blk2/2 mice. Bars represent mean 6 SEM of three mice per genotype; *p # 0.05.

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the Blk2/2 background to determine whether fixing the specificityof the gdTCR uncovered a requirement for Blk in gd lineagecommitment. Using the gdTCR Tg system, we found that loss ofBlk has no effect on ab/gd lineage choice or on the generation ofmature gd T cells (Fig. 6A–C). Interestingly, despite the presenceof wild-type numbers of thymic and peripheral gd T cells ingdTCR Tg Blk2/2 mice, the phenotype of these cells was alteredcompared with gd T cells from gdTCRTg Blk+/+ mice in that theirCD5 levels were significantly increased whereas their CD3 levelswere significantly decreased (Fig. 6D). Because CD5 levels directlycorrelate with TCR signal strength (33), these results suggestedthat, in the absence of Blk, gdTCR signal strength is augmented.

Blk is required for the development of IL-17–producing gd

T cell effectors

Although the total numbers of peripheral gd T cells in Blk2/2 andBlk+/+ mice were comparable, it is conceivable that the relativepercentages of specific gd T cell subsets, such as those expressinghigh levels of Blk, are reduced in Blk2/2 mice. To test this, weexamined the Vg usage of DN gdTCR+ cells from the fetal thymusand adult LNs by flow cytometric analysis using various Vg-specific mAbs. We found that the number of Vg52 gd thymocytesare significantly reduced in Blk2/2 E17 fetuses compared withBlk+/+ E17 fetuses (Fig. 5A, 5B). Likewise, there were signifi-cantly fewer Vg4+ gd T cells in the LNs of Blk2/2 mice thanin the LNs of Blk+/+ mice (Fig. 5D, 5E). Taken together, thesefindings demonstrated that Blk deficiency results in a selective lossof cells within the Vg4+ and Vg6+ gd T cell subsets.We reasoned that the changes in gdTCR repertoire observed in

Blk2/2 mice may affect gd T cell effector function. Because gdT cells from the Vg6/Vd1 gdTCR Tg mouse have the ability to

acquire different effector fates (18), which match those reportedfor this gd T subset following activation in vivo (34), we used ourgdTCR Tg system to assess the effects of Blk deficiency on gd

T cell effector function. To this end, we examined cytokine pro-duction by gd T cells following TCR activation and found that thepercentage of stimulated gd T cells producing IL-17 is drasticallyreduced in gdTCR Tg Blk2/2 mice compared with gdTCR TgBlk+/+ mice (Fig. 7A). In contrast, the percentages of stimulatedBlk2/2 gd T cells producing IFN-g and/or TNF-a were compa-rable to those of stimulated Blk+/+ gd T cells (Fig. 7A), suggestingthat there is a selective loss of IL-17–producing gd effectors ingdTCR Tg Blk2/2 mice.To investigate this further, we first determined whether Blk is

differentially expressed in IL-17–producing and IFN-g–producinggd T cells. It has recently been shown that expression of CCR6and IL-23R marks gd T cells of the IL-17 effector fate (15, 35–37), and expression of CD27 and CD122 marks those of the IFN-geffector fate (38, 39). Using our i.c. flow cytometric assay, wefound that CCR6+ DN gdTCR+ cells, in both the thymus and theLN, express relatively high levels of Blk, whereas thymic andperipheral CD27+ DN gdTCR+ cells express relatively low levelsof Blk (Fig. 7B). These data demonstrated that Blk is preferen-tially expressed in IL-17–producing gd T cells.We next quantified potential IL-17–producing gd effectors in

gdTCR Tg Blk2/2 mice and found a significant decrease (5- to 8-fold) in the number of CCR6+ gd T cells in both the thymus andLN of gdTCR Tg Blk2/2 mice relative to gdTCR Tg Blk+/+ mice(Fig. 7C). These findings indicated that, in the absence of Blk,Vg6+ gdTCRTg IL-17 effectors fail to develop. Moreover, becauseVg6+ fetal thymocytes in non-gdTCR Tg mice are monoclonal (3)and because we observed a loss in the number of Vg52 fetal

FIGURE 6. Effects of Blk deficiency on T cell development in gdTCR Tg mice. A, Phenotypic analysis of 4- to 6-wk-old gdTCR Tg Blk2/2 and gdTCR

Tg Blk+/+ mice. Dot plots show representative CD4 versus CD8 staining profiles on total thymocytes. Numbers in quadrants of the two-color plots represent

the percentage of cells in each quadrant. Adjacent dot plots show representative TCRgd versus CD3 staining on DN thymocytes and LN cells. Numbers

represent percentage of cells in each gate. B, Mean number6 SEM of DN (DN TCRgd+; gd lineage) and DP (ab lineage) thymocytes in gdTCRTg Blk2/2

and gdTCRTg Blk+/+ mice. Data represent five mice per genotype. C, Mean number6 SEM of DN gd T cells in the LNs of gdTCRTg Blk2/2 and gdTCR

Tg Blk+/+ mice. Data represent five mice per genotype. D, Comparison of CD5 and CD3 surface levels on DN TCRgd+ cells in the thymus and LNs of

gdTCR Tg Blk2/2 and gdTCR Tg Blk+/+ mice. Data are representative of four mice per genotype.

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thymocytes in non-gdTCR Tg Blk2/2 mice, our results suggestedthat Blk is required for the generation of Vg6+ fetal thymocytesdeveloping along the IL-17 effector fate pathway.In wild-type mice, IL-17 producers are found primarily within

the Vg6+ and Vg4+ gd T cell subsets (40–42). Notably, althoughVg6+ T cells are only generated in the fetal thymus, Vg4+ gd

T cells are generated in the late fetal and adult thymus (3). Be-cause of this difference in the timing of their development, wesought to determine whether loss of Blk affects the developmentof IL-17–producing gd T cells that arise in the postnatal thymus.Analysis of 4-wk-old Blk+/+ and Blk2/2 IL-23R-GFP.KI micerevealed a significant decrease in both the percentage and numberof IL-23R+ gd thymocytes (which are GFP+ in IL-23R-GFP.KImice) in Blk2/2 mice compared with Blk+/+ mice (Fig. 7D).Accordingly, significantly fewer IL-23R+ gd T cells were detectedin the LNs of Blk2/2 IL-23R-GFP.KI mice than in the LNs ofBlk+/+ IL-23R-GFP.KI mice (Fig. 7D). These findings indicatedthat the IL-17 gd effectors that are generated postnatally requireBlk for their development. However, because a small populationof IL-23R+ gd T cells can still be detected in Blk2/2 mice, theseresults also suggested that there are differences between subsets ofIL-17 gd effectors in the molecular requirements for their de-velopment.

DiscussionMany groups have used transcriptional profiling to gain a betterunderstanding of the genetic programs involved in gd T cell de-velopment and function. Likewise, we have used a genetic approach

to identify signaling molecules that are expressed in gd T cells butnot ab T cells as a means to elucidate the molecular basis for theenhanced signaling ability of the gdTCR. Global gene expressionanalysis identified Blk as a signaling molecule preferentially ex-pressed in gd T cells; however, subsequent analysis demonstratedthat its expression in gd T lineage cells does not provide a molec-ular mechanism by which the gdTCR can signal better than theabTCR. Instead, this study revealed that Blk plays unanticipatedroles in T lymphopoiesis and in the development of IL-17–pro-ducing gd T cell effectors.It is not surprising that we identified a gene involved in the

generation of IL-17–producing gd T cells, because our strategywas to compare the transcriptional profile of gd T cells with thatof CD8+ ab T cells, which are programmed to produce IFN-gduring an immune response. What is surprising is that we iden-tified Blk, an SFK whose expression was reported to be restrictedto B lineage cells (13). It is interesting to note that, in humans, Blkis expressed in thymocytes, indicating that its expression is notlimited to B cells (43). In this study, we show that murine Blk isexpressed in progenitor populations from both the BM and thy-mus, in immature DN thymocytes, in gd thymocytes, and in asmall subset of mature gd T cells that have the potential to pro-duce IL-17. Therefore, Blk has a much broader expression patternthan reported previously.Blk, because of its expression in T cell precursors, plays a role in

controlling thymus cellularity. Remarkably, this requirement forBlk activity is most evident when T cell development has notreached equilibrium, such as during the postnatal period or during

FIGURE 7. Effects of Blk deficiency on gd T cell effector function. A, Comparison of IL-17 versus IFN-g production and IFN-g versus TNF-a pro-

duction by DN gdTCR+ LN cells from gdTCR Tg Blk2/2 and gdTCR Tg Blk+/+ mice. LN cells from the two genotypes were in vitro stimulated with 1 mg/

ml immobilized anti-CD3 mAb (2C11) or 5 mg/ml immobilized hamster IgG. Forty-eight hours later, cells were harvested, and cytokine production was

assayed by i.c. flow cytometric analysis. Dot plots show representative i.c. staining for gated DN gdTCR+ cells. Numbers in the quadrants represent

percentage of cells in that quadrant. Data shown are representative of six mice per genotype. B, Histogram showing representative i.c. Blk staining on gated

CCR6+ (black histogram) and CD27+ (dashed dark gray histogram) DN gdTCR+ cells in the thymus and LN of Blk+/+ mice. Total gd thymocytes (light

shaded histogram) are shown as a staining control in the left histogram and B cells (light shaded histogram) are shown as a staining control in the right

histogram. C, Dot plots showing representative CD44 versus CCR6 staining on DN gdTCR+ thymocytes and LN cells from gdTCR Tg Blk2/2 (n = 4) and

gdTCR Tg Blk+/+ (n = 6) mice. Numbers represent percentage of cells in each gate. D, Histograms showing representative IL-23R (GFP+) staining on DN

gdTCR+ thymocytes and LN cells from Blk2/2 IL-23R-GFP.KI (n = 3) and Blk+/+ IL-23R-GFP.KI (n = 5) mice. Numbers represent percentage of cells in

each marker.

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thymic reconstitution following BM transfer into irradiated re-cipients. Notably, in young (2- to 4-wk-old) Blk2/2 mice, thedecrease in thymus cellularity results in lower thymic output, asevidenced by the reduction in the number of mature T cells inBlk2/2 mice compared with age-matched Blk+/+ mice. However,by 6 wk of age, we observed no difference between Blk+/+ andBlk2/2 mice in the total numbers of thymocytes or mature T cells.Because this is the age at which Blk2/2 mice were initially ana-lyzed (14); this may explain why these authors did not observe anydefects in T cell development.One way that Blk regulates thymus cellularity is by controlling

proliferation. Notably, we found that, in the absence of Blk, all DNthymocytes exhibited reduced proliferation, with those in the DN4subset displaying the greatest reduction. Remarkably, in wild-typemice, this is the only DN subset that does not express Blk. Toexplain this apparent paradox, we propose that Blk acts in a signal-ing pathway that not only induces proliferation but also regulates thecell’s fitness to respond to proliferative stimuli. Candidate re-ceptor signaling pathways include those of IL-7R, c-Kit, and Hed-gehog, all of which have been shown to activate SFKs (44–47) andto be involved in thymocyte proliferation (48–51).TCR signal strength plays a role in determining whether an

immature thymocyte chooses the ab or gd lineage fate. Specifi-cally, it has been shown, using gdTCR Tg mice, that a stronggdTCR signal favors commitment to the gd lineage, whereasa weak gdTCR signal favors commitment to the ab lineage (52,53). Paradoxically, although the thymic and peripheral gd T cellsin gdTCR Tg Blk2/2 mice had all the earmarks of cells that havereceived a strong TCR signal, ab/gd lineage commitment was notaffected, as evidenced by the comparable numbers of ab and gdlineage cells in the thymi of gdTCR Tg Blk2/2 and gdTCR TgBlk+/+ mice. This is in contrast with the results of previous studiesin which augmenting gdTCR signal strength consistently resultedin fewer ab lineage cells (18, 52, 53). An explanation for thesefindings is that TCR-induced Blk activity is not required at thestage when ab/gd lineage commitment occurs but instead is re-quired at a later developmental stage, after adoption of the gdlineage fate.The differential expression of Blk between gd thymocytes

destined to produce IL-17 and those destined to produce IFN-gsuggests that CD27 (38) and/or strong TCR signaling (39, 54),both of which are required for the development of IFN-g effectors,downregulate Blk expression. Moreover, Blk expression is re-tained in mature IL-17 gd effectors. This retention suggests thatBlk enforces the IL-17 effector differentiation program that wasestablished in the thymus and may explain why cytokine pro-duction by the different gd T cell effectors remains stable bothin vitro and in vivo (38).We also noted that Vg6+ gd T cells with the potential to produce

IL-17 are more dependent on Blk for their development than onesbearing other Vg gene segments. Moreover, this difference in theirrequirement for Blk may reflect a divergence in their respectivedevelopmental pathways and suggests that there may be differ-ences in their surface phenotype and functional ability.The mechanism(s) underlying the role of Blk in IL-17 effector

cell generation is currently unknown. One possible mechanism isthat Blk acts in a signaling pathway that initiates the genetic programfor IL-17–producing gd T cells. However, preliminary studies re-veal that IL-23R+ gd T cells from Blk2/2 mice express Rorc andproduce IL-17 following TCR stimulation (data not shown). An-other mechanism by which Blk may mediate development of IL-17–producing gd T cells is by regulating the gdTCR signalingthreshold. In a recent study (39), it was shown that IL-17–pro-ducing gd T cells develop in the absence of natural ligand,

suggesting that TCR engagement is not required for their gener-ation. However, under normal conditions, IL-17–producing gdT cells do develop in the presence of ligand (39). Because it isunlikely that gd thymocytes can avoid ligand encounter in a wild-type thymus, we propose that there are ligands with different af-finities for the gdTCR and that these ligands are expressed at dif-ferent levels within the thymus. Because Blk is a negative regulatorof gdTCR signaling, it would have an active role in establishingthe gdTCR signaling threshold in gd thymocytes. In this capacity,Blk may function to test the affinity/avidity of gdTCR–ligand in-teractions so that signals delivered by low-affinity/avidity interac-tions direct gd thymocytes to the IL-17 effector fate and thosedelivered by high-affinity/avidity interactions direct gd thymo-cytes to the IFN-g effector fate. Studies are under way to inves-tigate this possibility.In summary, we have found that Blk is not only expressed in gd

T cells but also is required for the development of wild-typenumbers of IL-17–producing gd T cells. The finding that B cellsand gd T cells express Blk is quite intriguing, especially in lightof the hypothesis that gd T cells may be the primordial Ag receptor-bearing lymphocyte (55). Thus, the discovery of Blk expression ingd lineage cells has broader implications in regards to the evolu-tion of AgR-coupled signal transduction.

AcknowledgmentsWe thank Jennifer Kieffer and Alyse Douglass for excellent technical ser-

vice, Karen Gentile and Dr. Frank Middleton from the SUNY Microarray

Core facility for service, and Drs. Mohamed Oukka, Paul Love, and Alex-

ander Tarakhovsky for mice. We also thank Dr. Paul Love for helpful advice

and critical review of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

References1. Chien, Y. H., R. Jores, and M. P. Crowley. 1996. Recognition by g/d T cells.

Annu. Rev. Immunol. 14: 511–532.2. Hayday, A. C. 2000. gd Cells: a right time and a right place for a conserved third

way of protection. Annu. Rev. Immunol. 18: 975–1026.3. Carding, S. R., and P. J. Egan. 2002. gd T cells: functional plasticity and het-

erogeneity. Nat. Rev. Immunol. 2: 336–345.4. Chen, Y. K., E. Chou, W. L. Fuchs, Havran, and R. Boismenu. 2002. Protection

of the intestinal mucosa by intraepithelial gd T cells. Proc. Natl. Acad. Sci. USA99: 1433–1443.

5. Jameson, J., K. Ugarte, N. Chen, P. Yachi, E. Fuchs, R. Boismenu, andW. L. Havran. 2002. A role for skin gd T cells in wound repair. Science 296: 747–749.

6. Sharp, L. L., J. M. Jameson, G. Cauvi, and W. L. Havran. 2005. Dendriticepidermal T cells regulate skin homeostasis through local production of insulin-like growth factor 1. Nat. Immunol. 6: 73–79.

7. Hiromatsu, K., Y. Yoshikai, G. Matsuzaki, S. Ohga, K. Muramori, K. Matsumoto,J. A. Bluestone, and K. Nomoto. 1992. A protective role of g/d T cells in primaryinfection with Listeria monocytogenes in mice. J. Exp. Med. 175: 49–56.

8. Ferrick, D. A., M. D. Schrenzel, T. Mulvania, B. Hsieh, W. G. Ferlin, andH. Lepper. 1995. Differential production of interferon-g and interleukin-4 inresponse to Th1- and Th2-stimulating pathogens by gd T cells in vivo. Nature373: 255–257.

9. Gao, Y., W. Yang, M. Pan, E. Scully, M. Girardi, L. H. Augenlicht, J. Craft, andZ. Yin. 2003. gd T cells provide an early source of interferon g in tumor im-munity. J. Exp. Med. 198: 433–442.

10. Girardi, M., E. Glusac, R. B. Filler, S. J. Roberts, I. Propperova, J. Lewis,R. E. Tigelaar, and A. C. Hayday. 2003. The distinct contributions of murineT cell receptor (TCR)gd+ and TCRab+ T cells to different stages of chemicallyinduced skin cancer. J. Exp. Med. 198: 747–755.

11. Hayes, S. M., and P. E. Love. 2002. Distinct structure and signaling potential ofthe gd TCR complex. Immunity 16: 827–838.

12. Laird, R. M., and S. M. Hayes. 2009. Profiling of the early transcriptional re-sponse of murine gd T cells following TCR stimulation. Mol. Immunol. 46:2429–2438.

13. Dymecki, S. M., J. E. Niederhuber, and S. V. Desiderio. 1990. Specific ex-pression of a tyrosine kinase gene, blk, in B lymphoid cells. Science 247: 332–336.

6526 ROLE OF Blk IN DEVELOPMENT OF IL-17–PRODUCING gd T CELLS

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Page 11: Unexpected Role for the B Cell-Specific Src Family Kinase B ... · PDF fileLymphoid Kinase in the Development of IL-17–Producing gd T Cells ... which encodes a B cell-specific

14. Texido, G., I. H. Su, I. Mecklenbrauker, K. Saijo, S. N. Malek, S. Desiderio,K. Rajewsky, and A. Tarakhovsky. 2000. The B-cell–specific Src-family kinaseBlk is dispensable for B-cell development and activation. Mol. Cell. Biol. 20:1227–1233.

15. Awasthi, A., L. Riol-Blanco, A. Jager, T. Korn, C. Pot, G. Galileos, E. Bettelli,V. K. Kuchroo, and M. Oukka. 2009. Cutting edge: IL-23 receptor gfp reportermice reveal distinct populations of IL-17–producing cells. J. Immunol. 182:5904–5908.

16. Sim, G. K., C. Olsson, and A. Augustin. 1995. Commitment and maintenance ofthe ab and gd T cell lineages. J. Immunol. 154: 5821–5831.

17. Laky, K., L. Lefrancois, E. G. Lingenheld, H. Ishikawa, J. M. Lewis, S. Olson,K. Suzuki, R. E. Tigelaar, and L. Puddington. 2000. Enterocyte expression ofinterleukin 7 induces development of gd T cells and Peyer’s patches. J. Exp.Med. 191: 1569–1580.

18. Laird, R. M., and S. M. Hayes. 2010. Roles of the Src tyrosine kinases Lck andFyn in regulating gdTCR signal strength. PLoS One 5: e8899.

19. Yin, Z., C. H. Chen, S. J. Szabo, L. H. Glimcher, A. Ray, and J. Craft. 2002. T-bet expression and failure of GATA-3 cross-regulation lead to default productionof IFN-g by gd T cells. J. Immunol. 168: 1566–1571.

20. Pennington, D. J., B. Silva-Santos, J. Shires, E. Theodoridis, C. Pollitt,E. L. Wise, R. E. Tigelaar, M. J. Owen, and A. C. Hayday. 2003. The inter-relatedness and interdependence of mouse T cell receptor gd+ and ab+ cells.Nat. Immunol. 4: 991–998.

21. Melichar, H. J., K. Narayan, S. D. Der, Y. Hiraoka, N. Gardiol, G. Jeannet, W. Held,C. A. Chambers, and J. Kang. 2007. Regulation of gd versus ab T lymphocytedifferentiation by the transcription factor SOX13. Science 315: 230–233.

22. Burkhardt, A. L., M. Brunswick, J. B. Bolen, and J. J. Mond. 1991. Anti-immunoglobulin stimulation of B lymphocytes activates src-related protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 88: 7410–7414.

23. Saouaf, S. J., S. Mahajan, R. B. Rowley, S. A. Kut, J. Fargnoli, A. L. Burkhardt,S. Tsukada, O. N. Witte, and J. B. Bolen. 1994. Temporal differences in theactivation of three classes of non-transmembrane protein tyrosine kinases fol-lowing B-cell antigen receptor surface engagement. Proc. Natl. Acad. Sci. USA91: 9524–9528.

24. DeFranco, A. L. 1997. The complexity of signaling pathways activated by theBCR. Curr. Opin. Immunol. 9: 296–308.

25. Silva-Santos, B., D. J. Pennington, and A. C. Hayday. 2005. Lymphotoxin-mediated regulation of gd cell differentiation by ab T cell progenitors. Sci-ence 307: 925–928.

26. van Oers, N. S., B. Lowin-Kropf, D. Finlay, K. Connolly, and A. Weiss. 1996. abT cell development is abolished in mice lacking both Lck and Fyn protein ty-rosine kinases. Immunity 5: 429–436.

27. Page, S. T., N. S. van Oers, R. M. Perlmutter, A. Weiss, and A. M. Pullen. 1997.Differential contribution of Lck and Fyn protein tyrosine kinases to intra-epithelial lymphocyte development. Eur. J. Immunol. 27: 554–562.

28. Petrie, H. T., R. Scollay, and K. Shortman. 1992. Commitment to the T cellreceptor-ab or -gd lineages can occur just prior to the onset of CD4 and CD8expression among immature thymocytes. Eur. J. Immunol. 22: 2185–2188.

29. Godfrey, D. I., J. Kennedy, T. Suda, and A. Zlotnik. 1993. A developmentalpathway involving four phenotypically and functionally distinct subsets of CD3–

CD4–CD8– triple-negative adult mouse thymocytes defined by CD44 and CD25expression. J. Immunol. 150: 4244–4252.

30. Ciofani, M., G. C. Knowles, D. L. Wiest, H. von Boehmer, and J. C. Zuniga-Pflucker. 2006. Stage-specific and differential notch dependency at the ab andgd T lineage bifurcation. Immunity 25: 105–116.

31. Michie, A. M., and J. C. Zuniga-Pflucker. 2002. Regulation of thymocyte dif-ferentiation: pre-TCR signals and b-selection. Semin. Immunol. 14: 311–323.

32. Porritt, H. E., L. L. Rumfelt, S. Tabrizifard, T. M. Schmitt, J. C. Zuniga-Pflucker,and H. T. Petrie. 2004. Heterogeneity among DN1 prothymocytes reveals mul-tiple progenitors with different capacities to generate T cell and non-T celllineages. Immunity 20: 735–745.

33. Azzam, H. S., A. Grinberg, K. Lui, H. Shen, E. W. Shores, and P. E. Love. 1998.CD5 expression is developmentally regulated by T cell receptor (TCR) signalsand TCR avidity. J. Exp. Med. 188: 2301–2311.

34. Hamada, S., M. Umemura, T. Shiono, H. Hara, K. Kishihara, K. Tanaka,H. Mayuzumi, T. Ohta, and G. Matsuzaki. 2008. Importance of murine Vd1gdT cells expressing interferon-g and interleukin-17A in innate protection againstListeria monocytogenes infection. Immunology 125: 170–177.

35. Haas, J. D., F. H. Gonzalez, S. Schmitz, V. Chennupati, L. Fohse, E. Kremmer,R. Forster, and I. Prinz. 2009. CCR6 and NK1.1 distinguish between IL-17Aand IFN-g–producing gd effector T cells. Eur. J. Immunol. 39: 3488–3497.

36. Martin, B., K. Hirota, D. J. Cua, B. Stockinger, and M. Veldhoen. 2009. In-terleukin-17–producing gd T cells selectively expand in response to pathogenproducts and environmental signals. Immunity 31: 321–330.

37. Sutton, C. E., S. J. Lalor, C. M. Sweeney, C. F. Brereton, E. C. Lavelle, andK. H. Mills. 2009. Interleukin-1 and IL-23 induce innate IL-17 productionfrom gd T cells, amplifying Th17 responses and autoimmunity. Immunity 31:331–341.

38. Ribot, J. C., A. deBarros, D. J. Pang, J. F. Neves, V. Peperzak, S. J. Roberts,M. Girardi, J. Borst, A. C. Hayday, D. J. Pennington, and B. Silva-Santos. 2009.CD27 is a thymic determinant of the balance between interferon-g- and in-terleukin 17-producing gd T cell subsets. Nat. Immunol. 10: 427–436.

39. Jensen, K. D., X. Su, S. Shin, L. Li, S. Youssef, S. Yamasaki, L. Steinman,T. Saito, R. M. Locksley, M. M. Davis, et al. 2008. Thymic selection determinesgd T cell effector fate: antigen-naive cells make interleukin-17 and antigen-experienced cells make interferon g. Immunity 29: 90–100.

40. Roark, C. L., J. D. French, M. A. Taylor, A. M. Bendele, W. K. Born, andR. L. O’Brien. 2007. Exacerbation of collagen-induced arthritis by oligoclonal,IL-17–producing g d T cells. J. Immunol. 179: 5576–5583.

41. Hamada, S., M. Umemura, T. Shiono, K. Tanaka, A. Yahagi, M. D. Begum,K. Oshiro, Y. Okamoto, H. Watanabe, K. Kawakami, et al. 2008. IL-17A pro-duced by gd T cells plays a critical role in innate immunity against listeriamonocytogenes infection in the liver. J. Immunol. 181: 3456–3463.

42. Shibata, K., H. Yamada, R. Nakamura, X. Sun, M. Itsumi, and Y. Yoshikai. 2008.Identification of CD25+ gd T cells as fetal thymus-derived naturally occurringIL-17 producers. J. Immunol. 181: 5940–5947.

43. Islam, K. B., H. Rabbani, C. Larsson, R. Sanders, and C. I. Smith. 1995. Mo-lecular cloning, characterization, and chromosomal localization of a humanlymphoid tyrosine kinase related to murine Blk. J. Immunol. 154: 1265–1272.

44. Venkitaraman, A. R., and R. J. Cowling. 1992. Interleukin 7 receptor functionsby recruiting the tyrosine kinase p59fyn through a segment of its cytoplasmictail. Proc. Natl. Acad. Sci. USA 89: 12083–12087.

45. Page, T. H., F. V. Lali, and B. M. Foxwell. 1995. Interleukin-7 activates p56lckand p59fyn, two tyrosine kinases associated with the p90 interleukin-7 receptorin primary human T cells. Eur. J. Immunol. 25: 2956–2960.

46. Timokhina, I., H. Kissel, G. Stella, and P. Besmer. 1998. Kit signaling through PI3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activationin mast cell proliferation. EMBO J. 17: 6250–6262.

47. Yam, P. T., S. D. Langlois, S. Morin, and F. Charron. 2009. Sonic hedgehogguides axons through a noncanonical, Src-family-kinase-dependent signalingpathway. Neuron 62: 349–362.

48. Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky,B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware, et al. 1994.Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180: 1955–1960.

49. von Freeden-Jeffry, U., N. Solvason, M. Howard, and R. Murray. 1997. Theearliest T lineage-committed cells depend on IL-7 for Bcl-2 expressionand normal cell cycle progression. Immunity 7: 147–154.

50. Agosti, V., S. Corbacioglu, I. Ehlers, C. Waskow, G. Sommer, G. Berrozpe,H. Kissel, C. M. Tucker, K. Manova, M. A. Moore, et al. 2004. Critical role forKit-mediated Src kinase but not PI 3-kinase signaling in pro-T and pro-B celldevelopment. J. Exp. Med. 199: 867–878.

51. Shah, D. K., A. L. Hager-Theodorides, S. V. Outram, S. E. Ross, A. Varas, andT. Crompton. 2004. Reduced thymocyte development in sonic hedgehogknockout embryos. J. Immunol. 172: 2296–2306.

52. Haks, M. C., J. M. Lefebvre, J. P. Lauritsen, M. Carleton, M. Rhodes,T. Miyazaki, D. J. Kappes, and D. L. Wiest. 2005. Attenuation of gdTCR sig-naling efficiently diverts thymocytes to the ab lineage. Immunity 22: 595–606.

53. Hayes, S. M., L. Li, and P. E. Love. 2005. TCR signal strength influences ab/gdlineage fate. Immunity 22: 583–593.

54. 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 gd T cells withrestricted TCR diversity. Proc. Natl. Acad. Sci. USA 106: 12453–12458.

55. Flajnik, M. F. 1998. Churchill and the immune system of ectothermic verte-brates. Immunol. Rev. 166: 5–14.

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