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Cell Development and Homeostasis Tδγ1 Interaction in γPLC−The Role of LAT

Lewis, Chih-wen Ou-Yang and Weiguo ZhangSarah A. Sullivan, Minghua Zhu, Steven Bao, Catherine A.

http://www.jimmunol.org/content/192/6/2865doi: 10.4049/jimmunol.1302493February 2014;

2014; 192:2865-2874; Prepublished online 12J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 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

The Role of LAT–PLCg1 Interaction in gd T CellDevelopment and Homeostasis

Sarah A. Sullivan, Minghua Zhu, Steven Bao, Catherine A. Lewis, Chih-wen Ou-Yang, and

Weiguo Zhang

LAT is a transmembrane adaptor protein that is vital for integrating TCR-mediated signals to modulate T cell development, ac-

tivation, and proliferation. Upon T cell activation, LAT is phosphorylated and associates with Grb2, Gads, and PLCg1 through its

four distal tyrosine residues. Mutation of one of these tyrosines, Y136, abolishes LAT binding to PLCg1. This results in impaired

TCR-mediated calcium mobilization and Erk activation. CD4 ab T cells in LATY136F knock-in mice undergo uncontrolled

expansion, resulting in a severe autoimmune syndrome. In this study, we investigated the importance of the LAT–PLCg1

interaction in gd T cells by crossing LATY136F mice with TCRb2/2 mice. Our data showed that the LATY136F mutation

had no major effect on homeostasis of epithelial gd T cells, which could be found in the skin and small intestine. Interestingly,

a population of CD4+ gd T cells in the spleen and lymph nodes underwent continuous expansion and produced elevated amounts

of IL-4, resulting in an autoimmune syndrome similar to that caused by ab T cells in LATY136F mice. Development of these

hyperproliferative gd T cells was not dependent on MHC class II expression or CD4, and their proliferation could be suppressed,

in part, by regulatory T cells. Our data indicated that a unique subset of CD4 gd T cells can hyperproliferate in LATY136F mice

and suggested that LAT–PLCg1 signaling may function differently in various subsets of gd T cells. The Journal of Immunology,

2014, 192: 2865–2874.

Themajority of T cells that traffic through lymphoid tissuesare ab T cells, whereas gd T cells represent only 1–5%of circulating lymphocytes. Rather, gd T cells populate

the gastrointestinal tract, skin, and other epithelial-rich tissues(1, 2). LAT, an important protein in the TCR-signaling pathway,is required for the development of both ab and gd T cells. LAT-deficient mice show an early block at the double-negative (DN)3stage during thymic development and lack mature ab and gd T cells(3). After T cell activation, LAT is phosphorylated on multiple ty-rosine residues. Among these tyrosine motifs, the four distal tyro-sines, 136, 175, 195, and 235 in murine LAT, are responsible forbinding PLCg1, Gads, and Grb2 and are required for TCR-mediatedcalcium mobilization, Erk activation, and cytokine production (4).Published studies (5–7) using mice with these residues mutated,

LATY136F and LAT3YF (Y175, Y195, and Y235 are all mu-tated), suggest a differential requirement for LAT signaling duringdevelopment and activation of ab and gd T cells. In LATY136Fmice, in which the PLCg1 binding site is mutated, ab T cell de-velopment is partially blocked at DN3. CD8 and gd T cells arenearly absent from secondary lymphoid organs. In contrast, CD4T cells hyperproliferate, resulting in a severe autoimmune syn-drome (5, 6). These CD4 T cells have an effector/memory-like

phenotype and produce large amounts of Th2 cytokines, such asIL-4. Correspondingly, serum IgE and IgG1 are elevated, and auto-

antibodies are detectable (5, 6). These data demonstrate that the

LAT–PLCg1 interaction plays important roles in the development

and homeostasis of ab T cells. The effect of this mutation on gd

T cell development and homeostasis has not been determined.In contrast, LAT3YF mice, in which the Gads and Grb2 binding

sites are mutated, display a strikingly different phenotype (7). Theyhave a complete block in ab T cell development at DN3, indi-cating the importance of signaling through Gads and Grb2 at thepre-TCR checkpoint. Surprisingly, gd T cells, which rearrangeboth g and d genes in the DN compartment, only have a partialdefect in their development in these mice. These gd T cells, ∼20%of which express CD4, are able to populate peripheral lymphoidtissues and instigate an autoimmune syndrome in 6-mo-old mice(7). Interestingly, mice devoid of Itk, a tyrosine kinase that phos-phorylates PLCg1, display a similar gd T cell phenotype. Itk2/2

mice have a markedly increased number of CD4 gd T cells in sec-ondary lymphoid tissues (8). These gd T cells are able to pro-duce Th2 cytokines and were identified as innate-like gd NKT cells,because they expressed the transcription factor PLZF (9, 10). Takenogether, these data suggest that ab and gd T cells have differentsignaling requirements during development and that dampenedTCR signaling in both ab and gd T cells can lead to a Th2-skewed phenotype.Because CD4+ ab T cells dominate the periphery of LATY136F

mice, it is difficult to study the effect of this mutation on gd T cells.

To specifically investigate the importance of LAT–PLCg1 signaling

in gd T cells, we crossed LATY136F mice, designated as LATm/m,

with TCRb2/2 mice to generate TCRb2/2LATm/m mice. These

mice revealed that abolishing the LAT–PLCg1 interaction resulted

in a partial block in gd T cell development. gd T cells could be

found in the skin and small intestine and had no obvious activated

phenotypes. Interestingly, a population of CD4+ gd T cells in the

spleen and lymph nodes underwent rapid proliferation and caused

Department of Immunology, Duke University Medical Center, Durham, NC 27710

Received for publication September 16, 2013. Accepted for publication January 13,2014.

This work was supported by National Institutes of Health Grants AI048674 andAI093717.

Address correspondence and reprint requests to Dr. Weiguo Zhang, Department ofImmunology, Jones Building, Room 112, Box 3010, Duke University Medical Cen-ter, Research Drive, Durham, NC 27710. E-mail address: zhang033@mc.duke.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: DETC, dendritic epidermal T cell; DN, doublenegative; IEL, intraepithelial lymphocyte; nTreg, natural regulatory T cell; Tcon,conventional T cell; Treg, regulatory T cell.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302493

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a severe autoimmune syndrome. Our data suggested that LATfunctions differently in maintaining the homeostasis of differentpopulations of gd T cells.

Materials and MethodsMice

LATm/m and LAT2/2 mice were described previously (3, 6), and TCRb2/2,MHCII2/2, and CD42/2 mice were purchased from The Jackson Labo-ratory (Bar Harbor, ME). Mice were housed in a specific pathogen–freefacility. All experiments with mice were conducted in accordance withNational Institutes of Health guidelines and were approved by the DukeUniversity Institutional Animal Care and Use Committee.

Flow cytometry and ELISA

Single-cell suspensions were stained with fluorescently conjugated Abs(BioLegend, San Diego, CA) in the presence of 2.4G2 (anti-FcgII/III re-ceptor). For intracellular staining, splenocytes were stained for surfacemarkers, fixed, permeabilized, and stained for IL-17, IL-4, IL-2, IFN-g,T-bet, EOMES, GATA3, and PLZF. For cytokine production, cells werestimulated with PMA (20 ng/ml) and ionomycin (0.5 mg/ml) for 4 h in thepresence of monensin. 7-aminoactinomycin D or a Live/Dead Staining kit(Invitrogen) was used to distinguish live cells from dead ones. Data wereacquired on a FACSCanto II (BD Biosciences) and analyzed usingFlowJo software (Tree Star, Ashland, OR). For the anti-dsDNA ELISAs,plates were coated with 5 mg/ml calf-thymus DNA in Reacti-Bind DNACoating Solution (Pierce). ELISA on serum Abs was done as previously de-scribed (11).

Isolation of intraepithelial lymphocytes and dendriticepidermal T cells

Small intestines were cut longitudinally and incubated in HBSS containing5% FBS and 5 mM EDTA. Cells were strained and resuspended in 45%Percoll (Amersham Biosciences) and isolated using a 45/70% Percollgradient. Ears were separated into ventral and dorsal halves and incubated in20 mM EDTA for 3 h at 37˚C. Epidermal sheets were removed, incubatedin trypsin, homogenized, and analyzed by FACS.

Cell purification

T cells from spleen and lymph nodes were isolated by negative depletionusing EasySep Biotin Purification kits (STEMCELL Technologies).Regulatory T cells (Tregs) (CD4+CD25+), non-Tregs (CD4+CD252), andgd T cells were sorted on a FACSVantage flow cytometer (BD Bio-sciences). A total of 1 3 106 T cells was transferred into recipient micevia i.v. injection.

Variable gene usage of g and d chains

gd T cells from TCRb2/2LATm/m mice were isolated from the spleen,stimulated for 24 h with PMA and ionomycin, and fused with TCR-deficient BW5147 cells to generate T cell hybridomas. PCR primers forVg1.1, Cg4, Vd2.2, Vd4, Vd5, Vd6.1, Vd6.2, Vd7, and Cd were used toamplify cDNAs from these hybridomas (12, 13). PCR products were pu-rified and sequenced. Sequences were analyzed on the international Im-MunoGeneTics information system (http://www.imgt.org).

ResultsEffect of the LATY136F mutation on gd T cell development

As previously reported, ablation of TCRb expression caused a blockin ab T cell development at the DN3 stage, but a small number ofdouble-positive thymocytes was still present in TCRb2/2 mice(14). Similar to TCRb2/2 mice, TCRb2/2LATm/m mice had ∼10%double-positive thymocytes (Fig. 1A). Analysis of the DN com-partment in TCRb2/2LATm/m mice revealed that, althoughthe percentages of DN1–DN4 subsets were similar to those inTCRb2/2 mice, the percentage of CD3+TCRgd+ DN thymocyteswas moderately reduced, from 15.6% in TCRb2/2 mice to 11.2%in TCRb2/2LATm/m mice (Fig. 1A). In addition, the numbers oftotal, DN, and CD3+TCRgd+ thymocytes were reduced inTCRb2/2LATm/m mice (Fig. 1B).

We subsequently analyzed whether LATm/m gd thymocytes wereproperly selected. CD5 is upregulated upon selection of gd cells,whereas CD25 is downregulated prior to their exit from the thy-mus (15–17). As shown in Fig. 1C, TCRgd+ thymocytes fromTCRb2/2LATm/m mice had impaired CD5 upregulation and CD25downregulation. The numbers of CD3+TCRgd+CD252 thymo-cytes in the DN compartment of TCRb2/2LATm/m mice werereduced significantly (Fig. 1B). These data indicated that gdthymocyte selection is impaired by the LATY136F mutation. Inaddition, overall surface expression of gd TCR was lower, andthere were more gd T cells that expressed an intermediate level ofthe TCR. gd T cells in adult thymuses and spleens express Vg1and Vg2. The percentages of gd thymocytes expressing Vg1+ andVg2+ were similar in TCRb2/2LATm/m and TCRb2/2 mice;however, the percentage of Vd6.3+ gd thymocytes was reduced inTCRb2/2LATm/m mice (Fig. 1D).It was suggested that gd T cells acquire their effector fates in the

thymus (18). CD27 is thought to be high on IFN-g–producing gdprogenitors and low on IL-17–producing progenitors (19). Furtherstudies indicated that TCRgd+CCR6+ thymocytes produce IL-17,whereas TCRgd+NK1.1+ thymocytes do not (20). The majority ofTCRgd+ in both TCRb2/2 and TCRb2/2LATm/m mice wereCD27hi and CCR62, indicating that they were not IL-17 producers(Fig. 1E). NK1.1 and the transcription factor PLZF identify apopulation of innate-like gd NKT cells (21). Analysis of gd+

thymocytes from TCRb2/2 and TCRb2/2LATm/m mice revealedthat the NK1.1+ population was reduced from 10.3% to 2.8%, andthe PLZF+ population was reduced from 17.1% to 2.6% (Fig. 1E),indicating that the development of innate-like gd NKT cells wasimpaired in the absence of LAT–PLCg1. We also examined thedevelopment of gd+ thymocytes that populate epithelial tissues.gd thymocytes that traffic to the epidermis tend to be T-bet+,whereas those expressing Vd4 go to the intestinal epithelial (22).Both the T-bet– and Vd4-expressing gd thymocyte populationswere reduced in TCRb2/2LATm/m mice compared with thosein TCRb2/2 mice (Fig. 1F). Together, these data indicated that,in the absence of the LAT–PLCg1 interaction, the developmentand selection of different gd thymocyte subsets were partiallyblocked.

gd T cells in the epithelial tissues of TCRb2/2LATm/m mice

gd T cells are highly enriched in epithelial, mucosal, and barriertissues, including the skin, lungs, and small intestine, where theyare thought to function in initial host defense (23). Epithelial gdT cells, which develop in the fetal and perinatal thymus, havea restricted repertoire, using Vg3 and Vg4 (12). We wanted todetermine whether gd T cells are present in epithelial-rich com-partments of TCRb2/2LATm/m mice.Immunofluorescent staining of ear sections from 3-mo-old mice

showed similar numbers and morphology of dendritic epidermalT cells (DETCs) present in TCRb2/2 and TCRb2/2LATm/m mice(Supplemental Fig. 1A). FACS analysis of cells isolated from earepidermal sheets confirmed similar percentages of DETCs in thesemice. All of these DETCs expressed Vg3 (Fig. 2A). Interestingly,although the surface expression of TCRb in LATm/m mice wasgreatly reduced (6), expression of TCRgd remained high on theseDETCs.Among the small intestine intraepithelial lymphocytes (IELs),

there are two subsets of gd T cells, derived either from the thymus(Thy1+) or extrathymically (Thy12) (24). In TCRb2/2LATm/m

mice, the percentages of both small intestine gd subsets werereduced (Fig. 2B), which could be attributed to a partial block indevelopment. In the Thy1.22 subset, the majority of T cells in

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both TCRb2/2 and TCRb2/2LATm/m mice were CD8a+; how-ever, in the Thy1.2+ subset, there was an increased percentage ofCD4+ T cells, even though the number of these cells was drasti-cally reduced. The surface expression of TCRgd for both subsetsin TCRb2/2LATm/m mice was similar to that in TCRb2/2 mice(Fig. 2B). Because the surface level of TCRgd was high, we wereable to analyze Vg and Vd usages by flow cytometry. The Thy1.22

subset in TCRb2/2LATm/m mice had similar usages of Vg1.1,Vg2, Vd6.3, and Vd4 to TCRb2/2 mice (Fig. 2C). The Thy1.2+

subset in TCRb2/2LATm/m mice seemed to be skewed, with in-creased Vg1.1+Vd6.3+ cells and reduced Vg2+ or Vd4+ cellscompared with control mice (Fig. 2C).Together, these data demonstrated that, in TCRb2/2LATm/m

mice, the development of IEL gd T cells was partially blocked;however, DETCs in the skin were not affected. Interestingly, un-like ab T cells in LATm/m mice, these gd T cells in the epithelialtissues maintained normal levels of surface TCR and were not

hyperproliferative, suggesting that LAT functions differently inab and gd T cells.

The LATY136F mutation on gd T cells in secondary lymphoidorgans

LATm/m mice have splenomegaly and lymphadenopathy at 4–6 wkof age, whereas LAT3YF mice show a similar syndrome at 6 mo.Comparatively, 4–6-wk-old TCRb2/2LATm/m mice had no ap-parent splenomegaly. However, at 8–12 wk, their spleens and lymphnodes appeared enlarged, and the spleen weight and number ofsplenocytes were increased dramatically (Fig. 3A, 3B). BecauseCD4 T cells in LATm/m mice downregulate the expression ofsurface TCR (5, 6), we used the T cell markers Thy1.2 and CD5 toidentify gd T cells in 4-, 6-, and 8–12-wk-old mice. In agreementwith a partial block in gd thymocyte development, 4-wk-oldTCRb2/2LATm/m mice had decreased percentages of splenic gdT cells (1.0% compared with 1.8% in control mice) (Fig. 3C). In-

FIGURE 1. Effect of the LATY136F mutation on gd development. (A) FACS analysis of thymocytes. (B) Numbers of different subsets of thymocytes. (C)

Expression of CD5, CD25, and TCRgd. Cells in (C)–(F) were gated on CD3+TCRgd+ DN thymocytes. (D) Vg and Vd usage. (E) Expression of CD27,

CCR6, NK1.1 and PLZF. (F) Expression of T-bet and Vd4. Mice analyzed were 4-wk-old. Data are representative of three or four individual experiments.

*p , 0.05, **p , 0.005, two-tailed t test.

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terestingly, at 6 wk of age, a second population of gd T cellsappeared in the spleens and lymph nodes of TCRb2/2LATm/m

mice. This new population expressed higher levels of CD5 thandid gd T cells in TCRb2/2 mice or those in 4-wk-old TCRb2/2

LATm/m mice. By 8–12 wk, these CD5hi T cells expanded tre-mendously and composed ∼30% of total splenocytes (Fig. 3C).Further analysis of CD5hi gd T cells revealed that the majority of

them (∼70%) expressed CD4. In contrast, very few gd T cells inTCRb2/2 mice were CD4+. These CD5hi cells in TCRb2/2LATm/m

mice had an effector/memory-like phenotype (CD44hiCD62Llo)(Fig. 3D). Reminiscent of hyperproliferative CD4+ ab T cells inLATm/mmice, CD5hi gdT cells in 12-wk-old TCRb2/2LATm/mmicedownregulated surface TCR expression; however, gd TCR surfaceexpression was only slightly reduced in 4-wk-old mice (Fig. 3E).Neither TCRb2/2 nor TCRb2/2LATm/m gd T cells from aged miceexpressed NK1.1, a marker for innate gd NKT cells (Fig. 3F).To investigate whether CD5hi gd T cells were derived from

CD5int gd T cells in the periphery, T cell–transfer experimentswere performed. Thy1.2+CD5int gd T cells purified from 4-wk-oldTCRb2/2 and TCRb2/2LATm/m mice and Thy1.2+CD5hi gd

T cells from 12-wk-old TCRb2/2LATm/m mice were adoptivelytransferred into Thy1.1+LAT2/2 mice, which lack T cells. Twelveweeks posttransfer, there was no conversion from Thy1.2+CD5int

T cells to Thy1.2+CD5hi T cells (Fig. 3G). CD5hi gd T cells re-mained CD4+, and CD5int gd T cells remained CD42. Moreover,CD5int gd T cells from TCRb2/2LATm/m mice did not expand asmuch as those from TCRb2/2 mice, suggesting that their homeo-static proliferation is impaired. In contrast, CD5hi gd T cells fromTCRb2/2LATm/m mice expanded much more than did those fromTCRb2/2 mice (6.9% versus 0.14%). These data suggested thatthese two gd subsets in TCRb2/2LATm/m spleens are likely derivedfrom two different thymic outputs: a conventional pool that ex-presses relatively normal surface TCR and are defective in homeo-static proliferation and a distinct subset of CD4+ gd T cells thatexpress low levels of TCR and hyperproliferate.

CD5hi LATm/m gd T cells have a diverse TCR repertoire

gd T cells that populate lymphoid tissues develop in late fetal andearly postnatal life. These cells typically display high levels ofjunctional diversity and commonly rearrange using Vg1.1, Vg1.2,

FIGURE 2. gd T cells in the skin

epidermis and IEL compartment. (A)

TCRgd, Vg3, and CD4 expression

on cells isolated from ear epidermal

tissues. (B) FACS analysis of IELs.

(C) Vg1.1, Vg2, Vd6.3, and Vd4 us-

age in Thy1.2+ and Thy1.22 gd IELs.

Mice analyzed were 8–12-wk-old.

Data are representative of three or

four individual experiments.

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and Vg2 (12). We investigated Vg and Vd usage and clonality ofCD5hi gd T cells in TCRb2/2LATm/m mice. Because TCRgd wasdownregulated from the cell surface, it was difficult to analyze Vgand Vd usage by flow cytometry. Instead, we generated hybrid-omas by fusing CD5hi gd T cells with TCR2 BW5147 cells. FACSanalysis of 40 hybridoma clones showed that they all were Vg1.1+

(Supplemental Fig. 1B).

To further analyze Vg and Vd usage, total RNAs were isolatedand used in PCR amplification of Vg and Vd regions. Sequencingof the PCR fragments indicated that these T cells were polyclonaland, in agreement with the FACS data, they indeed used Vg1.1.Their g-chain CDR3 lengths varied from 11 to 15 residues(Table I). Sequencing d fragments showed that they primarily usedVd5, Vd6.1, and Vd6.2. Interestingly, their d chain CDR3 lengths

FIGURE 3. Hyperproliferation of T cells

in the spleen and lymph nodes. (A) Spleens

from 12-wk-old TCRb2/2 and TCRb2/2

LATm/m mice. (B) Spleen weights and the

number of splenocytes and gd T cells in 4-

and 8–12-wk-old mice. Each symbol rep-

resents one mouse. Lines denote the average

value of cell numbers. (C) FACS analysis of

splenocytes. (D) TCRgd, CD4, CD44, and

CD62L expression on gd T cells from 12-

wk-old mice. (E) gd TCR expression on

Thy1.2+CD5+ cells. (F) NK1.1 expression

on gd T cells. (G) Adoptive transfer. A

total of 1 3 106 gd T cells was sorted

and transferred into LAT2/2 mice. Twelve

weeks later, splenocytes were analyzed by

FACS. FACS plot shown is representative

of three independent experiments. ***p ,0.001, two-tailed t test.

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varied greatly, from 12 to 22 residues. In addition, three clones(B14, D4, and D19) expressed two rearranged and in-frame dalleles. These data indicated that the hyperproliferative CD5hi gdT cells are polyclonal. They use Vg1.1 and different Vd segmentswith diverse junctional regions and CDR3 lengths.

The development of an autoimmune-like syndrome

As shown in Fig. 3, gd T cells in the spleens and lymph nodes ofTCRb2/2LATm/m mice underwent a large expansion, similar toab T cells in LATm/m mice. We next examined whether theseT cells were Th2 skewed and caused autoimmunity. Splenocytesfrom 4- or 12-wk-old mice were stimulated in vitro prior to in-tracellular staining. Similar to TCRb2/2 splenic gd T cells, ∼30%of CD5int gd T cells from 4-wk-old TCRb2/2LATm/m mice pro-duced IFN-g, and a small percentage of them produced IL-17 orIL-4. In contrast, ∼90% of CD5hi gd T cells in 12-wk-old TCRb2/2

LATm/m mice produced IL-4 (Fig. 4A). Further analysis revealedthat these CD5hi gd T cells downregulated T-bet and EOMES andupregulated GATA3, the master regulator of Th2 differentiation(Fig. 4B, 4C). Itk-deficient mice have increased gd T cells, whichexpress Vg1.1 and Vd6.3 and produce IL-4. These gd cells expressPLZF and are gd NKT cells (9, 10). Although TCRb2/2gd T cellshad a small population of cells expressing PLZF, TCRb2/2LATm/m

CD5hi gd T cells did not express PLZF, indicating that they werenot gd NKT cells (Fig. 4B).We next wanted to determine the effect of the hyperpro-

liferative gd T cells on B cell activation and maturation. Al-though the numbers of B cells were not significantly elevated inTCRb2/2LATm/m mice (data not shown), they did have an acti-vated phenotype, with upregulated expression of MHC class II andCD86 (Fig. 4D). We also assessed serum Ab levels by ELISA. Ourdata showed that the concentrations of IgG1 and IgE were sig-nificantly elevated in aged TCRb2/2LATm/m mice, which alsohad enhanced levels of anti-dsDNA Abs (Fig. 4E). Takentogether, these data suggested that hyperproliferative gd T cells inTCRb2/2LATm/m mice secrete Th2 cytokines, resulting in B cellactivation, class switching, and autoantibody production.Further evaluation of other organs showed the ability of CD5hi

gd T cells to infiltrate. In the livers of 4-wk-old TCRb2/2LATm/m

mice, the number of gd T cells was much reduced compared withTCRb2/2 mice (0.3% versus 4.3%), and most of them wereCD5int (Fig. 5A). However, in the livers of 12-wk-old mice, mostgd T cells were TCRgdloCD5hiCD4+ (Fig. 5A), and their numberswere drastically increased (Fig. 5B). These data indicated that, inaddition to excessive proliferation in the spleen and lymph nodes,CD5hi gd T cells also infiltrated into the liver.

Suppression of gd proliferation by Tregs

Next, we determined whether hyperproliferation of CD5hi gdT cells could be suppressed by natural Tregs (nTregs). A total of1 3 106 CD4+CD25+ Tregs or CD4+CD252 conventional T cells(Tcons) from congenic Thy1.1+ mice was adoptively transferredinto 4-wk-old TCRb2/2 and TCRb2/2LATm/m mice. Twelve weeksafter transfer, these mice were analyzed for development of theautoimmune syndrome. Donor cells (Thy1.1+) were clearly de-tected in these mice and had no apparent effect on gd T cells inTCRb2/2 mice (Fig. 6A). Conversely, TCRb2/2LATm/m micethat received Tregs had reduced percentages of CD5hi gd T cells(Fig. 6A) and much smaller spleens (Fig. 6B) compared with bothuninjected controls and mice that received Tcons. Interestingly,TCRb2/2LATm/m mice that received Tcons displayed an inter-mediate phenotype. They had slightly larger spleens than did miceinjected with Tregs, yet they had similar percentages of gd T cellsas uninjected mice (Fig. 6A, 6B). It is also interesting to note thatthe addition of Tregs allowed the persistence of CD5int gd T cells.Both Tregs and Tcons had no effect on the phenotype of gd T cellsbecause T cells still displayed high surface CD5 and CD4 and lowTCR expression (Supplemental Fig. 2A).We also examined the ability of Tregs to inhibit proliferation of

CD5hi gd T cells by transferring CFSE-labeled gd T cells, with orwithout Tregs, into LAT2/2 hosts. As shown in Fig. 6C, Tregsslowed the proliferation of CD5hi gd T cells from TCRb2/2LATm/m

mice. These results suggested that CD4+ Tregs and, to some extent,Tcons are able to inhibit the proliferation of CD5hi gd T cells.

MHC class II and CD4 are not required for the developmentof autoimmunity

Published studies (25–27) demonstrated that the absence of clas-sical MHC molecules has no effect on the development of gd

T cells; however, because most of the gd T cells in TCRb2/2

LATm/m mice are CD4+, we investigated whether these uncon-ventional CD4+ gd T cells are MHC class II restricted or requireCD4 signaling by crossing them with MHCII2/2 and CD42/2

mice. MHCII2/2TCRb2/2LATm/m mice had enlarged spleens(Supplemental Fig. 2B). Their gd T cells expressed CD4, down-regulated surface TCR (Fig. 7A), and overproduced IL-4 (Fig.7B). Interestingly, MHCII2/2TCRb2/2LATm/m mice actually hada lower percentage of gd T cells than did TCRb2/2LATm/m mice,12.9% versus 25.8% (Fig. 7A), suggesting that a proportion of thepolyclonal gd T cells might be selected on MHC class II. Still, inthe absence of MHC class II molecules, these mutant gd T cellshyperproliferated and induced splenomegaly. Interestingly, CD42/2

TCRb2/2LATm/m mice also developed splenomegaly and lymph-

Table I. Clonality of CD5hi gd T cells

Clone Vg1.1 CDR3 Length Junctional Sequence Vd Usage Vd CDR3 Length Junctional Sequence

A7 12 CAVWSGGTSWVKIF Vd5Dd2Jd1 12 CASGYRRDTGPPSFA8 15 CAVWAPSRSGTSWVKIF Vd6.2Dd1/2Jd1 19 CALSELRIWHIIGGIPDKLVFA9 12 CAVWPSGTSWVKIF Vd6.2Dd2Jd1 20 CALSELIWPPIGGIPATDKLVFA12 13 CAVWNRSGTSWVKIF Vd6.1Dd1/2Jd1 22 CALWERAPYGIYPYRRGATDKLVFB14 13 CAVWTRTGTSWVKIF Vd6.1Dd1/2Jd1 18 CALWELNSIWRDTSSDKLVF

Vd5Dd2Jd1 20 CASGYRGWHILLYRRDGNKLVFB30 12 CAVWIRSTSWVKIF Vd5Dd1/2Jd1 17 CASGSYGILGDTPTDKLVFB34 14 CAVWIWGSGTSWVKIF Vd5Dd2Jd1 14 CASGYLGDTSYGKLVFC14 14 CAVWIWGSGTSWVKIF Vd5Dd2Jd1 14 CASGYLGDTSYGKLVFC16 12 CAVWGSGTSWVKIF Vd6.1Dd2Jd1 13 CALWELGDVSDKLVFD4 13 CAVWVLSGTSWVKIF Vd5Dd1Jd1 20 CASGYRGWHILLYRRDGNKLVF

Vd6.1Dd2Jd1 13 CALWELGDPTDKLVFD15 12 CAVWIRSTSWVKIF Vd5Dd1/2Jd1 17 CASGSYGILGDTPTDKLVFD19 11 CAVWSGTSWVKIF Vd2.2Dd2Jd1 19 CALMERVRPIGGIRTTDKLVF

Vd6.2Dd2/2Jd1 20 CALSEYGIYFGGIRASTDKLVF

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adenopathy (Supplemental Fig. 2B). CD5hi gd T cells from thesemice expanded to make up almost 50% of the splenocytes (Fig. 7A)and overproduced IL-4 (Fig. 7B). These data indicated that CD4

is not required for the development, proliferation, or cytokineproduction of LATm/m gd T cells. In contrast, because CD4 de-ficiency enhanced their proliferation, CD4 may actually function

FIGURE 4. The development of an autoimmune syndrome in TCRb2/2LATm/m mice. (A) Cytokine production. Splenocytes were stimulated for 4 h with

PMA and ionomycin before intracellular staining for cytokine production. T cells were gated using CD5 and Thy1.2. (B) Intracellular staining for T-bet,

EOMES, GATA3, and PLZF. Shaded graphs represents B220+ cells; solid black line (TCRb2/2) and dashed black line (TCRb2/2LATm/m) are gated for gd

T cells. (C) Quantification of intracellular transcription factor levels by geometric mean fluorescent intensity (gMFI). (D) MHC class II and CD86 ex-

pression on B220+ B cells. Shaded graphs represent non–B cell controls. (E) Serum Ab titers of IgG1, IgE, and anti-dsDNA Abs. Data are representative of

four or five separate experiments using two or three mice in each cohort. Each dot represents one mouse. Lines denote the average value of serum Ab titers.

*p , 0.05, **p , 0.005, ***p , 0.001, two-tailed t test.

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to negatively regulate the development or proliferation of thesecells.

DiscussionMutation of Y136, the PLCg1 binding site in LAT, results ina severe block in thymocyte development of ab T cells. This

tyrosine residue is also indispensable for controlling CD4 T cellhomeostasis, because LATY136F mice bear hyperproliferativeCD4 T cells that are Th2 skewed (5, 6). Our data demonstratedthat, without the LAT–PLCg1 interaction, gd T cells had a partial

block in their development and selection. Consequently, the

amount of gd T cells in the thymus, intestine, and liver in 4-wk-old

FIGURE 5. Infiltration of T cells into the liver. (A) Representative FACS plots of gd T cells in the liver after Percoll isolation. (B) Total numbers of

T cells isolated from the liver of 12-wk-old mice.

FIGURE 6. Suppression of gd T cell proliferation by nTregs. (A) A total of 1 3 106 nTregs (CD4+CD25+) and Tcons (CD4+CD252) from congenic

Thy1.1+ mice was sorted and injected into 4-wk-old TCRb2/2 and TCRb2/2LATm/m mice. Twelve weeks later, mice were analyzed. (B) Spleen weights.

(C) gd T cells from 12-wk-old TCRb2/2LATm/m mice were negatively selected, labeled with CFSE and injected into LAT2/2 mice with or without nTregs.

One week later, CFSE dilution in T cells from these mice was analyzed. Shaded graphs are gated for B cells. Data are representative of two experiments.

*p , 0.05, **p , 0.005, two-tailed t test.

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mice was reduced, although the percentage of gd T cells in the

skin remained similar. Despite the developmental defect, gdT cells in the skin and intestine were not hyperactivated andhad normal levels of the TCR at their surface. However, a pop-ulation of CD4 gd T cells in the secondary lymphoid organs ofTCRb2/2LATm/m mice had a very similar phenotype to ab T cellsin LATm/m mice. These gd T cells, most of which were CD4+,underwent uncontrolled expansion and caused an autoimmunesyndrome. These data indicated that the LAT–PLCg1 interactionmay function differently in different subsets of gd T cells.An alternative explanation for the ability of gd T cells to

maintain homeostasis in the epithelium but hyperproliferate in thespleen may be due to temporal differences in development orreactivity. Epithelial gd T cells that develop in the fetus may notexpress TCRs that have high affinities for self-Ags. It is possiblethat the Ags that these T cells react to in the fetal thymus are notpresent in the skin and small intestine. Thus, these T cells may notbe constantly stimulated by self-Ags. Conversely, CD5 and CD25staining of TCRb2/2LATm/m thymocytes indicated that their post-natal selection is impaired. This may allow highly self-reactiveT cells into the circulation, where they are activated by self-Ags.Our results showed that secondary lymphoid tissues in TCRb2/2

LATm/m mice contain two distinct populations of gd T cells. TheCD5hi population expressed CD4, had low TCR levels, and rapidlyexpanded. Conversely, CD5int cells did not undergo uncontrolledexpansion. Over time, the CD5int T cells population actually di-minished, likely as a result of competition for cytokines. Transferexperiments also revealed that these CD5int T cells may havea defect in homeostatic proliferation or cell survival. A similarphenomenon was seen with CD8 T cells in LATm/m mice (28). Thedisparity in proliferation potential of these gd cells might be dueto a difference in self-reactivity or dependence on normal LATsignaling to maintain homeostasis.In LAT3YF mice, LAT interaction with Gads and Grb2 is dis-

rupted. LAT3YF mice have a complete block in ab T cell de-velopment, whereas LATm/m mice have a partial block. gd T cellsare able to develop in both of these mutant lines, suggesting that

they may require less TCR-mediated signaling during develop-ment. In LAT3YF and TCRb2/2LATm/m mice, gd T cells are ableto hyperproliferate in secondary lymphoid organs (7), although theLAT3YF disease is much less severe; the autoimmune syndromedoes not develop until 30 wk of age, and only ∼30% of T cellsproduce IL-4. LATm/m gd T cells induced splenomegaly as earlyas 8 wk, and ∼90% of these T cells produced IL-4. The gd thy-mocyte selection defect appears similar between these two knock-in lines, suggesting that the difference in autoimmune syndromeseverity is attributed to signaling differences in the periphery.Recruitment of Gads and Grb2 to LAT is important for PLCg1

function. Grb2 stabilizes the interactions between LAT and PLCg1(29). Furthermore, Gads binds SLP-76, which recruits Itk tophosphorylate PLCg1 (30, 31). The inability of LAT to bind Gadsand Grb2 in LAT3YF mice also may have ramifications for LAT–PLCg1 signaling. Therefore, the LAT3YF mutation may havemore of an impact on LAT function in TCR-mediated signalingthan does the LATY136F mutation. The residual function of theseLAT mutants is likely important in T cell expansion, thus ex-plaining the delayed and less severe phenotype in LAT3YF micecompared with TCRb2/2LATm/m mice.Finally, it is known that, in the absence of Tregs, autoimmunity

occurs because of the loss of tolerance in the periphery (32–34).nTregs are able to suppress the proliferation of conventional abT cells. Our data showed that nTregs were able to greatly in-hibit gd T cell expansion and prevent splenomegaly. Interest-ingly, Tcons also had some ability to dampen splenomegaly,perhaps by competing for limited cytokines. Although Treg-deficient mice have a defect in T cell tolerance, the absenceof nTregs in TCRb2/2 mice does not result in spontaneousautoimmunity. This indicates that the absence of Tregs alone isnot responsible for T cell hyperproliferation in TCRb2/2LATm/m

mice; rather it is caused by the absence of Tregs in combinationwith strong self-reactivity or abnormal TCR signaling. The cell-intrinsic mechanisms by which weakened LAT signaling in ab orgd T cells causes this type of autoimmunity remain to be fullyexplored.

FIGURE 7. gd T cell–mediated

autoimmunity is independent of MHC

class II or CD4. (A) Representative

FACS plots from 12-wk-old mice.

Lower panels are gated on CD5+

Thy1.2+ gd T cells. (B) Cytokine

production. Splenocytes were stim-

ulated with PMA and ionomycin for

4 h before intracellular staining. Plots

shown are gated on CD5+Thy1.2+ gd

T cells. Data are representative of

two experiments.

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AcknowledgmentsWe thank the Duke University Cancer Center Flow Cytometry and DNA

Sequencing facilities.

DisclosuresThe authors have no financial conflicts of interest.

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