development of innate cd4 and cd8 t cells in itk-deficient mice is

13
of March 17, 2018. This information is current as Distinct Pathways Cells in Itk-Deficient Mice Is Regulated by T + and CD8 + Development of Innate CD4 Schwartzberg and Leslie J. Berg Ng, Catherine C. Yin, Martha S. Jordan, Pamela L. Amanda L. Prince, Zachary Kraus, Shannon A. Carty, Caleb ol.1302059 http://www.jimmunol.org/content/early/2014/06/18/jimmun published online 18 June 2014 J Immunol Material Supplementary 9.DCSupplemental http://www.jimmunol.org/content/suppl/2014/06/18/jimmunol.130205 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 March 17, 2018 http://www.jimmunol.org/ Downloaded from by guest on March 17, 2018 http://www.jimmunol.org/ Downloaded from

Upload: vuonghanh

Post on 11-Jan-2017

230 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

of March 17, 2018.This information is current as

Distinct PathwaysCells in Itk-Deficient Mice Is Regulated by

T+ and CD8+Development of Innate CD4

Schwartzberg and Leslie J. BergNg, Catherine C. Yin, Martha S. Jordan, Pamela L. Amanda L. Prince, Zachary Kraus, Shannon A. Carty, Caleb

ol.1302059http://www.jimmunol.org/content/early/2014/06/18/jimmun

published online 18 June 2014J Immunol 

MaterialSupplementary

9.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/06/18/jimmunol.130205

        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

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 2: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

The Journal of Immunology

Development of Innate CD4+ and CD8+ T Cells inItk-Deficient Mice Is Regulated by Distinct Pathways

Amanda L. Prince,* Zachary Kraus,† Shannon A. Carty,‡ Caleb Ng,‡ Catherine C. Yin,*

Martha S. Jordan,‡ Pamela L. Schwartzberg,† and Leslie J. Berg*

T cell development in the thymus produces multiple lineages of cells, including innate T cells such as gd TCR+ cells, invariant

NKT cells, mucosal-associated invariant T cells, and H2–M3-specific cells. Although innate cells are generally a minor subset of

thymocytes, in several strains of mice harboring mutations in T cell signaling proteins or transcriptional regulators, conventional

CD8+ T cells develop as innate cells with characteristics of memory T cells. Thus, in Itk-deficient mice, mature CD42CD8+ (CD8

single-positive [SP]) thymocytes express high levels of the transcription factor eomesodermin (Eomes) and are dependent on IL-4

being produced in the thymic environment by a poorly characterized subset of CD4+ thymocytes expressing the transcriptional

regulator promyelocytic leukemia zinc finger. In this study, we show that a sizeable proportion of mature CD4+CD82 (CD4SP)

thymocytes in itk2/2 mice also develop as innate Eomes-expressing T cells. These cells are dependent on MHC class II and IL-4

signaling for their development, indicating that they are conventional CD4+ T cells that have been converted to an innate

phenotype. Surprisingly, neither CD4SP nor CD8SP innate Eomes+ thymocytes in itk2/2 or SLP-76(Y145F) mice are dependent

on gd T cells for their development. Instead, we find that the predominant population of Eomes+ innate itk2/2 CD4SP thymocytes

is largely absent in mice lacking CD1d-specific invariant NKT cells, with no effect on innate itk2/2 CD8SP thymocytes. In contrast,

both subsets of innate Eomes+itk2/2 T cells require the presence of a novel promyelocytic leukemia zinc finger–expressing, SLAM

family receptor adapter protein–dependent thymocyte population that is essential for the conversion of conventional CD4+ and

CD8+ T cells into innate T cells with a memory phenotype. The Journal of Immunology, 2014, 193: 000–000.

Thymic development produces a wide range of T cellsubsets with varying functions in immune responses. Inaddition to conventional naive CD4+ and CD8+ T cells,

which require prolonged activation and differentiation to acquireprotective effector functions, several subsets of T cells with innateeffector functions are now known to develop in the thymus (1, 2).This latter group includes several distinct categories of gd T cells,CD1d-specific invariant NKT (iNKT) cells, MR1-specific muco-sal-associated invariant T (MAIT) cells, H2–M3-specific CD8+

T cells, and Foxp3+ regulatory T cells, among others (1, 2). Al-though a thorough understanding of the signals giving rise to eachof these T cell lineages has not yet been achieved, recent studiesindicate a role for the strength of TCR signaling, extrinsic signalsprovided by cytokines, as well as components of intrinsic devel-opmental programming in this process (3–8).One important clue to dissecting the signals regulating T cell

lineage development has come from studies of genetically al-tered mice. In the absence of the Tec kinase Itk, as well as in

mice lacking the transcription factors Kr€uppel-like factor 2, inhib-itor of DNA-binding 3, and CREB-binding protein, conventional

CD8+ T cells developing in the thymus are converted into innate/

memory-like T cells expressing high levels of the effector-

promoting transcription factor eomesodermin (Eomes) (6, 8–10).

Although the involvement of Itk indicates a role for TCR signal-

ing in this process, further evidence supporting this conclusion

comes from studies demonstrating an identical phenotype in mice

expressing a mutant form of the adapter protein SLP-76, SLP-76

(Y145F), which lacks the ability to recruit Itk in response to

TCR stimulation (7). Taken together, these data indicate that in-

tact TCR signaling pathways are critical for the normal develop-

ment of conventional CD8+ T cells.Interestingly, impaired TCR signaling is not the only require-

ment for the development of innate/memory CD8+ thymocytes

expressing high levels of Eomes. Studies by Hogquist and col-

leagues (8) first demonstrated a requirement for exogenous IL-4 to

induce Eomes expression in CD8+ ab T cells (3, 6, 7). Thus, in the

absence of the IL-4Ra (CD124), Eomes is no longer expressed in

itk2/2 CD8+ T cells (8). Further evidence indicates that a subset of

T cells expressing the transcription factor promyelocytic leukemia

zinc finger (PLZF) are involved in this pathway and are the likely

source of the excess IL-4 (6–8).Data from our laboratory and others have shown that in the ab-

sence of Itk, there is an increase in PLZF+ gd T cells expressing the

Vg1.1 Vd6.3 TCR, a subset known as gd NKT cells (11, 12). In

general, it is thought that these gd NKT cells are responsible for the

excess IL-4 in the absence of Itk, and thus for the development of

Eomes+ innate/memory CD8+ T cells (13). Although it has been

demonstrated that gd NKT cells are responsible for the hyper-IgE

syndrome seen in itk2/2 mice (11, 12), it is not currently known

whether gd NKT cells are required to induce Eomes expression in

CD8+ T cells. Multiple cell types (e.g., ab iNKT cells, MAIT cells)

*Department of Pathology, University of Massachusetts Medical School, Worcester,MA 01655; †National Human Genome Research Institute, National Institutes ofHealth, Bethesda, MD 20892; and ‡Department of Pathology and Laboratory Med-icine, University of Pennsylvania, Philadelphia, PA 19104

Received for publication August 2, 2013. Accepted for publication May 8, 2014.

This work was supported by National Institutes of Health Grants AI084987 (to L.J.B.)and 5K08 AI101008 (to S.A.C.).

Address correspondence and reprint requests to Dr. Leslie J. Berg, Department ofPathology, University of Massachusetts Medical School, Albert Sherman Center,9-1049, 368 Plantation Street, Worcester, MA 01655. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: Eomes, eomesodermin; HSA, heat-stable Ag;iNKT, invariant NKT; MAIT, mucosal-associated invariant T; PLZF, promyelocyticleukemia zinc finger; SAP, SLAM family receptor adapter protein; SP, single posi-tive; WT, wild-type.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302059

Published June 18, 2014, doi:10.4049/jimmunol.1302059 by guest on M

arch 17, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Page 3: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

are capable of IL-4 production. Therefore, even though gd NKTcells are the most likely candidate for the excess IL-4 acting onCD8+ thymocytes, it remains possible that other cell types arecontributing to this process.Additionally, the effect of thymic IL-4 on the development of

conventional CD4+ ab T cells has not been addressed. Whereasnumerous studies have documented that itk2/2 mice have an in-creased frequency of activated CD4+ ab T cells, it is not knownwhether these cells are expressing Eomes similarly to itk2/2 CD8+

ab T cells (14). In this study, we examined itk2/2 CD4+ abthymocytes and found a substantial subset of conventional T cellsthat are upregulating Eomes in response to the environment.Using these innate/memory Eomes+ CD4+ and CD8+ thymocytesto track the environmental contribution to innate T cell develop-ment in itk2/2 mice, we demonstrate that this phenotype is not in-duced by gd T cells.

Materials and MethodsMice

Wild-type (WT) C57BL/6 mice were purchased from either Taconic Farms(Hudson, NY), The Jackson Laboratory (Bar Harbor, ME), or Charles RiverLaboratories International (Wilmington, MA). The itk2/2 mice were pre-viously described (15–17) and housed at the University of MassachusettsMedical School in accordance with the Institutional Animal Care and UseCommittee and in a specific pathogen-free environment. IL-4 reporter(4get) mice were a gift from Markus Mohrs (Trudeau Institute, SaranacLake, NY) and were crossed to itk2/2 mice at the University of Massa-chusetts Medical School. The cd1d2/2 mice were a gift from the labora-tory of Raymond Welsh and were crossed to itk2/2 mice at the Universityof Massachusetts Medical School. The mr12/2 and il152/2 mice werea gift from Joonsoo Kang and were also crossed to itk2/2 mice at theUniversity of Massachusetts Medical School. The sh2d1a2/2 mice werepreviously described (18) and were crossed to itk2/2 mice at the Universityof Massachusetts Medical School. H2dlAb1-Ea mice were purchased fromThe Jackson Laboratory (Bar Harbor, ME) and were crossed to itk2/2 miceat the University of Massachusetts Medical School. The tcrd2/2 mice werea gift from the laboratory of Raymond Welsh and were crossed to itk2/2

mice at the University of Massachusetts Medical School as previouslydescribed (11). SLP-76(Y146F) mice were previously described (19) andhoused at the University of Pennsylvania in accordance with their Insti-tutional Animal Care and Use Committee. The tcrd2/2 mice were pur-chased from The Jackson Laboratory and crossed with SLP-76(Y146F)mice to generate Y145F/tcrd2/2 mice. The il4ra2/2 mice were a gift fromKristin Hogquist (University of Minnesota, Minneapolis, MN) and were bredto itk2/2 mice at the National Institutes of Health in accordance with theirInstitutional Animal Care and Use Committee to generate itk/il4ra2/2 mice.

Cell preparation

Thymi were harvested from mice and stored in RPMI 1640 (Life Technol-ogies by Invitrogen, Grand Island, NY) supplemented with FBS, L-gluta-mine, penicillin, streptomycin, 2-ME, and HEPES (RPMI 10). Thymi wereprocessed using forceps and frosted microscope slides. Thymocytes werelysed with RBC lysis buffer prior to single-cell suspension with RPMI 10.

Cell stimulations

Cells were plated at 106–107 cells/well prior to stimulating with PMA(10 ng/ml) and ionomycin (1 mg/ml) for 6 h at 37˚C in the presence ofbrefeldin A and monensin.

Extracellular/intracellular staining

Cells were plated at 106–107 cells per well prior to washing with FACSbuffer (13 PBS supplemented with 2% FBS). Fc receptors were blockedusing supernatant from 2.4G2 hybridoma grown in the laboratory prior tostaining with the CD1d tetramer loaded with PBS57 (a gift from the Na-tional Institutes of Health). Cells were stained with various combinationsof the cell surface Abs against CD4 (RM4-5), CD8 (53-6.7 or 5H10), TCRb(Η57-597), TCRd (GL3), heat-stable Ag (HSA, CD24) (30-F1 or M1/69),CD44 (IM7), CD62L (MEL-14), CD124 (IL-4Ra) (mIL-4R-M1), CD122(IL-2Rb) (5H4 or TM-b1), and CXCR3 (CXCR3-173). Cells were thenpermeabilized using the eBioscience (San Diego, CA) Foxp3/transcriptionfactor staining kit according to the manufacturer’s protocol prior to stainingwith Abs against the transcriptions factors Eomes (Dan11mag) and PLZF

(D-9, IgG1: A85-1), as well as the cytokines IL-4 (BVD6-24G2) and IFN-g(XMG1.2). Abs were purchased from BD Biosciences (San Jose, CA),eBioscience, or Santa Cruz Biotechnology (Dallas, TX).

Statistical analysis

Statistics were analyzed using GraphPad Prism software (GraphPad Soft-ware, La Jolla, CA) using a Student t test or one-way ANOVA as applicable.

ResultsCD4+Eomes+ ab T cells develop in the absence of Itk

As previously described (15, 20), thymocytes from itk2/2mice havean increased frequency of CD42CD8+ (CD8 single-positive [SP])cells compared with WT thymocytes, leading to a decreased ratio ofCD4/CD8 SP thymocytes (Fig. 1A). Additionally, the predominantpopulations of CD8SP thymocytes in itk2/2 mice express highlevels of Eomes owing to increased IL-4 signaling (8, 15)(Fig. 1B). itk2/2 CD8SP thymocytes also express increased levelsof the IL-4Ra (CD124) and have a memory-like phenotypecharacterized by high expression of CD44, CD62L, IL-2Rb(CD122), and CXCR3 (Fig. 1B, 1D).To determine whether itk2/2 CD4+ ab T cells also underwent

altered development, we characterized CD4+CD82 (CD4SP) thy-mocytes from itk2/2 mice. For this analysis, we excluded invariantab NKT cells, which are known to have a memory-like phenotype(21). When conventional, mature (TCRbhiHSAlo) CD1d tetramer2

CD4SP thymocytes were analyzed, we found an increased pop-ulation of itk2/2 cells expressing Eomes compared with WTCD4SP thymocytes (Fig. 1C). Similar to the CD8SP thymocytes,Eomes+itk2/2 CD4SP thymocytes also expressed increased levelsof CD44, CD62L, IL-2Rb (CD122), IL-4Ra (CD124), and CXCR3(Fig. 1C, 1E). In addition to the upregulated expression of Eomes,CD44, CD62L, IL-2Rb, IL-4Ra, and CXCR3, itk2/2Eomes+

CD8SP thymocytes produce IFN-g upon ex vivo stimulation (15,20). This effector function is shared with itk2/2 CD4SP thymocytes(Fig. 2). Taken together, these data indicate that innate-like CD4+

ab T cells expressing Eomes develop in the absence of Itk.

IL-15 regulates the expansion of Eomes+ T cells

Previously, we and others demonstrated the importance of IL-15for the development and/or maintenance of itk2/2CD44hi periph-eral CD8+ T cells (15, 22). Additionally, we have observed a slightreduction in the overall thymic cellularity of itk2/2il152/2 mice(Supplemental Fig. 1A). Furthermore, as shown in SupplementalFig. 1B, itk2/2Eomes+ CD8SP thymocytes are substantially re-duced in number in the absence of IL-15. However, the presenceof a significant residual population of itk2/2Eomes+ CD8SP cells,even in the absence of IL-15, indicates that IL-15 is critical for themaintenance and/or expansion of these cells, rather than for theirdevelopment per se. In contrast, the frequency and number of itk2/2

Eomes+ CD4SP thymocytes increased dramatically in the absenceof IL-15 (Supplemental Fig. 1C). This increase is likely due to thereduced number of Eomes+itk2/2 CD8SP cells that would competewith the CD4+ T cells for survival cytokines, such as IL-7 or IL-4.These data indicate that CD4+Eomes+ T cells are independent ofIL-15 for their development, maintenance, and expansion. Thus,although both of these subsets of innate T cells in itk2/2 mice ex-press Eomes, they are differentially regulated by the cytokine IL-15.

Conventional CD4+ T cells promote the expansion anddevelopment of Eomes-expressing ab T cells

Eomes-expressing itk2/2CD8+ T cells are conventional ab T cellsthat are converted to this innate-like phenotype by IL-4 (6, 8). Todetermine whether Eomes+CD4+ T cells were also conventionalT cells that are converted to an innate-like phenotype, we exam-ined the development of these cells in itk2/2 mice lacking all

2 DISTINCT PATHS REGULATE INNATE CD4 AND CD8 T CELL DEVELOPMENT

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 4: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

MHC class II expression (H2dlAb1-Ea). Unexpectedly, we first ob-served that, in the absence of MHC class II, there is an overallreduction in the thymic cellularity of Itk-deficient mice lackingMHC class II (Fig. 3A). Furthermore, the frequency and numberof Eomes+CD8+ ab T cells were significantly decreased (Fig. 3B),

and lack of MHC class II expression led to a near disappearanceof itk2/2Eomes+ CD4SP thymocytes (Fig. 3C). These latter datasupport the conclusion that itk2/2Eomes+ CD4SP thymocytesare conventional CD4+ T cells that are converted to an innate-like phenotype. The reason for the reduced numbers of itk2/2

FIGURE 1. Mature Eomes+ CD8 and CD4 T cells develop in the absence of Itk. Thymocytes from WT and itk2/2 mice were isolated and stained with

CD1d-tetramer and Abs to CD4, CD8, TCRb, TCRd, HSA (CD24), CD44, CD62L, CD122 (IL-2Rb), CXCR3, CD124 (IL-4Ra), and Eomes. (A) CD4/

CD8 ratio of total thymocytes in WT versus itk2/2 mice. (B and C) Eomes versus CD44 staining of CD8SP (B) and CD4SP (C) thymocytes, gated as

indicated. The numbers indicate the percentages of CD44hiEomes+ cells in each subset. The graphs below show compilations of all data indicating per-

centages and absolute numbers of Eomes+ cells in each subset. Results are representative of more than five independent experiments (n = 20–22 mice/

group). Statistical analysis was done using a Mann–Whitney U test. (D and E) Histograms show staining of CD62L, CD122, CD124, and CXCR3 on mature

CD8SP (D) or mature CD4SP (E) thymocytes. WT thymocytes are shown in gray-filled histograms; itk2/2 thymocytes are shown in black. (D) WT and itk2/2

thymocytes are gated on TCRb+CD24lo CD8SP cells. (E) WT thymocytes gated on CD4SP TCRb+CD1d-tetramer2HSAlo thymocytes, itk2/2 thymocytes

gated on CD4SP TCRb+CD1d-tetramer2HSAloEomes+ thymocytes. Results are representative of at least three independent experiments.

The Journal of Immunology 3

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 5: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

innate-like CD8SP in mice lacking conventional ab CD4+ T cellsis unclear, but it may reflect an unforeseen contribution of IL-4from this conventional T cell subset. Alternatively, itk2/2 CD4SPthymocytes may promote the expansion of thymocytes duringthe double-negative to double-positive transition because we sawa significant decrease in thymic cellularity.

Eomes+ ab T cells develop independently of MAIT cells

The data shown above indicate that both conventional CD8+ andCD4+ ab T cells aberrantly upregulate Eomes expression in itk2/2

mice. For the CD8SP thymocytes, this altered development is de-pendent on excess IL-4 in the environment. One potential sourceof IL-4 is a subset of innate T, that is, MAIT cells. MAIT cellsexpress an invariant TCR (mouse Va19-Ja33, human Va7.2-Ja33)that recognizes the nonclassical MHC class Ib molecule MR1;furthermore, these cells preferentially home to the mesentericlymph node or gut lamina propria (23). Va19 transgenic cells canproduce copious amounts of IL-4, consistent with the finding thathuman Va7.2+ cells express the transcription factor PLZF, a keyregulator of cytokine production by iNKT cells (24, 25). As shownin Supplemental Fig. 2, a deficiency in MR1, which has beenshown to block the development of MAIT cells (23), had no effecton the numbers of itk2/2 total thymocytes or on the numbers ofCD8SP or CD4SP thymocytes expressing high levels of Eomes.

iNKT cells promote the development of Eomes+ ab T cells

A second potential source of excess IL-4 in itk2/2 mice isiNKT cells. Although mature iNKT cells predominantly produceIFN-g, with little IL-4 production, immature iNKT cells producehigh levels of IL-4 (26). Additionally, previous studies showedthat iNKT cell maturation in the absence of Itk is impaired,leading to increased proportions of the immature IL-4–producingsubset in these mice (27, 28). To test for a role of iNKT cells, wecrossed itk2/2 mice to CD1d-deficient mice and examined thethymocytes in these mice. In the absence of iNKT cells, althoughwe observed no overall decrease in thymic cellularity between

itk2/2 and itk2/2cd1d2/2 mice (Fig. 4A), we did observe a slightdecrease in the frequency and number of CD8SP Eomes+ cells(Fig. 4B). These data indicate that whereas iNKT cells do notappear to contribute to the development of Eomes+CD8+ thymo-cytes, iNKT cells may promote the expansion of itk2/2Eomes+

CD8+ thymocytes. In contrast, CD4SP Eomes+ cells were sub-stantially reduced in Itk/CD1d double-deficient mice when com-pared with Itk-deficient mice (Fig. 4C). Furthermore, Itk/CD1ddouble-deficient CD4SP thymocytes resembled WT and CD1d-deficient CD4SP thymocytes, with no statistical significance foundbetween these three groups (Fig. 4C). Detailed analysis re-vealed that the percentages and absolute numbers of total CD4SPthymocytes were identical between itk2/2 and itk2/2cd1d2/2

mice (Supplemental Fig. 3A, 3B), indicating that iNKT cellscontributed to the conversion of conventional CD4SP to Eomes+

CD4SP, and not to their development per se. These data show thatiNKT cells are significantly promoting Eomes upregulation inconventional CD4+ T cells, but iNKT cells are not contributingsignificantly to the development of itk2/2Eomes+CD8+ thymocytes.If iNKT cells are contributing significant amounts of IL-4 to

the thymic environment of itk2/2 mice, then IL-4Ra (CD124)expression should also be decreased on itk2/2 CD8SP and CD4SPthymocytes in the absence of iNKT cells, as IL-4 signaling isknown to induce a positive feedback loop leading to upregulationof the IL-4R (29). Although there was a slight decrease in thegeometric mean fluorescence intensity of IL-4Ra on mature Itk/CD1d double-deficient CD8SP and CD4SP cells compared withthose in itk2/2 mice, IL-4Ra was still highly expressed on itk2/2

mature SP thymocytes in the absence of iNKT cells (SupplementalFig. 3C, 3D). One possible explanation for these data is that theloss of IL-4 normally contributed by iNKT cells in itk2/2 mice isnot sufficient to impact CD124 expression, but it is enough toprevent Eomes upregulation. Alternatively, the absence of abiNKT cells may enhance the development of gd NKT cells, whichhas been demonstrated in WT mice (30). Indeed, we saw thatPLZF+ gd T cells were increased in both percentage and number

FIGURE 2. Eomes+ CD4SP thymocytes produce

IFN-g in response to stimulation. Thymocytes from

WT and itk2/2 mice were stimulated with 10 ng/ml

PMA and 1 mg/ml ionomycin for 6 h and then stained

with CD1d-tetramer and Abs against CD4, CD8, HSA,

IFN-g, and Eomes. Dot plots are gated on CD4SP

CD1d-tetramer2HSAlo thymocytes and show IFN-g

versus Eomes staining. Results are representative of

two independent experiments involving two to four

mice per group.

4 DISTINCT PATHS REGULATE INNATE CD4 AND CD8 T CELL DEVELOPMENT

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 6: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

in the absence of ab iNKT cells (Supplemental Fig. 3E, 3F),which may account for the maintained high expression of IL-4Ra.

Therefore, iNKT cells may regulate innate T cell development initk2/2 mice by a mechanism independent of IL-4 production.

FIGURE 3. CD4+Eomes+ T cells are MHC class II–dependent. Thymocytes from WT, itk2/2, H2dlAb1-Ea, and itk2/2/H2dlAb1-Ea mice were stained with CD1d-

tetramer and Abs to CD4, CD8, TCRd, TCRb, HSA, CD44, and Eomes. Dot plots show Eomes versus CD44 staining, and graphs show compilations of the

percentages and absolute numbers of Eomes+ cells. (A) Total thymic cellularity. Significant differences were seen betweenH2dlAb1-Ea and itk2/2/H2dlAb1-Eamice (p,0.005). (B) Gated on CD8SP TCRbhiHSAlo thymocytes. (C) Gated on CD4SP TCRd2TCRbhiHSAloCD1d-tetramer2 thymocytes. Results are representative of three

independent experiments (n = 6–8 mice/group). Statistical analysis was performed using a one-way ANOVA. **p , 0.005, ***p , 0.0005, ****p , 0.00001.

The Journal of Immunology 5

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 7: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

Eomes+ T cells develop independently of T gd cells

The data shown above indicate that iNKT cells are essential forthe development of innate Eomes+CD4+ ab T cells in itk2/2 mice.Nonetheless, the Itk/CD1d double-deficient mice still had a sub-stantial population of Eomes+CD8+ T cells. In addition to abiNKT cells, a closely related subset of NKT cells has been de-

scribed that expresses an invariant gd TCR, utilizing Vg1.1/Vd6.3(30, 31). Recent studies have reported a dramatic expansion in this

population in itk2/2 mice, as well as in other lines of mice that

develop Eomes+ innate CD8+ T cells, including SLP-76(Y145F)

mice (6, 7, 11, 12). gd NKT cells also produce copious amounts

of IL-4 in response to stimulation (11, 12) and have been

FIGURE 4. ab NKT cells promote the development of Eomes+ T cells. Thymocytes from WT, itk2/2, cd1d2/2, and itk2/2cd1d2/2 mice were stained with

CD1d-tetramer and Abs against CD4, CD8, TCRd, TCRb, HSA, CD44, and Eomes. Dot plots show Eomes versus CD44 staining, and graphs show com-

pilations of the percentages and absolute numbers of Eomes+ cells. (A) Total thymic cellularity. Significant differences were seen between WT and CD1d KO

mice (p , 0.05). (B) Gated on CD8SP TCRbhi HSAlo thymocytes. (C) Gated on CD4SP TCRd2TCRbhiHSAloCD1d-tetramer2 thymocytes. Results are from

four independent experiments (n = 7–9 mice/group). Statistical analysis performed using a one-way ANOVA. *p , 0.05, ***p , 0.0005, ****p , 0.0001.

6 DISTINCT PATHS REGULATE INNATE CD4 AND CD8 T CELL DEVELOPMENT

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 8: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

proposed as a possible cellular source of the excess IL-4driving innate T cell development in both itk2/2 and SLP-76(Y145F) mice (13).

To address the role of gd T cells, we crossed itk2/2 miceto TCRd-deficient (tcrd2/2) mice. To our surprise, thymocytesin the itk2/2 mice lacking gd T cells were indistinguishable

FIGURE 5. Eomes+ T cells develop independently of gd T cells. Thymocytes from WT, itk2/2, tcrd2/2, and itk2/2tcrd2/2 mice were stained with

CD1d-tetramer and Abs to CD4, CD8, TCRb, HSA, CD44, and Eomes. Dot plots show Eomes versus CD44 staining, and graphs show compilations of the

percentages and absolute numbers of Eomes+ cells. (A) Total thymic cellularity. No significant differences were detected. (B) Gated on CD8SP TCRbhi

HSAlo thymocytes. (C) Gated on CD4SP TCRbhiHSAloCD1d-tetramer2 thymocytes. Results are representative of two independent experiments (n = 4–7

mice/group). Statistical analysis was performed using a one-way ANOVA. *p , 0.05, **p , 0.005, ***p , 0.0005, ****p , 0.0001.

The Journal of Immunology 7

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 9: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

from those in single itk2/2 mice. Overall thymic cellularity andboth the CD8SP and CD4SP subsets had comparable proportionsand numbers of cells expressing Eomes when itk2/2 thymocyteswere compared with itk2/2tcrd2/2 thymocytes (Fig. 5). Althoughwe did see significant increases in the frequency and cell numberof ab iNKT cells in tcrd2/2 mice, there were no differences in ab

iNKT cells between itk2/2 and itk2/2tcrd2/2 mice (SupplementalFig. 4A, 4B). Thus, in contrast to recent studies in WT mice (30),it seems unlikely that itk2/2 ab iNKT cells are overcompensatingin the absence of itk2/2 gd NKT cells. Similar findings wereobserved in SLP-76(Y145F) mice that lacked gd T cells, such thatthe frequencies and numbers of Eomes+ CD8SP and CD4SP

FIGURE 6. CD4+ and CD8+ Eomes+ T cells are dependent on IL-4. Thymocytes from WT, itk2/2, il4ra2/2, and itk2/2il4ra2/2 mice were stained with

Abs to CD4, CD8, TCRb, HSA, CD44, and Eomes. Dot plots show Eomes versus CD44 staining, and graphs show compilations of the percentages and

absolute numbers of Eomes+ cells. (A) Total thymic cellularity. Significant differences were detected between WT and itk2/2 (p , 0.0001), WT and itk2/2

il4ra2/2 (p , 0.0005), itk2/2 and il4ra2/2 (p , 0.0005), and il4ra2/2 and itk2/2il4ra2/2 (p , 0.005). (B) Gated on CD8SP TCRbhiCD24 (HSA)lo

thymocytes. (C) Gated on CD4SP TCRbhiCD24 (HSA)lo thymocytes. Results are representative of three independent experiments (n = 3–6 mice/group).

Statistical analysis was performed using one-way ANOVA. ***p , 0.0001.

8 DISTINCT PATHS REGULATE INNATE CD4 AND CD8 T CELL DEVELOPMENT

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 10: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

thymocytes were similar in SLP-76(Y145F) mice and tcrd2/2

SLP-76(Y145F) mice (Supplemental Fig. 4C, 4D). Additionally,the absence of gd NKT cells did not influence the frequency ornumber of ab NKT cells in SLP-76(Y145F) mice (SupplementalFig. 4E). Interestingly, although gd T cells constitute half of thePLZF+ thymocytes in SLP-76(Y145F) mice, the total number ofPLZF+ thymocytes was not reduced in tcrd2/2 SLP-76(Y145F)mice (Supplemental Fig. 4F), leaving open the possibility that thecontribution of gd T cells to the development of innate-like thy-mocytes might be masked by a compensatory increase in otherPLZF+ populations. Thus, these data demonstrate that although gdNKT cells contribute significantly to the hyper-IgE syndrome seenin Itk-deficient mice (11, 12), these cells are not required for thedevelopment of Eomes+ ab T cells.

SLAM family receptor adapter protein–dependent PLZF+

CD1d-tetramer2 ab T cells develop in the absence of Itk

Previous studies have shown a dependence of itk2/2Eomes+ innateCD8SP thymocytes on IL-4 for their development (8). However,

we were unsure whether the same was true for itk2/2Eomes+ in-nate CD4SP thymocytes, because we observed differentialrequirements for IL-15 signaling (Supplemental Fig. 1) and thepresence of iNKT cells (Fig. 4) between these two subsets. Tofurther examine the dependence on IL-4 for the development ofitk2/2Eomes+ innate CD4SP thymocytes, Itk-deficient mice werecrossed to mice deficient in IL-4Ra (ΧD124). Although we sawreduced thymic cellularity in itk2/2 and itk2/2il4ra2/2 mice,these populations did not differ significantly from each other (Fig.6A). As previously seen for innate CD8SP thymocytes, theEomes-expressing population was eliminated in the absence of IL-4signaling (Fig. 6B). Eomes+ innate CD4SP thymocytes also failedto develop in the absence of IL-4 signaling (Fig. 6C). Thus, al-though Eomes+ innate CD8SP and CD4SP thymocytes do havesome differential requirements for their development, both pop-ulations require IL-4.The data presented above ruled out a role for well-defined IL-4–

producing CD4+ T cells populations, such as gd NKT cells andMR1-dependent MAIT cells, in the development of Eomes+ ab

FIGURE 7. IL-4–producing CD1d-

tetramer2PLZF+ ab T cells develop

in the absence of Itk. (A) Thymocytes

from WT or itk2/2 mice were stained

with CD1d-tetramer and Abs to CD4,

CD8, TCRd, TCRb HSA, Eomes, and

PLZF. Dot plots (left) show PLZF

versus Eomes staining on CD4SP

TCRd2TCRbhiHSAloCD1d-tetramer2

thymocytes. Graphs (right) show fre-

quencies and absolute numbers of

CD4SP TCRd2TCRbhiCD1d-tetra-

mer2HSAloPLZF+ thymocytes. Re-

sults are from independent experiments

(n = 20–23 mice/group). Statistical

analysis was performed using a Mann–

Whitney Student t test. ****p ,0.0001. (B) Itk KO mice were crossed

to IL-4 reporter (4get) mice as previ-

ously described (31). Thymocytes

from WT and Itk KO expressing the

IL-4 reporter (positive) or without the

transgene (negative) were harvested,

processed, and stained with CD1d

tetramer and with Abs against CD4,

CD8, TCRd, TCRb, HSA, and CD44.

Flow cytometry plots are gated on

CD4 SP TCRd2TCRbhi CD1d-tetra-

mer2HSAlo thymocytes. Results are

representative of four independent

experiments involving two to three

mice per group per experiment. (C and

D) Thymocytes from WT or Itk KO

mice were harvested, processed, and

stimulated with PMA and ionomycin

(right) or remained unstimulated (left)

in the presence of brefeldin A and

monensin for 5–6 h at 37˚C before

staining with CD1d tetramer and with

Abs against CD4, CD8, Eomes, PLZF,

IFN-g, and IL-4. (C) Gated on CD4SP

CD1d-tetramer2Eomes+ thymocytes.

(D) Gated on CD4SP CD1d-tetramer2

PLZF+ thymocytes. Results are repre-

sentative of two independent experiments

involving five to seven mice per group.

The Journal of Immunology 9

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 11: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

T cells in itk2/2 mice. Additionally, the data indicated that CD1d-dependent ab iNKT cells were not essential. This led us to con-sider whether itk2/2 mice might have an additional population ofPLZF+ cells capable of producing IL-4. Therefore, we examinedmature itk2/2 CD4SP ab thymocytes (TCRd2, TCRbhi, HSAlo,CD1d-tetramer2) and found an increased population of PLZF+

cells compared with WT mature CD4SP thymocytes (Fig. 7A).Furthermore, when Itk knockout mice were crossed to IL-4 re-porter mice that have a bicistronic promoter adding an IRES-GFPcomponent to the untranslated region of the IL-4 locus (32), wefound an increased population of mature CD4SP thymocytesexpressing IL-4 mRNA (Fig. 7B). IL-4 mRNA appears to only bepresent in cells expressing PLZF because CD4SP CD1d-tetramer2

thymocytes expressing PLZF produce IL-4 and IFN-g in responseto stimulation whereas CD4SP CD1d-tetramer2 thymocytes ex-pressing Eomes only produce IFN-g (Fig. 7C, 7D). Thus, in the

absence of Itk, a population of mature CD4SP PLZF+ ab T cellswith capabilities of producing IL-4 develops in the thymus.Eomes+ ab T cells in itk2/2 mice are dependent on SLAM

family receptor adapter protein (SAP) for their development (6,33), an observation that has been linked to the requirement forSAP in the development of PLZF+ cells in other mouse models(6). To determine whether itk2/2 CD4SP PLZF+CD1d-tetra-mer2 ab thymocytes were also dependent on SAP, we exam-ined Itk/SAP double-deficient mice. As shown in Fig. 8, SAPwas essential for the development of PLZF+ CD4SP CD1d-tetramer2 ab thymocytes in itk2/2 mice. Furthermore, all itk2/2

Eomes+ CD4SP thymocytes reverted to a conventional CD4SPphenotype in the absence of SAP. These data identify a novelpopulation of SAP-dependent PLZF+CD4+ T cells that is essen-tial for the development of innate-like Eomes+ ab T cells initk2/2 mice.

FIGURE 8. Development of all innate T cells in itk2/2 is dependent on SAP. Thymocytes from WT, itk2/2, sh2d1a2/2, and itk2/2 sh2d1a2/2 mice were

stained with CD1d-tetramer and Abs against CD4, CD8, TCRb, HSA, Eomes, and PLZF. (A) Total thymic cellularity. Significant differences were seen

between WT and itk2/2 mice (p , 0.005), WT and sh2d1a2/2 mice (p , 0.05), and WT and itk2/2 sh2d1a2/2 mice (p , 0.0005). (B) Dot plots show

PLZF versus Eomes staining on CD4SP TCRbhighHSAloCD1d-tetramer2 thymocytes. (C and D) Graphs show frequencies (C) and absolute numbers (D) of

CD4SP TCRbhiHSAloCD1d-tetramer2 cells expressing PLZF (left) or Eomes (right). Results are representative of four independent experiments (n = 10–12

mice/group). Statistical analysis was performed using a one-way ANOVA. ****p , 0.0001.

10 DISTINCT PATHS REGULATE INNATE CD4 AND CD8 T CELL DEVELOPMENT

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 12: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

DiscussionThe data presented in this study show that innate T cell devel-opment is not restricted to the CD8+ T cell subset in itk2/2 mice.

We demonstrate that a sizeable proportion of itk2/2 CD4SP thy-

mocytes develop as innate-like cells expressing high levels of

Eomes. Although there is a sizeable population of Eomes+ innate

itk2/2 CD4SP thymocytes, we found that only a small propor-

tion (5.5–11.4%) of these cells produce IFN-g upon stimulation.

However, this finding is similar to results obtained with mature

innate itk2/2 CD8SP thymocytes where only a portion (∼13%)

produce IFN-g upon stimulation, despite the fact that the vast

majority express Eomes (8, 15). We also saw a sizeable proportion

of Eomes2 CD4SP thymocytes that produced IFN-g (34). This

is most likely due to an increase in PLZF+ CD4SP thymocytes,

because PLZF allows ab NKT cells to produce both IL-4 and

IFN-g (25). Collectively, our data support the conclusion that the

Eomes+ CD4SP cells in itk2/2 mice are conventional MHC class

II–dependent cells that have been induced to upregulate Eomes by

the cell-extrinsic factor IL-4. When considering both the CD4+

and the CD8+ subsets of Eomes-expressing innate T cells in itk2/2

mice, we have identified two independent cell populations that are

critical for the dramatic conversion of conventional T cells into

innate T cells in these mice.First, we discovered that IL-4 signaling is crucial for the de-

velopment of Eomes+ innate CD4SP thymocytes, similarly towhat has been previously demonstrated for itk2/2 CD8SP thy-mocytes (8). Furthermore, we found that a SAP-dependent pop-ulation of PLZF+ T cells is essential for the development ofEomes+CD8+ T cells in itk2/2 mice, data that are consistent withseveral previous studies showing that both SAP and PLZF arenecessary for this process (6, 8). Second, our data reveal a sur-prising role for iNKT cells in the development of itk2/2Eomes+

CD4+ T cells but not CD8+ T cells. This latter finding indicatesthat the two subsets of Eomes+ innate T cells in itk2/2 mice havedistinct requirements and/or signaling pathways that regulate theirdevelopment. One possibility is that CD4SP thymocytes needa higher concentration of IL-4 to induce Eomes upregulationrelative to CD8SP thymocytes. If true, this might necessitate thecombined cytokine production of several PLZF+ populations toachieve this threshold. Although possible, this scenario seemsunlikely, as we observed no effect of eliminating gd T cells on theEomes+ CD4SP population, despite the fact that the itk2/2 gdNKT cells produce enough IL-4 to induce a hyper-IgE phenotypein these mice (11, 12). An alternative possibility is that differentcell populations reside in distinct compartments in the thymus, thuslimiting access of cytokines to cells in the immediate environment.Thus, Eomes+ CD4SP thymocytes in itk2/2 mice might colocalizewith iNKT cells to a greater extent than CD8SP thymocytes.However, although we find that iNKT cells do not appear to reg-ulate the development of itk2/2Eomes+ CD8SP thymocytes,iNKT cells may contribute to the expansion of this innate T cellpopulation. Furthermore, it appears that conventional CD4SP thy-mocytes may also have a role in the expansion of itk2/2Eomes+

CD8SP thymocytes. Our laboratory has previously described thatItk-deficient mice have a slight proliferative defect at the double-negative to double-positive transitions (35). Thus, SP thymocytesmay aid in the proliferation of developing thymocytes via cytokinesecretion.One unexpected finding from our studies was the lack of im-

portance of gd NKT cells in the development of innate Eomes+

CD4+ and CD8+ T cells in itk2/2 mice. Because this cell pop-ulation is greatly expanded in numbers in itk2/2 mice and isa significant producer of IL-4, it was a reasonable hypothesis

that these cells would contribute to, or possibly be completelyresponsible for, the IL-4 production that generates Eomes+ innateT cells. However, we observed no detectable change in eitherCD4SP or CD8SP thymocytes in itk2/2 mice lacking gd T cells,a result that was confirmed in SLP-76(Y145F) mice. Instead, ourdata suggest that the PLZF+ T cells responsible for convertingitk2/2 CD4+ and CD8+ thymocytes into innate Eomes-expressingcells are primarily ab TCR+ cells. Furthermore, for the develop-ment of itk2/2CD8+ innate T cells, our data rule out a role for theother known subsets of PLZF+ T cells, such as iNKT cells andMAIT cells. Although it is possible that these subsets could besufficient to induce the development of memory/innate-like thy-mocytes in some contexts, our findings indicate that these subsetsare not required. Instead, our data argue for a novel PLZF+CD4+

ab T cell subset capable of producing IL-4 that is expanded initk2/2 mice, similar to the situation for gd NKT cells.One interesting possibility is that the population of itk2/2

PLZF+CD4+ ab T cells responsible for inducing innate T celldevelopment is related to the “T-CD4” cells found in the thy-mus of mice engineered to express MHC class II proteins on theirthymocytes (36, 37). These T-CD4 cells develop as a result ofSAP-dependent thymocyte–thymocyte interactions that lead toupregulation of PLZF and to an innate capacity to secrete IL-4(38–40). Additionally, the CD8SP thymocytes in mice with T-CD4cells develop as innate cells expressing high levels of Eomes (40).Furthermore, these mice also have a population of CD4SP thy-mocytes expressing Eomes (40). Although there does appear to befew mature CD4SP thymocytes expressing Eomes in WT mice,we did find significant differences in a population of maturePLZF+CD4+ ab T cells between WTand SAP-deficient mice (p,0.0002 using a Mann–Whitney t test). Therefore, development ofPLZF+ thymocytes may be tightly regulated in WT mice to controlthe development of innate-like lymphocytes expressing Eomes.Furthermore, unlike mouse thymocytes, human thymocytes ex-press MHC class II molecules, correlating with a substantialnumber of Eomes+CD8+ T cells in human fetal thymus and spleen.Based on these findings, Min et al. (40) have suggested that theseinnate T cells may function in protective immunity during theperinatal period. Thus, it is possible that a normal developmentalpathway for human T cells has been revealed by the genetic ab-lation of key T cell signaling proteins and transcription factors inmice.

AcknowledgmentsWe thank Regina Whitehead and Sharlene Hubbard for technical assis-

tance and the National Institutes of Health Tetramer Core Facility for

use of the mCD1d tetramer.

DisclosuresThe authors have no financial conflicts of interest.

References1. Berg, L. J. 2007. Signalling through TEC kinases regulates conventional versus

innate CD8+ T-cell development. Nat. Rev. Immunol. 7: 479–485.2. Veillette, A., Z. Dong, and S. Latour. 2007. Consequence of the SLAM-SAP signaling

pathway in innate-like and conventional lymphocytes. Immunity 27: 698–710.3. Nayar, R., M. Enos, A. Prince, H. Shin, S. Hemmers, J.-K. Jiang, U. Klein,

C. J. Thomas, and L. J. Berg. 2012. TCR signaling via Tec kinase ITK andinterferon regulatory factor 4 (IRF4) regulates CD8+ T-cell differentiation. Proc.Natl. Acad. Sci. USA 109: E2794–E2802.

4. Moran, A. E., K. L. Holzapfel, Y. Xing, N. R. Cunningham, J. S. Maltzman,J. Punt, and K. A. Hogquist. 2011. T cell receptor signal strength in Treg andiNKT cell development demonstrated by a novel fluorescent reporter mouse. J.Exp. Med. 208: 1279–1289.

5. Qiao, Y., L. Zhu, H. Sofi, P. E. Lapinski, R. Horai, K. Mueller, G. L. Stritesky,X. He, H.-S. Teh, D. L. Wiest, et al. 2012. Development of promyelocyticleukemia zinc finger-expressing innate CD4 T cells requires stronger T-cell re-

The Journal of Immunology 11

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Page 13: Development of Innate CD4 and CD8 T Cells in Itk-Deficient Mice Is

ceptor signals than conventional CD4 T cells. Proc. Natl. Acad. Sci. USA 109:16264–16269.

6. Verykokakis, M., M. D. Boos, A. Bendelac, and B. L. Kee. 2010. SAP protein-dependent natural killer T-like cells regulate the development of CD8+ T cellswith innate lymphocyte characteristics. Immunity 33: 203–215.

7. Gordon, S. M., S. A. Carty, J. S. Kim, T. Zou, J. Smith-Garvin, E. S. Alonzo,E. Haimm, D. B. Sant’Angelo, G. A. Koretzky, S. L. Reiner, and M. S. Jordan.2011. Requirements for eomesodermin and promyelocytic leukemia zinc fingerin the development of innate-like CD8+ T cells. J. Immunol. 186: 4573–4578.

8. Weinreich, M. A., O. A. Odumade, S. C. Jameson, and K. A. Hogquist. 2010.T cells expressing the transcription factor PLZF regulate the development ofmemory-like CD8+ T cells. Nat. Immunol. 11: 709–716.

9. Weinreich, M. A., K. Takada, C. Skon, S. L. Reiner, S. C. Jameson, andK. A. Hogquist. 2009. KLF2 transcription-factor deficiency in T cells results inunrestrained cytokine production and upregulation of bystander chemokinereceptors. Immunity 31: 122–130.

10. Fukuyama, T., L. H. Kasper, F. Boussouar, T. Jeevan, J. van Deursen, andP. K. Brindle. 2009. Histone acetyltransferase CBP is vital to demarcate con-ventional and innate CD8+ T-cell development. Mol. Cell. Biol. 29: 3894–3904.

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

12. Qi, Q., M. Xia, J. Hu, E. Hicks, A. Iyer, N. Xiong, and A. August. 2009. En-hanced development of CD4+ gd T cells in the absence of Itk results in elevatedIgE production. Blood 114: 564–571.

13. Alonzo, E. S., and D. B. Sant’Angelo. 2011. Development of PLZF-expressinginnate T cells. Curr. Opin. Immunol. 23: 220–227.

14. Hu, J., and A. August. 2008. Naive and innate memory phenotype CD4+ T cellshave different requirements for active Itk for their development. J. Immunol. 180:6544–6552.

15. Atherly, L. O., J. A. Lucas, M. Felices, C. C. Yin, S. L. Reiner, and L. J. Berg.2006. The Tec family tyrosine kinases Itk and Rlk regulate the development ofconventional CD8+ T cells. Immunity 25: 79–91.

16. Schaeffer, E. M., G. S. Yap, C. M. Lewis, M. J. Czar, D. W. McVicar,A. W. Cheever, A. Sher, and P. L. Schwartzberg. 2001. Mutation of Tec familykinases alters T helper cell differentiation. Nat. Immunol. 2: 1183–1188.

17. Lucas, J. A., L. O. Atherly, and L. J. Berg. 2002. The absence of Itk inhibitspositive selection without changing lineage commitment. J. Immunol. 168:6142–6151.

18. Czar, M. J., E. N. Kersh, L. A. Mijares, G. Lanier, J. Lewis, G. Yap, A. Chen,A. Sher, C. S. Duckett, R. Ahmed, and P. L. Schwartzberg. 2001.Altered lymphocyte responses and cytokine production in mice deficient in theX-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP. Proc. Natl.Acad. Sci. USA 98: 7449–7454.

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

20. Broussard, C., C. Fleischacker, R. Horai, M. Chetana, A. M. Venegas,L. L. Sharp, S. M. Hedrick, B. J. Fowlkes, and P. L. Schwartzberg. 2006. Altereddevelopment of CD8+ T cell lineages in mice deficient for the Tec kinases Itkand Rlk. Immunity 25: 93–104.

21. Kronenberg, M., and I. Engel. 2007. On the road: progress in finding theunique pathway of invariant NKT cell differentiation. Curr. Opin. Immunol.19: 186–193.

22. Dubois, S., T. A. Waldmann, and J. R. M€uller. 2006. ITK and IL-15 support twodistinct subsets of CD8+ T cells. Proc. Natl. Acad. Sci. USA 103: 12075–12080.

23. Treiner, E., L. Duban, S. Bahram, M. Radosavljevic, V. Wanner, F. Tilloy,P. Affaticati, S. Gilfillan, and O. Lantz. 2003. Selection of evolutionarily con-served mucosal-associated invariant T cells by MR1. Nature 422: 164–169.

24. Kawachi, I., J. Maldonado, C. Strader, and S. Gilfillan. 2006. MR1-restrictedVa19i mucosal-associated invariant T cells are innate T cells in the gut laminapropria that provide a rapid and diverse cytokine response. J. Immunol. 176:1618–1627.

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

26. Gadue, P., and P. L. Stein. 2002. NK T cell precursors exhibit differential cy-tokine regulation and require Itk for efficient maturation. J. Immunol. 169: 2397–2406.

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

28. Au-Yeung, B. B., and D. J. Fowell. 2007. A key role for Itk in both IFNg andIL-4 production by NKT cells. J. Immunol. 179: 111–119.

29. Perona-Wright, G., K. Mohrs, K. D. Mayer, and M. Mohrs. 2010. Differentialregulation of IL-4Ra expression by antigen versus cytokine stimulation char-acterizes Th2 progression in vivo. J. Immunol. 184: 615–623.

30. Pereira, P., and L. Boucontet. 2012. Innate NKTgd and NKTab cells exert similarfunctions and compete for a thymic niche. Eur. J. Immunol. 42: 1272–1281.

31. Yin, C. C., O. H. Cho, K. E. Sylvia, K. Narayan, A. L. Prince, J. W. Evans,J. Kang, and L. J. Berg. 2013. The Tec kinase ITK regulates thymic expansion,emigration, and maturation of gd NKT cells. J. Immunol. 190: 2659–2669.

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

33. Horai, R., K. L. Mueller, R. A. Handon, J. L. Cannons, S. M. Anderson,M. R. Kirby, and P. L. Schwartzberg. 2007. Requirements for selection ofconventional and innate T lymphocyte lineages. Immunity 27: 775–785.

34. Prince, A. L., L. B. Watkin, C. C. Yin, L. K. Selin, J. Kang, P. L. Schwartzberg,and L. J. Berg. 2014. Innate PLZF+ CD4+ ab T cells develop and expand in theabsence of Itk. J. Immunol. 193: 673–687.

35. Lucas, J. A., M. Felices, J. W. Evans, and L. J. Berg. 2007. Subtle defects in pre-TCR signaling in the absence of the Tec kinase Itk. J. Immunol. 179: 7561–7567.

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

37. Choi, E. Y., K. C. Jung, H. J. Park, D. H. Chung, J. S. Song, S. D. Yang,E. Simpson, and S. H. Park. 2005. Thymocyte-thymocyte interaction for efficientpositive selection and maturation of CD4 T cells. Immunity 23: 387–396.

38. Li, W., M. H. Sofi, S. Rietdijk, N. Wang, C. Terhorst, and C.-H. Chang. 2007. TheSLAM-associated protein signaling pathway is required for development of CD4+

T cells selected by homotypic thymocyte interaction. Immunity 27: 763–774.39. Lee, Y. J., Y. K. Jeon, B. H. Kang, D. H. Chung, C.-G. Park, H. Y. Shin,

K. C. Jung, and S. H. Park. 2010. Generation of PLZF+ CD4+ T cells via MHCclass II-dependent thymocyte-thymocyte interaction is a physiological process inhumans. J. Exp. Med. 207: 237–246.

40. Min, H. S., Y. J. Lee, Y. K. Jeon, E. J. Kim, B. H. Kang, K. C. Jung, C.-H. Chang,and S. H. Park. 2011. MHC class II-restricted interaction between thymocytesplays an essential role in the production of innate CD8+ T cells. J. Immunol. 186:5749–5757.

12 DISTINCT PATHS REGULATE INNATE CD4 AND CD8 T CELL DEVELOPMENT

by guest on March 17, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from