TOWARDS UNDERSTANDING THE ROLE OF LYMPHOID ENHACER FACTOR-1 (Lef1) IN iNKT CELL DEVELOPMENT

Aditya Rao1,2, Tiffany Carr1,2, Mihalis Verykokakis1,2 and Barbara L. Kee1,2.

Dept. of Pathology1 and Committee on Immunology2, The University of Chicago, Chicago IL USA

ABSTRACT Invariant natural killer T (iNKT) cells are innate-like T cells that are activated by glycolipid antigens,

rapidly producing cytokines that impact anti-microbial immune responses, asthma, and autoimmunity.

Intrathymic expansion of iNKT cells and acquisition of multiple effector fates during thymic development

prior to foreign antigen stimulation are distinct features of the iNKT lineage. Interestingly, the number of

Th2-type effector iNKT cells (NKT2) varies across different mouse strains, and is correlated with

systemic alterations of the function of other immune cells. The molecular mechanisms that regulate

these important developmental processes are poorly understood. Here, we show that the transcription

factor Lymphoid Enhancer Factor-1 (LEF1) is required for the intrathymic expansion of iNKT cells and

NKT2 differentiation. To test whether LEF1 is sufficient to induce some aspects of iNKT cell

development, we will overexpress LEF1 in iNKT cells. To that end, we generated a retroviral vector that

can inducibly express LEF1 upon Cre recombination. Using this system, we will transduce hematopoietic

stem cells (HSC) from CD4-driven Cre transgenic mice and we will investigate iNKT cell development

from HSC-reconstituted mice overexpressing LEF1 in CD4+CD8+ cells.

Figure 1. iNKT cells differentiate into distinct lineages in the thymus that parallel the effector fates of CD4+ T-helper cells. CD1D-restricted postpositive selection iNKT cell progenitors can differentiate into multiple helper lineages analogous to those of CD4+ T cells. These include the IFNγ-producing TBET+ NKT1, the IL4-producing GATA3+ NKT2, or the IL17-producing RORγt+ NKT17 iNKT subsets. Steady state secretion of IL4 by NKT2 cells has been shown to condition bystander CD8+ T cells to adopt a more memory like phenotype, to stimulate peripheral B cells to produce IgE, and to stimulate thymic DCs to secrete CCL17 and CCL22.

Figure 4. Inducible Lef1 expression in vitro using the pMGfI1.1 Cre-inducible retroviral expression vector. A. Lef1 was subcloned into the pMGfI1.1 retroviral construct (pMGfI1.1-Lef1). Upon expression of Cre, GFP and the stop codon will be excised, allowing for expression of Lef1. B. Plat-E cells were transfected with pMGfI1.1 or pMGfI1.1-Lef1 retroviral construct, together with MIGR1-Cre or MIGR1. After 48 hours, cells were analyzed for expression of Lef1. All plots are gated on thy1.1+ cells.

Figure 6: MACS enrichment of BM progenitors for thy1.1+ cells. Bone marrow progenitors were retrovirally infected with either pMGfI1.1 or pMGfI1 .1-LEF1, then MACS-enr i ched w i th a -THY1.1-APC. The enriched cell population was retro-orbitally injected into irradiated CD45.1 Thy1.2 WT hosts.

Figure 5. Experimental scheme. CD4Cre mice were injected with 5FU (150mg/kg). After 3 days, bone marrow cells were harvested and cultured for 2 days. The c e l l s w e r e t h e n retrovirally infected with e i the r pMGfI1 .1 o r pMGfI1.1-LEF1. The next day, infected cells were MACS-enriched prior to retro-orbital injection into irradiated WT host mice. Mice will be analyzed 6-8 weeks post-reconstitution.

B

IgEproduction

CCL17production

CCL22production

DC

61.8

16.6

16.9

41.9

28.3

23.7

RORγt

PLZ

F

Ctrl Lef1∆/∆

iNKT1

iNKT2

iNKT17

60

40

20

0

80

Pe

rce

nta

ge

**

****

iNKT1

iNKT2

iNKT17

Ctrl Lef1∆/∆

iNKT1

iNKT2

iNKT17

0.335 0.0413

CD24

Tetr

am

er

Ctrl Lef1∆/∆

Ctrl0.8

0.6

0.4

0.2

0.0

****

Lef1∆/∆

Ce

ll n

um

be

r (x

10

6)

Total iNKTs

Figure 3: Lef1 directly regulates expression of Gata3. A. QPCR analysis of Gata3 mRNA in ST1, ST2 and ST3 iNKT cells from Control and Lef1Δ/Δ mice. Gata3 mRNA expression is shown relative to Hprt. B. GATA3 versus CD4 expression in ST1, ST2, and ST3 thymic iNKT cells from Control and Lef1Δ/Δ mice. C. ChIP from sorted ST1/ST2 Va14Tg iNKT thymocytes with anti-LEF1, followed by QPCR using primers spanning a TCF1/LEF1 binding site upstream of the Gata3b promoter. Numbers indicate average fold enrichment of the immunoprecipitated DNA relative to the b-globin locus.

0.766

5.23 45.1

1.67

LEF1

SSC

pMGfI1.1 pMGfI1.1-LEF1

-Cre

+Cre

Gata3

β-globin

6

4

2

0

8

10 *iNKT

Fold

enr

ichm

ent

Ctrl Lef1∆/∆

1.5

1.0

0.5

0.0Rel

ativ

e m

RN

A Gata3

ST1 ST2 ST3

2.0

22.360.2

9.44

6.2849.1

37.6

15.457.3

18.3

2.7138.9

50

6.7916.1

70.7

1.6714.9

79.1

GATA3

CD4

ST1 ST2 ST3

Ctrl

Lef1 ∆/∆

FUTURE DIRECTIONS • Analysis of thymuses in 4-6 weeks • We can test other candidate genes with our expression system

Figure 2. Lef1 is required for expansion of NKT2 cells. A. iNKT cells from the thymus of Lef1Δ/Δ and control mice, enriched with CD1D Tetramers. B. PLZF versus RORγt expression in Tetr+CD24lo iNKT cells from Control and Lef1Δ/Δ mice was used to resolve iNKT1 (PLZFloRORγt-), iNKT2 (PLZFhiRORγ t - ) , and iNKT17 (PLZFintRORγt+) cells. Numbers show percent of each population among total iNKT cells.

61.8

16.6

16.9

41.9

28.3

23.7

RORγt

PL

ZF

Ctrl Lef1∆/∆

iNKT1

iNKT2

iNKT17

60

40

20

0

80

Perc

enta

ge

**

****

iNKT1

iNKT2

iNKT17

Ctrl Lef1∆/∆

iNKT1

iNKT2

iNKT17

0.335 0.0413

CD24

Te

tra

me

r

Ctrl Lef1∆/∆

Ctrl0.8

0.6

0.4

0.2

0.0

****

Lef1∆/∆

Cell n

um

ber

(x10

6)

61.8

16.6

16.9

41.9

28.3

23.7

RORγt

PLZ

F

Ctrl Lef1∆/∆

iNKT1

iNKT2

iNKT17

60

40

20

0

80

Perc

enta

ge

**

****

iNKT1

iNKT2

iNKT17

Ctrl Lef1∆/∆

iNKT1

iNKT2

iNKT17

0.335 0.0413

CD24

Tetr

am

er

Ctrl Lef1∆/∆

Ctrl0.8

0.6

0.4

0.2

0.0

****

Lef1∆/∆

Cell n

um

ber

(x10

6)A

B

A

B C

8.52

15.5

4.5771.4

3.15

3.4792.5

0.842

6

32.9

11.150

2.14

0.853

3.9493.1

GFP

Thy1

.1

Thy1

.1 E

nric

hed

CD4Cre CD4Cre+LEF1

Harvest bone marrow

CD4Cre

5FU

pMGfI1.1 and pMGfI1.1-LEF1

retroviruses

Chimeric mouse

Infection of BM cells

CD45.2

WT

Irradiate

Inject infected BM cells

CD45.1

6-8 weeks

Harvest thymus

MACS sort of cells to enrich for thy1.1+ cells

Cd4-Cre

5’ LTR 3’ LTR GFP thy1.1 IRES Lef1

Age1 Not1

5’ LTR 3’ LTR thy1.1 IRES Lef1

CD44-

CD44hi

A

B