An Examination of miRNAs involved in T cell Programming Dr. Hilary (Billy) Sheppard Dunbar Laboratory.

An Examination of miRNAsinvolved in T cell Programming

Dr. Hilary (Billy) SheppardDunbar Laboratory

miRNAs are important for T celldifferentiation and function

Naive Tcm Tem TemRA

RA+RO-CCR7+27+28+

RA-RO+CCR7+27+/-28+/-62L+/-

RA-RO+CCR7-27+/-28+/-62L+/-

RA+RO-CCR7-27+/-28+/-62L+/-

CD8+ T cells are a heterogenous population

Adoptive Cellular Transfer

Naive Tcm Tem TemRA

IL 2

IL 7/IL 21

Growing Paired ClonesSingle cell sorting

1 X exp

ELA GIL NLV2 X exp

IL7/IL21 IL2 IL7/IL21 IL2IL7/IL21 IL2

IL2/IL7 IL2/IL7 IL2/IL7

ELA GIL NLV

ELA GIL NLVELA GIL NLV

Phenotype ofELA paired clones

62L

28

RO

CCR7

27

RA

IL2 IL7/21

IL2 IL7/21RO+/RA- 35 % vs 16 %RA+/RO- 13 % vs 39 %

28 + 35 % vs 82 %

CCR7+ 26 % vs 42 %62L 85 % vs 92 %

The experimental plan

Hypothesis – Specific miRNAs correlate with function and cell surface phenotype of Ag-specific T cells

• Assess miRNA profile in naive, Tcm and Tem

• Assess and compare miRNA profile in memory T cells grown in IL2 vs IL7 and 21

• Assess function/targets of miRNAs identified

Sorting T cell subsetsCD

45-R

A

CCR7

Naive Tcm Tem

hsa-miR-150_st

hsa-miR-26a_st

hsa-miR-16_st

hsa-let-7b_st

hsa-miR-92a_st

hsa-miR-768-3p_st

hsa-miR-191_st

hsa-miR-24_st

hsa-let-7a_st

hsa-miR-20a_st

hsa-miR-23a_st

hsa-miR-222_st

hsa-miR-19b_st

hsa-let-7d_st

hsa-miR-181a_st

hsa-miR-31_st

hsa-miR-106b_st

hsa-let-7g_st

hsa-miR-93_st

hsa-miR-1207-5p_st

hsa-miR-342-5p_st

hsa-miR-20a-star_st

hsa-miR-146b-5p_st

hsa-miR-149-star_st

hsa-miR-15b_st

hsa-miR-181b_st

hsa-miR-30c_st

hsa-miR-25_st

hsa-miR-574-3p_st

hsa-miR-194_st

hsa-miR-663_st

hsa-miR-1275_st

hsa-miR-20b_st

hsa-miR-423-3p_st

hsa-miR-125a-5p_st

hsa-miR-1183_st

hsa-miR-423-5p_st

hsa-miR-378_st

hsa-miR-210_st

hsa-miR-34b_st

hsa-miR-455-3p_st

hsa-miR-192_st

hsa-miR-1234_st

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

NTcmTem

Overview of Microarray Data

hsa-miR-150_st

hsa-miR-26a_st

hsa-miR-16_st

hsa-let-7b_st

hsa-miR-92a_st

hsa-miR-768-3p_st

hsa-miR-191_st

hsa-miR-24_st

hsa-let-7a_st

hsa-miR-20a_st

hsa-miR-23a_st

hsa-miR-222_st

hsa-miR-19b_st

hsa-let-7d_st

hsa-miR-181a_st

hsa-miR-31_st

hsa-miR-106b_st

hsa-let-7g_st

hsa-miR-93_st

hsa-miR-1207-5p_st

hsa-miR-342-5p_st

hsa-miR-20a-star_st

hsa-miR-146b-5p_st

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

NTcmTem

Top 45 hits

miR-92a

miR-31 miR-181a

miR-378

0

500

1000

1500

2000

2500

miR-150 miR-26a miR-342-3p

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

miR-24 miR-146a

miR-220

500

1000

1500

2000

2500

3000

Top hitsHighly expressed Down regulated Up regulated

*

* P = < 0.05

****

**P = < 0.01 N Tcm Tem

** **** **

*

** **

*

Biological RelevancemiR-92a Part of cluster involved in lymphocyte devt/proliferation

Expression related to proliferation potential?Bim = target, involved in AICD

miR-20a miR-92a miR-19b0

100

200

300

400

500

600

700

NTcmTem* ** *

AcknowledgementsLiam WilliamsCris Print, Vicky Fan

Anna Brooks, Dan Verdon, Vaughan Feisst – Dunbar Lab