JOURNAL CLUB, 28th september 2007

T-CELL ACTIVATION•T-CELLS ARE ABLE TO DISTINGUISH BETWEEN SELF AND NON SELF ANTIGENS (Ag)•T-CELL RECEPTOR (TCR) PLAY AN IMPORTANT ROLE, BECAUSE IT RECOGNIZES Ag PRESENTED BY APC IN MHC AND THIS LEADS TO THE INDUCTION OF INTRACELLULAR SIGNALING AND T-CELL RESPONSES•T-CELLS RESPONSIVENESS AND TCR SIGNALING VARY WITH T-CELL DEVELOPMENT

•miRNA WERE RECENTLY DISCOVERED TO BE DIFFERENTIALLY EXPRESSD DURING HEMATOPOIETIC DIFFRENTIATION AND THEY MAY BE INVOLVED IN CONTROLLING THE DEVELOPMENT OF IMMUNE SYSTEM

RESULTS

DN (CD4-, CD8-)

SP(CD4-, CD8+)(CD4+, CD8-)

DP(CD4+, CD8+)

IMMATURE T-CELLSDN1 (CD44+, CD25-)DN2 (CD44+, CD25+)DN3 (CD44-, CD25+)DN4 (CD44-, CD25-)

ORDER OFAPPEREANCE

DURING DEVELOPMENT

DINAMIC REGULATION OF miR181a EXPRESSIONIN T-CELLS MATURATION

miR181a EXPRESSION AUGMENTS TCR SIGNALING

TO ACCESS HOW miR181a AFFECTS TCR SIGNALING:

•INCREASED EXPRESSION OF miR181a IN 5C.C7 Ag PRIMED CD4+ T-CELLS•STIMULATED WITH MCC (AGONIST); TCR SIGNALING STRENGHT ASSESSED BY MESASURING Ca2+INCREASE

HIGH LEVEL OF miR181a AUGMENTS TCR-MEDIATED T-CELLS ACTIVATION

miR181a EXPRESSION AUGMENTS TCR SIGNALING

TCR SIGNAL INPUT=NUMB OFANTIGENIC pMHC COMPL. AT

T:APC INTERFACE

TCR SIGNAL OUTPUT=CALCIUM CONCENTR CHANGES

IN CYTOSOL

miR181a EXPRESSION INCREASES

T CELLS SENSITIVITY

miR181a CONVERTS ANTAGONIST INTO AGONIST

•MCC99R INDUCE CALCIUM INCREASE IN miR181a CELLS

•MCC99R ALONE STIMULATE EFFECTOR T-CELLS FUNCTION

•miR181a EXPRESSION DOESN’T ALTER CD4 AND TCR DENSITY ON T-CELLS SURFACE

•WHEN CD28 IS BLOCKED AND miR181a T-CELLS ARE TRETAED WITH MC99R, THEY CAN STILL RESPOND TO THE ANTAGONIST.

miR181a MODULATE TCR SENSITIVITY BY CONTROLLING INTRACELLULARSIGNALING MOLECULES, SUCH AS SHP1 (Lck NEGATIVE REGULATOR)

AND ERK1/2 PATHWAYS

USING UNPUBLISHED COMPUTER PROGRAMS, THEY FOUND TARGETS FOR mir181a:•Lck-SPECIFIC-TYROSIN POHOSPHATASE, SHP-2 AND PTPN22•ERK-SPECIFIC-PHOSPHATASE, DUSP5 AND DUSP6

miR181a REPRESSES MULTIPLE PHOSPHATASE IN T-CELLS

miRNA181a INCREASES THE BASAL PHOSPHORILATION LEVELS OF Lck AND ERK

miR181a EXPRESSION INHIBITS Lck/SHP1 INTERACTION

MECHANISM OF TCR SIGNALING

MULTITARGET REPRESSION IS REQUIRED FOR miR181a FUNCTION IN TCR SIGNALING

STRENGHT OF Ca2+ RESPONSE AND% OF T-CELLS ACTIVATED

EFFICACY AND SPECIFICITY OF shRNA CONSTRUCTS

CONTRIBUTION OF EACH PHOSPHATASETO Ag DISCRIMINATION

miR181a MODULATES NEGATIVE AND POSITIVE SELECTIONOF T-CELLS

TREATMENT WITH ANTAGOMIR RESTOREDPHOSPHATASE/KINASE INVOLVED IN TCR

SIGNALING IN DP CELLS

INHIBITION OF ERK ACTIVITY

CONCLUSIONS

•miRNAs ARE A NEW CLASS OF REGULATORY MOLECULES IN THE IMMUNE SYSTEM

miR181a IS AN INTRINSIC MODULATOR OF TCR SENSITIVITY IN T-CELL DEVELOPMENT

(ALTHOUGH OTHER COMPONENTS MAY CONTRIBUTE TO THESE CHANGES IN SENSITIVITY)

•miRNAs CAN MODULATE MULTIPLE TARGETSAS THEY EXERT A QUANTITATIVE REGULATIONTHAT ALLOWS A DISCRIMINATIVE SWITHC IN

Ag RECOGNITION

Paper discussionPaper discussion

What is the key experiment?What is the key experiment?

(the one confirming the statement in the title)(the one confirming the statement in the title)

What is the strongest point?What is the strongest point?

What is the weakest point?What is the weakest point?

and and What to do to strenghten it?What to do to strenghten it?

What is the take home message?What is the take home message?

(summarize it in a sentence)(summarize it in a sentence)

• POSITIVE

•Any cell that doesn’t recognize self MHC is deleted• Ensures functionalmatching of receptor,co-receptor and classof MHC•Lineage commitment

• NEGATIVE

• Any cell that reactsstrongly to selfantigen is deleted