徐子真徐子真2014.4.252014.4.25

AIHACLL 10%

Mechanisms of autoimmunity in CLL?Mechanisms of autoimmunity in CLL?

normal B cells

CLL cellsabnormally functioning

T cells

microenvironment

CLL complicated by autoimmune cytopeniCLL complicated by autoimmune cytopenia has been found to be associated with una has been found to be associated with unmutated IGVH genes, high ZAP-70 and Cmutated IGVH genes, high ZAP-70 and CD38 expression, stereotyped B cell receptD38 expression, stereotyped B cell receptors (BCRs), increased serum β 2 -microglors (BCRs), increased serum β 2 -microglobulin levels and a high number of regulatobulin levels and a high number of regulatory T cellsory T cells

reflect advanced and highrisk CLL rather treflect advanced and highrisk CLL rather than specific AIHA-related abnormalities in han specific AIHA-related abnormalities in malignant B CLL cells.malignant B CLL cells.

MicroRNAsCLL autoimmune disorders

whether CLL likely to be complicated with AIHA is associated with a distinct miRNA expression pattern?

Materials and methods

Patients:15 CLL cases who developed AIHA; 18 Patients:15 CLL cases who developed AIHA; 18 sex-, age- and clinical stage-matched patientssex-, age- and clinical stage-matched patients

AIHA : unexplained hemoglobin level <10g/dL or AIHA : unexplained hemoglobin level <10g/dL or hematocrit<30% ,DAT+ for either IgG or C3 and hematocrit<30% ,DAT+ for either IgG or C3 and the presence of >1 indirect marker of hemolysis: the presence of >1 indirect marker of hemolysis: high reticulocyte count, low serum haptoglobin lehigh reticulocyte count, low serum haptoglobin levels, increased serum lactate dehydrogenase or vels, increased serum lactate dehydrogenase or bilirubin level.bilirubin level.

Materials and methods

Isolation of B-cells and RNA extractionIsolation of B-cells and RNA extractionmiRNA profiling using multiplex real-time qmiRNA profiling using multiplex real-time q

uantitative RT-PCRuantitative RT-PCRRenilla luciferase reporter vectorRenilla luciferase reporter vectorPre-miRNA transfection and luciferase assPre-miRNA transfection and luciferase ass

ayayMeasurement of CD80 expression by fl ow Measurement of CD80 expression by fl ow

cytometrycytometry

Results

377 human miRNA

CLL associated with AIHA tends to cluster together

miRNAs expressed in CLL associated with AIHA

We inserted the 3 ’UTR regiWe inserted the 3 ’UTR region of CD80 in the 3 ’UTR of on of CD80 in the 3 ’UTR of the Renilla gene in the psiCthe Renilla gene in the psiCHECK-2 vector (modified psHECK-2 vector (modified psiCHECK-2 vector). iCHECK-2 vector).

Renilla luciferase assay redRenilla luciferase assay reduced at 24 h after transfectiuced at 24 h after transfection with pre-miR-146b-5p anon with pre-miR-146b-5p and modified psiCHECK-2 vecd modified psiCHECK-2 vector.tor.

Percentage of CD80-positive cells reduced after transfection with pre-miR-146b-5p.

CD80 and miR-146b-5p expression on CLL cells. (A) Protein levels were analyzed in cryopreserved CD19 + CD5+ CLL cells from patientswith ( n =5) and without ( n =12) associated AIHA. (B) Expression of miR-146b-5p in purified CD19 + CD5 + CLL cells.

CLL AIHA+

miR-29c miR-146b-5p miR-20a

CD80

B7-H3 immune escape

ZAP-70,IGVH gene

T cell activation

Auto-immunity

participates in the B–T cell synapse and has been associated with restoration of the APC capacity of CLL cells