1. 徐子真2014.4.25

2. AIHA CLL 10%

3. Mechanisms of autoimmunity in CLL? normal B cells CLL cells abnormally functioning T cells microenvironment

4. CLL complicated by autoimmune cytopenia has been found to be associated with unmutated IGVH genes, high ZAP-70 and CD38 expression, stereotyped B cell receptors (BCRs), increased serum β 2 -microglobulin levels and a high number of regulatory T cells CLL complicated by autoimmune cytopenia has been found to be associated with unmutated IGVH genes, high ZAP-70 and CD38 expression, stereotyped B cell receptors (BCRs), increased serum β 2 -microglobulin levels and a high number of regulatory T cells reflect advanced and highrisk CLL rather than specific AIHA-related abnormalities in malignant B CLL cells. reflect advanced and highrisk CLL rather than specific AIHA-related abnormalities in malignant B CLL cells.

5. MicroRNAs CLL autoimmune disorders whether CLL likely to be complicated with AIHA is associated with a distinct miRNA expression pattern?

6. Materials and methods Patients:15 CLL cases who developed AIHA; 18 sex-, age- and clinical stage-matched patients Patients:15 CLL cases who developed AIHA; 18 sex-, age- and clinical stage-matched patients AIHA : unexplained hemoglobin level 1 indirect marker of hemolysis: high reticulocyte count, low serum haptoglobin levels, increased serum lactate dehydrogenase or bilirubin level. AIHA : unexplained hemoglobin level 1 indirect marker of hemolysis: high reticulocyte count, low serum haptoglobin levels, increased serum lactate dehydrogenase or bilirubin level.

7. Materials and methods Isolation of B-cells and RNA extraction Isolation of B-cells and RNA extraction miRNA profiling using multiplex real-time quantitative RT-PCR miRNA profiling using multiplex real-time quantitative RT-PCR Renilla luciferase reporter vector Renilla luciferase reporter vector Pre-miRNA transfection and luciferase assay Pre-miRNA transfection and luciferase assay Measurement of CD80 expression by fl ow cytometry Measurement of CD80 expression by fl ow cytometry

8. Results 377 human miRNA CLL associated with AIHA tends to cluster together

9. miRNAs expressed in CLL associated with AIHA

10. We inserted the 3 ’UTR region of CD80 in the 3 ’UTR of the Renilla gene in the psiCHECK-2 vector (modified psiCHECK-2 vector). We inserted the 3 ’UTR region of CD80 in the 3 ’UTR of the Renilla gene in the psiCHECK-2 vector (modified psiCHECK-2 vector). Renilla luciferase assay reduced at 24 h after transfection with pre-miR- 146b-5p and modified psiCHECK-2 vector. Renilla luciferase assay reduced at 24 h after transfection with pre-miR- 146b-5p and modified psiCHECK-2 vector.

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

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

13. CLL AIHA+ miR-29cmiR-146b-5pmiR-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