徐子真2014.4.25. AIHA CLL 10% Mechanisms of autoimmunity in CLL? normal B cells CLL cells...
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Transcript of 徐子真2014.4.25. AIHA CLL 10% Mechanisms of autoimmunity in CLL? normal B cells CLL cells...
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徐子真徐子真2014.4.252014.4.25
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AIHACLL 10%
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Mechanisms of autoimmunity in CLL?Mechanisms of autoimmunity in CLL?
normal B cells
CLL cellsabnormally functioning
T cells
microenvironment
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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.
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MicroRNAsCLL autoimmune disorders
whether CLL likely to be complicated with AIHA is associated with a distinct miRNA expression pattern?
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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.
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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
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Results
377 human miRNA
CLL associated with AIHA tends to cluster together
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miRNAs expressed in CLL associated with AIHA
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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.
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Percentage of CD80-positive cells reduced after transfection with pre-miR-146b-5p.
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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.
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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