The granzyme B inhibitor PI-9 is differentially expressed in all main subtypes of pediatric acute lymphoblastic leukemias.
OBJECTIVE
The success of bone marrow transplantation in leukemia depends on graft-versus-leukemia (GvL) effects, mediated by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. These act by CD95L or granule cytotoxins, such as granzyme B (GrB) whose only known inhibitor is proteinase inhibitor 9 (PI-9). Since PI-9 protects cells from CTL, PI-9 may counteract GvL in leukemias. Our aim was to establish methods to analyze the expression and function of PI-9.


METHODS
We screened the most common pediatric ALL subsets, i.e. pre-B, common, and cortical T-ALL, for PI-9 expression, using a reverse transcription polymerase chain reaction (RT-PCR) approach which was correlated to semiquantitative and functional methods established in cell lines and patient probes.


RESULTS
In vitro, PI-9 mediated resistance towards CTL and inhibited GrB-induced clevage of caspase 3. In patient-derived ALL cells, PI-9 high and PI-9 low specimens were studied by flow cytometry, RT-PCR, in vitro GrB treatment and NK assay, demonstrating concordant results and PI-9-dependent target cell protection. Analysis of PI-9 in probes from ALL patients showed differential expression, but no correlation with immunotype.


CONCLUSIONS
Our data suggest that PI-9 in pediatric ALL is differentially expressed, without close correlation to subtype. Since PI-9 considerably alters GrzB and killer cell sensitivity, it may strongly influence the efficacy of GvL effects. The approaches applied here will allow evaluation of the expression and function of PI-9 in larger series of malignancies.
Classen
Carl Friedrich
CF
University Children's Hospital Ulm, Germany. carl.classen@medizin.uni-ulm.de
Ushmorov
Alexey
A
Bird
Phillip
P
Debatin
Klaus-Michael
KM
D045744:Cell Line, Tumor; D002648:Child; D005434:Flow Cytometry; D020544:Graft vs Leukemia Effect / Q000276:immunology*; D053804:Granzymes; D006801:Humans; D009447:Neuroblastoma; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000378:metabolism*; D015843:Serpins / Q000096:biosynthesis*; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*; D014162:Transfection
C102316:SERPINB9 protein, human; D015843:Serpins; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases