A human acute leukemia-derived T-cell line produces two inhibitor factors which suppress lymphocyte proliferation: characterization and purification of the molecules.
The crude supernatant of an CD1+/CD8+ T-cell clone (GI-CO-T-9) established from a long standing culture of an acute T-lymphoblastic leukemia (T-ALL) was shown to inhibit the responsiveness of normal PBMC to PHA. This clone does not produce TNF-alpha, TNF-beta, alpha-IFN, tau-IFN, IL-1, IL-2 and has no NK-like activity. The crude supernatant has been subjected to a multi-step chromatographic fractioning. Preparative gel permeation HPLC allowed us to recover two peaks of biologic activity in the range of 100-120 kDa and 75-85 kDa (referred to as "high molecular mass inhibitor factor", HMMIF, and "low molecular mass inhibitor factor", LMMIF, respectively). Both fractions were then subjected to anion exchange HPLC: HMMIF was recovered in fractions eluting at 0.04 M NaCl while LMMIF eluted at higher ionic strength (0.48 M NaCl). The fractions with biologic activity recovered from anion exchange HPLC have been subjected to hydrophobic interaction HPLC (HIC) for final purification. The highly purified material was characterized by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate (SDS) revealing single bands of 115 kDa and 80 kDa. The isoelectric points (pI), determined by flat-bed isoelectricfocusing, were 7.4 for HMMIF and 3.5-3.6 for LMMIF. Studies on temperature lability indicated that both proteins are stable for 3-4 hours at room temperature (RT), 24-36 hours at +4 degrees C and 7-10 days at -80 degrees C.
Montaldo
P G
PG
Pediatric Oncology Research Laboratory, G. Gaslini Children's Hospital, Genoa, Italy.
Lanciotti
M
M
Castagnola
E
E
Parodi
M T
MT
Cirillo
C
C
Cornaglia-Ferraris
P
P
Ponzoni
M
M
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007526:Isoelectric Point; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000276:immunology*; D008213:Lymphocyte Activation; D008970:Molecular Weight; D010835:Phytohemagglutinins / Q000494:pharmacology; D013491:Suppressor Factors, Immunologic / Q000096:biosynthesis* / Q000302:isolation & purification; D014407:Tumor Cells, Cultured / Q000276:immunology
D010835:Phytohemagglutinins; D013491:Suppressor Factors, Immunologic