A study of the proteins responsible for stimulating B-CLL-specific T-cell responses by autologous dendritic cells pulsed with tumour cell lysate.
We have previously shown that autologous dendritic cells (DCs) pulsed with tumour cell lysate and cultured with autologous T cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL) stimulate significant increases in proliferation, IFN-gamma secretion and specific HLA class II-restricted cytotoxicity to B-CLL targets. In this study, normal and tumour cell lysates were analysed by reducing SDS-PAGE, and protein bands of interest eluted from the polyacrylamide gel by electroelution. The eluted protein fractions and whole-cell lysate were then pulsed onto autologous DCs and cocultured with autologous T cells. Finally, the stimulatory fractions were sequenced. B-CLL cell lysates revealed protein bands at 65, 42, 35 and 25 kDa, while normal B-cell lysates only showed a single protein band at 65 kDa. Both the 65 kDa band and 42 kDa bands were capable of stimulating comparable amounts of IFN-gamma secretion and specific cytotoxicity as whole lysate, while the other protein bands were not. Sequencing of the 65 kDa fraction showed a dominant peptide that matched human serum albumin, while sequencing the 42 kDa fraction showed three dominant peptides which matched human actin and another unidentified protein. The significance of these findings remains unclear.
Goddard
Ruth
R
Derriford Combined Laboratory, Derriford Hospital, Plymouth, UK.
Prentice
Archie
A
Copplestone
Adrian
A
Kaminski
Edward
E
D000199:Actins / Q000276:immunology / Q000302:isolation & purification; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D016022:Case-Control Studies; D002457:Cell Extracts / Q000276:immunology; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000276:immunology* / Q000302:isolation & purification; D012709:Serum Albumin / Q000276:immunology / Q000302:isolation & purification; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014182:Transplantation, Autologous
D000199:Actins; D002457:Cell Extracts; D009363:Neoplasm Proteins; D012709:Serum Albumin; D007371:Interferon-gamma