Peptide loading of empty major histocompatibility complex molecules on RMA-S cells allows the induction of primary cytotoxic T lymphocyte responses.
The antigen processing-defective mutant cell line RMA-S expresses at the cell surface major histocompatibility complex (MHC) class I molecules devoid of peptide that can be efficiently loaded with exogenous immunogenic peptides. We now report that viral peptide-loaded RMA-S cells, unlike parental RMA cells, can induce primary cytotoxic T lymphocyte (CTL) responses in vitro, in a T helper cell-independent fashion. This was shown for an H-2Kb-binding peptide of Sendai virus nucleoprotein and an H-2Db-binding peptide of adenovirus type 5 E1A protein with responding spleen cells of C57BL/6 mice, the strain of origin of RMA and RMA-S cells. Primary Sendai peptide-induced CTL lyse both peptide-loaded and virus-infected cells. Pre-culture of RMA-S cells at low temperature (22 degrees - 26 degrees C), which increases the amount of empty MHC class I molecules at the cell surface, decreases the peptide concentrations required for the induction of primary CTL responses. Primary peptide-specific CTL responses induced by peptide-loaded RMA-S cells are CD4+ cell- and MHC class II+ cell-independent. CTL response induction is blocked by the presence of anti-CD8 monoclonal antibody during culture. Direct peptide binding studies confirm the efficient loading of empty MHC molecules on RMA-S cells with peptide and show 2.5-fold more peptide bound per RMA-S cell compared to RMA cells. An additional factor explaining the difference in primary response induction between RMA and RMA-S cells is related to the CD8 dependence of these responses. MHC class I molecules occupied with irrelevant peptides (a majority present on RMA, largely absent on RMA-S) may interfere in the interaction of the CD8 molecule with relevant MHC/peptide complexes. The results delineate a novel strategy of peptide based in vitro immunization to elicit CD8+ cytotoxic T cell responses.
De Bruijn
M L
ML
Division of Immunology, The Netherlands Cancer Institute, Amsterdam.
Schumacher
T N
TN
Nieland
J D
JD
Ploegh
H L
HL
Kast
W M
WM
Melief
C J
CJ
D000256:Adenoviridae / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry*; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D006183:H-2 Antigens / Q000378:metabolism*; D007111:Immunity, Cellular; D007156:Immunologic Memory; D066298:In Vitro Techniques; D008285:Major Histocompatibility Complex; D051379:Mice; D008969:Molecular Sequence Data; D010222:Parainfluenza Virus 1, Human / Q000276:immunology; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D013696:Temperature
D000956:Antigens, Viral; D006183:H-2 Antigens; D010455:Peptides