Peptide loading of empty major histocompatibility complex molecules on RMA‐S cells allows the induction of primary cytotoxic T lymphocyte responses

Abstract

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.

Tumor-infiltrating T lymphocytes: friends or foes?

“…The observed in vitro CTL priming was independent of CD4 þ T cells or MHC class II-expressing cells, but was dependent upon the level of MHC class I expression on the RMA-S cells. 104 A high expression level of MHC class I peptide complexes on the peptide-presenting cells was the decisive requirement for the induction of CD4…”

A rapid isolation procedure for dendritic cells from mouse spleen by centrifugal elutriation

“…APC activity in a primaiy T cell response. DC are the only cell type described, apart from the antigen processing defective cell line RMA-S, which are capable of inducing an anti-viral or peptide specific primary T cell response (Macatonia et aL, 1989;De Bruijn et al, 1991). To confirm that DC with this capacity were present in CE fraction 5, low density cells of CE fraction 5 obtained by discontinuous density gradient cen trifugation and DC isolated by the standard pro cedure were used to induce a primary peptidespecific cytotoxic T cell response.…”

A specific cytotoxic T‐lymphocyte epitope presentation system for antitumor immunity

“…Because of its deficiency of TAP, there are a huge amount of empty MHC I on RMA-S compared with its wild-type counterpart RMA cells. 36 RMA cell line was infected with the virus carrying TAP2-targeting shRNA to make RMA-91, a RMA-S equivalent that expresses decreased MHC I molecule on the cell surface. It would be expected that more peptides would bind to both RMA-S and RMA-91 than that to RMA cells when the cells were pulsed with exogenous peptides at low temperature.…”

“…OVA 257-264 /K b complex on RMA-S pulsed with an OVA 257-264 peptide was claimed to be one of the densest MHC class I molecules ever expressed on cells. 36 The cells with combined TAP disruption and peptide linked b2m delivery were more potent immunogens than that with peptide-linked b2m expression only, in correlation to OVA [257][258][259][260][261][262][263][264] Moreover, the cytotoxicity induced with OVA-presenting cells was specific for EG7 cells only, not for EL4 cells (Fig. 4b), excluding the possibility of NK cell-mediated nonspecific killing toward tumor cells.…”