Specific epitope‐induced conversion of CD8⁺ memory cells into effector cytotoxic T lymphocytes in vitro: presentation of peptide antigen by CD8⁺ T cells

Abstract

The requirements for the conversion of CD8+ memory T cells into effector class I major histocompatibility complex (MHC) Kd-restricted cytotoxic T (Tc) cells in vitro have been studied. Purified CD8+ splenocytes from influenza A/WSN-primed BALB/c (H-2d) mice stimulated with a synthetic nucleoprotein peptide 147-158 R156- (NPP) alone generated Tc cells specific for influenza virus-infected target cells. No additional requirements for accessory cells or their lymphokine products were necessary indicating that peptide antigen (Ag) in association with Kd was presented on CD8+ T cells. The evidence for presentation of NPP by CD8+ T cells was supported by the use of CD8+ memory T cells from semiallogeneic bone marrow radiation chimeras of P1----F1 type (H-2b----[H-2d x H-2b]F1). Memory CD8+ splenocytes from A/WSN-immune chimeras did not develop into secondary effector Tc cells as a result of a 4-day culture with NPP alone, however, were able to do so if NPP was presented by Kd-bearing Ag-presenting cells. In addition, these results exclude the possibility of direct recognition of free NPP molecules by the specific T cell receptor of CD8+ memory T cells. CD8+ memory splenocytes (H-2b) from chimeras were also able to develop into functionally active Tc cells as a result of presentation of Db-restricted synthetic peptide (NP 366-374) with a sequence derived from influenza virus nucleoprotein with high affinity for Db MHC class I molecules. Blockade of endogenously produced interleukin 2 (IL-2) activity by anti-IL-2 or anti-IL-2 receptor monoclonal antibody in the culture of CD8+ memory T cells during a 4-day NPP stimulation completely abolished Tc cell generation, indicating that the utilization of this lymphokine is absolutely required for the secondary Tc cell development. These findings demonstrate that CD8+ memory T cells per se are able to recognize the restimulating epitope as a result of its presentation by CD8+ T cells and develop into cytolytically active and highly specific Tc cells with no requirements for other cellular helper components or their lymphokine products.

Steric recognition of T‐cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes

“…CD8 T lymphocytes were further purified from mononuclear cells of RSV‐infected BALB/c mice with MACS sets (Miltenyi Biotech Co., Germany) to be re‐stimulated in vitro with peptide‐pulsed antigen‐presenting cells overnight for analysis by ELISPOT assays 41 …”

“…CD8 T lymphocytes were further purified from mononuclear cells of RSV-infected BALB/c mice with MACS sets (Miltenyi Biotech Co., Germany) to be re-stimulated in vitro with peptide-pulsed antigen-presenting cells overnight for analysis by ELISPOT assays. 41 Immunisation of BALB/c mice with variant epitopes for induction of specific CD8 T-lymphocyte responses Subsequent to second or third subcutaneous immunisation with a variety of synthetic peptides emulsified in Freund's adjuvants, spleen mononuclear cells were isolated from BALB/c mice to be re-stimulated in vitro with the immunised peptide or others for analysis of specific IFNc responses by the ELISPOT assay (Table 1). BALB/c mice were supplied by the animal facility at the College of Medicine, National Cheng Kung University in Taiwan.…”

Transfer of CD8+ T Cells into SCID Mice and Activation of Memory Virus‐Specific Cytotoxic T Cells

“…Oslo. Norway) as described [17], Ig splenocytes were depleted of MHC class II'. CD4* and asiato-GM! '…”

“…Three to 16 days after transfer of cells and inimuni/atton of SCID mice, spleens were removed and single-cell suspensions passed through Kicolt were prepared for -'Cr-release cytotoxicity assay. The target cells and the procedures have been described in detail eadier [17].…”

“…Our recent findings on the requirements for the conversion [17. IS] have implied that memory CD8^ T cells represent a population of cells that can be activated independently of collaboration with conventional Ag-presenting or helper cell populations.…”