Frequency Analysis of Simian Virus 40-specific Cytotoxic T Lymphocyte Precursors in the High Responder C57BL/6 Mouse Strain

Abstract

SUMMARYStudies in this laboratory have shown that long term simian virus 40 (SV40)-specific cytolytic T lymphocyte (CTL) cultures established from the spleens of high responder C57BL/6 (B6; H-2 b) mice exhibit a preference for the selection of H-2Db-restricted CTL clones. In this study, we have investigated the basis for this selection. Limiting dilution cultures were established using responder cells from the popliteal lymph nodes and the spleens of B6 mice immunized subcutaneously in the hind footpads or via the intraperitoneal route, respectively, with syngeneic SV40-transformed cells expressing a full length (1 to 708 amino acid residues) SV40 large T antigen. The relative frequency of CTL precursors (CTLp) able to expand in vitro in the presence of SV40-transformed stimulator cells and interleukin 2 and exhibit lytic activity against H-2 b cells expressing full length T antigen ranged from 1/1900 to 1/15000 in the popliteal lymph node and from 1/8000 to 1/55000 in the spleen. In these two experimental systems, CTLp restricted to H-2K b were apparently present at higher frequency than H-2Db-restricted CTLp. Furthermore, CTLp recognizing determinants within the amino-terminal or carboxy-terminal halves of T antigen were generated in approximately equal numbers. The relative affinity of SV40-specific CTL, assessed by inhibition with anti-Lyt 2 monoclonal antibody, indicated that CTL restricted to H-2D b interacted with their target with greater affinity than CTL restricted to H-2K b. These data suggest that the predominance of isolation of H-2Db-restricted CTL clones from long term in vitro cultures may be a function of the relative affinity of this population as a whole, rather than due to the immunodominance of this subpopulation during the in vivo response to SV40 T antigen.

In Vivo Expansion of the Residual Tumor Antigen-Specific CD8+T Lymphocytes That Survive Negative Selection in Simian Virus 40 T-Antigen-Transgenic Mice

“…Cultures were incubated at 37°C in 5% CO 2 for 5 h, after which the supernatants were collected and counted on a Packard Instruments Cobra II ␥-counter. Limiting dilution analysis was performed essentially as described previously (28). Briefly, splenocytes were obtained from groups of three to four naïve C57BL/6 mice or three to four SV11 or C57BL/6 mice that had been immunized 9 days earlier with rVV-ES-V. Pooled splenocytes from each group were serially diluted twofold and added (per dilution; range, 250,000 to 31,250 cells per well) to 48 wells of a 96-well microtiter plate in a total of 200 l of complete RPMI medium including 2 ϫ 10 3 ␥-irradiated (10,000 rads) B6/WT-19 cells, 1 ϫ 10 5 ␥-irradiated (3,000 rads) naïve C57BL/6 splenocytes, 5% (vol/vol) T-STIM culture supplement (Becton Dickinson Biosciences), 0.1 M methyl-␣-D-mannopyranoside (Sigma), and 0.25 U of recombinant interleukin-2.…”

“…Samples were considered positive when the corrected counts were higher than the mean counts per minute (ϩ 3 standard deviations of the mean) obtained using naïve C57BL/6 spleen cells. Frequencies were estimated using the minimal 2 method (65) as described previously (28).…”

Cytotoxic T cell response to Mengo virus in mice: effector cell phenotype and target proteins

“…This implies that non-structural proteins of Mengo virus are recognized very weakly if at all by Mengo virus-specific CTLs in these haplotypes. However, it has been previously shown that in vitro stimulation may select for certain populations of CTLs and obscure other populations (Jennings et al, 1988). Therefore, in our case, VP0-specific CTLs might have been preferentially expanded during in vitro stimulation with Mengo virus-infected cells, despite the presence of precursors specific for other polypeptides, e.g.…”

“…It is not clear which factors contribute to the selection of immunodominant peptides, but this could reflect differences in processing of individual peptides (Del Valet al, 1991) or in peptide binding affinity to MHC antigens (Jennings et al, 1988). This could also reflect deletions from the T cell receptor repertoire or peripheral regulation of CTL responses (H~immerling et al, 1993;Hill et al, 1993).…”

Differences in Immune Responses to Tumor Induced in Syngeneic Hosts by Injection of Hybrid and Parental Tumor Cells.

“…These CTL may recognize the MM antigens with restriction to MHC class I molecules interacting with Lyt-2.1 antigens which increase the avidity of the binding between the T cell receptors and MHC molecules (Dembic et al 1987;Doyle and Strominger 1987;Gennings et al 1988; Kuribayashi et al 1989). The CTL are thought to act in the classical manner.…”