Defective presentation of endogenous antigens by a murine sarcoma. Implications for the failure of an anti-tumor immune response

Abstract

MHC class I-restricted CTL play a central role in the immune response against methylcholanthrene (MCA)-induced sarcomas in mice. We, therefore, hypothesized that MCA-induced tumors may evade immune recognition by failing to present Ag to CD8+ CTL. Of a number of previously described MCA-induced sarcomas, one, MCA 101, fails to induce CTL, is nonimmunogenic, and grows rapidly and lethally in nonimmunosuppressed recipients. To better understand the nonimmunogenicity of MCA 101 we examined its ability to present foreign Ag to CTL. Unlike immunogenic sarcomas, MCA 101 failed to present endogenously synthesized influenza virus Ag to influenza virus-specific CTL. The deficiency in presentation of endogenous Ag by MCA 101 was attributed to a markedly reduced rate of synthesis of class I molecules because up-regulation of class I synthesis by IFN-gamma greatly increased the presentation of influenza A virus Ag. Despite low levels of cell surface class I expression, MCA 101 presented exogenous peptide Ag to anti-influenza CTL with efficiency similar to immunogenic MCA sarcoma cell lines. These findings could not be attributed to deficiencies in class I assembly or transport, as has been suggested by others who have studied mutant cells with defective Ag presentation. Furthermore, our studies suggest that some tumor cells can escape recognition by CTL and subsequent immune eradication by suppressing presentation of endogenous Ag.

Figure 1

Processing and presentation of endogenously generated viral Ag by cultured tumor cell lines. Effector cells were splenocytes generated by in vivo and in vitro stimulation with PR8. Tumor cell lines were infected with wild-type influenza A virus (PR8), followed by a 4-h incubation period to allow for elaboration of viral protein. Results of eight 4-h ⁵¹Cr-release assays are depicted here. Experimental values represent the average of triplicates, with a SE of the mean of less than 4%. These experimental values are then averaged. Error bars represent SE of the mean. Each tumor was included in the following number of experiments: MCA 101 in seven, MCA 102 in four, MCA 105 in two, MCA 106 in three, MC 38 in two, MCA 203 in two, MCA 205 in five, MCA 207 In four, RMA in two, and RMA-S in two. Average lysis for controls (i.e., uninfected cells) was <5% for each tumor depicted.

Figure 2

Processing and presentation of endogenous influenza virus proteins inserted into vaccinia vectors. In this experiment, MCA 101 (solid triangles) is compared with MCA 207 (solid circles). Polyclonal effectors used were splenocytes from mice immunized with PR8 virus in vivo and restimulated in vitro with wild-type PR8 influenza virus for targets infected with NS1, PA, and HA and primed with PR8 in vivo and synthetic peptide (aa 365-380) of NP in vitro for targets infected with NP and Control. Tumor lines were infected with vaccinia constructs containing the following influenza A genes: nucleoprotein (NP), nonstructural 1 (NS1), acidic polymerase (PA), and hemagglutinin (HA). The vesicular stomatitis virus gene for the Indiana nucleocapsid protein(Control) inserted into a vaccinia vector was used as a control. All experimental values represent the average of triplicates, with a SE of the mean of less than 4%. These results confirmed similar results obtained in five different experiments comparing MCA 101 and MCA 207, which used wild-type PR8 infection.

Figure 3

Recognition of MCA-induced tumor lines after exposure to synthetic peptide amino acids 365-380 of NP at varying concentrations. Effector cells were splenocytes from mice immunized with PR8 virus and restimulated in vitro with synthetic peptide (NP 365-380) at a concentration of 1 μM. All experimental values represent the average of triplicates, with a SE of the mean of less than 4%. Each value is representative of between two and five experiments; all had similar results.

Figure 4

SDS-PAGE analysis of immunoprecipitated class I molecules after pulsing cells with ³⁵S-methionine then chasing with cold methionine as described in Materials and Methods. Left lanes are with mock endo-H treatment where as right lanes show treatment with endo H. This experiment shows that class I molecules are present and transported in MCA 101. Although not readily appreciable in the present reproduction. appropriate quantities of β₂-microglobulin were found to co-precipitate with the H chain. Control cell line is RMA. This experiment was repeated with similar results.

Figure 5

⁵¹Cr-release assay in which tumors were pretreated with 200 U/ml of IFN-γ for 48 h before the assay. Cells not exposed to IFN-γ are shown in open circles. Cells were then infected with PR8 virus. ⁵¹Cr-labeled, then subjected to killing by splenocytes from mice immunized with PR8 virus in vivo and restimulated in vitro with NP peptide (365-380). Experimental values represent the average of triplicates, with a SE of the mean of less than 4%. This experiment was repeated with similar results.