Stromal Interferon-γ Signaling and Cross-Presentation Are Required to Eliminate Antigen-Loss Variants of B Cell Lymphomas in Mice

Abstract

To study mechanisms of T cell-mediated rejection of B cell lymphomas, we developed a murine lymphoma model wherein three potential rejection antigens, human c-MYC, chicken ovalbumin (OVA), and GFP are expressed. After transfer into wild-type mice 60–70% of systemically growing lymphomas expressing all three antigens were rejected; lymphomas expressing only human c-MYC protein were not rejected. OVA expressing lymphomas were infiltrated by T cells, showed MHC class I and II upregulation, and lost antigen expression, indicating immune escape. In contrast to wild-type recipients, 80–100% of STAT1-, IFN-γ-, or IFN-γ receptor-deficient recipients died of lymphoma, indicating that host IFN-γ signaling is critical for rejection. Lymphomas arising in IFN-γ- and IFN-γ-receptor-deficient mice had invariably lost antigen expression, suggesting that poor overall survival of these recipients was due to inefficient elimination of antigen-negative lymphoma variants. Antigen-dependent eradication of lymphoma cells in wild-type animals was dependent on cross-presentation of antigen by cells of the tumor stroma. These findings provide first evidence for an important role of the tumor stroma in T cell-mediated control of hematologic neoplasias and highlight the importance of incorporating stroma-targeting strategies into future immunotherapeutic approaches.

Targeting c-MYC with T-Cells

“…However, when 291PC cells were incubated with 100U/ml IFNγ for 24h prior to co-incubation and IFNγ was washed out before adding splenocytes, we observed a significant increase in IFNγ secretion. As shown previously, treatment of 291PC cells with IFNγ increases MHC I and II [25] and leaves c-myc RNA levels upregulated (Figure S3 left panel). IFNγ secretion could also be detected when splenocytes were stimulated with c-MYC protein in presence of anti c-MYC monoclonal antibody (9E10) but not if OVA protein was added instead of c-MYC.…”

“…This B-cell lymphoma line expresses the human c-MYC protein in a B-cell specific fashion [20]. As shown previously [25], when 1x10 5 291PC cells were subcutaneously inoculated into naïve C57BL/6 mice, animals died of local and - importantly - systemically progressive lymphoma. This demonstrated that the non-homologous regions of xenogenic c-MYC protein, when expressed by malignant B-cells, were not sufficient to induce a protective T-cell response.…”

“…In contrast to other studies that successfully used ATT to reject solid tumors of clinically relevant size [31] [40] and often utilize highly immunogenic tumors, i.e. regressor tumors, the 291PC lymphoma target cells do not display a regressor phenotype [25] [40]. To our surprise, we did not observe any difference in lymphoma growth after immunization with full-length c-MYC protein and subsequent challenge with 291 PC cells, even though we observed c-MYC-reactive CD4 + and CD8 + T-cells ex vivo .…”

“…Many models have demonstrated that high avidity T-cells will select for antigen loss variants within the tumor, if the malignant phenotype and growth of the tumor remain unaffected by shutting down antigen expression [37-39]. We have previously shown that antigen loss variants occur in the same model used for this study, when chicken ovalbumin (OVA) as a model antigen is targeted by specific OT-1 T-cells [25]. In this model, outgrowth of lymphomas expressing OVA is delayed and only OVA-negative lymphomas arise in immunocompetent hosts.…”

“…For tumor challenge experiments, 1x10 5 291PC cells [25] were injected subcutaneously into the abdominal flanks. Tumor growth was monitored 3 times per week using a sliding caliper.…”