The intrinsic immunogenic properties of cancer cell lines, immunogenic cell death, and how these influence host antitumor immune responses

Abstract

Modern cancer therapies often involve the combination of tumor-directed cytotoxic strategies and generation of a host antitumor immune response. The latter is unleashed by immunotherapies that activate the immune system generating a more immunostimulatory tumor microenvironment and a stronger tumor antigen-specific immune response. Studying the interaction between antitumor cytotoxic therapies, dying cancer cells, and the innate and adaptive immune system requires appropriate experimental tumor models in mice. In this review, we discuss the immunostimulatory and immunosuppressive properties of cancer cell lines commonly used in immunogenic cell death (ICD) studies being apoptosis or necroptosis. We will especially focus on the antigenic component of immunogenicity. While in several cancer cell lines the epitopes of endogenously expressed tumor antigens are known, these intrinsic epitopes are rarely determined in experimental apoptotic or necroptotic ICD settings. Instead by far the most ICD research studies investigate the antigenic response against exogenously expressed model antigens such as ovalbumin or retroviral epitopes (e.g., AH1). In this review, we will argue that the immune response against endogenous tumor antigens and the immunopeptidome profile of cancer cell lines affect the eventual biological readouts in the typical prophylactic tumor vaccination type of experiments used in ICD research, and we will propose additional methods involving immunopeptidome profiling, major histocompatibility complex molecule expression, and identification of tumor-infiltrating immune cells to document intrinsic immunogenicity following different cell death modalities.

Fig. 1. The main elements and forces of an ICD tumor response.

During cancer cell death by an ICD inducer (left panel), the release of DAMPs, chemokines and, cytokines, combined with the processing and presentation of tumor antigens on the cancer cell surface, will attract antigen-presenting cells like DCs. Following efferocytosis, the DCs migrate to the lymph nodes, where they cross-present the processed tumor antigens to NK and T lymphocytes. Combined with the effect of co-stimulatory factors such as CD80 and CD86, the lymphocytes become primed and activated in a process of cross-presentation. In turn, the now active CTLs and NK cells proliferate and travel to the tumor site, where they will recognize the tumor cells by expression of tumor antigenic epitopes, and induce their killing by secreting cytotoxic molecules involving perforins and granzymes and expressing death domain ligands such as FasL and TRAIL. The triforce of immunogenic cell death (right panel) consists of three main elements: inflammation, antigenicity (TAA, TNA, CTA, and MA), and adjuvanticity, with examples of each of these elements indicated in the small triangles on the edges.

Fig. 2. Timeline showing the occurrence of gp70/AH1-related and ICD research using CT26 cells.

The results from selected research papers are highlighted on this timeline, which dates from 1973, when MuLV DNA was first identified in mouse strains. The upper part (in red) highlights the gp70/AH1-centric work and some of the immunogenicity work which was initiated already in the seventies of the twentieth century. The ICD concept was launched only in the early 2000s. On the lower part of the timeline we have pointed out some key papers (in blue), in which CT26 cells were used to define new aspects of ICD. Finally, it was not until this year, 2020, that the immunodominant role of AH1 expression in CT26 cells was directly shown to influence the prototypic apoptotic ICD prophylactic tumor vaccination model (in purple).

Fig. 3. A role for antigen spread in the prophylactic tumor vaccination model.

Following prophylactic vaccination of mice with necroptotic AH1-deficient CT26 cells, the dying CT26 cancer cells are engulfed by dendritic cells during the process of efferocytosis and a transfer of TSAs to the immature DC takes place. In the lymph node, the now mature DCs cross-prime and activate T lymphocytes. Activated T cells kill tumor cells based on specific antigen recognition leading to the release of additional TSAs leading to additional specific T cells. Due to these cycles of antigen spread, a range of activated T lymphocytes with a greater TSA specificity than the original vaccination is generated. Of this pool of TSA-specific CTLs, some are likely to respond to AH1 expression on cancer cells during the challenge phase, and thereby prevent tumor outgrowth.

Fig. 4. Suggestive flowchart for the use of murine cancer cell lines in ICD in vivo studies.

When choosing a cancer cell line for the use in ICD assays in vivo, we suggest to first (Step 1) determine the expression levels of, e.g., known endogenous tumor antigens (TAA, such as gp70 and p15E retroviral antigens), MHC classes I and II molecule and immunostimulatory and immunoinhibitory molecules. Ideally, the cell lines should also first be tested in a titration vaccination setup using viable untreated cancer cells, before they are applied in an ICD vaccination experiment. Based on these expression profiles, cancer cell lines can be ranked according to their immunoprofile into those cell lines that are likely to be highly immunogenic (cyan arrows) versus low immunogenic (orange arrows). Whereas both high and low immunogenic cell lines are suitable for therapeutic tumor treatment models (Step 2), we would advise that the prophylactic tumor vaccination model is restricted to low immunogenic cell lines. Finally, when studying the level of tumor-infiltrating immune cells (Step 3), the difference between immune cells infiltrates in vehicle- versus ICD-treated tumors may vary between high and low immunogenic cell lines (gradient indicator shown in purple). In tumors established with a highly immunogenic cell line, even in the vehicle-treated background setting, there is likely to be a higher degree of immune cell infiltration than in a corresponding vehicle-treated low immunogenic tumor. Consequently, the relative difference is less pronounced between the amount of infiltrating immune cells in vehicle- and ICD-treated highly immunogenic tumors. On the other hand, in a tumor established with a low immunogenic cell line, we do not expect much if any immune cell infiltrate in a vehicle-treated setting, but once triggered with an ICD inducer, there would be a vast increase in immune cells resulting in relatively high immunogenicity following cell death treatment.