Cell death induced by cytotoxic CD8⁺T cells is immunogenic and primes caspase-3–dependent spread immunity against endogenous tumor antigens

Abstract

BackgroundElimination of cancer cells by some stimuli like chemotherapy and radiotherapy activates anticancer immunity after the generation of damage‐associated molecular patterns, a process recently named immunogenic cell death (ICD). Despite the recent advances in cancer immunotherapy, very little is known about the immunological consequences of cell death activated by cytotoxic CD8+T (Tc) cells on cancer cells, that is, if Tc cells induce ICD on cancer cells and the molecular mechanisms involved.MethodsICD induced by Tc cells on EL4 cells was analyzed in tumor by vaccinating mice with EL4 cells killedin vitroorin vivoby Ag-specific Tc cells. EL4 cells and mutants thereof overexpressing Bcl-XLor a dominant negative mutant of caspase-3 and wild-type mice, as well as mice depleted of Tc cells and mice deficient in perforin, TLR4 and BATF3 were used.Ex vivocytotoxicity of spleen cells from immunized mice was analyzed by flow cytometry. Expression of ICD signals (calreticulin, HMGB1 and interleukin (IL)-1β) was analyzed by flow cytometry and ELISA.ResultsMice immunized with EL4.gp33 cells killed in vitro or in vivo by gp33-specific Tc cells were protected from parental EL4 tumor development. This result was confirmed in vivo by using ovalbumin (OVA) as another surrogate antigen. Perforin and TLR4 and BATF3-dependent type 1 conventional dendritic cells (cDC1s) were required for protection against tumor development, indicating cross-priming of Tc cells against endogenous EL4 tumor antigens. Tc cells induced ICD signals in EL4 cells. Notably, ICD of EL4 cells was dependent on caspase-3 activity, with reduced antitumor immunity generated by caspase-3–deficient EL4 cells. In contrast, overexpression of Bcl-XLin EL4 cells had no effect on induction of Tc cell antitumor response and protection.ConclusionsElimination of tumor cells by Ag-specific Tc cells is immunogenic and protects against tumor development by generating new Tc cells against EL4 endogenous antigens. This finding helps to explain the enhanced efficacy of T cell-dependent immunotherapy and provide a molecular basis to explain the epitope spread phenomenon observed during vaccination and chimeric antigen receptor (CAR)-T cell therapy. In addition, they suggest that caspase-3 activity in the tumor may be used as a biomarker to predict cancer recurrence during T cell-dependent immunotherapies.

Cellular cytotoxicity is a form of immunogenic cell death

“…For instance, in the accompanying article published by the group of Julian Pardo in this issue, a key role for target-cell caspase-3 is revealed. 47 Overall, our findings and those in this companion article are mutually supportive and confirm our conclusions leading to ongoing work on the intrinsic molecular immunogenic mechanisms that follow cytotoxicity.…”

Elevated expression of FREM1 in breast cancer indicates favorable prognosis and high‐level immune infiltration status

“…[40][41][42] For instance, the CD8 + T cells representing the phenotype of lymphocytes with cytotoxicity mediate antitumor functions by inducing cell lysis via the release of IFN-gamma and Perforin/granzyme B complex, and they act as a reliable immune prognostic marker for the outcome of BC patients. 43,44 The processes of CD8 + T cells activation and maturation are in turn regulated through the cytokines produced by T-helper 1 (Th1) cells and tumor-specific antigens processed by dendritic cells. 45 Our results demonstrated that the synergistic increase of these cells is accompanied by increased expression of FREM1, suggesting that FREM1 may play a crucial role in regulating the functional integrity of adaptive antitumor immune response.…”

Immunogenicity of cell death driven by immune effectors

“…5 6 Conversely, the results from Pardo and colleagues appear to challenge the current literature in proposing that caspase 3 (CASP3), a key regulator of apoptosis with multipronged immunosuppressive effects including the ability to shut down type I interferon production by STING1, [8][9][10] is required for the immunogenicity of cancer cells succumbing to immune effectors. 5 This interpretation was based on the ability of chemical pan-caspase inhibitor (Q-VD-OPh) and a dominant-negative variant of CASP3, but not the overexpression of BCL2-like 1 (BCL2L1, a potent antiapoptotic molecule best known as BCL-X L ), to reduce (to some degree) immunological protection conferred by cancer cells dying on attack by antigen-specific CTLs. However, Q-VD-OPh also blocks CASP8, which is essential for the immunogenicity of cell death because of its key function in ICD-associated CALR exposure.…”

“…In line with this notion, BCL-X L overexpression had little effects on sensitivity to death by immune effectors, which correlated with a minimal influence on vaccination efficacy. 5 Thus, the impact of CASP3 on the efficacy of vaccination with cancer cells succumbing to CTLs and NK cells appears to be largely limited to its ability to precipitate cell death in this setting, which does not apply to ICD driven by other stimuli including radiation. [8][9][10] Early work from the late Jurg Tschopp and colleagues demonstrated that the lytic granules of CTLs contain high levels of CALR, which was interpreted as a safeguard mechanism to prevent PRF1 activation by Ca 2+ ions prior to granule exocytosis, largely relying on the ability of CALR to chelate Ca 2+ .…”