Abstract
Pre-apoptotic cancer cells release internalized calreticulin (CRT) to their surface prior to death, which acts as an ‘eat-me’ signal to local phagocytes. Chemotherapy and irradiation, which can induce immunogenic cell death through CRT translocation, can also result in local and/or systemic immune suppression in the host. To bypass the requirement of exposing the host to chemotherapy to induce translocation of CRT to the cell surface, murine acute myeloid leukemia (AML) cells (C1498), were engineered to constitutively express cell surface CRT (C1498.CRT). Vector control C1498 or C1498.CRT cells were inoculated intravenously (IV) into C57BL/6 mice. Significantly prolonged survival was observed in hosts harboring C1498.CRT versus vector control C1498 cells systemically. The survival benefit were abrogated in both Rag2-/- hosts or by depletion of T cells with anti-CD4 plus anti-CD8 antibodies, arguing that the immune-mediated effect of cell-surface CRT expression is dependent upon a functional adaptive immune system. More strikingly, systemic inoculation with C1498.CRT cells expressing the model SIYRYYGL (SIY) peptide antigen (C1498.SIY.CRT cells) resulted in almost complete protection from AML development (>90% long term survival vs. 10% of C1498.SIY vector control cells). All animals surviving a primary C1498.SIY.CRT challenge rejected a subsequent re-challenge with C1498.SIY cells, suggesting that CRT-expressing AML cells promote immunologic memory. Significantly enhanced expansion and unregulated IFNγ production were observed among SIY-specific T cell receptor transgenic CD8+ 2C T cells following their adoptive transfer into hosts bearing C1498.SIY.CRT AML cells versus vector control C1498.SIY cells. Interestingly, CRT expression on AML cells did not promote their in vivo phagocytosis by innate immune cells, specifically splenic CD8a+ dendritic cells known to engulf AML cells following their IV inoculation. IL-12 production by CD8α+CD11c+ dendritic cells which had engulfed C1498 and C1498.CRT cells in vivo was similarly induced, and cross-presentation of the SIY antigen to 2C T cells ex vivo by purified CD8a+DCs following in vivo exposure to C1498.SIY or C1498.SIY.CRT cells was also similar. In conclusion, it is clear that expression on CRT on the surface of AML cells leads to robust leukemia-specific T cell activation and expansion resulting in prolonged leukemia-specific survival in AML-bearing animals. Although a direct effect of CRT on innate immune cells, such as dendritic cells, is suspected, the molecular mechanism underlying the “CRT effect” remains unclear, and is being explored further through gene expression analysis in purified DCs which have engulfed CRT-expressing or control AML cells in vivo, as well as in animals genetically deficient in the putative CRT receptor, LRP, in dendritic cells. It will be of interest to analyze spontaneous CRT expression on AML cells from human samples and to correlate cell surface CRT expression with the presence or absence of spontaneous T cell responses to known AML antigens and with clinical outcomes.
Disclosures
No relevant conflicts of interest to declare.
The Regulation of Inflammation by Innate and Adaptive Lymphocytes