Apoptotic Blebs From Leukemic Cells as a Source of Tumor Associated Antigen for Monocyte-Derived Dendritic Cell Loading

Abstract 3283

Whereas in 80% of the patients suffering from acute myeloid leukemia (AML) complete remission is achieved, the 5-year survival rate is only 30% - 40% due to relapse of the disease. In order to increase overall survival of AML patients, novel or additional therapies are required. Due to their ability to ingest, process and subsequently present antigens to (naïve) lymphocytes in an immunogenic context, dendritic cells (DC) can initiate antigen-specific immunity. Therefore, DC have become a major subject of interest as mediators of anti-tumor immunity in a minimal residual disease setting in AML.

Since only few AML-associated antigens (LAA) have been characterized to date, the use of apoptotic tumor blast cells as a source of LAA could be instrumental, due to the presence of both known and unknown LAA. However, the immunogenicity of apoptotic cells remains questionable; e.g. apoptotic tumor cells may not activate DC adequately, but rather impair their function. Previously, it was shown in mice that apoptotic blebs, which detach from the main apoptotic cell during apoptosis, induce DC maturation, whereas the apoptotic cell remnant (ACR) does not.

Here, we analyzed monocyte-derived DC (MoDC) function in vitro, after co-culture with either human AML-derived ACR or blebs. Apoptosis of CFSE-labeled HL60 AML cells was induced by Mitoxantrone and ACR and blebs were isolated by differential centrifugation. The apoptotic fractions were subsequently added to allogeneic MoDC without the addition of a maturation stimulus (immature), or with the addition of the cytokine cocktail IL-1β, IL-6, PGE2, and TNFα, supplemented with TLR 7/8 ligand R848, 1 hour after initiation of the co-culture (maturing). Blebs were preferentially ingested compared to ACR by both immature and maturing MoDC (75% vs. 45%, and 75% vs. 40% CFSE positive MoDC (p =< 0.0001), respectively). Analysis of the capacity of mature MoDC to migrate towards the lymph node-homing chemokine CCL19, in a transwell migration system, demonstrated that MoDC migration was diminished by approximately 50% compared to unloaded mature MoDC, irrespective of the ingestion of blebs or ACR. However, the percentage of CFSE-positive MoDC which were able to migrate towards CCL19 was significantly higher when MoDC had ingested blebs, compared to ACR (18% vs. 7% (p = 0.05), respectively). Although the ingestion of blebs or ACR does not lead to significant changes in phenotypic maturation, CD40 ligation of immature MoDC that had taken up blebs, resulted in the secretion of twice the amount of the pro-inflammatory cytokines IL-6 and IL-12p70 as compared to MoDC that had ingested ACR (p = 0.05).

In conclusion, we show that blebs derived from human AML cells are preferentially ingested by both immature and maturing MoDC and that a higher percentage of these MoDC are able to migrate towards CCL19, compared to MoDC that have ingested ACR. Furthermore, compared to ACR, ingestion of blebs by MoDC leads to an increased production of the pro-inflammatory cytokines IL-6 and IL-12. Additional functional analyses are underway to determine whether ingestion of blebs also leads to a more efficient antigen presentation and subsequent anti-leukemic T cell activation.