(509e) Tumor Derived Exosomes As Local Modulators of Immunogenic Field Effects in Melanoma

Therapeutic modulation of immune checkpoints is one of the most recent clinical advances in the treatment of melanoma. Conceptually, immune checkpoints limit the clonal expansion of T cells. Antibodies against these immune checkpoints, such as anti-CTLA4 mAb, enable increased clonal expansion and enhance immune cell infiltration into the tumor microenvironment. Clinical results using anti-CTLA4 mAb demonstrate a durable clinical response but only in a subset of patients. Identifying the mechanisms of resistance is central to broadening the clinical benefit. Pre-clinical models indicate that the efficacy of immune checkpoint modulation depends inversely on tumor load [1]. We have previously identified that malignant melanocytes secrete a number of soluble factors that have the potential to alter the efficacy of immune cells that enter the tumor microenvironment [2]. The objective of this study was to characterize the biological influence of tumor-derived exosomes on immune cell function. In particular, we analyzed exosomes derived from three different cell models of melanoma: B16F0, a cell model of metastatic malignant melanoma; Cloudman S91, a cell model of immunogenic melanoma; and Melan-A, an immortalized melanocyte cell line. We used SEM and TEM to characterize the morphology of exosomes isolated from cell-conditioned media. The exosomes contained receptors derived from the parent cell as demonstrated by identification of interleukin 12 receptor beta 2 (IL-12Rβ2) on B16F0 exosomes by flow cytometry, western blot and immune-gold TEM. Furthermore, the profile of messenger RNA contained within B16F0-derived exosomes was compared against the parental cell using microarray gene expression analysis. Functional annotation and pathway enrichment methods were used to infer putative functional roles of exosomes from coding RNA. We also found that the coding RNA was functionally active as exosomes from B16F0 cells, but not Cloudman S91 and Melan-A exosomes, induced the synthesis of SHP2 in targeted immune cells. This is interesting as SHP2 is the negative regulator of T cell receptor signaling that is activated by the immune checkpoint CTLA4. Anti-CTLA4 mAb is intended to inhibit SHP2 activation. The SHP2 results also help explain the dependence of immune checkpoint modulation on tumor load observed in pre-clinical animal models. Collectively, the results suggest that exosomes help establish a density-dependent field effect by altering the activity of immune cells that enter the tumor microenvironment.

References

1. van Elsas A, Hurwitz AA, Allison JP. “Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation.” J Exp Med. 1999 Aug 2;190(3):355-66.

2. Kulkarni YM, et al. “A quantitative systems approach to identify paracrine mechanisms that locally suppress immune response to Interleukin-12 in the B16 melanoma model.” Integr Biol (Camb). 2012 Aug;4(8):925-36.