Assessment of IFN-γ and granzyme-B production by in "sitro" technology.

Tumor neantigens (TNAs) and tumor-associated antigens (TAAs) are crucial triggers of anticancer immune responses. Through major histocompatibility complex, such antigens activate T cells, which, by releasing interferon gamma (IFN-γ) and granzyme B (GRZB), act as crucial effectors against tumor onset and progression. However, in response to immune pressure, cancer cells use different strategies to favor the establishment of an immunosuppressive tumor microenvironment (TME). Elucidating the dynamics of tumor-host co-evolution provides novel opportunities for personalized cancer immunotherapies. The in sitro (in vitro+in situ) technology is an experimental approach involving the preparation of heterocellular cell suspensions from fresh tumors and their use in vitro. The in sitro experimental setup offers the possibility to (1) analyze immune-related parameters (e.g., quantification of cytokines released in the TME), (2) reveal the mechanism of action of drugs, and (3) unveil crucial cell-intrinsic and cell-extrinsic processes boosting anticancer immune responses. Nonetheless, the in sitro technology does not fully recapitulate the complexity of the tissue "in situ" nor models the patterns of infiltrating immune cell localization, and hence parallel experimentation should be scheduled. In this chapter we discuss in sitro technology to analyze and quantify IFN-γ and GRZB production by T cells either co-cultured with cancer cells in the presence of exogenous adjuvant stimuli (i.e., an antibody targeting the immune checkpoint programmed cell death protein 1, and recombinant calreticulin) and boosting with TAAs (i.e., the model SIINFEKL ovalbumin antigen). Specifically, we describe IFN-γ and GRZB quantification by flow cytometry, ELISA and ELISpot technologies.

Phenotypic Variation in Different Lesions of Mycosis Fungoides Biopsied Within a Short Period of Time From the Same Patient.

Phenotypic variants of mycosis fungoides (MF) include mainly the expression of cytotoxic markers by neoplastic cells (either α/ß or γ/δ cytotoxic). To manage the patient properly, distinction from other cutaneous cytotoxic natural killer/T-cell lymphomas is paramount. Particularly for cutaneous γ/δ T-cell lymphoma, distinction is often based on clinicopathologic correlation (presence of tumors at first diagnosis as opposed to patches only in MF). The authors report a case of cytotoxic MF characterized by expression of TCRγ in two of three biopsies performed within a time frame of 1 week. The patient presented with patches, plaques, and 1 tumor at the time of first diagnosis; thus, distinction from cutaneous γ/δ T-cell lymphoma was not possible on clinical grounds alone. The diagnosis of MF was established, thanks to the phenotypic variations revealed by the three biopsies, with 1 lacking expression of cytotoxic proteins (TIA-1 and granzyme B) and of TCRγ. This case shows the importance to perform several biopsies in cases of cutaneous lymphoma, as morphologic and phenotypic features are variable and information gathered from a single biopsy may result in a wrong diagnosis.