The major histocompatibility complex (MHC) class II-restricted antigen processing pathway presents antigenic peptides acquired in the endocytic route for the activation of CD4+ T cells. Multiple cancers express MHC class II, which may influence the anti-tumor immune response and patient outcome. Low MHC class II expression is associated with poor survival in diffuse large B-cell lymphoma (DLBCL), the most common form of aggressive non-Hodgkin lymphoma. Therefore, we investigated whether gamma-interferon-inducible lysosomal thiol reductase (GILT), an upstream component of the MHC class II-restricted antigen processing pathway that is not regulated by the transcription factor class II transactivator, may be important in DLBCL biology. GILT reduces protein disulfide bonds in the endocytic compartment, exposing additional epitopes for binding to MHC class II and facilitating antigen presentation. In each of four independent gene expression profiling cohorts with a total of 585 DLBCL patients, low GILT expression was significantly associated with poor overall survival. In contrast, low expression of a classical MHC class II gene, HLA-DRA, was associated with poor survival in one of four cohorts. The association of low GILT expression with poor survival was independent of established clinical and molecular prognostic factors, the International Prognostic Index and the cell of origin classification, respectively. Immunohistochemical analysis of GILT expression in 96 DLBCL cases demonstrated variation in GILT protein expression within tumor cells which correlated strongly with GILT mRNA expression. These studies identify a novel association between GILT expression and clinical outcome in lymphoma. Our findings underscore the role of antigen processing in DLBCL and suggest that molecules targeting this pathway warrant investigation as potential therapeutics.

T cell-mediated immunity has the ability to produce durable anti-melanoma responses resulting in improved survival of patients with advanced melanoma. Antigen presentation is a key determinant of T cell responses. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells. However, GILT expression in melanoma has not been defined. We evaluated GILT and MHC class II expression in human primary and metastatic melanomas and nevi using immunohistochemistry. GILT staining in melanocytes was observed in 70% of primary and 58% of metastatic melanomas versus 0% of nevi. When present, the GILT staining intensity in melanocytes was typically faint. Both GILT and MHC class II expression were increased in melanocytes of primary and metastatic melanomas compared with nevi. GILT staining in antigen presenting cells was detected in 100% of primary and metastatic melanomas versus 31% of nevi and was typically intense. GILT expression was increased in antigen presenting cells of primary and metastatic melanomas compared to nevi, whereas MHC class II had equivalent high expression in antigen presenting cells of all melanocytic lesions. GILT staining in keratinocytes was detected in 67% of primary melanomas versus 14% of nevi and 6% of metastatic melanomas. GILT, but not MHC class II, expression was increased in keratinocytes of primary melanomas compared to nevi and metastases. GILT expression is anticipated to result in improved presentation of melanoma antigens and more effective anti-melanoma T cell responses. GILT expression may be a biomarker of immune recognition of melanoma.

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In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non-small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the