Abstract 914: Differential expression of Cbl-b in tumor infiltrating immune cells in patients with solid tumors

Abstract Introduction: To mechanistically elucidate the differences in the cancer patient clinical response, several pre-clinical and clinical biomarker studies have suggested the existence of several novel intracellular immune checkpoints. Casitas B cell lymphoma (Cbl-b) is an intracellular immune checkpoint protein that impedes the T cell and NK cell function. Methods: Immunohistochemistry (IHC) analysis was performed on the tissue micro array (TMA) from five different cancer indications (n=25) and healthy tissue controls (n=5). TMAs were stained utilizing commercially available IHC antibodies for Cbl-b, CD8, CD56 and FoxP3, nuclei were stained with DAPI. For in vitro studies, T cells were activated with anti-CD3/CD28 and NK cells with recombinant human IL-2 and IL-15. SiRNA mediated Cbl-b silencing in primary T and NK cells was also confirmed by western blot analysis and the effects of Cbl-b silencing were evaluated by measuring IFN-γ, IL-2, TNF-α and Granzyme-B accumulation by ELISA. For single-cell analysis, we leveraged publicly available Gene Expression Omnibus (GEO) data to perform an in-depth single-cell analysis of CBLB expression in different immune cell-types. This workflow included quality control, normalization, dimensionality reduction, clustering and cell type annotation Results: Compared to healthy tissue controls, tumors have high number of Cbl-b positive immune cells. The proportion of Cbl-b positives CD8 T cells and CD56 NK cells varied between indications. Interestingly, FoxP3+ regulatory T (Treg) cells have low levels of Cbl-b. The IHC observations was complemented by scRNA sequencing data. Consistent with the published data, Cbl-b silenced immune cells (T and NK cells) showed rapid activation and cytokine production as assed by a significant increase in IFN-γ, IL-2, TNF-α and Granzyme-B levels. Furthermore, Cbl-b silenced T cells are dependent on TCR alone and become independent of CD28 co-stimulation. Interestingly, the basal level of Cbl-b expression is high on NK cells compared to T cells, but upon TCR activation Cbl-b expression in T cells was rapidly increased while in NK cells IL-2 and IL-15 cytokine activation did not alter the CBL-B expression as assessed by western blot. Conclusion: Previous studies have established the role of Cbl-b in suppressing T cell and NK cells function, however, differential expression of Cbl-b and Cbl-b expression kinetics in different immune cell type remained unknown. Using IHC method and scRNA sequencing, we found that the Cbl-b expression is high in CD8 and NK cells, and low in Treg cells. Furthermore, in vitro Cbl-b silencing and Cbl-b expression in TMA suggest that silencing or inhibiting Cbl-b in T cells and NK cell is a valid approach. Citation Format: Elli Narvi, Reetta Virtakoivu, Endrit Elbasani, Nelson Kibinge, Laura Ravanti, Anil K. Thotakura. Differential expression of Cbl-b in tumor infiltrating immune cells in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 914.