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ABSTRACTBacillus Calmette-Guérin (BCG), the nonpathogenic Mycobacterium bovis strain used as tuberculosis vaccine, has been successfully used as treatment for non-muscle invasive bladder cancer for decades, and suggested to potentiate cellular and humoral immune responses. However, the exact mechanism of action is not fully understood. We previously described that BCG mainly activated anti-tumor cytotoxic NK cells with upregulation of CD56 and a CD16+ phenotype. Now, we show that stimulation of human peripheral blood mononuclear cells with iBCG, a preparation based on BCG-Moreau, expands oligoclonal γδ T-cells, with a cytotoxic phenotype, together with anti-tumor CD56high CD16+ NK cells. We have used scRNA-seq, flow cytometry, and functional assays to characterize these BCG-activated γδ T-cells in detail. They had a high IFNγ secretion signature with expression of CD27+ and formed conjugates with bladder cancer cells. BCG-activated γδ T-cells proliferated strongly in response to minimal doses of cytokines and had anti-tumor functions, although not fully based on degranulation. BCG was sufficient to stimulate proliferation of γδ T-cells when cultured with other PBMC; however, BCG alone did not stimulate expansion of purified γδ T-cells. The characterization of these non-donor restricted lymphocyte populations, which can be expanded in vitro, could provide a new approach to prepare cell-based immunotherapy tools.

Therapy of metastatic melanoma advanced recently with the clinical implementation of signalling pathway inhibitors, such as vemurafenib, specifically targeting mutant BRAFV600E. In general, patients experience remarkable clinical responses under BRAF inhibitor (BRAFi) treatment but eventually progress within 6–8 months due to resistance development. Responding metastases show an increased immune cell infiltrate, including also NK cells, that, however, is no longer detectable in BRAFi-resistant lesions, suggesting NK cell activity should be exploited to prevent disease progression. Here, we examined the effects of BRAFi on the expression of ligands targeting activating NK cells receptors immediately after treatment onset, prior to resistance development. We demonstrate that BRAFV600E mutant melanoma cells cultured in the presence of vemurafenib, strongly decreased surface expression of ligands for NK activating receptors including the NKG2D-ligand, MICA, and the DNAM-1 ligand, CD155, and became significantly less susceptible to NK cell attack. NKG2D-ligand protein downregulation was due to a significant decrease in mRNA levels, already detectable 24 h after drug treatment. Interestingly, vemurafenib-induced MICA downregulation could be counteracted by treatment of melanoma cells with the histone deacetylase (HDAC) inhibitor (HDACi) sodium butyrate, that also upregulated the DNAM1-ligand, Nectin-2. HDACi treatment enhanced surface expression of NKG2D-ligands in the presence of BRAFi, accompanied by recovery of NK cell recognition, but only upon simultaneous drug application. These results suggest that co-administration of BRAFi and HDAC inhibitors as well as having direct effects on melanoma cell survival, could also synergise to improve NK cell recognition and avoid tumour immune evasion.

Immunotherapy, via intra-vesical instillations of BCG, is the therapy of choice for patients with high-risk non-muscle invasive bladder cancer. The subsequent recruitment of lymphocytes and myeloid cells, as well as the release of cytokines and chemokines, is believed to induce a local immune response that eliminates these tumors, but the detailed mechanisms of action of this therapy are not well understood. Here, we have studied the phenotype and function of the responding lymphocyte populations as well as the spectrum of cytokines and chemokines produced in an in vitro model of human peripheral blood mononuclear cells (PBMCs) co-cultured with BCG. Natural killer (NK) cell activation was a prominent feature of this immune response and we have studied the expansion of this lymphocyte population in detail. We show that, after BCG stimulation, CD56dim NK cells proliferate, upregulate CD56, but maintain the expression of CD16 and the ability to mediate ADCC. CD56bright NK cells also contribute to this expansion by increasing CD16 and KIR expression. These unconventional CD56bright cells efficiently degranulated against bladder cancer cells and the expansion of this population required the release of soluble factors by other immune cells in the context of BCG. Consistent with these in vitro data, a small, but significant increase in the intensity of CD16 expression was noted in peripheral blood CD56bright cells from bladder cancer patients undergoing BCG therapy, that was not observed in patients treated with mitomycin-C instillations. These observations suggest that activation of NK cells may be an important component of the anti-tumoral immune response triggered by BCG therapy in bladder cancer.