Relevance of Thymic Stromal Lymphopoietin on the Pathogenesis of Glioblastoma: Role of the Neutrophil.

Glioblastoma multiforme (GBM) is the most predominant and malignant primary brain tumor in adults. Thymic stromal lymphopoietin (TSLP), a cytokine primarily generated by activated epithelial cells, has recently garnered attention in cancer research. This study was aimed to elucidate the significance of TSLP in GBM cells and its interplay with the immune system, particularly focused on granulocyte neutrophils. Our results demonstrate that the tumor produces TSLP when stimulated with epidermal growth factor (EGF) in both the U251 cell line and the GBM biopsy (GBM-b). The relevance of the TSLP function was evaluated using a 3D spheroid model. Spheroids exhibited increased diameter, volume, and proliferation. In addition, TSLP promoted the generation of satellites surrounding the main spheroids and inhibited apoptosis in U251 treated with temozolomide (TMZ). Additionally, the co-culture of polymorphonuclear (PMN) cells from healthy donors with the U251 cell line in the presence of TSLP showed a reduction in apoptosis and an increase in IL-8 production. TSLP directly inhibited apoptosis in PMN from GBM patients (PMN-p). Interestingly, the vascular endothelial growth factor (VEGF) production was elevated in PMN-p compared with PMN from healthy donors. Under these conditions, TSLP also increased VEGF production, in PMN from healthy donors. Moreover, TSLP upregulated programed death-ligand 1 (PDL-1) expression in PMN cultured with U251. On the other hand, according to our results, the analysis of RNA-seq datasets from Illumina HiSeq 2000 sequencing platform performed with TIMER2.0 webserver demonstrated that the combination of TSLP with neutrophils decreases the survival of the patient. In conclusion, our results position TSLP as a possible new growth factor in GBM and indicate its modulation of the tumor microenvironment, particularly through its interaction with PMN.

Human Glomerular Endothelial Cells Treated With Shiga Toxin Type 2 Activate γδ T Lymphocytes.

The hemolytic uremic syndrome associated with diarrhea, a consequence of Shiga toxin (Stx)-producing Escherichia coli infection, is a common cause of pediatric acute renal failure in Argentina. Stx type 2a (Stx2a) causes direct damage to renal cells and induces local inflammatory responses that involve secretion of inflammatory mediators and the recruitment of innate immune cells. γδ T cells constitute a subset of T lymphocytes, which act as early sensors of cellular stress and infection. They can exert cytotoxicity against infected and transformed cells, and produce cytokines and chemokines. In this study, we investigated the activation of human peripheral γδ T cells in response to the incubation with Stx2a-stimulated human glomerular endothelial cells (HGEC) or their conditioned medium, by analyzing in γδ T lymphocytes, the expression of CD69, CD107a, and perforin, and the production of TNF-α and IFN-γ. In addition, we evaluated by confocal microscopy the contact between γδ T cells and HGEC. This analysis showed an augmentation in cellular interactions in the presence of Stx2a-stimulated HGEC compared to untreated HGEC. Furthermore, we observed an increase in cytokine production and CD107a expression, together with a decrease in intracellular perforin when γδ T cells were incubated with Stx2a-treated HGEC or their conditioned medium. Interestingly, the blocking of TNF-α by Etanercept reversed the changes in the parameters measured in γδ T cells incubated with Stx2a-treated HGEC supernatants. Altogether, our results suggest that soluble factors released by Stx2a-stimulated HGEC modulate the activation of γδ T cells, being TNF-α a key player during this process.

Glioblastoma cells potentiate the induction of the Th1-like profile in phosphoantigen-stimulated γδ T lymphocytes.

PURPOSE: γδ T lymphocytes are non-conventional T cells that participate in protective immunity and tumor surveillance. In healthy humans, the main subset of circulating γδ T cells express the TCRVγ9Vδ2. This subset responds to non-peptide prenyl-pyrophosphate antigens such as (E)-4-hydroxy-3-methyl-but-enyl pyrophosphate (HMBPP). This unique feature of Vγ9Vδ2 T cells makes them a candidate for anti-tumor immunotherapy. In this study, we investigated the response of HMBPP-activated Vγ9Vδ2 T lymphocytes to glioblastoma multiforme (GBM) cells. METHODS: Human purified γδ T cells were stimulated with HMBPP (1 µM) and incubated with GBM cells (U251, U373 and primary GBM cultures) or their conditioned medium. After overnight incubation, expression of CD69 and perforin was evaluated by flow cytometry and cytokines production by ELISA. As well, we performed a meta-analysis of transcriptomic data obtained from The Cancer Genome Atlas. RESULTS: HMBPP-stimulated γδ T cells cultured with GBM or its conditioned medium increased CD69, intracellular perforin, IFN-γ, and TNF-α production. A meta-analysis of transcriptomic data showed that GBM patients display better overall survival when mRNA TRGV9, the Vγ9 chain-encoding gene, was expressed in high levels. Moreover, its expression was higher in low-grade GBM compared to GBM. Interestingly, there was an association between γδ T cell infiltrates and TNF-α expression in the tumor microenvironment. CONCLUSION: GBM cells enhanced Th1-like profile differentiation in phosphoantigen-stimulated γδ T cells. Our results reinforce data that have demonstrated the implication of Vγ9Vδ2 T cells in the control of GBM, and this knowledge is fundamental to the development of immunotherapeutic protocols to treat GBM based on γδ T cells.