Baker, Carly

Abstract Background Immunotherapy has yet to make significant gains in glioblastoma (GBM) treatment, due in part to GBM-mediated immune suppression. Increasing evidence points to critical roles for tumor-derived extracellular vesicles (EVs) and immunosuppressive myeloid cells as key factors in this process. Methods Immunophenotyping of the tumor-immune microenvironment was performed using ultrasonic aspirate collected during GBM resection by high-dimensional flow cytometry. EVs collected from patient-derived GBM cell lines were used to condition myeloid cells collected from healthy donors to generate immunosuppressive myeloid cells. siRNA was used to knockdown TIGIT and/or NLRP3 expression prior to EV conditioning. T cell co-culture studies were performed with donor-matched T cells. Results Immune phenotyping of the tumor microenvironment and EV-conditioned myeloid cells revealed similar immunomodulatory protein expression across myeloid cell populations, with particularly elevated TIGIT expression. Knockdown of TIGIT reduced the immunosuppressive polarization of myeloid cells, resulting in improved T cell function. This finding proceeded in an NLRP3-dependent manner, with substantial co-expression of TIGIT and NLRP3 expression prior to knockdown, and concomitant knockdown of NLRP3 abrogating the effect of TIGIT knockdown. TIGIT expression correlated with increased IL-13 expression, and IL-13 blockade unmasked a pro-inflammatory myeloid cell phenotype. Conclusion TIGIT expression in myeloid cells in the GBM microenvironment is a functional marker of immunosuppressive activity, with TIGIT knockdown reducing IL-13 expression and unmasking the pro-inflammatory activity of NLRP3. This study bolsters our understanding of the immunosuppressive complexities of the GBM microenvironment and supports attenuation of immunosuppressive myeloid cell activity as a strategy to restore immune function in GBM.

Abstract Glioblastoma (GBM) is a deadly brain cancer with near-universal recurrence despite maximal treatment for which new innovations are sorely needed. Immunotherapy has yet to make significant gains in GBM treatment despite revolutionizing other cancer therapies, due in part to GBM-mediated immune suppression. This immune derangement proceeds through several mechanisms, but increasing evidence points to critical roles for tumor-derived extracellular vesicles (EVs) and immunosuppressive myeloid cells as key factors in this process. In the present study, we demonstrate broad expression of TIGIT across myeloid cell populations in the GBM microenvironment, a finding recapitulated by conditioning healthy monocytes with GBM-derived EVs. Further, knockdown of TIGIT expression reduced the immunosuppressive polarization of monocytes, resulting in improvement in T cell function. This finding proceeded in an NLRP3-dependent manner, with substantial co-localization of TIGIT and NLRP3 expression prior to knockdown. These findings point to a novel role for TIGIT expression in diverse myeloid cells in the GBM microenvironment as a marker of immunosuppressive activity and further indicate a hierarchy of immunomodulatory protein activity in these myeloid cells, with TIGIT knockdown unmasking the pro-inflammatory activity of NLRP3. This study bolsters understanding of the immunosuppressive complexities of myeloid cells in the GBM microenvironment, while lending further support to prevention or attenuation of immunosuppressive myeloid cell activity as a means of restoring immune function in GBM. Graphical abstract (Created in BioRender. Asad, M. (2025) https://BioRender.com/euiljoq Key Points Tumor-mediated immune suppression is a key barrier to the development of effective immunotherapies for GBM. TIGIT is broadly expressed in myeloid cells within the GBM microenvironment and can be induced by GBM-derived extracellular vesicles. Knockdown of TIGIT reduces immunosuppressive polarization of monocytes and enhances T cell activity via NLRP3 signaling, implicating TIGIT expression as a targetable modifier of immunosuppressive activity in GBM-associated myeloid cells. Importance of the Study Glioblastoma (GBM) remains a formidable clinical challenge, with poor prognosis and limited response to current immunotherapies. This study uncovers a novel immunosuppressive axis involving TIGIT expression in myeloid cells, which are key players in the GBM tumor microenvironment. By demonstrating GBM-derived extracellular vesicles induce TIGIT in healthy monocytes and TIGIT knockdown diminishes immunosuppressive polarization in an NLRP3-dependent manner, this study highlights TIGIT as both a marker and modulator of immune dysfunction in GBM. These findings introduce a functional hierarchy of immunoregulatory proteins in tumor-associated myeloid cells, positioning TIGIT as a potential checkpoint target. Restoring immune activity by disrupting this axis could enhance the efficacy of immunotherapy in GBM. Thus, this research not only advances our understanding of tumor-induced immune suppression but also opens a promising therapeutic avenue to reinvigorate anti-tumor immunity in a cancer type historically resistant to immunotherapeutic approaches.