TIGIT expression dictates the immunosuppressive reprogramming of myeloid cells in glioblastoma

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.

Keywords: NLRP3; TIGIT; extracellular vesicles; glioblastoma; immunotherapy; myeloid cell.

Figure 1:. Expression of immune modulatory molecules by suppressive monocytes of GBM patients.

For surveying the tumor-immune microenvironment in samples of ultrasonic aspirates collected during surgery, samples were processed for flow cytometric studies and the single cell suspensions, stained with various flow antibodies were run on Cytek aurora spectral flow cytometer. (A) UMAP analysis of CD45+ cells from GBM patients show various infiltrating immune cells. (B) Showing the cellular expression of immune modulatory molecules (TIGIT, PDL1, NLRP3, BTLA, CTLA4, ARG1) by infiltrating immune suppressive monocyte subsets, monocytic Myeloid Derived Suppressor Cells, mMDSCs (CD45⁺CD11b⁺CD15⁻HLA-DR^LoCD14 cells) and nonclassical monocytes (NCM, CD45⁺CD11b⁺CD15⁻HLA-DR⁺CD14⁻PD1⁺CD16 cells). (C) Histograms and a corresponding summary bar graph depicting the immune modulatory molecules expressing cell population in mMDSCs and NCM. (D) Box plot obtained from TCGA database illustrates the expression of TIGIT in normal and primary tumor samples obtained from GBM patients. (E) Kaplan-Meier curve of TCGA and GTEx datasets of human GBM, demonstrating the effect of TIGIT expression on patient survival in GBM. Data are presented as mean ± s.e.m. and were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons.

Figure 2:. Isolation and characterization of tumor-derived extracellular vesicles (TEVs) and induction of immune suppressive monocytes.

Extra cellular vesicles from human GBM primary cells lines were obtained by ultracentrifugation and characterized by Nano-sight and western blots. (A) Size distribution analysis of tumor-derived extracellular vesicles (TEVs) isolated from conditioned medium post ultracentrifugation. (B) Representative image depicting the shape and relative size of the heterogeneous TEVs, obtained from Nano-sight. (C) Western blot analysis of TEVs and whole cell lysate (WCL) for the presence of CD63, and CD9 as TEVs markers. (D) CD11b⁺ cells, isolated from healthy PBMCs and induced with control or different TEVs isolated from primary GBM cell lines (dBT114, dBT116 and dBT120) under hypoxic condition in serum free medium. After 72 hours, cells were collected and processed for flow cytometry and stained with various fluorochorm tagged antibodies. Flow cytometric analysis of monocytic Myeloid Derived Suppressor Cells (mMDSCs, CD45⁺CD11b⁺CD15⁻HLA-DR^LoCD14 cells and nonclassical monocytes (NCM, CD45⁺CD11b⁺CD15⁻HLA-DR⁺CD14⁻PD1⁺CD16 cells) isolated from healthy PBMCs and induced with control or different TEVs isolated from primary GBM cell lines. Data are presented as mean ± s.e.m. and were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons.

Figure 3:. Expression of immune modulatory molecules on TEVs induced peripheral monocytic Myeloid Derived Suppressor Cells (mMDSC) & non-classical monocytes (NCM).

CD11b⁺ cells, isolated from healthy peripheral blood and under hypoxic condition in a serum free medium, co-cultured for 72 hours with TEVs obtained from mentioned GBM cell lines. (A) TEVs induced CD11b⁺ monocytes were studied for the expression of various immune modulatory markers, TIGIT, PDL1, NLRP3, BTLA, CTLA4 & ARG1 on suppressive monocyte subsets (mMDSCs and NCMs). (B) Histograms depicting the expression levels of immune modulatory molecules on mMDSC and NCM populations cultured with control or different TEVs (dBT114, dBT116 and dBT120). The percentage of cells expressing each molecule within each subset is also shown correspondingly as a chart. (C) Immunofluorescence staining of TIGIT in suppressive monocytes treated with control or TEVs. (D) Western blot analysis of GBM whole cell lysate (WCL) for the presence of TIGIT. Data are presented as mean ± s.e.m. and were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons.

Figure 4:. Immune response by TIGIT⁺ suppressive monocytes.

Isolated peripheral blood CD11b⁺ monocytes were induced with TEVs in hypoxia chamber in serum free medium. After 72 hours of induction, cells were processed for flow cytometric studies. Single viable CD45⁺TIGIT cells were selected for the analysis. (A) UMAP analysis of mMDSCs and NCM treated with control or different TEV preparations (dBT114, dBT116, dBT120), highlighting the co-expression of various immune modulatory molecules (VISTA, PDL1, NLRP3, CTLA4, BTLA and ARG1) within TIGIT⁺ cell population. (B) Dotplot depicting the percentage of cells as a factor of size of the dot expressing different immune modulatory molecules within the TIGIT pockets of mMDSCs and NCMs treated with control or different EV preparations. Mean fluorescence intensity (MFI) shown corresponding to the color intensity of the dots for each analyte. (C) UMAP analysis of mMDSCs and NCM treated with control or different TEVs, highlighting the expression of various cytokines (IL17, IL13, IL10, IL5, IL4, IL18, IFNγ). (D) Dotplot depicting the percentage and MFI of TIGIT⁺ cells co-expressing different cytokines in mMDSCs and NCM treated with control or different EV preparations.

Figure 5.. Immune modulatory molecules expressing Immune suppressive monocytes post TIGIT knockdown.

Isolated CD11b⁺ Monocytic cells from healthy peripheral blood were knockdown by scramble or NLRP3-specific or TIGIT-specific siRNA and processed for flow cytometry post TEVs treatment for 72 hours under hypoxia in serum free condition. (A and B) Uniform Manifold Approximation and Projection (UMAP) plots showing the expression patterns of immune checkpoint molecules (Arg1, B7H3, BTLA, CTLA4, PD-L1, NLRP3, TIGIT, and VISTA) across different clusters by mMDSC population post TIGIT specific siRNA- (b) or scramble siRNA- (a) treated cells. Red color represents high expression, whereas gray indicates the total cellular population of mMDSCs. Violin plots depicting the percentage of mMDSCs expressing various immune modulatory molecules in TIGIT siRNA-treated and scramble siRNA-treated peripheral monocytes. (C and D) UMAP showing the expression of the same molecules by non-classical monocytes (NCM), after TIGIT siRNA-treated (d) and scramble siRNA-treated (c) peripheral monocytes. Violin plots depicting the percentage of NCMs expressing various immune modulatory molecules in TIGIT siRNA-treated and scramble siRNA-treated cells. Data are presented as mean ± s.e.m. and were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons.

Figure 6.. Immune modulatory milieu by immune suppressive monocytes post-TIGIT knockdown.

CD11b⁺ cells isolated from healthy peripheral blood mononuclear cells (PBMCs) were knockdown by TIGIT-specific or scramble siRNA under hypoxic condition in serum free medium. These healthy monocytes were co-cultured with TEVs for 72 hours and processed for flow cytometry. (A and B) UMAP plots showing the cellular distribution of mMDSCs based on their expression of various cytokines (IL17, IL13, IL10, IL5, IL4, IL1β, and IFNγ) producing cells in scramble-treated (a) and TIGIT-knockdown monocytes (b). Red color represents high expression, whereas gray indicates the total cellular population of mMDSC. Violin plots comparing the percentage of mMDSCs expressing these cytokines between the scramble-treated and TIGIT-knockdown groups. (C and D) UMAP showing the expression of the same cytokines producing cells, (IL17, IL13, IL10, IL5, IL4, IL1β, and IFNγ) among non-classical monocytes (NCM), in scramble-treated and TIGIT-knockdown groups. Violin plots comparing the percentage of NCMs expressing various given cytokines between the scramble-treated and TIGIT-knockdown groups. Data are presented as mean ± s.e.m. and were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons.

Figure 7.. Effect of TIGIT knockdown on T Cell Proliferation in the Presence of TEV-Induced MDSCs.

CD11b+ and CD3+ cells were isolated from healthy volunteer peripheral blood monocytes. CD11b cells were conditioned with TIGIT and NLRP3-specific siRNA in serum-free medium followed by priming with TEVs for under hypoxic conditions (1%) for 72 hours. Then, CD11b and T cells (T cells stained with cell trace yellow dye) were co-cultured for another 72 hours in RPMI-1640 medium with 10% serum at 37°C in 5% CO2. Finally, cells were processed for flow cytometry. (A) Representative flow cytometry plots and corresponding bar graphs showing CD3⁺ T cell proliferation (Cell Trace Yellow) in the absence of T cell activator, CD3/CD28 Dynbeads (inactive group), when T cells were cultured without conditioned monocytes Only T cell group), scramble treated group, TIGIT specific siRNA treated group (TIGIT), NLRP3 specific siRNA treated group (NLRP3) and both TIGIT along with NLRP3 siRNA treated group (T&N). (B) Representative flow cytometry plots showing IFNγ and IL-4 producing CD4+ T cells in CD3⁺ T cells and CD11b⁺ monocytes co-culture conditions, post scramble, TIGIT and/or NLRP3 knockdown. (C) IFNγ producing CD4+ T cells in the above-mentioned co-culture conditions in the given treatment groups. (D) IL-4 producing CD4+ T cells in CD3⁺ T cells and CD11b⁺ monocytes co-culture conditions, post scramble, TIGIT and/or NLRP3 knockdown. (E) Ratio of IFNγ producing CD4⁺ T cells and IL-4 producing CD4⁺ T cells in the given co-culture conditions. Data are presented as mean ± s.e.m. and were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons.