Immunotherapy reverses glioma-driven dysfunction of immune system homeostasis

Abstract

Background: Glioma-induced immune dysregulation of the hematopoietic system has been described in a limited number of studies. In this study, our group further demonstrates that gliomas interrupt the cellular differentiation programming and outcomes of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. HSPCs from glioma-bearing mice are reprogrammed and driven towards expansion of myeloid lineage precursors and myeloid-derived suppressor cells (MDSCs) in secondary lymphoid organs. However, we found this expansion is reversed by immunotherapy. Adoptive cellular therapy (ACT) has been demonstrably efficacious in multiple preclinical models of central nervous system (CNS) malignancies, and here we describe how glioma-induced dysfunction is reversed by this immunotherapeutic platform.

Methods: The impact of orthotopic KR158B-luc glioma on HSPCs was evaluated in an unbiased fashion using single cell RNAseq (scRNAseq) of lineage^- cells and phenotypically using flow cytometry. Mature myeloid cell frequencies and function were also evaluated using flow cytometry. Finally, ACT containing total body irradiation, tumor RNA-pulsed dendritic cells, tumor-reactive T cells and HSPCs isolated from glioma-bearing or non-tumor-bearing mice were used to evaluate cell fate differentiation and survival.

Results: Using scRNAseq, we observed an altered HSPC landscape in glioma-bearing versus non-tumor-bearing mice . In addition, an expansion of myeloid lineage subsets, including granulocyte macrophage precursors (GMPs) and MDSCs, were observed in glioma-bearing mice relative to non-tumor-bearing controls. Furthermore, MDSCs from glioma-bearing mice demonstrated increased suppressive capacity toward tumor-specific T cells as compared with MDSCs from non-tumor-bearing hosts. Interestingly, treatment with ACT overcame these suppressive properties. When HSPCs from glioma-bearing mice were transferred in the context of ACT, we observed significant survival benefit and long-term cures in orthotopic glioma models compared with mice treated with ACT using non-glioma-bearing HSPCs.

Keywords: dendritic cells; immunotherapy; myeloid-derived suppressor cells.

Figure 1

Differential gene expression of lineage-negative HSPCs from non-tumor-bearing and intracranial glioma-bearing mice via scRNAseq. scRNAseq analysis of HSPCs from non-tumor-bearing (n=4) and glioma-bearing (n=4) mice was performed. (A) UMAP dimensionality reduction and cluster visualization and (B) pie chart frequency depiction color coded to represent cluster ID. Clusters manually annotated based on pathway analysis of cluster marker genes. (C) Psuedotime visualization. HSPCs, hematopoietic stem and progenitor cells.

Figure 2

Expansion of immature and mature myeloid cells in bone marrow of intracranial glioma-bearing mice. (A–D) Bone marrow was collected from non-tumor-bearing, age-matched mice and glioma-bearing mice 28 days after implantation. Glioma-bearing mice have higher absolute counts of lin⁻cKit⁺Sca-1⁻CD16/32^hi GMPs relative to non-tumor-bearing, age-matched mice, while the frequencies of lin⁻cKit⁺Sca-1⁻CD16/32^lo CMPs, lin⁻cKit⁺M-CSFR⁺Flt3⁺ CDPs, and lin⁻cKit⁺Sca-1⁺M-CSFR⁺Flt3⁺ MDPs were similar between groups. Data represent mean±SD. *P<0.05 by Mann-Whitney t-test (n=5 biological replicates). (E–G) Bone marrow was collected from non-tumor-bearing, age-matched mice and glioma-bearing mice 28 days after implantation. Glioma-bearing mice have higher frequencies of CD11b+F4/80+ macrophages and CD11b+Gr-1+ MDSCs but possess similar frequencies of CD11c+MHC II+ DCs as non-tumor-bearing mice. (H) CD11b+Ly6G+ gMDSCs are expanded in glioma-bearing mice, while no difference in CD11b+Ly6C+ mMDSCs are observed. Data represent mean±SD. * **p<0.01, by Mann-Whitney t-test (n=5 biological replicates). I) % CD11b and Gr-1 expression on HSPC-derived cells after in vitro culture. ***p<0.001 by unpaired tudent’s t test (n=3 technical replicates). CDPs, common dendritic cell precursors; CMPs, common myeloid precursors; HSPCs, hematopoietic stem and progenitor cells; MDSCs, myeloid-derived suppressor cells.

Figure 3

MDSCs isolated from glioma-bearing mice possess greater suppressive capacity on T cell proliferation but similar T cell mediated tumor cell killing relative to MDSCs from non-tumor-bearing mice. (A–C) The 6.25×10³ bone marrow MDSCs were isolated from non-tumor-bearing (ntMDSC) and glioma-bearing mice (gMDSCs) and cocultured with 2×10⁵ T cells derived from mice primed with KR158B-luc RNA-pulsed DCs. T cells were gated on using CD3+ cells. When cocultured with non-tumor-bearing MDSCs, T cells possessed higher division indices than T cells cocultured with glioma-bearing MDSCs. (D and E) Splenocytes were cocultured with DCs electroporated with KR158B-luc RNA to expand tumor-specific T cells. On expansion, T cells were cultured at a 1:10 ratio with target KR158B-luc glioma cells and a 1:1 ration with ntMDSCs or gMDSCs for 24 hours. Similar frequencies of Annexin+Live/dead- and Annexin+Live/dead+ tumor cells were observed s when cocultured with glioma-bearing MDSCs relative to co-culture with non-tumor-bearing MDSCs. Cells were gated on CD45− tumor cells. Data represent mean±SD. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way analysis of variance (n=3 technical replicates). DCs, dendritic cells; MDSCs, myeloid-derived suppressor cells.

Figure 4

Components of ACT platform are capable of redirecting HSPC differentiation outcomes. (A and B) Expression of CD11b+ Gr-1+ cells after 3 days of in vitro culture in RPMI with 10% FBS either alone or conditioned with supernatants of activated T cells. Conditioned media was a 1:1 mix of 1 mL RPMI with 10% FBS and 1 mL of acellular supernatants generated from activated T cell cultures with cognate antigen-pulsed DCs (Wildes et al 201812). (C and D) Expression of CD11c+MHC II+ cells on HSPC-derived cells after in vitro culture. (E) HSPCs derived from C57BL/6 or IFNyR^−/− mice were cultured in RPMI with 10% FBS alone or supplemented with recombinant mouse IFNy and differentiation of CD11c+MHC II+ cells was evaluated 3 days later via flow cytometry.(F) scRNAseq expression of IFNyR1 from non-tumor-bearing and glioma-bearing HSPCs. (G) Visualization of IFNyR1 expression overlayed on UMAP projections. (H and I) HSPCs from non-tumor-bearing or glioma-bearing mice were harvested 28 days after implantation and flow cytometry was used to evaluate expression of IFNyR1 and IFNyR2 on lineage− cells. All data represent the mean±SD. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way analysis of variance (ANOVA) (n=3 technical replicates) for in vitro studies and Mann-Whitney t-test (n=10 biological replicates) for ex vivo studies. ACT, adoptive cellular therapy; HSPC, hematopoietic stem and progenitor cell.

Figure 5

Irradiation abrogates myeloid cell precursor and mature myeloid cell expansion and impacts IFNγR1 and IFNγR2 expression. (A–C) Mice were injected with KR158B-luc cells and 21 days later, half of the mice were subjected to 5 Gy total body irradiation. Seven days later, bone marrow was obtained from both groups and frequencies of IFNyR1+ and IFNyR2+ cells were evaluated on lineage− HSPCs. Mice subjected to total body irradiation were found to possess more IFNyR1+ and IFNyR2+ lineage- cells via flow cytometry. (D–F) A cohort of non-tumor-bearing mice were injected with KR158B-luc cells while a separate cohort remained non-tumor-bearing. Twenty-eight days after implantation, both cohorts were sacrificed and lineage− HSPCs were FACS sorted and then either non-tumor-bearing or glioma-bearing HSPCs were transferred into C57BL/6 that were subjected to 9 Gy total body irradiation the day prior. Twenty-eight days after HSPC transfer, bone marrow was collected and stained for flow cytometry. No significant differences in the frequencies of lin⁻cKit⁺Sca-1⁻CD16/32^hi GMPs and CD11b+Gr-1+ MDSCs between mice that received non-tumor-bearing HSPCs and those that received glioma-bearing HSPCs. All data represent the mean±SD. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Mann-Whitney t-test (n=5 biological replicates). HSPCs, hematopoietic stem and progenitor cells.

Figure 6

ACT abrogates GMP expansion and in context of glioma-bearing HSPCs, provides superior survival benefit relative to non-tumor-bearing HSPCs. (A) Glioma-bearing mice were treated with components ACT, and 21 days later, GMP frequency was evaluated via flow cytometry. (B) Mice treated with TBI, HSPCs, tumor RNA-pulsed DCs, and tumor-specific T cells were found to have significantly less GMPs compared with mice treated with TBI and HSPCs as well as mice treated with TBI, HSPCs, and tumor-specific T cells. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Mann-Whitney t-test (n=5 biological replicates). (C) After glioma-bearing mice were treated with our ACT platform using HSPCs from either non-tumor-bearing mice or glioma-bearing mice, survival was evaluated. (D). Survival curve of KR158B-luc-bearing animals treated with 5 Gy, ACT, and non-tumor-bearing or glioma-bearing HSPCs. (E). Survival curve of GL261-bearing animals treated with 5 Gy, ACT, and non-tumor-bearing or glioma-bearing-HSPCs. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Mantel-Cox log-rank test for survival experiments (n=7 biological replicates). ACT, adoptive cellular therapy; DCs, dendritic cells; GMP, granulocyte macrophage precursor; HSPCs, hematopoietic stem and progenitor cells; TBI, total body irradiation.