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Purpose:Immunotherapy has been demonstrably effective against multiple cancers, yet tumor escape is common. It remains unclear how brain tumors escape immunotherapy and how to overcome this immune escape.Experimental Design:We studied KR158B-luc glioma-bearing mice during treatment with adoptive cellular therapy (ACT) with polyclonal tumor-specific T cells. We tested the immunogenicity of primary and escaped tumors using T-cell restimulation assays. We used flow cytometry and RNA profiling of whole tumors to further define escape mechanisms. To treat immune-escaped tumors, we generated escape variant-specific T cells through the use of escape variant total tumor RNA and administered these cells as ACT. In addition, programmed cell death protein-1 (PD-1) checkpoint blockade was studied in combination with ACT.Results:Escape mechanisms included a shift in immunogenic tumor antigens, downregulation of MHC class I, and upregulation of checkpoint molecules. Polyclonal T cells specific for escape variants displayed greater recognition of escaped tumors than primary tumors. When administered as ACT, these T cells prolonged median survival of escape variant-bearing mice by 60%. The rational combination of ACT with PD-1 blockade prolonged median survival of escape variant glioma-bearing mice by 110% and was dependent upon natural killer cells and T cells.Conclusions:These findings suggest that the immune landscape of brain tumors are markedly different postimmunotherapy yet can still be targeted with immunotherapy.

Glioblastoma (GBM) is an incredibly aggressive and prevalent primary CNS tumor with dismal survival outcomes. GBM’s intra-tumor heterogeneity and lack of anti-tumor immune cell infiltration have proved to be formidable challenges to the development of effective therapies. These hurdles may be overcome by garnering a better understanding of the cell-cell interactions occurring within the tumor microenvironment (TME). Accounting for the complexity and the spatial orientation of the components within the TME may offer insight into the multitiered layers of immunosuppression capitalized on by GBM tumors, thus elucidating the factors responsible for poor response to therapy and uncovering potential therapeutic targets that may offer more favorable therapeutic results for immunotherapies. Current in vitro and in vivo models, however, sorely limit our ability to probe these distinct attributes while preserving the intricate nature of the TME. To bridge this gap, we have devised a novel organotypic brain slice culture (BSC) model using tumor-bearing C57BL/6J mice, allowing us to characterize the tumor milieu in situ. We have identified infiltration of myeloid-derived suppressor cells (MDSC) and microglia polarized to the M2 anti-inflammatory phenotype within the TME. Leveraging this platform, we sought to define further the factors contributing to the dynamic impacts of GBM immunosuppression, such as the tolerogenic response in dendritic cells (DC) upon interaction with the TME. We have demonstrated outcomes similar to those seen in vivo following the introduction of DCs to tumor-bearing BSCs, further supporting the physiological relevancy of this model, and have since worked to unravel elements responsible for this response. Citation Format: Alexandra Reid, Dan Jin, Bayli DiVita-Dean, Laura Falceto-Font, John Figg, Connor Francis, Catherine Flores. Leveraging organotypic brain slice models to enhance adoptive cellular therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4611.

BACKGROUND Our previous studies has developed an adoptive cellular therapy (ACT) against high grade gliomas in both human and murine systems. ACT significantly improves survival in murine models of CNS malignancies. Our studies have shown that efficacy is associated with the observed increase in tumor-associated dendritic cells (DC) which arise from transferred hematopoietic stem cells (HSC). HSC-derived DCs play a pivotal role in mounting the anti-tumor immune response. In this study, we have identified that treatment resistance may be due to glioma-driven dendritic cell dysfunction. We observed significant down regulation of co-stimulatory markers on tumor-associated dendritic cells in mice that escaped ACT and subsequent capacity to activate tumor-reactive T cells.Method: KR158B bearing mice received HSC and tumor reactive T cells one day before 9 Gy irradiation followed by 3 does of BMDC vaccine. Tumor associated DCs were sorted from tumors evaded from ACT and co-cultured with primary KR158B reactive T cells or escaped KR158B reactive T cells. T cell proliferation and interferon gamma were detected for comparison. PCR array was performed to identify dysregulated functional genes.Results and CONCLUSIONS Functional evidence demonstrated that dendritic cells from resistant tumor had significantly decreased capacity in activating tumor-reactive T cells against either primary KR158B glioma cells or T cells generated to target tumor cells that evaded ACT. Gene expression data showed this may be due to significant decreases in genes associated with co-stimulation, T cell engagement, and antigen presentation.

Immunotherapy has been demonstrably effective against various cancers, particularly those in the hematopoietic system and those with a high tumor-specific antigenic burden. Unfortunately, the development of immunotherapeutic strategies has proven more challenging against central nervous system (CNS) malignancies due to several unique characteristics of brain tumors that pose extraordinary barriers. To date, there is a lack of phase III trials demonstrating improved progression-free survival (PFS) and/or overall survival (OS) using immunotherapies in brain cancers. However, a better mechanistic understanding of current resistance to immunotherapies along with data from novel innovative techniques to overcome these barriers has been encouraging. This chapter gives an overview of current immunotherapies in the development of brain cancers. We will evaluate the present studies available in the clinical setting and any of their potential findings. The chapter will also discuss pertinent preclinical strategies whose translation for human use would potentially prove efficacious or provide invaluable scientific discovery.

Purpose: Immunotherapy has been demonstrably effective against multiple cancers, yet tumor escape is common. It remains unclear how brain tumors escape immunotherapy and how to overcome this immune escape. Experimental Design: We studied KR158B-luc glioma-bearing mice during treatment with adoptive cellular therapy (ACT) with polyclonal tumor-specific T cells. We tested the immunogenicity of primary and escaped tumors using T-cell restimulation assays. We used flow cytometry and RNA profiling of whole tumors to further define escape mechanisms. To treat immune-escaped tumors, we generated escape variant-specific T cells through the use of escape variant total tumor RNA and administered these cells as ACT. In addition, programmed cell death protein-1 (PD-1) checkpoint blockade was studied in combination with ACT. Results: Escape mechanisms included a shift in immunogenic tumor antigens, downregulation of MHC class I, and upregulation of checkpoint molecules. Polyclonal T cells specific for escape variants displayed greater recognition of escaped tumors than primary tumors. When administered as ACT, these T cells prolonged median survival of escape variant-bearing mice by 60%. The rational combination of ACT with PD-1 blockade prolonged median survival of escape variant glioma-bearing mice by 110% and was dependent upon natural killer cells and T cells. Conclusions: These findings suggest that the immune landscape of brain tumors are markedly different postimmunotherapy yet can still be targeted with immunotherapy.

BackgroundGlioma-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.MethodsThe 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.ResultsUsing 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.

INTRODUCTION Bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) give rise to the cellular components of the immune system. Unfortunately, immune reconstitution from HSPCs are negatively impacted by solid cancers, including high-grade gliomas. For example, an expansion of myeloid progenitor cells has been previously described across several cancers that originate outside the CNS. A similar expansion of MDSCs coupled with diminished T cell function has also been described in the peripheral blood of patients with newly-diagnosed GBM. Alterations in both lymphoid and myeloid compartments due to CNS malignancy led us to determine how intracranial gliomas impact HSPCs in both their capacity to reconstitute the immune compartment and in their cell fate determination. This is important to better understand the impact of gliomas on immunity and how we can leverage these findings to better develop cellular immunotherapeutics. METHODS HSPCs were isolated from bone marrow of C57BL/6 mice with orthotopic KR158B glioma, or age-matched naïve mice. Experiments were conducted to compare relative changes in: gene expression (RNA-sequencing), precursor frequencies, cell fate determination, and cellular function of cells derived from HSPCs of glioma-bearing mice. RESULTS RNA-sequencing revealed 700+ genes whose expression was significantly up- or downregulated in HSPCs from glioma-bearing mice, particularly those involved with stemness and metabolic activity. Importantly, HSPCs from glioma-bearing mice expressed upregulation of genes involved in myelopoiesis relative to naïve mice. This was coupled with an expansion of granulocyte macrophage precursors (GMPs), the progenitors to gMDSCs. Next, differentiation assays revealed that HSPCs from glioma-bearing mice had higher propensity of differentiating into MDSC under homeostatic conditions relative to controls both in vitro and in vivo. Furthermore, mice bearing intracranial gliomas possess an expansion of MDSCs which are more suppressive on T cell proliferation and hinders T cell-mediated tumor cell killing relative to MDSCs derived from naïve control mice.

Our lab previously published that adoptive hematopoietic stem cell (HSC) therapy administered in conjunction with aPD1 increases median survival in multiple models of CNS malignancies. Tumor-infiltrating lymphocytes (TIL) abundance is independently associated with better outcomes in many cancers. Impressively, approximately 25% of TILs are HSC-derived in early disease. Importantly, we show HSC therapy generates de novo TILs in a thymus-dependent fashion and offer evidence of altered central tolerance following aPD1 therapy, which may permit immune activation against a larger pool of tumor-associated antigens. Therapeutic HSCs were prepared from constitutive GFP-expressing (CD45.2+) and PD1KO (CD45.2+) mice and transferred into CD45.1+ hosts. Sublethal 5Gy total body irradiation was administered day 5 post-intracranial tumor implantation. The next day, 1E6 HSCs from a 1:1 – GFP:PD1KO cocktail were injected intravenously. Descriptive statistics are reported as mean ± SD. Transferred HSCs engraft, expand, differentiate, and migrate to form approximately 25% of the TIL compartment in only 15 days. We found that, 56.2% ± 20.3 of CD3+ TILs display a thymocyte-like CD4+CD8+ (DP) phenotype in early disease, and 43.8% ± 17.4 of these DP-TILs are HSC-derived. Interestingly, DP-TILs were virtually absent in advanced disease 29 days after treatment. At this stage, HSC-derived TILs most commonly fell within the single-positive (SP) TIL subsets, comprising 9.8% ± 5.6 of SP-CD8+ TILs. Notably, HSCPD1KO-derived cells were 4x more abundant amongst SP-CD8 TILs when compared to HSCGFP-derived cells (6.5% vs 1.6%, p < 0.05). SP-CD8 thymocytes were similarly enriched for HSCPD1KO progeny (41.1% vs 24.7%, p < 0.05) which may suggest PD-1 blockade directly promotes thymopoiesis. The privileged maturation of PD1-deficient HSCs is intriguingly reversed when GFP:PD1KO HSCs are co-transferred into non-tumor-bearing mice. Thus, we show the thymus is an active site where PD-1-blocking agents function, and that HSC + aPD1 therapy is uniquely positioned to leverage thymus-dependent anti-tumor immunity.

INTRODUCTION Our group has previously shown that Adoptive Cellular Therapy (ACT) is efficacious against CNS malignancies including medulloblastoma, brain stem glioma, and glioblastoma. In this study, we characterized the spatial distribution of immune cells within the tumor microenvironment after ACT and conducted regional genomic analysis in situ to obtain insights into the underlying cellular dynamics. This method enabled us to define specific differences in gene expression within the T cells in the treated versus untreated tumors including changes in gene expression of regulatory T cell (Treg) associated genes, such as Runx1 and TGF-β. We also found a significant increase in Batf3 expression associated with therapy. METHODS We performed in situ GeoMx Digital Spatial Profiling (DSP) and whole genome transcriptomics (RNA) of murine glioma KR-158B-luciferase tumors after adoptive cellular therapy. ACT was conducted in orthotopic KR158B tumor-bearing mice. Histological slides were sent to be stained for CD45, CD3, GFP (to mark hematopoietic stem cell-derived cells) and nuclei, and processed following GeoMx DSP workflow for RNA. Overall gene expression clustering profiles were conducted using a Principal Component Analysis (PCA), and relative gene expression differences were analyzed by unpaired t-tests. RESULTS/CONCLUSIONS We observed differential expression of a substantial number of genes in the ACT-treated group compared to control. Our findings also indicate that tumors treated with ACT cluster separately from control tumors in PCA analysis. Furthermore, we found downregulation of genes associated with the suppressive properties of regulatory T cells (Runx1 and TGF-β), and upregulation of dendritic cell characteristic genes (Batf3) and antigen presentation genes (H2-Ab1) in ACT-treated tumors vs control. These findings provide insights into the immune cell dynamics and specific genes that are differentially expressed within the tumor microenvironment. This could help improve current adoptive cellular therapies to treat brain tumors.

Our understanding of the relationship between the immune system and cancers has undergone significant discovery recently. Immunotherapy with T cell therapies and checkpoint blockade has meaningfully changed the oncology landscape. While remarkable clinical advances in adaptive immunity are occurring, modulation of innate immunity has proven more difficult. The myeloid compartment, including macrophages, neutrophils, and dendritic cells, has a significant impact on the persistence or elimination of tumors. Myeloid cells, specifically in the tumor microenvironment, have direct contact with tumor tissue and coordinate with tumor-reactive T cells to either stimulate or antagonize cancer immunity. However, the myeloid compartment comprises a broad array of cells in various stages of development. In addition, hematopoietic stem and progenitor cells at various stages of myelopoiesis in distant sites undergo significant modulation by tumors. Understanding how tumors exert their influence on myeloid progenitors is critical to making clinically meaningful improvements in these pathways. Therefore, this review will cover recent developments in our understanding of how solid tumors modulate myelopoiesis to promote the formation of pro-tumor immature myeloid cells. Then, it will cover some of the potential avenues for capitalizing on these mechanisms to generate antitumor immunity.

INTRODUCTION A major obstacle in efficacious therapeutics against high-grade gliomas has been an inability to overcome powerful regulatory mechanisms within the TME that hamper immune activation. Fortunately, immunotherapy has enabled the enhancement of immune activation within the TME through adoptive immunotherapy and checkpoint inhibition. We found that co-transfer of hematopoietic stem cells (HSCs) with either adoptive immunotherapy or PD-1 blockade significantly improves therapeutic outcomes by manipulating the cellular components that make up the TME in high-grade gliomas. HSC co-transfer with immunotherapy leads to increased in situ downregulation of multiple immune regulatory pathways, activation of tumor-reactive T cells, and significant reduction in the frequency of MDCs and TAMs within the TME. METHODS C57BL/6 mice with orthotopic KR158B-gliomas received either adoptive immunotherapy using activated tumor-reactive T cells or αPD-1, either with or without HSC co-transfer. After treatment of late-stage tumors, brains were sectioned and digital spatial profiling (NanoString GeoMx) of the TME was conducted in situ. Briefly, tumor-bearing brain sections were stained for nuclei, CD3, CD45, and GFP (HSC-derived cells); regions of interest (ROIs) containing 200 nuclei were selected and processed for whole genome sequencing. Areas rich in immune cells within the TME were chosen as ROIs and compared between groups. Results were corroborated with flowcytometry and PCR. RESULTS Mice that received HSCs with either adoptive cellular therapy or αPD-1 had reductions in expression of multiple regulatory markers in the TME including iNOS, TGFβ, and PD-L1. This was accompanied by reductions in the frequencies of MDSCs and TAMs. An increased relative abundance of activated CD8+ T cells within the TME was also observed. Interestingly, we found that HSC-derived cells provided rich amounts of dendritic cells at the TME when co-transferred with immunotherapy. Host-derived myeloid cells were significantly displaced from the TME in mice receiving HSC plus adoptive cellular therapy or αPD-1.

Interleukin-1 (IL-1) plays a key role in carcinogenesis and several IL-1-targeted therapeutics are under investigation for the treatment of pancreatic ductal adenocarcinoma (PDAC). We sought to broaden our understanding of how the family of IL-1 ligands and receptors impact the tumor immune landscape and patient survival in PDAC. Gene expression data and DNA methylation data for IL1A, IL1B, IL1RN, IL1R1, IL1R2, and IL1RAP was attained from The Cancer Genome Atlas (TCGA) database and cross validated using the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. Immune cell-type abundance was estimated using CIBERSORTx. Further confirmatory soluble protein analysis and peripheral blood immunophenotyping were performed on available tissue samples from our institution. 169 PDAC patients and 50 benign pancreatic TCGA-based samples were analyzed. IL1A (p 0.001), IL1RN (p 0.001), IL1R2 (p 0.001), and IL1RAP (p = 0.006) were markedly increased in PDAC tumor tissue compared to benign pancreatic tissue. Furthermore, expression of IL1A, IL1B and IL1R1 were positively correlated with gene expression of immune checkpoints PVR, CD274, CD47, CD80, and HLA-A/B/C (p 0.001). IL1B and IL1R1 were correlated to expression of PDCD1, CD86, CTLA4 and IDO1 (0.001). Low expression of IL1RN (p = 0.020), IL1R2 (p = 0.015), and IL1RAP (p = 0.003) and high expression of IL1B (p = 0.031) were correlated with increased patient survival. At the protein level, IL-1β was correlated with increased peripheral central memory CD4+ and CD8+ T-cells as well as decreased Th2 cells. These findings suggest that the IL-1 axis plays a complex and pivotal role in the host immune response to PDAC.

Immune checkpoint blockade using anti-PD-1 monoclonal antibodies has shown considerable promise in the treatment of solid tumors, but brain tumors remain notoriously refractory to treatment. In CNS malignancies that are completely resistant to PD-1 blockade, we found that bone marrow-derived, lineage-negative hematopoietic stem and progenitor cells (HSCs) that express C–C chemokine receptor type 2 (CCR2+) reverses treatment resistance and sensitizes mice to curative immunotherapy. HSC transfer with PD-1 blockade increases T-cell frequency and activation within tumors in preclinical models of glioblastoma and medulloblastoma. CCR2+HSCs preferentially migrate to intracranial brain tumors and differentiate into antigen-presenting cells within the tumor microenvironment and cross-present tumor-derived antigens to CD8+ T cells. HSC transfer also rescues tumor resistance to adoptive cellular therapy in medulloblastoma and glioblastoma. Our studies demonstrate a novel role for CCR2+HSCs in overcoming brain tumor resistance to PD-1 checkpoint blockade and adoptive cellular therapy in multiple invasive brain tumor models., Brain tumors are difficult to treat using existing immunotherapeutic strategies. Here, the authors show that in brain tumors resistant to PD-1 blockade, HSCs expressing CCR2+ can reverse treatment resistance and sensitizes mice to immunotherapy.

BackgroundColon cancer (CRC) is the second leading cause of cancer-related deaths in the US. CRC incidence is on the rise and there is an alarming increase in young onset CRC cases. Immune checkpoint inhibitors (ICIs) have yielded promising anti-tumor results in microsatellite instable high (MSI-high) patients, which represent only 15% of tumors. The remaining 85% are denoted as microsatellite stable (MSS) and are unresponsive to ICI. Using a murine glioma model, our group has previously found the combination of anti-PD-1 and a transfer of hematopoietic stem cells (HSCs) can sensitize mice that are resistant to anti-PD-1 alone. We evaluated survival after treatment with this combinatorial platform 3 or 5 days post-implantation in subcutaneous CRC-bearing mice and also phenotyped the splenic compartment of mice at endpoint.Methods1x106 MSS CRC cells, CT26, were subcutaneously injected into the right flank of BALB/cJ mice. 3 or 5 days later, HSCs were isolated from naïve BALB/cJ mice and injected through the tail vein into CT26-bearing mice and were also given 10 mg/kg anti-PD-1. Mice were given 3 additional doses of anti-PD-1 for a total of doses either every 3 or 5 days. Mice were sacrificed when tumors reached 1.5 cm at its widest point and spleens were excised and stained for flow cytometry.ResultsWhen mice were treated with HSC/anti-PD-1 3 days post-tumor implantation, we observed a statistically significant increase in survival in mice that received combinatorial HSCs and anti-PD-1 relative to no treatment control mice (p=0.0034, Mantel-Cox long-rank test) as well as mice that received HSCs alone (p=0.0462, Mantel-Cox log-rank test. In the same 3 day cohort, no differences in the frequency of T cell populations were observed. However, we found mice that received this combination therapy had a significant increase in the frequency of splenic CD11c+ MHC II+ dendritic cells (DCs) relative to no treatment control mice (p=0.0364, Mann-Whitney t test). When mice were treated with HSC/anti-PD-1 5 days post-tumor implantation, we found a statistically significant increase in survival of mice treated with combinatorial HSCs and anti-PD-1 compared to no treatment control mice (p=0.0024, Mantel-Cox log-rank test) and relative to mice that received HSC monotherapy (p=0.0462, Mantel-Cox log-rank test).ConclusionsThese results suggest combinatorial HSCs and anti-PD-1 represents a promising therapeutic axis in a murine model of MSS CRC. In addition, the increase in splenic DCs suggests the mechanism behind this anti-tumor response may be expansion of DCs within the periphery.Ethics ApprovalAll animal work approved through University of Florida IACUC # 201910777

BackgroundThe average overall survival for patients with brain tumors is only 8 to 12 months. For example, the 5-year survival rate for adults over 40 years old living with Glioblastoma multiforme, a type of brain tumor, is only 6%. The brain is also a common organ for metastasis deriving from tumors such as breast cancer. The field of cancer immunotherapy has been making efforts to develop strategies that can target these brain metastases more efficiently than the standard of care in the clinic. Immune checkpoint inhibitors such as anti-PD-1 (anti-programmed cell death-1) therapy are under investigation to treat brain metastases. However, several studies have shown brain metastases to be resistant to anti-PD-1 monotherapy. Our findings have shown that a hematopoietic stem cell (HSC) transfer can overcome this resistance in brain metastasis. Our goal is to elucidate and overcome the mechanism of resistance to anti-PD-1 therapy in brain metastasis.MethodsWe performed both orthotopic mammary fat pad and intracranial (IC) tumor implantations in C57/BL6J mice using a murine breast cancer cell line (E0771). Three days after tumor implantation, we administered the first of four doses of anti-PD-1 therapy delivered five days apart from each other. We measured survival and chemokine (from blood samples) differences between treatments.ResultsWe have observed that orthotopic mammary fat pad tumors are responsive to anti-PD-1 alone. Interestingly, when tumors derived from the same breast cancer cell line are implanted into the brain, they become non-responsive to anti-PD-1 monotherapy. We have also discovered that performing a hematopoietic stem cell transfer overcomes resistance to anti-PD-1 therapy in brain metastases (figure 1). Furthermore, in our preliminary studies we have found that both CD8+ and CD4+ T cells are required for the protective effects of anti-PD-1 against E0771 tumors.Abstract 243 Figure 1A hematopoietic stem cell transfer overcomes resistance to anti-PD-1 in brain metastasisConclusionsBrain metastasis are resistant to anti-PD-1 monotherapy. We have found that a hematopoietic stem cell transfer overcomes resistance to anti-PD-1 in brain metastasis. We hypothesize that, while peripheral T cells successfully remove breast tumors, they fail to mount anti-tumor responses in breast metastatic brain tumors. Therefore, the next step is to understand the mechanism of resistance to anti-PD-1 monotherapy in brain metastasis and to further elucidate the type of T cell immune responses required to overcome this resistance in brain metastasis.Ethics ApprovalIACUC Protocol #201910777

Glioblastoma (GBM) remains a deadly disease with an overall survival of 18 months. Despite advances in cancer immunotherapy, especially in the context of solid tumors derived outside of the central nervous system, GBM remains difficult to treat. This may be due, in part, to reduced T cell function and expansion of suppressive myeloid cells within the periphery. Here we sought to determine if GBM impacts the phenotype and gene expression of progenitor populations within the bone marrow where hematopoietic stem and progenitor cells (HSPCs) originate. Using RNAseq, we found HSPCs derived from intracranial glioma-bearing mice possess altered gene expression relative to HSPCs derived from non-tumor-bearing mice. In addition, we found glioma-bearing mice possess an expansion of myeloid-derived suppressor cells (MDSCs) within their bone marrow and are significantly more suppressive on T cell proliferation and T cell-mediated tumor cell killing than MDSCs isolated from non-tumor-bearing mice. We also determined HSPCs derived from glioma-bearing mice are more likely to differentiate into MDSCs than HSPCs derived from non-tumor-bearing mice. Interestingly, we found components of immunotherapy are capable of redirecting cell fate differentiation of glioma-bearing HSPCs. We determined HSPCs cultured in T cell supernatants are capable redirecting their differentiation from suppressive MDSCs towards stimulatory DCs. Finally, using two murine glioma models, we found adoptive cellular therapy using HSPCs derived from glioma-bearing mice is capable of providing a similar survival benefit as adoptive cellular therapy using HSPCs derived from non-tumor-bearing mice.

INTRODUCTION: The T cell repertoire of brain tumor bearing hosts has been previously described to be skewed to have increased regulatory phenotype relative to healthy hosts (Woroniecka et al 2018). We have found that hematopoietic stem cells (HSCs) isolated from tumor bearing mice are inefficient at engraftment and reconstitution of the hematopoietic compartment, giving rise to less live cells than HSCs derived from healthy controls. We found that CD4+ splenocytes and CD8+ bone marrow-derived cells and splenocytes that arise from HSCs of tumor bearing hosts are polarized towards a terminal memory phenotype relative to HSCs derived from healthy hosts. We believe that this dysregulation in T cell reconstitution is a major player in mounting immune responses against CNS malignancies. METHODS: Lineage negative HSCs were isolated from GFP transgenic healthy donors or DsRed transgenic tumor-bearing mice and adoptively transferred into C57BL/6 recipient cohort of lethally irradiated mice. HSCs are derived from DsRed or GFP transgenic mice to allow for tracking of HSC-derived populations. One month after transfer, mice are sacrificed and spleen, bone marrow, and blood are harvested and stained for flow cytometry. RESULTS & CONCLUSIONS: T cells derived from TB HSCs have a distinct phenotype compared to T cells from healthy HSCs, demonstrating intracranial brain tumors likely have an impact on HSC differentiation outcomes.

INTRODUCTION: Hematopoietic stem and progenitor cell (HSPC) transfer during adoptive T cell immunotherapy (ACT) prolongs median survival and generates 30% long-term cures for malignant brain tumors. We recently demonstrated during ACT that intratumoral HSPCs differentiate into immune-activating dendritic cells (DCs) through T cell-released IFN-γ. We also determined that CCR2+ HSPCs are the key immune-activating progenitor population. We previously utilized syngeneic transfer of naive HSPCs that were not endemic to the recipient tumor-bearing host. This is different from the clinical paradigm of autologous transfers. This is important because in peripheral cancers tumor-bearing host (TB) HSPCs possess considerable immunosuppressive potential. It remains unknown if this is true in brain tumor-bearing hosts, and it remains unknown how this would impact HSPC+ACT immunotherapy. We therefore evaluated the immunologic function of naïve or TB HSPCs during immunotherapy. METHODS: We utilized KR158B and GL261 intracranial gliomas to generate TB HSPCs. HSPC culture experiments were performed in RPMI alone or conditioned with T cell supernatants to study HSPC differentiation. In vivo treatment models utilized intracranial gliomas and intravenous transfer of HSPCs with ACT. Data from the immgen.org database was utilized for hypothesis generation. RESULTS: Brain tumors promoted a 20% expansion of HSPCs including granulocyte monocyte precursors (GMP) but a loss of DC progenitors. The immgen database revealed that GMPs express high levels of IFN-γR1 while DC progenitors express high levels of IFN-γR2, CCR2, and CXCR4, the chemotactic receptor for HPSC homing. We next determined by flow cytometry that TB HSPCs express 40% more IFN-γR1 and 90% more IFN-γR2 on DC progenitors. To determine the differentiation preference of these cells, we performed in vitro culture. After a 3 day culture, naïve HSPCs differentiated into 30% myeloid-derived suppressor cells (MDSCs; CD11b+Ly-6G/6C+) while TB HSPCs differentiated into 60% MDSCs. When both cells types were cultured in activated tumor-specific T cell supernatants containing IFN-γ, both HSPC types differentiated into 30% MDSCs, 80% MHCII+ antigen-presenting cells, and 20% CD11c+MHCII+ DCs. In vivo, ACT rescued intratumoral TB HSPCs to prolong median survival and generate long-term cures. Additionally, TB HSPC-derived cells in brain tumors maintained higher IFN-γR2 and displayed non-inferior differentiation into DCs when compared naïve HSPCs. We are now investigating the impact of PD-1 blockade on TB HSPC function. CONCLUSIONS: Gliomas exert an immunosuppressive pressure on HSPCs. However, ACT can overcome dysregulated TB HSPC programming and promote generation of DCs instead of MDSCs. A phase I trial evaluating the impact of HSPC transfer on adoptive immunotherapy in pediatric high-grade gliomas is underway at our center (ACTION; NCT03334305). Citation Format: Tyler J. Wildes, Catherine T. Flores, Bayli DiVita Dean, Adam Grippin, Kyle Dyson, Duane A. Mitchell. Adoptive cellular therapy overcomes tumor-induced dysregulation of myelopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1194.