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Table S1 and S2

Purpose:The success of checkpoint blockade against glioblastoma (GBM) has been disappointing. Anti–PD-1 strategies may be hampered by severe T-cell exhaustion. We sought to develop a strategy that might license new efficacy for checkpoint blockade in GBM.Experimental Design:We characterized 4-1BB expression in tumor-infiltrating lymphocytes (TIL) from human GBM. We implanted murine tumor models including glioma (CT2A), melanoma (B16), breast (E0771), and lung carcinomas intracranially and subcutaneously, characterized 4-1BB expression, and tested checkpoint blockade strategies in vivo.Results:Our data reveal that 4-1BB is frequently present on nonexhausted CD8+ TILs in human and murine GBM. In murine gliomas, 4-1BB agonism and PD-1 blockade demonstrate a synergistic survival benefit in a CD8+ T-cell–dependent manner. The combination decreases TIL exhaustion and improves TIL functionality. This strategy proves most successful against intracranial CT2A gliomas. Efficacy in all instances correlates with the levels of 4-1BB expression on CD8+ TILs, rather than with histology or with intracranial versus subcutaneous tumor location. Proffering 4-1BB expression to T cells licenses combination 4-1BB agonism and PD-1 blockade in models where TIL 4-1BB levels had previously been low and the treatment ineffective.Conclusions:Although poor T-cell activation and severe T-cell exhaustion appear to be limiting factors for checkpoint blockade in GBM, 4-1BB agonism obviates these limitations and produces long-term survival when combined with anti–PD-1 therapy. Furthermore, this combination therapy is limited by TIL 4-1BB expression, but not by the intracranial compartment, and therefore may be particularly well-suited to GBM.

The accepted paradigm for both cellular and antitumor immunity relies upon tumor cell kill by CD8+T cells recognizing cognate antigens presented in the context of target cell major histocompatibility complex class I (MHC I) molecules. Likewise, a classically described mechanism of tumor immune escape is tumor MHC-I downregulation. Here, we report that CD8+T cells maintain the capacity to kill tumor cells that are entirely devoid of MHC-I expression. This capacity proves to be dependent on interactions between T cell NKG2D and tumor NKG2D ligands (NKG2DL). Necessarily, tumor cell kill in these instances is antigen-independent, although prior T cell antigen-specific activation is required and can be furnished by myeloid cells or even neighboring MHC-replete tumors cells. These mechanisms are activein vivoin mice, as well asin vitroin human tumor systems, and are obviated by NKG2D knockout or blockade. Tumor cell killing following T cell NKG2D engagement is Fas-independent and appears to involve granzyme. These studies potentially obviate the long-advanced notion that downregulation of MHC-I is a viable means of tumor immune escape, and instead identify the NKG2D/NKG2DL axis as a novel therapeutic target for enhancing T cell-dependent anti-tumor immunity against MHC loss variants.

Background The nervous and immune systems interact in a reciprocal manner, both under physiologic and pathologic conditions. Literature spanning various CNS pathologies including brain tumors, stroke, traumatic brain injury and de-myelinating diseases describes a number of associated systemic immunologic changes, particularly in the T-cell compartment. These immunologic changes include severe T-cell lymphopenia, lymphoid organ contraction, and T-cell sequestration within the bone marrow. Methods We performed an in-depth systematic review of the literature and discussed pathologies that involve brain insults and systemic immune derangements. Conclusions In this review, we propose that the same immunologic changes hereafter termed ‘systemic immune derangements’, are present across CNS pathologies and may represent a novel, systemic mechanism of immune privilege for the CNS. We further demonstrate that systemic immune derangements are transient when associated with isolated insults such as stroke and TBI but persist in the setting of chronic CNS insults such as brain tumors. Systemic immune derangements have vast implications for informed treatment modalities and outcomes of various neurologic pathologies.

In chronic infections and in cancer, persistent antigen stimulation under suboptimal conditions can lead to the induction of T-cell exhaustion. Exhausted T cells are characterized by an increased expression of inhibitory markers and a progressive and hierarchical loss of function. Although cancer-induced exhaustion in CD8 T cells has been well-characterized and identified as a therapeutic target (i.e., via checkpoint inhibition), in-depth analyses of exhaustion in other immune cell types, including CD4 T cells, is wanting. While perhaps attributable to the contextual discovery of exhaustion amidst chronic viral infection, the lack of thorough inquiry into CD4 T-cell exhaustion is particularly surprising given their important role in orchestrating immune responses through T-helper and direct cytotoxic functions. Current work suggests that CD4 T-cell exhaustion may indeed be prevalent, and as CD4 T cells have been implicated in various disease pathologies, such exhaustion is likely to be clinically relevant. Defining phenotypic exhaustion in the various CD4 T-cell subsets and how it influences immune responses and disease severity will be crucial to understanding collective immune dysfunction in a variety of pathologies. In this review, we will discuss mechanistic and clinical evidence for CD4 T-cell exhaustion in cancer. Further insight into the derivation and manifestation of exhaustive processes in CD4 T cells could reveal novel therapeutic targets to abrogate CD4 T-cell exhaustion in cancer and induce a robust antitumor immune response.

Glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Despite extensive medical intervention, the median survival remains a bleak 15–17 months from time of diagnosis, calling for new therapeutic strategies. In recent years, immune checkpoint blockade (ICB) has garnered attention for its clinical successes in extending overall survival in many advanced-stage cancers. To date, ICB clinical trials have failed to proffer the same benefit in GBM patients, turning attention in the field toward understanding this shortcoming and employing new strategies. Genomic characteristics such as mutational burden, immunogenicity, and heterogeneity, along with immune infiltrate, CNS location, and immunosuppression (tumor-induced or iatrogenic), have been identified as critical components of ICB responsiveness. Promising results from recent clinical trials have rewarded efforts to understand the features of successful ICB and to restore enthusiasm for ICB as an encouraging therapeutic in the treatment of GBM.

BACKGROUND Immunotherapy has demonstrated efficacy in several cancers but has shown only modest effects in Glioblastoma (GBM). This is linked to the anti-inflammatory nature of the tumor microenvironment (TME) and the pro-tumor functions of brain native cells. Targeting stromal cells, such as tumor associated macrophages (TAMs) and neurons, is a promising approach. Re-analysis of human and murine brain single cell-RNAseq (scRNAseq) datasets shows Calmodulin Dependent Kinase Kinase 2 (CaMKK2) is highly expressed in both neurons and TAMs. Loss of CaMKK2 polarizes TAMs to an immunostimulatory phenotype and reduces pro-tumor neuronal functions. Thus, we hypothesize that CaMKK2 promotes the pro-tumor nature of the GBM TME and immunotherapy resistance. RESULTS Murine GBM was orthotopically implanted into wild-type and CaMKK2-/- mice. CaMKK2-/- mice exhibited significantly prolonged survival. To determine if anti-tumor immune function was enhanced, we probed the TME using flow cytometry and scRNAseq. CaMKK2-/- mice showed increased abundance of precursor exhausted, potentially immune checkpoint blockade (ICB) responsive, CD8 T cells. Furthermore, T cell depletion abrogated the survival benefit observed in CaMKK2-/- mice. Considering these T cell phenotypes, we treated CaMKK2-/- mice with ICB, and indeed they were sensitive. To determine if the CaMKK2-/- survival phenotype and ICB response depended on CaMKK2 expression in hematopoietic or in non-hematopoietic cells, we utilized a reciprocal chimera model. Loss of CaMKK2 in the non-hematopoietic cells was more vital for survival and ICB response than in hematopoietic cells, suggesting a potential novel and unique role for CaMKK2 in brain native cells - potentially neurons - in coordinating ICB resistance. CONCLUSIONS We find that CaMKK2 exacerbates mortality and drives ICB resistance by limiting the anti-tumor response in GBM via both hematopoietic and brain native cells. Our findings identify a novel therapeutic target for GBM, and a unique role for CaMKK2 in the TME.

Successful cancer immunotherapies rely on a replete and functional immune compartment. Within the immune compartment, T cells are often the effector arm of immune-based strategies due to their potent cytotoxic capabilities. However, many tumors have evolved a variety of mechanisms to evade T cell-mediated killing. Thus, while many T cell-based immunotherapies, such as immune checkpoint inhibition (ICI) and chimeric antigen receptor (CAR) T cells, have achieved considerable success in some solid cancers and hematological malignancies, these therapies often fail in solid tumors due to tumor-imposed T cell dysfunctions. These dysfunctional mechanisms broadly include reduced T cell access into and identification of tumors, as well as an overall immunosuppressive tumor microenvironment that elicits T cell exhaustion. Therefore, novel, rational approaches are necessary to overcome the barriers to T cell function elicited by solid tumors. In this review, we will provide an overview of conventional immunotherapeutic strategies and the various barriers to T cell anti-tumor function encountered in solid tumors that lead to resistance. We will also explore a sampling of emerging strategies specifically aimed to bypass these tumor-imposed boundaries to T cell-based immunotherapies.

Brain metastases affect nearly 20% of all cancer patients. Likewise, glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Current immunotherapeutic efficacy is hindered by immunosuppression present in the brain tumor microenvironment (TME). T-cells, critical for tumor clearance, take on a functionally exhausted phenotype. Importantly, two exhaustion states, progenitor (Tpe) and terminal (Tte), have been identified in models of chronic infection and cancer. This distinction is particularly relevant, as Tpe can remain responsive to immune checkpoint blockade (ICB), while Tte cannot. To date, the dynamics and characteristics of these exhausted populations in primary tumors and brain metastases remain unclear. Using intracranially implanted murine models of GBM (CT2A) and metastatic melanoma (B16F10), Tpe and Tte were identified by flow cytometry as PD1+SLAMF6+ and PD1+TIM3+, respectively. Functional differences between subsets were evaluated via intracellular staining of IFNγ, TNFα, IL2, CD107a, and Ki67. To determine the role of antigen, we performed adoptive lymphocyte transfers of tumor-specific and non-tumor-specific transgenic T-cells into a TRP2 or OVA overexpressing intracranial CT2A or B16 tumor, respectively. Tte displayed higher cytotoxic molecule expression than Tte, consistent with chronic infection models. Key exhaustion-associated transcription factors were identified in exhaustion subsets, including Tox, Tcf7, T-bet, and Eomes. Tox and Tcf7 expression identified Tpe within tumor-draining lymph nodes, suggesting a potential origin outside of the tumor, and the capacity for rescued function. We observed a decline in the Tpe population over time, with an associated rise in Tte within both tumor types. Notably, the ratio of Tpe to Tte was higher at all time points in B16F10 compared to CT2A. Tte appeared only in tumor-specific T-cells of the TME, further confirming the tumor-antigen dependence of T cell exhaustion. Tpe may arise outside the TME in tumor-specific T-cells. Further study may reveal a means and time window for rescuing T-cell function with ICB for brain tumors.

Major Histocompatibility Complex (MHC) Class I downregulation is a well described mechanism of tumor immune escape, posing a challenge for T cell based immunotherapies including immune checkpoint blockade (ICB). Recent studies, however, have demonstrated mixed roles of MHC Class 1 and the critical component beta-2-microglobulin (β2m) expression in cancer progression and ICB response, with some studies showing inactivation of antigen presentation to be associated with resistance to ICB and others showing low β2m expression to be associated with favorable prognosis. Glioblastoma (GBM) in particular expresses little or no MHC Class 1 and patients remain unresponsive to ICB. We thus sought to evaluate the role of MHC Class 1 in ICB, given that we have previously demonstrated that combination ICB with anti-PD-1 and co-stimulation with 4-1BB agonism has marked efficacy against intracranial murine glioma tumors in a CD8 T cell dependent manner. Surprisingly, in a CT2A murine glioma tumor line expressing the antigen TRP2 and lacking cell surface MHC I (CT2A-TRP2-β2mKO), the efficacy of combination 4-1BB and PD-1 therapy (ICB) was re-demonstrated in a CD8 dependent manner, independent of NK cells, CD4 T cells, and B cells. Furthermore, the efficacy of immunotherapy against intracranial CT2A-TRP2-β2mKO was demonstrated to be antigen dependent, with an adoptive lymphocyte transfer (ALT) of TRP2 TCR transduced T cells (TRP2 T cells) into a CD8KO mouse sufficient to eliminate CT2A-TRP2-β2mKO in the setting of ICB. Additionally, an ALT of TRP2 T cells did not kill CT2A-β2mKO tumors in the setting of ICB, while OT-1 mice whose CD8+ T cells primarily recognize OVA peptide with CT2A-TRP2-β2mKO tumors did not respond to ICB. In vitro studies revealed that TRP2 T cells demonstrated anti-tumor cytotoxicity against MHC Class I negative CT2A-TRP2-β2mKO tumor cells in the presence of TRP2 loaded bone marrow derived macrophages (TRP2 Mφ), but neither cell type was individually sufficient to induce tumor cell death, while the combination of TRP2 T cells and TRP2 Mφ demonstrated no cytotoxicity against CT2A-β2mKO tumors. Transwell experiments in which TRP2 Mφ and CT2A-TRP2-β2mKO tumor cells were separated by a 0.5µm membrane permeable to T cells but not Mφ or tumor cells revealed that contact between TRP2 Mφ and tumor cells was not necessary to induce T cell dependent killing. Indeed, tumor-bearing β2mKO bone marrow chimeras lacking MHC class 1 on hematopoeitically derived cells did not respond to ICB, highlighting the importance of antigen presentation from myeloid cells. The mechanism of killing was found to be dependent on interferon gamma (IFNγ), as IFNγKO mice did not respond to ICB. These findings demonstrate that tumors with low MHC Class 1 expression may still be targeted by T cell dependent immunotherapies such as ICB when antigen presentation can occur from myeloid cells. Citation Format: Emily Lerner, Vincent D'Anniballe, William Tomaszewski, Jonathan Perera, Xiuyu Cui, Jessica Waibl-Polania, Daniel Wilkinson, Michael D. Gunn, Peter E. Fecci, Karolina Woroniecka. CD8 T cell mediated killing of MHC class 1 negative tumors requires antigen presenting myeloid cells and interferon gamma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1378.

Cancer immunotherapy encompasses a variety of therapeutic modalities that aim to harness and/or enhance a patient’s own immune system to elicit anti-tumor responses. Promising outcomes from several clinical trials have led to recent FDA approvals and rendered immunotherapy as a standard of care for various types of cancer, including melanoma and lung cancers. In this chapter, we summarize the most recent therapeutic advances to various cancer immunotherapeutic modalities, including vaccines, viral-based therapies, chimeric antigen receptor (CAR) T cells, and checkpoint blockade. Additionally, we recap the challenges faced by cancer immunotherapy, including limitations to tumor immune access, tumor heterogeneity, and tumor-induced local and systemic immune-suppression. We also introduce the roles that nanotechnologies might play in enhancing the efficacy of cancer immunotherapy.

MHC-I downregulation is a well described mechanism of tumor immune escape. Thus, targeting tumors with low or no MHC-I expression remains a significant challenge for T cell-based immunotherapies, including immune checkpoint blockade (ICB). We previously demonstrated that the combination of PD-1 blockade and 4-1BB agonism has marked efficacy against intracranial murine CT2A glioma in a CD8 T cell-dependent manner. Surprisingly, this combination therapy remained effective in a β2 microglobulin knockout CT2A line overexpressing TRP2 (CT2A-TRP2-β2mKO). These tumors lack MHC-I but retain the Trp2 rejection antigen. Remarkably, the persisting efficacy remained dependent on CD8 T cells, but independent of NK cells, CD4 T cells, and B cells. Furthermore, the efficacy was demonstrated to be antigen-specific in vivo, as adoptively transferred Trp2-specific T cells prolonged survival of mice bearing CT2A-TRP2-β2mKO tumors, but not those bearing CT2A-β2mKO, which lacked the TRP2 antigen. To analyze the mechanism driving antigen-dependent killing of tumor cells lacking MHC-I, we performed a series of in vitro cytotoxicity experiments using Trp2-specific T cells in the presence or absence of Trp2 antigen-loaded bone marrow-derived macrophages (BMDM). These studies demonstrated that in the absence of BMDM, Trp2-specific T cells efficiently killed MHC-I-expressing CT2A-Trp2, but failed to kill MHC-I deficient CT2A-Trp2-B2mKO tumors. However, killing was restored in the presence of Trp2 loaded BMDMs; the latter of which possessed little to no cytotoxic effect alone. These findings suggest a novel role for myeloid populations in mediating the influence of ICB on T cell function and challenge the traditional model of T cell tumor killing.

Purpose: The success of checkpoint blockade against glioblastoma (GBM) has been disappointing. Anti–PD-1 strategies may be hampered by severe T-cell exhaustion. We sought to develop a strategy that might license new efficacy for checkpoint blockade in GBM. Experimental Design: We characterized 4-1BB expression in tumor-infiltrating lymphocytes (TIL) from human GBM. We implanted murine tumor models including glioma (CT2A), melanoma (B16), breast (E0771), and lung carcinomas intracranially and subcutaneously, characterized 4-1BB expression, and tested checkpoint blockade strategies in vivo. Results: Our data reveal that 4-1BB is frequently present on nonexhausted CD8+ TILs in human and murine GBM. In murine gliomas, 4-1BB agonism and PD-1 blockade demonstrate a synergistic survival benefit in a CD8+ T-cell–dependent manner. The combination decreases TIL exhaustion and improves TIL functionality. This strategy proves most successful against intracranial CT2A gliomas. Efficacy in all instances correlates with the levels of 4-1BB expression on CD8+ TILs, rather than with histology or with intracranial versus subcutaneous tumor location. Proffering 4-1BB expression to T cells licenses combination 4-1BB agonism and PD-1 blockade in models where TIL 4-1BB levels had previously been low and the treatment ineffective. Conclusions: Although poor T-cell activation and severe T-cell exhaustion appear to be limiting factors for checkpoint blockade in GBM, 4-1BB agonism obviates these limitations and produces long-term survival when combined with anti–PD-1 therapy. Furthermore, this combination therapy is limited by TIL 4-1BB expression, but not by the intracranial compartment, and therefore may be particularly well-suited to GBM.

Here we present a 14-color flow cytometry panel for the evaluation of 13 myeloid and lymphoid populations within murine glioblastoma samples. Reagents, processing protocols, and downstream analyses were thoroughly validated and optimized to resolve the following populations: T cells (CD4, CD8, CD3), B cells (B220), NK cells (NK1.1), neutrophils (Ly6G), classical and non-classical monocytes (Ly6c, CD43), macrophages (F4/80, CD11b), microglia (CD45-lo, CD11b), and dendritic cells (DCs) (CD11c, MHC class II). In addition, this panel leaves Alexa Fluor 488/FITC open for the inclusion of fluorescent reporters or congenic marker staining.

BACKGROUND Glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Median survival remains a bleak 15-17 months from time of diagnosis, and current immunotherapeutic efficacy continues to be hindered by the robust immunosuppression present in the GBM microenvironment. T cells, critical for tumor clearance, are particularly affected, and many take on a functionally exhausted phenotype within the tumor. Importantly, two exhaustion states, progenitor and terminal, have been identified in models of chronic infection and cancer. This distinction is particularly relevant, as progenitor exhausted T cells can respond favorably to immune checkpoint blockade, while terminally exhausted T cells are resistant. To date, the dynamics and characteristics of these exhausted populations in GBM remain unclear. RESULTS In an orthotopic murine model of GBM, progenitor and terminal exhausted CD8 T cells were identified by flow cytometry as PD1+SLAMF6+ and PD1+TIM3+, respectively. Using a time-course approach, we detected progenitor exhaustion by day 8 in the tumor, but not in draining lymph nodes. Additionally, we show that the frequency of progenitor exhaustion is highest during early tumor progression, while terminal exhaustion is the most abundant in more advanced tumors ( >14 days). Functional differences between subsets were evaluated via intracellular staining of IFNγ, TNFα, granzyme B, and Ki67. Terminally exhausted T cells displayed higher cytotoxic molecule expression than progenitor exhausted T cells, similar to what has been documented in melanoma models. CONCLUSIONS Our findings identify T cell exhaustion subsets within GBM that require further investigation and may be relevant to overcome current barriers to immunotherapeutic efficacy.

Objective Glioblastoma (GBM) is the most lethal primary brain tumor in adults. Although immunotherapy has been successful in some cancers, it has shown limited success in GBM. This is partially due to poor T cell infiltration, and because many T cells found in tumors are either exhausted or Tregs. Re-analysis of publicly available data has shown that CaMKK2 is more highly expressed in advanced brain tumors, and high CaMKK2 expression results in worse survival outcomes in GBM. Additionally, in survival studies that we performed, CaMKK2−/− mice showed an impressive survival phenotype that was dependent on the presence of CD8+ tumor infiltrating lymphocytes (TILs). The objective of our study was to investigate what is different about T cells found in the TME of CaMKK2−/− mice, and if this can explain the observed survival benefit. Methods We performed flow cytometry to determine the identities of T cells found in the tumors of CaMKK2−/− and WT mice. Results/Conclusions Our experiments show that there was a greater percentage of CD4+ TILs relative to CD8+ TILs in CaMKK2−/− mice, and this increase in percentage was due to increased accumulation of CD4+ TILs. Additionally, we found that there was greater accumulation of CD4+ and CD8+ effector memory TILs and a greater ratio of effector memory TILs to Tregs in CaMKK2−/− mice. The increased accumulation of the CD8+ effector memory TILs is particularly interesting because it is not simply explained by an increased accumulation of cells, as is the case with the CD4+ TILs. The increased accumulation of CD4+ TILs and greater ratio of effector memory TILs to Tregs is expected to have an anti-tumor effect. These findings make CaMKK2 a promising target to treat GBM.

Objective: Glioblastoma is the most common and aggressive primary brain tumor with a 5 year prognosis of less than 5% survival. Efforts to extend this survival through therapeutic advancement have only led to modest improvements. Immunotherapy has shown promise in other cancers but only has limited effect in GBM, partially due to the dense infiltration of pro-tumor myeloid cells. Calmodulin-dependent Kinase Kinase 2 (CaMKK2) is highly expressed in myeloid cells and high expression is associated with a more severe tumor grade and worse survival in GBM. Preliminary studies show that CaMKK2 KO mice display extended median survival and tumor clearance. We hypothesize that CaMKK2 may drive myeloid cells’ pro-tumor function. Methods: To investigate this, immune cells were isolated from tumor-bearing CaMKK2 KO, LysMcre x CaMKK2fl/fl and CaMKK2-eGFP reporter mice. Multi-parameter flow was used to detect CaMKK2 and phenotype the tumor microenvironment (TME). The presence of CaMKK2 expressing myeloid cells in the TME was confirmed by confocal microscopy. Results/Conclusions: CaMKK2 is preferentially expressed in myeloid cells in naïve and tumorous mice. CaMKK2 deletion upregulated MHC-II in macrophages which is expected to promote an antitumor response. Upregulation was not seen in naive CaMKK2 KO mice suggesting it is a product of tumor-exposure. Conditional deletion of CaMKK2 in bone-marrow derived macrophages (BMDM) did not lead to this MHC-II upregulation suggesting that it is not a BMDM-intrinsic phenotype but results from CaMKK2 deletion in another cell-type. In summary, CaMKK2 deletion induced macrophages to a more immunostimulatory phenotype with expected anti-tumor effect, making CaMKK2 a promising therapeutic target for GBM.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults, with the median survival time for GBM patients being only about 15 months. Although immunotherapy treatments have been successful in more immunogenic tumors, it has shown very limited success in GBM. This could be because it is difficult for T cells to infiltrate the tumor, and the tumors have a very immunosuppressive tumor microenvironment (TME) that can often consist of up to about 30 percent tumor associated macrophages (TAMs) by mass. Thus, two ways of possibly improving immunotherapy treatments on GBMs would be to increase the number of tumor infiltrating lymphocytes (TILs) present, and to target tumor-supportive cells such as TAMs. Calmodulin Dependent Protein Kinase Kinase 2 (CaMKK2) is a gene that has been shown to be highly expressed in myeloid cells and is known to alter their phenotype. Recent studies in breast cancer have shown that myeloid cells adopt a more immunostimulatory phenotype in murine knockout CaMKK2 models. However, it is not well understood how CaMKK2 causes this immunosuppressive effect and warrants further exploration. The data from our experiments help to answer this question. High-dimensional flow cytometry with 15 parameters was used in order to determine the identities of immune cells found in the tumor of wildtype and CaMKK2-/- mice. The results of our experiments show that there appear to be more CD4+ TILs present in the TME of knockout mice and that there is an increase in MHC II expression on TAMs found in knockout mice. Since CD4+ TILs recognize peptides presented on MHC II molecules, and there is an increase in the number of CD4+ TILs and expression of MHC II on TAMs in knockout mice, we hypothesize that a stronger immune response could be instigated against the tumor. Therefore, it can be concluded that a possible reason that knocking out CaMKK2 leads to a less immunosupressive TME is because MHC II expression on TAMs is upregulated, and the number of CD4+ TILs in the TME is increased. Whether or not the increase in MHC II expression causes the increase in the number CD4+ TILs requires further exploration. This makes CaMKK2 a promising therapeutic target to treat tumors with immunosuppressive TMEs such as GBM. Citation Format: Molly A. Chakraborty, William H. Tomaszewski, Jessica Waibl Polania, Lauren S. Riley, Luigi Racioppi, Luis A. Sanchez-Perez, John H. Sampson. CaMKK2 knockout in mice challenged by orthotopic GBM leads to upregulation of MHC II on TAMs and an increase in accumulation of CD4+ TILs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2667.

Glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Median survival remains a bleak 15–17 months from time of diagnosis, calling for new therapeutic strategies. Current immunotherapeutic efficacy continues to be hindered by the robust immunosuppression of the GBM tumor microenvironment (TME). T cells, critical for tumor clearance, are particularly affected and many take on an exhausted phenotype within the tumor. We have previously observed significantly worse exhaustion in models of glioma relative to other solid tumor models, suggesting a TME-specific phenomenon. In an orthotopic murine model of GBM, T cell exhaustion was identified using surface expression of inhibitory ligands, PD1, TIM3, and LAG3. Functional status was examined using intracellular flow cytometry for IFNy, TNFa, and IL-2. We consistently observed T cell exhaustion among antigen-experienced tumor-specific CD8+ T cells, suggesting that tumor-imposed exhaustion is dependent upon T cells encountering their antigen. It remains undetermined, however, which cell within the GBM microenvironment provokes a uniquely severe T cell exhaustion phenotype. Using CRISPR-Cas9 gene editing, we modified tumor cells to lack antigen-presentation capacities and revealed persistence of T cell exhaustion. In contrast, when the same capacity was removed from infiltrating, hematopoietic cells, T cells in the GBM microenvironment were less exhausted and retained their function. This suggests antigen-presenting cells (APC) are critical for T cell exhaustion within GBM. Our findings uncover an important role for APCs in eliciting T cell exhaustion, which will provide novel interactions for targeted immunotherapies.

Introduction: While key regulators of early NK cell development and differentiation have been identified, few studies have looked at transcription factor (TF) dynamics and regulatory interactions during subsequent stages of NK cell maturation. Epigenetic landscapes are highly dynamic during cellular differentiation, with TFs playing an important role in the establishment and activation of specific DNA elements, such as enhancers, and subsequent programming of gene expression. ETS1 is a TF that is expressed in adult immune tissues and is critical for the development of lymphoid cells. A role for ETS1 has been described in early NK cell development by activating core transcriptional regulators such as T-BET and ID2. However, despite its continual expression in subsequent stages of NK maturation, the role of ETS1 in NK maturation is not well characterized. Methods and Results: We used FACS to isolate purified human NK cells at various maturation stages as established previously (Freud et. al. Cell Reports, 2016, 16:379-91), ranging from intermediate precursors (Stage 3) through to fully developed and mature peripheral NK cells (Stage 6). Epigenetic programming of cells during lineage maturation allows us to identify critical TFs that are active at each stage of development. We employed Illumina EPIC/850K methylation arrays and RNA sequencing to interrogate epigenetic changes at regulatory elements and TF dynamics at multiple stages along the NK developmental axis. Analysis of TF recognition motifs within hypomethylated regions revealed strong enrichment of specific motif sequences implicating T-box (T-BET and Eomes), along with RUNX and ETS TF families in specific programming of epigenetic patterns during NK development. In studying the expression of TFs that potentially bind these motifs, ETS1 exhibited the highest and most consistent expression throughout NK development. Interestingly, despite consistently high expression, ETS motifs were continually programmed throughout NK maturation, including a significant degree of modification between tonsillar Stages 4A to 4B, where NK cells acquire the ability to produce IFN-γ and significantly gain cytotoxic capability and functional maturity. Among the genes that are upregulated at this stage is the NK-cell-specific gene, NKp46. The progressive hypomethylation of regulatory regions enriched in ETS motifs led us to believe that ETS1 has a continuous role in full NK cell maturation. To test our hypothesis, we developed a novel genetically engineered mouse line with a NK cell intermediate stage-specific conditional deletion of Ets1 mediated by NKp46-driven Cre expression, NKp46-Cre-Ets1fl/fl (NKp46-Ets1fl/fl). This allowed us to study the role of ETS1 in the transition between immature and mature NK cell stages in vivo. Using a comprehensive NK cell development panel for multi-color flow cytometry, we found a drastic reduction of total NK cells in NKp46-Ets1fl/fl mice (n=7) compared to the Ets1fl/fl (n=7) and the NKp46-Cre (n=7) controls in bone marrow (3.2x104 ± 5.9x103, 2.9x105 ± 5.7x104, 2.6x105 ± 8.0x104 total NK cells respectively; p= 0.0007), spleen (3.1x104 ± 7.2x103, 1.2x106 ± 2.4x105, 1.5x106 ± 7.7x105 total NK cells respectively; p= 0.0091) and blood (21 ± 6, 385 ± 35, 185 ± 35 NK cells/uL whole blood respectively; p= 0.0001). Supporting our hypothesis, we indeed observed that while CD11b-/CD27+/- immature NK cell populations in our model are unaltered, the loss of ETS1 is associated with a decrease in CD11b+/CD27+/- mature NK cell populations. Conclusions: Our findings demonstrate that in addition to its role in early NK establishment, persistent ETS1 expression is important in intermediate differentiation stages in both human and murine NK cell development. This constitutes a significant step forward in understanding the role of ETS1 as a master transcriptional regulator in the entire NK cell developmental axis. Current studies are ongoing to dissect the mechanism by which ETS1 affects NK cell development and function in the NKp46-Ets1fl/fl mice. (*EH and JW are recipients of Pelotonia Graduate and Undergraduate student fellowships respectively and contributed equally to this work. This work was partly supported by OCRA, NIH R01 CA159296, NIH R01 CA208353, P01CA95426, R35 CA197734 and OSUCCC Leukemia Tissue Bank and Genetically Engineered Mouse Modeling Core supported by P30CA016058) Disclosures No relevant conflicts of interest to declare.