IL-1 and IL-1ra are key regulators of the inflammatory response to RNA vaccines.

The use of lipid-formulated RNA vaccines for cancer or COVID-19 is associated with dose-limiting systemic inflammatory responses in humans that were not predicted from preclinical studies. Here, we show that the 'interleukin 1 (IL-1)-interleukin 1 receptor antagonist (IL-1ra)' axis regulates vaccine-mediated systemic inflammation in a host-specific manner. In human immune cells, RNA vaccines induce production of IL-1 cytokines, predominantly IL-1ß, which is dependent on both the RNA and lipid formulation. IL-1 in turn triggers the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6). Unlike humans, murine leukocytes respond to RNA vaccines by upregulating anti-inflammatory IL-1ra relative to IL-1 (predominantly IL-1α), protecting mice from cytokine-mediated toxicities at >1,000-fold higher vaccine doses. Thus, the IL-1 pathway plays a key role in triggering RNA vaccine-associated innate signaling, an effect that was unexpectedly amplified by certain lipids used in vaccine formulations incorporating N1-methyl-pseudouridine-modified RNA to reduce activation of Toll-like receptor signaling.

Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity.

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

Dexamethasone premedication suppresses vaccine-induced immune responses against cancer.

Glucocorticosteroids (GCS) have an established role in oncology and are administered to cancer patients in routine clinical care and in drug development trials as co-medication. Given their strong immune-suppressive activity, GCS may interfere with immune-oncology drugs. We are developing a therapeutic cancer vaccine, which is based on a liposomal formulation of tumor-antigen encoding RNA (RNA-LPX) and induces a strong T-cell response both in mice as well as in humans. In this study, we investigated in vivo in mice and in human PBMCs the effect of the commonly used long-acting GCS Dexamethasone (Dexa) on the efficacy of this vaccine format, with a particular focus on antigen-specific T-cell immune responses. We show that Dexa, when used as premedication, substantially blunts RNA-LPX vaccine-mediated immune effects. Premedication with Dexa inhibits vaccine-dependent induction of serum cytokines and chemokines and reduces both the number and activation of splenic conventional dendritic cells (cDC) expressing vaccine-encoded antigens. Consequently, priming of functional effector T cells and therapeutic activity is significantly impaired. Interestingly, responses are less impacted when Dexa is administered post-vaccination. Consistent with this observation, although many inflammatory cytokines are reduced, IFNα, a key cytokine in T-cell priming, is less impacted and antigen expression by cDCs is intact. These findings warrant special caution when combining GCS with immune therapies relying on priming and activation of antigen-specific T cells and suggest that careful sequencing of these treatments may preserve T-cell induction.

Immunization associated with primary tumor growth leads to rejection of commonly used syngeneic tumors upon tumor rechallenge.

The recent success of multiple immunomodulating drugs in oncology highlights the potential of relieving immunosuppression by directly engaging the immune system in the tumor bed to target cancer cells. Durable responses to immune checkpoint inhibitors experienced by some patients may be indicative of the formation of a T cell memory response. This has prompted the search for preclinical evidence of therapy-induced long-term immunity as part of the evaluation of novel therapeutics. A common preclinical method used to document long-term immunity is the use of tumor rechallenge experiments in which tumor growth is assessed in mice that have previously rejected tumors in response to therapy. Failure of rechallenge engraftment, typically alongside successful engraftment of the same tumor in naive animals as a control, is often presented as evidence of therapy-induced tumor immunity. Here, we present evidence that formation of tumor immunity often develops independent of therapy. We observed elevated rates of rechallenge rejection following surgical resection of primary tumors for four of five commonly used models and that such postexcision immunity could be adoptively transferred to treatment-naïve mice. We also show that tumor-specific cytolytic T cells are induced on primary tumor challenge independent of therapeutic intervention. Taken together these data call into question the utility of tumor rechallenge studies and the use of naïve animals as controls to demonstrate therapy-induced formation of long-term tumor immunity.

Inhibition of the dipeptidyl peptidase DPP4 (CD26) reveals IL-33-dependent eosinophil-mediated control of tumor growth.

Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33- and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited.

High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies.

As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.

Amyloid fibrils activate B-1a lymphocytes to ameliorate inflammatory brain disease.

Amyloid fibrils composed of peptides as short as six amino acids are therapeutic in experimental autoimmune encephalomyelitis (EAE), reducing paralysis and inflammation, while inducing several pathways of immune suppression. Intraperitoneal injection of fibrils selectively activates B-1a lymphocytes and two populations of resident macrophages (MΦs), increasing IL-10 production, and triggering their exodus from the peritoneum. The importance of IL-10-producing B-1a cells in this effective therapy was established in loss-of-function experiments where neither B-cell-deficient (µMT) nor IL10(-/-) mice with EAE responded to the fibrils. In gain-of-function experiments, B-1a cells, adoptively transferred to µMT mice with EAE, restored their therapeutic efficacy when Amylin 28-33 was administered. Stimulation of adoptively transferred bioluminescent MΦs and B-1a cells by amyloid fibrils resulted in rapid (within 60 min of injection) trafficking of both cell types to draining lymph nodes. Analysis of gene expression indicated that the fibrils activated the CD40/B-cell receptor pathway in B-1a cells and induced a set of immune-suppressive cell-surface proteins, including BTLA, IRF4, and Siglec G. Collectively, these data indicate that the fibrils activate B-1a cells and F4/80(+) MΦs, resulting in their migration to the lymph nodes, where IL-10 and cell-surface receptors associated with immune-suppression limit antigen presentation and T-cell activation. These mechanisms culminate in reduction of paralytic signs of EAE.

Inflammation and hyperglycemia mediate Deaf1 splicing in the pancreatic lymph nodes via distinct pathways during type 1 diabetes.

Peripheral tolerance is partially controlled by the expression of peripheral tissue antigens (PTAs) in lymph node stromal cells (LNSCs). We previously identified a transcriptional regulator, deformed epidermal autoregulatory factor 1 (Deaf1), that can regulate PTA expression in LNSCs of the pancreatic lymph nodes (PLNs). During the pathogenesis of type 1 diabetes (T1D), Deaf1 is spliced to form the dominant-negative isoform Deaf1-Var1. Here we show that Deaf1-Var1 expression correlates with the severity of disease in NOD mice and is reduced in the PLNs of mice that do not develop hyperglycemia. Inflammation and hyperglycemia independently drive Deaf1 splicing through activation of the splicing factors Srsf10 and Ptbp2, respectively. Inflammation induced by injection of activated splenocytes increased Deaf1-Var1 and Srsf10, but not Ptbp2, in the PLNs of NOD.SCID mice. Hyperglycemia induced by treatment with the insulin receptor agonist S961 increased Deaf1-Var1 and Ptbp2, but not Srsf10, in the PLNs of NOD.B10 and NOD mice. Overexpression of PTBP2 and/or SRSF10 also increased human DEAF1-VAR1 and reduced PTA expression in HEK293T cells. These data suggest that during the progression of T1D, inflammation and hyperglycemia mediate the splicing of DEAF1 and loss of PTA expression in LNSCs by regulating the expression of SRSF10 and PTBP2.

Mechanisms of action of therapeutic amyloidogenic hexapeptides in amelioration of inflammatory brain disease.

Amyloid fibrils composed of peptides as short as six amino acids are effective therapeutics for experimental autoimmune encephalomyelitis (EAE). Immunosuppression arises from at least two pathways: (1) expression of type 1 IFN by pDCs, which were induced by neutrophil extracellular traps arising from the endocytosis of the fibrils; and (2) the reduced expression of IFN-γ, TNF, and IL-6. The two independent pathways stimulated by the fibrils can act in concert to be immunosuppressive in Th1 indications, or in opposition, resulting in inflammation when Th17 T lymphocytes are predominant. The generation of type 1 IFN can be minimized by using polar, nonionizable, amyloidogenic peptides, which are effective in both Th1 and Th17 polarized EAE.

Gene related to anergy in lymphocytes (GRAIL) expression in CD4+ T cells impairs actin cytoskeletal organization during T cell/antigen-presenting cell interactions.

GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.

Regulation of IL-10 expression by upstream stimulating factor (USF-1) in glioma-associated microglia.

Understanding the local CNS immune response to neoplasms is essential in the development of immune-based treatments for malignant brain tumors. Using rodent glioma models, we have recently found tumor-associated microglia/macrophages (MG/MP) to be less responsive to known MG/MP activators such as CpG, LPS and IFN-gamma. To understand the mechanism of MG/MP suppression, nuclear extracts from rodent intracranial C6 gliomas, C6 glioma-associated MG/MP, normal brain, and normal MG/MP were obtained and studied using Electrophoretic Mobility Shift Assay (EMSA). Among the nuclear factors studied (AP-1, IRF, USF-1 and Stat-1) only USF-1, which is constitutively expressed in most cells, was down-regulated in tumor-associated MG/MP, but not normal MG/MP. Because tumor-associated MG/MP had higher expression of IL-10 (but not TNF-alpha or TGF-beta), we evaluated the role of USF-1 on IL-10 expression. siRNA mediated inhibition of USF-1 expression in primary MG/MP cultures resulted in up-regulation of IL-10 mRNA but not TNF-alpha or TGF-beta. These findings suggest that USF-1 may play a role in IL-10 regulation in MG/MP in brain tumors.

Microglia function in brain tumors.

Microglia play an important role in inflammatory diseases of the central nervous system (CNS). These cells have also been identified in brain neoplasms; however, as of yet their function largely remains unclear. More recent studies designed to characterize further tumor-associated microglia suggest that the immune effector function of these cells may be suppressed in CNS tumors. Furthermore, microglia and macrophages can secrete various cytokines and growth factors that may contribute to the successful immune evasion, growth, and invasion of brain neoplasms. A better understanding of microglia and macrophage function is essential for the development of immune-based treatment strategies against malignant brain tumors.

Impaired capacity for upregulation of MHC class II in tumor-associated microglia.

Immunotherapy for malignant gliomas is being studied as a possible adjunctive therapy for this highly fatal disease. Thus far, inadequate understanding of brain tumor immunology has hindered the design of such therapies. For instance, the role of microglia and macrophages, which comprise a significant proportion of tumor-infiltrating inflammatory cells, in the regulation of the local anti-tumor immune response is poorly understood. To study the response of microglia and macrophages to known activators in brain tumors, we injected CpG oligodeoxynucleotide (ODN), interferon-gamma (IFN-gamma), and IFN-gamma/LPS into normal and intracranial RG2 glioma-bearing rodents. Microglia/macrophage infiltration and their surface expression of MHC class II B7.1 and B7.2 was examined by flow cytometry. Each agent evaluated yielded a distinct microglia/macrophage response: CpG ODN was the most potent inducer of microglia/macrophage infiltration and B7.1 expression, while IFN-gamma resulted in the highest MHC-II expression in both normal and tumors. Regardless of the agent injected, however, MHC-II induction was significantly muted in tumor microglia/macrophage as compared with normal brain. These data suggest that microglia/macrophage responsiveness to activators can vary in brain tumors when compared with normal brain. Understanding the mechanism of these differences may be critical in the development of novel immunotherapies for malignant glioma.

Microglia cyclooxygenase-2 activity in experimental gliomas: possible role in cerebral edema formation.

PURPOSE: Cerebral edema is responsible for significant morbidity and mortality in patients harboring malignant gliomas. To examine the role of inflammatory cells in brain edema formation, we studied the expression cyclooxygenase (COX)-2, a key enzyme in arachidonic acid metabolism, by microglia in the C6 rodent glioma model. EXPERIMENTAL DESIGN: The expression of COX-2 in primary microglia cultures obtained from intracranial rat C6 gliomas was examined using reverse transcription-PCR, Western analysis, and prostaglandin E(2) (PGE(2)) enzyme immunoassay. Blood-tumor barrier permeability was studied in the same tumor model using magnetic resonance imaging. RESULTS: In contrast to C6 glioma cells, microglia isolated from intracranial C6 tumors produced high levels of PGE(2) through a COX-2-dependent pathway. To test whether the observed microglia COX-2 activity played a role in brain edema formation in gliomas, tumor-bearing rats were treated with rofecoxib, a selective COX-2 inhibitor. Rofecoxib was as effective as dexamethasone in decreasing the diffusion of contrast material into the brain parenchyma (P = 0.01, rofecoxib versus control animals), suggesting a reduction in blood-tumor barrier permeability. CONCLUSIONS: These findings suggest that glioma-infiltrating microglia are a major source of PGE(2) production through the COX-2 pathway and support the use of COX-2 inhibitors as possible alternatives to glucocorticoids in the treatment of peritumoral edema in patients with malignant brain tumors.