Nociceptor neurons affect cancer immunosurveillance.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems1-5. Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)-one such nociceptor-produced neuropeptide-directly increases the exhaustion of cytotoxic CD8+ T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8+ T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8+ T cells, Ramp1-/- CD8+ T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1-deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1-expressing CD8+ T cells were more exhausted than their RAMP1-negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8+ T cells.

Transcriptional neural-like signaling contributes to an immune-suppressive tumor microenvironment.

Tumor innervation has recently been documented and characterized in various settings and tumor types. However, the role that nerves innervating tumors play in the pathogenesis of cancer has not been clarified. In this study, we searched for neural signaling from bulk RNA sequencing from The Cancer Genome Atlas (TCGA) dataset and looked for patterns of interactions between different cell types within the tumor environment. Using a presynapse signature (PSS) as a probe, we showed that multiple stromal cell types crosstalk and/or contribute to neural signals. Based on the correlation and linear regression, we hypothesized that neural signals contribute to an immune-suppressive tumor microenvironment (TME). To test this hypothesis, we performed in vitro dorsal root ganglion (DRG)/macrophage coculture experiments. Compared to the M2 macrophage monoculture, the DRG/M2 macrophage coculture prevented anti-inflammatory M2 to pro-inflammatory M1 polarization by LPS stimulation. Finally, a survey of different TCGA tumor types indicated that higher RNA neural signature is predictive of poor patient outcomes in multiple tumor types.

ICOS agonism by JTX-2011 (vopratelimab) requires initial T cell priming and Fc cross-linking for optimal T cell activation and anti-tumor immunity in preclinical models.

Immunotherapy checkpoint inhibitors, such as antibodies targeting PD-1 and CTLA-4, have demonstrated the potential of harnessing the immune system to treat cancer. However, despite encouraging results particularly with respect to survival, only a minority of patients benefit from these therapies. In clinical studies aimed at understanding changes in the immune system following immunotherapy treatment, ICOS (Inducible T cell CO-Stimulator) was shown to be significantly up-regulated on CD4+ T cells and this was associated with clinical activity, indicating that ICOS stimulatory activity may be beneficial in the treatment of solid tumors. In this report, we describe the generation of specific, species cross-reactive, agonist antibodies to ICOS, including the humanized clinical candidate, JTX-2011 (vopratelimab). Preclinical studies suggest that the ICOS stimulating antibodies require Fc receptor cross-linking for optimal agonistic activity. Notably, the ICOS antibodies do not exhibit superagonist properties but rather require T cell receptor (TCR)-mediated upregulation of ICOS for agonist activity. Treatment with the ICOS antibodies results in robust anti-tumor benefit and long-term protection in preclinical syngeneic mouse tumor models. Additional benefit is observed when the ICOS antibodies are administered in combination with anti-PD-1 and anti-CTLA-4 therapies. Based on the preclinical data, JTX-2011 is currently being developed in the clinical setting for the treatment of solid tumors.

Novel PET probes specific for deoxycytidine kinase.

UNLABELLED: Deoxycytidine kinase (dCK) is a rate-limiting enzyme in the deoxyribonucleoside salvage pathway and a critical determinant of therapeutic activity for several nucleoside analog prodrugs. We have previously reported the development of 1-(2'-deoxy-2'-(18)F-fluoro-beta-D-arabinofuranosyl)cytosine ((18)F-FAC), a new probe for PET of dCK activity in immune disorders and certain cancers. The objective of the current study was to develop PET probes with improved metabolic stability and specificity for dCK. Toward this goal, several candidate PET probes were synthesized and evaluated in vitro and in vivo. METHODS: High-pressure liquid chromatography was used to analyze the metabolic stability of (18)F-FAC and several newly synthesized analogs with the natural D-enantiomeric sugar configuration or the corresponding unnatural L-configuration. In vitro kinase and uptake assays were used to determine the affinity of the (18)F-FAC L-nucleoside analogs for dCK. The biodistribution of selected L-analogs in mice was determined by small-animal PET/CT. RESULTS: Candidate PET probes were selected using the following criteria: low susceptibility to deamination, high affinity for purified recombinant dCK, high uptake in dCK-expressing cell lines, and biodistribution in mice reflective of the tissue-expression pattern of dCK. Among the 10 newly developed candidate probes, 1-(2'-deoxy-2'-(18)F-fluoro-beta-L-arabinofuranosyl)cytosine (L-(18)F-FAC) and 1-(2'-deoxy-2'-(18)F-fluoro-beta-L-arabinofuranosyl)-5-methylcytosine (L-(18)F-FMAC) most closely matched the selection criteria. The selection of L-(18)F-FAC and L-(18)F-FMAC was validated by showing that these two PET probes could be used to image animal models of leukemia and autoimmunity. CONCLUSION: Promising in vitro and in vivo data warrant biodistribution and dosimetry studies of L-(18)F-FAC and L-(18)F-FMAC in humans.

Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells.

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.

Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers.

The use of fluorescently labeled major histocompatibility complex multimers has become an essential technique for analyzing disease- and therapy-induced T-cell immunity. Whereas classical major histocompatibility complex multimer analyses are well-suited for the detection of immune responses to a few epitopes, limitations on human-subject sample size preclude a comprehensive analysis of T-cell immunity. To address this issue, we developed a combinatorial encoding strategy that allows the parallel detection of a multitude of different T-cell populations in a single sample. Detection of T cells from peripheral blood by combinatorial encoding is as efficient as detection with conventionally labeled multimers but results in a substantially increased sensitivity and, most notably, allows comprehensive screens to be performed. We obtained proof of principle for the feasibility of large-scale screening of human material by analysis of human leukocyte antigen A3-restricted T-cell responses to known and potential melanoma-associated antigens in peripheral blood from individuals with melanoma.

Quantitative PET reporter gene imaging of CD8+ T cells specific for a melanoma-expressed self-antigen.

Adoptive transfer (AT) T-cell therapy provides significant clinical benefits in patients with advanced melanoma. However, approaches to non-invasively visualize the persistence of transferred T cells are lacking. We examined whether positron emission tomography (PET) can monitor the distribution of self-antigen-specific T cells engineered to express an herpes simplex virus 1 thymidine kinase (sr39tk) PET reporter gene. Micro-PET imaging using the sr39tk-specific substrate 9-[4-[(18)F]fluoro-3-(hydroxymethyl)-butyl]guanine ([(18)F]FHBG) enabled the detection of transplanted T cells in secondary lymphoid organs of recipient mice over a 3-week period. Tumor responses could be predicted as early as 3 days following AT when a >25-fold increase of micro-PET signal in the spleen and 2-fold increase in lymph nodes (LNs) were observed in mice receiving combined immunotherapy versus control mice. The lower limit of detection was approximately 7 x 10(5) T cells in the spleen and 1 x 10(4) T cells in LNs. Quantification of transplanted T cells in the tumor was hampered by the sr39tk-independent trapping of [(18)F]FHBG within the tumor architecture. These data support the feasibility of using PET to visualize the expansion, homing and persistence of transferred T cells. PET may have significant clinical utility by providing the means to quantify anti-tumor T cells throughout the body and provide early correlates for treatment efficacy.

Molecular imaging of lymphoid organs and immune activation by positron emission tomography with a new [18F]-labeled 2'-deoxycytidine analog.

Monitoring immune function with molecular imaging could have a considerable impact on the diagnosis and treatment evaluation of immunological disorders and therapeutic immune responses. Positron emission tomography (PET) is a molecular imaging modality with applications in cancer and other diseases. PET studies of immune function have been limited by a lack of specialized probes. We identified [(18)F]FAC (1-(2'-deoxy-2'-[(18)F]fluoroarabinofuranosyl) cytosine) by differential screening as a new PET probe for the deoxyribonucleotide salvage pathway. [(18)F]FAC enabled visualization of lymphoid organs and was sensitive to localized immune activation in a mouse model of antitumor immunity. [(18)F]FAC microPET also detected early changes in lymphoid mass in systemic autoimmunity and allowed evaluation of immunosuppressive therapy. These data support the use of [(18)F]FAC PET for immune monitoring and suggest a wide range of clinical applications in immune disorders and in certain types of cancer.

Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain.

It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.c.) tumors underwent total body irradiation (TBI) to induce transient lymphopenia, followed by the adoptive transfer of gp100(25-33)-specific CD8+ T cells (Pmel-1). Pmel-1 cells were transduced to express the bioluminescent imaging (BLI) gene luciferase. Following adoptive transfer, recipient mice were vaccinated with hgp100(25-33) peptide-pulsed dendritic cells (hgp100(25-33)/DC) and systemic interleukin 2 (IL-2). This treatment regimen resulted in significant reduction in tumor size and extended survival. Imaging of T cell trafficking demonstrated early accumulation of transduced T cells in lymph nodes draining the hgp100(25-33)/DC vaccination sites, the spleen and the cervical lymph nodes draining the CNS tumor. Subsequently, transduced T cells accumulated in the bone marrow and brain tumor. BLI could also detect significant differences in the expansion of gp100-specific CD8+ T cells in the treatment group compared with mice that did not receive either DC vaccination or IL-2. These differences in BLI correlated with the differences seen both in survival and tumor infiltrating lymphocytes (TIL). These studies demonstrate that peripheral tolerance to endogenous TAA can be overcome to treat tumors in the brain and suggest a novel trafficking paradigm for the homing of tumor-specific T cells that target CNS tumors.

Non-invasive imaging of adaptive immunity using positron emission tomography.

Non-invasive monitoring of adaptive immunity in infection, cancer, and autoimmunity remains a major challenge. Current techniques to monitor lymphocytes involve numeric and functional determinations of immune cells isolated from the peripheral blood (most often) and tissue (rarely). Invasive measurements are prone to sampling errors and are poorly reflective of the dynamic changes in the location, number, and movement of lymphoid cells. These limitations indicate the need for non-invasive whole-body imaging methodologies that allow longitudinal, quantitative, and functional analyses of the immune system in vivo. Positron emission tomography (PET), a clinically based whole-body imaging modality, has the potential to revolutionize diagnostics and therapeutic monitoring in both clinical and pre-clinical settings. This review discusses studies using PET to image adaptive immune responses in small animal models. We address the challenges inherent in assessing whole-body immunity with PET and recent developments that can improve its performance. Finally, we discuss work to translate PET immune imaging into clinical practice.

Positron emission tomography with computed tomography imaging of neuroinflammation in experimental autoimmune encephalomyelitis.

2-[(18)F]Fluoro-2-deoxy-d-glucose positron emission tomography ([(18)F]FDG PET) detection of the up-regulated glycolysis associated with malignant transformation is a noninvasive imaging technique used extensively in cancer diagnosis. Although striking similarities exist in glucose transport and metabolism between tumor cells and activated immune cells, the potential use of [(18)F]FDG PET for the diagnosis and evaluation of autoimmune disorders has not been systematically investigated. Here we ask whether [(18)F]FDG PET in conjunction with computed tomography (CT) could be used to monitor a complex autoimmune disorder such as murine experimental autoimmune encephalomyelitis (EAE) and whether this approach is sensitive enough to evaluate therapeutic interventions. We found that (i) coregistration of metabolic (i.e., microPET) and high-resolution anatomical (i.e., CT) images allows serial quantification of glycolysis with [(18)F]FDG in various spinal column segments; (ii) [(18)F]FDG PET/CT can detect the increased glycolysis associated with paralysis-causing inflammatory infiltrates in the spinal cord; and (iii) the [(18)F]FDG measure of glycolysis in the spinal cord is sensitive to systemic immunosuppressive therapy. These results highlight the potential use of serial [(18)F]FDG PET/CT imaging to monitor neuroinflammation in EAE and suggest that similar approaches could be applied to the diagnosis and evaluation of other autoimmune and inflammatory disorders in animal models and in humans.

Visualization of a primary anti-tumor immune response by positron emission tomography.

Current methodologies that monitor immune responses rely on invasive techniques that sample tissues at a given point in time. New technologies are needed to elucidate the temporal patterns of immune responses and the spatial distribution of immune cells on a whole-body scale. We describe a noninvasive, quantitative, and tomographic approach to visualize a primary anti-tumor immune response by using positron emission tomography (PET). Bone marrow chimeric mice were generated by engraftment of hematopoietic stem and progenitor cells transduced with a trifusion reporter gene encoding synthetic Renilla luciferase (hRluc), EGFP, and Herpes virus thymidine kinase (sr39TK). Mice were challenged with the Moloney murine sarcoma and leukemia virus complex (M-MSV/M-MuLV), and the induced immune response was monitored by using PET. Hematopoietic cells were visualized by using 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG), a radioactive substrate specific for the sr39TK PET reporter protein. Immune cell localization and expansion were seen at the tumor and draining lymph nodes (DLNs). 2-[(18)F]fluoro-2-deoxy-D-glucose ([(18)F]FDG), which is sequestered in metabolically active cells, was used to follow tumor growth and regression. Elevated glucose metabolism was also seen in activated lymphocytes in the DLNs by using the [(18)F]FDG probe. When M-MSV/M-MuLV-challenged mice were treated with the immunosuppressive drug dexamethasone, activation and expansion of immune cell populations in the DLNs could no longer be detected with PET imaging. The method we describe can be used to kinetically measure the induction and therapeutic modulations of cell-mediated immune responses.

The NIT domain: a predicted nitrate-responsive module in bacterial sensory receptors.

A nitrate- and nitrite-sensing (NIT) domain as found in the NasR protein, has been detected in various receptor components of signal transduction pathways in different bacterial lineages. Cellular functions controlled by receptors that contain this novel domain include regulation of gene expression (transcription anti-terminators and histidine kinases), cell motility (chemotaxis receptors) and enzyme activity (diguanylate cyclases and phosphodiesterases).

ANTAR: an RNA-binding domain in transcription antitermination regulatory proteins.

An RNA-binding domain exemplified by the AmiR protein was detected in various response regulators of the two-component systems and in one-component sensory regulators from different bacterial lineages, including important pathogens and symbionts. Transcription antitermination appears to be a common mechanism for controlling gene expression by this family of regulators.