Type I interferon activates MHC class I-dressed CD11b+ conventional dendritic cells to promote protective anti-tumor CD8+ T cell immunity.

Tumor-infiltrating dendritic cells (DCs) assume varied functional states that impact anti-tumor immunity. To delineate the DC states associated with productive anti-tumor T cell immunity, we compared spontaneously regressing and progressing tumors. Tumor-reactive CD8+ T cell responses in Batf3-/- mice lacking type 1 DCs (DC1s) were lost in progressor tumors but preserved in regressor tumors. Transcriptional profiling of intra-tumoral DCs within regressor tumors revealed an activation state of CD11b+ conventional DCs (DC2s) characterized by expression of interferon (IFN)-stimulated genes (ISGs) (ISG+ DCs). ISG+ DC-activated CD8+ T cells ex vivo comparably to DC1. Unlike cross-presenting DC1, ISG+ DCs acquired and presented intact tumor-derived peptide-major histocompatibility complex class I (MHC class I) complexes. Constitutive type I IFN production by regressor tumors drove the ISG+ DC state, and activation of MHC class I-dressed ISG+ DCs by exogenous IFN-ß rescued anti-tumor immunity against progressor tumors in Batf3-/- mice. The ISG+ DC gene signature is detectable in human tumors. Engaging this functional DC state may present an approach for the treatment of human disease.

Anti-TIGIT antibody improves PD-L1 blockade through myeloid and Treg cells.

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

Alum-anchored intratumoral retention improves the tolerability and antitumor efficacy of type I interferon therapies.

Effective antitumor immunity in mice requires activation of the type I interferon (IFN) response pathway. IFNα and IFNß therapies have proven promising in humans, but suffer from limited efficacy and high toxicity. Intratumoral IFN retention ameliorates systemic toxicity, but given the complexity of IFN signaling, it was unclear whether long-term intratumoral retention of type I IFNs would promote or inhibit antitumor responses. To this end, we compared the efficacy of IFNα and IFNß that exhibit either brief or sustained retention after intratumoral injection in syngeneic mouse tumor models. Significant enhancement in tumor retention, mediated by anchoring these IFNs to coinjected aluminum-hydroxide (alum) particles, greatly improved both their tolerability and efficacy. The improved efficacy of alum-anchored IFNs could be attributed to sustained pleiotropic effects on tumor cells, immune cells, and nonhematopoietic cells. Alum-anchored IFNs achieved high cure rates of B16F10 tumors upon combination with either anti-PD-1 antibody or interleukin-2. Interestingly however, these alternative combination immunotherapies yielded disparate T cell phenotypes and differential resistance to tumor rechallenge, highlighting important distinctions in adaptive memory formation for combinations of type I IFNs with other immunotherapies.

Public Health Dashboards in Overdose Prevention: The Rhode Island Approach to Public Health Data Literacy, Partnerships, and Action.

As the field of public health rises to the demands of real-time surveillance and rapid data-sharing needs in a postpandemic world, it is time to examine our approaches to the dissemination and accessibility of such data. Distinct challenges exist when working to develop a shared public health language and narratives based on data. It requires that we assess our understanding of public health data literacy, revisit our approach to communication and engagement, and continuously evaluate our impact and relevance. Key stakeholders and cocreators are critical to this process and include people with lived experience, community organizations, governmental partners, and research institutions. In this viewpoint paper, we offer an instructive approach to the tools we used, assessed, and adapted across 3 unique overdose data dashboard projects in Rhode Island, United States. We are calling this model the "Rhode Island Approach to Public Health Data Literacy, Partnerships, and Action." This approach reflects the iterative lessons learned about the improvement of data dashboards through collaboration and strong partnerships across community members, state agencies, and an academic research team. We will highlight key tools and approaches that are accessible and engaging and allow developers and stakeholders to self-assess their goals for their data dashboards and evaluate engagement with these tools by their desired audiences and users.

Variable processing and cross-presentation of HIV by dendritic cells and macrophages shapes CTL immunodominance and immune escape.

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8⁺ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.

Different antigen-processing activities in dendritic cells, macrophages, and monocytes lead to uneven production of HIV epitopes and affect CTL recognition.

Dendritic cells (DCs), macrophages (MPs), and monocytes are permissive to HIV. Whether they similarly process and present HIV epitopes to HIV-specific CD8 T cells is unknown despite the critical role of peptide processing and presentation for recognition and clearance of infected cells. Cytosolic peptidases degrade endogenous proteins originating from self or pathogens, exogenous Ags preprocessed in endolysosomes, thus shaping the peptidome available for endoplasmic reticulum translocation, trimming, and MHC-I presentation. In this study, we compared the capacity of DCs, MPs, and monocyte cytosolic extracts to produce epitope precursors and epitopes. We showed differences in the proteolytic activities and expression levels of cytosolic proteases between monocyte-derived DCs and MPs and upon maturation with LPS, R848, and CL097, with mature MPs having the highest activities. Using cytosol as a source of proteases to degrade epitope-containing HIV peptides, we showed by mass spectrometry that the degradation patterns of long peptides and the kinetics and amount of antigenic peptides produced differed among DCs, MPs, and monocytes. Additionally, variable intracellular stability of HIV peptides prior to loading onto MHC may accentuate the differences in epitope availability for presentation by MHC-I between these subsets. Differences in peptide degradation led to 2- to 25-fold differences in the CTL responses elicited by the degradation peptides generated in DCs, MPs, and monocytes. Differences in Ag-processing activities between these subsets might lead to variations in the timing and efficiency of recognition of HIV-infected cells by CTLs and contribute to the unequal capacity of HIV-specific CTLs to control viral load.

SOMAScience: A Novel Platform for Multidimensional, Longitudinal Pain Assessment.

Chronic pain is one of the most significant health issues in the United States, affecting more than 20% of the population. Despite its contribution to the increasing health crisis, reliable predictors of disease development, progression, or treatment outcomes are lacking. Self-report remains the most effective way to assess pain, but measures are often acquired in sparse settings over short time windows, limiting their predictive ability. In this paper, we present a new mobile health platform called SOMAScience. SOMAScience serves as an easy-to-use research tool for scientists and clinicians, enabling the collection of large-scale pain datasets in single- and multicenter studies by facilitating the acquisition, transfer, and analysis of longitudinal, multidimensional, self-report pain data. Data acquisition for SOMAScience is done through a user-friendly smartphone app, SOMA, that uses experience sampling methodology to capture momentary and daily assessments of pain intensity, unpleasantness, interference, location, mood, activities, and predictions about the next day that provide personal insights into daily pain dynamics. The visualization of data and its trends over time is meant to empower individual users' self-management of their pain. This paper outlines the scientific, clinical, technological, and user considerations involved in the development of SOMAScience and how it can be used in clinical studies or for pain self-management purposes. Our goal is for SOMAScience to provide a much-needed platform for individual users to gain insight into the multidimensional features of their pain while lowering the barrier for researchers and clinicians to obtain the type of pain data that will ultimately lead to improved prevention, diagnosis, and treatment of chronic pain.

A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells.

BACKGROUND: Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions. RESULTS: In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes. CONCLUSION: By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.

Proteomic analysis of lipid raft-enriched membranes isolated from internal organelles.

The mitochondria-associated membrane (MAM) is a sub-region of the endoplasmic reticulum (ER) that facilitates crosstalk between the ER and mitochondria. The MAM actively influences vital cellular processes including Ca(2+) signaling and protein folding. Detergent-resistant microdomains (DRMs) may localize proteins to the mitochondria/MAM interface to coordinate these events. However, the protein composition of DRMs isolated from this region is not known. Lipid-raft enriched DRMs were isolated from a combined mitochondria/MAM sample and analyzed using two-dimensional reversed-phased tandem mass spectrometry. Strict post-acquisition filtering of the acquired data led to the confident identification 250 DRM proteins. The majority (58%) of the identified proteins are bona fide mitochondrial or ER proteins according to Gene Ontology annotation. Additionally, 74% of the proteins have previously been noted as MAM-resident or -associated proteins. Furthermore, ∼20% of the identified proteins have a documented association with lipid rafts. Most importantly, known internal LR marker proteins (inositol 1,4,5-trisphosphate receptor type 3, erlin-2, and voltage-dependent anion channel 1) were detected as well as most of the components of the mitochondrial/MAM-localized Ca(2+) signaling complex. Our study provides the basis for future work probing how the protein activities at the mitochondrion/MAM interface are dependent upon the integrity of these internal lipid-raft-like domains.

Impact of anatomic site on antigen-presenting cells in cancer.

Checkpoint blockade immunotherapy (CBT) can induce long-term clinical benefits in patients with advanced cancer; however, response rates to CBT vary by cancer type. Cancers of the skin, lung, and kidney are largely responsive to CBT, while cancers of the pancreas, ovary, breast, and metastatic lesions to the liver respond poorly. The impact of tissue-resident immune cells on antitumor immunity is an emerging area of investigation. Recent evidence indicates that antitumor immune responses and efficacy of CBT depend on the tissue site of the tumor lesion. As myeloid cells are predominantly tissue-resident and can shape tumor-reactive T cell responses, it is conceivable that tissue-specific differences in their function underlie the tissue-site-dependent variability in CBT responses. Understanding the roles of tissue-specific myeloid cells in antitumor immunity can open new avenues for treatment design. In this review, we discuss the roles of tissue-specific antigen-presenting cells (APCs) in governing antitumor immune responses, with a particular focus on the contributions of tissue-specific dendritic cells. Using the framework of the Cancer-Immunity Cycle, we examine the contributions of tissue-specific APC in CBT-sensitive and CBT-resistant carcinomas, highlight how these cells can be therapeutically modulated, and identify gaps in knowledge that remain to be addressed.

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.

A team effort: natural killer cells on the first leg of the tumor immunity relay race.

Recent work by Böttcher and colleagues defines a new role for Natural Killer cells in the anti-tumor immune response, arriving early into the tumor microenvironment before passing the baton to DC1 dendritic cells. DC1 dendritic cells subsequently activate CD8+ T cells resulting in effective anti-tumor immunity. This work highlights the cooperative nature of anti-tumor immunity set in motion by Natural Killer cells, and immune evasion by tumors through their exclusion.

A real-time killing assay to follow viral epitope presentation to CD8 T cells.

The ability of cytotoxic T lymphocytes (CTL) to clear virus-infected cells requires the presentation of viral peptides intracellularly processed and displayed by major histocompatibility complex class I. Assays to measure CTL-mediated killing often use peptides exogenously added onto target cells--which does not account for epitope processing--or follow killing of infected cells at a single time point. In this study we established a real-time fluorogenic cytotoxic assay that measures the release of the Glucose-6-phosphate-dehydrogenase by dying target cells every 5 min after addition of CTL. It has comparable sensitivity to (51)chromium-based killing assay with the additional advantage of incorporating the kinetics of epitope presentation. We showed that HIV infection of immortalized or primary CD4 T cells leads to asynchronous killing by two CTL clones specific for epitopes located in different proteins. Real-time monitoring of killing of virus-infected cells will enable identification of immune responses efficiently preventing virus dissemination.