Successful Anti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL-12.

Anti-PD-1 immune checkpoint blockers can induce sustained clinical responses in cancer but how they function in vivo remains incompletely understood. Here, we combined intravital real-time imaging with single-cell RNA sequencing analysis and mouse models to uncover anti-PD-1 pharmacodynamics directly within tumors. We showed that effective antitumor responses required a subset of tumor-infiltrating dendritic cells (DCs), which produced interleukin 12 (IL-12). These DCs did not bind anti-PD-1 but produced IL-12 upon sensing interferon γ (IFN-γ) that was released from neighboring T cells. In turn, DC-derived IL-12 stimulated antitumor T cell immunity. These findings suggest that full-fledged activation of antitumor T cells by anti-PD-1 is not direct, but rather involves T cell:DC crosstalk and is licensed by IFN-γ and IL-12. Furthermore, we found that activating the non-canonical NF-κB transcription factor pathway amplified IL-12-producing DCs and sensitized tumors to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.

An In Vivo Model of Human Macrophages in Metastatic Melanoma.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome, and high-dimensional analyses of patient samples reveal the heterogeneity of the immune TME. Macrophages infiltrate TMEs and generally associate with tumor progression, but the underlying mechanisms are incompletely understood. Because experimental systems are needed to elucidate the functional properties of these cells, we developed a humanized mouse model reconstituted with human immune cells and human melanoma. We used two strains of recipient mice, supporting or not supporting the development of human myeloid cells. We found that human myeloid cells favored metastatic spread of the primary tumor, thereby recapitulating the cancer-supportive role of macrophages. We next analyzed the transcriptome of human immune cells infiltrating tumors versus other tissues. This analysis identified a cluster of myeloid cells present in the TME, but not in other tissues, which do not correspond to canonical M2 cells. The transcriptome of these cells is characterized by high expression of glycolytic enzymes and multiple chemokines and by low expression of gene sets associated with inflammation and adaptive immunity. Compared with humanized mouse results, we found transcriptionally similar myeloid cells in patient-derived samples of melanoma and other cancer types. The humanized mouse model described here thus complements patient sample analyses, enabling further elucidation of fundamental principles in melanoma biology beyond M1/M2 macrophage polarization. The model can also support the development and evaluation of candidate antitumor therapies.

External proficiency testing improves inter-scorer reliability of polysomnography scoring.

PURPOSE: This study evaluated whether or not polysomnography (PSG) inter-scorer reliability (ISR) across sleep centres could be improved by external proficiency testing (EPT), or by EPT combined with method alignment training. METHODS: Experienced scorers form 15 sleep centres were randomised to the following: (1) a control group, (2) a group that received a self-directed intervention of EPT reports (EPTPassive) or (3) a group that received an active intervention of method alignment training and EPT reports (EPTActive). Respiratory, arousal and sleep scoring ISR from sixteen PSG fragments were compared between groups across time. RESULTS: Among 30 scorers, there were no ISR changes in controls between baseline (BL) and 6 months (6 m). Both EPT groups showed ISR improvement from BL to 6 m for respiratory, arousal and sleep scoring (p < 0.05). Respiratory scoring back-transformed mean (95CI) proportion of specific agreement (PSA) for the EPTPassive group improved from 0.78 (0.72-0.84) to 0.80 (0.74-0.86) and for the EPTActive group from 0.80 (0.74-0.85) to 0.82 (0.76-0.88). Arousal scoring PSA for the EPTPassive group improved from 0.72 (0.66-0.77) to 0.74 (0.69-0.79) and for the EPTActive group from 0.71 (0.65-0.76) to 0.77 (0.72-0.82). Sleep scoring kappa for the EPTPassive group improved from 0.64 (0.58-0.69) to 0.73 (0.68-0.77) and for the EPTActive group from = 0.75 (0.71-0.80) to 0.80 (0.76-0.85). Overall, poorer performers achieved greater improvement. CONCLUSION: External proficiency testing produced modest, statistically significant PSG inter-scorer reliability improvements among experienced scorers across sleep centres, with potential to improve clinical management of individual patients and increase research study statistical power.

Bugs, Bumps, and Bacteria: The Role of Virtual Collaborative Learning Network in Pediatric Dermatology.

Patients in rural and underserved areas face significant barriers in accessing specialty care due to unavailability of services, geographic isolation, travel burden, and other cultural and socioeconomic factors.1 Pediatric dermatology is among the top three subspecialties that provides routine care for pediatric patients, however, shortage and maldistribution of pediatric dermatologists have remained a major hurdle for those living in remote and isolated areas.2 Pediatric dermatologists cluster in urban areas with high-patient volume and estimated wait times for new patients that often exceed 13 weeks, making access one of the major drivers of inequity for rural patients.2-4.

Diabetes Control in a Student-Run Free Clinic During the COVID-19 Pandemic.

Student run free health clinics (SRFCs) provide medical care to vulnerable populations in communities throughout the United States. The COVID-19 pandemic had a significant impact on the delivery of healthcare services and demanded a rapid adjustment in care delivery methods in both resource-rich and resource-poor settings. The aim of this study is to evaluate the impact of the pandemic on the management of chronic disease, specifically diabetes. Patients with diabetes who received care continuously throughout the pre-pandemic (face-to-face) and pandemic (telehealth) study periods at MedZou Community Health Center, a SRFC located in central Missouri, were evaluated. This sample of patients (n = 29) was evaluated on six quality measures including annual eye exams, blood pressure, hemoglobin A1c, chronic kidney disease monitoring, flu vaccination, and statin therapy. Overall diabetes care, as measured by the number of quality measures met per patient, decreased by 0.37 after the onset of the pandemic. The median COVID-era ranks were not statistically significantly different than the pre-pandemic ranks (z = 1.65, P = 0.099). Fewer patients received an influenza vaccination the year following the onset of the pandemic (10.3%) compared to the year before the pandemic (37.9%; difference in proportions 0.276, 95% CI 0.079, 0.473; p = 0.005). No other individual measures of diabetes care statistically differed significantly in the year after the pandemic began. Twenty-six (90%) patients received diabetes care using telehealth after the onset of the pandemic. Diabetes care using telehealth in a SRFC may be an acceptable alternative model when face-to-face visits are not feasible. Observed decreases in diabetes-related clinical quality measure performance warrant further study.

Impact of the COVID-19 Pandemic & Telehealth Implementation in a Student Run Free Clinic.

Student run free clinics (SRFCs) fill a void in healthcare access for many communities and have been subject to unprecedented shifts in care delivery brought about by the coronavirus disease 2019 (COVID-19) pandemic. Our single-center institution serving uninsured patients in central Missouri switched from in-person visits to strictly telehealth visits with the onset of the pandemic. This study investigated the impact of the pandemic and the switch to telehealth on the clinic return rates by ethnicity, race, gender, rurality, and age. The pandemic led to a 47.4% reduction in the number of monthly patient encounters. Of the established SRFC population (N = 309), only 87 patients (28.2%) returned for a telehealth visit during the COVID-19 pandemic. Older patients (≥ 45 years old) were more likely to return (OR 1.71, 95% CI 1.02-2.85) for care via telehealth after the onset of the pandemic than younger patients (< 45 years old). No differences in the likelihood of returning for a telehealth visit were identified by race, ethnicity, gender, or rurality. Telehealth offers an effective solution to the complex problems faced by SRFCs during the COVID-19 pandemic and has not added barriers to care with regards to race, ethnicity, gender, or rurality at our SRFC.

PD-1 blockade induces responses by inhibiting adaptive immune resistance.

Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8(+) T cells (termed adaptive immune resistance). Here we show that pre-existing CD8(+) T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8(+) T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8(+) T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance.

Characterization of abscopal effects of intratumoral electroporation-mediated IL-12 gene therapy.

Intratumoral electroporation-mediated IL-12 gene therapy (IT-pIL12/EP) has been shown to be safe and effective in clinical trials, demonstrating systemic antitumor effects with local delivery of this potent cytokine. We recently optimized our IL-12 gene delivery platform to increase transgene expression and efficacy in preclinical models. Here we analyze the immunological changes induced with the new IT-pIL12/EP platform in both electroporated and distant, non-electroporated lesions. IT-pIL12/EP-treated tumors demonstrated rapid induction of IL-12-regulated pathways, as well as other cytokines and chemokines pathways, and upregulation of antigen presentation machinery. The distant tumors showed an increase in infiltrating lymphocytes and gene expression changes indicative of a de novo immune response in these untreated lesions. Flow cytometric analyses revealed a KLRG1hi CD8+ effector T-cell population uniquely present in mice treated with IT-pIL12/EP. Despite being highly activated, this population expressed diminished levels of PD-1 when re-exposed to antigen in the PD-L1-rich tumor. Other T-cell exhaustion markers appeared to be downregulated in concert, suggesting an orchestrated "armoring" of these effector T cells against T-cell checkpoints when primed in the presence of IL-12 in situ. These cells may represent an important mechanism by which local IL-12 gene therapy can induce a systemic antitumor immune response without the associated toxicity of systemic IL-12 exposure.

Positive airway pressure for sleep-disordered breathing in acute quadriplegia: a randomised controlled trial.

RATIONALE: Highly prevalent and severe sleep-disordered breathing caused by acute cervical spinal cord injury (quadriplegia) is associated with neurocognitive dysfunction and sleepiness and is likely to impair rehabilitation. OBJECTIVE: To determine whether 3 months of autotitrating CPAP would improve neurocognitive function, sleepiness, quality of life, anxiety and depression more than usual care in acute quadriplegia. METHODS AND MEASUREMENTS: Multinational, randomised controlled trial (11 centres) from July 2009 to October 2015. The primary outcome was neurocognitive (attention and information processing as measure with the Paced Auditory Serial Addition Task). Daytime sleepiness (Karolinska Sleepiness Scale) was a priori identified as the most important secondary outcome. MAIN RESULTS: 1810 incident cases were screened. 332 underwent full, portable polysomnography, 273 of whom had an apnoea hypopnoea index greater than 10. 160 tolerated at least 4 hours of CPAP during a 3-day run-in and were randomised. 149 participants (134 men, age 46±34 years, 81±57 days postinjury) completed the trial. CPAP use averaged 2.9±2.3 hours per night with 21% fully 'adherent' (at least 4 hours use on 5 days per week). Intention-to-treat analyses revealed no significant differences between groups in the Paced Auditory Serial Addition Task (mean improvement of 2.28, 95% CI -7.09 to 11.6; p=0.63). Controlling for premorbid intelligence, age and obstructive sleep apnoea severity (group effect -1.15, 95% CI -10 to 7.7) did not alter this finding. Sleepiness was significantly improved by CPAP on intention-to-treat analysis (mean difference -1.26, 95% CI -2.2 to -0.32; p=0.01). CONCLUSION: CPAP did not improve Paced Auditory Serial Addition Task scores but significantly reduced sleepiness after acute quadriplegia. TRIAL REGISTRATION NUMBER: ACTRN12605000799651.

Intratumoral delivery of an HPV vaccine elicits a broad anti-tumor immune response that translates into a potent anti-tumor effect in a preclinical murine HPV model.

Therapeutic cancer vaccines have met limited clinical success. In the setting of cancer, the immune system is either tolerized and/or has a limited tumor-specific T cell repertoire. In this study, we explore whether intratumoral (IT) vaccination with an HPV vaccine can elicit quantitative and qualitative differences in immune response as compared to intramuscular (IM) vaccination to overcome immune resistance in established tumors. We report that IT administration of an HPV-16 E7 peptide vaccine formulated with polyinosinic-polycytidylic acid [poly(I:C)] generated an enhanced antitumor effect relative to IM delivery. The elicited anti-tumor effect with IT vaccination was consistent among the vaccinated groups and across various C57BL/6 substrains. IT vaccination resulted in an increased frequency of PD-1hi TILs, which represented both vaccine-targeted and non-vaccine-targeted tumor-specific CD8+ T cells. Overall, the CD8+/Treg ratio was increased within the tumor microenvironment using IT vaccination. We also assessed transcriptional changes in several immune-related genes in the tumor microenvironment of the various treated groups, and our data suggest that IT vaccination leads to upregulation of a broad complement of immunomodulatory genes, including upregulation of interferon gamma (IFNγ) and antigen presentation and processing machine (APM) components. IT vaccine delivery is superior to traditional IM vaccination routes with the potential to improve tumor immunogenicity, which has potential clinical application in the setting of accessible lesions such as head and neck squamous cell carcinomas (HNSCCs).

Assessing Tumor-infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcinoma In Situ, Metastatic Tumor Deposits and Areas for Further Research.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Assessing Tumor-Infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method from the International Immuno-Oncology Biomarkers Working Group: Part 2: TILs in Melanoma, Gastrointestinal Tract Carcinomas, Non-Small Cell Lung Carcinoma and Mesothelioma, Endometrial and Ovarian Carcinomas, Squamous Cell Carcinoma of the Head and Neck, Genitourinary Carcinomas, and Primary Brain Tumors.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

IL-23 is critical for induction of arthritis, osteoclast formation, and maintenance of bone mass.

The role of IL-23 in the development of arthritis and bone metabolism was studied using systemic IL-23 exposure in adult mice via hydrodynamic delivery of IL-23 minicircle DNA in vivo and in mice genetically deficient in IL-23. Systemic IL-23 exposure induced chronic arthritis, severe bone loss, and myelopoiesis in the bone marrow and spleen, which resulted in increased osteoclast differentiation and systemic bone loss. The effect of IL-23 was partly dependent on CD4(+) T cells, IL-17A, and TNF, but could not be reproduced by overexpression of IL-17A in vivo. A key role in the IL-23-induced arthritis was made by the expansion and activity of myeloid cells. Bone marrow macrophages derived from IL-23p19(-/-) mice showed a slower maturation into osteoclasts with reduced tartrate-resistant acid phosphatase-positive cells and dentine resorption capacity in in vitro osteoclastogenesis assays. This correlated with fewer multinucleated osteoclast-like cells and more trabecular bone volume and number in 26-wk-old male IL-23p19(-/-) mice compared with control animals. Collectively, our data suggest that systemic IL-23 exposure induces the expansion of a myeloid lineage osteoclast precursor, and targeting IL-23 pathway may combat inflammation-driven bone destruction as observed in rheumatoid arthritis and other autoimmune arthritides.

Disparities in the use of telehealth at the onset of the COVID-19 public health emergency.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic resulted in an unprecedented expansion in telehealth, but little is known about differential use of telehealth according to demographics, rurality, or insurance status. METHODS: We performed a cross-sectional analysis of 7742 family medicine encounters at a single USA institution in the initial month of the COVID-19 public health emergency (PHE). We compared the demographics of those using telehealth during the PHE to those with face-to-face visits during the same time period; we also compared the demographics of those using full audio-video to those using audio-only. RESULTS: The likelihood of any telehealth visit in the first 30 days of telehealth expansion was higher for women, those age 65 years and older, self-pay patients, and those with Medicaid and Medicare as primary payers. The likelihood of a telehealth visit was reduced for rural residence and Black or other races. Among all telehealth visits, the likelihood of a full audio-video telehealth visit was reduced for patients who were older, Black, from urban areas, or who were self-pay, Medicaid, or Medicare payer status. DISCUSSION: Significant disparities exist in telehealth use during the COVID-19 PHE by age, race, residence and payer.

Conditionally Active, pH-Sensitive Immunoregulatory Antibodies Targeting VISTA and CTLA-4 Lead an Emerging Class of Cancer Therapeutics.

Immune checkpoints and other immunoregulatory targets can be difficult to precisely target due to expression on non-tumor immune cells critical to maintaining immune homeostasis in healthy tissues. On-target/off-tumor binding of therapeutics results in significant pharmacokinetic and pharmacodynamic problems. Target-mediated drug disposition (TMDD) significantly limits effective intratumoral drug levels and adversely affects anti-tumor efficacy. Target engagement outside the tumor environment may lead to severe immune-related adverse events (irAEs), resulting in a narrowing of the therapeutic window, sub-optimal dosing, or cessation of drug development altogether. Overcoming these challenges has become tractable through recent advances in antibody engineering and screening approaches. Here, we review the discovery and development of conditionally active antibodies with minimal binding to target at physiologic pH but high-affinity target binding at the low pH of the tumor microenvironment by focusing on the discovery and improved properties of pH-dependent mAbs targeting two T cell checkpoints, VISTA and CTLA-4.

Effects of HLA single chain trimer design on peptide presentation and stability.

MHC class I "single-chain trimer" molecules, coupling MHC heavy chain, ß2-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation.

Toll-Like Receptor 4 Agonist Injection With Concurrent Radiotherapy in Patients With Metastatic Soft Tissue Sarcoma: A Phase 1 Nonrandomized Controlled Trial.

Importance: Metastatic soft tissue sarcomas (STSs) have limited systemic therapy options, and immunomodulation has not yet meaningfully improved outcomes. Intratumoral (IT) injection of the toll-like receptor 4 (TLR4) agonist glycopyranosyl lipid A in stable-emulsion formulation (GLA-SE) has been studied as immunotherapy in other contexts. Objective: To evaluate the safety, efficacy, and immunomodulatory effects of IT GLA-SE with concurrent radiotherapy in patients with metastatic STS with injectable lesions. Design, Setting, and Participants: This phase 1 nonrandomized controlled trial of patients with STS was performed at a single academic sarcoma specialty center from November 17, 2014, to March 16, 2016. Data analysis was performed from August 2016 to September 2022. Interventions: Two doses of IT GLA-SE (5 µg and 10 µg for 8 weekly doses) were tested for safety in combination with concurrent radiotherapy of the injected lesion. Main Outcomes and Measures: Primary end points were safety and tolerability. Secondary and exploratory end points included local response rates as well as measurement of antitumor immunity with immunohistochemistry and T-cell receptor (TCR) sequencing of tumor-infiltrating and circulating lymphocytes. Results: Twelve patients (median [range] age, 65 [34-78] years; 8 [67%] female) were treated across the 2 dose cohorts. Intratumoral GLA-SE was well tolerated, with only 1 patient (8%) experiencing a grade 2 adverse event. All patients achieved local control of the injected lesion after 8 doses, with 1 patient having complete regression (mean regression, -25%; range, -100% to 4%). In patients with durable local response, there were detectable increases in tumor-infiltrating lymphocytes. In 1 patient (target lesion -39% at 259 days of follow-up), TCR sequencing revealed expansion of preexisting and de novo clonotypes, with convergence of numerous rearrangements coding for the same binding sequence (suggestive of clonal convergence to antitumor targets). Single-cell sequencing identified these same expanded TCR clones in peripheral blood after treatment; these T cells had markedly enhanced Tbet expression, suggesting TH1 phenotype. Conclusions and Relevance: In this nonrandomized controlled trial, IT GLA-SE with concurrent radiotherapy was well tolerated and provided more durable local control than radiotherapy alone. Patients with durable local response demonstrated enhanced IT T-cell clonal expansion, with matched expansion of these clonotypes in the circulation. Additional studies evaluating synergism of IT GLA-SE and radiotherapy with systemic immune modulation are warranted. Trial Registration: ClinicalTrials.gov Identifier: NCT02180698.

Ineffective CD8(+) T-cell immunity to adeno-associated virus can result in prolonged liver injury and fibrogenesis.

Chronic viral hepatitis depends on the inability of the T-cell immune response to eradicate antigen. This results in a sustained immune response accompanied by tissue injury and fibrogenesis. We have created a mouse model that reproduces these effects, based on the response of CD8(+) T cells to hepatocellular antigen delivered by an adeno-associated virus (AAV) vector. Ten thousand antigen-specific CD8(+) T cells undergo slow expansion in the liver and can precipitate a subacute inflammatory hepatitis with stellate cell activation and fibrosis. Over time, antigen-specific CD8(+) T cells show signs of exhaustion, including high expression of PD-1, and eventually both inflammation and fibrosis resolve. This model allows the investigation of both chronic liver immunopathology and its resolution.