Neoantigen-specific cytotoxic Tr1 CD4 T cells suppress cancer immunotherapy.

CD4+ T cells can either enhance or inhibit tumour immunity. Although regulatory T cells have long been known to impede antitumour responses1-5, other CD4+ T cells have recently been implicated in inhibiting this response6,7. Yet, the nature and function of the latter remain unclear. Here, using vaccines containing MHC class I (MHC-I) neoantigens (neoAgs) and different doses of tumour-derived MHC-II neoAgs, we discovered that whereas the inclusion of vaccines with low doses of MHC-II-restricted peptides (LDVax) promoted tumour rejection, vaccines containing high doses of the same MHC-II neoAgs (HDVax) inhibited rejection. Characterization of the inhibitory cells induced by HDVax identified them as type 1 regulatory T (Tr1) cells expressing IL-10, granzyme B, perforin, CCL5 and LILRB4. Tumour-specific Tr1 cells suppressed tumour rejection induced by anti-PD1, LDVax or adoptively transferred tumour-specific effector T cells. Mechanistically, HDVax-induced Tr1 cells selectively killed MHC-II tumour antigen-presenting type 1 conventional dendritic cells (cDC1s), leading to low numbers of cDC1s in tumours. We then documented modalities to overcome this inhibition, specifically via anti-LILRB4 blockade, using a CD8-directed IL-2 mutein, or targeted loss of cDC2/monocytes. Collectively, these data show that cytotoxic Tr1 cells, which maintain peripheral tolerance, also inhibit antitumour responses and thereby function to impede immune control of cancer.

Post-immunotherapy CTLA-4 Ig treatment improves antitumor efficacy.

Immune checkpoint therapies (ICT) improve overall survival of patients with cancer but may cause immune-related adverse events (irAEs) such as myocarditis. Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin fusion protein (CTLA-4 Ig), an inhibitor of T cell costimulation through CD28, reverses irAEs in animal models. However, concerns exist about potentially compromising antitumor response of ICT. In mouse tumor models, we administered CTLA-4 Ig 1) concomitantly with ICT or 2) after ICT completion. Concomitant treatment reduced antitumor efficacy, while post-ICT administration improved efficacy without affecting frequency and function of CD8 T cells. The improved response was independent of the ICT used, whether CTLA-4 or PD-1 blockade. The frequency of Tregs was significantly decreased with CTLA-4 Ig. The resulting increased CD8/Treg ratio potentially underlies the enhanced efficacy of ICT followed by CTLA-4 Ig. This paradoxical mechanism shows that a CTLA-4 Ig regimen shown to reduce irAE severity does not compromise antitumor efficacy.

Pancreatic Epithelial IL17/IL17RA Signaling Drives B7-H4 Expression to Promote Tumorigenesis.

IL17 is required for the initiation and progression of pancreatic cancer, particularly in the context of inflammation, as previously shown by genetic and pharmacological approaches. However, the cellular compartment and downstream molecular mediators of IL17-mediated pancreatic tumorigenesis have not been fully identified. This study examined the cellular compartment required by generating transgenic animals with IL17 receptor A (IL17RA), which was genetically deleted from either the pancreatic epithelial compartment or the hematopoietic compartment via generation of IL17RA-deficient (IL17-RA-/-) bone marrow chimeras, in the context of embryonically activated or inducible Kras. Deletion of IL17RA from the pancreatic epithelial compartment, but not from hematopoietic compartment, resulted in delayed initiation and progression of premalignant lesions and increased infiltration of CD8+ cytotoxic T cells to the tumor microenvironment. Absence of IL17RA in the pancreatic compartment affected transcriptional profiles of epithelial cells, modulating stemness, and immunological pathways. B7-H4, a known inhibitor of T-cell activation encoded by the gene Vtcn1, was the checkpoint molecule most upregulated via IL17 early during pancreatic tumorigenesis, and its genetic deletion delayed the development of pancreatic premalignant lesions and reduced immunosuppression. Thus, our data reveal that pancreatic epithelial IL17RA promotes pancreatic tumorigenesis by reprogramming the immune pancreatic landscape, which is partially orchestrated by regulation of B7-H4. Our findings provide the foundation of the mechanisms triggered by IL17 to mediate pancreatic tumorigenesis and reveal the avenues for early pancreatic cancer immune interception. See related Spotlight by Lee and Pasca di Magliano, p. 1130.

Molecular Pathways and Cellular Subsets Associated with Adverse Clinical Outcomes in Overlapping Immune-Related Myocarditis and Myositis.

Immune checkpoint therapies (ICT) can induce life-threatening immune-related adverse events, including myocarditis and myositis, which are rare but often concurrent. The molecular pathways and immune subsets underlying these toxicities remain poorly understood. To address this need, we performed single-cell RNA sequencing of heart and skeletal muscle biopsies obtained from living patients with cancers treated with ICTs and admitted to the hospital with myocarditis and/or myositis (overlapping myocarditis plus myositis, n = 10; myocarditis-only, n = 1) or ICT-exposed patients ruled out for toxicity utilized as controls (n = 9). All biopsies were obtained within 96 hours of clinical presentation. Analyses of 58,523 cells revealed CD8+ T cells with a cytotoxic phenotype expressing activation/exhaustion markers in both myocarditis and myositis. Furthermore, the analyses identified a population of myeloid cells expressing tissue-resident signatures and FcγRIIIa (CD16a), which is known to bind IgG and regulate complement activation. Immunohistochemistry of affected cardiac and skeletal muscle tissues revealed protein expression of pan-IgG and complement product C4d, which were associated with the presence of high-titer serum autoantibodies against muscle antigens in a subset of patients. We further identified a population of inflammatory IL1B+TNF+ myeloid cells specifically enriched in myocarditis and associated with greater toxicity severity and poorer clinical outcomes. These results provide insight into the myeloid subsets present in human immune-related myocarditis and myositis tissues and nominate new targets for investigation into rational treatments to overcome these high-mortality toxicities. See related Spotlight by Fankhauser et al., p. 954.

Other Secondary Headaches: Odontogenic Pain and Other Painful Orofacial Conditions.

This article discusses extremely common odontogenic pain conditions, which may occasionally present to the neurology clinic mimicking headache, and other uncommon orofacial pain conditions, which may do the same. Typical presentations, investigative strategies, and management are discussed, as well as highlighting key diagnostic criteria and the importance of involving oral or dental specialists where diagnostic uncertainty exists.

A Modular Trial of Androgen Signaling Inhibitor Combinations Testing a Risk-Adapted Strategy in Patients with Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: To determine the efficacy and safety of risk-adapted combinations of androgen signaling inhibitors and inform disease classifiers for metastatic castration-resistant prostate cancers. PATIENTS AND METHODS: In a modular, randomized phase II trial, 192 men were treated with 8 weeks of abiraterone acetate, prednisone, and apalutamide (AAPA; module 1) and then allocated to modules 2 or 3 based on satisfactory (≥50% PSA decline from baseline and <5 circulating tumor cell/7.5 mL) versus unsatisfactory status. Men in the former were randomly assigned to continue AAPA alone (module 2A) or with ipilimumab (module 2B). Men in the latter group had carboplatin + cabazitaxel added to AAPA (module 3). Optional baseline biopsies were subjected to correlative studies. RESULTS: Median overall survival (from allocation) was 46.4 [95% confidence interval (CI), 39.2-68.2], 41.4 (95% CI, 33.3-49.9), and 18.7 (95% CI, 14.3-26.3) months in modules 2A (n = 64), 2B (n = 64), and 3 (n = 59), respectively. Toxicities were within expectations. Of 192 eligible patients, 154 (80.2%) underwent pretreatment metastatic biopsies. The aggressive-variant prostate cancer molecular profile (defects in ≥2 of p53, RB1, and PTEN) was associated with unsatisfactory status. Exploratory analyses suggested that secreted phosphoprotein 1-positive and insulin-like growth factor-binding protein 2-positive macrophages, druggable myeloid cell markers, and germline pathogenic mutations were enriched in the unsatisfactory group. CONCLUSIONS: Adding ipilimumab to AAPA did not improve outcomes in men with androgen-responsive metastatic castration-resistant prostate cancer. Despite the addition of carboplatin + cabazitaxel, men in the unsatisfactory group had shortened survivals. Adaptive designs can enrich for biologically and clinically relevant disease subgroups to contribute to the development of marker-informed, risk-adapted therapy strategies in men with prostate cancer.

ICOS costimulation in combination with CTLA-4 blockade remodels tumor-associated macrophages toward an antitumor phenotype.

We have previously demonstrated synergy between ICOS costimulation (IVAX; ICOSL-transduced B16-F10 cellular vaccine) and CTLA-4 blockade in antitumor therapy. In this study, we employed CyTOF and single-cell RNA sequencing and observed significant remodeling of the lymphoid and myeloid compartments in combination therapy. Compared with anti-CTLA-4 monotherapy, the combination therapy enriched Th1 CD4 T cells, effector CD8 T cells, and M1-like antitumor proinflammatory macrophages. These macrophages were critical to the therapeutic efficacy of anti-CTLA-4 combined with IVAX or anti-PD-1. Macrophage depletion with clodronate reduced the tumor-infiltrating effector CD4 and CD8 T cells, impairing their antitumor functions. Furthermore, the recruitment and polarization of M1-like macrophages required IFN-γ. Therefore, in this study, we show that there is a positive feedback loop between intratumoral effector T cells and tumor-associated macrophages (TAMs), in which the IFN-γ produced by the T cells polarizes the TAMs into M1-like phenotype, and the TAMs, in turn, reshape the tumor microenvironment to facilitate T cell infiltration, immune function, and tumor rejection.

The future of targeting cytotoxic T-lymphocyte-associated protein-4: Is there a role?

The 2022 yearly Think Tank Meeting in Siena, Tuscany (Italy), organized by the Italian Network for Tumor Biotherapy (NIBIT) Foundation, the Parker Institute for Cancer Immunotherapy and the World Immunotherapy Council, included a focus on the future of integrating and expanding the use of targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). The conference members exchanged their views on the lessons from targeting CTLA-4 and compared the effect to the impact of blocking Programmed cell death protein 1 (PD1) or its ligand (PDL1). The increasing experience with both therapeutic approaches and their combination suggests that targeting CTLA-4 may lead to more durable responses for a sizeable proportion of patients, though the specific mechanism is not entirely understood. Overcoming toxicity of blocking CTLA-4 is currently being addressed with different doses and dose regimens, especially when combined with PD1/PDL1 blocking antibodies. Novel therapeutics targeting CTLA-4 hold the promise to reduce toxicities and thus allow different combination strategies in the future. On the whole, the consent was that targeting CTLA-4 remains an important strategy to improve the efficacy of cancer immunotherapies.

Exercise Training Reduces the Inflammatory Response and Promotes Intestinal Mucosa-Associated Immunity in Lynch Syndrome.

PURPOSE: Lynch syndrome (LS) is a hereditary condition with a high lifetime risk of colorectal and endometrial cancers. Exercise is a non-pharmacologic intervention to reduce cancer risk, though its impact on patients with LS has not been prospectively studied. Here, we evaluated the impact of a 12-month aerobic exercise cycling intervention in the biology of the immune system in LS carriers. PATIENTS AND METHODS: To address this, we enrolled 21 patients with LS onto a non-randomized, sequential intervention assignation, clinical trial to assess the effect of a 12-month exercise program that included cycling classes 3 times weekly for 45 minutes versus usual care with a one-time exercise counseling session as control. We analyzed the effects of exercise on cardiorespiratory fitness, circulating, and colorectal-tissue biomarkers using metabolomics, gene expression by bulk mRNA sequencing, and spatial transcriptomics by NanoString GeoMx. RESULTS: We observed a significant increase in oxygen consumption (VO2peak) as a primary outcome of the exercise and a decrease in inflammatory markers (prostaglandin E) in colon and blood as the secondary outcomes in the exercise versus usual care group. Gene expression profiling and spatial transcriptomics on available colon biopsies revealed an increase in the colonic mucosa levels of natural killer and CD8+ T cells in the exercise group that were further confirmed by IHC studies. CONCLUSIONS: Together these data have important implications for cancer interception in LS, and document for the first-time biological effects of exercise in the immune system of a target organ in patients at-risk for cancer.

Inflammation induced by tumor-associated nerves promotes resistance to anti-PD-1 therapy in cancer patients and is targetable by interleukin-6 blockade.

While the nervous system has reciprocal interactions with both cancer and the immune system, little is known about the potential role of tumor associated nerves (TANs) in modulating anti-tumoral immunity. Moreover, while peri-neural invasion is a well establish poor prognostic factor across cancer types, the mechanisms driving this clinical effect remain unknown. Here, we provide clinical and mechniastic association between TANs damage and resistance to anti-PD-1 therapy. Using electron microscopy, electrical conduction studies, and tumor samples of cutaneous squamous cell carcinoma (cSCC) patients, we showed that cancer cells can destroy myelin sheath and induce TANs degeneration. Multi-omics and spatial analyses of tumor samples from cSCC patients who underwent neoadjuvant anti-PD-1 therapy demonstrated that anti-PD-1 non-responders had higher rates of peri-neural invasion, TANs damage and degeneration compared to responders, both at baseline and following neoadjuvant treatment. Tumors from non-responders were also characterized by a sustained signaling of interferon type I (IFN-I) - known to both propagate nerve degeneration and to dampen anti-tumoral immunity. Peri-neural niches of non-responders were characterized by higher immune activity compared to responders, including immune-suppressive activity of M2 macrophages, and T regulatory cells. This tumor promoting inflammation expanded to the rest of the tumor microenvironment in non-responders. Anti-PD-1 efficacy was dampened by inducing nerve damage prior to treatment administration in a murine model. In contrast, anti-PD-1 efficacy was enhanced by denervation and by interleukin-6 blockade. These findings suggested a potential novel anti-PD-1 resistance drived by TANs damage and inflammation. This resistance mechanism is targetable and may have therapeutic implications in other neurotropic cancers with poor response to anti-PD-1 therapy such as pancreatic, prostate, and breast cancers.

An efficient strategy for evaluating new non-invasive screening tests for colorectal cancer: the guiding principles.

OBJECTIVE: New screening tests for colorectal cancer (CRC) are rapidly emerging. Conducting trials with mortality reduction as the end point supporting their adoption is challenging. We re-examined the principles underlying evaluation of new non-invasive tests in view of technological developments and identification of new biomarkers. DESIGN: A formal consensus approach involving a multidisciplinary expert panel revised eight previously established principles. RESULTS: Twelve newly stated principles emerged. Effectiveness of a new test can be evaluated by comparison with a proven comparator non-invasive test. The faecal immunochemical test is now considered the appropriate comparator, while colonoscopy remains the diagnostic standard. For a new test to be able to meet differing screening goals and regulatory requirements, flexibility to adjust its positivity threshold is desirable. A rigorous and efficient four-phased approach is proposed, commencing with small studies assessing the test's ability to discriminate between CRC and non-cancer states (phase I), followed by prospective estimation of accuracy across the continuum of neoplastic lesions in neoplasia-enriched populations (phase II). If these show promise, a provisional test positivity threshold is set before evaluation in typical screening populations. Phase III prospective studies determine single round intention-to-screen programme outcomes and confirm the test positivity threshold. Phase IV studies involve evaluation over repeated screening rounds with monitoring for missed lesions. Phases III and IV findings will provide the real-world data required to model test impact on CRC mortality and incidence. CONCLUSION: New non-invasive tests can be efficiently evaluated by a rigorous phased comparative approach, generating data from unbiased populations that inform predictions of their health impact.

Is there benefit to adding ezetimibe to a statin for the secondary prevention of CVD?

YES. In patients with known cardiovascular disease (CVD), ezetimibe with a statin decreases major adverse cardiovascular events (MACE) but has no effect on all-cause and cardiovascular mortality, compared to a statin alone (strength of recommendation [SOR], A; meta-analysis of randomized controlled trials [RCTs] including 1 large RCT). In adults with atherosclerotic CVD (ASCVD), the combination of ezetimibe and a moderate-intensity statin (rosuvastatin 10 mg) was noninferior at decreasing cardiovascular death, major cardiovascular events, and nonfatal stroke, but was more tolerable, compared to a high-intensity statin (rosuvastatin 20 mg) alone (SOR, B; 1 RCT).

Immune checkpoint therapy-current perspectives and future directions.

Immune checkpoint therapy (ICT) has dramatically altered clinical outcomes for cancer patients and conferred durable clinical benefits, including cure in a subset of patients. Varying response rates across tumor types and the need for predictive biomarkers to optimize patient selection to maximize efficacy and minimize toxicities prompted efforts to unravel immune and non-immune factors regulating the responses to ICT. This review highlights the biology of anti-tumor immunity underlying response and resistance to ICT, discusses efforts to address the current challenges with ICT, and outlines strategies to guide the development of subsequent clinical trials and combinatorial efforts with ICT.

Scientist Spotlights in Secondary Schools: Student Shifts in Multiple Measures Related to Science Identity after Receiving Written Assignments.

Based on theoretical frameworks of scientist stereotypes, possible selves, and science identity, written assignments were developed to teach science content through biographies and research of counter-stereotypical scientists-Scientist Spotlights (www.scientistspotlights.org). Previous studies on Scientist Spotlight assignments showed significant shifts in how college-level biology students relate to and describe scientists and in their performance in biology courses. However, the outcomes of Scientist Spotlight assignments in secondary schools were yet to be explored. In collaboration with 18 science teachers from 12 schools, this study assessed the impacts of Scientist Spotlight assignments for secondary school students. We used published assessment tools: Relatability prompt; Stereotypes prompt; and Performance/Competence, Interest, and Recognition (PCIR) instrument. Statistical analyses compared students' responses before and after receiving at least three Scientist Spotlight assignments. We observed significant shifts in students' relatability to and descriptions of scientists as well as other science identity measures. Importantly, disaggregating classes by implementation strategies revealed that students' relatability shifts were significant for teachers reporting in-class discussions and not significant for teachers reporting no discussions. Our findings raise questions about contextual and pedagogical influences shaping student outcomes with Scientist Spotlight assignments, like how noncontent Instructor Talk might foster student shifts in aspects of science identity.

How dental teams can help patients with temporomandibular disorders receive general dental care: An International Delphi process.

BACKGROUND: Many patients with temporomandibular disorders (TMD) find it difficult to undergo dental care due to challenges caused by their condition, previous temporomandibular joint surgery or invasive dental procedures, and the impact of comorbid conditions. Managing routine dental care for some patients with TMD can be seen as challenging by some dental practitioners. OBJECTIVE: The objective of this study was to work with patients experiencing TMD and clinicians to co-produce recommendations aimed at helping general dentists to provide routine dental care for patients with TMD. METHODS: A modified Delphi process was used to co-produce recommendations. Six patients experiencing TMD, patient advocates and seven clinicians took part, including international TMD clinicians. Two meetings were held with patient participants, mediated by a trained facilitator. Recommendations suggested by patient participants were distributed to clinicians who were asked to add additional suggestions, but not to modify patients' recommendations unless to aid clarity. Additional themes were identified from the existing literature, and the recommendations were then reviewed by the International Network for Orofacial Pain and Related Disorders Methodology (INfORM) consortium. RESULTS: Recommendations were given to support patients before, during and after dental treatment. Participants identified specific and practical recommendations to help patients with TMD receive routine dental care, but also emphasised the need for professionals to listen sensitively to patients' concerns and work with patients in an empathetic and non-judgmental way. CONCLUSION: These recommendations, co-developed with patients experiencing TMD, should help dental professionals to provide supportive general dental care for patients with TMD.

Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)-mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4-mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

A phase 2 study of nivolumab combined with ibrutinib in patients with diffuse large B-cell Richter transformation of CLL.

Richter transformation (RT) is a rare complication of chronic lymphocytic leukemia (CLL) that has dismal outcomes. Upregulation of PD-1/PD-L1 drives immunological evasion in patients with RT. We hypothesized that combining nivolumab, a PD-1 blocking antibody, with the BTK inhibitor (BTKi) ibrutinib could potentiate tumor-cell killing. We conducted an investigator-initiated phase 2 clinical trial to assess the efficacy of combined nivolumab and ibrutinib in patients with diffuse large B-cell lymphoma (DLBCL) RT and CLL. Patients included were ≥18 years of age with adequate hepatic and renal function. Patients received nivolumab every 2 weeks of a 4-week cycle for a maximum of 24 cycles. A standard dose ibrutinib was initiated from cycle 2 onward and continued daily until progression. For patients who were already on ibrutinib at the time of study entry, the same was continued while nivolumab was initiated. A total of 24 patients with RT with a median age of 64.5 years (range, 47-88) were enrolled. Ten patients (42%) had received prior treatment for RT and 13 patients (54%) had received a prior BTKi. A total of 10 patients (42%) responded with a median duration of response of 15 months. The median overall survival was 13 months. Four of 24 (17%) patients had checkpoint inhibition-related immunological toxicities. In the CLL cohort, 10 patients were enrolled, of whom 3 patients converted from partial to complete remission; 1 patient had a grade 2 immunological toxicity. Combined nivolumab and ibrutinib is an active regimen for patients with DLBCL RT with an overall response rate of 42%. Given the limited treatment options for patients with RT, checkpoint inhibition provides a potential therapeutic option. This trial is registered at www.clinicaltrials.gov as #NCT02420912.

Immune checkpoint therapy: Forging ahead.

Spatiotemporal immune monitoring in clinical trials and reverse translation will help to determine optimal combination immune therapies to cure cancer.