Phosphodiesterase-5 inhibition collaborates with vaccine-based immunotherapy to reprogram myeloid cells in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistant to immunotherapy. Although immune recognition can be enhanced with immunomodulatory agents including checkpoint inhibitors and vaccines, few patients experience clinical efficacy because the tumor immune microenvironment (TiME) is dominated by immunosuppressive myeloid cells that impose T cell inhibition. Inhibition of phosphodiesterase-5 (PDE5) was reported to downregulate metabolic regulators arginase and iNOS in immunosuppressive myeloid cells and enhance immunity against immune-sensitive tumors including head and neck cancers. We show for the first time that combining a PDE5 inhibitor, tadalafil, with a mesothelin-specific vaccine, anti-PD1, and anti-CTLA4 yields antitumor efficacy even against immune-resistant PDAC. To determine immunologic advantages conferred by tadalafil, we profiled the TiME using mass cytometry and single-cell RNA analysis with Domino to infer intercellular signaling. Our analyses demonstrated that tadalafil reprograms myeloid cells to be less immunosuppressive. Moreover, tadalafil synergized with the vaccine, enhancing T cell activation including mesothelin-specific T cells. Tadalafil treatment was also associated with myeloid-T cell signaling axes important for antitumor responses (e.g., Cxcr3, Il12). Our study shows that PDE5 inhibition combined with vaccine-based immunotherapy promotes pro-inflammatory states of myeloid cells, activation of T cells, and enhanced myeloid-T cell crosstalk to yield antitumor efficacy against immune-resistant PDAC.

Multimodal immune phenotyping reveals microbial-T cell interactions that shape pancreatic cancer.

Microbes are an integral component of the tumor microenvironment. However, determinants of microbial presence remain ill-defined. Here, using spatial-profiling technologies, we show that bacterial and immune cell heterogeneity are spatially coupled. Mouse models of pancreatic cancer recapitulate the immune-microbial spatial coupling seen in humans. Distinct intra-tumoral niches are defined by T cells, with T cell-enriched and T cell-poor regions displaying unique bacterial communities that are associated with immunologically active and quiescent phenotypes, respectively, but are independent of the gut microbiome. Depletion of intra-tumoral bacteria slows tumor growth in T cell-poor tumors and alters the phenotype and presence of myeloid and B cells in T cell-enriched tumors but does not affect T cell infiltration. In contrast, T cell depletion disrupts the immunological state of tumors and reduces intra-tumoral bacteria. Our results establish a coupling between microbes and T cells in cancer wherein spatially defined immune-microbial communities differentially influence tumor biology.

CyTOF protocol for immune monitoring of solid tumors from mouse models.

Techniques for robust immune profiling of mouse tumor and blood are key to understanding immunological responses in mouse models of cancer. Here, we describe mass cytometry (cytometry by time-of-flight) procedures to facilitate high-parameter profiling of low-volume survival blood samples and end-of-study tumor samples. We employ live-cell barcoding systems to mark all cells from each tumor and blood to improve cost-effectiveness and minimize batch effects. For complete details on the use and execution of this protocol, please refer to Charmsaz et al. (2021).1.

Profiling of syngeneic mouse HCC tumor models as a framework to understand anti-PD-1 sensitive tumor microenvironments.

BACKGROUND AND AIMS: The treatment of hepatocellular carcinoma (HCC) has been transformed by the use of immune checkpoint inhibitors. However, most patients with HCC do not benefit from treatment with immunotherapy. There is an urgent need to understand the mechanisms that underlie response or resistance to immunotherapy for patients with HCC. The use of syngeneic mouse models that closely recapitulate the heterogeneity of human HCC will provide opportunities to examine the complex interactions between cancer cells and nonmalignant cells in the tumor microenvironment. APPROACH AND RESULTS: We leverage a multifaceted approach that includes imaging mass cytometry and suspension cytometry by time of flight to profile the tumor microenvironments of the Hep53.4, Hepa 1-6, RIL-175, and TIBx (derivative of TIB-75) syngeneic mouse HCC models. The immune tumor microenvironments vary across these four models, and various immunosuppressive pathways exist at baseline in orthotopic liver tumors derived from these models. For instance, TIBx, which is resistant to anti-programmed cell death protein 1 therapy, contains a high proportion of "M2-like" tumor-associated macrophages with the potential to diminish antitumor immunity. Investigation of The Cancer Genome Atlas reveals that the baseline immunologic profiles of Hep53.4, RIL-175, and TIBx are broadly representative of human HCCs; however, Hepa 1-6 does not recapitulate the immune tumor microenvironment of the vast majority of human HCCs. CONCLUSIONS: There is a wide diversity in the immune tumor microenvironments in preclinical models and in human HCC, highlighting the need to use multiple syngeneic HCC models to improve the understanding of how to treat HCC through immune modulation.

Integrated T cell cytometry metrics for immune-monitoring applications in immunotherapy clinical trials.

Mass cytometry, or cytometry by TOF (CyTOF), provides a robust means of determining protein-level measurements of more than 40 markers simultaneously. While the functional states of immune cells occur along continuous phenotypic transitions, cytometric studies surveying cell phenotypes often rely on static metrics, such as discrete cell-type abundances, based on canonical markers and/or restrictive gating strategies. To overcome this limitation, we applied single-cell trajectory inference and nonnegative matrix factorization methods to CyTOF data to trace the dynamics of T cell states. In the setting of cancer immunotherapy, we showed that patient-specific summaries of continuous phenotypic shifts in T cells could be inferred from peripheral blood-derived CyTOF mass cytometry data. We further illustrated that transfer learning enabled these T cell continuous metrics to be used to estimate patient-specific cell states in new sample cohorts from a reference patient data set. Our work establishes the utility of continuous metrics for CyTOF analysis as tools for translational discovery.

Systemic inhibition of PTPN22 augments anticancer immunity.

Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a novel small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8+ T cells and macrophage subpopulations toward MHC-II expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD1-PDL1 axis in the setting of PTPN22 inhibition could be further leveraged with PD1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.

Diffuse retinal pigment epithelial disease in an adult with cystic fibrosis.

An adult with cystic fibrosis presented for decreased vision in the right eye of several months' duration. Biomicroscopy and fluorescein angiography demonstrated bilateral large subretinal drusenoid deposits concentrated temporal to the fovea, as well as a small subretinal hemorrhage associated with focal leakage of fluid within the macula in the right eye. Optical coherence tomography demonstrated subretinal fluid consistent with choroidal neovascular membrane in the right eye with the absence of fluid in the left eye. The retinal manifestations of bilateral diffuse drusen associated with choroidal neovascular membrane may be consistent with autosomal dominant drusen, age-related macular degeneration, and type II glomerulonephritis, or alternatively may be a unique finding associated with cystic fibrosis. To the authors' knowledge, these findings have not previously been described in association with cystic fibrosis.

Quantitative assessment of photoreceptor recovery in atypical multiple evanescent white dot syndrome.

Multiple evanescent white dot syndrome (MEWDS) is part of a spectrum of disease entities known as acute zonal occult outer retinopathy-complex that share photoreceptor disruption as a cardinal feature. Optical coherence tomography (OCT) allows for qualitative assessment of the integrity of the photoreceptor layer via examination of the junction between the photoreceptor inner and outer segments. A patient with atypical MEWDS who demonstrated disruption of the inner segment/outer segment junction during the acute phase of the disease is described. The change in photoreceptor architecture and abnormalities on fundus autofluorescence imaging and microperimetry were restored to normal following resolution of the disease. Using a novel OCT-based method of quantitatively measuring photoreceptor outer segment length, the authors show that photoreceptor outer segment length appears decreased acutely with restoration to normal following disease resolution. OCT can assess photoreceptor recovery, both qualitatively and quantitatively, in patients with MEWDS features.

Nature and risk of neovascularization in the fellow eye of patients with unilateral retinal angiomatous proliferation.

PURPOSE: To determine the nature and risk of neovascularization in the fellow eyes of patients with unilateral retinal angiomatous proliferation (RAP), a neovascular form of age-related macular degeneration (AMD). METHODS: A consecutive series of 52 patients diagnosed with unilateral RAP were studied retrospectively. Clinical biomicroscopic examination, fluorescein angiography, and indocyanine green angiography were used to evaluate all patients for the development of neovascular manifestations in the fellow eye. RESULTS: Neovascularization developed in the fellow eye in 52 patients over the follow-up period (range, 2-36 months). All patients developed neovascular manifestations of RAP in the fellow eye. Twenty-one patients (40%) developed a RAP lesion within 1 year; 29 (56%), within 2 years; and 52 (100%), within 3 years. At the time of diagnosis of neovascularization in the fellow eye, 8 patients (15%) had a stage I RAP lesion, 36 (70%) had a stage II RAP lesion, and 8 (15%) had a stage III RAP lesion. Other characteristic findings in these patients included the presence of preretinal, intraretinal, and subretinal hemorrhages in 49 patients (94%) and pigment epithelial detachments in 41 patients (79%). CONCLUSIONS: In patients diagnosed with unilateral RAP lesions, the form of neovascularization that develops in the fellow eye is virtually always RAP. The annual and accumulative risk of neovascularization in the fellow eye is higher in patients with RAP than in those with other forms of neovascular AMD. These new findings enhance our understanding of the clinical spectrum of RAP in terms of its natural course and visual prognosis and may possibly offer useful information to establish future treatment options.

Retinal choroidal collateral circulation after radial optic neurotomy correlated with the lessening of macular edema.

PURPOSE: To evaluate patients who had undergone radial optic neurotomy for central retinal vein occlusion for the presence of retinal choroidal collateral circulation and to correlate these collaterals with changes in macular thickness during follow-up. Radial optic neurotomy is designed to release proposed pressure within the scleral canal. METHODS: Retrospective review of patients undergoing radial optic neurotomy. The patients had a baseline examination including ophthalmoscopy, fluorescein angiography, and optical coherence tomography. At an interval follow-up at approximately 3 months the patients were reevaluated with ophthalmoscopy, optical coherence tomography, and indocyanine green angiography. RESULTS: There were 6 patients, and the mean age was 68.3 years. The mean time from onset of the central retinal vein occlusion to the radial optic neurotomy was 2.3 months. One patient had no collateral vessels, three patients had significant collaterals, and two patients had moderate-caliber collaterals. The mean central macular thickness preoperatively was 1,021 microm and the mean central macular thickness postoperatively was 733 microm. The change in macular thickness was highly correlated with the degree of development of collaterals from the retinal to the choroidal circulation (P = 0.008, Spearman's rho = 0.93). CONCLUSION: Although all patients had a radial optic neurotomy a significant determinant in reduction of macular edema was the presence of retinal-choroidal collateral circulation. This suggests that there may be additional mechanisms, other than simple release of alleged pressure in the scleral canal, for any observed effects from radial optic neurotomy.

Optical coherence tomography of branch retinal vein occlusion.

PURPOSE: To evaluate the incidence of serous retinal detachment (SRD) secondary to a branch retinal vein occlusion (BRVO) by using optical coherence tomography (OCT). METHODS: Fourteen eyes of 14 patients with a BRVO underwent a detailed history, ophthalmoscopic examination, and fluorescein angiographic evaluation. They were also studied with OCT. RESULTS: The 14 patients included eight women and six men with a mean age of 73.6 +/- 10.5 years (range, 55-90 years). Four eyes were found to have cystoid macular edema by fluorescein angiography, whereas 10 cases were detected by OCT. SRD involving any portion of the macula was found in 10 (71.4%) of the 14 eyes, and SRD extending into the fovea was found in six (42.9%) eyes. Two (14.3%) of the 14 patients also showed a subfoveal hemorrhage that appeared to have gravitated inferiorly through the SRD to the dependent portion of the detachment. CONCLUSIONS: That few patients with SRD secondary to a BRVO discovered by ophthalmoscopy have been reported in the literature would suggest that this is an uncommon complication. The authors found with OCT that SRD commonly occurs in BRVO. In addition, subretinal hemorrhage may occur in the context of BRVO, and the authors propose that blood gravitates through the subretinal fluid to settle behind the retina.

Indocyanine green angiography-guided photodynamic therapy for treatment of chronic central serous chorioretinopathy: a pilot study.

BACKGROUND: Most patients with central serous chorioretinopathy (CSC) have spontaneous resolution of exudative macular detachments and a good visual prognosis. Patients with CSC have a primary choroidal hyperpermeability problem evident as multifocal areas of hyperpermeability during indocyanine green (ICG) angiography. A small percentage of patients develop chronic or progressive disease with widespread decompensation of the retinal pigment epithelium and severe vision loss. There is no known treatment for this variant of the disorder. PURPOSE: To study ICG-guided photodynamic therapy (PDT) with verteporfin as a potential treatment for patients with chronic CSC. METHODS: Twenty eyes of 15 patients were studied with fluorescein angiography, optical coherence tomography, and ICG angiography to diagnose the maculopathy, monitor the detachments, and localize the choroidal hyperpermeability of the disorder. PDT with ICG guidance was applied to areas of choroidal hyperpermeability, and the patients were observed to determine the anatomic and functional outcomes. RESULTS: Photodynamic therapy guided by ICG was associated with complete resolution of exudative macular detachments in 12 patients and incomplete resolution in the remaining eight eyes. The vision improved in six eyes and remained unchanged in 14 eyes during a mean follow-up of 6.8 months. Six weeks after treatment, the mean visual acuity improved by 0.55 lines, an amount that was marginally significant. There was a significant inverse correlation between the baseline visual acuity and the amount of improvement in acuity at 6 weeks. No patient had any treatment-related side effects. CONCLUSIONS: Indocyanine green angiography-guided PDT with verteporfin seems to aid in the resolution of exudative detachments in patients with chronic CSC. This treatment was associated with a rapid reduction in subretinal fluid and improvement in visual acuity. Although the follow-up time and number of patients in this pilot study were limited, the encouraging results and lack of complications suggest that further study is indicated.