Supplementary Methods from CD73 on Tumor Cells Impairs Antitumor T-Cell Responses: A Novel Mechanism of Tumor-Induced Immune Suppression

Supplementary Figures 1-7 from CD73 on Tumor Cells Impairs Antitumor T-Cell Responses: A Novel Mechanism of Tumor-Induced Immune Suppression

Fig. S8 IFN-α plus DD renders tumor dendritic cells less suppressive.

AbstractPurpose:Immunotherapy treats some cancers, but not ovarian cancer. Regulatory T cells (Tregs) impede anti-ovarian cancer immunity but effective human Treg-directed treatments are lacking. We tested Treg depletion with denileukin diftitox (DD) ± IFNα as ovarian cancer immunotherapy.Patients and Methods:Mice with syngeneic ID8 ovarian cancer challenge were treated with DD, IFNα, or both. The phase 0/I trial tested one dose-escalated DD infusion for functional Treg reduction, safety, and tolerability. The phase II trial added IFNα2a to DD if DD alone failed clinically.Results:DD depleted Tregs, and improved antitumor immunity and survival in mice. IFNα significantly improved antitumor immunity and survival with DD. IFNα did not alter Treg numbers or function but boosted tumor-specific immunity and reduced tumor Treg function with DD by inducing dendritic cell IL6. DD alone was well tolerated, depleted functional blood Tregs and improved immunity in patients with various malignancies in phase 0/I. A patient with ovarian cancer in phase 0/I experienced partial clinical response prompting a phase II ovarian cancer trial, but DD alone failed phase II. Another phase II trial added pegylated IFNα2a to failed DD, producing immunologic and clinical benefit in two of two patients before a DD shortage halt. DD alone was well tolerated. Adding IFNα increased toxicities but was tolerable, and reduced human Treg numbers in blood, and function through dendritic cell–induced IL6 in vitro.Conclusions:Treg depletion is clinically useful but unlikely alone to cure ovarian cancer. Rational treatment agent combinations can salvage clinical failure of Treg depletion alone, even when neither single agent provides meaningful clinical benefit.

In high-transmission regions, we expect parasite lineages within complex malaria infections to be unrelated due to parasite inoculations from different mosquitoes. This project was designed to test this prediction. We generated 485 single-cell genome sequences from fifteen P. falciparum malaria patients from Chikhwawa, Malawi-an area of intense transmission. Patients harbored up to seventeen unique parasite lineages. Surprisingly, parasite lineages within infections tend to be closely related, suggesting that superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Both closely and distantly related parasites comprise an infection, suggesting sequential transmission of complex infections between multiple hosts. We identified tetrads and reconstructed parental haplotypes, which revealed the inbred ancestry of infections and non-Mendelian inheritance. Our analysis suggests strong barriers to secondary infection and outbreeding amongst malaria parasites from a high transmission setting, providing unexpected insights into the biology and transmission of malaria.

Malaria patients can carry one or more clonal lineage of the parasite, Plasmodium falciparum , but the composition of these infections cannot be directly inferred from bulk sequence data. Well-defined, complete haplotypes at single-cell resolution are ideal for describing within-host population structure and unambiguously determining parasite diversity, transmission dynamics and recent ancestry but have not been analyzed on a large scale. We generated 485 near-complete single-cell genome sequences isolated from fifteen P. falciparum patients from Chikhwawa, Malawi, an area of intense malaria transmission. Matched single-cell and bulk genomic analyses revealed patients harbored up to seventeen unique lineages. Estimation of parasite relatedness within patients suggests superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Our single-cell analysis indicates strong barriers to establishment of new infections in malaria-infected patients and allows high resolution dissection of intra-host variation in malaria parasites.

Malaria patients can carry one or more clonal lineage of the parasite, Plasmodium falciparum. Well-defined, complete haplotypes at single-cell resolution are ideal for describing within-host population structure and unambiguously determining parasite diversity, transmission dynamics and recent ancestry but have not been analyzed on a large scale. We generated 485 near-complete single-cell genome sequences isolated from fifteen P. falciparum patients from Chikhwawa, Malawi, an area of intense malaria transmission. Matched single-cell and bulk genomic analyses revealed patients harbored up to seventeen unique lineages. Current statistical approaches were unable to accurately reconstruct infection composition from bulk sequence data. Analysis of parasite relatedness within patients suggests superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Our single-cell analysis indicates strong barriers to establishment of secondary infections, providing new insights into the biology and transmission of malaria.

Murine mammary stem/progenitor cell isolation has been routinely used in many laboratories, yet direct comparison among different methods is lacking. In this study, we compared two frequently used digestion methods and three sets of frequently used surface markers for their efficiency in enriching mammary stem and progenitor cells in two commonly used mouse strains, C57BL/6J and FVB. Our findings revealed that the slow overnight digestion method using gentle collagenase/hyaluronidase could be easily adopted and yielded reliable and consistent results in different batches of animals. In contrast, the different fast digestion protocols, as described in published studies, yielded high percent of non-epithelial cells with very few basal epithelial cells liberated in our hands. The three sets of markers tested in our hands reveal rather equally efficiency in separating luminal and basal cells if same fluorochrome conjugations were used. However, the tendency of non-epithelial cell inclusion in the basal cell gate was highest in samples profiled by CD24/CD29 and lowest in samples profiled by CD49f/EpCAM, this is especially true in mammary cells isolated from C57BL/6J mice. This finding will have significant implication when sorted basal cells are used for subsequent gene expression analysis.

Single-cell genomics is a powerful tool for determining the genetic structure of complex communities of unicellular organisms. Patients infected with the malaria-causing parasite, Plasmodium falciparum , often carry multiple, genetically distinct parasites. Little is known about the diversity and relatedness of these lineages. We have developed an improved single-cell genomics protocol to reconstruct individual haplotypes from infections, a necessary step in uncovering parasite ecology within the host. This approach captures singly-infected red blood cells (iRBCs) by fluorescence-activated cell sorting (FACS) prior to whole genome amplification (WGA) and whole genome sequencing (WGS). Here, we demonstrate that parasites in late cell cycle stages, which contain increased DNA content, are far superior templates for generating high quality genomic data. Targeting of these cells routinely generates near-complete capture of the 23Mb P. falciparum genome (mean breadth of coverage 90.7%) at high efficiency. We used this approach to analyze the genomes of 48 individual cells from a polyclonal malaria infection sampled in Chikhwawa, Malawi. This comprehensive dataset enabled high-resolution estimation of the clonality and the relatedness of parasite haplotypes within the infection, long-standing problems in malaria biology.

The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry

Breast cancer incidence increases during aging, yet the mechanism of age-associated mammary tumorigenesis is unclear. Mammary stem cells are believed to play an important role in breast tumorigenesis, but how their function changes with age is unknown. We compared mammary epithelial cells isolated from young and old mammary glands of different cohorts of C57BL6/J and BALB/c mice, and our findings revealed that old mammary glands were characterized by increased basal cell pool comprised of mostly CD49fhi cells, altered luminal-to-basal cell ratio, and irregular ductal morphology. More interestingly, basal stem cells in old mice were increased in frequency, but showed a functional decline of differentiation and increased neoplastic transformation potential. Gene signature enrichment analysis revealed a significant enrichment of a luminal cell gene expression signature in the basal stem cell-enriched population from old mice, suggesting some luminal cells were expressing basal markers. Immunofluorescence staining confirmed the presence of luminal cells with high CD49f expression in hyperplastic lesions implicating these cells as undergoing luminal to basal phenotypic changes during aging. Whole transcriptome analysis showed elevated immune and inflammatory responses in old basal stem cells and stromal cells, which may be the underlying cause for increased CD49fhi basal-like cells in aged glands.

BackgroundA close relationship between aging, inflammation, and prostate cancer is widely accepted. Aging is accompanied by a progressive increase in pro-inflammatory cytokines, including interleukin 17 (IL-17), a key pro-inflammatory cytokine that becomes dysregulated with age. However, the contribution of IL-17 to age-related prostate tumorigenesis remains unclear. The aim of this study was to investigate the role of age-related IL-17 dysregulation in prostate tumorigenesis.MethodsSerum and splenic T-lymphocytes from young GPAT-1 knock-out aging-mimic T cell mice as well as young and aged wild-type mice were collected. shRNA was used to knock down the IL-17 receptor in LNCaP prostate cancer cells and RWPE-1 non-transformed prostate epithelial cells, which were then exposed to the mouse sera or conditioned media from stimulated T-lymphocytes. NF-B activation, NF-B target gene expression, and cell proliferation were all measured in these cells by luciferase assay, qPCR, Western blot analysis, and MTT assay, respectively.ResultsT-lymphocyte-secreted IL-17 from aging-mimic mice induced NF-B activity and target gene expression in LNCaP and RWPE-1 cells. It also promoted proliferation of these cells.ConclusionAging-mimic T cell mice produce increased levels of IL-17, which stimulates the pro-inflammatory NF-B pathway in prostate epithelial cells. NF-B increases inflammation, carcinogenesis and metastatic potential in the prostate. These findings provide evidence that the dysregulation of cytokine production seen in aged T cells may directly contribute to the increased risk for prostate cancer in the elderly. Prostate 75: 449-462, 2015. (c) 2015 Wiley Periodicals, Inc.

Regenerative endodontic procedures are stem cell-based treatments for immature teeth with pulp necrosis. The translation of regenerative endodontic procedures into treating mature teeth depends, among other factors, on the availability and delivery of mesenchymal stem cells (MSCs) into the root canal system. The aim of this clinical study was to evaluate whether evoked bleeding from the periapical tissues elicits the influx of MSCs into the root canal system in mature teeth with apical lesions. Participants included in this study (N = 20) were referred for endodontic treatment of mature teeth with apical lesions. Following chemomechanical debridement, intracanal bleeding from the periapical tissues was achieved, and intracanal blood samples were collected. A positive blood aspirate was also collected in the cartridges during local anesthesia. Total RNA was isolated and used as a template in quantitative reverse transcription polymerase chain reactions using MSC-specific arrays. Data were analyzed with the Wilcoxon signed-rank test, and correlation between gene expression and sex or age was tested with Spearman's rank correlation coefficient test. In addition, MSCs were isolated from an intracanal bleeding sample and subjected to flow cytometry and quantitative osteogenesis assay. Last, the presence and distribution of MSCs within periradicular lesions were evaluated with immunohistochemistry (n = 4). The MSC markers CD73, CD90, CD105, and CD146 were significantly upregulated, with median fold change values of 2.9, 31.7, 4.6, and 6.8, respectively. Conversely, the negative marker for MSCs, CD45, was significantly downregulated (median, -2.7). There was no correlation with age, sex, tooth type, or treatment for any of the evaluated genes. Isolated intracanal cells coexpressed MSC markers and demonstrated robust mineralizing differentiation potential. Finally, immunohistochemical analysis revealed that MSCs were found compartmentalized mainly within vasculature structures located in periapical lesions. Collectively, findings indicate that the evoked-bleeding technique delivers MSCs into the root canal system in mature teeth with apical lesions.

Abstract Tregs hinder anti-tumor immunity, and depleting them treats cancers effectively in mice. By contrast, Treg depletion in human trials has limited efficacy. In a phase I trial of advanced stage carcinomas (breast, lung, melanoma, ovarian, bladder), we showed that the IL-2/diphtheria fusion toxin denileukin diftitox (DT) significantly depleted functional CD4+CD25hiFoxp3+ Tregs in blood with improved T cell function (increased Ki-67 and interferon-γ) with minimal effects on other blood mononuclear cells. One patient with stage IV ovarian carcinoma experienced significant reduction of metastatic tumor burden with denileukin diftitox at 12 μg/kg. Additional work showed that Treg depletion effects could last up to 4 weeks, and that weekly DT at 12 μg/kg eventually depleted anti-tumor and tumor-specific CD8+ T cells. Based on these data, we conducted a phase II clinical trial of epithelial ovarian carcinomas FIGO stage III-IV failing standard treatments, using DT as a single agent at 12 μg/kg by intravenous infusion every 3-4 weeks. In this trial, DT significantly depleted blood and tumor microenvironmental Tregs with only grade I-II toxicities, but with minimal clinical efficacy in 28 consecutive patients. This trial was halted for futility according to the Simon 2-stage design. Interferon-α alone does not treat ovarian cancer, but we now show that it significantly improves clinical and immune DT-mediated Treg depletion efficacy in human ovarian cancer. In the ID8 mouse ovarian carcinoma model, DT modestly increased survival and anti-tumor immunity. Interferon-α alone did not affect Treg numbers or function, but enhanced CD8+ T cell anti-tumor immunity. Interferon-α plus DT increased mouse survival significantly over either individual drug. In type I IFNR-/- mice unable to mediate interferon-α signals, interferon-α directly increased adoptively transferred IFNR+CD8+ T cell function independent of CD4+ T cell help. When combined with DT, IFN-α reduced Treg function without further numerical Treg reduction by indirect effects on tumor microenvironmental dendritic cells. In vitro studies identified IFN-α-driven dendritic cell IL-6 as a mechanism for reducing Treg function. When three ovarian cancer patients failed DT alone in the phase II trial, two had clinical and immune benefit by adding pegylated interferon-α2a in a separate trial, with acceptable toxicities. This trial was halted due to a lack of further DT. These studies demonstrate that DT depletes Tregs in distinct human carcinomas but is unlikely to be clinically effective as a single agent. We identified novel IFN-α mechanisms that improve Treg depletion effects using FDA-approved agents that can be rapidly translated. More selective Treg depletion agents and rationale combinations with other agents could improve clinical efficacy further. Citation Format: Suzanne Thibodeaux, Vincent Hurez, Shawna Wall, Srilakshmi Pandeswara, Benjamin Daniel, Aijie Liu, Lishi Sun, Leslie Wood, Weiping Zou, Tyler Curiel. Interferon-α enhances clinical benefits of regulatory T cell depletion in ovarian cancer through direct T cell effects and by inducing bystander IL-6. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-259. doi:10.1158/1538-7445.AM2014-LB-259

Melanomas are vascular tumors with a high incidence of BRAF mutations driving tumor proliferation. Complete inhibition of vascular endothelial growth factor (VEGF) signaling has potential for enhanced antitumor efficacy.