Efficacy of a Dual-Epitope Dendritic Cell Vaccine as Part of Combined Immunotherapy for HER2-Expressing Breast Tumors.

Previous work from our group and others has shown that patients with breast cancer can generate a T cell response against specific human epidermal growth factor 2 (HER2) epitopes. In addition, preclinical work has shown that this T cell response can be augmented by Ag-directed mAb therapy. This study evaluated the activity and safety of a combination of dendritic cell (DC) vaccination given with mAb and cytotoxic therapy. We performed a phase I/II study using autologous DCs pulsed with two different HER2 peptides given with trastuzumab and vinorelbine to a study cohort of patients with HER2-overexpressing and a second with HER2 nonoverexpressing metastatic breast cancer. Seventeen patients with HER2-overexpressing and seven with nonoverexpressing disease were treated. Treatment was well tolerated, with one patient removed from therapy because of toxicity and no deaths. Forty-six percent of patients had stable disease after therapy, with 4% achieving a partial response and no complete responses. Immune responses were generated in the majority of patients but did not correlate with clinical response. However, in one patient, who has survived >14 y since treatment in the trial, a robust immune response was demonstrated, with 25% of her T cells specific to one of the peptides in the vaccine at the peak of her response. These data suggest that autologous DC vaccination when given with anti-HER2-directed mAb therapy and vinorelbine is safe and can induce immune responses, including significant T cell clonal expansion, in a subset of patients.

T-cell expression of AhR inhibits the maintenance of pTreg cells in the gastrointestinal tract in acute GVHD.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that affects the function and development of immune cells. Here, we show that recipient mice receiving AhR-/- T cells have improved survival and decreased acute graft-versus-host disease (aGVHD) in 2 different murine allogeneic bone marrow transplant (BMT) models. We also show that CD4+ T cells lacking AhR demonstrate reduced accumulation in secondary lymphoid tissue because of low levels of proliferation 4 days after BMT. Additionally, we found a significant increase in the quantity of peripherally induced regulatory donor T (pTreg) cells in the colon of recipients transplanted with AhR-/- T cells 14 days after transplant. Blockade of AhR using a clinically available AhR antagonist greatly enhanced the in vitro generation of inducible Treg (iTreg) cells from naïve CD4+ human T cells. We have identified AhR as a novel target on donor T cells that is critical to the pathogenesis of aGVHD.

Assembly-based inference of B-cell receptor repertoires from short read RNA sequencing data with V'DJer.

MOTIVATION: B-cell receptor (BCR) repertoire profiling is an important tool for understanding the biology of diverse immunologic processes. Current methods for analyzing adaptive immune receptor repertoires depend upon PCR amplification of VDJ rearrangements followed by long read amplicon sequencing spanning the VDJ junctions. While this approach has proven to be effective, it is frequently not feasible due to cost or limited sample material. Additionally, there are many existing datasets where short-read RNA sequencing data are available but PCR amplified BCR data are not. RESULTS: We present here V'DJer, an assembly-based method that reconstructs adaptive immune receptor repertoires from short-read RNA sequencing data. This method captures expressed BCR loci from a standard RNA-seq assay. We applied this method to 473 Melanoma samples from The Cancer Genome Atlas and demonstrate V'DJer's ability to accurately reconstruct BCR repertoires from short read mRNA-seq data. AVAILABILITY AND IMPLEMENTATION: V'DJer is implemented in C/C ++, freely available for academic use and can be downloaded from Github: https://github.com/mozack/vdjer CONTACT: benjamin_vincent@med.unc.edu or parkerjs@email.unc.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Characterization of cell lines derived from breast cancers and normal mammary tissues for the study of the intrinsic molecular subtypes.

Five molecular subtypes (luminal A, luminal B, HER2-enriched, basal-like, and claudin-low) with clinical implications exist in breast cancer. Here, we evaluated the molecular and phenotypic relationships of (1) a large in vitro panel of human breast cancer cell lines (BCCLs), human mammary fibroblasts (HMFs), and human mammary epithelial cells (HMECs); (2) in vivo breast tumors; (3) normal breast cell subpopulations; (4) human embryonic stem cells (hESCs); and (5) bone marrow-derived mesenchymal stem cells (hMSC). First, by integrating genomic data of 337 breast tumor samples with 93 cell lines we were able to identify all the intrinsic tumor subtypes in the cell lines, except for luminal A. Secondly, we observed that the cell lines recapitulate the differentiation hierarchy detected in the normal mammary gland, with claudin-low BCCLs and HMFs cells showing a stromal phenotype, HMECs showing a mammary stem cell/bipotent progenitor phenotype, basal-like cells showing a luminal progenitor phenotype, and luminal B cell lines showing a mature luminal phenotype. Thirdly, we identified basal-like and highly migratory claudin-low subpopulations of cells within a subset of triple-negative BCCLs (SUM149PT, HCC1143, and HCC38). Interestingly, both subpopulations within SUM149PT were enriched for tumor-initiating cells, but the basal-like subpopulation grew tumors faster than the claudin-low subpopulation. Finally, claudin-low BCCLs resembled the phenotype of hMSCs, whereas hESCs cells showed an epithelial phenotype without basal or luminal differentiation. The results presented here help to improve our understanding of the wide range of breast cancer cell line models through the appropriate pairing of cell lines with relevant in vivo tumor and normal cell counterparts.

Entinostat induces antitumor immune responses through immune editing of tumor neoantigens.

Although immune-checkpoint inhibitors (ICIs) have been a remarkable advancement in bladder cancer treatment, the response rate to single-agent ICIs remains suboptimal. There has been substantial interest in the use of epigenetic agents to enhance ICI efficacy, although precisely how these agents potentiate ICI response has not been fully elucidated. We identified entinostat, a selective HDAC1/3 inhibitor, as a potent antitumor agent in our immune-competent bladder cancer mouse models (BBN963 and BBN966). We demonstrate that entinostat selectively promoted immune editing of tumor neoantigens, effectively remodeling the tumor immune microenvironment, resulting in a robust antitumor response that was cell autonomous, dependent upon antigen presentation, and associated with increased numbers of neoantigen-specific T cells. Finally, combination treatment with anti-PD-1 and entinostat led to complete responses and conferred long-term immunologic memory. Our work defines a tumor cell-autonomous mechanism of action for entinostat and a strong preclinical rationale for the combined use of entinostat and PD-1 blockade in bladder cancer.

Stable CD8+ T cell memory during persistent Trypanosoma cruzi infection.

CD8(+) T cell responses to persistent infections caused by intracellular pathogens are dominated by resting T effectors and T effector memory cells, with little evidence suggesting that a T central memory (T(CM)) population is generated. Using a model of Trypanosoma cruzi infection, we demonstrate that in contrast to the T effector/T effector memory phenotype of the majority of T. cruzi-specific CD8(+) T cells, a population of cells displaying hallmark characteristics of T(CM) cells is also present during long-term persistent infection. This population expressed the T(CM) marker CD127 and a subset expressed one or more of three other T(CM) markers: CD62L, CCR7, and CD122. Additionally, the majority of CD127(high) cells were KLRG1(low), indicating that they have not been repetitively activated through TCR stimulation. These CD127(high) cells were better maintained than their CD127(low) counterparts following transfer into naive mice, consistent with their observed surface expression of CD127 and CD122, which confer the ability to self-renew in response to IL-7 and IL-15. CD127(high) cells were capable of IFN-gamma production upon peptide restimulation and expanded in response to challenge infection, indicating that these cells are functionally responsive upon Ag re-encounter. These results are in contrast to what is typically observed during many persistent infections and indicate that a stable population of parasite-specific CD8(+) T cells capable of Ag-independent survival is maintained in mice despite the presence of persistent Ag.

Characterization of Donor Variability for γδ T Cell ex vivo Expansion and Development of an Allogeneic γδ T Cell Immunotherapy.

Gamma delta (γδ) T cells recently emerged as an attractive candidate for cancer immunotherapy treatments due to their inherent cytotoxicity against both hematological and solid tumors. Moreover, γδ T cells provide a platform for the development of allogeneic cell therapies, as they can recognize antigens independent of MHC recognition and without the requirement for a chimeric antigen receptor. However, γδ T cell adoptive cell therapy depends on ex vivo expansion to manufacture sufficient cell product numbers, which remains challenging and limited by inter-donor variability. In the current study, we characterize the differences in expansion of γδ T cells from various donors that expand (EX) and donors that fail to expand, i.e., non-expanders (NE). Further, we demonstrate that IL-21 can be used to increase the expansion potential of NE. In order to reduce the risk of graft vs. host disease (GVHD) induced by an allogeneic T cell product, αß T cell depletions must be considered due to the potential for HLA mismatch. Typically, αß T cell depletions are performed at the end of expansion, prior to infusion. We show that γδ T cell cultures can be successfully αß depleted on day 6 of expansion, providing a better environment for the γδ T cells to expand, and that the αß T cell population remains below clinically acceptable standards for T cell-depleted allogeneic stem cell products. Finally, we assess the potential for a mixed donor γδ T cell therapy and characterize the effects of cryopreservation on γδ T cells. Collectively, these studies support the development of an improved allogeneic γδ T cell product and suggest the possibility of using mixed donor γδ T cell immunotherapies.

Machine-Learning Prediction of Tumor Antigen Immunogenicity in the Selection of Therapeutic Epitopes.

Current tumor neoantigen calling algorithms primarily rely on epitope/major histocompatibility complex (MHC) binding affinity predictions to rank and select for potential epitope targets. These algorithms do not predict for epitope immunogenicity using approaches modeled from tumor-specific antigen data. Here, we describe peptide-intrinsic biochemical features associated with neoantigen and minor histocompatibility mismatch antigen immunogenicity and present a gradient boosting algorithm for predicting tumor antigen immunogenicity. This algorithm was validated in two murine tumor models and demonstrated the capacity to select for therapeutically active antigens. Immune correlates of neoantigen immunogenicity were studied in a pan-cancer data set from The Cancer Genome Atlas and demonstrated an association between expression of immunogenic neoantigens and immunity in colon and lung adenocarcinomas. Lastly, we present evidence for expression of an out-of-frame neoantigen that was capable of driving antitumor cytotoxic T-cell responses. With the growing clinical importance of tumor vaccine therapies, our approach may allow for better selection of therapeutically relevant tumor-specific antigens, including nonclassic out-of-frame antigens capable of driving antitumor immunity.

Immuno-PET imaging of tumor-infiltrating lymphocytes using zirconium-89 radiolabeled anti-CD3 antibody in immune-competent mice bearing syngeneic tumors.

The ability to non-invasively monitor tumor-infiltrating T cells in vivo could provide a powerful tool to visualize and quantify tumor immune infiltrates. For non-invasive evaluations in vivo, an anti-CD3 mAb was modified with desferrioxamine (DFO) and radiolabeled with zirconium-89 (Zr-89 or 89Zr). Radiolabeled 89Zr-DFO-anti-CD3 was tested for T cell detection using positron emission tomography (PET) in both healthy mice and mice bearing syngeneic bladder cancer BBN975. In vivo PET/CT and ex vivo biodistribution demonstrated preferential accumulation and visualization of tracer in the spleen, thymus, lymph nodes, and bone marrow. In tumor bearing mice, 89Zr-DFO-anti-CD3 demonstrated an 11.5-fold increase in tumor-to-blood signal compared to isotype control. Immunological profiling demonstrated no significant change to total T cell count, but observed CD4+ T cell depletion and CD8+ T cell expansion to the central and effector memory. This was very encouraging since a high CD8+ to CD4+ T cell ratio has already been associated with better patient prognosis. Ultimately, this anti-CD3 mAb allowed for in vivo imaging of homeostatic T cell distribution, and more specifically tumor-infiltrating T cells. Future applications of this radiolabeled mAb against CD3 could include prediction and monitoring of patient response to immunotherapy.

Molecular Subtype-Specific Immunocompetent Models of High-Grade Urothelial Carcinoma Reveal Differential Neoantigen Expression and Response to Immunotherapy.

High-grade urothelial cancer contains intrinsic molecular subtypes that exhibit differences in underlying tumor biology and can be divided into luminal-like and basal-like subtypes. We describe here the first subtype-specific murine models of bladder cancer and show that Upk3a-CreERT2; Trp53L/L; PtenL/L; Rosa26LSL-Luc (UPPL, luminal-like) and BBN (basal-like) tumors are more faithful to human bladder cancer than the widely used MB49 cells. Following engraftment into immunocompetent C57BL/6 mice, BBN tumors were more responsive to PD-1 inhibition than UPPL tumors. Responding tumors within the BBN model showed differences in immune microenvironment composition, including increased ratios of CD8+:CD4+ and memory:regulatory T cells. Finally, we predicted and confirmed immunogenicity of tumor neoantigens in each model. These UPPL and BBN models will be a valuable resource for future studies examining bladder cancer biology and immunotherapy.Significance: This work establishes human-relevant mouse models of bladder cancer. Cancer Res; 78(14); 3954-68. ©2018 AACR.

Endogenous retroviral signatures predict immunotherapy response in clear cell renal cell carcinoma.

Human endogenous retroviruses (hERVs) are remnants of exogenous retroviruses that have integrated into the genome throughout evolution. We developed a computational workflow, hervQuant, which identified more than 3,000 transcriptionally active hERVs within The Cancer Genome Atlas (TCGA) pan-cancer RNA-Seq database. hERV expression was associated with clinical prognosis in several tumor types, most significantly clear cell renal cell carcinoma (ccRCC). We explored two mechanisms by which hERV expression may influence the tumor immune microenvironment in ccRCC: (i) RIG-I-like signaling and (ii) retroviral antigen activation of adaptive immunity. We demonstrated the ability of hERV signatures associated with these immune mechanisms to predict patient survival in ccRCC, independent of clinical staging and molecular subtyping. We identified potential tumor-specific hERV epitopes with evidence of translational activity through the use of a ccRCC ribosome profiling (Ribo-Seq) dataset, validated their ability to bind HLA in vitro, and identified the presence of MHC tetramer-positive T cells against predicted epitopes. hERV sequences identified through this screening approach were significantly more highly expressed in ccRCC tumors responsive to treatment with programmed death receptor 1 (PD-1) inhibition. hervQuant provides insights into the role of hERVs within the tumor immune microenvironment, as well as evidence that hERV expression could serve as a biomarker for patient prognosis and response to immunotherapy.

A Strong B-cell Response Is Part of the Immune Landscape in Human High-Grade Serous Ovarian Metastases.

PURPOSE: In high-grade serous ovarian cancer (HGSOC), higher densities of both B cells and the CD8+ T-cell infiltrate were associated with a better prognosis. However, the precise role of B cells in the antitumor response remains unknown. As peritoneal metastases are often responsible for relapse, our aim was to characterize the role of B cells in the antitumor immune response in HGSOC metastases. EXPERIMENTAL DESIGN: Unmatched pre and post-chemotherapy HGSOC metastases were studied. B-cell localization was assessed by immunostaining. Their cytokines and chemokines were measured by a multiplex assay, and their phenotype was assessed by flow cytometry. Further in vitro and in vivo assays highlighted the role of B cells and plasma cell IgGs in the development of cytotoxic responses and dendritic cell activation. RESULTS: B cells mainly infiltrated lymphoid structures in the stroma of HGSOC metastases. There was a strong B-cell memory response directed at a restricted repertoire of antigens and production of tumor-specific IgGs by plasma cells. These responses were enhanced by chemotherapy. Interestingly, transcript levels of CD20 correlated with markers of immune cytolytic responses and immune complexes with tumor-derived IgGs stimulated the expression of the costimulatory molecule CD86 on antigen-presenting cells. A positive role for B cells in the antitumor response was also supported by B-cell depletion in a syngeneic mouse model of peritoneal metastasis. CONCLUSIONS: Our data showed that B cells infiltrating HGSOC omental metastases support the development of an antitumor response. Clin Cancer Res; 23(1); 250-62. ©2016 AACR.

Antibacterial properties of common herbal remedies of the southwest.

Curanderismo, widely practiced in the southwest, is an alternative medical system that has been neglected by scientific research. This project analyzed the antibiotic properties of 23 common herbal remedies used in South Texas to treat wounds and infections. Ethanolic tinctures and aqueous extracts of each plant were prepared and applied to blank diffusion disks. These disks were desiccated and used in Kirby-Bauer disk diffusion susceptibility tests on three bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Control disks contained solvent only. The efficacy of the tinctures and aqueous extracts was compared to that of commercially prepared antibiotic diffusion disks. No inhibition was observed with the aqueous extracts. The various tincture-saturated disks produced zones of clearance ranging from 1 to 5 mm. Ten plants consistently inhibited bacterial growth of Staphylococcus aureus. None of the plants tested produced consistent inhibition of the two Gram-negative species, Pseudomonas aeruginosa and Escherichia coli. No zones of clearance were produced by the solvent-only control disks. The zones of clearance produced by commercial antibiotics were, on average, larger and more uniform than those produced by the tincture disks. Thus, it appears that some of the herbal remedies used in folk medicine are potentially effective antibacterial agents against Staphylococcus aureus.

Peptide/MHC tetramer-based sorting of CD8⁺ T cells to a leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire.

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8⁺ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer⁺ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor ß (TCRß) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCRß clonotypes in a patient with a UNC-CDK4-1 tetramer⁺ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRß repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCRß clonotypes represented >17% of the entire TCRß repertoire-far in excess of the UNC-CDK4-1 tetramer⁺ frequency-indicating that the recurrent TCRß clonotypes identified from UNC-CDK-4-1 tetramer⁺ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1-driven clonal T-cell expansion. Mapping recurrent TCRß clonotype sequences onto TCRß repertoires can help confirm or refute antigen-specific T-cell expansion in vivo.

Drug-induced cure drives conversion to a stable and protective CD8+ T central memory response in chronic Chagas disease.

In this study, we document the development of stable, antigen-independent CD8+ T cell memory after drug-induced cure of a chronic infection. By establishing a system for drug cure of chronic Trypanosoma cruzi infection, we present the first extensively documented case of total parasite clearance after drug treatment of this infection. Cure resulted in the emergence of a stable, parasite-specific CD8+ T cell population with the characteristics of central memory cells, based upon expression of CD62L, CCR7, CD127, CD122, Bcl-2 and a reduced immediate in vivo CTL function. CD8+ T cells from treated and cured mice also expanded more rapidly and provided greater protection following challenge than those from chronically infected mice. These results show that complete pathogen clearance results in stable, antigen-independent and protective T cell memory, despite the potentially exhausting effects of prior long-term exposure to antigen in this chronic infection.