bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence of ongoing immune surveillance to cancer-associated phosphopeptides in healthy individuals
Authors: Lulu Amanda M , Cummings Kara L. , Engelhard Victor H. 
Published: January 2023

Abstract
Dysregulated protein phosphorylation plays a major role in cancer cell malignancy. We showed that phosphopeptides are presented by MHC-I molecules, representing a new class of cancer neoantigens. We found that healthy donors with no evident prior cancer had robust CD8+ T cell responses to a cohort of HLA-B7+ leukemia-associated phosphopeptides. Here we report that CD8+CD45RO+ T cells purified from blood of different healthy donors showed responses to 2–25% of HLA-A2+ and 0–23% of HLA-B7+ cancer-associated phosphopeptides, confirming that individuals with no evident cancer history have pre-existing phosphopeptide specific immune memory. Six of 59 HLA-A2+ phosphopeptides were “immunodominant,” with memory responses evident in the majority of analyzed donors. This suggests that these phosphopeptides result from processes common in cellular transformation or viral infection. In contrast, no immunodominant HLA-B7+ phosphopeptides were identified among the 168 analyzed, suggesting that mechanisms driving their expression in healthy donors are less common. Robust day 14 responses were observed against a small cohort of HLA-B7+ phosphopeptides in 2 donors. Responses to some of these phosphopeptides could be detected ex vivo and were evident over 3 years, suggesting they reflected chronic antigen exposures. In contrast, responses to 2 phosphopeptides evident ex vivo diminished or disappeared over 14 days in culture, suggesting a recent acute immune response composed of terminal effectors unable to persist long term. These results provide evidence of ongoing immune surveillance in healthy individuals to phosphopeptides known to be expressed on cancers.
Tumor microenvironment dictates function of tumor-expressed PD-L1
Authors: Klement John David , Redd Priscilla S , Lu Chunwan , Yang Dafeng , Liu Kebin 
Published: January 2023

Abstract
To evade immune clearance by cytotoxic T lymphocytes (CTLs), tumors express immunosuppressive checkpoints such as PD-L1. Tumor-expressed PD-L1 is thought to suppress CTL effector functions by binding CTL-expressed PD-1. To investigate the molecular consequences of CTL engagement with tumor PD-L1, we generated PD-L1 knockouts in murine sarcoma, breast and colorectal cancer cell lines by Crispr/Cas9. Surprisingly, PD-L1 expression conferred no protection from CTL-mediated cytotoxicity in an in vitro co-culture model. Similarly, loss of PD-L1 had no effect on in vivo growth following subcutaneous or orthotopic challenge in immunocompetent mice. However, tumor PD-L1 facilitated metastasis to the lung in both spontaneous and experimental metastasis models. Bulk RNA-seq of tumor-bearing lungs revealed global enrichment in interferon-stimulated gene expression following loss of tumor PD-L1. Flow cytometric analysis and single-cell RNA-sequencing localized this signature to an inflammatory macrophage population that expressed PD-1 and selectively expanded following injection of PD-L1 deficient cell lines. In vitro and in vivo experimentation showed that engagement of macrophage PD-1 by tumor PD-L1 reduced the expression of interferon-stimulated genes and antigen processing machinery. We propose a model by which tumor-expressed PD-L1 regulates CTL function indirectly in the lung microenvironment by suppressing the recruitment and activation of CTLs through myeloid cells.
Single cell sequencing identifies novel metastasis-associated macrophage subsets in the lung
Authors: Huggins Danielle , LaRue Rebecca , Knutson Todd P , Schwertfeger Kathryn L 
Published: January 2023

Abstract
Macrophages are among the most abundant immune cell type found within primary mammary tumors and their metastatic lesions. Within the lung, a common site of metastasis, two major subsets of macrophages have been characterized under homeostatic conditions. However, how the complexity and heterogeneity of macrophage subsets changes with the development and progression of metastatic lesions remains unknown. We aim to identify macrophage populations that can be targeted to either suppress pro-tumorigenic function or enhance anti-tumorigenic potential in order to improve patient outcomes. In the present study, macrophages were isolated from the lungs of mice bearing 4T1-Luc mammary gland tumors in which metastatic lesions were evident. Cells were sequenced using the 10X Genomics single cell sequencing platform and at least seven distinct macrophage clusters were identified displaying unique gene expression signatures. These include clusters exhibiting upregulation of genes related to antigen presentation (H2-Aa, Cd74) and interferon signaling (Isg15, Ifitm3). Interestingly, one cluster demonstrates an expression profile highly concordant to lipid-associated macrophages found within atherosclerotic plaques (Lgals3, Trem2). Gene set enrichment analysis of this cluster reveals enrichment for genes implicated in hallmark pathways involved in anti-tumorigenic immune responses as well as for pathways related to cholesterol and metabolism. Understanding the unique localization and regulation of these cells will aid in the development of novel approaches for selectively targeting them to promote polarization toward a more anti-tumorigenic phenotype.
High-dimensional profiling of tumor-infiltrating CD4+T cells in Human cancer: Are they all tumor-specific?
Authors: Li Shamin , Simoni Yannick , Zhuang Summer , Heit Antje , Tan Iain , Tan WS Daniel , Newell Evan 
Published: January 2023

Abstract
Tumor-specific T cells act as major actors in underpinning effective immune checkpoint-blockade therapies in cancer, but most of the studies focus on CD8+ tumor-infiltrating lymphocytes (TILs). Although roles of CD4+ T cells and especially Tregs in cancer have been established, the basis for their phenotypic heterogeneity in human tumor infiltrates is not well determined. Here we profile ex-vivounexpanded CD4+ TILs in human lung and colorectal cancer using single-cell and mass-cytometry analysis. Surprisingly, up to 80% of CD4+ TILs are composed of non-Treg cells (FoxP3−). Non-Treg CD4+ TILs are highly heterogeneous and consist of different populations of effector (CD45RO+), cytotoxic (Granzyme B+), senescent (CD57+) and exhausted cells at different levels (PD-1+/++, CTLA-4+/−, TIGIT+, CD39+/−). Recently, we showed that the surface marker CD39 can accurately discriminate between bystander (CD39−) and tumor-specific (CD39+) CD8+ TILs. Across all tumors tested, more than 90% of Tregs cells expressed CD39. However, frequencies of CD39+ among non-Treg CD4+ TILs varied dramatically from 0% to 90% and strongly correlated (r2=0.8) with the frequency of CD39+CD8+ TILs in our cohort of patients. These observations suggest the presence of bystander CD4+ TILs and a high degree of coordination between the tumor-specific CD4 and CD8 T cell responses. Using in-vitroassay, we aim to confirm the presence of these bystander CD4+ TILs specific for cancer-unrelated antigens (HCMV, EBV, Influenza). Overall, our findings could highlight that not all CD4+ TILs are tumor-specific and could suggest measuring CD39 expression as a straightforward way to quantify or isolate tumor-specific T cells, thus opening new diagnostic and therapeutic avenues.
In silico assessment of somatic mutation generated T cell epitopes in various subsets of Acute Myeloid Leukemia
Authors: Loo Christopher P 
Published: January 2023

Abstract
The mechanisms of targeted-immunotherapy are still at the beginning stages for blood tumors. In the current era of big data and genomic sequencing, evolving bioinformatic tools are need for the validation and prediction of current and future immunotherapies. In particular, many neoepitope prediction programs do not consider germline context or the potential for co-occurrence of two or more somatic variants on the same mRNA transcript in the context of DNA-seq of complementary tumor and normal patient samples. Without consideration of these phenomena, existing approaches are likely to produce both false positive and false negative results, resulting in an inaccurate and incomplete picture of the cancer neoepitope landscape. In this study, we investigate the neoepitope landscape in largest-to-data dataset on acute myeloid leukemia (AML) from the Beat AML program (lead by OHSU), a cohort of 562 patients. Using a novel computational pipeline Neoepiscope (derived by Reid Thompson, MD PhD, OHSU) to chiefly address this issue for single nucleotide variants (SNVs) and insertions/deletions (indels), and herein illustrate how germline and somatic variant phasing affects neoepitope prediction. We hypothesize that in silico neoepitope prediction will accurately predict viable novel peptides that can be used for target immunotherapy in AML.
Multiplex Immunofluorescence Image Cytometry Combined with Spatially-Resolved UMAP Defines Novel Immune Prognostic Biomarkers in Metastatic Melanoma
Authors: Giraldo-Castillo Nicolas A , Berry Sneha , Stein Julie , Green Benjamin , Nguyen Peter , Soni Abha , Succaria Farah , Ates Deniz , Ogurtsova Aleks , Xu Haiying , Danilova Ludmila , Baras Alex , Taube Janis 
Published: January 2023

Abstract
Multiplex IF (mIF) provides a detailed characterization of spatial relationships and complex cell phenotypes in the tumor microenvironment. However, the data-analysis and visualization is complex and time-consuming. Here, we developed a platform to analyze mIF data through flow cytometry workflows (image cytometry), while maintaining spatial information, and applied it to tissue microarrays of metastatic melanoma specimens (n=93; 6-plex mIF panel: PD-1, PD-L1, CD163, CD8, FoxP3, Sox10/S100). Then, we used a UMAP-based approach driven by cell-to-cell distances (rather than fluorescence intensity) to display and analyze geographic organization and cell interactions. Our pipeline provided equivalent results to the digital pathology gold standard with faster run times (5-fold reduction) and higher reproducibility. We identified key prognostic immune variables, including CD8PD1Low and CD8PD1Neg cells which associated with longer overall survival (OS, both p&lt;0.01), and CD163PDL1Neg cells which associated with shorter OS (p=0.001). The spatial UMAPs showed that PD-L1 and PD-1 intensities were spatially encoded, and their expression on distinct cell subsets was organized in geographic clusters. Specifically, PD-L1Hi cells co-located to areas of CD8 cells, and PD-1Hi cells were observed near dense collections of tumor cells. Spatial UMAP subtraction analysis (survivors vs. non-survivors at 5 years) identified geographic and co-expression signatures associated with improved prognosis, i.e. CD8-driven PD-L1 expression and lacking CD163PDL1Neg macrophages. These data demonstrate the use of image cytometry and spatial UMAPs for improved visualization and interpretation of single-cell, spatially-resolved mIF data.
Myeloid Response Score (MRS): A Simple Signature That Reveals the Myeloid Contexture and Predicts the Prognosis of Hepatocellular Carcinoma
Authors: Wu Chong , Lin Jie , Weng Yulan , Zeng Dan-Ni , Zheng Limin 
Published: January 2023

Abstract
Myeloid cells, including monocytes/macrophages (Mo/Mϕ), neutrophils, and dendritic cells, can mediate divergent immune responses according to different pathological conditions. In cancer, myeloid cells play important roles in disease progression and therapy, suggesting that appraising the overall and exact intratumoral myeloid response may provide valuable information that may help to predict cancer prognosis and tailor more precise therapies. In this study, we screened biomarkers that cover major myeloid subtypes in human tumors, then constructed and validated a simple prognostic signature, called Myeloid Response Score (MRS), based on a dataset from a multi-center cohort of 1,177 HCC patients. Using multiplex immunofluorescence staining, we observed a shift of predominant myeloid population from CD169+, CD169−CD11b−CD14+, and CD169−CD11b−CD163+ Mo/Mϕs in MRSlow HCC to neutrophils and CD11b+CD169− Mo/Mϕs in MRShigh tumor. Transcriptomic and flow cytometric analyses showed that MRShigh HCC exhibited patterns of CD8+ T cell exhaustion including impaired cytotoxicity and increased PD-1 expression, suggesting highly suppressive immune microenvironment. The “climb trajectory” of MRSs between HCC tumors were depicted using the TCGA dataset, and was linked to CD8+ T cell dysfunction and poorer survival of patients. Moreover, MRS-based nomograms were highly discriminative, accurate, and useful in predicting HCC prognosis and we provide additional evidence that MRS may associate with the efficacies of post-recurrence sorafenib and transcatheter arterial chemoembolization (TACE) treatments. Together, we constructed and validated a new myeloid signature with valuable immunological and clinical potential.
Exploring the main mechanisms of anti-tumor and immunotherapy-related adverse events for safer and effective anti-CTLA-4 immunotherapy
Authors: Du Xuexiang , Liu Mingyue , Zhang Yan , Tang Fei , Zheng Pan , Liu Yang 
Published: January 2023

Abstract
Targeting CTLA-4 has shown remarkable long-term benefit and thus remains a valuable tool for cancer immunotherapy if the immunotherapy-related adverse effects (irAEs) can be brought under control. Here, we systematically studied the immunotherapeutic effect of anti-human CTLA-4 mAbs and suggest that Fc/Fc receptor mediated intra-tumor Treg depletion rather than the widely held mechanism of checkpoint blockade contribute to the cancer immunotherapeutic effect, which raised the reappraisal of CTLA-4 checkpoint blockade in cancer immunotherapy. Meanwhile, an animal model, which recapitulates clinical irAE and cancer immunotherapeutic effect (CITE), would be valuable for developing safer CTLA-4-targeting reagents. We established such an irAEs model and found that the Ipilimumab (or TremeIgG1) induced severe irAEs especially when combined with an anti-PD-1 antibody; whereas HL12/HL32 induced comparable CITE with very mild irAE under the same conditions. Furthermore we tried to uncouple the undesired irAEs from the beneficial CITE by exploring the mechanism of irAEs of anti-CTLA-4 antibodies in our irAEs model, and found that while irAE-prone Ipilimumab and TremeIgG1 rapidly direct cell surface CTLA-4 for lysosomal degradation, the non-irAE-prone antibodies we generated, HL12 or HL32, dissociate from CTLA-4 after endocytosis and allow CTLA-4 recycling to cell surface, increasing the pH sensitivity of TremeIgG1 by introducing designed tyrosine-to-histidine mutations prevents antibody-triggered lysosomal CTLA-4 downregulation and dramatically attenuates irAE. Our data establish a new paradigm for cancer research that allows for abrogating irAE while increasing CITE of anti-CTLA-4 antibodies.
A tumor-infiltrating IL23R-expressing CD4+ Treg population represents a safe and potent immunotherapeutic candidate in mice
Authors: Wight Andrew E , Sido Jessica Margaret , Degryse Sandrine , Oseghali Oba , Ao Lin , Nakagawa Hidatoshi , Kim Hye-Jung , Cantor Harvey 
Published: January 2023

Abstract
Interleukin 23 receptor (IL23R) is a signaling protein normally implicated in gut T cell development, but which has also been correlated with poor cancer prognoses. Previous studies have found that interfering with this pathway results in improved cancer outcomes in mice. These studies found that blocking IL23R was more effective than neutralizing IL23, suggesting that an IL23R-expressing cell was responsible for the tumor-protective effects observed in targeting IL23R. Here, we show that Treg-specific targeting of IL23R provides potent tumor immunity that explains previous successes with anti-IL23R therapy in cancer. We show that there is indeed a tumor-infiltrating Treg subset, marked by IL23R expression, that is enriched for markers of potent suppressive activity. Moreover, we find that targeting their IL23R expression is correlated with Treg conversion and increased IFNγ expression on tumor-infiltrating CD8 T cells. Preliminary results suggest that this Treg conversion is associated with enhanced IL12 sensitivity as a result of targeting the IL23R chain. In keeping with the tumor-specific expression pattern of IL23R, we did not find these changes in systemic T cells in the tumor-bearing mice. Ultimately, these Treg-specific IL23R-deleted mice do not display overt autoimmune disease at even advanced ages, suggesting this approach could represent a safe avenue of cancer immunotherapeutic research.
Characterization of novel CD25 antibodies that deplete human regulatory T cells in humanized mouse model
Authors: Buszko Maja A , Ward Kirstin , Christie Darah , Dziegelewski Michael , Blair David , Shevach Ethan 
Published: January 2023

Abstract
Regulatory T cells (Tregs) are key players in the suppression of immune responses and in the maintenance of immune homeostasis. Due to their central role in pathogenesis of multiple conditions, Tregs are an attractive target for immunomodulatory drug development. Tregs exhibit an activated phenotype and share the expression of many activation antigens with activated conventional T cells (Tconv). Our goal in this project was to generate and characterize monoclonal antibodies (mAbs) that would specifically react with human Tregs. mAbs were raised in mice by immunization with purified, in vitro expanded human Tregs. In contrast to widely available anti-CD25 mAbs that equally bound both activated Tregs and activated Tconv, we identified several mAbs that bound CD25 on expanded Tregs, but failed to bind to CD25 on expanded Tconv. To determine whether a similar selective binding could be demonstrated in vivo, we used the xeno-GVHD model in which NOD-SCID-γc−/− (NSG) mice are engrafted with human peripheral blood mononuclear cells (PBMCs). Several of the mAbs bound to CD25 expressed on Tregs, but not to CD25 expressed on activated Tconv. To test the effect of in vivo administration of the Treg specific mAbs, one of the clones, 2B010, was selected and injected intravenously (i.v) 2 weeks after PBMCs engraftment. Injection of 2B010 resulted in markedly reduced Tregs frequencies and numbers, much less depletion of CD25+ activated Tconv, and no reduction of CD25+CD8+ T cells. Further studies will address the effect of these mAbs at reversing the Treg-mediated protection of xeno-GVHD and enhancing tumor immunity in reconstituted NSG recipients bearing transplanted tumors.
Early Suppression of Immune Regulatory Mechanisms Reduces Tumor Progression in a Murine Model of Urothelial Cancer
Authors: Eaton Valerie L , Fantini Damiano , Yu Yanni , Chiang Ming-Yi , Meeks Joshua , Podojil Joseph R , Miller Stephen D 
Published: January 2023

Abstract
Urothelial cancer (UC) is the fifth most common cancer in the US. While cancer cells may be recognized initially by the immune system, this response is reduced as the tumor escapes immune surveillance over time. An understanding of the immune responses early in cancer progression could identify new therapies that can suppress tumor progression. To that end, we used a murine model of bladder cancer induced by the carcinogen BBN, which replicates the clinical presentation of UC in humans. RNAseq analysis of bladder tumors from mice exposed to BBN revealed an increase in immune regulatory signatures as early as 2 – 4 weeks post-initiation of exposure. These findings were confirmed by FACS analysis of spleen and bladders of mice at 0.5, 1, 2, 3 and 5 months post-initiation of exposure to BBN. The data show a bi-phasic pattern in the immune response, where there is an early increase in the number of Treg and Teff (CD4+ and CD8+) cells within the bladder at 0.5 – 1 months post-initiation of BBN exposure. The Treg and CD8+ T cells populations appear to decrease at 2 months and then increase at 3 and 5 months (corresponding to development of detectible tumor development). To determine if early suppression of Tregs affects tumor development, we depleted Tregs in wildtype C57BL/6 mice with anti-CD25 or diphtheria toxin treatment of Foxp3-DTR mice during the first month of BBN exposure. The data show that the absence of functional Tregs during the first month results in sustained Teff activity at 4 months. The increased Teff cells were found to induce a decrease in overall bladder weight, tumor development, and consequently contained fewer infiltrating immune cells. These findings suggest that early inhibition of Tregs allows for long-lived killing of tumor cells.
The human antibody repertoire genetically encodes for both productive and non-productive targeting solutions against the same broadly neutralizing antibody epitope on influenza virus
Authors: Sangesland Maya , Ghebremichael Musie , Ronsard Larance , Lingwood Daniel 
Published: January 2023

Abstract
A major obstacle to universal influenza vaccine development centers on the ability to elicit high titer responses against functionally conserved but immunologically subdominant features of this virus. Using a transgenic mouse vaccine model wherein antibodies develop with human CDRH3 diversity but are constrained to user defined antibody V genes, we have recently demonstrated that the human VH gene IGHV1-69 encodes for germline B cell receptors (BCRs) that naturally engage a conserved broadly neutralizing epitope on the hemagglutinin (HA) stalk of Group 1 influenza A viruses (IAV). Importantly, this VH endowment enables elicitation of an immunodominant bnAb response against IAV following vaccination with an engineered HA vaccine nanoparticle. However, IGHV1-69 is polymorphic with ~15% of the global population homozygous for alleles that bear a single nucleotide polymorphism (SNP) in CDRH2 (F54L) which confers germline binding to the bnAb target. To test the consequences of this, we applied our transgenic vaccine model to compare the capacity of IGHV1-69 vs IGHV1-69 SNP to elicit bnAb responses following immunization with our HA nanoparticle. We found that while both VH forms endow germline BCR targeting solutions to the same IAV bnAb site, only the WT form is productive following immunization. Germline competition within heterozygous animals (F54/L54) suggests that the capacity for pathway expansion arises via differences in the germline-endowed affinity for the same bnAb target. While globally, most humans utilize at least one copy of IGHV1-69 and would be predicted to support a bnAb response to our vaccine antigen, certain ethnicities show heightened usage of IGHV1-69 SNP and may therefore be limited by non-productive responses.
Fusion protein (M2e-HA2-NP) adjuvanted with γ-PGA/Alum induce the cross-reactivity against heterologous influenza A viruses
Authors: Kwak Chaewon , Nguyen Quyen Thi , Poo Haryoung 
Published: January 2023

Abstract
The epidemic and pandemic influenza viruses result in substantial morbidity and mortality in humans. Vaccination is the most effective way to prevent influenza virus infection. In this study, we cloned the conserved regions of influenza A virus, extracellular domain of matrix protein 2 (M2e), hemagglutinin 2 (HA2) and nucleoprotein (NP). Then, we generated fusion (M2e-HA2-NP) protein as a universal vaccine antigen candidate. γ-PGA/Alum (PA) was used as an adjuvant to enhance the efficacy of fusion protein. In mice challenged with influenza viruses, fusion protein (15 μg) provided 100% protection against A/Puerto Rico/8/1934 (H1N1; PR8) or A/California/04/09 (pH1N1; CA04) and 80% protection against H3N2 (a reassortant virus carrying HA and NA genes of A/Hong Kong/1/68). In dose-sparing effect experiments, low dose (1 or 5 μg) of fusion protein with PA provided the same protection comparable to 15 μg of fusion protein against H1N1, pH1N1 and H3N2. Humoral and cellular immunities against heterologous influenza viruses were significantly enhanced in mice vaccinated with fusion protein mixed with PA compared with alum-adjuvanted fusion protein. Taken together, the fusion protein adjuvanted with PA may be a good vaccine candidate showing heterosubtypic cross-reactivity.