bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multi-Omics Profiling of Tumor Infiltrating CD8+ T Cells Specific to Neoantigens and Tumor-Associated Antigens in Skin Cancer
Authors: Kong Lingyang , Huang Alexander , Jiang Ning 
Published: November 2024

Abstract
The objective is to characterize the immunological response towards skin cancer, particularly Merkel cell carcinoma (MCC), by conducting a detailed multi-omics analysis of CD8+ T cells infiltrating tumors, specifically those recognizing neoantigens and tumor-associated antigens. We utilized a high-throughput DNA-barcoded Tetramer-associated T cell antigen receptor sequencing (TetTCR-SeqHD) technique to profile T cell receptor (TCR) specificity, gene expression, and surface protein expression at the single-cell level. Our methodology included a neoantigen prediction pipeline using patient sequencing data, optimizing the identification of individualized neoantigens. Initial analyses were performed on melanoma patient samples, extracting tumor-antigen-specific T cells from peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs). Our results show distinct TCR clonotypes and their dynamics, providing insights into the immune landscape and disease progression in skin cancer. These findings highlight the potential of multi-omics profiling in understanding the intricate interactions between the immune system and tumor cells, offering promising avenues for advancing skin cancer therapeutics and personalized medicine. Through this research, we aim to contribute significantly to the field of cancer immunology, demonstrating the profound implications of multi-omics approaches in deciphering the multifaceted immune responses within the tumor microenvironment.
Type 2 innate lymphoid cells fuel breast cancer development by inhibiting anti-cancer immunity
Authors: Naef Pascal , Rodriguez Jacob , Savage Hannah , Adam Isam , Villalta Armando , Lawson Devon , Kessenbrock Kai 
Published: November 2024

Abstract
The tumor microenvironment is a crucial regulator of breast cancer (BC) development. It is composed of acellular and cellular factors, among others immune cells like type 2 innate lymphoid cells (ILC2s). The molecular mechanisms of how ILC2s regulate BC immunity remain largely unknown.
To reveal the phenotype of ILC2s in BC, we used the polyoma middle T antigen transgenic mouse model, mimicking luminal BC development. ScRNA sequencing and flow cytometry experiments showed that ILC2s (expressing Gata3, Rora, Il2ra, and Il7ra) accumulated during disease development and that the ST2, cKit, and Neuropilin-1 expression on ILC2s increased with tumor burden. Additionally, PMA/Ionomycin restimulated ILC2s expressed stable interleukin-5 and -13 levels. We detected the same ILC2 phenotype in the tumors of BRCA1mutp53-/- mice, which mimic the basal BC subtype. To assess the functional relevance of ILC2s in BC, we co-injected ILC2s and the BC cell line VO orthotopically into FVB mice and measured the tumor growth. While 85% of the mice that were co-injected with VO cells and ILC2s developed a tumor, 50% of the VO-only recipients rejected the tumor cells. ILC2-co-injection induced an increased infiltration of Gr-1+CD11b+ neutrophils, which could potentially inhibit anti-cancer immunity.
In summary, we show that ILC2s accumulate, get activated, and promote an immune-inhibitory environment. Further experiments will reveal if ILC2s can be used as targets to treat BC patients.
TGF-β signaling limits effector function capacity of NK cell anti-cancer immunity in human bladder cancer
Authors: Wong Joshua , Rodrigues Rossi Gustavo , Guimaraes Fernando 
Published: November 2024

Abstract
NK cells are important innate immune cells that have unique abilities to recognize and eliminate cancer cells, particularly through antibody dependent cellular cytotoxicity (ADCC). However, NK cell-based responses in bladder cancers to targeted antibody therapeutics are typically immunosuppressed, and these mechanisms in this disease are largely unknown. Single cell RNA sequencing (scRNA-seq) and high-dimensional flow cytometry were used to investigate the phenotype of tumor-infiltrating NK cells in samples from patients with bladder cancer. NK cells within bladder cancer displayed reduced expression of FcγRIIIa/CD16, the critical Fc receptor involved in ADCC-mediated cytotoxicity, on both transcriptional and protein levels. Transcriptional signatures of transforming growth factor (TGF)-β-signaling, a pleiotropic cytokine known for its immunosuppressive effects, were upregulated in tumor-infiltrating NK cells. We further validated TGF-β mediated CD16 downregulation on NK cells in vitro, which was accompanied by a transition into a tissue residency phenotype. CD16 downregulation was also abrogated by TGF-βR signaling inhibition, which could also restore the ADCC ability of NK cells subject to TGF-β effects. This study highlights how TGF-β-rich bladder cancers inhibit NK cell-mediated ADCC by downregulating CD16. TGF-β inhibition represents new avenues to reverse immunosuppression and enhance the tumoricidal capacity of NK cells.
Natural killer cells display unique, non-cytotoxic profiles in human endometrial cancer
Authors: Eisele Caprice , Riedinger Courtney , Haight Paulina , Kowalski Jesse , Mundy-Bosse Bethany , Porrett Paige , Collins Patrick , Cosgrove Casey , Freud Aharon 
Published: November 2024

Abstract
Endometrial cancer (EC) responds variably to immune checkpoint blockade, warranting study of the tumor microenvironment (TME). Natural killer (NK) cells are cytotoxic innate lymphocytes that can directly kill tumor cells, yet their diversity, locations, and functions in the EC TME are unknown. CITE-seq analysis of fresh EC tumor samples revealed multiple NK cell populations in the TME, with distinct patterns of surface activating and inhibitory receptor expression and less cytolytic granule expression by tumor-derived conventional NK (cNK) and tissue-resident NK (trNK) cells compared to autologous blood cNK. Ex vivo flow cytometry revealed significantly lower expression of cytolytic granules by TME-derived NK cell subsets (granzyme B MFI: blood cNK 1.5e5 +/- 5.4e4, tumor cNK 7.9e4 +/- 2.9e4 p&lt;0.05, tumor trNK 3.7e4 +/- 1.4e4 p&lt;0.05, n=10; perforin MFI: blood cNK 1.3e4 +/- 3.1e3, tumor cNK 1.0e4 +/- 3.2e3 p=0.19, tumor trNK 3.5e3 +/-1.3e3 p&lt;0.01, n=8). To identify NK cell subsets within the TME, we performed immunohistochemistry using CD3, CD56, granzyme B, and CD103 to demarcate cNK and trNK, respectively. We observed granzyme B+ cells primarily in the stroma (78.6 +/- 3.7% Stroma, 21.4 +/- 3.7% Glands, n=10, p&lt;0.05) and CD103+ cells primarily among tumor cells (15.6 +/- 4.5% Stroma, 84.3 +/- 0.64% Glands, n=10, p=0.07). These data suggest that both cNK and trNK cell cytotoxicity is suppressed in the EC TME and ongoing work seeks to elucidate the mechanisms.
Role of the Afferent Lymph as an Immunological Conduit to Analyze Tissue Antigenic and Inflammatory Load
Authors: Nanaware Padma , Clement Cristina , Khan Zohaib , Mota Ines , Stern Lawrence , Santambrogio Laura 
Published: November 2024

Abstract
The lymphatic fluid is the conduit by which part of the tissue “omic’ is transported to the draining lymph node for immunosurveillance. Following cannulation of the pre-nodal cervical and mesenteric afferent lymphatics we analyzed the lymph proteomic composition and determined that its tissue specificity is reflected in the dendritic cell-MHCII-eluted immunopeptidome harvested from the cervical or mesenteric nodes respectively. Additionally, since the mesenteric lymph drains directly from the lymphatics underlying the intestinal barrier, we performed the same analysis following inflammatory disruption of the gut barrier to map the antigenic and inflammatory load draining to the mesenteric lymph nodes, in both mice and human subjects with inflammatory bowel diseases. The gastrointestinal tissue damage was reflected in microbiome-derived by-products, components of the bacterial wall and other immunomodulators, including molecules of the gut-brain axis, found in the afferent lymph following damage to the gut epithelium. Our data points to the relevance of the lymphatic fluid to probe the tissue-specific antigenic and inflammatory load transported to the draining lymph node for immunosurveillance.
PD-1/cDC1 axis in the tumor draining lymph node regulates stem-like CD8+ T cell differentiation
Authors: Hor Jyh Liang , Schrom Edward , Germain Ronald 
Published: November 2024

Abstract
Stem-like CD8+ T cells constitute the major proliferative cytotoxic CD8+ T cell subset during checkpoint therapy. While it is increasingly appreciated that tumor draining LNs (tdLN) serve as reservoirs from which intratumoral stem-like CD8+ T cells can be replenished, how activated CD8+ T cells interact with the tdLN environment and differentiate into stem-like T cells remain unresolved.
Using multiplexed 3D tissue microscopy, we identified that during late differentiation (Days 4-8+), TCF-1+ PD-1+ stem-like CD8+ T cells formed distinct clustering niches with XCR1+ dendritic cells (cDC1), which serve as sites of ongoing antigen presentation in the tdLN that drive continuous stem-like T cell expansion. Intriguingly, PD-1 expression among polyclonal TCF-1+ stem-like cells is positively correlated with tetramer binding, suggesting that the PD-1/cDC1 axis promotes selective enrichment and expansion of high affinity stem-like cells during effector differentiation. In vivo blockade of PD-1/PD-L1 signaling resulted in the loss of high affinity stem-like clones in the tdLN, along with a phenotypic shift towards terminally differentiated effector cells.
Our findings raise questions about whether prolonged use of PD-1 checkpoint therapy in cancer patients interferes with maintenance of a key precursor population necessary for the robust effector generation needed to achieve full tumor regression.
This work was supported by the Intramural Research program of NIAID, NIH.
Targeting Pim2 improves effector function and longevity of T cells in cancer immunotherapy through regulating autophagy
Authors: Wu Yongxia , Tian Linlu , Pugel Allison , Yu Xue-Zhong 
Published: November 2024

Abstract
The Pim kinase family is critically involved in tumorigenesis, yet its role in primary T cells is not well known. We reported that Pim2, distinct from the other two isoforms, inhibits T-cell responses to alloantigen. To study how Pim2 impacts T-cell immunity against cancer, we used murine models of adoptive cell therapy (ACT) and hematopoietic cell transplantation. Pim2 deficiency enhanced the ability of antigen specific CD8 T cells and polyclonal T cells in controlling solid tumor and leukemia growth, respectively. Pim2 deficiency increased cytokine production and metabolic activities of CD8 T cells in vitro and promoted effector but constrained exhausted CD8 subsets in tumor. Pim2-/- CD8 T cells had increased Tcf1 expression and longevity after ACT. Mechanistically, Pim2 binds P62 and constrains T-cell activation through facilitating autophagy. Pim2 deficiency in T cells attenuated LC3 lipidation, P62 degradation as well as autophagosome formation. Augmentation of autophagy via Atg5 overexpression or metformin treatment reduced effector differentiation and anti-tumor function of Pim2-/- T cells. Importantly, a Pim2-specific inhibitor improved murine T-cell immunity against melanoma and leukemia and increased activity of human melanoma-specific T cells and CD19 CAR-T cells. Taken together, Pim2 restrains T-cell anti-tumor immunity through regulating autophagy, and blocking Pim2 enhances effector differentiation and persistence of T cells after cancer immunotherapy.
RNA sequencing methods enable immune cell gene expression profiling and clonality determination
Authors: Song Chen , Janzen Evan , Devoe Tessa , Erijman Ariel , Naishadham Gautam , Langhorst Bradley 
Published: November 2024

Abstract
The adaptive immune system defends the human body from pathogens through B/T cell functions in various organs. Abnormality of these cells may reflect immunological disorder, presence of neoplastic tissue, or response to infectious agents. High-throughput sequencing technology has facilitated understanding gene expression and related cell functions. Still, an in-depth understanding of B and T cell phenotype and clonotype profiling is an emerging application that has yet to take full advantage of sequencing technology for disease diagnosis and prognosis.
Here, we share results of RNA sequencing of immune system tissues, including healthy donor peripheral blood mononuclear cells (PBMC), lymph nodes, bone marrow, spleen, thyroid, thymus, and colon, as well as diseased PBMC and colon samples. Sequencing libraries were constructed and sequenced for B and T cell-specific gene expression and full-length immune repertoire profiling. We have identified tissue-specific B/T cell phenotype and clonality patterns related to tissue functions and disease progression. RNA-seq enables highly multiplex immunophenotyping and clonality determination for high-abundant clones that correlate with Immune-seq, with the latter targeted approach providing more in-depth clonality signatures. Integrating both RNA sequencing and immune repertoire sequencing approaches allows accurate phenotype and clonotype determination for the immune landscape in various tissues.
Multiplexed ImmunoSpot™ enables streamlined assessment of antigen-specific B cells; lessons toward maximizing cell utilization and increasing sample throughput
Authors: Kirchenbaum Greg , Chepke Jack , Becza Noemi , Yao Lingling , Liu Zhigang , Lehmann Paul 
Published: November 2024

Abstract
There are few techniques capable of detecting rare antigen-specific B cells while also providing information regarding their abundance, Ig class/subclass usage and functional affinity. The ImmunoSpot™ assay approach is suited for such antigen-specific B cell assessments since it provides information at single-cell resolution for individual ASC. Moreover, recently introduced innovations in the implementation of this assay modality have opened the door to testing larger sample sets. Towards this objective, multiplexed B cell FluoroSpot assays reduce the cell material requirement through parallel assessment of multiple Ig classes or IgG subclasses. Nevertheless, accurate determination of ASC frequencies, especially in the context of diverse antigen panels and complex exposure histories, is best achieved through evaluating multiple cell inputs to accommodate the inherent variability present within and amongst individual donors. The scope of this research effort was to further optimize the assay pipeline to facilitate high-throughput suitable assessment of antigen-specific ASC. This goal has been achieved through introducing advancements in the image analysis software component, improving both the time requirement and precision of downstream data analysis. Collectively, implementation of these assay optimizations serves to increase the feasibility of making high-throughput assessment of antigen-specific ASC using the ImmunoSpot™ approach a routine practice in immune monitoring.
Integration of single-cell transcriptomic and chromatin accessibility on heterogenicity of human peripheral blood mononuclear cells utilizing microwell-based single-cell partitioning technology
Authors: Hatami Elham , Song Hye-Won , Huang Hongduan , Zhang Zhiqi , McCarthy Thomas , Kim Youngsook , Li Ruifang , Bao Quyen , Nguyen Rosary , Narayan Punya , Wang Larry , Ayer Aruna 
Published: November 2024

Abstract
Single-cell RNA sequencing (scRNA-Seq) deepens our understanding of cellular development and heterogeneity. However, limitations exist in unraveling cell states and gene regulatory programs. Chromatin state profiles assess gene expression potential and offer insights into transcriptional regulation. Integrated with gene expression data, chromatin accessibility region (CAR) profiles establish fundamental gene regulatory logic for cell fate. ATAC-seq (Assay for Transposase-Accessible Chromatin using Sequencing) is a highly potent approach for profiling genome-wide CARs.
To investigate the power of the multiomics assay in identifying differentiated gene regulations, we conducted multiomic snATAC-seq+ snRNA-seq on PBMCs from two different donors, by utilizing the gentle and robust microwell-based single-cell partitioning technology. The assay showed high sensitivity and specificity metrics (&gt;10,000 median unique fragments/cell, &gt;0.7 fragments in peak score). Integrative analysis across donors revealed enriched transcription factor motifs and fragment coverage tracks in distinct cell types, correlating significantly with gene expression data. These findings highlight mRNA's intricate connections with CARs in immune cell development.
Our study underscores the power of multiomic analysis in analyzing heterogeneity of PBMC cell populations and offers a toolkit to identify gene regulation specific to diverse cell types, enhancing our comprehension of epigenetic diversity.
Next-Level Efficiency: StraightFrom Spleen Isolation Kits - Standardizing Isolation of Untouched T cells with Minimal Hands-On Time
Authors: Kostadinovska Konstantina , Flade Sebastian , Borczewski Amelie , Neufeld Elina , Dhana Ermanila , Winkels Gregor , Baranska Anna 
Published: November 2024

Abstract
Isolation of T cells from mouse spleen is essential for various research fields. Studies using magnetically separated cells help to understand the immune system, develop new vaccines and advance immunotherapy. Thus, selecting a reliable cell isolation method is critical for consistent experimental results.
Simultaneous spleen dissociation and labeling of unwanted cells with an antibody cocktail was performed using the GentleMACS dissociator. Subsequent automated magnetic labeling of cells and isolation of untouched T cells (CD4, CD8 or Pan T) was performed using the autoMACS NEO Separator. Target cell purity, yield, cell activation status and functionality were assessed.
Untouched CD4, CD8 or Pan T cells obtained with our StraightFrom Spleen workflow were of the same high purity and yield as conventional more time-consuming protocols, which require preparation of single cell suspension and various centrifugation steps prior to enrichment. Flow cytometric analysis of isolated cells revealed no expression of the activation markers CD25 or CD69 in non-stimulated cells even after overnight cultivation. However, significant upregulation was observed after stimulation. In addition, isolated cells showed excellent proliferation in culture.
We have developed a simple and fast cell isolation method that significantly accelerates the isolation of functional cells, providing an attractive alternative to the laborious multi-step protocols used in academic and industrial laboratories.
Molecular Pixelation: Spatial proteomics of single-cells by next generation sequencing
Authors: Glavas Steven 
Published: November 2024

Abstract
Molecular Pixelation (MPX) is a novel single cell spatial proteomics panel able to simultaneously quantify protein abundance, the spatial distribution, and colocalization of targeted cell surface proteins on up to 1,000 individual cells.
MPX creates 3D spatial maps of cells by imprinting spatial information on antibody oligonucleotide conjugates using a DNA reagent we call DNA-pixels. These DNA-pixels form over 1,000 connected spatial zones per single cell producing graphs that reconstruct the cell surface in silico; forming a single cell surface map of 80 proteins. By applying spatial statistics on these cell surface graph representations, we uncover both known and novel patterns of protein spatial polarization and co-localization associated with vital immune processes such as intercellular communication and mobility.
In our recent paper we show how MPX can be used to monitor the cell surface effects of treatment or stimulation, such as the spatial restructuring of the T cell surface proteins upon the formation of the uropod, the hind part of a migrating T cell.
With high throughput and multiplex, MPX is a powerful tool for investigating the intricate spatial organization of proteins in single cells, paving the way for new discoveries in immune system research.
Making a stabilized HLA-E complex with native properties.
Authors: Blicher Thomas , Mortensen Tobias , Brix Liselotte 
Published: November 2024

Abstract
Objective
The non-classical, human MHC-I molecule HLA-E is functionally monomorphic and therefore a highly attractive target in immunotherapy against cancers and infectious diseases. However, HLA-E is characterized by low stability due to its biological role as a rapid-response system to monitor HLA-I expression. The inherent instability makes production of HLA-E difficult and severely limits its use as a reagent, which in turn negatively impacts the understanding of HLA-E biology. Here, we present native-like, stabilized forms of HLA-E, which brings production of HLA-E in line with that of classical MHC-I molecules.
Methods
Disulfide bridges were introduced in HLA-E at selected locations deemed to not interfere with recognition of HLA-E by NK or T cell receptors. The stability of the modified HLA-E molecules was monitored using thermal denaturation, and conservation of native HLA-E properties was assessed by in vitro TCR-binding assays and through staining of NK cells.
Results
The modified HLA-E molecules exhibited increased stability, increased refolding yields, both or neither. The most stable HLA-E variant exhibited thermal stability comparable to complexes of HLA-A*02:01, while retaining native-like recognition by both NK and T cell receptors.
Summary
Stabilized forms of HLA-E have been generated and their native-like properties validated using in vitro assays and NK cell staining. This enables production of reagents for the study HLA-E biology.
Profiling antigen-reactive T cells using high throughput single-cell T cell receptor sequencing.
Authors: Papalexi Efthymia , Hariadi Bryan , Curca Crina , Roco Charles , Rosenberg Alexander 
Published: November 2024

Abstract
Recent advances in single-cell sequencing allow for simultaneous profiling of T cell receptors (TCRs) and transcriptomes leading to the characterization of key T cell subsets with antigen recognition and killing capabilities. Despite their success, these methods rely on microfluidic devices or plate-based protocols with limited sensitivity and throughput (1,000s-10,000s of cells, 1-2 samples) making the study of disease-relevant T cells time consuming and costly.
To overcome these limitations, we developed a split pool combinatorial barcoding technology to simultaneously characterize the TCRs alongside the full transcriptomes of up to 1 million T cells from up to 96 unique samples. Here, we profiled hundreds of thousands of human T cells stimulated with antigens from a variety of diseases. Applying our method, we performed deep characterization of antigen-reactive TCR clonotypes and assessed their sequence similarities. In addition, we performed transcriptome-based clustering analyses and identified highly proliferative and cytotoxic T cell subsets. Finally, we correlated T cell activation and cytotoxicity states to clonal expansion frequencies in order to identify TCRs with increased killing potential.
In summary, we present a highly flexible and scalable combinatorial barcoding technology to allow researchers to profile up to 1 million T cells in one experiment and investigate their functional responses during infection, cancer, autoimmunity, or therapeutic interventions.