bluuebunny/crossref_metadata_2025_split huggingface dataset:
The importance of distinguishing cell-cell complexes from singlets in flow cytometry and single-cell sorting: a case for an image-based cell sorter
Authors: Burel Julie G , Pomaznoy Mikhail , Arlehamn Cecilia S Lindestam , Seumois Gregory , Vijayanand Pandurangan , Sette Alessandro , Peters Bjoern 
Published: January 2023

Abstract
Our recent work highlights that care needs to be taken when interpreting single-cell data originating from flow cytometry acquisition or cell sorting: We found that doublets involving T cells bound to another immune cell are indeed very often present in the live singlet gate of human samples acquired by flow cytometry, in PBMC as well as in tissues. This hidden ‘contamination’ of cell-cell complexes generates atypical gene signatures of mixed cell lineage in what is assumed to be single cells, which can lead to data misinterpretation, such as the description of novel immune cell types with dual lineage properties. Based on the example of T cell-monocyte complexes, we investigated experimental and data analysis strategies to help distinguishing between singlets and cell-cell complexes in non-imaging flow cytometry and single-cell sorting. We found robust molecular signatures in both T cell-monocyte and T cell-B cell complexes that can distinguish them from singlets at both protein and mRNA level. Imaging flow cytometry with appropriate gating strategy (matching the one used in cell sorting) and direct microscopy imaging after cell sorting were the two preferred methods to validate the presence of cell-cell complexes in suspicious dual-expressing cells. In parallel, we also investigated the molecular profile of T cells and monocytes present in a doublet and found that their association is not random, but enriched for LFA1/ICAM1 interactions and gene expression profiles associated with disease-specific immune responses. Taken together, these data highlight the importance of systematically investigating for the presence of cell-cell complexes in single-cell data derived from non-imaging flow cytometry.
Adaptive optics permits label-free intravital imaging of tissue resident and infiltrating immune cells in the mouse retina
Authors: Chu Colin J , Joseph Aby , Feng Guanping , Dholakia Kosha , Schallek Jesse B 
Published: January 2023

Abstract
The eye is ideally suited for unperturbed longitudinal intravital imaging of immune responses. No confounding inflammatory surgery, externalization or depressurization is required for access as it is uniquely transparent. Directly imaging immune cells in vivo would greatly advance our biological understanding but it is not currently possible in man, and only feasible in mice using exogenous fluorescent labeling or transgenic modification which can result in physiologically unrepresentative results.
We discover both tissue resident and infiltrating immune cells can be visualized in the retina using a custom adaptive optics scanning light ophthalmoscope (AOSLO) employing phase contrast detection of reflected 796nm light. This is analogous to scatter characteristics observed in flow cytometry, with motile cells visible within the laminated structure of the retina.
Anesthetized mice underwent intraocular injection of LPS to induce acute inflammation before repeated imaging at the same anatomical location across intervals from 6 hours to 2 months. Leukocyte rolling within post-capillary venules and tissue infiltration was visible without exogenous contrast. We confirmed 83% of this population were neutrophils by co-localization following tail vein injection of Alexa Fluor-647 anti-Ly6G antibody. Tracking the same location revealed reduced motility (24 vs 6 hours) despite cellular persistence preceding resolution of inflammation. Microglia could be imaged including their highly motile processes, confirmed using healthy eyes from Cx3cr1GFP/+ reporter mice.
This is the first report of label-free immune cell imaging within the mouse retina and the approach could feasibly translate to existing human AOSLO platforms.
ctDNA for early cancer diagnosis in Pacific Communities
Authors: Sika-Paotonu Dianne , Anitelea Toni , Faatoese Adam , Laing Bridie , Puni Ron , Vaipuna Tevita , Guilford Parry 
Published: January 2023

Abstract
Circulating tumour DNA (ctDNA) refers to small fragments of tumour DNA found in the blood circulation, recognised by the presence of mutations in cancer genes. These DNA fragments can be isolated and quantified to obtain data about the cancer’s size and progression. Since gaining access to and obtaining biopsy samples from solid cancers in people is not always possible, the possibility of utilizing a simple blood sample to allow detection and monitoring of cancer growth is highly desirable. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer diagnostics and surveillance and also lead to improved cancer health outcomes for Pacific Peoples. The purpose of this work was to support engagement with Pacific communities and share, and discuss and explore current research efforts, that included the potential utilization of ctDNA to improve the diagnosis, care and health outcomes of Pacific Peoples. A Pacific Fono (gathering) was held in the Hutt Valley region to promote appropriate knowledge translation activity, engagement and meaningful dialogue between researchers and the Pacific communities regarding current research works and findings of direct relevance and significance to Pacific communities. The Pacific community response to this research outreach effort was overwhelmingly positive and encouraging. Discussions were respectful and informative with shared information being exchanged between researchers and those in attendance. Feedback included expressed support for regarding the ctDNA research and for other areas of research focus that would be of benefit to Pacific communities. Requests from Pacific community members were also made for future similar events to be held.
ViaKrome Fixable Viability Dyes: A new approach to dead cell discrimination for intracellular multicolor flow cytometry application
Authors: Dupas Nathalie , Dempsey Nancy , Bowers Rita , Burgaud Mégane , Miloud Tewfik 
Published: January 2023

Abstract
The ability to stain live cells with a viability dye and preserve that staining pattern after fixation is critical for intracellular immunophenotyping by flow cytometry. Excluding dead cells from the data allows cleaner separation and identification of cell populations. All commercially available viability dyes are based on NHS ester activated functional groups crosslinking with primary amines on proteins. These functional groups are susceptible to hydrolysis, necessitating the use of non-aqueous solvents, and additional reagent handling steps to protection from water vapor, such as aliquoting the stock solution and reducing freeze-thaw cycles. Here, we propose a new thiol reactive fluorescent dye that covalently binds free thiols present on cellular proteins at a neutral pH. Live cells, impermeable to the FVD, will be weakly stained by the covalent binding of proteins express on the cell surface while dead cells whose membranes have lost structural integrity, will be strongly stained by the covalent binding of intracellular and membrane expressed proteins.
Contrary to current dyes, the ViaKrome dyes are soluble in aqueous solution and do not require DMSO for re-suspension. Hence, we show that the stability of dye ensure robust and reproducible experimental data.
To further increase the ease of use, this new thiol based chemistry is available in a range of dyes excited by the common lasers used in flow cytometry. This choice provides flexibility for multicolor panel design. The compatibility of the ViaKrome dyes, i.e ViaKrome 405, ViaKrome 561, ViaKrome 638 and ViaKrome 808 will be presented in different workflow set-up such as 18-colors immunphenotyping panels and functional assays using PBMCs and whole blood.
Graft vs. host disease leads to elimination of tissue-resident innate lymphoid cells following experimental and clinical allogeneic transplantation
Authors: Vinci Paola , Garcia-Martinez Elena , O’Connor Margaret H. , Egorova Anastasiya , Kuttiyara Jason , Arnhold Viktor , Ponce Doris M. , Hanash Alan M. 
Published: January 2023

Abstract
Innate lymphoid cells (ILCs) are associated with protective mucosal responses after allogeneic bone marrow transplantation (allo-BMT). However, the specific in vivo roles of ILCs after BMT remain unclear. We thus sought to characterize the kinetics and significance of ILC reconstitution after allo-BMT.
In a sex-mismatched acute graft-vs-host-disease (GVHD) model, depletion of recipient ILCs induced significantly worse systemic signs of GVHD, as well as increased thymic injury and intestinal pathology, indicating a contributory role for ILCs in tissue protection post-BMT.
Analysis of ILCs in distinct GVHD target organs after T-cell-depleted BMT demonstrated a high frequency of NK cells and ILC1s in the liver, a higher proportion of ILC2s in lungs, and a higher proportion of ILC3s in the small intestine post-transplant in the absence of GVHD. For all tissues analyzed, the majority of ILCs were host-derived. In contrast, in the presence of GVHD, all ILC subsets were significantly reduced in all tissues analyzed. Furthermore, ILC precursors were significantly reduced in the bone marrow of mice with GVHD, suggesting that alloreactive immunity can impair ILC reconstitution by targeting both mature ILCs and their developmental niche.
To examine the clinical relevance of these findings, human ILCs were analyzed by FACS of lamina propria lymphocytes isolated from patient duodenal biopsy specimens obtained to evaluate symptoms of acute GI GVHD. Consistent with experimental findings, duodenal ILC frequencies were significantly lower in patients with histologic evidence of GVHD.
In conclusion, tissue-resident ILCs contribute to tissue protection after allo-BMT, but GVHD leads to ILC elimination and impairs their reconstitution.
Ablation of IRF4 in donor T cells eliminates GVHD while partially preserving GVT in experimental bone marrow transplantation
Authors: Guo Yang , Chen Wenhao 
Published: January 2023

Abstract

Objective
We discovered that the transcription factor IRF4 governs T cell function in heart transplantation (Immunity 47:1114). Here, using a bone marrow (BM) transplantation model, we studied the role of IRF4 in regulating T cell function in graft-vs-host disease (GVHD) and graft-vs-tumor (GVT).

Methods
9 Gy irradiated Balb/c recipients were i.v. injected: 1) with 5 × 106 T cell-depleted CD45.1+ B6 BM cells; 2) with or without 0.1 × 106 Balb/c A20-luciferase lymphoma cells; and 3) with or without 1 × 106 CD45.2+ wild type (WT), Irf4−/−, or Irf4−/−Foxp3sf/Y (Treg-deficient Irf4−/−) B6 donor T cells. GVHD, GVT, survival, and donor T cell states were determined.

Results
In the absence of A20 infusion, all recipients without T cell transfer (n=10), or transferred with Irf4−/− (n=10) or Irf4−/−Foxp3sf/Y (n=5) donor T cells survived long-term (&gt;60 days post BM transplantation). Recipients transferred with WT donor T cells had developed lethal GVHD [mean survival time (MST) = 9.5±2 days; n =8]. Among A20 infused groups, recipients without T cell transfer died due to lymphoma progression (MST=22±2.80 days; n =15). Transfer of Irf4−/− donor T cells led to a moderate GVT effect and significantly prolonged survival (MST=33.4 ± 11.22 days; n =15; p&lt;0.001 vs no T cell). Transferred WT donor T cells exerted potent GVT activity but caused lethal GVHD (12.56 ± 6.02 days; n = 18). Mechanistically, Irf4−/− donor T cells developed into effector T cells, but barely infiltrated into GVHD tissues and were prone to apoptosis.

Conclusions
IRF4 ablation in donor T cells eliminates GVHD in a Treg independent manner while partially preserving GVT. This reveals the possibility to separate GVHD and GVT effects by modulating transcription factors in T cells.
PRMT5 promotes STAT-1 mediated T cell inflammatory response and is a therapeutic target for acute graft-versus-host disease
Authors: Snyder Katiri Jenna , Zitzer Nina C. , Gao Yandi , Choe Hannah K. , Sell Natalie E. , Neidemire-Colley Lotus , Devine Raymond , Behbehani Gregory , Pietrzak Maciej , Vaddi Kris , Baiocchi Robert , Ranganathan Parvathi 
Published: January 2023

Abstract

Introduction
T cell mediated acute graft-versus-host disease (aGVHD) is the main cause of non-relapse mortality in allogeneic hematopoietic cell transplant recipients. Protein arginine methyltransferase 5 (PRMT5) is a post-translational modifier and promotes activation of memory TH cells. We investigate mechanisms by which PRMT5 regulates T cell function and propose PRMT5 inhibition as a therapeutic strategy for aGVHD.

Materials and Methods
PRMT5 expression and function was evaluated in T cells of healthy and aGVHD mice and humans. We assessed T cell proliferation and effector function, using C220, a novel PRMT5 inhibitor. We tested effects of PRMT5 inhibition using in vivo mouse models of aGVHD, where mice received T cell depleted bone marrow + allogeneic splenocytes. In graft-versus-leukemia (GVL) experiments, P815 tumor cells were administered. Mice were treated with C220 or vehicle and monitored for survival and clinical aGVHD scores.

Results
PRMT5 expression and function is upregulated in T cells of mice and humans with aGVHD. Inhibition of PRMT5 reduces T cell proliferation, perturbs cell cycle and downregulates STAT-1 mediated interferon response. Administration of C220 improved survival in aGVHD mouse models and maintained tumor specific CTL response contributing to retention of Graft versus leukemia (GVL) effect.

Conclusions
Inhibition of PRMT5 using C220, down-regulates T cell proliferative and effector response, induces cell-cycle arrest, reduces interferon response and perturbs signaling pathways. C220 shows potent biological activity in vivo by reducing aGVHD clinical severity and prolonging survival in mouse models without compromising GVL. Therefore, PRMT5 is a novel and druggable target for aGVHD.
Recipient vitamin A levels influence the risk of graft-versus-host disease after experimental allogeneic stem cell transplantation
Authors: Pan Pan , Taylor Brian M , Zhu Haojie , Zhou Dian , Chen Xiao 
Published: January 2023

Abstract
Graft-versus-host disease (GVHD) remains a major complication after allogeneic hematopoietic stem cell transplantation (HSCT). Retinoic acid (RA), the active metabolite of vitamin A, has been implicated in GVHD pathogenesis. However, whether recipient vitamin A levels modulate GVHD risk remains controversial based upon recent clinical data. To gain insights into this clinically relevant question, Balb/c mice were kept on diets containing normal (VAN) or excessive (VAH) amounts of vitamin A. Lethally irradiated VAN and VAH mice were then transplanted with bone marrow and splenic T cells from MHC-mismatched C57BL/6 mice to induce GVHD. There was no significant difference in the expansion of donor CD4 and CD8 T cells in the spleen and CD4 T cells in the mesenteric lymph nodes between the two groups early after HSCT. There was also no significant difference in the expression of gut-homing molecule α4β7 on donor T cells in VAN versus VAH mice. However, there was a significant reduction in serum LPS levels in VAH mice compared with those of VAN mice in later stage of GVHD development, indicating a better maintained intestinal barrier integrity. Gene expression of proinflammatory cytokines including IFN-γ and TNF-α was also significantly decreased in the colon tissues of VAH mice compared with that of VAN mice. Despite reduced intestinal inflammation, VAH mice unexpectedly died significantly faster than VAN mice after transplantation. Collectively, our studies indicated that higher vitamin A levels are associated with accelerated GVHD-associated mortality after experimental HSCT. Therefore, some caution should be exercised when considering high dose vitamin A supplementation for GVHD prevention as proposed in recent clinical trials.
Intestinal dysbiosis predicts lower gastrointestinal tract acute graft-versus-host (aGVHD) disease development
Authors: da Silva Marina Burgos , Ponce Doris M. , Gomes Antonio L.C. , Moore Gillian , Slingerland John B. , Pamer Eric , Taur Ying , Peled Jonathan U. , Van Den Brink Marcel R.M. 
Published: January 2023

Abstract

Introduction
aGVHD is a major complication after hematopoietic cell transplantation (HCT) and is driven by alloimmune targeting of intestine, liver, and skin. As aGVHD has been linked to intestinal dysbiosis and the majority of the intestinal microbiota biomass resides within the colon, we hypothesized that certain microbiota features confer protection to the lower GI tract.

Methods
We evaluated 16S stool profiles of 849 samples from 215 recipients of unmodified allo-HCT. Patients were classified in four groups according to aGVHD organ involvement: upper GI only (UGI, n =53), lower GI with/without upper GI (LGI, n=56), no GI involvement (non-GI, n=30) and no GVHD (n=76). Samples were grouped into pre-onset (day −20 to 0) and post-onset (day 1 to 20) aGVHD.

Results
Pre-onset, LGI patients showed a decreased strict anaerobes to facultative anaerobes (A/F) ratio vs. non-GI (p=0.036) and a low A/F ratio was associated with reduced overall survival (p=0.004) from aGVHD onset. After aGVHD onset, LGI patients had reduced microbial alpha diversity, abundance of predicted butyrate-producing (BP) bacteria and A/F ratio vs. non-GI patients (p=0.017, p=0.003 and p=0.020, respectively). LGI patients also had less BP bacteria, members of genus Blautia, and lower A/F ratio (p=0.0.017, p=0.008 and p=0.048 respectively) vs. UGI patients.

Conclusions
LGI patients had distinct microbial features consistent with dysbiosis, when compared to UGI and non-GI patients. Importantly, we observed microbiota disruption in the 20 days preceding aGVHD onset, suggesting a predictive marker of LGI aGVHD and an association with overall survival, which have potential practical implications for GVHD risk stratification and therapeutic interventions.
Tc17 cells cause gut graft-versus-host disease through induction of dysbiosis and depletion of CX3CR1hi phagocytes
Authors: Song Qingxiao , Wang Xiaoning , Wu Xiwei , Qin Hanjun , Zhao Dongchang , Jenq Robert R , Van Den Brink Marcel RM , Riggs Arthur D. , Martin Paul J , Chen Yuanzhong , Zeng Defu 
Published: January 2023

Abstract
Th/Tc17 but not Th/Tc1 cells are resistant to steroid-induced apoptosis or inhibition of cytokine production, but the role of Th/Tc17 in pathogenesis of steroid-resistant intestinal graft versus host disease (SR-Gut-GVHD) remains controversial. Here, we show a reduction of IFN-g-producing Th/Tc1 cells and expansions of IL-22-producing Th17 and especially Tc17 cells in the gut tissues of mice with SR-Gut-GVHD. IL-22 from Tc17-dervied from IFN-g−/− donor CD8+ T cells augments host-tissue production of RegIIIg, leading to dysbiosis. IL-22 also augments host tissue production of IL-18 that sustains Tc17 expansion. In the absence of donor-derived IFN-g, host-tissue PD-L1 depletes donor-derived CX3CR1hi intestinal mononuclear phagocytes (MNP), leading to increased bacterial translocation and loss of IL-18 binding protein that neutralizes IL-18. Depletion of CX3CR1hi MNP in the colon is also observed in mice with SR-Gut-GVHD. Taken together, our results indicate that depletion of donor-derived CX3CR1hi MNP in the colon plays an important role in Tc17-mediated gut GVHD pathogenesis. These results call for caution in clinical testing of IL-22 agonists or IFN-g antagonist in patients with SR-Gut-GVHD, and they offer a new avenue toward understanding mechanisms that account for SR-Gut-GVHD.
Long term time-course monitoring of NK cell-mediated ADCC using the Celigo Image Cytometer
Authors: Chan Leo , Wucherpfennig Kai W , De Andrade Lucas 
Published: January 2023

Abstract
Antibody-dependent cell-mediated cytotoxicity (ADCC) assay has been widely performed for immuno-oncological research. Traditionally, ADCC assays are conducted by measuring the amount of released Chromium, calcein AM, or Lactate dehydrogenase (LDH) molecules after the target cancer cells are killed by effector/antibody pair. These methods can be inaccurate due to indirect measurement of supernatant at the end point of 4 hour, however, there is a need to characterize the effects of antibody-dependent cytotoxicity for longer than 72 hours. In this work, we demonstrated the time-course monitoring of NK cell-mediated ADCC of A375 cells in the presence or absence of IL2 and antibodies for 76 hours. First, A375-ZsGreen cells were seeded into a 96-well plate with and without target antibodies. Next, NK cells with and without IL2 were added to the wells at 10:1, 5:1, 2:1, and 1:1 E:T ratios. The co-culture was allowed to incubate for 76 hours and the image cytometer was used to scan and analyze at times from 0 to 76 hours. The software was able to count individual cells as well as confluence % at each time point by segmenting highly fluorescent objects in the images. We were able to show time-dependent ADCC killing with decrease in ZsGreen positive cells as the read-out. In addition, the uniformity of cell killing can be observed in the Celigo obtained whole well images. One of the most important findings was that there was regrowth of target cells after the initial 30 hours of cell killing, indicating the activated NK cells without antibodies did not eradicate the cancer cells. Therefore, the ability to perform long term time-course monitoring of ADCC is highly important to better characterize the effects of target antibodies on cell killing function.
Immune pressure against ovarian cancer depends on antigen-specific CD69+CD103+TRM T cells
Authors: Galindo Carmen Maria Anadon , Yu Xiaoqing , Gonzalez Ricardo A Chaurio , Payne Kyle K , Biswas Subir , Costich Tara Lee , Perez-Sanz Jairo , Mandal Gunjan , Martin Alexandra L , Harro Carly , Powell Chase , Mine Jessica A , Wang Zhitao , Conejo-Garcia Jose 
Published: January 2023

Abstract
The relationship between stem-like, tissue resident memory (TRM) and exhausted T cells at tumor beds is incompletely understood. We found that more than 50% of the CD8+ T cells in human ovarian carcinomas are TRM cells. RNA seq of TRM and their re-circulating counterparts infiltrating multiple human ovarian carcinomas showed a very distinctive phenotype, characterized by co-upregulation of effector and exhaustion markers, along with increased clonality and marked proliferative and lipid metabolism signatures. Interestingly, both populations showed very little overlap in TCR repertoire. Single cell analysis of TRM population showed that this is a heterogeneous population composed of different clusters defined by gene expression and TCR repertoire. A distinctive cluster shows higher expression of cytotoxic mediators and exhaustion markers, along with higher clonality and higher proliferation rate, with a TCR repertoire shared by CD103+TCF7+ stem-like cells. Single cell ATACseq showed that TRM and their counterpart have different chromatin structure and, within both populations also appear variables epigenetic landscapes. In addition, the presence of TRM is correlated with better prognosis, while tumor antigen-specific TRM T cells transferred into syngeneic tumor-bearing mice were more effective at delaying tumor growth than their tumor antigen-specific re-circulating counterparts. Together, our data indicate that productive immune pressure against malignant progression depends on a subset of CD8+ stem-like T cells differentiating into a narrow cluster of TRM T cells of ~300 TCR clones, which represent true tumor antigen-specific effector lymphocytes.