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We describe retrospective data from the largest series of patients (n=142) with multiple sclerosis (MS) from Pakistan. Mean age at onset was 27 years, with a female to male ratio of 1.45:1. The disease onset was polysymptomatic in 75% patients. Motor weakness was the most common onset symptom (70%), followed by sensory symptoms (45%). Optico-spinal type of MS was seen in only 3% of patients The courzse was relapsing-remitting (RR) in 81%, primary progressive (PP) in 21%, and secondary progressive (SP) in 4% of patients. Almost three-fourths of the patients were moderately (45%) or severely (31%) disabled at the time of evaluation. Two-thirds of patients with severe disability had a mean disease duration of only 5.2 years. In conclusion, MS is not uncommon in Pakistan, and many patients were found to have severe disability despite short disease duration. [ABSTRACT FROM AUTHOR]

Despite the curative potential of allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia (AML), its efficacy is limited by intrinsic resistance of cancer cells to donor-derived T-cell cytotoxicity. Using a genome-wide CRISPR screen, we identified the SOCS1-JAK1-STAT1 pathway as a mediator of AML susceptibility to T cells. SOCS1 knockdown in AML cells sensitized them to killing by allogeneic T cells, whereas SOCS1 overexpression in AML cells induced resistance to T-cell anti-leukemic activity. Mechanistically, SOCS1 protected AML cells from T-cell killing by antagonizing IFNγ-JAK1-induced ICAM-1 expression. Furthermore, primary AML cells with lower SOCS1 expression correlated with better overall survival in patients, especially those with a lower exhausted CD8+ T-cell score. Thus, this study reveals SOCS1 and its downstream mediators as a potential targetable pathway to enhance T cell-based immunotherapy for AML.

Background: Development of cancer treatments such as antibody-based therapy relies on several factors across the drug-target axis, including the specificity of target expression and characterization of downstream signaling pathways. While existing tools for analyzing and visualizing transcriptomic data offer evaluation of individual gene-level expression, they lack a comprehensive assessment of pathway-guided analysis, relevant for single- and dual-targeting therapeutics. Here, we introduce VIBE (VIsualization of Bulk RNA Expression data), an R package which provides a thorough exploration of both individual and combined gene expression, supplemented by pathway-guided analyses. VIBE's versatility proves pivotal for disease stratification and therapeutic targeting in cancer and other diseases., Results: VIBE offers a wide array of functions that streamline the visualization and analysis of transcriptomic data for single- and dual-targeting therapies. Its intuitive interface allows users to evaluate the expression of target genes and their associated pathways across various cancer indications, aiding in target and disease prioritization. Metadata, such as treatment or number of prior lines of therapy, can be easily incorporated to refine the identification of patient cohorts hypothesized to derive benefit from a given drug. We demonstrate how VIBE can be used to assist in indication selection and target identification in three user case studies using both simulated and real-world data. VIBE integrates statistics in all graphics, enabling data-informed decision-making., Conclusions: VIBE facilitates detailed visualization of individual and cohort-level summaries such as concordant or discordant expression of two genes or pathways. Such analyses can help to prioritize disease indications that are amenable to treatment strategies such as bispecific or monoclonal antibody therapies. With this tool, researchers can enhance indication selection and potentially accelerate the development of novel targeted therapies with the goal of precision, personalization, and ensuring treatments align with an individual patient's disease state across a spectrum of disorders. Explore VIBE's full capabilities using the vignettes on the GitLab repository (https://gitlab.com/genmab-public/vibe )., Competing Interests: IK, SA, ICK, LM, CK, BH, and FB are all employees and shareholders of Genmab. The authors are employees of Genmab and received funding from their company to produce this work., (Copyright © 2025 Khatri, van Asten, Moreno, Higgs, Klijn, Blokzijl and Kolder.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a second-line treatment with curative potential for leukemia patients. However, the prognosis of allo-HSCT patients with disease relapse or graft-versus-host disease (GvHD) is poor. CD4 + or CD8 + conventional T (Tconv) cells are critically involved in mediating anti-leukemic immune responses to prevent relapse and detrimental GvHD. Hence, treatment for one increases the risk of the other. Thus, therapeutic strategies that can address relapse and GvHD are considered the Holy Grail of allo-HSCT. CD3 + CD4 - CD8 - double-negative T cells (DNTs) are unconventional mature T cells with potent anti-leukemia effects with "off-the-shelf" potential. A phase I clinical trial demonstrated the feasibility, safety, and potential efficacy of allogeneic DNT therapy for patients with relapsing acute myeloid leukemia (AML) post-allo-HSCT. Here, we studied the impact of DNTs on the anti-leukemic and GvHD-inducing activities of Tconv cells. DNTs synergized with Tconv cells to mediate superior anti-leukemic activity. Mechanistically, DNTs released soluble factors which activated and evoked potent anti-leukemic activities of Tconv cells. In contrast, DNTs suppressed GvHD-inducing activities of Tconv cells in a CD18-dependent manner by mediating cytotoxicity against proliferative Tconv cells. The seemingly opposite immunological activities of DNTs were dictated by the presence or absence of AML cells. Collectively, these results support the potential of DNTs as an adjuvant to allo-HSCT to address both disease relapse and GvHD., Competing Interests: Declarations. Consent for publication: All of the authors concur with the submission of this manuscript. Competing interests: L.Z. has financial interests (e.g., holdings/shares) in WYZE Biotech Co Ltd and previously received research funding and consulting fee/honorarium from the Company. L.Z., J.B.L and H.K. are inventors of several patents related to DNT cell technology, and L.Z. received license fees. The remaining authors declare no conflict of interest., (© 2025. The Author(s).)

Meta-researchers commonly leverage tools that infer gender from first names, especially when studying gender disparities. However, tools vary in their accuracy, ease of use, and cost. The objective of this study was to compare the accuracy and cost of the commercial software Genderize and Gender API, and the open-source gender R package. Differences in binary gender prediction accuracy between the three services were evaluated. Gender prediction accuracy was tested on a multi-national dataset of 32,968 gender-labeled clinical trial authors. Additionally, two datasets from previous studies with 5779 and 6131 names, respectively, were re-evaluated with modern implementations of Genderize and Gender API. The gender inference accuracy of Genderize and Gender API were compared, both with and without supplying trialists' country of origin in the API call. The accuracy of the gender R package was only evaluated without supplying countries of origin. The accuracy of Genderize, Gender API, and the gender R package were defined as the percentage of correct gender predictions. Accuracy differences between methods were evaluated using McNemar's test. Genderize and Gender API demonstrated 96.6% and 96.1% accuracy, respectively, when countries of origin were not supplied in the API calls. Genderize and Gender API achieved the highest accuracy when predicting the gender of German authors with accuracies greater than 98%. Genderize and Gender API were least accurate with South Korean, Chinese, Singaporean, and Taiwanese authors, demonstrating below 82% accuracy. Genderize can provide similar accuracy to Gender API while being 4.85x less expensive. The gender R package achieved below 86% accuracy on the full dataset. In the replication studies, Genderize and gender API demonstrated better performance than in the original publications. Our results indicate that Genderize and Gender API achieve similar accuracy on a multinational dataset. The gender R package is uniformly less accurate than Genderize and Gender API., Competing Interests: JLW is supported by grants from the National Cancer Institute (NCI), the American Association for Cancer Research (AACR), and Brown Physicians Incorporated through his institution. He reports consulting for Westat and The Lewin Group, outside the submitted work. He reports ownership of HemOnc.org LLC; shares have no monetary value. He is the Chief Technology Officer of HemOnc.org LLC; this position is uncompensated. SM is supported by grants from the NCI and the AACR through his institution. He has received money from the National Institutes of Health (NIH) and the Federation of American Societies for Experimental Biology (FASEB), personal fees from the Disney Corporation, and hospitality from the Schmidt Family Foundation. ADV, IK, CGH, and EGDU declare that no competing interests exist., (Copyright: © 2024 VanHelene et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Abstract: Allogeneic double-negative T cells (DNTs) are a rare T-cell subset that effectively target acute myeloid leukemia (AML) without inducing graft-versus-host disease in an allogeneic setting. A phase 1 clinical trial demonstrated the feasibility, safety, and potential efficacy of allogeneic DNT therapy among patients with relapsed AML. However, the molecular mechanisms of DNT-mediated cytotoxicity against AML remain elusive. Thus, we used a flow cytometry-based high throughput screening to compare the surface molecule expression profile on DNTs during their interaction with DNT-susceptible or -resistant AML cells and identified a tumor necrosis factor α (TNFα)-dependent cytotoxic pathway in DNT-AML interaction. TNFα secreted by DNTs, upon encountering susceptible AML targets, sensitized AML cells to DNT-mediated killing, including those otherwise resistant to DNTs. Mechanistically, TNFα upregulated ICAM-1 on AML cells through a noncanonical JAK1-dependent pathway. DNTs then engaged with AML cells more effectively through an ICAM-1 receptor, lymphocyte function-associated antigen 1, leading to enhanced killing. These results reveal a TNFα-JAK1-ICAM-1 axis in DNT-mediated cytotoxicity against AML to improve therapeutic efficacy., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Identifying cancer risk groups by multi-omics has attracted researchers in their quest to find biomarkers from diverse risk-related omics. Stratifying the patients into cancer risk groups using genomics is essential for clinicians for pre-prevention treatment to improve the survival time for patients and identify the appropriate therapy strategies. This study proposes a multi-omics framework that can extract the features from various omics simultaneously. The framework employs autoencoders to learn the non-linear representation of the data and applies tensor analysis for feature learning. Further, the clustering method is used to stratify the patients into multiple cancer risk groups. Several omics were included in the experiments, namely methylation, somatic copy-number variation (SCNV), micro RNA (miRNA) and RNA sequencing (RNAseq) from two cancer types, including Glioma and Breast Invasive Carcinoma from the TCGA dataset. The results of this study are promising, as evidenced by the survival analysis and classification models, which outperformed the state-of-the-art. The patients can be significantly (p-value<0.05) divided into risk groups using extracted latent variables from the fused multi-omics data. The pipeline is open source to help researchers and clinicians identify the patients' risk groups using genomics., (© 2024. The Author(s).)

Abstract: Allogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 Epstein-Barr virus transformed B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, that is translated from unconventional open reading frames, for example long noncoding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The high cost of acquiring and annotating samples has made the "few-shot" learning problem of prime importance. Existing works mainly focus on improving performance on clean data and overlook robustness concerns on the data perturbed with adversarial noise. Recently, a few efforts have been made to combine the few-shot problem with the robustness objective using sophisticated meta-learning techniques. These methods rely on the generation of adversarial samples in every episode of training, which further adds to the computational burden. To avoid such time-consuming and complicated procedures, we propose a simple but effective alternative that does not require any adversarial samples. Inspired by the cognitive decision-making process in humans, we enforce high-level feature matching between the base class data and their corresponding low-frequency samples in the pretraining stage via self distillation. The model is then fine-tuned on the samples of novel classes where we additionally improve the discriminability of low-frequency query set features via cosine similarity. On a one-shot setting of the CIFAR-FS dataset, our method yields a massive improvement of 60.55% and 62.05% in adversarial accuracy on the projected gradient descent (PGD) and state-of-the-art auto attack, respectively, with a minor drop in clean accuracy compared to the baseline. Moreover, our method only takes 1.69× of the standard training time while being ≈ 5× faster than thestate-of-the-art adversarial meta-learning methods. The code is available at https://github.com/vcl-iisc/robust-few-shot-learning.

Summary: T-cell receptors (TCRs) on T cells recognize and bind to epitopes presented by the major histocompatibility complex in case of an infection or cancer. However, the high diversity of TCRs, as well as their unique and complex binding mechanisms underlying epitope recognition, make it difficult to predict the binding between TCRs and epitopes. Here, we present the utility of transformers, a deep learning strategy that incorporates an attention mechanism that learns the informative features, and show that these models pre-trained on a large set of protein sequences outperform current strategies. We compared three pre-trained auto-encoder transformer models (ProtBERT, ProtAlbert, and ProtElectra) and one pre-trained auto-regressive transformer model (ProtXLNet) to predict the binding specificity of TCRs to 25 epitopes from the VDJdb database (human and murine). Two additional modifications were performed to incorporate gene usage of the TCRs in the four transformer models. Of all 12 transformer implementations (four models with three different modifications), a modified version of the ProtXLNet model could predict TCR-epitope pairs with the highest accuracy (weighted F1 score 0.55 simultaneously considering all 25 epitopes). The modification included additional features representing the gene names for the TCRs. We also showed that the basic implementation of transformers outperformed the previously available methods, i.e. TCRGP, TCRdist, and DeepTCR, developed for the same biological problem, especially for the hard-to-classify labels. We show that the proficiency of transformers in attention learning can be made operational in a complex biological setting like TCR binding prediction. Further ingenuity in utilizing the full potential of transformers, either through attention head visualization or introducing additional features, can extend T-cell research avenues., Availability and Implementation: Data and code are available on https://github.com/InduKhatri/tcrformer., (© The Author(s) 2023. Published by Oxford University Press.)

Although absence of interleukin-7 (IL-7) signaling completely abrogates T and B lymphopoiesis in mice, patients with severe combined immunodeficiency caused by mutations in the IL-7 receptor α chain (IL-7Rα) still generate peripheral blood B cells. Consequently, human B lymphopoiesis has been thought to be independent of IL-7 signaling. Using flow cytometric analysis and single-cell RNA sequencing of bone marrow samples from healthy controls and patients who are IL-7Rα deficient, in combination with in vitro modeling of human B-cell differentiation, we demonstrate that IL-7R signaling plays a crucial role in human B lymphopoiesis. IL-7 drives proliferation and expansion of early B-cell progenitors but not of pre-BII large cells and has a limited role in the prevention of cell death. Furthermore, IL-7 guides cell fate decisions by enhancing the expression of BACH2, EBF1, and PAX5, which jointly orchestrate the specification and commitment of early B-cell progenitors. In line with this observation, early B-cell progenitors of patients with IL-7Rα deficiency still expressed myeloid-specific genes. Collectively, our results unveil a previously unknown role for IL-7 signaling in promoting the B-lymphoid fate and expanding early human B-cell progenitors while defining important differences between mice and humans. Our results have implications for hematopoietic stem cell transplantation strategies in patients with T- B+ severe combined immunodeficiency and provide insights into the role of IL-7R signaling in leukemogenesis., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Patients with relapsed/refractory T-cell malignancies have limited treatment options. The use of chimeric antigen receptor (CAR)-T cell therapy for T-cell malignancies is challenging due to possible blast contamination of autologous T-cell products and fratricide of CAR-T cells targeting T-lineage antigens. Recently, allogeneic double-negative T cells (DNTs) have been shown to be safe as an off-the-shelf adoptive cell therapy and to be amendable for CAR transduction. Here, we explore the antitumor activity of allogeneic DNTs against T-cell malignancies and the potential of using anti-CD4-CAR (CAR4)-DNTs as adoptive cell therapy for T-cell malignancies., Methods: Healthy donor-derived allogeneic DNTs were ex vivo expanded with or without CAR4 transduction. The antitumor activity of DNTs and CAR4-DNTs against T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma (PTCL) were examined using flow cytometry-based cytotoxicity assays and xenograft models. Mechanisms of action were investigated using transwell assays and blocking assays., Results: Allogeneic DNTs induced endogenous antitumor cytotoxicity against T-ALL and PTCL in vitro, but high doses of DNTs were required to attain therapeutic effects in vivo. The potency of DNTs against T-cell malignancies was significantly enhanced by transducing DNTs with a third-generation CAR4. CAR4-DNTs were manufactured without fratricide and showed superior cytotoxicity against CD4 + T-ALL and PTCL in vitro and in vivo relative to empty-vector transduced-DNTs. CAR4-DNTs eliminated T-ALL and PTCL cell lines and primary T-ALL blasts in vitro. CAR4-DNTs effectively infiltrated tumors, delayed tumor progression, and prolonged the survival of T-ALL and PTCL xenografts. Further, pretreatment of CAR4-DNTs with PI3Kδ inhibitor idelalisib promoted memory phenotype of CAR4-DNTs and enhanced their persistence and antileukemic efficacy in vivo. Mechanistically, LFA-1, NKG2D, and perforin/granzyme B degranulation pathways were involved in the DNT-mediated and CAR4-DNT-mediated killing of T-ALL and PTCL., Conclusions: These results demonstrate that CAR4-DNTs can effectively target T-ALL and PTCL and support allogeneic CAR4-DNTs as adoptive cell therapy for T-cell malignancies., Competing Interests: Competing interests: LZ has financial interests (eg, holdings/shares) in Wyze Biotech Co Ltd and previously received research funding and consulting fee/honorarium from the Company. LZ and JL are inventors of several DNT cell technology-related patents and intellectual properties. JL, IK, and LZ are inventors of a patent related to this study., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Monitoring of innate myeloid cells (IMC) is broadly applied in basic and translational research, as well as in diagnostic patient care. Due to their immunophenotypic heterogeneity and biological plasticity, analysis of IMC populations typically requires large panels of markers. Currently, two cytometry-based techniques allow for the simultaneous detection of ≥40 markers: spectral flow cytometry (SFC) and mass cytometry (MC). However, little is known about the comparability of SFC and MC in studying IMC populations., Methods: We evaluated the performance of two SFC and MC panels, which contained 21 common markers, for the identification and subsetting of blood IMC populations. Based on unsupervised clustering analysis, we systematically identified 24 leukocyte populations, including 21 IMC subsets, regardless of the cytometry technique., Results: Overall, comparable results were observed between the two technologies regarding the relative distribution of these cell populations and the staining resolution of individual markers (Pearson's ρ=0.99 and 0.55, respectively). However, minor differences were observed between the two techniques regarding intra-measurement variability (median coefficient of variation of 42.5% vs. 68.0% in SFC and MC, respectively; p<0.0001) and reproducibility, which were most likely due to the significantly longer acquisition times (median 16 min vs. 159 min) and lower recovery rates (median 53.1% vs. 26.8%) associated with SFC vs. MC., Discussion: Altogether, our results show a good correlation between SFC and MC for the identification, enumeration and characterization of IMC in blood, based on large panels (>20) of antibody reagents., Competing Interests: JJMvD and AO are chairmen of the EuroFlow scientific foundation, which receives royalties from licensed patents, which are collectively owned by the participants of the EuroFlow Foundation and are exclusively used for continuation of the EuroFlow collaboration and sustainability of the EuroFlow consortium. JJMvD and AO report an Educational Services Agreement from BD Biosciences San José, CA and a Scientific Advisor Agreement with Cytognos/BD Biosciences; all related fees and honoraria are for the involved university departments at Leiden University Medical Center and University of Salamanca. JJMvD, AO, CT and KvdP are listed as coinventors on the patent “Means and methods for multiparameter cytometry-based leukocyte subsetting” PCT/NL2020/050688, filing date 5 November 2019, owned by the EuroFlow scientific consortium, that formed the basis for part of the antibody combination described in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest., (Copyright © 2023 van der Pan, Khatri, de Jager, Louis, Kassem, Naber, de Laat, Hameetman, Comans, Orfao, van Dongen, Díez and Teodosio.)

Despite their low abundance in the tumor microenvironment (TME), classical type 1 dendritic cells (cDC1) play a pivotal role in anti-cancer immunity, and their abundance positively correlates with patient survival. However, their interaction with CD4 + T-cells to potentially enable the cytotoxic T lymphocyte (CTL) response has not been elucidated. Here we show that contact with activated CD4 + T-cells enables human ex vivo cDC1, but no other DC types, to induce a CTL response to cell-associated tumor antigens. Single cell transcriptomics reveals that CD4 + T-cell help uniquely optimizes cDC1 in many functions that support antigen cross-presentation and T-cell priming, while these changes don't apply to other DC types. We robustly identify "helped" cDC1 in the TME of a multitude of human cancer types by the overlap in their transcriptomic signature with that of recently defined, tumor-infiltrating DC states that prove to be positively prognostic. As predicted from the functional effects of CD4 + T-cell help, the transcriptomic signature of "helped" cDC1 correlates with tumor infiltration by CTLs and Thelper(h)-1 cells, overall survival and response to PD-1-targeting immunotherapy. These findings reveal a critical role for CD4 + T-cell help in enabling cDC1 function in the TME and may establish the helped cDC1 transcriptomic signature as diagnostic marker in cancer., (© 2023. The Author(s).)

Mass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations., Competing Interests: Authors JD and AO are chairmen of the EuroFlow scientific foundation, which receives royalties from licensed patents, which are collectively owned by the participants of the EuroFlow foundation, to be exclusively used for the continuation of the EuroFlow collaboration and sustainability of the EuroFlow consortium, originally supported by the European Commission (EU-STREP Project LSHB-CT-2006-018708). Authors JD and AO report an Educational Services Agreement from BD Biosciences (San José, CA) and a Scientific Advisor Agreement with Cytognos; all related fees and honoraria are for the involved university departments at Leiden University Medical Center and University of Salamanca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van der Pan, Kassem, Khatri, de Ru, Janssen, Tjokrodirijo, al Makindji, Stavrakaki, de Jager, Naber, de Laat, Louis, van den Bossche, Vogelezang, Balvers, Lamfers, van Veelen, Orfao, van Dongen, Teodosio and Díez.)

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated ( vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings., Competing Interests: JD and AO report to be chairmen of the EuroFlow scientific foundation, which receives royalties from licensed patents, which are collectively owned by the participants of the EuroFlow Foundation. These royalties are exclusively used for continuation of the EuroFlow collaboration and sustainability of the EuroFlow consortium. JD and AO report an Educational Services Agreement from BD Biosciences (San José, CA) and a Scientific Advisor Agreement with Cytognos; all related fees and honoraria are for the involved university departments at Leiden University Medical Center and University of Salamanca. AH-D is an employee of Cytognos (Salamanca, Spain). Lastly, JD, CT, AO, JA, WB, KP, MB, AD, DD, and AH-D, are listed as (co)inventors on the patent “Means and methods for multiparameter cytometry-based leukocyte subsetting” (NL2844751, filing date 5 November 2019), owned by the EuroFlow scientific consortium, which describes the flow cytometry panels developed in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van der Pan, de Bruin-Versteeg, Damasceno, Hernández-Delgado, van der Sluijs-Gelling, van den Bossche, de Laat, Díez, Naber, Diks, Berkowska, de Mooij, Groenland, de Bie, Khatri, Kassem, de Jager, Louis, Almeida, van Gaans-van den Brink, Barkoff, He, Ferwerda, Versteegen, Berbers, Orfao, van Dongen and Teodosio.)

The development of autologous chimeric antigen receptor T (CAR-T) cell therapies has revolutionized cancer treatment. Nevertheless, the delivery of CAR-T cell therapy faces challenges, including high costs, lengthy production times, and manufacturing failures. To overcome this, attempts have been made to develop allogeneic CAR-T cells using donor-derived conventional CD4 + or CD8 + T cells (T convs ), but severe graft-versus-host disease (GvHD) and host immune rejection have made this challenging. CD3 + CD4 - CD8 - double-negative T cells (DNTs) are a rare subset of mature T cells shown to fulfill the requirements of an off-the-shelf cellular therapy, including scalability, cryopreservability, donor-independent anticancer function, resistance to rejection, and no observed off-tumor toxicity including GvHD. To overcome the challenges faced with CAR-T convs , we evaluated the feasibility, safety, and efficacy of using healthy donor-derived allogeneic DNTs as a CAR-T cell therapy platform. We successfully transduced DNTs with a second-generation anti-CD19-CAR (CAR19) without hampering their endogenous characteristics or off-the-shelf properties. CAR19-DNTs induced antigen-specific cytotoxicity against B cell acute lymphoblastic leukemia (B-ALL). In addition, CAR19-DNTs showed effective infiltration and tumor control against lung cancer genetically modified to express CD19 in xenograft models. CAR19-DNT efficacy was comparable with that of CAR19-T convs . However, unlike CAR19-T convs , CAR19-DNTs did not cause alloreactivity or xenogeneic GvHD-related mortality in xenograft models. These studies demonstrate the potential of using allogeneic DNTs as a platform for CAR technology to provide a safe, effective, and patient-accessible CAR-T cell treatment option.

The IMGT database profiles the TR germline alleles for all four TR loci (TRA, TRB, TRG and TRD), however, it does not comprise of the information regarding population specificity and allelic frequencies of these germline alleles. The specificity of allelic variants to different human populations can, however, be a rich source of information when studying the genetic basis of population-specific immune responses in disease and in vaccination. Therefore, we meticulously identified true germline alleles enriched with complete TR allele sequences and their frequencies across 26 different human populations, profiled by "1000 Genomes data". We identified 205 TRAV, 249 TRBV, 16 TRGV and 5 TRDV germline alleles supported by at least four haplotypes. The diversity of germline allelic variants in the TR loci is the highest in Africans, while the majority of the Non-African alleles are specific to the Asian populations, suggesting a diverse profile of TR germline alleles in different human populations. Interestingly, the alleles in the IMGT database are frequent and common across all five super-populations. We believe that this new set of germline TR sequences represents a valuable new resource which we have made available through the new population-matched TR (pmTR) database, accessible via https://pmtrig.lumc.nl/ ., (© 2022. The Author(s).)

To mount an adequate immune response against pathogens, stepwise mutation and selection processes are crucial functions of the adaptive immune system. To better characterize a successful vaccination response, we performed longitudinal (days 0, 5, 7, 10, and 14 after Boostrix vaccination) analysis of the single-cell transcriptome as well as the B-cell receptor (BCR) repertoire (scBCR-rep) in plasma cells of an immunized donor and compared it with baseline B-cell characteristics as well as flow cytometry findings. Based on the flow cytometry knowledge and literature findings, we discriminated individual B-cell subsets in the transcriptomics data and traced over-time maturation of plasmablasts/plasma cells (PB/PCs) and identified the pathways associated with the plasma cell maturation. We observed that the repertoire in PB/PCs differed from the baseline B-cell repertoire e.g., regarding expansion of unique clones in post-vaccination visits, high usage of IGHG1 in expanded clones, increased class-switching events post-vaccination represented by clonotypes spanning multiple IGHC classes and positive selection of CDR3 sequences over time. Importantly, the Variable gene family-based clustering of BCRs represented a similar measure as the gene-based clustering, but certainly improved the clustering of BCRs, as BCRs from duplicated Variable gene families could be clustered together. Finally, we developed a query tool to dissect the immune response to the components of the Boostrix vaccine. Using this tool, we could identify the BCRs related to anti-tetanus and anti-pertussis toxoid BCRs. Collectively, we developed a bioinformatic workflow which allows description of the key features of an ongoing (longitudinal) immune response, such as activation of PB/PCs, Ig class switching, somatic hypermutation, and clonal expansion, all of which are hallmarks of antigen exposure, followed by mutation & selection processes.

The double negative T cell (DNT) is a unique subset of T cells with potent anti-leukemic potential. Previously, DNT therapy has been shown to effectively target AML cells in patient-derived xenograft (PDX) models. Further, a recently completed phase I/IIa clinical study demonstrated the safety, feasibility, and potential efficacy in AML patients that relapsed after allogeneic hematopoietic stem cell transplantation. However, the persistence and durability of DNT-mediated anti-leukemic response is less well understood. In this study, we characterized the in vivo persistence of DNTs in PDX models. Further, we improved the efficacy and durability of DNT-mediated activity with phosphoinositide 3-kinase delta (PI3Kδ) inhibition. Mechanistically, DNTs treated with the PI3Kδ inhibitor, Idelalisib (Ide), exhibited early memory phenotype with superior viability and proliferative capacity but less cell exhaustion. Collectively, the findings from this study support the use of Ide-treated DNTs to improve its therapeutic outcome.

We study the effects of strain on the monomer and dimer diffusion mechanisms and island morphology during the growth of Cu on a biaxially strained Cu(100) substrate. We find an approximately linear dependence of the activation barriers on strain. In particular, while hopping is favored for compressive and/or small (<2%) tensile strain, for greater than 2% tensile strain, the exchange mechanism is favored. We then present the results of temperature-accelerated dynamics simulations of submonolayer growth at 200 K. For the case of 2% compressive strain we find that, as in previous kinetic Monte Carlo simulations of Cu/Ni(100) growth, the competition between island growth and multi-atom relaxation ("pop-out") events leads to an island morphology with a mixture of open and closed steps. At slightly higher coverage, island coalescence then leads to elongated islands. However, annealing leads to a significant decrease in the number of open steps. In contrast, for the case of 8% tensile strain, only one large strongly anisotropic island is formed. Surprisingly, we find that despite the large strain, the island anisotropy is not due to energetics but is instead due to anisotropic attachment barriers that favor the exchange-mediated attachment of monomers to corners over close-packed step-edges. An explanation for the asymmetry in attachment barriers is provided. Our results provide a new general kinetic mechanism for the formation of anisotropic islands in the presence of isotropic diffusion and tensile strain.

Immunoglobulin (IG) loci harbor inter-individual allelic variants in many different germline IG variable, diversity and joining genes of the IG heavy (IGH), kappa (IGK) and lambda (IGL) loci, which together form the genetic basis of the highly diverse antigen-specific B-cell receptors. These allelic variants can be shared between or be specific to human populations. The current immunogenetics resources gather the germline alleles, however, lack the population specificity of the alleles which poses limitations for disease-association studies related to immune responses in different human populations. Therefore, we systematically identified germline alleles from 26 different human populations around the world, profiled by "1000 Genomes" data. We identified 409 IGHV, 179 IGKV, and 199 IGLV germline alleles supported by at least seven haplotypes. The diversity of germline alleles is the highest in Africans. Remarkably, the variants in the identified novel alleles show strikingly conserved patterns, the same as found in other IG databases, suggesting over-time evolutionary selection processes. We could relate the genetic variants to population-specific immune responses, e.g. IGHV1-69 for flu in Africans. The population matched IG (pmIG) resource will enhance our understanding of the SHM-related B-cell receptor selection processes in (infectious) diseases and vaccination within and between different human populations., (© 2021. The Author(s).)

Antigen-specific serum immunoglobulin (Ag-specific Ig) levels are broadly used as correlates of protection. However, in several disease and vaccination models these fail to predict immunity. In these models, in-depth knowledge of cellular processes associated with protective versus poor responses may bring added value. We applied high-throughput multicolor flow cytometry to track over-time changes in circulating immune cells in 10 individuals following pertussis booster vaccination (Tdap, Boostrix ® , GlaxoSmithKline). Next, we applied correlation network analysis to extensively investigate how changes in individual cell populations correlate with each other and with Ag-specific Ig levels. We further determined the most informative cell subsets and analysis time points for future studies. Expansion and maturation of total IgG1 plasma cells, which peaked at day 7 post-vaccination, was the most prominent cellular change. Although these cells preceded the increase in Ag-specific serum Ig levels, they did not correlate with the increase of Ig levels. In contrast, strong correlation was observed between Ag-specific IgGs and maximum expansion of total IgG1 and IgA1 memory B cells at days 7 to 28. Changes in circulating T cells were limited, implying the need for a more sensitive approach. Early changes in innate immune cells, i.e. expansion of neutrophils, and expansion and maturation of monocytes up to day 5, most likely reflected their responses to local damage and adjuvant. Here we show that simultaneous monitoring of multiple circulating immune subsets in blood by flow cytometry is feasible. B cells seem to be the best candidates for vaccine monitoring., Competing Interests: AD, CT, JD, AO, MP-A and MB report inventorship of the patent “Means and methods for multiparameter cytometry-based leukocyte subsetting” (NL2844751, filing date 5 November 2019) (21), owned by the EuroFlow Consortium. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Diks, Khatri, Oosten, de Mooij, Groenland, Teodosio, Perez-Andres, Orfao, Berbers, Zwaginga, van Dongen and Berkowska.)

The pandemic of Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 has induced global eagerness to develop vaccines and therapeutics for treating COVID-19, including neutralizing antibodies. To develop effective therapeutic antibodies against SARS-CoV-2, it is critical to understand the interaction between viral and host's proteins. The human ACE2 ( h ACE2) protein is the crucial target for the SARS-CoV's Spike protein that allows the virus to adhere to host epithelial cells. X-ray crystal structures and biophysical properties of protein-protein interactions reveal a large interaction surface with high binding-affinity between SARS-CoV-2 and h ACE2 (18 interactions), at least 15-fold stronger than between SARS-CoV-1 and h ACE2 (eight interactions). This suggests that antibodies against CoV-1 infection might not be very efficient against CoV-2. Furthermore, interspecies comparisons indicate that ACE2 proteins of man and cat are far closer than dog, ferret, mouse, and rat with significant differences in binding-affinity between Spike and ACE2 proteins. This strengthens the notion of productive SARS-CoV-2 transmission between felines and humans and that classical animal models are not optimally suited for evaluating therapeutic antibodies. The large interaction surface with strong affinity between SARS-CoV-2 and h ACE2 (dG-12.4) poses a huge challenge to develop reliable antibody therapy that truly blocks SARS-CoV-2 adherence and infection. We gauge that single antibodies against single epitopes might not sufficiently interfere with the strong interaction-synapse between Spike and h ACE2 proteins. Instead, appropriate combinations of high-affinity neutralizing antibodies against different epitopes might be needed, preferably of IgA-class for optimal and prolonged activity at epithelial layers of respiratory and intestine tracts., (Copyright © 2020 Khatri, Staal and van Dongen.)

Pancreatic ductal adenocarcinoma (PDAC) is generally incurable due to the late diagnosis and absence of markers that are concordant with expression in several sample sources (i.e., tissue, blood, plasma) and platforms (i.e., Microarray, sequencing). We optimized meta-analysis of 19 PDAC (tissue and blood) transcriptome studies from multiple platforms. The key biomarkers for PDAC diagnosis with secretory potential were identified and validated in different cohorts. Machine learning approach i.e., support vector machine supported by leave-one-out cross-validation was used to build and test the classifier. We identified a 9-gene panel (IFI27, ITGB5, CTSD, EFNA4, GGH, PLBD1, HTATIP2, IL1R2, CTSA) that achieved ∼0.92 average sensitivity and ∼0.90 average specificity in distinguishing PDAC from healthy samples in five training sets using cross-validation. These markers were also validated in proteomics and single-cell transcriptomics studies suggesting their prognostic role in the diagnosis of PDAC. Our 9-gene classifier can not only clearly discriminate between better and poor survivors but can also precisely discriminate PDAC from chronic pancreatitis (AUC = 0.95), early stages of progression [Stage I and II (AUC = 0.82), IPMA and IPMN (AUC = 1), and IPMC (AUC = 0.81)]. The 9-gene marker outperformed the previously known markers in blood studies particularly (AUC = 0.84). The discrimination of PDAC from early precursor lesions in non-malignant tissue (AUC > 0.81) and peripheral blood (AUC > 0.80) may assist in an early diagnosis of PDAC in blood samples and thus will also facilitate risk stratification upon validation in clinical trials., (Copyright © 2020 Khatri and Bhasin.)

Various factors including diet, age, geography, culture and socio-economic status have a role in determining the composition of the human gut microbiota. The human gut microbial composition is known to be altered in disease conditions. Considering the important role of the gut microbiome in maintaining homeostasis and overall health, it is important to understand the microbial diversity and the functional metagenome of the healthy gut. Here, we characterized the microbiota of 31 fecal samples from healthy individuals of Indian ethnic tribes from Ladakh, Jaisalmer and Khargone by shotgun metagenomic sequencing. Sequence analysis revealed that Bifidobacterium and Prevotella were the key microbes contributing to the differences among Jaisalmer, Khargone and Ladakh samples at the genus level. Our correlation network study identified carbohydrate-active enzymes and carbohydrate binding proteins that are associated with specific genera in the different Indian geographical regions studied. Network analysis of carbohydrate-active enzymes and genus abundance revealed that the presence of different carbohydrate-active enzymes is driven by differential abundance of genera. The correlation networks were different in the different geographical regions, and these interactions suggest the role of less abundant genera in shaping the gut environment. We compared our data with samples from different countries and found significant differences in taxonomic composition and abundance of carbohydrate-active enzymes in the gut microbiota as compared to the other countries., Competing Interests: The authors have declared that no competing interests exist.

The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.

Background: Some of the spore-forming strains of Bacillus probiotics are marketed commercially as they survive harsh gastrointestinal conditions and bestow health benefits to the host., Results: We report the composite genome of Bacillus clausii ENTPro from a commercially available probiotic Enterogermina® and compare it with the genomes of other Bacillus probiotics. We find that the members of B. clausii species harbor high heterogeneity at the species as well as genus level. The genes conferring resistance to chloramphenicol, streptomycin, rifampicin, and tetracycline in the B. clausii ENTPro strain could be identified. The genes coding for the bacteriocin gallidermin, which prevents biofilm formation in the pathogens Staphylococcus aureus and S. epidermidis, were also identified. KEGG Pathway analysis suggested that the folate biosynthesis pathway, which depicts one of the important roles of probiotics in the host, is conserved completely in B. subtilis and minimally in B. clausii and other probiotics., Conclusions: We identified various antibiotic resistance, bacteriocins, stress-related, and adhesion-related domains, and industrially-relevant pathways, in the genomes of these probiotic bacteria that are likely to help them survive in the harsh gastrointestinal tract, facilitating adhesion to host epithelial cells, persistence during antibiotic treatment and combating bacterial infections.

The species Bacillus badius is one of the oldest members of the genus Bacillus isolated from faeces of children and was classified based on its ability to form endospores [8]. In 16S rRNA gene sequence and phylogenetic analysis, Bacillus badius DSM 23 T shared low similarity (93.0%) and distant relationship with B. subtilis, the type species of the genus Bacillus indicating that it does not belong to this genus. Additional strains of the species, B. badius DSM 5610, DSM 30822 and B. encimensis SGD-V-25 (which has been recently reclassified as a member of this species) were included in the study to consider intraspecies diversity. Detailed molecular phylogenetic and comparative genome analysis clearly showed that the strains of B. badius were consistently retrieved outside the cluster of Bacillus sensu stricto and also distantly related to the genera Domibacillus and Quasibacillus. Further, the data from biochemical reactions (inability to ferment most carbohydrates), polar lipids profile (presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an aminophosphoglycolipid) and fatty acids supported the molecular analysis. Thus the four B. badius strains; DSM 23 T , DSM 5610, DSM 30822 and SGD-V-25 displayed sufficient demarcating phenotypic characteristics that warrant their classification as members of a novel genus and single species, for which the name Pseudobacillus badius gen. nov. comb. nov. is proposed with Pseudobacillus badius DSM 23 T (= ATCC 14574 T ) as the type strain. Additionally, based on our findings from phenotypic, chemotaxonomic and genotypic parameters, Bacillus wudalianchiensis DSM 100757 T was reclassified as Pseudobacillus wudalianchiensis comb. nov., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The genus Chiltana Shakila-Mushtaq & Akbar, 1995 is redescribed and redefined based on the types and new material from Pakistan. Chiltana includes two species, C.acarinata sp. n. and C.baluchi Shakila-Mushtaq & Akbar, 1995 (the type species), both from Chiltan, Balochistan, Pakistan. A key to the species of the genus is provided. Nomenclatorial remarks on original publication, author, and date of Chiltana are given.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a 5-year survival rate of <8%. Its dismal prognosis stems from inefficient therapeutic modalities owing to the lack of understanding about pancreatic cancer pathogenesis. Considering the molecular complexity and heterogeneity of PDAC, identification of novel molecular contributors involved in PDAC onset and progression using global "omics" analysis will pave the way to improved strategies for disease prevention and therapeutic targeting. Meta-analysis of multiple miRNA microarray datasets containing healthy controls (HC), chronic pancreatitis (CP) and PDAC cases, identified 13 miRNAs involved in the progression of PDAC. These miRNAs showed dysregulation in both tissue as well as blood samples, along with progressive decrease in expression from HC to CP to PDAC. Gene-miRNA interaction analysis further elucidated 5 miRNAs (29a/b, 27a, 130b and 148a) that are significantly downregulated in conjunction with concomitant upregulation of their target genes throughout PDAC progression. Among these, miRNA-29a/b targeted genes were found to be most significantly altered in comparative profiling of HC, CP and PDAC, indicating its involvement in malignant evolution. Further, pathway analysis suggested direct involvement of miRNA-29a/b in downregulating the key pathways associated with PDAC development and metastasis including focal adhesion signaling and extracellular matrix organization. Our systems biology data analysis, in combination with real-time PCR validation indicates direct functional involvement of miRNA-29a in PDAC progression and is a potential prognostic marker and therapeutic candidate for patients with progressive disease.

Functional aptamers displaying agonistic or antagonistic properties are showing great promise as modulators of immune responses. Here, we report the development of a polyethylene glycol-modified (PEGylated) DNA aptamer as a cross-species (murine and human) CD200R1 agonist that modulates inflammatory responses in vivo. Specifically, DNA aptamers were discovered by performing independent SELEX searches on recombinant murine and human CD200R1. Aptamer motifs identified by next generation sequencing (NGS) were subsequently compared, leading to the discovery of motifs common to both targets. The CD200R1 DNA aptamer CCS13 displayed the highest agonistic activity toward CD200R1 in terms of suppressing the induction of cytotoxic T-lymphocytes (CTLs) in both human and murine allogeneic-mixed lymphocyte cultures (allo-MLCs). A 20-kDa polyethylene glycol (PEG) chain was covalently attached to the 5' end of this aptamer, and the resulting conjugate was shown to block inflammatory responses in murine models of skin graft rejection and house-dust-mite-induced allergic airway inflammation. Importantly, this agonistic aptamer does not suppress CTL induction in 5-day allo-MLCs with responder cells derived from CD200R1 -/- mice, indicating that its mode of action is directly linked to CD200R1 activation. This study suggests that one can derive agonistic DNA aptamers that can be verified as immuno-modulators in murine models with outcomes potentially translatable to the treatment of human conditions., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Previously, we reported that Zika virus (ZIKV) causes ocular complications such as chorioretinal atrophy, by infecting cells lining the blood-retinal barrier, including the retinal pigment epithelium (RPE). To understand the molecular basis of ZIKV-induced retinal pathology, we performed a meta-analysis of transcriptome profiles of ZIKV-infected human primary RPE and other cell types infected with either ZIKV or other related flaviviruses (Japanese encephalitis, West Nile, and Dengue). This led to identification of a unique ZIKV infection signature comprising 43 genes (35 upregulated and 8 downregulated). The major biological processes perturbed include SH3/SH2 adaptor activity, lipid and ceramide metabolism, and embryonic organ development. Further, a comparative analysis of some differentially regulated genes (ABCG1, SH2B3, SIX4, and TNFSF13B) revealed that ZIKV induced their expression relatively more than dengue virus did in RPE. Importantly, the pharmacological inhibition of ABCG1, a membrane transporter of cholesterol, resulted in reduced ZIKV infectivity. Interestingly, the ZIKV infection signature revealed the downregulation of ALDH5A1 and CHML, genes implicated in neurological (cognitive impairment, expressive language deficit, and mild ataxia) and ophthalmic (choroideremia) disorders, respectively. Collectively, our study revealed that ZIKV induces differential gene expression in RPE cells, and the identified genes/pathways (e.g., ABCG1) could potentially contribute to ZIKV-associated ocular pathologies.

We previously showed that congenic bone marrow transplantation (BMTx) post myeloablation augmented tissue allograft survival in association with increased regulatory T (Treg) cells of both host and bone marrow donor origin. Regulatory B (Breg) cells can also modulate T-cell immunity and B cells may be implicated in the development of Treg cells. Accordingly, we explored the effect of B-cell depletion in vivo on augmented graft survival post BMTx. C57BL/6 mice received BALB/c skin allografts followed 7 days later by myeloablation using cyclophosphamide and busulphan. Mice then received T-cell-depleted bone marrow from CD45.1 congenic donors, and ongoing immunosuppression with rapamycin (to day 28 after BMTx). Control mice received cyclophosphamide and busulphan followed by rapamycin, but not congenic bone marrow. At different times post BMTx, mice received B-cell-depleting antibody treatment, and the effect on both skin graft survival, and induction of Treg cells was assessed. BMTx resulted in significantly prolonged skin graft survival versus control mice, in association with attenuated donor-specific alloreactivity relative to controls, increased splenic Treg cells and significantly diminished anti-donor IgG. In mice receiving infusion of B-depleting antibodies for 12 days from day 15 post BMTx, both graft survival and Treg cell activity were diminished, particularly for functional Treg cells of donor origin. Adoptive transfer of Breg cells from mice harvested at 15 days post BMTx prolonged survival in naive transplanted mice and increased Treg cell levels. Thus, autologous BMTx augmentation of graft survival is dependent in part upon a population of Breg cells that can modulate the function of donor-derived Treg cells., (© 2018 John Wiley & Sons Ltd.)

Injections of a crude fetal sheep liver extract (FSLE) containing fetal hemoglobin, MPLA, and glutathione (GSSH) reversed cytokine changes in aged mice. To investigate the role of fetal hemoglobin we derived mice with homzygous deletions for either of the two major βchains, Hgbβ ma KO or Hgbβ mi KO. Hgbβ mi is the most prominent fetal Hgbβ chain, with Hgbβ ma more prominent in adult mice. Mice lacking another fetal Hgb chain, HgbεKO, died in utero. CHO cells transfected with cloned Hgb chains were used to produce proteins for preparation of rabbit heteroantibodes. Splenocytes from Hgbβ ma KO mice stimulated in vitro with Conconavalin A showed a higher IL-2:IL-4 ratio than cells from Hgbβ mi KO mice. Following immunization in vivo with ovalbumin in alum, Hgbβ ma KO mice produced less IgE than Hgbβ mi KO mice, suggesting that in the absence of Hgbβ mi KO mice had a predeliction to heightened allergic-type responses. Using CHO cells transfected with cloned Hgb chains, we found that only the fetal Hgb chain, Hgbε, was secreted at high levels. Secretion of Hgbβ ma or Hgbβ mi chains was seen only after genetic mutation to introduce the two N-linked glycosylation sites present in Hgbε, but absent in the Hgbβ chains. We speculated that a previously unanticipated biological function of a naturally secreted fetal Hgb chain may be partly responsible for the effects reported following injection of animals with fetal, not adult, Hgb. Mice receiving injections of rabbit anti-Hgbε but not either anti-Hgbβ ma or anti-Hgbβ mi from day 14 gestation also showed a bias towards the higher IL-2:IL-4 ratios seen in Hgbβ mi KO mice.

In previous studies we had reported that the immunosuppressive cell membrane bound molecule CD200 is released from the cell following cleavage by matrix metalloproteases, with the released soluble CD200 acting as an immunosuppressant following binding to, and signaling through, its cognate receptor CD200R expressed on target cells. We now show that although the intracellular cytoplasmic tail (CD200 C-tail ) of CD200 has no consensus sites for adapter molecules which might signal the CD200 + cell directly, cleavage of the CD200 C-tail from the membrane region of CD200 by a consensus γ-secretase, leads to nuclear translocation and DNA binding (identified by chromatin immunoprecipitation followed by sequencing, Chip-sequencing) of the CD200 C-tail . Subsequently there occurs an altered expression of a limited number of genes, many of which are transcription factors (TFs) known to be associated with regulation of cell proliferation. Altered expression of these TFs was also prominent following transfection of CD200 + B cell lines and fresh patient CLL cells with a vector construct containing the CD200 C-tail . Artificial transfection of non CD200 + Hek293 cells with this CD200 C-tail construct resulted in altered expression of most of these same genes. Introduction of a siRNA for one of these TFs, POTEA, reversed CD200 C-tail regulation of altered cell proliferation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chronic Lymphocytic Leukemia B cells (CLL) are malignant cells which retain at least some functions of normal B cells. Paramount amongst the latter is that when such cells are appropriately stimulated, they are able to present antigens, including any potential tumor antigens, making them excellent choices as a candidate tumor vaccine. We show that following stimulation of CLL cells with Phorbol myristic acetate, IL-2, the TLR7 agonist imiquimod (P2I) and ionomycin (P2Iio), markedly increased expression of CD54 and CD83 was seen, indicative of B cell activation and a transition to antigen-presenting cells. However, this occurred in the context of augmented expression of the known immunoregulatory molecule, CD200. Accordingly we explored the effect of stimulation of CLL cells with P2Iio, followed by coating of cells with a non-depleting anti-CD200mAb, on the ability of those cells to immunize PBL in vitro to become cytotoxic to CLL cells, or to protect NOD-SCIDγc null (NSG) mice from subsequent CLL tumor challenge. Our data indicate that this protocol is effective in inducing CD8 + CTL able to lyse CLL cells in vitro, and decrease tumor burden in vivo in spleen and marrow of mice injected with CLL cells. Pre-treatment of mice with a CD8 depleting antibody before vaccination with P2Iio/anti-CD200 coated cells abolished any protection seen. These data suggest a potential role for blockade of CD200 expression on CLL cells as a component of a tumor vaccination strategy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chemosensory systems (CSS) are among the most complex organizations of proteins functioning cooperatively to regulate bacterial motility and other cellular activities. These systems have been studied extensively in bacteria, and usually, they are present as a single system. Eight CSS, the highest number in bacteria, have been reported in Myxococcus xanthus DK1622 and are involved in coordinating diverse functions. Here, we have explored and compared the CSS in all available genomes of order Myxococcales. Myxococcales members contain 97 to 476 two-component system (TCS) proteins, which assist the bacteria in surviving and adapting to varying environmental conditions. The number of myxobacterial CSS ranges between 1 and 12, with the largest number in family Cystobacteraceae and the smallest in Nannocystaceae CheA protein was used as a phylogenetic marker to infer evolutionary relatedness between different CSS, and six novel CSS ("extra CSS" [ECSS]) were thus identified in the myxobacteria besides the previously reported Che1 to Che8 systems from M. xanthus Che1 to Che8 systems are monophyletic to deltaproteobacteria, whereas the newly identified ECSS form separate clades with different bacterial classes. The comparative modular organization was concordant with the phylogeny. Four clusters lacking CheA proteins were also identified via CheB-based phylogenetic analysis and were categorized as accessory CSS (ACSS). In Archangium , an orphan CSS was identified, in which both CheA and CheB were absent. The novel, accessory, and orphan multimodular CSS identified here suggest the emergence of myxobacterial CSS and could assist in further characterizing their roles. IMPORTANCE This study is focused on chemosensory systems (CSS), which help the bacterium in directing its movement toward or away from chemical gradients. CSS are present as a single system in most of the bacteria except in some groups, including Myxococcus xanthus , which has 8 CSS, the highest number reported to date. This is the first comprehensive study carrying out a comparative analysis of the 22 available myxobacterial genomes, which suggests the evolutionary diversity of these systems. We are interested in understanding the distribution of CSS within all known myxobacteria and their probable evolution., (Copyright © 2018 American Society for Microbiology.)

Small RNA (sRNA)-mediated regulation of gene expression is a major tool to understand bacterial responses to environmental changes. In particular, pathogenic bacteria employ sRNAs to adapt to the host environment and establish infection. Members of the Burkholderia cepacia complex, normally present in soil microbiota, cause nosocomial lung infection especially in hospitalized cystic fibrosis patients. We sequenced the draft genome of Burkholderia cenocepacia KC-01, isolated from the coastal saline soil, and identified several potential sRNAs in silico . Expression of seven small RNAs (Bc&#95;KC&#95;sr1-7) was subsequently confirmed. Two sRNAs (Bc&#95;KC&#95;sr1 and Bc&#95;KC&#95;sr2) were upregulated in response to iron depletion by 2,2'-bipyridyl and another two (Bc&#95;KC&#95;sr3 and Bc&#95;KC&#95;sr4) responded to the presence of 60 µM H 2 O 2 in the culture media. Bc&#95;Kc&#95;sr5, 6 and 7 remained unchanged under these conditions. Expression of Bc&#95;KC&#95;sr2, 3 and 4 also altered with a change in temperature and incubation time. A search in the Rfam and BSRD databases identified Bc&#95;Kc&#95;sr4 as candidate738 in B. pseudomallei D286 and assigned Bc&#95;Kc&#95;sr5 and 6 as tmRNA and 6S RNA, respectively. The novel sRNAs were conserved in Burkholderiaceae but did not have any homologue in other genera. Bc&#95;KC&#95;sr1 and 4 were transcribed independently while the rest were part of the 3' UTR of their upstream genes. TargetRNA2 predicted that these sRNAs could target a host of cellular messages with very high stringency. Intriguingly, regions surrounding the translation initiation site for several enzymes involved in Fe-S cluster and siderophore biosynthesis, ROS homeostasis, porins, transcription and translation regulators, were among the suggested putative binding sites for these sRNAs.

Two novel Gram-staining positive, rod-shaped, moderately halotolerant, endospore forming bacterial strains 5.5LF 38TD and 5.5LF 48TD were isolated and taxonomically characterized from a landfill in Chandigarh, India. The analysis of 16S rRNA gene sequences of the strains confirmed their closest identity to Bacillus thermotolerans SgZ-8T with 99.9% sequence similarity. A comparative phylogenetic analysis of strains 5.5LF 38TD, 5.5LF 48TD and B. thermotolerans SgZ-8 T confirmed their separation into a novel genus with B. badius and genus Domibacillus as the closest phylogenetic relatives. The major fatty acids of the strains are iso-C 15:0 and iso-C 16:0 and MK-7 is the only quinone. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The digital DNA-DNA hybridization (DDH) and ortho average nucleotide identity (ANI) values calculated through whole genome sequences indicated that the three strains showed low relatedness with their phylogenetic neighbours. Based on evidences from phylogenomic analyses and polyphasic taxonomic characterization we propose reclassification of the species B. thermotolerans into a novel genus named Quasibacillus thermotolerans gen. nov., comb. nov with the type strain SgZ-8 T (=CCTCC AB2012108 T =KACC 16706 T ). Further our analyses also revealed that B. encimensis SGD-V-25 T is a later heterotypic synonym of Bacillus badius DSM 23 T ., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Inactivation of retrotransposons is accompanied by the emergence of centromere-binding protein-B (CENPB) in Schizosaccharomyces , as well as in metazoans. The RNA interference (RNAi)-induced transcriptional silencing (RITS) complex, comprising chromodomain protein-1 (Chp1), Tas3 (protein with unknown function), and Argonaute (Ago1), plays an important role in RNAi-mediated heterochromatinization. We find that whereas the Ago1 subunit of the RITS complex is highly conserved, Tas3 is lost and Chp1 is truncated in Schizosaccharomyces cryophilus and Schizosaccharomyces octosporus We show that truncated Chp1 loses the property of heterochromatin localization and silencing when transformed in Schizosaccharomyces pombe Furthermore, multiple copies of CENPB, related to Tc1/mariner and Tc5 transposons, occur in all Schizosaccharomyces species, as well as in humans, but with loss of transposase function (except Schizosaccharomyces japonicus ). We propose that acquisition of Tc1/mariner and Tc5 elements by horizontal transfer in S. pombe (and humans) is accompanied by alteration of their function from a transposase/endonuclease to a heterochromatin protein, designed to suppress transposon expression and recombination. The resulting redundancy of RITS may have eased the selection pressure, resulting in progressive loss or truncation of tas3 and chp1 genes in S. octosporus and S. cryophilus and triggered similar evolutionary dynamics in the metazoan orthologues., (© 2017 Upadhyay et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Significant barriers to transplantation exist for individuals who are pre-sensitized to donor antigen and have high titres of donor-reactive antibody. We report the effect of autologous bone marrow transplantation (BMTx) after myeloablation in pre-sensitized mice along with the use of monoclonal antibodies (mAbs) to tumour necrosis factor-receptor super family 25 (TNFRSF25), expressed on regulatory T (Treg) cells. C57BL/6 mice, which had been sensitized earlier with BALB/c skin allografts, received secondary BALB/c grafts after the primary grafts had been rejected. Subsequently, recipient mice underwent myeloablation with cyclophosphamide and busulphan and were injected with T-cell-depleted bone marrow from CD45.1 congenic donors (BMTx). Recipient mice underwent immunosuppressive treatment with rapamycin. A subgroup of mice was also treated with mAbs to TNFRSF25. Control mice were pre-sensitized mice that received cyclophosphamide and busulphan followed by rapamycin. BMTx-treated mice had significantly prolonged skin graft survival versus control mice. These mice also showed attenuated donor-specific mixed lymphocyte co-culture responses relative to controls, increased splenic Treg cells and markedly diminished serum anti-donor IgG. Infusion of anti-TNFRSF25 mAbs further augmented graft survival and increased graft-infiltrating Treg cells. These mAbs also expanded murine and human Treg cells in vitro with the capacity to attenuate mixed lymphocyte co-cultures using fresh peripheral blood mononuclear cells. Overall, this study delineates the roles of autologous BMTx and anti-TNFRSF25 mAbs in expanding Treg cells and attenuating alloimmune responses in pre-sensitized mice., (© 2016 John Wiley & Sons Ltd.)

Purpose: We investigated whether miRNAs in exosomes from EMT6 or 4THM tumor-bearing mice played a role in regulating inflammatory cytokine expression and/or metastasis in WT mice injected with EMT6 and/or 4THM tumor cells., Methods: EMT6 tumors in BALB/c CD200R1KO mice resolve following surgical resection of localized tumor and immunization with irradiated EMT6 cells along with CpG as adjuvant. Wild-type (WT) animals treated in the same fashion develop pulmonary and liver metastases within 20 days of surgery. DLNs from CD200R1KO mice contain no tumor cells detectable at limiting dilution. In contrast, 4THM tumor cells injected into CD200R1KO show increased metastasis compared with WT mice. Transfer of serum exosomes from 4THM tumor-bearing mice to WT animals increased metastasis of EMT6 tumors, an effect attenuated by anti-IL-6 antibody. We compared miRNA expression in exosomes from the serum of 4THM/EMT6 WT or CD200R1KO tumor-bearing mice, and the effects of antagomirs to miRNAs on tumor growth., Results: Complex changes in miRNA expression were observed in the isolated exosomes. Some miRNAs, including miR155, have been reported to potentiate inflammatory responses and augment inflammatory cytokine expression. Expression of miR155 increased in exosomes from 4THM relative to EMT6 tumor bearers, and antagomirs to miR155 attenuated tumor growth and metastasis, and improved survival, following infusion into WT mice. Antagomirs to the miR205 family were thought to affect metastasis by targeting epithelial-to-mesenchymal transition (EMT), increased growth and metastasis in both 4THM and EMT6 tumor-bearing mice, and decreased survival, with some modulation of inflammatory cytokine production., Conclusions: Multiple pathways are implicated in differential metastasis of EMT6/4THM, and targeting these may have clinical utility in human breast cancer.

The probiotic yeast, Saccharomyces boulardii (Sb) is known to be effective against many gastrointestinal disorders and antibiotic-associated diarrhea. To understand molecular basis of probiotic-properties ascribed to Sb we determined the complete genomes of two strains of Sb i.e. Biocodex and unique28 and the draft genomes for three other Sb strains that are marketed as probiotics in India. We compared these genomes with 145 strains of S. cerevisiae (Sc) to understand genome-level similarities and differences between these yeasts. A distinctive feature of Sb from other Sc is absence of Ty elements Ty1, Ty3, Ty4 and associated LTR. However, we could identify complete Ty2 and Ty5 elements in Sb. The genes for hexose transporters HXT11 and HXT9, and asparagine-utilization are absent in all Sb strains. We find differences in repeat periods and copy numbers of repeats in flocculin genes that are likely related to the differential adhesion of Sb as compared to Sc. Core-proteome based taxonomy places Sb strains along with wine strains of Sc. We find the introgression of five genes from Z. bailii into the chromosome IV of Sb and wine strains of Sc. Intriguingly, genes involved in conferring known probiotic properties to Sb are conserved in most Sc strains.

Cell-surface CD200 expression by mouse EMT6 breast tumor cells increased primary tumor growth and metastasis to the draining lymph nodes (DLN) in normal (WT) BALB/c female recipients, while lack of CD200R1 expression in a CD200R1-/- host negated this effect. Silencing CD200 expression in EMT6siCD200 tumor cells also reduced their ability to grow and metastasize in WT animals. The cellular mechanisms responsible for these effects have not been studied in detail. We report characterization of tumor infiltrating (TILs) and draining lymph node (DLN) cells in WT and CD200-/- BALB/c mice, receiving WT tumor cells, or EMT6 lacking CD200 expression (EMT6siCD200 cells). Our data show an important correlation with augmented CD8+ cytotoxic T cells and resistance to tumor growth in mice lacking exposure (on either host cells or tumor) to the immunoregulatory molecule CD200. Confirmation of the importance of such CD8+ cells came from monitoring tumor growth and characterization of the TILs and DLN cells in WT mice challenged with EMT6 and EMT6siCD200 tumors and treated with CD8 and CD4 depleting antibodies. Finally, we have assessed the mechanisms(s) whereby addition of metformin as an augmenting chemotherapeutic agent in CD200-/- animals given EMT6 tumors and treated with a previously established immunotherapy regime can increase host resistance. Our data support the hypothesis that increased autophagy in the presence of metformin increases CD8+ responses and tumor resistance, an effect attenuated by the autophagy inhibitor verteporfin.

Myxobacteria are members of δ-proteobacteria and are typified by large genomes, well-coordinated social behavior, gliding motility, and starvation-induced fruiting body formation. Here, we report the 10.33 Mb whole genome of a starch-degrading myxobacterium Sandaracinus amylolyticus DSM 53668(T) that encodes 8,962 proteins, 56 tRNA, and two rRNA operons. Phylogenetic analysis, in silico DNA-DNA hybridization and average nucleotide identity reveal its divergence from other myxobacterial species and support its taxonomic characterization into a separate family Sandaracinaceae, within the suborder Sorangiineae. Sequence similarity searches using the Carbohydrate-active enzymes (CAZyme) database help identify the enzyme repertoire of S. amylolyticus involved in starch, agar, chitin, and cellulose degradation. We identified 16 α-amylases and two γ-amylases in the S. amylolyticus genome that likely play a role in starch degradation. While many of the amylases are seen conserved in other δ-proteobacteria, we notice several novel amylases acquired via horizontal transfer from members belonging to phylum Deinococcus-Thermus, Acidobacteria, and Cyanobacteria. No agar degrading enzyme(s) were identified in the S. amylolyticus genome. Interestingly, several putative β-glucosidases and endoglucanases proteins involved in cellulose degradation were identified. However, the absence of cellobiohydrolases/exoglucanases corroborates with the lack of cellulose degradation by this bacteria., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)