bluuebunny/crossref_metadata_2025 huggingface dataset:
Early life selection of gut microbiota specific T cells
Authors: Ruiz Daniel Fernando Zegarra , Kim Dasom , Norwood Kendra , Majumdar Shubhabrata , Wu Wan-Jung , Kim Myunghoo , Bettini Matthew , Diehl Gretchen
Published: January 2023

AIRE-dependent expression of tissue-specific antigens by medullary thymic epithelial cells allows for the selection of non-autoreactive T cells, supporting self-tolerance. Although gut microbiota is known to educate the immune system, balancing effector and regulatory T cells responses, it remains unknown how bacteria-specific T cells are selected. Here we studied how thymic T cell development is altered after colonization with model intestinal microbes. Colonization of mice at weaning induced thymic expansion of bacteria-specific naive T cells. Remarkably, bacterial DNA was found in the thymus of colonized mice and 16s rDNA sequencing identified bacterial DNA from a broad range of intestinal microbes in the thymus of steady-state mice. We identified enrichment of CX3CR1+ dendritic cells (CX3+ DCs) after microbial colonization and depleting these cells or inhibiting their antigen-presentation suppressed induction of bacteria-specific T cells. Further, depletion of the gut microbiota with antibiotics reduced positive selection, bacterial 16s, and CX3+ DCs in the thymus. In older mice, we did not observe microbial DNA in the thymus, altered CX3+ DCs, or expansion of microbiota specific T cells, indicating a developmental window where intestinal microbes can regulate thymic output. Using models of microbiota-specific T cell immunity or pathogen infection, we demonstrate thymic microbiota-specific T cells can differentiate into intestinal effector T cells, offering protection or mediating pathology. Together, our data suggests trafficking of bacteria to the thymus by intestinal DCs drives T cell selection of microbiota-specific T cells to shape peripheral immunity against pathogens and the microbiota itself.
The Role of Innate Lymphoid Cells in Immune Regulation and Fetal Protection During Allogeneic Pregnancies
Authors: Chung Douglas , Wong Emily , Han SeongJun , Elford Alisha , Garcia-Batres Carlos , Ohashi Pamela S
Published: January 2023

Maternal immune tolerance against paternal alloreactive antigens expressed by the fetus is essential in the development of successful pregnancies. Disturbances of immunoregulatory mechanisms by genetic modifications or viral infections results in aberrant placental development, intrauterine growth restriction (IUGR), and fetal loss. Mechanisms of immune regulation during pregnancy has not been fully elucidated. Our lab and others were the first to uncover a novel subset of regulatory innate lymphoid cells (ILCregs) that suppressed immune responses in chronic viral infections, organ transplant rejection, and cancer. In this study, we identified a subset of decidual NK1.1+ ILC1s in healthy murine pregnancies that expressed surface markers and intracellular cytokines reminiscent of an ILCreg phenotype. Interestingly, these characteristics were more abundant early in gestation compared to mid-late gestation. Furthermore, depletion of NK1.1+ ILC1s using neutralizing antibodies induced fetal destruction in allogeneic (C57BL/6J female X BALB/c male), but not in syngeneic pregnancies (C57BL/6J female X C57BL/6J male). This highlights the importance of allogenicity in fetal destruction in the absence of NK1.1+ ILC1s. Decidual ILC1s have been shown to promote trophoblast invasion, placental angiogenesis, and in utero fetal growth during pregnancy. However, we are one of the first groups to thoroughly investigate potential immunosuppressive role of decidual ILC1s to prevent immune-mediated fetal destruction. Further elucidation of immune tolerance mechanisms during pregnancy may uncover novel therapeutic targets for pregnancy-related complications such as preeclampsia and spontaneous miscarriages.
Role of the microbiota in the development of intraepithelial CD4+CD8aa+ T cells
Authors: Barragan Luisa Cervantes , Royer Charlotte
Published: January 2023

The small intestine contains CD4+CD8aa+ double-positive intraepithelial T lymphocytes (DP IELs) which originate from intestinal CD4+ T cells through downregulation of the transcription factor ThpoK and have regulatory functions. We have previously found that development of DP IEL depend on the presence of Lactobacillus reuteri. This species induced DP IELs in germ-free mice and conventionally-raised mice lacking these cells. L. reuteri did not shape DP-IEL-TCR repertoire, but generated indol derivatives of tryptophan that activated the aryl-hydrocarbon receptor in CD4+ T cells. Monocolonization of germ-free mice with L. reuteri resulted in the induction of DP IEL, however reconstitution with a complex microbiota + L. reuteri induce a much larger DP IEL population suggesting that additional bacterial species are required for optimal DP IEL development. In this study we have aimed at elucidating additional bacterial species from the intestinal microbiota that contribute to the development of DP IEL.
Targeting MAIT cells by vaccination with S. enterica serotype Typhimurium to prevent lung infections
Authors: Paynich Mallory L , Hartmann Nadine , Riffelmacher Thomas , Kronenberg Mitchell
Published: January 2023

Nearly all currently licensed vaccines elicit antibody responses to induce long-lasting immunity. For intracellular pathogens, to some extent out of reach for antibodies, protective vaccination will require not just antibody production, but also T cell-mediated responses. Mucosal Associated Invariant T (MAIT) cells recognize microbial vitamin B metabolites presented by the MHC-I-like molecule, MR1, via a semi-invariant TCR. These innate-like T cells are present at low levels in mice in mucosal tissues, especially the lung, as well as other sites such as blood, liver and spleen. Previous studies have demonstrated that Salmonella enterica serotype Typhimurium potently expands long-lived MAIT cells to nearly 40% of lung αβ T cells. Using this model, we determined that expansion of MAIT cells requires infection with intracellular bacteria, as an invasion mutant of S. enterica does not induce MAIT cell expansion. Following treatment with an attenuated strain of S. enterica, MAIT cells expand into short-term effector memory (CD127−KLRG1+) and memory precursor (CD127+KLRG1−) populations that differ in cytokine, transcription factor, and metabolic profiles. Following adoptive transfer, CD127+ MAIT cells, but not KLRG1+ cells, protect mice from infection with a different bacterium, Streptococcus pneumoniae, a leading cause of pneumonia and other diseases. Further, vaccination with S. enterica limits S. pneumoniae-induced mortality in mice. These studies demonstrate that some intracellular bacteria induce distinct, long-lived MAIT cell populations, and that these cells are a promising target for vaccines against lung pathogens.
Aiolos and T-bet cooperate to repress the ILC3 transcriptional program in human innate lymphocytes
Authors: da Silva Cristiane Sécca , Cella Marina , Gamini Ramya , Collins Patrick , Zhao Shanrong , Peng Vincent , Robinette Michelle , Schettini Jorge , Zaitsev Konstantin , Gordon Willian , Bando Jennifer K. , Yomogida Kentaro , Cortez Victor , Fronick Catrina , Fulton Robert , Lih Ling Lin , Gilfillan Susan , Flavell Richard A , Shan Liang , Artyomov Maxim N. , Bowman Michael , Oltz Eugene M , Jelinsky Scott , Colonna Marco
Published: January 2023

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes devoid of antigen-specific receptors that provide an immediate source of cytokines in early immune responses and are enriched in mucosal tissues. Human ILC3s that produce the cytokine interleukin-22 and express the transcription factor RORγt have been shown to be able to convert into ILC1-like cells that produce mainly IFN-γ and express T-bet, in vitro. Whether this conversion also occurs in vivo remains unclear. In the present study we found that ILC3s and ILC1s in human tonsils represented the ends of a spectrum that included additional discrete subsets with intermediate transcriptional signatures. This finding strongly suggests that the ILC3 to ILC1-like conversion also occurs in vivo in humans. Gene expression analysis showed that the transcription factors Aiolos and T-bet were induced in the subsets closer to the canonical ILC1 subset. Chromatin studies indicated that Aiolos and T-bet cooperate to repress regulatory elements active in ILC3s. Ectopic expression of Aiolos and T-bet in the ILC3-like cell line MNK-3 repressed the production of IL-22 and induced IFN-γ. We conclude that Aiolos and T-bet cooperate to repress the ILC3 transcriptional program therefore contributing to the ILC3 to ILC1-like conversion.
TREM2/β-catenin attenuates macrophage pyroptosis to promote bacterial eradication of pyogenic bacteria
Authors: Wei Liwen , Wang Yi , Liang Siping , Wu Yongjian , Ming Siqi , Wu Minhao
Published: January 2023

Triggering receptors expressed on myeloid cells 2 (TREM2) inhibits inflammatory responses, but promotes macrophage-mediated bacterial clearance both in vivo and in vitro. However, the underlying mechanism remains unclear. Our study demonstrated that deficiency of TREM2 significantly enhanced four common pyogenic bacteria-induced macrophage pyroptosis, including Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae and Escherichia coli. Blockage of pyroptosis by knocking out Caspase-1 or knocking down gasdermin D (GSDMD) enhanced macrophage-mediated pyogenic bacteria clearance. And treatment with Caspase-1 inhibitor relieved TREM2 deficiency-mediated suppression of pyogenic bacteria clearance. In addition, TREM2 enhanced β-catenin stability via promoting its phosphorylation at S675. Moreover, silencing of β-catenin enhanced pyroptosis and impaired Pseudomonas aeruginosa clearance, while overexpression of β-catenin inhibited pyroptosis and restored bacterial elimination in TREM2-silenced macrophages. Notably, in vitro studies suggested that TREM2/β-catenin suppressed NLRP3 and NLRC4 inflammasome activation at priming and/or assembly stage. Collectively, our study provides a novel mechanism of TREM2-mediated anti-pyogenic bacterial immunity and explores the relationship between pyroptosis and bacterial eradication.
Phenotypic and functional characterization of lung resident lymphocytes of BCG vaccinated mice
Authors: McAllister Madeline K , Tripathi Deepak , Radhakrishnan Rajesh Kumar , Thandi Ramya Sivangala , Paidipally Padmaja , Vankayalapati Ramakrishna
Published: January 2023

Mycobacterium tuberculosis (Mtb) remains to be a leading cause of morbidity and mortality, causing an estimated 1.3 million deaths annually. It is known that intranasal Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccination in mice has proven to provide superior protection against pulmonary TB, as compared to parenterally administered BCG. In the current study, we determined the phenotype and function of lung-resident lymphocytes in intranasally BCG vaccinated mice. C57BL/6 mice were vaccinated intranasally with live-attenuated BCG and lung lymphocytes were isolated and stained for immunophenotyping via flow cytometry at 24, 48, and 72 hours post-vaccination. At 48 hours post-vaccination, we have observed an expansion of CD69+, CD103+, and CD69+CD103+ lymphocytes within the lung of vaccinated mice. Studies are underway to determine the phenotype and function of lung-resident lymphocyte subsets and their expansion capacity at three months after BCG vaccination and Mtb infection.
Androgen receptor activity underlies sex differences in lung-resident ILC2 functional responses during influenza virus infection
Authors: Kovats Susan , Turner Sean , Karlik Anna , Miller Reegan , Ainsua-Enrich Erola , Kadel Sapana
Published: January 2023

Lung-resident group 2 innate lymphoid cells (ILC2s) produce type 2 cytokines and facilitate tissue repair in response to alarmins such as IL-33 released during respiratory virus infection, but they also may be functionally suppressed by type 1 cytokines. Lung ILC2s express notably high levels of intracellular androgen receptor (AR) compared to ILC1s, T and B cells. To test the hypothesis that females and males show differential ILC2 responses in influenza virus (IAV) infection, we analyzed the numbers and functional responses of lung ILCs in IAV infected mice. On days 3–10 post-infection, lungs of female mice contained greater numbers of ILC2s and ILC1s compared to males. However, the female ILC2s were preferentially suppressed at the peak of infection, with a dampened type 2 program manifest as attenuated proliferation, decreased IL-5 and amphiregulin production, reduced expression of GATA-3 and IL-33R and increased surface IFNGR. IFNG levels in the lung were comparable between sexes at day 7 post-infection, suggesting intrinsic differences in ILC2 responses to interferons. Indeed, naïve female ILC2s showed higher expression of IFNGR and higher phospho-STAT1 levels following stimulation by IFNG. ILC2s in naive male mice with lymphocyte-restricted AR deficiency displayed levels of IFNGR comparable to female mice, suggesting AR activity underlies sex differences in intrinsic IFNGR expression. Early life orchiectomy revealed that endogenous androgens decreased ILC2 numbers but protected males from suppression of ILC2s in IAV infection. Taken together, our data show that AR activity preserves canonical ILC2 function in males during IAV infection and may help to explain the documented human gender differences in immunity to IAV.
Lung resident memory B cells are a common and functionally significant component of lung adaptive immunity
Authors: Barker Kimberly A , Shenoy Anukul T , Stauffer-Smith Nicole , Arafa Emad Isam , de Ana Carolina Lyon , Barron Alex , Aihara Fumiaki , Martin Ian , Zhong Xuemei , Kepler Thomas B , Browning Jeffrey L , Jones Matthew , Quinton Lee , Mizgerd Joseph P.
Published: January 2023

Resident memory B cells (BRM) in influenza-recovered mouse lungs were recently described, but whether other types of infections elicit these cells is unknown. The relevance of BRM in human lungs and to lung immune defenses also remains unexplored. Using flow cytometry and immunofluorescence, we found that respiratory pneumococcal exposures in mice elicited lung BRM without concurrent tertiary lymphoid structure formation. Additionally, flow cytometry analysis of normal human lung tissue showed that human lungs are enriched compared to human blood for B cells bearing a resident memory phenotype. These findings indicate that lung BRM are a common feature of antigen-experienced lungs. Multiple mouse models were used to address the contributions of B cell immunity to anti-pneumococcal lung defenses. Mice exposed to a low virulence pneumococcal strain 4 weeks previously were well-protected from a serotype-mismatched pneumococcal challenge. When previously exposed mice were depleted of circulating B cells (but not lung B cells) with anti-CD20 treatment before the challenge infection, there was no effect on the acquired lung immunity. However, a genetically engineered mouse strain allowed effective depletion of lung B cells bearing PD-L2 (a mouse memory B cell marker) from previously exposed mice, and doing so before the virulent pneumococcal challenge resulted in substantial defects in bacterial clearance compared to mice with lung B cells intact. These results provide the first direct evidence of a role for lung BRM in anti-bacterial lung immunity. Notably, this defense was pneumococcal serotype-independent, distinguishing it from the serotype-specific immunity elicited by current pneumococcal vaccines.
Novel computational analytics of clinical flow cytometry data identifies difficult-to-resolve leukemia cells for precision diagnosis
Authors: Qian Yu , Nguyen Quang Vinh , Putzel Preston , Bevins Nicholas , Bui Jack D , Wang Huan-You , Smyth Padhraic , Scheuermann Richard H
Published: January 2023

Motivation

Diagnosis of leukemia relies on accurate identification of leukemic cell populations. Flow cytometry (FCM) is a primary diagnostic assay routinely used in clinical practice. The assay workflows consist of multiple manual gating steps performed by technicians, followed by interpretation by hematopathologists. Challenges to this process include technical variability in manual gating, difficulty in identification of the atypical leukemic cells, and the growing number of antigens used for diagnosis.

Methods

Instead of conducting ad hoc analysis of individual samples, our proposed computational approach leverages preexisting clinical FCM data to improve robustness of the computational identification of leukemic cells. Instead of separating cell population identification and sample classification into two steps, our machine learning classification method optimizes them simultaneously, producing gating locations that are recognizable to hematopathologists.

Results

Our study consists of 10-color FCM data from blood or bone marrow samples of 129 random subjects for chronic lymphocytic leukemia (CLL) diagnosis. Our initial automated gating analysis rendered an accuracy of 89% matched the diagnosis of the hematopathologist. In the remaining cases with discrepant results, the misclassified CLL cells had atypical molecular phenotypes, making them difficult to identify. In our improved pipeline, we demonstrate that these atypical types of CLL cells can be clearly captured using a non-linear embedding dimensionality reduction step.

Conclusion

The results demonstrate the power of a novel computational analysis pipeline for improving the identification of aberrant leukemia cells for precision diagnosis.
Evaluation of Peripheral Blood Leukocyte Subsets in Systemic Lupus Erythematosus (SLE) Enabled by a Microfluidic Cell Separator
Authors: Olferiev Mikhail , Greenman Dina , Kulnarni Nitin , Kou Peng Meng , Smith Kyle C , Crow Mary K
Published: January 2023

Although granulocyte signatures have been associated with SLE severity, their role in SLE has been poorly understood due to the suboptimal methods for granulocyte recovery from peripheral blood. An automated microfluidic technology (Sorterra™ prototype) was used to isolate all leukocytes with minimal platelet (PLT) or red blood cell (RBC) contamination to assess the cell subsets.

Leukocytes were isolated from peripheral blood collected from healthy donors (HD) and SLE patients (3 female, 3 male in each group) using Sorterra, RBC lysis, or Ficoll®. Viability and recovery of each leukocyte subset was evaluated by cell counter and flow cytometry.

Sorterra recovered 92±4% of leukocytes from HD and SLE samples with negligible RBC/PLT/microparticle (MP) contamination, whereas Ficoll isolated 35±15% of leukocytes, losing nearly all the granulocytes and retaining a substantial level of RBC/PLT/MP. With RBC lysis, up to 65% of neutrophils and 41% of eosinophils were lost in the process. Sorterra recovered more monocytes (~80%) than Ficoll (p<0.05). The recovery of lymphocytes by Sorterra (~90%) was not different from Ficoll or RBC lysis. The recovery of T and B cell subsets, NK, and NKT cells, was also not different among the isolation methods. The viability of Sorterra isolated leukocytes was significantly higher than the Ficoll isolated cells.

The Sorterra prototype was superior to RBC lysis and Ficoll in recovering total leukocytes with high viability and ease of use and provided a more consistent leukocyte isolate from HD and SLE patients than other methods. The ability to recover granulocytes in their native state is unmatched by existing technologies and enables future studies to better elucidate the role of granulocytes in SLE development.
DNA-scaffolded biomaterials enable modular and tunable presentation of proteins to control immune cell therapies
Authors: Huang Xiao , Williams Jasper Z. , Chang Ryan , Li Zhongbo , Gai Eric , Patterson David M. , Wei Yu , Lim Wendell A. , Desai Tejal
Published: January 2023

Advanced biomaterials can be used to spatially and temporally control immune cell activities to improve the efficacy and safety of cell therapies. Precise immune cell modulation demands multi-modal display of functional proteins on biomaterials with a high level of control. Here, we developed biodegradable immune cell engaging particles (ICEp) with synthetic short DNA as scaffolds for efficient and tunable protein loading. We demonstrate the precise ratiometric control of anti-CD3 and anti-CD28 antibodies on this biodegradable platform can impact the quality of ex vivo expanded human primary T cells. Furthermore, the high-density presentation of antigens, difficult to achieve by other chemistries, is necessary for controlling the activity of engineered T cells in vivo. Antigen-presenting ICEp injected intratumorally can provide a local priming signal for systemically administered AND-gate chimeric antigen receptor (CAR) T cells that recognize dual antigens: the first antigen on ICEp activates CAR expression to target the second antigen on tumor cells. This leads to local tumor clearance while sparing uninjected tumors that model potentially cross-reactive healthy tissues. This modularly functionalized biomaterial thus provides a flexible platform to achieve sophisticated control of cell-based immunotherapies.
Individualized expansion of T cells from CLL patients for immunotherapy
Authors: Yuan Dennis Jinglun , Fernandes Stacey M , Brown Jennifer R , Kam Lance C
Published: January 2023

T cell therapy has shown significant promise in treating blood cancers, yet high proportions of T cells from patients are either unresponsive to stimulation methods or show wide variability in regards to which activation conditions induce optimal expansion. In this work, we explore how expansion of T cells from CLL patients using scaffolds with tunable physical parameters (rigidity, fiber dimensions, pore size) is correlated to patient biomarker data, as well as exhaustion and functional profiles of patient T cells, to gain insight on personalizing T cell production for immunotherapy.

Polymer fiber scaffolds are fabricated via electrospinning with PDMS/PCL polymer blend and coated with activating antibodies to CD3 and CD28 to stimulate T cells isolated from CLL patients with pre-annotated mutation burdens and disease progression. Exhaustion surface markers, memory/effector phenotyping, and cytokine secretion are measured via immunostaining before and after expansion.

Preliminary data has shown that decreasing fiber diameters (from 6 to 1 μm) enhanced T cell expansion and increased the ratio of effector memory to central memory phenotypes. We observe that higher CLL Rai stage leads to decrease in proliferative potential. Interestingly, cells from each CLL patient in our initial study showed different patterns of expansion for the same set of scaffold compositions. Machine learning methods can identify clinical markers that predict exhaustion and functional profiles from starting cell populations as well as expansion with different biomaterial parameters. Collectively, insight into predicting how patient T cells can be robustly expanded in a personalized approach is powerful for translating T cell therapies to more patients.
One step ultra-high-throughput sequencing of the paired antibody VH:VL and TCRβ:α repertoires using cell lysate resistant xenopolymerase in emulsion
Authors: Tanno Hidetaka , McDaniel Jonathan , Stevens Christopher , Voss William , Li Jie , Durrett Russell , Lee Jiwon , Gollihar Jimmy , Tanno Yuri , Delidakis George , Pothukuchy Arti , Ellefson Jared , Goronzy Jörg , Maynard Jennifer , Ellington Andrew , Ippolito Gregory , Georgiou George
Published: January 2023

Antibodies and T cell receptors (TCRs) have critical roles in adaptive immunity and thus the determination of their sequences is essential for understanding immune responses in infections or vaccinations, for the discovery of therapeutic antibodies, and for the engineered T cell therapies. Antibodies are composed of variable heavy (VH) and light (VL) chains, and TCRs are composed of variable TCRα/γ and TCRβ/δ chains. Since those chains are encoded by two different transcripts, the determination of functional sequences requires single-cell sequencing. Emulsification of single-cells followed by barcoding of the transcripts or fusion of VH:VL or TCRβ:α transcripts using overlap extension RT-PCR allows high-throughput single-cell sequencing; however, because cell lysate in emulsion inhibits the RT-PCR reaction, these methods require complex microfluidics devices to bypass the inhibition which most of biomedical laboratories cannot access. We discovered recently engineered xenopolymerase, RTX, has an exceptional cell lysate resistance that can overcome this limitation. By employing this unique feature of RTX, we have developed a one-step emulsion-based ultra-high-throughput sequencing method for paired antibody VH:VL and TCRβ:α repertoires not requiring microfluidics or any other specialized equipment. Using this methodology, we obtained high yields (5,000 to >20,000/sample) of paired VH:VL or TCRβ:α clonotypes at low cost. As a demonstration, we applied the methodology to the peripheral blood plasmablasts and T follicular helper (TFH) cells from a seasonal influenza vaccine recipient and discovered high-affinity influenza-specific antibodies and TCRβ:α.
StraightFrom®MicroBeads: Fast T cell isolation for CAR T cell manufacturing without density gradient centrifugation
Authors: Lennartz Simon , Lock Dominik , Kolbe Carolin , Winkels Gregor , Assenmacher Mario , Kaiser Andrew
Published: January 2023

In recent years, CAR T cell mediated immunotherapy has shown clinical efficacy in the fight against cancer. Increased research efforts to address several challenges associated with CAR T cell therapy for solid tumor treatment for instance, such as poor efficacy and unwanted side effects, are required to progress current approaches. Fast, efficient and flexible CAR T workflows can offer the opportunity to progress such research.

At Miltenyi Biotec we have established an optimized CAR T cell research workflow, combining cell isolation, culture and analysis tailored for CAR T cell generation that can be transferred into clinical setting.

Using our StraightFrom® MicroBeads, the fundamental step of isolating high quality T cells as starting material to generate functional CAR T cells, can be performed directly from whole blood, buffy coat and blood products without density gradient centrifugation. Thereby, experimental procedure is accelerated and allows for automation on platforms such as the MultiMACS™ Cell24 Separator Plus and the MultiMACS™ X, whilst maintaining the quality, functionality and downstream compatibility of the isolated T cells, resulting in an ideal starting population for the generation of fully functional CAR T cells.

In conclusion, our innovative tools can reduce experimental time and increase the reproducibility and quality of data obtained in your CAR T cell research efforts in immuno-oncology and immunotherapy.
Multiomic characterization of T-cell populations at the single-cell level utilizing sensitive dextramers and BD® AbSeq on the BD Rhapsody™ Single-Cell Analysis system
Authors: Sakofsky Cynthia , Jacobsen Kivin , Lopez-Salmeron3 Vadir , Nakamoto Margaret , Brix Liselotte , Mortimer Stefanie
Published: January 2023

Adoptively transferred antigen-specific T cells have shown great efficacy in treatment of some virus-associated diseases and malignancies. A major driver of the development of adoptive T-cell therapy has been our ability to successfully characterize the functional status and antigen specificity of T cells. However, this has been limited by inefficient detection of antigen-specific T cells possibly due to their low frequency and low binding affinities to known MHC-peptide complexes. Here, we aim to combine two powerful technologies, advanced dCODE™ Dextramer® from Immudex and single-cell multiomics analysis using the BD Rhapsody™ Single-Cell Analysis system, to detect and characterize disease-specific CD8+ T cells within thousands of PBMCs. Currently, we are able to identify over 350 mRNAs alongside a panel of over 20 BD® AbSeq cell surface protein markers which can be associated with T cell activation states. These data can be used to define T-cell phenotypes alongside antigen specificity of enriched CD8+ dextramer+ cells from a PBMC population. This study outlines our ability for high-resolution T-cell profiling that has broader implications and utility in immuno-oncology, infectious diseases and autoimmunity.
Evaluation of oligonucleotide conjugated antibodies as reporter molecules in single-cell assays
Authors: Takahashi Chikara , Au-Yeung Amelia , Gutierrez Johnny , Toghi-Eshghi Shadi , Mathews Rod , O’Gorman William
Published: January 2023

Multiplexing in conventional flow and mass cytometric analysis has been constrained by both the number of available reporter molecules as well as signal spillover between these molecules. Monoclonal antibodies(mAbs) conjugated to oligonucleotides have been used in the design of ultrasensitive immunoassays but the multiplexing potential of these reagents have only recently been realized. The Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) method is established and uses oligo-conjugated mAbs and single-cell RNA sequencing technology to simultaneously enable the quantitation of both mRNA and protein molecules. Using the BD Rhapsody™ platform and BD® AbSeq, we examined the expression of 399 genes and 40 proteins in human PBMCs. The ability of single cell RNA-seq (scRNA-seq) to differentiate major and minor subsets of immune cells was evaluated. As in conventional cytometric analysis, immune cell populations could be identified based on the expression of canonical cell surface proteins and we compared the frequency of immune cell populations identified via scRNA-seq to CyTOF technology. Additionally, genes expected to be up- or down-regulated between established major populations such as T- and B-cells were observed thereby encouraging further analysis of gene expression in minor sub-populations. Overall, immune cell frequencies were found to be highly concordant between technologies. Single-cell “multi-omics” in which mRNA, proteins, and immune cell clonality are simultaneously measured on single cells thus stands poised to innovate drug development by providing unprecedented depth in the analysis of cells expressing therapeutic targets and driving disease pathogenesis.
Functional anomaly of CD8 T cells at earlier differentiation stages during HIV infection as established by analysis of synaptic interface and degranulation pattern
Authors: Anikeeva Nadia , Steblyanko Maria , Kuri-Cervantes Leticia , Buggert Marcus , Betts Michael R , Sykulev Yuri
Published: January 2023

It is thought that HIV infection could lead to a global defect in CD8+ T cell function. However, the effect of the infection on CD8+ T cells at various stages of differentiation has not been well studied. To evaluate the functional activity of CD8+ T cells from HIV infected people at various differentiation stages, we assessed their ability to form synaptic interfaces using planar lipid bilayers containing anti-CD3 antibody and soluble ICAM-1 ligand. We analyzed the dynamics of the T cell/bilayer interface formation, size of adhesion area, and kinetics of T-cell degranulation, parameters that are linked to the efficiency of CD8+ T cell functional activity. Unexpectedly, we observed that purified naïve CD8+ T cells (TN) and transitional memory (TTM) from HIV-infected individuals developed mature synapses whereas the same subsets from HIV uninfected individuals did not. While the kinetics of degranulation of naïve T cells was slower, both TN and TTM cells reached a similar extent of degranulation. The fraction and kinetics of CD27-CD45RO+ T cell degranulation were similar to that observed for TTM cells. Treatment with ART led to increase in the fraction of degranulating CD27-CD45RO+ T cells. As opposed to memory cells from healthy donors, a sizable fraction of CD27-CD45RO+ T cells from infected individuals develop lamellopodia and a larger adhesion area after synapse formation. These data suggest that less differentiated CD8+ T cells from HIV-infected people revealed functional anomaly that is likely mediated by aberrant functions of integrins and cytoskeleton causing changes in T-cell plasticity as a result of continuous inflammation during chronic HIV infection.
Development of two novel microglia-specific monoclonal antibodies to discriminate microglia from other CNS-resident macrophages by flow cytometry
Authors: Zahner Sonja Petra , Wilkinson Nicole , Nguyen Chau , Ellsworth Jessica Lynn , Stepniak Dariusz
Published: January 2023

Although microglia are the predominant resident macrophages of the central nervous system (CNS), other macrophages can be found in the CNS. As both populations express CD11b, microglia are generally distinguished from peripheral macrophages by lower expression of CD45. This can introduce bias in the analysis of flow cytometric data and make cell sorting challenging. Other microglia associated markers such as Aif1/Iba1 are less cell specific and therefore of limited use to dissect microglia from other brain residing macrophages. Recent single cell transcriptome studies have identified more specific signature genes of microglia, including surface molecule Tmem119 and transcription factor Sall1.

To allow robust discrimination between microglia and other brain resident/infiltrating macrophages based on the expression of a single marker by flow cytometry, we have developed monoclonal rat antibodies against mouse Tmem119 and Sall1, respectively. Both clones, V3RT1GOsz (Tmem119) and NRNSTNX (Sall1), were validated by flow cytometric analysis of fresh mouse brain. Here we show that both antibodies can be used to distinguish microglia from other macrophages by flow cytometry without reliance on CD11b or CD45 expression. The availability of these specific antibodies against microglia signature molecules will contribute significantly to the ability to isolate mouse microglia and their advanced characterization.
Immuno-phenotyping of tumor-specific CD8 T cells using high-dimensional mass cytometry
Authors: Abel Brian , Fehlings Michael , Nardin Alessandra , Newell Evan , Yadav Mahesh
Published: January 2023

There is strong evidence that tumor-specific T cells play a central role in tumor rejection. immunoSCAPE leverages the high-dimensional immune profiling capabilities of CyTOF for the identification of antigen-specific T cells to support the development of immunotherapy strategies. By applying CyTOF in conjunction with combinatorial peptide-MHC tetramer staining and high-performance dimensional analysis tools, we broadly and sensitively map T cell reactivity against MHC-class I epitopes while concurrently performing in-depth characterization of these rare antigen-specific T cells.

Here, we have applied our unique capabilities to detect and characterize circulating neoantigen-specific T cells in lung carcinoma patient undergoing atezolizumab treatment. Out of 782 neoantigen candidates, T cell reactivity against 13 unique neoantigens was detected within patients who responded to atezolizumab treatment, while T cells within non-responder patients recognized 7 neoantigens. Additionally, neoantigen-specific CD8 T cells from treatment responders exhibit a differentiated effector phenotype similar to CMV or EBV-specific T cells, whereas neoantigen-specific CD8 T cells from non-responders display a memory-like phenotype, suggesting that the properties of tumor-reactive T cells may be associated with clinical response to anti-PD-L1 treatment.

By providing insight into the nature and function of tumor-specific T cells, immunoSCAPE’s unique high-dimensional immune profiling platform is a valuable tool to guide the development of novel immunotherapy strategies through assessment of drug biological activity, definition of mechanism of action, and identification of biomarkers of patient response.
Evaluation of regulatory immune cells that favor engraftment following haploidentical peripheral blood stem cell transplantation for sickle cell disease
Authors: Bhat Deepali K , Gangaplara Arunakumar , Saxena Ankit , Pfeiffer Ruth , McCoy Philip , Fitzhugh Courtney D
Published: January 2023

Haploidentical hematopoietic stem cell transplantation is one of the most widely available curative options for patients with sickle cell disease (SCD). Our previous non-myeloablative haploidentical peripheral blood stem cell transplantation (PBSCT) trial showed improved engraftment with post-transplant cyclophosphamide, but with a high incidence of allograft rejection. Here, our aim was to evaluate mechanisms of engraftment in SCD patients who underwent PBSCT. We analyzed and compared the following cells (Treg, Tr1, Th1, Th17, NK, Breg, pmnMDSCs, mMDSCs, eMDSCs, mDCs, and pDCs) in peripheral blood samples obtained from 20 transplanted patients at baseline (BSL), post-transplant day (PT-D) 30, 60, 100, and 180 using flow cytometry. Because we previously showed that 20% donor myeloid chimerism (DMC) is sufficient to reverse SCD, we classified patients at each time point as engrafted (DMC ≥20%) and rejected (DMC< 20%). Early MDSC frequencies were significantly elevated in engrafted patients (PT-D30). Patients were also evaluated based on engrafted/rejected at the end of study and mMDSC and Th17 cell frequencies were significantly elevated at PT-D30 and PT-D60 respectively in engrafted patients. All other cell populations were not statistically different. Tregs showed an elevated trend in engrafted patients and exhibited direct proportionality in patients with varying DMC levels. Our results reveal that eMDSC and mMDSC frequencies were elevated in engrafted patients and decreasing Tregs were associated with decreasing DMC. Taken together, eMDSCs, mMDSCs and Tregs may have a synergistic association in favoring the development of a tolerogenic immune milieu that promotes stable mixed chimerism after PBSCT in SCD patients.
Dimethyl Fumarate ameliorates cGVHD development by reducing Tfh and Breg differentiation
Authors: Gong Huanle , Ma Shoubao , Yang Bingyu , Wu Depei
Published: January 2023

Our previous study demonstrated that Dimethyl Fumarate (DMF), an activator of Nrf2, could significantly prevent acute graft-versus-host disease (aGVHD) development by inhibiting Treg differentiation after allogeneic hematopoietic stem cell transplantation. However, whether the DMF regulated anti-oxidant response also plays an important role in the setting of chronic GVHD remains unknown.

In this study, we firstly immunized B6 mice with NP-OVA in CFA and found that DMF remarkably inhibited Tfh differentiation. Next, we established SLE-cGVHD (DBA2 to B6D2F1) model to assess the role of DMF in cGVHD development. DMF treatment significantly reduced proteinuria and dsDNA production, leading to a markedly decreased cGVHD clinical score. Moreover, DMF treatment significantly enhanced the percentage of IL-10 positive Breg cells, but not Tregs in cGVHD.

Taken together, we found that DMF treatment significantly inhibited cGVHD by reducing Tfh and Breg cells. Further studies are needed to investigate the detailed mechanisms of DMF in the regulation of Tfh and Breg differentiation.
Inhibiting fatty acid synthesis (FAS) can prevent and treat chronic graft versus host disease (cGVHD)
Authors: Aguilar Ethan George , Rhee Stephanie , Thangavelu Govindarajan , Loschi Michael , Paz Katelyn , Saha Asim , Ly Joanna , Sage Peter , Sharpe Arlene H , Maillard Ivan , Murphy William J , Serody Jonathan S , Jin Suji , Wagers Sara Bolivar , Nicholls Jemma , Hill Geoffrey , MacDonald Kelli PA , Compeer Ewoud , Dustin Michael , O’Connor Roddy , Sparwasser Tim , Blazar Bruce R
Published: January 2023

GVHD remains the primary complication of allogeneic hematopoietic stem cell transplantation. Considerable interest exists in understanding how metabolism affects immune cell function. We show targeting FAS represents an attractive strategy to ameliorate cGVHD. Acetyl-CoA carboxylase 1 (ACC1) catalyzes the first step of FAS, and can be pharmacologically inhibited with either Soraphen A or 5-(Tetradecyloxy)-2-furoic acid (TOFA). FAS is critical for T cell effector responses, a finding we confirmed using an in vivo multiorgan system cGVHD model with bronchiolitis obliterans, a model dependent on germinal centers (GC), IgG2c secretion, lung deposition and fibrosis. Compared to wild type T cells, cGVHD mice receiving CD4CreACC1fl/fl allogeneic T cells had reduced cGVHD severity assessed by pulmonary function test and immune phenotyping. As thymic regulatory T cell (tTreg) infusion can prevent and treat cGVHD, we examined tTreg FAS needs. Inhibiting FAS in sorted tTreg via drug (Soraphen A; TOFA) or genetic (FoxP3CreACC1fl/fl) strategies increased oxidative and glycolytic metabolism measured by Seahorse assay, that correlated with superior in vitro suppressor function. These findings were extended in vivo where transfer of FoxP3CreACC1fl/fl tTreg, at day 28 following disease establishment, decreased cGVHD severity. Daily treatment of cGVHD mice from day 28 to day 56 with TOFA reversed cGVHD. An in vitro assay in which T follicular regulatory cells suppress T follicular helper and GC B cell class switching showed that inhibiting tTreg FAS can increase their suppression of GC responses. These data provide evidence that inhibiting FAS can prevent or treat cGVHD and enhance Treg function, providing a new approach to ameliorate cGVHD.
Prophylactic antibiotic treatment exacerbates Graft vs. Host Disease-induced bone marrow failure and spleen hypoplasia
Authors: McDaniel Brianyell T , Enriquez Josue , Furr Kathryn , Grisham Matthew
Published: January 2023

Acute graft vs. host disease (aGVHD) is a major complication following hematopoietic stem cell transplantation. Preclinical and clinical studies suggest aGVHD may be potentiated by gut damage and translocation of intestinal microbiota following toxic, pre-transplant conditioning protocols. We recently reported that aGVHD-induced bone marrow (BM) failure and splenic hypoplasia develops in the absence of gut damage suggesting that intestinal bacteria may not be required for disease pathogenesis.

Objective

Determine whether prophylactic gut decontamination with broad spectrum antibiotics (Abx) affects the onset and/or severity of aGVHD-induced BM and spleen damage.

Methods

Syngeneic (Bl6) or allogeneic (Balb/c) CD4+CD25−T cells (5×106 cells) were injected into NK cell-depleted Bl6 RAG1−/− recipients. Prior to T cell transfer, RAG1−/− mice received water (ab libitum) containing aspartame (Asp) or an Abx cocktail containing Asp, neomycin and vancomycin for 7 days prior to and following T-cell transfer.

Results

Treatment of allogeneic mice with Abx reduced colonic bacterial load by more than 20-fold when compared to their Asp-treated counterparts. Abx treatment also resulted in large and significant reductions in BM- and spleen-residing T cells and myeloid cells as well as circulating erythrocytes, platelets and hematocrit when compared to Asp treated mice. These Abx-induced alterations were associated with significant increases (~4-fold) in serum IL-6 levels compared to Asp-treated mice.

Conclusions

Prophylactic Abx treatment exacerbates aGVHD-induced BM failure and spleen hypoplasia.
iNKT cell modulation of tumor microenvironment (TME) and antitumor immunity: the role of antitumorigenic and protumorigenic iNKT cells
Authors: Zhang Hui , Nguyen Jasmine , Pham Michael , Smith Anita
Published: January 2023

Preclinical and clinical data indicate that iNKT cells play important roles in antitumor immunity. High number of iNKT cells in the PBL of cancer patients usually correlates with a favorable prognosis. We found that compared to wild-type (WT) mice, depletion of iNKT cells did not affect tumor progression or metastasis in mice bearing melanoma or Lewis lung cancer; restoring iNKT cells by transferring thymocytes into iNKT KO mice dramatically enhanced antitumor immunity. The underlying mechanism is no known. Herein, we report progress on the mechanistic study. Thymocyte transfer only reconstituted iNKT cells in liver, spleen and blood, but not in LN of iNKT KO mice. This suggests that two functionally opposite iNKT subsets exist in the immune system, and thymocyte transfer only restored antitumorigenic iNKT, but not protumorigenic iNKT cells. Transferring IFNγ KO thymocytes diminished iNKT reconstitution-induced antitumor immunity; transferring IL-17A KO thymocytes enhanced iNKT reconstitution-enhanced antitumor immunity. Compared to WT mice or iNKT KO mice, iNKT reconstitution in iNKT KO mice up regulated the expression of XCL1, FLT3L and CCL5, down regulated IL-6, TGFβ1, TGFβ2, COX2 expression, increased IRF8+ cDC1 cells and Siglec F− DC, and decreased αSMA+ FAP+ cancer-associated fibroblasts (CAF) and MDSC in the tumor. Taken together, these results support that antitumorigenic iNKT cells produce IFNγ dominant cytokine and facilitate NK/cDC1 infiltration and activation in the tumor to induce antitumor immunity; protumorigenic iNKT cells induce CAF to produce TGFβ, which induces a myeloid cell-mediated inhibitory TME. The balance between antitumorigenic and protumorigenic iNKT cells shapes the TME and antitumor immunity.
LEF1 activates a central memory-like program in human NKT cells
Authors: Ngai Ho , Tian Gengwen , Courtney Amy N , Balzeau Julien , Liu Bin , Guo Linjie , Di Pierro Erica J , Metelitsa Leonid S
Published: January 2023

Vα24-invariant natural killer T cells (NKTs) possess potent antitumor activity that can be exploited for use in cancer immunotherapy. We recently demonstrated that the CD62L+ NKT subset persists longer and mediates more potent antitumor activity in vivo than its CD62L− counterpart; as such, CD62L+ NKTs are crucial to maximizing the therapeutic efficacy of NKTs. Key factors involved in transcriptional control of the CD62L+ central memory-like program in NKTs remain largely undefined. To address this gap, we performed comparative gene expression analysis in CD62L+ and CD62L− NKTs and found that LEF1, which encodes a mediator of Wnt/β-catenin signaling, was the top overexpressed gene in the positive subset. We also found that LEF1 protein expression was largely restricted to CD62L+ primary human NKTs and was associated with increased expression of Wnt target genes. While CRISPR-Cas9-mediated knockout of LEF1 expression did not impact CD62L+ cell numbers after initial ex vivo expansion, it did significantly reduce the frequency of these cells after antigenic restimulation. On the other hand, treatment with Wnt/β-catenin signaling activator TWS119 or Wnt3a ligand increased CD62L+ NKT cell frequency. Combined treatment of TWS119 with IL-21, which selectively protects the CD62L+ subset from apoptosis, resulted in significantly higher CD62L+ cell numbers than each single agent alone. These results identify LEF1 as a key transcriptional activator of the central memory-like program in CD62L+ NKTs and pave the way for targeted pharmacological enhancement of NKT cell-based cancer immunotherapy.