bluuebunny/crossref_metadata_2025 huggingface dataset:
Gimap5-dependent inactivation of GSK3β is required for CD4+ T cell homeostasis and prevention of immune pathology
Authors: Patterson Andrew R. , Endale Mehari , Lampe Kristin , Flagg Aron , Woodgett James , Bleesing Jack , Hoebe Kasper
Published: January 2023

GTPase of immunity-associated protein 5 (Gimap5) has been linked with lymphocyte survival, autoimmunity and colitis, but its mechanisms of action are unclear. Here we show that Gimap5 is essential for inactivation of glycogen synthase kinase-3β (GSK3β) following T cell activation. In the absence of Gimap5, constitutive GSK3β activity constrains c-Myc induction as well as NFATc1 nuclear import, thereby reducing the frequency of CD4+ T cells that complete cell cycle. Additionally, Gimap5 facilitates phosphorylation of GSK3β at Ser389 and its nuclear translocation. Impairment in the latter steps of GSK3β regulation by Gimap5 is associated with increased DNA damage and reduced survival of activated CD4+ T cells. Importantly, pharmacological targeting and genetic deletion of GSK3β in mice can override Gimap5-deficiency in CD4+ T cells, limiting DNA damage and enhancing T cell proliferation and survival. Additionally, this ameliorates immune-mediated pathology in vivo caused by loss of Gimap5. Finally, we show that a human patient with a GIMAP5 loss-of-function mutation has lymphopenia and impaired T cell proliferation. T cells from this patient exhibit impaired proliferation, but can be rescued in vitro with GSK3 inhibitors. Given the restricted expression of Gimap5 in lymphocytes, we propose that its control of GSK3b activity is an important checkpoint in lymphocyte proliferation and function.
Helios expression defines a phenotypically distinct population of Treg cells
Authors: Thornton Angela M. , Korty Patricia E. , Kim Yong Chan , Martens Craig , Shevach Ethan M.
Published: January 2023

The transcription factor Helios is expressed by 65–75% of Treg and we have proposed that it is a marker of thymic derived Treg (tTreg). In order to study the differences between Helios+ and Helios− Treg, we have generated a Helios-GFP/Foxp3-RFP double reporter mouse. Helios+ Treg expressed a more activated phenotype and had higher suppressive capability in vitro. Both subsets were equivalent in their ability to suppress IBD in vivo, but Helios+ Treg were more protective against disease induced by the transfer of splenocytes from scurfy mice. Both subsets expressed a highly demethylated Foxp3 TSDR, with slightly higher demethylation in the Helios+ Treg subset. Upon transfer to normal mice, both Helios+ and Helios− Treg cells maintained equal Foxp3 stability. However, upon transfer to lymphopenic mice, the percentage of cells from the Helios− subset that maintained Foxp3 expression was significantly lower than that of the Helios+ subset. Upon transfer to both normal and lymphopenic mice, Foxp3+Helios+ Treg maintained stable expression of Helios, while 30% of the Helios− Treg acquired low levels of Helios expression upon transfer to normal mice and 70–80% of the Helios− Treg acquired Helios expression when transferred to lymphopenic mice. Gene expression profiling of the two populations demonstrated 1000 differentially expressed genes. Additionally, TCR repertoire deep sequencing analysis revealed little to no overlap of the two populations. Taken together, our data indicate that Helios expression can differentiate two distinct populations of Treg with overlapping functions, most likely representing tTreg (Helios+) and stable peripherally-induced pTreg (Helios−).

Supported by the Intramural Research Program of the NIH, NIAID.
Performance of the Syphilis Serology Algorithm Diagnosis
Authors: Balada-Llasat Joan-Miquel
Published: January 2023

Syphilis is a chronic, sexually transmitted disease caused by Treponema pallidum. The clinical manifestations of syphilis are often non-specific. Serology tests are used for diagnosis. The aim of this study was to evaluate the reverse algorithm used at The Ohio State Wexner Medical Center (OSUWMC) for the diagnosis of syphilis. The algorithm consist of a treponemal test (Bio-Rad BioPlex Syphilis IgG) a multiplex flow immunoassay (MFI), which utilizes treponemal antigens coated on microspheres to detect IgG to T. pallidum. If reactive, a nontreponemal test, rapid plasma reagin (RPR), is used to detect antibodies against cardiolipin-cholesterol-lecithin antigens, though not specific they are present in most patients with syphilis. If results are reactive with MFI but nonreactive with RPR, a second treponemal-specific antibody test using T. pallidum particle agglutination (TP-PA) is used. Sera submitted at OSUWMC between 01/1/2013 and 10/13/2017 were tested for syphilis using the described algorithm. A total of 70,849 specimens were tested using the MFI assay, of these 96.5% were nonreactive (n= 68,394), 3.3% were reactive (n=2,318) and 0.2% produced an equivocal result (n= 137). Of the MFI reactive results, 1.5% confirmed reactive with RPR (n=1,031) and 1.8% (n=1,287) were nonreactive and considered discrepant. The discrepant specimens were tested by TP-PA, 0.6% (n=438) were confirmed nonreactive and 1.2 % (n=849) were reactive. Furthermore, 137 specimens that produced an equivocal result by the MFI were tested by RPR and TP-PA, 133 were nonreactive and 4 were reactive. In summary, only 2% (n=1,424) of the specimens were discrepant and required a third test for resolution.
Intracerebral Mycobacterium tuberculosis infection induces robust myeloid and lymphocytic immunity in the central nervous system
Authors: Hernandez Gianna L. , Ritter Anna , Mergaert Aisha Mergaert , Rayasam Aditiya , Gerhart Christian , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Central nervous system tuberculosis (CNSTB) is a rare, highly devastating manifestation of extra-pulmonary tuberculosis. To date, research has focused on pulmonary tuberculosis, with no significant advancements being made in understanding the mechanisms associated with CNSTB. This study uses a small animal model of CNSTB that permits us to investigate the immune responses following CNS Mycobacterium tuberculosis infection. We use intracerebral infection of mice with Mtb H37rv which has been fluorescently tagged with tdTomato, allowing us to track it in vivo, and analyze the cellular composition of the subsequent immunological response in the brain. Starting at 7 days post-infection (PI), we show a robust infiltration of immune cells into the brain including the infiltration of CD11chigh dendritic cells, CD11b+ monocytes, and an IFNγ dominated T cell response. Mtb-containing granulomatous lesions develop starting at 1 week and continue to form until 7 weeks PI. Granuloma composition in the brain is dominated by CD11chigh dendritic cells, CD11b+ monocytes, CD4+ T cells and to a lesser extent, Tmem119+ microglia. We show robust immune cell infiltration in the choroid plexus during infection indicating a potential route for secondary dissemination and immune cell infiltration into the brain. We also discovered that anti-Mtb immunity in the brain develops as early as 1 week following intracerebral inoculation of Mtb, earlier than following peripheral inhalation infection in the lung. Finally, robust anti TB immunity leads to infection control in the brain at 7 weeks post-infection. This study provides new knowledge into the pathogenesis of CNSTB, and could provide possible treatment strategies to lessen its destructive outcome.
Oxidant sensor cation channel TRPM2 activates neutrophil extracellular trap formation by autophagy and plays a protective role in pneumoseptic Klebsiella pneumoniae infection
Authors: Tripathi Jitendra Kumar , Sharma Atul , Sukumaran Pramod , Mishra Bibhuti , Singh Brij , Sharma Jyotika
Published: January 2023

Neutrophil extracellular trap (NET) formation represents an important antimicrobial function of neutrophils, which partly depends on reactive oxygen species (ROS) and autophagy activation. However, the factors governing these processes remain unknown. In this study, we demonstrate that nonselective calcium permeable cation channel Transient receptor potential melastatin 2 (TRPM2), senses ROS and contributes to the NET formation. The NET forming function of TRPM2 in response to ROS stimulation requires autophagy activation through ADP ribose, AMPK phosphorylation and p38 MAPK activation. TRPM2 deficient neutrophils fail to activate this pathway with a block of NET release and a concomitant impairment of antimicrobial capacity. Trpm2−/− mice were found to be highly susceptible to pneumonic infection with Klebsiella pneumoniae owing to an impaired NET formation and high bacterial burden despite increased neutrophil infiltration in their lungs. Taken together our data identifies a key role of TRPM2 in NET formation by ROS sensing and links it to autophagy activation via AMPK/p38 pathway. Our study also provides evidence for a protective anti-microbial role of TRPM2 in pneumonic bacterial infection thus positing TRPM2-mediated NET formation as a valuable target for modulating NETs and related pathologies.
Suboptimal stimulation with staphylococcal enterotoxin C1 induces immunosuppressive CD4+CD25+ regulatory T cells by differential expression of FOXP3 isoforms
Authors: Lee Juyeun , Park Nogi , Park Joo Youn , Yoon Sunghyun , Park Juw Won , Pruett Stephen B. , Seo Keun Seok
Published: January 2023

Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis and preventing excessive tissue damage upon immune activation. However, pathogens could exploit Tregs for their successful pathogenesis. We demonstrated that immunosuppressive CD4+CD25+FOXP3+ Tregs were induced by staphylococcal enterotoxin (SE) produced by an important human pathogen, Staphylococcus aureus. CD4+CD25+FOXP3+ T cells were induced from stimulation with SEC1 at a high (1 μg/ml) and low (1 ng/ml) concentrations that induced optimal stimulation and suboptimal stimulation, respectively. However, CD4+CD25+FOXP3+ T cells induced from suboptimal stimulation were functionally immunosuppressive, not from optimal stimulation. Immunosuppressive CD4+CD25+FOXP3+ T cells induced from suboptimal stimulation showed typical Treg surface markers such as CTLA-4, GITR, TNFR2, and CD45RO and produced immunomodulatory cytokines such as TGF-β and IL-10. However, suppression was mainly mediated by galectin-1 in a contact-dependent manner. We found that CD4+CD25+ Tregs from suboptimal stimulation highly express FOXP3 isoform lacking exon 2 (ΔE2) and partially lacking exon3 (ΔpE3) preferably localized to the nucleus, compared to those from optimal stimulation. Lentiviral transduction of FOXP3 isoforms (full length, ΔE2, ΔpE3) to Jurkat T cells did not result in immunosuppressive function. By contrast, when cultured in the media generated from suboptimal T cell stimulation, transduced Jurkat T cells became more immunosuppressive in an order of ΔE2 ΔpE3, and full length FOXP3. These results suggest that soluble factors generated from suboptimal T cell proliferation play an important role in induction of immunosuppressive Tregs.
Cold-induced stress impairs the production of Th1 protective cytokines during co-culturing of dendritic cells and naïve T helper cells of Chlamydia muridarum infected mice.
Authors: Belay Tesfaye
Published: January 2023

The purpose of this study was to determine how cold-induced stress affects the maturing of mouse bone marrow-derived dendritic cells (BMDCs) and to see the dynamics of cytokines production associated with T helper subtypes. Quantitative real time PCR and ELISA were used to measure gene expression and production of cytokines in co-culture of BMDC and Naïve CD4+ T cells. Culturing BMDC in vitro stimulated with LPS in the presence of norepinephrine (NE) or Feroterol, β-AR agonist resulted in a marked increase (75 pg/ml) of IL-10 compared to that of stressed mice. Treatment with β-AR antagonist, ICI 118,551 resulted in lowering production of IL-10 in BMDC of nonstressed (control) mice. In contrast, IL-12 production in stressed group was significantly low (132 vs 239 pg/ml) compared to that of control mice. Naïve T cells co-cultured with BMDC pretreated with NE or Feroterol showed decreased IL-12 production compared to controls. In contrast IL-4 production was increased with pretreatment of NE or Feroterol (64 vs 28 pg/ml) compared to control group. Increased IL-4 production in the genital tracks of mice compared to control (114 vs 0 pg/ml) was observed. The relative expression of GTAT 3 in T cells co-cultured with BMDC of stressed mice was high (2.8 vs 0.49) compared to control mice. The same gene expression trend of IL-4 in stressed mice group (1.8 versus 0.12) compared to control group was observed. In control mice, T-bet expression was (1.78 vs 1.0) and interferon gamma (2.12 versus 1.0) compared to that of stressed mice group. Our results demonstrate that stress influences the activation and differentiation of DCs stimulated by LPS shifting the Th1/Th2 ratio and impairing the production of Th1 protective cytokines during Chlamydia muridarum infection.
T-independent IFNγ and B cells synergize to prevent mortality associated with disseminated Chlamydia muridarum genital infection
Authors: Poston Taylor , O’Connell Catherine , Girardi Jenna , Sullivan Jeanne E. , Nagarajan Uma , Marinov Anthony , Scurlock Amy , Darville Toni
Published: January 2023

CD4 T cells and antibody are required for optimal acquired immunity to C. muridarum genital tract infection, and T cell-mediated IFNγ production is necessary to clear infection in the absence of humoral immunity. However, the role of protective T cell-independent immune responses against primary infection remain unclear. We addressed this problem by inoculating wild-type and immune-deficient mice with a plaque-purified strain (CM001) isolated from C. muridarum Nigg stock capable of enhanced extragenital replication. Genital CM001 inoculation resulted in transient dissemination to the lungs and spleen in wild-type mice prior to clearance. However, CM001 genital infection induced lethality in STAT1−/− and IFNG−/− mice, where IFNγ signaling is absent, and in Rag1−/− mice that lack T and B cells but are competent for innate IFNγ signaling. In contrast, muMT, nude, and Tcra−/− mice survived. These data collectively indicate that IFNγ prevents lethal CM001 infection in the absence of T cells and suggest a B cell co-requirement for protection. Adoptive transfer of convalescent immune sera, but not naïve IgM antibody, to Rag1−/− mice significantly increased survival time and transfer of naïve B cells completely rescued Rag1−/− mice against CM001 lethality. Protection was associated with a significant reduction in the lung chlamydial burden of genitally infected mice. These data reveal an important synergism between T-independent B cell responses and innate IFNγ in chlamydial host defense, and suggest cooperative interactions between T-independent IgG and IFNγ are essential for limiting extragenital dissemination.
Staphylococcus aureus colonization is influenced by cutaneous interferon production.
Authors: Sirobhushanam Sirisha , Parsa Navya , Sarkar Mrinal K. , Gudjonsson Johann E. , Kahlenberg Joann Michelle
Published: January 2023

Cutaneous inflammation is a recurrent manifestation of systemic lupus erythematosus (SLE), a severe autoimmune disease, and is triggered upon exposure to ultraviolet B (UVB) light. The exaggerated inflammatory response of SLE epidermis to UVB exposure is thought to be, in part, secondary to chronic exposure to type I interferons (IFN). S. aureus colonization has been found to stimulate cutaneous IFN. Indeed, our analysis of 54 SLE patients swabbed on their chest, nares and any active lesions demonstrated colonization of SLE patients at twice the rate (61%) of normal adult population (~30%). The rate increased further (67%) when active cutaneous lesions were sampled. We have also identified differences in expression of cutaneous IFN production in SLE patients, in particular IFNκ, and thus sought to investigate the role of IFNs in S. aureus adhesion to better define their impact on colonization. Immortalized keratinocytes (NTERTs), were grown to confluence, exposed to 20 mJ/cm2 UVB (310 nm) and 24hrs later exposed to ~107 cells of S. aureus. Following washing, the number of adherent cells were then determined by serial dilution and plating. Our results show that treatment with UVB increased S. aureus adhesion to WT-NTERTs. An increase in adhesion was observed in treatment of WT-NTERTs with 5ng/ml IFNα for 24 hours as well. NTERTs lacking IFNκ (IFNκ KO) unlike WT NTERTs exhibited lower S. aureus adhesion after UVB exposure which was reversed when functional IFNκ was added to growth media. These results suggest that increased IFN production in SLE skin may contribute to enhanced S. aureus colonization in these patients. The impact of S. aureus colonization on SLE skin remains to be determined.
Anatomically remote education of B cells is required for colonic health
Authors: Surana Neil , Kasper Dennis L.
Published: January 2023

Mucosa-associated lymphoid tissues contain roughly 80% of all immune cells and produce virtually all of the body’s IgA. Although the majority of IgA-secreting cells educated within a mucosal site home back to the same anatomic region, some cells are also found in distant mucosal tissues. These observations underlie the notion of a common mucosal immune system, which holds that anatomically unrelated mucosal sites are functionally connected by a shared immune system. However, the ontological basis of this separation between site of immune education and functionality has remained elusive. Here we show that mice lacking Peyer’s patches (PPs)—small-intestinal lymphoid tissue covered by antigen-sampling M cells—have no immunologic defect in the small-intestinal lamina propria. Surprisingly, the primary immunological abnormality in PP-deficient mice was a reduction in colonic B cells, including plasmablasts but not plasma cells. Adoptive transfer experiments conclusively demonstrated that PP-derived cells preferentially give rise to colonic—but not small-intestinal—B cells and plasmablasts. Finally, these PP-derived colonic B cells were critical for restraining colonic inflammation. Thus, PPs bridge the small-intestinal and colonic immune systems and provide a clear example of immune education being required in an anatomic compartment distinct from the effector site. Our findings, which highlight that the majority of fecal IgA is produced by colonic plasmablasts that originate from PPs, will help inform design of mucosal vaccines.
B cell-intrinsic epigenetic modulation of local and systemic antibody response by gut microbiota through catabolic short-chain fatty acids
Authors: Sanchez Helia N. , Gan Huoqun , Moroney Justin B. , Daw Cassidy C. , Taylor Julia , Zan Hong , Casali Paolo
Published: January 2023

Immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM), as initiated by AID, and plasma cell differentiation, as mediated by Blimp-1, are critical for the maturation of the antibody response. This, as we have suggested, can be modulated by epigenetic modifications arising from environmental factors, such as the gut microbiota. Here we show that the gut microbiota regulates host immune responses through the production of metabolites such as short chain fatty acids (SCFAs). Butyrate and propionate are two of the major metabolic products generated by gut microbiota through processing of dietary fibers. They effectively dampened local and systemic IgG, IgA and IgE responses through B cell-intrinsic modulation of Aicda and Prdm1 gene expression, and, therefore, CSR/SHM and plasma cell differentiation. This SCFA-mediated reduction of class-switched B cells and plasma cells in PP, MLNs and the spleen curtailed the antibody response to T-dependent and T-independent antigens. It also virtually abrogated the allergic IgE responses to house dust mite and peanut as well as the lupus IgG autoantibody response. Mice fed fiber-free diets showed reduced levels of gut SCFAs and increased titers of serum IgG, IgA, and IgE, which were reversed by the administration of exogenous butyrate and propionate. SCFA-mediated modulation of B cell was not due to their G-protein coupled receptor signaling or their use as an energy source. Rather, it resulted from their HDAC inhibitory activity, which upregulated select miRNAs silencing Aicda and Prdm1 mRNAs. Thus, the gut microbiota modulates B cell differentiation and antibody responses through B cell-intrinsic epigenetic regulation, by fiber processing metabolic products.
Absence of B Cell Function Impairs Protective Mucosal Immunity to Brucellosis
Authors: Goodwin Zakia I. , Clapp Beata , Yang Xinghong , Hoffman Carol , Pascual David
Published: January 2023

Mucosal vaccination with a live attenuated B. melitensis double-mutant (BMDM) confers sterilizing protection against pulmonary challenge with wild-type (wt) BM 16M. Although T cell-dependent immunity is essential for protection, we queried the role of B cells in this response. We hypothesized that B cells enable BMDM to persist in the lungs for stimulation of robust adaptive responses. Using BALB/c B cell-deficient (μMT) mice, early and long-term immunity subsequent mucosal vaccination was examined. Groups of BALB/c and μMT mice were orally primed, nasally boosted, and 4 wks later, splenic and lung lymphocytes were antigen-pulsed to determine cytokine responses. μMT mice showed significantly reduced pulmonary proinflammatory responses by three-fold relative to BALB/c mice. Nasal challenge with wt BM 16M of BMDM-vaccinated mice resulted in impaired protective immunity in μMT mice evidenced by the significantly increased brucellae colonization of lungs and spleens. To determine the types of myeloid cells involved in brucellae recognition, μMT mice nasally vaccinated with a mCherry variant of BMDM showed a 23-fold reduction in lung mCherry expression relative to similarly vaccinated BALB/c mice at 5 days post-immunization. mCherry expression associated with various myeloid and B cells. Interestingly, although antigen-presenting cells (APCs) increased similarly in both groups of mice, mCherry expression in lung neutrophils and monocyte-derived macrophages in BALB/c mice was 23- and 4-fold enhanced, respectively, to μMT mice. We believe B cell retention of BMDM mutant enhances APC activity within the lungs facilitating the development of potent local T cell responses. Work supported by NIH R01 AI123244 and AI124244-03S1.
Lactobacillus reuteri induces gut intraepithelial CD4+CD8αα+ T cells
Authors: barragan LUISA CERVANTES , Chai Jiani , Tianero Ma Diarey , Di Luccia Blanda , Ahern Philip , Merriman Joseph , Cortez Victor S. , Caparon Michael G. , Donia Mohamed S. , Gilfillan Susan , Cella Marina , Gordon Jeffrey , Hsieh Chyi Song , Colonna Marco
Published: January 2023

The small intestine contains CD4+CD8αα+ 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. DP IELs are absent in germ-free mice, suggesting that their differentiation depends on microbial factors. We found that DP IEL numbers in mice varied in different vivaria, correlating with 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, allowing ThPOK downregulation and differentiation into DP IELs. Thus, L. reuteri together with a tryptophan-rich diet can reprogram intraepithelial CD4+ T cells into immunoregulatory T cells.
Intracellular pairing of TCRα and TCRβ mRNA at the single-cell level using DNA Origami
Authors: Ulrich Peaches Rebecca , Schoettle Louie , Anderson Karen S. , Blattman Joseph
Published: January 2023

T cell receptors (TCRαβ) are heterodimeric proteins that recognize MHC:peptide ligands. Coding sequences of TCRαβ is determined by semi-random and imprecise joining of variable, diversity, and joining gene segments. Complementarity determining region 3 (CDR3) spans the joint between these segments and typically makes the most contact with peptide during TCR recognition, largely determining specificity. A problem in identifying TCR from cells for clinical applications has been development of high-throughput methods for obtaining CDR3 sequence information from TCRαβ pairs from individual cells. We developed novel DNA origami nanostructures to simultaneously capture TCRαβ mRNA from single cells. T cells are transfected with the origami, and TCRαβ mRNA are co-captured onto bowtie-integral probes. Individual TCR sequences are then reverse transcribed and appended to origami-specific unique bowtie oligonucleotide barcodes that can be matched in downstream next-generation high-throughput sequencing. We have validated this approach using P14 TCR transgenic T cells, expressing known TCRαβ sequences. Following the synthesis of DNA origami with TCRα and TCRβ capture probes, transfection of P14 CD8+ T cells with the scaffold resulted in the specific capture and amplification of TCR sequences. Validation using Sanger sequencing confirmed capture of barcoded TCRα and TCRβ CDR3 sequences. We have developed an approach to single cell analysis of multiple mRNA species. Using a custom bowtie dual-barcoding system, we captured and re-isolated barcoded TCR sequences from individual cells. Although our focus is on the TCR repertoire, this technology is highly adaptable and can capture sequence information of any two genes of interest.
Autofluorescence Imaging of T cell Activation
Authors: Walsh Alex J. , Mueller Katie , Jones Isabel , Heaster Tiffany M. , Saha Krishanu , Skala Melissa C.
Published: January 2023

T cells can have different activities based on receptor expression and cytokine production. Current methods to classify and assess immune cell behavior include flow cytometry and immunohistochemistry, which require immune cell labeling and tissue fixation. A non-invasive method for determining T cell behavior is needed to study immune cell behaviors in tumors and evaluate novel immunotherapies. Activated T cells require high rates of glycolysis to maintain immune activities. Therefore, we are developing optical metabolic imaging (OMI) to assess the metabolic profile of T cell subtypes and activation states using cells isolated from human blood. OMI probes the fluorescence intensity and lifetime of the metabolic coenzymes NAD(P)H and FAD, to quantitate the redox state of the cell through the optical redox ratio (NAD(P)H fluorescence intensity divided by the sum of NAD(P)H and FAD fluorescence intensity) and co-enzyme binding. Our results show that the optical redox ratio is increased in activated populations of unsorted T cells, and of CD8+ T cells, consistently across four different donors. Single-cell analysis of the unsorted, unactivated T cell populations revealed a small portion of cells in an activated state. Inter-donor heterogeneity highlights the variability of immune responses between patients. These results indicate that OMI is a powerful tool for assessing T cell subtype and behavior. OMI utilizes the autofluorescent properties of NAD(P)H and FAD, and thus is contrast agent free, non-damaging, and requires no genetic manipulation. Therefore, OMI can be used to image T cell interactions with tumors in time-course studies of tumor development or assess the efficacy of immunotherapy.
Analysis of T cells made easy - Complete workflows improve the isolation and analysis of T cells
Authors: Evaristo Cesar , Pitzka Christina , Siemer Ramona , Foerster-Marniok Anna , Gotot Janine , Dose Christian , Richter Anne
Published: January 2023

T cells play a central role in immunity to pathogens and tumors, but also in autoimmunity. T cell research has generated knowledge that led to multiple clinical breakthroughs, such as the generation of T cell vaccines and tumor immunotherapy approaches. However, additional work is required to fully understand T cell biology, harness their therapeutic potential and control immunopathologies. Cutting edge experimental protocols that interrogate and manipulate T cell biology often require prior purification of T cell populations. Strategies to simplify and accelerate T cell purification are highly desirable to save time, reduce bias and allow complex experiments to be performed.

We established workflows combining automated tissue dissociation with T cell isolation. We developed new CD4, and CD8 T cell specific enrichment reagents that significantly accelerated the isolation protocol. T cells were isolated to purities above 90%, reducing time of downstream analysis. Importantly, culture of isolated T cells did not induce activation as measured by CD69 or CD25 upregulation, as compared to controls. Furthermore, magnetic isolation did not affect proliferation or cytokine expression following in vitro stimulation. Finally, T cell isolation could be fully automated and multiple samples could be processed in parallel.

Our new workflow greatly reduces time of downstream analysis while preserving cell phenotype and functional properties. We believe these innovative tools significantly shorten time-consuming experiments and can be used to increase reproducibility and the quality of data obtained in T cell research.
Effect of MHC clustering on antigen recognition and T cell responses
Authors: Anikeeva Nadia , Fisher Nicholas , Blanchette Craig , Sykulev Yuri
Published: January 2023

MHC proteins that present peptide ligands to T cells form nano-scale clusters on the cell membrane of antigen-presenting cells. While MHC clustering is thought to influence antigen presentation to T cells and may depend on cell type and pathophysiological conditions, how the extent of MHC clustering controls productive TCR engagement and T cell responses has not been systematically studied. To evaluate the effect of MHC clustering on T cell responses, we utilized nano-scale discoidal membrane mimetics to capture and present pMHC ligands at various densities. We examined the binding parameters of these model membrane clusters interacting with live CD8+ T cells and their impact on the triggering of TCR signaling. Our data show that not just the level of expression of pMHC ligands but the extent of their clustering controls antigen recognition and responses by T cells. Variations in the density of pMHC with MHC clusters regulate CD8-MHC and TCR-pMHC interactions, each contributing in a unique way to antigen recognition and the quality of TCR-mediated signaling. Along with well-documented co-clustering of TCR and CD8 on activated, but not naïve T cells, these findings provide new understanding of how the immune receptor clustering controls the specificity and effectiveness of antigen-specific responses by naïve and activated T cells during acute and chronic viral infection.
Utilizing the Multi-parameter Capability of the ZE5 to Monitor T-Cell Exhaustion Following Immunotherapy.
Authors: Green Angela M. , Brampton Chris , Alexander Matt , Talaga Yasha , Jordon Eliabeth
Published: January 2023

The immune systems as evolved sophisticated mechanisms to distinguish between self and non-self, and dangerous and non-dangerous signals leading to appropriate and controlled immune responses against infection and cancer or tolerance to avoid attacking the host body. One pathway of immune suppression is T-cell exhaustion which shuts down effector T-cells. T-cells or T-lymphocytes, are an immune cell that help protect the body from infection and fight cancer. T-cell exhaustion is exploited by chronic viral infections and cancers to escape being targeted by the immune system. It results in a sustained expression of suppressive cell surface markers and effector function in these cells, meaning they are no longer able to function to remove the cancerous and infected cells.

Recently, researchers have focused on using monoclonal antibody drugs to block these suppressive cell surface markers and re-invigorate the T-cells, restoring their effector capability to clear viral infection and cancer cells from the body.

In this study, we use a 15-parameter panel to examine in-depth changes to the T-cell phenotype, exhaustion and cellular subset identity markers before and after chronic stimulation and in combination with a monoclonal antibody drug treatment targeting the receptor Programmed Cell Death 1 (PD-1).
Comprehensive assessment of immune cells in mouse and human atherosclerosis by single cell RNA sequencing and mass cytometry
Authors: Winkels Holger , Ehinger Erik , Vassallo Melanie , Buscher Konrad , Dinh Huy , Kobiyama Kouji , Hamers Anouk , Cochain Clement , Vafadarnejad Ehsan , Saliba Antoine Emmanuel , Zernecke Alma , Bala Pramod Akula , Ghosh Amlan , Michel Nathaly Anto , Hoppe Natalie , Hilgendorf Ingo , Zirlik Andreas , Hedrick Catherine , Ley Klaus , Wolf Dennis
Published: January 2023

Atherosclerosis, an inflammatory disease of large arteries, is – through its clinical manifestations stroke and myocardial infarction – globally the leading cause of morbidity and mortality. Although cells of the innate and adaptive immune system are found in atherosclerotic plaques, their phenotypic and functional diversity is poorly understood. Here, we applied single cell RNA-sequencing (scRNAseq) and mass cytometry (CyTOF) to assess leukocyte diversity in depth, thus defining an immune cell atlas in atherosclerosis.

Single cell transcriptional profiling of aortic leukocytes from 20-week old chow (CD) and western diet (WD) fed Apoe−/− and Ldlr−/− mice revealed 11 phenotypically different leukocyte clusters, whilst WD changed their abundance. Gene set enrichment analysis of single cells established that multiple pathways, e.g. for lipid metabolism or cytokine secretion, pertained to particular leukocyte clusters. Applying a novel 35-marker CyTOF panel confirmed the phenotypic diversity of aortic leukocytes. Among lymphocytes, we detected three principal B-cell subsets defined by scRNAseq, CyTOF, and flow cytometry. These B cell subsets harbor distinct surface marker expression, functional gene pathways, and ex vivo cytokine production. Finally, we used leukocyte cluster gene signatures to assess leukocyte frequencies in 121 human plaques by a transcriptomic deconvolution strategy revealing similar immune cell complexity.

The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunological mechanisms and cell-type specific pathways, and may result in novel diagnostic risk stratification tools.
Oligo-conjugated antibodies (Ab-seq) and massively parallel single cell sequencing reveal the high parameter correlation of protein and mRNA expression in individual immune cells.
Authors: Martin Jody , Fan H. Christina , Shum Eleen
Published: January 2023

Single-Cell mRNA sequencing has enhanced our knowledge of the cell populations found in individual tissues or disease states. However, low correlation between mRNA and protein expression levels hinders our understanding of biology and disease. Traditional single cell protein assays are limited in the number of parameters that can be achieved within one experiment.

Here we use oligo-conjugated antibodies (Ab-seq) in combination with massively parallel single cell mRNA sequencing on the BD Rhapsody™ platform to simultaneously measure the protein and mRNA content of individual human blood cells (PBMCs). To accomplish this, a 30+ parameter Ab-seq panel against immune relevant cell surface markers was paired with the BD Rhapsody™ Immune Response panel (a targeted gene expression panel consisting of 399 targets). The Multi-omics data from this single workflow experiment provides a measurement of both gene expression and protein expression. The digital measurements are rendered free of PCR bias through the use of the unique molecular indices (UMIs).

Our results show that protein expression detected with Ab-seq is highly sensitive and specific. Protein expression patterns correlate well with results from flow cytometry data on the same samples. Ab-seq allowed the robust detection of expressed genes even when their cognate mRNA transcripts have low abundance. Certain markers cannot be easily distinguished at the mRNA level (i.e. isoforms CD45RA and CD45RO) but are easily distinguished at the protein level. Our study shows successful analysis of protein and mRNA expression data within a single workflow, and should enable the further elucidation single cells within different tissues, developmental time points, and disease states.
Multicolor Immunophenotyping using Flow Cytometry: Evaluation of Multiple Methods for Instrument Optimization
Authors: Bradford Jolene A. , Shallice Matthew , Diz Ramiro , Oxford Carol , Punj Manik , Alford Kate , Donaldson Phil , Seredick Barbara , Buller Gayle , Sardina Patricia
Published: January 2023

A basic tool of the Immunologist, multicolor flow cytometry is a technology that uses multiple fluorescent markers to identify and characterize populations of cells. To obtain high quality fluorescent flow cytometry data, a well-optimized instrument is required. The most common type of detector is the photo-multiplier tube (PMT), which amplifies signal from emitted light photons by applying a voltage to the PMT. As the voltage is increased the fluorescent signal is increasingly separated from background, providing greater resolution of the positive signal. However, at a certain voltage, the increasing separation of fluorescent signal from background will plateau and the separation of fluorescent signal from background will remain constant. This is called the minimum voltage required; an ideal minimal voltage will amplify dim signal above background, but is not so high that the fluorescent signal exceeds the upper range of PMT linearity. In most cases, adjusting voltages above the minimal voltage does not increase the separation, while voltages below the minimal voltage requirement compromise detection of dim fluorescent signal. A variety of methods have been proposed to optimize the PMT voltage, most using a technique called the voltage walk, or voltration. This study compares a variety of techniques and calculations, using different types of hard-dyed beads that are detected in all channels but do not include specific fluorophores used for typical experimentation; both cells and antibody-capture beads labeled with fluorophores specific to a detector; Electronic Noise (EN) of the PMT, and methods combining these approaches. For Research Use Only. Not for use in diagnostic procedures.
The utilization of ctDNA technology as an early diagnostic tool for Cancer detection in Tonga
Authors: Sika-Paotonu Dianne , Vaea Kolinisiupeli , ‘Ulufonua Lisiate Kilipeni , ‘Akau’ola Siale , Aho George , Tuipulotu Amelia Afuha’amango , Toumoua Seventeen , Lilo Filimone , Fotu Latu , Guilford Parry
Published: January 2023

Circulating tumour DNA (ctDNA) refers to small fragments of tumour DNA found in the blood circulation. These DNA fragments can be isolated and quantified to obtain data about a cancer’s size and progression. The possibility of utilizing a simple blood sample to allow early detection and monitoring of cancer growth is a highly desirable application for this ground breaking technology. Significant health inequalities exist for Pacific peoples with cancer living in New Zealand and in the Pacific region, where the burden of cancer remains a public health concern. The application of this simple ctDNA method of detecting and monitoring cancer to enhance the precision of early cancer diagnostics and surveillance to improve cancer health outcomes for Pacific peoples in Tonga was investigated. Consultation was sought with senior Government officials, Medical, Nursing, Health and Community research staff concerning the development and implementation of ctDNA as a diagnostic tool within the health care setting throughout Tonga. Preliminary assessment of the on-the-ground laboratory requirements needed for the implementation of the ctDNA technology was also undertaken. Strong support for the development of ctDNA as an early diagnostic tool within the clinical setting was expressed. A key feature of the ctDNA technology within this resource constrained environment was the anticipated detection of cancers at an earlier stage with a greater chance of being treatable given that most cancer presentations in Tonga are late. This work sought to explore and progress the implementation of ctDNA as an early cancer diagnostic tool within the Pacific setting, and remains a highly feasible early cancer detection tool within Tonga.
Magnetic Delivery of Antibody-Coated Beads to Live Lymph Node Tissue Slices
Authors: Belanger Maura C. , Pompano Rebecca R.
Published: January 2023

Lymph nodes have a high degree of spatial organization, characterized by dynamic zones of cells secreting changing sets of cytokines. Experimental measurement of the local distribution of secreted cytokines in live tissue would provide new insights into the dynamics of cell-cell communication, and may help inform spatially targeted therapies. As a step towards bead-based immunoassays for cytokine detection in situ, here we report our findings for delivering beads uniformly to live tissue, a significant technical challenge due to a dense tissue matrix and potential internalization by endocytic cells. We utilized live slices of lymph node tissue ex vivo to allow access to the interior of the node while maintaining structural integrity and dynamics. Lymph node tissue viability post slicing was confirmed by flow cytometry (80% viable by PI exclusion). We conjugated capture antibodies to magnetic beads using carboxylic acid-amine linker chemistry; linkage was confirmed by a secondary antibody. To test penetrance into a dense matrix, beads with various diameters were delivered to 2 % agarose gel by magnetic force. Large (4 and 6 μm) beads were excluded, while smaller beads (0.22, 0.56 and 1.0 μm) were delivered successfully. Small antibody-conjugated beads also penetrated into live, 300-μm thick lymph node tissue slices, but penetrated only 40 μm deep and were localized to the sinuses. Covalent conjugation of polyethylene glycol (PEG) alongside antibody allowed beads to penetrate up to 70 μm into the tissue and provided a more uniform distribution. Having delivered beads successfully, future work will implement a complete multi-step immunoassay in the tissue.
Early Treatment of Experimental Autoimmune Encephalomyelitis with Cannabidiol Suppresses Tc1 Cells and Reduces Neuroinflammation
Authors: Nichols James Matthew , Kummari Evangel , Sherman Jessica , Szafran Brittany , Kaplan Barbara
Published: January 2023

Multiple sclerosis (MS) is a neurodegenerative, T cell-mediated, autoimmune disease. In this study, we examined use of the phytocannabinoid, cannabidiol (CBD), in the experimental autoimmune encephalomyelitis (EAE) model to determine CBD’s potential as an oral therapy for MS. We hypothesized that 75mg/kg of oral CBD for 5 days after initiation of disease would reduce EAE severity through suppression of either the early peripheral immune or late neuroimmune response. EAE was induced in C57BL/6 mice at two different magnitudes, and peripheral inflammatory and neuroinflammatory responses were measured at days 3, 10, and 18. Th1, Th17, Tc1, Tc17, Tregs, and myeloid derived suppressor cells (MDSC) were identified in lymph node and spleen by flow cytometry to determine if CBD altered these cell populations in the secondary lymphoid tissues. Additionally, neuroinflammation was identified in brain and spinal cord using hematoxylin and eosin (H&E) and CD3 staining to detect cellular infiltrates and T cells, respectively. Our results show a clear correlation between reduction in the percentage of MOG-35–55 specific Tc1 cells in the spleen at day 10, and reduced neuroinflammation and clinical scores at day 18 in EAE mice treated with CBD; however, this correlation was not seen in the milder form of disease. These results suggest CBD’s ability to reduce neuroinflammation in EAE might be due to early suppression of the Tc1 phenotype in the peripheral inflammatory response, and that CBD’s ability to suppress disease is dependent on the magnitude of the inflammatory response. Furthermore, this study highlights the effectiveness of oral CBD as a treatment for EAE and suggests that CBD might be effective for treatment of other T cell-mediated diseases.
Delta-Like Ligand 4 (DLL4)/Notch axis regulates EAE development by modulating T cell trafficking across the blood brain barrier
Authors: Walter Fruzsina R. , Hsu Martin , Gilpin Trey E. , Sandor Matyas , Fabry Zsuzsanna , Karpus William J.
Published: January 2023

The Notch receptor system has been suggested to play a critical role in the regulation of chemokine receptor expression on encephalitogenic T cells and subsequent T cell trafficking to the central nervous system (CNS). Here we tested whether Delta-Like Ligand (DLL) 4 (DLL4)/Notch axis regulates EAE development by modulating T effector cell trafficking mechanisms across the blood-brain barrier (BBB). Primary cultures of brain microvessel endothelial cells were established and migration of splenocytes was tested in the presence of DLL4 blocking antibody. Our data show that anti DLL4 blocks transendothelial migration of non-activated T cells, but does not influence the migration of anti CD28 and CD3-activated splenocytes. Additionally, activation of brain microvessel endothelial cells with TNFα abrogated the blocking effect of anti DLL4 in transendothelial migration of T cells. We also demonstrate that blockade of DLL4 function inhibits the development of a progressive model of clinical experimental autoimmune encephalomyelitis (EAE), in C57Bl6 mice. Based on these results, we propose that endothelial DLL4 engagement with Notch expressing T cells is critical in strengthening the neuroinflammation and blockage of DLL4 could provide novel therapies in early phases of CNS neuroinflammatory diseases.
A non-redundant role for RORα in TH17 cell development and TH17-driven inflammatory disorders
Authors: Solt Laura A. , Wang Ran , Amir Mohammed , Chaudhari Sweena M. , Campbell Sean , Bassette Molly A. , Kamenecka Theodore M.
Published: January 2023

Dysregulated TH17 immune responses have been associated with the pathogenesis of several autoimmune diseases. While full development of TH17 cells requires both nuclear receptors RORα and RORγt, most of the work surrounding TH17 cells has focused on the lineage defining transcription factor RORγt. However, TH17 cells are not absent in RORγ−/− mice, suggesting that RORα plays an important role. Despite this, RORα is considered redundant to RORγt and little is known about its function in TH17 cells. The aim of this study was to determine whether RORα played a significant role in TH17 cell development and TH17-driven inflammatory disorders.

Using molecular, genetic, and pharmacological approaches our data suggests that RORα has non-redundant functions to RORγt driving TH17 cell development. Loss or overexpression of RORα in CD4+ T cells significantly decreased or increased IL-17A, respectively, despite no change in RORγt expression. Using two mouse models of autoimmunity known to be TH17 driven [experimental autoimmune encephalomyelitis (EAE) and adoptive transfer model of colitis], we found that loss of RORα significantly inhibited the development of EAE and colitis, largely as a consequence of decreased frequencies of pro-inflammatory TH17 cells and increased frequencies of anti-inflammatory Foxp3+ T regulatory cells. Finally, using a RORα-selective small molecule that we developed, we found that modulation of RORα activity significantly inhibited the development of autoimmunity. Our data suggests that RORα plays a non-redundant role to RORγt in TH17 cell development. Studies focusing on assessing RORα activity alone or along with RORγt may lend novel insight into future therapeutic design for TH17-mediated autoimmunity.