bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a New Lentivirus Restriction Counteracted by Vpr
Authors: Nicodemos Rodrigo A Matus , Ambrozak David , Ransier Amy , Darko Samuel , Douek Daniel C , Koup Richard A
Published: February 2023

Lentiviruses encode non-structural accessory proteins that function to counteract host restriction factors. The lentivirus protein Vpr is known to block G2 to M transition of the cell cycle and degrades various host DNA repair proteins, including UNG2. The reason why Vpr induces these cellular changes in the infected cell is unknown.

We explored this question by direct infection of primary resting and activated CD4 T cells with a CCR5-tropic replication-competent GFP reporter virus. We measured GFP expression by flow cytometry and virus expression by ELISA. We also performed bulk and scRNAseq of sorted GFP+ cells to measure host mRNAs. Additionally, we used a Uracil-qPCR to quantify provirus uracil versus thymidine incorporation. Lastly, we stimulated infected resting cells with and without Vpr and measured cellular proliferation.

We detected resting GFP+ cells 3 to 4 days after infection. Transcriptome analysis of resting GFP+ versus activated GFP+ revealed a pathway for dNTP production in resting CD4 T cells where deoxyuracil is present instead of thymidine. We confirmed provirus uracil incorporation using the Uracil-qPCR assay. Stimulation of infected resting CD4 T cells showed infected cells are preventing from dividing by Vpr. Lastly, we found replication-competent virus was only produced from the initially infected parent cells instead of the divided daughter cells.

We conclude HIV can directly infect primary resting CD4 T cells. HIV-infected resting CD4 T cells incorporate uracils instead of thymidine. After T cell stimulation, Vpr prevents infected cells from dividing because it counteracts an innate lentivirus restriction mechanism against the integrated provirus through UNG2 recognition of the incorporated uracil.
Sodium pyruvate affects influenza A virus infection in vivo
Authors: Reel Jessica Morgan , Lupfer Christopher R
Published: February 2023

Pyruvate is a key metabolite for energy synthesis. After the production of pyruvate through glycolysis, the molecule then gets shuttled into the mitochondria. Once there, it is modified for use in the TCA cycle. While it is a key metabolite for the cell, it has been shown to have anti-inflammatory properties in vitro. Sodium pyruvate has been shown to have effects on metabolic pathways and their influence on the immune response to diseases. Previously we found that infecting mouse bone marrow derived macrophages (BMDM) with influenza A virus (IAV) and treating those macrophages with sodium pyruvate dampened the immune response compared to untreated controls and other infections. We found that subcutaneous injections in vivo, while increasing activity, did not show noticeable differences between placebo and sodium pyruvate treated groups during IAV infection. However, nebulizing sodium pyruvate significantly improved proportional weight loss compared to saline controls. Sodium pyruvate treated groups were found to lose less weight and consume more chow over the course of infection. Nebulizing C57BL/6J mice with sodium pyruvate decreased viral titer and pro-inflammatory cytokines 7 days post-infection as well. Conclusively, nebulizing sodium pyruvate ameliorates IAV infection in vivo.
TRIMming viral infection: identification and characterization of the novel antiviral TRIM7
Authors: Sullender Meagan E , Pierce Linley , Rodgers Rachel , Schriefer Lawrence , Dunlap Bria F , Doench John G , Orchard Robert C , Baldridge Megan T
Published: February 2023

Noroviruses (NoVs) are the leading cause of acute viral gastroenteritis worldwide, causing up to 700 million infections and 200,000 deaths annually, and treatment is limited to supportive care as there are currently no targeted antivirals nor vaccines against NoVs. Therefore, it is critical to discover novel antivirals and develop targeted interventions in order to improve outcomes.

Here we have used a novel genome-wide CRISPR activation screening approach to identify host genes that can inhibit murine norovirus (MNoV) infection or replication. A substantial number of high scoring gene candidates acted in interferon and immune regulation signaling networks, but several were not previously associated with antiviral activity or immunity. TRIM7, the strongest screen hit, was a little studied E3 ubiquitin ligase associated with metabolism and some cancers, yet its expression served as a powerful antiviral in an MNoV-infected cell. Since our initial screen, TRIM7 has begun to emerge as a strategic player in both positive and negative regulation of the innate immune response during infection.

We have since characterized the antiviral mechanism of action of TRIM7 against MNoV in vitro, and have found TRIM7 to be acting early in the viral life cycle to limit viral protein production. Co-immunoprecipitation and in vitro viral evolution experiments have identified MNoV NS6 (viral protease) and NS7 (RNA-dependent RNA polymerase) as important targets of the antiviral activity of TRIM7, and also point to key residues of interest associated with MNoV resistance to TRIM7. Finally, we have investigated the relevance of TRIM7 during in vivo infection using knock-in mouse lines to determine tissue specificity and potency of the antiviral effect.
Antigen receptor on a new lymphocyte represents a key immune player in autoimmune diseases
Authors: Ahmed Rizwan , Omidian Zahra , Giwa Adebola , Ananth Kusuma , Karakus kagan Ege , Aljack Mahamoud , Majety Neha , Yang Angela , Macdonald Alana , Tyagi Sanjana , Zhang Hao , Rabb Hamid , Jie Chunfa , Donner Thomas , Hamad Abdel
Published: February 2023

PURPOSE:

We have recently identified a previously unknown lymphocyte that is a dual expresser (DE) of productively rearranged and surface-expressed TCRαβ and BCR (surface immunoglobulin, Ig) (Ahmed et al, Cell, 2019: 177:11583). Importantly, a single immunoglobulin heavy-chain, IGHV clonotype (clone-x) predominates DEs that encodes a potent autoantigen (x-autoantigen) in its CDR3 region. The x-autoantigen (as a soluble intact x-mAb) cross-activate autoreactive T cells. The goal of this study is to investigate the properties of x-mAb reactive T cells and examining the mechanisms of how x-mAb recognizes and activates the tolerant autoreactive T cells in autoimmune diseases particularly in T1D.

METHODS:

We used EBV immortalized DE clone as a source of x-mAb and FACS-based protocols to identify x-mAb-responsive autoreactive T cells and their functional properties. ImmunoSEQ assay used to characterize TCR repertoires.

RESULTS:

Preliminary data show that x-mAb potentially binds and activates a subset of autoreactive T cells in T1D compared to HC subjects. Additionally, x-mAb-reactive T cells exhibits an activated and antigen experienced phenotype, including expression of CD45RO, CD44, and CD69. TCRVβ repertoire analysis shows that x-mAb reactive T cells are enriched for public clonally expanded TCRs in T1D patients. Further, x-mAb activates the autoreactive T cell through by crosslinking directly to their T cell receptor (TCR).

CONCLUSIONS:

DE cells in T1D patients secretes a public x-mAb that binds and activate specific subset of autoreactive T cells predominated by few clonotypes that express public TCRs. Our results are revealing previously unknown mechanism that appears to be a play critical role in pathogenesis of T1D.
IL-17 receptor A signaling on B cells promotes B-cell responses to TLR7 and enhances autoreactive germinal center B-cell development
Authors: Liu Shanrun , Sun Chiao-Wang , Lu Changming , Ryan Thomas M , Hsu Hui-Chen , Mountz John D
Published: February 2023

IL-17 receptor A (IL-17RA) expression has been identified in B cells and macrophages. There was low expression of IL-17RA on T cells. Surprisingly, we previously showed that IL-17RA signaling through NF-κB p65/p50 has a complex effect on both B and T-cell localization and germinal center (GC) architecture, making it difficult to dissect the specific effects of IL-17RA on B cells. To isolate the effects of the absence of IL-17RA specifically on B cells, we have generated a B-cell specific IL-17RA knockout (Il17rafloxed/floxed x Cd19.Cre) BXD2 mice. Flow cytometry confirmed the loss of IL-17RA expression in only CD19+ B cells but not CD3+ T cells or CD11b+ macrophages. There was a 2-fold decreased spontaneous GC B cell population in Il17rafloxed/floxed x Cd19.Cre BXD2, compared to Il17rawt/floxed x Cd19.Cre BXD2 mice. Interestingly, the abnormal activation and expansion of T-follicular helper cell (Tfh) phenotype in BXD2-Il17ra−/− mice was abrogated in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. In the BXD2-Il17ra−/− mice, B cells were anergic to TLR7 stimulation and exhibited a strong stimulus-specific transcription repressor p50/p50 homodimer and a lower NF-κB phospho-p65. To demonstrate that this anergy is due specifically to IL-17RA on B cells but not other cells, we have identified that there was a lower TLR7-induced CD69 in vitro in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. Taken together, our results suggest that IL-17RA signaling directly regulates B cell autoimmune phenotype in the BXD2 mice through an enhancement of B-cell responses stimulated by the NF-κB signaling pathway. A deficiency of such signal enforces B cell anergy and prevents autoreactive GC formation.
PhIP-Seq analysis reveals autoantibodies for novel antigens in the mouse model of Coxsackievirus B3 infection
Authors: Rasquinha Mahima T , Lasrado Ninaad , Larman Benjamin H , Reddy Jay
Published: February 2023

Myocarditis is a frequent occurrence in children and adolescents that can lead to dilated cardiomyopathy, and viruses are commonly suspected in those affected. Experimentally, Coxsackievirus B3 (CVB) infection in mice occurs in viral and non-viral phases, but in continuum, and histologically, the disease features have similarities with that of human disease. Mechanistically, by using traditional methods, autoantibodies have been demonstrated for various antigens in post-infectious myocarditis leading to a suggestion that autoimmunity could be an underlying mechanism. However, their role continues to be enigmatic, raising a question that whether autoantibody repertoires are sufficiently characterized. Here, we describe a versatile and high-throughput, phage-immunoprecipitation sequencing platform that permitted us to comprehensively evaluate antibody reactivities in serum samples harvested from mice infected with CVB. By screening the phage-displayed virus library (VirScan), antibody reactivity occurred specifically to CVB, and thus, validating our methodology. Similar analysis with the human library revealed antibody reactivity for antigens conserved in both humans and mice, suggesting a role for them in the viral pathogenesis. Alternatively, detection of these autoantibodies may have a diagnostic value in the evaluation of myocardial damage, including cardiac autoimmunity as might occur in CVB infection.
L-type amino acid transporter 1 is essential for Th2 cell-mediated allergic inflammation
Authors: Kaminuma Osamu , Miura Kento , Yamasaki Norimasa , Ogata Sawako , Saeki Mayumi , Hiroi Takachika , Iwata Yohei , Sugiura Kazumitsu , Mori Akio , Endou Hitoshi , Hayashi Keitaro
Published: February 2023

L-type amino acid transporter 1 (LAT1), a transporter of large neutral amino acids, is rarely expressed in resting T cells, whereas its expression is remarkably upregulated upon activation. We have proven the essential role of T cells in the development of allergic inflammation in various target organs, though the contribution of LAT1 to T cell function in allergic diseases has not been clarified. Here, we investigated the effect of a LAT1 inhibitor, JPH203, on antigen-induced inflammatory responses in the skin, lungs, and nasal mucosa of immunized mice or mice transferred with in vitro-differentiated antigen-specific Th2 cells. Consistent with the augmented expression of LAT1 in skin-accumulated CD4+ T cells of atopic dermatitis patients, antigen-induced skin inflammation with eosinophil infiltration and Th2 cytokine production developed in immunized and Th2 cell-transferred mice was suppressed by JPH203 treatment. Essentially the same efficacy of JPH203 was seen in antigen-induced nasal eosinophilic inflammation accompanied by nasal hyperresponsiveness evoked in both mice. Remarkably, antigen-induced bronchial hyperresponsiveness was suppressed, whereas lung accumulation of eosinophils or antigen-specific T cells was not affected, by JPH203 in Th2 cell-transferred mice. The blockade of LAT1-mediated glycolysis, oxidative phosphorylation, and activating transcription factor 4-regulated amino acid starving responses in cytokine production and proliferation of Th2 was suggested to be involved in the efficacy of JPH203. LAT1 is essential for the pathogenesis of activated Th2 cell-mediated allergic inflammation and would be a promising target for the development of new means to treat allergic diseases.
Phytochemical Effects on FcepsilonRI and MrgprX2-mediated Activation of Human Mast Cells
Authors: Kulka Marianna
Published: February 2023

Phytochemicals and herbal extracts are currently used in food supplements as a means of preventing and treating disease. Although phytochemicals have been studied for their potentially beneficial effects on coronary heart disease and their ability to prevent atherosclerosis, certain cancers and inflammatory disease, very little attention has been focused on the potential health benefits of phytochemicals in atopic disease. Mast cells synthesize and store several proinflammatory mediators and are centrally important in atopic diseases such as asthma. Our team has been characterizing the effect of several phytochemicals (flavonoids and polysaccharides) on mast cell activation and pro-inflammatory mediator production when mast cells were activated via the high affinity Fc epsilon receptor I (FceRI) or mas-related G protein coupled receptor, MrgprX2. We show that quercetin and resveratrol decrease mast cell degranulation of both bone marrow-derived mast cells and the human mast cell line (LAD2) by at least 50% (at 10 ug/mL) when the cells were activated via FceRI with IgE and antigen. However, these flavonoids had no effect on LAD2 cells activated via MrgprX2. Polysaccharides isolated from seaweed inhibited IgE/Ag and MrgprX2 activated degranulation of LAD2 cells (by 50%, p<0.01) with some polysaccharides specific to only MrgprX2 activation. These results suggest that mast cell activation can be differentially modulated by phytochemicals.
Characterization of the mast cell surface proteome leads to the identification of CD98hc as a critical surface molecule for optimal mast cell function
Authors: Piliponsky Adrian Martin , Saha Siddhartha , Samanas Nyssa , Shubin Nicholas J , Niino Kerri , Bhise Gauri , Acharya Manasa , Seo Albert , Camp Nathan , Deutsch Gail , James Richard
Published: February 2023

Mast cells are known for their involvement in many distinct pathological conditions, suggesting that mast cells recognize and respond to various stimuli and thus require a rich cell surface protein repertoire. Mast cell surface proteomes have not been comprehensively characterized.

In this study, we aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease.

We enriched for glycosylated surface proteins expressed in murine bone marrow-derived mast cells (BMCMCs) and identified them using mass spectrometry analysis. This approach resulted in the identification of 1270 proteins in BMCMCs, 403 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins are represented by small GTPases, receptors and transporters. Novel surface proteins in mast cells was validated by qPCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs).

Among the novel surface proteins, we found that CD98 heavy chain (CD98hc) encoded by the Slc3a2 gene, was highly expressed in mast cells. Slc3a2 gene disruption by CRISPR/Cas9 gene editing resulted in a significant reduction in CD98hc expression, mast cell degranulation, adhesion and proliferation.

Our study indicates that we can use glycoprotein enrichment coupled with mass spectrometry to identify novel surface molecules in mast cells. Moreover, we found that CD98hc plays an important role in mast cell function.
IL-33 sensitizes mast cells to ATP-induced cytokine secretion
Authors: Pryor Destiny T. , Straus David
Published: February 2023

Mast cells mediate inflammatory responses that promote hypersensitivity reactions and allergic disease. Mast cell activation can be induced by danger-associated molecular patterns such as IL-33 and ATP released by cell necrosis. IL-33 and ATP are elevated in the asthmatic lung, but how these factors act together is poorly understood. Using mouse bone marrow-derived mast cells, we show that IL-33 greatly sensitizes mast cells to ATP. Short term (30 minute) stimulation with IL-33 followed by its removal by washing, increased subsequent ATP-induced cytokine secretion up to 15-fold. This was consistent for ATP-induced IL-6, TNF, and IL-13. IL-33 did not significantly alter mast cell sensitivity to ATP as judged by IC50 calculations, and had no effect on ATP-induced degranulation. We therefore propose IL-33 enhances ATP-induced transcription factor activation without altering calcium-mediated degranulation. We hypothesize that an IL-33-ATP cascade occurs during allergic inflammation and functions as an amplification loop promoting chronic disease.
Internalization of benzylisoquinoline alkaloids by resting and activated bone marrow-derived mast cells utilizes energy-dependent mechanisms
Authors: Alam Syed Benazir , Kulka Marianna
Published: February 2023

Berberine (BBR), a benzylisoquinoline alkaloid, binds to heparin and targets glycosaminoglycan-rich granules of myeloid-derived mast cells, but, the mechanism of berberine’s internalization is poorly understood. Our data showed that 84±3.3% of highly granulated bone marrow-derived mast cells (BMMC) internalized BBR (10 ug/mL) after 45 hr. Methanol fixation completely blocked this process, suggesting an energy-dependent active transport mechanism, and 3-mtheyladenine (600 uM) inhibited internalization by ~43%, indicating that lysosomal self-degradation via the autophagy pathway was involved. BBR incorporation into lipoplexes did not improve BBR internalization, but, rather decreased it by ~30% (1 ug/mL Lipo/BBR; n=3, p<0.05), suggesting that BBR internalization may utilize clathrin-dependent endosomal endocytosis. Interleukin-3 (IL-3)-mediated activation of BMMC augmented BBR internalization (~54% by 40 ng/mL IL-3) in a concentration-dependent manner, further suggesting that clathrin-mediated endocytosis as well as active cell division, have a profound role in BBR internalization. Altogether, our data suggest that internalization of BBR in resting and IL-3 activated BMMC, utilizes clathrin-dependent pathway. Our findings provide important new mechanistic insights into the internalization of benzylsoquinoline compounds by resting and activated mast cells and open new avenues for designing organelle-targeted nanotherapeutics.
Intrinsic B cell TLR-BCR coengagement induces mature and protective antibody responses in the absence of T cells
Authors: Rivera Carlos Eduardo , Chupp Daniel P , Yan Hui , Fernandez Maria J , Zan Hong , Simon Raphael , Xu Zhenming , Casali Paolo
Published: February 2023

Somatic hypermutation (SHM) and class-switch DNA recombination (CSR) together with plasma cell and memory B cell differentiation are central to the maturation of the antibody response. Prompted by our finding that CSR and plasma cell differentiation can be induced in vitro by a TLR ligand together with BCR crosslinking, we hypothesized that TLR and BCR coengagement can induce mature antibody responses in the absence of T cells. To test this hypothesis, we injected Tcrβ−/−Tcrδ−/− mice, which are devoid of T cells, with NP-LPS or Salmonella Typhimurium flagellin, which coengages B cell TLR4 or TLR5 with BCR. These mice generated NP or flagellin-specific IgG class-switched antibodies and plasma cells. The IgG response to NP evoked formation of germinal center microclusters, a high frequency of point-mutations in IgH V186.2 (region which encodes NP-binding BCRs), including the affinity-enhancing Trp33Leu mutation that underpins the production of high-affinity anti-NP antibodies, and generation of memory B cells. The T cell-independence of the class-switched antibody response was further emphasized by the anti-NP IgG response in immune-deficient NSG mice engrafted with purified mouse B cells. It was mimicked by the specific TLR5-dependent IgG response to S. Typhimurium flagellin in Tcrβ− /−Tcrδ−/− mice. The LPS-immunized and flagellin-immunized Tcrβ−/−Tcrδ−/− mice generated LPS-specific and flagellin-specific IgG antibodies that neutralized E. coli and S. Typhimurium in vitro and protected them from lethal infection in vivo. Thus, TLR-BCR coengagement can induce a class-switched, hypermutated and protective antibody response in the absence of T cells.
Genetic regulation of immune homeostatic lung leukocyte populations influences respiratory virus induced disease in collaborative cross mice
Authors: Hampton Brea K , Jensen Kara L , Whitmore Alan C , Gralinski Lisa E , Leist Sarah R , Linnertz Colton L , Maurizio Paul , Menachery Vineet D , Morrison Clayton R , Noll Kelsey E , Plante Kenneth S , Shäfer Alexandra , Shaw Ginger D , West Ande , de Villena Fernando Pardo-Manuel , Baric Ralph S , Heise Mark T , Ferris Martin T
Published: February 2023

Immune homeostasis is the state that the immune system maintains in the absence of insult. Perturbation of immune homeostasis can impact autoimmunity/allergy and adversely affect infectious responses. To date, much of the analysis of immune homeostasis has focused on systemic immunity, but it is also likely to be important in an organ specific manner. Since the lungs are a major site of environmental exposure and infection, we used the Collaborative Cross (CC) mouse genetic reference population to study the genetic regulation of the breadth of baseline immune cell populations in the lung and identify loci regulating these cells at homeostasis. We found that all 54 immune cell phenotypes measured showed strong genetic variation in cell type abundances. We identified 28 quantitative trait loci associated with variation in 24 immune cell populations or the relationship between cell populations. Further, we identified significant associations between 20 of these loci and responses to either influenza A virus (IAV) or Severe acute respiratory syndrome associated coronavirus (SARS-CoV) disease in the same strains of mice. Notably, a locus mapped for variation in Ly6C+ monocyte abundance was associated with SARS-CoV weight loss, as well as titer at days 2 and 4 post-infection. This locus is also associated with influenza-induced disease, as measured by weight loss post-infection. Our analysis highlights the strong genetic control of immune homeostasis, and the key role that immune homeostasis plays in contributing to downstream infectious responses. In particular, our analysis suggests that the abundance of a variety of lung leukocyte populations prior to infection could serve as predictors of immune responses to respiratory viruses.
Engagement of ICOS in tissues promotes establishment of CD8+ tissue-resident memory T cells
Authors: Peng Changwei , Huggins Matthew A , Wanhainen Kelsey M , Lu Hanbin , Georgiev Hristo , Wang Haiguang , Mittelsteadt Kristen L , Hogquist Kristin A , Campbell Daniel J , Silva Henrique Borges da , Jameson Stephen C
Published: February 2023

Resident memory T cells (TRM) provide localized protective immunity in non-lymphoid tissues, but the signals and interactions within non-lymphoid tissues involved in generating TRM are incompletely defined. Inducible T-cell costimulator (ICOS) is a costimulatory molecule with well elaborated roles in regulating differentiation of CD4+ T cell populations, its function in CD8+ T cells is largely unexplored. With acute infection models, we found that ICOS-deficient CD8+ T cells showed minimal defects in the initial effector expansion and the generation of recirculating memory subsets. However, consistent with its high expression in TRM, ICOS deficiency significantly compromised the establishment of CD8+ TRM cells in diverse non-lymphoid tissues. We further confirmed that rather than pre-programming the TRM fate during initial T cell priming, the interaction between ICOS ligand (ICOSL) and ICOS in the non-lymphoid tissues during CD8+ T cell recruitment is critical for efficient tissue retention and acquisition of tissue residency signatures. ICOS signaling through PI3K pathway is key for TRM generation and ICOS stimulation promotes downregulation of KLF2, a transcription factor that regulates T cell trafficking, in CD8+ T cells after entering non-lymphoid tissues. Altogether, our data illustrate that engagement of ICOS is one of the critical local costimulatory cues that promote the establishment of tissue-resident populations, with potential implication for therapeutic manipulation.
The actin-disassembly protein glia maturation factor γ enhances actin remodeling and B cell antigen receptor signaling at the immune synapse
Authors: Deretic Nikola , Bolger-Munro Madison , Choi Kate , Gold Michael R
Published: February 2023

Signaling by the B cell antigen receptor (BCR) initiates actin remodeling. The assembly of branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, allowing B cells to form membrane protrusions that can scan the surface of antigen-presenting cells (APCs). The resulting Arp2/3 complex-dependent actin retrograde flow promotes the centripetal movement and progressive coalescence of BCR microclusters, which amplifies BCR signaling. Glia maturation factor γ (GMFγ) is an actin disassembly-protein that releases Arp2/3 complex-nucleated actin filaments from actin networks. By doing so, GMFγ could either oppose the actions of the Arp2/3 complex or support Arp2/3 complex-nucleated actin polymerization by contributing to the recycling of actin monomers and Arp2/3 complexes. We now show that depleting GMFγ in human B cell lines impairs the ability of B cells to spread on antigen-coated surfaces, decreases the coalescence of BCR microclusters into a central cluster at the B cell-APC contact site, and reduces APC-induced BCR signaling. These effects of depleting GMFγ are similar to what occurs when the Arp2/3 complex is inhibited. This suggests that GMFγ cooperates with the Arp2/3 complex to support BCR-induced actin remodeling and amplify BCR signaling at the immune synapse.
Single cell analysis of iNKT cell phenotype, functionality and metabolism
Authors: Kane Harry , LaMarche Nelson , Brenner Michael , Lynch Lydia
Published: February 2023

iNKT cells have been identified as potent regulators of diverse physiological responses ranging from tumor immunity to homeostatic regulation of adipose tissue. Recent studies have also established that iNKT cells are heterogeneous, and include distinct NKT1, NKT2, NKT17 and NKT10 subsets. To date, these subsets have been broadly characterized using various transcriptomic-based approaches, providing a basis for further analysis and exploration. However, many studies characterizing the iNKT cell transcriptome have been limited to resting iNKT cells, or have focused on iNKT cell development, and thus have not captured the dramatic changes in iNKT phenotype and function that have been observed following antigen exposure. Here, we took advantage of single cell RNA-Sequencing to characterize adipose and splenic iNKT cells before and after activation, and performed integrated analysis of novel and published data to construct a comprehensive overview of the iNKT cell response to antigen. We find that all iNKT cells undergo rapid and profound transcriptional remodeling in response to αGalCer, and identify conserved features of iNKT cell activation, including dynamic reprogramming of cellular metabolism. We also find differences between activated iNKT cell subsets, and we demonstrate that oxidative metabolism functionally segregates NKT2 and NKT17 cells from NKT1 cells. Furthermore, we find that key features of iNKT cell activation are blunted in regulatory iNKT cell populations, which are enriched in adipose tissue, and we identify a conserved network of gene signatures associated with NKT10 cells. Overall, this study provides novel insights into the biology of iNKT cells, and the interplay between their biology and metabolism.
LINGO2 expression on T cells promotes anti-helminth immunity
Authors: Belle Nicole Maloney , Pastore Christopher , Jean Erin , Herbert DeBroski
Published: February 2023

T helper type 2 (TH2) cells are critical for clearance of parasitic helminth infections. Their coordinated efforts harness cellular and molecular pathways to expel worms, resolve intestinal inflammation and drive tissue repair. Yet the mechanisms that control TH2 cell activation, maintenance, and downregulation remain unknown. We recently demonstrated that Leucine rich repeat and Ig domain containing 2 (LINGO2) can function as a receptor for the mucosal reparative cytokine, Trefoil factor 3, to limit epithelial damage, colitic disease and immunopathology. Because LINGO2 is broadly expressed in both hematopoietic and non-hematopoietic cell lineages, this study specifically addressed whether LINGO2 expression in the T cell compartment served a biologically important function in host protection against the parasitic helminth Trichuris muris. Data show that mice selectively deficient for LINGO2 in T lymphocytes (CD4Cre+ LINGO2flox/flox) had significantly higher numbers of adult worms in the cecum, parasite eggs in the stool and colon immunopathology compared to CD4Cre+ or LINGO2flox/flox controls. Intriguingly, after Trichuris infection, CD4Cre+ LINGO2flox/flox mice had significantly fewer GATA3+ TH2 cells and expressed significantly increased interferon gamma levels in the mesenteric lymph node. Taken together, this work highlights a previously unrecognized cell-intrinsic role for LINGO2 in controlling T cell responses that critically shapes the outcome of helminth infection.
A novel SLP-76 signaling pathway T-cells that controls cancer immunotherapy
Authors: Tu Thai Hien , Hulea Laura , Rudd Christopher E.
Published: February 2023

The receptors and signaling pathways that regulate T-cell activation are central in the control of immune responses against tumors. In addition to proximal kinases such as p56lck and downstream immune cell adaptors, we have identified a new signaling pathway in T-cells that involves the direct regulation of the nuclear pore complex (NPC) by the binding of immune adaptor SLP-76 to SUMO-RanGAP1 of the NPC (Mol Cell 2015 59(5):840–9). This interaction represents a second checkpoint for NFATc1 nuclear entry and accounts for some 40–50% of transcription factor entry into the nucleus. RanGAP1 binds to a motif in SLP-76 involving the lysine at residue 56 where its mutation ablates complex formation. In this new study, we report the generation of K56E SLP-76 knock-in (KI) mice which are markedly impaired in the control of tumor growth. This loss of control was accompanied by a marked reduction in the presence CD8+ effector TILs and in the production of interferon-g1 and CD8 cytolytic effector molecules, perforin and granzyme B (GZMB). Further, K56E T-cells showed an impairment in metabolism. Seahorse analysis showed a >70% decrease in glycolysis in response to TCR ligation due to this single mutation in a single adaptor protein. Moreover, K56E T-cells showed a decrease in the expression of Glut1 and in c-Myc translocation into the nucleus of T-cells. By contrast, the expression of other mediators such as PPARα and CPT-1A in fatty acid oxidation were unaffected. Our findings identify a novel SLP-76-RanGAP1 signaling pathway in T-cells that controls transcription factor entry into the nucleus and which modulates immunity against tumors.
The role of tyrosine phosphatase SHP-1 in regulatory T cell function and immune tolerance
Authors: Gu QinLei , Parlak Mahmut , Lorenz Ulrike
Published: February 2023

Our immune system relies on multiple levels of controls to balance its response to danger while maintaining self-tolerance. The functional interactions between conventional T cells (Tcon) and regulatory T cells (Treg) are crucial in this process. Previous studies from our lab revealed that the tyrosine phosphatase SHP-1 plays a critical role in immune tolerance, and while its regulatory role in Tcon cells is widely studied, the role of SHP-1 in Treg is much less understood.

Here, we investigated the role of SHP-1 in Treg cells by using a genetic mouse model carrying a Treg-specific deletion of SHP-1 to address two questions: (I) how SHP-1 affects the activity of Treg cells; and (II) how SHP-1-deficient Treg cells shape the overall immune response.

(I) A characterization of SHP-1-deficient Treg cells demonstrated an increase in the expression of a subset of Treg signature markers compared to SHP-1-sufficient Treg cells. Upon stimulation, SHP-1 deficiency in Tregs increases IL-17 cytokine expression. Moreover, we observed that SHP-1 decreases AKT phosphorylation in Treg cells, consistent with previous studies in Tcon cells from us and others. Finally, we found that SHP-limits Treg suppressive activity in vitro.

(II) Next, we asked whether SHP-1 deficiency in Treg cells affects the overall immune system and found a significant increase in CD44hi CD4+ and CD8+ T cells, indicating a secondary in vivo effect of SHP-1-deficient Treg cells on SHP-1-sufficient Tcon cells. Using murine models of autoimmune diseases, we are currently addressing the underlying mechanism of this phenotype.

In summary, our studies suggest an important regulatory role of SHP-1 in Treg activity and function thereby additionally contributing to the overall immune balance.
A superior dead cell removal platform using Akadeum’s BACS microbubbles
Authors: Grimley Edward C , Roussey Jon , Petlakh-Co Nadia , Wegner Casey , McNaughton Brandon
Published: February 2023

Single cell sequencing (SCS) is one of the most commonly used analytical techniques for cellular characterization and has become a mainstay in immune profiling. As SCS has gained in popularity and improved in sensitivity, it has become apparent that ambient nucleic acids can have a major impact on the quality and reproducibility of obtained SCS results. Since the bulk of ambient nucleic acids in a sample come from dead or dying cells that have lost membrane integrity, one way to dramatically reduce ambient nucleic acids and decrease background noise in SCS is to remove dead cells from the sample prior to sequencing.

To address the challenge presented by contaminating dead cells, Akadeum has developed a Buoyancy Activated Cell Sorting (BACS™) microbubble approach for depleting dead cells from biological samples. Leveraging the power of gravity to drive self-isolation, Akadeum’s BACS microbubbles represent some of the gentlest isolations available; therefore, they are ideally suited for applications where cell viability is integral to success of the experiment. Additionally, BACS microbubbles from Akadeum are quick and easy to use since they require only minimal manipulation and do not rely on any extra equipment or consumables.

Depletion of dead cells is achieved through the selective capture of cells with exposed phosphatidylserine with Annexin V conjugated BACS microbubbles. Once mixed with the sample, the Annexin V BACS microbubbles capture dead cells and float them to the sample surface for removal leaving behind healthy and untouched cells. Samples exposed to the Annexin V BACS microbubbles demonstrated significantly reduced dead cell populations and ultimately yielded more reliable results.
Detection and characterisation of lung-resident memory T cells in human and non-human primates
Authors: Humphries Duncan Charles , O‘Connor Richard , Chabaud-Riou Martine , Boudet Florence , Finlayson Keith , Dhaliwal Kevin , Pavot Vincent
Published: February 2023

Rationale: Lung-resident memory T cells (TRM) play a critical role in protecting against respiratory infection. Maintained in a quiescent state, TRM are poised for rapid effector function upon reactivation with cognate antigen. Kronoscan is a clinic-ready, fibre-based, optical imaging system developed by the University of Edinburgh, capable of measuring both fluorescence intensity and lifetime. The aim of this study is to develop a staining protocol to enable the detection of human and non-human primate (NHP) pulmonary TRM populations in situ.

Methods:

Human lungs (deemed non-suitable for transplantation) following ex vivo lung perfusion (EVLP) and NHP lungs (cynomolgus macaque obtained from Charles River Laboratories) were enzymatically digested to achieve a single cell suspension. Cells were stained with cross-reactive antibodies prior to flow cytometric analysis. Fluorescence intensity and lifetime imaging was also performed using Kronoscan on MACs-enriched, antibody-stained, pulmonary T cells.

Results:

Human and NHP TRM were primarily identified using CD69 and CD103 and co-expressed a number of chemokine receptors (CXCR3, CCR10), adhesion molecules (CD49a, CD49d) and the immune checkpoint inhibitor PD-1. Differences in phenotype were observed between CD4+ and CD8+ TRM. TRM could be identified via fluorescence intensity and lifetime imaging using Kronoscan.

Conclusions:

A cross-reactive antibody panel capable of detecting human and NHP pulmonary TRM was successfully developed. Fibre-based fluorescence lifetime imaging may provide a valuable tool for assessing pulmonary TRM populations in situ, reflecting the level of immunity following infection and vaccination.
An increasingly important role for salivary antibodies in public health surveillance
Authors: Augustine Swinburne A. J. , Eason Tarsha , Wade Tim , Griffin Shannon , Dufour Al
Published: February 2023

Rapid, sensitive, and specific salivary IgG antibody tests can reveal past infections that could assist in the determination of the true prevalence of microbial infections in population studies. Multiplex immunoassays combining IgM, IgA, and IgG antibody responses against multiple pathogens simultaneously, can provide a wealth of information to delineate acute from chronic infections. Previously, we developed and applied salivary antibody multiplex immunoassays against a range of environmental pathogens including noroviruses (GI.1 and GII.4), hepatitis A virus, Helicobacter pylori, Campylobacter jejuni and Toxoplasma gondii to examine infections associated with recreating in contaminated waters. Here, we employ the methods to study saliva samples collected from individuals recreating at an Iowa, USA, riverine beach. Results showed that nearly 80% of beachgoers had salivary antibodies to at least one of the targeted pathogens at the beginning of the study. Most of these exposures were to noroviruses (GI.1: 59.4%, GII.4: 58.8%) and T. gondii (22.8%). Of the individuals who returned the requested samples for each collection period, 6.1% immunoconverted to one or more pathogen, mostly to noroviruses (GI.1: 3.82%, GII.4: 2.3%) and T. gondii (1.5%). Outcomes of this effort illustrate that this rapid immunological assay serves as a viable, high-throughput, noninvasive and cost-effective tool for providing valuable information on the occurrence of known and emerging pathogens (e.g., SARS-CoV-2) in population surveillance studies.
Improved inter-instrument consistency and high precision for CHO cell bioprocessing using the Cellaca&[trade] MX high-throughput cell counter
Authors: Bell Jordan , Huang Yongyang , Hoover Willis , Kuksin Dmitry , Qiu Jean , Chan Leo
Published: February 2023

CHO cell bioprocessing is a common application for producing biologics, antibodies, and proteins for therapeutic products. An important aspect of the CHO bioprocess is the characterization of a cell culture’s concentration and viability to ensure that the cells are in optimal condition for production. Traditionally, CHO cells have been measured using a manual hemocytometer or automated cell counter with trypan blue staining. However, these methods have limitations in throughput and instrument-to-instrument consistency.

Here, we evaluate the performance of the Cellaca™ MX high-throughput cell counter for implementation into the CHO cell bioprocess. We investigate the precision, instrument-to-instrument consistency, linearity, and proportionality following ISO cell counting standard 20391-2:2019. We demonstrate close agreement between multiple Cellaca™ MX instruments using both CHO cells with Trypan Blue (5 instruments) and beads (32 instruments). We also report system-wide precision, which includes variation between multiple counts, consumables, instruments, and days (in the case of beads). Furthermore, we include the results of several comparison experiments in which samples were counted using Cellaca™ MX, hemocytometer, “Cell Counter V” (a competing instrument), as well as the Celigo® Imaging Cytometer. Finally, we demonstrate the use of the ISO cell counting standards to evaluate the linearity, precision, and proportionality index of the Cellaca™ MX. These results show that Cellaca™ MX can count trypan blue-stained CHO cells in brightfield in less than 1 min for 24 samples, and that the consistency, comparability, and precision of the Cellaca™ MX are significantly improved over the traditional methods.
Metabolite-Based Microparticles Enhance the Metabolic Fitness of T cells
Authors: Wu Christopher , Inamdar Sahil , Acharya Abhinav P. , Curtis Marion
Published: February 2023

Adoptive Cell Transfer (ACT) therapies have recently fostered excitement for the treatment of hematogenous cancers. However, efficacy has largely been stunted in the treatment of solid tumors, especially for ovarian cancer, in part due to nutrient restriction within the tumor microenvironment. We recently demonstrated that intracellular delivery of biodegradable microparticles (MPs) made from polymers of alpha-ketoglutarate modulated dendritic cell energy metabolism and immunophenotype, altering regulatory and helper T cell frequencies. To determine if continuous metabolite release from MPs would rescue T cell dysfunction in nutrient-poor, immunosuppressive conditions, we generated MPs of polymerized glycolytic and tricarboxylic acid cycle central-carbon metabolites. Flow cytometric analysis and confocal microscopy indicated that FITC-conjugated MPs were efficiently loaded into the cytoplasm of activated primary murine T cells via membrane adsorption or electroporation. The MPs were retained intracellularly and sustained proliferation of mouse T cells for over five days in vitro. Glycolysis and mitochondrial respiration stress tests using an Agilent Seahorse XFe96 Analyzer demonstrated that primary mouse CD8 T cells utilized the MPs as a metabolic substrate. In particular, MPs increased metabolic function, as indicated by increased spare respiratory capacity, glycolysis, and glycolytic capacity. The results of this study provide preliminary evidence in support of utilizing sustained-releasing particles of metabolic substrates for the metabolic reprogramming of immune cells used in ACT therapies. This strategy has potential to improve or expand the efficacy of existing ACT therapies for solid tumors.
Analysis of soluble immune checkpoint proteins using quantitative multiplex microbead-based immunoassays
Authors: Lie Wen-Rong , McDonald John , Russell Donna , Banerjee Munmun , Zimmermann Daniel , Kornmeier Christine , Pepin Danielle
Published: February 2023

Immune-checkpoint receptors play critical roles in immune regulations and maintenance of immune homeostasis. Recently, a series of soluble immune checkpoint proteins have been found in circulation, and are putative biomarkers in infection, cancer, and autoimmune diseases. We have developed two multiplex immuno-oncology (I/O) panels. Human I/O Checkpoint Protein Panel 1 (17-plex) consists of 17 targets: BTLA, CD27, CD28, CD40, CD80, CD86, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, TLR-2. Human I/O Checkpoint Protein Panel 2 (31-plex) consists of 31 targets: 4-1BBL, CD73, APRIL, Arginase-1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, BAFF, CD25, CD30, CD40L, 4-1BB, CD226, E-Cadherin, FGL1, Gal-1, Gal-3, Granulysin, Granzyme B, IDO1, MICA, MICB, Nectin-2, Nectin-4, OX40, Perforin, CD155, Siglec-7, and Siglec-9. Using these multiplex arrays, we have simultaneously quantitated the expression levels of the above 48 key immune regulators in cancer serum samples and in cell culture supernatants. Soluble checkpoint protein signatures generated from this multiplex approach revealed differential expression of sBTLA, sCD27, sCD40, sCD86, sHVEM, sPD-1, sTIM-3, sTLR-2, sCD40L, Gal-3, Gal-1, FGL1, BAFF in the colorectal or breast cancer serum samples, compared to healthy serum controls. Heatmaps demonstrate the detection of checkpoint proteins in the conditioned media collected from human tumor cell lines and stimulated PBMCs. In summary, our results demonstrate that these two multiplex immunoassays we developed are useful research tools for the simultaneous quantitation of circulating immune checkpoint proteins, as well as for its potential application in biomarker discovery and translational research.
Identification and function of a CD4+/CD8αβ+ T cell population that is predictive of GVHD development in a xenogeneic transplant model
Authors: Hess Nicholas J , Hematti Peiman , Gumperz Jenny E , Capitini Christian M.
Published: February 2023

Graft-vs-host disease (GVHD) is mediated by donor reactive T cells that have a hierarchical classification based on CD4 and CD8 expression. While CD4 and CD8 lineages are thought to have fixed expression, CD4+/CD8αβ+ double positive (DP) T cells have been reported in cases of human cancers and autoimmune diseases though the lack of a suitable model system has hindered their research. In this study, we transplanted primary human graft tissue into non-conditioned immunodeficient mice and observed the development of a human DP T cell population that was not present in the starting grafts. This DP T cell population developed irrespective of graft tissue (peripheral blood, bone marrow or umbilical cord blood), accessory cells (transplantation with isolated T cells) and immunodeficient mouse strain (NSG and NBSGW). Furthermore, an increase in the percentage of DP T cells in the blood of these mice is correlated and predictive of GVHD development. We also observed that DP T cells are functionally active with significantly elevated IFNγ and TNFα secretion compared to CD4 and CD8 single positive T cells. DP T cell also display elements of the cytotoxic machinery including NKG2D and perforin/granzyme expression. Interestingly, transplantation of isolated CD4+ cells did not result in the development of DP T cells while a robust population developed after transplantation of isolated CD8+ T cells. DP T cells were also identified in primary clinical samples taken from HSCT patients with their clinical relevance to GVHD currently under investigation. In conclusion, this ongoing study has identified a novel human DP T cell that arises from the CD8+ T cell population, is functional active and is predictive of GVHD in a xenogeneic transplant model.