bluuebunny/crossref_metadata_2025 huggingface dataset:
Aryl hydrocarbon receptor regulation of T follicular helper cells during respiratory viral infection
Authors: Houser Cassandra L
Published: February 2023

The aryl hydrocarbon receptor (AHR) is a ligand-regulated transcription factor that binds structurally diverse molecules from the environment, including pollutants, dietary factors, and chemicals from microorganisms. AHR-binding compounds alter adaptive immune responses, but the cellular components affected and governing mechanisms are not fully defined. For instance, AHR activation affects antibody responses; a process for which T follicular helper cells (Tfh cells) are critical. We recently discovered that AHR activation alters the percentage and number of Tfh cells during influenza A virus (IAV) infection. Yet, how the AHR modulates Tfh cells is not known. The present work shows that AHR-mediated changes to Tfh cells are due to changes in CD4+ T cell proliferation and BCL6 expression, an essential transcription factor for Tfh cell differentiation. Utilizing conditional AHR knockout mice, we show that changes to Tfh cells require AHR expression in CD4+ T cells. The AHR regulates gene expression directly, via its DNA-binding domain (DBD), and indirectly via other signaling molecules. To delineate which pathways it uses to modulate Tfh cells, we used mice with a mutated AHR DBD and demonstrate that it requires its cognate DBD to regulate Tfh cells during infection. This supports that AHR modulates Tfh cells by directly regulating gene expression. Overall, these findings provide new information about how AHR signaling influences humoral immunity to a common respiratory pathogen. Given that exposure to AHR-binding pollutants impacts immune responses to infections and vaccines, better understanding of the mechanisms that control Tfh cell responses has broad reaching impact on immune defenses and immune-mediated diseases.
CD40L acts as an allosteric activator of integrins for signal transduction independent of inside-out signaling
Authors: Shimoda Michiko , Takada Yoko , Maverakis Emanual , Felding Brunhilde , Takada Yoshikazu
Published: February 2023

CD40L plays a major role in immune response and is a target for inflammatory disease therapy. Besides CD40, CD40L binds to several integrins but their role in signaling is unclear. We showed that integrins αvβ3 and α5β1 bind to the CD40L trimeric interface through classical ligand binding site of integrins (site 1). Several CD40L mutants from HIGM1 (hyper-IgM syndrome type 1) patients were clustered in trimeric interface and defective in integrin binding, and in NF-κB and B cell activation, but still bound CD40 and acted as antagonists of CD40L signaling. Thus, integrins play a critical role in CD40L signaling. Previous studies showed that CX3CL1 and sPLA2-IIA can activate integrins by binding to a recently identified allosteric site (site 2) of integrins. Using cell-free assays, we found that soluble (s)-CD40L activated soluble integrins, α4β1, αvβ3 and α5β1, and induced their binding to known specific ligands independent of inside-out signaling. Also, sCD40L activated cell-surface integrins in CHO cells lacking CD40. Finally, sCD40L bound to a cyclic peptide derived from site 2. These findings indicate that CD40L acts as an allosteric activator of integrins by binding to site 2. Docking simulation predicted that the integrin site 2-binding site of CD40L is located outside of CD40L trimer. Importantly, four HIGM1 mutations are clustered in the predicted site 2-binding site of CD40L. The K143T and G144E mutants were the most defective in integrin activation, indicating that integrin binding occurs around these residues of the predicted site 2-binding site. We propose that allosteric integrin activation by CD40L plays a role in CD40L signaling and that defective integrin site 2-binding may be causal for defective CD40L signaling in HIGM1.
Lin28b promotes innate functions in CD8+ T cells through rapid chromatin remodeling
Authors: Watson Neva B , Patel Ravi K , Oyesola Oyebola O , Laniewski Nathan , Smith Norah L , Mon Kristel Yee , Tabilas Cybelle , Daly Ciaran , West Jessica D , Luyen Vu Tien , Maymi Viviana I , Nzingha Kito , Wesnak Samantha P , Scheible Kristin M , Wojno Elia D Tait , Grimson Andrew W , Rudd Brian D
Published: February 2023

Lin28b serves as a master regulator of fetal lymphopoiesis and promotes the development of more innate-like lymphocytes in early life. However, it is still unclear how Lin28b alters the function of CD8+ T cells and protects the host against infection. In this report we examined how Lin28b transcriptionally and epigenetically programs neonatal CD8+ T cells for innate immune defense. We found that murine neonatal CD8+ T cells possess innate-like transcriptomes, are more responsive to innate cytokines in the absence of antigen and produce a broader spectrum of cytokines compared to their adult counterparts. This unique program of bystander activation in early life corresponded with enhanced innate-like protection against irrelevant strains of bacterial, helminth and virus infections. To understand how this diversity of immune function is mediated, we used flow cytometry and scRNAseq and discovered that the neonatal CD8+ T cell pool is composed of many subsets of cells expressing distinct cytokines. ATACseq analysis revealed that neonatal CD8+ T cells are programmed to display innate-like properties and produce a diverse array of cytokines, which may be due to their ability to undergo extensive chromatin remodeling upon cytokine stimulation. These unique properties may be driven by high Lin28b expression in fetal progenitor cells, as adult cells expressing Lin28b initiated a program of bystander activation and immune protection that was analogous to neonates. Collectively, our data suggests that Lin28b enables neonatal CD8+ T cells to undergo a more diverse program of bystander activation, allowing them to deploy a bet-hedging immune strategy and protect the host against a rapidly changing pathogenic environment.
Modular approach to generating long-lived effector/memory (Teff/Tem) T cells to prolong malaria immunity
Authors: Gbedande Komi , Ibitokou Samad , Brown Michael G. , Degli-Esposti Mariapia A , Stephens Robin
Published: February 2023

Immunity to Plasmodium infection or vaccination is known to decay. In mouse models, both B and T cells are required for immunity; however, decay of protection correlates with loss of malaria-specific T cells, not antibody. Whole parasite vaccines, such as the infection and drug cure protocol, protect. However, sterile protection from P. chabaudi or vaccination last less than 200 days. Therefore, we have reverse-engineered a vaccine strategy that first induces a long-lived protective antibody response using live parasite vaccination, and then boosts Teff and Tem response to prolong protection. Cytomegalovirus (CMV), is a known chronic vaccine vector that induces T cell mediated immunity and shows promise against SIV, tuberculosis and liver-stage malaria. Therefore, we used chronic vaccination with MCMV to test our hypothesis that promoting longer generation of Teff in mice would prolong protection by a live vaccine. MCMV encoding P. chabaudi MSP-1 epitope B5 (MCMV-B5) was used to follow the malaria- specific T cell response. Upon infection of mice with MCMV-B5, both adoptively transferred B5 TCR Tg T cells and MCMV-specific CD4 T cells proliferated and were maintained over 2.5 months. Coinciding with this protection, the MCMV vector worked to prolong the protection generated by live malaria vaccine. In investigating the mechanisms of protection, we found that IFN-γ induced by MCMV prolongs the protection, potentially through promotion of IL-12 producing and antigen presenting cell function. Our findings suggest the methods and the mechanisms to effectively prolong immunity to Plasmodium infection based on CMV mechanism to redress the problem of short-lived protection to malaria infection and vaccines.
TRAF3 in T cells restrains negative regulators of LAT to promote TCR/CD28 signaling
Authors: Arkee Tina , Hostager Bruce , Houtman Jon , Bishop Gail A
Published: February 2023

The adaptor protein TRAF3 plays an important role in T cell biology. T cell conditional TRAF3−/− mice, despite normal T cell numbers, have poor responses to immunization and infection, and T cells from these mice have defective TCR/CD28 signaling. TRAF3 associates with, and enhances signaling by, the TCR/CD28 complex upon engagement of both TCR and CD28. The molecular mechanism by which TRAF3 interacts with and impacts TCR/CD28-mediated complexes and function remains an important knowledge gap. We investigated how TRAF3 is recruited to, and regulates, the TCR/CD28 complex. Our work revealed that TRAF3 recruitment did not require direct binding of TRAF3 to CD28; rather, TRAF3 associates with the CD28-binding protein Linker of activated T cells (LAT). The TRAF3-LAT association required the TRAF3 TRAF-C domain and a newly identified TRAF2/3 binding motif in LAT. TRAF3 enhanced TCR/CD28 signaling via inhibition of the LAT-associated negative regulatory protein Dok1, which is phosphorylated and inactivated by the tyrosine kinase Breast tumor kinase (Brk/PTK6). In TRAF3−/− T cells, there was increased plasma membrane localization of Protein tyrosine phosphatase 1B (PTP1B), which dephosphorylates Brk at an activating tyrosine residue, resulting in altered Brk activation. Consistent with these findings, inhibiting Brk activity in WT T cells inhibited basal and TCR/CD28-mediated Src family kinase activation, while inhibiting PTP1B activity in TRAF3−/− T cells rescued such activation. These results reveal a new mechanism for the enhancement of TCR/CD28-mediated signaling by TRAF3. We are currently investigating the role of TRAF3 in T cell metabolic reprogramming, which requires effective TCR/CD28 signaling.
LSECtin (CLEC4G) inhibits T-cell activation and proliferation in the presence of TCR and CD28 signaling
Authors: Wang Jinghua , Manick Brian , Renelt Mark , Gerassenkov Tatsiana , Bi Ming , Kalabokis Vassilios , Person Anthony , Wu Guoping
Published: February 2023

The liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin), also known as CLEC4G, belongs to the calcium-dependent lectin family, acts as a co-inhibitor of human T cell immunity, and mediates tumor immune escape in melanoma. However, the influence of LSECtin on T cell activation remains controversial. In this study, we demonstrate that the LSECtin protein inhibits human T cell activation and proliferation through the butyrophilin family receptor, BTN3A1. LSECtin markedly suppressed human T cell proliferation in the presence of T cell receptor signaling. Furthermore, LSECtin significantly reduced IFN-γ, IL-2, IL-17 and TNF-a cytokine and IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10, and TARC/CCL17 chemokine production in activated human T cells. Interestingly, a functional ELISA binding assay revealed that LSECtin bound to BTN3A1, LAG-3 and CD44, but very weakly bound to BTN3A3. Anti-BTN3A1 neutralizing antibody attenuated the binding of LSECtin to BTN3A1. BTN3A1 is expressed on both resting and activated human T cells, and pretreatment with an anti-BTN3A1 antibody partially attenuated the ability of LSECtin to inhibit IL-2 secretion from human T cells, suggesting that BTN3A1 acts as a receptor for LSECtin to deliver a negative signal to T cells. Thus, we show that LSECtin/BTN3A1 represents a novel negative regulatory pathway on T cells and may serve as a unique immune checkpoint for developing new immunotherapy strategies for the treatment of immune-related diseases, including cancers.
Single Cell Accessibility Profiling of Murine DN3 Thymocytes
Authors: Collins Patrick , Lee Bella , Stevens Patrick , Nganga Vincent K , Rincon Mercedes , Bassing Craig , Oltz Eugene M
Published: February 2023

Heterogeneity is critical for immune system health, else our defenses are easily evaded by pathogens. Likewise, epigenetic diversity likely governs lineage decisions during development. Some enhancers appear to open prior to the transcription of proximal genes, which we hypothesized would create gradients of epigenetic activity in developmentally intermediate, but otherwise uniform, cells. To evaluate regulatory diversity in a fixed, well defined population, we performed single cell accessibility measurements on Rag-deficient double negative 3 (DN3) thymocytes. Computationally defined models predicted epigenetic stratification within this homogenous population. Sequence-based imputations further separated the DN3 cells via enrichment for lineage-defining transcription factor motifs, suggesting the epigenetic spectra reflects early versus late DN3 stages. As antigen receptor (AgR) loci recombine within DN3 cells, we also examined if single cell accessibility of AgR promoters and recombination signal sequences could predict pre-selection T Cell Receptor Beta (TCRb) repertoire. Compared to algorithms that use bulk measurements of chromatin composition or DNA sequence, single cell accessibility had slightly improved accuracy in predicting Vb segment choice. Collectively, our results illustrate the application, and perhaps complexities, of interpreting single cell accessibility data, and identify possible regulatory dynamics within an otherwise homogenous DN3 population.
Proteomic analysis of microglia during LPS-induced acute neuroinflammation.
Authors: Cartier Anna , Moyron-Quiroz Juan , Huang Tse Shun , Tam Miguel
Published: February 2023

Dysregulation of microglia function is associated with neuroinflammation. A major limitation in understanding microglial contribution to cellular processes and their role in disease has been the lack of tools to specifically distinguish these cells from other myeloid cells. We generated and validated a rat monoclonal antibody against a selective microglia marker P2RY12. We utilized LPS administration as a model for neuroinflammation to assess alterations in P2RY12 expression, a homeostatic protein. Mice were injected with either PBS, low or high dosages of LPS daily for 4 days after which microglia were harvested from the brain. Isolated cells were co-stained with CD11b, CX3CR1, and P2RY12 as general markers for microglia. Flow cytometric quantification using live cells showed a significant increase in CD11b+ expression in both low and high dose LPS treated mice compared to the control untreated group. Low and high dose LPS injections led to a significant reduction in P2RY12 and CX3CR1 expression in these cells. These data are consistent with a decrease in P2RY12 expression under inflammatory conditions. We assessed microglial proteomic profile using BioLegend’s proprietary technology, TotalSeq™, which allows simultaneous analysis of both the transcriptome and proteome at the single-cell level. We utilized a panel of 200 antibody-oligonucleotide conjugates, including P2RY12, to analyze differentially expressed proteins under normal and LPS-induced inflammation in mice. These studies help elucidate the diversity and physiology of microglia, and potentially aid in the discovery of novel markers in response to neuroinflammation.
Human-type artificial lymphoid tissues (organoids) induce antigen-specific immune responses upon antigen-stimulation
Authors: Watanabe Takeshi
Published: February 2023

We had previously reported that the immunologically active artificial lymphoid tissues (aLTs) could be stably generated in mice by implantation of lymphoid stromal cell-embedded biocompatible scaffold (collagen sponge) first in 2004 (Suematsu and Watanabe) as well as later by applying the chemokine/lymphokine-trapped gel-embedded scaffold in 2016 (Kobayashi and Watanabe). The generation of the functional human-type aLTs might be beneficial in future for clinical application as the immunotherapy against cancers as well as the normalization of diseases caused by malfunction of human immune system. It may be applied for the evaluation of vaccines. We first established the human stromal cell lines suitable for the formation of the human aLTs. Then, spheroids composed of the human stromal cells were prepared. The mixtures of stromal cell spheroids and human peripheral blood lymphocytes (PBMCs) were transplanted into renal subcapsular spaces of the immunodeficient NOG-dKO mice. After 2–3 weeks, human type aLTs (hPBMC-aLTs) were stably formed. The human type aLTs are composed of the clusters of human B and T cells including scattered human DC cells. The human type aLTs were immunized with the inactivated varicella zoster virus (VZV) or with NP-conjugated ovalbumin (NP-OVA) in adjuvant. Anti-VZV or anti-NP-hapten specific antibody-producing cells were strongly induced in the human-aLTs. IFNg was produced from human T cells in aLTs upon the immunization. These data indicate that the immunologically functional human aLTs could be successfully generated. We are currently investigating anti-cancer activity of the human aLTs as well as attempting to construct them in vitro system.
Immunophenotypic and functional characterization of circulating cytotoxic T lymphocytes and natural killer cells using the BD FACSymphony™ A1 Cell Analyzer
Authors: Baracho Gisele Vanessa , Kara Nihan , Rigaud Stephanie , Widmann Stephanie , Tyznik Aaron
Published: February 2023

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells have become attractive therapeutic targets for cancer immunotherapy because of their efficient mechanisms for killing cancerous cells. Both effector CTLs and mature NK cells are endowed with secretory granules containing pre-formed cytolytic proteins that, once released at the immunological synapse, initiate a cascade of reactions leading to the apoptosis of the target cell. CTLs and NK cells may also promote cell killing through death-receptor-mediated and cytokine-induced cell death mechanisms. Here we show a 16-color flow cytometry panel allowing clear identification of CTLs, NK cells and related cell subsets in whole blood. We also describe an optimized intracellular staining protocol for analysis of granules content, including granzyme B, granzyme K and perforin. Optimal resolution of cell populations was achieved by employing a selection of BD Horizon Brilliant™ Reagents and BD Horizon™ Red 718 Reagent, a new small molecule fluorochrome option for the red laser. Cell fixation and permeabilization were carried out directly in whole blood using the BD Phosflow™ Reagents, adding convenience to the workflow and practical analysis of cell frequencies. The data also showed the expression of selected activation and inhibitory surface receptors. These results combined with the analysis of intracellular proteins contribute to characterization of the functional status of cytotoxic cells.

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Proteomic stabilization of Cynomolgus Macaque blood with PROT-1 for flow cytometric analysis of activated innate and adaptive immune cells after extended storage
Authors: Williams Jamie , Genell Caroline A , Nuti Sandra , Delamere Patricia A , Maier Curtis C , Marshall Nikki B
Published: February 2023

Flow cytometry is a widely used, and rapidly evolving, immunophenotyping application; however, limited advancements around sample stability and preservation continue to strain scientists in resource-limited settings. As flow cytometric samples are labile, data acquisition is generally performed within 24 hours, with traditional fixation methods, to minimize impact to the quality of the analysis. Here, we describe the development of an optimized proteomic stabilization method and 13-color immunophenotyping panel that allows for long-term storage of Cynomolgus Macaque whole blood and retrospective phenotyping of resting and activated populations of innate and adaptive immune cells including T cells, NK cells, B cells, monocytes and dendritic cells. Taken together, these methods highlight a key advancement around flow cytometry sample stability and preservation, allowing for greater resource flexibility without compromising quality of the analysis.
Sertoli cell induction of Tregs: a critical component of Sertoli cell immune-regulation in transplantation
Authors: Hibler Taylor , Kaur Gurvinder , Dufour Jannette
Published: February 2023

Sertoli cells (SC) are immunoregulatory testicular cells that protect and nurture germ cells, which develop after central tolerance is established and are classified as non-self by the immune system. This ability to protect germ cells makes SC a promising alternative to immunosuppressive therapy in transplantation; indeed, they have been co-transplanted with various tissues in animal models with significant success. It has been suggested that regulatory T cells (Tregs) play a role in SC graft survival and protection of germ cells. The goal of this study was to determine if SC promote graft survival through induction of Tregs. To test this, B6 SC were transplanted into BALB/c mice in absence of immunosuppressive drugs. 100% of SC grafts survived to day 20, while 100% of controls (nonimmune privileged cells) rejected by day 20. Analysis of the grafts through immunohistochemistry and flow cytometry revealed that SC grafts had significantly increased Tregs compared to rejecting control grafts. To determine if Tregs were critical for SC survival, B6 SC were transplanted into BALB/c mice depleted of Tregs. 100% of SC grafts without Tregs rejected within 20 days. Excitingly, 57% of SC grafts in Treg depleted mice survived. Flow cytometry revealed that all of the surviving grafts had Tregs, indicating that SC can induce Tregs from naïve T cells and that these Tregs are critical for SC graft survival. The presence of Tregs in surviving pig SC grafts was also shown in a xenogeneic model. Interestingly, the xenograft model showed a significant decrease in T effector cells, indicating that SC are both inducing Tregs and inhibiting effector cells. In summary, SC can survive as both allo- and xenografts without immunosuppression through the activation of Tregs.
Immunosuppression by cyclosporin A via inhibition of TCR-dependent but NFAT-independent functions
Authors: Otsuka Shizuka , Melis Nicolas , Gaida Matthias M , Dutta Debjani , Weigert Roberto , Ashwell Jonathan D
Published: February 2023

Inhibitors of calcineurin phosphatase activity (CNIs) such as cyclosporin A (CsA) are widely used to treat tissue transplant rejection and graft-versus-host disease (GVHD), for which CNI inhibition of NFAT-dependent gene expression is thought to be the major mechanism. We recently reported that CNIs also inhibit TCR-proximal signaling by preventing calcineurin-mediated dephosphorylation of LckS59, an inhibitory modification. Here we have utilized mice expressing LckS59A, which cannot accept a phosphate at residue 59, to explore the biological significance of CsA-mediated inhibition of TCR signaling. We find that whereas CsA inhibited NFAT activation normally, TCR-signaling activation in T cells expressing LckS59A was resistant to CsA. Importantly, although upregulation of NFAT-dependent effector cytokines such as IL-2 and IFN-g was inhibited, CsA was largely ineffective in suppressing acute GVHD when the allo-aggressive T cells expressed LckS59A. Two important T cell effector pathways were found to be resistant to CsA in LckS59A T cells from acute GVHD mice: upregulation of the cytolytic protein perforin in CD8+ T cells and induction of antigen specific LFA-1/ICAM-mediated T cell:DC (dendritic cell) adhesion as detected by 2-photon imaging. These results demonstrate that therapeutic effective treatment of aGVHD by CsA requires inhibition of TCR-associated calcineurin and proximal signaling.
Blockade of the immune checkpoint B7-H3 alters glucose metabolism in non-small lung cancer cells
Authors: Zhao Qi , Yang Shuo
Published: February 2023

B7-H3 an immune-checkpoint molecule, a transmembrane protein, is overexpressed in non-small cell lung cancer (NSCLC), making it an attractive therapeutic target. An anti-B7-H3 antibody omburtamab has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. Elicitation of cellular mechanism behind the anti-B7-H3 blockade is important to reveal the immune-independent functions of B7-H3. Warburg effect is one feature of metabolic dysregulation during malignant transformation. To investigate the effect of anti-B7-H3 blockade in the regulation of glucose metabolism in cancer cells, we measured dynamic ECAR and OCR as indicatives of glycolysis and oxidative respiration in NSCLC cells. We demonstrated that anti-B7-H3 antibody blockade switched cell metabolism from glycolysis to oxidative phosphorylation. The alteration of glucose metabolism probably was associated with cellular ROS-mediated pathway. These results clearly showed that, in addition to immune-regulatory function, B7-H3 possessed immune-independent functions in NSCLC. The study is supported by the Science and Technology Development Fund, Macau SAR (FDCT/0015/2918/A1).
Inhibigens™ drive pro-tumor responses and may act through non-classical inhibitory mechanisms
Authors: DeVault Victoria L , Starobinets Hanna , Arnold Julie , Rinaldi Stephanie , Odeh Osaruese , Nguyen Cindy , Flechtner Jessica B , Lam Hubert
Published: February 2023

The ATLAS™ platform screens a patient’s tumor mutanome with autologous T cells and antigen presenting cells to identify targets for cancer immunotherapy. Tumor mutations that lead to increased T cell responses are deemed stimulatory (neoantigens), while those rendering decreased responses are defined as Inhibigens. In B16F10 mouse melanoma, therapeutic neoantigen vaccination resulted in durable protection, while Inhibigen co-administration resulted in a remarkable reversal of protection and abrogation of anti-tumor responses. The Inhibigen-related pro-tumor effects were not reduced by αPD-1 therapy, and administration of αCTLA-4 resulted in modest benefit, suggesting that Inhibigen-specific responses can overpower current standard-of-care immunotherapies. The tumor microenvironment of Inhibigen-vaccinated mice was immune cold, with dramatic decreases in infiltrating T cells and myeloid cells relative to controls. Inhibigen effects were not associated with classical inhibitory mechanisms such as an overabundance of CD4+CD25+Foxp3+Tregs, MHC competition or downregulation. In addition, T cell proliferation appeared unaffected by Inhibigen administration. Time course experiments revealed that Inhibigen-specific phenotypes (e.g. defective T cell IFNγ production) occur as early as 4 days post vaccination, suggesting deficiencies related to T cell priming. RNAseq analyses showed distinct changes in T cell transcriptional patterns between neoantigen and Inhibigen-experienced T cells. Taken together, these data reveal a potential non-classical inhibitory mechanism by which responses to naturally occurring cancer mutations may promote tumor growth and reverse beneficial anti-tumor immune responses.
Novel immunotherapeutic Albumin-Flt3L enhances antigen-specific immunity and tumor control following chemoradiation: An analysis of underlying mechanism
Authors: Kung Yu Jui , Lam Brandon , Lin John , Wu T.C. , Hung Chien-Fu
Published: February 2023

CD8+ cells are essential to anti-cancer immunity following chemoradiation and immunotherapy. The induction of tumor specific T cells depends on the presentation of tumor associated antigens/neoantigens. This generates a bottleneck to effective antitumor immunity, where only antigens efficiently presented on MHCI can serve as CD8 T cell targets. To bypass this, we have developed a novel immunotherapeutic by fusing albumin to Flt3L (Alb-Flt3L), a cytokine known to enhance cross-presenting cDC1s, and described its function. Previously, we observed that MC38-tumor bearing mice treated with Alb-Flt3L + radiotherapy engenders neoantigen-specific T cells by tetramer staining. Now we have expanded this analysis to query the breadth and depth of CD8+ TILs by TCRb sequencing. Sorted and sequenced ADPGKneo-specific CD8 T cells encompassed a multitude of unique rearrangements, suggesting polyclonal neoantigen coverage, and bulk CD8+ cells showed a greater Diversity Index in Alb-Flt3L + radiotherapy treated mice compared to controls. We also sought to investigate the mechanism of tumor control following treatment with Alb-Flt3L + chemoradiation. The role of Batf3, a cDC1 transcription factor, type-I-interferons, cytokines essential to immune activation following chemoradiation, and pDCs, an Alb-Flt3L-tropic cell type also capable of cross-presentation, were investigated using knockout mice or antibody depletion in tumor bearing mice treated with cisplatin. This study confirmed the role of these factors in Alb-Flt3L-mediated responses, as worse tumor control was observed in the absence of Batf3, pDCs, or type-I-interferon signaling. Altogether, we were able to characterize mechanisms mediating Alb-Flt3L’s antitumor effects.
Determining Adenosine Deaminase 1 (ADA-1) Impact on Immune Memory and Durability as a Molecular Adjuvant in a SARS-CoV-2 DNA Vaccine Formulation.
Authors: Cusimano Gina , Gary Ebony N. , Bell Matt , Connors Jennifer R , Tursi Nicholas J. , Zhang Shiyu , Canziani Gabriela , Warner Bryce , Ali Ali R. , Taramangalam Bhavani , Chaiken Irwin C. , Wootton Sarah , Weiner David , Kobasa Darwyn , Kutzler Michele A. , Haddad Elias K.
Published: February 2023

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease of 2019 (COVID-19), has infected millions of people causing a global pandemic. SARS-CoV-2 vaccines candidates have demonstrated acute immunogenicity and protection however, it has yet to be demonstrated whether natural or vaccine induced immunity against SARS-CoV-2 induces long term, protective immunity. In this study we sought to understand if adenosine deaminase (ADA), as a molecular adjuvant, can enhance immune memory and durability in the context of a SARS-CoV-2 DNA vaccine. Mice were immunized with a plasmid encoding for SARS-CoV-2 spike alone or in combination with plasmid encoded ADA. Subsequent B and T cell responses were measured until d60pi. In mice co-immunized with ADA, there were increased concentrations of spike receptor binding domain (RBD)-specific IgG in the sera which were found to bind RBD at an increased affinity as well as exhibit increased neutralization capability against SARS-CoV-2 pseudotyped viruses. Additionally, ADA co-immunized mice exhibited increased frequency of RBD specific memory B cells. In regard to T cell responses, mice co-immunized with ADA exhibited increased spike-specific IFN-γ, TNF-a and IL-2 as measured by flow cytometry and ELISpot. The ADA-enhanced anti-spike antibody durability over time was associated with increased frequencies of T follicular helper cells (TFH). Preliminary analysis supports that co-immunization with pADA impacts viral load in a SARS-CoV-2 infection model. These data suggest that ADA enhances immune memory and durability and supports further study with translational focus for enhancement of vaccines.
BCG-derived peptides induce T cell cross-reactivity to Sars-CoV-2 homologues
Authors: Eggenhuizen P J , Ng Boaz H , Chang Janet , Fell Ashleigh L , Ooi Joshua D
Published: February 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently-discovered beta coronavirus capable of infecting humans and causing Coronavirus Disease 2019 (COVID-19). COVID-19 causes a variety of pathologies that are still being understood. While vaccines specific for COVID-19 are being developed and deployed throughout the world, another vaccine called Bacillus Calmette–Guérin (BCG) vaccine is being trialled to test whether it has a protective effect against COVID-19. Epidemiological studies have identified a negative correlation between BCG vaccination regime and COVID-19 severity and mortality. However, the mechanism by which a vaccine for a bacterium can protect against a virus is not well understood. Here we have identified a number of homologous peptide sequences shared between BCG and SARS-CoV-2. Using in vitro human T cell recall assays, we found that pre-sensitisation to BCG-derived peptides can induce T cell cross-reactivity to SARS-CoV-2 peptides. These findings assist in explaining the molecular mechanism behind the protective effect conferred by BCG vaccination in COVID-19, and supports the clinical trials exploring BCG vaccine as an integral part of the global effort to vaccinate against COVID-19.
Effect of SARS-CoV-2 vaccine on the breastmilk antibody response among lactating healthcare workers
Authors: Stafford Lauren Stewart , Luaces Vivian Valcarce , Neu Joseph , Cacho Nicole , Parker Leslie , Burchfield David , Li Nan , Larkin Joseph
Published: February 2023

In 2019, a deadly virus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, emerged. Two mRNA-based COVID-19 vaccines were approved for use in December 2020 in the US and have begun providing immunity to those receiving the vaccination. Certain vaccines given to pregnant and lactating mothers provide immunity to infants through transmission across the placenta and umbilical cord (IgG) and breast milk (IgA). Breastmilk produced by mothers with a history of COVID-19 infection has found to be a source of anti-SARS-CoV-2 IgA and IgG. This study aimed to determine the presence of specific SARS-CoV-2 IgA in the breastmilk of lactating women after the COVID-19 vaccine administration. As such, lactating healthcare workers who received the SARS-CoV-2 mRNA vaccine (Pfizer-BioNtech or Moderna) made up a sample group. Plasma and breast milk were collected at three time points (pre-vaccination, 14–28 days post first dose of vaccine, and 7 days post second dose of vaccine). SARS-CoV-2 specific IgA (breastmilk) and IgG (plasma) concentration were then measured by ELISA. We found consistent secretion of SARS-CoV-2 specific IgA in breast milk after COVID-19 vaccination. The second dose of the SARS-CoV-2 vaccine was necessary to elicit IgA response in breastmilk detectable by ELISA. There is a correlation of the level of SARS-CoV-2 specific IgG in blood with SARS-CoV-2 specific IgA in breastmilk. Our results show that the mRNA-based COVID-19 vaccines from Pfizer-BioNTech and Moderna induce SARS-CoV-2 specific IgA secretion in breastmilk. Further studies are needed to determine the duration of this immune response and a possible passive immunity transfer of SARS-CoV-2 specific IgA to breastfeeding infants.
T cells in relapsing-remitting multiple sclerosis demonstrate diminished expression of LAG-3
Authors: Jones Britta E , Maerz Megan D , Somasundaram Ashwin , Buckner Jane H
Published: February 2023

The co-inhibitory receptor lymphocyte activation gene 3 (LAG-3) is an immune checkpoint molecule that negatively regulates T cell activation, proliferation, and homeostasis. Blockade or deletion of LAG-3 in autoimmune-prone backgrounds or induced-disease models has been shown to exacerbate disease. We observed decreased surface expression of LAG-3 on T cells in subjects with rheumatoid arthritis (n=35), type 1 diabetes (n=105), or relapsing-remitting multiple sclerosis (RRMS; n=121) as compared to age and sex-matched healthy subjects (n=123). Interestingly, decreased LAG-3 expression was observed across CD4 and CD8 T cell subsets. T cell receptor stimulation on a subset of our initial cohort revealed decreased surface and intracellular LAG-3 as well as decreased LAG3 transcript in subjects with RRMS. Decreased LAG-3 in subjects with RRMS was associated with diminished IFNγ production and intracellular cleaved caspase 3; the addition of a LAG-3 blocking antibody further underscored this relationship. Despite the decreased level of LAG-3, CD8 T cells from RRMS subjects were less proliferative than healthy subjects after a low-dose viral peptide stimulation as well as after blocking LAG-3; another indication of defective T cell activation associated with LAG-3 expression. In T cells from RRMS subjects, we observe a global dysregulation of LAG-3 expression that stems from decreased transcription and persists after T cell stimulation. These findings further support the potential clinical benefits of a LAG-3 agonist in the treatment of human autoimmunity.
Two lineages of memory B cell development regulated by type I interferon and IL-4R are perturbed in systemic lupus erythematosus
Authors: Gao Min , Liu Shanrun , Lu Changming , Chen Jake Y , Chatham W Winn , Hsu Hui-Chen , Mountz John D
Published: February 2023

Abnormal expansion of type I interferon (IFN) stimulated B cells and T-bet+CD11c+IgD−CD27− DN2 atypical memory B cells are both hallmarks of SLE. To determine how they are interrelated, we have carried out single cell transcriptomics analysis using B cells derived from 3 SLE patients (anti-DNA+ and anti-Smith+) and 3 healthy controls (HCs)(all African Americans). We have identified that elevation of IL4R in pre-switched IGHM+/IGHD+ B cells is the opposing signature of type I IFN stimulated genes (ISGs) and DN2 B cells (TBX21, ITGAX, FCRL3, FCRL5, and ZEB2). In HCs, IGHM+/IGHD+ B cells expressed elevated IL4R. Further, activated naïve B cells (IGHM+IGHD+IGHG+) exhibited upregulation of the germinal center-oriented classical memory B-cell signature genes, CD27, TNFRSF13B (the gene encoding TACI), and GPR183 (the gene encoding EBI2). Flow cytometry analysis confirmed that down-regulation IL-4R and upregulation of intracellular IFN-β in naïve IgD+CD27− B cells correlated with the accumulation of atypical CD21−IgD−CD27−B cells and a reduction in the percentage of classical IgD−CD27+memoryB cells in SLE patients (n=47). In vitro exposure of SLE B cells to IL-4 prior to treatment with anti-Ig+IL-21+IFN-gamma+TLR7+BAFF stimulation significantly enhanced B-cell development into CD27+CD38+ plasmablasts/plasma cells as well as IgD−CD27+memoryB cells but it significantly blocked the development of DN2 B cells. Our results show that upregulation of type I IFN and down-regulation of IL-4R signaling in naïve B cells lead to the accumulation of atypical memory B cells in SLE. Type I IFN and IL-4R signaling in naïve B cells induce the development of distinct lineages of atypical versus classical memory B cells, respectively.
Human monoclonal autoantibodies in autoimmune basal ganglia encephalitis target dopamine receptors and act as drug agonists
Authors: Menendez Chandra M , Zuccolo Jonathan , Reim Sean , Swedo Susan , Kovoor Abraham , Cunningham Madeleine W
Published: February 2023

Neuropsychiatric diseases in autoimmune basal ganglia encephalitis (BGE) are associated with anti-neuronal autoantibodies which target the basal ganglia. BGE includes the group A streptococcal sequelae Sydenham chorea (SC) and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). In SC, autoantibodies against dopamine receptors 1 and 2 (D1R and D2R) are elevated, and IgG in SC sera and cerebrospinal fluid (CSF) induce excessive dopaminergic D2R signaling and dopamine release. D1R autoantibody reactivity targeting the basal ganglia is not defined. Here we demonstrate specific IgG targeting of D1R in multiple neuropsychiatric PANDAS cohorts by serum, CSF, and human-derived mAbs. Anti-D1R human mAbs and serum autoantibodies induced D1R second messenger signaling in a dose-dependent manner. The results suggest that D1R autoantibodies enhanced antibody-mediated IgG signaling activity in the presence of the neurotransmitter dopamine. D1R autoantibodies sensitized D1R and exacerbated endogenous dopamine signaling in disease. Agonistic D1R signaling was inhibited by synthetic D1R peptide epitopes derived from human D1R extracellular loops which identified the immunodominant epitopes and antibody specificity. Taken together, D2R autoantibodies in BGE lead to excessive dopamine release and chorea, while D1R autoantibodies act like dopamine on receptors leading to allosteric enhancement and neuropsychiatric disease. Collectively, our novel findings suggest dopamine receptor autoantibodies in basal ganglia disorders act like drugs and mediate hyperactive dopaminergic receptor signaling.
Role of B-cell endogenous interferon beta in lupus nephritis
Authors: Alduraibi Fatima Khalid , Hsu Hui-Chen , Fatima Huma , Chatham W. Winn , Mountz John D
Published: February 2023

Lupus nephritis (LN) is histologically evident yet early diagnosis can be challenging since some patients do not exhibit overt clinical manifestations until advanced stages. The objective of the present study is to determine if B-cell expression of interferon beta (IFNβ) can be a unique systemic lupus erythematosus (SLE) clinical prognostic marker and if specific histopathologic features of LN can be identified in patients with elevated B-cell expression of IFNβ. A total of 80 patients who met the ACR/EULAR classification criteria of SLE were recruited. LN was identified in 41% (N=33). Multivariate regression analysis indicated a significant positive correlation between naïve B-cell IFNβ with race (African American > European American; P=0.006) and LN class (P<0.0001). Patients with elevated serum anti-Smith (P=0.01) and anti-DNA (P=0.013) exhibited significantly higher naïve B-cell IFNβ. B-cell IFNβ was further compared with renal histopathology microscopy findings on light, electron microscopy, and immunofluorescence (IF) (N=23). Patients with a higher percentage of IFNβ in naïve B cells showed increased segmental scaring (P=0.046). These patients also exhibited aggressive LN class (P=0.14), membranous pattern (P=0.15), and global sclerosis (P=0.18), although the differences were not significant. B-cell IFNβ did not show significant correlation with IgG, IgM, IgA, and C1q, and C3 in situ staining. The present study suggests that B-cell endogenous IFNβ may be developed into a unique target-based marker for the development and severity of LN. IFNβ blockade may be developed into an individualized therapy for this subset of SLE patients.
Genetic studies play an important role in the correct diagnosis of congenital immunodeficiency conditions
Authors: Fazal Nadeem , Nasrullayeva Gulnara , Mammadova Vafa
Published: February 2023

More than 350 kinds of PID (Primary Immunodeficiency disorders) related gene defects have been found, which lead to multiple clinical manifestations including repeated infection or opportunistic bacterial infections, autoimmune and malignant tumors. The 6-year-old boy was born from third pregnancy and not related parents. His oldest brother died at the age of 3 from unknown disease, second sibling, 11 years old, is alive. Until the age of 3 patient developed normally, when he started exhibiting swelling in the lymph node and temperature increased to 38–40°C. Lymph node biopsy showed tuberculosis. Bilateral pneumonia and the swollen right ankle, right and left elbow with purulent abscesses in the ankles and elbows. Surgical treatment gave a short-term relieve. At the age of 5, the disease recurred and all treatments were ineffective. CBC, biochemical, serological, immunological analysis and X-ray, US, MRT were done. Genetic sequence analysis of the 207 genes listed in the Genes Analyzed section (Invitae Primary Immunodeficiency Panel) was carried out. On objective examination, the anterior cervical lymph node and the axillary lymph nodes were swollen on both sides. Aplasia of tonsils, “high palate”, hepatomegaly and splenomegaly were detected. Lab results showed severe anemia and high CRP (60 mq/l), however, HbsAg, HCV, Anti-Syphilis, Brucella antibodies were negative. Genetic examination: heterozygous mutations in CASP10, CLPB, CSF3R, CTPS1, IL17RC, LRBA, NLRP12, RFX5 genes were found. Diagnostic genetic testing to clarify the severity of disease and assistance with a personalized treatment may help management of patient.
A novel variant in CoQ biosynthesis highly prevalent in Papua New Guinea children increases mortality following bacterial pneumonia
Authors: Walker Emma C , Todd Elizabeth M. , Ramani Rashmi , Anaya Edgar , Javati Sarah , Matlam John-Paul , Pomat William , Morley Sharon Celeste
Published: February 2023

To identify immune variants predisposing to severe pneumonia, we performed whole exome sequencing in a pediatric population highly susceptible to acute lower respiratory infections, identifying a candidate novel variant in the CoQ biosynthetic pathway. To evaluate the effect of this variant on immune function during bacterial pneumonia, we generated a mouse line using CRISPR-Cas9 that expresses the homologous variant in the enzyme COQ6. Interestingly, we found that the variant does not result in CoQ deficiency, as other known variants in biosynthetic proteins do, however intra-tracheal S. pneumoniae infection leads to increased bacteremia and mortality in mutant mice. Mechanistic studies show that mutant macrophages have reduced pneumococcal killing in vitro, showing an intrinsic defect in innate immune function conferred by the COQ6 mutation. Variant macrophages have decreased mitochondrial respiratory capacity both at baseline and following stimulation with LPS, as well as an inability to induce mitochondrial reactive oxygen species (mROS) in response to stimulation despite increased mROS at baseline. Thus, the novel variant in a CoQ biosynthetic enzyme leads to changes in macrophage mitochondrial function and an intrinsic inability to kill internalized bacteria. As alveolar macrophages are the first responders in the lung to bacterial challenge, the inability of these macrophages to mount a sufficient immune response leads to the observed increase in mortality following bacterial pneumonia. This work describes a novel susceptibility locus to severe childhood pneumonia, and also represents the first known pathogenic variant in a CoQ biosynthetic protein that does not cause pathology resulting from CoQ deficiency.
Crosstalk of TDP-43 and Optineurin in in vitro and ex vivo Models of Amyotrophic Lateral Sclerosis
Authors: Munitic Ivana , Prtenjaca Nikolina , Rob Matea , Peradinovic Josip , Buratti Emanuele
Published: February 2023

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease marked by protein aggregation and neuroinflammation. Protein aggregates in >95% of ALS cases are cytoplasmic and contain ubiquitinated and phosphorylated TAR DNA binding protein 43 kDa (TDP-43). Mutations in a ubiquitin-binding adaptor protein optineurin have recently been found in a subset of ALS cases. Its mutations are thought to act by loss of function, leading to disbalanced inflammatory signaling and/or impaired disposal of aggregated proteins by autophagy. Here we addressed the putative link between TDP-43 proteinopathy and loss of optineurin and/or its function. To this end we generated optineurin knockout (KO) neuronal and microglial cell lines by CRISPR/Cas9 technology, and (2) primary cells from a mouse optineurin insufficiency model lacking the ubiquitin-binding region (Optn470T). Elevated TDP-43 protein levels were found in optineurin-deficient BV2 microglial cell line, and in primary Optn470T bone marrow-derived macrophages (BMDM) and microglia. No differences were detected at TDP-43 mRNA levels, arguing for post-translational regulation. Elevated TDP-43 protein levels were not caused by autophagy blockade, and were specific to myeloid cells as they were not observed in neuronal NSC-34 and Neuro2A optineurin-deficient cell lines. Upon lipopolysaccharide (LPS) stimulation, TDP-43 levels increased in WT BV2 cells and BMDM, but in BV2 KO and OPTN470T BMDM they remained at the same elevated state as in basal conditions. Our results show that optineurin directly influences basal TDP-43 protein levels in myeloid but not in neuronal cells. Further studies are needed to determine if this could be a mechanistic link to protein aggregation in ALS.