bluuebunny/crossref_metadata_2025 huggingface dataset:
MLL1-COMPASS establishes developmental patterning of the memory CD8 T cell compartment and ensures regenerative immunity.
Authors: Getzler Adam , Frederick Megan A. , Diao Huitian , Milner Justin , Belanger Simon , Tsuda Shanel , Kim Jihye , Crotty Shane , Goldrath Ananda W , Pipkin Matthew E.
Published: February 2023

CD8+ T cells with stem cell-like qualities (TSTEM) provide central memory following resolved intracellular infections, and serve as progenitor cells that sustain effector-like cell development during chronic infections and tumors, both naturally and in response to immunotherapeutic PD-1 blockade. However, the chromatin regulatory factors (CRFs) and transcriptional programs that establish TSTEM cell differentiation are unclear. Using an RNA interference screen of all CRFs we discovered that Mixed Lineage Leukemia 1 (MLL1) was required in activated CD8 T cells to promote Id3-GFP reporter expression and to coordinately restrain PD-1, LAG3 and TIM3 expression. Single cell RNA-seq analysis demonstrated Kmt2a (MLL1) was preferentially expressed in P14 CD8 T cells enriched with TSTEM gene expression during lymphocytic choriomeningitis virus (LCMV) infection. P14 CD8 T cells depleted of MLL1 failed to establish TSTEM-like cells, terminally differentiated prematurely, failed to sustain responses to chronic LCMV infection and tumors, and failed to develop central memory subsets during acute LCMV infection. MLL1 preferentially bound transcriptional start sites (TSSs) in CD8 T cells, and was essential for global accumulation of H3K4me3, H4K16ac, and RNA Polymerase II CTD-phosphorylation during TCR stimulation. These results suggest that MLL1-COMPASS organizes TSS activity to establish transcriptional programs that delineate TSTEM and terminal differentiation.
Candida albicans-induced EphA2 signaling promotes kidney injury and sepsis during invasive candidiasis
Authors: Millet Nicolas , Solis Norma , Swidergall Marc
Published: February 2023

Pattern recognition receptors (PRRs) play key roles in fungal recognition and initiation of host immune responses. Recently, we identified that the ephrin type-A receptor 2 (EphA2) is a key β-glucan receptor on oral epithelial cells and neutrophils which orchestrates innate immune responses thereby preventing oral Candida infection. However, the contribution of this PRR in the pathogenesis of invasive candidiasis (IC) remains unknown. Although no differences in renal fungal burden could be observed, EphA2-deficient mice were more resistant to C. albicans infection suggesting that EphA2 deficiency enhances renal host tolerance to lethal fungal challenge. By testing renal cytokines, serum NGAL, a marker of renal injury, and the sepsis marker TREM-1, we found that EphA2 deficiency altered the pro- and anti-inflammatory response, reduced renal injury, and sepsis during IC. Epha2−/− mice decreased neutrophil and monocyte recruitment during IC. However, dendritic cells lacking EphA2 migrated more efficient to infected tissue. Since EphA2 is highly expressed on both, hematopoietic and stromal cells, we generated bone marrow chimeric mice. Both Epha2+/+ mice reconstituted with Epha2−/− BM and Epha2−/− mice reconstituted with Epha2+/+ BM were more resistant to IC compared to Epha2+/+ mice reconstituted with Epha2+/+BM. However, these chimeric mice were more susceptible than Epha2−/− mice reconstituted with Epha2−/− BM suggesting that EphA2 deficiency within cells of both compartments, the hematopoietic and the stromal, is required for full protection against IC. Collectively, our data highlight a detrimental role of EphA2 in promoting kidney injury and sepsis during lethal fungal infection.
Harnessing Regulatory T cells to Suppress Pathological Immune Response during Cryptococcus neoformans Meningoencephalitis
Authors: Xu Jintao , Goughenour Kristie , Zhao Ziyin , Olszewski Michal A
Published: February 2023

Central nervous systems (CNS) infections are marked by high mortality or frequently lasting neurologic disorders in the survivors. Cryptococcus neoformans meningoencephalitis (CM) is one of the leading causes of infectious neurological death. Many CM patients develop severe neurological symptoms despite evidence of ongoing immune response and fungal eradication, suggesting a dysregulated pathological host response. Our objective is to test whether and how regulatory T cells (Treg) maintain immune balance in CM and whether they could be used therapeutically to limit inflammatory CNS damage.

Using a murine CM model, we found Foxp3+ Treg massively accumulate in the infected brain. They acquire a Th1-like phenotype, expressing transcription factor T-bet, IFN-γ, and chemokine receptor CXCR3. This phenotype is critical for Treg accumulation in the CM brain. Treg depletion using anti-CD25 antibody results in deteriorated neurological symptoms and increased mouse mortality. Mechanistically, we found that brain-infiltrating Treg express immune suppressive molecules, such as IL-10 and tissue repair molecules, such as Amphiregulin compared to effector T cells. Single-cell RNA seq analysis showed broad cross-talk between brain Tregs with infiltrating T cell, monocyte-derived cells, and brain resident glial cells. Treg depletion resulted in increased activation of pathological T cells and monocyte-derived cells, promoting brain immunopathology during CM. Selectively enhancing Treg through low-dose IL-2 treatment improves mice survival and neurological function readouts. Taken together, our findings suggest that Treg limit pathological CNS inflammation and may provide the therapeutic opportunity for CM patients.
Characterization of beta2-adrenergic receptor homozygous knockout stress mouse model during Chlamydia muridarum genital infection
Authors: Belay Tesfaye
Published: February 2023

The relationship of stress in chlamydia genital disease pathogenesis and its influence on the host immune response remains unknown. Beta2-adrenergic receptor(β2-AR), the major receptor of the stress hormone, norepinephrine (NE), is known to impair the function of immune cells. This study was undertaken to determine whether cold-induced stress (CIS) results in differential weight loss, stress hormone level production, and gene expression of β-AR subtypes in the β2-AR knockout (KO) and wildtype (WT) C57BL/J6 mice. CIS was induced by immersing mice in cold water for 5 minutes daily for 21 days, as per previously established protocols in our lab. CIS in 21 days resulted in average body or spleen weight loss of 1.88 or 0.018 gm, respectively, to β2-AR KO and 1.7 or 0.015 gm to WT C57BL/6J compared to their respective non-stressed group. Furthermore, β2-AR deficiency lead to a significant enhancement in C. muridarum shedding from days 9–15 following intravaginal infection, but no statistically significant difference between the stressed (4000 IFU/mL) and non-stressed (4200 IFU/ml) of β2-AR KO was obtained. β2-AR OK and WT stressed mice showed about 65–70 ng/mL production of NE; however, the intensity of C. muridarum infection was low in the KO than the WT (p <0.05). Relative fold-change in mRNA in splenic CD4+ T cells of stressed β2-AR OK was non-detectable. However, a slight upregulation of β1- and β3-AR was observed primarily in CD4+ T cells of β2-AR KO mice. Overall, the β2-AR and the WT C57BL/6J are related in many ways, and we predict to have similar physiological conditions suitable to compare pathogenies. Immunity, histopathology, and infertility.
The purinergic receptor P2RX7 promotes the accumulation of effector CD4+ T cells in the lung parenchyma during hypervirulent mycobacterial infection
Authors: Carvalho Igor Santiao , Bonfim Caio César Barbosa , de Almeida Santos Gislane , Melo Bruno M , Hirata Mario H , Alves-Filho José Carlos C , Alvarez José M , Lassounskaia Elena L , Silva Henrique Borges da , D‘Império-Lima Maria Regina
Published: February 2023

CD4+ T cells are crucial to control pulmonary tuberculosis, but also favor uncontrolled inflammation in severe disease. Effective CD4+ T cell response to tuberculosis is related to tissue location: Lung-resident CD4+ T cells display higher effector function in comparison to circulating cells. Among the factors favoring T cell residency in non-lymphoid tissues, extracellular ATP sensing via P2RX7 is crucial for the generation of virus-specific resident memory CD8+ T cells. Yet, its role in CD4+ T cell non-lymphoid tissue residency has not been defined. Moreover, mycobacterial infection induces high extracellular ATP levels due to lung tissue damage, which is increased in response to hypervirulent strains. Here, we used P2rx7 ablation models to test the role of P2RX7 in CD4+T cell function in tuberculosis. In response to the hypervirulent Mycobacterium bovis MP287/03 strain, lung-resident WT CD4+ T cells express higher P2RX7 levels compared to circulating CD4+ T cells. Compared to WT mice, P2RX7-deficient (P2rx7−/−) mice display reduced numbers of antigen-specific (I-AbESAT-64-17) and IFN-γ-producing CD4+ T cells in the lung parenchyma. Using cell adoptive transfers, while recipient CD4-deficient mice transferred with CD4+ WT T cells are protected, mice that received CD4+P2rx7−/− T cells develop severe tuberculosis. Mechanistically, CD4+P2rx7−/− T cells have poorer accumulation and IFN-γ production in the lung parenchyma; moreover, they show reduced proliferation in situ. This defect in proliferation is exclusively observed in the lung, with no differences observed in draining lymph nodes. Together, our results show that P2RX7 promote the accumulation of protective CD4+ T cells in the lung during severe tuberculosis.
Mucosal LTA1 Elicits Lung Th17 cells with reduced plasticity towards Th1 cells and resistance to FK506.
Authors: Yang Haoran , Iwanaga Naoki , Norton Elizabeth B. , Kolls Jay K
Published: February 2023

Objective:

Based on our previous work, we identified a vaccine composed of the A1 domain of heat-labile toxin from E. coli (LTA1) adjuvant and Outer membrane protein (Omp) X from K2 strain of Klebsiella pneumoniae (K. pneumoniae), which can elicit serotype independent protection against K1 strain by generating lung Th1 and Th17 cells. To better understand the characteristics of these Th17 cells, we investigated the TCR repertoire, plasticity, and resistance to FK506.

Methods:

Naïve or immunized lung cells were collected and stained with Anti-Mouse TCR Vβ Screening Panel for FACS. Naïve CD4+ cells were isolated from spleens and cultured under Th17 differentiation conditions in vitro. Immunized lung Th17 cells were purified. To assess plasticity towards Th1, cells were cultured w/wo IL-12 and analyzed by FACS.

To assess resistance to calcineurin inhibitors, the naïve/immunized CD4+ cells were treated w/wo FK506 both in vivo and ex vivo, analyzed by FACS.

Results and Conclusions: The LTA1-OmpX elicited CD4+ cells were oligoclonal with skewing towards TCRVβ14+ cells.

After incubation with IL-12, 57.73 ± 3.9% of the in vitro differentiated Th17 cells showed plasticity towards Th1 cells whereas in immunized lung Th17 cells only showed 6.25 ± 1.6%. Lung Th17 cells also showed substantially reduced STAT4 phosphorylation and surface IL-12rβ2 expression compared to in vitro Th17 cells.

Furthermore, lung Th17 cell IL-17A expression was resistant to FK506 treatment both in vivo and ex vivo, which potentially indicated that this vaccine strategy may confer protection in the setting of transplant immunosuppression.
Complex immunogenomic control of serum thyroid hormone levels in fetuses and piglets challenged with Porcine Reproductive and Respiratory Syndrome Virus
Authors: Van Goor Angelica G , Pasternak Alex , Walugembe Muhammed , Hamonic Glenn , Dekkers Jack , Harding John C. S. , Lunney Joan K
Published: February 2023

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) causes respiratory disease in piglets and reproductive disease in sows. However, the interaction between host immunity and hormone homeostasis is not completely understood but piglet and fetal serum thyroid hormone (i.e., T3 and T4) levels decrease rapidly in response to PRRSV infection. Our objective was to estimate genetic parameters, identify QTL, and uncover nearby candidate genes for T3 and/or T4 levels of piglets and fetuses challenged with PRRSV2. Serum from 5-week old pigs (N=1792) at 11 days post infection (dpi) with PRRSV2 were assayed for T3 levels (piglet_T3). While serum from fetuses (N=1267) at 12 or 21 dpi from sows (N=150) challenged with PRRSV2 in late gestation were assayed for T3 (fetal_T3) and T4 (fetal_T4) levels. Animals were genotyped for 60K or 650K SNPs, heritabilities were estimated and genome wide association studies were performed for each trait separately. We found all three traits to be moderately heritable (18–28%) and the significant QTL (N=37) across several chromosomes collectively explained 24–34% of the genetic variation. A shared QTL identified for all traits on chromosome 6 contained the IL12RB2 and IFI44 genes. The largest QTL identified for both piglet_T3 and fetal_T3 on chromosome 5 contained IL17RA. A large QTL identified for piglet_T3 on chromosome 7 contained 9 MHC genes (e.g., SLA-DMA, SLA-DOA, SLA-DQA, SLA-DRA, and MHC-IA7). In conclusion, our data support the concept that there are complex interactions between thyroid hormone levels and multiple immunogenetic pathways during PRRSV infection, and that there is potential to select for pleiotropic QTL to simultaneously improve host immunity for reproductive and respiratory PRRS.
Unveiling the immunological attributes of chicken tumor necrosis factor-α (TNF-α) using new sets of monoclonal antibodies specific for poultry TNF-α
Authors: Lu Mingmin , Lee Youngsub , Lillehoj Hyun S
Published: February 2023

Tumor necrosis factor-α (TNF-α) is a type II transmembrane protein with either membrane-bound or soluble forms and is a prototypical member of TNF superfamily. TNF-α is a pleiotropic cytokine associated with the regulation of systemic inflammation and host defense. Chicken TNF-α (chTNF-α) is a long missed avian ortholog and the immunological properties of chTNF-α remains largely unknown compared to the mammalian counterpart. Here, we report on the functional characterization and immunomodulatory roles of chTNF-α using a panel of new anti-chTNF-α mouse monoclonal antibodies (mAbs) that we developed. A chTNF-α-specific antigen-capture ELISA was developed using compatible mAb partners via the screening and validation of ten different mAbs (3G11, 4C4, 4F3, 6A52, 6E6, 10E8, 12G6, 12H7, 14H2 and 15G7). Employing 3G11 and 12G6 as capture and detection antibodies, respectively, the levels of native chTNF-α in the circulation of Clostridium perfringens-, Eimeria- or dual C. perfringens/Eimeria-infected chickens were determined. Furthermore, intracellular expressions of chTNF-α in primary cells or cell lines derived from chickens were validated in immunocytochemistry and flow cytometry assays using both 3G11 and 12G6 mAbs. Importantly, both 3G11 and 12G6 neutralized chTNF-α-induced nitric oxide production in chicken HD11 cells in vitro. Collectively, our results provide a better understanding of the functional characteristics of chTNF-α and these anti-chTNF-α mAbs will serve as valuable immunological tools for the fundamental and applied studies in avian species.
Active Influenza D coinfection reduces Influenza A disease severity in mice
Authors: Robinson Ema , Shepardson Kelly M. , Skelton Raegan M. , Hernandez Emiliano , Huber Victor C. , Rynda-Apple Agnieszka
Published: February 2023

Influenza D virus (IDV), the only member of the fourth genus of the Orthomyxoviridae family, primarily infects bovines. While no symptoms have been identified in humans, studies find seropositivity rates up to 95% among people with close contact to cattle. This suggests that IDV may cocirculate in human populations with Influenza A viruses (IAV). Clinical data indicate that coinfections with some mild respiratory viruses, such as rhinovirus, can reduce IAV severity. The current study investigates whether and how the immune response to preceding IDV infection affects the outcome of subsequent IAV. Previously we reported that IDV stimulates strong IFN-β production in mice and in human epithelium (A549 cells). We extend these findings by showing that IDV productively infects respiratory tract of wild-type (WT) mice and induces recruitment of innate and adaptive immune cells to the lungs. IDV-IAV coinfected mice exhibit a stronger IFN-β response early in IAV infection compared to mice infected with IAV alone. Consistently, IDV-infected WT mice showed less weight loss and improved survival after IAV. To better understand the role of type I IFN signaling in host response during IDV and IAV coinfection, we used mice deficient in either subunit of type I IFN receptor (Ifnar1−/−, Ifnar2−/−). While IDV-infected WT mice exhibit only slight weight loss occurring mid-infection, Ifnar1−/− mice exhibit dramatic weight loss mid-infection but recover fully within 3 days. In contrast to its effect in WT mice, IDV exacerbated IAV-induced mortality and lengthened the duration of symptoms in Ifnar1−/− and Ifnar2−/− mice. Our results show that active IDV infection elicits a type I IFN-dependent protection against IAV-associated weight loss and mortality.
Endogenous Type I interferon signaling plays a pivotal role in restricted SARS-CoV-2 infection in human ACE2 transgenic mice
Authors: Kempaiah Kempaiah Rayavara , Drelich Aleksandra , Hsu Jason C , Tat Vivian , Tseng Chien-Te K
Published: February 2023

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become pandemic, resulting in severe morbidity and mortality around the world. COVID-19 pathogenesis includes lung inflammation, cytokine storm and organ failures, but the mechanism underlying the disease remains to be studied. To determine what role(s) the well-characterized type I interferon (IFN-I)-mediated innate antiviral responses might play against SARS-CoV-2 infection, we treated hACE2 transgenic (Tg) mice, AC70 line, with mouse IFN-I receptor-specific monoclonal antibody prior to challenge with 106TCID50 of SARS-CoV-2 (US-WA-1/2020). Both IFN-blocked and-unblocked animals developed clinical signs of disease with body weight loss from day 3 and 100% mortality between 4 and 5 days post-infection with SARS-CoV-2 at high dose (106 TCID50). Although live virus was detected in the lungs and brain of both groups, live virus was detected only in the kidneys, liver, spleen, heart, and gastrointestinal tract of the IFN-blocked group. Elevated expression of proinflammatory cytokines and interferons including IFN-λ were detected in all organs tested in both groups. Immunostaining for SARS-CoV-2 spike protein along with profound histopathological observations in the lungs and brain of both groups show severe COVID-19 disease pathogenesis. Our results show that AC70 Tg mice are highly permissive to SARS-COV-2 infection and develop severe COVID-19. Furthermore, we show type I IFN-mediated signaling pathways play a critical role in restricting viral spread and are ideal for development of medical countermeasures (MCMs) against COVID-19.
House dust mite proteins reduce the spread of Respiratory Syncytial Virus in the BEAS-2B bronchial epithelial cell line.
Authors: Rife Maria , Freiman Andrew , Hillyer Philippa , Rabin Ronald L
Published: February 2023

House dust mites (HDM) are a predominant causative agent of airway hypersensitivity and asthma. The HDM group 1 allergens include Der p 1, a cysteine protease that contributes to sensitization and symptom exacerbation. Respiratory syncytial virus (RSV) infection of infants and small children can lead to severe pneumonitis. RSV activation of TLRs and RIG-I-like receptors induces proinflammatory cytokines and chemokines, which exacerbate disease, and type I/III interferons (IFNs) which induce expression of antiviral IFN stimulated genes (ISGs). Since RSV in children is coincident with HDM exposure, we used BEAS-2B human bronchial epithelial cells to explore the effects of HDM on RSV infection

BEAS2B cells were exposed to HDM extract or Der p 1 alone before, during, or after RSV infection. Surprisingly, HDM extract or Der p 1 decreased RSV infection in a dose and time-dependent manner. In a viral entry assay, HDM extract reduced the area of foci, rather than their number, suggesting that one or more HDM proteins attenuate cell to cell spread. Preliminary experiments point to a role for the cysteine protease activity of Der p 1. We also measured expression of a panel of representative proinflammatory mediators and ISGs. Compared to RSV alone, Der p 1 increased expression of some proinflammatory mediators, while HDM extract decreased expression of a panel of ISGs. In the absence of RSV, HDM extract increased proinflammatory mediators while Der p 1 did not affect gene expression. These results were unchanged following heat treatment at 65°C for 1 h. Elucidating mechanisms by which HDM proteins enhance protection against RSV may reveal novel antiviral mediators that locally control RSV infection.
Murine Th9 cell-mediated allergic inflammation shows different steroid responsiveness depending on target organs
Authors: Miura Kento , Koyama Teidai , Yamasaki Norimasa , Ogata Sawako , Saeki Mayumi , Hiroi Takachika , Mori Akio , Kaminuma Osamu
Published: February 2023

Th9 cell has been implicated in the development of allergic inflammation, though the sensitivity of Th9 cell-mediated responses to steroid therapy remains unclear. Here, allergen-induced bronchial and nasal inflammatory responses and their steroid responsiveness were comparatively investigated in ovalbumin (OVA)-specific Th9 cell-transferred mice. BALB/c mice were transferred with in vitro-differentiated Th9 cells and challenged by intratracheal or intranasal injection of OVA. The significant nasal hyperresponsiveness accompanied by nasal infiltration of eosinophils as well as allergen-specific T cells was induced in Th9 cell-transferred mice upon intranasal allergen challenge. These responses were strongly suppressed by the treatment with dexamethasone. Substantial accumulation of eosinophils in the lungs and bronchial hyperresponsiveness (BHR) were induced by intratracheal OVA challenge. In contrast with the nasal responses, neither cellular infiltration in the lungs nor BHR in Th9 cell-transferred mice was affected by dexamethasone treatment. In addition to the possible contribution of Th9 cells to the development of allergic inflammation in various tissues, different steroid responsiveness of Th9 cell-mediated responses among target organs was suggested.
Glycogen synthase kinase 3 (GSK3) is a critical regulator of allergen-mediated mast cell activation
Authors: Crozier Robert W.E. , Hamstra Sophie I. , Fajardo Val A. , MacNeil Adam J.
Published: February 2023

Mast cells are granulated immune sentinels responsible for perpetuating allergic inflammatory events. Allergen-induced FcɛRI signaling leads to the activation of MAPK, PLC and PI3K/Akt signaling cascades, which are responsible for mediating a biphasic mast cell response, characterized by degranulation in the early phase and sustained release of pro-inflammatory mediators in the late phase. Glycogen synthase kinase 3 (GSK3) is a constitutively active serine/threonine kinase and a major convergence point for several highly conserved signaling cascades, including the PI3K/Akt pathway. Due to its central role in regulating downstream pro-inflammatory signals, GSK3 has become a therapeutic target in inflammatory pathologies, however, the role that GSK3 plays in allergen-induced FcɛRI signaling has yet to be characterized. Thus, the objective of this study was to determine the functional role of GSK3 in allergen-activated mast cells. Sensitized murine bone marrow-derived mast cells were incubated with the GSK3 inhibitor CHIR99021 and stimulated with allergen. CHIR99021 treatment significantly inhibited degranulation dose-dependently to 63% (10 μM, p<0.001) and 43% (20 μM, p<0.001) of the control. Release of cytokines TNF (p<0.01), IL-6 (p<0.001), IL-13 (p<0.01) and chemokines CCL1 (p<0.001), CCL2 (p<0.05) and CCL3 (p<0.001) were inhibited following treatment at 20 μM. Finally, inhibition of GSK3 was found to significantly reduce downstream phosphorylation of JNK (10 & 20 μM, p<0.05), while ERK and p38 remained unaffected. These results are the first to characterize GSK3 as a central regulator of allergen-induced FcɛRI signaling, making it an intriguing target to attenuate mast cell functionality in pathological contexts.
GC-Tfh cell induction by Langerhans cells in steady-state is antigen-dose dependent and IL-6 independent.
Authors: Bouteau Aurélie , Zurawski Sandra , Zurawski Gerard , Igyártó Botond Z
Published: February 2023

We previously compared the contributions of different skin dendritic cell subsets in the induction of humoral immune responses. We found that antigen delivery without adjuvants to Langerhans cells (LCs) through the c-type lectin receptor Langerin, unlike cDC1, induces germinal center T follicular helper cells (GC-Tfhs), GC B cells, and high antibody titers.

We also showed that targeting LCs with higher antigen dose induced significantly lower B cell responses and antibody titers. To better understand the role of antigen dose in this response, we assessed the T cell responses following targeting of LCs with different amounts of antigen. In accordance with the B cell responses, we found that GC-Tfh cell formation was also antigen dose-dependent. Using two-complementary approaches targeting IL-6-deficient LCs and targeting LCs in mice treated with neutralizing anti-IL-6 antibody, we showed that LCs induced GC-Tfh cells’ formation is an IL-6 independent fashion.

In summary, this study shows that targeting LCs at steady-state induces the formation of GC-Tfh cells through an antigen-dose dependent and IL-6 independent mechanism.
Role of PRMT5 on splicing events in T cell biology
Authors: Sengupta Shouvonik , West Kelsi O , Agosto-Rosa Laura , Lynch Kristen W , Watson Robert , Patrick Kristin , Guerau-De-Arellano Mireia
Published: February 2023

Protein Arginine Methyltransferase (PRMT) 5 catalyzes symmetric dimethylation of arginine, a post-translational modification involved in cancer and embryonic development. The mechanisms by which PRMT5 modulates T helper (Th) cell activity are still not completely understood. Here we find that PRMT5 in T cells promotes spliceosome component methylation and influences splicing in resting and activated cells. RNAseq of resting and activated wildtype (WT) and PRMT5-knockout (KO) CD4 T cells show the breadth of pathways affected by knockout of PRMT5. Importantly, metabolic processing, signaling, cell cycle and protein localization were affected. We also used a novel software, Modeling Alternative Junction Inclusion Quantification (MAJIQ), to extract information on the classes of alternative splicing (AS) events – Exon skipping, Intron retention, Alternative 5′ splice site and Alternative 3′ splice site. In our samples, we noticed a bias towards exon skipping. We took a closer look at the number of alternative junctions (AJ) arising from these events and the location of the local splice variants (LSV). Analysis of WT and KO T cells showed 9071 AJs upon T cell activation and 2136 AJs dependent on PRMT5. This work demonstrates that PRMT5 modulates the gene expression splicing profile of T cells.
Depletion of microRNA-183-96-182 miRNA cluster in lymphocytes suppresses anti-dsDNA autoantibody production and IgG deposition in kidney in C57BL/6-Faslpr/lpr mice
Authors: Wang Zhuang , Heid Bettina , Ren Jingjing , Edwards Michael R , Cecere Thomas E. , Lu Ran , Khan Deena , Kirsch David , Reilly Christopher M. , Dai Rujuan , Ahmed S. Ansar
Published: February 2023

The miR-183-96-182 (miR-183C) is a highly conserved miRNA cluster among species. Our previous work found a significant upregulation of miR-183C in the splenic cells of three different murine models of systemic lupus erythematosus (SLE). Current studies revealed that miR-183C miRNAs are critically involved in immunity and autoimmunity.

In this study, we found that inhibition of miR-182 alone or miR-183C in vitro with antagomirs significantly reduced lupus-related inflammatory cytokine IFN-γ and IL-6 in activated splenocytes from MRL or MRL/lpr mice. To further characterize the pathogenic role of miR-182 and miR-183C in lupus in vivo, we developed B6-lpr mice with conditional depletion of miR-182 or miR-183C in CD2+ lymphocyte. We found that depletion of either miR-182 or miR-183C in the lymphocytes of B6/lpr mice had no obvious effect on T and B cell development as similar percentage of CD4+. CD8+, CD19+, as well as Tregs, follicular helper T (TFH), germinal center B (GCB), and plasma cells were observed in the miR-182−/− and miR-183C−/− and their respective control. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C−/− mice when compared to age-matched controls and the B6/lpr mice with miR-182 or miR-183C deficiency have significantly reduced IgG deposition in the kidneys. Meanwhile, there was reduced IFN production in ex vivo activated splenocytes from the knockout mice. Furthermore, we demonstrated that miR-182 and miR-183C regulated the inflammatory response in splenocytes via targeting forkhead box O1 (Foxo1). Together, our data suggest a potential therapeutic effect of targeting miR-183C in lupus.
Interleukin-2-Inducible T-cell Kinase (ITK) signaling regulates non-canonical regulatory T cells
Authors: Mammadli Mahinbanu , Harris Rebecca , May Adriana , Gentile Teresa , Karimi Mobin A
Published: February 2023

Regulatory T cells (Tregs) play an indispensable role in maintaining immunological tolerance to self-antigens and foreign antigens in suppressing excessive immune responses deleterious to the host by exerting a suppressive program towards a wide variety of target immune cells.

Here we showed that ITK signaling pathways control the increased frequency of “Non-Canonical” CD4+CD25−FoxP3+ Tregs (ncTregs) population as well as canonical Tregs CD4+CD25+FoxP3+ expression significantly. Using several animals models we demonstrated that transplanting ncTregs and cTregs from ITK deficient mice ameliorated WT T cells mediated Graft versus Host Disease (GVHD). Furthermore, our data showed that adaptively transferring ncTregs and cTregs from ITK deficient mice significantly reduces proinflammatory cytokine production and T cell proliferation in GVHD induced recipient mice. Furthermore, our data uncover that adaptatively transferring ncTregs and cTregs from ITK deficient mice to GVHD induced recipient mice reduced tissue damage which induced by WT T cells. Finally, we have recently developed a novel peptide inhibitor which enhance Tregs expression in both mice and human. This novel peptide inhibitor also reduced cytokine production in mice and primary GVHD patient samples. Our data demonstrated that targeting specifically SLP76:ITK signaling could be used as a therapeutic strategy not only limit to GVHD but also treat multiple T cells mediated autoimmune disorders.
CD22/Lyn-mediated suppression of trophoblast antigen-specific B cells underlies fetomaternal tolerance
Authors: Rizzuto Gabrielle , Brooks Jeremy , Fisher Susan , Zikherman Julie , Erlebacher Adrian
Published: February 2023

The placenta releases allogeneic trophoblast antigens into maternal circulation during pregnancy. Maternal antigen presenting cells present these antigens to T cells but fail to induce strong immunogenic responses, even in the presence of danger signals. While this observation illustrates the phenomenon of fetomaternal tolerance, its cellular and molecular basis remains largely unknown. By studying the pathways mediating adaptive immune responses to a surrogate trophoblast antigen in mice, we found that maternal B cells, and not regulatory T cells, directly underlie the impairments in CD4 T cell activation. Trophoblast-antigen specific B cells, rather than dendritic cells, are the main presenters of trophoblast antigen to CD4 T cells. The B cells are themselves profoundly suppressed, thus limiting their ability to activate cognate CD4 T cells. Mechanistically, B cell suppression occurs as the result of CD22/Lyn inhibitory signaling, likely induced by sialic acid residues that coat the trophoblast-derived antigen. These results not only identify maternal B cells as key players in fetomaternal tolerance but also provide a basis for further work on B cell responses during healthy and pathologic pregnancies. More broadly, these results suggest that exploiting antigen-intrinsic properties such as sialylation may provide new therapeutic ways to ignite or inhibit antigen-specific responses outside the context of pregnancy.
Therapeutic Vaccine (TheraVac) in combination with cGAMP cures more differentiated melanomas in mice
Authors: Alam Md Masud , Yang De , Tross Debra , Oppenheim Joost J
Published: February 2023

We have identified a combination of immunotherapeutics, termed TheraVac, capable of curing multiple large established mouse tumors, but it failed to cure melanoma in mice. TheraVac consists of an immunostimulating arm containing an agonist (HMGN1) for TLR4 and an agonist (R848) for TLR7/8 that synergize to activate tumor-infiltrating dendritic cells (DCs) and upregulate the polarization of Th1 immune responses. The second arm uses an immune checkpoint blockade inhibitor, αPDL-1, to diminish tumor associated immunosuppression. Here, we investigated supplementation of TheraVac by a STING agonist, cGAMP, because they synergize in activating DC’s and produced more immunostimulating IL-12p70 and TNFα cytokines. We therefore treated five different tumor types including melanin producing B16F1, M3, and M4 mouse melanomas that are homologous to human melanomas with intratumoral delivery of cGAMP and TheraVac. This eradicated 80% of the B16F1, 80% of the M3 and 60% of the M4 tumors, but still failed to cure the non-melanin producing undifferentiated M1 and M2 tumors. Biopsies of M3 tumor treated with TheraVac plus cGAMP showed a significant increase of CD8+ CTL’s infiltrating into the tumor. Importantly, there was a marked increase of both CD4 and CD8 memory T cells and generation of functional tumor specific CTLs in tumor-draining lymph nodes. The resultant tumor-free mice were selectively resistant to subsequent challenge with the same tumors, indicating long-term tumor specific protective immunity. Overall, our findings have important implications for clinical trials with a combination of immunotherapeutics to cure melanin producing human melanomas, without the need for exogenous tumor antigens and no obvious toxic effects in mice.
αPD-1 and extended half-life IL-2 combination therapy clears established GL261 gliomas in an MHC class I independent fashion
Authors: Tritz Zachariah Peter , Ayasoufi Kathy , Malo Courtney Sarah , Himes Benjamin , Khadka Roman , Yokanovich Lila T , Goddery Emma , Fain Cori , Hansen Michael , Jin Fang , Wang Chensu , Moynihan Kelly , Irvine Darrell J , Wittrup K Dane , Parney Ian , Johnson Aaron J
Published: February 2023

Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12 months post diagnosis despite aggressive treatment. Immunotherapy approaches for GBM have been attempted but the unique immune environment of the brain and the immunosuppressive features of GBM complicate these efforts. Even αPD-1 checkpoint blockade, highly useful in the context of melanoma, has proved ineffective in the treatment of GBM. In order to promote the efficacy of αPD-1 against the GL261 murine model of GBM, we supplemented this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated dangerously high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. While neither αPD-1 nor MSA-IL-2 are effective monotherapies against the GL261 model tumor, we have found this combination immunotherapy to provide durable tumor clearance and to create immunologic memory capable of resisting GL261 rechallenge. Most strikingly, this combination therapy cleared established GL261 tumors even in mice incapable of MHC class I restricted antigen presentation. Instead, therapeutic efficacy was abrogated by the depletion of CD4 T cells. Independence of MHC class I restricted antigen presentation makes this combination immunotherapy using αPD-1 checkpoint blockade both highly novel and translatable to clinical use.
Fomitopsis cajanderi extracts have differential tumoricidal actions on human MCF7 and U-937 cancer cells and inhibit TNF secretion by U-937 monocytes
Authors: Wenner Cynthia A , Froehlich Olivia R , Sitkoff Anna
Published: February 2023

This study aimed to determine if hot ethanol and aqueous extracts from the Fomitopsis cajanderi mushroom species are cytotoxic to human cancer cell lines and modulate TNF secretion by human monocytes. The hot ethanol extract was made via a 2 hour Soxhlet extraction of F. cajanderi fruiting body in ethanol using a cold finger condenser. The hot aqueous extract was made via a 2 hour decoction in water at 100°C. Human MCF7 mammary adenocarcinoma and U-937 histiocytic lymphoma cells were treated with increasing concentrations of extract or solvent controls and were assessed for cytotoxicity by XTT colorimetric assay. Effects of the aqueous extract on TNF secretion by PMA-differentiated, LPS-stimulated U-937 cells were determined by TNF ELISA. Glycosyl content and linkage analysis of the aqueous extract were assessed by the Complex Carbohydrate Research Center (Athens, Georgia, USA). The hot ethanol extract induced concentration-dependent cytotoxicity of MCF7 and U-937 cells, with stronger U-937 tumoricidal effects. The hot aqueous extract was not cytotoxic to MCF7 cells, but had cytotoxic effects on undifferentiated U-937 cells at high concentrations. The aqueous extract, found to contain beta glucans, inhibited TNF secretion by LPS-stimulated U-937 cells in a concentration-dependent manner. Hot ethanol and aqueous F. cajanderi extracts showed differential tumoricidal actions on human cancer cells. The ethanol extract was cytotoxic to the human cancer cells tested. The aqueous extract contained beta glucans reported to be immunomodulatory, and inhibited U-937 TNF secretion. These results warrant further research of F. cajanderi extracts as novel adjunctive anti-cancer and anti-inflammatory agents.
Clearance of established oropharyngeal and genital HPV tumors by mucosal vaccination via induction of multiple cytotoxic effector populations
Authors: Hegde Venkatesh L , Sierra Gloria , Nookala Sita , Yanamandra Ananta , O’Hara Madison , Sastry Jagannadha K
Published: February 2023

High-risk type human papillomaviruses (HPV) are the underlying cause of >5% of all cancers including cervical and Head & Neck cancers in which virus-encoded E6/E7 oncoproteins serve as tumor-specific antigens. Despite the success of prophylactic HPV vaccine, therapeutic strategies targeting the E6/E7 have been ineffective in established HPV+ tumors due mainly to inadequate antigen-specific immunity. Therefore, enhancing the therapeutic potential HPV vaccines to achieve curative efficacy is an unmet clinical need. We present here preclinical evidence for efficient clearance of immunocompetent HPV tumors at the vaginal (TC-1) and oral (mEER) mucosal tissues by intranasal vaccine using HPV16 E6 and E7 short peptides along with the combination of clinically relevant adjuvants QS-21 and CpG-ODN. This vaccine exhibited high therapeutic efficacy with >80% mice with oral or vaginal tumors showing regression as well as significant extended survival advantage over untreated mice or mice treated with vaccine containing single adjuvants. The analysis of immune correlates by multicolor flow cytometry showed robust increases in the overall as well as antigen-specific (E7 tetramer+) cytotoxic CD8 T cells expressing Granzyme B and/or IFNg in both intra-tumoral and systemic compartments in vaccine-treated mice. Furthermore, the intranasal vaccine containing the combination of adjuvants induced significant frequencies of a unique subpopulation of NK cells expressing CD11c, granzyme B and IFNg, termed NKDC. Our results suggest that mucosal HPV E6/E7 peptide vaccine formulation containing QS-21+CpG-ODN adjuvants may provide an effective therapeutic strategy for the treatment of patients with HPV+ oropharyngeal and cervical cancers.
The comparison of two SARS-CoV-2 spike protein antigens in TLR-adjuvanted subunit vaccines.
Authors: Partlow Haley A , Davison Clara J. , Lathrop Stephanie K , Evans Jay T.
Published: February 2023

Adjuvanted subunit vaccines offer improved safety and efficacy over whole inactivated or live attenuated vaccines by incorporating recombinant proteins with highly selective innate immune activators. Selection of an antigen in subunit vaccines can alter immune responses, making it an important consideration. For severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the spike (S)-protein aids in receptor recognition and membrane fusion. Previous studies of SARS-CoV-2 and the closely related SARS-CoV-1 define the receptor binding domain (RBD) portion of the S-protein as the target of most neutralizing antibodies (nAbs). Here the recombinant full spike trimer (FST) and the isolated recombinant RBD are compared as antigens in an adjuvanted subunit vaccine. Induction of Th1-biased CD4+ and CD8+ cellular responses are believed to be important for durable antiviral immunity. Therefore, we used synthetic agonists of the toll-like receptor (TLR)4 or TLR7/8 as vaccine adjuvants for their ability to promote Th1 cellular immunity. We found that a TLR4 agonist was effective at adjuvanting both RBD and FST, while a TLR7/8 ligand provided superior adaptive immune responses with FST. In addition, increased induction of nAbs were demonstrated when vaccinating mice with FST. Thus, we find that early selection and optimization of adjuvant and antigen combinations is critical for the induction of humoral (including nABs) and cell-mediated immunity to SARS-CoV-2 antigens.
Combinations of alum and synthetic toll-like receptor agonists as adjuvants for CoVID-19 vaccines
Authors: Davison Clara J , Partlow Haley A , Lathrop Stephanie K , Siram Karthik , Abdelwahab Walid , Burkhart David J , Evans Jay
Published: February 2023

Aluminum-based adjuvants (“alum”) are the most widely-used adjuvants for human vaccines, with an extensive history of safety and efficacy. Alum is known to elicit high antibody titers by driving a Th2-based immune response, which may not be optimal for many viral infections, such as SARS-CoV-2. Therefore, combining the strong safety and efficacy attributes of alum with a Th1-polarizing adjuvant could improve immunity against viral antigens. Here we test the combination of alum with synthetic TLR4- and TLR7/8 -ligands in vaccines against SARS-CoV-2. The combination of alum and TLR agonists resulted in efficient adjuvantation of both humoral and cell-mediated immunity. Alum adsorption studies showed low association between the small, positively-charged receptor binding domain (RBD) antigen and aluminum hydroxide (Alhydrogel) and high adsorption to negatively charged aluminum phosphate (Adju-phos). However, Adju-phos and Alhydrogel both enhanced immunity to the SARS-CoV-2 RBD antigen, suggesting antigen adsorption may not be required for the immune enhancing effects of alum. These data demonstrated that adjuvants combining alum with a TLR agonist result in an improved anti-viral immune response. However, the ability of alum to adsorb to an antigen may not predict its ability to effectively adjuvant.
STAT4 influences the expression of IL-10 in Th17 cells
Authors: Anderson Ashlyn E , Shin Boyoung , Bryars Morgan , Maynard Craig , Harrington Laurie
Published: February 2023

Autoimmune diseases such as Multiple Sclerosis (MS), Rheumatoid Arthritis (RA) and Crohn’s Disease (CD) affect more than twenty million people in the United States. While the exact etiology of most autoimmune diseases is unknown, single nucleotide polymorphisms in the STAT4 locus are associated with an elevated risk of developing various autoimmune diseases. Specifically, in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), STAT4 is essential for the development of neuroinflammation. Interestingly, STAT4 is a transcription factor known to be important for Th1 cell differentiation, however we and others have demonstrated that many Th1 associated molecules such as IL-12, IFNγ and Tbet are dispensable for EAE. These data lead us to postulate that STAT4 functions in another effector CD4 T cell, Th17 cells, to drive neuroinflammation. Using an adoptive transfer model, we find that STAT4 is required in a Th17-mediated EAE. Transcriptional profiling of the adoptively transferred Th17 cells reveals that STAT4 influences the expression of over 200 genes, one of them being the immunosuppressive cytokine IL-10. Strikingly, we find an indirect relationship between STAT4 and IL-10 in Th17 cells as well as T regulatory 1 (Tr1) cells. We hypothesize that STAT4 regulates the balance between Th17 and Tr1 cells in the context of autoimmune inflammation. Together, these data illustrate a novel role for STAT4 in controlling the functional and plastic properties of Th17 cells, and this may provide a promising therapeutic target for patients affected by Th17 driven autoimmune diseases.
Single cell profiling identifies IgMMDA-LDL-producing human B cells and a novel role for CD24
Authors: Pattarabanjird Tanyaporn , Nguyen Anh Tram , McSkimming Chantel , Dinh Huy , Marshall Melissa A , Ghosheh Yanal , Gulati Rishab , Durant Chistopher , Vallejo Jenifer , Saigusa Ryosuke , Drago Fabrizio , Taylor Angela M , Tsimikas Sotirios , Miller Yury , Ley Klaus , Hedrick Catherine C. , McNamara Coleen A
Published: February 2023

Objectives:

IgMs that inactivate oxidation specific epitopes (IgMOSE) on phospholipids such as low density lipoprotein (LDL) were shown to confer athero-protection. We developed a 24 antibodies mass cytometry panel (CyTOF) to identify the human B cell subtype producing IgM to malondialdehyde modified LDL (IgMMDA-LDL), a predominant IgMOSE in humans. We also utilized humanized mice model to characterize a role of CD24 mediated IgM production.

Methods:

Peripheral blood mononuclear cells from healthy donors and subjects with coronary artery disease (CAD) undergoing quantitative coronary angiography were used to perform high dimensional analysis using CyTOF, RNAseq and Abseq as well as adoptive transfer into humanized mice model.

Results:

CD20+CD27+IgM+ cells (B27+IgM+) spontaneously produced IgM and produced IgMMDA-LDL in response to MDA stimulation when injected into humanized mice; an effect significantly augmented in B27+IgM+ cells with high expression of CD24 (B27+IgM+CD24hi). CD24 expression also enhanced splenic and bone marrow trafficking of B27+IgM+ cells. Blocking CD24 with a mAb reduced CCR6 expression, increased CCL20-induced CCR6 internalization and impaired migration to the spleen leading to lower IgM production. Lastly, single cell protein and transcriptome sequencing of PBMCs from 60 CAD subjects revealed enhanced CCR6 and IgM signaling in B27+IgM+CD24hi cells in subjects with low compared to high CAD severity.

Conclusions:

Identification of IgMMDA-LDL-producing cells in humans has the potential to allow for the development of therapeutics aimed at cellular targeting that may allow for enhancing production of IgM to the many OSE produced during inflammatory states such as atherosclerosis.