bluuebunny/crossref_metadata_2025 huggingface dataset:
A Protein Subunit Filovirus Vaccine Facilitates Maternal Immunization in Mice
Authors: Williams Caitlin , Wong Teri Ann S. , Lehrer Axel T.
Published: February 2023

Ebola virus disease (EVD) is highly pathogenic and virulent and has caused large scale outbreaks in Central and West Africa. To date there are no known correlates of protection however, recently the FDA and EMA have approved a vaccine, Ervebo. This vaccine utilizes a replication competent viral vector which may be contraindicated in immunocompromised individuals, pregnant women, and infants. Our lab has developed a recombinant protein subunit vaccine, a platform that is generally considered safe for immunocompromised, pregnant women, and infants. Our vaccine, when properly adjuvanted, demonstrates protection in non-human primates and may also induce maternal immunization. Maternal Immunization is the phenomena of inducing passive immunity via maternal antibody. This occurs via transfer of IgG using the FcRn. This receptor binds the Fc portion of IgG in a pH dependent manner which facilitates transcytosis of IgG across the placental, mammary tissue, as well as gut epithelial layer. In rodents, maternal IgG is transferred across the gut epithelia throughout the nursing period. This project demonstrates in mice the immunogenicity of a protein subunit vaccine comprised of Ebola glycoprotein and adjuvant administered during gestation as well as the transfer of antigen specific maternal antibody to offspring through milk. To investigate maternal immunization we vaccinated female Swiss Webster mice during gestation and measured antigen specific IgG levels in both the mothers and pups as well as in the milk. Antigen specific maternal antibody titers found in pup sera mimic those found in fully immunized adult females weeks after weaning, indicating the potential for maintaining an antigen specific humoral response in neonates.
Intravenous BCG protects SIV+ macaques from tuberculosis.
Authors: Larson Erica C , Rodgers Mark A , Ellis Amy L , Ameel Cassaundra L , Gleim Janelle L , Gubernat Abigail K , Balgeman Alexis , Moriarty Ryan V , Darrah Patricia A , Maiello Pauline , Roederer Mario , Seder Robert A , O’Connor Shelby L , Scanga Charles A
Published: February 2023

Tuberculosis is the most common cause of death due to infection in people living with HIV (PLHIV). BCG, a live attenuated Mycobacterium bovis strain given intradermally to infants, is the only licensed vaccine to prevent TB. However, intradermal BCG offers little protection from pulmonary TB in adults and safety concerns limit its use in PLHIV. Recently, intravenous (IV) BCG has been shown to provide striking protection from TB in rhesus macaques. Given this dramatic success, we tested whether IV BCG could protect macaques with a pre-existing SIV infection using our established model of SIV/Mtb coinfection in Mauritian cynomolgus macaques (MCM). MCM were intrarectally infected with SIVmac239 and 5 months later were vaccinated with BCG at 8 ×107 CFU delivered IV. To prevent disseminated BCG disease in immunocompromised animals, four weeks after IV vaccination animals were treated with an 8-week regimen of isoniazid/rifampin/ethambutol (HRE). Four weeks after HRE cessation, animals were challenged with low-dose Mtb Erdman. SIV+ MCM exhibited no signs of BCG disease before or after HRE treatment. Following BCG vaccination, a rapid and sustained increase in airway T cells was observed in both SIV+ and SIV-naïve animals. Increased antibody titers to Mtb lysate were observed in plasma and airways following IV BCG. PBMC responses to Mtb-specific stimuli by IFNγ ELISpot indicated rapid and early clearance of Mtb in vaccinated animals regardless of SIV infection. All SIV-naïve (n=7) and 7 out of 10 SIV+, vaccinated animals were free of TB and without culturable bacilli in their tissues at 12 weeks post challenge. Flow cytometric analysis of PBMCs and tissues obtained at necropsy are underway to further define immune correlates of protection.
In-vivo engineering of B cells elicits memory retention and allows for secretion of broadly neutralizing antibodies in mice
Authors: Nahmad Alessio David , Lazzarotto Cicera , Zelikson Natalie , Kustin Talia , Tenuta Mary , Huang Deli , Reuveni Inbal , Horovitz-Fried Miriam , Dotan Iris , Rosin-Arbesfeld Rina , Nemazee David , Voss James , Stern Adi , Tsai Shengdar , Barzel Adi
Published: February 2023

A potential single-shot HIV therapy may be transplanted engineered B cells allowing strong secretion of broadly neutralizing antibodies (bNAbs). However, extensive, and expensive ex-vivo manipulations performed in specialized facilities hinders clinical potential of this approach. Furthermore, allogeneic B cell therapy necessitates MHC-II compatibility to receive mandatory T-cell help.

To overcome these limitations, we engineer B cells in-vivo. In particular, we demonstrate that a single, systemic dose of dual AAV, one coding for CRISPR/Cas9 and another coding for a bNAb donor cassette, allows for site specific integration in B cells. Following immunizations, we show memory retention and bNAb secretion at high titers. Antibodies secreted by the engineered B cells were found to be of multiple isotypes and IgGs could neutralize autologous and heterologous pseudoviruses. We found engineered B cell subsets in the spleen and blood. We detected homing of in-vivo engineered cells to germinal centers and bone marrow. Biodistribution of the donor AAV over time and as compared to a CRISPR- group, indicated expansion of engineered B cells in lymphatic tissues. We determined minimal CRISPR/Cas9 off-target cleavage, using unbiased, highly sensitive, CHANGE-Seq analysis. Finally, we diminished on-target, non-productive double-strand breaks at undesired tissues by expressing Cas9 from a B cell specific promoter.

Eliciting a specific, neutralizing serological response to hypervariable viruses is a long-standing challenge in medicine. B cell engineering provides an opportunity to express therapeutic antibodies to generate an adaptive and evolving immunity.
Regulation of Inflammatory Bowel Disorder Inflammation by ST8Sia6
Authors: Crotts Sydney B , Friedman David J , Shapiro Michael J , Rajcula Matthew , Shapiro Virginia Smith
Published: February 2023

Inflammatory bowel disorders (IBD), including Crohn’s disease and colitis, have a prevalence between 10–20%. IBD patients experience a reduced quality of life due to symptoms such as diarrhea, abdominal cramps, anal bleeding. The exact etiology of IBD is incompletely understood, but the immune response plays a critical role. Innate immune cells integrate the sum of stimulatory and inhibitory signals to determine overall activation. Solidifying the roles of pertinent proteins and their interactions is critical in mapping the immune response and understanding the implications of clinical treatment of IBD. ST8Sia6 is a sialic acid transferase which adds terminal a2,8 disialic acids to cell surface glycoproteins, and we have found that ST8Sia6 generates ligands for Siglec-2 and Siglec-E on innate immune cells, including colonic macrophages and neutrophils. These siglecs are inhibitory sialic acid-binding membrane receptors expressed on several immune cell subsets. Restraining ST8Sia6 should therefore enhance the immune response by reducing siglec-mediated inhibition. To examine this, our lab has generated a novel ST8Sia6 knockout (KO) mouse. At baseline these mice are healthy, but we have found that ST8Sia6 KO mice challenged with dextran sodium sulfate (DSS)-induced colitis lose weight and succumb to colonic inflammation significantly faster than their wildtype (WT) counterparts, particularly at low doses. ST8Sia6 KO mice exhibit increased histological damage and immune infiltration of the colon and small bowel as compared to WT controls. This data demonstrates a role for ST8Sia6 in regulating the hose immune response to inflammatory bowel disorders.
Loss of IL17 signaling in lupus-prone Ets1 knockout mice leads to worsened autoimmunity combined with increased susceptibility to Staphylococcal skin infections
Authors: Battaglia Michael , Sunshine Alex , Luo Wei , Jin Richard , Garris Matthew , Stith Alifa , Lindemann Matt , Miller Lloyd S , Sinha Satrajit , Wohlfert Elizabeth , Garrett-Sinha Lee Ann
Published: February 2023

Ets1 is a lineage-specific transcription factor that regulates B and T cell functions in development and disease. Mice that lack Ets1 (Ets1 KO) develop autoimmune disease with high levels of autoantibodies. Naïve CD4+ T cells isolated from Ets1 KO mice differentiate more readily to IL17 producing Th17 cells. To determine if IL17 production contributes to the observed autoimmunity in Ets1 KO mice, we crossed Ets1 KO mice to mice lacking the IL17 receptor A subunit (IL17RA KO) to generate double knock out (DKO) mice. We found that the absence of IL17RA signaling did not ameliorate the autoimmune phenotype of Ets1 KO mice. Instead, DKO animals exhibited worse symptoms with increases in activated B cells, autoantibody titers, T helper 2, and T follicular helper cells. DKO mice showed aspects of immunodeficiency, developing spontaneous skin lesions colonized by Staphylococcus xylosus. When DKO mice were infected with Staphylococcus aureus they were unable to clear the bacteria, indicating a general immunodeficiency to Staph bacteria. Gene expression analysis revealed diminished expression of the Skint gene family in DKO skin. Skint genes play important roles in the maintenance of TCRγδ+ dendritic epidermal T cells (DETC) and in skin wound healing. DKO mice had reduced numbers of DETC in the skin, which may lead to increased susceptibility to Staph infections. We propose that Staph skin infections in DKO mice leads to enhanced immune activation and enhanced autoimmunity. Our studies suggest that therapies targeting IL17RA signaling in autoimmune diseases may result in increased autoimmunity or to increased susceptibility to skin Staph infections.
Utility of immunological and neuronal markers in blood to identify relapse in Multiple Sclerosis patients
Authors: Gawde Saurabh , Axtell Robert C.
Published: February 2023

Multiple sclerosis is a demyelinating disease caused by immune cell infiltration into the Central Nervous System (CNS). Relapsing Remitting Multiple Sclerosis (RRMS) is characterized by episodes of worsening disability (relapses) followed by complete or partial recovery (remissions). Currently, MRI scans are used to conclusively identify relapses, but there is a need for an inexpensive, non-invasive test for monitoring relapses and remission status in MS patients. Serum neurofilament light (NFL) has emerged as a biomarker for relapse in MS. However, a test using NFL alone has poor sensitivity and specificity to identify an MS relapse. Our goal was to identify a panel of biomarkers that could improve the accuracy of distinguishing relapse and remission in MS patients. We used a multiplexed, sensitive assay to measure levels of blood proteins in two cohorts of RRMS patients – a cross-sectional cohort and a longitudinal cohort. In the cross-sectional patient cohort, we found five serum proteins were elevated in relapse, including NFL. Furthermore, we identified 94 serum proteins that were significantly reduced in relapse compared to remission, including IFN responsive chemokines CXCL9 and CXCL11. For the longitudinal cohort, pairwise comparisons found that five plasma proteins were elevated in relapse compared to remission, including NFL. Nine plasma proteins were significantly reduced in relapse compared to remission, including the IFN responsive chemokines CXCL9 and CXCL10. In conclusion, our studies provide evidence that a panel including NFL and IFN responsive chemokines in the blood may serve as biomarkers to distinguish relapse and remission in RRMS patients.
Inflammatory response within the nervous system in Tat induced HIV-associated sensory neuropathy
Authors: Cao Ling , Higley Jane , Healy Ryan , Harrison Benjamin
Published: February 2023

HIV sensory neuropathy (HIV-SN) affects 30–50% of patients infected with HIV. The role of Tat in HIV-SN has not been studied extensively. Previously, using the doxycycline (DOX) inducible HIV-1 Tat transgenic (iTat) mice, we showed that Tat expression induced neuropathy-like behaviors. Here, we further investigated the role of inflammatory responses within dorsal root ganglia (DRG) and lumbar spinal cord in the development of peripheral neuropathy. To induce Tat expression, DOX was given to iTat mice daily (i.p.) for 14 days. Lumbar spinal cord and DRG tissues were collected at days 0 (no injection), 3, 7, 14, 21, 28 and 35 following the initial injection of DOX. Vehicle control mice received pH-matched phosphate-buffered saline injections. qRT-PCR was performed with DRG tissues. Tat expression induced either transient or progressive upregulation of RNA levels of CCL2, CCL3, CCL4, CCL5, CXCL10, and IL-1beta. Lumbar spinal cord tissues underwent NanoString RNA analysis using a panel of 200+ inflammation-related genes. The patterns of the changes of these genes over time were clustered via the Short Time-series Expression Miner program. Out of the 4 patterns identified, the most significant one was a cluster of genes that were downregulated over time upon Tat induction. Gene ontology term enrichment analysis of this gene cluster via the PantherGo tool further identified a set of genes (IL-6, MAPK2K4, IL-10, HRAS1, BCL2L1, RHOA, JUN, and CREB1) that prevent/inhibit neuronal cell death/apoptosis. In all, both upregulation of selected pro-inflammatory cytokines within DRG, and down regulation of inflammation-related, neuronal survival genes within the spinal cord, could contribute to Tat-mediated peripheral neuropathy.
Obesity effects serum levels of SARS-CoV-2-specific antibodies in COVID-19 patients
Authors: Blomberg Bonnie , Reidy Lisa , Romero Maria , Diaz Alain , Cray Carolyn , Kahl Kristin , Frasca Daniela
Published: February 2023

SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona Virus-2), cause of COVID-19 (Coronavirus Disease of 2019), represents a significant risk to people living with pre-existing conditions including increased inflammatory responses and consequent dysfunctional immunity.

In these studies we evaluated the effects of obesity, a condition associated with chronic systemic inflammation, on the secretion of SARS-CoV-2-specific IgG antibodies in the blood of COVID-19 patients. Results showed that SARS-CoV-2 IgG antibodies are negatively associated with Body Mass Index (BMI) in COVID-19 obese patients, as expected based on the known effects of obesity on humoral immunity, as we have previously published for the response to the influenza vaccine. Antibodies in COVID-19 obese patients are also negatively associated with serum levels of pro-inflammatory and metabolic markers of inflammaging and pulmonary inflammation, such as SAA (serum amyloid A protein), CRP (C-reactive protein) and ferritin, but positively associated with NEFA (nonesterified fatty acids). These results altogether help to identify an inflammatory signature with strong predictive value for immune dysfunction that could be targeted to improve humoral immunity in individuals with obesity as well as with other chronic inflammatory conditions.
HIV infection drives immune activation of angiogenic T cells
Authors: Li Tong , Zhu Ziang , Chen Jinya , Kumar Jai , Kumar Princy , Catalfamo Marta
Published: February 2023

In people living with HIV (PLWH), immune activation and systemic inflammation are associated with cardiovascular risk independently of the traditional risk factors or duration of antiretroviral treatment and CD4 counts. Endothelial inflammation and injury play a critical role in the pathogenesis of cardiovascular risk/disease. Recent reports had shown that T cells can promote vascular repair by secretion of proangiogenic factors. Because of these functional properties these T cells are called angiogenic T cells (Tang) and are defined by expression of CXCR4 and CD31. Accordantly, we found that in vitro, TCR stimulated CD8 Tang from healthy volunteers induced secretion of proangiogenic cytokines including angiopoietin-2, fibroblast growth factor, and vascular endothelial growth factor (VEGF) by HUVECs. Because HIV chronic infection drives T cell immune activation, we hypothesized that HIV infection alters Tang’s proangiogenic function. To address this question, we studied the phenotype and function of Tang in PBMCs from PLWH and healthy volunteers. We found that PLWH had a significant decrease in the proportion of both CD4 and CD8 Tang when compared to healthy controls, and this difference was more striking in the CD8 Tang. CD8 Tang from PLWH had an activated phenotype and showed an increased expression of granzymes (A and B) when compared to healthy controls. In PLWH, the frequency of GZA+GZB+CD8 Tang was positively associated with 10yr cardiovascular risk score. In addition, upon TCR stimulation, CD8 Tang from PLWH showed a diminished ability to secrete VEGF. These results suggest that in the context of HIV infection, CD8 Tang possesses a pro-inflammatory potential and reduced proangiogenic function to promote endothelial repair.
Patient-reported comorbidity affects symptom duration in non-hospitalized patients with mild to moderate COVID-19 in a clinical study
Authors: Haran John P , Zheng Yan , Alonzo-Palma Norma , Wingertzahn Mark
Published: February 2023

Background:

Most COVID-19 cases in the US are managed via outpatient care, yet the outpatient natural history of COVID-19 is not well understood. We assessed outpatient natural history of COVID-19 as part of a clinical food study evaluating KB109 safety and its effects on select health measures when combined with supportive self-care (SSC) vs SSC alone. KB109 is a novel synthetic glycan developed to support immune system homeostasis through gut microbiome modulation. The SSC-alone arm permitted COVID-19 natural history exploration, the focus of this abstract.

Methods:

Adult patients were randomized 1:1 to SSC alone or SSC+KB109. Randomization was stratified by site, age subgroup, and comorbidity status. Signs/symptoms of COVID-19 and other end points were evaluated over a 35-d study period. Patients self-assessed 8 COVID-19–related cardinal symptoms; 5 additional symptoms were collected (13 overall symptoms).

Results:

Overall, 350 patients were randomized (SSC alone, n=176). Interim results are presented; full analysis expected in March 2021. In the SSC-alone interim results (n=89), 36.0% of patients self-reported ≥1 comorbidity. Median time to resolution of the 8 cardinal symptoms was 12d in patients with no comorbidities vs 27d in patients with ≥1 comorbidity (HR=1.708 [0.875, 3.332]). Median time to resolution of the 13 overall symptoms was 14d in patients with no comorbidities vs 27d in patients reporting ≥1 comorbidity (HR=1.800 [0.953, 3.400]). The most common comorbidities were hypertension (19.1%) and chronic lung disease (9.0%).

Conclusions:

This is the first clinical study to show ≥1 comorbidity is associated with a protracted time to resolution of symptoms in non-hospitalized patients with mild to moderate COVID-19.
Stearoyl-CoA desaturase mediated monounsaturated fatty acid availability supports humoral immunity
Authors: Zhou Xian , Zhu Xingxing , Li Chaofan , Shapiro Virginia , Hitosugi Taro , Sun Jie , Copland John A. , Zeng Hu
Published: February 2023

Immune cells can metabolize glucose, amino acids, and fatty acids (FAs) to generate energy. The role of different FA species, and their impacts on humoral immunity remains poorly understood. Here we report that proliferating B cells require monounsaturated FAs (MUFA) to maintain mitochondrial metabolism and mTOR activity, and to prevent excessive autophagy and endoplasmic reticulum (ER) stress. Immune challenges increase systemic MUFA availability in vivo. Furthermore, B cell extrinsic Stearoyl-CoA desaturase (SCD) activity generates MUFA to support early B cell development and germinal center (GC) formation in vivo during immunization and influenza infection. Thus, SCD-mediated MUFA production is critical for humoral immunity.
Type I interferon enhanced double negative 2 B cell differentiation and lupus autoantibody production
Authors: Lu Changming , Liu Shanrun , Gao Min , Hsu Hui-Chen , Mountz John D
Published: February 2023

Systemic Lupus Erythematosus (SLE) is characterized by high level of type I interferon (IFN) and abnormal expansion of effector B cells including IgD−CD27−double negative (DN), CD21−T-bet+DN2 B cells, and antibody secreting cells (ASCs). It remains unclear as to the role of type I and type II interferon (IFN) to promote DN2 B cell differentiation. The in vivo effects of type I IFN signaling in promoting DN B cells were accessed using the BXD2 mouse model of lupus which exhibit a low Th1 response, compared to normal B6 mice. Compared to normal B6 mouse, there was a significantly elevated percent of DN B cells in the BXD2 mice, but this was reduced in BXD2 mice that are deficient in IFNAR1 (BXD2-Ifnar1−/−). Compared to wild-type BXD2 mice, there was a lower level of IgG autoantibodies specific to DNA, histone and Smith (Sm) in BXD2-Ifnar1−/− mice. BXD2-Ifnar1−/− B cells presented impaired differentiation into the DN and the DN2 populations under an in vitro stimulation condition in the presence of IFNb and a TLR7 agonist. To determine if type I IFN stimulation can direct differentiation of healthy B cells to these effector B cells without the influence of type II IFN, B cells purified from healthy individuals were stimulated under a DN stimulation condition (anti-Ig+IL-21+BAFF+TLR7) in the presence and absence of IFNb. We found that IFNb significantly increased the percentage and of DN and DN2 B cells, but decreased the percentage of CD27+CD38+ plasmablasts/plasma. Under the same condition, IFNb increased the secretion of autoantibodies specific to DNA, histone and Sm. Our results suggest that type I IFN signaling stimulates DN2 B cell differentiation and can promote the abnormal expansion of these B cells in vivo independent of type II IFN.
Epigenetic regulation of IgA class-switching by miR-146a through selective downregulation of Smad2, Smad3 and Smad4
Authors: Casali Paolo , Moroney Justin B , Daw Cassidy , Morales Grecia , Chupp Daniel P , Zan Hong
Published: February 2023

IgA is the predominant antibody isotype at mucosal surfaces. It plays a critical role in mucosal homeostasis and provides a first line of defense in immune protection. Class switch DNA recombination (CSR) to IgA is initiated by TGF-β, which induces Smad2, Smad3, Smad4 and Runx3 binding to the Iα promoter. This promotes germline Iα-Cα transcription, thereby increasing accessibility to the Igα locus. Epigenetic modifications, such as microRNAs, have been shown to modulate the antibody response, although the role of microRNAs in B cell differentiaton processes, such as guiding CSR to specific Ig isotypes, remains unexplored. miR146a has been shown to regulate T cell, dendritic cell and macrophage functions in innate and adaptive immune responses. Here we show that miR146a, which targets Smad2, Smad3 and Smad4 3′UTRs and is the most abundant miRNA in resting B cells, is significantly downregulated in B cells undergoing CSR to IgA. Deletion of miR146a, as in miR146a−/− B cells, frees Smad2, Smad3 and Smad4 from transcriptional repression, thereby increasing recruitment of these transcription factors to the Iα promoter. This results in increased germline Iα-Cα transcription and CSR to IgA without alterations to Aicda and Prdm1 expression. Accordingly, miR146a−/− mice displayed an elevated gut, systemic IgA responses and kidney IgA deposition, a hallmark of hyper IgA nephropathy. This was recapitulated in μMT/miR146a−/− mixed bone marrow chimera mice, further demonstrating the B cell-intrinsic role of miR146a in the modulation of CSR to IgA. Thus, by silencing Smad2, Smad3 and Smad4 transcription, miR-146a plays an important B cell intrinsic role in the modulation of the IgA response.
Hyper MAPK/ERK signaling in DCs causes increased LPS-induced TNFa at the post-transcriptional level
Authors: Minichino Danielle E , Behrens Edward
Published: February 2023

Background:

Langerhans Cell Histiocytosis (LCH) is an inflammatory disease driven by abnormal dendritic cells (DCs) with hyper-MAPK/ERK signaling, usually due to Braf-V600E mutation. Since DCs are poised with Toll like Receptors (TLRs), which utilize MAPK/ERK signaling to incite an immune response, we hypothesize that LCH cells have a hyper-TLR response.

Methods:

We use an animal model of LCH (CD11c-Cre:BRAFV600E-flox; which expresses Braf-V600E in DCs) to measure serum TNFa and tnfa transcripts in CD11c+ splenocytes after LPS injection. Cultured BMDCs were pre-treated with/without a V600E-inhibitor before LPS stimulation. We measured intracellular TNFa, secretion and reuptake of TNFa, tnfa transcripts, NFkB signaling, TNFa degradation, and TACE activity.

Results:

LCH mice have increased LPS-induced circulating TNFa compared to WT. LCH-BMDCs have reversible LPS-induced increase in TNFa secretion and intracellular accumulation, but decreased tnfa and tlr4 transcripts and NFkB signaling compared to WT. There is no difference in TACE activity, TNFa reuptake, or protein degradation.

Conclusions:

Hyper-MAPK/ERK signaling in DCs causes a hyper LPS-induced TNFa response, despite diminished LPS signaling. Increased intracellular TNFa indicates a cell intrinsic, post-transcriptional mechanism. Current experiments are analyzing translation of TNFa, which is implicated by MAPK/ERK signaling. These data indicate novel effects of Braf-V600E on LPS-induced TNFa production, and current studies are investigating whether this is a more global phenomenon.
Distinct chromatin modifications at the Il33 locus in mast cells contribute to sex dimorphic susceptibility to EAE, an autoimmune CNS demyelinating disease
Authors: Brown Melissa Ann , Yang Yuchen
Published: February 2023

Many autoimmune diseases, including multiple sclerosis (MS), exhibit a striking female bias, but the underlying mechanisms remain poorly understood. Using the SJL mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which recapitulates this sex dimorphism, we previously demonstrated that preferential IL-33 expression contributes to male protection. IL-33 expression by mast cells in PLP139–151-immunized males activates type 2 innate lymphoid cells, which in turn drives a non-pathogenic Th2 response to myelin peptide. Testosterone directly activates Il33 in male but not female mast cells, but this male-specific response is also observed with other modes of activation, suggesting there are constraints on the chromatin landscape that limit Il33 expression in females. Here we use chromatin immunoprecipitation (ChIP) and ATAC-seq to examine chromatin modifications at the Il33 locus. At steady state, ChIP assays reveal activating H3K4me3 and H3K9ac histone modifications at the promoter and conserved non-coding sequences within the Il33 gene are most prevalent in male-derived cells, while H3K27me3 repressive marks dominate in females. Increased basal Il33 chromatin accessibility assessed by ATAC-seq is also evident in males. Together, these results indicate that while testosterone can directly stimulate IL-33 production, it also exerts effects on Il33 during development conferring a higher potential for expression in males by altering the chromatin landscape. RNAseq analyses of IgE-activated mast cells revealed a broad range of sex-biased gene expression, indicating that sex hormone influences during development drive epigenetic changes and may be a common mechanism to explain sex dimorphism in immunity.
Mixed Lineage Kinase 3 Promotes Dexamethasone-Induced Thymocyte Apoptosis
Authors: Kumar Sandeep , Singh Sunil Kumar , Viswakarma Navin , Sondarva Gautam , Nair Rakesh Sathish , Rana Basabi , Rana Ajay
Published: February 2023

Dexamethasone (DEX) is a corticosteroid known to regulate T-lymphocyte growth/differentiation and used in the clinic as an anti-inflammatory and immunosuppressant agent. The glucocorticoid hormones, including DEX, can induce thymocyte apoptosis. It has been reported that DEX-induced thymocyte apoptosis is associated with an early stage ceramide generation. Since we reported earlier that ceramide induces Mixed Lineage Kinase 3 (MLK3) activation and therefore, we probed whether MLK3 inhibition could attenuate DEX-induced thymocyte apoptosis. Here, we report that genetic loss of MLK3, a member of MAP3K family attenuates DEX-induced apoptosis in thymocytes. To understand any differences in thymic T cell surface markers at the basal level between WT and MLK3−/− mice, we determined protein expression of various surface proteins involved in T cell activation and apoptosis. Flow cytometry analyses indicated that the genetic loss of MLK3 increases CD27 expression on thymic CD4+, CD8+, and CD4+CD8+ T cells; however, CD28 expression was mainly increased on CD8+ and CD4+CD8+ T cells due to loss of MLK3. Interestingly, genetic loss of MLK3 paradoxically regulated CD70 expression on thymic and splenic T cells. Furthermore, 4–5 weeks old WT and MLK3−/− mice were challenged with DEX for 24 h, and apoptosis in thymic T cells was determined. The flow cytometry results showed a significant decrease in the DEX-induced apoptosis in thymic CD4+, CD8+, and CD4+CD8+ T cells, isolated from MLK3−/− mice. Taken together, these results suggest that MLK3 could be a potential target for DEX-induced thymocyte apoptosis and its inhibitors can be used to mitigate thymocyte apoptosis.
Highly protective T-h17-polarized responses against Influenza A Virus develop in the absence of CD4 T cell-intrinsic T-bet and Eomes expression
Authors: Dhume Kunal , Finn Caroline M , McKinstry Karl Kai
Published: February 2023

Though bulk CD4 T cell responses against influenza A virus (IAV) are characterized as Th1, expression of the transcription factor T-bet (Tbx21), the ‘master regulator’ of Th1 fate, is not required for mice to clear IAV. While the Tbx21−/− CD4 T cells develop some Th17 characteristics in the infected lung, they still retain relatively high Th1 functionality including robust IFNγ production. Here, we show that the transcription factor Eomesodermin (Eomes) is required for the residual Th1 identity of Tbx21−/− cells by comparing Tbx21−/− and double Tbx21−/−Eomes− /− CD4 T cells in an adoptive transfer model of IAV infection. The Tbx21−/−Eomes−/− cells not only lose Th1 attributes but develop much stronger Th17 attributes versus Tbx21−/− cells. The elaboration of Th17 responses by Tbx21−/− Eomes−/− cells is not a default effector state but requires receipt of both IL-6 and TGFβ in IAV-infected lungs. Similar Th17 identity in the absence of Th1 attributes are seen analyzing CD4 T cells in Tbx21−/−Eomes−/− mice infected with IAV, and this response correlates with protection as CD4 T cell depletion abrogates viral control. To more stringently test the protective capacity of Th17 responses, we transferred in-vitro Th17 primed WT or Tbx21−/−Eomes−/− effectors to unprimed WT hosts and challenged with lethal IAV. Both populations protected, but while WT Th17 cells gained Th1 characteristics in vivo, Tbx21−/−Eomes−/− cells strengthened their Th17 functionality remarkably without any shift towards Th1 identity. Our observations thus show that prototypical Th17 cells, that cannot gain Th1 attributes through mechanisms of plasticity often seen during pathogen responses, can mediate strong protection against IAV infection.
Helicobacter pylori infected gastric epithelial cells drive dendritic cells towards a tolerogenic phenotype.
Authors: Hashimi Marziah , Schearer Julia , Sebrell Thomas , Gentry Andrew B , Bimczok Diane
Published: February 2023

The interactions of dendritic cells (DCs) with the gastric epithelium are thought to play a role in shaping DC function and thus the adaptive immune response to Helicobacter pylori (H. pylori) infection. We have shown that DCs are recruited to the H. pylori-infected gastric epithelium, positioning them for uptake of bacterial antigen from the gastric lumen. Using a gastric organoid model of H. pylori infection, we sought to define how the interactions between gastric epithelial cells and DCs regulate the antigen-presenting cell capacity of DCs. Supernatants were collected from organoids microinjected with H. pylori (infected) or PBS (mock), and monocyte-derived dendritic cells (MoDCs) were incubated with these supernatants. Flow cytometry data showed that the supernatant from infected organoids drove the DCs toward a tolerogenic state, as the expression of CD40, CD83, and CD86 was significantly decreased compared to incubation with supernatants from mock infected organoids, whereas CCR7 expression was increased consistent with a more tolerogenic phenotype. DCs treated with supernatants from infected, but not mock-infected organoids suppressed the proliferation of naïve autologous T cells. We also assessed T cell polarization using a cytokine array and found an increase in Th1, Th17 and Treg cytokines, but a decrease in Th2 cytokines in T cells that were challenged with DCs pre-treated with supernatants from infected organoids compared to mock-infected organoids. In summary, we have shown that H. pylori-infected gastric epithelium drives the DCs toward a tolerogenic state, resulting in reduced expression of maturation markers and CD4+ T cell proliferation while maintaining the DCs’ ability to induce T cell cytokine secretion.
In vitro expanded human regulatory T cells (tTreg) and CD4+ effector T cells (Teff) display distinct metabolic demands and response to lactate.
Authors: Hippen Keli L , Zhang Yigang , Aguilar Ethan George , Rhee Stephanie , Wagers Sara Bolivar , O‘Connor Roderick , Rathmell Jeffrey C , Blazar Bruce R
Published: February 2023

Thymus-derived regulatory T cells (tTreg) have been used as a cellular therapeutic to prevent or treat autoimmunity, graft rejection, and graft versus host disease (GVHD) in humans. Metabolic pathways affect tTreg differentiation, expansion and suppressive function. Recent experiments challenge the initial paradigm that tTregs depend on fatty acid oxidation (FAO) and glycolysis. Murine studies also show that central memory and effector tTreg have distinct metabolic profiles, but human studies to date use pooled populations of tTreg. The goal of the current study was to identify and compare the metabolic pathways critical for naïve and memory human tTreg, and to determine whether these pathways differ from naïve human CD4 T cells. We found that memory tTreg have higher rates of glycolysis and oxidative phosphorylation (OXPHOS) than naïve tTreg, both at rest and after stimulation. Activated naïve and memory tTreg had high basal OXPHOS, but neither glucose nor FAO were major substrates. Consistent with this, culture in low glucose did not affect expansion of either naïve or memory tTreg, but greatly decreased that of CD4 Teff. Extracellular lactate increased tTreg basal OCR and, in contrast to the lactate-dependent inhibition of CD4 T cells, enhanced tTreg expansion. This is consistent with differential expression of a class of lactate transporters (monocarboxylate transporters, or MCT) where Treg have increased expression of MCT1/2, which favor import whereas Teff have increased expression of MCT4, which favors export. These data demonstrate that naïve and memory tTreg should be analyzed separately, and provide a molecular insight into why tTreg, but not Teff, function in a low glucose, high lactate environment.
Activation of B10 cells via gut microbiota in the pathogenesis of systemic lupus erythematosus
Authors: Prakash Meeta B , Luo Xin M , Mu Qinghui , Edwards Michael R
Published: February 2023

Systemic Lupus Erythematosus(SLE), a disease of various genetic and environmental origins, is often characterized by immune complexes causing inflammation and damage in various organ systems, such as the brain, kidneys, lung, joint, and skin. Current treatment for SLE include steroidal and nonsteroidal anti-inflammatory drugs, antimalarial agents, and immunosuppressive medications. These treatments primarily reduce symptoms, and patients may develop side effects with long-term care. The gut microbiome has an important role in various metabolic and immunological diseases. A previous study found that increased Lactobacillus colonization restored the mucosal barrier, and increased abundance in these bacteria attenuated SLE symptoms through reversing a ‘leaky gut’. This allows for potential therapies of SLE with probiotics. In preliminary trials, vancomycin treatment during active disease killed gram-positive bacteria, but spared Lactobacillus, and attenuated symptoms. Surprisingly, significantly decreased amounts of CD10+CD19+ B cells that produce IL-10 (B10) were found when vancomycin was given before the onset of lupus. Vancomycin treatment before disease onset significantly removed bacterial DNA from the gut and circulation, causing exacerbation of clinical symptoms. Oral gavage of activated B10 cells in vancomycin treated mice rescued the negative effects of vancomycin treated mice at the pre-disease stage. This work suggests that a form of oral tolerance induced by bacterial DNA-mediated expansion of B10 cells suppress disease onset in the autoimmune-prone MRL/lpr mouse model. Future studies are warranted to further define the mechanism behind bacterial DNA promoting B10 cells.
Specific coping strategies in JSLE depression and anxiety – the untold story of brave soldiers
Authors: Poole Brian D , Silva Beatriz , Figueiredo-Braga Margarida
Published: February 2023

Background and aims: Patients with Juvenile-onset Systemic lupus erythematosus (jSLE) have to overcome the burden of unpredictable and disabling disorder. Coping strategies influence patient’s ability to cope with negative emotions, treatment exigencies and physical impairment related with the disease. Our aim was to explore the relationship between coping mechanisms and the presence of anxiety and depression.

Methods:

Thirty jSLE patients answered the Brief Coping Orientation to Problems Experienced (COPE) questionnaire, the Hospital Anxiety and Depression Questionnaire (HADS) and the Subjective Happiness Scale (SHS).

Results:

Clinically relevant anxiety (63.3%) and depressive symptoms (13.3%) were present in the jSLE patients. Our population showed higher coping scores for problem-focused (0,7±0,2), emotion-focused (0,6±0,2) and social supported coping (0,5±0,2), than avoidant coping (0,3± 0,1). Higher depression scores and lower subjective happiness levels correlated with higher anxiety scores (r=0.673, p=0.000 and r=0.592, p=0.001, respectively). Problem focused coping was associated with higher self-reported happiness (r=0.564, p=0.002) and avoidant coping was positively correlated with higher number of anxious symptoms (r=0.560, p=0.002).

Conclusions:

These results underscore the relevance of psychological assessment in jSLE patients. Adaptive coping strategies led to greater happiness, while avoidant coping strategies were associated with higher anxiety. Teaching of adaptive coping mechanisms, in combination with other interventions are likely to enhance the patients’ ability to respond to the multiple physical, social and emotional challenges imposed by this autoimmune disorder.
STING pathway mediated immunologic consequences of DNA damage repair gene losses in high-grade serous ovarian tumors
Authors: Shakfa Noor E. , Lightbody Elizabeth , Li Deyang , Wilson-Sanchez Juliette , Afriyie-Asante Afrakoma , Chenard Stephen , Hamade Ali , Tyryshkin Kathrin , Koti Madhuri
Published: February 2023

High grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy with high rates of chemotherapy resistance and poor outcomes. Our previous studies have demonstrated the variable tumor immune microenvironment states that associate with platinum chemotherapy response. We further showed the significance of the interferon induced chemokine CXCL10 as a key mediator of tumor infiltrating immune cell recruitment. Using the ID8-Trp53−/− murine model of HGSC, we demonstrated the potential of Stimulator of Interferon Genes (STING) pathway activation in enhancing response of HGSC tumors to carboplatin chemotherapy and sensitizing them to immune checkpoint blockade. Notably, CXCL10 production via IFN1 is also governed by genes that regulate cellular DNA damage repair pathways. Evolving evidence indicates a role of BRCA1 and PTEN genes in mediating cellular IFN1 responses. Losses in the function of these genes is widely prevalent in a large proportion of HGSC tumors, where tumors with BRCA1 mutations have higher CD8+ T cell infiltration in contrast to those with loss of PTEN. We hypothesized that HGSC tumors with loss of PTEN expression can be rendered susceptible to immune mediated killing via activating the STING pathway. Tumors generated from ID8-Trp53−/−; Brca1−/− cells and those from ID8-Trp53−/−; Pten−/− cells in C57BL6 mice showed significant immunologic differences. STING agonist treatment significantly increased chemosensitivity and improved overall response in mice implanted with ID8-Trp53−/−; Pten−/− cells compared to those treated with carboplatin alone, altering immune responses. This study is foundational to guide rationalistic combinations of STING pathway activating therapies in HGSC.
Targeted Chemotherapy and Checkpoint Blockade Mediate Synergistic Protection Against BCR-ABL+ B Cell Leukemia
Authors: Venkatesh Hrishi , Tracy Sean I. , Hekim Can , Harris Lynn-Heltemes , Farrar Michael A.
Published: February 2023

Immunotherapy using anti-PDL1/anti-CTLA4 checkpoint blockade has been successfully employed for cancers with many non-synonymous mutations. Whether checkpoint blockade-based approaches can be adapted to cancers with low mutational burden, such as BCR-ABL+ B cell leukemia, is still unclear. We have previously demonstrated that the endogenous anti-tumor T cell response, although important for initial control of leukemia, is unable to control leukemia by itself. We show here that leukemia induces an exhaustion phenotype in activated T cells, as well as an associated loss of effector function, that could contribute to the inability to clear leukemia. However, checkpoint blockade therapy using aPDL1 and aCTLA4 by themselves had no impact on survival in leukemic mice. Combining aPDL1 blockade with targeted chemotherapy using the BCR-ABL inhibitor nilotinib had a significant synergistic therapeutic effect. The effectiveness of the aPDL1-nilotinib dual therapy was primarily dependent on CD4 T cells, indicating a key role for endogenous anti-tumor CD4 T cells in leukemia clearance. Indeed, a significant proportion of long-term surviving nilotinib + aPDL1-treated mice were protected against a secondary leukemia re-challenge; indicative of an effective memory T cell responses. Thus, combination therapeutic approaches involving checkpoint blockade can elicit potent anti-tumor CD4 T cell responses that induce long-term protection against leukemia. Future studies will explore the mechanisms by which CD4 T cells orchestrate protective anti-leukemia responses.
Obese microenvironments attenuate the efficacy of endogenous and CD19-directed CAR T-cells
Authors: Ank Raira Silva , Henry Curtis J.
Published: February 2023

Background:

Hematological malignancies represent the largest class of cancers in pediatric patients. Despite successes in treating primary diseases, patients with relapsed B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL) have significantly lower survival outcomes. This has led to the use of immunotherapies, notably chimeric antigen receptor (CAR) T-cells, to treat relapse. Unfortunately, only 50% respond to CAR-T therapy, with relapse common in less than a year.

Objective:

Given that obesity is associated with poorer survival outcomes in pediatric patients with B-cell malignancies and promotes a loss of immune homeostasis, we hypothesize that obesity compromises the function of endogenous and engineered T-cells in B-cell malignancies.

Results:

Using in vitro models of adiposity and diet-induced obesity mouse models to study the impact of obesity on primary T-cell function, we observe defects in proliferation, cytokine production, and the upregulation of cytolytic mediators when cultured in adipocyte but not in bone marrow cell or unconditioned media. The introduction of CD19-direct CARs did not overcome adipocyte-induced defects in T-cell function, which resulted in increased PD-1 surface expression, compromised cytokine production, and defective killing of human CD19-expressing B-ALL cells. Interestingly, human B-ALL cells cultured in adipocyte-secreted factors downregulated CD19, suggesting that adipocytes promote B-ALL pathogenesis by compromising T-cell function and reducing the surface expression of antigens on target cells.

Conclusion:

Our studies are the first to demonstrate the inhibitory impact of the adipocyte secretome on CAR T-cell function in models of B-ALL.
Single-cell mapping of human endometrial cancer reveals tumor-specific instruction of tissue-infiltrating leukocytes
Authors: Tong Huan , Feng Hao , Wan Xiaoping
Published: February 2023

Background:

Endometrial cancer (EC) is a hormone-related carcinoma with increased morbidity among female patients of all backgrounds. For second-and third-line treatment, evaluation of tumor DNA mismatch-repair function by determining microsatellite instability (MSI) status is essential to choose proper targeted therapies. Pembrolizumab, a PD-1 immune checkpoint inhibitor, is an effective agent for patients with high-MSI endometrial cancer. Advanced or recurrent ECs identified as MSS are more sensitive to Pembrolizumab plus Lenvatinib, a receptor tyrosine kinase inhibitor (TKI), which slows the growth of endometrial cancer cells. Unrevealing the complex immune microenvironment of EC and the crosstalk between leukocytes and tumor cells is essential for providing an immunotherapeutic strategy.

Method:

the leukocyte landscape of endometrial carcinoma was mapped using bioinformatic analysis and high-dimensional single-cell profiling (CyTOF), which contained 42 biomarkers as the panel.

Result:

The heterogeneous composition of tissue-resident and invading immune cells within endometrial cancer and adjacent normal tissues permitted a clear distinction. NK cells, monocyte-derived macrophages, and neutrophils were enriched in adjacent normal tissue. A Th9 subtype of T cells with CCR5 and CD38 positive expression was recruited to the invasive margin. PD1 was elevated for these Th9 cells. Intriguingly, PD1+ESRneg T cells, Treg cells, and ESR+ leukocytes such as ESR+ granulocytes and Siglec1+CCR5+CD40+ESRhi macrophage were enriched in the infiltrating immune cells.

Conclusion:

Defining the specific immunological signature of EC can facilitate the rational design of targeted immunotherapy strategies.