bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-cell transcriptome reveals the developmental progression of human ILCs from the bone marrow to lymph nodes
Authors: Hashemi Elaheh , McCarthy Colleen , Malarkannan Surammaniam
Published: August 2023

Innate lymphoid cells (ILCs) are largely tissue-resident and do not express clonotypic receptors. In humans, ILCs are primarily described within the mucosal tissues. However, their development and functions in the human draining lymph nodes (LN) and bone marrow (BM) are unknown. Here, we performed single-cell RNA sequencing to unravel the tissue-specific transcriptional profiles of 47,287 ILCs within the human abdominal and thoracic LNs and compared them with that of BM and spleen. We found a higher frequency of CD127+ ILCs in LNs than in BM or spleen. We identify different stages of early progenitors in BM including CD34+HSCs and NFIL3+ITGA4+TCF7+ EILP. Our data indicated BM ILC progenitor cells differentiate into a unique subset of IL7R+KIT+PLZF+ progenitor ILCs (pILCs) that are predominantly present in LNs. Transcriptomically, ILC3 are closer to pILCs compared to ILC1 and ILC2. Using unsupervised clustering, we defined three clusters of ILC1, six clusters of ILC3, and only a minimal presence of ILC2 in the LNs. ILC1 and ILC3 have clusters with a high level of heat shock protein genes including, HSPA1A, HSPA1B, and DNAJB and clusters possibly trafficking from BM with high level of chemokine receptor, CXCR4. ILCs from draining LNs are polyfunctional and can produce cytokines in response to cytokines in vitro. Our study is the first characterization of human ILCs in LNs that provides an in-depth understanding of ILC-mediated immunity in humans.
Primary immunodeficiency disorders (PID) or inborn errors of immunity : preliminary work West Africa Côte d’Ivoire
Authors: Siransy Liliane K , Adou A Honore , Kira Jokebed , Moussa N Salimata , Sery Jocelyne , Oura Doris
Published: August 2023

The immune system is a particularly sophisticated protection system. As a true guardian of our health, the immune system defends our body against external or internal aggressions. Sometimes, the immune system is not able to fight because it lacks or does not function properly causing an ineffective or absent immune response. Although more than 400 diseases exist, there is no data available in our country.

We analyze through a retrospective study carried out in the pediatric department at Hospital University Center, in Abidjan, between February and April 2022, involving 93 PID suspect children, the epidemiological, clinical, paraclinical and therapeutic aspects.

The average age of our patients was 2 years with extremes of 22 days to 15 years, with a male predominance: sex ratio of 1.45. The predominant geographical origin was Abidjan (85.32%). Consanguinity was found in 12.9%, death in siblings was found in 3.49% of patients. The most common clinical manifestations were repeated respiratory infections (60.71%), fever (16%), severe infections at least twice a year (13.04%), persistent fungal infections (12.90), thrive retardation in 48.7%. Abnormalities such as hyperleukocytosis (42.4%), neutropenia (29.23%), lymphopenia (16.44%), thrombocytopenia (38.36%) and mostly anemia (62.36%) were noted. Phagocytosis deficiency combined immune deficiencies and humoral deficiencies were respectively found at 95.16%, 89.78%.79.03%.

This work draws attention to the importance of PID which are often underdiagnosed, with absent or delayed treatment and poor prognosis. It is important to build a cohort with confirmed data to better support children with PID in our country.

no support
Proliferation, Plasticity, and Migration: Exploring the synergistic relationship between eosinophils and TH2 cells in development of HES-like syndrome
Authors: Trusiano Brie L , Allen Irving C
Published: August 2023

Aberrancies in eosinophil proliferation, activation and chemotaxis can lead to acute eosinophilic leukemia, paraneoplastic eosinophilia associated with T cell lymphoma and carcinomas, and Hypereosinophilic Syndrome (HES), respectively. HES is an idiopathic disease affecting humans and veterinary patients characterized by persistent eosinophilia (>1,500 cells/uL) and eosinophilic infiltration of solid organs leading to increased morbidity and mortality. Previous studies have demonstrated the role of NF-κB inducing kinase, NIK, an upstream regulator of the noncanonical NF-κB signaling pathway, in development of a HES-like syndrome due to aberrant TH2 polarization in the Nik−/−murine model. However, the intricacies of eosinophilic granulopoiesis, eosinophil/neutrophil plasticity, and chemotactic ability has not been assessed during development of HES-like syndrome in Nik−/− mice. Here we show that loss of NIK potentially enhances eosinophilic chemotaxis to CCL11, enhances eosinophilic/neutrophilic plasticity, and affects granulopoiesis in the bone marrow and spleen beginning at the level of eosinophilic metamyelocytes, despite potentially decreasing the proliferative potential of these cells. Taken together, these results suggest that NIK and the noncanonical NF-κB signaling pathway plays a role in dictating the hematopoietic and chemotactic potential of eosinophils and highlights a synergistic relationship with TH2 cells and their respective cytokines during development of HES-like syndrome in Nik−/− mice. Overall, these findings warrant further study to better characterize the synergistic relationship described above to better understand development of HES and HES-like diseases.

Virginia-Maryland College of Veterinary Medicine and the Institute for Critical Technology and Applied Science
Highly controlled nutritional interventions uncover defined immune and microbiome responses in human
Authors: Link Verena M , Hall Kevin , Belkaid Yasmine
Published: August 2023

Previous studies linked diet to numerous physiological functions including the control of the immune system and composition of the microbiome. Here, we report the first human study using a well-controlled cross-over experiment of a vegan, low-fat diet and a ketogenic, low-carbohydrate diet in a clinical setup.

We assess the impact of these dietary interventions via complementary approaches including protein expression via SOMAScan assay, gene expression via RNA-seq, cell population composition via flow cytometry, and metabolomics in the blood, as well as fecal microbiome composition via metagenomics sequencing. Based on functional enrichment analysis of RNA-seq, a vegan diet had a more pronounced impact on the innate immune system than a ketogenic diet as evidenced by enhanced monocyte response, immune activation and upregulation of TNFa, INFa, and INFg responses. On the other hand, a ketogenic diet had a more pronounced impact on B and T cell response. Proteomic analysis uncovers a stronger response to the ketogenic diet in female compared to male participants. Finally, a ketogenic diet alters the microbiome composition significantly more than the vegan diet with enrichment in many microbes including bile-tolerant gut Bacteroides taxa. Pathway analysis in the microbiome and metabolomics data showed changes in amino acid biosynthesis pathways, with many pathways down-regulated in the ketogenic diet. Together this study represents the first comprehensive multidimensional exploration of how diet can impact the immune system and microbiome in human.
Bio-active lipids protect against immune-related adverse events due to immune checkpoint blockade therapy
Authors: Saminathan Priyanka , Mathews Ian T , Henglin Mir , Liu Mingyue , Mercader Kysha , Chee Serena J D W , Campbell Allison , Tiwari Saumya , Watrous Jeramie , Dicker Martina , Dao Khoi , Najhawan Mahan , Quach Lily , Nguyen Thien-Tu Catherine , Nadig Namratha , Fang Camille , Vijayanand Pandurangan , Joosten Leo A B , Decker Roy , Patel Abijit , Netea Mihai , Long Tao , Zheng Pan , Kronenburg Mitchell , Patel Sandip Pravin , Ottensmeier Christian , Kaech Susan M , Cheng Susan , Jain Mohit , Sharma Sonia
Published: August 2023

Introduction

Immune checkpoint blockade (ICB) therapy has been revolutionary in its ability to treat advanced malignancies. Yet many patients receiving ICB develop Immune related adverse events (IRAEs), a leading cause for patients to discontinue treatment. Analyzing patients who develop IRAEs can help advance our knowledge of the molecular drivers of these poorly understood off target toxicities.

Methods

Our recent study of plasma from patients undergoing ipilimumab (anti-CTLA4) or pembrolizumab (anti-PD1) therapy for melanoma, lung cancer or other solid tumors was assessed using high-resolution liquid chromatography-tandem mass spectrometry.

Results

We uncovered a novel protective mechanism related to a class of circulating bio-active lipids that suppress ICB-related IRAEs. Significant reduction of bio-active lipids in circulation was associated with increased ICB-mediated IRAEs. Mouse-models (both DSS-colitis and humanized models) were used to show that supplementation with these lipids ameliorated colonic inflammation without impacting ICB-driven tumor regression. We also uncovered a significant correlation between increasing neutrophil counts and decreased bioactive lipids in circulation.

Conclusion

These results uncover a previously unidentified regulatory mechanism whereby the identified lipids in circulation specifically suppress deleterious inflammation during ICB therapy, while preserving anti-tumor immunity, suggesting that supplementation of bio-active lipids can be developed as a new therapeutic strategy to improve clinical outcomes in cancer immunotherapy.

Supported by grants from NIH (R01CA256133-02)
Impaired local VD3 metabolism contributes to tissue neutrophilic inflammation in chronic rhinosinusitis with nasal polyps
Authors: Xiao Qiao
Published: August 2023

Objective

Vitamin D (VD) possesses immunomodulatory properties, but its role in chronic rhinosinusitis with nasal polyps (CRSwNP) remains poorly studied. Herein, we aim to explore the regulation of VD3 function and its effect on local inflammation in CRSwNP.

Methods

25-hydroxyvitamin D3 (25VD3) levels in serum and tissue lysates were detected by ELISA. The expression of VD receptor (VDR), cytochrome P450 family 27 subfamily B member 1 (CYP27B1), the enzymes converting 25VD3 to the active 1,25VD3, and their expression regulation in human nasal epithelial cells (HNECs) were studied by RT-PCR, western blotting, immunofluorescence. HNECs and polyp tissue explants were treated with 1,25VD3. Tissue ECP+ eosinophils and MPO+ neutrophils were stained and quantified by immunofluorescence.

Results

25VD3 levels in serum and nasal tissue lysates were decreased in CRSwNP patients compared to control subjects. The expression of VDR and CYP27B1 were reduced in CRSwNP. VDR and CYP27B1 expression in HNECs were downregulated by IFN-γ and poly (I:C). 1,25VD3 suppressed neutrophilic inflammation in HNECs and polyp tissues. Tissue 25VD3 levels negatively correlated with neutrophilic inflammation in CRSwNP.

Conclusion

Reduced 25VD3 level and decreased CYP27B1 and VDR expression may impair local VD signaling activation, and thus exaggerate neutrophilic inflammation in CRSwNP.
CD4 cells and primary immune cells as a veritable factor on reproductive hormones and fertility
Authors: Amadi Emmanuel Chike , Akanno Faith Chisom
Published: August 2023

Immune system contributes to infertility, where Regulatory T cells play major role. Paucity of research in this crucial area, deems pertinent refocusing attentions on it. So, immune cells as veritable factors on fertility & reproductive hormones [Follicles stimulating hormone (FSH); Luteinizing hormone (LH); Prolactin (PRL); Estradiol (EZ); Progesterone (PROG); Testosterone (TESTO)] were investigated at a Teaching Hospital in Enugu, Nigeria between February – October 2022 on a pilot scale. Patients on doctor’s provisional diagnosis of infertility were examined for their hormones’ levels, CD4 & primary immune cells (PIC) counts. So far, seven patients were investigated. Results were analyzed using Paired Students t-test at 5% (0.05) alpha level; & p value of test statistics used for confirmation. All the patients have low CD4 counts (Range: 157–490). Our hypotheses were largely rejected: (i.e. Yes) that CD4 cells & PIC have impact on the hormones; p values confirmed negative correlative association between CD4 & the six hormones; all the PIC have impact on PRL; except on EZ, neutrophil (N) has very significant impact on all the hormones; except on FSH, lymphocytes (L) has very significant impact on all the hormones; only on PRL that all the PIC have impact; except on PRL & TESTO, WBC (T) has no significant impact on the hormones; only N & L have significant impact on the PROG; only WBC (T), N & L have impact on TESTO; except L none of the PIC has significant impact on the EZ. Monocyte, Eosinophil & Basophils has impact only on FSH, LH & PLT. So, under no hormonal abnormality, (except PRL in one patient), the work strongly suggests that the inability to conceive resides on the low CD4 cells, plus impacts of it & PIC on hormones; hence, they are veritable factors.

None
Genome engineering of HMC-1.2 cells renders a human mast cell line with a single KIT mutation: an improved preclinical model for mastocytosis research
Authors: Falduto Guido H , Bandara Geethani , Luker Andrea , Bai Yun , Pfeiffer Annika , Lack Justin , Metcalfe Dean , Olivera Ana
Published: August 2023

The HMC-1.2 human mast cell (huMC) line is often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. HMC-1.2 cells express constitutively active KIT, an essential growth factor receptor for huMC survival and function, due to the presence of two oncogenic mutations (D816V and V560G). However, systemic mastocytosis is commonly associated with a single D816V-KIT mutation. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9 engineering to reverse the V560G mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single monoallelic D816V-KIT variant. Transcriptome analyses predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable. Phosphorylation levels of ERK1/2, AKT and STAT5 were also similar between HMC-1.2 and HMC-1.3 cells. However, HMC-1.3 cells were more susceptible to KIT, JAK2, and BCL2 inhibitors than HMC-1.2. Our study reveals that the additional V560G-KIT oncogenic variant modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases neoplasia, suggesting that huMCs with a single D816V-KIT (HMC-1.3) variant may represent an improved preclinical model for mastocytosis.

This work was supported by the Division of Intramural Research of the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID).
Potential role for a Nematode Pan Allergen (NPA) in Tropical Pulmonary Eosinophilia (TPE)
Authors: Katru Samuel Christopher , Balakrishnan Anand Setty , Hadadianpour Azadeh , Smith Scott Alan , Kalyanasundaram Ramaswamy
Published: August 2023

Tropical Pulmonary Eosinophilia (TPE) is an asthma-like hyper-responsive syndrome characterized by accumulation of a high number of eosinophils in the lungs leading to severe respiratory morbidity in patients infected with lymphatic filariasis (LF), a helminth parasitic infection that affects 51 million people in 47 different countries worldwide. Subjects showing TPE also have circulating microfilariae (larval stages of LF) and a high titer of parasite-specific IgE in their blood. IgE and eosinophils are thought to play a major role in TPE pathology. However, specific antigens that are released from microfilariae responsible for generating the IgE antibodies in TPE are not known. Therefore, in this study, we screened a phage displayed cDNA expression library of Wuchereria bancrofti microfilaria with human monoclonal IgE antibodies generated from B cells isolated from TPE patients. The iterative screening identified WbNPA (Nematode Pan Allergen) and WbTCTP (Translationally Controlled Tumor Protein) as IgE antibody binding antigens. Subsequent analysis showed that rWbNPA and rWbTCTP can result in the release of histamine from mast cells in an IgE-dependent and independent manner. rWbNPA can induce accumulation of eosinophils in the peritoneal cavity of mice and can trigger the secretion of IL-2 and TNF-α from alveolar macrophages. These results suggest that WbNPA and WbTCTP have a potential role in the pathology of TPE in LF infections.
Sex-dependent influence of LMAN1 on lung function and allergen-induced airway hyperresponsiveness
Authors: Sauber Faith , Swaby Lindsay , Tigno-Aranjuez Justine , Miller Madelyn H , Zhang Bin
Published: August 2023

We recently reported LMAN1 as a regulatory receptor capable of downregulating NF-kB signaling in response to house dust mite (HDM) in vitro. In this follow up work, we have subjected WT and LMAN1 KO mice to a HDM model of allergic airway inflammation and assessed lung function and airway hyperreactivity (AHR). Given our previous in vitro findings, we had expected worsened disease in the absence of LMAN1. Interestingly, when mice were stratified by sex, we observed that while female, HDM-treated LMAN1 KO mice exhibited signs of enhanced airway narrowing and closure and enhanced AHR compared to their WT counterparts, male, HDM-treated LMAN1 KO mice showed the opposite trend. These sex-dependent effects appeared to be localized to the peripheral airways. We additionally characterized the type and extent of immune cell infiltration into the airway and highlight those features which might account for the gender-biased effects of LMAN1 on lung function in the setting of allergic asthma.
IFNγ, IL-4, and IL-13 upregulate IL-31 receptor alpha in airway smooth muscle cells to induce airway hyperresponsiveness in asthma
Authors: Akkenapally Santhoshi Vani , Yombo Dan J K , Geereddy Bhanuprakash Reddy , Madala Satish K
Published: August 2023

Asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR), inflammation, and goblet cell hyperplasia. AHR is a major cause of hospitalization, morbidity, and mortality in allergic asthma. Both Th1 and Th2 cytokines, including IFN-γ, IL-4, and IL-13 have been shown to induce AHR; however, the underlying mechanisms remain unclear. We observed a significant increase in the expression of IL-31RA, but not its cognate ligand IL-31 during house dust mite- and Schistosoma mansoni soluble egg antigen-induced allergic asthma. In support of this, IFN-γ and Th2 cytokines, IL-4 and IL-13, upregulated IL-31RA but not IL-31 in airway smooth muscle cells (ASMC). Importantly, the deficiency of IL-31RA was sufficient to attenuate contractility of ASMC and allergen-induced AHR but had no effect on inflammation and goblet cell hyperplasia. Similarly, the loss of IL31RA attenuated both IFNγ and IL13-induced AHR with limited changes in inflammation and goblet cell hyperplasia. Importantly, suggesting a mechanism for direct regulation of ASMC contractility, IL-31RA increased muscarinic acetylcholine receptor 3 (CHRM3) expression and calcium signaling. Together, these results suggest an important role for IL-31RA in the regulation of ASMC contractility and AHR distinct from airway inflammation and goblet cell hyperplasia in asthma.

This research was supported by NIH NHLBI grants 1R01HL157176 and 5R01HL134801
IL-17A deficiency alleviates PM-induced allergic asthma by suppressing Th17 cell secretion cytokines and mucus overproduction
Authors: Jee Youngheun , Yang Jiwon , Kim Hyo Jin , Dinh Duong Thi Thuy , Udayanganie Olupathage Indrachapa , Ko Mi-Ok
Published: August 2023

Particulate matter (PM) aggravates airway and lung inflammatory response in allergic asthma by directly inducing Th17 cells to secrete Th17 cell-associated inflammatory factors and chemokines. IL-17A is a key Th17 cell secretion pro-inflammatory cytokine. In this study, we investigated the role of IL-17A in PM-induced allergic asthma in mice. 7–8 weeks old wild type (WT) mice, and similarly IL-17A knockout (KO) mice, were divided into four experimental groups (healthy control, PM only, OVA only, OVA+PM treatment groups) and treated accordingly. Inflammatory cell and eosinophil infiltration were increased in the PM-induced WT mice compared to WT healthy control, but were decreased in KO mice compared to PM-induced WT mice. Among toll-like receptors (TLRs) that play an important role in innate immune response, TLR2/4/7 expression was significantly increased in PM-induced WT mice, but was reduced in KO mice. Furthermore, we observed decrease in mRNA expression of IL-13, IL-17A, IL-22, and TGF-β among cytokines of Th2 and Th17 cells in KO mice. PM-induced WT mice facilitated the overt histopathological symptoms of allergic asthma such as mucus overproduction and goblet cell hyperplasia while KO mice showed mitigated such responses compared to PM-induced allergic asthma WT mice. These results prove that IL-17A plays a key role in pathogenesis of PM-induced allergic asthma.

This research was funded by the Ministry of Education.
Dynamic chromatin accessibility licenses STAT5- and STAT6-dependent innate-like function of Th9 cells to promote allergic inflammation
Authors: Schwartz Daniella M , Son Aran , Meylan Francoise , Gomez-Rodriguez Julio , Kaul Zenia , Sylvester McKella , Kitakule Moses , Falduto Guido H , Wang Chujun , Manthiram Kalpana , Qi Chen-Feng , Gurram Rama , Zhu Jinfang (Jeff) , Schwartzberg Pamela , Milner Joshua , Guererrio Pamela
Published: August 2023

Allergic diseases are a major global health issue, causing significant morbidity and mortality. Interleukin-9 (IL-9) producing T helper 9 (Th9) cells promote allergic inflammation, yet Th9 effector functions are incompletely understood because the heterogeneity and instability of Th9 cells makes them challenging to study. Although the significance and mechanisms of Th9 instability are unknown, T cell receptor (TCR) activation is hypothesized to play a key role. Conversely to this paradigm, we found that resting Th9 cells did not require TCR restimulation for IL-9 production, which was induced by STAT5- and STAT6-dependent paracrine cytokines. This mechanism was seen in circulating T cells from allergic patients and was restricted to recently activated cells. Analysis of IL-9+ T cells from allergic subjects revealed a transcriptional program that was enriched for activation pathways and unstable over time, with IL9 amongst the least stable genes. Prolonged resting of cells decreased the accessibility of STAT5-dependent IL9 enhancers, inactivating the locus. In vivo, Th9 cells induced airway inflammation via TCR-independent, STAT-dependent mechanisms. In allergic patients, Th9 expansion was associated with STAT activation and responsiveness to JAK inhibitors. These findings suggest that Th9 instability is a negative checkpoint on TCR-independent inflammation that breaks down in allergic disease, and that JAK inhibitors should be considered for allergic patients with a Th9 signature.

Supported by the NIAID intramural research program (2018-2022), University of Pittsburgh start-up funds (2022-current), and the National Psoriasis Foundation (Robertson Fellowship, 2018-2021; Translational Grant, 2022-current)
Defining complex T-cell signatures linked to rhinovirus infection in the lower airways of children with severe asthma
Authors: Bryant Naomi , Muehling Lyndsey M , Teague W Gerald , Woodfolk Judith A
Published: August 2023

Rhinovirus (RV) infection is a risk factor for the development of asthma and acute wheezing episodes. RV-specific Th1 cells are expanded in the blood of adults with asthma, even in the absence of infection. Such cells are further amplified during acute infection and resemble those CCR5+ T cells found in the lower airways of children with severe asthma. We posit that in early childhood, pathogenic T cells induced by repeated RV exposures are sustained by cues in the inflamed airways and serve to drive chronic inflammation. Little is known about the complexity and function of T cells in the lower airways of children with severe asthma, or their relation to RV, due to challenges in obtaining lung specimens and low T-cell numbers in the airway lumen. We leveraged high-dimensional single-cell phenotyping, marker enrichment modeling for designation of complex molecular signatures, and the novel machine learning algorithm, T-REX, to discriminate rare pathogenic T cells in the airways that are linked to RV infection in children aged 1–15 years who had severe asthma (RV PCR+, n=3; RV PCR-, n=8). Our novel workflow identified activated (CD38+), proliferating (Ki-67+) and tissue-resident (CD69+) CCR5+CD4+ T cells (CD45RO+ICOS+CD95+CD27+PD-1+TCF-1+) in the lower airways of all children; however, this cell population was markedly expanded in RV+ children (median of 10% vs 1% of total T cells) and was over 9-fold enriched in the airways compared with blood. Notably, these cells were inversely correlated with inhaled corticosteroid dose. Our findings suggest that the expansion and persistence of RV-induced lung-resident T cells contribute to chronic airway inflammation in children with severe asthma.

Supported by grants from NIH/NIAID (RO1 AI077653, R21 AI160334)
Prophylactic and therapeutic use of synthetically glycosylated antigens to limit allergic airway disease
Authors: Medellin Jorge E Gomez , Ung Trevor Wai-Ming , Kurtanich Trevin Robert , Reda Joseph William , Wang Thomas , Chang Kevin , Cao Shijie , Swartz Melody A , Hubbell Jeffrey
Published: August 2023

Asthma is a disease involving chronic airway inflammation affecting more than 300 million people worldwide. There is currently no cure for asthma. While effective, corticosteroids cause broad immune suppression and long-term debilitating disease. Novel immune modulatory therapeutics leave patients vulnerable to infections. Therefore, there is a current need for new therapeutics that target the underlying causes of the disease without triggering detrimental side effects.

Through synthetic glycosylation, our platform targets hepatic antigen presenting cells and leverages the liver’s tolerance-inducing mechanisms to limit inflammatory T cell responses, while promoting regulatory T cell (Treg) responses, thus preventing broad immunosuppression. In a model of experimental allergic airway inflammation, prophylactic treatment with synthetically mannosylated OVA (pManOVA) ameliorated the allergic response to inhaled OVA. Our treatment reduced lung and airway eosinophilia, systemic levels of IgE and OVA-specific IgG1, and airway mucus. Furthermore, pManOVA treatment reduced OVA specific lung TH2 cells, and exhibited reduced production of IL-4, IL-5 and IL-13 upon antigen specific restimulation. Treatment with pManOVA was accompanied by induction of OVA-specific Tregs, which negatively correlated with features of allergic airway disease. In previously sensitized mice, therapeutic intervention with pManOVA reduced airway and lung eosinophilia, B cell and CD4+ T cell inflammation. Altogether, these data suggest a potential role for prophylactic and therapeutic intervention with synthetically glycosylated allergens in ameliorating disease severity in a model of allergic airway inflammation.
CD3ζ ITAM Diversity Determines Chimeric Antigen Receptor Signaling and Function
Authors: Majumdar Shubhabrata , Echelibe Hilda , Bettini Matthew L
Published: August 2023

One of the major challenges facing CD28-based CAR-T immunotherapy is a lack of long-term T-cell persistence in patients. CD28 CARs exhibit strong signaling patterns that result in an acute tumor-killing response. However, based on studies with the conventional TCR, strong signaling favors terminal differentiation of T-cells that get exhausted faster while weaker CAR signaling favors more stem-like memory T-cell states with longer persistence. Hence attenuation of CAR signaling strength is a viable strategy for long term survival of CD28-CAR T-cells. Previous work has suggested that reducing CD3 ITAM diversity to single ITAM sequences while keeping the number of ITAMs same, attenuates TCR signaling strength to different extents. Based on this, we have designed modified ITAM diversity restricted CD28 based anti-CD19 CARs with the goals of optimizing CAR signaling and studying the non-redundant signaling properties of the individual ITAMs in the context of CARs. Our approach is different from reducing ITAM multiplicity as there is evidence suggesting that ITAM numbers might be important for CAR sensitivity. We hypothesize that the individual ITAM non-redundancy and the reduction in ITAM diversity will allow for optimization of CAR T cell signaling and improve T-cell persistence while maintaining sensitivity to low ligand densities. We have observed that our modified CARs can kill CD19+ leukemia cells in vitro. We observe functional differences in terms of cytokine production, proliferation, AICD and exhaustion. We have also observed differences in signal strength based on ITAM usage. These observations support the non-redundancy of ITAM signaling and open the possibility of optimizing CAR persistence and function in patients.
Modulating the ER and Golgi stress response in T cells with hydrogen sulfide signaling to enhance the anti-tumor immune response
Authors: Oberholtzer Nathaniel , Chakraborty Paramita , Mills Stephanie Ann , Choi Seungho , Mehrotra Shikhar
Published: August 2023

Adoptive cell therapy (ACT) continues to emerge as a novel therapeutic strategy for treating cancer. New approaches to improve ACT protocols are needed to enhance in vivo persistence of adoptively transferred tumor antigen-specific T cells and overcome tumor-induced immunosuppression and cellular stress. Here, we show that hydrogen sulfide (H2S) can be used to promote the antitumor immune response using an H2S donor compound and by enhancing endogenous production of H2S with overexpression of Cbs, a key H2S-producing enzyme. Expansion of tumor antigen-specific T cells with exogenous H2S promoted a central memory phenotype with enhanced antioxidant capacity. H2S-treated T cells and T cells overexpressing Cbs also displayed enhanced reactivity to tumor antigen, with an increase in production of cytolytic cytokines and increased overall protein translation. In in vivo models of melanoma and lymphoma, antitumor T cells conditioned ex vivo with exogenous H2S demonstrated superior tumor control upon ACT. Proteomics analysis revealed an increase in protective thiol modifications on ER-associated proteins involved in regulating ER stress in T cells treated with H2S. Further, treatment of antitumor T cells with H2S resulted in resistance to Golgi and ER stress. Golgi stress and overall Golgi mass were identified as important determinants of T cell function, with T cells possessing high Golgi mass exerting more robust tumor control when adoptively transferred. Overall, these data show that antitumor T cells exposed to H2S possess an enhanced antitumor phenotype that allows them to exert greater tumor control in ACT. Further, these studies identify the status of the Golgi in T cells as an important determinant of their antitumor capacity.

R01 CA236379
mTOR signals regulate T cell exhaustion and T cell responses during PD-1 targeted immunotherapy in chronic viral infection
Authors: Ando Satomi , Araki Koichi
Published: August 2023

Antigen-specific CD8 T cells gradually have impaired functions in chronic infection and cancers, which is termed T cell exhaustion. Since T cell exhaustion is associated with programmed death 1 (PD-1), the blockade of PD-1 pathway reinvigorates exhausted CD8 T cells, resulting in better control of infection and cancers.

Stem-like CD8 T cells are important for maintenance of antigen-specific CD8 T cells during chronic infection and cancers because they have self-renewal ability and provide effector-like CD8 T cells which possess transiently cytotoxicity. PD-1 targeted therapies enhance functional T cells response by promoting differentiation of stem-like CD8 T cells toward effector-like CD8 T cells.

mTOR is a serine/threonine kinase that regulates cellular functions and proliferation. However, the role of mTOR during T cell exhaustion and PD-1 targeted therapies remains elusive.

Here, we found that mTOR inhibition enhanced accumulation of stem-like CD8 T cells at the early phase of infection, leading to improved efficacy of PD-1 blockade. In contrast, after the establishment of chronic LCMV infection, mTOR inhibition suppressed the transition to effector-like CD8 T cells from stem-like CD8 T cells. Mechanistically, a cell intrinsic mTOR signal was critical for differentiation of stem-like CD8 T cells into effector-like CD8 T cells during chronic infection as well as PD-1 targeted therapy. Thus, simultaneous treatments of PD-1 blockade with mTOR inhibition failed to induce effector-like CD8 T cells, resulting in reduced efficacy of PD-1 targeted therapy. Our data demonstrate that mTOR signals regulate T cell exhaustion and PD-1 targeted therapy and have important implications for combination cancer therapies with PD-1 blockade.

Supported by grants from NIH (R01 AI139675)
A Novel Multicolor Enzyme-linked Immunosorbent Spot Assay for the Functional Assessment of CAR T Cell Products
Authors: Iraguha Thierry , Omili Destiny , Gebru Etse , Hardy Nancy M , Luetkens Tim , Rapoport Aaron P , Atanackovic Djordje
Published: August 2023

Introduction

Chimeric antigen receptor (CAR) T cell therapies are powerful anti-tumor immunotherapies and CD19-targeted CAR T cells have revolutionized the treatment of B cell lymphomas. Unfortunately, recurrent disease after CAR T cell therapy remains a clinical problem with up to 50% early relapses after CD19-targeted CAR T cells. Therefore, there is a critical and unmet need to identify biomarkers associated with durable responses to guide the optimization of CAR T cell therapies.

Methods

We developed a novel multicolor enzyme-linked immunosorbent spot (ELISpot) assay for the functional assessment of CAR T Cell products on a single-cell level combining the numerical assessment of CAR T cell products with their functional characterization.

Results

We first used a standard single-cell interferon (IFN)γ ELISpot assay to measure CD19-targeted CAR T cell responses to CD19 protein-coated beads or to beads coated with an irrelevant target. Beads coated with anti-CD3/anti-CD28 antibodies were used as a positive control. We then developed a multicolor ELISpot assay simultaneously measuring the secretion of combinations of different cytokines on a single-CAR T cell level. We identified IFNγ/TNFα/Granzyme B as the most relevant combination of cytokines and we used our novel multicolor ELISpot assay to functionally and numerically characterize two clinical-grade CAR T cell products.

Conclusions

We have developed a novel multicolor, single-cell ELISpot for the quantitative and functional assessment of CAR T cell products. The clinical value of our novel assay will be assessed in clinical studies correlating the pre-infusion assessment of CAR T cell products with the patients’ outcome.

Supported by the Kehlert Foundation (to D. A) by the Maryland Department of Health’s Cigarette Restitution Fund Program and by the National Cancer Institute - Cancer Support Grant (CCSG) P30CA134274.
Reinvigorated exhausted T cells exhibit distinct features within 24 hours after PD-1 therapy
Authors: Leng Xuebing , Nivelo Luis , Buchness Lance , Ristin Svetlana , Rafie Christine , Villarino Alejandro , Stelekati Erietta
Published: August 2023

CD8 T cells are potent cytolytic immune cells. However, persistent antigens during chronic infections and cancers drive differentiation of exhausted T cells (TEX). TEXdo not generate optimal immune protection. Immunotherapy such as anti-PD-L1 provides a partial TEX reinvigoration. Some TEX regain effector functions, enter cell cycle and start proliferation by PD-1 therapy. However, the underlying molecular and epigenetic mechanisms of TEX reinvigoration are not well understood. Moreover, a key unknown question is why only some TEX gain reinvigoration. Here, we used proliferation as a surrogate for TEX reinvigoration. A transgenic mouse model carrying a cell cycle reporter (Fucci) was used. Fucci LCMV-specific CD8 T cells were transferred to mice that were then infected with chronic LCMV to induce exhaustion. Recipients were treated with anti-PD-L1 at d21 p.i. Fucci staining (cell cycle) and CTV dilution (cell division) allowed us to isolate reinvigorated TEX before first cell division and ask what molecular signals induce TEX reinvigoration. Thus, we found that TEX reinvigoration starts as early as 24 hours after a single anti-PD-L1. Proliferating cells upon treatment contained an increased progenitor-like population and a reduced terminally exhausted population compared to control, whereas non-proliferating cells were similar between groups. Single cell transcriptomics data further identified unique transcriptional signatures in reinvigorated TEX upon anti-PD-L1 compared to untreated TEX at 48 hours post treatment. Therefore, we identified the molecular signatures of TEX that have the potential to become reinvigorated upon immunotherapy and suggest novel molecular targets to enhance the potency of PD-1 therapy.
Sensing and alarm function of vaccine-elicited SIV-gag specific CD8 TRM in the reproductive mucosa of rhesus macaques
Authors: Joag Vineet , Bimber Benjamin , Quarnstrom Clare , Bollimpelli Venkata Satish , Schenkel Jason M , Bertogliat Mario , O’Flanagan Stephen , Soerens Andrew , Stolley Michael , Hunter Eric , Amara Rama Rao , Masopust David
Published: August 2023

Resident memory T cells (TRM) constitute a recently identified lymphocyte lineage that occupies non-lymphoid tissues (NLT) without recirculating. In murine models, upon antigenic rechallenge, TRM trigger antiviral responses in neighboring innate and adaptive immune cells, and recruit immune effectors from circulation. Collectively this is referred to as a ‘sensing and alarm’ function. However, the full range of TRM functions has not been assessed, and non-human primate/human TRM function remains almost entirely uncharacterized. Here we established a prime-boost vaccine modality in rhesus macaques to generate abundant SIV-gag-specific CD8 TRM in the female reproductive tract (FRT) and 14 other NLTs. Upon vaginal challenge with CM9 peptide from the SIV gag protein, CD8 TRM reactivation induced expression of CD69 and granzyme B in SIV-gag specific CD8 T cells throughout the rhesus FRT, in situ proliferation (Ki67 expression), and rapid antiviral and IFN response gene expression in essentially all hematopoietic and non-hematopoietic cells. Upregulation of effector genes in CD8 T cells, CD4 T cells, NK cells, and ILCs peaked at 24h and persisted at 48h. CITE-Seq analysis revealed that mucosal CD4 T cells expressed antiviral genes including HIV restriction factors and had reduced expression of the HIV-co-receptor CCR5. Increased numbers of vaginal T cells, memory B cells, and plasma cells at 24h coincided with increased expression of chemokines and VCAM-1 on endothelial and stromal cells and a concomitant reduction in circulating T and B cells. These data demonstrate that CD8 TRM trigger local activation and the mobilization of innate, cellular, and humoral immune responses to the site of antigen exposure in non-human primates.
PP4 deficiency contributes to DEP exacerbated epithelial barrier dysfunction and airway inflammation in asthmatic patients
Authors: Yang Feng-Ming
Published: August 2023

Diesel exhaust particles (DEP) emitted from motor vehicle or heavy-duty industries is thought to be the major source of air pollutants that lead to the rapid increase and worsening of allergic disease, such as asthma. In asthma, exposure to DEP has been linked to aberrant epithelial barrier integrity, which leads to the promotion of allergen delivery into the subepithelia and induced alarmins (IL-33, IL-25 and thymic stromal lymphopoietin (TSLP)) release to promote Th2-mediated inflammation. However, it is still not known the detail mechanism of airway epithelial barrier in response to DEP. In this study, we fund that PP4 expression was decreased in DEP-challenged human bronchial epithelial cells and air-pollution animal model. Moreover, silencing PP4 decreased E-cadherin and barrier integrity through and activated Src kinase and increased expression of Slug. Sequentially, transactivating the EGFR phosphorylation led to increased alarmin synthesis. Overexpression of PP4 wild type, but not phosphatase mutant, prevented DEP-induced loss of airway epithelial E-cadherin expression and decreased alarmin release, suggested that phosphatase activity is critical to maintain epithelial integrity and alarmin production. Primary human bronchial epithelial cells (HBECs) from mild and severe asthmatic patients showed a significantly low TJ integrity in ALI cultures in response to DEP, compared with HBECs from healthy subjects. Our data demonstrate that exposure to DEP causes a significant reduction in expression of PP4 leading to decreased epithelial barrier function might accelerate the allergen-induced airway inflammatory response in asthma.

Supported by grants from (MOST 110-2320-B-038-065-MY3) from the Ministry of Science and Technology of Taiwan.
Murine chorioamnionitis causes a dampened innate immune response that persists into infancy
Authors: Knobbe Jessica , Bermick Jennifer
Published: August 2023

Chorioamnionitis is infection or inflammation of the chorion, the amnion, and the placenta. Chorioamnionitis is associated with 40–70% of preterm births and increases the risk of many immune-mediated neonatal sequelae. Chorioamnionitis also increases the risk of developing wheezing and asthma later in life. With this in mind, we sought to characterize the innate immune dysfunction associated with chorioamnionitis. We hypothesized that neonatal and infant mice exposed to chorioamnionitis would display dampened innate immune responses to bacterial pathogens. To test this, genetically homogenous C57Bl/6 dams were injected IP with LPS or PBS on E18.5. Pups were born naturally and euthanized at P4–6 (neonate) or P12–14 (infant). Splenic F4/80+ cells were isolated, then unstimulated or stimulated with heat-killed Staph epidermidis, Staph aureus, or Escherichia coli for 24 hours. IL-1β, IL-6, IL-10, IL-12, KC, MCP-1, MIP-1α, and TNF-α were quantified in cell supernatants. We observed that E. coli-stimulated F4/80+ cells from chorioamnionitis-exposed infant mice displayed lower concentrations of IL-6 (p<0.0001) and MCP-1 (p=.0057) compared to controls. S. epidermidis-stimulated F4/80+ cells from chorioamnionitis-exposed infant mice also displayed lower concentrations of MCP-1 (p=.0024) compared to controls. This suggests that murine chorioamnionitis impacts the developing immune system, resulting in a dampened innate immune response to bacterial pathogens that persists into infancy.

Supported by grants from the NIH (T32GM139776, R01AI141673).
Multi-parametric quantification of monocytes using live-cell analysis
Authors: McBain Kirsty A , Trigg Jasmine , Lovell Gillian , Bevan Nicola , Dale Tim
Published: August 2023

Monocytes play critical roles in innate immunity by migrating to inflamed tissue, where they clear micro-organisms and apoptotic cells, repair injured tissues, and recruit other immune cells. These highly plastic cells can change their functional phenotypes in response to a variety of cellular signals, including the Akt signaling pathway. Here we provide robust in vitro assays for the kinetic evaluation of monocytes and exemplify how these further our understanding of their biological roles.

The Incucyte® Live-Cell Analysis System was used to acquire phase and fluorescence images of monocytic cell lines, which were automatically analyzed using integrated software. To assess immune cell activation, RAW264.7 monocytes were treated with lipopolysaccharide (LPS) and phenotypic morphological changes were quantified using label-free analysis. These data showed a concentration-dependent increase in activated cells as they become larger and flatter, with a maximal response of 56% for 100 ng/mL LPS at 33 hours. To monitor Akt activity, we used cells expressing the Incucyte® Kinase Akt Green/Red biosensor, a genetically encoded fluorescent reporter whose subcellular localization is dependent on Akt activation. Cytokine analysis was performed using the iQue® Advanced Flow Cytometer and a custom Qbeads® PlexScreen Kit. LPS induced a rapid concentration-dependent activation of Akt (EC50 1.31 ng/mL) and stimulated release of inflammatory proteins TNFα and CCL3 (MIP-1α), consistent with LPS-inducing activation via the release of pro-inflammatory agents.

These data exemplify that live-cell analysis, alongside advanced analytical methods, is a powerful approach enabling multi-parametric quantification of immune cells.
Intracellular complement protein C3 promotes cell survival via Factor B (FB)
Authors: Sahu Sanjaya Kumar
Published: August 2023

Intracellular complement proteins such as C3 and FB play protective role during stress-induced cell death. C3−/−and Cfb−/− mice had worse Pseudomonas aeruginosa (P.a.)-induced acute lung injury at 24 h post infection compared to wildtype (WT) mice. However, the mechanism by which C3 exerts these functions is unclear.

We performed CRISPR-induced deletion of C3 and CFB in BEAS-2B cells, a human tracheobronchial airway epithelial cell line and assessed key findings in primary human airway cells. We also tested our observations in bone-marrow derived macrophages (BMDM) from Cfb−/− versus WT mice. Measures of acute lung injury were quantified in C3−/−, Cfb−/− and WT mice infected with PA57, a clinical P.a. strain.

We observed that C3 and FB-deficient airway cells have increased death overtime compared to their parent clones in the setting of both oxidative stress and P.a. infection in vitro. An increased autophagic stress response, along with increased apoptosis, was observed in both C3 and FB deficiency. On further evaluation, both C3 and FB deficiency resulted in significantly lower AKT-mTOR signaling in P.a. infection. FB deficiency resulted in lower phospho-P70 S6 kinase levels, and higher LC3-II levels in P.a. infection. A similar phenotype was observed in infected BMDMs from Cfb−/− compared to WT mice. FB-deficient BEAS-2B cells and Cfb−/− mouse BMDMs had higher intracellular bacterial burden compared to FB-sufficient controls, suggesting that a lack of intracellular FB altered intracellular processing of P.a. The protective effect of C3 was present even when its secretion was inhibited but was abolished in FB deficiency.

We conclude that intracellular complement protein C3 plays an essential role in cell survival via FB.