bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for intrathymic and neuroimmune alterations in a mouse model of neglect-related early life stressors
Authors: Choe Jamie Y , Donkor Michael , Vann Philip , Sumien Nathalie , Thorpe Roland J , Jones Harlan P
Published: August 2023

Environmental exposures during the impressionable early life period can influence biological and behavioral development. These exposures accumulate from birth and comprise the exposome, which impacts health and disease. Neglect is a major source of early life stress (ELS) and accounts for 80% of reported child abuse in the United States. Forms of neglect vary (e.g., physical, medical, emotional, nutritional) and with increased concerns for abuse during the COVID-19 pandemic, we require a deeper understanding of how ELS affects immune system programming/development. T cells undergo a stringent selection process in the thymus to achieve self-nonself discrimination capacity and can be identified based on double-positive (DP) or single-positive (SP) CD4/CD8 expression status. We utilize a maternal separation with early weaning paradigm to study the effects of ELS on anxiety/depressive behavior and intrathymic T cell distribution. Conditions of neglect-related stress were simulated based on scheduled dam-pup separation (physical neglect) and early weaning to a high carbohydrate diet (malnutrition). Pups were subjected to one of two distinct ELS conditions: (1) daily separation with early weaning (MSEW) or (2) early weaning alone (EW) at postnatal day (PD) 14. Control pups experienced no separation and were maintained on the dam’s milk to the standard wean date (PD21). Body metrics and tissues were collected at PD21. Behavioral phenotypes were assessed at PD28–35. The results demonstrated significant stress effects and revealed an altered distribution of thymocytes within DP and SP compartments. This study provides preliminary causal evidence and provokes questions about ELS-mediated consequences on T cell immunity.

NIH/NIA T32 AG020494 NIH/NIMHD 5U54MD006882-10 UNTHSC MIG Dept. Intramural Grant
New generation of fluorescent probes for cell-based measurements of caspase Activation and mitochondrial superoxide
Authors: Mandavilli Bhaskar S , Beacham Daniel , Hu Yi-Zhen , Yang Jongtae , Chen Aimei
Published: August 2023

Cell health and stress readouts are critical indicators of altered or impaired function in normal and diseased states of cells, and work has been underway to develop improved small molecule sensor dyes compatible with traditional imaging and High Content Analysis (HCA) interrogation of apoptotic and mitochondrial stress pathways.

The CellEvent™ Caspase Green dye effectively reports caspase activation, but suffers complications in assay configuration when attempting to multiplex with the Green Fluorescent Protein (GFP), calcein, or other 488 laser line tools in fluorescence microscopy. Here, we describe the testing and functional characterization of a new candidate molecule for measuring apoptosis in living cells. Our sensor is comprised of a fluorogenic reporter dye that is liberated from a DEVD peptide substrate by caspase activation, with ex/em spectra of 590/610 nm, permitting easy multiplex with GFP or calcein stained neurons in both traditional and HCA microscopy configurations. MitoSOX™ Green that has specificity to superoxide like MitoSOX™ Red dye. In addition, it has better spectral properties and works with GFP filters in microscopy, and allows for measurement of mitochondrial superoxide in RFP-expressing cells. MitoSOX™ Green Mitochondrial Superoxide Indicator also localizes to mitochondria of live cells and selectively reports superoxide generation, while ignoring other ROS and RNS species in ex vivo testing. With an Excitation/Emission profile in the GFP/FITC microscopy channel, a series of comparative studies in immortalized and neural cells are shown, highlighting photostability, specificity and signal amplitude from the dye.

These reagents are research use only, not for diagnostic purposes
Re-evaluating the role of coreceptor: LCK association in T cell receptor signalling
Authors: Zhang Justin Boxuan , Jones Claerwen M , Nguyen Angela , Thiele Daniel , Zareie Pirooz , La Gruta Nicole L
Published: August 2023

The CD4 and CD8 coreceptors of T cells are essential for TCR signalling. The current knowledge describes that the coreceptors serve to deliver the tyrosine kinase LCK to the CD3 complex to phosphorylate immunoreceptor tyrosine activation motifs (ITAMs) to initiate TCR signalling. Recently, CD8 was shown to prevent signalling of reverse docking TCRs by sequestering LCK away from CD3, thus restricting TCR docking orientation to peptide MHC complexes and explaining the inability of these unconventional TCRs to signal during immune responses. However, how the association of LCK with the coreceptor shapes antigen-specific TCR repertoires and affects T cell activation in vivo has not been demonstrated. We utilise LCKfree mice (in which LCK cannot bind the CD4 or CD8 coreceptors) to study the role of coreceptor-associated LCK in TCR signalling. We found that the LCKfree mutation specifically reduced CD4 T cell development while not affecting CD8 T cell output. After influenza A infection, epitope specific CD8 T cell reponses in LCKfree mice are numerically and functionally intact, whereas CD4 T cell responses are substantially reduced. Single-cell RNA sequencing data showed minor gene expression differences between WT and LCKfree activated CD8 and CD4 T cells. Contrary to our hypothesis, epitope-specific TCR analysis revealed a decreased TCR diversity in CD8 TCR repertoires in LCKfree mice, biased towards the dominant clones. In contrast, CD4 repertoire show distinct TCR usage compared to WT. In summary, we found that coreceptor: LCK association is not necessary for the generation of functional MHC restricted TCR reperotires, but it is important for the development, functions, and TCR repertoire of CD4 T cells.

Supported by grants from ARC (M07001-3237405)
A multi-level study of the effects of glucocorticoids on human T cells
Authors: Bhatt Brinda , Dell’Orso Stefania , Jenkins Lisa , Maity Tapan K , Howe Katherine N , Gadkari Manasi , Franco Luis M
Published: August 2023

Glucocorticoids (GCs) have been the cornerstone of immunosuppressive therapies for the past 70 years, yet the mechanisms by which they regulate human immunity remain poorly understood. Specifically, there remains a need for direct insight into how GCs regulate the functions of human T cells. We have previously demonstrated that response to GCs in humans is highly cell type-dependent. In this project, we combine high-throughput techniques to study the effects of GCs on human CD4+ and CD8+ T cells, in vivo and in vitro. As part of a clinical study in 20 healthy volunteers, T cells were purified before and 2 and 4 hours after a dose of the GC methylprednisolone. RNA-seq was performed to identify GC-responsive genes. We identified a strong transcriptional response in T cells, where increased transcript abundance predominates. While there is a high degree of overlap of GC-responsive genes and in response directionality, CD4+ T cells show a stronger and more unique response than CD8+ T cells. To understand the functional genomic consequences of GC treatment in primary T cells, we developed a protocol to simultaneously assay gene expression (RNA-seq), chromatin accessibility (ATAC-seq), and glucocorticoid receptor binding (CUT&RUN) on cells treated in vitro. We are also performing experiments using tandem mass tagging (TMT) and mass spectrometry to identify which of the differentially expressed transcripts correspond to protein-level changes. Integrating data obtained at the transcript, chromatin, and protein levels will provide us with a means to identify biologically relevant effects of glucocorticoids and will guide the selection of assays to determine the functional consequences of those effects on human T cells.
SATB1 is a master gatekeeper that limits virus-specific CD8+ T cell differentiation
Authors: Turner Stephen John , Lee Jason , Russ Brendan
Published: August 2023

Special AT-rich sequence binding protein-1 (SATB1) is a chromatin binding protein that has key roles in ensuring appropriate chromatin structure and remodelling during T cell development. While a role for SATB1 in both CD4+ T regulatory and CD4+ TH2 lineage specific function has been described, little is known about its role during virus-specific CD8+ T cell differentiation. Here we demonstrate that SATB1 down regulation is associated with naïve CD8+ T cell activation and subsequent differentiation. Genome wide SATB1 binding indicates that SATB1 is critical for maintaining the naïve CD8+ T cell transcriptional programs prior to activation. Overexpression of SATB1 upon CD8+ T cell activation results in major restraint of virus-specific CD8+ cell differentiation resulting in lower expression of inflammatory chemokine receptors, checkpoint molecules such as PD-1 and limits effector CD8+ T cell infiltration into infected tissues. Moreover, SATB1 over expression maintains high levels of TCF-1 expression and maintenance of the naïve T cell program. Our data suggest that SATB1 expression within naïve CD8+ T cells acts as gatekeeper for maintaining the naïve T cell program, and that down regulation upon activation is required for optimal effector CD8+ T cell differentiation.

Supported by grants from the Australian Research Council (DP170102020).
CD25 recruits inhibitory phosphatases for feedback control of B-cell receptor signaling
Authors: Sun Ruifeng , Lee Jaewoong , Robinson Mark E , Kume Kohei , Cosgun Kadriye Nehir , Chan Lai N , Leveille Etienne , Geng Huimin , Vykunta Vivasvan , Shy Brian , Marson Alexander , Meffre Eric , Müschen Markus
Published: August 2023

Successful initiation of the B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers are both marked by sharp increases of CD25 surface-expression. Likewise, oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma induces CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. We discovered that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells feedback regulates BCR-signaling or its oncogenic mimics.

Recapitulating phenotypes of genetic ablation of PKCD, SHIP1 and SHP1, conditional CD25-deletion suppressed early B-cell development but induced hyperactivation of antigen-experienced B-cells and autoimmunity. Additionally, B-cell-specific CD25 deletion resulted in formation of spontaneous germinal centers and expansion of autoreactive B cells.

Biochemical and interactome studies revealed that BCR-signaling induced PKCd-dependent CD25-phosphorylation of the CD25 cytoplasmic tail (S268) to recruit inhibitory phosphatases to the BCR complex. Upon CD25-deletion, SHIP1 and SHP1 were no longer recruited and activated to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca2+-oscillations induced anergy and negative selection during early B-cell development, as opposed to proliferation and autoantibody production in antigen-experienced B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to prevent chronic BCR signaling and safeguard B cell tolerance.
Gata3
ZsG-fl: a novel murine Gata3 reporter allele revealed a unique expression profile of GATA3 controlled genes in ILC2 and Th2 under in vivo condition
Authors: Gurram Rama Krishna , Wei Danping , Yu Qiao , Xi Chen , Cui Kairong , Hu Gangqing , Zheng Mingzhu , Chung Hyunwoo , Butcher Matthew J , Zhao Keji , Liu Chengyu , Khillan Jaspal S , Zhu Jinfang (Jeff)
Published: August 2023

The transcription factor GATA3 expression is essential for T helper-2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) mediated type 2 immune responses. However, most of the studies were done in in vitro system, and such functions cannot be easily verified in vivo because the expression of the markers for identifying Th2 and ILC2s depends on GATA3. We have generated a novel GATA3 reporter mice strain carrying Gata3ZsG-flallele. This mouse carries a ZsGreen-T2A cassette at the translation initiation site along with exon 4 flanked by two loxP sites. Later, an inducible Gata3 deletion system was created by crossing Gata3ZsG-flmice with a tamoxifen-inducible Cre (CreERT2) system. This reporter mice allowed us to visualize Gata3 deleted Th2 cells and ILC2s in vivo. Transcriptome comparison of Gata3 KO ILC2s and Th2 cells revealed common and distinct expression patterns of genes under the control of the GATA3 protein. Interestingly, this study also identified some GATA3-controlled genes specifically expressed under in vivo conditions but not in vitro. Therefore, transcriptome comparison of Gata3 KO ILC2s and Th2 cells in vivo provided valuable information to investigate the GATA3-controlled type 2 immune responses.

This research was supported by the Intramural Research Programs of NIAID, NIH.
Clonally-Expanded, Thyrotoxic Autoimmune Mediator CD8+ T cells Driven by IL21 Contribute to Checkpoint Inhibitor Thyroiditis
Authors: Lechner Melissa G , Zhou Zikang , Scott Lauren N , Ortega Jessica , Huang Nicole , Chen Ho-Chung , Hoang Aline , Tafti Rana Yakhshi , Kim Kristy , Hugo Willy , Famini Pouyan , Drakaki Alexandra , Ribas Antoni , Angell Trevor E , Su Maureen A
Published: August 2023

Autoimmune toxicity occurs in up to 60% of patients treated with immune checkpoint inhibitor (ICI) cancer therapy and is an increasing clinical challenge with the expanding use of these treatments. Previous human studies into the cause of immune related adverse events (IRAEs) have often evaluated only circulating immune cells rather than affected tissues. Here, we directly sampled thyroid specimens from subjects with ICI-thyroiditis, one of the most common IRAEs, to determine driving mechanisms. We compared immune infiltrates in ICI-thyroiditis to those in subjects with spontaneous autoimmune Hashimoto’s thyroiditis (HT) or no thyroid disease. Single cell RNA sequencing revealed a dominant, clonally expanded population of thyroid-infiltrating cytotoxic CXCR6+ CD8+ T cells (“CD8+ autoimmune mediators”) present in ICI-thyroiditis, but not HT or healthy subjects. Furthermore, we identified a crucial role for interleukin 21, a cytokine secreted by intrathyroidal T follicular (Tfh) and T peripheral helper (Tph) cells, as a driver of these thyrotoxic CD8+ autoimmune mediators. In the presence of IL21, human CD8+ T cells acquired the autoimmune mediator phenotype with upregulation of cytotoxic molecules (IFNg, granzyme); the chemokine receptor CXCR6; and thyrotoxic capacity. We validated these findings in vivo using a novel mouse model of IRAEs, and further demonstrated that genetic inhibition of IL21 signaling protected ICI-treated mice from thyroid autoimmunity. Taken together, our data reveal novel mechanisms by which IL21+Tfh/Tph cells drive thyrotoxic CD8+ autoimmune mediators for the development of IRAEs in humans. Furthermore, these studies highlight IL21 as a potential therapeutic target in patients to prevent IRAEs.

Supported by grants from the NIH NIDDK (K08 DK129829), the American Thyroid Association, and the Aramont Charitable Foundation.
Inhibition of Janus Kinase pathway corrects IFN-driven inflammation and epithelial dysfunction in Sjogren’s disease
Authors: Gupta Sarthak , Yamada Eiko , Nakamura Hiroyuki , Khavandgar Zohreh , Martin Daniel , Tandon Mayank , Alevizos Ilias , Jang Shyh-Ing , Perez Paola , Dominick Kalie , Pranzatelli Thomas , Baer Alan , Chiorini John , Warner Blake
Published: August 2023

Background/Purpose

A systematic investigation of the role of interferons (IFNs) and Janus kinases (JAK) - signal transducer and activator of transcription (STAT) pathway at the cellular and tissue level in humans is lacking.

Methods

PBMC and minor salivary gland (MSG) biopsies were obtained from healthy volunteers (HVs) and SjD patients that met the 2016 American College of Rheumatology classification criteria on IRB approved protocols.

Results

RNA sequencing (RNAseq) on MSGs found enrichment of JAK-STAT pathway genes in SjD (n=36) compared to HV (n=13). A composite score of 21-IFN stimulated genes (ISGs) was elevated in SjD and positively correlated with clinical variables of SjD (focus scores). Immunofluorescence showed increased expression of JAK3 in infiltrating lymphocytes. Single cell RNAseq (scRNAseq) of MSGs from SjD (n=12) and HVs (n=17) showed upregulated JAK-STAT pathway genes in infiltrating T and B lymphocytes; antigen presenting cells; and endothelial cells. Ex-vivo studies showed baseline elevation of intracellular phosphorylated STAT1 and STAT3 levels in PBMCs in SjD (n=21) vs HVs (n=11). Stimulation with IFNb further increased the pSTAT1 signaling and JAK inhibition with tofacitinib led to normalization of JAK-STAT signaling. HV and SjD-derived primary salivary gland epithelial cells (pSGEC) showed elevated basal CXCL10 in patients with SjD (n=5) vs HV (n=5); and JAK inhibitors (tofacitinib, baricitinib) blocked IFNb driven ISG expression.

Conclusion

Our findings pinpoint cells responsive to JAK inhibition and illustrate in patient tissues that JAK inhibitors may be beneficial in SjD. These data will serve as biological endpoints for clinical trials [NCT04496960].

This research was supported by the Intramural Research Programs of the National Institute of Arthritis and Musculoskeletal and Skin Disease and National Institute of Dental and Craniofacial Research
In vitro generation of disease-specific autoantibodies produced by B cells from patients with NMOSD and MOGAD
Authors: Payumo Rosah May Palermo , Kim Yeseul , Kim Soyeon , Han Sang-Min , Park Kevin , Park Hyewon , Kim Ha-Eun , Kim Su-Hyun , Hyun Jae-Won , Kim Ho Jin
Published: August 2023

The main pathogenic feature of neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein associated disease (MOGAD) is the presence of autoantibodies against water channel, aquaporin-4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG), respectively. However, the underlying mechanisms in which the B cells from these patients produce autoantibodies is largely unknown.

Our study aims to establish a human culture system that could characterize the contribution of B cells in the production of these autoantibodies.

PBMCs were obtained from NMOSD (n=30) and MOG (n=10) patients. B cells were differentiated into plasmablasts in vitro with different stimuli resembling the mechanism related with NMOSD and MOGAD development. After 6d culture, cell proliferation and plasmablast frequency were assessed. Quantification of total IgG levels and detection of AQP4-IgG or MOG-IgG were determined on D13.

Our data showed that the presence of R848 (TLR7/8), regardless of sCD40L or cytokines, resulted in B cell proliferation in NMOSD (ave: 78.2% ± 18.5 SEM) and MOGAD patients (ave: 90.4% ± 5.2 SEM). Moreover, in vitro generation of plasmablasts require sCD40L and R848 in NMOSD (ave: 45.6 ± 21.1 SEM) and MOGAD (ave: 54.4% ± 11.5 SEM). Further culture until D13 resulted in IgG secretion by the plasmablasts generated from NMOSD (ave: 18,150.07 ng/mL) and MOGAD (ave: 17,959.25 ng/mL). For all the conditions that generated plasmablasts, autoantibodies were detected to be reactive with either AQP4-transfected cells in NMOSD (10/30 patients) or MOG-transfected cells in MOGAD (6/10 patients).

This in vitro model have important implications for therapy selection and understanding the mechanisms of NMOSD and MOGAD development.

This study was supported by the International Cooperation Education Program of National Cancer Center Korea (NCCRI·NCCI 52210-52211).
Humanized BAFF transgenic mice develop autoimmune manifestations
Authors: Li Zhiying , Liang Juan , Zhang Yuxi , Xu Shuxin , Liu Qinglong , Tong Huan , Ku Cunxiang , Zhao Jing , Xiang Gao
Published: August 2023

The B cell activating factor(BAFF) is a B cell survival factor that supports autoreactive B cells’ survival and prevents their depletion. Excessive BAFF can increase autoreactive B cells, driving autoimmunity. BAFF is commonly overexpressed in Systemic Lupus Erythematosus(SLE) and is strongly involved in the pathogenesis of the disease. Mice with BAFF deficiency results in the lack of mature B cells, whereas mice that overproduce BAFF have high numbers of mature B cells and antibodies, including autoantibodies, and develop an autoimmune disease similar to SLE in humans. BAFF has been shown to be the pivotal target in autoimmune diseases. In this study, we report the generation of two transgenic mouse strains with overexpression of human BAFF (B6-hBAFF, T036791 and T036794), which could be used to study anti-BAFF therapies.

Overexpression of human BAFF led to significantly increased IgG, IgA, and IgM levels since 6 weeks of age compared with wild-type mice, which continued to increase gradually as the transgenic mice aged. These mice secreted a high level of anti-DNA autoantibodies, along with an increased ratio of B cells/CD45+ in the spleen and lymph node compared with wild-type mice, indicating overreactive B cells. Proteinuria started at 6 weeks of age with an increase in protein/creatinine ratio. This was accompanied by the infiltration of inflammatory leukocytes in the kidney at 15 weeks of age, suggesting local kidney inflammation. Taken together, the humanized BAFF transgenic mice autoimmune manifestations with enhanced overreactive B cells, providing a valuable tool supporting preclinical efficacy studies of drugs targeting B cells for the development of autoimmune disease therapies.
Selective inhibition of estrogen receptor alpha restores regulatory T-cell function in autoimmune hepatitis
Authors: Longhi Maria Serena , Graham Jonathon J , Gao Li , Zhang Wei , Zhang Lina , Nastasio Silvia , Kalbasi Ahmadreza , Bonder Alan , Patwardhan Vilas , Robson Simon C
Published: August 2023

Dysfunction in regulatory T-cells (Tregs) is a hallmark of autoimmune hepatitis (AIH), a serious hepatopathy often leading to end-stage liver damage and transplantation, if not treated. Treg impairment in AIH has been linked to defective response to aryl hydrocarbon receptor (AhR) activation. AhR acts as a modulator of toxin and adaptive immunity. We have previously shown that altered response of Tregs to AhR activation derives from heightened levels of estrogen receptor alpha (Erα), an AhR non-canonical binding partner. In this study we investigated whether Erα selective antagonism modulates Treg function and response to AhR in AIH.

Experiments were performed on immunomagnetically purified Tregs, exposed to the Erα-specific antagonist methyl-piperidinopyrazole (MPP), in combination with or without unconjugated bilirubin (UCB), a known AhR endogenous ligand. In healthy controls (HC), UCB boosted Treg ability to suppress T-responder cell proliferation, and this was further increased when UCB was combined with MPP. In AIH, addition of MPP significantly ameliorated Treg ability to suppress, when exposed to UCB. Stimulation of Tregs with UCB and MPP boosted CYP1A1, CD39 and IL-10 levels both in AIH and HC. Analysis of Treg transcriptome indicated that treatment of Tregs with MPP increased the levels of CD73, an ectoenzyme that works in tandem with CD39, to ultimately generate adenosine.

In conclusion, selective inhibition of Erα restores Treg ability to respond to AhR resulting in increased CD39 levels and ameliorated suppressive function in AIH. This could provide a new therapeutic strategy to boost and maintain immunotolerance in AIH.

Supported by grants from NIH (R01 DK124408, R01 DK108894)
ANATOMY OF THE IMMUNE RESPONSE TO FRANCISELLA TULARENSIS LVS INFECTION IN THE LUNGS
Authors: Okoye G Donald , Kumar Amrendra , Scott Algood Holly M , Joyce Sebastian
Published: August 2023

The respiratory mucosa is under constant immune surveillance because of its vulnerability to infectious diseases. Infection of the lungs with the live vaccine strain (LVS) derived from Francisella tularensis (Ft) subspecies holarctica models pulmonary tularaemia-like disease in mice. Current evidence suggest many immune cells and cytokines respond to Ft LVS infection, but how these various immune system components are integrated to mount a protective response remains unclear. Hence, in a transcriptomics study at single cell resolution, we characterized the acute immune landscape in the lungs of C57BL/6 mice at day 0 and day 7 post intranasal inoculation with Ft LVS. Defining features of the immune response include a robust type 1 immune response characterized by the accumulation of inflammatory neutrophils and the expansion of innate-like effector lymphocytes, primarily interferon-γ producing NKT1, NK, and effector CD8+ T cells. Increased accumulation of MAIT17 over MAIT1 cells is another feature, which is in line with previous findings linking MAIT cells and IL-17 to LVS immune response. Surprisingly, a significant proportion of MAIT17 cells either maintained or upregulated type 1 inflammatory markers. Similarly, MAIT1 cells maintained or upregulated Type 3 inflammatory markers. Additionally, a highly active MAIT cell subset has increased Nr4a1 expression, which encodes Nur77, suggesting a T cell receptor-mediated activation. By contrast, NKT1 cells poorly upregulated Nr4a1 expression but induced Il18r1 expression suggesting, a cytokine-mediated activation. Consequently, we predict that unconventional NKT & MAIT cells integrate innate cues to control tularaemia-like disease caused by Ft LVS infection in the mouse.

VUSM MSTP NIGMS of the National Institutes of Health T32GM007347 (GDO); IO1 BX001444, BX001610, BX00xxx, IK6 BX004595 & RO1 AI137082; IBX000915A (HMA)
Cell-specific P2RX7 promotes the longevity of lung tissue-resident helper CD4 T cells in response to influenza infection
Authors: Santiago-Carvalho Igor , de Gois Macedo Bruna , Vardam-Kaur Trupti , van Dijk Sarah , Salgado Caio Loureiro , da Silva Henrique Borges
Published: August 2023

Development of universal influenza (flu) vaccines requires better knowledge of lung-resident immune responses. That includes flu-specific CD4 T cells, which establish long-term lung residency and induce a diversified local protective response to subsequent infection. Tissue-resident helper (Trh) cells are a subset of lung-resident CD4 T cells that share phenotypic and functional features with follicular helper cells (Tfh). Trh cells serve to amplify lung-resident immune responses, and express high levels of the extracellular ATP (eATP) sensor P2RX7. Considering that eATP levels in the lung increase in response to flu infection, we used T cell-specific P2RX7-knockout (KO) mice to test the role of P2RX7 for CD4 T cell responses to flu. During acute phase of infection, we found that P2RX7 promotes the accumulation of flu-specific CD4 T cells inside the lung tissue. Moreover, P2RX7 promoted this establishment via induction of CXCR3 upregulation. Despite no specific defects in the Trh pool at the acute phase, T cell-specific P2RX7-KO led to a progressive decay of Trh cells. Consequently, lungs of T cell-P2RX7-KO mice had reduced lung B cell responses. Our single-cell RNAseq of lung flu-specific CD4 T cells showed reduced expression of cell survival and mitochondrial genes in P2RX7-KO Trh cells. Our findings suggest that lung eATP sensing by CD4 T cells induces lung-resident CD4 T cell accumulation in response to flu, impacting the survival of specific lung CD4 T cell subsets.

National Institute of Allergy and Infectious Diseases (NIAID) (H.BdS.: R00 AI139381, R01 AI170649).
Upper respiratory tract resident memory CD8 T cells limit viral transmission and provide durable protection
Authors: Michalets Sarah , Uddbäck Ida , Williams M Elliott , Mattingly Cameron , Kost Kirsten N , Lawrence Laurel A , Lowen Anice C , Scharer Christopher D , Kohlmeier Jacob E
Published: August 2023

An effective vaccine against respiratory viruses should not only limit disease in infected individuals, but also restrain viral dissemination among the population. Furthermore, directing vaccine responses to conserved viral epitopes can offer cross-strain protection and potentially limit viral escape from pre-existing immunity. Although studies have demonstrated that intranasal vaccination can generate populations of resident memory CD8 T cells (TRM) throughout the respiratory tract that can rapidly clear a secondary infection and limit immunopathology, the ability of CD8 TRM to limit natural respiratory virus transmission has not yet been evaluated. To address this gap in knowledge, we developed a model of parainfluenza virus transmission whereby a bioluminescent Sendai virus can be used in conjunction with in vivo imaging to track transmission dynamics longitudinally across murine cohorts. Recombinant influenza and adenoviral vectors expressing the immunodominant Sendai virus nucleoprotein CD8 T cell epitope enable the establishment of Sendai virus-specific CD8 TRM throughout the respiratory tract without inducing Sendai-specific antibodies. Using these models, we demonstrate that virus-specific CD8 TRM can inhibit the establishment of infection in immunized hosts and prevent respiratory virus transmission. Additionally, we show that protection against respiratory virus transmission is mediated through TRM localized in the upper respiratory tract and is durable for at least six months. These results indicate that vaccines which induce localized T cell immunity at the initial site of respiratory virus infection can contribute to the generation of herd immunity and limit viral spread in the population.

This project is supported by NIH grants R35 HL150803 and U01 HL139483.
CD11b+Ly6g+CD69+CD74+ cells produce interferon-α in Mycobacterium tuberculosis infected alcohol diet-fed mice
Authors: Mukherjee Tanmoy , Tripathi Ashutosh , Radhakrishnan Rajesh Kumar , Vankayalapati Abhinav , Vankayalapati Ramakrishna , Tripathi Deepak
Published: August 2023

Chronic alcohol consumption is associated with significant mortality and morbidity in people affected by Mycobacterium tuberculosis (Mtb) infection. Our Lab previously reported that alcohol diet increased mortality in young mice infected with Mtb in an Interferon-α (IFN-α) dependent manner. We also found CD11b+Ly6g+ cells are major source for IFN-α. In the current study, using single cell RNA sequencing, we further characterized IFN-α producing CD11b+Ly6g+ cells in the lungs of Mtb infected alcohol-diet fed mice. Our analysis revealed ‘6’ distinct clusters in CD11b+Ly6g+ cell population based on gene signatures. We identified cluster ‘6’, through gene-set scoring and expression of interferon related genes such as IRF1, IRF5 to be responsible for type I interferon production. Pseudotime analysis along with KEGG enrichment, revealed transcriptome of cluster ‘6’ to be in a separate trajectory compared to the core of the cluster, suggesting a differentiated phenotype. Similarly, we identified CD69 and CD74 as a surface marker for this unique cell population which was subsequently verified with t-sne analysis using flow cytometry. We are currently characterizing the cellular mechanisms regulating the expansion of these cells in Mtb infected alcohol-diet fed mice to be able to target them for effective early Interferon-α neutralization.
Intracellular pH controls death and proliferation of lymphocytes in immune response and tumor cells
Authors: Zeng Wei-ping , Yang Shuangshuang , Zhou Baohua
Published: August 2023

Cancer and highly proliferating normal cells have high demand for energy but paradoxically switch energy production from the highly efficient mitochondrial respiration to the low efficient cytosolic glycolysis. This phenomenon is known as the Warburg effect, but the reason for which is not fully understood. This study found that low intracellular pH (pHi) induced apoptosis, whereas high pHi enhanced proliferation, of normal lymphocytes. In tumor cells, low pHi also induced apoptosis whereas high pHi maintained high rates of proliferation. In the draining mediastinal lymph nodes (MLNs) of mice sensitized and challenged with ovalbumin, lymphocytes had dramatically higher pHi and became more heterogeneous than lymphocytes in the non-draining lymph nodes. The pHi of the lymphocytes was inversely related to mitochondrial energetic activities, and the rise of the lymphocyte pHi in the MLNs was accompanied by the reduction of mitochondrial energetic activities. Furthermore, high lymphocyte proliferation in the MLNs powered by strong mitochondrial energy led to low pHi thereby predestined the lymphocytes to death by apoptosis. These findings support a proposed explanation for the Warburg effect as a substitution for mitochondrial respiration that allows the highly proliferating cells to avoid low pHi-induced apoptosis.

None
Sphingosine 1-phosphate / Acetyl CoA Carboxylase1 Axis regulates the Differentiation and Function of Tumor-associated Myeloid-Derived Suppressor Cells
Authors: Chakraborty Paramita , Choi Seungho , Kassir Mohamed Faisal , Oberholtzer Nathaniel , Hedley Zachariah T , Beeson Gyda C , Chatterjee Shilpak , Ogretmen Besim , Mehrotra Shikhar
Published: August 2023

The presence of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME) poses a serious obstacle to cancer treatment. The bioactive lysophospholipid sphingosine 1-phosphate (S1P), which is produced by sphingosine kinases (SphK1 or SphK2), regulates tumor and T cell survival and activity. Thus, it has been targeted to improve the outcome of anti-cancer therapeutics. However, S1P has not yet been investigated in terms of how it regulates myeloid cell activity. The present study demonstrates that deletion of SphK2-mediated S1P in MDSCs favorably modifies the tumor microenvironment and renders infiltrating myeloid cells with antigen presenting phenotype, leading to increased ability to control tumor growth. Furthermore, in preclinical prostate and melanoma cancer models, the combination of PD1 antibody and SphK2 pharmacological inhibitor (ABC294640) dramatically reduced tumor growth and increased the survival of tumor bearing mice. Mechanistically, the S1P pulldown and subsequent MS-based proteomics analysis revealed that S1P binds to various proteins involved in metabolic and ribosomal biogenesis pathways. Notably, both the isoforms of Acetyl CoA Carboxylase, the rate-limiting fatty acid metabolic enzyme, binds to S1P. Furthermore, investigations using deletion, overexpression, and site-directed mutagenesis demonstrated that ACC1 activity is necessary to preserve the immunogenic capacity of SphK2 KO MDSCs. This study highlights the clinical value of manipulating MDSC suppressive activity by targeting the S1P/ACC1 axis to boost T cell-mediated tumor control and highlights the crucial function that S1P plays as an immunoregulatory mediator in MDSCs.

Supported grants from NIH (R01 CA250458, R01 CA236379)
Poly(lactic acid)-based immunomodulatory nanoparticles promote survival in a polymicrobial mouse model of sepsis
Authors: Truong Nhu , Dharmaraj Shruti , Cottingham Andrea , Shaw Jacob , Lasola Jackline Joy , Scotland Brianna L , Pearson Ryan M
Published: August 2023

Sepsis is a life-threatening multifactorial organ dysfunction caused by a dysregulated host response to infection. Despite being recognized as a global health priority, current therapeutic approaches for sepsis have yet to demonstrate significant improvements in patient outcomes. Our group previously demonstrated that cargo-less immunomodulatory nanoparticles (iNPs) could suppress innate immune cell proinflammatory responses and increase survival in an LPS-induced endotoxemia mouse model. In the present work, we examine the impact of poly(lactic acid)-based iNP delivery on survival and comprehensively characterize the host immune response using a clinically relevant and lethal mouse model of polymicrobial sepsis, cecal ligation and puncture (CLP). iNPs, when administered intraperitoneally in combination with broad spectrum antibiotics following CLP surgery, resulted in a significant improvement in disease symptoms and survival compared to CLP + antibiotics only. We also observed that iNP treatment reduced the levels of proinflammatory cytokines in the peritoneal fluid. Analysis of immune cell populations suggested that iNP treatment modulated macrophage, myeloid-derived suppressor cell, and inflammatory monocyte populations both locally and systemically. Our study demonstrates that local administration of iNPs promotes survival through a multimodal immunomodulatory mechanism to improve sepsis outcomes.

This research was supported by startup funds from the University of Maryland School of Pharmacy, National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM142752, and the Shock Society Faculty Research Award awarded to Ryan Pearson. Nhu Truong is supported by a PhRMA Foundation Pre-Doctoral Fellowship from Washington, DC. This publication was supported by funds through the National Cancer Institute - Cancer Center Support Grant (CCSG) - P30CA134274, and Maryland Department of Health’s Cigarette Restitution Fund Program
IL-6 suppresses vaccine responses in neonatal mice by enhancing IL-2 activity on T follicular helper cells
Authors: Parvathaneni Swetha , Yang Jiyeon , Lotspeich-Cole Leda , Sakai Jiro , Lee Robert C , Akkoyunlu Mustafa
Published: August 2023

The inability of neonates to develop CD4+CXCR5+PD-1+ T follicular helper (TFH) cells contributes to their weak vaccine responses. In previous studies, we measured diminished IgG responses when IL-6 was co-injected with a pneumococcal conjugate vaccine (PCV) in neonatal mice. This is in sharp contrast to adults, where IL-6 improves vaccine responses by downregulating the expression of IL-2Rβ on TFH cells and protecting them from the inhibitory effect of IL-2. In this study, we found that splenic IL-6 levels rapidly increased in both adult and neonatal mice following immunization, but the increase in neonatal mice was significantly more than that of adult mice. Moreover, immunized neonatal TFH cells expressed significantly more IL-2 as well as its receptors, IL-2Rα and IL-2Rβ, than the adult cells. Remarkably, IL-6 co-injection with PCV vaccine further increased the production of IL-2 and the expression of its receptors by neonatal TFH cells, whereas excess IL-6 had totally opposite effect in immunized adult mice. Underscoring the role of IL-6 in activating the IL-2 mediated suppression of vaccine responses, immunization of IL-6 knock-out neonates led to improved antibody responses accompanied by expanded TFH cells as well as lower levels of IL-2 and IL-2 receptors on TFH cells. Moreover, CpG containing PCV improved TFH response in neonates by suppressing the expression of IL-2 receptors on TFH cells and inhibiting IL-2 activity. These findings unveil age-specific differences in IL-6 mediated vaccine responses and highlight the need to consider age-related immunobiological attributes in designing vaccines.

This study was funded by US FDA, Perinatal Health Center of Excellence (PHCE) grant to Mustafa Akkoyunlu as well as intramural grants from US FDA.
Design and Preclinical Evaluation of a Universal SARS-CoV-2 mRNA Vaccine
Authors: Langston Laura Katherine , Xu Renhuan , Zhu Huabin , Qin Jane , Jeon Ju , Xu Jiangsheng , Mou Stephanie
Published: August 2023

Due to the rapid mutations of severe acute respiratory syndrome coronavirus (SARS-CoV-2), an effective vaccine against SARS-CoV-2 variants is needed to prevent coronavirus disease 2019 (COVID-19). T cells, in addition to neutralizing antibodies, are an important component of naturally acquired protective immunity, and a number of studies have shown that T cells induced by natural infection or vaccination contribute significantly to protection against several viral infections including SARS-CoV-2. However, it has never been tested whether a T cell-inducing vaccine can provide significant protection against SARS-CoV-2 infection in the absence of preexisting antibodies. In this study, we designed and evaluated lipid nanoparticle (LNP) formulated mRNA vaccines that induce only T cell responses or both T cell and neutralizing antibody responses by using two mRNAs. One mRNA encodes SARS-CoV-2 Omicron Spike protein in prefusion conformation for induction of neutralizing antibodies. The other mRNA encodes over one hundred T cell epitopes (multi-T cell epitope or MTE) derived from non-Spike but conserved regions of the SARS-CoV-2. We show immunization with MTE mRNA alone protected mice from lethal challenge with the SARS-CoV-2 Delta variant or a mouse-adapted virus MA30. Immunization with both mRNAs induced the best protection with the lowest viral titer in the lung. These results demonstrate that induction of T cell responses, in the absence of preexisting antibodies, is sufficient to confer protection against severe disease, and that a vaccine containing mRNAs encoding both the Spike and MTE could be further developed as a universal SARS-CoV-2 vaccine.
Antigen presenting cell targeted T cell DNA vaccine inducing strong and specific cellular responses across multiple T cell epitopes of SARS-COV-2
Authors: Norheim Gunnstein , Kuczkowska Katarzyna , Stubsrud Elisabeth , Bjerkan Louise , Jørgensen Marthe , Husbyn Hannah , Bøthun Anne , Dürkoop Mirjam , Sekelja Monika , Namouchi Amine , Lindahl Torbjørn , Granum Stine , Chellappa Stalin , Skarshaug Renate , Klinger Mark , Ebert Peter , Osborne Edward , Fredriksen Agnete Brunsvik , Pedersen Mikkel Wandahl
Published: August 2023

The severe respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 has continuously evolved with successive new virus variants of concern (VOC). A key challenge is to develop vaccines that can prevent infection and/or protect against severe disease caused VOCs that evade vaccine induced Spike neutralizing antibodies. T cell responses likely contribute to the efficacy of approved vaccines against VOCs and provide the rationale for the development of T-cell based vaccines. VB10.2210 is a T-cell based vaccine that was developed using Nykode’s easily adaptable DNA plasmid (pDNA) vaccine platform. VB10.2210 pDNA encodes homodimers consisting of i) a targeting unit that binds chemokine receptors on antigen-presenting cells, ii) a dimerization unit, and iii) an antigenic unit consisting of a selection of validated immunogenic SARS-CoV-2-specific T cell epitopes. The T cell epitopes were identified by Adaptive using T cell receptor sequencing of more than 6500 samples from COVID-19 individuals representing diverse geographies. The vaccine candidate was designed with broad HLA coverage and contains a diversity of both MHCI and MHCII T-cell epitopes across multiple viral proteins and known VOCs. In vitro characterization of the VB10.2210 pDNA showed that intact protein was expressed and secreted in human cell culture. The immunogenicity of the vaccine candidate was evaluated in transgenic HLA-A2.1, C57BL/6 and BALB/c mice. Preclinical data in these three mouse models demonstrate that VB10.2210 consistently induced strong, broad, dose-dependent, and persistent T cell responses across multiple T cell epitopes. VB10.2210 is currently being evaluated for safety and immunogenicity in a Phase I clinical trial (NCT05069623).
Relatively preserved functional capacity with standard COVID-19 vaccine regimen in People Living with HIV asymptomatic or with mild immunodeficiency
Authors: Hsiao Sung-Han , Hung Chen-Yiu , Huang Chung-Guei , Chen Tse-Ching , Chang Chia-Shiang , Huang Yu-Lin , Lin Yung-Chang , Lin Chun-Yen , Dutta Avijit , Huang Ching-Tai
Published: August 2023

People living with HIV (PLWH) are at a higher risk of sever disease with SARS-CoV-2 virus infection. COVID-19 vaccines are effective in most PLWH, with significant reduction in the risk of severe disease or death when the standard 2-shot regimen is taken. However, suboptimal immune response to the standard course of vaccination is a concern for PLWH, especially for those with moderate to severe immunodeficiency. An additional dose is recommended as part of the extended primary series. In Taiwan, this additional dose is endorsed even for asymptomatic or mild immunodeficiency with CD4 counts >200/mm3 and suppressed virology. We aimed to investigate the immune responses with the additional dose in PLWH asymptomatic or with mild immunodeficiency. We collected peripheral blood mononuclear cells (PBMC) from 72 PLWH asymptomatic or with mild immunodeficiency and from 362 non-HIV subjects, after two and three shots respectively. Two shots elicited lower responses in PLWH, although the difference was statistically insignificant. They have comparable levels of neutralizing and anti-S antibodies and comparable effector CD4+ and CD8+ T cell responses. The 3rd shot boosted the SARS-CoV-2 immunity significantly more in PLWH compared to the non-HIV people. Upon in vitro stimulation with extracted Wuhan strain SARS-CoV-2 proteins, CD8+ T cells from PLWH after 3 shots has more durable effector responses than the non-HIV controls with extended time of stimulation. This subtle difference between PLWH and non-HIV people implied immune exhaustion with two shots in non-HIV people. Slightly compromised immunity in PLWH indeed preserved the functional capacity for further response to the 3rd shot or natural infection.

The Research Center for Emerging Viral Infections receives support from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), Taiwan and NSTC 111-2634-F-182-001 from the National Science and Technology Council, Taiwan. This work was supported by Projects CMRPG3M1811 (CTH) from the Medical Research Project Fund, Chang Gung Memorial Hospital, Taiwan.
Adjuvant nanocellulose with different physicochemical property can induce immune adjuvant effects without posing cytotoxicity
Authors: Wang Lingzhen
Published: August 2023

Objective

To analyze the in vitro effectiveness of cellulose nanocrystals (CNC) in inducing pro-inflammatory and immune adjuvant effects in THP-1 cells, a monocyte cell line isolated from the peripheral blood of an acute monocytic leukemia patient.

Methods

THP-1 cells were counted, and their viability was calculated followed by the quantification of IL-1 β production.

Results

We observed the following: 1) CNCs were not cytotoxic to the cell lines, and 2) a 280 nm length of CNC induced the strongest immune response within our selected size spectrum.

Conclusion

This study provides a preliminary demonstration of the optimal length range CNCs that could induce most adjuvant effects without posing cytotoxicity.

This research didn’t receive funding support.
Investigating the potential of silver nanocubes and gold nanocages as protein subunit vaccine carriers
Authors: Yavuz Emine , Walters Adam , Chudasama Bhavnesh , Han Shunping , Qin Yue , Al-Jamal Khuloud
Published: August 2023

Metal nanoparticles have been exploited as vaccine delivery platforms for various diseases. In particular, Ag and Au NPs of different size and shape have been shown to promote delivery of antigens and act as efficient adjuvants. Yet, there is limited description of cuboidal Ag nanocubes (AgNCs) and Au nanocages (AuNCs) as antigen carriers for vaccination. Here, we first investigated the intrinsic protein binding ability of AgNCs and AuNCs, using ovalbumin (OVA) as a model. Next, the effect of AgNCs and AuNCs on bone marrow-derived dendritic cells (BMDCs) in terms of cytotoxicity and maturation was assessed. Finally, we investigated humoral and cellular immune responses of AgNCs-OVA and AuNCs-OVA following intramuscular immunization and their prophylactic effects in B16F10-OVA mice tumor model. In terms of OVA loading efficiency, AgNCs were more potent than AuNCs, despite their non-porous form. Both cubic nanomaterials were found not to induce BMDCs maturation in vitro or DCs maturation in spleen. Following the administration of nanovaccine doses, serum IgG responses were comparable between groups. However, there were significant alterations in relative frequencies of lymphocyte sub-populations. Overall, AgNCs-OVA and AuNCs-OVA immunization via intramuscular route did not alter tumour growth. This study has demonstrated the intrinsic immunomodulatory properties of AgNCs and AuNCs, preceding their potential use as nanocarriers.

Supported by grants from British Council (Newton Fund, 337313), Wellcome Trust (WT103913), and TUBITAK.