bluuebunny/crossref_metadata_2025 huggingface dataset:
Ssu72 phosphatase directly binds to ZAP-70, thereby providing fine-tuning of TCR signaling and preventing autoimmunity
Authors: ko jaesung , Jeong Dongjin , Koh Jaemoon , Jung Hyeryeon , Jung Kyeong Cheon , Jeon Yoon Kyung , Kim Hye Young , Yi Eugene C , Lee Ho , Lee Chang Woo , Chung Doo Hyun
Published: January 2023

ZAP-70 is required for initiation of TCR signaling. However, the mechanism by which ZAP-70 regulates the fine-tuning of TCR signaling remains elusive. Here, we found that the phosphatase Ssu72, originally known to regulate RNA polymerase II activity in the nucleus, contributed to the fine-tuning of TCR signaling by binding to ZAP-70. Affinity purification–mass spectrometry and an in vitro assay demonstrated specific interaction between Ssu72 and ZAP-70 in T cells. Upon TCR stimulation, Ssu72-deficient T cells increased the phosphorylation of ZAP-70 and downstream molecules, and exhibited hyper-responsiveness, which was restored by reducing ZAP-70 phosphorylation. Cd4-CreSsu72fl/fl mice showed a defect in the thymic development of invariant NKT cells and reductions in CD4+ and CD8+ T cell numbers in the periphery, but more CD44hiCD62Llo memory T cells and fewer CD44loCD62Lhinaïve T cells compared with wild-type mice. Furthermore, Cd4-CreSsu72fl/fl mice developed autoimmunity spontaneously at 6 months. In conclusion, Ssu72 regulates the fine-tuning of TCR signaling by binding to ZAP-70 and regulating its phosphorylation, thereby preventing autoimmunity.
Aberrant NKG2D receptor expression on CD 4 T cells were considerably reduced by accumulated effect of traditional Thai massage in Thai elderly
Authors: SORNKAYASIT KANDA , PHOKSAWAT WISITSAK , LEELAYUWAT CHANVIT , EUNGPINICHPONG WICHAI , JUMNAINSONG AMONRAT
Published: January 2023

Natural killer group 2, member D (NKG2D) is a potent activating receptor of the immune system that can express on most of T cells. The NKG2D expression on CD 4 T cells have elucidated as pathogenic immune cells in chronic inflammatory diseases also elderly. A possible effect of traditional Thai massage (TTM) on immunity via inflammatory response and homeostasis was supposed. TTM is an exclusive technique of deep tissue massage by acupressure along ten meridian lines and combined muscle stretching. Thus, this study aimed to investigate the effect of TTM on pathogenic immune cells in elderly. A randomized crossover trial was performed in 12 Thai elderly participants aged 67.7±5.12 years. Each of them underwent 6 sessions of 1-hour TTM/rest (once weekly) for 5 weeks with a 30-day washout period. The immunophenotyping was specifically investigated on cell surface markers using monoclonal antibody conjugated with different fluorochromes staining by flow cytometry. The results showed that the participants had risen NKG2D expression on CD 4 T cells before having TTM. The pathogenic immune cells were considerably reduced after having the sessions accumulated of TTM. The pairwise comparison between before and accumulated effect of NKG2D expressions on CD4+ T cells, CD4+28+ T cells, and CD4+28null T cells were significantly decreased (p=0.003, p=0.005, and p=0.005, respectively) in the TTM period whereas no significantly change was found during the control period. We conclude that TTM might promote immunity by selectively decreasing pathogenic immune cells in elderly. However, the mechanism should be clarified in the future.
A PI(4,5)P2-derived “gasoline engine model” for the sustained B cell receptor activation
Authors: Liu Wanli
Published: January 2023

To efficiently initiate activation responses against rare ligands in the microenvironment, lymphocytes employ sophisticated mechanisms involving signaling amplification. Recently, a signaling amplification mechanism initiated from phosphatidylinositol (PI) 4, 5-biphosphate [PI(4,5)P2] hydrolysis and synthesis for sustained B cell activation has been reported. Antigen and B cell receptor (BCR) recognition triggered the prompt reduction of PI(4,5)P2 density within the BCR microclusters, which led to the positive feedback for the synthesis of PI(4,5)P2 outside of the BCR microclusters. At single molecule level, the diffusion of PI(4,5)P2 was slow, allowing for the maintenance of a PI(4,5)P2 density gradient between the inside and outside of the BCR microclusters and the persistent supply of PI(4,5)P2 from outside to inside of the BCR microclusters. Here, we review studies that have contributed to uncovering the molecular mechanisms of PI(4,5)P2-derived signaling amplification model. Based on these studies, our recent work supported a “gasoline engine model” in which the activation of B cell signaling inside the microclusters is similar to the working principle of burning gasoline within the engine chamber of a gasoline engine.
Impact of Epstein-Barr Virus LMP2A-dependent BTK/STAT3 activation on resting and activated murine B cells
Authors: Swanson-Mungerson Michelle Anne , Del Toro Rosalinda , Zanotti Brian , Incrocci Ryan
Published: January 2023

Epstein-Barr virus (EBV) latently infects over 90% of the population and is associated with the development of numerous autoimmune diseases and B cell lymphomas. Therefore, understanding the impact of latency proteins on B cell survival and function is vital. One EBV latency protein, LMP2A, acts as a BCR mimic to promote B cell survival, increase B cell activation, and accelerate tumor development in murine mouse models. Our laboratory identified that LMP2A activates the PI3K/BTK/STAT3 pathway in human B cell lymphomas to increase IL-10 production that promotes survival. However, the ability of LMP2A to activate BTK and STAT3 in non-transformed B cells is unknown. To test this possibility, we returned to the LMP2A transgenic mouse model as a source of resting B cells that express LMP2A. To determine if LMP2A activates STAT3 in the mouse model, resting B cells from wildtype and LMP2A-transgenic mice were isolated and STAT3 phosphorylation was assessed by Western blot analysis. Our data indicate that LMP2A-postitive B cells express higher levels of phosphorylated STAT3 than wildtype controls. We further analyzed whether the LMP2A-dependent activation of STAT3 resulted in an increase in STAT3-dependent targets. Flow cytometric analysis indicates that the addition of STAT3 inhibitors to activated LMP2A-expressing B cells blocked the LMP2A-dependent increase in STAT3 targets in comparison to wildtype controls. These data suggest that LMP2A increases the activation of the oncogene STAT3 in resting B cells, which may contribute to the development of EBV-associated tumors and autoimmune diseases.
B cell-intrinsic STAT3 regulates the germinal center dark zone
Authors: Chodisetti Sathi Babu , Fike Adam , Bricker Kristen , Domeier Phillip P , Choi Nicholas M , Rahman Ziaur
Published: January 2023

Germinal centers (GCs) are sites for antibody diversification, somatic hypermutation (SHM) and antigen specific B cell selection. GCs are anatomically divided into two distinct areas known as dark zone (DZ) and light zone (LZ) where SHM and clonal selection occurs, respectively. Although previous studies have suggested a B cell-intrinsic role of STAT3 in GC maintenance and antibody responses, how STAT3 signaling in B cells may maintain GCs for B cell selection is not clear. Using immunization and infection models, we show that STAT3 in B cells regulates the GC DZ, resulting in decreased high affinity antibody production. STAT3 deficient DZ B cells demonstrate a decreased cell proliferation and increased apoptotic activity. Further, STAT3 deletion in B cells led to defects in plasma cell formation and B cell memory response. Using GC B cell specific STAT3 conditional knockout mice and GC B cell culture system, we show that STAT3 deficient GC B cells display low amounts of FOXO1, a critical transcription factor required for GC DZ program. Transcriptomic analysis of GC B cells from STAT3 deficient and sufficient mice reveals STAT3-mediated gene regulation of multiple cellular pathways known to be involved in GC DZ maintenance. In conclusion, STAT3 signaling in B cells controls the GC DZ, which, in turn, helps maintain GCs for high-affinity antibody production. Our findings identify STAT3 as a novel target for enhancing humoral immunity as well as for the treatment of diseases involving abnormal GC activity.
The Notch-Myc signaling axis sets the division-differentiation clock in marginal zone B cells
Authors: Gaudette Brian T. , Roman Carly , Atria Daniela Gomez , Maillard Ivan , Allman David
Published: January 2023

Lymphocyte differentiation is often tightly linked to mitosis. Clonal bursts due to antigen- or TLR-driven responses increase numbers of responding cells and may also facilitate changes in gene expression and chromatin needed for effector cell differentiation. Alternatively, to guard against rapid infection, lymphocyte pools may also contain cells poised for effector differentiation with minimal proliferation. Using cell cycle inhibitors or induced mutation of CDK1, we directly compared the impact of arresting mitosis on early plasma cell (PC) differentiation for naive follicular and marginal zone (MZ) B cells. MZ B cells reside in the marginal sinus of the spleen where they are positioned to respond rapidly to blood borne microbes. Whereas PC differentiation from follicular B cells occurred after only 4 or more divisions and was highly dependent on mitosis, MZ B cells yielded PCs much faster and despite full cell cycle blockade. Furthermore, short-term withdrawal of Notch signaling in MZ B cells in vivo caused rapid and robust down-regulation of large numbers of established Myc-regulated genes, and resulted in division/differentiation kinetics that mirrored follicular B cells. Altogether these results suggest that ongoing Notch signaling establishes a differentiation-poised state in MZ B cells needed for rapid division-independent antibody responses.
PI3K-dependent Reprogramming of Hexokinase Isoforms Regulates B Lymphocyte Metabolism
Authors: Marshall Aaron J , Mejia Edgard , Hou Sen , Sher Affan , Hatch Grant , Aliani Michel
Published: January 2023

The PI3K signalling pathway is known to regulate B cell metabolic programming upon activation, but the mechanisms involved are not well understood. Here we find that the PI3K pathway controls reprogramming of hexokinases (HKs), the enzymes that convert glucose into glucose-6-phosphate as a key rate-limiting step of glycolysis and other metabolic pathways. In primary mouse B cells, PI3K pathway inhibition substantially impaired the activation-induced increase in extracellular acidification rates (ECAR), a measure of glycolysis. In contrast, B cells isolated from PI3Kdelta gain-of-function mutant mice exhibit elevated ECAR. We find that B cell activation substantially elevates protein levels of HK2 and HK3 isoforms, but not HK1, in a PI3K-dependant manner. PI3K or mTOR inhibition significantly reduced induction of HK2 and HK3 expression, whereas Akt inhibition did not affect HK isoform expression. In human B lymphoma cells, HK isoforms differ significantly in their degree of mitochondrial localization, with HK1 being mitochondrial, HK3 being cytoplasmic and HK2 present in both mitochondria and cytoplasm. To assess whether HK isoforms have unique non-redundant functions, HK2-deficient B lymphoma cells were generated and were found to exhibit decreased ECAR as well as significant changes in metabolomic profile, despite normal expression and localization of HK1 and HK3. Taken together, our study reveals that PI3K-dependant reprogramming of hexokinase isoforms can impact on B cell glycolysis and other metabolic pathways. Studies in progress are examining the functional importance of HK2 in antibody responses using mice with B cell-specific deletion of this enzyme.
Desmosterol, an intermediate of cholesterol biosynthesis, modulates B cell activation and functions
Authors: Gauronskas Phillip Joseph , Waseem Tayab , McDonald Jeffrey , Fernandez-Hernando Carlos , Galkina Elena
Published: January 2023

Introduction

Atherosclerosis is a disease of large/medium size vessels attributed to a hyperlipidemic environment and a low-grade chronic inflammation. Normally, macrophages uptake modified low-density lipoprotein (mLDL), resulting in excess intracellular desmosterol, an immediate precursor to cholesterol, which promotes anti-inflammatory signaling. In contrast, mLDL uptake by macrophages in atherosclerosis produces foam cells that play an atherogenic role. While it has been previously shown that B cells can uptake mLDL, it remains unclear how mLDL uptake modulates B cell phenotype.

Methods

Murine B cells were treated with 1 mM of desmosterol for 12/24 hrs, cholesterol efflux genes were analyzed using qPCR. CFSE-labeled splenocytes were incubated with LPS/BAFF, treated with 1 mM of desmosterol for 72 hrs, and analyzed via FACS. B cells isolated from transgenic mice ooverexpressing DHCR24, a dehydrocholesterol reductase, were analyzed for desmosterol content by lipidomics, Ca2+ flux by FACS, and DNP-Ficoll induced Igs production in vivo by ELISA.

Results

Desmosterol uptake results in an increased expression of cholesterol efflux genes and attenuated B cell proliferation. Overexpression of DHCR24 leads to a decreased intracellular desmosterol, hyper-response of B cells shown by increased Ca2+. DNP-Ficoll immunization of B cell-deficient mice populated with either DHCR24- or WT B cells induce more anti-IGM specific Abs in the DHCR24-B cell recipients.

Conclusions

These results suggest desmosterol is synthesized and sensed by B cells, and its accumulation regulates B cell activation thresholds in vitro and in vivo. These novel data offer new insights for B cell biology and atherosclerosis pathology.
A histone code for inducible transcription in immune responses
Authors: de Paz Alexia Martinez , Beguelin Wendy , Sahin Merve , Leslie Christina S. , Melnick Ari M. , Josefowicz Steven Z.
Published: January 2023

Complex organisms have been subjected to intense selective pressure by host-pathogen coevolution to adapt robust immune responses to pathogen sensing. These responses occur by inducing specific genes within the context of large genomes condensed into chromatin. Despite general description of the chromatin changes that occur across inducible genes, little is known of how specific chromatin features, including histone post-translational modifications (PTM), functionally contribute to transcriptional output of responsive genes.

We have described two “signaling-to-chromatin” pathways leading to phosphorylation of histone H3 at residues S28 and S31 that are “read out” by chromatin regulatory factors to augment transcription. H3S28 phosphorylation drives early chromatin activation preceding transcription. This modification delineates expansive chromatin architectural domains containing responsive genes. We are pursuing the hypothesis that H3S28 phosphorylation increases chromatin interaction dynamics within the compartment to favor specific enhancer-promoter interactions and concentrate transcription factors. H3.3S31 is co-transcriptionally phosphorylated along the bodies of induced genes, and coordinately stimulates co-activators and ejects repressors to augment transcription.

We hypothesize that these stimulation-dependent histone phosphorylations act synergistically as a dedicated histone code selectively employed at stimulation responsive genes in macrophages and B cells, with potential for involvement in diverse cell types.

Our data suggest that co-option of these signaling pathways may contribute to lymphomagenesis, and their disruption could represent novel therapeutic strategies.
Assessment of function of neutrophil-derived properdin in a novel functional assay
Authors: Menon Smrithi Sugumaran , Galwankar Neeti , Moore Sara R. , Ferreira Viviana P.
Published: January 2023

The alternative pathway (AP) of complement is an essential immune effector mechanism. Properdin (P), the only positive regulator of the AP, is required for stabilization of AP enzymatic convertases. P also binds to certain surfaces, in vitro, activating complement by recruiting a de novo C3bBb convertase. P is present in serum as dimers (P2), trimers (P3) and tetramers (P4) in a 1:2:1 ratio, with tetramers the most active, followed by trimers and dimers. Activated neutrophils release properdin from secondary granules into the cellular microenvironment. However, the distribution of neutrophil-secreted P oligomers (which would correlate with P function), and whether this distribution modulates local inflammatory processes, are unknown. We developed a novel ELISA-based assay that detects functional differences between P oligomers, based on the immobilization of P and assessment of its ability to generate C3 convertases leading to C3b deposition. Pure P2, P3, and P4 were functionally distinct in this assay and serum-derived P behaved similar to pooled P2-P4, as expected. Surprisingly, P from PMA-activated neutrophil supernatants had significantly lower activity than serum P (p<0.0001), and varied between healthy donors. To understand whether serum is required for enhancing P function (i.e. by allowing P polymerization), neutrophil supernatants were pre-incubated with P-depleted serum, followed by measurement of P function by ELISA. No increase in neutrophil-derived P function was observed. Additional studies are underway to understand the mechanisms involved in the lower function of neutrophil-derived P, with the aim of contributing to understanding molecular mechanisms of AP regulation in inflammatory microenvironments.
Transcriptional regulation of IL10 gene in human macrophages
Authors: Kwon Geunho , Kang Kyuho
Published: January 2023

IL-10 is an anti-inflammatory cytokine with a crucial role in immune homeostasis. Dysregulated production of IL-10 has been linked to certain autoimmune and inflammatory diseases. IL-10 is strongly induced by TLR ligands and suppressed by IFN-γ as part of the synergistic activation of inflammatory genes. However, the molecular mechanisms that regulate expression of the IL10 gene, especially in human macrophages, are incompletely understood. By using epigenomic analysis of ATAC-seq and ChIP-seq analysis we have identified human macrophage-specific enhancers in the IL10 gene locus. In these putative enhancers, we observed that chromatin accessibility and histone acetylation are increased by LPS stimulation. We also found that p300 was frequently recruited to these macrophage-specific cis-regulatory elements (CREs) in LPS-stimulated macrophages. One of these potential LPS-induced enhancers, CRE+6, is located 6kb upstream of the IL10 transcriptional start site and contains transcription factor binding motifs and autoimmune disease-associated SNPs. CRE+6 showed constitutive binding of the macrophage-lineage-determining factor PU.1 and LPS-inducible binding of AP-1 and STAT3. LPS stimulation increased eRNA transcription from CRE+6. The functional relevance of CRE+6 eRNA was confirmed by eRNA-targeted knockdown experiments. Strikingly, the mechanism by which IFN-γ suppresses IL10 expression appeared to be decommissioning of enhancers, as IFN-γ strongly suppressed TLR-induced positive enhancer marks and eRNA transcription. Our study implicates that crucial enhancer elements cooperate with the core promoter in LPS- and IFN-γ-mediated regulation of IL-10 gene transcription in human macrophages.
MicroRNAs of the miR-17~92 family inhibit macrophage activation and inflammation
Authors: Zhang Xiang , Hu Xiaoyu
Published: January 2023

Production of pro- and anti-inflammatory cytokines by macrophages is tightly controlled by multiple layers of mechanisms including those involving miRNA-mediated regulation of post-transcriptional events. By genetically deleting all three individual miRNA clusters in the miR-17~92 family, we found that this family of miRNAs inhibit macrophage activation by promoting expression of a key anti-inflammatory cytokine IL-10 and thus suppress production of pro-inflammatory mediators such as TNF. Consistent with the heightened inflammatory responses in vitro, myeloid-specific ablation of miR-17~92 family miRNAs in vivo led to exacerbated obesity under the condition of a high-fat diet and poor survival in the DSS-induced colitis model. Moreover, miRNA-deficient adipose tissue macrophages and intestinal macrophages exhibited enhanced levels of TNF and reduced levels of IL-10. Mechanistically, miR-17~92 family miRNAs sustained IL-10 production in macrophages by promoting transcription of the Fos gene encoding a key transcription factor driving Il10 expression. The positive effects of miR-17~92 family miRNAs on Fos expression are secondary to negative regulation of Fos by transcription factor YY1 that is a direct target of miR-17~92 family miRNAs. Taken together, these results identified miR-17~92 family miRNAs as crucial regulators of the balance between pro- and anti-inflammatory cytokines and illustrated key roles for three clusters of the miR-17~92 family in cooperatively regulating macrophage activation and inflammatory responses.
CARD19 interacts with MICOS complex proteins and protects against mitochondrial dysfunction in macrophages
Authors: Rios Kariana E , Beauregard Chelsi , Zhou Ming , Conrads Thomas P , Schaefer Brian C
Published: January 2023

CARD19 is a mitochondrial protein of unknown function; gene expression databases indicate that CARD19 is highly expressed in myeloid cells. We have observed that Card19 −/− mice injected with LPS display elevated levels of some pro-inflammatory cytokines relative to Card19 +/+ mice. Because CARD19 is a mitochondrial protein and mitochondrial dysfunction is frequently associated with immune dysregulation and chronic inflammation, we sought to identify the function of CARD19 in macrophages. We demonstrated via super resolution microscopy that endogenous CARD19 colocalizes with mitochondrial markers in bone marrow derived macrophages (BMDMs) and has a punctate distribution. Through immunoprecipitation and mass spectrometry analyses, we determined that CARD19 interacts with MIC19 and other components of the mitochondrial contact site and cristae organizing system (MICOS) complex. We further demonstrated that CARD19 colocalizes with MIC19 in mitochondrial sub-domains via super resolution microscopy. We then measured outcomes of mitochondrial stress in BMDMs. Consistent with previous reports of MICOS deficiencies, we found that Card19 −/− BMDMs have a modestly decreased oxygen consumption rate (OCR) measured by a Seahorse extracellular flux analyzer relative to Card19 +/+ BMDMs. Additionally, Card19 −/− BMDMs display elevated levels of mitochondrial reaction oxygen species (mROS) and a population of mitochondria with a decreased mitochondrial membrane potential, as measured by flow cytometry. Based on these data, we propose that CARD19 may be a previously unknown regulator of MICOS function which potentially links this complex to other mitochondrial and/or non-mitochondrial CARD proteins in macrophages.
Sex-dimorphic trajectories in aging macrophage populations
Authors: Benayoun Bérénice A , Lu Ryan , Sampathkumar Nirmal Kumar
Published: January 2023

The existence of human supercentenarians reveals a surprising predictor for exceptional longevity: being female. Not only are 33 out of 34 living supercentenarians women, women are also more resistant to most diseases responsible for age-related morbidity in the US. However, because most molecular aging studies generally opt to use only one sex, sex-driven differences in aging remain poorly understood. A key compartment that can actively respond to sex-specific inputs throughout life is the immune system. Indeed, the majority of age-related diseases share common inflammatory mechanisms, a phenomenon described as “inflamm-aging”. Macrophages play an important role in the inflammatory response throughout life, and are considered major mediators of this phenomenon. Thus, to unbiasedly dissect sex differences in immune aging, we generated ‘omics’ data from 4 and 20 months old female and male mice. Intriguingly, we found that transcriptional aging in primary macrophage populations varies strongly between sexes, with up to 20-fold more aging changes in female vs. male cells. Pathways specifically downregulated in females with aging included lysosome, inflammation and phagolysosome. We confirmed experimentally that metabolic preferences of macrophages are indeed directly modulated in this context (e.g. glycolytic preference for male-derived cells). Our results support the notion that there are functional differences in aging trajectories in the immune system of female vs. male mice. Our research could provide new insights into the molecular underpinnings of sex-dimorphism in aging and disease.
Templated mutagenesis in B cell non-Ig genes
Authors: Navarrete Karla , Dale Gordon A. , Woodruff Matthew C. , Scharer Christopher D. , Boss Jeremy M. , Sanz Ignacio , Jacob Joshy
Published: January 2023

The immunoglobulin genes of germinal center B cells (GCB) undergo somatic hypermutation in order to generate antibody diversity. Additionally, several non-immunoglobulin (non-Ig) genes are known to accumulate mutations in the germinal center. There are two methods by which mutations are introduced at the Ig and non-Ig loci. The first is through the accumulation of pseudorandom point mutations as a result of activation-induced cytidine deaminase (AID)-dependent double stranded breaks (DSB). The second is through the process of gene conversion, which involves the use of a template sequence to repair a DSB resulting in the template being copied at the site of repair. Findings from our lab suggest that some mutations in the non-Ig genes of human GCB arise through a gene conversion-like mechanism, referred to as templated mutagenesis. We sorted 4 B cell populations, including light zone and dark zone GCB from a young human tonsil and sequenced ~500bp sections from non-Ig genes including FAS, RHOH, and BTG1 via Illumina MiSeq. Previously, our lab has developed a computational pipeline called TRACE, that is able to match mutation clusters within an input sequence to partially homologous donor templates elsewhere in the genome. TRACE-identified donor templates are statistically likely to have been used in a templated mutagenesis event which led to the mutation of the gene. Through this analysis, we found that unlike gene conversion observed in chickens, human templated mutagenesis occurs between sequences on different chromosomes. Furthermore, donor template sequences tend to originate in introns and intergenic regions, not exons. These findings have opened an avenue into the study of non-Ig templated mutagenesis in human B cells.
RIP Kinase death pathways regulate B cell homeostasis and splenic architecture
Authors: McLennan Alexandra L. , Parthasarathy Raksha , Hägglöf Thomas , Mattke Aiden , Dudley Elizabeth , Leadbetter Elizabeth A.
Published: January 2023

Receptor-interacting protein kinase (RIPK) 1, RIPK3, and Caspase-8, are essential adapter proteins in death receptor-induced apoptosis and programmed necrosis. Apoptosis and necroptosis are crucial for normal cell development, homeostasis, and immune responses. RIP kinases and Caspase-8 form parts of multiprotein complexes like the Death-Inducing Signaling Complex (DISC) and the necrosome, thus mediating signaling events in these cell death pathways. We compared RipK3−/−, RipK1−/−RipK3−/−Caspase-8−/−, RipK3−/−Caspase-8−/−, and RIPK1 kinase-dead (RIPK1kd) mice to define the role of RIP kinase inflammatory pathways and Caspase-8 in immune responses and lymphocyte development.

Preliminary studies show that lack of RIPK1 and Caspase-8, but not RIPK3, leads to the accumulation of Marginal Zone (MZ) B cells. Evidence from mixed-bone marrow chimera experiments reveals an intrinsic requirement for RIPK1 and caspase 8 in normal B cell differentiation post-T2 B cell status. Immunofluorescence showed splenic architecture disorganization in the T cell zone and MZB populations in RIPK-deficient mice. Surprisingly, humoral immune responses to T-dependent and T-independent antigens were largely normal, but immunization-induced splenic GC formation was diminished in mice lacking both Caspase-8 and RIPK3. Elucidation of cell development/death in the context of RIPK1 (whose ubiquitination status influences cellular homeostasis) could lead to better priming of B cells for chemotherapy-induced apoptosis. Previous studies also suggest human RIP kinases are potential therapeutic targets for treating a wide range of autoimmune, inflammatory, and neurodegenerative diseases.
Myocardial associated B cells are a dynamic subgroup of circulating B cells, in equilibrium with splenic and circulating B cells, but with organ specific features
Authors: Rocha-Resende Cibele , Yang Wei , Adamo Luigi
Published: January 2023

Study objective

Recent evidence shows that subgroups of circulating B cells accumulate intravascularly in the naïve murine heart. However, the timing of their appearance and their organ specificity remain unclear.

Methods and Results

We performed a systematic analysis of B cells isolated from the myocardium and other organs, from mid development to early adulthood. We found that B cells are present in the developing heart already at E13.5. Myocardial associated B cells were mostly intravascular and in close association with the endothelium. Their phenotype changed rapidly. Through flow cytometry and proteogenomics analysis we found that heart associated B cells were initially almost exclusively CD11b+ and CD11b−CD21−CD23− but became mostly CD11b−CD21+CD23+ cells at 5 weeks of age. Importantly, comparative analysis of B cells isolated from perfused hearts, livers, lungs, spleen and peripheral blood at different time points showed that the B cells isolated from the heart were in equilibrium with both splenic/circulating B cells and B cells isolated from other organs, but they had specific features. This finding was confirmed through simultaneous 10X single cells sequencing of hashtag labelled B cells.

Conclusions

Taken together these observations highlight that myocardial associated B cells are part of a tightly regulated population of circulating B lymphocytes that exists in multiple organs and is in dynamic equilibrium with the spleen and peripheral blood B cells. Further work will be needed to identify the molecular mechanisms that lead to the accumulation of specific subsets of circulating B cells in peripheral organs and to investigate their functional significance.
The class II peptide editor, H2-M, affects the development and repertoire of B-1 cells
Authors: Ghosh Debopam , Pham Tho D , He Xiao , O’mara Mary E , Kantor Aaron B , Nguyen Khoa , Sengupta Deepanwita , Eisenlohr Laurence C , Jensen Peter E , Herzenberg Leonore A , Boyd Scott D , Ghosn Eliver E.B. , Mellins Elizabeth D
Published: January 2023

Non-classical major histocompatibility complex class-II (MHCII) protein H2-M edits peptides bound to conventional MHCII (pMHCII) in favor of stable pMHCII complexes. Although the impact of H2-M deficiency on pMHCII expression and T cell activation is well-studied, information on how this protein affects the development and phenotypic profile of APCs is lacking. We found that the absence of H2-M in C57BL/6 (B6) mice, resulted in a loss of surface expression and peptide-cargo of MHCII molecules in B cells across lymphoid organs. Lower pMHCII expression reduced MHCII association with BCR, affecting the integrated BCR signaling. Importantly, in H2-M deficient B6 mice, compared to the wildtype mice, frequency and abundance of B-1 cells, but not conventional B-2 cells, was reduced in the spleen and peritoneum. This H2-M mediated effect on the B-1 cell population was only evident in the B6 background (I-Ab), but not in BALB/c (I-Ad/I-Ed), indicating an MHCII haplotype-dependent phenomenon. A decrease in B-1 cell number also was evident in immature B-1 cells, emphasizing that H2-M deficiency affects B-1 cell development. In H2-M KO mice compared to WT B6, B-1 cells display a significantly lower self-renewal capacity and a higher rate of apoptosis. Furthermore, H2-M deficiency alters the B-1 BCR repertoire, selecting for B-1 cells specific for highly abundant epitopes, but not for low-frequency epitopes. Collectively, these data identify the impact of H2-M/MHCII interaction as a regulator of BCR signaling that influences the selection, maturation, and maintenance of mature B-1 cell clones in the periphery.
Alteration in TNFα mediates impaired antigen-specific humoral responses in sepsis survivors
Authors: Rana Minakshi , La Bella Andrea , Lederman Rivka , Ochani Mahendar , Volpe Bruce T. , Sherry Barbara , Diamond Betty
Published: January 2023

Sepsis survivors show impaired responsiveness to antigen (Ag), which is linked to increased susceptibility to infection. Using the cecal ligation and puncture (CLP) model of sepsis, we showed that CLP surviving mice have a reduced antibody response to the T-dependent Ag, NP-CGG, compared to mice that underwent a sham procedure, but an intact response to the T-independent Ag, NP-Ficoll. CLP mice have reduced TNFα levels due to sustained vagus nerve activity. We hypothesized that reduced TNFα production might lead to disruption in follicular dendritic cell (FDC) function and impaired germinal center (GC) responses. FDCs play a key role in GC responses and TNFα is critical for FDC clustering and maturation. Immunofluorescence staining in spleens showed a reduced number of FDC clusters and reduced binding of immune complexes on FDCs in CLP mice compared to sham mice. NP-specific GC B cells sorted from CLP mice immunized with NP-CGG exhibit reduced TNFα, AICDA, and BCL6 mRNA expression compared to sham mice. To confirm the role of the vagus nerve in impaired FDC clusters, CLP mice were subjected to bilateral subdiaphragmatic vagotomy 2 weeks post-CLP. These mice exhibit increased anti-NP IgG levels in response to immunization with NP-CGG compared to non-vagotomized CLP mice. FDC are radioresistant cells; therefore, we lethally irradiated sham and CLP mice and reconstituted them with bone marrow cells from naive mice that never experienced sepsis. CLP mice still show an impaired Ag-specific response confirming involvement of FDCs and vagus nerve signals. In summary, our data suggest that altered vagus nerve activity, low TNFα, and disruption in FDC function contributes to reduced Ag-specific humoral responses in sepsis survivors.
2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Gimap5 Expression to Ameliorate Concanavalin-Induced T Cell-Mediated Liver Injury
Authors: Cannon Alkeiver S , Miranda Kathryn , Wilson Kiesha , Nagarkatti Prakash S. , Nagarkatti Mitzi
Published: January 2023

GTPase of the immune-associated protein 5 (Gimap5) has been implicated in immune cell maintenance and development. A mouse model comparable to autoimmune hepatitis in humans was used to study the alterations induced in the gene expression profile of immune cells upon administration of environmental toxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). In this study, we tested the hypothesis that TCDD is capable of modulating gene expression to ameliorate autoimmune hepatitis severity. Mice were injected intravenously with 12.5 mg/kg concanavalin A (ConA) and treated intraperitoneally one hour after challenge with vehicle or 10 μg/kg TCDD. TCDD-treated mice showed lower levels of liver injury enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as compared to vehicle-treated mice. Histopathological analysis revealed a reduction in liver damage in the TCDD-treated group as compared to the vehicle-treated control. Furthermore, flow cytometric analysis of liver-infiltrating mononuclear cells demonstrated that following treatment with TCDD, there was an increase in FoxP3+CD4+ Tregs as well as a decrease in proinflammatory Tbet+ Th1 and RORγt+ Th17 T helper cell subtypes after induction of the disease. Single cell RNA-sequencing was conducted on liver mononuclear cells to further assess the changes induced by TCDD. The gene expression profile showed that TCDD treatment significantly upregulated the expression of vital genes such as Gimap5, a maintenance gene for normal liver function and survival of T cells. In summary, our data suggests that TCDD is capable of inducing Gimap5 expression in autoimmune hepatitis, which in turn modulates the progression of this disease.
IL-4 modulates reconstituted human T cells as a regulatory factor to mitigate GVHD in humanized mice model
Authors: Kuo Tzu-Jiun Tzu-Jiun , Sun Cheng-Pu , Tao Mi-Hua
Published: January 2023

Allogeneic hematopoietic cell transplantation is an essential treatment in hematological diseases. However, graft-versus-host disease (GVHD) is the leading cause of the mortality and treatment failures in transplant recipients. In humanized mice engrafted with human peripheral-blood mononuclear cells (PBMCs), the rapid development of lethal xenogeneic GVHD is also a major model limitation that hinders long-term and precise immune analysis. Therefore, the study aims to suppressed GVHD symptoms in human PBMCs-engrafted humanized mice model for the study of human immune responses. We investigated whether supplementary of human IL-4 that modulate naïve T cells maturing and differentiating can lead to GVHD amelioration. Adeno-associated virus (AAV) vectors were used to express human cytokines in humanized mice. We first demonstrated that rAAV vectors allow sustained expression of human IL-4 in vivo. After injection of PBMCs, GVHD symptoms observed in NSG mice were significantly suppressed in human IL-4 expressing recipients, with the survival of originally less than two months to more than 7 months without body weight loss. Long-term engraftment of human T cells were also observed. Among the reconstituted human cells, CD4+ T cells were the dominant expanding subpopulation, comparing to CD8+ dominated cell proliferation in mice with GVHD development. In addition, significantly high levels of human IgG in humanized mice sera (1 mg/ml, comparable to human’s) were detected 5 months after PBMC transplantation, suggesting the potential use for long-term study of human humoral immune responses. In conclusion, human IL-4 expression by AAV vectors is an efficient strategy to ameliorate GVHD in PBMC-engrafted humanized mice model.
Senescence-associated β-galactosidase activity and other markers of senescence are present in human peripheral blood mononuclear cells during healthy aging
Authors: Dewald Hannah K , Martínez-Zamudio Ricardo Iván , Vasilopoulos Themistoklis , Herbig Utz , Fitzgerald-Bocarsly Patricia
Published: January 2023

Aging is associated with a decline in the immune system, termed “immunosenescence”. Cellular senescence is defined by persistent cellular growth arrest and overall loss of function. If immune cells undergo cellular senescence in vivo and whether cellular senescence has a role in immunosenesence remains controversial due to the lack of specific markers to reliably identify these cells in blood. Freshly isolated human peripheral blood mononuclear cells from younger and older donors were fluorescently labeled for senescence-associated β-galactosidase (SA-βGal). CD8+ T cells were sorted based on SA-βGal and analyzed by qRT-PCR, RNA sequencing, and microscopy. We found a higher percentage of cells with high SA-βGal in the older vs. younger cohort. This increase was apparent in NK cells, CD4+ and CD8+ T cells. CD8+ T cells with high SA-βGal displayed increased transcripts of p21, p16INK4a, and inflammatory cytokines. They also had greater DNA damage response foci and p16INK4a protein. SA-βGal increased as T cells matured, which was consistent regardless of age. Intriguingly, a portion of T effector cells did not exhibit a senescent phenotype and maintained low SA-βGal. RNA sequencing of CD8+ T cells revealed alterations in gene expression were associated with SA-βGal and cells with low, medium, and high SA-βGal shared similar profiles, independent of age. The presence of p16INK4a, DNA damage, and inflammatory cytokines demonstrate that SA-βGal is a strong indicator of cellular senescence. SA-βGal can be leveraged to further identify senescent immune cells and elucidate cellular senescence’s role in immunosenesence. Understanding this connection in healthy aging will further our understanding of premature aging seen chronic diseases.
Cellular Phenotypes in Cancer Patient Spleen and Peripheral Blood
Authors: Talmadge James E , Cole Kathryn , Ly Quan , Hollingsworth Tony , Cox Jesse , Stromnes Ingunn M , Padussis James , Foster Jason , Vargus Luciano
Published: January 2023

Adoptive cellular therapy (ACT) has progressed; however, it remains ineffective for most solid tumors. Further, neoadjuvant patients frequently lack tumor tissue sufficient to isolate and expand tumor infiltrating lymphocytes (TIL’s) that are the primary source of T-cells for ACT. However, patients with tumors near the splenic vasculature often undergo a splenectomy, providing a cellular resource for ACT. We document herein that spleens provide a high frequency and number of CD8+ T-cells, with low expression of exhaustion markers (program death ligand-1 (PD-1), T-cell immunoglobulin and mucin domain containing-3 (TIM-3), and lymphocyte activation gene-3 (LAG-3)) relative to the peripheral blood (PB). The memory phenotypes of these CD8+ T-cells (predominantly central and transitional) support their potential for expansion and their high frequency of PD-1 expression indicates tumor specificity as PD-1, is a surrogate for tumor specificity. Further, spleens have a low frequency of myeloid derived suppressor cells (MDSCs) subsets vs. patient PB. Our preliminary studies with spleens from 19 abdominal tumor patients revealed that the spleens, relative to the PB from the same patients, have a significantly higher frequency of CD8+ and PD-1+CD8+ T-cells, including central and transitional memory T-cells. Further, the CD8+PD-1+ T-cells in the spleen also have a low expression of TIM-3 and LAG-3 suggesting minimal exhaustion as supported by ELISPOT analysis of tumor specific CD8+ T-cells responses. Based on our studies we suggest that resected spleens from cancer patients including pancreatic ductal adenocarcinoma (PDAC) patients can provide a valuable source for ACT due to their high frequency of CD8+PD-1+ T-cells.
Development of regulatory IL-10-producing ILCs during type 2 inflammation
Authors: Nidetz Natalie F. , McGee Michael C , Limper Candice B , Ye Kaixiong , Islam Rezwanul , August Avery , Huang Weishan
Published: January 2023

Innate lymphoid cells (ILCs) are important for mucosal homeostasis and host defense against infectious pathogens. ILC subsets ILC1, 2, and 3, classified similar to T helper cells Th1, Th2, and Th17, are well characterized and known to promote both protective and harmful inflammatory responses. However, the immunosuppressive roles of ILCs are less understood. Regulatory ILCs that produce the immunomodulatory cytokine IL-10 have been recently defined, but the mechanisms regulating their development and function are not fully understood. Similar to other ILCs, regulatory ILCs were thought to be a pre-existing population, however unlike ILC1, 2, and 3 which all differentiate from ILC precursor cells (ILCPs), regulatory ILCs were thought to differentiate exclusively from upstream precursors, common helper-like innate lymphoid precursors (CHILPs) and common lymphoid cell precursors (CLPs). In our animal facilities, we found no evidence of pre-existing tissue resident IL-10+ ILCs in our investigations. In contrast, we find that co-stimulation of IL-33 and a γ-chain cytokine (IL-2, IL-4, or IL-7) robustly induces IL-10+ILCs that exhibit immunosuppressive functions in vitro and in vivo. Further, in addition to CLPs and CHILPs, we find that downstream ILC2Ps can develop into functionally suppressive IL-10+ ILCs following cytokine stimulation. Together, our data support an alternative pathway of IL-10-producing regulatory ILC development and regulation of inflammatory responses.
Dysregulated TGF-β Signaling Leads to Cell-Intrinsic Changes in T cell Development that Promote Allergic Inflammation
Authors: Haque Tamara , Weissler Kate , Frischmeyer-Guerrerio Pamela
Published: January 2023

Variants in genes in the Transforming Growth Factor Beta (TGFb) signaling pathway are associated with allergic diseases suggesting that this cytokine may be a central player in allergic disease. The role of TGFb in preserving T cell mediated self-tolerance is well-appreciated but how disruption in this pathway promotes allergic inflammation is not clear. TGFb signals through its receptor TGFβRI and II, leading to phosphorylation and nuclear translocation of Smad proteins 2 and 3. We previously demonstrated that patients with Loeys-Dietz Syndrome (LDS), an autosomal dominant disorder caused by mutations in TGFBR1 and TGFBR2 are highly predisposed to developing allergic disease. Both LDS patients and mice harboring a knock-in allele (Tgfbr1mut) of an LDS mutation known to cause severe disease in humans exhibit increased levels of total and food-specific IgE, consistent with their allergic tendencies. These serologic changes were associated with increased numbers of T follicular helper (Tfh) cells which promote memory B cell and plasma cell formation, and decreased T follicular regulatory (Tfr) cells, which suppress humoral immune responses, in LDS patients and mice. Using a mixed bone marrow chimera model we demonstrated that these alterations in T cell development are cell-intrinsic. Furthermore, OVA specific OTII cells harboring the Tgfbr1mut were more likely to accumulate in the peyer’s patch, differentiate into Tfh cells, and less likely to upregulate Foxp3 in response to orally ingested OVA. Tgfbr1mut OTII cells also produced more IL-4 and less IL-10 in response to OVA. These findings suggest that TGF-β signaling plays an important role in T cell development and function that, when disrupted, promotes allergic inflammation.
CD8 T cell fate is programmed by a miRNA-mediated mechanism
Authors: Pobezinsky Leonid
Published: January 2023

CD8 T cells are the cytotoxic effectors of the adaptive immune response, clearing virally infected and cancerous cells within the host. CD8 T cells acquire their cytotoxic function by differentiating into cytotoxic T lymphocytes (CTLs). Once the CTLs have cleared the antigen, the majority of responding cells will die during contraction, while a small population of the antigen-specific responders will remain, differentiating into long-lived memory cells that provide potent protection to the host upon re-encounter with the antigen. However, during chronic infection and cancer, CD8 T cells are often diverted into the so-called exhausted state, in which CTLs are losing their function. As such, the ability to program the differentiation of CD8 T cells into a particular lineage is an important therapeutic strategy. We have identified a global post-transcriptional mechanism that directs the fate of differentiating CD8 T cells. Specifically, we have found that let-7 miRNAs are downregulated upon T cell activation, allowing T cells to differentiate into CTLs. Moreover, we have found that prolonged expression of these miRNAs programs CD8 T cells to become memory cells, while complete loss induces exhaustion. Thus, these findings may have important implications for the improvement of current immunotherapies targeting CD8 T cell fate.