bluuebunny/crossref_metadata_2025 huggingface dataset:
PGD2/DP1 signaling deficiency ameliorates experimental autoimmune encephalitis
Authors: Zheng Jian , Perlman Stanley
Published: January 2023

D-prostanoid receptor 1 (DP1) is a myeloid cell-expressing receptor of prostaglandin D2 (PGD2), the most abundant prostaglandin in the brain. PGD2/DP1 signaling, which triggers G protein activation and cAMP production, is associated with neuroprotection after ischemia, astrogliosis and demyelination in mice. In our previous study, PGD2/DP1 signaling was found to suppress mouse hepatitis virus (MHV)-induced central nervous system (CNS) inflammasome activation through up-regulating PYDC3 expression in CD11b+ cells. In this study, we now investigated the role of PGD2/DP1 signaling in active experimental autoimmune encephalitis (EAE). In contrast to their enhanced susceptibility to virus-induced CNS inflammation, DP-1−/− mice showed resistance to MOG35–55 peptide-induced EAE, which was characterized by milder demyelination and improved remyelination compared to wild type mice. The amelioration of CNS injury resulted from less infiltration of leukocytes and of activation of microglia and macrophages in the brain and spinal cord. On the other hand, in draining lymph nodes (DLN) of immunized DP-1−/− mice, myeloid cells (macrophages, neutrophils and dendritic cells (DCs)) exhibited highly pro-inflammatory phenotypes, which correlated with activation-induced apoptosis of MOG35–55 peptide-specific T cells. Finally, conditional deletion of DP-1−/− in either microglial (CX3CR1+ cells) or macrophage/monocyte/granulocyte (Lyz2tm1+ cells) failed to ameliorate EAE, suggesting involvement of a different DP1-expressing cell. Ongoing research is directed at determining this critical cell type.
GTPase-activating protein (RASAL1) associates with ZAP-70 of the TCR and negatively regulates T-cell proliferation and anti-tumor immunity
Authors: Rudd Christopher E , Thaker Youg Raj , Raab Monika , Strebhardt Klaus
Published: January 2023

T-cell activation is needed for responses to antigen and controls responses to foreign antigen and cancer neo-antigens in immunotherapy. The full range of signaling events that inhibit T-cell activation and limit anti-tumor reactivity is unclear. Previous studies have identified the importance of phosphatases and E-3 ligases as inhibitors of activation. In this study, we show by a combination of tandem affinity chromatography, co-precipitation and proximity ligation analysis (PLA) that RASAL1, a novel GTPase-activating protein (GAP), binds to ZAP-70 of the TCR complex and inhibits anti-CD3 activation of ERKs and proliferation in T-cells. RASAL1 inhibited via two pathways where it binds and inhibits ZAP-70 activity, and acts as a GAP to inhibit the p21ras-ERK pathway. As a negative regulator, its expression is induced as a consequence of T-cell activation, where it reduced in vitro responses to anti-CD3 and to antigenic peptides presented by dendritic cells (DCs), while having no effect on T-cell dwell times. Further, we show that siRNA knock-down of RASAL1 expression increased in vivo CD4+ T-cell responses to peptide antigen while reducing the pulmonary metastasis of B16 melanoma and the growth of solid EL-4 lymphoma tumors. This anti-tumor effect was accompanied by a marked increase in CD8+ tumor-infiltrating T-cells (TILs) expressing effector molecules granzyme B (GZMB) and interferon γ-1 (IFNγ1). These findings identify RASAL1 as a new negative regulator of T-cell activation and tumor immunity.
FOXP3, CD48 and autophagy confer the protection of CD4 and CD8 human T cells from T cell receptor restimulation-induced cell death
Authors: Voss Kelsey , Lott Nathaniel M , Lake Camille , Bauman Bradly M , Dorjbal Batsukh , Luthers Christopher R , Arjunaraja Swadhinya , Dalgard Clifton L , Snow Andrew L
Published: January 2023

The proliferation and contraction of activated effector T cells must be carefully coordinated to maintain immune homeostasis. This is achieved in part through restimulation-induced cell death (RICD), a pre-programmed apoptosis program triggered by antigen restimulation through the T cell receptor (TCR). Forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) also constrain conventional T cell (Tcon) responses, but can resist RICD themselves despite frequent TCR stimulation. We previously showed that FOXP3 protects Tregs from RICD by suppressing SLAM-associated protein (SAP), a key adaptor protein that amplifies TCR signal strength. Mysteriously, FOXP3 expression is also transiently induced in human Tcons after activation, with a kinetic expression profile that correlates inversely with acquired RICD sensitivity. Hence we asked whether FOXP3 protects expanding human effector T cells from premature RICD by modulating SAP expression. Our results show that although siRNA-mediated FOXP3 knockdown sensitizes early effector CD4 and CD8 T cells to RICD, SAP expression remains unaffected. Unlike late stage effector T cells, a low level of RICD in expanding Tcons was entirely dependent on de novo transcription, and knockdown of SAP failed to reduce death. Subsequent RNA-Seq analyses revealed that CD48, a SLAM family receptor, was markedly reduced upon FOXP3 knockdown. Blockade or knockdown of CD48 also increased RICD in CD4 and CD8 T cells. We now show that CD48 protects early effector T cells both by promoting autophagy and upregulation of pro-survival genes such as BATF. These findings implicate FOXP3 as the central governor of a distinct transcriptional program that promotes RICD resistance early in the effector T cell response.
PD-L2 generates a signal through the PD-1 co-receptor distinct from that of PD-L1
Authors: Srinivasamani Anupallavi , Liu Qinying , Curran Michael
Published: January 2023

Immune checkpoint blockade of the PD-1/PD-L1/PD-L2 signalling axis yields potent anti-tumor responses by disinhibiting T cell effector function. Mouse and human data demonstrating an inhibitory role for PD-L1 has resulted in FDA approval of anti-PD-L1 antibodies; however, PD-L2 remains sparsely studied despite being the higher affinity of the two ligands for PD-1. Recent findings demonstrate that PD-L2 is widely expressed by human malignancies. PD-L1 and PD-L2 have also been shown to induce distinct conformational changes on engaging human PD-1.

We utilized a Jurkat T cell based bioluminescent assay of T cell activation and PD-1 inhibition to determine the unique mechanism of action of human PD-L2. In this system, human PD-L2 generates a solely co-inhibitory signal, but at reduced inhibitory potential relative to PD-L1. We find that unlike murine PD-L2, human PD-L2 appears to have a dedicated co-inhibitory function as we find no significant engagement of the human homolog of its murine co-stimulatory partner RGMb. Reverse phase protein array analysis of human PD-L1 and PD-L2 engagement of PD-1 on Jurkat T cells revealed significant differences in downstream T cell signalling generated by each ligand. We also find that human PD-L1 and PD-L2 have disparate impacts on proliferation wherein PD-L2 supports a preferential arrest of T cells in the S-phase of the cell cycle. We observed that combination blockade of PD-L1 and PD-L2 improves on the blockade of PD-L1 alone resulting in increased production of IL-2 and IFNγ in primary human mixed lymphocyte reactions. Our findings address critical gaps in our knowledge about the basic function of PD-L2 and will inform future attempts to manipulate the PD-1 co-inhibitory circuit.
Indole-3-carbinol ameliorates colonic inflammation in DSS-treated, Helicobacter muridarum-infected mice
Authors: Alkarkoushi Rasha Raheem , Singh Udai P. , Chatzistamou Ioulia , Bam Marpe , Hui Yvonne , Nagarkatti Mitzi , Nagarkatti Prakash , Testerman Traci L.
Published: January 2023

Enterohepatic Helicobacter species are epidemiologically linked to increased inflammatory bowel disease; however, little research has been done to elucidate potential contributions of individual species. We hypothesized that Helicobacter muridarum (Hm) would alter the course of DSS-induced colitis and the response to indole-3-carbinol (I3C), an anti-inflammatory phytochemical. We treated Hm-infected C57BL/6 mice with1% DSS +/− 40 mg/kg I3C and measured inflammatory biomarkers. We found that H. m exacerbated DSS-induced colitis and increased the percentage of Th17 cells in the mesenteric lymph nodes and spleen and increased IL-17 in colonic tissue compared to the DSS group. Also, we found that Hm bacteria itself produced inflammation and pathology. I3C, on the other hand, ameliorated colitis and shifted the Treg/Th17 balance in DSS+H.m-infected mice. We found that I3C treatment of DSS + H.m-infected mice decreased the expression of pro-inflammatory IL17 and RORC as well as increased anti-inflammatory Foxp3 when compared to the untreated group. The decreased expression of RORC correlated with increased miR-let7a-2 and miR-29a-3p expression and increased FoxP3 correlated with decreased miR-874 expression following I3C treatment. Moreover, I3C reduced the abundance of certain taxa, such as Clostridiales, Actinobacteria, and Erysipelotrichales, and increased the abundance of Ruminococcus. In summary, H. muridarum causes baseline inflammation and exacerbates colitis via microRNA-mediated increases in Th17 cells, while I3C ameliorates colitis via increased Treg populations.
MiR-146a is a negative regulator of IL-33-stimulated mouse mast cells
Authors: Kee Sydney Ann , Taruselli Marcela T , Ryan John J , Qayum Amina Abdul
Published: January 2023

Interleukin-33 (IL-33) is an inflammatory cytokine that promotes allergic disease by activating ILC2, Th2, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. The IL-33 receptor, ST2, shares signaling cascades with the TLR family, but homeostatic control of ST2 function is poorly understood. MicroRNA-146a (miR-146a) is induced by LPS and suppresses TLR4 signaling in macrophages. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow derived mast cells (BMMC). Induction required MyD88, Akt, and NFkB, since antagonizing these pathways decreased miR-146 expression. BMMC transfected with a miR-146a antagomir or derived from miR-146a KO mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. Our data further suggest that miR-146a may act by targeting IRAK proteins, because mir-146a KO BMMC have increased TRAF6, IRAK1, and IRAK4 expression. In vivo, miR-146a expression in plasma exosomes was elevated after intraperitoneal injection of IL-33. Also mast cell deficient c-Kitw-sh mice acutely reconstituted with miR-146a KO BMMC intraperitoneally had elevated plasma IL-6 levels in comparison to their WT counterparts after IL-33 challenge. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.
Transcription factor Blimp-1 regulates sebocyte homeostasis and cytokine production
Authors: Hung Kuo-Hsuan , Wu Yen-Fei , Lin Kuo-I
Published: January 2023

Blimp-1, encoded by Prdm1, is a master transcriptional factor that governs the differentiation fate of many cell types, including epidermal keratinocytes. Previous results from our laboratory showed that inducible deletion of Prdm1 in mouse epidermis spontaneously induces skin inflammation. Epidermis composes of not only epidermal keratinocytes, but also epidermal appendages, including hair follicles, sebaceous glands and sweat glands. Sebaceous glands release the “oil” contents that lubricate the skin. Our previous results also showed that Blimp-1 is required for the maintenance of the homeostasis of terminally differentiated sebocytes. Here, we aim to investigate the potential function of Blimp-1 in inflammation and lipid metabolism in sebocytes. We depleted Blimp-1 by siRNA in SZ95, the immortalized human sebocyte cells. We found accumulated and enlarged lipid droplets in Blimp-1 depleted cells. Consistently, gas chromatography–mass spectrometry (GC-MS) analyses showed that the levels of several long-chain fatty acids, including 1,3-dipalmitoyl-2oleoylglycerol, 1-palmitoyl-2oleoyl-3-lineoleoyl-rac-glycerol, and triolein, were accumulated in Blimp-1-depleted SZ95 cells, linking with the significant increases in the production of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8 and IL-18. Thus, Blimp-1 served as a pivotal mediator in the regulation of the inflammatory responses in epidermis, likely also through affecting the lipid metabolism and cytokine production in sebocytes.
Using single-cell RNA-seq to lineage trace effector to memory transitions in CD4+ T cells during malaria
Authors: Soon Sioe Fei Megan , Engel Jessica , Lee Josh , James Kylie , Lonnberg Tapio , Svensson Valentine , Teichmann Sarah , Haque Ashraful
Published: January 2023

Hallmark features of adaptive CD4+ T-cells include differentiation into multiple effector T helper (Th) fates and subsequent emergence of memory states. Due to the heterogeneous nature of CD4+ T-cells, determining possible developmental relationships between effector and memory states in vivo has remained a challenge. Previously we employed single-cell RNA-sequencing (scRNA-seq) to delineate the molecular trajectories of Plasmodium-specific TCR transgenic CD4+ T cells during the first week of experimental malaria. We observed a fate bifurcation as cells progressed from an intermediate, activated state to either effector Th1 or T follicular helper (Tfh) cells. My current project tests the hypothesis that CD4+ Th cells directly convert into memory CD4+ T cells during malaria, and aims to define the underlying transcriptomic processes. Tracking endogenous TCR sequences via TraCeR analysis firstly confirmed that sibling clones populated both Th1 and Tfh fates. Next, by comparing transcriptomes, and performing computational lineage tracing of the scRNA-seq data, we observed a direct transition from Th1 and Tfh effector states to memory states. This was characterised by gradual transcriptomic quiescence during the first month of infection, as well as transcriptomic coalescence of Th1 and Tfh fates, but with retention of Th1/Tfh bifurcated structure in memory cells. Finally, premature cure of infection with anti-malarial drugs altered transcriptomic pathways taken by cells towards those with better Th1 recall and germinal centre Tfh phenotypes, respectively. Thus, by examining transcriptional dynamics using scRNA-seq, I am tracing the molecular pathways taken by effector CD4+ T cells towards functional memory states.
Th2 cytokines and specific donor alloantigen activated CD4+CD25+T cells reverse chronic rejection: role of IL-4 and IL-5 in promoting antigen-specific Treg
Authors: Hall Bruce Milne , Rakesh Prateek , Tran Giang , Hall Rachael , Bedi Sukandeep , Robinson Catherine Margaret , Sharland Alexandra , Wang Chaunmin , Hodgkinson Suzanne Jean , Verma Nirupama Darshan
Published: January 2023

Naïve CD4+CD25+Foxp3+T regulatory cells (tTreg) activated by specific alloantigen (Ag) and IL-4 express IL-5Ralpha. We examined if IL-5 treatment activated alloAg specific Treg to prevent chronic rejection in F344 rats grafted with Lewis hearts. 5000units rIL-5 (rIL-5Rx) was given daily from 7d after grafting when rejection had started. Changes in CD4+CD25+T cells were assessed by FACS, RT-PCR of Il5ra and response to alloAg in MLC.

Sham Rx rats fully rejected their grafts by 28d while IL-5Rx for 10d reversed rejection and grafts survived 60d (p<0.01). There were rejection episodes after stopping IL-5 but continued IL-5Rx reduced this. Pre-treatment with anti-CD25 or anti-IL-4 mAb abolished the benefit of rIL-5Rx, all grafts rejected by 28d. IL-5 treated hosts had increased CD4+CD25+T cells, 6–8% vs 3–4% in controls. After 10d of rIL-5Rx, host CD4+CD25+T cells responded to Lewis but not to F344 or third-party PVG alloAg in MLC, showing activation of alloAg-specific Treg. CD4+CD25+T cells from rats treated for 50d with rIL-5 had more il5ra, remained FOXP3+ and in MLC rIL-5 enhanced responses to Lewis but not other alloAg. These findings are consistent with IL-4 and alloAg activating tTreg to induce Ag-specific Treg that suppress rejection.

To demonstrate rIL-4 and specific alloantigen activated naïve tTreg to suppress rejection, these cells were prepared ex vivo. 5×106 of these IL-4 alloAg activated tTreg, given after rejection commenced, reversed rejection. 5×106 fresh naïve tTreg or 5×106 tTreg activated by third-party PVG alloAg had no effect on rejection.

IL-5 prevented heart graft rejection through hosts’ CD25+Treg that had been activated by alloAg and IL-4. IL-5 therapy may have potential to prevent chronic allograft rejection.
Activation-dependent Posttranscriptional Regulation of CD40L is Required for an Optimal Germinal Center (GC) response
Authors: Narayanan Bitha , De Maio Diego Prado , Voskoboynik Katie , La Porta James , Xie Ping , Covey Lori R
Published: January 2023

We have previously shown that regulation of CD40L occurs in part through a posttranscriptional mechanism of activation-induced mRNA stability. This pathway is engaged at extended times of activation and is mediated by the binding of a polypyrimidine tract binding protein (PTBP1) complex to the 3′UTR of the CD40L mRNA. This process leads to sustained expression of CD40L on CD4 T cells at a time when overall transcript levels are low. To assess the role of this regulatory pathway on an ongoing immune response we engineered a novel knock-in mouse (CD40LΔ5) that lacked the PTBP1 stability element and challenged mice with NP-KLH and SRBCs. Examination of splenic subsets prior to immunization revealed no difference in number or ratio between mutant and WT mice. However, after immunization there were significant decreases in T-dependent antibodies (Abs) and these differences were exacerbated with secondary challenge. In contrast, there were no differences in Ab response to a T-independent antigen. In total, CD40LΔ5 mice displayed significant changes in multiple aspects of the GC response including reduced levels of plasma cells, memory cells and GL7+ B cells. However, there were no observed differences in the number of Tfh cells between mutant and WT mice. Evaluation of GC structure in splenic sections revealed highly disordered GCs in CD40LΔ5 compared to WT mice at 8 days following immunization with SRBCs. Therefore, we hypothesize that the T cell activation-induced stabilization of the CD40L message and the subsequent surface expression of the protein is critical for development of a robust GC response and this occurs on one level by directly impacting the structural architecture of the GC.
Characterization and transcriptional profiling of tissue-resident memory CD4+ T cells
Authors: Nguyen Quynh P , Deng Tianda , Goldrath Ananda W
Published: January 2023

In response to acute infection, naive T cells proliferate and differentiate into effector cells, which aid in clearing pathogens, and long-lived memory cells, which provide protection from reinfection. CD4+ T cells play a key role in this response because they mediate the activity of innate cell, B cell, and CD8+ T cell immunity. Memory T cells have classically been categorized into central memory cells (TCM), which circulate between blood and secondary lymphoid organs, or effector memory cells (TEM), which can enter non-lymphoid tissues. Recently, evidence for a third subset of memory cells has emerged, called tissue-resident memory T cells (TRM), which populate barrier surfaces such as skin and intestinal mucosa. TRM serve as sentinels at the sites of pathogen exposure, coordinating the initial response and providing a significant boost to immunity. The ontogeny and regulation of CD8+ TRM differentiation have been characterized in recent years, but less is known about CD4+TRM within the context of antiviral immunity. Using bulk and single-cell RNA-seq and ATAC-seq, we characterize the transcriptional and epigenetic profiles of viral-specific CD4+ T cells in the small intestine. We demonstrate that antigen-specific CD4+ TRM have a transcriptional profile distinct from circulating populations. CD4+ TRM can also be distinguished from circulating memory CD4+ T cells by expression of CD69, Ly6C, and CD27 cell-surface receptors. Additionally, we examine the role of transcriptional regulators, including Bcl6, Prdm1, and Id2/Id3, in CD4+ TRM development. A better understanding of CD4+ TRM will allow us to harness the protective capacity of this memory population and modulate activity in infection or inflammatory diseases.
The role of Aquaporin-4 in T cell activation
Authors: Nicosia Michael , Beavers Ashley M , Farr George W , McGuirk Paul R , Pelletier Marc , Valujskikh Anna
Published: January 2023

Aquaporins (AQPs) are membrane channels that mediate a variety of biological processes via water and other small molecule movement. Several aquaglyceroporins have been shown to regulate key immune functions. However, the role of AQP4 in immune cells is poorly characterized. We recently reported that AQP4 is expressed by both naïve and memory CD4 and CD8 T cells. Furthermore, AQP4 blockade with a small molecule inhibitor, AER-270, reduced T cell activation and proliferation leading to a significant prolongation of fully MHC-mismatched murine heart allograft survival. The goal of this study was to determine the mechanisms by which AQP4 facilitates T cell activation and TCR signaling. Wild-type (WT) but not AQP4−/− T cells treated with AER-270 were protected from lysis in a hypoosmotic shock assay demonstrating AQP4 requirement for water transport in T cells. Following αCD3/αCD28 stimulation, AQP4−/− T cells demonstrated inferior activation compared to WT T cells. Similar decreased activation was observed after treatment of WT T cells with AER-270. Consistent with reduced T cell activation, AQP4 inhibition diminished Ca2+flux in WT but not AQP4−/− T cells in response to TCR crosslinking. AQP4 inhibition also resulted in altered pattern of protein tyrosine phosphorylation at 2 min following TCR crosslinking. These results indicate that despite T cell expression of several water channels, AQP4 function is critical for optimal T cell activation and functions. Our study identifies a novel role of water channels in T cell biology and suggests AQP4 inhibition as a promising strategy to modulate T cell alloresponses following transplantation.
MAP kinase phosphatase (Mkp)-1 Controls Program Death-Ligand (PD-L)1 Expression during Sepsis
Authors: Kirk Sean G , Batty Abel J , Wang Xiantao , Li Jinhui , White William M. , Ackerman William E. , Nelin Leif D. , Buhimschi Irina , Hafner Markus , Liu Yusen
Published: January 2023

Mkp-1 is a critical negative regulator of the inflammatory response, and plays a crucial role for bacterial clearance in an animal model of sepsis. Mkp-1−/− mice exhibit augmented inflammation, increased bacterial burden, elevated organ damage, and increase mortality relative to Mkp-1+/+ mice following E. coli infection. To understand the functions of Mkp-1, we performed RNA-seq with mouse livers. Under normal conditions, 357 genes were differentially expressed between Mkp-1+/+ and Mkp-1−/− mice. However, following E. coli infection > 5,400 genes displayed differential expression between the two groups. Among genes upregulated by E. coli infection and exacerbated by Mkp-1−/− are PD-L1 and CTLA-4, two key immune checkpoint proteins. Interestingly, the expression of PD-L1 and CTLA-4 were highly correlated with the expression of IL-6, IL-10, and STAT3. qRT-PCR and Western blot analyses verified E. coli infection-induced PD-L1 expression and further exacerbation in Mkp-1−/− mice. Examination of the RNA-seq dataset revealed a clear interferon signature. ELISA analyses on the serum revealed low IFN-β levels in uninfected Mkp-1+/+ and Mkp-1−/− mice, increased IFN-β levels in E. coli-infected Mkp-1+/+ mice, and further augmented IFN-β levels in E. coli-infected Mkp-1−/− mice. Similarly, Mkp-1−/− bone marrow-derived macrophages also produced more IFN-β following stimulation with either LPS or heat-killed E. coli than Mkp-1+/+ macrophages. Our studies indicate that expression of immune check point proteins, PD-L1 and CTLA-4, are controlled by Mkp-1 during microbial infection. Our studies also suggest that exaggerated expression of type I interferons and PD-L1 contribute to the phenotypes of Mkp-1−/− mice in the model of sepsis.
Suppression of Aiolos and Ikaros expression by lenalidomide reduces human ILC3–ILC1/NK cell transdifferentiation
Authors: Mazzurana Luca , Forkel Marianne , Rao Anna , Van Acker Aline , Kokkinou Efthymia , Ichiya Tamaki , Almer Sven , Höög Charlotte , Friberg Danielle , Mjösberg Jenny
Published: January 2023

The Ikaros family of transcription factors (TFs) are important regulators of lymphocyte function. However, their roles in human innate lymphoid cell (ILC) function remain unclear. Here, we found that Ikaros (IKZF1) is expressed by all ILC subsets, including NK cells, in blood, tonsil and gut, while Helios (IKZF2) is preferentially expressed by ILC3 in tonsil and gut. Aiolos (IKZF3) followed the expression pattern of T-bet and Eomes, being predominantly expressed by ILC1 and NK cells. Differentiation of IFN-γ-producing ILC1 and NK cells from ILC3 by IL-1β plus IL-12-stimulation was associated with upregulation of T-bet and Aiolos. Selective degradation of Aiolos and Ikaros by lenalidomide suppressed ILC1 and NK cell differentiation and expression of ILC1 and NK cell-related transcripts (LEF1, PRF1, GRZB, CD244, NCR3, IRF8). In line with reduced ILC1/NK cell differentiation we observed an increase in the expression of the ILC3-related TF Helios, as well as ILC3 transcripts (TNFSF13B, IL22, NRP1 and RORC) and in the frequency of IL-22 producing ILC3 in cultures with IL-1β and IL-23. These data suggest that suppression of Aiolos and Ikaros expression inhibits ILC1 and NK cell differentiation while ILC3 function is maintained. Hence, our results open up for new possibilities in targeting Ikaros family TFs for modulation of type 1/3 immunity in inflammation and cancer.
Development of functional mannose binding lectin is delayed in extremely low birth weight infants
Authors: Dean Melinda M , Lopez Genghis H , Liley Helen G , Heatley Susan L , Minchinton Robyn M
Published: January 2023

Background

Mannose binding lectin (MBL) is a liver derived plasma pattern recognition molecule that plays an important role in innate immune defence. Genetically driven deficiencies in MBL level and function are associated with increased frequency and severity of infection, and increased prevalence of autoimmune and chronic inflammatory diseases. Extremely low birth weight infants (ELBWI) are at increased risk of acquiring infection and innate immunity is particularly important in this group. We assessed functional MBL level and capacity to activate the complement cascade in ELBWI.

Methods

ELBW infants (< 1500 g, n=141) were recruited from the Mater Mothers’ Hospital (informed parental consent, ethics #492M). Whole blood was collected into EDTA coated tubes at birth (cord blood), and at D7 and D28 postpartum (heel prick). Functional MBL level and C4 deposition capacity were determined by ELISAs. MBL genotype was determined by PCR. MBL oligomerisation was determined by western blot. Full birth weight infants (n=190) and healthy adult blood donors (n=236) were used as comparators (unpaired t-test P<0.05).

Results

Compared to both full term neonates and adults, new born ELBW infants had significantly reduced MBL level (both P<0.001) and C4 deposition capacity (both P<0.001). Mannan binding and C4 deposition capacity of MBL in ELBW neonates increased over the first 28 Days, reaching comparative function to full term neonates by D7. Increased mannan binding and C4 deposition was associated with increased oligomerisation of MBL.

Conclusions

Development of functional MBL was delayed in ELBW infants. MBL oligomerisation was associated with increased capacity to activate the complement cascade.
Therapeutic effects of a phospholipase D1 inhibitor against polymicrobial sepsis
Authors: Bae Yoe-Sik , Lee Sung Kyun , Kim Ye Seon , Lee Ha Young
Published: January 2023

In this study, we found that a phospholipase D1-selective inhibitor (VU0155069), significantly increased survival rate against cecal ligation and puncture (CLP)-induced sepsis. Pathological events caused by CLP such as lung inflammation and widespread leukocyte apoptosis were markedly inhibited by the PLD1 inhibitor. The PLD1 inhibitor also strongly inhibited the production of proinflammatory cytokines especially IL-1β in CLP mice and in LPS-stimulated splenocytes. VU0155069 also significantly blocked IL-1β production, caspase-1 activation, and pyroptosis caused by several inflammasome-activating signals (LPS+MSU, NIG, dsDNA) in mouse bone marrow-derived macrophages (BMDM). However, VU0155069 did not affect LPS-induced activation of signaling such as MAPK (ERK, p38 MAPK, JNK), Akt, NF-kB, and NLRP3 expression in BMDM. The PLD1 inhibitor also failed to affect mitochondrial ROS generation and calcium increase caused by LPS or ATP, and subsequent ASC oligomerization caused by several inflammasome-activating signals. The PLD1 inhibitor inhibited caspase-1 activity caused by LPS+NIG in BMDM, which is not a direct effect on caspase-1. Our results suggest that the PLD1 inhibitor shows anti-septic activity as well as inflammasome-inhibiting effects.
Dynamic interaction of PSGL-1 with the T cell receptor regulates the extent of T cell activation
Authors: Otero Dennis C , Ma Jiadai , Bradley Linda
Published: January 2023

We previously identified PSGL-1 (P-selectin glycoprotein-1) as a T cell-intrinsic, cell surface checkpoint inhibitor during chronic viral infection. In this model, we found that cross-linking PSGL-1 with antibody reduced signaling through the T cell receptor (TCR) in exhausted T cells and further upregulated PD-1 expression. However, the function of PSGL-1 in the early activation of naïve T cells has not been fully investigated. We found that, crosslinking PSGL-1 on naïve T cells augments TCR signaling, including enhanced Erk and Akt phosphorylation and Nur77 expression. In addition, T cells expressed more activation markers, including PD-1, proliferated to a greater extent, and secreted more cytokines when PSGL-1 was crosslinked compared to TCR stimulation alone, indicating a role as a costimulatory molecule. Strikingly, we have discovered that PSGL-1 co-localizes with the TCR upon stimulation. CD3 and PSGL-1 can be found in the same micro vesicles following anti-CD3 crosslinking, indicating a close connection between PSGL-1 and the TCR. However, the effects of PSGL-1 ligation are similar to what is seen in T cells that lack PSGL-1 expression, except with respect to PD-1 expression. We propose that decreased induction of PD-1 with PSGL-1 deficiency supports greater effector T cell responses. Further studies will address the function of PSGL-1 in cytoskeletal rearrangement and the formation of the immune synapse. Altogether, these data indicate that the extent of T cell activation is regulated by an association of PSGL-1 with the TCR.
Key roles for the innate immune response to genotoxic stress in neurological disease
Authors: Lukens John R , Bellinger Calli E , Frost Elizabeth L , Shaw Mariah E , Bolte Ashley C , Lammert Catherine R
Published: January 2023

Mounting evidence indicates that DNA damage accumulation in the brain centrally contributes to a number of neurodevelopmental, psychiatric, and neurodegenerative disorders. Yet, little is currently known about the specific molecular pathways that the brain relies on to safeguard itself from DNA insults or how altered regulation of these pathways leads to neurological disease. In recent years, a number of new innate immune signaling pathways that respond to genotoxic stress have been identified. While many of these genomic sensors are highly expressed in the central nervous system (CNS), their roles in CNS homeostasis and disease remain poorly understood. In our recent unpublished studies, we have identified a novel DNA damage sensor that centrally contributes to both neurodevelopmental and neurodegenerative disease. Deletion of this genomic sensor in neurodevelopment was found to cause excessive DNA damage accumulation throughout the brain, abnormal CNS maturation, and the development of motor dysfunction, anxiety, and autism-related behaviors. Moreover, engagement of this DNA damage response in the adult brain was found to drive neurodegenerative disease. Interestingly, we observe that DNA damage-induced cytokine responses are not directly responsible for driving neurological disease in our model. In contrast, our findings using genetic knockout mouse strains, indicate that immune-based genomic sensors influence CNS function and health by regulating cell death signaling. Our studies demonstrate that DNA damage surveillance by the innate immune response coordinates the expulsion of genetically compromised cells from the brain and reveal novel roles for immune-based genomic sensors in CNS disease.
The E3 ligase VHL promotes the differentiation of follicular helper T cells through glycolytic-epigenetic pathways
Authors: Zhu Yangyang , Zhao Yanxia , Aki Daisuke , Liu Yun-Cai
Published: January 2023

Follicular helper T (Tfh) cells are essential for germinal center formation and effective humoral immunity, which undergo different stages of development to become fully polarized. However, the detailed mechanisms of their regulation remain unsolved. Here we found that the E3 ubiquitin ligase VHL was required for Tfh cell development and function upon acute virus infection or antigen immunization. VHL acted through hypoxia-inducible factor (HIF)-1α-dependent glycolysis pathway to positively regulate early Tfh cell initiation. The enhanced glycolytic activity due to VHL deficiency was involved in the epigenetic regulation of ICOS expression, a critical molecule for Tfh cell development. By using the RNAi-based screen, we identified the glycolytic enzyme GAPDH as the key target for the reduced ICOS expression via m6A modification during Tfh cell initiation. Our results thus demonstrated that VHL-HIF-1α axis played an important role during the initiation of Tfh cell development through glycolytic-epigenetic reprogramming. Here we will discuss how immune signals and cell metabolic programs are linked to instruct Tfh cell initiation and function, which can provide some insight into rational vaccine design against human infectious diseases and therapeutic intervention of autoimmune diseases.
Production of qualitatively different antibodies by T-bet+ IgM memory cells and plasmablasts
Authors: Lange Rachel Elizabeth , Levack Russell , King Rodney , Winslow Gary
Published: January 2023

We are investigating the genesis of CD4 T cell-independent T-bet+ IgM+ plasmablasts and memory B cells during intracellular bacterial infection. Although the plasmablasts are generated early during ehrlichial infection, and the memory cells become established within 30 days, it is not known if the populations are related lineally. We showed previously that a high frequency of plasmablast-derived IgM was polyreactive, that is, these IgM bound structurally diverse antigens. Polyreactive IgM were particularly abundant among those Abs that bound the ehrlichial outer membrane protein, OMP-19. Here we addressed whether polyreactivity is also characteristic of memory cell-derived IgM. Antibodies were cloned from sorted CD11c+ T-bet+ IgM memory B cells by single cell Ig sequencing and expressed in HEK cells as chimeric human IgG1. Supernatants were tested by ELISA for reactivity to OMP-19, as well as for double-stranded DNA and insulin. Preliminary data showed that approximately 10% (29 out of 284 samples) of antibodies were OMP-19 specific, but unlike those generated by the plasmablasts, the memory cell-derived antibodies did not exhibit polyreactivity. These data reveal that T-bet+ IgM plasmablasts and memory cells produce distinctly different Igs, which may indicate that the two populations have different origins, or that the populations undergo different selection during their differentiation. Ongoing studies will help resolve these different interpretations.
B cell activation and differentiation employ mTORC1 rather than Xbp1 to initiate distinct segments of the unfolded protein response prior to antibody secretion
Authors: Gaudette Brian M , Jones Derek D , Wilmore Joel R , Allman David
Published: January 2023

Little is known about how activated B cells build biosynthetic pathways and organelle structures needed for subsequent robust antibody secretion. The dominant model holds that newborn plasma cells adapt to increased antibody synthesis by activating the unfolded protein response (UPR) under the control of the transcription factor Xbp1. By analyzing gene expression with either activating or plasma cell-inductive signals we identified qualitatively distinct programs of UPR-affiliated genes preceding the onset of antibody secretion. Genetic loss of function experiments revealed that initial transcription of these loci required the mTORC1 kinase adaptor Raptor, but not Xbp1. Furthermore, single cell RNA-seq analysis of immature and long-lived plasma cells revealed that expression of activation-associated UPR genes correlated with apoptotic gene expression in a subset of plasma cells and in cycling plasma blasts. By contrast, UPR genes associated with true plasma cell induction were expressed broadly across steady state plasma cells including in cells experiencing high levels of pro-survival signaling. We conclude that B cells utilize mTORC1 to activate specialized UPR programs necessary to prepare for and optimize plasma cell function.
Expression of a core gene module of anergy governs maternal CD8+ T cell hypofunction after pregnancy
Authors: Porrett Paige , Xu Rong , Wherry E. John
Published: January 2023

Maternal exposure to fetal antigen during pregnancy results in T cell alloimmunization that impacts immunologic responses later in postpartum life. To study the mechanisms of memory T cell differentiation during pregnancy, we transferred 1x106 CD45.2 OVA-specific OT-1 CD8 T cells into CD45.1 mice. Mice were either 1) mated with Act-mOVA males (pOVA), 2) received an OVA skin graft from an Act-mOVA donor (gOVA) or 3) had no antigen exposure (naïve). Sixty days after cell transfer (~30 days after delivery in pOVA mice and ~30 days after skin graft rejection in gOVA mice), OT-1 cells were recovered for functional, phenotypic, and transcriptional analysis. Whereas gOVA OT-1 T cells differentiated into PD-1intCD127+CD62L+ central memory cells with enhanced recall capacity, pOVA OT-1 T cells persisted as hypofunctional PD-1hi long-lived effectors (CD127-CD62L-). pOVA OT-1 cells were hypoproliferative during recall challenge [Fold expansion over naïve: 4.6 (gOVA) vs. 1.5 (pOVA); p<0.001] and demonstrated impaired cytotoxicity and cytokine production [%IFNγ+TNFα+ = 46±6% (gOVA), vs. 7±6% (pOVA); p<0.001]. Transcriptional profiling using RNA-seq revealed 400 differentially expressed genes (FDR<0.05) between gOVA and pOVA OT-1 T cells; these included anergy-inducing genes such as master regulators of T cell hypofunction (i.e. Tox), repressors of the IL-2 locus (i.e. Aiolos), modulators of calcium responses (i.e. CD38), and enzymes which mediate ubiquitination of signaling molecules of the MAP kinase pathway (i.e. Deltex1). Altogether, pregnancy alloimmunization primes a program of hypofunction in long-lived maternal CD8+ T cell that is shared with other models of anergy.
Microbial exposure expands a Ly6C+ subpopulation of long lived naive CD8 T cells with a rapid effector function via type I intereferons
Authors: Jergovic Mladen , Nikolich-Zugich Janko
Published: January 2023

Previously we have described human ‘memory T cells with a naïve phenotype’ which were increased in human subjects recovering from acute West Nile Virus infection (Pulko et al., Nature Immunology, 2016). These cells had a naïve phenotype but exhibited some signs of basal activation and displayed a rapid effector function upon polyclonal stimulation. These findings in humans prompted us to investigate effects of bystander infections on naïve CD8 T cells in mice, and here we have utilized viral (West Nile Virus) and bacterial (Listeria monocytogenes) models but most importantly we investigated naïve T cells under non specific pathogen free (SPF) conditions in B6 mice cohoused with pet shop mice hosting a variety of infections. We describe a novel population of naïve CD8 T cells characterized by expression of a single memory marker Ly6C with similar properties to human ‘memory T cells with a naïve phenotype’. Under basal conditions Ly6C is expressed on ~20% on naïve T cells and is transiently up regulated during acute infection but only on CD5hi cells with higher affinity for self peptide-MHC I complexes. Under conditions of ‘non SPF microbiome’ this population is expanded long term and enriched in the lymph nodes. We show that Ly6C+ naïve CD8s are enriched in the lymph nodes and receive improved homeostatic signals from lymph node stromal cells which lead to upregulation of survival factors such as BCL-2 and extend the lifespan of Ly6C+ cells. Our mechanistic studies have identified type I interferons as the only inflammatory signal required for the induction of this population. However this effect is partially indirect as type I interferon induced expression of Ly6C on CD8 T cells is prevented by MHC I blockade.
STAT1 restrains peripheral naive CD8+ T cell responsiveness to homeostatic cytokines by regulating mTORC1 signaling
Authors: Kye Yoon-Chul , Yun Cheol-Heui , Cho Jae-Ho
Published: January 2023

Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, South Korea In a steady-state, naïve CD8+ T cells are kept to be quiescent in response to homeostatic cues such as self-ligands and cytokines while preserving a robust responsiveness to foreign antigens. Although such a steady-state quiescence involves multiple regulators, an exact nature of the regulators and underlying mechanisms are still incompletely understood. Here we showed that the signal transducer and activator of transcription 1 (STAT1), a key transcription factor downstream of type I and II interferon receptor signaling, plays a cell-intrinsic role in regulating naïve CD8+ T cell responsiveness to tonic homeostatic cytokines. Thus, mice lacking STAT1 led to abnormalities in the composition and number of naïve and effector/memory phenotype among CD8+ T cells. The observed alterations were not due to dysregulation in thymic development but rather resulted from enhanced proliferative capacity of peripheral mature STAT1-deficient naïve CD8+ T cells in a cell-autonomous manner. In vitro analyses revealed that two key cytokines, IL-7 and type I interferon, serve as a major driver to induce abnormal proliferation of STAT1-deficient but not wild-type CD8+ T cells depending on a pathway that is sensitive to rapamycin, indicating a role of mTORC1 signaling. Based on these findings, we suggest that, under steady-state, naïve CD8+ T cells that are continuously exposed to homeostatic cytokines actively restrain their responsiveness to the “Goldilocks” levels – that is, not too strong to induce activation/proliferation and not too weak to lose a survival ability – presumably via blocking mTORC1 pathway, therefore maintaining a functional state of quiescence before encountering a cognate foreign antigen.
Candida albicans secrete a small cysteine-rich protein Sel1 to trigger TLR2/4-dependent inflammatory response during invasive infection
Authors: Xiao Hui , Wang Wenjuan , Deng Zihou , Li Tiantian , Wu Hongyu , Chen Jiangye
Published: January 2023

Despite being a commensal for healthy individuals, Candida albicans can rapidly switch to pathogenic mode featured with hyphal development, altered cell wall composition and secreted virulence factors, thereby causing mucosal or disseminated candidiasis in immunocompromised patients. The host relies on a diversity of pattern-recognition receptors, particularly Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) to sense invading fungal pathogens and launch immune defense mechanisms. However, the complex interplay between fungus and host innate immune system remains incompletely understood. Here we report that Candida albicans upregulate the expression of a small secreted cysteine-rich protein Sel1 upon encountering limited nitrogen and abundant serum, two hallmarks of human bloodstream, leading to marked proinflammatory response. Notably, Sel1 was able to trigger the activation of signaling pathways NF-kB and MAP kinases, leading to the expression of a myriad of proinflammatory cytokines and chemokines in macrophages and dendritic cells. Comprehensive genetic and biochemical analyses revealed that both TLR2 and TLR4 are required for the recognition of Sel1, and TLR2/4-deficient macrophages were blunted in response to Sel1 stimulation. Further, SEL1-deficient mutant of C. albicans was less efficient in eliciting immune response in vivo, thus causing severer morbidity and mortality in bloodstream infection model. Together, our results reveal a previous unsuspected mechanism underlying TLRs-mediated immune recognition of C. albicans, hence open a new avenue to tackle candida infection and inflammation.
Neonatal granulocytic myeloid-derived suppressor cells possess phagocytic properties during bacterial infection
Authors: Seman Brittany G. , Vance Jordan K. , Witt Michelle R. , Robinson Cory M.
Published: January 2023

Neonates exhibit high susceptibility to bacterial sepsis due to immature immunity in transition from the fetal-maternal environment. Myeloid-derived suppressor cells (MDSCs) are immunosuppressive and more abundant in neonates than at older ages, suggesting a possible role for these cells in susceptibility to infection. MDSCs have been well characterized for their suppression of T cell activity in neonates. Further, although they are related to other myeloid-derived professional phagocytes, their direct response to bacteria during infection has not been rigorously examined. We aimed to thoroughly determine the phagocytic capacity of human MDSCs during bacterial infection. Nearly half of neonatal sepsis deaths in the U.S. are caused by Escherichia coli. Using CD66+ MDSCs isolated from human umbilical cord blood, we compared the ability of MDSCs to phagocytose E. coli to CD14+ monocytes using three approaches: flow cytometry, fluorescence microscopy, and bacterial recovery assays. We found that MDSCs internalize bacteria within acidic compartments in a dose-dependent manner. However, the rate of phagocytosis and killing by MDSCs is significantly reduced compared to monocytes, suggesting that MDSCs are less efficient at microbial uptake and elimination. Overall, we show that granulocytic MDSCs possess the ability to clear bacteria, although with reduced efficiency relative to monocytes. Work is ongoing to further characterize these mechanisms and understand how MDSCs exposed to bacteria interact with other immune cells to control host response during infection. This understanding of MDSC activity during bacterial infection may direct the use of novel therapies to improve neonatal immunity and disease outcome.