bluuebunny/crossref_metadata_2025 huggingface dataset:
Immune cell composition at the feto-maternal interface in health and disease
Authors: Mihalic Zala Nikita , Raftopoulou Sofia Nikita , Santiso Ana Sanchez , Maitz Kathrin Stefanie , Kindler Oliver , Valadez-Cosmes Paulina , Heinemann Akos , Wadsack Christian , Kargl Julia
Published: February 2023

Objectives:

We hypothesize that innate and adaptive leukocyte infiltration, function and localization within the placenta is altered in health and disease and that immune cell interactions play a crucial role in maintaining immune tolerance at the fetal-maternal interface.

Methods:

To evaluate the complexity and differences of the immune landscape in decidua basalis we examine tissue samples from healthy women and women who developed gestational diabetes (GDM). We use flow cytometry (18-plex), fluorescent multiplex immunohistochemistry and gene expression analysis.

Results:

Preliminary data suggest abundant innate immune cell infiltration (macrophages and neutrophils) as well as accumulation of T-cell and NK cell subsets at the fetal-maternal interface. In gestational diabetes mellitus pregnancies, we were able to observe lower T cell infiltration when compared to healthy, which was not seen in obese pregnant women. However, in both groups (obese and GDM) there was a trend for lower T regulatory cell infiltration. For monocyte and macrophage populations a trend towards lower immune cell infiltration was observed in both obese and GDM pregnancies when compared to healthy. 70–80% of macrophages show M2 polarization, which suggest that they have an important role in anti-inflammatory processes during pregnancies. We could observe also abundant neutrophil infiltration (up to 20% of all leukocytes), although there was no difference between heathy and diseased pregnancies.

Conclusion:

Our study comprehends a detailed picture of the immune landscape at the fetal-maternal interface in normal and GMD pregnancies, which will aid our understanding of possible dysfunctional immune mechanisms.
Atlas of Human IL-6-induced Signaling in Peripheral Blood Mononuclear Cells in Health and Disease
Authors: Kothari Hema , McSkimming Chantel , Drago Fabrizio , Williams Corey M , Zunder Eli R , McNamara Coleen A
Published: February 2023

Objective:

IL-6 is implicated in the development of coronary artery disease (CAD), autoimmune (AI) disorders, and cytokine storm syndrome (CSS). IL-6 inhibitors are effective in treating AI disorders and are being tested for CAD and CSS. While some studies have reported on IL-6-induced STAT signaling in humans, a comprehensive map of IL-6 signaling in human immune cells is currently lacking. We developed a 32-antibody custom mass cytometry (CyTOF) panel to characterize IL-6 signaling across all major human immune cell subsets, and applied it to identify IL-6-induced immune signatures linked with unstable atherosclerotic plaque.

Methods:

Blood cells from healthy donors and CAD subjects undergoing virtual histology-intravascular ultrasound imaging were stimulated with IL-6, stained and ran in CyTOF. Unsupervised analytical tools (SPADE, UMAP, and Leiden clustering) were used to identify immune cell subsets and IL-6-induced intracellular phosphorylation status.

Results:

IL-6 induced STAT1 and STAT3 activation in CD4 and CD8 naïve T cell subsets and CD4 memory T subsets. Notably, we identified that IL-6 also activates STAT5 within the CD4 and CD8 naïve T subsets. IL-6 induced a much more robust activation of STAT1 as compared to STAT3 and STAT5. Other cell types such as CD14+ monocytes, and CD11c+ and CD123+ dendritic cells also showed IL-6-induced STAT activation. IL-6-induced phosphorylation of STAT1 and STAT3 in a novel PD-1+CD27−CD127lowCD4+ effector memory T cell subtype was associated with higher CAD burden and unstable plaque features.

Conclusions:

Findings are significant for mechanistic insights into IL-6-induced inflammation and may enable discovery of new approaches to reduce inflammation in CAD and other pathologies.
A Novel approach towards Drug Design
Authors: Sika-Paotonu Dianne
Published: February 2023

Acute Rheumatic Fever (ARF) is the body’s autoimmune reaction to an untreated Group A Streptococcal (GAS) throat or skin infection. When recurrent and/or severe ARF episodes occur and are left untreated, this can trigger a condition known as Rheumatic Heart Disease (RHD) - permanent cardiac damage that can result and a major cause of morbidity and mortality worldwide. Without a GAS vaccine, painful monthly injections of Benzathine Penicillin G (BPG) are given intramuscularly to prevent GAS infections that may lead to further ARF episodes and trigger RHD if untreated. These injections are known as secondary prophylaxis and are given for at least 10 years, or sometimes a lifetime. In Aotearoa/New Zealand and the Pacific Region, Indigenous Māori and Pacific children and young people remain disproportionately affected by ARF and RHD. Work is currently underway towards a new Penicillin for ARF/RHD prevention which is more appropriate for children and young people who require these injections. Ideally, a longer lasting Penicillin that is less painful to be given is needed. The purpose of this work was to incorporate the Kakala Indigenous Pacific framework within research efforts involving Indigenous Māori and Pacific communities impacted by ARF and RHD. Application of the Indigenous Kakala research framework supported inclusivity, respect, reciprocity and ensured a strong focus on fostering respectful relationships between researchers and vulnerable communities, appropriate to cultural contexts for Pacific Peoples in Aotearoa/New Zealand. This highlights the importance of Indigenous frameworks and their application within Immunology based research work efforts to improve engagement and applicability within cultural settings.
TIM-3 drives temporal differences in restimulation-induced cell death sensitivity in effector CD8+ T cells in conjunction with CEACAM1
Authors: Lake Camille , Voss Kelsey , Bauman Bradly M. , Pohida Katherine , Jiang Timothy , Dveksler Gabriela , Snow Andrew L.
Published: February 2023

Immune homeostasis depends upon effective clearance of pathogens while simultaneously preventing immunopathology in the host. Restimulation-induced cell death (RICD) is one such mechanism where by activated T cells receive subsequent antigenic stimulation, reach a critical signal threshold through the T cell receptor (TCR), and commit to apoptosis. Many details of this process remain unclear, including the role of proteins that influence the TCR signaling cascade. Here we characterize the role of T cell immunoglobulin and mucin domain containing 3 (TIM-3) in RICD regulation. TIM-3 protected newly activated CD8+ effector T cells from premature RICD during clonal expansion. Surprisingly, however, we found that TIM-3 potentiated RICD in late-stage effector T cells. The presence of TIM-3 increased proximal TCR signaling and pro-apoptotic protein expression in late-stage effector T cells, with no consistent signaling effects in newly activated cells. We found that TIM-3 was expressed on the surface of newly activated effector T cells, but remained largely intracellular in late-stage effector cells. Consistent with this, TIM-3 required a ligand to prevent early RICD only immediately after activation. We found that CEACAM1, a known ligand of TIM-3, protected newly activated cells from premature RICD, with no measurable effects in late-stage effectors. Indeed, CEACAM1 enabled TIM-3 surface expression on T cells, implying a co-dependency for these proteins in protecting expanding T cells from premature RICD. Our findings suggest that co-signaling proteins like TIM-3 and CEACAM1 can alter RICD sensitivity at different stages of the effector T cell response, with important implications for checkpoint blockade therapy.
Tumor Necrosis Factor Alpha inhibits humoral immunity by regulating the antibody secreting cell bone marrow niche
Authors: Aaron Tonya , Fooksman David R
Published: February 2023

Humoral immunity is a critical component of the immune memory, and its longevity is partly dependent on the survival of antibody-secreting cells (ASC). Previous studies have shown that several types of infections can deplete pre-existing antibody titers, but the exact mechanism of this process is unclear. Most, if not all, forms of infection induce inflammation including Tumor Necrosis Factor Alpha (TNFa). Here we show that TNFa-mediated inflammation depletes pre-existing antibody titers by limiting ASC retention in the bone marrow (BM) survival niche. Acute recombinant TNFa (rTNFa) treatment induces ASC egress from the BM into the blood and limits engraftment of adoptively transferred ASCs into the BM of recipient mice. To determine if the TNFa-mediated ASC egress from the BM was cell-intrinsic or –extrinsic, we generated BM chimeras with wild-type and TNFa receptor knockout mice. We found that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells induces ASC egress from the BM in a cell-extrinsic manner. Treatment with rTNFa did not induce ASC death in the BM, but it increased glucose uptake and expression of CXCR4, a receptor involved in ASC retention in the BM. In contrast, mobilized ASCs in the blood had reduced glucose uptake, CD98 expression, and CD93 expression suggesting that ASC metabolism is highly dependent on the BM niche. Our findings demonstrate that inflammation alters ASC physiology in both cell-intrinsic and extrinsic manners, leading to reduced antibody titers and long-term immune memory.
Treg-expressed CTLA-4 depletes CD80/CD86 by trogocytosis, releasing free PD-L1 on antigen-presenting cells
Authors: TEKGUC MURAT , WING JAMES BADGER , OSAKI MOTONAO , LONG JIA , SAKAGUCHI SHIMON
Published: February 2023

Foxp3-expressing CD4+CD25+ regulatory T cells (Tregs) are engaged in the maintenance of immunological self-tolerance and homeostasis. They constitutively and highly express the immune checkpoint receptor CTLA-4, whose Treg-specific deficiency causes severe systemic autoimmunity. As a key mechanism of Treg-mediated suppression, CTLA-4 downregulates the expression of CD80/CD86 costimulatory ligands on antigen-presenting cells (APCs). Here we showed that CTLA-4 facilitated the immune synapse formation and conjugation of Tregs and APCs. These conjugates thus provided stable platforms whereby Tregs were able to deplete CD80/CD86 proteins on APCs by uptaking them via CTLA-4-dependent trogocytosis. The depletion occurred even with Tregs expressing a mutant CTLA-4 lacking the cytoplasmic portion required for its endocytosis. The CTLA-4-dependent trogocytosis of CD80/CD86 also accelerated in vitro and in vivo passive transfer of other membrane proteins and lipid molecules from APCs to Tregs without their significant reduction on the APC surface. Furthermore, CD80 downregulation or blockade by the Treg-expressed membrane CTLA-4 or its solubilized form, respectively, disrupted cis-CD80/PD-L1 heterodimers and increased free PD-L1 on dendritic cells. Taken together, Tregs can exert dual suppressive effects on T-cell immune responses by converting highly stimulatory (CD80highfree PD-L1low) DCs to non-stimulatory/inhibitory (CD80lowfree PD-L1high) DCs via CTLA-4-dependent trogocytosis. Immune checkpoint blockade therapies with the combination of anti-CTLA-4 and anti-PD-1/PD-L1 antibodies may therefore synergistically hinder Treg-mediated immune suppression, thereby effectively enhancing tumor immunity.
Regulatory T cells suppress antigen-specific CD8+ T cells in vivo by modulating the immune-synapse
Authors: Mansoori Mohammad Nizam , Kamenyeva Olena , Kabat Juraj , Shevach Ethan M
Published: February 2023

T Regulatory cells (Tregs) plays an important role in regulating self and foreign antigens related immune response by different mechanisms. Recent studies have shown that antigen-specific Treg suppress CD4+ effectors T cells in vitro and in vivo by depleting peptide-MHC-II complexes from the DC surface. These, results raise questions as to how Treg suppress CD8+ T effectors, particularly in vivo. To explore Treg mediated suppression of CD8+ T cells, we generated CD4+ iTregs from OT-II mice specific for Ova323–339 in association with I-Ab and determined their capacity to suppress CD8+ T cells from OT-I mice specific for Ova257–264 (SIINFEKL) in association with H-2Kb. CD4+ OT-II iTreg suppressed the in vitro proliferation of OT-I T cells in the presence of dendritic cells (DCs) pulsed with both Ova323–339 and Ova257–264 or with DCs singly pulsed with each peptide. Surprisingly, the expansion of OT-I CD8+ T cells in vivo was not suppressed by OT-II iTreg when the two peptides were presented on separately pulsed DCs, but was suppressed when both peptides were presented on the same DCs. OT-II Tregs depleted the Kb-SIINFEKL complex from the DC surface by a process of trogocytosis in vitro only in presence of their cognate antigen. Imaging experiments in vivo depict a close interaction between Tregs and CD8+ T cells suggesting that suppression of CD8+ T cells in vivo requires close proximity between Treg and responder CD8+ T cell and that pMHCI and pMHCII complexes may be closely associated in the immune synapse.
Dynamic Adoption of Anergy by Antigen-Exhausted CD4+ T Cells
Authors: Kadam Pallavi , Trefzer Anne , Wang Shu-Hung , Pennavaria Stefanie , Lober Benedikt , Akcabozan Batuhan , Kranich Jan , Brocker Thomas , Nakano Naoko , Irmler Martin , Beckers Johannes , Straub Tobias , Obst Reinhard
Published: February 2023

Exhausted immune responses to chronic diseases represent a major challenge to global health. To explore their cell fate and plasticity in exhaustion, we analyzed CD4+T cells in a mouse model with regulatable antigen presentation. When the cells are driven through the effector phase, and are then exposed to different levels of persistent antigen, they lose their Th1 functions, upregulate exhaustion markers, resemble naturally anergic cells and become unable to help B cells and, at the highest dose, undergo apoptosis. Mechanistically, TCR signaling pathways are modulated with increasing dose and time. Ca2+ fluxes were found to be more robust to persistent antigen presentation while the MAPK and Akt pathways are easily desensitized. Transcriptomic analyses show the dynamic adjustment of gene expression and the accumulation of TCR signals over a period of weeks. Upon antigen removal, the cells recover their functionality while losing exhaustion and anergy markers. Preliminary data indicate that antigen-exhausted CD4+T cells retain their plasticity for at least 30 days and are able to adjust to different levels of antigen. Our data suggest a dynamic response of CD4+ T cells to different levels of persisting antigen and contribute to a better understanding of chronic disease.
Prenatally Educated Friendly NK Cells Suppress Allospecific T cell Responses and Expand Foxp3+CD4+Treg Cells.
Authors: Kang Hee Kap , Otto Katherine C , Turner Lucas E , Redden Catherine R , Skital Veronica M , Alhajjat Amir A , Scorletti Federico , Shaaban Aimen F
Published: February 2023

Prenatal allospecific tolerance hinges on the developmental selection of NK cells expressing a “friendly” phenotype that is not cytotoxic to allogeneic donor cells. Prenatally educated friendly NK cells (fNK’s) that express a tolerant alloreceptor phenotype toward the donor cells also exert suppressive effects on T cell alloimmunity in vivo and in vitro through direct cell contact. Little is known about the mechanisms regulating this suppression. Therefore, we hypothesized that fNK’s suppress induction of allospecific T cell responses through TGF-beta expression.

To challenge this hypothesis, fNK cells were stimulated with PMA/Ionomycin (1ug/ml each) for 6h and supplemented with brefeldin A for the final 4 hours. TGF (LAP) was detected by intracellular flow cytometry. Following stimulation fNK’s from engrafter mice expressed significantly higher levels of TGF-beta when compared to fNK’s from naïve mice. Furthermore, when naïve splenocytes and allogeneic targets were co-cultured with fNK’s from engrafter mice, an expansion in the frequency of regulatory T cells was noted. Conversely, no change in Treg frequency was seen when co-cultured with fNK’s from naïve mice.

From these results, we conclude that prenatally educated friendly NK cell exert antigen-specific suppressive effects on T cell immunity through TGF-b expression and Treg expansion.
Epigenetic and transcriptional programs of CD4+ T cell anergy
Authors: Titcombe Philip J , Morales Milagros Silva , Mueller Daniel L
Published: February 2023

T cell tolerance is critical to avoid autoimmunity and resolve inflammation. CD4+ T cells that recognize self-antigens in the periphery can exert active suppression as regulatory T cells (Treg) or enter an inactive state known as anergy. Recent evidence suggests that anergy occurs naturally within a subpopulation of polyclonal CD4+ T cells and can generate precursors to Foxp3-expressing Treg cells. We aimed to identify transcription factors underlying T cell anergy induction and its potential plasticity. Polyclonal anergic CD4+ T cells from untreated wildtype mice were sorted based on previously reported markers and compared to naïve, effector-memory, and Treg cell populations identified within the same samples. Paired epigenome and transcriptome analysis revealed a unique anergy signature driven by bZIP family (Batf, Nfil3), NFAT family (Nfatc1), and EGR family (Egr3, Egr4) transcription factors. Following tolerization by repeated peptide infusions, antigen-specific CD4+ T cells from Batf-deficient mice demonstrated exaggerated expansion and minimal Foxp3+ Treg cell differentiation. Our data suggest that a unique transcription factor network, including BATF, generates and maintains peripheral CD4+ T cell anergy.
Alternative splicing of Bax controls cell death and innate immune responses in macrophages
Authors: Wagner Allison , Weindel Chi , Watson Robert , Patrick Kristin
Published: February 2023

To defend against invading pathogens, the macrophage innate immune response requires rapid, robust gene expression reprogramming. Innate immune genes that are induced as part of this response are regulated at many steps. While transcriptional activation of innate immune genes has been extensively studied, the mechanisms driving their post-transcriptional regulation (i.e. alternative splicing) needs further investigation. To begin learning how pre-mRNA splicing changes impact the innate immune response in macrophages, we examined how loss of a splicing regulatory protein called SRSF6 (serine arginine rich splicing factor 6) impacts gene expression. We discovered a novel phenotype whereby loss of SRSF6 increased basal expression of many interferon stimulated genes (ISGs). Consistent with mitochondrial dysfunction, a phenotype that we and others have previously associated high basal ISG expression, we measured an increase in apoptosis and macrophage depolarization in SRSF6 knockdown macrophages compared to scramble controls. Subsequent bioinformatic analysis of RNA-seq data from SRSF6 knockdown macrophages identified an alternative splicing event in the apoptosis activation factor BAX. While Bax isoforms have previously been linked to cell death regulation, no one has reported a role for Bax isoforms in modulating mtDNA-driven innate immune responses. Our data is consistent with a model whereby upregulation of a Bax isoform promotes pore formation in mitochondria which 1) causes leakage of mtDNA into the cytosol where it is detected by cGAS, eliciting type I interferon production and 2) primes macrophages to undergo programmed cell death.
MER Tyrosine Kinase Mediates Efferocytosis Through a Novel β2 Integrin-Activating Signalling Pathway.
Authors: Heit Bryan , Tasnim Tarannum , Le Lam Austin
Published: February 2023

Efferocytosis – the phagocytic removal of apoptotic cells – is a central component of tissue homeostasis and contributes to inflammatory and autoimmune conditions including atherosclerosis and multiple sclerosis. MER tyrosine kinase (MERTK) is the predominant or sole efferocytic receptor in multiple tissues, and is the predominant efferocytic receptor on macrophages. MERTK is an opsonin-dependent receptor tyrosine kinase that triggers a poorly understood signaling pathway to mediate apoptotic cell engulfment. Using immunoprecipitation, mass spectrometry, and super-resolution microscopy, we have identified a pre-formed receptor complex on the macrophage plasma membrane comprised of ~100 nm clusters of MERTK, β2 integrins, and multiple signaling molecules including Src-family kinases, PI3-kinases, and the integrin regulatory proteins ILK and FAK. MERTK is unable to mediate efferocytosis in the absence of β2 integrins or their opsonins, while β2 integrins require activation via MERTK signaling to induce the engulfment of apoptotic cells. Using FRET microscopy, we determined that MERTK directly induces the conformational change of β2 integrins from the low to high-affinity form via a PI3-kinase, FAK and ILK-dependent signaling pathway. Activation of this pathway then drives the coalescence of the MERTK/β2 integrin clusters into a larger synapse through which efferocytosis occurs. The identification of the MERTK signaling pathway and the role of β2 integrins in this pathway provides new insights into the function of this critical homeostatic receptor and provides new insights into how MERTK mutations and signaling defects may contribute to inflammatory and autoimmune diseases.
Specialized pro-resolving lipid mediators stimulate mitochondrial metabolism in macrophages
Authors: Calderin Ernesto Pena , McNally Lindsey , Hill Bradford G. , Hellmann Jason
Published: February 2023

Chronic inflammation and sustained recruitment of classically activated macrophages (CAMs) with unchecked pro-inflammatory cytokine production is associated with disease development (e.g., atherosclerosis). Pro-inflammatory actions of CAMs are fueled by enhanced glycolysis and disruptions in the TCA cycle. Conversely, alternatively activated macrophages (AAMs) display an intact TCA cycle and mitochondrial oxidative phosphorylation (OXPHOS). Specialized pro-resolving lipid mediators (SPMs) stimulate resolution, in part, through actions on specific G-protein coupled receptors (GPCRs) in macrophages (e.g., RvD1□Fpr2). Whether SPMs exert pro-resolving effects by altering macrophage metabolism is unknown. We found that 1 hr treatment of SPMs (e.g., RvD1, RvD2, RvE1, and MaR1) enhanced basal (vehicle vs 1nM SPM; 6.11±0.1 vs RvD1 7.76±0.6 vs RvD2 9.48±0.9 vs RvE1 7.20±0.5 vs MaR1 8.73±0.9 pmol/min/μg protein; P<0.05; n=3), maximal (vehicle vs 0.1nM SPM; 9.06±1.1 vs RvD1 14.37±1.1 vs RvD2 16.54±2.7 vs RvE1 12.32±1.5 vs MaR1 13.28±1.3 pmol/min/μg protein; P<0.05; n=3), and ATP-linked respiration (vehicle vs 1nM SPM; 2.83±0.2 vs RvD1 4.50±0.3 vs RvD2 4.39±0.2 vs RvE1 4.71±0.3 vs MaR1 4.20±0.2 pmol/min/μg protein; P<0.05; n=3). Stimulation of mitochondrial metabolism with RvD1 was receptor mediated as Fpr2-deficient macrophages display no significant changes in metabolism following treatment (vehicle vs 1nM RvD1; basal 6.74±0.6 vs 6.52±0.2; maximal 11.54±1.2 vs 13.26±1.0; ATP-linked 5.84±0.8 vs 5.33±0.2 pmol/min/μg protein). Collectively, these data suggest that SPMs, despite having unique GPCRs, stimulate OXPHOS in macrophages, which could underlie their common pro-resolving properties.
Mast cell-specific inactivation of Fosb exacerbates release of pro-inflammatory mediators in models of systemic anaphylaxis and lipopolysaccharide-induced sepsis
Authors: Duque-Wilckens Natalia , Maradiaga Nidia C , Szu-ying Yeh , Nestler Eric J. , Robison Alfred J. , Moeser Adam J.
Published: February 2023

Mast cells (MC) are innate immune cells that can orchestrate diverse physiologic and pathologic responses. This is due to the MC capacity to respond to a variety of stimuli and selectively release a diverse repertoire of mediators including prestored granule mediators, cytokines, growth factors and neurotransmitters. This vast heterogeneity in MC functions implies that their transcriptional profile needs to be extensively regulated, but the underlying mechanisms are not understood. Our previous data showed that MC expression of FosB and ΔFosB, two transcription factors formed by alternative splicing of the FosB gene, is markedly upregulated in response to a variety of stimuli, including IgE-antigen activation and stress, but the role of MC-specific FosB and ΔFosB in MC function remains unexplored. Here we crossed Mcpt5-Cre mice with a Cre-dependent floxed FosB mouse strain (Mcpt5-Cre;FosB (fl/fl)) to assess the role of MC-specific FosB expression. Bone marrow derived mast cells from Mcpt5-Cre;FosB (fl/fl) mice exhibited increased IgE-antigen mediated histamine release. Using a passive systemic anaphylaxis model, we next found that Mcpt5-Cre;FosB (fl/fl) mice exhibited greater serum histamine levels, and more severe hypothermia and clinical scores, compared with Mcpt5-Cre-negative controls. Mcpt5-Cre;FosB (fl/fl) mice also showed exacerbated hypothermia and serum IL-6 levels in response to lipopolysaccharide (LPS) (1 mg/Kg) injection. Together, these data demonstrate that MC Fosb expression exerts a negative regulation on MC mediator release and associated inflammatory responses. Ongoing studies using CUT&RUN-sequencing aim to identify the down_stream targets of FosB and ΔFosB in MCs.
STAT3 and IL-10 inhibit interferon signaling in type 1 conventional dendritic cells
Authors: Chrisikos Taylor T , Zhou Yifan , Denne Nina , Brooks Athena , Watowich Stephanie S
Published: February 2023

Conventional dendritic cells (cDCs) are the primary antigen presenting cells of the immune system, and thus control adaptive immunity. Type 1 cDCs (cDC1s) are crucial for mounting protective T cell responses against tumors and viruses. Recently, we reported that interleukin (IL)-10 inhibits polyinosinic-polycytidylic acid (poly I:C; a toll-like receptor 3 agonist) induced maturation of cDC1s in a signal transducer and activator of transcription (STAT) 3-dependent manner. In addition, using a tumor vaccine approach, we demonstrated that STAT3-deficient (Stat3Δ/Δ) cDC1s are more capable of inducing anti-tumor immunity than STAT3-sufficient (Stat3fl/fl) cDC1s. To further investigate how STAT3 and IL-10 inhibit cDC1 function, we performed RNA-sequencing using Stat3Δ/Δ and Stat3fl/fl cDC1s treated with IL-10, poly I:C, or both IL-10 and poly I:C. Gene Set Enrichment Analysis revealed that although many inflammatory signaling pathways are enriched in cDC1s upon treatment with poly I:C, only the interferon-alpha (IFN-α) response pathway is inhibited by IL-10 and STAT3. Furthermore, Ingenuity Pathway Analysis identified IFNs, and components of IFN receptor signaling, such as STAT1, as the primary targets of IL-10/STAT3-mediated inhibition. Therefore, pathway analyses suggest IL-10 and STAT3 inhibit poly I:C-induced IFN signaling in cDC1s. Indeed, IL-10 was found to inhibit poly I:C-induced accumulation of phosphorylated STAT1 by immunoblot. Moreover, investigations into specific IFNs determined that IFN-β and IFN-γ, but not IFN-λ, induce IFN-stimulated genes in cDC1s. Currently, studies are underway to further dissect the role of IFNs in cDC1 function and IL-10/STAT3-mediated inhibition of poly I:C-induced maturation.
Hypoxia-inducible factor 1α is a negative regulator of IRF5 in hypoxic macrophages
Authors: Son Myoungsun , Peng Travis , Du Shin-Yi , Diamond Betty
Published: February 2023

Patients with severe Covid-19 exhibit a low level of oxygen in affected tissue and blood and a low interferon level in blood. It is necessary to understand cell function during hypoxia to understand the pathophysiology of Covid-19 infection. We investigated aspects of human monocyte activation under hypoxic conditions. HMGB1 is an alarmin released by stressed cells. Under normoxic conditions, HMGB1 activates IRF5 and NF-κB in monocytes, leading to type 1 interferon expression and the inflammatory cytokines TNFa and IL-1b, respectively. When hypoxic monocytes are activated by HMGB1, they produce pro-inflammatory cytokines but fail to produce type 1 interferon as HIF-1α functions as a direct transcriptional repressor of IRF5. Moreover, exposure to Dimethyloxaloylglycine (DMOG), which stabilizes HIF-1α, exhibits the same transcriptional program as hypoxia. These findings have implications for the pathogenesis of Covid-19 showing that a hypoxic microenvironment that favors macrophage production of inflammatory cytokines but not interferon.
Mechanism of differentiation and unique repertoire of IgM secreting B-1 plasma cells
Authors: Smith Fauna Leah , Savage Hannah P , Baumgarth Nicole
Published: February 2023

Natural antibodies are broadly reactive, mostly IgM antibodies that provide a first line of defense against pathogens. Distinct populations of fetal/neonatal-derived IgM-secreting B-1 cells, including non-terminally differentiated B-1 and B-1 plasma cells (PC) are present in bone marrow (BM) and spleen. B-1PC are transcriptionally and phenotypically similar to foreign antigen-induced, long-lived conventional PC, prompting us to hypothesize that both receive similar differentiation signals. To determine the extent to which natural IgM-producing B-1PC depend on T cell help, we studied TCRβδ−/− mice lacking either all T cells, or depleted of CD4 T cells by anti-CD4 mAb treatment. Both showed significantly reduced numbers of BM but not spleen B-1PC and slightly increased populations of B-1 cells. Consistent with those data, reconstitution of RAG-deficient mice with B-1 and CD4+ T cells, but not with B-1 cells alone, restored the BM B-1PC compartment and significantly enhanced serum IgM levels. B-1PC did not bind to PtC-containing liposomes and depletion of B-1 PC had minimal effect on the number of anti-PtC IgM ASC. In contrast, most anti-influenza natural IgM ASC were B-1PC, consistent with reduced serum levels influenza-binding natural IgM in TCRβδ−/− mice. Together, the study reveals that in contrast to previous assumptions, some natural IgM production is dependent on CD4 T cell help, suggesting that multiple mechanisms regulate induction of natural IgM secretion.
Sex dictates organ-specific differences in the production and activation of plasmablasts and plasma cells
Authors: Pioli Peter , Crone Michael , Kornas Matthew , Renner Patrick , Pioli KimAnh
Published: February 2023

In humans and mice, sex dichotomies exist in regards to physiological immune responses as well as pathological autoimmune responses. However, very little information exists in regards to how sex patterns the production and function of antibody-secreting plasmablast (PB) and plasma cell (PC) populations. Using the Prdm1-enhanced yellow fluorescent (eYFP) reporter mouse strain, we compared the percentages and numbers of PBs and PCs in the bone marrow (BM), spleen (SPL) and thymus (THY) of young (3 months old) female and male mice. While PB/PC generation was equivalent in the BM and SPL of both sexes, the female THY had significantly increased percentages and numbers of both PBs and PCs when compared to males. This correlated with the overall increase in thymopoiesis present in females. Characterization of THY PBs/PCs demonstrated increased expression of canonical B cell activation markers such as CD69 and MHC II when compared to their BM and SPL counterparts. In some aspects, these differences were sex-based in origin. αCD45 intravenous antibody labeling suggested that THY PBs/PCs were locally generated and not a consequence of immigration from the periphery. As such, female THY demonstrated increased numbers of a germinal center B (GCB) cell-like population expressing both GL7 and CD95(Fas). THY B cells have been previously shown to participate in the regulation of T cell selection and we hypothesized that THY PBs/PCs would be generated in a T cell dependent manner. Indeed, administration of αCD40L blocking antibodies ablated THY GCB, PB and to some extent PC production. In summary, female mice are skewed towards increased THY PB/PC generation. The production of these cells happens locally and is dependent on CD40L-based T cell interactions.
Meta-analysis of the CD56-negative NK cell subset indicates altered functional responses and unique KIR regulation
Authors: Cocker Alexander , Parham Peter
Published: February 2023

Chronic infections such as HIV-1, HCV, and some herpesviruses, expand a subset of NK cells that lack CD56 expression (CD56neg) and are less responsive to HLA-negative targets than CD56+CD16+ (CD56dim) cells. The 39 research papers that have reported on CD56neg NK cells have considered that this subset comprises anergic, dysfunctional or adaptive NK cells. Our objective was to assess these possibilities through meta-analysis of these papers, with inclusion of new data extracted from openly deposited CyTOF datasets (n=7).

Data on CD56neg NK cells was extracted from papers. CyTOF datasets were downloaded and markers expressed by CD56neg cells identified. Meta-analysis was undertaken following the Cochrane guidelines using Random Effect Model assuming study variability.

We found through analysis of “healthy” control groups (n=30) that the CD56neg subset represents 5.42% of peripheral blood NK cells, and confirmed that HIV-1 infection expands this population by 9.00%. Four studies presenting comparable functional data report decreased IFNγ and CD107a CD56neg cell responses to HLA-lacking targets compared to CD56dim cells (−5.99% and −4.24% respectively). Comparison to CD56dim marker expression showed no difference in Ki67 implying CD56neg cells are not anergic. The CD56neg subset also has a decreased proportion of CD57+NKG2C+ cells so are not adaptive NK cells. Lower KIR expression and range of KIR combinations were seen in CD56neg cells indicating their reduced responsiveness is linked to altered KIR:HLA signalling capacity. The expansion of this subset may be due to disrupted HLA expression induced by chronic viral infections, or may be an adaptation to long-term infection to mediate ongoing activation of the NK compartment.
Reciprocal regulation of mTORC1 and ribosomal biosynthesis determines cell cycle progression in activated T cells
Authors: Rosenlehner Teresa , Pennavaria Stefanie , Straub Tobias , Kranich Jan , Obst Reinhard
Published: February 2023

Anabolic metabolism of antigen-stimulated T cells is coordinated by mTOR which is activated by TCR and IL-2 signals. We show that mTORC1 regulates both the exit of quiescence and later divisions as Raptor-deficient T cells proceed 3x slower through each cell division in vivo. We noticed that following stimulation the amount of RNA per cell increases dramatically (10–40x) and is reduced to ~50% in Raptor-, but not Rictor-deficient T cells. RNAseq data indicate that this applies to all RNA polymerase I- and II-dependent biotypes. Using Coulter volume measurements, RNA-, FISH- and imaging-flow cytometry we determined that following stimulation the cell volume increases five-fold. It consists of the increase of nuclear volume and nucleolar rRNA synthesis on day 1 and a further increase of cytoplasmic volume on day 2. Raptor-deficiency delays and diminishes the growth of these cellular compartments, decreases ribosomal biosynthesis and thus the cell volume. Inhibitors of rRNA transcription block cell cycle progression at two checkpoints, of which only the G1/S transition is regulated by Raptor. We found that mTORC1 activity depends on ongoing rRNA synthesis in early activation and regulates the further synthesis rate. Altogether our data show that mTOR-adjusted cell physiology and the biosynthesis of ribosomal ‘hardware’ are reciprocally regulated during clonal T cell expansion.
Circulating KLRG1+ memory CD8 T cells support immune responses in non-lymphoid tissues
Authors: Huggins Matthew A , Peng Changwei , Wanhainen Kelsey M , Gress Abigail R , Jameson Stephen C , Hamilton Sara E
Published: February 2023

The memory T cell population must be durable and capable of responding with rapid effector functions during re-encounter with pathogens. This is accomplished by forming a memory CD8 T cell pool containing multiple distinct subsets of cells that combine varying functional and homing characteristics. At steady-state, memory T cells expressing KLRG1 are restricted to circulation and patrol the host from within the vasculature. These cells convey robust protection during acute rechallenge with pathogens delivered intravenously. Given their localization pattern, we questioned whether KLRG1+ memory T cells participate in immune responses within non-lymphoid tissues. Here, we find that during local and systemically administered infections, KLRG1+ memory cells alter their trafficking pattern and enter infected non-lymphoid tissues. Following intranasal influenza infection, antigen-specific KLRG1+ memory cells proliferate and extravasate into the lung parenchyma. Additionally, during acute LCMV infection, similar findings were observed in all non-lymphoid tissues assayed, including small intestine, kidney, and salivary gland, indicating this is not a unique phenomenon, but rather a consistent trait of these cells. Unexpectedly, progeny from KLRG1+ cells remained within non-lymphoid tissues after acute infection is cleared, downregulating markers of circulation (CX3CR1, KLRG1) and acquiring markers of tissue residency (CD69, CD103). This work challenges the concept that KLRG1+ memory cells are terminally differentiated and demonstrates unappreciated plasticity within this subset to support immunity within non-lymphoid tissues.
Signature of Enhanced Effector Function Defines Early Life Regulatory T Cells
Authors: Connors Thomas , Bradley Marissa , Verma Shivali , Waldman Erik , Jang Minyoung , Grunstein Eli , Maurrasse Sarah E , Pethe Kalpana , Dogra Pranay , Farber Donna L
Published: February 2023

The induction of self-tolerance and regulation of inflammatory immune responses is critical during early life when a myriad of primary antigenic challenges are encountered. Regulatory T Cells (Tregs) promote self-tolerance and function to modulate immune responses utilizing a variety of immunosuppressive mechanisms. Tregs are known to be disproportionately more abundant during early life, suggesting an essential role during immune development. We performed bulk RNA sequencing and multi-parameter flow cytometry on Tregs isolated from adult and pediatric (1 – 3 years of age) tissue (tonsils) and blood to discern the defining characteristics of early life Tregs. Early life Tregs were found to have an increased expression of genes associated with cell cycle (Ki67), effector and suppressive function (ICOS/CTLA4/IL10/GZMB/GZMA) and chemokine signaling (CCR5/CCR9) compared to adults. Importantly, distinctions in canonical effector function genes were apparent across all early life Tregs and not restricted to tissue site. Interestingly, CCR9, essential in gut-specific homing, was upregulated in both tissue and blood early life Tregs, suggesting a biasing towards intestinal immune homeostasis during childhood. We further explored the upregulation of genes related to cell cycle and found that early life Tregs exhibit higher proliferative capacity compared to both adult Tregs and early life CD4 conventional T cells, revealing a potential effector mechanism unique to early life. Early life Tregs are defined by a signature of augmented effector functionality enhancing their ability to respond to the abundant immunological challenges of infancy and childhood.
Unique abilities of human memory T cell subsets are associated with both specialized gene expression modules and epigenetic priming.
Authors: Rose James R , Rahmberg Andrew , Scharer Christopher , Boss Jeremy M
Published: February 2023

Memory T cells are a diverse set of cells that respond to immunologic challenge more quickly and with greater efficacy than their naïve counterparts. Their potential in the resting and stimulated state has not been explored for each of the subsets. To define their capabilities, RNA-seq and ATAC-seq data assessing the transcriptome and chromatin accessibility potential of both resting and activated human memory T cells was analyzed. Clustering gene expression modules identified a core set of genes highly expressed in both resting central memory and effector memory cells (but not in naïve or terminally differentiated effector cells). Memory subsets also expressed unique gene modules enabling specialized functions such as cell cytotoxicity, migration, or self-renewal. Subset differences in gene expression were reflected in differentiated chromatin accessibility and enhanced variation around binding motifs for key T cell differentiation transcription factors such as Tbet, EOMES, and LEF1. Upon activation memory T cells showed substantial rapid upregulation of genes including cytokines which were absent in activated naïve T cells. These differences in gene expression from naïve T cells were often preceded by areas of open chromatin in a resting memory state, suggesting mechanisms of epigenetic priming for rapid recall or augmentation of effector T cell function in memory. In summary, memory T cell subsets are distinct in both resting gene expression functionalities and differentiated epigenetic landscape—both of which enable unique, rapid, and effective response to antigen.
FCRL1 inhibits ERK: Implications on B cell homeostasis and humoral immunity.
Authors: DeLuca Jenna M , Murphy Maegan K , Wilson Timothy J
Published: February 2023

FCRL1 is a tyrosine-phosphorylated receptor found on the surface of B cells and some dendritic cells. FCRL1 co-localizes with BCR signaling complexes following BCR engagement and augments BCR-induced calcium flux. By examination of phosphotyrosine-dependent interactions of FCRL1 with intracellular signaling partners, we have identified interactions between FCRL1-Tyr281 and GRAP, GRB2, SHIP-1, and SOS1, with a requirement for GRB2 to mediate binding of FCRL1 to SHIP-1 and SOS1. Examination of MAP kinase signaling downstream of FCRL1 revealed that FCRL1 suppresses ERK phosphorylation in B cells at a resting state and following BCR engagement. In vivo, naïve FCRL1 knockout mice accumulate more B cells in the spleen, including immature, follicular and marginal zone cells, implicating a potential role for FCRL1 in B cell selection and maturation. By contrast, after immunization with thymus-dependent and independent antigens, FCRL1 knockout mice show impaired production of antigen-specific antibodies indicating an essential role for FCRL1 in humoral immune responses.
Genomic Screening of B cell differentiation by using CRIPSR/Cas9
Authors: Zuo Zhihong , Kania Anna K. , Patterson Dillon G. , Scharer Christopher D. , Boss Jeremy M.
Published: February 2023

The differentiation of naïve B cells to antibody secreting plasma cells plays a crucial role in humoral immunity. This process is regulated by multiple factors including, transcription factors and epigenetic modifiers. Type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems can be used to identify genes that influence a phenotype of interest. Herein, we established this system to identify novel regulators of plasma cell differentiation. To accomplish this, we transduced hematopoietic stem cells derived from mice expressing Cas9 specifically in B cells (Rosa26-Cas9, Cd19-Cre) with lentiviral based gRNAs targeting Ptprc (CD45), Irf8 and Bach2 and transplanted the stem cells into congenically disparate hosts. Hematopoietic stem cells are enriched with lineage depletion kit and then sorted by C-kit and Sca-1, followed by culture for 24 hours and spin-infection with corresponding lentivirus. After another 24-hour recovery, these cells are transferred to radioactively immune system destroyed hosts. Following immune constitution, B cells were isolated and the deletion of CD45, IRF8 and BACH2 was confirmed. Consistent with previous findings, deletion of IRF8 and BACH2 augmented plasma cell formation in ex vivo differentiation assays. Additionally, we used a genome-wide CRISPR gRNA lentiviral library targeting 18,424 genes to screen for factors that influenced B cell differentiation to LPS, IL-2 and IL-5. In conclusion, the CRISPR/Cas9 system allows us to rapidly generate genetic deletions to explore B cell differentiation in vivo and to discover novel factors that regulate plasma cell formation.
Stroma-derived Notch signals drive transdifferentiation of follicular B cells into marginal zone B cells during lymphopenia
Authors: Atria Daniela Gomez , Londregan Jennifer , Gaudette Brian T. , Perkey Eric , Kelly Samantha , Ludewig Burkhard , Siebel Christian W. , Ryan Russell J. H. , Pear Warren S. , Allman David , Maillard Ivan
Published: February 2023

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain poorly understood. To evaluate the homeostasis of the mature B cell pool during lymphopenia, we developed an adoptive transfer model of follicular B (FoB) cells into Rag2−/− mouse recipients. We report that highly purified FoB cells transdifferentiate into marginal zone (MZ)-like B cells when transferred into Rag2−/− lymphopenic hosts, but not into wild type hosts. In lymphopenic spleens, transferred B cells gradually lost their FoB phenotype and acquired multiple characteristics of MZ B cells, as judged by their cell surface phenotype (sIgMhi sIgDlow CD21hi CD1dhi CD23low), expression of integrins and chemokine receptors, positioning close to the marginal sinus, and functional ability to rapidly generate plasma cells. Initiation of FoB to MZ B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by Ccl19-Cre+ fibroblastic stromal cells, a pathway also required for MZ B cell development in steady-state conditions. We postulate that stromal Notch ligands are critical to regulate the size and composition of the splenic B cell pool via interactions with Notch2 receptors in B cells. Thus, FoB cells are not locked in their FoB cell fate, but are are endowed with plastic transdifferentiation potential in response to increased stromal Dll1 availability in lymphopenic environments.
Redox regulation of autophagy in thymic stromal cell function.
Authors: Semwal Manpreet Kaur , Hester Allison K , Cepeda Sergio , Xiao Yangming , Udeaja Chioma , Griffith Ann V.
Published: February 2023

T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs the progenitors along a well-characterized program of differentiation. However, the biology of the TSCs comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the H202 quenching enzyme catalase (CAT) in TSCs, and found that CAT deficiency results in high H202 levels in this population, eventually leading to thymic atrophy. Our current studies address the possibility that high H202 levels serve physiological functions in TSCs in the young, steady state thymus. TSCs exhibit high basal levels of autophagy at the steady state, which is critical for self-antigen presentation and T cell selection. The mechanisms governing high basal autophagy in TSCs are unknown, however autophagy is induced by many stressors, including high H202 levels. Our data indicates that catalase overexpression targeted to mitochondria in transgenic mice (mCAT Tg) results in diminished autophagy in TSCs and causes diminished negative selection in the thymus, eventually leading to autoimmunity. The effects on negative selection in mCAT Tg mice are rescued by increased basal autophagy on the beclin 1 knock-in (Becn1F121A/F121A) background. Our results suggest that the high basal autophagy level required in TSCs for T cell negative selection is promoted by physiologically low levels of catalase expression in the steady state thymus.