bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophil elastase activates MMPs in macrophages and promotes adhesion and cytokine expression through the integrin – Srk pathway.
Authors: Krotova Karina , Khodayari Nazli , Oshins Regina , Aslanidi George , Brantly Mark
Published: January 2023

There are a number of diseases characterized by the presence of neutrophil elastase (NE) activity in tissues. It is generally accepted that presence of NE actively contributes to a pathological process, but the precise mechanism remains to be elucidated.

We hypothesize that one NE action is activation of an inflammatory response in macrophages (Mφ). First, we show that in response to free NE exposure, Mφ release profound proteolytic activity composed of several MMPs which could contribute to extracellular matrix (ECM) degradation. Second, NE treatment also upregulated inflammatory cytokine expression, such as that of TNFα, IL-6, IL-1b, and IL-8. Therefore, Mφ activated by NE can contribute to tissue destruction on several levels, including release of proteolytic activity and by supporting chronic inflammation through expression of chemokines and cytokines.

We also demonstrate that NE increases Mφ adhesion which can be attenuated by antibodies against integrin subunits. In support of our hypothesis of integrin involvement, we show an activation of the Srk kinase family in response to NE, which is a hallmark of integrin signaling pathway activation. Moreover, pretreatment of Mφ with specific Srk kinase inhibitor PP2 completely blocked cytokine production in response to NE.

In summary, these data propose the mechanism of macrophage activation by NE and also suggest the inhibition of Srk can be potential therapeutic target to reduce NE-induced inflammation in lungs.
CD11c+ cells are required for lymphocyte trafficking into previously infiltrated pancreatic islets during type 1 diabetes.
Authors: Sandor Adam , Lindsay Robin S. , Gebbert Matthew , Bradley Brenda , Haskins Kathryn , Jacobelli Jordan , Friedman Rachel Sharon
Published: January 2023

Type 1 diabetes (T1D) results from the T cell mediated autoimmune attack of the insulin producing beta cells within the pancreatic islets. Although inhibition of T cell trafficking has been used therapeutically for other autoimmune diseases, the requirements for T cell trafficking to the islets are unclear. We show that short-term CD11c+ cell depletion in non-obese diabetic (NOD) mice with previously infiltrated islets reduced lymphocyte trafficking to the islets. Initial CD11c+ cell antigen presentation in the lymph node is known to be critical for initial T cell trafficking to the islets, but the role of CD11c+ cells in lymphocyte trafficking to infiltrated islets is unclear. CD11c+ cells include cells that are highly efficient antigen presenters; however, we show entry of both activated or naïve T cells into previously infiltrated islets were not reliant on cognate antigen. There were also no changes in vascular adhesion molecule expression or adhesion to the vasculature after CD11c depletion. We also show that chemokine receptor signaling is necessary for T cell trafficking to previously infiltrated islets. Based on these data, we hypothesize that CD11c+ cells facilitate the process of extravasation into infiltrated islets through chemokine production. CD11c+ cells in the islets express high levels of CXCL9 and CXCL16, chemoattractants for T lymphocytes. CXCR3 and CXCR6, the receptors for CXCL9 and CXCL16 respectively, are present on T cells in the islets. We therefore explored the importance of these myeloid expressed chemokines in T cell trafficking to the islets. Targeting CD11c+ cell chemokine production may be useful therapeutically for the treatment of T1D by preventing T cell entry into remaining or transplanted islets.
Gut pathobiont translocation induces lymphocyte migration to internal organs in autoimmunity
Authors: Fine Rebecca , Vieira Silvio Manfredo , Ruiz Daniel Fernando Zegarra , Kriegel Martin A.
Published: January 2023

Gut commensal translocation to distant sites can drive autoimmunity. We have evidence that a gut pathobiont, Enterococcus gallinarum, translocates spontaneously to internal organs in the lupus-prone (NZWxBXSB)F1 mouse model to induce systemic autoimmune disease. Lymphocyte migration during commensal translocation to secondary lymphoid organs is not well characterized but may contribute to understanding host-microbiota interactions in autoimmunity. We thus investigated if gut-imprinted lymphocytes migrate to non-gut organs that are progressively colonized by a pathobiont in the (NZWxBXSB)F1 model. We longitudinally characterized gut homing markers on lymphocytes isolated from various tissues of autoimmune-prone F1 mice and monocolonized C57BL/6 mice. In F1 males, circulating CD4+α4β7+ and CD4+CCR9+ cells were increased with age. These subpopulations were reduced after administration of oral antibiotics that deplete the microbiota including E. gallinarum. Next, we monocolonized non-autoimmune prone C57BL/6 mice with E. gallinarum to track lymphocyte migration specifically due to this pathobiont that we have shown to translocate to liver and spleen, induce Th17 responses and autoantibody production. Aged monocolonized C57BL/6 mice harbored greater Peyer’s patch and splenic CD4+ T cells as well as splenic CD4+α4β7+ T cells, supporting that gut-imprinted lymphocytes follow E. gallinarum to non-gut sites. In summary, we linked gut-imprinted lymphocyte migration to internal organs in the setting of systemic autoimmunity that is induced by a translocating pathobiont. Ongoing studies are aimed at determining if these T cells are commensal-reactive and if blockade of gut homing can abrogate autoimmunity.
Histone Demethylase Jmjd3 Controls T-Cell Trafficking and Persistence by Interacting with Klf2 for Expression of Pdlim4
Authors: Fu Chuntang , Li Qingtian , Zou Jia , Xing Changsheng , Zhu Motao , Tan Peng , Lin Meng , Wang Rongfu
Published: January 2023

Jmjd3, a histone H3K27 demethylase, plays a critical role in gene expression and T-cell differentiation, but its role and mechanisms in T cell trafficking remain poorly understood. Here we have shown that Jmjd3 deficiency in CD4+ T cells resulted in the accumulation of T cells in the thymus, but a reduced number in secondary lymphoid organs. To understand the molecular mechanisms of Jmjd3 action, we identified PDLIM4 as a significantly downregulated target gene in Jmjd3-deficient CD4+ T cells by gene profiling and ChIP-seq analyses. Further investigation shows that Jmjd3 bound to the promoter and gene body regions of PDLIM4 gene and regulated PDLIM4 expression by interacting with zinc finger transcription factor KLF2. Furthermore, we showed that PDLIM4 was involved in filamentous actin (F-actin) remodeling, thus functioning as a key regulator of T cell trafficking. Our findings have identified PDLIM4 as a novel Jmjd3 target gene that affects T-cell trafficking, and provided insights into the molecular mechanisms by which Jmjd3 regulates genes participate in T-cell trafficking.
Effect of insulin receptor expressing T cell infiltration in islets on beta cell functionality
Authors: Nandedkar Neha D. , Rhoades Emily L. , Gupta Nirdesh , Al-Dieri Ali G. , Bretz James , Hafner Hannah , Gregg Brigid , McInerney Marcia F.
Published: January 2023

T cells have a major role in the progression of type 1 diabetes (T1D) in human and the non-obese diabetic (NOD) mouse model. Activated T cells have increased insulin receptor (IR) expression on their surface. When T cells with high density of IR from NOD mice were transferred into young irradiated non-diabetic NOD mice, recipient mice developed insulitis and diabetes. Irradiated non-diabetic NOD mice that received T cells with low to negative density of IR had neither insulitis nor diabetes. In order to understand the effect of IR expressing T cells in T1D development, our lab has developed a novel transgenic mouse model, C57BL/6-CD3FLAGmIR/mfm, genetically modified to express FLAG tagged mouse insulin receptor (mIR) on T cells.

It was observed that FLAG-mIR expressing T cells can chemotax to the islet leading to insulitis. However, C57BL/6-CD3FLAGmIR/mfm mice were not hyperglycemic at any age. Metabolic studies were performed to evaluate if FLAG-mIR expressing T cell infiltration in the transgenic mice has effect on beta cell functionality. Impaired in vitro islet insulin secretion on treatment with non-glucose beta cells secretagogue and abnormal glucose tolerance in the transgenic mice resemble the pathological changes during prediabetes in human and NOD mice. Islet and Metabolic changes were also found in immunocompromised RAG mice that received transgenic mice splenocytes confirming that the transgenic phenotype can be passaged to immunocompromised RAG mice in a short time frame. This study provides a new perspective to understand the pathology of T1D and design a novel therapeutic approach targeting IR overexpressing T cells.
Inhibitor of MyoD Family A (I-mfa) regulates HSPC and myeloid differentiation
Authors: Houser Jeremy S. , Onopiuk Marta , Ratliff Michelle , Ngo Peter , Webb Carol , Tsiokas Leonidas , Humphrey Mary Beth
Published: January 2023

Hematopoietic stem and progenitor cell (HSPC) populations are capable of expanding in response to immunological insult or homeostatic turnover. Regulation of the fate decision between self-renewal and differentiation allows for both maintenance of blood cell populations and sustained hematopoiesis across the entire life-span. I-mfa, a small cytosolic protein, has been shown to inhibit lineage-specific transcription factors as well as regulate β-catenin/Wnt signaling. We have previously shown that I-mfa-null mice have increased myeloid cells resulting in increased osteoclasts and hypothesized that I-mfa regulates myeloid lineage differentiation and may affect HSPC populations.

In order to test this, bone marrow (BM) from I-mfa−/− mice was analyzed by flow cytometry, showing increases in both long-term and short-term hematopoietic stem cells compared to WT controls, an effect which increased with age. Additionally, both common myeloid precursors (CMP) and granulocyte-macrophage precursors (GMP) were increased in I-mfa−/− mice. Conditional knockout of I-mfa in all hematopoietic lineages using Vav1-Cre lead to similar findings, while conditional knockout in myeloid cells with LysM-Cre showed only increases in myeloid progenitors. These data indicate that I-mfa has separate roles in regulating HSPC expansion and myeloid cell differentiation. Mechanistically, M-CSF-stimulated I-mfa−/− BM had significantly increased transcription of β-catenin signaling target genes Ccnd1, Axin2, and Myc, as well as myelopoietic transcription factors Spi1, Irf8, and Klf4, compared to WT. Further studies are being conducted to determine the effect of I-mfa on quiescence, Wnt ligand sensitivity, and BM reconstitution capacity.
The pro-inflammatory microRNA-155 promotes FLT3-ITD-induced myeloproliferative disease through inhibition of interferon signaling
Authors: Wallace Jared A. , O’Connell Ryan
Published: January 2023

microRNA-155 (miR-155) is a pro-inflammatory microRNA that functions within a variety of immune cell types to direct proper immune responses. However, miR-155 has also emerged as an oncogene in a number of cancers, and overexpression of miR-155 in the bone marrow is sufficient to induce a lethal myeloproliferative disease in mice. This suggests that miR-155 could be a critical link between inflammation and cancer. Activating mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor known as FLT3-ITD are common in human myeloid malignancies and confer a poor prognosis. Interestingly, FLT3-ITD aberrantly activates well known inflammatory pathways, including NF-kB and STAT5, and drives increased expression of miR-155. We utilized a mouse model of FLT3-ITD-induced myeloproliferative neoplasm to examine the role of miR-155 in promoting disease outcomes in this setting.

We found that miR-155-deficient FLT3-ITD mice exhibit decreased myeloid expansion in the bone marrow, blood, and spleen resulting in a less severe myeloproliferative disease. Furthermore, our data indicate that miR-155 is needed to maintain a robust hematopoietic stem and progenitor cell (HSPC) and myeloid progenitor pool in FLT3-ITD mice by promoting proliferation of these stem cell compartments. Further analysis of the HSPCs and myeloid progenitor cells revealed that FLT3-ITD mice lacking miR-155 had an increased response to interferon, known to have a growth-suppressive effect on hematopoietic cells. These findings demonstrate an interesting link between inflammatory pathways and hematopoietic malignancy, and suggest that miR-155 inhibition could have therapeutic efficacy in FLT3-ITD induced myeloproliferative diseases and/or myeloid leukemia.
A novel approach to enhance T cell recovery after bone marrow transplant
Authors: Wimberly Kymberly L. , Semwal Manpreet , Xiao Yangming , Griffith Ann
Published: January 2023

Cytoablative treatments such as chemotherapy and radiation are commonly used during allogeneic hematopoietic stem cell (HSC) transplants. After cytoablation, T cells must be replaced to generate effective immune responses. Cytoablative therapies damage the thymic stromal cells (TSC) comprising the lymphopoietic microenvironment necessary for T cell development. Damage to TSCs causes delayed thymic recovery, and corresponding delays in T cell recovery in HSC transplant patients, which directly contribute to increased susceptibility to infection and malignant relapses. However, the mechanisms that cause TSCs to be particularly sensitive to cytoablative therapies are not completely understood. Substantial cellular damage is caused by reactive oxygen species (ROS) generated during radiation and chemotherapy treatments. Our recent work identified a deficiency in the antioxidant enzyme catalase in TSCs, indicating that this population may be particularly sensitive to ROS. We hypothesize that oxidative damage to TSCs during cytoablative therapy is exacerbated by catalase insufficiency, and that this is an important contributor to delayed thymus recovery after treatment. If so, an optimized regimen of antioxidant supplementation during cytoablative therapy may represent a novel approach to enhance immunity after HSC transplant. We have tested this hypothesis using dietary and genetic supplementation of catalase activity in mice receiving bone marrow transplants after irradiation. Our preliminary data support the notions that catalase deficiency increases TSC sensitivity to radiation, and that thymus recovery after bone marrow transplant can be improved by supplementing catalase activity in mice.
Niche-specific regulation of hematopoietic stem and progenitors by NR4A1
Authors: Mumau Melanie , Emerson Stephen , Punt Jennifer
Published: January 2023

Hematopoietic stem and progenitor cells (HSPCs) have the potential to generate all cells in the blood. Although the differentiation of HSPCs depends on their interactions with the niche, the exact mechanisms that control lineage specification remain unclear. Adult HSPC development predominates in the bone marrow, but also occurs at extramedullary sites including the spleen and there favors myelopoiesis. To explore how the spleen regulates myeloid differentiation, we transplanted bone marrow-derived, myeloid-biased HSPC subsets, including multipotent progenitor populations MPP2s and MPP3s, and monitored their development. We discovered that MPP2s, not MPP3s, contributed ~2-fold more to the total cell number in the spleen than in the bone marrow. At this same time point, ~25% of the Lin−Sca1+cKit+ population in the spleen, but not the bone marrow, were MPP2-derived. These data indicate that for specific HSPC populations, the splenic environment promotes their differentiation and self-renewal. Since we recently defined a role for the nuclear receptor, NR4A1, in regulating myeloid progenitor differentiation, we wondered if NR4A1 also affected HSPCs. Using NR4A1GFP mice, we discovered that HSPCs expressed higher levels of NR4A1 in the spleen compared to the bone marrow. Almost all NR4A1+ HSPCs in the spleen co-expressed VLA-4, a known HSPC niche retention factor. The few NR4A1+ HSPCs (~10%) in the bone marrow also expressed VLA-4 suggesting that NR4A1 affects the location of HSPCs either within the local niche or via mobilization to distant sites like the spleen. Together, our data highlight the importance of the splenic microenvironment in HSPC maintenance, differentiation, and localization, in part by the influence of NR4A1.
Full Length RAG2 Expression Enhances the DNA Damage Response in Pre-B Cells
Authors: Byrum Jennifer , Rodgers William A. , Rodgers Karla
Published: January 2023

V(D)J recombination of lymphocyte antigen receptor genes occurs via formation of DNA double strand breaks (DSBs) through activity of RAG1 and RAG2. The co-existence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. It is not known how developing immune cells respond to DNA damage in a manner that achieves optimal balance between continued cell development with genomic integrity. Studies from our lab show full length RAG2 exports from the nucleus and enriches at the centrosome within minutes following DNA damage in pre-B cells, exhibiting a response of RAG2 upon DNA damage. The exact mechanism by which RAG2 relocalization occurs is not known, however, mass spectrometry results identified three centrosomal proteins that interact with RAG2 strictly in the presence of DNA DSBs. To determine how RAG2 may affect cell viability upon genotoxic stress, we examined various cell responses versus time following irradiation of cells expressing either full length or mutants of RAG2. Our results indicate that RAG2 plays an active role in bolstering DNA damage responses to genotoxic stresses. Specifically, cells expressing full length RAG2, versus mutant forms, show an increase in the ability to cull damaged cells from the cell population, with evidence of more robust cell division several days after cells were subjected to genotoxic insult. Overall, we propose that while RAG2 functions to cleave DNA during V(D)J recombination, it also performs an alternate function to reinforce the cell’s response to DNA damage, thus facilitating continued development of immune cells to help generate a robust immune system.
Maturation of cord blood natural killer (NK) cells differs among neonates
Authors: Prince Amanda , Watkin Levi , Davis Valerie , Orange Jordan , Aagaard Kjersti , Mace Emily
Published: January 2023

The source, timing of colonization, and stability of the neonatal microbiome is largely unknown. We and other groups recently demonstrated that infant colonization may begin in utero, challenging the notion that neonates are born sterile. However, neonates are particularly vulnerable to viral infection partially due to dampened responses from NK cells and delayed maturation of adaptive immunity. Given the degree of variability in neonatal vulnerability to infection, we hypothesized that intrauterine exposure to microbes enables a greater diversity of NK cells in the neonates, relative to the adult, to allow for immune tolerance. Here, we performed multi-parametric flow cytometry analyses to dissect the normal range and expression of human NK cell developmental markers from umbilical cord (UC) blood (n=37). NK cells were identified as CD45+ CD3− CD56+ using FlowJo X and Cytobank. UC blood had a lower frequency of mature NK cells than adult peripheral blood. Furthermore, examination of NK cell maturation via spanning analysis of density events (SPADE) highlighted inter-individual diversity of neonatal NK cell subsets. Diversity of NK cells subsets between neonates and adults was significantly different with higher diversity in neonates (Simpson’s Diversity Index, p = 0.008). Our findings suggest diversity in stages of NK cell development between individual neonates that may reflect early immune exposure. Given ours and others recent work demonstrating that the intrauterine environment is not sterile, we speculate that microbial exposures during gestation may be a driving difference in the diversification NK cell populations in human infants and hence enable immune tolerance at the risk of neonatal viral vulnerability.
RAG2 interactions with H3K4me3 are regulated by Thr490 proximal to the RAG2 PHD region.
Authors: Rodgers William A. , Byrum Jennifer , Rodgers Karla
Published: January 2023

Association of RAG2 with the epigenetic marker H3K4me3 through a plant homeodomain (PHD) in the RAG2 non core domain is important for relieving auto inhibition of the RAG recombinase activity for V(D)J recombination. However, little is known regarding the properties of RAG2 that regulate its PHD-dependent interactions with H3K4me3. Accordingly, we measured the localization and dynamics of an N-terminal GFP fusion protein of full length RAG2 (GFP-FL); a T490A mutant of full length RAG2 (GFP-T490A) that does not undergo a regulatory phosphorylation proximal to the PHD region of RAG2; and GFP-labeled core domain of RAG2 (GFP-Core). The proteins were expressed in RAG2−/− pro-B cells, and the H3K4me3 was labeled using monoclonal antibody. Cross-correlation analysis showed that GFP-FL and GFP-Core exhibited similar colocalization with H3K4me3, which was significantly less than colocalization of GFP-T490A with H3K4me3. Blocking H3K4 demethylation using 2,4-pyridinedicarboxylic acid (PDA) caused GFP-FL/H3K4me3 colocalization to approach that of GFP-T490A and H3K4me3, whereas GFP-Core/H3K4me3 colocalization was unchanged. Super resolution imaging showed that RAG2 interactions with H3K4me3 were restricted to interfaces with H3K4me3-enriched puncta, and that GFP-T490A exhibited greater overlap with the H3K4me3-enriched puncta than GFP-FL. Finally, measurement of fluorescence recovery after photobleaching demonstrated that GFP-T490A had a lower mobile fraction and rate of diffusion than GFP-FL in conditions where RAG2 interactions with H3K4me3 were enhanced. These data suggest that RAG2 interactions with H3K4me3 are regulated by Thr490 proximal to the PHD region, such as by a phosphoryation/dephosphorylation cycle.
Ikaros regulates epigenetic and transcriptional programs in progenitor B cell leukemia
Authors: Schjerven Hilde , Ayongaba Etapong F. , Rodriguez Princess , Frietze Seth
Published: January 2023

Hematopoietic cell development is tightly controlled by a network of transcriptional and epigenetic regulators, many of which are mutated or have altered expression in hematological malignancies. Ikaros is a transcription factor that is critical for the proper development of several hematopoietic lineages, and essential for the B-cell lineage. It is recognized as a critical tumor suppressor in precursor B-cell lineage acute lymphoblastic leukemia (pre-B ALL), and is emerging to play roles also in other hematopoietic malignancies. In pre-B ALL, Ikaros mutations are particularly prevalent in the Ph+ (BCR-ABL1+) and ‘Ph-like’ subgroups of leukemia, and Ikaros mutations correlate with poor prognosis. To study the mechanisms of Ikaros tumor suppressor function, we developed a mouse model and a human model system with selective perturbation of Ikaros in Ph+ pre-B ALL cells. This has revealed conserved deregulated genes and pathways, and underscored the role of Ikaros in regulating progenitor-restricted gene programs. Furthermore, our recent studies have highlighted the role of Ikaros in regulation of chromatin structure, and revealed a novel role in epigenetic regulation. It is challenging to therapeutically target mutations in a tumor suppressor factor. It is therefore important to understand the mechanism of action and downstream targets to elucidate targetable vulnerabilities of the Ikaros-mutant leukemic cells. Investigating the tumor suppressor role of Ikaros in developing B cells also sheds light on its role in normal B cell development. We are using our newly established models of Ikaros-mutated pre-B ALL to study the underlying molecular mechanisms and downstream targets, and will present our results to-date at the meeting.
The Differential Role of GM-CSF and Flt3L on Myelopoiesis of Lineage Negative Bone Marrow Cells
Authors: Oh Sehwa , Choi Eun Young
Published: January 2023

During the inflammatory response, multiple cytokines drive myelopoiesis to expand innate cells. Of those cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and FMS-like tyrosine kinase 3 ligand (Flt3L) are known cytokines secreted during the immune response and drive myeloid and dendritic cell (DC) development. Though GM-CSF and Flt3L have been used for bone marrow differentiation culture, their specific role in myeloid and DC differentiation derived from lineage negative hematopoietic cells has not been studied. To compare the kinetics of myeloid and DC development under GM-CSF and Flt3L culture, we sorted and cultured lineage negative bone marrow (Lin− BM) cells from B6 mice in vitro using GM-CSF- or Flt3L-containing media. We found that Flt3L had the potential to produce both cDC1 (XCR1+) and cDC2 (CD11b+) DCs from the Lin− BM cells, whereas GM-CSF lacked the potential to produce XCR1+ cells. Flt3L culture preferentially differentiated Lin− BM cells into immature (MHCIIlo CD11cint) and mature (MHCII+ CD11chi) DCs, and monocytic Ly6C+ myeloid cells. However, GM-CSF culture condition influenced differentiation towards immature and mature cDC2s, and Ly6C+ monocytes and Ly6Ghi granulocytes. Flt3L and GM-CSF conditions were found to preferentially differentiate not only dendritic cell subsets but also myeloids. This study demonstrates the different roles of GM-CSF and Flt3L on myeloid and DC differentiation during in vitro Lin− BM culture.
HSC-requirement for macrophage regeneration is tissue-specific
Authors: Ghosn Eliver , Kuipers Hedwich , Waters Jeffrey , Sosa Jasmine , Phillips Megan , Herzenberg Leonore A.
Published: January 2023

The current paradigm posits that a single long-term hematopoietic stem cell (LT-HSC) is capable of regenerating all components of the immune system. However, recent studies have overturned this paradigm by showing that tissue-resident macrophages (in brain, skin, and liver) develop from fetal progenitors in the yolk sac that emerge separate from, and prior to, the LT-HSC.

Recently, we have identified novel subsets of tissue-resident macrophages in the mouse peritoneum, namely Small and Large Peritoneal Macrophages (SPM and LPM). Here, using Runx1 lineage-tracing assays, we show that, similar to brain microglia, both SPM and LPM emerge at around embryonic day 8 (E8), prior to, and independent of, the conventional LT-HSC. However, after E11, the fetal liver LT-HSC can be called upon to regenerate both SPM and LPM, but not microglia. Single-cell high-dimensional phenotypic and gene expression assays reveal that LT-HSC-derived SPM and LPM are phenotypically and functionally comparable to their E8-derived yolk sac counterpart. After birth, and throughout adulthood, both peritoneum and liver, but not the brain, call upon LT-HSCs in the bone marrow to continuously regenerate their fetal-derived tissue-resident macrophages.

These results suggest that the requirement for LT-HSCs to regenerate macrophages is tissue-specific. In rodents, both peritoneum and liver, but not the brain, might be exposed to injuries and other exogenous insults that could require constant regeneration of their tissue-resident macrophages. Collectively, these findings add a new layer to the complex developmental landscape of the myeloid lineage and challenge the current notion that tissue-resident macrophages develop independently of the LT-HSCs.
Matrix protein Tenascin-C expands and reversibly blocks maturation of eosinophil progenitors
Authors: Doan Ton C. , Jeong Brian M. , Coden Mackenzie , Abdala-Valencia Hiam , Berdnikovs Sergejs
Published: January 2023

While eosinophil depletion is a sought after strategy in treating allergic disease, little is known about the local tissue factors regulating eosinophils. In disease, eosinophils encounter provisional extracellular matrix (ECM) glycoproteins such as Tenascin-C (TNC), which strongly correlates with eosinophil expansion in tissues. While restricted in healthy adult lungs, TNC is a hematopoietic niche component in bone marrow stroma and is expressed de novo during wound healing or in pathologic epithelial remodeling in asthma. RNA-Seq shows that exposing naïve murine eosinophils to TNC upregulates immaturity markers (CD34, CD117, and Sca-1) and suppresses IL-5Rα. Thus, we hypothesized that TNC represents a factor in the provisional allergic tissue microenvironment that could support in situ eosinophil progenitor expansion. In murine bone marrow-derived cultures, TNC: 1) 3-fold expanded the available Lin−Sca1+ early precursor pool; 2) downregulated IL-5Rα expression on Lin− CD45+ c-kit+ cells during the eosinophil lineage commitment phase; 3) served as a reversible eosinophil maturation block, as TNC withdrawal rescued maturation and increased final eosinophil yield. Moreover, TNC knockout mice lack in situ lung expansion of Lin− c-kit+ CD34+ common myeloid progenitors and Lin− Siglec-F+ Sca-1+ cells in an allergic inflammation model as well as exhibit accelerated lung eosinophil maturation ex vivo. Adding TNC to lung homogenates cultured in IL-5 ex vivo suppressed eosinophil maturation in a manner consistent with TNC block of eosinophil maturation in bone marrow-derived cultures. Together, our results stress the local tissue factor potential to promote in situ expansion and eosinophil persistence in allergic disease.
An adjuvant-free mouse model of anaphylaxis to saline-soluble wheat protein
Authors: Gangur Venugopal , Jin Yining , Ng Perry KW. , Olson Eric
Published: January 2023

Wheat is a major food that can trigger life-threatening systemic anaphylaxis. Mechanism of sensitization to wheat anaphylaxis is not completely understood at present. For example, whether or not skin exposure to saline-soluble wheat protein (SSWP) can cause sensitization is unknown. Here we tested the hypothesis that transdermal exposure (TDE) to SSWP from durum wheat results in IgE response and clinical sensitization for anaphylaxis. Groups of Balb/c mice weaned onto a plant free diet received six TDEs with SSWP (1 mg/mouse/week) or saline over a 6 week period without adjuvant. Blood was analyzed for IgE responses by ELISA, and anaphylaxis and mucosal mast cell protease (mMCP-1) release upon challenge were quantified. Spleen tissue was analyzed for cytokines using a protein microarray system. Results showed significant IgE response, hypothermia shock response and elevation of mMCP-1 upon challenge. Spleen analysis showed significant elevations of IL-4/5/7/10/17B,E,F, and TSLP and significant reductions of IL-1a and IL-6. The following cytokines were not significantly altered: IL-1b/2/9/13/17A/22/23, IFNg and TNFa.
Engineered ovalbumin-expressing regulatory T cells protect from chicken egg white-induced allergy in vivo
Authors: Abdeladhim Maha , Zhang Aihong , Kropp Laura E. , Kim Yong Chan , Mitre Edward E.S. , Scott David W.
Published: January 2023

Allergy is a major public health concern, the main treatment for which is symptomatic relief with anti-inflammatory drugs. A key clinical challenge is to induce specific tolerance in order to control allergen-specific memory B and T cells. Our lab recently developed antigen-specific regulatory T cell therapies as a treatment for adverse responses in hemophilia (Kim et al. BLOOD 125: 1107, 2015; Yoon et al. BLOOD 129: 238–245, 2017). In this report, we created a chimeric antigen receptor (CAR) approach in which we engineered the target protein antigen, ovalbumin (OVA), linked with the transmembrane and signal transduction domains, CD28-CD3ζ to directly target B cells to induce tolerance in an allergy model. We named this receptor “BAR” for B-cell Antibody Receptor. Murine Tregs, retrovirally transduced with a BAR containing OVA or control cells, were successfully expanded in vitro and tested in a standard murine OVA-alum allergy model with high titered circulating anti-OVA IgE antibodies. We first confirmed that intravenously injected OVA-BAR Tregs did not directly lead to temperature drops indicative of anaphylaxis. Forty eight hours later, mice were challenged intraperitoneally with 200μg of OVA to induce an anaphylactic reaction, and temperature taken immediately again after challenge for 30 minutes. Our results demonstrated that OVA-BAR Tregs protected mice from hypothermia, whereas the control BARs or PBS-treated mice showed temperature drops indicative of anaphylaxis. These results were repeated and confirmed using human Tregs (CD4+CD25hiCD127low) transduced and expanded and injected into mice. These data provide proof of principle that engineered OVA-BAR Tregs may be useful to treat IgE based allergies.
Revisiting the role of B7-CD28 co-stimulation in thymic negative selection: B7-CD28 co-stimulation enforces central tolerance by clonal deletion and Treg generation as independent cellular processes
Authors: Watanabe Masashi , Hodes Richard J.
Published: January 2023

Self-Ag-reactive T cells are eliminated from the conventional mature T cell repertoire by clonal deletion and/or fate diversion to the Treg during thymocyte development as a consequence of strong recognition of self-peptide-MHC complex by the TCR, a process termed negative selection. However, the role of the B7-CD28 costimulatory pathway in this process has not been fully elucidated. We analyzed the role of B7-CD28 interaction in thymic negative selection using pMHCII tetramer binding to directly measure the endogenous T cell repertoire specific for distinct self-Ags. Ablation of expression of a self-Ag or of the Aire gene resulted in increase in the number of self-peptide-specific CD4SP thymocytes, consistent with the physiologic process of ongoing negative selection. We found that absence of B7 also resulted in an increase in numbers of conventional CD4SP (non-Treg) thymocytes specific for some but not all self-Ags. Observed increases in conventional CD4SP were far in excess of the concomitant decrease in the number of Treg that occurred in the absence of B7. In addition, the identity of B7-expressing cell populations required for clonal deletion was distinct from the requirements for Treg generation. Thus, the increase of CD4SP cell number specific for self-Ag was largely due to defect in B7 dependent clonal deletion, but not simply conversion of “want-to-be Treg” to conventional CD4SP cells. We propose that 1) thymic clonal deletion occurs through B7-CD28 dependent and independent pathways; 2) B7-CD28 dependent negative selection is mediated by clonal deletion and/or fate diversion to the thymic Treg lineage; and 3) B7-CD28 dependent clonal deletion is a largely independent cellular process from B7-CD28 dependent Treg generation.
Increased antioxidant activity mitigates age-associated dysfunction of thymic stromal cells in aged mice.
Authors: Cepeda Sergio , Xiao Yangming , Hester Allison K. , Venables Thomas , Griffith Ann
Published: January 2023

Age-associated thymic atrophy results in diminished production of new T lymphocytes and a concomitant decrease in responsiveness to new pathogens and vaccines. In addition to loss of thymic size with age, critical stromal functions, including tissue-restricted antigen (TRA) expression, are diminished with age. We previously identified deficiency of the hydrogen peroxide quenching enzyme catalase (CAT) in thymic stromal cells as a cause of thymic atrophy during aging, and established that thymic atrophy can be mitigated by genetic or dietary complementation of catalase antioxidant activity. Ongoing studies indicate that, in addition to increasing thymic cellularity, life-long overexpression of catalase in mitochondria of transgenic mice (mCAT Tg) results in increased TRA expression and decreased numbers of T cells binding self-peptide MHCII tetramers in aged mice. We also find mitigated acquisition of an aged global gene expression signature in aged mCAT Tg mice relative to non-transgenic littermates. Together, these results suggest that complementing catalase activity in thymic stromal cells mitigates age-associated declines in thymic function.

Supported by N.I.H. R01AI121367 and N.I.H. R01AI121367-S
The Role of B7-CD28 and CD40L-CD40 costimulatory signaling in thymic Invariant NKT Cell subset development
Authors: Alkhaleel Farrah , Watanabe Masashi , Hodes Richard J.
Published: January 2023

Invariant Natural Killer T (iNKT) cells are a unique subset of NKT cells that express a highly restricted αβ T cell receptor (TCR) and are selected in the thymus by recognition of Glycolipid/CD1d complex expressed by double-positive thymocytes. We previously demonstrated that thymic iNKT cell development is highly sensitive to B7-CD28 costimulatory interactions, with substantially decreased numbers of thymic iNKT cells in B7 and CD28 knockout mice. Advances in iNKT subset identification have allowed the detailed study of three major iNKT subsets, NKT1, NKT2, and NKT17, defined by characteristic transcription factor expression. We have analyzed the requirement for B7-CD28 and CD40-CD40L costimulatory signaling for development of thymic iNKT subsets. Comparison of BALB/c wild-type, B7 knockout, and CD28 knockout mice showed a substantial decrease in number of all thymic iNKT subsets in B7 and CD28 knockouts, demonstrating a critical role for B7-CD28 signaling in development of all thymic iNKT subsets. In contrast, CD40 knockout and CD40L knockout mice showed a substantial selective decrease in NKT2 cells with less effect on NKT1 and NKT17 subsets. These findings identify distinct requirements for B7-CD28 costimulation in thymic development of all iNKT subsets and for CD40L-CD40 costimulation selectively in NKT2 subset development. Further experiments are currently in progress examining the cellular requirements for B7 and CD40 expression in NKT development using conditional B7 and CD40 knockouts and complementary bone marrow mixed chimera approaches.
LUBAC-mediated signals in thymic epithelial cells prevent early thymic atrophy and immunodeficiency
Authors: Jain Reema , Sheridan Julie M. , Heinlein Melanie , Kupresanin Fiona , Hall Cathrine , Rickard James , Bouillet Philippe , Walczak Henning , Strasser Andreas , Silke John , Gray Daniel H.D.
Published: January 2023

Thymic epithelial cells (TECs) govern the differentiation of haematopoietic precursors into functional T cells. Defects in this process can lead to immunodeficiency and autoimmune disease. Distinct signals from members of the TNF receptor superfamily have key roles in TEC proliferation and differentiation, yet it remains unclear how these stimuli are integrated to support thymus function. We find that components of the linear ubiquitin chain assembly complex (LUBAC) that act downstream of TNFR signaling to regulate NFkB activity are essential for TEC homeostasis and thymic function. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, induced early thymic atrophy shortly after birth, with severe loss of tissue in 2-month-old adult mice attended by gross defects in peripheral T cell populations. The requirement for HOIL-1 or HOIP extended to all major TEC subsets, including those expressing the autoimmune regulator, AIRE. In contrast, loss of a third LUBAC component in SHARPIN-deficient mice (Shcpdm/cpdm) had a milder impact, reducing TECs in the thymic medulla that require CD40 signals for their maintenance. Ablation of TNF or Caspase 8 and RIPK3 in Shcpdm/cpdm mice failed to rescue these defects, indicating that CD40/SHARPIN signals are not required to prevent TNF-induced cell death. These differential roles for LUBAC components in TEC correlate with their function in linear ubiquitylation and reveal a critical signaling hub in the maintenance of the thymic microenvironment
Two-photon imaging reveals distinct contributions of Aire+ medullary thymic epithelial cells and dendritic cell subsets to central tolerance induction
Authors: Ehrlich Lauren I. , Lancaster Jessica N. , Thyagarajan Hiran Maye
Published: January 2023

Following positive selection, thymocytes migrate into the medulla where they encounter diverse tissue-restricted antigens (TRAs) that induce central tolerance. Both medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present TRAs to induce thymocyte negative selection and regulatory T cell induction. However, their relative contributions to tolerance remain unresolved, partially because the genetic models that have been used to address this question can interfere with thymocyte-stromal cell crosstalk, potentially resulting in secondary defects in stromal differentiation that independently alter central tolerance. Thus, we developed a 2-photon microscopy approach to directly observe thymocytes as they undergo tolerance induction in response to interactions with DCs versus Aire+ mTECs presenting various forms of a TRA in an intact thymic stromal environment. We find that both mTECs and DCs present transmembrane TRAs with equivalent efficiency to CD8SP and CD4SP thymocytes. However, in the context of a secreted TRA, DCs play a greater role than mTECs in presenting antigen to CD4SP, but not CD8SP thymocytes. Furthermore, distinct DC subsets differentially contribute to TRA presentation to CD4SP versus CD8SP thymocytes, with a major role for Sirpα+DCs in cross-presentation of TRAs. In the context of a polyclonal T cell repertoire responding to endogenous antigens, DCs play a more significant role than mTECs in activating CD4SP cells. In summary, we find that in an intact thymic microenvironment, multiple DC subsets and Aire+ mTECs cooperate to present self-antigens to auto-reactive thymocytes during the establishment of tolerance against diverse medullary TRAs.
Catalase expression mediates redox regulation of autophagy and promiscuous gene expression in thymic stromal cells
Authors: Hester Allison K. , Semwall Manpreet , Xiao Yangming , Almutairi Abdulaziz , Cepeda Sergio , Venables Thomas , Griffith Ann V.
Published: January 2023

T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs progenitors along a well-characterized differentiation program. However, the biology of stromal cells 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 peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen species (ROS) levels in this population, eventually leading to thymic atrophy in aged mice. Here, we find that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), both ROS levels and stromal function decline in young mice relative to non-transgenic littermates. TSC transcriptome analysis reveals decreased expression of tissue-restricted antigen (TRA) and autophagy pathway genes in mCat Tg mice. Analysis of autophagy flux reporter mice ubiquitously expressing the RFP-EGFP-LC3 fusion transgene also indicates diminished autophagic flux in mCAT Tg mice, particularly in TSCs. Stromal TRA expression and autophagic flux are required for self-antigen presentation, and therefore promote negative selection of potentially auto-reactive T cells. We propose that oxidative stress generated by low catalase levels in stromal cells promotes these key physiological functions in the young, steady state thymus; in contrast however, the resulting accumulated oxidative damage ultimately impairs function in the aged thymus.
Newly generated CD4+ T cells acquire metabolic quiescence after thymic egress.
Authors: Ge Qing , Zhang Shusong
Published: January 2023

Mature naive T cells circulate through the secondary lymphoid organs in an actively enforced quiescent state. Impaired cell survival and cell functions could be found when T cells have defects in quiescence. One of the key features of T cell quiescence is low basal metabolic activity. It remains unclear at which developmental stage that T cells acquire this metabolic quiescence. We compared mitochondria among CD4 single positive (SP) T cells in the thymus, CD4+ recent thymic emigrants (RTEs), and mature naive T cells in the periphery. The results demonstrate that RTEs and naive T cells had reduced mitochondrial content and mitochondrial reactive oxygen species when compared to SP thymocytes. This down-regulation of mitochondria requires T cell egress from the thymus and occurs early after young T cells enter the circulation. Autophagic clearance of mitochondria, but not mitochondria biogenesis or fission/fusion, contributes to mitochondrial down-regulation in RTEs. The enhanced ASK1/MAPKs and reduced mTOR activities in RTEs relative to SP thymocytes may be involved in this mitochondrial reduction. These results indicate that the gain of metabolic quiescence is one of the important maturation processes during SP-RTE transition. Together with functional maturation, it promotes the survival and full responsiveness to activating stimuli in young T cells.
Time of ART initiation in perinatally HIV-infected children impacts on HIV-specific T cell functionality
Authors: Rinaldi Stefano , Cotugno Nicola , Pallikkuth Suresh , Pahwa Rajendra , Palma Paolo , Pahwa Savita
Published: January 2023

Early initiation of antiretroviral therapy (ART) in vertically HIV-infected children provides an opportunity to limit the size of HIV reservoir. However, how the time of ART initiation impacts host HIV-specific immune responses is still poorly understood. In this study, we analyzed HIV-specific CD4 and CD8 T cell functionality in vertically HIV-infected children (age range 2.3–17.5yrs) enrolled at Bambino Gesù Children Hospital under suppressive ART and durable viral control (plasma HIV-RNA<50cp/mL). Children were designated as early treated (ET, n=8, ART initiated at age <6mo) or late treated (LT, n=7, ART initiated at age >1 year) with longer duration of ART in ET (p<0.05). Antigen-specific (CD40L+CD4+/CD107a+CD8+) T cells were evaluated in cryopreserved PBMC by flow cytometry for intracellular cytokines (IL2, IFN g, TNFα, IL21, Perforin, Granzyme B) following 12hr stimulation with GAG PTE peptides. Differences between groups were determined by Mann-Whitney t tests (P≤0.05).

Frequencies of GAG-specific CD8 and CD4 T cells were not different between ET and LT. Boolean analyses revealed that CD8 T cells of LT had a higher frequency of Granzyme B+ cells compared to ET whereas CD4 T cells were qualitatively superior in ET compared to LT and exhibited higher proportions of IL2, IFNg and TNFα producing T cells and enrichment of polyfunctional T cells.

Our results suggest that time of ART initiation in HIV-infected children has a long-term impact on the quality but not the quantity of the host HIV-specific T-cell immune responses. Larger studies are needed to confirm these results and to further evaluate their role in future strategies aiming at functional cure.