bluuebunny/crossref_metadata_2025 huggingface dataset:
Lnk/Sh2b3 modulates bioenergetic metabolism of activated CD8 T cells and control the development of Type 1 Diabetes
Authors: Pant Tarun , Foda Bardees , Geurts Aron , Chen Yi-Guang
Published: August 2023

Recent genome-wide association studies (GWAS) have shown a strong correlation between a genetic polymorphism in the SH2B3 gene and the development of autoimmune diseases, including type 1 diabetes (T1D). Lnk/Sh2b3 is an adaptor protein interacting with various intracellular proteins involved in JAK/STAT cytokine signaling of different cell types, including CD8 T cells. Additionally, Sh2b3-deficient CD8 T cells are hypersensitive to IL-15 stimulation. However, how reduced Sh2b3 function affects the molecular mechanisms that activate and sustain CD8 T cells promoting T1D development remains elusive. We hypothesized that genetic deletion of Sh2b3 in the non-obese diabetic (NOD) mouse would impact T1D incidence by altering T cell activation. NOD. Sh2b3−/− mice developed diabetes faster than wild-type controls. Additionally, Sh2b3-deficient CD8 T cells were hyperresponsive to IL-15 stimulation, promoting the generation of the activated CD44 high CD8 T cell population. Intriguingly, IL-15-stimulated Sh2b3−/− splenocytes possessed significantly higher mitochondrial respiratory capacity (SRC) and ATP synthesis than the wild-type counterparts. Furthermore, CD8 T cells of NOD. Sh2b3−/− mice showed increased expression of genes associated with mitochondrial biogenesis and oxidative metabolisms in response to in vitro IL-15 stimulation. Overall, our study supports a role of Sh2b3 in T1D development by regulating mitochondrial biogenesis and metabolism of IL-15-stimulated CD8 T cell. Our study provides new insight into the role of Sh2b3 in activating and sustaining the CD8 T cell response in the pathogenesis of T1D.

Funded by NIH DK097605

Funded by NIH DK097605
Knockout CD73 reduces thymus-derived lymphocytes and clinical phenotype of Foxp3+Treg deficient autoimmune scurfy mice
Authors: Liu Yuying , Okeugo Beanna , Armbrister Shabba A , Daniel Rhea C , Saleh Zeina M , Rhoads J Marc
Published: August 2023

CD73 is ubiquitously expressed and regulates critical functions across multiple organ systems. The combination of CD39 and CD73 degrade ATP to adenosine that shifts ATP-driven pro-inflammatory immune cell activity toward an anti-inflammatory state. This immunological switch is a major mechanism for Tregs to control inflammation. In the Foxp3+Treg deficient scurfy (SF) mouse, a mouse model of human IPEX syndrome, we showed that a specific probiotic exhibits therapeutic effects via probiotic-derived adenosine. We generated CD73KOSF mice by removing host CD73, aiming to identify a specific probiotic-CD73 effect. CD73KOSF mice were generated by breeding heterozygous-Foxp3sf/J females to CD73KO male mice with genotyping confirmation. We unexpectedly observed that KO of CD73 in SF mice reduced clinical severity. CD73KOSF mice showed reduced ear thickness, increased ear size, improved deformed ears, and reduced scurfy skin compared to SF mice. SF mice had increased CD4+ and CD8+ T cells compared to WT mice; KO of CD73 in SF mice significantly reduced the numbers of these cells in the spleen and blood to normal. However, the inflammatory cytokines and chemokines in plasma and lymphocyte infiltration in the liver of CD73KOSF were similar to those in SF mice. We further identified that KO of CD73 in SF mice reduced the numbers of CD3+T and DP CD4+CD8+ T cells in the thymus compared to those in SF mice, indicating that reducing clinical phenotype specifically scurfy tails and deformed ears may mainly be due to reduced central and peripheral lymphoproliferation. This study provides data to aid further discovery of CD73 as a target to improve dermatitis-one of the most common symptoms in Treg-deficiency-associated primary immune deficiencies.

Supported by grants from NIH/NIAID (R03AI153725)
Modulation of murine lupus by Aconitate Decarboxylase 1
Authors: Martinez Eduardo Patino , Nakabo Shuichiro , Claybaugh Dillon , Romero Aracely , Blanco Luz P , Kaplan Mariana J
Published: August 2023

Mitochondrial synthesis of itaconate by myeloid cells (monocytes and macrophages) is mediated by the TCA cycle enzyme Aconitate Decarboxylase 1 (Acod1). Upregulation of Acod1–itaconate pathway, occurs after activation of various Toll-like receptors (TLRs) or cytokine stimulation (TNF or type I Interferons). While various anti-inflammatory effects of the Acod1 – Itaconate pathway have been previously reported (e.g. decreases in IL-6 and IL-1β synthesis) the contribution of impairments in endogenous itaconate synthesis in systemic lupus erythematosus (SLE) remain poorly characterized. First, we explored and characterized Acod1 expression in bone marrow or monocyte-derived macrophages (BMDM and MDM, respectively) following in vitro imiquimod (IMQ) treatment. Then, we investigated the role of endogenous itaconate modulation in the development and severity of TLR-7 induced lupus using the IMQ model. 10-week-old wild type and Acod1−/− mice were treated with topical IMQ cream for 5 weeks to induce an SLE phenotype. We found that Acod1 protein expression is induced in BMDM and MDM after 12 hours of IMQ stimulation, and this induction is partially dependent on IFNRa1 signaling. Moreover, Acod1−/− BMDM treated with IMQ for 12 h displayed higher mRNA expression of il6, il1b, il18, ccl2, cxcl2, and cxcl9, together with an increase in pro-IL-1β protein levels. IL-6 and IL-1β production were also increased. After 5 weeks of in vivo IMQ topical treatment, Acod1−/− mice have a higher concentration of circulating anti-dsDNA autoantibodies but a decrease in splenomegaly compared to the treated WT mice. These results, demonstrate a putative role for endogenous Acod1-itaconate pathway in the regulation of autoimmunity.

This work is support by the NIH/NIAMS Intramural Research Program
Combining activity-based protein profiling and flow cytometry to elucidate the role of lipid-metabolizing enzymes in cell heterogeneity
Authors: McSorley Caitlin , Murtagh Olivia , Hsu Ku-Lung
Published: August 2023

Cell metabolism is mediated by intracellular enzymes that supply essential and specific molecular cues needed to initiate the differentiation of homogenous cell populations into heterogenous cell populations. Intracellular enzymes involved in the metabolism of lipids are of particular interest as lipids play key roles in cell signaling and differing lipid-metabolizing enzyme activity may produce varying signaling profiles, and thus, a varying cell phenotype. To understand how these enzymes regulate function at the single-cell level, we aim to develop methodology that combines activity-based protein profiling (ABPP) and flow cytometry to allow for the separation of heterogenous cells into new populations based on enzyme activity. Our initial studies have focused on targeting serine hydrolases, as this family of enzymes includes numerous intracellular lipases involved with the metabolism of lipids. We have synthesized a modified activity-based fluorescent probe containing a triazole urea warhead known to label active serine hydrolases in live cells. Based on the fluorescent profile generated by measuring single cell fluorescence using flow cytometry, the probe targeted a select population of cells when compared to a control probe and unlabeled cells. This probe-labeled population was concentration dependent as higher concentration of probe labeled more enzyme, suggesting that a different population of cells could be collected between two different concentrations. After these cells are sorted based on the differing fluorescent signals, these new subsets will be analyzed via targeted lipidomics and proteomics methods to quantity any differences in the probed intracellular enzyme expression.

This work was supported by the National Science Foundation CAREER Award no. 1942467.
A new antibody for the study of human CD157 expression and function
Authors: Kassmer Susannah H , Divekar Anagha , Chen Weihao , Yang Xifeng
Published: August 2023

CD157 is an ectoenzyme that has both cyclic ADP-ribose hydrolase and ADP-ribosyl cyclase activities. In the human hematopoietic system, CD157 is prevalently expressed by cells of the myelomonocytic lineage, and interacts with extracellular matrix components and regulates leukocyte diapedesis via integrin-mediated signaling. CD157 is implicated in the pathophysiology of several neurological disorders.

Although it lacks a cytoplasmatic domain, CD157 is able to transduce intracellular signals by establishing functional and structural crosstalk with β1 (CD29) and β2 (CD18) integrins. CD157 ligation causes integrin– dependent cytoskeletal remodeling and increase in F-actin content. Activation of downstream signaling pathways leads to cell polarization, with CD157 migrating prevalently to the rear of the cells whereas F-actin is localized at the opposite pole. However, the molecular and cellular functions of CD157 still remain poorly understood.

Here, we describe the generation of a monoclonal antibody specific for human CD157 (clone W21007F) that can be used in flow cytometry, immunofluorescence and blocking of CD157 mediated adhesion of monocytes to fibronectin. Treatment of the pro-monocytic, myeloid leukemia cell line U937 with clone W21007F induces cell polarization and a marked increase in filamentous actin, with CD157 clustering at the uropod. The majority of F-Actin fibers are located at the opposite end of CD157, and CD157 co-localizes with integrin beta-1 in distinct membrane domains. The reference clone SY/11B5 and isotype control antibody do not show this effect. In conclusion, anti-human CD157 (clone W21007F) is a valuable tool for the study of CD157 function and expression in human cells.
Staphylococcus aureus promotes increase IL-1 mediated immunopathology and delayed healing in cutaneous leishmaniasis
Authors: Amorim Camila Farias , Lovins Victoria , Singh Tej P , Novais Fernanda O , Harris Jordan , Carvalho Lucas P , Carvalho Edgar M , Beiting Daniel P , Grice Elizabeth A , Scott Phillip
Published: August 2023

The chronic infection by Leishmania braziliensis causes a skin barrier (ulcer) breach and is susceptible to secondary bacterial infections. The development of the ulcer is associated with a dysregulation of the skin microbiome, which we hypothesized impacts host gene expression, exacerbates the inflammatory response, and prolongs the resolution of the disease. We conducted an integrative multi-omics meta-analysis of the skin microbiome, host gene expression, and clinical metadata of patients. We found that a high bacterial burden was associated with an increased inflammatory response and delayed healing. 16S-seq analysis of lesion swabs revealed that while patients exhibit several distinct skin microbiomes, a microbiome dominated by Staphylococcus is the most common. Furthermore, the patients with a dominant lesional Staphylococcus microbiome were delayed in healing compared to those with a heterogeneous microbiome. Since we found that S. aureus was a common isolate from the ulcers, we carried out dual RNA-seq analysis on the lesion biopsies to quantify S. aureus transcriptional abundances and associated them with host gene expression. High S. aureus transcript abundance was associated with delayed healing and increased expression of IL-1α and IL-1β. These cytokines were critical for modulating disease outcome since pathology was reduced when we neutralized either in L. braziliensis-infected mice colonized with S. aureus. These results indicate that an altered skin microbiome develops in cutaneous leishmaniasis, influencing responsiveness to anti-parasitic drug treatment. They suggest that host-directed therapies designed to reduce the bacterial burden in leishmanial lesions may be therapeutic.

NIH (AI143790)
Distinct lymphatic remodeling is associated with healing and non-healing murine cutaneous leishmaniasis
Authors: Fry Lucy , Bowlin Anne , Roys Hayden , Weinkopff Tiffany
Published: August 2023

Cutaneous leishmaniasis (CL) is the spectrum of diseases caused by Leishmania parasites endemic to the tropical and subtropical regions of the world causing 1–2 million new cases each year. Based on the species of infecting parasite and the host immune status, CL can be self-healing or lead to permanent disfiguration. We showed a critical role for the lymphatics in wound healing during L. major infection in C57BL/6 mice, a self-healing model of CL that replicates most human disease. Specifically, lymphangiogenesis, mediated by vascular endothelial growth factor-A (VEGF-A)/vascular endothelial growth factor receptor-2 (VEGFR-2) signaling, is required for efficient lesion resolution. The lymphatic vasculature regulates the immune response to infection, transporting soluble antigen and antigen presenting cells to the lymph nodes and engaging an active role by modulating inflammatory immune cell exit from the site of infection. Here, we investigate the role of the lymphatics in mice infected with L. amazonensis and L. mexicana, species causing non-healing CL. We found that the mediators of lymphangiogenesis like VEGF-A and VEGF-D are elevated during infection with L. amazonensis and L. mexicana, but they are either delayed or not sustained over time which compromises lymphangiogenesis and increases vessel dilation compared to naïve mice. During Leishmania infection we find increased lymphatic vessel dilation is associated with enhanced disease severity. Taken together, the current work suggests lymphangiogenesis is attenuated later during infection with species causing non-healing CL suggesting impaired lymphatic remodeling contributes to persistent lesions in patients with CL.

COBRE UAMS CMPHIR
γδ T Cells Contribute to Immunopathology During Neonatal Sepsis in an Age-Dependent Manner
Authors: Witt Lila T , Greenfield Kara , Knoop Kathryn
Published: August 2023

Bacterial bloodstream infections (BSIs) remain a significant contributor to neonatal mortality, despite improved hygienic practices in the NICU. Although the understanding of the neonatal immune system has increased greatly over the past decade, why neonates, but not older infants, are susceptible to severe adverse immunopathology – sepsis – in response to bacteremia remains poorly understood. In the present study, we find that postnatal day 5 (P5) neonatal γδ T cells are highly inflammatory during a mouse model of E. coli BSI and are associated with mortality. Furthermore, γδ T cells in P5 mice rapidly produce pro-inflammatory cytokines, such as IFN-γ, IL-17A, and TNFα, and increase their expression of cytotoxic molecules such as granzyme and perforin. Interestingly, blockade of the γδ TCR in vivo is associated with increased survival and decreased pro-inflammatory cytokine production in P5 pups. Signaling and activation via the γδ TCR was confirmed using Nur77-GFP mice, with P5 γδ T cells significantly upregulating expression of Nur77 during in vivo infection. Intriguingly, E. coli BSI in P15 pups results in markedly different γδ T cells responses compared to neonates, despite similar systemic bacterial burden. During bacterial bloodstream infection, P15 pups do not experience mortality, and unergo minimal activation of γδ T cells and production of IFN-γ, IL-17A, and TNFα. Furthermore, BSI infection in P15 Nur77-GFP mice revealed lack of TCR signaling in P15 γδ T cells at this age. In conclusion, the present study reveals rapid age-dependent changes to γδ T cell responses during early life and suggests neonates may be uniquely susceptible to γδ T cell-mediated immunopathology during bacterial bloodstream infection.

T32 AI170478-01
EnvZ/OmpR two-component system induces antibiotic resistance in Salmonella enterica Serovar Enteritidis in response to β-lactams
Authors: Ko Duhyun , Choi Sang Ho
Published: August 2023

Outer membrane porins (OMPs) of Gram-negative bacteria including Salmonella enterica serovar Enteritidis allow the entry of small hydrophilic antibiotics. S. Enteritidis is a human pathogen causing food-borne illnesses, and infection by S. Enteritidis often results in severe invasive disease and requires antibiotic treatments. Although compositional modulation of OMPs is frequently observed in multidrug-resistant S. Enteritidis, little is known about the mechanisms by which the pathogen modulates OMP composition to enhance antibiotic resistance. Here, we found that the S. Enteritidis strain with an active state of EnvZ/OmpR two-component system is highly resistant to various antibiotics, especially β-lactams. Transcriptome analysis showed that the active state of EnvZ/OmpR induces differential expression of multiple OMP genes, including SEN1522, SEN2875, ompD, and ompW. Interestingly, the cellular level of response regulator OmpR determines the expression of the four OMP genes, and phosphorylation of OmpR is not necessary for the regulation of ompD and ompW. Phenotypic analysis revealed that ompD is a key OMP gene responsible for the EnvZ/OmpR-mediated antibiotic resistance in S. Enteritidis. Notably, EnvZ/OmpR responds to the presence of β-lactams and provides S. Enteritidis with direct benefits for survival. Altogether, this study suggests that EnvZ/OmpR remodels OMP composition of S. Enteritidis in response to β-lactams and enhances antibiotic resistance.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF-2022R1A6A3A01086815).
CD8+ T cells require CCR5 expression to mediate immunopathology in cutaneous leishmaniasis
Authors: Sacramento Lais A , Amorim Camila Farias , Lombana Claudia , Scott Phillip
Published: August 2023

Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis by a mechanism dependent on degranulation and lysis of Leishmania-infected cells, culminating in NLRP3 activation and IL-1b release. Here, we sought to identify chemokine receptors involved in CD8+ T cell migration to the lesion that could be employed as a treatment target to ameliorate disease severity. A transcriptional study identified the profile of chemokine receptors that predicts treatment failure in Leishmania braziliensis patients and identifies possible targets that may be involved in CD8+ T cell migration. Using murine models of cutaneous leishmaniasis, we found that around 20% of CD8+ T-cells express CCR5 in lesions at the peak of the disease. In previous studies, we found that CD8+ T cells transferred to Rag1−/− mice mediated increased NLRP3 and IL-1b dependent disease. Therefore, to test if CCR5 expression on CD8+ T cells was involved in their migration to lesions, we transferred wild-type or CCR5−/− CD8+ T cells to L. braziliensis infected Rag1−/− mice. While infected Rag1−/− mice reconstituted with wild-type CD8+ T cells developed severe pathology, Rag1−/− mice that received CCR5−/− CD8+ T cells developed smaller lesions accompanied by a significant reduction in the number of CD8+ T cells in the lesions. To test whether CCR5 blockade would control disease severity, we used maraviroc (MVC), a selective inhibitor of CCR5 approved by the FDA. MVC treatment significantly reduced lesion development without affecting parasite number in our murine models. Collectively, these results demonstrate that cytolytic CD8+ T cells migrate to leishmania lesions in a CCR5-dependent manner, and MVC treatment efficiently prevents CD8+ T-cell mediated pathology.

R01 AI106842 and U01 AI08865006
Gut regulatory T cells mediate immunological tolerance in Salmonella-infected super-spreader hosts
Authors: Di Luccia Blanda , Massis Liliana Moura , Ruddle Sarah , Narasimhan Ramya , Pham Trung Hoang Minh , Vilches-Moure Josè , Amieva Manuel R , Monack Denise M
Published: August 2023

Salmonella Typhimurium is an enteric pathogen that causes gastroenteritis in humans and Typhoid fever-like symptoms in mice. Our lab has shown that oral infection of 129X1/SvJ mice with Salmonella Typhimurium results in ~25% of mice becoming persistent high-shedders (>108 CFU/g feces). Because these mice can transmit infection to a naïve host, we refer to them as super-spreaders (SS). Unexpectedly, SS mice are asymptomatic despite a persistent high-pathogen burden in the gut. We hypothesized that SS mice are disease resistant due to a gut-specific immune state that facilitates homeostasis between host and pathogen. To examine this, we performed extensive immuno-phenotyping of SS mice, which revealed a distinct type-1 immune state in the gut. We found that IFNγ producing effector CD4 T cells were the dominant population to accumulate in SS guts. Additionally, a population of Tbet+ Tregs were found only in the gut of SS mice, suggesting these cells may enable the SS asymptomatic state. Accordingly, specific ablation of Tregs resulted in a dramatic weight loss, gut inflammation, and a compromised epithelial barrier in SS mice. Bacterial burden was unchanged in Treg depleted mice, demonstrating that morbidity was not caused by Salmonella burden but likely due to immune pathology. Indeed, depletion of effector CD4+ T cells in Treg-deficient SS mice reversed this morbidity, confirming the disease state was directly caused by immune pathology. These results demonstrate that in SS mice Tregs play an essential tolerogenic role maintaining intestinal homeostasis. We are currently investigating the immunological mechanisms triggering this effect and whether this new immune-adapted state may impact infections with other enteric pathogens.
Giardia duodenalis potentiates macrophage IL-10 production in response to LPS
Authors: Singer Steven M , Miskovsky Eleanor , Darmadi Matthew , Menegas Samantha , Shapiro Danielle , Fink Marc , Angelova Vanessa V
Published: August 2023

Giardia duodenalis is one of the world’s most common intestinal parasites, with an estimated 280 million cases globally each year. Infections can cause diarrhea and cramps, although they are often subclinical. Interestingly, Giardia can have a protective effect and reduce the incidence of fatal diarrhea in co-infections with other enteric pathogens. In this study, we investigate the anti-inflammatory response to Giardia via the macrophage galactose lectin-type 1 (MGL1) receptor. Our reasons for choosing this receptor are three-fold: it is expressed on macrophages and some dendritic cells and plays a role in immune response to other pathogens, it has been shown to induce anti-inflammatory effects in the DSS colitis model, and it demonstrably binds galactose-containing carbohydrates, like the N-Acetylgalactosamine (GalNAc) present in the cell wall of Giardia cysts. We show that peritoneal macrophages isolated from wildtype C57BL/6 mice produce markedly more of the anti-inflammatory cytokine IL-10 when stimulated with lipopolysaccharide (LPS) and Giardia extract compared to LPS alone. The potentiated IL-10 response to Giardia is lost in peritoneal macrophages isolated from MGL1 receptor knockout mice or in extracts centrifuged at 10,000 X g. Elucidating this anti-inflammatory pathway not only promises a better understanding of giardiasis, which disproportionately affect lower-middle income communities. also might present opportunities to mimic the parasite in the treatment of a wide array of inflammatory diseases.

Supported by a grant from NIH (R15 AI109591).
Analysis of naturally acquired immune responses against the Plasmodium falciparum malaria vaccine candidate PF3D7_1136200
Authors: Garza Rolando , Reers Ashley , Reyes Raphael , Ssewanyana Isaac , Greenhouse Bryan , Bol Sebastiaan , Bunnik Evelien
Published: August 2023

Humoral immunity is critical for protection against Plasmodium falciparum malaria. In endemic areas, individuals acquire protective immunity against malaria through repeated exposure to P. falciparum parasites. Understanding these naturally acquired immune responses would be highly informative for the development of improved vaccination strategies against malaria. Antibody responses against the uncharacterized protein PF3D7_1136200 correlate with protection from malaria, however, the development of these antibody responses over time and their role in protecting against malaria remain unknown. Here, we compared PF3D7_1136200-specific antibody and B cell responses to that of other P. falciparum antigens in Ugandan individuals with different immune status: immune adults (n=24) and children with high (n=10) or low (n=8) susceptibility to malaria. In all groups, serum antibody reactivity against MSP1, MSP3, AMA1, VFT, Pf41, and Pf113 was dominated by IgG. In contrast, serum IgG antibody reactivity against PF3D7_1136200 was much lower, while IgM reactivity was at a similar or higher level compared to these antigens. In line with the low serum IgG reactivity against PF3D7_1136200 among adults, we exclusively observed IgM+ memory B cells (MBCs) against PF3D7_1136200, while IgG+ MBCs were more abundant than IgM+ MBCs for all other antigens. These results indicate that PF3D7_1136200 has a low antigenic profile, similar to that of other favorable P. falciparum vaccine candidates, and suggest potential shielding of antigen from the host immune system. Together with the genetic conservation of PF3D7_1136200, these characteristics highlight that Pf3D7_1136200 has potential as a novel vaccine candidate against malaria.

Supported by grants from NIH (R01 AI153425, P30 CA054174-20)
Memory phenotype Tfh cells are homeostatically maintained and are important for the development of germinal centers and B cell responses to virus infection
Authors: Wang Ping , Symonds Alistair , Li Suling
Published: August 2023

Memory phenotype (MP) CD4 T cells that develop in the absence of overt antigen stimulation are important for both innate and adaptive immune responses. Although pathogen specific MP CD4 T cells have been found to function effectively against infection, it is not known if MP Tfh cells can also be developed in the absence of overt antigen stimulation. Here, we demonstrate that MP Tfh cells develop in the absence of overt antigen stimulation. In addition to FR4+, a mark found in pathogen induced memory Tfh cells, MP Tfh expressed high levels of Egr2. The development of MP Tfh are defective in CD2-Egr2/3−/− mice. MP Tfh cells share similar features in phenotypes and epigenetics with antigen induced memory Tfh cells and can effectively induce germinal center formation and B cells responses. Thus, MP Tfh cells regulated by Egr2/3 pathway are important component of adaptive immunity.

Medical Research Council UK (MR/N00096X/1), Barts Charity (MGU0463)
Prevention of thymus involution rescues old mice from fatal Toxoplasma gondii infection
Authors: Morales-Sanchez Abigail , Cowan Jennifer , Vacchio Melanie , Zhao Yongge , Jankovic Dragana , Sen Ranjan , Bosselut Remy , Bhandoola Avinash
Published: August 2023

Thymic epithelial cells (TEC) make up the thymic microenvironments that support the generation of a functionally competent and self-tolerant T-cell repertoire. Infections, starvation, and aging, among other factors, cause thymus involution with loss of T-cell generation and decreased frequency of naive T-cells in secondary lymphoid organs. However, the causes and consequences of thymus involution are not clearly understood. Since thymus function declines with involution, we need a better understanding how the thymus controls T cell development. We found that constitutive overexpression of Myc (cMycTg) in TEC prevents thymus involution, causing a dramatic increase in thymus size in adult mice (Cowan et al, 2019). In addition, cMycTg mice restored frequencies of CD4+ and CD8+ naive T-cell populations to the levels observed in young adult mice. In unpublished work, we have found that prevention of thymus involution rescues aging mice from lethal Toxoplasma gondii infection. During the acute infection, old mice showed a significantly decreased frequency of parasite-specific T-cells and increased serum levels of IFNγ, IL6 and TNFα. Furthermore, antibody depletion of T-cells in aging mice challenged with Toxoplasma gondii prolonged their survival. These findings identify a role for thymic involution in the age-associated mortality of Toxoplasma gondii infection. We are now investigating the mechanisms responsible for the pathogenic immune responses seen in aged individuals to understand how improved thymic function modifies immune responses.

AMS is supported by the patronage of the Children’s Hospital of Mexico.

SCS, JEC and AB are supported by the NIH Intramural Cancer Research Training Award to post-doctoral fellows.
Identification of immunosuppressive and tumor-promoting neutrophils in bone marrow of human and human immune system mice
Authors: Li Yan , Liu Wei , Che Keying , Lu Chun , Wei Jia
Published: August 2023

Neutrophils in cancer patients were identified as a specialized heterogeneous population that suppress anti-tumor responses and accelerate tumor progression. However, the developmental stage and the source of this population remain elusive. To address this issue, we defined human neutrophil development based on previous publications and demonstrated that immature neutrophils predominate tumor tissues of patients. On the quest to identify the origin of immature neutrophils, we unexpectedly discovered that immature neutrophils collected from bone marrow of healthy people and human immune system (HIS) mice are naturally immunosuppressive and tumor promoting, whereas the mouse counterpart is not. To further investigate the linkage between peripheral and bone marrow immature neutrophils, we created a NCG-Gfi1−/− strain, which overcomes the issue of poor human neutrophil engraftment in periphery. NCG-Gfi1−/− HIS mice manifest similar kinetics of immature neutrophil expansion as cancer patients both in bone marrow and periphery after tumor cell inoculation. Single cell analysis of immature neutrophils cross human tumor, bone marrow and HIS mouse bone marrow samples revealed FCN1 as a common marker for this heterogeneous population. Finally, we proved that elimination of bone marrow immature neutrophils with Flt3L treatment reduced tumor associated neutrophils and delays tumor growth in NCG-Gfi1−/− HIS mice. In summary, human immunosuppressive and tumor-promoting neutrophils are predominantly immature neutrophils and targeting this population in bone marrow may represent a novel strategy for cancer immunotherapy

This study was supported by grants from the National Key Research and Development Program of China (2019YFA0802900), and National Natural Science Foundation of China (32070942, 32122035, 32000669).
Breast cancer remodels the bone marrow immune microenvironment to favor bone metastasis
Authors: Teijeiro Ana , Belkaid Yasmine
Published: August 2023

Bone is the most common site of breast cancer (BC) metastasis, occurring in 70% of patients, and an indication of short-term prognosis. Understanding the environmental and host factors involved in the induction of metastasis is essential to develop therapies that extend patient’s lifespan. Bone colonization by cancer cells is a stepwise process that relies on interactions between cancer cells and the cells that comprise the bone microenvironment, such as osteoclasts, osteoblasts, hematopoietic cells, immune cells, and adipocytes. Calcium, growth factors, and chemokines facilitate bone metastasis. However, the role of immune cells in bone metastasis remains poorly understood. Further, how physiological stressors such as nutrition contribute to pro- or anti-metastatic bone immunity remains unclear. Using mouse models of BC, we found that BC profoundly transforms the bone marrow (BM) immune microenvironment to support metastasis. BC induces bone lymphoid cell loss, with significant reduction in B cells, T cells and NK cells. On the other hand, mammary tumors are associated with enhanced monocytes and eosinophils within the BM. These results support the idea that BC cells crosstalk with the bone and remodel the immune microenvironment to favor tumor cell colonization. We have previously showed that caloric restriction (CR) induces T cell migration to the BM and remodels the bone microenvironment, favoring T cell function and a protective environment. Of note, CR-mediated targeting of the bone microenvironment restores the immune homeostatic pool and contributes to an anti-tumor bone immunity. Thus, nutritional interventions my represent potential therapeutic strategies to target bone metastasis.

This work was supported in part by intramural funds of NIAID, NIH. A.T. is supported by the EMBO Postdoctoral Fellowship Programme (ALTF 1014-2021).
Mass cytometry analysis of mouse nonclassical monocyte heterogeneity in breast tumors
Authors: Valentin-Guillama Gabriel A , Alkan Fulya , Alkan Hilmi , Alimadadi Ahmad , Korkaya Hasan , Hedrick Catherine C
Published: August 2023

Monocytes have emerged as important regulators of cancer progression. Our previous studies demonstrated the importance of nonclassical Ly6Clo monocytes in regulating cancer metastasis in mouse models. Here, we aimed to study monocyte heterogeneity in two different breast cancer tumors in mice using either 4T1 or EMT6 cells. 4T1 tumors exhibit an increased ability to metastasize in the lung in a BALB/c mouse breast cancer model compared to the less aggressive EMT6 tumor model. We evaluated 35 surface markers of tumor-infiltrated monocytes and dendritic cells in tumors isolated from each model using CyTOF mass cytometry. Analysis of monocytes and dendritic cells in the tumors using the Seurat package and the FlowSom algorithm identified 12 cell subsets. Significant differences were discovered between the two tumor types in frequencies of four of 12 identified subsets. However, only one of these subsets, a nonclassical monocyte subset, was higher in the less metastatic EMT6 tumors. This subset was 5-fold higher in EMT6 tumors versus more aggressive 4T1 tumors. This Ly6Clo nonclassical monocyte subset showed high expression of CD169 and CX3CR1, CD86 and CD80. These differentially expressed markers suggest a functional importance in this subset in migration to the tumor and antigen presentation, both of which are anti-tumoral functions. Ongoing studies are aimed towards understanding the role of this non-classical monocyte population in regulating breast cancer metastasis.

This work was supported by NIH R01 CA202987, and P01 HL136275
Blood monocyte-derived CD169+ macrophages contribute to antitumor immunity against glioblastoma
Authors: Kim Hyun-Jin , Park Jang Hyun , Kim Hyeon Cheol , Kim Chae Won , Kang In , Lee Heung Kyu
Published: August 2023

Infiltrating tumor-associated macrophages (TAM) are known to impede immunotherapy against glioblastoma (GBM), however, TAMs are heterogeneous, and there are no clear markers to distinguish immunosuppressive and potentially immune-activating populations. Here we identify a subset of CD169+ macrophages promoting an anti-tumoral microenvironment in GBM. Using single-cell transcriptome analysis, we find that CD169+ macrophages in human and mouse gliomas produce pro-inflammatory chemokines, leading to the accumulation of T cells and NK cells. CD169 expression on macrophages facilitates phagocytosis of apoptotic glioma cells and hence tumor-specific T cell responses. Depletion of CD169+ macrophages leads to functionally impaired antitumor lymphocytes and poorer survival of glioma-bearing mice. We show that NK-cell-derived IFN-γ is critical for the accumulation of blood monocyte-derived CD169+ macrophages in gliomas. Our work thus identifies a well-distinguished TAM subset promoting antitumor immunity against GBM, and identifies key factors that might shift the balance from immunosuppressive to anti-tumor TAM.

This work was supported by Samsung Science and Technology Foundation (SSTF-BA1902-05), Republic of Korea. This work was also supported by the National Research Foundation of Korea grant (NRF-2021M3A9H3015688).
1,25-Dihydroxy Vitamin D Enhances the Differentiation of Intestinal Stem Cells Ex Vivo and In Vivo
Authors: Shaikh Nisar Ali F N U , Liu Chenfan , Yin Yue , Zhang Xiao-Bing , Baylink David J , Galea Dan , Das Bhaskar , Tang Xiaolei
Published: August 2023

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestines. Current therapies aim at suppressing inflammation to prevent further damage. However, healing already damaged intestinal epithelia is still an unmet medical need. In this regard, epithelial healing is achieved via intestinal stem cells (ISCs) that are located in the intestinal crypts. Considering the literature reports that the active vitamin D (1,25[OH]2D) is essential for the maintenance of Lgr5+ ISCs, the active proliferating ISCs, we recently proposed to deliver locally high 1,25(OH)2D concentrations to the proximity of intestinal crypts to promote intestinal stem cell functions. This local delivery was devised to avoid potential side effects associated with systemically high 1,25(OH)2D concentrations. Using 5-Bromo-20-deoxyuridine (BrdU) to label the actively proliferating ISCs, our previous data indicated that the locally delivered 1,25(OH)2D enhanced the migration and differentiation of Lgr5+ ISCs. However, although sparse in the crypts, other proliferating cells may be labeled with BrdU. This current study used a unique mouse strain to precisely trace Lgr5+ ISCs and consolidated our previous finding that the locally delivered 1,25(OH)2D augmented ISC differentiation. Besides, we generated pure Lgr5+ ISC lines that could be induced to differentiate on demand. Our data showed that, in vitro, 1,25(OH)2D enhanced the differentiation of Lgr5+ ISCs. Moreover, by knocking down the vitamin D receptor (VDR), we demonstrated that the 1,25(OH)2D functions in ISCs are mediated through VDR. Accordingly, our findings suggest that locally high 1,25(OH)2D concentrations are a promising strategy for intestinal epithelial healing.

This study was partially supported by the National Institute of Health (NIH, USA) grant (1R21AI142170); the US Army Medical Research and Materiel Command grant (W81XWH-15-0240); and the GI Foundation of Loma Linda University Innovation grant (RIG-GI 681207-2967).
A computational pipeline for drug discovery in immunometabolism against autoimmune diseases
Authors: Puniya Bhanwar Lal , Bessell Brandt , Zhao Zhongyuan , Loecker Josh , Aghamiri Sara Sadat , Mohanty Sabyasachi , Amin Rada , Helikar Tomas
Published: August 2023

We developed a computational pipeline to study immunometabolism for drug discovery in immune mediated diseases. Our pipeline integrates RNA-seq and proteomics data to build genome scale metabolic models and integrate these models with external drug databases and patient data to perform metabolic flux analyses for identifying drug targets and repurposing. We applied our pipeline to explore the metabolism of B cells in the context of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We constructed metabolic models of B cells, performed enzymatic inhibition of targets of existing drugs and compounds, and integrated the model’s results with differentially expressed genes in RA and SLE patients. Using a computational scoring method, we ranked the inhibited targets based on their ability to stimulate the reactions of differentially expressed genes in diseases to have metabolic flux in opposite directions of their up and down-regulation. As a result, we identified 25 B cell drug targets against RA and 23 B cell drug targets against SLE. We validated these targets by comparing them against the current treatment options for these diseases. For RA, five of the identified targets are genes that are already targeted by existing drugs used to treat RA. Our top-ranked targets for RA are adenine phosphoribosyltransferase, galactosidase beta 1, and aldehyde dehydrogenase 5 family member A1. For SLE, the top-ranked new targets are geranylgeranyl diphosphate synthase 1, glutathione-disulfide reductase, and galactosidase beta 1. Our pipeline provides a streamlined approach for identifying drug targets and predicting repurposable drugs and can facilitate the investigation of novel indications for diseases to improve global health.

Supported by Layman Seed grant from University of Nebraska Foundation, and NIH grant R35GM119770.
In vitro assay development to assess successful removal of T and B cell epitopes through Seismic’s IMPACT platform
Authors: Newton Andita P , Pellerin Alex , Anderson Jordan , Rollins Nathan , Hollfelder Melissa , Peckner Ryan , Higginson-Scott Nathan , Otipoby Kevin , Mascanfroni Ivan
Published: August 2023

Proteases derived from human pathogens can specifically cleave IgG into F(ab’)2 and Fc fragments. IdeS, an IgG cleaving enzyme derived from Streptococcus pyogenes, has shown clinical proof of concept, and is approved for use before kidney transplantation. Due to the immunogenic nature of these proteases, dosing is limited by high prevalence of pre-existing antibodies and the induction of anti-drug antibodies after dosing. Therefore, to mitigate the impact of the immune system on our enzyme, we identify and remove putative T and B cell epitopes using Seismic’s IMPACT platform leveraging machine learning.

To experimentally validate in silico predictions, we developed two complementary in vitro assays using intravenous immunoglobulin (IVIg) to assess removal of B cell epitopes. First, we assess how mutating the wild type (WT) protease sequence impacts pre-existing antibodies in a competitive binding assay followed by evaluating global binding of pre-existing antibodies to our protease. Additionally, we developed an in vitro PBMC proliferation assay to determine the success of T cell epitope removal. Using flow cytometry, we quantitively assess CD4 proliferation via bromodeoxyuridine (BrdU) incorporation.

We demonstrate examples of molecules designed through our IMPACT platform to reduce both T and B cell epitopes. The B cell epitope assays show a decrease in competition as compared to the WT protease while achieving a reduction in IVIg binding. These data suggest that these in vitro assays can be used to screen for successful invisibilization of molecules of interest.
PPARd restricts intratumoral Treg function through negatively regulating the CIITA-MHCII axis
Authors: Yang Qiyuan , Liang Yuqiong , Zheng Ye
Published: August 2023

Regulatory T cells (Treg) have been associated with poor clinical prognosis of multiple types of cancers due to their suppression of anti-tumor immunity. Although Treg depletion can lead to the rejuvenation of the patient’s immune response to tumors, systemic Treg depletion induces autoimmune diseases such as type-1 diabetes and inflammatory bowel disease. Therefore, understanding how Treg cells adapt to the tumor microenvironment (TME) and how they suppress anti-tumor immunity is critical for the development of novel Treg-based cancer treatment. Peroxisome proliferator activated receptor δ (PPARd) is a member of the nuclear hormone receptor family, and plays a crucial regulatory role in fatty acids metabolism. Here, we uncover that ablation of PPARd in Treg leads to accelerated tumor growth with a more immuno-suppressive TME. Mechanistically, the loss of PPARd in Treg leads to upregulation of a set of genes involved in the antigen presentation pathway, including CIITA and MHCII. Deleting CIITA in PPARd deficient Tregs boosts anti-tumor immunity and inhibits tumor growth, indicating CIITA is a key regulator downstream of PPARd in Tregs. Moreover, overexpressing CIITA in Treg enhances its suppressive function, while knocking out MHCII reduces Treg suppressive function. Our study demonstrated that PPARd restrains tumor Treg function by downregulating the CIITA-MHCII pathway, and identified a previously unrecognized MHCII+ tumor Treg subset as a potent suppressor of anti-tumor immunity. Targeting this Treg subset could open a new avenue for boosting anti-tumor immunity while mitigating autoimmunity risk.

Q.Y. was supported by a NOMIS fellowship.

Y.Z. was supported by the NOMIS Foundation and the National Institutes of Health (R01-AI107027, R01-AI1511123, R21-AI154919, and P30-CA014195).
CRISPR-Cas9 screen identifies potential novel regulators of CD8+ T cell dysfunction
Authors: Holcomb Erin A , Jiang Long , Tate Akshay , Pearson Ashley N , Wang Zhuwen , Jungles Kassidy Mikala , James Jadyn , Huber Amanda , Green Michael
Published: August 2023

CD8+ T cells directly target and kill cancer cells and are required for effective antitumor immunity and response to immunotherapy. However, CD8+ T cells may become dysfunctional and exhausted when exposed to chronic antigen stimulation during cancer. Dysfunction is characterized by increased expression of inhibitory receptors as well as impaired effector function, yet the genetic mechanisms controlling dysfunction remain poorly understood. To address this gap, we have developed an ex vivo restimulation model that recapitulates features of CD8+ T cell dysfunction seen within the tumor microenvironment. By coupling this system with genome-wide CRISPR-Cas9 loss of function screening, we have identified genetic factors which may play a novel role in the progression of CD8+ T cells to a dysfunctional state. Deletion of select factors restored CD8+ T cell survival and effector function during chronic restimulation conditions in vitro, including tumor cell killing capability. Future studies will focus on further characterizing the phenotypic and functional roles of these regulators, as well as translating this model to primary human CD8+ T cells. In summary, we have uncovered novel potential regulators of CD8+ T cell dysfunction and targeting these genes may improve antitumor immunity and immunotherapy efficacy.
Molecular circuitry of T-cell Development and Leukemogenesis
Authors: Tousinas Georgios , Tracy Melissa , Arnovitz Stephen , Friedman David J , Emmanuel Akinola Olumide , Khazaie Khashayarsha , Gounari Fotini
Published: August 2023

T-cell Acute lymphoblastic leukemia (T-ALL) is an aggressive disease with only a 10% 5-year survival rate for patients over 60, due to genomic instability. We have shown that β-catenin and TCF-1 cooperate in DP thymocytes to promote genomic instability leading to lymphomagenesis. We have also established that TCF-1 and HEB cooperate in the DP stage. Now, we aim to determine the molecular underpinnings of T-ALL and thymic development, by elucidating the interplay of all three factors, β-catenin, TCF-1, and HEB.

Aberrant activation of β-catenin promotes the development of Lymphoma/Leukemia equivalent to the human disease T-ALL and blocks thymocyte development in the early DP stage. Ablation of TCF-1 in thymocytes with activated β-catenin rescues from lymphomagenesis, but the developmental block persists. Antithetically, developmental progression is achieved only when HEB is conditionally knocked out. In an effort to explain this phenomenon, we found that genes associated with the transition of DP thymocytes from DPsmall to CD69+ are downregulated by activating β-catenin (e.g. Runx-3, Ccr-7, TOX-2, etc.) and revert upon deletion of HEB.

This is associated with changes in the binding of TCF-1 to DNA. Genomic sites that acquire TCF-1 binding upon β-catenin activation forfeit this binding when HEB is deleted, while at the same time novel TCF-1 binding sites emerge. These new binding sites are significantly more enriched for the conserved TCF-1 motif than TCF-1-bound sites in WT thymocytes. This observation promotes the notion that HEB guides TCF-1 towards alternative binding sites, thus altering gene expression.

These findings highlight the complex interactive relationship of TCF-1 and HEB in both normal development and disease.

NIH/NIAID R01 AI147652-01A1