bluuebunny/crossref_metadata_2025 huggingface dataset:
Small molecule drug inhibition of PD-1 transcription is as effective as anti-PD-1 biologic blockade in cancer therapy
Authors: Rudd Christopher E , Rothstein David , Taylor Alison
Published: January 2023

Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway(s) that regulate PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoform α and β) is a serine-threonine kinase implicated in cellular processes. We have shown that GSK-3 is a key upstream kinase that regulates PD-1 expression in CD8+ T cells. GSK-3 inactivation increased Tbx21 transcription for enhanced Tbet expression, and its suppression of Pdcd1 transcription in CD8+ cytolytic T-cells (CTLs) (Taylor et al 2016 Immunity 44, 274–86). Here, we show that GSK-3 inhibitor blockade of pcdc1 (PD-1) transcription with a small molecule inhibitor (i.e. SB415286) is as effective as anti-PD-1 and PDL-1 blocking antibodies in the control of pulmonary metastasis of B16 melanoma, intra-dermally injected B16 and EL4 lymphoma solid tumors, and in the ex vivo pre-treatment of T-cells before adoptive transfer into mice carrying tumors. In an analysis of knock out mice, GSK-3α/β−/− mice, which had greatly reduced PD-1 expression on T-cells, showed the same reduction in B16 pulmonary metastasis as Pdcd1−/− mice. Further, the ex vivo treatment of T-cells with SB415286, anti-PD-1, or in combination followed by adoptive transfer, had identical effects in inhibiting EL4 lymphoma growth. In all tumor models, GSK-3 inactivation markedly inhibited Pdcd1 transcription and PD-1 expression on tumor infiltrating T-cells (TILs), while increasing Tbx21 (Tbet) transcription and the presence of CD8+ TILs expressing CD107a+ (LAMP1) and granzyme B (GZMB). Our findings define for the first time that a next generation approach using small molecule inhibition of PD-1 expression is as effective as anti-PD-1/PL1 biologics in cancer therapy.
Tumor infiltrating T cells acquire exhaustion-associated epigenetic programs
Authors: Youngblood Ben A , Ghoniem Hazem , Moustaki Ardiana , Dogra Pranay , Abdelsamed Hossam Aly , Fan Yiping
Published: January 2023

Antigen-specific CD8 T cells play a critical role in controlling chronic infections and cancer, but progressively lose their effector functions during prolonged antigen exposure. Repression of CD8 T cell effector functions, commonly referred to as T cell exhaustion, limits the ability of the immune system to purge the chronic pathogen from the host. It has recently become recognized that CD8 T cell exhaustion programs can be reinforced and heritably maintained. Therefore in order to develop and/or improve current therapeutic approaches that utilize host antigen-specific T cells to treat chronic infections or cancer a major challenge for the field is to identify mechanisms that stabilize T cell exhaustion. Using the LCMV model system of chronic viral infection we determined that Dnmt3a mediated de novo DNA methylation plays a causal role in establishing CD8 T cell exhaustion. We then performed whole-genome methylation profiling of WT and Dnmt3a cKO CD8 T cells from chronically infected animals and identified Dnmt3a-dependent DNA methylation programs in genes, including interferon gamma and Tcf7, that are coupled to the progressive decline in effector function and developmental plasticity of the antigen specific cell. Lastly, we extended these findings to the tumor setting using a syngeneic mouse tumor model. We found that tumor-infiltrating PD-1hi CD8 T cells acquire exhaustion-associated de novo DNA methylation programs. These results have significant implications for therapeutic strategies that utilize reactivation of host pathogen-specific CD8 T cells to control chronic viral infections or cancer and provide a nucleotide-resolution map of epigenetic programs progressively acquired during T cell exhaustion.
Assessing the infection rates and pathogenesis of Borrelia burgdorferi in wild rodents and ticks in Western Maryland
Authors: Kuchinsky Sarah Catherine , Elliott Hannah , Brown Drew , Granger Mallary , Lloyd Nicole , Nyachieo Fred , Weakland Amy , Wygal William , Taylor Rebekah
Published: January 2023

Lyme disease is the most prevalent vector-borne disease in the United States. The causative agent is the spirochete Borrelia burgdorferi. Many rodents are reservoir species that carry B. burgdorferi, through which immature ticks (Ixodes sp.) readily transmit the bacteria. Human cases of Lyme disease have dramatically increased over the past 10 years due to tick range expansion. Incidence has expanded from its New England origin into the Midwest and Mid-Atlantic. We hypothesized that Western Maryland is a prime candidate for B. burgdorferi expansion. To prepare, we sought to (1) determine the incidence of B. burgdorferi infection in wild rodent and tick populations in Western Maryland, (2) identify simple and quick methods for diagnosing B. burgdorferi infection in the field, and (3) gain insight into the pathogenesis of B. burgdorferi in wild rodent populations. DNA was extracted from 67 rodent blood samples and 202 ticks, collected from sites in Western Maryland. Approximately 35.14% of these samples tested positive for B. burgdorferi by PCR. Blood samples were tested for the presence of anti-B. burgdorferi antibody using SNAP 3Dx tests, a rapid ELISA-based veterinary diagnostic. Interestingly, the SNAP 3Dx tests were successful in identifying B. burgdorferi infection in both wild mice and chipmunks, suggesting that the capture antibody used in these canine tests is cross-reactive. Finally, whole B. burgdorferi bacteria were identified on frozen sections of wild mouse spleen by immunofluorescence. Bacteria were found throughout the red pulp and in splenic germinal centers of infected mice. Studies are underway to further characterize the infected cells in these samples.
Reduced inflammation and increased endothelial activation in children with asymptomatic Plasmodium falciparum parasitemia in a low transmission area
Authors: Okafor Elizabeth Chioma , Shabani Estela , Park Gregory S. , Opoka Robert O. , John Chandy C.
Published: January 2023

Few studies investigate asymptomatic parasitemia (AP) and researching the mechanism by which these children remain healthy can provide novel information to understand the pathogenesis of Plasmodium falciparum malaria. We hypothesized children with AP to have higher levels of pro-inflammatory cytokines, angiogenic growth factors, and markers of endothelial activation compared to children without parasitemia. Healthy children were recruited from the community as part of a study on cognitive and neurological deficits in children with central nervous system malaria in Kampala, Uganda. Plasma was prepared from blood samples acquired at enrollment and tested using magnetic bead assay. Nested PCR was performed to detect P. falciparum DNA. Of the 211 community children (CC) samples tested, 73 CC had AP and 138 CC without parasitemia. CC with AP had lower levels of pro-inflammatory cytokines and chemokines IFN-γ (p<0.0001), IL-1β (p=0.0001), IL-8 (p=0.0015), MIP-1α (p=0.01), IL-12p70 (p=0.0002), and RANTES (p=0.0001), and higher levels of TNF-α (p=0.05), MIP-1β (p=0.04), and IP-10 (p=0.01) when compared to CC without parasitemia. CC with AP had lower levels of angiogenic growth factors G-CSF (p<0.0001) and FGF-basic (p<0.0001), and higher levels of markers of endothelial activation ICAM-1 (p=0.0007), VCAM-1 (p<0.0001), and VWF (p=0.0006). In this area of low malaria transmission, children with parasitemia have suppression of these pro-inflammatory cytokines, chemokines and angiogenic factors, but an increase in endothelial activation. Endothelial activation may be an early uniform response to P. falciparum, but symptoms may be controlled by ability to suppress the inflammatory response.
Innate immunity does not contribute to the resistance of mice from different vendors to P. yoelii infection
Authors: Ganusov Vitaly V , Bejnood Aram
Published: January 2023

Nearly 500,000 individuals die annually from Plasmodium infections. Factors contributing to susceptibility to Plasmodium infections remain poorly understood. It was recently observed that C57BL/6 mice from different vendors (NCI, Harlem, Taconic, Jackson) have different susceptibility to Plasmodium yoelii infection: Jackson/Taconic mice generally have lower peak parasitemia in the blood and better survival than NIC/Harlem mice (Villarino et al. PNAS 2016). Bioinformatic analysis suggested that high mortality of susceptible mice could arise due to a higher expression of basigin, a protein required by Plasmodium falciparum to invade red blood cells in humans (Stough et al. Front Microbiol 2017). We applied a series of mathematical models of different levels of complexity to experimental data on P. yoelii dynamics in mice from these 4 vendors. Our analysis suggests that there are no statistically significant differences in the P. yoelii dynamics during the first 10–14 days post infection, and that the estimated rates of parasite replication were independent of the vendor type. In contrast, we found large differences in parameters for Plasmodium-specific adaptive immune response suggesting that resistant mice mounted a faster and a more efficient response. Our results thus suggest that higher expression of basigin is unlikely to explain higher susceptibility of NCI/Harlem mice to P. yoelii infection and that innate immunity controlling parasite replication during the first 2 weeks of infection unlikely to be different between susceptible and resistant mice. In contrast, a faster generation of the adaptive immunity in resistant mice was likely to be responsible for better parasite control.
Evaluation of the pathogenesis of Chlamydia induced reactive arthritis
Authors: Volin Michael V , Ahmed Farah , Zanotti Brian , Manam Srikanth , Murthy Ashlesh K
Published: January 2023

We hypothesized that reactive arthritis can be induced in the knees and ankles of wild-type C57BL/6 mice following infection with Chlamydia muridarum through the involvement of inflammatory mediators. Reactive arthritis is a chronic form of arthritis, which can also result in conjunctivitis and inflammation of the genital, urinary, or gastrointestinal systems. C. muridarum is a gram-negative obligate intracellular pathogen which can infect the epithelium of the cervix, urethra, and rectum. The exact pathological mechanism of reactive arthritis is unknown. It is understood to result from CD4+ and CD8+ T cells, and factors including TNF-alpha and perforin. Five groups of C57BL/6J mice were used including a positive control group, CD4+ T cells knockout mice, TNF-alpha knockout mice, perforin knockout mice and TNFRSF knockout mice. Mice from each group were intra-vaginally infected with 5×104 inclusion forming units of C. muridarum, marked as day 0. The mice were swabbed a few days after infection to confirm C. muridarum. The mice from each group were euthanized on day 7 and day 14, and sera was collected and analyzed for cytokine expression using an antibody array. Ankles collected from C57BL/6J positive control mice were histologically processed and graded to determine pathology. C57BL/6J positive control mice showed increased synovial lining thickening 14 days following infection. Cytokine expression from TNF-alpha knockout mice, CD4+ T cells knockout mice, perforin knockout mice, and TNFRSF knockout mice was generally reduced. The results show that chlamydial induced reactive arthritis can be detected in the ankles of C57BL/6J mice and suggest that TNF-alpha and CD4+ T cells may be involved in this inflammatory process.
Role for skin-associated microbiota in development of endogenous anti-melanocyte immunity in vitiligo
Authors: Vujkovic-Cvijin Ivan , Wei Maria , Restifo Nicholas P , Belkaid Yasmine
Published: January 2023

Despite major advances in immunotherapeutic approaches designed to bolster endogenous immune responses against tumors, metastatic melanoma remains associated with high mortality and rapid progression. Development of novel approaches toward enhancing endogenous immune responses against melanoma constitutes a critical step toward the management of disease. Of note, initiating immune responses against non-mutated melanocyte-specific protein antigens has shown efficacy in augmenting immunotherapies. Vitiligo is an autoimmune disease characterized by immune-mediated destruction of melanocytes, though the etiology of this disease remains poorly understood. Microbes have been recently found to have a profound effect on immune processes of the skin, including those that are associated with vitiligo progression. Identifying skin-resident microbes that initiate or exacerbate anti-melanocyte immunity, as well as the precise pathways that mediate these processes, may provide a novel framework for the development of immunotherapies to augment anti-tumor immune responses and efficacy of existing therapies. Thus, we have recruited a cohort of individuals with vitiligo and profiled their skin microbiomes at various body sites. We have found that vitiligo subjects harbor a skin microbiota that is unique from healthy subjects, and have isolated numerous vitiligo-associated bacteria. Furthermore, we have adapted a mouse model of vitiligo to understand mechanisms of host-microbiome interactions in this setting. Interrogation of this host-microbiome relationship may lead to the identification of novel targets and pathways by which to treat vitiligo and augment melanoma immunotherapies.
Gut dysbiosis contributes to autoimmune pathogenesis in lupus-prone mice
Authors: Choi Seung-Chul , Mohmadzadeh Mansour , Morel Laurence
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease mediated by a combination of genetic and environmental factors. We have used a congenic model of the disease, the B6.Sle1.Sle2.Sle3 (TC) mice, which share 95% of their genome with B6 controls. TC and B6 mice have a different distribution of gut bacteria based on 16S rDNA sequencing. Fecal transfers into germ free (GF) B6 mice showed that microbiota from aged TC mice was sufficient to induce the production of anti-dsDNA IgG. TC microbiota expanded germinal center (GC)-like organization in Peyer’s patches and TFH cells in the mesenteric lymph node. Co-housing TC and B6 mice for 6 months starting at weaning revealed significant interactions between disease status and the microbiota compared with independently housed mice from both strains. Remarkably, the frequency of TFH cells was decreased in co-housed TC mice, corresponding to a drastic reduction of autoantibodies and amelioration of renal disease. Co-housed TC and B6 mice showed a similar fecal metabolome profile, which was significantly different from those of independently housed mice. Studies to characterize the role of specific metabolites related to disease pathogenesis are ongoing. Finally, treatment of TC mice with a combination of antibiotics significantly delayed anti-dsDNA IgG induction and expanded Treg and TFR cells. Overall, our results suggest that the dysregulated gut microbiota in lupus contributes to disease pathogenesis either directly or indirectly, through the production of critical metabolites.
Inherited CD55 Deficiency in Patients with Early Onset Protein-Losing Enteropathy and Thrombosis
Authors: Comrie William A , Ozen Ahmet , Ardy Rico Chandra , Su Helen , Boztug Kaan , Lenardo Michael
Published: January 2023

Studies of monogenic gastrointestinal diseases have revealed molecular pathways crucial for gut homeostasis and enabled the development of targeted therapies. We studied 11 patients with abdominal pain and diarrhea caused by protein-losing enteropathy with primary intestinal lymphangiectasia, hypoproteinemia, malabsorption, bowel inflammation, recurrent infections, and angiopathic thromboembolic disease. DNA sequencing identified autosomal recessive mutations in the gene encoding CD55/Decay accelerating factor, leading to loss of protein expression. CD55 regulatory function including complement deposition, costimulation, and anaphylatoxin responses were assessed by shRNA and CRISPR-mediated gene suppression and rescue of CD55 expression using lentiviruses. In all patients, Patient T lymphocytes and CD55-deficient cell lines displayed uncontrolled complement activation causing C3d deposition and generation of C5a. Genetic reconstitution of CD55 or treatment with a complement-inhibitory therapeutic antibody normalized complement activation. Patient T lymphocytes produced increased tumor necrosis factor, which was associated with a decreased ratio of the anti-coagulatory protein thrombomodulin to the pro-coagulatory protein tissue factor on endothelial cells. Additionally, CD55 costimulation by CD97 and contingent production of interleukin-10 were defective in patient T lymphocytes. Altogether, CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and PLE (CHAPLE) disease is caused by abnormal complement activation due to autosomal recessive loss-of-function mutations in CD55, and may be effectively treated with complement-inhibitory therapeutics.
Dominant-activating Gαi2 mutations cause human immunodeficiency and autoimmunity by causing defective leukocyte migration and altered T cell activation
Authors: Ham Hyoungjun , Lamborn Ian , Zhang Yu , Berchiche Yamina , Koval Alexey , Jing Huie , Mandl Judith N. , Happel Corinne , murdock Heardley M , Masutani Evan , McElwee Joshua J , Matthews Helen F , Druey Kirk M , Mustillo Peter J , Germain Ronald N , Sakmar Thomas , Katanaev Vladimir , Su Helen
Published: January 2023

Heterotrimeric Ga subunits are the essential molecular switches that activate intracellular signaling in response to G-protein coupled receptor (GPCR) ligation. Gai2, one of 16 human Ga subunits serving nearly 800 GPCRs, plays critical roles in leukocyte biology; however, how Gai2 modulates human immunity in vivo is unknown. We found a dominant missense mutation in GNAI2, the gene encoding the heterotrimeric G-protein Gai2, in a patient with life-threatening, multisystem autoimmunity and susceptibility to mucocutaneous bacteria and virus infections. The mutant proteins impaired immunity by uncoupling chemokine receptor signaling and thereby interfering with leukocyte chemotaxis in vitro and to sites of inflammation in vivo. They also augmented T cell effector responses and diminished the requirement of co-stimulation for T cell activation by constitutively signaling downstream co-stimulatory pathways. These results identify essential roles of Gai2 in maintaining immune-competence and self-tolerance in humans and highlight Gai2-responsive pathways as potential candidates for new therapeutics for autoimmune diseases.
B cell-Intrinsic Defects of Wiskott-Aldrich Syndrome Protein-Deficiency in generating humoral memory responses
Authors: Dai Rongxin , Li Wenyan , Zhao Hongyi , Zhao Qin , Shu Zhou , Sohn Hae-Won , Pierce Susan K. , Zhao Xiaodong , Song Wenxia
Published: January 2023

Patients with Wiskott-Aldrich syndrome protein (WASP)-deficiency exhibit severe immunodeficiency, including a lack of long-lasting humoral protection. Our published data reveal that T follicular helper cells in both WAS patients and WASP-deficient mice are impaired. This study demonstrates a B cell-intrinsic role of WASP in generating antigen-specific memory B cells and humoral recall responses. While the serum IgG levels in patients with WAS are equivalent to those in age-matched healthy controls, the number and percentage of isotype-switched CD27+ memory B cells and the serum level of tetanus toxin-specific IgG post immunization in the patients are significantly lower than those in controls. Furthermore, the peripheral blood of the patients exhibits a substantial increase in atypical memory B cells that resemble B cells expanded in persistent viral and parasite infections. In immunized WASP knockout mice, the percentages of antigen-specific germinal center, isotype-switched memory B cells, and plasmablasts in the spleen all are decreased, while the percentages of total germinal center B cells and plasmablasts are unchanged, compared to wild-type mice. In bone marrow chimera mice, where only B cells lack WASP expression, WASP-deficient B cells show reductions in the antigen-specific germinal center and memory subsets similar to those seen in WASP knockout mice. However, the percentages of total germinal center and isotype-switched B cells in the WASP knockout chimera mice are equivalent to or higher than those in the wild-type chimera. These results indicate that WASP-deficient B cells play a critical and cell-intrinsic role in developing the humoral immune deficiency in WAS.
Immunodeficiency, centromeric instability and facial dysmorphism Syndrome in a Saudi boy with an unusual presentation diagnosed by whole exome sequencing
Authors: Alonazi Noufa Afri
Published: January 2023

Immunodeficiency, centromeric instability and facial dysmorphism (ICF syndrome) is a rare autosomal recessive disorder. It is characterized by variable reduction in serum immunoglobulin, sometimes combined with defective cellular immunity.

We present 18 month old boy, a product of emergency C-section due to fetal distress at term with good APGAR score and low birth weight. He presented at the age of one year with history of fever, vomiting and abdominal distention complicated by colonic perforation required Laparotomy and Ileostomy. He was born to a consanguineous healthy parent (first degree cousins). Clinical examination showed failing to thrive infant dysmorphic, with low-set ears, hypertelorism, micrognathia and flat nasal bridge. No lymphadenopathy or hepatosplenomegaly. Normal muscle tone and reflexes with delayed gross motor activities. Investigations showed normal full blood count and differential, with no positive cultures, normal IgG with low IgM and IgA. Lymphocyte subset analyses and antibodies titer to Pneumococcal and Tetanus were normal.

Chromosomal analysis revealed 46 XY normal male karyotype. However, pericentric breaks of chromosome 1 and 16 are observed in the majority of metaphasis, which is consistent with Immunodeficiency, centromeric instability and facial anomalies syndrome. Whole exome sequencing revealed a homozygous c2506G>A mutation in DNMT3B gene. This mutation was previously reported in only one of the three patients reported from Saudi Arabia.

In conclusion, this patient had an unusual presentation for the ICF syndrome with colonic perforation which was previously not reported. In communities with high incidence of consanguinity, the possibility of ICF should be kept in mind is such cases.
The role of miR17-92 in regulating thymus fate during development
Authors: Mendoza Maria Gracia Garcia , Bain Virginia , Reeh Kaitlin A. , Manley Nancy , Martin James F. , Richie Ellen R
Published: January 2023

The thymus develops bilaterally from third pharyngeal pouch (3rd pp) endoderm, which is initially patterned into separate thymus and parathyroid specific domains. The transcription factors FOXN1 and GCM2 are expressed in 3rd pp thymus-fated or parathyroid-fated domains, respectively. They are required for organ development, but do not specify organ fate. We are investigating the molecular pathways required to generate a functional thymus from 3rd pp endodermal progenitors. We recently reported that Tbx1, which is required for parathyroid development, negatively regulates thymus development by suppressing Foxn1 expression. We are currently using gain-of-function and loss-of-function genetic models to test the hypothesis that Tbx1 expression in 3rd pp endoderm is regulated by microRNA (miR) 17-92. We find that TBX1 expression is expanded and FOXN1 expression is reduced in the 3rd pp endoderm of miR17-92nullembryos and that thymus organogenesis is severely impaired. We are currently targeting deletion of floxed miR17-92 to the endoderm using Sox17Cre to determine if the null phenotype is endoderm intrinsic. Similarly, Sox17Cre is being used to activate an R26miR17-92OE allele to determine the consequences of overexpressing miR17-92 in the 3rd pp endoderm. These studies will clarify the role of miR17-92 in regulating Tbx1 expression and provide insight into molecular mechanisms that regulate 3rd pp patterning and thymus organogenesis.
Deletion of ABCB7 arrests lymphocyte development
Authors: Lehrke Michael Jonathan , Wolf Natalie , Chung Ji Young , Arocha Sinibaldo Romero , Shapiro Michael , Shapiro Virginia Smith
Published: January 2023

Patients with iron overload disorders often develop lymphocyte deficiencies, indicating iron homeostasis is critical for lymphopoiesis. Loss of ABCB7, a mitochondrial iron transporter, leads to mitochondrial iron accumulation and rapid hematopoietic failure. We found that ABCB7 is required for lymphopoiesis, as CD2-icre ABCB7 cKO mice have a severe block in both T and B cell development. The block in T cell development occurs at the DN3 stage. This DN3 block is not due to aberrant β-selection, proliferation, or viability. The block in B cell development occurs at the Fr. C to Fr. C′ transition. Pre-BCR expression was reduced in developing B cells and they exhibited reduced proliferation compared to WT B cells. Because loss of ABCB7 causes mitochondrial iron accumulation, which is toxic to cells, we hypothesized that lymphoid cells increase heme synthesis in order to detoxify the accumulating iron. We found that HO-1, a surrogate marker for intracellular heme levels, was elevated in these lymphocytes. Currently, we are examining the mechanism responsible for the block in lymphocyte development upon loss of ABCB7.
Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells
Authors: Campfield Brian Thomas , Eddens Taylor John , Henkel Matthew , Majewski Martin , Horne William , Chaly Yury , Gaffen Sarah L , Hirsch Raphael , Kolls Jay K
Published: January 2023

Follistatin-like protein 1 (FSTL-1) possesses several newly identified roles in mammalian biology, including autoimmunity and IL-17 driven inflammation, though the mechanism underlying FSTL-1 influence on IL-17 mediated cytokine production is unknown. To examine the role of FSTL-1 in IL-17 signaling, we utilized parallel in vitro bone marrow stromal cell models of FSTL-1 attenuation. Microarray identified FSTL-1 regulated genes that could influence IL-17 dependent production of IL-6 and G-CSF, wherein we discovered that FSTL-1 modulates Il17rc gene expression. Specifically, FSTL-1 was necessary for Il17rc gene transcription, IL-17RC surface protein expression and IL-17-dependent cytokine production. In FSTL-1 KO BMSCs, ectopic Il17rc expression rescued IL-17 stimulated cytokine, and ectopic Fstl1 expression rescued Il17rc expression as well as IL-17 stimulated cytokines. Mechanisms influencing FSTL-1 dependent Il17rc transcript abundance were examined using Actinomycin D and nascent mRNA labeling approaches and identified that FSTL-1 regulates Il17rc gene transcription. Taken together, this work identifies a mechanism by which FSTL-1 influences IL-17 driven inflammation and reveals a novel function for FSTL-1 as a modulator of gene expression. Thus, enhanced understanding of the interplay between FSTL-1 and IL-17 mediated inflammation may provide insight into potential therapeutic targets of IL-17 mediated diseases.
Regulatory effects of immune system thyroid stimulating hormone β-subunit splice variant (TSHβv) on thyroid hormone secretion in mice.
Authors: Klein John R.
Published: January 2023

It has been known for over three decades that thyroid stimulating hormone (TSH) is made by cells of the immune system, yet little is known about the biological significance of that. Studies in my laboratory identified a novel splice variant of the TSH β-subunit (TSHβv) that is produced by bone marrow (BM) hematopoietic cells and peripheral leukocytes in humans and mice. As shown here, recombinant mouse TSHβ (rTSHβ) binds in a dose-dependent manner to the TSH receptor (TSHR) on the mouse AM macrophage cell line, and circulating T4 levels are suppressed 38% in mice 24 hrs after injection of rTSHβv. Moreover, TSHβ-producing leukocytes traffic to the thyroid during systemic L. monocytogenes infection. TSHβv is suppressed in mouse macrophages following transfection with short-hairpin RNA TSHβv (shRNA-TSHβv) expression plasmids, and is suppressed in BM cells transduced with shRNA-TSHβv lentivirus pseudoparticles. These findings collectively suggest that binding of immune system TSHβv to the TSHR on thyroid follicular cells may serve to modulate thyroid hormone output, thus providing a novel mechanism for regulating host metabolism during times of immune stress. Experiments are underway in mice to understand the in vivo involvement of immune system TSHβv in healthy conditions, and during chronic inflammation such as in autoimmune thyroiditis.
VLA4 expression by B cells enables B cell-restricted antigen presentation to support CD4 T cell driven central nervous system autoimmunity
Authors: Parker Harp Chelsea R. , Archambault Angela S. , Sim Julia , Carrero Javier A. , Russell John , Wu Gregory F.
Published: January 2023

Clonal expansion along with immunoglobulin (Ig) class-switching and secretion result from B cell antigen presentation to cognate CD4 T cells. The presence of oligoclonal Ig in the cerebrospinal fluid of most patients with multiple sclerosis raises the important question of where cognate B:T cell interactions occurs during disease. Previously we showed that increasing the frequency of MOG-specific B cells is sufficient to drive EAE in mice expressing MHCII solely by B cells. We have now established a system in which EAE reliably occurs upon expression of MHCII using Tamoxifen-induced temporal regulation in MOG-specific B cells. While B cells express MHCII in the central nervous system (CNS) in these models, whether or not B cells acquire and present antigen in the CNS compartment is unknown. Large clusters of B cells rapidly formed in the subarachnoid space of the spinal cord during EAE in our model, suggesting homing to the CNS is important for antigen presentation. Genetic regulation of B cell MOG-specificity and expression of MHCII and Very Late Antigen-4 (VLA-4) was used to determine the dependence of passive EAE susceptibility on B cell access to the CNS. Mice in which VLA-4 expression was abrogated simultaneously with MHCII expression by MOG-specific B cells were subjected to passive EAE. Disease exclusively mediated by B cell antigen presentation was dependent on VLA-4 expression by B cells. MOG-specific CD4 T cell hybridomas did not detect endogenous MOG presented by B cells harvested from the periphery of mice prior to the onset of disease. Taken together, our findings indicate that B cells require access to CNS myelin targets in order to propagate antigen-specific CD4 T cell-dependent neuro-inflammation.
Adjuvant-induced rheumatoid arthritis in Lewis rats alters dopamine utilization and cytokine concentration in five brain regions
Authors: Yang Han , Stinson Amelia J. , Carcia Carlos A.
Published: January 2023

Word Rheumatoid arthritis (RA) is a chronic autoimmune disease which can cause inflammation in joints, bone damage, as well as disability. A reported symptom of RA includes cognitive dysfunction. This study aimed to measure brain dopamine utilization and inflammatory markers in an adjuvant-induced Lewis Rat model of RA. Arthritic intensity was scored based on physical examination of the four paws. Twenty-eight days after the adjuvant injection, control and RA group were sacrificed and the frontal lobe of the cerebral cortex (Cx), hippocampus (Hip), hypothalamus (Hypo), amygdala (Amg), and cerebellum (Cbl) dissected using the stereotaxic atlas of Paximos and Watson as a guide. High performance liquid chromatography (HPLC) was used to analyze the concentration of dopamine, DOPAC, and homovanillic acid (HVA). Pro-inflammatory cytokines IL-1β, IL-6, and IL-23, and the anti-inflammatory IL-10 were measured by enzyme-linked immunosorbent assays (ELISA) in tissue homogenates. The results indicate RA significantly (p<0.05) decreased the dopamine content in Cbl, Amg, Hip and Cx; DOPAC significantly decreased in Amg and Cbl; HVA significantly decreased in Cbl. RA increased the concentration of pro-inflammatory cytokines and the anti-inflammatory cytokine IL-10 in the Cbl. IL-1β increased in Hyp, and Amg with a concomitant decrease in IL-10. IL-6 and IL-23 increased in Cx, Hip, Hypo, and Amg with a concomitant decrease in IL-10. This data provides evidence that adjuvant-induced RA alters the inflammation status and dopamine utilization of these brain regions. The results are useful in the development of novel drug therapies for the millions of RA patients at risk of developing mood changes, memory loss, and other cognitive deficits.
Autoimmune disease severity is suppressed by type I interferon signaling in radioresistant cells
Authors: Riding Rebecca L. , Richmond Jillian M. , Fukuda Keitaro , Harris John E.
Published: January 2023

Type I interferons broadly modulate immune responses and can drive both anti-inflammatory and immunostimulatory programs. Vitiligo is an autoimmune disease where melanocytes are destroyed by autoreactive CD8+ T cells. We have previously shown in mice and human patient tissue that IFNγ and the induced downstream chemokines, CXCL9 and CXCL10 drive disease pathogenesis. Since type I interferon signaling can drive expression of multiple overlapping IFNγ target genes, we wanted to determine the role of type I interferon in disease progression. Interestingly, we found that type I interferon-deficient mice developed accelerated and severely enhanced vitiligo compared to WT control mice. These results suggest that in contrast to IFNγ, IFNAR signaling acts to suppress disease. We subsequently found that IFNAR signaling is not required on autoreactive CD8+ T cells or other radiosensitive immune cells but rather it is required on radioresistant cells to suppress vitiligo. In radioresistant IFNAR-deficient mice, autoreactive CD8+ T cells have significantly reduced levels of PD-1 expression which correlates to increased production of the effector cytokines TNFα and IFNγ. Generation of conditional knockout mice showed that epidermal radioresistant Langerhans cells and keratinocytes do not require IFNAR to suppress disease and we are currently investigating the role of IFNAR signaling on epidermal melanocytes. These studies will not only enhance our understanding of the role of IFNAR in promoting tissue specific tolerance, but will have broad insights into the role of IFNAR in other organ–specific autoimmune diseases, such as multiple sclerosis, in which IFNβ is an available treatment.
Metabolic Functions of the Murine Lupus Susceptibility Gene Pbx1
Authors: Roach Tracoyia A. , Titov Anton , Soh Sujung , Robusto Brian , Morel Laurence
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an autoimmune disease that affects many organ systems. Poorly characterized genetic factors contribute to SLE, in part through the production of autoreactive or inflammatory T cells. Pre-B cell leukemia homeobox 1 (Pbx1) is a transcription factor whose Pbx1-d dominant negative splice isoform is overexpressed in CD4T cells of lupus patients as well as in the NZM2410 lupus-prone mouse as compared to the normal Pbx1-b isoform. Based on gene expression studies comparing murine CD4 T cells overexpressing Pbx1-d to controls, we hypothesize that Pbx1-d enhances cellular metabolism in T cells through the HIF1α and mTORc1 pathways. CD4 T cells expressing Pbx1-d present a higher cellular metabolism and show a higher mTORc1 activation than normal control T cells. Using mesenchymal stem cells, we showed that transfection of Pbx1-d was sufficient to increase glycolysis, a pathway linked to T cell activation. We found that Ddit4, an mTORc1 inhibitor, shows a lower expression in the Pbx1-d-expressing CD4 T cells than in normal T cells. We also discovered that Pbx1-d preferentially binds to the promoter of Ddit4, as well as Egln1 and Egln3, two HIF1a inhibitors. These results suggest that a mechanism by which the Pbx1-d allele contributes to lupus pathogenesis is to enhance CD4 T cell metabolism. Future work will define how Pbx1 controls the immune system and how the function of this transcription factor is linked to cellular metabolism.
Identification of Btnl2 as a Key Gene in Suppressing Autoimmunity: New Players in SLE pathogenesis
Authors: Khan Shaheen , Hwang Sun-Hee , Belobrajdic Katherine , Viswanathan Kasthuribai , Wakeland Benjamin , Casco Jose , Wakeland Edward K.
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disease with extensive clinical heterogeneity, affecting mainly females. It is initiated with breach in immune tolerance and as a result one of the main characteristics of SLE is the production of autoantibodies to a wide spectrum of self-antigens. Sle1 is a potent susceptibility locus for spontaneous systemic autoimmunity derived from the NZM2410 mouse strain. The NZW-derived suppressive modifier locus, Sles1, specifically prevents the spontaneous loss in tolerance mediated by the B6.Sle1 congenic. Fine mapping combined with phenotypic characterization of series of recombinant mouse strains have mapped Sles1 to an approximately 638 Kb segment on murine chromosome 17. This revealed extensive epistatic gene interactions within the Sles1 region and suggested Btnl2 and H2 genes as the strongest candidates. We generated BAC transgenic mice by introducing BAC harboring B6 allele of Btnl2 alone or Btnl2 and H2 combined allelic region on to Sle1.Sles1 mice to identify the gene driving the suppressive phenotype. Phenotypic characterization of aging BAC transgenic mice is completed and results strongly indicate Btnl2 as the key gene within the 638 Kb region driving the suppression. Additionally we have performed targeted resequencing on B6 and B6.Sles1 mice and have identified functional polymorphisms within the Btnl2 gene between the two mice strains. We are currently generating and analyzing RNA-seq data on our BAC transgenic mice to gain insight in to the mechanisms on how Btnl2 is suppressing autoimmunity. This study confirms Btnl2 as the key driver gene in suppressing loss of tolerance in SLE pathogenesis.
Dietary Tryptophan Modulates Autoimmunity in a Mouse Model of Lupus
Authors: Brown Josephine Michelle , Choi Seung-Chul , Kanda Nathalie , Morel Laurence
Published: January 2023

Both kynurenine and serotonin are synthesized from dietary tryptophan via indolamine-2,3-dioxygenase (IDO) and tryptophan hydroxylase (tph1) respectively. SLE patients have altered serum and PBMC tryptophan metabolites relative to healthy controls such that there is a depletion of both tryptophan and serotonin and an accumulation of kynurenine. Mass spectrometry analyses showed that SLE B6.Sle1.Sle2.Sle3 congenic (TC) mice on a standard diet that were positive for serum autoantibodies have the same serum tryptophan metabolite alterations reported in SLE patients as compared to B6 controls. Additionally, TC mice have an altered fecal tryptophan metabolite profile relative to B6 controls, and antibiotic treatment restores this profile, suggesting a microbial involvement. Based on these findings, we sought to understand how altering dietary tryptophan would impact autoimmunity in lupus-prone TC mice. Congenic mice consuming low (0.1%) or no (0%) tryptophan for 6 months were protected from autoimmunity whereas those consuming supplemented tryptophan (1%) had exacerbated autoimmunity, including anti-dsDNA IgG production that was correlated with serum kynurenine levels. Furthermore, enzyme expression within the tryptophan catabolic pathway was altered in total CD4+ T cells from SLE congenic mice relative to B6 controls. These data show an altered tryptophan metabolism in lupus prone mice associated with the gut microbiome and with changes in enzyme expression within the tryptophan catabolic pathway in immune cells. Further we showed that manipulation of tryptophan metabolites through diet modulates autoimmunity in these lupus-prone mice.
Noc4L dictates Treg activation in vivo
Authors: Zhou Xuyu
Published: January 2023

Regulatory T cells (Treg) are a specialized CD4+ T cell lineage that plays a central role in controlling self-immune tolerance. It is well documented that antigen triggered Tregs activation is crucial for their suppressive function; however, the underlying molecular mechanism is poorly understood.

The ribosome plays a universally conserved role in catalyzing the translation of all mRNAs in every cell across all kingdoms of life. It is therefore not surprising that the ribosome is one of the most complex and elaborately formed “molecular machines” in the cell, whose biogenesis is extraordinarily orchestrated. Although ribosome biogenesis is considered as a conserved housekeeping process, emerging of evidence support a new hypothesis that different cell types might actually have various of the repertoire of ribosome biogenesis factors (RBFs), which are required to generate specialized ribosome and confer the translation of selected mRNAs.

Here, we focused on the conserved ribosome protein Noc4L based on the observations that Noc4L was highly expressed in activated CD4+CD25+ T cells compared with the resting Treg cells in vitro. Treg specific knockout Noc4L mice developed a lethal autoimmune phenotype, resembling Treg-deficient “scurfy” mice. Interestingly, Noc4L defection did not globally affect the overall protein translation in Tregs, but a rather select detriment of the expression of the genes related to Treg activation. Together, these results demonstrated a central role for the ribosome biogenesis factor Noc4L in controlling Tregs activation. The specialized ribosome is a new layer in the regulation of gene expression of Tregs.
Generation of a mouse model of extrathymically-derived regulatory T cells in type 1 diabetes
Authors: Holohan Daniel R. , Dong Shen , Porter Caroline , Rozenblatt-Rosen Orit , Regev Aviv , Van Gool Frederic , Bluestone Jeffrey A.
Published: January 2023

Regulatory T cells (Tregs) are an immunosuppressive population that were initially identified as uniformly and stably expressing the transcription factor Foxp3. Tregs have now been further distinguished based on whether they developed in the thymus (thymically-derived Tregs, or tTregs) or in the periphery (peripherally-derived Tregs, or pTregs). Previous studies have shown that deletion of CNS1, a conserved non-coding DNA response element in the Foxp3 locus, leads to selective impairment of pTreg generation without disrupting tTreg generation in the B6 background. However, the role of pTregs versus tTregs in autoimmunity is unclear. Using CRISPR-Cas9, we removed the CNS1 region in the NOD mouse model of spontaneous type 1 diabetes. Based on in vitro Treg generation assays, removal of the CNS1 region impairs Foxp3 induction in naïve NOD CD4+ T cells. However, it did not lead to statistically significant differences in the percent of Tregs found in various tissues, including the spleen, thymus, gut, lymph nodes, and pancreatic islets of non-diabetic mice aged 8–13 weeks. Single-cell RNA seq comparing a 12-week old NOD mouse to a CNS1 KO NOD mouse also did not reveal strong differences in gene expression in the pancreatic islet, gut, or pancreatic lymph node, although flow cytometry analysis does reveal a small but significant decrease in RORyt+ Tregs and an increase in Helios+ Tregs in the gut of CNS1 KO NOD mice. CNS1 KO NOD mice are also being compared to normal NOD mice for type 1 diabetes onset and incidence. These preliminary data suggest that extrathymically-derived Tregs do not play a large role in this mouse model of type 1 diabetes.
Migration Kinetics and Migratory Morphology of Mycobacterium-Infected CD11c-eYFP Murine Bone Marrow Derived Cells
Authors: Gilpin Trey E.
Published: January 2023

Central nervous system tuberculosis (CNSTB) is the most serious manifestation of extrapulmonary tuberculosis infections that represent 5–10% of all cases. Although migration of mycobacterium infected cells from the lung to the CNS is critical in bacterial dissemination into the CNS, very little is known about the migratory phenotypic changes of mycobacterium infected cells and overall the mechanism of dissemination into the brain is poorly understood. Mycobacterium tuberculosis is an obligate pathogenic bacterial species with it’s only mode of transportation being within innate immune cells. Dendritic cells (DCs) are potent antigen presenting cells that migrate long distances and through interactions with T-cells, initiate the adaptive immune response. Our lab has previously shown DCs are able to leave Mycobacterial granulomatous lesions from the lung with bacteria intact. Here we used live cell imaging of an under-agarose migration assay and cell tracking to define the kinetics of CD11c-eYFP DC migration during Mycobacterial infection. We found that migration of mycobacterium infected cells was impaired towards CCL19 chemokine and infected cells portrayed a migratory morphology distinct from uninfected DCs. With the use of CD11c-eYFP-LifeAct-RFP reporter mice, we characterized actin polymerization dynamics within infected CD11c cells. As actin polymerization plays a central role in cell migration, morphology, and pathogen internalization, to understand this process during mycobacterial infection will lead to novel therapeutic therapies to modulate dissemination into the CNS.
CD8 T cells are recruited to the central nervous system after peripheral infections and adopt a tissue resident memory phenotype
Authors: Urban Stina Lisa , Pewe Lecia L. , Harty John T.
Published: January 2023

The central nervous system (CNS) is classically viewed as an immune privileged site; however recent advances in the field highlight the importance between the interaction of the peripheral immune system and the CNS in maintaining tissue homeostasis. Tissue resident memory (Trm) CD8 T cells have been described in almost every organ and are shown to provide a first line of defense against secondary invading pathogens. Trm cell generation generally involves active infection at the site of tissue residence. Using multicolor flow cytometry and 2-photon microscopy, we describe a population of CD8 T cells isolated from the CNS after peripheral systemic infections. At an effector time point, intravascular (IV) stain negative antigen-specific cells isolated from the CNS express high levels of chemokine receptors CXCR6 and CXCR3, adhesion molecules CD11a and CD49a and the Trm marker CD69. This CD8 population acquires CD103 expression and persists long term. Peripherally-induced CNS CD8 T cells are highly dynamic both inside and outside the blood vessels. Within the blood vessels, CD8 T cells can be seen flowing free, sticking to the walls of the vessels and crawling along the blood vessels with and against the flow of blood. Outside the vessels, CD8 T cells are seen surveying the tissue with multiple mechanisms including moving along the blood vessels, traveling long distances in apparently random fashion or remaining stationary. The purpose of systemically-induced Trm-like CD8 T cell populations remains to be determined; however, it is possible that that these cells are similar to conventionally-derived Trm populations and may provide the first line of defense should an invading pathogen reach the CNS.