bluuebunny/crossref_metadata_2025 huggingface dataset:
Human skin mucosal associated invariant T (MAIT) cells show increased pro-inflammatory cytokine expression following burn trauma
Authors: Labuz Daniel , Fleming Irma D. , Lewis Giavonni , Thompson Callie M. , Leung Daniel T
Published: February 2023

The role of T cells in burn trauma is an understudied research area explored primarily in mice, with sparse data in humans. Patients surviving an initial burn injury are at a high risk of infection, sepsis, and death. Unconventional T-cells such as mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that respond rapidly to pathogens. Circulating MAIT cells are primarily interferon (IFN)-gamma and tumor necrosis factor (TNF) producing when presented with a microbial-derived ligand on MHC class-1 related protein (MR1) or activated by cytokines interleukin (IL)-12 & IL-18. In this study, we obtained discarded burn (n=6) and normal (from discarded autograft, n=4) skin tissue from burn patients and compared unconventional T cell functionality by flow cytometry and single-cell RNA-sequencing (scRNA-seq). We did not find any significant differences between the frequency of total MAIT cells between burn injury or normal skin, 1.51 ± 1.42 and 2.98 ± 4.40, respectively. There is a significant higher level of CD38 expression in MAIT cells from burn injury tissue and a significant higher level of CD69 expression in MAIT cells from normal skin tissue. Skin MAIT cells in a burn injury show significantly higher levels of IFN-g and TNF before and after PMA-ionomycin stimulation compared to normal skin MAIT cells, which mainly produce IL-6 & IL-17A, suggesting that skin MAIT cells of burn patients show a MAIT cell phenotype that is more responsive to infection. We are characterizing these MAIT cell subtypes in further detail with scRNA-seq and anticipate presentation of that data at time of the conference. Our findings provide the means towards an understanding of the role that skin MAIT cells play in skin of patients with burn trauma.
Mouse lung tissue resident memory CD8 T cells exhibit unique metabolic profiles
Authors: Sportiello Michael John
Published: February 2023

Tissue resident memory T cells (TRM) confer protection against a variety of illnesses ranging from infections to cancers. As demonstrated in the skin, transfer of TRM from immune to naïve hosts is sufficient to prevent herpes simplex virus type 1 (HSV) associated pathology. Comparably, the presence of antigen-specific TRM in a mouse model of melanoma can limit tumor growth. In the lungs, TRM offer critical for protection against influenza, even in the absence of specific antibodies. In the skin, the functions of different CD8 T cell subsets have been linked with distinct metabolic pathways. Skin TRM display a unique metabolic profile to other CD8 memory cells subsets. They undergo enhanced fatty acid oxidation and oxidative phosphorylation as compared with circulating memory cells and naïve cells. However, it is unclear if this programming is universal between all TRM subsets and holds true in the lung. We hypothesize that lung TRM with different integrin profiles will utilize unique metabolic programs. Metabolic profiles of lung CD8 T cells were assessed using a variety of functional metabolic assays including glucose analogue uptake, fatty acid uptake, and oxidative phosphorylation as assessed by fluorescent dyes measured by flow cytometry. Nonfunctional assays including neutral lipid storage, mitochondrial mass, and mRNA transcript levels of key metabolic genes were also assessed via flow cytometry using fluorescent dyes, while transcripts were quantified via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Our findings indicate that lung TRM show enhanced fatty acid oxidation, more neutral lipid storage, and less glucose uptake than their non-TRM T cells in our influenza mouse model.
Tumour immune evasion mechanism in non-small cell lung cancer by inducing Blimp-1 in CD4+CD25+Foxp-3+ T regulatory cells
Authors: Finotto Susetta , Tausche Patrick , Hohenberger Katja , Neurath Laura , Trufa Denis I , Geppert Carol I , Rieker Ralf J. , Sirbu Horia , Finotto Susetta
Published: February 2023

Immunotherapy improves the life expectancy of patients with lung cancer by targeting the immune inhibitory T cell surface markers resulting in the activation of anti-tumor T cell survival and suppression of tumor cell growth.

The transcription factor repressor Positive Regulatory Domain Containing Protein 1 (encoded in humans by the Prdm1 gene) also known as B lymphocytes-induced maturation protein-1 (Blimp-1) affects the homeostasis and function of CD4+ and CD8+ T cells as well as T regulatory cells (T regs).

We recently reported that Blimp-1 is induced in the lung tumoral region of patients with lung ADC. To further investigate whether Blimp-1 could be a candidate gene involved in the regulation of the anti-tumor immune-response in lung cancer, we started to investigate peripheral blood mononuclear cells (PBMCs) isolated from healthy control subjects and NSCLC patients for Blimp-1 expression in immunosuppressive CD4+CD25+Foxp3+ T regs. The PBMCs were cultured with and without antiCD3/CD28 antibodies, without and with TGF-beta, to mimic the tumor microenvironment, for 4 days and subsequently analyzed by flow cytometry using antibodies against Blimp-1, Foxp3, CD4 and CD25(IL-2R alpha chain).

Here we found, by trend, induced CD4+Blimp1+CD25+Foxp3+ Tregs in unstimulated PBMCs from patients with lung cancer. Moreover, TGF-beta further induced these Blimp1+CD4+CD25+Foxp3+ Tregs in PBMCs obtained both from healthy controls and from NSCLC patients.

Taken together these data indicate that the tumor microenvironment induces Blimp-1 in immunosuppressive CD4+CD25+Foxp3+ Tregs via TGF-beta.

Thus, current immunotherapy should be combined to TGF-beta inhibitors to target CD4+CD25+Blimp1+Foxp-3+ T regulatory cells.
The Role of Truncated SOS1 in Acute Lymphoblastic Leukemia
Authors: Harris Lynn M Heltemes , Hubbard Grey , LaRue Rebecca , Miller Jeffrey , Knutson Todd , Farrar Michael A.
Published: February 2023

Progenitor B cell acute lymphoblastic leukemia (B-ALL) remains the most common form of childhood cancer and has a poor survival in adult patients. Therefore, efforts to identify further driver mutations in B-ALL are critical. Previously, we identified a truncated form of SOS1 that cooperated with constitutively active STAT5b to induce leukemia. Expression of truncated Sos1 transcript correlated inversely with overall survival. Using a new model of B-ALL characterized by heterozygous expression of Pax5 and Ebf1, we identified increased expression of a truncated form of Sos1 transcript in RNA-Seq analyses. Using western blot analyses, we confirmed the over-expression of smaller SOS1 protein. To identify the truncated Sos1 starting site, we performed 5′ RACE and found a small region of a Riken gene fused to Sos1 at the junction of exon 8 and 9. Further, there is an almost 3 kb truncation of the 3′ untranslated region of Sos1. Next, we used a 10X Genomics approach to identify gene arrangements that showed clear evidence of a linkage between exon 9 of Sos1 and 3′ region of Map4k3. WGS on three independent leukemia samples identified an inversion event between Sos1 and a region downstream of Cdkl4, the location of the RIKEN we identified earlier. These results provide evidence that the truncated Sos1 arises from a micro-inversion event. SOS1 is known to play a critical role in the RAS-ERK signaling pathway and is important in PreBCR signaling. We examined both pERK and pp38. Surprisingly, we found reduced levels of pERK correlated with increased pSTAT5b. Thus, novel chromosomal rearrangements generate a truncated form of SOS1 that plays an important role in transformation of progenitor B cells in B-ALL.
LPS Induces GM-CSF Production by Breast Cancer MDA-MB-231 Cells via Long-Chain Acyl-CoA Synthetase 1
Authors: Alrashed Fatema , Thomas Reeby , Al-Roub Areej , AL-Mulla Fahd , Ahmad Rasheed
Published: February 2023

Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a monomeric glycoprotein that has been implicated in the tumor growth and progression of different types of cancer. GM-CSF is produced by various non-immune cells including MDA-MB-231 in response to various stimuli. However, the role of lipopolysaccharide (LPS) in the regulation of GM-CSF in MDA-MB-231 breast cancer cells so far remains unclear. Herein, we asked whether LPS could induce GM-CSF production in MDA-MB-231 cells, and if so, which signaling pathway was involved. MDA-MB-231 cells were treated with LPS or tumor necrosis factor alpha (TNF-α; positive control), and GM-CSF expression levels were determined by qRT-PCR, ELISA, and confocal microscopy. Phosphorylation of the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-kB) signaling proteins were evaluated by flow cytometry. Our results show that LPS induces GM-CSF expression at both mRNA and protein levels in MDA-MBA-231 cells. Inhibition of acyl-CoA synthetase 1 (ACSL1) activity in the cells with triacsin C significantly reduces the secretion of GM-CSF. Furthermore, the inhibition of ACSL1 activity significantly blocks the LPS-mediated phosphorylation of p38 MAPK, MEK1/2, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and nuclear factor-κB (NF-kB) in the cells. These findings provide the first evidence that LPS induces ACSL1-dependent GM-CSF gene expression in MDA-MB-231 breast cancer cells, which requires the activation of p38 MAPK, MEK1/2, ERK1/2, JNK, and NF-kB.
Unleashing ILC2-dependent anti-tumor immunity to melanoma
Authors: Belz Gabrielle T , Jacquelot Nicolas
Published: February 2023

Group 2 innate lymphoid cells (ILC2) are essential to maintain tissue homeostasis. In cancer, ILC2 can harbor both pro- and anti-tumorigenic functions but the mechanisms underpinning the execution of paradoxical outcomes is not known, nor how it affects clinical outcomes for tumors such as melanoma. We begin to unravel how ILC2 infiltrate human melanoma tumors and execute their anti-tumor potential through a variety of cytokines such granulocyte-macrophage colony-stimulating factor (GM-CSF) to coordinate the recruitment and activation of eosinophils to enhance the anti-tumor response. We identify cytokine-inhibitory receptor circuits that concurrently both promote and limit ILC2 function. Significantly, by understanding these hubs can break the negative impact of ILC2 through specific targetting to limit ILC2 promotion of tumor growth. Together, our results identified ILC2 as a critical immune cell type involved in melanoma immunity and revealed an as yet untapped synergistic approach to harness ILC2 function for anti-tumor immunotherapies.
IL-2 decreased PU.1 and PD-1 expression in NK-cells in NSCLC patients
Authors: Hohenberger Katja , Trufa Denis I , Geppert Carol-Immanuel , Rieker Ralf J , Sirbu Horia , Finotto Susetta
Published: February 2023

The transcription factor PU box binding-1 (PU.1), which is encoded by the SPI1 gene, is required for the development of most myeloid cell lineages as well as B- and T cells. Consequently, an alteration of the SPI1 gene and thus PU.1 can have severe impact on the immune system. However, the role of PU.1 in cancer is not yet fully understood.

In this study, we were looking for a hint on the influence of PU.1 expression on various cell types during non-small cell lung cancer (NSCLC). To start the investigation, we isolated peripheral blood mononuclear cells (PBMCs) from patients with NSCLC as well as from healthy controls. The PBMCs were cultured with different cytokines for 4 days and subsequently analyzed by flow cytometry. Furthermore, RNA was isolated from the PBMCs and qPCR was performed.

The qPCR results showed induced PU.1 mRNA levels in NSCLC patients in relation to GAPDH, compared to control patients. By flow cytometry, we could detect higher PU.1 expression in CD56+ Natural killer (NK) cells from lung cancer patients compared to the healthy donors. Moreover, NK-cells from lung cancer patients, in contrast to healthy controls, expressed the immunosuppressive protein Programmed Death 1 (PD-1). Furthermore, PBMCs from NSCLC patients that were cultured with aCD3/aCD28 antibodies, showed suppression of PU.1 as well as PD-1 in NK-cells, which was even more prominent after addition of IL-2.

These preliminary data suggest, that conditions inducing T cell activation like IL-2 and antiCD3/antiCD28 stimulation, can downregulate both the transcription factor PU.1 and PD-1 in NK-cells in tumor patients. Thus, improvement of current immunotherapy could use additional IL-2, which targets PU-1 as well as PD-1 in NK cells in lung cancer.
Tumor-resident memory CD8+ T cells have a pivotal role in cancer immunity and immunotherapy.
Authors: Corgnac Stéphanie , Mami-Chouaib Fathia
Published: February 2023

CD8+ resident memory T (TRM) cells expressing the CD103 integrin frequently accumulate in human lung tumors and are associated with a favorable prognosis. However, the contribution of this T-cell subset to anti-tumor immunity and to the response to immune checkpoint blockade (ICB) immunotherapy has not been established. Using quantitative multiplex immunofluorescence staining on a cohort of non-small-cell lung cancer (NSCLC) patients treated with PD-1-targeted immunotherapy, we show that an increased density of CD103+CD8+ cells in tumors is associated with greatly improved outcome. CD103+CD8+ cells co-expressed CD49a and CD69, and display a molecular profile characterized by expression of PD-1 and CD39 exhaustion markers, and T-bet, AhR and phosphorylated-STAT-3 transcriptions factors, which confer them a Tc1/Tc17 profile. Moreover, CD103+CD8+ tumor TRM cells, but not CD103−CD8+ (non-TRM) tumor-infiltrating counterparts, demonstrate enhanced proliferation and cytotoxicity toward autologous cancer cells, and display oligoclonal expansion of specific TCRβ clonotypes. Human lung tumors are also infiltrated by CD103+CD4+ T cells likely suggesting a role for this resident population in orchestrating the antitumor T-cell response. Moreover, data obtained in pre-clinical mouse tumor models demonstrate that the absence of CD103 on CD8+ T cells impact the antitumor immune responses. These results explain why CD103+CD8+ tumor-reactive TRM cells are associated with better outcome in ICB-treated patients.
Elucidating the roles of PIK3IP1/TrIP in PI3-kinase regulation, T cell differentiation and T cell function in vivo
Authors: Murter Benjamin M , banerjee hridesh , Szymczak-Workman Andrea , Kane Lawrence P
Published: February 2023

Background:

The cell surface protein TrIP (Transmembrane Inhibitor of PI3K, gene name: Pik3ip1) functions to downregulate PI3K signaling in T cells and can act as a negative regulator of T cell immune responses. This negative immune regulation was recently reported to promote anti-tumor immunity, implicating TrIP as a potential immunotherapy target. Additionally, TrIP was shown to regulate Th1 vs. Th17 cell fate decisions, suggesting a role in Th17-driven autoimmune disease.

Methods:

Using a conditional TrIP knockout mouse model we developed, we have begun to interrogate how TrIP expression regulates the effector function of CD8+ T cells and its impact on tumor immune landscape and overall tumor burden. In parallel, we have assessed the effects of TrIP conditional knockout in T cells on progression of experimental autoimmune encephalomyelitis (EAE) in vivo, and also analyzed the effects on Th17 skewing in vitro.

Results:

We have found that TrIP knockout in CD8+ T cells leads to lower tumor burden compared to WT counterparts. We have also found that the absence of TrIP expression in T cells leads to lower clinical scores and slower disease progression of EAE.

Conclusions/Future Directions: We describe preliminary data showing that the absence of TrIP expression on CD8 T cells promotes anti-tumor immunity and have begun to explore its effects on the tumor microenvironment. Expanding on this work, we are developing a P14-transgenic TCR system for use with gp33-expressing tumor lines to investigate potential differences in T cell trafficking to the tumor. Our preliminary data also demonstrates that TrIP knockout T cells lead to a resistance to EAE, prompting further exploration of the role of TrIP in this disease model and in multiple sclerosis.
Genetically engineered CD4 T cells as a novel immunotherapy against drug resistant Klebsiella pneumoniae infections.
Authors: Hoffmann Joseph P , Iwanaga Naoki , Yang Haoran , Chen Kong , Norton Elizabeth B. , Rangel-Moreno Javier , Kolls Jay K , McCombs Janet E.
Published: February 2023

With mortality rates ranging from 30% to 70%, carbapenem-resistant Klebsiella pneumoniae (Kp) is a significant public health threat. With growing incidence of drug resistance, it is imperative to develop alternative strategies to combat these severe infections. One novel immunotherapy is adoptive T cell transfer therapy using T cells genetically modified to express a T cell receptor (TCR) specific to an antigen of interest. Though successful against cancers and viral infections, this strategy has yet to be utilized for bacterial infections. Our study’s objective was to validate adoptive transfer as an immunotherapy against Kp infections using CD4 T cells from immunized mice. Further, we sought to identify TCRs specific to Kp epitopes for the generation of engineered Kp specific T cells. CD45.1 WT mice were intratracheally vaccinated with outer membrane protein X (OmpX) adjuvanted with heat-labile enterotoxin A1 twice at three weeks apart. Lung and splenic CD4 T cells from immunized mice were transferred into CD45.2 WT mice, followed by a challenge with Kp ST258 C4 strain. Immunized CD4 T cells were sequenced by single cell TCRseq to identify Kp specific TCR sequences. OmpX immunization resulted in a robust population of Kp specific Th17 cells in the lungs. Adoptive transfer of lung, but not splenic T cells from immunized mice significantly reduced bacterial burdens in challenged mice. Additionally, we identified 7 enriched TCR sequences that may be Omp specific. We have confirmed the efficacy of adoptive T cell transfer therapy against Kp infections using T cells from immunized mice. Moving forward we will validate our TCR sequences as Kp specific to genetically engineer both primary and cell line T cells for future therapeutic studies.
The role of monocytes in UVB-mediated acute skin damage in lupus prone mice
Authors: Maz Mitra Padideh , Shi Hong , Wolf-Fortune Sonya , Estadt Shannon N , Kahlenberg J Michelle
Published: February 2023

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder with manifestations including skin, joint, and kidney inflammation, that is characterized by production of autoantibodies and sensitivity to ultraviolet (UV) light. The precise mechanisms governing UVB-mediated inflammation are incompletely understood; thus, our study objective is to delineate specific innate immune cell populations contributing to UV-mediated inflammation in lupus-prone mice. To that end, lupus-prone NZM mice, lupus-prone iNZM mice (knockout of alpha chain of type I interferon receptor), and wild type Balb/c mice were treated with 100mJ of UV light daily for 5 days. 24 hours after the final dose, skin samples were harvested from each, and CyTOF analysis was performed, revealing skewing of monocyte and macrophage populations in the skin of lupus prone versus wild type mice in a type I IFN dependent fashion. Subsequently, RNA sequencing was performed on skin samples of UV-irradiated NZM and Balb/c mice, which showed upregulation of known inflammatory monocyte chemoattractant genes in lupus prone mice after UV exposure compared to wild type mice. We further characterized monocyte populations recruited to the skin of NZM, iNZM, and Balb/c mice 24 and 72 hours after a single 300mJ dose of UV light by flow cytometry. Our data show a type I IFN dependent, transient predominance of inflammatory Ly6c hi monocytes in lupus prone versus wild type mice at 24 hours after UV exposure. By 72 hours after UV exposure, proportions of inflammatory monocytes in the lupus prone mice decrease. Future studies will be directed at understanding how these inflammatory monocytes differentiate in the context of a type I IFN rich environment and how this contributes to disease.
STAT4 in Conventional Dendritic Cells Regulates Pathogenic T Cell Differentiation and Inflammation in Experimental Autoimmune Encephalomyelitis
Authors: Alakhras Nada Salem , Kaplan Mark H
Published: February 2023

Multiple sclerosis (MS) is an inflammatory demyelinating disease affecting the central nervous system (CNS). Signal transducer and activator of transcription 4 (STAT4) is a major regulator of inflammatory in protective immunity and autoimmunity. STAT4 is activated downstream of two cytokines known to mediate the pathogenic T cell differentiation in autoimmune diseases, IL-12 and IL-23. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, germ-line deletion of STAT4 in mice results in resistance to the development of EAE. In this study, we investigated the contribution of innate myeloid cells to the phenotype observed in STAT4−/− mice in EAE. Using conditional mutant mice, we immunized mice deficient in STAT4 in myeloid cells and found that mice deficient in STAT4 in CD11c+ cells, but not Lyz2 + cells, are resistant to disease. STAT4fl/flCD11cCre mice are resistant to EAE induction and have inefficient differentiation of the pathogenic IL-17A, GM-CSF, and IFN-γ secreting CD4+ T cells. More importantly, STAT4fl/flCD11cCre mice have a significant decrease of migrated MHCIIhigh+ CD11chigh+CD26+ conventional dendritic cells (cDCs) to the CNS, suggesting a critical role of STAT4 on cDC migration to the CNS. Overall, our results identify a functional requirement of STAT4 in cDCs during EAE induction and propose a potential therapeutic target for autoimmune neuroinflammation diseases.
Card9 promotes pathogenic neutrophil responses that induce Th17-mediated autoimmune arthritis and spondylitis
Authors: Napier Ruth J , Vance Emily E , Lee Ellen J , Rosenzweig Holly L
Published: February 2023

Caspase recruitment domain-containing protein 9 (Card9) promotes anti-fungal immunity following C-type lectin receptor Dectin-1 activation by b-glucans. CARD9 has also been implicated in the pathogenesis of T cell-mediated inflammatory diseases including rheumatoid arthritis and ankylosing spondylitis, but how Card9 controls non-infectious inflammation is poorly understood. Using genetically susceptible SKG mice that develop b-glucan (zymosan)-induced, Th17-mediated autoimmune arthritis and spondylitis we found that Card9-deficiency (Card9−/− SKG mice) conferred complete protection from disease. Card9 promoted arthritis via a Dectin-1-mediated mechanism, as the Dectin-1-specific ligand (curdlan) induced disease and bacterial/fungal ligands specific for Dectin-2 or Mincle did not. At 8 weeks post-zymosan Th17 cells and neutrophils were dramatically reduced in Card9−/− SKG mouse joints, indicating an interplay between Card9, neutrophils and Th17 cells in promoting arthritis. Importantly, pre-arthritic Card9−/− SKG mice had reduced neutrophils in lavage fluid 4 hours post-zymosan compared to SKG mice, but no changes in dendritic cells or macrophages. Lavage fluid neutrophils from Card9−/− SKG mice had similar Dectin-1 expression to SKG mice, yet decreased activation (retention of CD62L) and degranulation of primary (CD63) and secondary (CD66b). Akin to Card9−/− SKG mice, neutrophil-deplete SKG mice had decreased Th17 cells and delayed arthritis onset, supporting a pathogenic function for neutrophils in induction of arthritis. These data suggest that Card9 functions downstream of Dectin-1 to promote pathogenic neutrophil responses necessary to induce Th17-mediated arthritis and spondylitis.
PD-L1 on macrophages protects from severe collagen induced arthritis
Authors: Wood Megan Kay , Talor Monica V , Won Taejoon , Cihakova Daniela
Published: February 2023

Immune related adverse events linked to severe inflammatory arthritis have been identified in 10–43% of patients receiving immune checkpoint inhibitor therapy, aCTLA-4, aPD-1 and aPD-L1, indicating the importance of immune inhibitory pathways in maintaining homeostasis in the synovium. We found that immune checkpoint, PD-L1, is primarily expressed in the joint compared to other immune checkpoints. Furthermore, blocking of PD-L1 during collagen induced arthritis (CIA) resulted in more severe arthritis. Mice synovial macrophages have the highest PD-L1 expression level at homeostasis and macrophage PD-L1 expression is further increased during CIA. Similarly, in human synovial fluid, macrophages have the highest frequency of PD-L1 expression. We hypothesize that the expression of PD-L1 by macrophages is essential for synovial homeostasis and regulation of joint inflammation. We selectively deleted PD-L1 in macrophages using the cre/flox system and induced CIA analyzing disease severity using flow cytometry. LyzcrePD-L1fl/fl showed more severe CIA with a greater change in paw thickness, higher disease and histology score and a higher frequency of CD4 and CD8 T cells in the synovium. Interestingly, female LyzcrePD-L1fl/fl mice have higher arthritis severity to LyzcrePD-L1fl/fl males. This indicates the importance of sex specific PD-L1 expression by synovial macrophages during CIA. Our project has high translational potential since joint specific PD-L1 expression could be modulated for therapeutic purposes in humans with rheumatoid arthritis or check point inhibitors induced arthritis.
Elucidating the role of IL-27 in Sjögren disease-like autoimmunity in the non-obese diabetic mouse model
Authors: Debreceni Ivy L , Chen Yi-Guang , Lieberman Scott M
Published: February 2023

Sjögren’s is an autoimmune disease targeting lacrimal and salivary glands leading to debilitating ocular and oral complications. Lack of understanding of the immunopathogenic mechanisms of disease has hindered development of effective therapies. Non-obese diabetic (NOD) mice spontaneously develop gland inflammation with features similar to humans providing a model to study immune signals that drive this autoimmune process. IL-27 is elevated in serum and saliva of Sjögren’s patients and is required for disease development in NOD mice. However, the mechanisms by which IL-27 contributes to Sjögren’s is not fully elucidated. Here, we define the requirements of IL-27 signaling in effector T cell subsets in the context of lacrimal gland autoimmunity.

IL-27 is a heterodimeric cytokine (EBI3 + p28) and binds a heterodimeric receptor (gp130 + IL-27Rα). IL-27Rα is expressed on CD8 and CD4 T cells, and both T cell subsets play pathogenic roles in lacrimal gland disease development. We utilized IL-27 KO and IL-27Rα KO NOD mice and an adoptive transfer model, to determine which T cell subsets required intact IL-27 signaling to mediate lacrimal gland inflammation in NOD-SCID recipient mice. Neither CD4 nor CD8 T cells sort-purified from IL-27Rα KO donors transferred disease. In contrast, while CD4 effector T cells from IL-27 KO NOD mice transferred disease, IL-27 KO CD8 T cells did not. Together these data demonstrate a T cell-intrinsic requirement for IL-27 signaling in both effector T cells subsets and suggest that CD8 T cells may also produce IL-27 to drive lacrimal gland inflammation. Flow cytometric and gene expression analyses to further define the immunological pathways driven by IL-27 in the context of lacrimal gland autoimmunity are underway.
TNF plays a crucial role in inflammation by signaling via T cell TNFR2
Authors: Alam Muhammad S
Published: February 2023

TNF, produced largely by T and innate immune cells, is potently pro-inflammatory, as are cytokines such as IFN-γ and IL-17 produced by Th1 and Th17 cells, respectively. Analysis of single-cell RNA-Seq datasets from multiple sclerosis (MS) patients identified CSF-infiltrating CD4+ T cells that had inflammatory gene signatures including high levels of TNF. This prompted us to ask if TNF is upstream of Th skewing toward inflammatory phenotypes. We found that exposure of naive human or mouse CD4+ T cells to TNF and TGF-β generated Th17 cells that express low levels of IL-17 (RORγt+IL-17lo) and high levels of inflammatory markers, independently of IL-6 and STAT3. This was mediated by the non-death TNF receptor TNFR2, which was also found to contribute to the generation of inflammatory Th1 cells. Single-cell RNA-seq of CNS-infiltrating CD4+ T cells in mouse experimental autoimmune encephalomyelitis (EAE) found an inflammatory gene expression profile similar to MS. Notably, TNFR2-deficient CD4+ T cells produced fewer inflammatory mediators and were less pathogenic in EAE and colitis. IL-1β, a Th17-skewing cytokine, induced TNF and pro-inflammatory GM-CSF in both human and mouse T cells, which was prevented by disruption of TNFR2 signaling, demonstrating IL-1β can function indirectly via the production of TNF. Thus, TNF is not just an effector cytokine but also an initiator of inflammatory Th differentiation, raising the possibility that targeting TNFR2 may diminish the generation of pathogenic Th cells in diseases in which TNF, IFN-γ, and IL-17 contribute.
Single-cell clonotype tracing of tissue Th17 cells during CNS autoimmunity reveals the conversion of intestinal homeostatic cells to pathogenic cells
Authors: Schnell Alexandra , Huang Linglin , Singer Meromit , Singaraju Anvita , Bollhagen Alina , Thakore Pratiksha I , Dionne Danielle , Delorey Toni M , Pawlak Mathias , Irizarry Rafael , Regev Aviv , Kuchroo Vijay K
Published: February 2023

At homeostasis, most Th17 cells are found in the lamina propria of the intestine, where they contribute to tissue homeostasis by inhibiting microbiota from tissue invasion and promote barrier functions. Recent studies in humans and mice have implicated intestinal Th17 cells in extra-intestinal autoimmune diseases, including multiple sclerosis, but the mechanism in which intestinal Th17 cells mediate these dichotomous functions remains unknown. Here, we combined single-cell RNA- and TCR-seq (scRNA/TCR-seq) with fate mapping of tissue Th17 cells to profile over 84,000 tissue Th17 cells and characterize their heterogeneity, plasticity, and migration at homeostasis and during CNS autoimmunity. We discovered a homeostatic Th17 cell population, that is induced by the intestinal microbiota, is present in both lymphoid organs and the intestine, and expresses IL-17. Joint scRNA- and TCR-seq showed that during EAE this homeostatic population gives rise to a pathogenic Th17 cell population, that migrates specifically through the draining lymph nodes and the spleen to the CNS, and highly expresses GM-CSF and IFNγ, but expresses little or low levels of IL-17. This conversion of homeostatic Th17 cells into encephalitogenic Th17 cells depended on IL-23R signaling. Our work characterizes Th17 heterogeneity, plasticity, and migration during homeostasis and CNS autoimmune disease, identifies a mechanism by which intestinal Th17 cells influence the generation of an autoimmune disease-inducing population, and provides further evidence for a direct relationship between the IL-17-producing and GM-CSF- and IFNγ- producing pathogenic T cells.
STAT3 gain-of-function variants and T cell-mediated inflammation
Authors: Toth Kelsey A , Saucier Nermina , Agbigbe Tejiri , Schmitt Erica , Keppel Molly P , Klechevsky Eynav , Kim Brian , Cooper Megan A
Published: February 2023

STAT3 is a transcription factor that plays a role in the proliferation, survival, and function of many different cell types. In T cells, STAT3 is required for the polarization and function of Th17 cells and can inhibit development of peripheral Tregs. STAT3 polymorphisms have been linked to several common autoimmune disorders where the balance between Th17 and Tregs has been disrupted. Patients with germline gain-of-function variants in STAT3 present with a syndrome of early-onset poly-autoimmunity. We generated two mouse models of STAT3 GOF by CRISPR/Cas9. In these models, mice have a single point mutation, p.G421R or p.T716M, which are two of the exact amino acid substitutions identified in patients. Heterozygous STAT3 GOF mice do not show spontaneous disease or constitutive activation of STAT3 and instead display delayed STAT3 de-phosphorylation following IL-6 stimulation. We hypothesized that STAT3 GOF would enhance Th17 responses and found increased Th17 polarization from naïve STAT3 GOF T cells in vitro. To test whether this increased disease susceptibility in vivo, we treated WT and STAT3 GOF mice with topical imiquimod. Compared to WT, STAT3 GOF mice have more severe disease as demonstrated by ear swelling and epidermal thickness, as well as increased numbers of Th17 cells in peripheral organs at day 7 and in the skin at day 14. No difference was observed in IL-17+ γδ T cell populations in these organs at either time point. STAT3 GOF mice crossed to Rag 1 −/− or IL-22−/− show decreased disease severity, indicating that this response may be mediated by adaptive lymphocytes and IL-22 signaling. These data indicate that STAT3 GOF can exacerbate disease by enhancing Th17 responses that may drive autoimmunity.
Follicular T cells are clonally and transcriptionally distinct in B cell-driven autoimmune disease
Authors: Akama-Garren Elliot H , van den Broek Theo , Simoni Lea , Castrillon Carlos , van der Poel Cees , Carroll Michael C
Published: February 2023

Pathogenic autoantibodies contribute to tissue damage and clinical decline in autoimmune disease. Follicular T cells are central regulators of germinal centers, although their role in epitope spreading towards autoantigens remains unclear. We performed single cell RNA and T cell receptor (TCR) sequencing of follicular T cells in autoantibody-mediated disease, allowing for analyses of paired transcriptomes and unbiased TCRab repertoires at single cell resolution. A minority of clonotypes were preferentially shared amongst autoimmune follicular T cells, and clonotypic expansion was associated with differential gene signatures in autoimmune disease. Antigen prediction using algorithmic and machine learning approaches revealed convergence towards shared specificities between non-autoimmune and autoimmune follicular T cells. However, differential autoimmune transcriptional signatures were preserved even amongst follicular T cells with shared predicted specificities. These results demonstrate that follicular T cells are phenotypically distinct in B cell-driven autoimmune disease, providing potential therapeutic targets to modulate autoreactive epitope spreading.

Supported by grants from NIH (R01AI130307, R01AR074105, T32GM007753).
Functional SARS-CoV-2-specific immune memory persists after mild COVID-19
Authors: Rodda Lauren Barbara , Netland Jason , Shehata Laila , Pruner Kurt B. , Morwaski Peter A. , Thouvenel Christopher D. , Takehara Kennidy K. , Eggenberger Julie , Hemann Emily A , Waterman Hayley R. , Fahning Mitchell L. , Chen Yu , Hale Malika , Rathe Jennifer , Stokes Caleb , Wrenn Samuel , Fiala Brooke , Carter Lauren , Hamerman Jessica A , King Neil P. , Gale Michael , Jr.  , Campbell Daniel J. , Rawlings David J , Pepper Marion
Published: February 2023

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is causing a global pandemic, and cases continue to rise. Most infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that could contribute to immunity. We performed a longitudinal assessment of individuals recovered from mild COVID-19 to determine whether they develop and sustain multifaceted SARS-CoV-2-specific immunological memory. Recovered individuals developed SARS-CoV-2-specific immunoglobulin (IgG) antibodies, neutralizing plasma, and memory B and memory T cells that persisted for at least 3 months. Our data further reveal that SARS-CoV-2-specific IgG memory B cells increased over time. Additionally, SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral function: memory T cells secreted cytokines and expanded upon antigen re-encounter, whereas memory B cells expressed receptors capable of neutralizing virus when expressed as monoclonal antibodies. Therefore, mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity.
A distinct non-enzymatic role for Indoleamine-2,3-dioxygenase (IDO)2 vs. IDO1 in autoimmune arthritis
Authors: Peng Weidan , Merlo Lauren MF , DuHadaway James B , Montgomery James D , Murray Peter J. , Yeh Syun-Ru , Prendergast George C , Muller Alexander J , Mandik-Nayak Laura
Published: February 2023

IDO1 and IDO2 are closely related immune modulating enzymes encoded by linked genes. Whereas IDO1 is best known for its immunoregulatory role in tumor immune evasion, IDO2 acts as a proinflammatory effector of B cell-mediated autoimmunity. Given their opposing roles in inflammatory responses, interpretation of results obtained using IDO1 or IDO2 single knockout (ko) mice have been complicated by the expression of the other enzyme. Using single and double knockout (dko) mice and the KRN arthritis model, we distinguished differential roles for IDO1 and IDO2 in autoreactive B vs. T cell responses driving arthritis. Both autoreactive T and B cell responses and severity of arthritis were decreased in IDO2, but not IDO1, ko mice. Despite a similar attenuation of disease, only autoreactive B cell responses were reduced in dko mice, demonstrating that IDO2 directly mediates autoreactive B cell responses, while autoreactive T cell responses are indirectly affected by IDO1. Like IDO1, IDO2 was originally identified as a tryptophan catabolizing enzyme. However, we recently showed that the enzymatic activity of IDO2 is not required to drive arthritis. To define the non-enzymatic mechanism mediating IDO2 function, we identified IDO2-interacting proteins using a yeast two-hybrid screen. Specific interaction between IDO2, but not IDO1, and several candidate proteins was confirmed using overexpressing cell lines and co-IP/Western. Using site-directed mutagenesis and an in vitro IDO2-expression model, we identified potential regions outside of the catalytic site on IDO2 that mediate these interactions, providing the first evidence for the non-enzymatic mechanism by which IDO2 mediates proinflammatory B cell responses.
Mechanisms of TRAF3 Regulation of TLR Signaling in B Lymphocytes
Authors: Ybarra Tiffany K. , Bishop Gail A
Published: February 2023

TRAF3 is an adaptor protein that binds to receptors of the TNFR superfamily and exerts multiple regulatory functions in signaling pathways. However, functions are cell type and context dependent. B cell TRAF3 inhibits homeostatic NFκB2 activation, CD40/BAFFR signaling and other pro-survival signaling pathways; restraint of survival is a B cell unique TRAF3 function. Loss of function mutations of human TRAF3 are detected in B cell lymphoma and multiple myeloma. In addition to homeostatic survival, we found that TRAF3 inhibits B cell TLR signaling, a pathway implicated in B cell lymphomagenesis and autoimmunity. Numerous reports describe TLR function in myeloid cells, but specific TLR signaling pathways in B cells are under-studied. B cell TRAF3 inhibits TLR-mediated cytokine and Ab production, and Ig class switching. Our working hypothesis is that B cell TRAF3 regulates the expression, recruitment and/or post-translational modification of TLR signaling proteins. Preliminary data show that B cell TRAF3 associates with signaling proteins MyD88 and IRAK1, and the kinase Syk following TLR stimulation. We are currently investigating how this association normally restrains the functions of these proteins in promoting TLR signals to B cells. We expect that information gained will provide new insights into the molecular mechanisms contributing to the regulation of B cells by TRAF3 in TLR signaling, potentially informing selection and development of more targeted therapies for B cell malignancies and autoimmunity.
Dysregulation of microRNA expression in chronic periodontitis
Authors: Banks Jonathan Matthew , Naqvi Raza Ali , Valverde Araceli , Bambila Maria F. , Martínez Gloria , Chapa Gabriela , Nares Salvador , Naqvi Afsar R.
Published: February 2023

Objective:

Assess microRNA (miRNA) expression in healthy and diseased gingival tissue of human subjects and identify potential miRNA-target gene interactions and biological pathways relevant to periodontal pathogenesis.

Methods:

Gingival biopsies were obtained from subjects meeting the criteria for “healthy” or “diseased” according to the current AAP guidelines. Total RNA (including miRNA) was isolated using miRNeasy kit (Qiagen). Global human miRNA expression was quantified using microRNA microarray (LC Sciences μParaflo® technology platform). Potential gene targets and biological pathways of differentially expressed miRNAs were predicted using bioinformatics analysis.

Results:

Our results show significantly different expression levels of miRNAs when comparing inflamed and healthy gingival biopsies. Among differentially expressed miRNAs, 35 were upregulated and 29 were downregulated in diseased tissue. Using pathway analysis, these miRNAs were predicted to influence the following pathways: ECM-receptor interaction, cell adhesion molecules (CAMs), MAPK signaling pathway, and cAMP signaling pathway. Genes associated with these pathways were scanned for potential miRNA binding sites. We identified IKBKB as a target gene, an endogenous inhibitor of NF-kappa-B and MAPK signaling, which possesses multiple binding sites for candidate miRNAs including hsa-miR-195-5p.

Conclusion:

Our findings reveal human miRNA expression profiles are significantly different in healthy and diseased gingival tissues. Post-transcriptional regulation of gene expression by miRNAs can modulate inflammatory biological pathways, suggesting their integral role in periodontal pathogenesis.
Long noncoding RNAs RN7SK and GAS5 Regulate Macrophage Innate Immune Function and Polarization
Authors: Naqvi Raza Ali , Ahmad Imran , Valverde Araceli , Naqvi Afsar R
Published: February 2023

Objective:

Macrophages (Mϕ) are polarized in two phenotypes: classically activated M1 phenotype and alternatively activated M2 phenotype to marginalize the pathogenic/non-pathogenic threat. Burgeoning reports are now elucidating the regulatory role of lncRNAs in various immune process. In this study, we aimed to identify the lncRNAs that mediate Mϕ function and polarization.

Methods:

CD14+ monocytes isolated from PBMCs and differentiated to M1 and M2Mϕ, using GM-CSF or M-CSF, respectively. Expression of 88 different lncRNAs was examined by RT-qPCR. Cells transfected with siRNAs targeting RN7SK, GAS5, IPW and ZFAS1 were examined for Mϕ polarization markers and innate immune functions (phagocytosis and antigen uptake/processing).

Results:

Our results show differential expression of seventeen lncRNAs in M2Mϕ compared to monocytes including RN7SK, GAS5, IPW and ZFAS1. Knockdown of RN7SK and GAS5 showed downregulation of M2 surface markers (CD163, CD206 or Dectin) and concomitant increase in M1 markers (MHC II or CD23). M1/M2 markers were not impacted by IPW or ZFAS1 knockdown. mRNA levels of polarization markers were not effected by RN7SK or GAS5 RNAi. We observed enhanced antigen uptake and processing both in M1/M2 Mϕ transfected with siRNA targeting GAS5 and RN7SK but not IPW and ZFAS1. In addition, knockdown of RN7SK significantly augmented labelled E.coli uptake by M1/M2 Mϕ, while no significant difference was noticed in GAS5 silenced cells.

Conclusion:

This study clearly deciphered that remarkable changes in lncRNA occur during monocyte to Mϕ differentiation, polarization and regulate integral innate functions of Mϕ. Manipulation of lncRNAs could be tested in therapeutic reversal of pro and anti-inflammatory pathologies.
Glycolysis Inhibition Modulates Unique Metabolic and Immune Pathways Across Multiple Tissue Compartments
Authors: Wells Ann , Wilson John , Roopenian Derry C , Chang Chih-Hao , Carter Gregory W.
Published: February 2023

Inhibition of glycolysis via 2-deoxyglucose (2DG), a competitive inhibitor of glucose-6-phosphate, has recently shown efficacy in attenuating tumor growth and reducing autoimmune pathology; however, the mechanism of this efficacy is somewhat unclear. To elucidate how 2DG longitudinally modulates pathways without the influence of immunity in diseased status, wild-type C57BL/6J mice were treated orally with 2DG for 96-hours or 4-weeks, after which the transcriptional profiles of 9 tissues (heart, hippocampus, hypothalamus, kidney, liver, prefrontal cortex, skeletal muscle, small intestine, and spleen) were analyzed using unsupervised clustering and weighted gene correlation network analysis. Principal component analysis showed mice clustered by tissue. Tissues grouped together according to their summary gene expression for modules correlated to time, treatment and/or tissue, and two pathway groups, immune and metabolic, emerged. However, within each tissue cluster, pathways for each module were uniquely altered by time, treatment and/or tissue, according to ANOVA. Furthermore, while most pathways identified were grouped into either immune or metabolic, there was little overlap of specific pathways identified between tissues. These results show that 2DG has a systemic impact altering immune and metabolic pathways, but in a tissue-specific manner. Ongoing work includes identifying genes that are potentially central to pathway alterations for follow-up analysis.
P2X3 is a Female-Dominant Activator of Mast Cells
Authors: Kee Sydney Ann , Haque Tamara , Dailey Jordan , Ryan John J
Published: February 2023

Asthma is characterized by increased mucus production, airway hyperresponsiveness and airway inflammation, all promoted by mast cell activation. There is a clear bias for greater incidence and acuity of asthma among females, but the reasons for this are not clear. In seeking FDA-approved drugs that might be repurposed to suppress mast cell function, we found that the selective serotonin reuptake inhibitor fluoxetine was able to suppress IgE mediated-mast cell degranulation and cytokine secretion in vitro, and protect against anaphylaxis in vivo. Fluoxetine effects appear to be mediated by an off-target interaction with the ATP receptor P2X3. Interestingly, male mice and mast cells cultured from them showed little fluoxetine responsiveness. These data coincided with little detectable P2X3 expression on male mast cells. Our data support the theory that P2X3 is a female-dominant mast cell activator that can be targeted with fluoxetine to reduce allergic inflammation. Female-biased P2X3 expression may also contribute to sexual dimorphism in allergic disease.