bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen-presenting cells from naive mice constitutively present the T cell epitope of SERCA2a and induce inflammatory cytokine production in antigen-specific T cells
Authors: Arumugam Rajkumar , Yalaka Bharathi , Massilamany Chandirasegaran , Ali Shihabudeen , Riethoven Jean-Jack , Sun Xinghui , Reddy Jay
Published: January 2023

We recently reported that sarcoplasmic/endoplasmic reticulum (SR) calcium-ATPase2a (SERCA2a) 971–990 induces atrial myocarditis by generating autoreactive T cells in A/J mice. However, it was unknown as to how the antigen-sensitized T cells could recognize SERCA2a 971–990, since SERCA2a-expression is confined to SR. Here, we present evidence that antigen-presenting cells (APCs) from naïve animals constitutively present SERCA2a 971–990 and stimulate T cells antigen-specifically. By using major histocompatibility complex (MHC) class IIdextramers for SERCA2a 971–990, we created a panel of antigen-specific T cell hybridomas and demonstrated that splenocytes from naïve A/J mice stimulated the hybridoma cells without exogenous supplementation of SERCA2a 971–990, and such an effect was pronounced with unirradiated cells as compared to irradiated cells. We recapitulated this phenomenon by using SERCA2a 971–990-specific, primary T cells, and the T cell responses were verified to be MHC-restricted. While T cells exposed to mononuclear cells obtained from heart and liver responded similar to splenocytes, endothelial cells (ECs) generated from the corresponding organs displayed opposing effects in that the T cell responses were suppressed with the heart-ECs, but not from liver. Finally, SERCA2a 971–990-reactive T cells exposed to APCs were found to produce inflammatory cytokines namely, interferon-γ, granulocyte macrophage colony stimulating factor and interleukin-17. Our data suggest that the constitutive expression of SERCA2a 971–990 by APCs can trigger pathogenic responses under the conditions of autoimmunity that may have implications on endothelial dysfunction.
Biomarker discovery in pre-Type 1 Diabetes; Th40 cells as a predictive risk factor
Authors: Vaitaitis Gisela , Waid Dan , Wagner David H
Published: January 2023

The incidence of Type 1 Diabetes (T1D) is on the rise. Quests to understand T1D etiology as well as how to predict onset are ongoing and, in many ways, those goals intertwine. CD4+CD40+ T cell (Th40) percentages are expanded in T1D subjects compared to controls. Antibodies to β-cell antigens are considered risk factors for T1D development but risk of seroconversion to antibody-positivity declines after age 10. While genetic components associate with T1D, not all T1D subjects have those components and not all subjects with those components develop T1D. Therefore, more robust methods for prediction of T1D are needed. Can high Th40 cell percentages be a biomarker in addition to other markers?

We assessed Th40 cell percentages, along with other parameters, in blood collected from TrialNet preT1D subjects and stratified them according to their percentages. Th40-low versus Th40-high preT1D subjects demonstrate patterns of CD4 T cell levels that parallel those seen between control and T1D. Cytokine patterns are significantly different between the Th40 groups and a CD4+CD8+ population, described previously in Rheumatoid Arthritis, is more common in Th40-high subjects. Subjects experiencing impaired glucose tolerance have significantly higher Th40 cell percentages than controls. PreT1Ds expressing T1D associated HLA DR4/DR4 or DQ8/DQ8 are more likely found among Th40-high subjects. Interestingly, DR4/DR4 subjects were significantly older compared to all other preT1Ds, suggesting that this haplotype together with a high Th40 cell percentage may represent someone who will onset after age 30, which is reported for 42% of T1D cases. It will be important to determine if preT1D subjects with high Th40 cell percentages will develop overt T1D.
Agonist-induced 4-1BB activation ameliorates the pathogenesis of Sjögren’s syndrome-like sialadenitis in non-obese diabetic mice
Authors: Zhou Jing , Yoo Bora , Yu Qing
Published: January 2023

4-1BB is a member of the tumor-necrosis factor receptor (TNFR) superfamily. As a potent costimulatory molecule, it is critically involved in both enhancement and suppression of immune responses in a disease context-dependent manner. The role of 4-1BB in the development and onset of Sjögren’s syndrome (SS), an autoimmune disease as a result of immune system attacking self-exocrine glands with dryness of mouth and eyes as major clinical symptoms, still remains unknown. To elucidate the precise function of 4-1BB in the pathogenesis of SS, we intraperitoneally administered an agonistic anti-4-1BB monoclonal antibody to female non-obese diabetic (NOD) mice, a well-defined mouse model of this disorder, during the stage prior to SS disease onset. We observed that anti-4-1BB treatment significantly increased the salivary flow rate, indicating an effective amelioration of SS clinical manifestation. Administration of anti-4-1BB antibody markedly suppressed the leukocyte infiltration of the submandibular glands (SMGs) and reduced the percentage of CD4 T cells among total infiltrating leukocytes, without altering that of CD8 T cells and B cells with statistical significance. The decrease in the frequency of CD4 T cells was accompanied by a reduced expression of T-bet but not IL-4 in the SMGs, suggesting an inhibitory effect of 4-1BB activation on type 1 T helper cells. In addition, treatment with this agonistic anti-4-1BB also caused a significant reduction in the level of serum antinuclear antibodies. Taken together, our findings imply that 4-1BB plays a protective role against the pathogenesis of SS-like sialadenitis and its agonists could serve as promising therapeutic agents for controlling this autoimmune disorder.
Type 1 alveolar epithelial cell-derived chemokine CCL19 promotes airway inflammation
Authors: Nakano Hideki , Lyons-Cohen Miranda , Nakano Keiko , Karcz Tadeusz , Thomas Seddon Y , Whitehead Gregory , Cook Donald N
Published: January 2023

Allergic asthma is largely driven by the actions of allergen-specific CD4+T helper cells. Following allergen inhalation, these cells are induced by the interaction of naïve CD4+ T cells with antigen-bearing dendritic cells (DCs) in lung-draining lymph nodes. The molecular mechanisms underlying the induction of T helper cells have been well studied, and include the migration of allergen-bearing DCs to lung-draining lymph nodes in a manner dependent on the chemokine receptor, CCR7. However, comparatively little is known regarding how these effector T cells become activated following their subsequent encounter with allergen in the lung. This knowledge would be useful in the design of novel therapies for asthma, especially for neutrophilic asthma, which is refractory to inhaled glucocorticoids. We recently noticed that lung DCs migrating to lymph nodes originate from the large airways, whereas DCs associated with small airways, parenchyma and subpleural areas are non-migratory. Video confocal analysis of precision cut lung slices revealed that these non-migratory DCs form tight interactions with OVA-specific T cells and stimulate T cell receptor signaling, as measured by activation of a Nur77-gfpreporter gene. Furthermore, Ccl19, which encodes a CCR7-ligand, is highly expressed by type 1 alveolar epithelial cells, and in mouse model of asthma, airway inflammation was significantly diminished in CCL19-deficient mice compared with WT mice. Together, our findings suggest that CCL19 produced by type I epithelial cells acts to position lung DCs in the distal area of the lung where they interact with effector T cells, thereby promoting allergic airway inflammation.
β-catenin negatively regulates IL-6 and IL-8 expression at transcriptional level and induces reactivity in human astrocytes
Authors: Robinson KaReisha , Narasipura Srinivas , Al-Harthi Lena
Published: January 2023

HIV invades the brain during acute infection, setting the stage for persistent neuroinflammation despite combined antiretroviral therapy (cART) and leads to HIV-Associated Neurocognitive Disorders (HAND), which occurs in ~50% of HIV-infected individuals. Our lab is focused on understanding the role of Wnt/β-catenin signaling in HAND. Here, we evaluated the impact of β-catenin on inflammatory mediators associated with neuroinflammation, chemotactic molecules, and regulation of A1 (proinflammatory)/A2 (protective/repair) phenotypes of astrocytes. We demonstrate that knockdown (KD) of β-catenin in normal human astrocytes (NHAs) significantly induced IL-6 and IL-8 at the transcription and protein levels and conversely, induction of β-catenin significantly downregulated these two molecules. These findings are intriguing given that no role for β-catenin to date is associated with IL-6 and IL-8 regulation. Further, KD of β-catenin induced three genes associated with A1 phenotype by 2.4–6.4 fold. These findings indicate that β-catenin expression in astrocytes is a critical regulator of anti-inflammatory responses and its disruption can potentially mediate persistent neuroinflammation.
In vivo imaging of caspase-1 activation enables efficient and rapid detection of various inflammatory disorders
Authors: Lee Jaewon , Ko Young ji , Yang Eunjeong , Jang Nayoon , Kim Hye sun , Kwon Ick chan , Cho Nam-hyuk
Published: January 2023

Inflammasome plays a critical role in diverse inflammatory diseases, including cancers and Alzheimer’s disease. It can be activated by a various stimuli and induces secretion of IL-1β after cleavage by active caspase-1, an executing enzymatic component of the inflammasome complex. Therefore, direct imaging of active caspase-1 in vivo may provide enormous advantages for diagnosis, drug discovery, and therapeutic monitoring in various inflammatory disorders. Here, we developed a new imaging probe for in vivo visualization of active caspase-1. For this, we synthesized a caspase-1-specific cleavable peptide bridging a near-infrared (NIR) fluorescence (Cy5.5) dye and a hydrophobic quencher. This novel caspase-1 probe can be efficiently delivered into cells and specifically emit fluorescence upon caspase-1 activation as measured by in vitro and in vivo inflammatory models. We demonstrated efficient in vivo imaging of caspase-1 activation in early stage of various inflammatory conditions in mice models, including endotoxin stimulation, inflammatory bowel disorder, transplanted cancer, and Alzheimer’s disease. Efficient and rapid detection of caspase-1 activity in vivo can revolutionize the current paradigm for diagnosis and therapeutics in various inflammatory disorders.
The anti-inflammatory effects of the Chromogranin A hormone cleavage product catestatin on human monocytes and macrophages
Authors: Muntjewerff Elke M. , Christoffersson Gustaf , Nicolasen Mara J.T. , Mahata Sushil K. , van den Bogaart Geert
Published: January 2023

Objective

High levels of the prohormone chromogranin A (CgA) have been detected in the plasma of patients with various cancers and inflammatory diseases such as rheumatic arthritis, inflammatory bowel disease as well as type 1 and 2 diabetes mellitus (T2DM). In contrast, T2DM and hypertensive patients show reduced levels of the CgA-derived peptide catestatin (CST). Here we show the anti-inflammatory effects of CST on human macrophages and phagocyte migration.

Method

Mouse phagocyte (monocytes and granulocytes) migration was imaged in the cremaster muscle of mice superperfused with buffer containing CST or the chemokine MIP-2. Human blood monocytes were used for in vitro migration to CCL2 and/or CST and macrophage differentiation assays. Macrophages were treated with CST and polarized towards pro-inflammatory or anti-inflammatory phenotypes. Afterwards, cytokine production was measured by ELISA (Il-10, TNF-a, IL-6) and cells were phenotyped by EM and RNA sequencing.

Results

In vivo and in vitro imaging shows that CST works as a chemo-attractant for monocytes. Interestingly, the combination of CST with the well-known chemoattractant CCL2 results in an anti-inflammatory effect. Moreover, CST treatment of inflammatory macrophages inhibited TNF-α and IL-6 production. Additionally, several proinflammatory gene levels in CST treated macrophages were decreased.

Conclusions

CST influences monocyte migration and promotes M1-M2 polarization by decreasing expression of pro-inflammatory cytokines and increasing anti-inflammatory cytokine expression. These factors are important for tissue homeostasis, which makes CST an interesting therapeutic target for treatment and/or diagnosis of various metabolic and immune diseases.
Trauma Induces Emergency Hematopoiesis through IL-1/MyD88-Dependent Production of G-CSF
Authors: Fuchs Anja G , Monlish Darlene , Ghosh Sarbani , Chang Shin-Wen , Bochicchio Grant V , Schuettpelz Laura G , Turnbull Isaiah R
Published: January 2023

The acute inflammatory response to infection or injury dramatically increases the hematopoietic demand on the bone marrow to replace effector leukocytes consumed in the inflammatory response. In the setting of infection, pathogen-associated molecular patterns induce emergency hematopoiesis, activating hematopoietic stem and progenitor cells to proliferate, thereby providing progeny for accelerated myelopoiesis. Sterile tissue injury due to trauma also increases leukocyte demand; however, the effect of sterile tissue injury on hematopoiesis is not well described. We used a mouse model of multisystem injury to investigate the effects of these injuries on bone marrow progenitor frequencies and phenotypes. We find that tissue injury alone induces emergency hematopoiesis in mice subjected to polytrauma. This process is driven by IL-1/MyD88-dependent production of G-CSF. G-CSF induces expansion of hematopoietic progenitors including hematopoietic stem cells and multipotent progenitors and increases the frequency of myeloid-skewed progenitors. These data provide the first comprehensive description of injury-induced emergency hematopoiesis and identify an IL-1/MyD88/G-CSF dependent pathway as the key regulator of emergency hematopoiesis after injury.
Reduced Expression of the DNA-Binding Protein ARID3a, a Mediator of Human Hematopoiesis, in Hematopoietic Progenitors Contributes to Age-Related Transcriptional Changes
Authors: Ratliff Michelle , Garton Joshua W , James Judith A , Webb Carol
Published: January 2023

Population studies estimate that 40% of European and US populations will be over the age of 60 by 2050. Aged individuals exhibit impaired immune responses consisting of poor responses to vaccines and increases in myeloid lineage cells. The reasons for decreased immunity in the elderly haven’t been completely elucidated, making studies of the mechanisms leading to these poor responses important. One possibility is that defects in early hematopoiesis may contribute to poor immune responses in the elderly. Previous studies in our lab indicated that inhibition of the DNA-binding protein ARID3a in hematopoietic cultures of human cord blood led to hematopoietic developmental skewing toward myeloid lineage rather than lymphoid lineage progenitors. Furthermore, we previously reported that numbers of ARID3a-expressing hematopoietic stem cells (HSCs) can be highly variable in some patient subsets as well as in healthy individuals. ARID3a expression levels in hematopoietic progenitor cells have not previously been analyzed in relationship to age. We hypothesized that alterations in ARID3a expression levels in early hematopoietic progenitors could contribute to poor immune responses observed in elderly people. Our data suggest that hematopoietic progenitors obtained from peripheral blood of aged healthy persons show decreased frequencies of ARID3a-expressing HSCs in comparison to HSCs from young individuals. Furthermore, lineage committed progenitor numbers were also altered in aged persons compared young people. Ongoing analyses to investigate the gene expression data in young and aged HSCs in relation to ARID3a expression will assess potential genes regulated by ARID3a that contributes to lineage commitment and development.
Glucocorticoids control allergic inflammation via Foxp3+regulatory T cells
Authors: Nguyen Quang Tam , Min Booki
Published: January 2023

Asthma is a chronic inflammatory disease in the lung characterized by airway inflammation and airway hyper-responsiveness (AHR). Synthetic glucocorticoids such as Dexamethasone (Dex) are a mainstay treatment option for the clinical management of asthma. However, the target cells that mediate their therapeutic effects and the underlying mechanisms remain unclear. Utilizing a cockroach antigen-induced allergic inflammation model in mice, we sought to test the roles of Foxp3+ regulatory T cells (Tregs) during Dex-mediated regulation of allergic inflammation. Administration of Dex significantly reduced the development of airway inflammation and AHR. Accumulation of eosinophils and effector CD4 T cells expressing Th2 cytokines in the lung were dramatically reduced following Dex administration. To our surprise, the Dex-induced reduction was completely abolished in the absence of Tregs. Inflammatory cell accumulation, cytokine production, and AHR development remained unchanged in this condition, suggesting that Tregs may be an important target cell of Dex in vivo. Indeed, adoptive transfer of in vitro generated Tregs into Treg-depleted mice fully restored Dex effects. We performed an RNA-sequencing analysis comparing gene expression profiles in lung infiltrating effector CD4 T cells from mice treated with control, Dex, and Dex with Treg depletion. Genes upregulated or downregulated by Dex treatment remained unchanged by Treg depletion. Taken together, our results uncover a novel function of Tregs during Dex-mediated treatment of allergic inflammation.
Metabolic differences between Th2 and Th17 cells in airway inflammation
Authors: Contreras Diana C , Cephus Jacqueline , Newcomb Dawn , Rathmell Jeffrey C
Published: January 2023

Asthma is an airway inflammatory disease that is mediated by T effector (Teff) cells, specifically Th2 and Th17 cells. As disease increases in severity, there is a shift towards a higher Th17 response. Treatment of asthmatic patients include the use of glucocorticoid (GC) steroids. These drugs are effective at controlling inflammation in mild cases but as disease severity increases towards neutrophilic disease there is resistance by Th17 cells to effects of the drugs. A key therapeutic objective is to identify either alternative treatments for asthma or targets that make Th17 cells more susceptible to GCs. Studies have shown cells which have increased glycolysis and oxidative phosphorylation are more resistant to the effects of GCs. In this study we show, that in the murine model of airway inflammation there are differences in the expression of metabolic proteins between Th2 and Th17 cells that are present in the lung. This has been seen in cells that have higher amounts glucose uptake. These results imply that the higher glucose utilization of Th17 cells and their metabolic flexibility may contribute to their resistance to the effects of GCs. We also show by CyTOF of PBMCs that individuals with asthma have increased expression of metabolic associated proteins compared to control individuals in their CD4 cells. These data support the metabolic shift observed in our in vitro studies.
The function of the circulating innate lymphoid cells is dysregulated in patients with mild asthma
Authors: Drake Li Y , Bachman Kay , Kita Hirohito
Published: January 2023

Group 2 innate lymphoid cells (ILC2s) play crucial roles in type 2 immunity in the lungs and in the development of asthma pathology. While ILC2s are residents in mucosal tissues, they also circulate in peripheral blood. Evidence suggests that blood ILC2s can be used as an indirect but useful biomarker to monitor the activity of ILC2s in mucosal tissues, such as the lungs. To investigate both the innate immune response and the corresponding ILC2 cell levels in the peripheral blood of asthma patients, we collected peripheral blood mononuclear cells (PBMCs) from 11 adult patients with mild asthma (MA) and 12 adult healthy control (HC) subjects. The innate type 2 immune responses of PBMCs and the number of blood ILC2s were examined by in vitro culture and flow cytometry. We found that in response to IL-25 or IL-33 stimulation, PBMCs from MA subjects produced significant higher levels of IL-5 than PBMCs from HC subjects. However, there were no significant differences in ILC2 numbers or proportions between MA and HC groups. These results suggest that the function, but not the number, of blood ILC2s may serve as a general biomarker for human asthma.
Prolonged activation in CD4+ T cells results in extensive mitochondrial remodeling despite the metabolic dominance of aerobic glycolysis
Authors: Akkaya Billur , Roesler Alexander , Theall Brandon P , Al Souz Jafar A , Miozzo Pietro , Traba Javier , Smelkinson Margery , Kabat Juraj , Dorward Dave , Pierce Susan K , Akkaya Munir
Published: January 2023

Activation of CD4+ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling using a combination of conventional strategies and a novel high-resolution imaging method. We show that for four days following activation, mouse CD4+ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed increased reactive oxygen species production. Despite elevated ROS production, it was successfully counterbalanced by antioxidant enzymes, hence the mitochondria did not display signs of oxidative stress as evidenced by the healthy cristae structure and overall morphology as well as the maintained mitochondrial reserve. Moreover, prolonged activation of CD4+ T cells led to increases in mitochondrial content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation, CD4+ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling, resulting in increased mitochondrial activity.
A cognate interaction between T cell-expressed BTLA and B cell-expressed HVEM regulates mucosal immunity in the gut
Authors: Stienne Caroline , Moshayedi Aref , Chappell Elizabeth , Ware Carl F. , Sedy John R.
Published: January 2023

Mucosal immunity in the gut is achieved in part through cellular connections between lymphoid cells such as regulatory-T cells (Treg), T follicular helper cells (Tfh), T follicular regulatory cells (Tfr) and Germinal Center B cells (GC B). Btla (B and T Lymphocyte Attenuator) is a receptor mainly expressed on B and T cells that is activated by its ligand Hvem (Herpesvirus entry mediator; Tnfrsf14) to inhibit cellular activation. However, the role of these specific immune checkpoints in the regulation of mucosal immunity in the gut is still unknown.

In mice containing genetic ablation of Btla in T cells, we observed an increased frequency of Tfh and GC B cells in Peyer’s Patches (PP), and a decreased frequency of Treg and Tfr cells in these tissues. Interestingly, in mice with a B-cell-specific deletion of the Btla ligand Tnfrsf14 (Hvem), we observe similar increases in GC B cells and Tfh cells and decreased in Treg. Moreover, these mice are characterized by an increase of mucosal IgA bound to bacteria in fecal pellets, indicating a functional outcome of Btla regulation of the GC. Finally, treating wild-type mice with an antibody agonist for Btla increases the frequency of regulatory T cells in PP demonstrating the potential therapeutic effect of activating Btla inhibitory signaling in mucosal immunity.

Together, we show that T cell-expressed Btla interacts with B cell-expressed Hvem to regulate GC responses in Peyer’s Patches. A disruption of the balance effector T cell/regulatory T cell is responsible for many inflammatory autoimmune diseases. It will be crucial to understand the role of Btla and its ligand in these populations in mucosal tissues to understand how immunity is regulated, and to develop potential novel therapeutics to treat disease.
The skin: a site of extralymphoid IgM production
Authors: McGettigan Shannon E , Wilson R. Paul , Dang Van Duc , Kumar Anil , Nikbakht Neda , Stohl William , Debes Gudrun F
Published: January 2023

Antibodies are critical to cutaneous host defense and inflammation, but the mechanisms by which skin antibodies are maintained are poorly described. In this study, we found extralymphoid antibody production in unperturbed human and mouse skin. IgM was the predominant isotype produced by plasma cells in healthy murine skin and was also secreted in healthy human skin. In mice, cutaneous IgM plasma cells were independent of T cells and microbiota. Importantly, chronic inflammation caused the massive accumulation of IgM secreting cells that was dependent on APRIL or BAFF in skin but not in other sites. In addition, skin immunization caused cutaneous accumulation of both T cell-dependent and -independent antigen-specific IgM secreting cells. Thus, we reveal the skin as a novel niche for plasma cells with important implications for the regulation of cutaneous antibodies. We propose that skin plasma cells supply natural and adaptive IgM to the cutaneous environment, thereby supporting homeostatic skin barrier functions in addition to providing defense against pathogen intrusion.
Pressure enabled drug delivery of anti-CEA CAR-T cells increases intra-hepatic CAR-T tumor penetration and therapeutic index in a murine model of liver metastasis
Authors: Hardaway John C , Chai Louis F. , Lopes Mikayla , Rabinowitz Benjamin , Guha Prajna , Jaroch David , Cox Bryan , Knight Robert , Zeldis Jerome , Espat N. Joseph , Katz Steven C
Published: January 2023

Introduction

We have demonstrated safety and clinical activity with anti-CEA CAR-T (Sorrento/TNK Therapeutics, Inc.) regional infusions for treatment of liver metastases (LM) using a pressure-enabled drug delivery device (PEDD™, TriSalus™ Life Sciences, Inc.) to increase CAR-T penetration. To further explore the potential benefit of PEDD, we developed a pre-clinical murine LM model that enables correlation of infusion pressure with response.

Methodology

Mice with CEA+ LM were infused with anti-CEA CAR-T into the portal vein with specified rate and pressure levels. Flow cytometry (FC), bioluminescence imaging (BLI), and serum liver function tests (LFTs) were conducted at multiple time points.

Results

Flow-pressure relationship was established with ΔP of 7.23 mmHg vs 2.33 mmHg (p=0.01) in high-pressure (10 mL/min, HP) and low-pressure (1 mL/min, LP) cohorts. FC assessment of hepatic non-parenchymal cells 1d after infusion demonstrated 15.9% CD3+CAR+ in HP, compared to 5.1% in LP (p=0.0004). Tumor BLI at day 3 (geometric mean relative to baseline) showed significantly enhanced therapeutic effect in HP, −18% relative signal intensity (RSI), compared to +148% RSI in LP (p=0.05) or +178% RSI in controls (p=0.04). LFTs were similar in LP and HP groups with AST 302 vs 437 U/L (p=0.60) and total bilirubin 0.25 vs 0.7 mg/dL (p=0.33), respectively.

Conclusions

HP delivery correlated with enhanced CAR-T delivery and control of tumor growth, without an increase in liver toxicity. Follow up to this proof of concept study will investigate multiple infusions with combinatorial approaches with the goal of durable tumor eradication. An ongoing clinical study will further explore CAR-T PEDD in patients.
Mouse Models for Immunology Research available from The Jackson Laboratory Repository
Authors: Valenzuela Alicia , Fancher Karen , Rockwood Stephen , Lutz Cathleen
Published: January 2023

The Jackson Laboratory Repository (JAX) serves as a centralized facility for the development, distribution and cryopreservation of mouse models of human biology and disease. JAX distributes more than 10,500 strains to the scientific community, many with applications in immunology research, including mouse models for autoimmune disease, infectious disease, immunodeficient platforms for PDX studies, and multi-purpose “tool strains” (such as CRISPR cas9-expressing lines), recombinase expressing strains, as well as conditional and inducible expression lines.

One of our newest strains is a conditional knock-out (KO) of STING/MYPS, a pathogen sensor involved in the activation of IL6 and IFNb. Additions to our extensive set of interleukin alleles include a SMART-17A knock-in (KI) expressing human NGFR on IL17A expressing cells and a KI/KO of Il9-expressing iCre/EYFP. The latest reporter and cre-expressing strains include an inducible cre KO/KI to Lyz2, and a KI/KO strain with dual reporters of chemokine Ccr2RFP and Cx3cr1GFPactivity, and two strains expressing the INDIA apoptosis reporter, one with widespread conditional expression and the other with expression in germinal center B cells. KOMP mice, offered via the MMRRC portion of the JAX Mouse Repository, provide a growing number of targeted immunological mutations.

Donating a strain to the Repository fulfills NIH’s requirements for sharing mice. Researchers wishing to have mice considered for inclusion in the Repository are encouraged to submit strains: www.jax.org/donate-a-mouse.

This work is supported by NIH, HHMI and private foundations.
Novel therapeutic strategies to mitigate erectile dysfunction in a rat pelvic trauma model
Authors: Hertz Alexandria M , George Evalyn , Hibbert Joseph , Reed-Maldonado Amanda , Brand Timothy , Salgar Shashikumar
Published: January 2023

Introduction

Battlefield pelvic injuries can result in erectile dysfunction (ED) in young soldiers. We investigated whether phosphodiesterase-5-inhibitor (PDE5i), low-energy shock wave (LESWT) and/or mesenchymal stem cell (MSC) therapy could improve erectile function (EF) after pelvic injury.

Methods

Lewis rats 10–12 weeks old were divided into nine groups (n=6/group) - Sham, Injury (control), PDE5i, LESWT, MSC, LESWT+PDE5i, LESWT+MSC, MSC+PDE5i, and LESWT+PDE5i+MSC. Bilateral cavernous nerve crush injury and internal pudendal bundle ligation were performed. Bone marrow-derived MSCs (2x106cells) were injected in to the corpora cavernosum. Rats received LESWT (1000 pulses at 0.06 mJ/mm2, 3Hz, 3 times/week) or sildenafil (PDE5i) in drinking water (~10 mg/kg/day) for four weeks. At 6th week intracavernous pressure (ICP; mmHg) was recorded as EF outcome measure.

Results

Pelvic injury (control group) showed decrease (p<0.001) in EF based on maximum ICP (88±16), average ICP (81±11), and change in ICP (81±13) compared to sham (maximum137±25; average 125±13.0; change 55±24). EF was improved (P<0.001) with PDE5i+LESWT therapy, maximum ICP (131±14), average ICP (116±9), and change in ICP (63±15) compared to control. LESWT and MSC alone improved EF (P<0.05). RT-PCR demonstrated lower (21–87%) BCL-2, eNOS, epiregulin and connective tissue growth factor relative gene expression in control group compared to sham. MSC or LESWT+PDE5i treatment showed increased (69–87%) epiregulin (marker for smooth muscle) expression compared to control; eNOS was markedly higher in LESWT+PDE5i group. Histology/Molecular studies are ongoing.

Conclusion

LESWT, MSC and/or PDE5i combination therapy appears to be promising in EF recovery.
Use of modified peptides for live tracking of peptide-MHCII complexes during antigenspecific T cell-dendritic cell interactions
Authors: Al Souz Jafar A , Akkaya Munir , Kamdar Rahul , Kamenyeva Olena , Kabat Juraj , Shevach Ethan M , Akkaya Billur
Published: January 2023

The recognition of peptide-MHC class II complexes (pMHCII) presented by dendritic cells (DCs) is critical for the activation of CD4+ T cells, and proper detection of such complexes is essential for understanding T cell-DC interactions. However, current detection methods for pMHCII complexes using antibodies may block cellular interactions, limiting their use, while conventional tags, such as GFP, are too large to be loaded onto MHCII without disrupting its function. To probe pMHCII complexes, we constructed a variant of the ovalbumin peptide (OVA323–339) linked to a tetracysteine tag (CCPGCC), which can be treated with organoarsenic compounds to induce fluorescence. This modified peptide retained its functionality as it was able to stimulate T cells to the same degree as the unmodified peptide. We also analyzed signal specificity both at the pMHCII level, by using MHCII−/− APCs, and at a broader level by quenching endogenous tetracysteine expression, and confirmed that our fluorescent signal was derived from the peptide modification. We then used microscopy to visualize pMHCII movement after interaction of pulsed DCs with OVA-specific T cells in vitro and subsequently in vivo, and detected a gradual increase of pMHCII complexes at the synapse. Interestingly, we also observed a transfer of fluorescent signal from DCs to antigen-specific T cells, but not to polyclonal T cells, indicating acquisition of pMHCII, which we were able to quantify. Taken together, our results show a non-disruptive approach to assay pMHCII, and provide a window to observe intricate T cell-DC interactions in real-time.
REAlease® technology: Controlled release of antibody-fluorochrome conjugates for maximal flexibility in flow sorting applications
Authors: Evaristo Cesar , Pankratz Jennifer , Schmachtenberg Sabine , Jansen Nicole , Krauthäuser Susanne , Kinkhabwala Ali , Schlegel Karolina , Siewert Christiane , Bartholomäus Alina , Meiler Svenja , Yushchenko Dmytro A. , Dose Christian
Published: January 2023

Multi-parameter immunofluorescent labeling is the method of choice for the flow-based sorting of targeted cell populations from heterogeneous samples. However, existing antibody-fluorochrome conjugates used for flow sorting limit the types of downstream analyses that can be applied to the isolated cells, as their continued presence on the cells blocks specific cellular epitopes and restricts the choice of downstream fluorescence detection channels. Herein, we present a new type of antibody-fluorochrome conjugates that facilitates a highly specific multi-parameter cell staining for flow sorting as well as subsequent complete release of the conjugate probes from the sample. These new conjugates rely on recombinantly engineered antibody fragments that individually possess low epitope binding affinities. A tailor-made covalent conjugation chemistry allows for their multimerization as well as fluorochrome labeling, generating fluorescent probes comparable to conventional antibody conjugates. Importantly, upon addition of a release reagent, the antibody-fluorophore conjugates can be rapidly released from the cell surface. Accordingly, previously blocked epitopes are free for re-labeling, which provides maximal flexibility for renewed epitope targeting in downstream applications. The versatility of this technology is presented in multi-parameter panels enabling clear distinction and discrimination of different cell populations. The applicability of the new REAlease® antibody fluorochrome conjugates is also demonstrated by flow sorting of human naïve regulatory T cells.
Comprehensive Benchmarking of T-cell epitope prediction tools
Authors: Paul Sinu , Peters Bjoern
Published: January 2023

Computational prediction tools are commonly used to select T cell epitope candidates in several immunological applications including vaccine design, cancer neoantigen identification, development of diagnostics and removal of unwanted immune responses against protein therapeutics. Most of these prediction tools are based on machine learning algorithms trained on MHC binding data or naturally processed MHC ligand data. The ability of currently available tools to predict T cell epitopes has not been comprehensively evaluated. In this study, we use a recently published dataset that systematically defined T cell epitopes recognized in vaccinia virus infected mice to evaluate the performance of all currently publicly available computational T cell epitope prediction tools. Using the same data set across all prediction methods provides an unbiased performance evaluation. The derived performance metrics allow immunologists to rationally decide on which prediction method to apply for their work at hand.
Development of PureQuant Real-Time PCR-Based Assay for Assessment of Immune Cell Identity and Purity in CAR-T Workflows
Authors: pradhan suman , Guzman Jerry , Landon Mark , Dargitz Carl , Lakshmipathy Uma
Published: January 2023

Significant progress has been made in harnessing the power of immune system, in particular, T cells, to treat certain kinds of cancers. One of the key challenges in developing immune cells as therapeutic agents is the accurate estimation of their identity and purity. Current methods used for the characterization of immune cell types rely on flow cytometry. Flow cytometry can accurately estimate CD8+ T lymphocytes and other surface markers, but the method is challenging to implement in a GMP manufacturing environment posing logistical challenges such as requirement for live cells, variability leading to difficult in standardizing and high throughput. In addition, cytometric methods are not accurate for specific intracellular targets that positively identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Therefore, there is an emerging need for alternative assay methods. Methylation state is known to be unique for specific cell types and can thus be used as an identifier in heterogeneous population of cells. Exploiting differences in cell type-specific methylation signatures, we developed assay kits that quantify the percentage of Treg and Th17 by detecting methylation status of FoxP3 and IL17A via qPCR of bisulfite converted genomic DNA. In contrast to flow analysis, sample requirement is minimal and the assay works well with fresh/frozen cells or genomic DNA. This assay has been implemented to accurately identify and estimate different T cell population in Chimeric Antigen Receptor (CAR)-modified T cells. The combination of accuracy, low sample requirement and flexibility provides an ideal measurement system for confirmation of identify and purity of T cell types critical for therapeutic applications.
Buoyant microbubbles as alternatives or adjuncts to magnetic bead methods for high-purity cell isolation
Authors: Younger John G , Roussey Jon , Bao Jianjun , Co Nadia Petlakh , Jones Thomas , Ostruszka Leo
Published: January 2023

Widely used in immunologic applications, magnetic bead systems have significant limitations including cumbersome workflows, generation of high-shear and high-compressive forces during routine use, and limited performance in low and high volume samples. Buoyancy-activated cell sorting (BACS) overcomes these issues by using low-density microbubbles in lieu of magnets and iron beads to perform isolation. Here we report methods for three common uses: removal of extraneous RBCs prior to FACS, isolation of high-purity B lymphocytes from murine spleens, and improvement of a magnetic NK separation system by jointly applying BACS to remove contaminating NKT cells. RBC removal from leukopheresis-derived PBMC was performed with anti-CD235a-conjugated microbubbles. B cell isolation was performed with streptavidin microbubbles and a negative selection cocktail of biotinylated anti-murine antibodies. NK cell isolation was performed on PBMC using Miltenyi human CD56 MicroBeads and an additional purification step using streptavidin microbubbles and an anti-CD3 antibody. Each experiment used Akadeum microbubbles and a one-minute trituration to combine microbubbles with cell suspensions. RBC microbubbles provided 98.3 ± 1.0% removal of RBCs with 11.3 ± 0.9% nonspecific binding. B cells isolated with microbubbles were 91.8 ± 2.8% pure with a yield of 94.2 ± 4.3%. NK purification with magnetic microparticles produced 80.5 ± 0.7% purity. Additional purification with microbubbles increased purity to 90.8 ± 1.3%. Microbubbles are a flexible and simpler alternative to magnetic cell isolation systems, delivering comparable performance and, when used in conjunction with magnetic beads, superior purity.
Physical cognate pairing of mouseTCR alpha-beta VDJ-rearrangements with phenotype directly from FACS sorted cells
Authors: Brown Brittany E , Byrne-Steele Miranda , Pan Wenjing , Hou Xiaohong , Depinet Mollye , Eisenhower Mary , Han Jian
Published: January 2023

Phenotypic characterization of single cells paired with immune receptor information can provide valuable insight into T cell function, which may be beneficial for the development of personalized therapies. In order to comprehensively analyze T cell function and specificity, it is necessary to integrate antigen receptor sequencing with phenotypic markers at the single cell level. We therefore developed a sensitive technology called iPair+™ that enables the simultaneous amplification and sequencing of paired mouse TCR alpha and beta chains with functional gene markers in a single, sorted immune cell via a combination of flow cytometry, multiplex PCR, and next generation sequencing. We utilized iPair+™ to study CD4 and CD8 T cells isolated from treated and non-treated mice from a breast cancer model. CDR3 diversity, clonality, and frequency were analyzed together with 80+ phenotypic genes. When cells isolated from both treated and non-treated mice were compared, we found gene expression levels changed significantly with treatment. Our data demonstrates that genes that were upregulated prior to treatment were associated with cancer growth, while genes upregulated after treatment were associated with T cell activation and function in cancer repression or growth inhibition. Our study indicates that iPair+ technology could reveal important insights into T cell function, clonal development, and immunotherapy response during treatment.
Investigating the human immune response with Myriad RBM’s TruCulture system and the NanoString nCounter Autoimmune Profiling Panel
Authors: Elliott Nathan A , Hwang Shen-An , Bailey Michael , Bailey Christina , LaBrie Samuel , Warren Sarah
Published: January 2023

TruCulture is a whole blood collection and culturing system designed for in vitro modeling of human immune responses. TruCulture retains all circulating immune cells, soluble factors, and Fc receptor expressing cells within a closed culture environment. The NanoString nCounter gene expression platform enables direct, enzyme-free counting of mRNA molecules from samples such as blood and biofluids with a total assay time to results around 24 hours. The Human Autoimmune Profiling Panel assays 770 genes (35 unique pathways) involved in autoimmunity. Additionally, the panel includes a cell profiling feature for measuring the relative abundance of immune cell types as well as gene targets for over 30 approved and investigational therapies. Whole blood from healthy donors is collected and exposed to potent immunostimulatory agents (SEB or TNF-a) in TruCulture tubes. Cell pellets are collected for total RNA isolation and nCounter gene expression analysis. In parallel, culture supernatants are analyzed for secreted cytokines using the Myriad OptiMap profiling system. Unsupervised hierarchical analysis of transcriptional profiles differentiates blood samples based on treatment groups. Both single gene and pathway level analysis identified significant differences between the treatment groups. Replicate TruCulture blood tubes produce gene expression data with high technical reproducibility (R2 > 0.96). The data also demonstrates excellent correlation of RNA targets and protein abundance for key cytokines from the same blood sample. These results indicate the feasibility of combining TruCulture blood culture with NanoString gene expression analysis for the study of relevant pathological conditions from clinical samples.
Microchip platform for imaging-based efficacy testing of cells and reagents for immunotherapy
Authors: Onfelt Bjorn , Guldevall Karolin , Brandt Ludwig , Olofsson Karl , Carannante Valentina , Sandström Niklas , Wiklund Martin
Published: January 2023

Driven by a wave of new and improved methods for single cell analysis, e.g. flow cytometry, mass cytometry and single cell sequencing, it is increasingly recognized that there is a vast heterogeneity between individual cells within traditional cell classes. However, most of these techniques give a static picture of the molecular content in individual cells and provides limited information about plasticity or how the molecular content is translated into function, and it is easy to miss rare cell populations with functions important for mediating clinical effects. Current experimental approaches require large amounts of material, often impossible to obtain in clinical settings. Therefore, techniques that allow detailed functional evaluation of immune cells with single cell resolution from small samples are critically needed to advance assessment of therapies. We have developed a microchip platform for assessing cellular dynamics and function, with focus on assays for determining heterogeneity in cytotoxic response between individual NK or T cell. This microchip approach allows detailed characterization of thousands of individual cells within larger populations. By mounting an ultrasonic transducer on the chip we can also generate tumor spheroids that serve as model systems for studies of how NK and T cells attack solid tumors and respond to changes in the tumor microenvironment. Here I present this platform and give a few examples of how it has been utilized to generate mechanistic understanding of the cytotoxic process, classify cells based on response and to test efficacy of drugs for immuno-oncology and cells expanded for therapy.
Mapping leucocyte populations in mouse lymphoid tissues and blood using a 28-color panel on the BD FACSymphonyTM System
Authors: Baracho Gisele , Widmann Stephanie , Sisouvanthong Chad , Tyznik Aaron , Saksena Suraj
Published: January 2023

Multiparameter flow cytometric analysis of single cells allows the enumeration and characterization of heterogeneous cell populations in a variety of infections, blood cancers, immunodeficiencies and other disorders. Supported by advances in reagent development and instrumentation, this approach has increased dimensionality in single-cell data collection, and accelerated the discovery and testing of new therapies. Here we show a broad immunophenotyping panel that identifies the major leucocyte populations and various sub-populations from different mouse tissues. The panel can also be divided into focused modules for specific analysis of T cell, B cell or myeloid/natural killer cell lineages. BD FACSymphony combined with BD Horizon Brilliant Dyes allowed for high resolution identification of known leucocyte populations. Dimensionality reduction with t-SNE and automated clustering algorithms simplified cell analysis, enabling a faster and more efficient comparison of cell phenotypes across the tissues. Importantly, automated cell clustering analyses, while recognizing the predicted cell populations, also identified cell subsets missed during supervised analysis of the dataset. Thus, we describe a comprehensive single-cell profiling application capable of identifying well-characterized leucocyte populations and that has the potential to discover unique cell phenotypes.