bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-8 Reprograms Monocyte-derived Macrophages into M2-like States in Human Glioblastoma
Authors: Medina Stephanie , Hayes Madeline , Brockman Asa , Ihrie Rebecca , Irish Jonathan
Published: November 2024

Glioblastoma (GBM), the most common primary brain tumor in adults, remains incurable and has not yet benefitted from immunotherapies. Microglial and hematopoietic macrophages are the most abundant immune cells in GBM and play contrasting roles in the disease. Our prior studies revealed a signature of CD32 and HLA-DR protein expression and STAT3 signaling in hematopoietic macrophages associated with poor GBM patient survival and neural stem cell niche contact (PMC10371245). We hypothesized that GBM cells may secrete factors to remodel the cellular microenvironment and generate this signature macrophage subset from myeloid cells. In a series of experiments, 1) IL-8 was the primary cytokine expressed in vivo and ex vivo by human GBM cells (immunohistochemistry, cytokine arrays), 2) IL-8 was sufficient to instruct healthy monocytes to adopt the CD32+ HLA-DR+ signature of in vivo GBM macrophages (spectral flow cytometry, mass cytometry), and 3) IL-8 was required for primary GBM cell supernatant to polarize healthy macrophages into CD163+ CD206+ CD32+ HLA-DR+ M2-like cells in ex vivo 6-day cultures (α-IL-8 blocking antibodies, spectral flow cytometry). This ex vivo system will now be used to test the function of the GBM signature macrophages and to explore targeting of IL-8 to improve GBM therapy.
Novel Oral Treatment for Atopic Dermatitis: Allosteric Modulation of GABAA Receptor Signaling
Authors: Meyer Michelle , Webb Daniel , Lee Majia , Kowalczyk Ethan , Stafford Douglas , Arnold Leggy
Published: November 2024

Background: This study explores the development of a groundbreaking oral treatment for atopic dermatitis (AD) by investigating the effects of allosteric modulators targeting the gamma-aminobutyric acid type A (GABAA) receptor outside the brain.

Methods: To induce the AD phenotype, mice strains including Swiss Webster, Balb/c, and C57/BL6 received Calcipotriol (MC903) applied to both ears. The experimental groups were treated with either the novel GABAA receptor ligands, vehicle, or positive control compounds. Over a seventeen-day period, various parameters such as animal weight, ear thickness, and scratching behavior were monitored. Post-study, mice were euthanized, and gene expression and histology of their ears were examined. Statistical analysis employed a 2-way ANOVA.

Results: Our findings indicate a significant reduction in the induction of inflammatory genes especially for the thymic stromal lymphopoietin (TSLP) in response to the MC903-induced AD phenotype when treated with GABAA receptor ligands. A notable reduction in scratching behavior was observed, especially when treated after the establishment of the AD phenotype.

Conclusion: The administration of GABAA receptor ligands demonstrated a substantial reduction in ear inflammation, ear thickness, and ear scratching in the murine model for AD. This research provides promising insights into the development of a novel and effective oral treatment for AD.
IL-10 mitigates inflammation within the CNS during West Nile virus neuroinvasive disease
Authors: Halasah Nadeem , Kallingal Isha , Durrant Douglas
Published: November 2024

West Nile virus (WNV) causes a severe CNS infection in humans, primarily in the elderly and immunocompromised. Prior studies revealed that IL-10, primarily through the suppression of antiviral and proinflammatory cytokine production, predominately has a negative role in WNV immunity. However, in the absence of a critical DC-subset, IL-10-expressing lymphocyte infiltration was reduced in the WNV-infected CNS and excessive inflammation and immunopathology caused increased susceptibility to WNND. These results suggest a neuroprotective role of IL-10. Preliminary findings demonstrate CD8+ T cells as major IL-10 producers at day 12 post-infection compared with animals deficient in CD8a+DEC-205+ DCs. We intend to validate these observations by assessing IL-10 production in infiltrating cells expressing CD4, CD11b, and CD11c. Additionally, we will administer anti-IL-10 monoclonal antibody (mAb) post-WNV infection to neutralize IL-10's effects within the CNS. Monitoring survival and viral burden after intracranial injection of aIL-10 mAb or control IgG, our study seeks to elucidate IL-10's protective role in WNND by mitigating inflammation.
The molecular program of CD4+ regulatory and effector T cell expansion in vitro
Authors: Moro Alejandro , Yu Aixin , Nivelo Luis , Villarino Alejandro , Malek Thomas
Published: November 2024

Adoptive Treg therapy is being advanced to treat autoimmunity. To discern the molecular pathways underlying Treg expansion in vitro, we evaluated the chromatin landscape and gene expression profiles of Treg and Teff cells in response to TCR/CD28/IL-2 priming followed by IL-2 expansion over 12-days. Although initially similar over the first 6 days, Tregs showed somewhat higher STAT5 activation and chromatin opening, leading to optimal expansion on day 6, which was 2-fold greater than Teff cells. After day 6, Tregs, but not Teff cells, showed substantial reduction in expansion. At day 9 and 12, more marked changes in gene expression and chromatin accessibility at promoter regions were seen between Treg and Teff cells. PCA revealed for RNAseq and ATACseq a clockwise movement, where the day 12 cells expressed a profile that reverts toward the input populations. Temporal integration of differentially expressed genes and chromatin accessibility revealed 6 patterns of responses, 4 of which were similar between Treg and Teff cells. The glycolytic activity in Treg and Teff cells during expansion until day 6 was similar but then they declined more rapidly in Tregs. Hence, these differences in gene accessibility and expression and glycolysis likely contribute to the more rapid decay in Treg expansion in response to IL-2. Thus, Tregs are programmed temporally for more limited expansion in vitro that may reflect a mechanism to finely tune Treg numbers to maintain homeostasis in vivo.
Umbilical Cord Mesenchymal Stromal/Stem Cells Interplay with Th-17 Cell Response Pathway
Authors: Alsabri Sami , Najar Mehdi , Rahmani Saida , Faour Wissam , Lombard Catherine , Fayyad-Kazan Hussein , Sokal Etienne , Merimi Makram , Fahmi Hassan
Published: November 2024

Umbilical cord derived mesenchymal stromal/stem cells (UC-MSCs) were assessed, as an immunomodulatory therapeutics, for their dynamic interaction with the Th-17 immune response pathway. UC-MSCs were able to modulate lymphocyte response by promoting a Th-17-like profile. Such modulation depended on the cell ratio of the co-cultures as well as the presence of an inflammatory setting. UC-MSCs significantly increased the expression of IL-17A and RORγt but differentially modulated IL-23R expression by T cells. In parallel, the secretion profile of the fifteen factors (1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-22, IL-21, IL-23, IL-25, IL-31, IL-33, INF-γ, sCD40, and TNF-α) involved in the Th-17 immune response pathway was substantially altered during these co-cultures. Upregulation of cytokines and regulatory mediators demonstrates the capacity of UC-MSCs to sense and actively respond to tissue challenges. Protein network and functional enrichment analysis indicated that several biological processes, molecular functions and cellular components linked to distinct Th-17 signaling interactions are involved in several trophic, inflammatory and immune network responses. These immunological changes and interactions with the Th-17 pathway are likely critical to tissue healing and may help to identify molecular targets that will improve therapeutic strategies involving UC-MSCs.
Label-free single cell metabolic imaging of neutrophil activation
Authors: Datta Rupsa , Miskolci Veronika , Britt Emily , Fan Jing , Huttenlocher Anna , Skala Melissa
Published: November 2024

Upon activation, neutrophils combat infiltrating pathogens through a variety of effector functions, including oxidative burst, phagocytosis, and neutrophil extracellular traps (NETs). Metabolic pathways within neutrophils can be pharmacologically affected to modify immune outcomes. However, there is a lack of techniques to assess neutrophil metabolism in a label-free, non-destructive manner at single cell level. To address this, human neutrophil metabolism was investigated using two-photon microscopy of intrinsic fluorophores reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, and flavin adenine dinucleotide, FAD. We image the optical redox ratio (ORR), [NAD(P)H / (NAD(P)H + FAD)] and the fluorescence lifetimes of NAD(P)H. Single-cell metabolic profiles of neutrophils were generated using an automatic cell segmentation pipeline. We found that activation of neutrophils either pharmacologically or by pathogen infection induced a significant metabolic change characterized by an increase in ORR and decrease in NAD(P)H lifetime, indicating an increase of the NAD(P)H pool, which suggests increases in glycolysis and the pentose phosphate pathway. Neutrophil activation was confirmed by measuring effector functions including oxidative burst. Importantly, identical metabolic changes were detected in the neutrophils of transgenic zebrafish larvae following activation, confirming the validity of these findings in vivo.
Deficiency of Myeloid PP4 in mice exacerbates Neutrophil Hyperactivation during Sepsis
Authors: Yang Feng-Ming
Published: November 2024

We previously demonstrated that serine/threonine phosphatase PP4 functions as a negative regulator of innate immunity, prevents hyperactivation of Toll-like receptors (TLRs) signaling in sepsis. Because the recruitment of monocytes to sites of inflammation is critical for host defense, we then explored the role of PP4 in myeloid lineage cells. Mice without PP4 only in myeloid cells were more susceptible to sepsis due to serious tissue damage. Importantly, CLP induced myeloid PP4 deficiency mice produced significantly more proinflammatory cytokines and chemokines, which contributed to dysregulated neutrophil recruitment. Neutrophils isolated from mice without PP4 showed an increase in NETs formation in response to subsequent RANTES stimulation. Mechanistically, PP4 regulated RANTES production via TBK1/IRF3 axis in macrophages. Finally, RNA-seq analysis of the livers obtained from myeloid PP4 deficiency mice relative to wildtype controls identified 1849 differentially expressed genes. Of these, 628 were upregulated and 1221 were downregulated. Moreover, 25 significantly enriched GO terms and 1 significantly enriched KEGG pathways were identified, involving multicellular organismal homeostasis, regulation of phosphorylation and inflammation response, and cytokine-cytokine receptor interaction, which might also play roles in the process of sepsis. Thus, our data provide a remarkable new resource of regulatory pathways for understanding PP4 function at the organism level.
Proteostasis maintenance by macrophage trogocytosis
Authors: Kimura Yoshitaka , Philip Naomi , Chavan Arun , Medzhitov Ruslan
Published: November 2024

Cells are frequently exposed to a wide variety of stresses that perturb cellular fitness. Stressed cells autonomously drive the defense mechanism to maintain their homeostasis, such as DNA repair program upon DNA damage and chaperone induction and protein degradation pathway upon protein misfolding. However, how cell-to-cell interaction contributes to the maintenance of cellular fitness has been elusive. Here, we show that macrophages interact with proteotoxically stressed cells and promote their proteostasis. Cells undergoing protein aggregate accumulation are detected by macrophages, and a part of their body is taken up via trogocytosis. In this process, protein aggregates in stressed cells are transferred to macrophages, leading to the reduction of protein aggregate load in stressed cells. Mechanistically, the macrophage detection and uptake of protein aggregates requires the stressed cells to express membrane-associated isoform of a macrophage growth factor CSF1 (mCSF1), which is upregulated by endoplasmic reticulum stress response pathways. The analysis of mCSF1-deficient mice in systemic proteotoxicity model reveals mCSF1 suppresses cellular damage in tissues, suggesting the trogocytosis-mediated removal of protein aggregates contributes to cellular homeostasis in vivo. These findings point to the fundamental cellular stress responses to induce full-blown proteostasis through the communication to immune cells in cooperation with autonomous responses.
A novel anti-inflammatory function of Thy1 stromal cells in colitis
Authors: Torres-Huerta Alvaro , Boger-May Antonia , Ruley Haase Katelyn , Reed Theodore , Mysore Rajashekara Arpitha , Hiller Morgan , Mayer Lauren , Frech Maddy , Kenney Kaylee , Kane Grace Rowe , Elliott Kyle , Schmidt Annie Cate , Boone David
Published: November 2024

Background: Inflammatory Bowel Disease (IBD) is a complex immune disorder affecting the gastrointestinal tract. Stromal cells play a crucial role modulating the immune response. We evaluated the function of the Thy1 positive stromal population, specifically elucidating its role in the context of the innate immune system within the framework of IBD.

Methods: We created a mouse model that develops 100% penetrant, early-onset colitis in the absence of adaptive immunity. Transgenic mice expressing the TNFAIP3 (A20) gene under the villin promoter, (Villin-tgA20), crossed with RAG1-/- mice (villin-tgA20 x RAG1-/-; aka TRAG) develop innate colitis. To study ILCs and Thy1 functions in colitis we used IL2Rg-/- and Thy1-/- mice, crossed with TRAG (IL2Rg-/- x TRAG, and Thy1-/- x TRAG). IL2Rg-/- x TRAG, and Thy1-/- x TRAG were injected with anti-Thy1 antibody and assessed for prevention of colitis.

Results: In absents of ILCs (IL2Rg-/- x TRAG) and Thy1 (Thy1-/- x TRAG) colitis persists. Treatment with anti-Thy1 antibody prevents colitis in TRAG and IL2Rg-/- x TRAG, but not in Thy1-/- x TRAG, mice. Colitis in TRAG, IL2Rg-/- x TRAG, and Thy1-/- x TRAG, is characterized by an abundance of neutrophils and inflammatory monocytes. In the colon of IL2Rg-/- x TRAG mice we observed a Thy1 positive population that expressed podoplanin but not CD31. Thus, Thy1 is a functional molecule and stimulation of stromal cell Thy1 induces an anti-inflammatory response that prevents IBD.
IL-33-mediated mast cell function is female dominant
Authors: Maldonado Tania , Ryan John , Kee Sydney , Ching John
Published: November 2024

Allergic asthma is a common chronic disease characterized by airway inflammation and bronchoconstriction. Asthma is more common and more severe in women than in men. One component of allergic inflammation is IL-33, a cytokine released by airway epithelium when these cells are damaged by allergens. Using two models of IL-33-mediated inflammation, we have found that IL-33 elicits a stronger response in female mice than males. These differences are reversed when using castrated mice. Castrated females show reduced IL-33 responses than controls, while castrated males show enhanced responses. IL-33 is a potent mast cell activator in these models. We find that mast cells cultured from female mice respond more strongly to IL-33 than male mast cells. These data support the hypothesis that IL-33-mediated inflammation exhibits female-dominant sexual dimorphism. Further this dimorphism is linked to sex hormones and may be partly explained by IL-33 effects on mast cells.
IL-27 signalling increases lung injury in murine beta-coronavirus and Influenza infection
Authors: Worley Matthew , Gong Henry , Carver Kyle , Deng Jane
Published: November 2024

Introduction: IL-27 is elevated during COPD, ARDS and high levels are predictive of severe SARS-CoV-2 infection. Despite the association with severe pulmonary outcomes, little is understood on how it affects lung injury or cellular recruitment early in viral pulmonary infection.

Methods: C57BL/6 (WT) and IL-27ra-/- mice were infected with 500,000PFU of murine beta-coronavirus (MHV-1) or 500PFU of Influenza (H1N1) and evaluated for survival. At days 3 and 5 post infection kinetics of cellular recruitment, viral load and markers of lung injury were evaluated.

Results: IL-27ra-/- mice have increased survival compared to the WT mice when infected with MHV-1 (p= 0.0184) and H1N1 (p=0.0305). Despite no differences in viral load, the IL-27ra-/- had decreased lung injury at days 3 and 5 post infection (p<0.05) for both MHV-1 and H1N1. IL-27ra-/- mice had significant neutrophilia during infection at day 3 and 5 (p<0.05) but with altered neutrophil phenotypes (adhesion, activation and maturation markers) including decreased expression of TLR3, TLR4 and CD49d (p<0.05 for all).

Conclusion: The IL-27ra-/- mice are resistant to pulmonary infection with MHV-1 and H1N1 with increased survival but no differences in viral load. Despite high pulmonary neutrophilia in both infections, the IL-27ra-/- mice paradoxically have decreased lung injury. This study highlights IL-27 as a potential early target for altering neutrophil phenotypes to limit lung injury during respiratory viral infections.
Activation of S1P/SK2 pathway in classical monocytes enhances anti-PD-1 immunotherapy
Authors: Krieg Carsten , Ball Lauren , Orgetmen Besim , Hardiman Gari , Gan Wenjian , Robinson Mark , Guglietta Silvia
Published: November 2024

Tumor-associated macrophages (TAMs) often impede immunotherapy success by promoting an immune-suppressive environment in the tumor. Depleting TAMs directly has shown limited success in clinics due to challenges in precise targeting and avoiding toxic side effects. An alternative approach involves targeting circulating monocytes (CMs), the precursors of TAMs. In inflammatory conditions like cancer, CMs replace exhausted tissue-resident macrophages. Our study found that anti-PD-1 responders had elevated CM numbers before treatment, correlating with increased anti-tumorigenic macrophages in tumors. Analyzing CMs revealed higher sphingosine 1-phosphate (S1P) levels in responder patients, crucial for immune cell bioenergetics. Preliminary experiments using S1P/SK2 treatment improved CM function, supporting T cell proliferation and enhancing checkpoint inhibition therapy response. Exploring the S1P/SK2 pathway's impact on CM-derived macrophages and in vivo behavior is ongoing. Targeting the S1P/SK2 pathway to reprogram CM for anti-tumorigenic macrophage generation epigenetically shows promise in enhancing cancer immunotherapy efficacy, with potential implications for treating diverse diseases beyond cancer.
The Role of Recirculating Thymic T regulatory Cells in Regulation of de novo T reg Development and Central Tolerance
Authors: Joo Sookyong , Owen David , Farrar Michael
Published: November 2024

AIRE+ medullary thymic epithelial cells (mTECS) play a key role in establishing immune tolerance. These cells peak 2-3 weeks after birth and then steadily decline as mice get older. The mechanism by which AIRE+ mTECs decline remains unclear. We observed that a subset of recirculating thymic T regulatory cells (RT-Tregs) accumulates in the thymus at the same time that AIRE+mTECs decline. Using scRNA-Seq we found that RT-Tregs are quite diverse and may have cytotoxic potential. We propose that RT-Tregs play an essential role in balancing the stringency of T cell selection in the thymus by regulating mTEC abundance. To test this hypothesis we injected diphtheria toxin (DT) into the thymus of FOXP3-DTR mice, which selectively deplete RT-Tregs, but not de novo generated thymic Tregs or peripheral Tregs. This treatment led to a substantial increase in the number of AIRE+ mTECs. Using previously published datasets, we observed that the TCR diversity of conventional T cells (Tconv) in lymphoid organs of AIRE-/- mice was significantly increased relative to that of WT mice. We are currently testing whether depletion of RT-Tregs results in the opposite phenotype - a less diverse Tconv repertoire. We propose that early in life AIRE+ mTECs promote self-tolerance. RT-Tregs then act in a feedback loop to signal that peripheral tolerance has been established and eliminate AIRE+ mTECs resulting in a more diverse TCR repertoire that may be more self-reactive but also more pathogen-reactive.
Autoimmune diseases (AIDs) in Côte d’Ivoire West Africa
Authors: Siransy Liliane , Moussa Y Salimata , Doris Fleur Oura Brou , Adou Honoré , Sery Jocelyne , Dasse Sery Romuald
Published: November 2024

Introduction

Autoimmune diseases (AIDs) are until now very well unknown in Africa and particularly in subsaharien countries. They were even considered non-existent .

The objective of this work is to estimate the prevalence, describe the epidemiological, clinical and biological aspects of AID in our country.

Methods

This was a descriptive retrospective study conducted from January 1, 2010 to December 31, 2020, on the files of patients hospitalized in the rheumatology department of the University Hospital of Cocody in Abidjan, Côte d’Ivoire.

Results

We studied 116 patients including 104 female patients (90%) and 12 male patients (10%). Most systemic autoimmune diseases have been observed in patients aged from 31 to 45 years.

The average age was 40.02 +/- 15.07 years (extreme 12 and 72 years). The main diagnoses retained were systemic lupus erythematosus (34%), rheumatoid arthritis (22%), mixed connectivites (27%), sclerodermia(13%) .

Of the 116 patients, 82% were not tested, 4% were negative and 14% were found positive for antinuclear antibodies, the most common biomarker in autoimmunity.

Conclusion

AIDs are prevalent in our countries with various presentation in our patients and they occur in young adults. Autoimmune disease are prevalent in our country and increasing. Further studies and a national regsitry of autoimmune disease need to be established to understand the disease and its increase in african environnement.
Mitochondrial transcription is essential for the potent antitumor stem memory T cells
Authors: Kumaresan Soundharya , Rangel Rivera Guillermo , Knochelmann Hannah , Smith Aubrey , Ruffin Ayana , Cole Anna , Wittling Megen , Wyatt Megan , Lowe Michael , Lesinski Gregory , Paulos Chrystal
Published: November 2024

Adoptive T cell transfer (ACT) therapy is an effective approach to treat cancer patients, where expanding the patient’s T lymphocytes and redirecting them to recognize and regress solid tumors. Unfortunately, ACT can fail in some patients due to exhaustion or impaired metabolic fitness during T cell expansion. We discovered through transcriptomic analysis that the novel mitochondrial transcription regulator REXO2 induced by regulating PI3Kδ signaling in vitro - generated stem memory T cells (TSCM) - is responsible for the antitumor potency of TSCM cells. This finding is novel, as REXO2 has never been studied in immune cells and has only recently been appreciated in regulating heart muscles during embryonic development. We found that knocking out REXO2 via CRISPR in TSCM cells impaired their mitochondrial transcription and morphology in vitro. Moreover, they had reduced metabolic fitness marked by poor spare respiratory capacity, reduced mitochondrial potential and increased production of reactive oxygen species post tumor antigen recognition. Of clinical importance, REXO2 knockout in TSCM cells impaired the antitumor potency when infused into mice. This work also has translational potential, as REXO2 could be induced in human TIL or CAR T cell products by expanding them in the presence of PI3Kδ inhibitor. Our findings reveal that REXO2 is a promising target to bolster the antitumor activity of next generation TIL and CAR T cell products to treat cancer patients.
Dysfunction of exhausted T cells is enforced by MCT11-mediated lactate metabolism
Authors: Peralta Ronal , Delgoffe Greg
Published: November 2024

CD8+ T cells are critical mediators of antitumor immunity but differentiate into a state of dysfunction, known as exhaustion, after experiencing persistent TCR stimulation and metabolic stress in the tumor microenvironment (TME). The TME creates an immunosuppressive metabolic environment via reduced levels of vital nutrients and oxygen combined with increased catabolic waste products. How Texh cells survive and function in the face of this altered metabolite balance remains unclear. Here we show Texh cells upregulate a newly characterized monocarboxylate transporter, MCT11, enabling uptake of lactic acid which can drive their dysfunction. MCT11 expression requires both the core exhaustion program induced through chronic activation and metabolic stress signals, like hypoxia. Conditional deletion of MCT11 in T cells reduced lactic acid uptake in Texh cells and improved effector function and response to immunotherapy. Further, a monoclonal antibody targeting MCT11 reduced lactate uptake specifically in Texh cells, which, when used therapeutically in tumor-bearing mice, resulted in significantly reduced tumor growth, inducing complete regressions. Our data support a model in which the exhaustion program upregulates MCT11, rendering exhausted T cells sensitive to toxic lactic acid. MCT11 represents an attractive target to edit in therapeutic T cells or block using antibodies to render Texh cells impervious to lactic acid, allowing for tumor eradication.
B cell Maturation Antigen Impacts Microglia and Macrophage Phenotypes in Neuro-autoimmune disease.
Authors: Kumar Gaurav , Massey Kaylea , Ko Rose , Axtell Robert
Published: November 2024

BAFF and APRIL, crucial to B cell function, play a role in multiple sclerosis (MS), with therapeutic blockade linked to worsened disease. These cytokines signal through the receptors: BAFFR, TACI, and BCMA. BCMA stands out in MS due to elevated soluble levels in the cerebrospinal fluid. Our prior study using EAE revealed increased disease severity in BCMA knockout mice. Additionally, BCMA showed divergent roles in response to anti-CD20 and TACI-Fc treatments in EAE, enhancing anti-CD20 therapy responsiveness but diminishing TACI-Fc efficacy. The contrasting effects of BCMA on these treatments led us to explore its function in cell types beyond B cells. Here, we report that BCMA is highly expressed on microglial cells, and infiltrating Ly6C+ myeloid cells in the CNS of wildtype mice with EAE. Additionally, we found that BCMA-/- mice exhibited altered CD86 expression on microglial cells and elevated iNOS on infiltrating macrophages, suggesting a role in skewing towards an M1 phenotype in the CNS during EAE. In the lymph nodes, we also observed a similar effect on the M1-type macrophages in BCMA-/- mice compared to BCMA+/+ mice. These findings offer evidence of a novel role for BCMA in modulating microglia and macrophage function, providing additional insights into the intricate role that BAFF and APRIL play in neuroautoimmune diseases.
DB-2304, a Duality Immune Modulating Antibody‒Drug Conjugate (DIMAC) targeting BDCA2, displays strong potency in the suppression of pDC functions.
Authors: Li Xi , Li Bing , Yao Jun , Liu Shenxue , Shi Rong , Zhang Yu , Zhu Zhongyuan , Qiu Yang , Hua Haiqing
Published: November 2024

Study Objective: BDCA2 is specifically expressed on plasmacytoid dendritic cells (pDCs), whose overproduction of type I IFN is crucial in SLE pathogenesis. Combining anti-BDCA2 mAB with glucocorticoids (GCs) has shown clinical efficacy. Previous studies revealed superior anti-inflammatory effects of anti-BDCA2 antibody‒drug conjugate (ADC), motivating the development of DB-2304, an anti-BDCA2 ADC using a novel glucocorticoid receptor agonist as a payload. This study assessed the preclinical efficacy and safety of DB-2304.

Methods: A novel BDCA2 mAB, Hu033-03, was developed and conjugated with the DIMAC payload to generate DB-2304. The inhibitory potency of DB-2304 was evaluated in human PBMCs in vitro. The PK and safety profile and pDC function suppression were characterized in cynomolgus monkeys.

Results: Hu033-03 exhibited high affinity for BDCA2, inducing rapid internalization upon binding. DB-2304 showed enhanced suppression of IFNa production and a broader spectrum of proinflammatory cytokines in human PBMCs without cytotoxicity. Reduction in pDC cytokine production capability and surface BDCA2 level occurred in an ADC serum concentration-dependent manner. DB-2304 exhibited a favorable safety profile, with an HNSTD of 125 mg/kg in cynomolgus monkeys.

Conclusions: DB-2304 has demonstrated potent in vitro and in vivo anti-inflammatory effects on type I IFN genes and proinflammatory cytokines in pDC. These findings strongly support future clinical investigations of DB-2304.
Preclinical evaluation of Janus Kinase inhibitors in atopic dermatitis: Insights from an oxazolone-induced mouse model
Authors: Zhang Xiaotuan , Wang Jingjing , Zhang Ya , Lu Dan , Gu Qingyang , Zhang Qiyao
Published: November 2024

Atopic dermatitis (AD) is a prevalent inflammatory skin condition affecting a substantial proportion of the population. However, the lack of robust preclinical evidence of Janus kinase (JAK) inhibitors in AD treatment necessitates the development of more effective therapies, including those repurposed from other indications. Thus, we aimed to establish a preclinical evaluation system targeting JAK inhibitors to explore their effectiveness and mechanism of action in AD mouse model.

We utilized an oxazolone (OXA)-induced AD mouse model to assess the effects of JAK inhibitors. Ear thickness, IgE and MPO levels were measured regularly. At the end of study, spleen weights were measured, and ear tissues were processed for cytokine analysis and pathological evaluation. Additionally, flow cytometry was utilized to analyze immune cells in auricular draining lymph nodes (DLN).

Following OXA challenge, temporal changes in the immune system were observed, characterized by increased pro-inflammatory cytokines, immune cell infiltration in auricular DLN, and elevated IgE and MPO levels. Treatment with JAK inhibitors led to a reduction in AD-associated features and resulted in decreased immune cell infiltration and alterations in specific molecular features.

Our study successfully established a preclinical mouse model for AD, facilitating a comprehensive understanding of skin inflammation, immunomodulation, and the pharmacodynamics of test articles in the context of AD.
Inhibition of p38α MAPK-MK2 axis alleviate inflammatory response in various inflammation-related disease models
Authors: Han Tian , Han Tian , Fu Donglin , Hu Zhilong , Xia Yuanfeng , Yang Fanglong
Published: November 2024

Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MK2) is activated by p38 MAPK and regulates stability of mRNAs encoding inflammatory cytokines. Preclinical studies indicate that targeting MK2 may be a much safer approach than blocking p38a MAPK activation because that dissect the pathway downstream of p38α MAPK without affecting additional cellular functions. 141-A was identified as a highly potent and selective p38α MAPK-MK2 allosteric inhibitor that exhibited nanomolar-level inhibition on p38α MAPK activation of MK2 while sparing the p38α MAPK activation of ATF2 and PRAK. 141-A potently reduced various cytokine production (TNFα, IL-1β, IL-6, IL-8 and IL-17) in human peripheral blood mononuclear cells (PBMC) and displayed excellent efficacy in adjuvant-induced arthritis (AIA), psoriasis and MSU-induced air pouch rat models with 1/6 of the systematic exposure of CDD450. The compound exhibits favorable physicochemical and pharmacokinetic properties, along with an excellent preclinical toxicity profile, which may enhance therapeutic efficacy without causing side effects. 141-A is currently under IND-enabling studies and P1 trial is planned in early 2025.
CD11d, an NK cell ally in their fight against tumours
Authors: Bourel Capucine , Al Khaldi Maher , Dubuissez Marion , Chabot-Roy Geneviève , Mullins-Dansereau Victor , Guimont-Desrochers Fanny , Coderre Lise , Lombard-Vadnais Félix
Published: November 2024

NK cells participate in viral clearance and tumour control. They differentiate from bone marrow precursors and undergo a series of guided differentiation steps to acquire various functional properties. We previously showed that pre-mNK cells represent one of these precursors and that a locus on mouse chromosome 7 is linked to pre-mNK abundance. As pre-mNK cells are associated with a heightened anti-tumour activity, we sought to identify genes within this locus that influence their number, and could thus affect NK cell-mediated tumour growth. Through a candidate gene-based approach, we identify Itgad, encoding for CD11d, a member of the ß2 integrin family. We find that the absence of CD11d affects the expression of the other ß2 integrin subunits, particularly on NK cells. This led us to investigate the functional outcome of the loss of CD11d expression in NK cells. Although NK cells from CD11d-KO mice show unabated expression of effector proteins in vitro, their anti-tumour activity is impaired in vivo. Indeed, the growth of NK-sensitive RMA-S lymphoma is significantly accelerated in CD11d-KO mice. This increase in tumour growth was associated with a reduction in the number of NK cells in the tumours, suggesting a role for ß2 integrins in NK cell migration. Altogether, our candidate gene-based approach determined that expression of ß2 integrin subunits are co-regulated in NK cells and revealed an important role for CD11d in NK cell-mediated anti-tumour activity.
Single-cell RNA sequencing unveils metabolic and transcriptional dynamics in CD8+ T cells of Autoimmune Hepatitis murine model, offering insights into potential therapeutic targets.
Authors: Dolasia Komal , Centa Monica , Giotti Bruno , Ughetta Mimi , Alexandropoulos Konstantina , Tsankov Alexander
Published: November 2024

Autoimmune hepatitis (AIH) is a chronic liver disorder characterized by immune-mediated hepatocellular damage, potentially leading to liver failure, and necessitating liver transplantation if uncontrolled. Current treatments, primarily immunosuppressive drugs, are hampered by variable efficacy, significant adverse effects, and risks of non-responsiveness or relapse, underscoring the need for novel therapeutic strategies. In AIH, the inflammatory liver infiltrate predominantly consists of α/β T cells; moreover, recent findings highlight a significant contributory role of CD8+ T cells in AIH pathogenesis. Utilizing Traf6ΔTEC mice that exhibit a liver-centric autoimmune response characterized by mTEC (murine thymic epithelial cells) depletion due to conditional Traf6 deletion in thymic epithelial cells, we conducted a single-cell RNA sequencing (scRNA-seq) analysis of CD8+ T cells. This analysis elucidated diverse cellular states and functional dynamics within these cells and a preponderance of exhausted CD8+ T cells. Furthermore, flow cytometry was employed to confirm the presence of identified CD8+ T cell subtypes. Our study identified unique metabolic profiles and transcription factors differentially expressed in CD8+ T cells, shedding light on potential disease-driving mechanisms. These insights lay the groundwork for the discovery of new therapeutics aimed at modulating specific metabolic and transcriptional pathways in autoimmune hepatitis.
B Cell Leukemia Elicits a Suppressive Tr1-like CD4-T cell Response that can be circumvented with PDL1-blockade
Authors: Venkatesh Hrishi , Tracy Sean , Qiu Yinjie , Knutson Todd , Farrar Michael
Published: November 2024

Although inhibitory receptor (IR)hi CD4-T cells are associated with relapse in B cell leukemia (B-ALL), how they facilitate relapse is not understood. Using a murine B-ALL model, we show that leukemia induced IRhi CD4-T cells express genes associated with suppressive FOXP3- Tr1 CD4-T cells. These CD4-T cells do not have epigenetic silencing of effector cytokines, suggesting a functional state distinct from exhaustion. While anti-PDL1 or nilotinib (targeted therapy) alone had a minimal impact on survival, concurrent treatment with anti-PDL1 and nilotinib had a significant CD4-T cell-dependent synergistic effect. In contrast, IL10R blockade and nilotinib had a minimal impact on survival. IL10R blockade did improved the effector function of leukemia-specific CD4-T cells, but also impairing their long-term maintenance. Treatment with nilotinib and anti-PDL1 promoted the expansion of leukemia-specific CD4-T cells with lower IL10 expression and higher expression of helper molecules (CD40L, CCL4). The expansion of these CD4-T cells correlated with an increase in GzmB+ CD8-T cells, and resulted in a memory response that protected mice from a leukemia re-challenge. Nilotinib and IL10R blockade improve CD4-T cell function while also impairing their persistence, resulting in sub-optimal leukemia control. In contrast, nilotinib and anti-PDL1 induce CD4-T cells with a lower level of IL10 and improved helper function-this results in a potent and durable anti-leukemia T cell response.
NAC1 deficient regulatory T cell support tumor growth by CD36 mediated metabolic adaptation in tumor microenvironment.
Authors: Kumar Anil
Published: November 2024

In regulatory T cells, NAC1 enhances the acetylation of FoxP3 and protect from proteasomal degradation. Elevated levels of FoxP3+ Tregs within the tumor microenvironment (TME) showed a positive correlation with poor prognosis in various cancer patients. To address this dichotomy, we studied the role of NAC1 deletion on tumor growth. Here we report that NAC1 deficiency support tumor growth by enhanced infiltration of NAC1 deficient Tregs cells. This study confirmed that NAC1 deficient intra-tumoral Tregs are metabolically more able to adapt to acidic and nutrient deficient tumor microenvironment through enhanced FoxP3 expression mediated increased lipid metabolism and mitochondrial biogenesis. These results revealed that the NAC1-FoxP3-CD36–PGC1a signal sustains survival and functional fitness in intratumoral Treg cells by modulating mitochondrial fitness, lipid metabolism and oxidative phosphorylation driven protection from lipotoxicity and metabolizing lactate in the TME. These results highlight the unexplored NAC1-FoxP3-CD36–PGC1a modulated metabolic adaptation, which allows intratumoral Treg cells to utilize lactate and fatty acid in tumors. In adoptive Tregs transfer experiment we confirmed that Treg specific NAC1 deficiency is sufficient and a key factor to support tumor initiation and growth. This study that targeting metabolic adaptation in intratumoral Treg cells would be a hopeful strategy for reprogramming the TME.
Comparative Analysis of PD-1, PD-L1 and CTLA-4 Expression in Infiltrating Leukocytes in Tumor Tissue and Peripheral Blood in Patients with Renal Cell Carcinoma.
Authors: Gonzalez Garza Raquel , Salinas Carmona Mario , Gutierrez Gonzalez Adrian , Flores Gutierrez Juan , Castro Corona Maria , Mejia Torres Manuel
Published: November 2024

Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of kidney cancer. Despite the availability of treatment options, the overall prognosis for ccRCC remains poor, mainly for the acquired resistance to conventional therapies. Current evidence indicates that the immune system is indirectly involved in tumor progression, where T cells play a critical role in antitumor immunity. In addition, the functional state of infiltrating leukocytes predicts response to therapy in most types of cancer. In the present study, we describe the nature of infiltrating leukocytes in tumor tissue and the exhaustion-associated markers PD-1, PD-L1 and CTLA-4 in blood and renal tissue obtained from patients diagnosed with ccRCC. Our results indicated that within the tumor microenvironment, the main source of inhibitory molecule PD-L1 are the infiltrating monocytes whereas the most expressed inhibitory marker in lymphoid cells are PD-1 in CD8+ cells, but CTLA-4 in CD4+ cells. More importantly, the phenotype remains consistent across central and peripheral tissues, suggesting that exhaustion-associated markers could be measured in blood, which is a less invasive alternative to conventional histopathologic evaluation.
Digital PCR as a novel way to assess chimerism of humanized immune system mice
Authors: Smith Nicholas , Hansen Sarah , Livingston Robert , Smith Steve , Roesch Paula , MacBride Megan
Published: November 2024

Humanized immune system (HIS) mice contain human and murine immune cells. Analysis of chimerism in HIS mice is typically accomplished via flow cytometry, but this requires collecting a 75 µl blood sample and access to a flow cytometer. Repeated bleeding can negatively affect the health of HIS mice, limiting serial analysis. Digital PCR offers a novel method for chimerism analysis, which uses much smaller blood volumes and can be performed on clotted or frozen samples, sample types unsuitable for flow cytometry. A digital PCR assay directly comparing the presence of three human genes and one murine gene was developed. This assay was validated in the huNOG-EXL HIS mouse engrafted with CD34+ hematopoetic stem cells from 3 unique donors via blind comparison with chimerism data from flow cytometry. At 10 weeks post engraftment, the digital PCR assay showed excellent correlation for all three human genes against chimerism as measured by flow cytometry of the peripheral blood. This assay represents a new, user-friendly, rapid tool for the analysis of HIS mice and may permit serial chimerism measurement using 10 μL whole blood. This assay may be useful to investigate other mouse-human chimera models, including mice harboring functional human hepatocytes, tumors, and other cells.
Development of a T-cell activation panel using image cytometry in rheumatoid arthritis patient samples
Authors: Chan Leo , Stuckey Tyanna , Pierce Mackenzie , Musgrove Blessing , Hernandez Veronica , Lin Bo , Rapson Sean , McDonald James , Reading Julian
Published: November 2024

Rheumatoid arthritis (RA) is an autoimmune disease that results in cartilage damage and bone loss due to chronic inflammation of joints. Treatments for RA include disease-modifying antirheumatic drugs and nonsteroidal anti-inflammatory drugs, as well as the inhibition of activated T-cells. Consequently, there is a need to screen RA patient samples rapidly and effectively to monitor treatment efficacy, disease progression, immune response, and predictive markers. In this work, we developed and demonstrated a multiplex detection method to monitor T-cell activation in a panel consisting of CD3, CD69, CD25, and CD279 (PD-1) surface markers using the Cellaca™ PLX Image Cytometer (Revvity). First, a dosage titration of PHA-P was performed on PBMCs to stimulate T-cell activation. Once stimulated, CD3 positive T-cells were evaluated for CD69, CD25, and the checkpoint molecule CD279. After 24 hours, the percent surface marker positive T-cells and the level of marker expression (median fluorescent intensity values) were used to determine optimal PHA-P dosage. Results showed concentration dependent increases in the expression levels with a calculated EC50 value for PHA-P ranging from 20.16-30.78 µg/mL with a 95% CV based on the CD279 titration curve. The proposed image cytometric surface marker detection method may prove to be an efficient tool to rapidly screen RA patient samples for T-cell activation in the setting of clinical disease treatment or progression.
Functional single-cell screening of chimeric antigen receptor (CAR)-T cells based on multiple cytokines using Nanovial Technology
Authors: Kuo Wei-Ying , Iyer Neha , Liang Jesse , Bosler Lucienne , Wu Alex , Padgett Sean , Di Carlo Dino , de Rutte Joseph
Published: November 2024

CAR-T therapies, while potent against some cancers, face challenges including toxicities and tumor relapse, particularly in solid tumors with hostile TMEs and sub-optimal CAR designs. A major obstacle is the lack of efficient functional screening methods for CAR-T cells, crucial for T-cell-based immunotherapy progress.

Our study introduces a novel functional screening approach using hydrogel microparticles (Nanovials) to analyze millions of individual CAR-T cells based on antigen specificity and functional cytokine secretion. We modified Nanovials with Mesothelin (MSLN) antigen, enriching and binding specific CAR-T cells. Additionally, Nanovials were adapted with antibodies to detect key cytokines (IFN-gamma, GranzymeB) from activated CAR-T cells, identified through fluorescent markers. This setup allows for analyzing and sorting CAR-T cells with unique functional traits using standard flow cytometers.

Using the Nanovial assay we distinguished MSLN CAR-T variants by cytokine profiles, revealing minor CAR engineering differences and enabling early functional assessment. This technique identified distinct CAR-T cell subpopulations within patient samples, surpassing traditional bulk analysis methods like ELISA and cell killing assays.

In summary, our Nanovial-based method offers a scalable, efficient solution for CAR-T cell functional screening, enhancing CAR design and improving therapeutic manufacturing quality control.
Role of Tregs in maintaining alloreactive Tconv hypofunction in transplantation tolerance
Authors: Cassano Alexandra , Mora Cartin Ricardo , Wang Peter , Wang Ying , McIntosh Christine , Andrade Michael , Chong Anita , Alegre Maria-Luisa
Published: November 2024

Whether Tregs are necessary to maintain Tconv dysfunction long-term after tolerance has been achieved remains to be established. Our preliminary data indicate that complete, transient depletion of Tregs using diphtheria toxin (DT) in Foxp3-DTR mice at the maintenance phase of tolerance precipitated the rejection of previously tolerized cardiac allografts and resulted in de novo production of graft-specific antibodies. These Tconvs showed a trend toward renewed cytokine production after Treg depletion. RNA-sequencing of graft-specific Tconvs after Treg depletion in tolerant mice demonstrated transcriptional distinction from Tconvs in tolerant mice that retain Tregs. Conversely, graft-reactive Tconvs from tolerant mice infected with Listeria monocytogenes or receiving an injection of plasmids encoding IFNb and IL-6 to break tolerance, were transcriptionally similar to Tconvs from unmanipulated tolerant mice. GSEA revealed that B cell mediated immune response related pathways were enriched in differentially expressed genes between graft-specific Tconvs from tolerant and Treg-depleted mice. Tconvs from Treg-depleted mice expressed genes associated with Tfh cell differentiation compared to both tolerant and naïve graft-reactive Tconvs. This indicates a unique state of potential reinvigoration and implicates Tregs in actively maintaining dysfunction of graft-specific Tconvs, and suggests that graft-reactive Tconvs may become Tfh cells when reinvigorated by Treg depletion.
Comparing the immunogenicity of different SARS-CoV-2 spike vaccine platforms in mice
Authors: Lew Min Han , Sanchez Sarah , Penaloza-Macmaster Pablo
Published: November 2024

Comparing the immunogenicity of different SARS-CoV-2 spike vaccine platforms in mice

Min Han Lew1Y, Sarah Sanchez1Y, Pablo Penaloza-MacMaster1

1Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.

YEqual contribution.

Multiple vaccine platforms have been deployed during the COVID-19 pandemic, and it is still unclear how these differ in terms of their immunogenicity and efficacy. Here, we compare the immunogenicity of different vaccine platforms in C57BL/6 mice, including Ad5-, mRNA- and protein-based regimens expressing the SARS-CoV-2 spike antigen. After vaccination with different vaccine vectors, immune responses were measured using enzyme-linked immunosorbent assays (ELISA) and tetramer binding assays to measure antibody responses and CD8 T cell responses to the SARS-CoV-2 spike protein, respectively. Immunization with Ad5 vectored vaccines elicited the highest CD8 T cell and antibody response relative to other vaccine platforms, but only when administered into Ad5 seronegative mice. However, when administered into Ad5 seropositive mice, mRNA vaccines elicited the highest immune response. These data suggest that the relative efficacy of adenovirus- or mRNA-based vaccines depends on whether the host has pre-existing immunity to the adenovirus vector itself.