bluuebunny/crossref_metadata_2025 huggingface dataset:
Unmasking Arterial-resident Autoreactive B cells involved in Atherosclerosis and Peripheral Artery Disease Progression
Authors: Taylor Joshua A , Gearhart Patricia J , Maul Robert W
Published: August 2023

Atherosclerosis activates the immune system, including B cells which secrete antibodies that bind antigens released by necrotic cells within plaques and may promote inflammation. To identify these antibodies, we isolated activated B cells from atherosclerotic ApoE-deficient mice aortas and human tibial arteries with peripheral artery disease post-amputation. Single-cell sequencing of the B cell receptors identified 35 mouse and 48 human overrepresented disease-relevant heavy and light chain pairings, which were sequentially expressed as antibody. Epitope determination was performed by human proteome array, and 14 mouse and 21 human protein targets were found with significant affinity and specificity. Mouse antigens were scrutinized by immunohistochemistry to confirm presence in inflamed plaques, and expression of these proteins were used to interrogate autoreactive antibody titers in diseased sera by ELISA. ApoE-deficient mice were immunized with the 6 most promising antigens. Serum antibody measurement showed strong, rapid, and early onset of antigen-specific antibody responses in the immunized mice compared to an adjuvant only control group. Strikingly, we saw decreased plaque accumulation in the immunized animals, suggesting antigen responses may clear pathogenic antigens before disease develops. Likewise, human antigen ELISAs comparing antigen-specific serum antibody titers to patient Framingham score for cardiovascular risk supported a role for two antigens in disease. To confirm these antigens as potential biomarkers, we will characterize the emergence of their antibody counterparts using a historical library of serum samples from individuals enrolled in the Baltimore Longitudinal Study on Aging.

This work is directly funded by the National Institutes of Health – National Institute on Aging’s Intramural Research Program in the Lab of Molecular Biology and Immunology
Selective targeting of auto reactive B cells using bispecific molecules
Authors: Griffith Amber Simone , Apley Kyle , Downes Grant , Ross Patrick , Dekosky Brandon , Farrell Mark , Berkland Cory , Kendall Peggy
Published: August 2023

Type 1 diabetes occurs when autoreactive T cells attack insulin-producing cells in pancreatic islets. B cells promote disease by acting as essential antigen presenting cells. Targeted immune therapy for treatment and prevention of autoimmune diseases is preferable to drugs that cause global immunosuppression. We therefore investigated the efficacy of three constructs designed to specifically target and neutralize anti-insulin B cells. Construct one has a 4-arm PEG backbone with insulin attached to 2 of the arms (insulin-PEG). Construct two has insulin attached to 2 of the PEG arms and a trisaccharide molecule that acts as a ligand for inhibitory receptor CD22 on the remaining 2 PEG arms (insulin-PEG-CD22L). Construct three consists of insulin directly conjugated to CD22L (insulin-CD22L). In vitro experiments using transgenic anti-insulin B cells demonstrate that insulin-PEG unexpectedly promotes excessive proliferation and upregulation of CD86 in anti-CD40 treated anti-insulin B cells. Addition of CD22L (insulin-PEG-CD22L) restores anti-insulin B cell responses to baseline but has no overall benefit. Conversely, insulin-CD22L blunts the effects of anti-CD40 stimulation, reducing proliferation by 15% and IgM surface expression by 50% . CD22L alone has no effect on stimulated anti-insulin B cells. Preliminary in vivo data using 2-photon imaging shows that insulin-CD22L binds anti-insulin B cells in spleen, pancreatic lymph nodes and pancreatic islets within 30 minutes of injection. Thus, this novel insulin-CD22L construct directs the dampening effect of CD22 specifically to anti-insulin B cells that promote T1D, making it a promising therapeutic intervention that may also be useful when paired with anti-T cell treatments.

P20GM1133117 T32GM008545 5T32AI7163-43
Pro-resolution lipid mediator maresin-1 reduces inflammation, promotes neuroprotection, and prevents disease progression in multiple sclerosis model of autoimmunity
Authors: Zahoor Insha , Nematullah Mohammad , Mir Sajad , Ahmed Mohammad Ejaz , Rattan Ramandeep , Shailendra Giri
Published: August 2023

Multiple sclerosis (MS) is the most common inflammatory neurodegenerative disease in young adults, resulting in neurological defects and disability. The endogenous mechanisms for resolving inflammation are intact but become defective in patients, resulting in a lack of resolution mediators and unresolved inflammation. Because DHA metabolism is impaired in MS, we hypothesize that supplementing its downstream metabolite maresin 1 (MaR1) will alleviate inflammation and demyelination in animal model of MS called experimental allergic encephalomyelitis (EAE). EAE was induced in SJL mice, followed by treatment with MaR1 (300ng, 200ul in PBS/animal/day ip) on day 6 after disease induction. Monitoring the disease course, recall response by ELISA, cytokine expression analysis by qPCR and western blotting, and immune profiling by flow cytometry were used to assess the effect of MaR1 treatment in EAE. The neuroprotective effect of MaR1 was also assessed using single molecule array (SIMOA), histopathology, and IHC. Statistical analysis was done using Graph-Pad Prism. Restoration of MaR1 had a protective effect on neurological deficits, prevented disease progression, and reduced disease severity in EAE by reducing immune cell infiltration (CD4+IL17+ and CD4+IFN+) into the CNS (P<0.001). It significantly reduced the proinflammatory cytokine IL17 (P<0.01) and promoted an anti-inflammatory response via IL10 and IL4 (P<0.001). Furthermore, it exerted neuroprotective effects as evidenced by higher MBP expression in the brain from the MaR1 treated group. Overall, MaR1 supplementation has anti-inflammatory and neuroprotective effects in preclinical animal models, implying that it could be a new therapeutic molecule in MS.

Supported by grants from National Multiple Sclerosis Society (US) Research Grant (RG-2111-38733, RG-1807–31964 and RG-1508– 05912), the US National Institutes of Health Grant (NS112727 and AI144004), and Henry Ford Hospital Internal Grant (A10270 and A30967) to SG.
Amelioration of Collagen-Induced Arthritis in Rats by a Monoclonal Antibody against the Novel Epitope of Cysteine Protease from P. gingivalis
Authors: Tsay Gregory J , Yeh Yung-Ju , Xue Ting-Yin , Peng Hsin-Yi , Hsieh Fei-Hung , Wu Ya-Wun , Chen Shih-Yao , Chen Yeh , Chen Hui-Chen , Chang Han , Tsai Chung-Hao
Published: August 2023

Background

The use of biological diseases-modifying anti-rheumatic drugs (bDAMRD) including anti-tumor necrosis factor-α (TNF-α) has revolutionized the therapy of rheumatoid arthritis (RA). However, approximately 30–40% of patients with RA do not response to anti-TNF therapy when used as the first-line bDMARD.

Objective

The aim of the study is to develop a new, novel monoclonal antibody for the therapy of RA.

Methods

Monoclonal antibody development: BALB/c mice were immunized with OVA-conjugated peptide for CIA. Spleen cells were collected for fusion with myeloma cells. The rats were divided into 6 groups: Normal control, CIA, IgG1(15 mg/kg)-treated CIA, low-mAb (3 mg/kg)-treated CIA, High-mAb (9 mg/kg)-treated CIA, and Enbrel (4.5 mg/kg)-treated CIA.

Results

The monoclonal is only reacted to the immunized peptide and is IgG1 Kappa, high melting temperature with Tm=4.8, High binding to FCγRIIb and FCγR III, and low dissociation constant (Kd=19.6×10−9) of Ag-mAb affinity. The mAb stains mainly on cytoplasm with several coarse discrete dots on SW982 cells. After treatment with this mAb in CIA, the mice have showed dose-dependent effects, improving articular index and ankle circumference, reduced inflammation and synovium proliferation, restored joint space narrowing, and cartilage on immunohistochemistry. The therapeutic results in the mAb-treated group has shown to be excellent in CIA animal RA model.

Conclusion

We’ve generated a novel monoclonal antibody which has excellent therapeutic effects in the therapy of RA animal model. This study is the first time to demonstrate that targeting on the different signal pathway of CIA can have a promising effect on RA therapy.
A KIR x CD8 targeting bispecific modulator enhances regulatory CD8 T cell functions, and reduces inflammation in models of autoimmune disease
Authors: Gardell Jennifer L , Boster Daniel , Bowser Justin , Fasnacht Rachael , Fitzpatrick Kristin , Hermansky Gleda , Julien Susan , Maurer Meghan , McMahan Catherine , Meengs Brent , Patton Daniel , Pham Minh , Therriault Jon , Swiderek Kristine M , Crane Courtney
Published: August 2023

INTRODUCTION

We have characterized a subset of CD8 T cells (CD8 Treg) with immunosuppressive characteristics in inflammatory disease settings. CD8 Treg activation results in their oligoclonal expansion and elimination of pathogenic CD4 T cells. Here we describe a bispecific CD8 Treg modulator that activates autoimmune patient derived CD8 Treg resulting in the cytolytic elimination of pathogenic CD4 T cell in vitro, ex vivo, and in vivo.

METHODS

We tested target and cell binding of a novel bispecific CD8 Treg modulator by Octet and flow cytometry. The functional impact was evaluated using cytotoxicity assays and supernatant analysis in vitro, using Celiac patient derived biopsies ex vivo, and in acute GVHD studies using human PBMCs.

RESULTS

We evaluated the efficacy and mechanism of action of a novel bispecific CD8 Treg modulator directed toward CD8 and inhibitory KIR2DL1/2/3. The modulator enhanced cytolytic CD8 Treg functions in vitro using autoimmune patient PBMCs, increased activated CD4 T cell death, and decreased pro-inflammatory cytokines. In Celiac patient derived duodenal tissues, the modulator increased CD8 Treg prevalence and reduced epithelial cell death after gliadin peptide stimulation. In acute GVHD studies, CD8 Treg modulator treated mice had CD8 Treg expansion in a dose dependent fashion and reduced inflammation.

CONCLUSIONS

A novel KIR x CD8 targeting bispecific modulator restores cytolytic function of CD8 Treg, resulting in a decrease of pathogenic CD4 T cells. We postulate that the bispecific CD8 T cell modulator can reduce autoreactive CD4 T cell prevalence to reestablish immune balance and may represent a selective and broadly applicable therapeutic approach for the treatment of autoimmune disease.
Agonistic anti-PD-1 antibodies downregulate autoantibody development in lupus model mice
Authors: Tokumaru Yosuke , Suzuki Kensuke , Tajima Masaki , Ohkura Iori , Honjo Tasuku , Ohta Akio
Published: August 2023

T follicular helper (Tfh) cells provide B cell help and promote antibody development. Since Tfh cells may be responsible for the development of self-reactive antibodies, this CD4+ T cell subset represent a potential target in the treatment of autoimmune diseases. We have developed anti-PD-1 agonist antibodies that can stimulate the immunosuppressive activity of PD-1. In this study, we sought to inhibit Tfh cells that express PD-1 at high levels and to downregulate antibody production using the PD-1 agonists. PD-1 agonist antibody suppressed IL-21 production from human Tfh-like cells. The co-treatment with PD-1 agonist antibody significantly reduced antigen-specific antibodies in mice immunized with 4-hydroxy-3-nitrophenylacetyl ovalbumin (NP-OVA) and dampened germinal center responses. Chronic GVHD by the transfer of CD8+-depleted splenocytes from C57BL/6 mice into BDF1 mice induces lupus-like symptoms including the induction of autoantibodies and immune complex-mediated nephritis. PD-1 agonist antibody alleviated the induction of anti-dsDNA Ab in the lupus model along with the suppression of germinal center formation. In conclusion, agonistic anti-PD-1 antibodies can effectively prevent autoantibody production and represent unique therapeutic agents in the treatment of autoimmune diseases.
Loss of the MLL3 tumor suppressor accelerates breast tumor onset via HIF1a-induced CCL2-mediated recruitment of CCR2+ regulatory T cells
Authors: Boutet Marie , Erler Piril , Nishitani Kenta , Couturier Nicole , Zhang Zheng , Barbieux Emeline , Guillen Erik , Suzuki Masako , Lauvau Gregoire , Guo Wenjun
Published: August 2023

Understanding how specific cancer mutations modulate the tumor microenvironment is essential for developing new precision cancer therapies. The histone methyltransferase MLL3 is a tumor suppressor that is frequently mutated together with p53 and PI3-kinase in breast cancer. Herein, using a mouse mammary-stem-cell-based tumor model and CRISPR gene editing, we recapitulated this genetic setting and revealed that Mll3 loss promotes tumorigenesis through enhancing tumor-intrinsic aggressiveness and establishing an immunosuppressive microenvironment. Specifically, HIF1a was upregulated in Mll3-mutant tumors and controlled proliferation and stem cell activity of the tumor cells. HIF1a also transcriptionally upregulated expression of the chemokine CCL2 by tumor cells. CCL2 further contributed to rapid CCR2-dependent accumulation of activated, proliferative KLRG1+Foxp3+ regulatory T (Treg) cells at very early stages of tumor initiation, promoting faster tumor onset. These results link MLL3 loss to HIF signaling, CCL2 and Treg cells, and the establishment of an early immune-suppressive microenvironment, a finding that may open new therapeutic opportunities for MLL3-mutant cancers.

Department of Defense Breakthrough awards BC161696 and BC200410
Characterizing the role of IL-33/ST2 axis in regulating anti-tumor function by CD8 T cells during tumor metastasis in the omentum
Authors: Desai Sweta , Meza-Perez Selene , Liu Mingyong , Randall Troy D
Published: August 2023

The omentum, a visceral adipose tissue, is a frequent site of metastasis for gastric and ovarian cancers. Advanced ovarian cancer almost always metastasizes to the omentum. Currently available therapies do not control tumor growth long-term and there is a frequent recurrence of tumor metastasis. In mice, tumor cells injected intraperitoneally implant in the omentum and grow progressively despite tumor-specific CD8 T cells. The lack of effective immunity is due in part, to rapid Treg recruitment in the omentum, which provides an immunosuppressive environment to support tumor growth. The Tregs in the omentum are a specialized population called visceral adipose tissue associated Tregs (VAT-Tregs). A portion of the omental VAT-associated Tregs express ST2, a receptor for Interleukin-33 (IL-33), suggesting that local IL-33 production may contribute to Treg-mediated suppression in the omentum. We also detected persistent, continuously expanding, tumor-specific CD8 T cells in the omentum. Given that IL-33 can also enhance effector function by CD8 T cells and promote their persistence in the presence of chronic antigen stimulation, we are investigating how IL-33 regulates the balance of Treg-mediated immune-suppression and CD8-mediated tumor cell killing in the context of omental adipose tissue.

R01CA216234
PD-L1 couples with LTβR signaling to accelerate regulatory T cell lymphatic migration and tumor metastasis
Authors: Piao Wenji , Wu Long , Pettit Sarah , Bromberg Jonathan S
Published: August 2023

Introduction

Tregs in the tumor environment inhibit anti-tumor immunity and are critical targets for anti-tumor strategies. We previously showed that Tregs use surface lymphotoxin (LTαβ) and PD-1 to signal LTβR and PD-L1 on lymphatic endothelial cells, thereby promoting Treg lymphatic migration. Most tumor cells express LTβR and PD-L1, yet tumor interactions with LTαβ and PD-1 on Tregs are poorly studied. We hypothesized that PD-L1 couples with LTβR signaling on tumors to regulate migration and tumor metastasis.

Methods

Wild type and CRISPR/Cas9 LTβR or PD-L1 knockout B16F10 melanoma were used for signaling and cell migration. Specific LTβR classical and nonclassical NFκB blocking peptides were used.

Results

PD-L1 bound to LTβR in resting B16F10, and blocking LTβR-nonclassical NFκB signaling increased melanoma PD-L1 expression. RNASeq analysis of melanoma revealed that gene expression regulated by LTβR-nonclassical NFκB signaling was increased by PD-L1 depletion, while genes driven by PD-L1 signaling remained unaltered by LTβR depletion. Melanoma cocultured with Tregs had enhanced migration, while combined blockade of PD-L1 and LTβR nonclassical NFκB pathways synergistically blocked tumor migration. In vivo, PD-L1 blockade combined with LTβR classical or nonclassical NFκB blocking peptides inhibited tumor growth and metastases, and enhanced host survival.

Conclusions

PD-L1 couples with LTβR-nonclassical NFκB signaling to regulate tumor growth and migration. Blocking both arms of tumor LTβR-NFκB-signaling enhanced immune checkpoint blockade efficacy and mouse survival. These observations provide a rational strategy to modulate Treg activities to prevent tumor spread.

supported by NIH R37 AI062765 and Emerald grant 2022
Antigen-specific CD4 T cell polarization is altered by immune checkpoint blockade in pancreatic ductal adenocarcinoma
Authors: Cruz Eduardo , Burrack Adam L , Miller Ebony , Stromnes Ingunn
Published: August 2023

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with poor response to conventional immune checkpoint inhibitors. Early data by our group suggests CD4 T cell depletion abrogates response to a novel combination therapy that is transiently curative in a murine model of PDA. Given the importance of CD4 T cells in PDA control, we now seek to describe CD4 T cell dynamics in PDA at an antigen-specific resolution. To this end, we developed a novel tetramer reagent towards a recently identified MHC class II model epitope. Through this, we observe antigen-specific T-cell polarization that is distinct from the bulk CD4 population both in the spleen and in the tumor microenvironment. Tumor-specific CD4 T cells also displayed differential polarization during combination therapy, suggesting positive response to therapy is mediated in part by TCR-dependent stimulation of the CD4 compartment.
Exogenous catalase treatment enhances CD8+ T cell functionality and hinders T cell exhaustion
Authors: Shah Rushil H , Aksoylar Halil-Ibrahim , Pal Rinku , Boussiotis Vassiliki A , Patsoukis Nikolaos
Published: August 2023

ROS are small short-lived oxygen-containing molecules which are highly chemically reactive and involved in the regulation of biological processes like T cell activation and proliferation. The tumor microenvironment (TME) is a metabolically hostile environment with a deficiency in nutrients like glucose and glutamine, accumulation of tumor-driven metabolic waste, and extreme hypoxia. Hypoxia results in impaired mitochondrial function and generation of excess levels of ROS, which have been known to promote T cell exhaustion. Here, we investigated how exogenous treatment of antioxidant catalase altered CD8+ T cell effector responses and T cell exhaustion using OT-I TCR transgenic mice. During CD8+ T cell effector differentiation in vitro by stimulation with cognate antigen followed by culture with IL-2, addition of catalase significantly enhanced T cell activation and effector differentiation. Catalase-treated effector cells had higher expression of critical activation markers including CD69 and CD25, increased expression of the stemness marker TCF-1, and enhanced secretion of effector cytokines IFNγ and TNFα. During induction of T cell exhaustion in vitro by co-culture with B16 melanoma expressing ovalbumin (B16-Ova), catalase-treated OT-I T cells had significantly increased cytotoxic activity, enhanced expression of CD25 and lower co-expression of PD-1 and TIM-3 relative to non-treated OT-I T cells. Our findings uncover a new role for catalase in altering the fate of CD8+ effector T cells and potentially a new therapeutic tool for managing T cell exhaustion.

NIH (R01 CA212605)
Unique Immunoregulation of Lung Cancer by CD8+ T Cells
Authors: Kratzmeier Christina , Khalil May , Banerjee Anirban , Krupnick Alexander Sasha
Published: August 2023

We hypothesize that immunoregulation of lung cancer differs from that of other malignancies. To study this hypothesis in greater detail, we utilized a primary carcinogenesis model of 3-MCA-induced flank fibrosarcoma or urethane-mediated lung cancer in B6 wild-type or CD8+ T cell deficient mice. CD8+ T cell deficiency increased sarcoma tumor growth (199±65 vs. 511±138 mm3 for B6 vs B6CD8−/−mice respectively p = 0.03) but decreased the growth of lung cancer (1.7±0.2 vs. 0.6±0.13 cm3 for B6 vs B6CD8−/−mice respectively p = 0.006). We validated this surprising finding in multiple other tumor models including B16 melanoma, EG7 lymphoma and LLC lung cancer. For all non-lung cancer tumors, the presence of CD8+ T cells decreased tumor growth. In direct contrast, the presence of CD8+ T cells accelerated the growth of lung cancer. Cytokine analysis revealed a significant CD8+ T cell-dependent increase of Th-1 polarizing cytokines, IFN-γ and TNF-α, in LLC lung cancer-bearing but not B16 melanoma-bearing mice. Similar to CD8+ T cell deficiency, neutralization of IFN-γ and TNF-α in B16 melanoma-bearing mice increased tumor growth but ameliorated the growth of lung cancer. In addition, we noted an IFN-γ and TNF-α-dependent expansion of CD8+Foxp3+ T cells in the tumor infiltrating lymphocytes of lung cancer-bearing mice, but not melanoma-bearing mice. Therefore, contrary to the accepted dogma, an increase in Th-1 polarizing cytokines, mediated in some fashion by lung cancer associated CD8+ T cells, facilitates rather than ameliorates the growth of this malignancy. Our data suggests that immunoregulation of lung cancer is unique and may require novel immunomodulating strategies that may not necessarily apply to other malignancies.

This project was funded by P01 AI116501, R01 AI145108, I01BX002299
Archaeal and bacteriophage derived Cas proteins demonstrate memory T cell response in humans
Authors: Bajgain Pratima , Sauna Zuben E , Simhadri Vijaya
Published: August 2023

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a powerful gene editing tool with clinical applications in the pipeline. In vivo delivery of Cas proteins could induce immune responses which would limit the utility of the technology. Previously, we and others have demonstrated pre-existing immunity to Cas9 proteins derived from Staphylococcus aureus (Sa) and Streptococcus pyogenes (Sp) in human populations. As these Cas proteins are of microbial origin this finding was not surprising. There are numerous naturally occurring alternatives to Cas9 and many of these are being leveraged for gene editing. Alternatives to Cas9 that have successfully been used in gene editing technologies include Cas12a (derived from Acidaminococcus sp), Cas14a (derived from DPANN, a super-phylum of archaea) and Casϕ (derived from a huge bacteriophage). It has been postulated that humans would not have pre-existing immunity to some of these Cas proteins. In the comparative study described here, we evaluated pre-existing antibodies and memory T-cell responses to Cas9, Cas12a, Cas14a and Casϕ from the same 18 healthy donors for all proteins. Pre-existing antibodies were identified using ELISA and memory T-cell responses were measured using the ELISpot assay. All four Cas proteins tested positive for both assays in at least some donors. It has been speculated that pre-existing immunity to Cas9 proteins is due to human exposure to the pathogenic bacteria they are derived from. Consequently, Cas14a and Casϕ should not be associated with pre-existing immunity. However, our results show that anti-Cas14a and anti-Casϕ antibodies were detected in ~50% of the study population and these donors also showed memory T-cell responses.
Human Memory T cells show differential metabolic preferences
Authors: Akdogan Bagdeser , Rose James R , Rahmberg Andrew R , Scharer Christopher D , Boss Jeremy M
Published: August 2023

Metabolic reprogramming regulates survival and function of immune cells, including T cells. Previous studies revealed that memory T cells are quiescent populations that primarily use OXPHOS for their metabolic needs. However, antigen rechallenge induces metabolic reprogramming including the engagement of aerobic glycolysis in which glucose is converted into lactate rather than oxidized into mitochondria. Here, we compared the metabolic characteristics of human naïve and memory T cells (TCM, TEM, and TEMRA) that are characterized by the cell surface proteins CCR7, and CD45RA. T cells were enriched from human PBMC samples, and then the subsets were sorted by flow cytometry. RNA-seq libraries constructed from each population were sequenced. Comparison of metabolism related transcript levels of the subsets revealed a distinct metabolic state in each cell type. Seahorse assays, mitochondrial staining flow cytometry assays and the SCENITH (Single Cell ENergetIc metabolism by profilIng Translation inHibition) assays were performed to assess the metabolisms of the individual populations. Our data indicated that resting CD4 and CD8 memory T cell populations have higher glycolytic and fatty acid capacity than the naïve T cells, consistent with the RNA-seq data. Resting naïve T cells depend on mitochondrial ATP production more than the memory T cells. However, when the cells are stimulated, the glycolytic capacity increased in all cell types and mitochondrial dependence decreased without a significant difference between subsets. Overall, our data highlights how memory T cell subsets retain greater metabolic capacity than naive T cell, potentially contributing to more efficient secondary responses.
Lactose differentially modulates functionalities of antigen-presenting cells and T cells
Authors: Sur Meghna , Mone Kiruthiga , Zhang Guolong , Reddy Jay
Published: August 2023

Lactose is a milk sugar that appears to have immunomodulatory properties, but conflicting reports are available regarding its effects on inflammatory response and Treg cell functions. To delineate these aspects, we sought to test the hypothesis that lactose alters T-cell responses by modulating the functionalities of antigen-presenting cells (APCs). First, using the mouse macrophage cell line (RAW 264.7), we noted that lactose could reduce LPS-induced oxidative stress (P>0.05) without altering viability. Second, while the expression of MHC class II molecule was unchanged, an inverse relationship was noted with the costimulatory molecules [downregulated CD40 (P>0.005) and CD80 (P>0.005), and upregulated CD86 (P>0.005)], suggesting that lactose-mediated effects on APCs are heterogeneous and may involve unique regulatory elements for individual gene expression. Third, by testing the effects of lactose in the sorted mouse primary T cells, lactose moderately modulates T cell activation stimulated by anti-CD3 and anti-CD28, including IL-2 and IFN-γ production, but the cell viability was unaffected. However, upon the addition of IL-2, the reduced T cell activation was no longer apparent as evaluated by CFSE staining of CD4 and CD8 T cells, indicating that lactose’s effects on T cells may involve an IL-2-dependent mechanism. Taken together, our data support the notion that lactose could exert its effects on T cells directly or indirectly by modulating the APC functions that may have implications on health and disease.
C-terminally phosphorylated p27 drives cancer-promoting inflammation
Authors: Razavipour Seyedehfatemeh , McIntosh Alec , Bagheri Amir , Yoon Hyunho , Shin Miyoung , Van Boven Derek , Yi Chunling , Slingerland Joyce
Published: August 2023

p27 normally inhibits cell cycle progression and cell growth. In cancers, deregulated p27 acts as an oncogene to promote metastasis when phosphorylated by PI3K-activated kinases at T157 and T198 (p27pTpT). We showed p27pTpT transcriptionally co-activates cJun and induces STAT3 activation. Here, we pursue the hypothesis that p27pTpT transcriptionally co-activates STAT3 target genes to expand cancer stem cells (CSC) and promote cancer inflammation.

In triple negative breast cancer (TNBC) lines, we showed that p27pTpT increases stem cell properties, including sphere formation and expression of stem cell transcription factors MYC, OCT4, NANOG, and KLF4 through STAT3. ChIP-seq/RNA-seq showed p27 recruits STAT3 to gene promoters to induce profiles of p27/STAT3 co-target genes, including MYC and JAG1, that increase tumor-initiating stem cells (TISC) in vivo. This work revealed a novel role for p27pTpT as a driver of CSC expansion.

Since CSCs mediate treatment resistance, in part, by promoting inflammation, we next tested if p27pTpT drives cancer inflammation. p27/STAT3 co-target genes associate strongly with inflammatory pathways. ATAC-seq/ ChIP-seq showed p27pTpT increased chromatin accessibility to co-recruit p27/STAT3 to promoters of oncogenic, proinflammatory genes (IL-6, NF-κB/RELA, VEGFA) and to mediators of immune evasion. p27pTpT also increased STAT3 activation, p65 (NF-κB subunit), and IL-6 protein levels on WB, and increased CCL2 secretion on ELISA. Notably, both p27 knockdown and STAT3 inhibition decreased p27pTpT-driven CCL2 secretion.

Together, these data indicate that when p27 is C-terminally phosphorylated, it acquires a novel oncogenic role to promote STAT3-driven CSC expansion and cancer inflammation.
Characterization and clinical significance of T follicular helper cells in follicular lymphoma
Authors: Yang Zhi-Zhang , Tang Xinyi , Kim Hyo Jin , Mukherjee Prithviraj , Bhardwaj Vaishali , Mondello Patrizia , Ansell Stephen
Published: August 2023

T follicular helper (TFH) cells are a subset of CD4+ T cells that reside within the follicles of secondary lymphoid organs and play an essential role in the germinal center (GC) reaction. To examine the role TFH cells in follicular lymphoma (FL), we first characterized TFH cells using single cell analysis with the high-throughput technologies of mass cytometry (CyTOF) and the Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). Clustering analysis revealed that TFH cells form subsets with unique phenotypes, and the expression of many common markers including CXCR5, ICOS, IL-21, TOX2, CD57, GZMK, XCL1, XCL2, CXCL13 and CD40L varies on TFH subsets. We found that TFH cells are phenotypically more heterogenous in FL than benign tonsil tissues by both proteomic (n=82) and transcriptomic (n=8) profiling. Each FL patient showed different dominant TFH subsets, in contrast to tonsil donors which were highly homogenous. Some TFH subsets were only present in FL and absent in tonsil tissues, suggesting the existence of FL-specific TFH cells. Cytokines (IL-2, IL12, IL-17 and IL-21) and activated B cells upregulated TFH markers including PD-1, CXCR5 and IL-21 to induce the development of TFH subsets such as IL-21+ or IL-21+IFN-g+ TFH cells, which was inhibited by rapamycin, an immunosuppressor. We observed that the TFH cell frequency significantly correlated with the number of lymphoma B cells. Futhermore, while TFH cells as a whole showed no correlation with prognosis, TFH subsets such as CD57+ TFH cells were significantly associated with an inferior patient outcome. Taken together, our results indicate that TFH cells are highly heterogenous with different tumor-specific TFH subsets present in FL and associated with patient outcomes in FL.
The metabolite derived from high-salt diet-induced gut microbiota modulates brain tumor environment
Authors: Kim Chae Won , Park Jang Hyun , Kim Hyun-Jin , Kang In , Lee Heung Kyu
Published: August 2023

The Westernized diet refers to consuming high amounts of saturated fat, sugar, and salt, and low amounts of dietary fiber. Such a diet is currently thought to contribute to non-communicable diseases such as autoimmune diseases, heart disease, and cancer. A high-salt diet (HSD) is known to affect various organs, including the cardiovascular, renal, and central nervous systems. Recently, several studies reported the effect of HSD on cancer due to the enhanced osmolarity of tumor microenvironment. However, it has been known that the concentration of sodium in brain is tightly regulated. That is, there may be another effect of HSD to tumor environment in brain. In addition, the effects of HSD on brain tumor have not been elucidated. In this study, we observed an increased tumor burden in mice who received the high-salt diet. Mice on a high-salt diet also showed a change in their gut microbiota. The specific bacteria significantly increased in HSD group compared to normal-salt diet (NSD) group. This bacteria is known to produce several metabolites, and we measured these metabolites in cecum of NSD and HSD group. Among them, the one metabolite increased in HSD group and we supplemented this metabolite to mice with brain tumor. Then, we observed the exacerbation of brain tumor post the supplement compared to control group. Therefore, we speculated that the HSD exacerbated brain tumor via the increased production of metabolite by altered gut microbiota.
Liver Metastases Potentiate Cancer Progression
Authors: Pearson Ashley N , Holcomb Erin , James Jadyn , Jungles Kassidy Mikala , Nedzesky Nicholas , Schaefer Grace , Tate Akshay , Liu Meilan , Wang Zhuwen , Huber Amanda , Green Michael
Published: August 2023

Metastases are the primary cause of cancer mortality, and different sites of metastasis are associated with different prognostic outcomes. In particular, liver metastases are associated with poor overall survival and increased disease progression. It is known that liver metastases suppress systemic antitumor immunity to limit immunotherapy efficacy on primary tumors through hepatic siphoning and deletion of CD8+ T cells, induction of T regulatory cells, and depletion of dendritic cells. However, it remains unclear how liver metastases promote subsequent disease progression and the metastatic cascade. To investigate how liver metastases reprogram the immune system to potentiate disease progression, we developed a preclinical polymetastatic murine model of liver and lung metastases. Using B16F10 melanoma in syngeneic C57Bl/6 mice, we observed that the presence of liver metastases increased the lung metastatic burden. We hypothesized that liver metastases potentiate the formation of a tumorigenic, immunosuppressive premetastatic niche in distant sites. We conducted single cell RNA (scRNA) sequencing and flow cytometry immune profiling of the liver, peripheral blood, and lungs of mice with or without liver metastases. Together, these data suggest an association between Ly6g+Ly6c− neutrophils and melanoma progression in mice with liver metastases. Future efforts will focus on defining the contribution of neutrophils to disease progression with the ultimate goal of understanding and addressing the immunosuppression promoted by liver metastases.
Premalignancy induced B cell-centered immune aggregates for immune-prevention against triple negative breast cancer
Authors: Zhou Xian , Zeng Jianhao , Riddick Alexys T , Engelhard Victor H , Zong Hui
Published: August 2023

Immune surveillance is believed to be a critical mechanism to eradicate premalignant mutant cells to prevent cancer. However, the cellular organization and mechanisms for immune surveillance remain largely unknown because premalignant mutant cells are “invisible” in patient samples and conventional animal models. To gain a glimpse into this process, our lab developed a genetically engineered mouse model recapitulating human basal-like breast cancer through unequivocal labeling of premalignant p53-Brca1 mutant cells with GFP. Using this model, we discovered prominent immune aggregates in close proximity to mutant ducts, which eventually disperse at the tumor stage. These immune aggregates manifest tertiary lymphoid structure (TLS)-like organization, enriched with activated B cells co-clustered with T cells and CD11b+ myeloid-derived cells. To fully characterize these TLS-like aggregates, we are profiling the dynamic changes in immune composition and activation status from premalignancy to malignancy. In parallel, to investigate the molecular mechanism behind their organization, we are studying the relevant chemokines, like CXCL13, and corresponding source cells. Ultimately, we plan to perform functional experiments to determine if these TLS-like structures are responsible for controlling the malignant progression of breast cancer. Our work provides direct evidence of how immune response against premalignant cells before breast cancer forms.

Supported by grants from NIH (R01CA256199-01A1)
Aging, Immunity and Cancer
Authors: Blomberg Bonnie , Frasca Daniela , Diaz Alain , Antoni Michael B
Published: August 2023

Our studies have shown that breast cancer patients receiving CBSM (Cognitive Behavioral Stress Management) have less stress and inflammation and better immune response, e.g. antibody response to influenza vaccine. More recently immune (B lymphocyte, antibodies) and metabolic (higher) conditions were characterized. We previously showed the stress reduction group had better long term (11 year) survival. Currently we are further analyzing negative and positive contributors to immune response in cancer patients including age and body mass.

Supported by grants from NIH (R01 AI)", Florida Breast Cancer and SCCC
Immune Spatial Differences Between Short and Long-term Surviving Glioblastoma Patients
Authors: Najem Hinda , McCortney Kathleen , Steffens Alicia , Tripathi Shashwat , Walshon Jordain , DeCuypere Michael G , Brandenburg Raymond , Hoerich Daniel , James Charles David , Lesniak Maciej S , Sonabend Adam M , Chandler James P , Tate Matthew , Burks Jared K , Horbinski Craig , Heimberger Amy B
Published: August 2023

Introduction

Immune surveillance, distribution, interactions, and antigen presentation within the tumor microenvironment (TME) may influence anti-tumor reactivity and survival.

Methods

Dichotomized newly diagnosed treatment-naïve IDH1 wild-type glioblastoma, negative MGMT promotor methylation (65%), short-term (STS; ≤ 6 months; mean age 66 years; n=10) or long-term (LTS; >2 years; mean age 50 years; n=10) survivors, were profiled using high dimensional fluorescence multiplex staining spatial analysis of the TME using a panel of 26 markers.

Results

The amounts of specific immune cells (CD4+, CD8+, FOXP3+, CD11c+, CD20+, CD68+, CD205+, NKG2D+) were similar between cohorts, except STS were enriched with CD163+ expressing macrophages and LTS had more P2RY12+ microglia throughout the TME. There was minimal MHC-I expression and no difference in MHC-II on the myeloid populations (CD68+, CD11c+, CD163+, P2RY12+, and/or in combination) in both cohorts. TIM-3 was the only immune regulator substantially expressed on P2RY12+ microglia and the other myeloid populations, irrespective of survival. Perivascular clustering of CD11c+CD163+ cells was enriched in STS cases. The TME in LTS cases demonstrated adaptive immune activation processes, including CD11c+P2RY12+CD205+ microglia interacting with CD45RO− naïve, non-exhausted (PD-1−, LAG3−, Tim3−) CD8+ T cells expressing the LCK+ immune synaptic marker.

Conclusion

Immune activation interactions between CD8+ T cells and microglia in the TME may help determine STS versus LTS in GBM patients.
PLA2G2A and ANXA1 are correlated with NLRP3 in colitis-associated colorectal cancer
Authors: Yoon Chanjin , Yang Chul-Su
Published: August 2023

Inflammation inspires the development of cancer and contributes to all stages of tumorigenesis. Inflammatory bowel disease (IBD) is involved with an elevated risk of colitis-associated colorectal cancer (CAC). NLRP3 inflammasome is linked to progression and survival in colorectal cancer; however, the underlying NLRP3 inflammasome mechanism in CAC remains unclear. Nlrp3 mRNA is highly expressed in AOM/DSS induced-CAC. Using bioinformatics analysis, we identified candidate genes involved with CAC and NLRP3 in AOM/DSS induced-CAC mice model. These genes are altered in AOM/DSS induced-CAC compared with low-grade dysplasia and are associated with Inflammatory response. We found both Pla2g2a and Anxa1 expression are elevated in AOM/DSS induced-CAC mice and were predicted to interact with Nlrp3 in the STRING database. Furthermore, we found that Pla2g2a and Anxa1 mRNA expression is positively correlated with Nlrp3 expression in colorectal cancer tissues. Our data suggest that PLA2G2A or ANXA1 may regulate NLRP3 in CAC and provide a novel therapeutic approach to treatment.

This work was supported by the NRF grant funded by the Korea government (MSIP) (2019R1I1A2A01064237).
The influence of sex on the immune microenvironment in male and female breast cancer
Authors: Hargrove-Wiley Ebony , Fingleton Barbara
Published: August 2023

Even when accounting for multiple clinical parameters, male breast cancer patients have significantly lower survival rates than females. Males and females differ in their levels of sex hormones androgens and estrogens. These sex hormones have varying influences on immune cell populations, which in turn may affect disease progression. Thus, the sex of the patient and the levels of sex hormones are likely important for anticancer immunity. Here, we assessed sex differences in the immune landscape of primary and metastatic tumors. We used flow cytometry to determine the immune profile of mammary tumors, spleens, and lungs, and histochemistry to evaluate metastasis in transgenic MMTV-PyMT and in mice orthotopically injected with sex-specific MMTV-PyMT-derived cell lines in male and female cohorts. In transgenic mice, males had greater proportions of CD4+ T-helper and total myeloid cells in all organs analyzed. The splenic and pulmonary immune profiles of tumor-bearing males were more similar to cancer-free littermates than in females. Interestingly, males trended towards increased metastatic colonization in the lungs. Sex-associated immune profiles differed between transgenic and orthotopic mice. In mice bearing male-derived R221a PyMT tumors, females had decreased intratumoral neutrophils and elevated natural killer cells with increased splenic B-cells in comparison to males. Overall, our data supports a sex-dependent immune response in mammary carcinoma that is, in part, regulated by the hormonal environment of the host. With emerging therapeutics targeting the tumor immune microenvironment, characterization of immune profiles is critical for optimizing their use in all breast cancer patients.

Supported by grants from METAvivor Research and Support Inc. and the National Science Foundation Graduate Research Fellowship Program under Grant No. 1937963.
The role of Th2 in immune surveillance of NAFLD-associated liver metastasis
Authors: Chan Ting Hei Thomas , Tang Wenshu , Zhou Jingying
Published: August 2023

Non-alcoholic fatty liver disease (NAFLD) is an increasing global epidemic associated with chronic inflammation, metabolic disorders, immunosuppression and importantly, increased incidence of primary and metastatic liver cancers. We have recently demonstrated that the immunosuppressive nature of NAFLD livers represented by increased myeloid-derived suppressor cells and dysregulated natural-killer T cells, resulted in aggressive tumorigenesis and immunotherapy resistance of primary liver cancer. Given the importance of metabolic and immune imbalance in the formation of metastatic-prone liver microenvironment, we continued to dissect the cellular complexity of NAFLD livers in metastatic liver cancers. Through bulk and single-cell RNA-sequencing (scRNA-seq) dataset analysis from mice and patients with NAFLD or liver metastasis (LM), we found that the CD4+ T helper-2 (Th2) population and the regulon activity of its master transcription factor GATA3 were also remarkably reduced in NAFLD or LM compared to healthy controls. Interestingly, increased hepatic metastasis was observed in mice with NAFLD induced by high-fed-high-carbohydrate-diet compared to controlled mice by orthotopic LM mouse models, accompanied with reduced hepatic Th2 cells which negatively correlated with increased triglyceride and cholesterol levels. Taken together, our preliminary findings suggested a potential immunosurveillance role of hepatic Th2 cells that may be dysregulated by lipid metabolic alterations in NAFLD. Therapeutic approaches to restore hepatic Th2 cells may therefore provide new perspective and therapeutic approaches to reduce liver metastasis in the era of NAFLD epidemic. The project is supported by RGC/GRF14104820.

The project is supported by RGC/GRF14104820.
A novel ion channel-mediated mechanism facilitates the inhibitory effect of exosomes on anti-tumor immune responses
Authors: Chimote Ameet A , Alshwimi Abdulaziz O , Bhati Jay , Langevin Scott M , Wise-Draper Trisha M , Khodoun Marat V , Conforti Laura
Published: August 2023

Head and neck squamous cell carcinomas (HNSCC) are aggressive cancers that are associated with high morbidity and mortality. Anti-tumor immune responses rely on the ability of cytotoxic T and NK cells to infiltrate the solid tumor microenvironment and kill the cancer cells. However, solid tumors utilize multiple mechanisms to evade these cells including the production of exosomes, which inhibit immune cell function and present a major block to immunotherapy. The mechanisms by which the tumor-derived exosomes (TEX) inhibit immune cell functionality are poorly understood. We isolated TEX from a HNSCC patient tumor-derived primary cancer cell line by ultracentrifugation and characterized them by electron microscopy and flow cytometry. We exposed healthy donor (HD) peripheral blood mononuclear cells (PBMCs) to TEX, followed by activation, and conducted preliminary single-cell studies by measuring transcriptome (NanoString), secretome of 32 secreted proteins (Isoplexis) and immune cell function (electrophysiology). Transcriptomic studies revealed that TEX reduced the abundance of cytotoxic cells, and inhibited immune responses associated with cytotoxicity, chemokines, and NK and T cell functions. In CD8+ T and NK cells, TEX reduced polyfunctionality (the ability to secrete multiple cytokines from a single cell). ELISA showed that TEX reduced IFN-γ secretion by CD8+ T and NK cells. KCa3.1 channels regulate the Ca2+ signaling necessary for these immune effector functions, but the effect of TEX on KCa3.1 activity is not known. Electrophysiology revealed that TEX inhibited KCa3.1 channel activity in HD CD8+ T cells. Overall, our data suggest that KCa3.1 channels mediate the inhibitory effect of TEX on anti-tumor immunity.

Bridge funds by the Dept of Internal Medicine, University of Cincinnati to Dr. Laura Conforti Pilot grant from the University of Cincinnati Cancer Center to Dr. Laura Conforti