bluuebunny/crossref_metadata_2025 huggingface dataset:
iNKT cell stimulation by glycolipid ligands modified from α-galactosylceramide results in differential interleukin-2 secretion profiles
Authors: Schafer Carolina E. , Santibáñez Álvaro , Gutiérrez-González Matías , Toro Jessica , Marcelain Katherine , Howell Amy R. , Porcelli Steven A. , Carreño Leandro J.
Published: January 2023

Invariant natural killer T (iNKT) cells are a specialized group of unconventional T cells that recognize glycolipids presented by the surface protein CD1d, expressed by most antigen-presenting cells. This results in the rapid secretion of a wide array of cytokines without the need for prior antigen immunization, thus making iNKT cells an attractive target for immunotherapy. Marine sponge sphingolipid α-galactosylceramide (α-GalCer) is a potent agonist for iNKT cell activation, characterized for inducing both Th1- and Th2-like cytokine secretion profiles. There is an increasing interest for the development of Th1- and Th2-polarizing ligands for more focused immunotherapeutic applications. Our aim is to evaluate the iNKT cell activation ability of C6” modified α-GalCer derivatives. We generated and isolated clones from iNKT cell hybridomas derived from a partially humanized mouse, consisting of a knock-in of the human CD1d gene. We selected by flow cytometry iNKT cell populations reactive to α-GalCer presented in the context of the human CD1d molecule. Additionally, we evaluated activation of iNKT cells by C6” modified α-GalCer derivatives and obtained a diverse interleukin-2 response depending on the structural modifications of the ligands and the individuality of the iNKT cell clones. In several cases, this response was more potent than that induced by stimulation with α-GalCer. Our results highlight differences between mouse and human lipid antigen presentation, as well as differences in the magnitude of the cytokine response amongst clonal populations. Our ongoing work aims to elucidate the mechanism for this observation by performing Next Generation Sequencing of the TCR chains of the responding clones.
MHC class I and II genes dependent hypersensitivity to nevirapine and efavirenz in Indian HIV-1 patients
Authors: Dalal Bhavik Ashok , Shankarkumar Aruna , Pazare Amar
Published: January 2023

OBJECTIVE

The present study demonstrates and investigates the involvement of differences in human leukocyte antigens (HLA) class I and class II genes in the risk of hypersensitive reaction to nevirapine and efavirenz among Indian HIV- 1 infected patients.

METHODS

Thirty-three HIV-positive patients treated with efavirenz and nevirapine were studied. The HLA class I and II molecular typing in both Non-tolerant and tolerant patients were performed using the sequence-specific primers (SSP), the Olerup HLA-A-B-C and DQ-DR SSP Combi Tray. Odds ratios (OR) with 95 % confidence intervals (CI) were calculated to assess patients’ increased risk of developing hypersensitivity in comparison with Tolerant.

RESULTS

Thirteen patients (39.39%), developed a hypersensitive reaction thus requiring the drug to be discontinued. AnHLA-B*40 and HLA-C*15 allelesrevealed a significant association in 8/13 (61.53%) p = 0.028, OR= 9.6; 95% CI=1.04–222.49 and 7/13 (53.84%)p = 0.05, odds ratio (OR)= 8.75; 95% CI= 0.92–205.44 of hypersensitivity reaction in HIV patients compared with only 4% in Tolerant group, respectively. MHC class I genes, HLA-A*24 allele and MHC class II genes, DRB1*10:01:01:01 allele showed significant association with skin rash in 5/13 (38.46%) and 4/13 (30.76%) respectively (p<0.05). One patient developed potentially life-threatening condition i.e. Steven Johnson Syndrome.

CONCLUSION

A strong significant associations were seen between the MHC genes alleles (HLA-B*40 and HLA-C*15) and cART drug (nevirapine and efavirenz) induced skin rash in the Indian population. It can be utilized to avoid a subset of hypersensitivity reaction in HIV infected patients in Indian population.
Identification of novel subsets amongst dendritic cells and macrophages generated from the culture of bone marrow
Authors: In Hyunju , Na Hye Young , Ryu Seul Hye , Sohn Moah , Choi Wanho , Shin Hyun soo , Park Ji Soo , Park Chae Gyu
Published: January 2023

Dendritic cells (DCs) play pivotal roles in various immune responses by sampling and presenting antigens to naïve T cells and inducing the subsequent T-cell activation or tolerance. In vitro culture systems have been widely used to study DC biology. The culture of bone marrow (BM) with the aids of a hematopoietic cytokine, Fms-like tyrosine kinase 3 ligand (FLT3L), yields plasmacytoid DCs (pDCs) and classical DCs (cDCs), whereas the culture of BM in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) produces BM-derived DCs and macrophages considered to possess inflammatory phenotypes. In the present study, various antibodies including DC-specific monoclonal antibodies were utilized to carefully describe the subsets of cells present in the cultures of BM with GM-CSF or FLT3L. We identified new subsets of DCs and macrophages: 3 different subsets of DCs and macrophages were characterized from the CD11c+ MHCIIhi cells in the BM culture with Flt3L by flow cytometric analyses. The newly discovered subsets of DCs and macrophages present in the BM culture require further investigation to distinguish and elucidate their phenotypes and functions in details.
Characterizing the MHC-II immunopeptidome of HIV using a cell-free antigen processing system and peptide:MHC-specific antibodies
Authors: Sengupta Srona , Douglass Jacqueline , Boronina Tatiana , Wu Fengting , Moskovljevic Milica , Reed Madison , Yu Jeanna , Tabdili Yasmine , Siliciano Robert , Sadegh-Nasseri Scheherazade
Published: January 2023

The breadth and specificity of CD4+T cell responses in HIV-1 infection are critical determinants of viral load and an individual’s ability to control infection. Most studies of CD4+T cell responses against HIV have relied on indirect evidence from peptide-pulsing analyses. Direct studies of antigen presentation have been hampered by the technical difficulties inherent in isolating peptide:MHC (p:MHC) complexes from HIV-infected cells. This has made it challenging to understand the HIV immunopeptidome, particularly for MHC-II epitopes. Using a cell-free antigen processing system, we have generated a near complete map of potential DR1-restricted HIV-1 epitopes. To confirm that epitopes identified using this system are actually presented by HIV-infected cells, we generated novel reagents termed single-chain diabodies (scDbs), or bispecific antibodies, which contain one Fab fragment against a p:MHC and another against CD3. These antibodies enable the use of CD8+ or CD4+T cells as readouts for antigen presentation. In proof of concept experiments, scDbs directed against immunodominant HIV MHC-I epitopes enhance cytokine production by CD8+ T cells, confirming presentation of these peptides. Preliminary experiments showed similar results with scDbs targeting MHC-II Gag epitopes on dendritic cells overexpressing the relevant peptides. We are now profiling the use of scDbs to detect antigen presentation in HIV-infected macrophages and CD4+T cells, the two target cells of infection. By establishing an immunopeptide map and tools to detect HIV peptide presentation, we can begin to address the kinetics, pathways, and cell types contributing to HIV antigen presentation on MHC-II, which will inform improved vaccine therapies.
Biodegradable Nanoparticles Containing Antigen and Vitamin D3 Efficiently Induce Antigen-specific Immune Suppression
Authors: Lee Chong-Kil , Jeong Seong-Un , Jung Ho-Hyun , Kim Sang-Hyun , Kim Kyungjae
Published: January 2023

Biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) encapsulated with antigens can efficiently deliver the antigens to antigen-presenting cells such as dendritic cells (DCs). Active form of vitamin D, 1α,25-dihydroxyvitamin D3 (VD3), is known to have various immunosuppressive effects. Encapsulation of VD3 within NPs would enhance the delivery of VD3 to APCs, while preventing the potential systemic side effects of VD3. In this study, we examined the effects of PLGA NPs encapsulated with ovalbumin (OVA) and VD3. PLGA NPs containing OVA and VD3 (NP[OVA+VD3]) were prepared by W1/O/W2 double emulsion solvent evaporation method. Bone marrow-derived DCs treated with NP[OVA+VD3] were decreased in the expression of co-stimulatory molecules, and produced higher levels of immunosuppressive cytokines, while producing lower levels of pro-inflammatory cytokines, compared with DCs treated with NP[OVA]. Addition of NP[OVA+VD3] to allogeneic MLR reduced the proliferation of allogenic T cells. In addition, NP[OVA+VD3]-treated DCs efficiently generated CD4+CD25+Foxp3+Treg cells from CD4+CD25− T cells. Furthermore, mice injected i.v. with NP[OVA+VD3] were suppressed in inducing OVA-specific cytotoxic T cells. These results show that phagocytic delivery of antigen and VD3 to APCs using biodegradable NPs efficiently suppresses antigen-specific immune responses.
Chronic immune barrier dysregulation among women with a history of violence victimization
Authors: Kohlmeier Alison , Haddad Lisa B , Li Tiger Zheng-Rong , Brookmeyer Kathryn A , Baker James M , Chen Cheng Y , Kersh Ellen N , Herbst-Kralovetz Melissa M. , Hogben Matthew , Ofotokun Igho , Kohlmeier Jacob E
Published: January 2023

We explored the association between violence victimization and increased risk for acquiring sexually transmitted infections (STI) in women by measuring cellular immune barrier properties from the female reproductive tract (FRT). STI-negative participants reporting repeated prior victimization occurrences through the lifetime trauma and victimization history (LTVH) instrument were more likely to exhibit alterations in barrier homeostasis and the composition of critical immune mediators irrespective of demographic parameters or presence of bacterial vaginosis. By combining cellular data with mixed-effect linear modeling, we uncovered differences in local T cells, MHCII+ antigen presenting cells, and epithelial cells indicative of altered trafficking behavior, increased immunosuppressive function, and decreased barrier integrity at sites of STI exposure that correlate with LTVH score. These data evidence a biological link between a history of violence victimization and risk of STI acquisition through immune dysregulation in the female reproductive tract.
Synergy between T-dependent antibodies and IL-10 limits susceptibility to inflammatory bowel disease
Authors: Landuyt Ashley E , Klocke Barbara J , Schoeb Trenton R , Maynard Craig L
Published: January 2023

Inflammatory bowel diseases (IBD) are chronic autoinflammatory conditions in which the immune system loses tolerance to gastrointestinal microbes. The role of humoral immunity in IBD is largely undefined. We examined the potential of T-dependent antibodies to prevent intestinal inflammation using mouse models of IBD. For this, we used mice without inducible T cell costimulator ligand (ICOSL). Icosl−/− mice produce significantly fewer T-dependent antibodies. When tested, we found Icosl−/− mice had similar IgA binding but significantly reduced IgG binding to mucosally-associated microbes. Additionally, Icosl−/− mice have more CD4 T cells producing IL-10, an immunosuppressive cytokine, in the colon. We hypothesized that T-dependent antibodies and IL-10 cooperatively promote intestinal homeostasis. To test this, we temporarily eliminated IL-10 producing cells from adult mice. Icosl−/−, but not wild type or IgA−/− mice, rapidly developed colitis under these conditions. This colitis could be prevented if mice were first depleted of Gram-positive or anaerobic bacterial flora. We further investigated the synergy between these T-dependent antibodies and IL-10 utilizing mice with a codeletion of CD4 T cell-derived IL-10 (Il10cKO) and ICOSL. This codeletion resulted in colitis before 28 days of age. However, fostering Icosl−/− Il10cKO mice on wild-type mothers delayed this early-onset inflammation. This suggests T-dependent antibodies conveyed through breastmilk are protective against early-onset colitis. Collectively, our results suggest that IL-10 and T-dependent antibodies toward mucosa-associated Gram-positive anaerobes continually cooperate to maintain intestinal homeostasis throughout the lifespan.
Deficiency in B Cell Maturation Antigen reveals gender differences in Experimental Autoimmune Encephalomyelitis
Authors: Kumar Gaurav , Ko Rose M , Axtell Robert C.
Published: January 2023

Multiple sclerosis (MS) is immune mediated demyelinating disease of the central nervous system (CNS) leading to neuronal damage. The disease is heterogeneous with respect to onset and clinical course, being more prevalent in women than in men with a ratio of 3:1. Further sex differences have been observed with males having a more severe and debilitating disease course compared to female. The complex interplay of immune cells, sex hormones or genetic differences could be contributing to these clinical phenomena, which remains to be elucidated. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, several reports have demonstrated no sex differences in disease onset or severity in C57BL/6 mice. Herein we report that deficiency in B Cell Maturation Antigen (BCMA) in C57BL/6 mice reveals a sex difference in EAE. BCMA plays a prominent role in B cell function, however, the role BCMA plays in MS and EAE is unknown. Our initial goal of this project was to determine the impact BCMA deficiency has on EAE. In doing so we confirmed that EAE disease was not different in males and females from BCMA+/+mice. Strikingly in the BCMA−/− mice, there was a significant increase in EAE disease severity in the males compared to females. The increased EAE in the BCMA−/− male mice was associated with increased demyelination and infiltration of inflammatory T cells and macrophages into CNS. Also, we observed decreased splenic transitional B cells in male BCMA−/− mice compared to females. Altogether, our data demonstrate that male BCMA−/− have elevated inflammatory responses compared to females leading to increased neuro-inflammation. Further experiments are underway to define the specific mechanisms behind the sex differences in BCMA deficient mice.
GMCSF-neuroantigen vaccines engender FOXP3+ regulatory T cell responses through low-efficiency self-antigen recognition events integrated through diminished CD40L-CD40 signaling
Authors: Moorman Cody D , Mannie Mark D
Published: January 2023

Neuroantigen (NAg)-specific tolerogenic vaccines that induce myelin-specific FOXP3+ regulatory T cells (Tregs) may be an effective treatment for Multiple Sclerosis. Previous studies showed that fusion proteins comprised of GM-CSF and NAg (GMCSF-NAg) are potent tolerogens that inhibit several rodent models of experimental autoimmune encephalomyelitis. GMCSF-NAg-induced T cell responses were investigated in vivo by use of 2D2-FIG mice, which have a FOXP3 GFP reporter (FIG) and a transgenic TCR (2D2) which recognizes myelin oligodendrocyte glycoprotein peptide 35–55 (MOG) as a low-efficiency antigen and neurofilament medium peptide 13–37 (NFM) as a high-efficiency antigen. A single subcutaneous vaccination of 2D2-FIG mice with GMCSF-MOG resulted in high numbers and percentages of Tregs in the blood, spleen, and lymph nodes, whereas GMCSF-NFM elicited a dominant conventional T cell (Tcon) response. To determine how low and high-efficiency TCR responses regulate cell lineage fate, we investigated TCR-induced CD40L expression. GMCSF-NFM, but not GMCSF-MOG, induced significant CD40L expression when cultured with 2D2 responder T cells. Pretreatment of 2D2-FIG mice with an agonistic anti-CD40 monoclonal antibody prevented GMCSF-MOG induction of Tregs and favored Tcon responses. MOG35–55 was a partial TCR antagonist that blocked NFM stimulation of CD40L expression in 2D2 T cell cultures. Similarly, GMCSF-MOG was functionally dominant and induced Tregs in 2D2-FIG mice when mixed with an equimolar dose of GMCSF-NFM. These data indicated that low-efficiency antigen recognition is a key parameter for the induction of Treg responses and is in part controlled through CD40L-CD40 signaling pathways to determine T cell lineage fate.
Identification of markers of relapse in a relapsing-remitting mouse model of multiple sclerosis
Authors: Chase Carol Ann , Forsthuber Thomas
Published: January 2023

Approximately 85 percent of individuals newly diagnosed with multiple sclerosis have the relapsing-remitting form of the disease characterized by attacks of neurologic symptoms that are both unpredictable in occurrence and duration. The lack of markers that reliably predict the occurrence of multiple sclerosis relapses hinders the ability for proactive therapeutic intervention. To address the urgent need for biomarkers of relapse we investigated proteome changes over the disease course of relapsing-remitting experimental autoimmune encephalomyelitis (EAE) in SJL mice as a preclinical model of the disease using a high-throughput quantitative proteomic technique. In this study, we utilized this established proteomic technique as well as bioinformatics tools to prioritize key proteins that were expressed differentially at different stages of disease including remission and relapse. Principal component analysis of protein expression shows that the remission and relapse phases of disease cluster separately, indicating distinguishable variation between the two disease states. Importantly, statistical testing identified proteins with differential expression in the CNS at different stages of disease, several of which are CNS specific. We are seeking to detect corollary changes in these CNS-specific proteins in the serum, pointing to a minimally invasive means of monitoring disease progress and measuring drug efficacy. Our studies will provide proof-of-concept for identifying homologous human biomarkers to guide treatment in individual patients. Furthermore, our results may provide insights into mechanisms that contribute to disease pathology and offer additional therapeutic targets.
Gemcitabine promotes tumor cell-derived inflammatory responses leading to immunosuppression
Authors: Ding Chuanlin , Yan Jun
Published: January 2023

Some chemotherapeutic agents can selectively deplete MDSC in vivo. However, recent studies suggest that Gemcitabine (GEM) and 5-Fluorouracil (5-FU) treatment decrease the anticancer efficacy by activating the inflammasome pathway in MDSC. In addition, chemotherapy can modify the tumor microenvironment by induction of chronic inflammation. Therefore, regulations of MDSC by chemotherapy still remain elusive. Here, we explored the mechanisms by which multi-dose clinical regimen of GEM treatment induce an ongoing inflammation leading to the enhanced immunosuppressive activity of monocytic MDSC (M-MDSC). We found that repeated GEM treatment promoted the expansion and differentiation of immunosuppressive M-MDSC although tumor development was delayed in E0771 tumor-bearing mice. Accordingly, IFN-γ-producing CD4 and CD8 T cells were significantly decreased in the TME of GEM-treated mice. The conditioned medium of GEM-treated tumor cells (GEM-CM) promoted the differentiation of immunosuppressive mouse BM Ly6Chigh cells. Tumor-derived soluble factors, such as GM-CSF and soluble ICAM-1(sICAM-1), were up-regulated upon chemo-drug treatment. Blocking of GM-CSF or ICAM-1 with neutralizing Abs could partially reduce immunosuppression of Ly6Chigh cells induced by GEM-CM. Enhanced production of reactive oxygen species (ROS) and activation of NF-κB were observed in GEM-treated tumor cells. Treatment with the inhibitor of NF-κB signaling could abrogate GEM-induced hyperexpression of GM-CSF in E0771 cells. Together, our results define an undesired effect of GEM-induced ROS which leads to NF-κB activation, up-regulation of GM-CSF, and subsequent differentiation of immunosuppressive myeloid cells.
Tumor exosome mediated immune regulation in colorectal cancer
Authors: Subramanian Subbaya , Zhao Xianda , Yaun Ce
Published: January 2023

Colorectal cancer (CRC) remains the third most common cause of cancer-related deaths in the United States. Most (~85%) of CRC tumors are nonimmunogenic, i.e. they lack a significant number of tumor-infiltrating T cells, and are typically unresponsive to the current immune checkpoint inhibitor-based therapies. T cells isolated from nonimmunogenic, microsatellite stable (MSS) CRC have lower levels of CD28, which provides a costimulatory signal required for T-cell activation, trafficking, proliferation, differentiation, and cytotoxic activity. CRC tumors have elevated levels of miR-503 and its cluster member miR-424, both directly target CD28 in vitro. Our data also showed that these miRNAs are present in extracellular vesicles (EVs) secreted by CRC cells, which are taken up by T cells and affect its proliferation and function in the tumor microenvironment. Further, EVs with these immune suppressive miRNAs support tumor progression in CRC mouse models. Strikingly, in orthotopic models of CRC, mice preconditioned or treated with modified EVs (EVs lacking functional miR-503 and/or miR-424 (Δ503/424-EVs)) are protected against tumor formation and have a significantly reduced tumor burden, while mice receiving control EVs or PBS solution all form tumors. Our results provide preclinical evidence that treating CRC patients with modified EVs (lacking immunosuppressive miRNAs) will significantly improve antitumor immune response and reduce tumor burden. These results provide insights on how cancer cells modulate and suppress the immune response, provide novel targets, and form the basis for a new anti-cancer therapeutic strategy.
Butyrophilin 3A1 is a Dynamic T Cell Regulator in Ovarian Cancer
Authors: Payne Kyle K , Gonzalez Ricardo Chaurio , Sanz Jairo Perez , Galindo Carmen Maria Anadon , Biswas Subir , Mine Jessica A , Perales-Puchalt Alfredo , Tsiganov Evgenii , Costich Tara Lee , Harro Carly M , Marrs Aubrey K , Lajoie Jason , Li Kun-Po , Ophir Michael , Eskiocak Ugur , Schmidt Michael , Gabrilovich Dmitry I , Conejo-Garcia Jose R
Published: January 2023

Ovarian carcinoma microenvironmental T cells exert clinically relevant pressure against malignant progression; however current immunotherapies rarely induce ovarian cancer regression. Here we investigate CD277-containing butyrophilin 3A1 (BTN3A1), a poorly investigated immunoregulatory pathway driven by myeloid and tumor cells in ovarian tumor beds. We show that BTN3A1 is overexpressed in ovarian cancer and is associated with a significant survival disadvantage in these patients (n=200). Concomitantly, ectopic expression of BTN3A1 on APCs inhibits αβ T cell proliferation and Th1 cytokine production. Proteomic analyses and binding assays demonstrate that BTN3A1 interacts with the CD45 phosphatase and elements of the TCR. Consequently, TCR ligation in the presence of BTN3A1 inhibits the segregation of CD45 from the immune synapse and blunts downstream signaling by antagonizing the phosphorylation of CD3Zeta, Lck, and Zap70. We developed fully human αCD277 antibodies which rescue αβ T cell proliferation and Th1 cytokine responses, while driving the infiltration of T cells into tumor beds, delaying ovarian tumor progression in novel BTN3A1+ humanized mice and xenograft studies. Paradoxically, αCD277 antibodies promote the activation of γδ T cells by driving a conformational transformation of BTN3A1. Thus, co-transfer of γδ and Ag-specific αβ T cells in the presence of αCD277 antibodies synergize to further impair malignant progression in vivo. Overall, we show that BTN3A1 drives αβ T cell dysfunction in ovarian cancer, while αCD277 antibodies transform this molecule from immunosuppressive to immunostimulatory by rescuing αβ T cells and activating γδ T cells, thus dynamically unleashing T cell-driven antitumor immunity.
Human intrinsic immunity factor SLFN11 promotes cell death upon DNA damages through cleavage of distinct type II tRNAs and inhibition of proteins syntheses related to DNA damage responses and repairs
Authors: Li Manqing , Kao Elaine , Malone Dane , Gao Xia , Wang Jean Y. J. , David Michael
Published: January 2023

Previously we showed that as an intrinsic immunity factor, human SLFN11 inhibits HIV viral proteins syntheses by modulating tRNAs abundances and exploiting the biased codon usage of the virus. Transcriptome analysis in vast collection of cancer cell lines revealed that SLFN11 is the only gene whose expression significantly sensitizes tumor cells to DNA damaging agents (DDAs). We hereby report that SLFN11 preferentially inhibits translation of genes involved in DNA damage response and repair, including ATR and ATM, upon DDAs treatment based on their distinct codon usages without disrupting early DNA damage response signaling. Type II tRNAs, which include all serine and leucine tRNAs, are cleaved in a SLFN11-dependent manner in response to DDAs. Genes with high TTA (Leu) codon usage such as ATR display utmost susceptibility to translational suppression by SLFN11 due to the extreme low abundance of cognate tRNA-Leu-TAA. Specific attenuation of tRNA-Leu-TAA with antisense oligonucleotides (ASO) sufficed to ablate ATR protein expression and restore DDA sensitivity of SLFN11-deficient tumor cells. Our study uncovered a novel mechanism of DNA damage response regulation mediated by SLFN11-dependent tRNA cleavage and codon-specific translational inhibition. The immunomodulation of SLFN11 expression and activation can be a potent novel approach to overcome the wide-spreading resistance of tumor cells to chemo-/radio-therapies.
IFN-γ inhibit ALDHbr breast cancer cells in murine 4T1 tumor model
Authors: Shi Guilan , Zhuang Xiufen , Wu Yanhong , Heller Richard
Published: January 2023

Background

Due to the heterogeneity of breast cancer cells, cytotoxic treatments targeting proliferative cancer cells could not eradicate all cancer cells. Compelling evidence suggests that breast cancer stem cells (BCSCs) have a crucial impact in current shortcomings of cancer therapy because they are largely responsible for tumor initiation, relapse, metastasis, and chemo-resistance. Cytokines in the tumor microenvironment can regulate the self-renewal and survival of BCSCs in a variety of ways.

Methods and Results

We have utilized in vitro and mouse xenograft models (immunocompetent Balb/c mice and thymus-deficient nude mice (Balb/c nude)) to examine the interaction between breast cancer stem cells (BCSCs) and T cells or cytokine IFN-γ. In vitro assay, such as migration and invasion and sphere-forming, we demonstrate that CD8+ T lymphocytes regulate aldehyde dehydrogenase (ALDH) expressing in BCSCs (ALDHbr 4T1 cells) may through cytokine IFN-γ. Utilizing immunocytochemistry, western blotting and gene expression microarray of ALDHbr vs ALDHdim 4T1 cells, we identified cytokine IFN-γ decreased ALDH1A protein expression and regulated the cancer stem cell population. In various in vivo-like screening assays, depletion of either CD4+ or CD8+ T cells associated with increased proportions of BCSCs.

Conclusion

The collective results suggest that IFN-γ as a highly secreted cytokine may affect cancer stem cells in mouse breast cancer model.
Complete workflows allow comprehensive tumor microenvironment analysis and culture of cell subsets of limited tumor patient samples
Authors: Evaristo Cesar , Siemer Ramona , Criado-Moronati Elvira , Baranska Anna , Gert Philipp , Agorku David , Hardt Olaf , Vorholt Daniela , Singh Aparajita , Dose Christian , Heemskerk Bianca , Richter Anne
Published: January 2023

Immunotherapy has proven clinical efficacy and tremendous potential. But clinical benefit is experienced by only a subset of patients, such that additional research is necessary to improve outcomes. In particular, analyze steady-state anti-tumor immunity and monitor the effects of therapy on the tumor microenvironement. However, TIL numbers can be very low. Flow cytometry phenotyping different cell populations requires dividing limited tumor material into multiple samples, reducing the number of cells available for analysis. Therefore, innovative tools and workflows are needed to maximize the quality of data obtained from limited tumor patient samples.

We established workflows combining tissue storage, dissociation, cell isolation and analysis. We developed new reagents for magnetic cell sorting, incorporating novel technology enabling the removal of magnetic beads and antibody fragments (REAlease®). Tumor-infiltrating (TI) T-, B-, Myeloid cells, cancer-associated fibroblasts and tumor cells were sequentially isolated and characterized by flow cytometry, multi-parameter imaging and transcriptomics. Importantly, purification of TI T cells with high viability was essential for high-resolution immune profiling by single cell transcriptomics.

In conclusion, we optimized workflows for the comprehensive analysis of the tumor microenvironement. Sequential isolation of the different cell types maximized the number of cells available for analysis. These workflows reduce experimental time and allow the performance of more complex experimental setups. We believe the use of these innovative tools and workflows can significantly increase the quality of the data obtained in immunooncology and immunotherapy research.
Combination of anti-CD40 and anti-PD1 revert M2 polarization to limit tumor growth in a genetically engineered blander cancer mouse model
Authors: Verdeil Gregory , Leblond Marine , Imbratta Claire , Speiser Daniel E
Published: January 2023

Bladder cancer (BCa) represents the fourth most common cancer in men with a poor patient prognosis for advanced disease. About 75% of patients present non-muscle invasive BCa (NMIBC) and 25% present muscle invasive BCa (MIBC) or metastatic disease. Although BCG immunotherapy is a common treatment of BCa, the dynamics of immune cell infiltration and its relevance for disease outcome are still not well understood. We took advantage of a genetic based mouse model of MIBC to obtain a profound understanding of the immune contexture over tumor development. P53Fl/Fl/PTENFl/Fl mice were injected with Cre recombinase-expressing adenoviral vectors directly into bladder by micro-surgery. We observed the presence of NMIBC, MIBC and the development of metastases at 5, 9 and 13 weeks after vector injection, respectively. As tumor evolves, we observed a transition from an anti-tumoral microenvironment (MHCIIhigh TAM, CD4 and CD8 TILs) to a pro-tumoral one (MHCIIlow TAM and MDSC), going along with an increased expression of PD-1 by T cells and accumulation of regulatory T cells. After testing various immunotherapy tretaments on mice with MIBC, we found out that combination of αCD40+αPD1 increases CD8 TILs infiltration and IFNg production, re-polarized TAM toward a M1-like phenotype and increased the survival of the mice.
Therapeutic targeting of the immunosuppressive axis in ovarian cancer using dietary supplements
Authors: Bollino Dominique , Webb Tonya J
Published: January 2023

Ovarian cancer is the deadliest gynecologic malignancy worldwide and mortality rates have remained steady despite advances in treatment. A major obstacle for current therapies is overcoming tumor immunosuppression, therefore targeting inhibitory factors produced in the tumor microenvironment could enhance antitumor responses. Natural Killer T (NKT) cells, a subset of T cells that recognize glycolipid antigens presented by CD1d, are important mediators of antitumor immune responses. We have previously shown that ovarian cancers shed the ganglioside GD3, which binds with high affinity to CD1d, thereby suppressing NKT cell activation. In addition to GD3, we have identified vascular endothelial growth factor (VEGF) as another immunosuppressive factor secreted by ovarian cancers. We found that VEGF inhibition results in decreased GD3 expression in human ovarian cancer cell lines and restoration of NKT cell responses. In addition, we found that treatment with VEGF can directly impair NKT cell functional responses. To therapeutically target the VEGF-GD3 signaling cascade, mouse and human ovarian cancer cell lines were treated with triptolide, a phytochemical which has demonstrated potent antitumor activity in vitro and in vivo. Treatment with triptolide reduces VEGF secretion by ovarian cancer cell lines and results in a concomitant decrease in the expression of GD3 synthase. In addition, pre-treatment of ovarian cancer cells with triptolide improves the ability of NKT cells to recognize and lyse the cancer cells. Thus, we have identified a novel therapeutic strategy that can be used to block the production of immunosuppressive factors, thereby restoring NKT cell-mediated antitumor immune responses in ovarian cancer.
The NOD-like receptor NLRP12 plays a critical role in hepatic inflammation and cancer
Authors: Zaki Hasan , Udden SM Nashir
Published: January 2023

Hepatocellular carcinoma (HCC) is a deadly human cancer associated with chronic inflammation. The cytosolic NOD-like receptor NLRP12 has emerged as a negative regulator of inflammation, but its role in HCC is unknown. Cancer genomics database analysis showed that NLRP12 expression is significantly reduced in human HCC and around 2% HCC patients carry mutations in NLRP12. We investigated the role of NLRP12 in HCC using mouse models of HCC induced by carcinogen diethylnitrosamine (DEN). Nlrp12−/− mice were highly susceptible to DEN-induced HCC with increased inflammation, hepatocyte proliferation, and tumor burden. Consistently, Nlrp12−/− tumors showed higher expression of proto-oncogenes cJun and cMyc and downregulation of tumor suppressor p21. Interestingly, antibiotics treatment dramatically diminished tumorigenesis in Nlrp12−/− mouse livers. Signaling pathway analyses demonstrated higher JNK activation in Nlrp12−/− HCC and cultured hepatocytes during stimulation with microbial pattern molecules. Inhibition of JNK activation or NLRP12 overexpression reduced proliferative and inflammatory responses of Nlrp12−/− hepatocytes. In summary, this study demonstrated for the first time that NLRP12 negatively regulates HCC pathogenesis via downregulation of JNK-dependent inflammation and proliferation of hepatocytes.
Humanized PD-1/PD-L1 (HuPD) mice facilitate the direct functional comparison of immune checkpoint inhibitors in vivo
Authors: Barham Whitney Jo , Harrington Susan M. , Liu Xin , Dong Haidong
Published: January 2023

Though anti-PD-1/L1 antibody therapies have generated unprecedented responses in a limited number of patients, the biological and immunological parameters that define their effective use are still largely unknown. The absence of appropriate pre-clinical models has limited our ability to compare the mechanistic differences between the approved PD-1/L1 antibodies in vivo and to determine the context in which each might be most effective. To address this critical need, we have produced humanized PD-1 and PD-L1 mice in which the extracellular domains (antibody binding sites) of mouse PD-1 and PD-L1 are replaced with the sequences of human PD-1 and PD-L1. The PD-1 and PD-L1 in the resulting “HuPD” mice can be recognized by FDA-approved antibodies, while the intracellular domains are kept intact for normal downstream signals. Following characterization of the new mouse model, in our first in vivo treatment studies, HuPD mice were immunized with OVA/Poly I:C in the context of FDA-approved PD-1/L1 antibodies and antigen-specific T cell production was quantified. Nivolumab, Pembrolizumab, and Atezolizumab all increased effector CD8+T cell production against the OVA antigen as expected given their immune stimulating function. Surprisingly, OVA-tetramer staining was unable to detect antigen-specific CD8+T cells in the spleens of immunized mice treated with Avelumab. This may reflect the unique ability of Avelumab to induce ADCC, and indicates that our model is capable of distinguishing key differences among the antibodies in vivo. Ongoing studies will identify how these differences might be exploited to render the most effective results for patients in a variety of tumor types and immune environments.
A novel TNFR2 antibody induces T cell co-stimulation and promotes durable anti-tumor immunity
Authors: Sampson James F , Fulton Ross B , Kurella Vinodh B , Richards Jennifer M , Camblin Adam J , Wong Christina S , Koshkaryev Alexander , Lulo James E , Qiu James A , Kumar Sandeep , Luus Lia , Jiao Yang , Suchy James , Grabow Stephanie , Paragas Violette , Razlog Maja , Xi Lihui , Muda Marco , Drummond Daryl C , Tam Eric M , Raue Andreas
Published: January 2023

TNFR2, an emerging therapeutic target in immuno-oncology, is upregulated upon T cell activation and is expressed by tumor-infiltrating effector and regulatory T (Treg) cells. We generated a novel monoclonal antibody, Y9, specific to murine TNFR2 and investigated its mechanism of action. In vitro, Y9 stimulation of purified T cells increased proliferation and effector function, indicating that Y9 acts as an agonist and can provide co-stimulation.

In vivo, Y9 treatment of mice with established tumors resulted in complete tumor clearance across a variety of models. The activity of Y9 on immune cells was confirmed by its decreased activity in mice depleted of NK or CD8+ T cells. Unlike the proposed Treg depletion mechanism of other therapeutic antibodies, depletion of Treg cells is not the primary mechanism of action of Y9 treatment. Instead, decreased TNFR2 and other co-inhibitory receptor surface expression was observed following treatment. Y9 activity depended on FcγR binding, which facilitated enhanced agonist activity.

Based on this evidence, we developed MM-401, a human antibody targeting TNFR2. MM-401 has agonistic activity; upon incubation of MM-401 with human CD4+ and CD8+ T cells, we observed upregulation of activation markers and cytokine production comparable to MEDI6469 (anti-OX40). We also observed that MM-401 promotes antibody-dependent cellular cytotoxicity (ADCC) in an NK cell-mediated in vitro assay and a reduction in the number of Treg cells in human ovarian cancer ascites. These data suggest that MM-401 could also promote anti-tumor immunity by mediating ADCC, as well as by direct co-stimulation of T cell responses, and justifies the continued development of MM-401 as a novel cancer immunotherapy.
The TNFR1/mTNFα signaling axis initiates tumor-promoting inflammation and is associated with poor outcome in neuroblastoma
Authors: Tomolonis Julie Anne , Ngai Ho , Courtney Amy , Metelitsa Leonid S
Published: January 2023

Neuroblastoma (NB) is a poorly differentiated, aggressive pediatric solid tumor for which half of high-risk cases have no identified genetic alteration. While such high-risk tumors are often infiltrated by macrophages that generate an inflammatory gene signature predictive of poor outcome, the initiation mechanism of this pro-tumorigenic inflammation remains unknown.

In our analysis of clinical outcome-linked NB gene expression data, high tumor expression of TNFR1 was strongly correlated with poor event-free survival (5-year EFS 15% vs 70%, bonf p=9.3e-07), highlighting the potential importance of TNFα signaling in NB. Using TNFα knockout human NB cell lines, we determined that monocytes, but not NB cells, produce TNFα during in vitro co-culture. Furthermore, using a TNFα converting enzyme inhibitor to prevent secretion of soluble TNFα, we found that the membrane-bound form of TNFα (mTNFα) expressed on monocytes was critical for NF-κB pathway activation and downstream production of IL-6, a known NB growth factor. Neutralization of TNFα or its receptor TNFR1, but not TNFR2, in a monocyte-NB co-culture prevented NF-κB activation in monocytes, reducing IL-6 production by 50% compared to controls (p <0.01).

Our results demonstrate that TNFR1-expressing NB cells activate monocytes via contact-dependent mTNFα signaling, leading to NF-κB activation and production of IL-6, a known driver of NB progression and therapy resistance. These findings reveal a novel, druggable mechanism of tumor-promoting inflammation in NB.
Regulation of PD-L1 expression in stimulated antigen specific CD8+ T cells by immunoglobulin 4 (IgG4)
Authors: Daftarian Pirouz M , Ouerkaxi Wushouer
Published: January 2023

Background

Many studies have focused on finding a correlation between the responsiveness to immune checkpoint (IC) therapies and the expression level of IC markers. Here, we report the studies on the expression of co-inhibitory molecules on healthy donor peripheral mononuclear cells (PBMC). We co-cultured PBMCs from different donors with recall antigen peptide to analyze the responses to PD-1 blockade antibody treatment. We have used our previously reported optimized recall antigen assay in which the expanded antigen specific CD8+ T cells (TET+) were measured as a readout after the peptide stimulation. Interestingly, the IgG4 appears to reduce the TET+ cell population. To further study this potential effect of IgG4, we conducted a series of immune-profiling studies by comparing TET+ gated and TET− CD8+ gated T cells.

Results and conclusion

Healthy donor PBMCs were stimulated with recall peptide alone, or peptide plus either PD-1 blockade, or IgG4 and evaluated the PD-1, PD-L1, Tim-3, and Lag-3 expressions in either T cells or in monocytes. We then compared such expressions in TET+ and TET− CD8+ T cells. Unexpectedly, IgG4 isotype control treatment was associated with upregulated PD-L1 expression only in the TET+ CD8+ T cells. To confirm whether the decrease of TET+ population in our assay was caused by the up regulation of PD-L1, we blocked the PD-1 signal transduction pathway by treating the cells with a validated PD-1 blocking antibody. As a result of PD-1 blockade, the expansion of TET+ cell population was recovered by anti-PD-1 blocking treatment. Taken together, these results suggest that IgG4 treatment with recall peptide up regulates PD-L1 expression and therefore reduces the expansion of antigen specific CD8+ T cells in some donors.
Effects of NLRP1 risk polymorphisms on IL-18 levels and gene expression
Authors: Poole Brian D , Westhoff Laura Judge , Gill Erin , Nishiguchi Michelle
Published: January 2023

Background

NLRP1 is a central component of the inflammasome. Several polymorphisms in the gene are linked to increased risk for autoimmune and autoinflammatory diseases. We examined the effects of these risk polymorphisms on cytokine levels and NLRP1 expression levels in human volunteers.

Materials and Methods

NLRP1 was genotyped at the SNPs rs2670660 and rs12150220 using TaqMan on peripheral blood cells. Homozygous individuals were selected for further study.

Serum IL-18 and IL-1B levels were quantified using ELISA. NLRP1 gene expression was measured semiquantitatively by SybrGreen real-time PCR.

Results

The risk variants at rs2670660 and rs12150220 were almost never found separately. There was no case in which a volunteer with homozygous risk SNP variant also had a homozygous protective variant at the other, indicating very strong linkage (p<2.33 × 10-13).

Volunteers with the risk haplotype (AAAA) had significantly higher levels of serum IL-18 than volunteers with the protective haplotype (GGTT) (0.439 ng/μL compared to 0.152 ng/μL, p=0.024)

A trend towards increased NLRP1 expression was seen in the risk haplotype compared to the protective (4.4-fold increase, p=0.101).

Discussion

The risk haplotype showed increased production of IL-18 even in non-autoimmune individuals, with a trend towards higher NLRP1 expression. This suggests that the risk variants lead to higher NLRP1 activity, and therefore a pro-inflammatory phenotype. Because of the strong linkage of the AAAA and GGTT genotypes, it was impossible to differentiate which SNP was responsible for these effects. Further studies with increased numbers of volunteers should allow an increased understanding of the contribution of NLRP1 risk variants to autoimmune disease
The role of factor Y in primary biliary cirrhosis
Authors: Cho Min Jeing , Dho So Hee , Shin Saeam , Lee Ji Yeon , Lee Yeong Un , Lee Kyung-a , Kim Lark Kyun
Published: January 2023

Primary biliary cirrhosis (PBC) is an autoimmune disease which is characterized by bile duct injuries result from excessive immune responses. In female over the age of 40, the incidence of PBC has been estimated to be one out of 1000 people. Liver transplantation is required for patients who progress to end-stage PBC. However, risk factors and causes of PBC remain elusive.

We performed whole-exome sequencing in a family with four sisters diagnosed with PBC and one healthy half-sister. Additionally, Sanger sequencing was conducted with 62 unrelated PBC patient specimens. Here, we identified a potential PBC causative gene factor Y, a regulator of cell death and inflammation. We investigated whether factor Y plays a role in PBC using factor Y null human macrophage cell line generated by CRIPSR/Cas9 technology. We found ablation of factor Y induces inflammatory cytokines compared with control cells upon treatment with LPS or TNF. Surprisingly, molecules secreted by factor Y null cell line induced fibrosis in human hepatic stellate cell, LX-2. These results indicate that factor Y is a potential target for treatment of PBC.
Critical role of macrophages in checkpoint blockade induced autoimmune diabetes in the NOD mouse
Authors: Hu Hao , Zakharov Pavel , Unanue Emil R
Published: January 2023

We confirmed that the PD-1/PD-L1 regulatory pathway is one control mechanism of autoimmune diabetes in NOD mice: administration of anti-PD-1 but not anti-CTLA-4 antibodies rapidly induced diabetes in young mice showing a control phase established very early in the autoimmune process. We confirmed that both CD4 and CD8 T cells and resident macrophages were essential for the PD-1 regulation to take effect.

We performed an unbiased single-cell RNA sequencing analysis of islets after anti-PD-1 treatment. We identified a subpopulation of highly activated macrophage showing an IFNγ signature with high level of CXCL9 and PD-L1 and a monocyte signature distinguishable by the markers Ly6c and CCR2. Flow cytometry analysis of islets upon anti-PD-1 treatment identified a predominance of the monocyte derived macrophages compared to the islet resident macrophages. Depleting blood monocytes by clodronate liposomes inhibited the recruitment of the monocyte derived macrophages and prevented the development of acute diabetes induced by anti-PD-1 treatment. To analyze the pathogenicity of this monocyte-derived macrophages, we performed cytotoxicity assays by co-culturing macrophages stimulated with LPS&IFNγ and a β-cell line Min6: the highly activated macrophage killed β-cells in a manner dependent on IFNγ and inducible nitric oxide.

In sum, our study shows highly activated macrophage derived from monocytes are cytotoxic for beta cells and promoting diabetes progression. Our findings also indicate that a consequence of checkpoint blockade is the recruitment and activation of cytocidal macrophages derived from monocytes. Targeting monocyte-derived macrophages may prevent some of the checkpoint blockade related immune-adverse effects.