bluuebunny/crossref_metadata_2025 huggingface dataset:
Influenza vaccination administered in the tumor induces an inflamed tumor microenvironment and reduces tumor growth
Authors: Newman Jenna H , Chesson Brent , Herzog Nora , Bommareddy Praveen , Aspromonte Salvatore , Pepe Russell , Estupinian Ricardo , Aboelatta Mones , Buddhadev Stuti , Schenkel Jason , Thomas Paul , Rudra Jai , Silk Ann , Zloza Andrew
Published: January 2023

Immunotherapy has revolutionized cancer treatment; however, a significant fraction of patients exhibit resistance to immunotherapy, in part due to the lack of an inflamed (“hot”) tumor microenvironment. Seeking to recruit immune cells to tumors lacking immune cell infiltration (“cold” tumors), we observed that intratumoral injection of heat-inactivated influenza, but not live influenza, slows growth of B16-F10 melanoma in C57BL/6 mice (p<0.001). With clinical translation in mind, we next tested whether FDA-approved seasonal influenza vaccines could likewise reduce tumor growth. Mice administered influenza vaccines by intratumoral injection exhibited smaller tumors than PBS-injected controls (p<0.001) and, importantly, were protected against future live influenza challenge in the lung. Vaccinated mice harbored a greater proportion of intratumoral dendritic cells, overall T cells, and tumor-reactive CD8+ T cells, and exhibited a more inflamed tumor signature associated with clinical response to immunotherapy. Furthermore, we observed that intratumoral influenza vaccination results in systemic responses, reduces metastasis, and synergizes with PD-L1 blockade to induce tumor regression. While all unadjuvanted vaccine formulations tested slowed tumor growth, a formulation containing a squalene-based adjuvant failed to do so due to induction of B regulatory cells in the tumor. Adjuvant removal or B cell or IL-10 depletions rendered anti-tumor effects to the adjuvanted vaccine (p<0.01, p<0.01, and p<0.001, respectively). Future research will focus on further understanding underlying mechanisms and translating these findings to the clinic as an immunotherapy for cancer.
Targeting DAMP-induced inflammation to prevent breast cancer metastasis and improve anti-tumor immunity
Authors: Frazier Victoria N , Holl Eda , Boczkowski David , Sullenger Bruce , Nair Smita K
Published: January 2023

Biomarkers associated with systemic inflammation correlate with poor prognosis in cancer patients. Circulating cell-free DNA (cfDNA) is released passively and actively by both tumor cells and host cells and cfDNA levels in blood correlate with tumor burden in many tumor types. Notably, studies in breast cancer have shown that inhibition of cfDNA has a significant effect in reducing invasion and migration in vitro and metastatic disease in vivo. We have previously demonstrated that nucleic acid binding polymers (NABPs) bind and inhibit the proinflammatory effects of cfDNA in a murine model of pancreatic cancer.

We observed that levels of cfDNA in blood increase with tumor burden in an orthotopic immunocompetent murine model of breast cancer. Moreover, treatment of mice with the NABP, PAMAM-G3, following surgical removal of primary tumor results in decreased lung metastasis. This decrease in lung metastasis is associated with reduction in the levels of cfDNA in blood.

In addition, treatment of mice with NABPs in an intravenous experimental metastasis breast cancer model results in reduced seeding of tumor cells in lungs and lung metastasis. Analysis of immune infiltrates in lungs of PAMAM-G3- and control-treated mice demonstrates NABP-mediated transition of immune cell composition in lungs from innate to adaptive immune cells. Collectively, these data suggest that NABPs can be used to target cfDNA and inhibit DAMPs associated pathological inflammation. The use of NABPs is an innovative approach to treat cancer progression and metastasis, particularly in a clinical setting following standard of care surgical tumor resection.

This work is funded by the Department of Defense Breast Cancer Research Program award (PI, Smita Nair).
Glucocorticoid signaling regulates CD8+T cell responses in cancer
Authors: Acharya Nandini , Madi Asaf , Zhang Huiyuan , Klapholz Max , Christian Elena , Dixon Karen O , Mangani Davide , Xia Junrong , Singer Meromit , Rozenblatt-Rosen Orit , Regev Aviv , Kuchroo Vijay K , Anderson Ana C
Published: January 2023

Identifying signals in the tumor microenvironment (TME) that promote CD8+T cell dysfunction can inform the development of novel approaches to improve anti-tumor T cell responses. Here, we identified Nr3c1, the gene encoding the glucocorticoid receptor (GR), as one of the genes preferentially expressed by dysfunctional CD8+TILs. Glucocorticoids (GCs) are steroid hormones and ligands for the GR. Despite their widespread use, surprisingly little is known regarding the molecular circuitry by which glucocorticoids suppress immune responses. We demonstrate that glucocorticoids are synthesized de novo in the TME and activation of glucocorticoid signaling promotes features of dysfunction, including increased expression of checkpoint receptors like Tim3, PD1, Lag3 and TIGIT, and dampened effector function including decreased production of pro-inflammatory cytokines in both murine and human CD8+T cells. Deletion of GR in CD8+T cells in vivo resulted in loss of dysfunctional phenotype in CD8+TILs concomitant with improved tumor growth control. We thus identified glucocorticoid signaling as a component of immune suppression within the TME and uncovered a molecular mechanism by which it suppresses T cell responses. Glucocorticoids are the mainstay for treating the immune-related adverse events (IRAEs) that develop in patients receiving immune checkpoint blockade. Thus, having a better understanding of the mechanisms downstream of GCs could inform the application of glucocorticoids for suppressing immune responses.
Pharmacologic inhibition of liver X receptors enhances T cell anti-tumor function in triple negative breast cancer
Authors: Carpenter Katherine Joyce , Abuirqeba Suomia , Majidi Shabnam , Sengupta Monideepa , Valfort Aurore-Cecile , Chatterjee Arindam , DiPaolo Richard J , Flaveny Colin A.
Published: January 2023

While immunotherapies have demonstrated success in solid malignancies, they have limited efficacy in triple negative breast cancer (TNBC). The liver-X-receptors (LXR) are modulators of metabolism and are known to inhibit pro-inflammatory (Th17 and Th1) and increase anti-inflammatory (Tregs) T-cell differentiation. Colorectal and hematologic cancer cells have also been demonstrated to suppress immune function through production of endogenous LXR agonists. Here we show that targeted suppression of LXR activity using the inverse agonist, SR9243, induces immune-mediated tumor destruction in an orthotopic TNBC model. FACs analysis on tumors revealed a significant increase in Th1 and cytotoxic CD8+ T-cells and a decrease in tumor promoting myeloid derived suppressor cells (MDSCs) in response to SR9243. Intratumor immune cell activation coincided with enhanced dendritic cell (DC) lymph node homing and activity. SR9243 also enhanced Th1 CD4+ and CD8+ cell proliferation in the presence of immune-suppressive tumor conditioned media and cancer cell lysates an in vitro model of the tumor-microenvironment. Metabolic analysis of immune cells exposed to LXR-ligands revealed that LXR activation by tumor-produced ligands or synthetic agonists blocks T-cell glycolytic activity essential to immune cell proliferation and activation in response to tumor antigens. Similarly, we observed that LXR activation disrupts de novo cholesterol synthesis which has been shown to be vital to immune synapse formation between T-cells and DCs. Our results suggest that LXR inverse agonists can be used as immunotherapy drugs to treat TNBC by blocking tumor-induced dysregulation of T-cell metabolism.
Tumor-intrinsic PD-L1 reduces actin cytoskeleton polymerization to promote mTORC1 signals driving tumor stemness
Authors: Kari Suresh Chandra , Kancharla Aravind , Gupta Harshita B. , Risinger April , Curiel Tyler J
Published: January 2023

Tumor-intrinsic PD-L1 signals contribute to tumor virulence and pathology, but mechanisms are poorly understood. We previously showed that tumor-intrinsic PD-L1 increases mTORC1 and tumor initiating cell (TIC) generation and function in ovarian cancer and melanoma. We used CRISPR/Cas9 (KO) and shRNA (lo) to reduce PD-L1 in mouse melanoma (B16) and ovarian cancer (ID8agg), and human ovarian cancer (ES2). PD-L1lo reduced mTORC1 and TIC as expected, but unexpectedly increased actin polymerization and filopodia, and altered cell morphology. The actin depolymerizing agents Latrunculin-A or Cytochalasin-D reversed actin polymerization in PD-L1lo ID8agg cells and increased mTORC1 (p-rpS6) without affecting mTORC2 (pAktS473). Latrunculin-A and Cytochalasin-D increased TIC numbers and stemness gene expression (Oct4) in PD-L1lo ID8agg cells with no effect on control ID8agg cells. Similar actin effects were seen in PD-L1KO B16 and PD-L1KO ES2. To confirm TIC function, Latrunculin-A increased tumorospheres (TIC self-renewal function) in PD-L1lo, but not control ID8agg cells. The mTORC1 inhibitor rapamycin reduced TIC numbers and functions with no effect on actin polymerization, suggesting mTORC1 is downstream of actin polymerization. Rptorlo ID8agg cells (with low mTORC1 signaling) did not exhibit the PD-L1lo actin polymerization, nor did Latrunculin-A increase Rptorlo ID8agg TIC numbers, tumorospheres, or stemness genes, confirming the flow of signals is PD-L1 to actin to mTORC1 to TIC (stemness). Our data define a highly novel tumor virulence control mechanism of cell-intrinsic PD-L1 through inhibiting actin polymerization suggesting new drug discovery targets. Preliminary data show some similar effects of tumor PD-1.
Attenuated Listeria vaccine for cancer immunotherapy expands Vγ2Vδ2 T cells in rhesus monkeys that retain their central memory phenotype and HMBPP responsiveness
Authors: Morita Craig T , Al-Janabi Mohanad Hameed Nada , Workalemahu Grefachew , Wang Hong
Published: January 2023

Human γδ T cells expressing Vγ2Vδ2 TCRs monitor foreign- and self-isoprenoid metabolites to mediate immunity to microbes and tumors. Bisphosphonate treatment of tumors increases isopentenyl pyrophosphate that is sensed by butyrophilin 3A1, allowing Vγ2Vδ2 cells to kill tumors independent of MHC expression or tumor mutational burden. In clinical trials, adoptive immunotherapy with Vγ2Vδ2 cells has few side effects but has resulted in only a few partial and complete remissions. Direct immunization of cancer patients with either a bisphosphonate or bromohydrin pyrophosphate and IL-2, expands Vγ2Vδ2 cells but they rapidly contract, terminally differentiate, and lose responsiveness. Monkeys immunized with zoledronic acid also lost responsiveness. We sought to develop a live bacterial vaccine for Vγ2Vδ2 cells. Listeria monocytogenes (Lm) were attenuated by actA deletion with a mutant PrfA protein (G155S) that increased their virulence. Immunization with attenuated Lm expanded Vγ2Vδ2 cells on secondary immunization that persisted after a third immunization. Vγ2Vδ2 cells retained central memory phenotypes, expressed inflammatory chemokine receptors, and produced IFN-γ without induction of anergy. Expression of PrfA (G155S) was critical because immunization with ΔactA Lm with wild-type PrfA resulted in minimal Vγ2Vδ2 T cell expansions. Similarly, despite high HMBPP levels, immunization with ΔlytB ΔactA prfAwt Lm also resulted in minimal expansions. Thus, Lm stimulation of Vγ2Vδ2 T cells is dependent on their virulence rather than their HMBPP levels. Unlike zoledronic acid, immunization with ΔactA prfAG155S Lm induces large expansions of Vγ2Vδ2 T cells that retain central memory phenotypes and responsiveness.
Immune pressure against ovarian cancer depends on antigen-specific TRM T cells
Authors: Galindo Carmen Maria Anadon , Sanz Jairo Perez , Biswas Subir , Payne Kyle K. , Yu Xiaoqing , Costich Tara Lee , Gonzalez Ricardo Chaurio , Conejo-Garcia Jose R
Published: January 2023

Conventional memory T cells classically include central memory T (TCM) cells, residing in lymphoid organs, and effector memory T (TEM) cells, circulating through various tissues. Recently, a novel population of memory T cells has gained interest, namely tissue resident memory T (TRM) cells, which persist in tissues and do not recirculate. It is described that these cells reside in human tumors playing a role in tumor-specific T-cell responses. We found by flow cytometry that between 50%–80% of the CD8+ T cells in human ovarian carcinomas are CD103+CD69+TRM cells. RNA-seq of TRM and their re-circulating counterparts from 7 different human carcinomas showed a very distinctive phenotype, characterized by co-upregulation of effector (GZMB, IFNG) and exhaustion (PD-1, TIM3) markers, along with transcription factors and signaling molecules likely involved in the acquisition of the TRM phenotype. Unexpectedly, we found very little overlap between the TCR repertoire of both populations in multiple tumors, and TRM T cells consistently showed significantly higher clonality. Tumor antigen-specific TRM T cells intratumorally transferred into syngeneic mice were more effective at delaying tumor growth compared with their tumor antigen-specific recirculating counterparts. Finally, both the acquisition and the maintenance of a TRM phenotype within the CD8 T cell compartment at tumor beds are dependent on exposure to tumor cognate antigen. Together, our data indicate that TRM CD8+ T cells, but not their CD103− counterparts, represent tumor antigen-specific effector lymphocytes actively exerting anti-tumor immune pressure in the ovarian cancer microenvironment.
Epigenetic targeting of Brd4 constrains CD8+ T cell differentiation during infection and cancer
Authors: Milner J Justin , Toma Clara , Omilusik Kyla , Nguyen Hong , Yu Bingfei , Delpoux Arnaud , Yoshida Tomomi , Quon Sara , Dey Anup , Hedrick Stephen M , Ozato Keiko , Crotty Shane , Pipkin Matthew E , Goldrath Ananda W
Published: January 2023

Memory CD8+ T cells afford long-lived, durable protection against infection and malignancy. Despite the critical relevance of memory T cells to vaccination and immunotherapies, the transcriptional and epigenetic signals instructing memory T cell fate remain unclear. Here, we utilized a pooled RNA interference screen to evaluate the activity of transcription factors and chromatin modifiers governing memory T cell differentiation in vivo. The epigenetic ‘reader,’ Brd4 of the BET protein family, emerged as a top regulator of memory T cell differentiation. Through RNA interference, small molecule inhibition and inducible genetic deletion, we established a central role for Brd4 in mediating CD8+ T cell proliferation, differentiation and function during acute viral infection. Brd4 was required for optimal expression of fate-specifying transcription factors, and a deficiency of Brd4 activity resulted in diminished formation of effector memory and CD103+ tissue-resident memory CD8+ T cells. Given that BET inhibition has emerged as a powerful approach for suppressing tumor growth, we also evaluated how BET inhibition and Brd4-deficiency influence T cell activity during cancer. Brd4 was required for intratumoral accumulation and function of T cells in a mouse model of melanoma, and BET inhibition suppressed T cell-mediated control of tumor growth. However, epigenetic targeting of Brd4 constrained terminal differentiation of T cells within tumors, biasing tumor-resident T cells towards a reprogrammable TCF1-expressing phenotype. These studies establish a novel role for Brd4 in CD8+ T cell biology and provide insight for immunotherapy approaches designed to leverage the dynamic activity of CD8+ T cells in tumors.
Therapeutic antibody discovery in lung tumors by B-cell receptor sequencing
Authors: Kramer Kevin J , Raju Nagarajan , Greenplate Allison , Pilewski Kelsey , Massion Pierre , Georgiev Ivelin
Published: January 2023

Malignancies of the lung are expected to be responsible for over 25% of all cancer-associated mortalities in the United States in 2019. Clinical treatment of lung cancer is complicated by both poor detection of early disease activity and relapse or unresponsiveness to administered therapy. Taken together, these realities of patient outcome underscore the need for alternative therapeutic strategies. Research efforts in the cancer immunology field focus primarily on T cells in the tumor microenvironment, however, there is evidence that B cells may impart a clinical benefit in patients. The formation of tertiary lymphoid structures and antibody secretion in lung tumors associate with positive clinical outcomes yet remain understudied and poorly characterized. In an effort to expand upon these observations, we isolated B cells from cryopreserved human lung cancer tissue and recovered B-cell receptor (BCR) sequences by paired heavy and light chain single cell RNA sequencing. From these experiments, we identified clonally expanded B-cell populations and convergent BCR sequences shared between different patients. We further demonstrated that recombinant antibodies derived from lung cancer patients bind cultured lung cancer cell lines in a dose-dependent manner. This preliminary data supports the notion that tumor-resident B-cells secrete functional antibodies that may additionally be tumor-reactive. Future characterization of these antibodies includes antigen identification and the measurement of in vitro and in vivo cytotoxic activity. Human patient-derived antibodies not only could serve as a novel source for therapeutic and diagnostic agents but may also help inform vaccine design in lung cancer indications.
Microbial LPS enhances PD-1 therapy during chronic LCMV infection
Authors: Penaloza-MacMaster Pablo A , Wang Yidan , Eitzinger Simon , Bhattacharyya Mitra , Penaloza-MacMaster Pablo
Published: January 2023

CD8 T cells are critical for controlling intracellular pathogens, but during chronic viral infection, these cells become exhausted and unable to control virus. Programmed cell death-1 (PD-1) blockade therapy partially improves exhausted CD8 T cells during chronic viral infections. However, its limited efficacy in models of chronic infection has precluded its licensure to treat chronic infection. Interestingly, prior studies show that certain microbiota can improve PD-1 therapy, but the specific microbial components that mediate this positive effect remain unknown. In this study, we interrogated whether microbial LPS could enhance PD-1 therapy during chronic viral infection. Using the model of chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, we show that sublethal doses of LPS followed by PD-1 therapy results in synergistic improvement of virus-specific CD8 T cells in blood and tissues (~10-fold greater compared to PD-1 therapy alone). We show that the improvement of PD-1 therapy by LPS is dependent on reinforced B7/CD28 costimulation and is mediated by a dendritic cell intrinsic mechanism. Although LPS has safety concerns, prior studies show that low LPS doses (≤4 ng/kg) are well-tolerated in humans, demonstrating that there is a threshold of natural resistance to this microbial product. Altogether, our data suggest an unexpected role for LPS/TLR4 signaling in modulating response to PD-1 therapy. Our findings may be important for improving immune checkpoint therapy against chronic infections and for understanding how microbial products modulate responses to therapy.
Monocytes and macrophages limit systemic infection and modulate the CD4 T cell response during Zika virus infection in mice
Authors: Wang Ying-Ting , Hattakam Sararat , Young Matthew Perry , Regla-Nava Jose Angel , Shresta Sujan
Published: January 2023

Monocytes and macrophages are antiviral innate immune effector cells that also serve as cellular hosts of Zika virus (ZIKV). To investigate the precise role of monocytes and macrophages during ZIKV infection in vivo, monocytes and macrophages were depleted with clodronate liposomes before ZIKV infection in wild-type C57BL/6 mice, and viral burden and ZIKV-specific immune responses were assessed. Chlodronate liposometreated mice had higher level of ZIKV RNA in the serum and spleen relative to control liposome-treated mice at early time points after infection. At later time points after infection, chlodronate liposome-treated mice developed an impaired ZIKV-specific Th1 response in terms of both magnitude and quality, whereas they exhibited enhanced Tfh, germinal center B cell, plasma cell, and neutralizing antibody responses as compared to control liposome-treated mice. These data indicate that monocytes and macrophages limit ZIKV dissemination and regulate the balance between Th1 versus Tfh responses during ZIKV infection. Thus, monocytes and macrophages contribute to both the innate control of ZIKV infection and regulation of the adoptive immune response.
Identification of specific T lymphocytes against to an IFNg inducible lysosomal thiol reductase (GILT)-dependent, class II MHC-associated epitope in GILT−/− mice
Authors: He Xiao , Dai Hu , Reyes-Vargas Eduardo , Jensen Peter E
Published: January 2023

IFNγ inducible lysosomal thiol reductase (GILT) is the only thiol reductase localized in lysosomes and late endosomes, with its optimal activity at acidic pH. GILT is constitutively expressed in professional APC, further upregulated by IFNγ, and participates in Ag processing by catalyzing disulfide (S-S) bond reduction in proteins thus facilitating unfolding of native S-S bond containing Ag for further processing. It was reported that GILT−/− mice mounted significantly reduced T cell proliferative responses to S-S bond-containing protein Ags in vitro, but responded normally to a non-S-S bond containing protein. In addition, GILT−/− APC showed a selective deficiency in presenting some cysteine-containing epitopes within a model protein Ag, hen egg white lysozyme. Using HPLC-MS/MS, we first identified one group of unique GILT-dependent, class II MHC-associated peptides from B6 mice, but not from GILT−/− mice. Binding of these GILT dependent, class II MHC-associated peptides to I-Ab proteins was confirmed in vitro. Furthermore, T cells specific to one of the unique GILT-dependent peptides, BP7, was detected in GILT−/− mice, but not found in B6 mice. BP7 peptide specific T cell hybridoma clones were established as well. Taken together, our results indicate that GILT−/− mice have a deficiency in generation of certain T cell epitopes due to loss of GILT activity. Missing of GILT dependent epitopes might lead to survival of the T cells specific to these GILT-dependent epitopes (escaping from the negative selection) in thymus of GILT−/− mice. We are in progress of cloning the BP7 specific TCRs from T cell hybridoma clones. The potential role of the BP7 specific T lymphocytes in autoimmunity will be examined in BP7 specific T cell retrogenic mice.
Mast Cells in Patients with Hereditary α-Tryptasemia Promote HLA-DR Expression and a Th2-Polarizing Microenvironment in the Gastrointestinal Tract
Authors: Shirley James Forrest , Drourr Joshua , Edwards W. Taylor , Tuna Kubra , Ryan Lisa K , Alli Abdel , Tang Ying , Glover Sarah C.
Published: January 2023

Hereditary α-tryptasemia (HαT) is a recently identified, genetic disorder characterized by multisystem comorbidities resulting from increased monoallelic α-tryptase copy number (CN) at TPSAB1 (α-TPSAB1). Affected individuals frequently exhibit heterogeneous multisystem symptoms and comorbidities which negatively impact daily functioning and quality of life. In the cohorts examined thus far, functional dyspepsia and irritable bowel syndrome, were most frequently reported. However, the mechanism by which increased α-TPSAB1 CN produces GI disease is unknown.

Tryptase is almost exclusively produced by mast cells (MC). In the gut, MC play important roles in immune responses, leukocyte recruitment, neuroimmune inflammation, and tissue repair. MC can also promote T-cell activation and polarization via cytokine secretion, co-stimulatory marker expression and MHC II antigen presentation. MCs also release exosomes, nanosized vesicles secreted by most cell types present in biological fluids such as blood, cerebrospinal fluid, and urine, which have a reported influence on T cell differentiation.

In early experiments, we found increased numbers of GI MCs, activated T cells, and urinary exosomes in HαT patients compared to healthy donors. Thus, we evaluated MC surface antigen expression, exosome proteomics, and cytokine expression of exosome-treated T cells. We found increased HLA-DR and FcɛR1α expression in HαT-patient GI MCs, increased Th2 associated signaling proteins in HαT urinary exosomes, and elevated Th2 cytokine expression in HαT-exosome treated T cells. These results suggest that HαT GI morbidity involves aggravated MC hyperplasia and antigen presentation which promotes T cell activation and Th2 polarization.
Intestinal epithelial cells differentiated from iPS cells (iPS-IECs) suppressed bacterial translocation in an organ model of alcohol-induced intestinal epithelial dysfunction
Authors: Ito Ichiaki , Jacob Sam , Suzuki Sumihiro , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Microbiota translocated from the intestine causes infectious complications in alcoholics, frequently. In the present study, the effect of iPS-IEC transplantation on gut-associated bacterial translocation was investigated in an organoid model of intestinal epithelial burrier (IEB) dysfunction in patients with alcohol use disorder. iPS-IECs (a mixture of Paneth cells, goblet cells, enteroendocrine cells, and enterocytes) were differentiated from iPS cells, originated from human peripheral CD38+ hematopoietic stem cells. Human gut organoids (HGO) were generated from iPS cells in 3D cultures for 2 weeks. HGO were alcoholized with 0.2% ethanol for 2 weeks (A/HGO). iPSIECs (5 × 103 cells) were co-cultured with 100 A/HGO for 3 days. IEB functions of A/HGO were tested by severities of Enterococcus faecalis (E. faecalis) invasion into the organoids. The gene expressions of tight junction proteins and intestinal cell markers of A/HGO were determined by real-time PCR and Western blotting. In the results, E. faecalis added to culture media invaded the A/HGO, while the pathogen did not invade the HGO in the same cultures. However, the pathogen did not invade the A/HGO co-cultured with iPS-IECs. Reduced expression of tight junction protein mRNAs was shown in A/HGO, while these mRNAs were expressed in A/HGO co-cultured with iPS-IECs. LGR5 and NOTCH1 gene expressions in A/HGO were shown to be a minimum, while these genes were expressed sufficiently in A/HGO co-cultured with iPS-ICs. These results indicate that the alcohol-induced IEB dysfunction of A/HGO is significantly improved in cultures added with iPSIECs.
IFNγ and poly(I:C) primed MSCs enhances the therapeutic effects on DSS induced colitis via enhancing indoleamine 2, 3-dioxygenase and inducing regulatory T cell phenotype
Authors: Lim Ji-Young , Ryu Da-Bin , Lee Sung-Eun , Park Gyeongsin , Min Chang-Ki
Published: January 2023

Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the colon and small intestine caused by chronic and excessive inflammation. Mesenchymal stem cells (MSCs) are pluripotent cells with immunosuppressive properties, currently studied as a potential treatment for IBD. We previously demonstrated that among various TLR ligands, MSCs treated with poly(I:C), which is a TLR3 ligand, more profoundly induced IDO, which is a therapeutically relevant immunosuppressive factor, without any observable phenotype change in vitro. The goal of the study was to determine whether poly(I:C) can enhance the therapeutic efficacy of bone marrow-derived MSCs for the treatment of IBD. To compare the therapeutic effects between naïve MSCs and primed MSCs by poly(I:C) stimulation on murine colitis, mice (C57BL6) were administered with 2.5% dextran sodium sulfate (DSS) in drinking water for 5 days and injected with MSCs intraperitoneally on days 1 and 3 following DSS ingestion. The disease activity index (DAI) score and body weight loss were significantly improved in the primed MSCs treated mice group. Also the pathologic severity of colon were more recovered in primed MSCs treated group. The frequency of T cells in spleen and mesenteric lymph node was markedly decreased in primed MSCs treated group. Foxp3 expressing regulatory T cells were more expanded in primed MSCs treated group. Treatment of primed MSCs reduced the expression of proinflammatory cytokines in colon tissue.

In conclusion, we demonstrate that primed MSCs with poly(I:C) improve their efficacy in treating DSS-induced colitis, and this effect at least partly depends on the enhancement of their immunosuppressive activity through increasing their production of IDO.
Ulcerative Colitis-associated E. coli pathobionts potentiate colitis in susceptible hosts
Authors: Yang Hyungjun , Mirsepasi-Lauridsen Hengameh Chloé , Struve Carsten , Allaire Joannie M. , Bosman Else , Sivignon Adeline , Vogl Wayne , Ma Caixia , Reid Gregor , Li Xiaoxia , Petersen Andreas Munk , Jacobson Kevan , Gouin Sébastien , Barnich Nicolas , Yu Hongbing , Krogfelt Karen Angeliki , Vallance Bruce A
Published: January 2023

Ulcerative colitis (UC) is chronic inflammatory condition linked to intestinal microbial dysbiosis, including the expansion of E. coli strains related to extra-intestinal E. coli. These “pathobionts” exhibit pathogenic properties, but their potential to promote UC is unclear due to the lack of suitable animal models. Here, we established a mouse model using a representative UC pathobiont strain (p19A), and mice lacking single immunoglobulin and toll-interleukin 1 receptor domain (SIGIRR), a deficiency increasing susceptibility to gut infections. p19A was found to adhere to the cecal mucosa of Sigirr−/− mice, causing modest inflammation. Moreover, it dramatically worsened DSS induced colitis, in concert with adherence to, and penetration of the inflamed mucosa. This pathogenicity was lost in a p19A strain lacking the adhesin FimH; following treatment with FimH antagonists, or was attenuated when using a p19A strain lacking a-hemolysin genes. Thus UC pathobionts can worsen the course of colitis in susceptible hosts.
Identification of novel loci controlling IBD susceptibility utilizing the genetic diversity of wild-derived mice
Authors: Lahue Karrie Godburn , Crothers Jessica , Lavoie Brigitte , Fang Qian , Mawe Gary , Krementsov Dimitry
Published: January 2023

Inflammatory bowel disease (IBD) is a complex disease that imposes a significant and growing health burden in industrialized nations. Recent genetic studies have identified multiple genetic associations with IBD, but the mechanisms underlying these associations are poorly understood. Animal models are needed to bridge this gap in knowledge, but conventional laboratory mouse strains lack the genetic diversity of human populations. To more accurately model human genetic diversity and to identify new genetic loci that control susceptibility to IBD, we utilized a panel of chromosome (Chr) substitution (consomic) strains, carrying chromosomes from the wild-derived and genetically divergent PWD/PhJ (PWD) strain on the commonly used C57BL/6J (B6) background. Two models of IBD were used, TNBS- and DSS-induced colitis. Multiple disease severity parameters were evaluated, including weight loss, disease activity, fecal Lipocalin-2 production, and histopathology. Strikingly, compared with B6 mice, wild-derived PWD mice were highly susceptible to TNBS-induced colitis, but resistant to DSS-induced colitis. Using consomic mice, we found several PWD-derived loci that exhibited significant effects on IBD susceptibility. Most pronounced was a locus on Chr2, which resulted in very high susceptibility to both DSS and TNBS colitis, while several other PWD-derived loci were protective. Altogether, our experiments identified novel IBD susceptibility loci, harboring genes that control susceptibility to various aspects of IBD pathogenesis, and underscoring the utility of wild-derived mouse genetics. Future studies will identify these genes and their functions, to generate new insights into IBD etiology and pathophysiology.
Persistent IL-1b Signaling Aggravates Murine Enteropathogen, Citrobacter rodentium Infection in Mice
Authors: Yeoh Beng San , Saha Piu , Golonka Rachel M , Vijay-Kumar Matam
Published: January 2023

The inflammasome cytokine IL-1b is an important mediator of the inflammatory responses against invading pathogens. However, it is not clear whether sustained IL-1b signaling following the loss of its endogenous inhibitor, secretory IL-1 receptor antagonist (sIL-1Ra), could improve mucosal immunity against the enteropathogen, Citrobacter rodentium (CR). In light of increased prescription of rec-sIL-1Ra (aka Anakinra) to treat inflammatory bowel disease, we undertook this study. At basal levels, sIL-1Ra-deficient (sIL-1RaKO) mice displayed leukocytosis and elevated inflammatory marker, i.e. serum and fecal lipocalin 2. Furthermore, bone marrow-derived macrophages and neutrophils from sIL-1RaKO mice generated higher levels of iNOS and nitrite, and ROS and NETs, respectively. These results collectively suggest sIL-1RaKO mice have low-grade chronic inflammation. Based on these results, we hypothesized that sIL-1RaKO mice with persistent IL1-b signaling could efficiently clear CR infection than their WT littermates. Oral challenge of CR (1×109 CFU/mouse) resulted in luminal colonization, which peaked at day 7 post-infection, in both groups; however, sIL-1RaKO mice displayed a higher CR burden and an exacerbated infection. The aggravated course of infection was further visualized by inoculating sIL-1RaKO mice with bioluminescent CR. Histologic analysis revealed that transmissible colonic hyperplasia was more pronounced in sIL-1RaKO mice. Interestingly, basal ileal Paneth’s cell-specific antimicrobial proteins (Ang4 and Reg3g) were significantly reduced in sIL-1RaKO mice. Collectively, our results demonstrate that a balanced mucosal IL-1b signaling is required to counter and clear enteropathogen infection.
AS101 ameliorates experimental autoimmune uveitis by regulating Th1 and Th17 responses and inducing Treg cells
Authors: Bing So Jin , Shemesh Itay , Chong Wai Po , Jittayasothorn Yingyos , Silver Phyllis B , Sredni Benjamin , Caspi Rachel R
Published: January 2023

AS101 is an organotellurium compound with multifaceted immunoregulatory properties that is remarkable for its lack of toxicity. We tested the therapeutic effect of AS101 in experimental autoimmune uveitis (EAU), a model for human autoimmune uveitis. Unexpectedly, treatment with AS101 elicited Treg generation in vivo in otherwise unmanipulated mice. Mice immunized for EAU with the retinal antigen IRBP and treated with AS101 developed attenuated disease, as did recipients of retina-specific T cells activated in vitro in the presence of AS101. In both settings, eye-infiltrating effector T cells were decreased, whereas regulatory T (Treg) cells in the spleen were increased. Mechanistic studies in vitro revealed that AS101 restricted polarization of retina-specific T cells towards Th1 or Th17 lineage by repressing activation of their respective lineage-specific transcription factors and downstream signals. Retina-specific T cells polarized in vitro towards Th1 or Th17 in the presence of AS101 had impaired ability to induce EAU in naïve recipients. Finally, AS101 promoted differentiation of retina-specific T cells to Tregs in vitro independently of TGF-β. We conclude that AS101 modulates autoimmune T cells by inhibiting acquisition and expression of effector function and by promoting Treg generation, and suggest that AS101 could be useful as a therapeutic approach for autoimmune uveitis.
IL-22 and Reg3γ overexpression affects Experimental Autoimmune Encephalomyelitis Severity
Authors: Eken Ahmet , Yetkin Mehmet Fatih , Okus Fatma Zehra , Cakir Mustafa , Karayigit Mehmet Onder , Erdem Serife , Haliloglu Yesim , Oukka Mohamed , Altuntas Hamiyet Donmez , Mirza Meral , Canatan Halit
Published: January 2023

IL-22 belongs to IL-10 family cytokines and is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ, iNKT, Th17, and Th22 cells. IL-22 receptor, however, is expressed primarily by non-hematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knock out mice were previously shown to develop experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis (MS), how IL-22 manipulation in adult mice would affect EAE course has not been studied. In this study, we over-expressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to assess the impact of IL-22 modulation on EAE course. IL-22 overexpression significantly but marginally decreased EAE scores, demyelination and reduced infiltration of IFNγ+IL-17A+ Th17 cells into central nervous system (CNS). In contrast, neutralization of IL-22 exacerbated EAE scores. Surprisingly, overexpression of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22, significantly exacerbated EAE scores and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+ Th17 cells to CNS. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptides in the pathogenesis of CNS inflammation in a murine model of MS.
Lactobacillus reuteri reduces the severity of experimental autoimmune encephalomyelitis in mice by modulating gut microbiota
Authors: Liu Yuying , He Baokun , Hoang Thomas K , Tian Xiangjun , Taylor Christopher M , Blanchard Eugene E , Luo Meng , Bhattacharjee Meenakshi B , Freeborn Jasmin , Park Sinyoung , Couturier Jacob , Lindsey J William , Tran Dat Q , Rhoads J Marc
Published: January 2023

The gut microbiome plays an important role in immune function and has been implicated in multiple sclerosis (MS). However, how to modulate microbiota to prevent or treat MS remain largely unknown. Probiotic Lactobacillus reuteri DSM 17938 (L. reuteri) has anti-inflammatory effects in mouse models of neonatal necrotizing enterocolitis and Treg-deficiency-induced autoimmune disease. L. reuteri in these models modulated gut microbiota and microbiota-associated metabolites to inhibit TH1 and TH2 differentiation and their-associated cytokines, significantly ameliorating the severity of these diseases. An in vitro study indicated that L. reuteri inhibits the differentiation of naïve CD4+T cells into TH17 and TH1 cells. We determined the effect of L. reuteri on experimental autoimmune encephalomyelitis (EAE) mouse model, a widely used animal model of MS, a model which is primarily mediated by TH17 and TH1 cells. We discovered that L. reuteri treatment reduced TH1/TH17 cells and their associated cytokines IFN-γ/IL-17 in EAE, inhibiting the development of EAE. We also showed that the loss of diversity in gut microbiota induced by EAE was largely restored by L. reuteri treatment. Taxonomy-based analysis of gut microbiota showed that the “beneficial” genera Bifidobacterium, Prevotella, and Lactobacillus were negatively correlated with EAE clinical severity scores, whereas the genera Anaeroplasma, Rikenellaceae, and Clostridium were positively correlated. Notably, L. reuteri treatment altered the relative abundances of these EAE-associated taxa in mice. In conclusion, probiotic L. reuteri changed gut microbiota to modulate immune responses in EAE, making it a novel candidate in future studies to modify the severity of MS.
The cytoplasmic domain of CTLA-4 control autoimmunity via inducing regulatory T cells
Authors: Kim Gil-Ran , Lim Sangho , Lee Jae-Ung , Choi Je-Min
Published: January 2023

Regulatory T cells (Tregs) have critical roles for maintaining immune tolerance and homeostasis. Recently, it is revealed that CTLA-4 is an essential membrane protein for the suppressive function of Treg. However, the molecular mechanism of cytoplasmic domain of CTLA-4 (ctCTLA-4) and its role in Treg function and differentiation is not clearly understood. In this study, we utilized ctCTLA-4 peptide and its various mutant forms conjugated with dNP2, a cell-permeable peptide, to investigate the mechanism of ctCTLA-4 in Treg function and differentiation. We found that the ctCTLA-4 convert naïve T cells to Foxp3-positive cells in T helper 17 cell differentiation condition in vitro, while the lysine-rich motif mutant form could not. The dNP2-ctCTLA-4 also significantly inhibits experimental autoimmune encephalomyelitis (EAE) in vivo, by reducing CNS-infiltrating Th17 cells with significantly increased Treg proportion. As similar with in vitroresults, lysine-rich motif mutant form could not increase Treg proportion and inhibit EAE. To elucidate its molecular mechanisms, we conducted HuProt protein microarray to identify lysine-rich motif dependent binding partner of ctCTLA-4 related with increasing Tregs. With the results, we found that ctCTLA-4 decreased a phosphorylation of MAPK in T cell stimulation conditions resulting enhanced nuclear localization of Smad2/3, a key transcription factor for Foxp3 expression. These results suggest that ctCTLA-4 has a critical molecular mechanism of inducing Tregs to maintain peripheral immune tolerance to control autoimmunity.
The effect of Chinese herbal medicine compound, CS029, in suppression of dendritic cells and its therapeutic application in experimental autoimmune encephalomyelitis
Authors: Song Ying-Chyi , Yen Hung-Rong
Published: January 2023

Dendritic cell (DC) is a kind of professional antigen-presenting cells that play a crucial role in the activation of naive T cell response. Previous studies have demonstrated that controlling DCs can affect the development of autoimmune diseases. Multiple sclerosis (MS) is an inflammatory autoimmune disease, in which the immune system attacks myelinated axons in the central nervous system (CNS). Although several immunomodulatory drugs have been developed to treat MS, the current drugs have no satisfactory efficacy. Therefore, it is necessary to develop new drugs to improve the treatment of MS. We aimed to develop new immunomodulatory agents for the treatment of MS from Chinese herbal medicine (CHM). Among the CHM, we identified an alkaloid CHM compound (CS029), which could reduce inflammatory cytokine production through the TLR-dependent and -independent activation of DC. Moreover, we demonstrated that CS029 could inhibit antigen presentation ability of DC in vitro. In a murine experimental autoimmune encephalomyelitis (EAE) model, we demonstrated that the mean clinical score decreased 6-fold by CS029 treatment (50 mg/kg) compared with the vehicle control. Furthermore, we identified that CS029 treatment could significantly decrease the infiltration of DCs, T cells and total immune cells into the CNS. Therefore, we suggested that CS029 treatment can be used as an immunosuppressive agent to inhibit DC functional maturation and then lead to the suppression of autoreactive T cell response, blockade of immune cells trafficking into CNS, and further mitigation of the symptoms of MS. This study elucidated the role of DCs in multiple sclerosis by CHM treatment, which may shed light on the development of new therapeutic approaches.
Efficacies of anti-CD20 antibody and Atacicept are differentially impacted by B Cell Maturation Antigen in neuro-autoimmunity
Authors: Kumar Gaurav , Ko Rose M , Axtell Robert C.
Published: January 2023

B cells have emerged as a therapeutic target for multiple sclerosis (MS). Depleting B cells with anti-CD20 antibodies are effective in treating MS. Yet, TACI-Ig treatment, which blocks BAFF and APRIL (two cytokines important for B cell development and function), paradoxically increased disease activity in MS patients. The stark differences in clinical outcomes with these therapies demonstrate that B cells have both inflammatory and regulatory effects in MS. B cell Maturation Antigen (BCMA), a receptor that binds BAFF and APRIL, plays a prominent role in B cell function. The role BCMA plays in MS remains enigmatic and therefore we explored the impact BCMA deficiency has on the development and progression of the experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. We found that EAE induced with MOG35–55 was severe in BCMA−/− mice compared to BCMA+/+ mice. The increased disease was associated with increased CNS inflammation and a skewing of B-cells toward a mature/inflammatory phenotype. Also, for the first time, we observed that BCMA deficiency directly affects the infiltration of inflammatory myeloid cells during EAE. We found that anti-CD20 treatment significantly reduced EAE in BCMA−/− mice but had no effect on BCMA+/+ mice. Conversely, we found that TACI-Ig treatment had no effect on BCMA−/− mice but did reduce EAE in BCMA+/+ mice. Our study demonstrates that BAFF and APRIL constrain the development and function of inflammatory B-cells through BCMA. We further demonstrate that BCMA inhibits inflammatory B cell responses, yet promotes inflammatory responses in macrophages. Finally, our data provide clues into the paradoxical results of clinical trials with anti-CD20 and TACI-Ig in MS patients.
The significance of spontaneously immortalized B lymphocytes from patients with AIDS-related non-Hodgkin’s lymphoma and HIV-infected individuals
Authors: Zhuang Ke , Zhang Yongxi , Zhou Li , Qi Xiaoying , Xu Xiqiu , Meng Fengzhen , Xu Zhigao , Liu Jinbiao , Shao Liang , Xiong Yong , Ke Hengning , Gui Xien
Published: January 2023

Reliable tumor biomarkers for risk assessment, screening, detection, diagnosis, and prognosis remain an unmet need for patients with AIDS-Related Non-Hodgkin’s Lymphoma (AIDS-NHL) and its high risk population–HIV/AIDS patients. In this study, we generated spontaneously immortalized B lymphocyte cell (SIBC) lines by culturing peripheral blood mononuclear cells (PBMCs) derived from AIDS-NHL and HIV/AIDS patients, then evaluated their clinical significance for detection, diagnosis, and prognosis. From 34 AIDS-NHL and 71 HIV/AIDS patients, we generated 14 AIDS-NHL-SIBC and 6 HIV-SIBC lines, respectively. Among 14 AIDS-NHL-SIBC lines, 12 SIBCs were derived from patients with poor prognoses. All SIBCs presented main markers typical of memory B-cells (CD3−CD20+CD27+) and the majority of cells expressed NHL related immunologic proteins (BCL-2, MUM-1. Ki67, etc.). AIDS-NHL-SIBCs exhibited the typical biological characteristics of tumor cells, including monoclonal Ig gene rearrangement, abnormal chromosome, and growth of xenograft tumors in NOD/SCID mice, while HIV-SIBCs possessed partial tumor cell properties, but no xenograft tumors. These findings suggest that the AIDS-NHL-SIBC lines are representative of the malignant B-lymphocytes circulating in AIDS-NHL patients (CTC), while HIV-SIBC might be a CTC precursor in HIV/AIDS patients. These data provide evidence of a potentially valuable diagnostic and prognostic biomarker for AIDS-NHL patients, and present a basis for early screening of NHL among HIV/AIDS patients.