bluuebunny/crossref_metadata_2025 huggingface dataset:
Separate molecular pathways mediate anti-tumor versus tumor-promoting aspects of Poly-I:C signaling in cancer microenvironments
Authors: Theodoraki Marie-Nicole , Sarkar Saumendra N , Mogundo Francesmary , Edwards Robert P , Kalinski Pawel
Published: January 2023

Background

We have previously shown that Poly-I:C, a frequently used adjuvant, induces both the CTL-attracting chemokines and Treg attractants. Here we evaluated the molecular pathways, which lead to the induction of chemokines by poly-I:C in TME and different types of tumor-associated cells, in order to develop improved adjuvants which selectively attract the desirable effector cells rather than suppressive cells.

Methods

Isolated cells or human cancer biopsies were cultured for 0.5–48 hours in the absence or presence of one of two synthetic TLR3 ligands Poly-I:C (non-selective activator of TLR3 and helicases) rintatolimod (selective TLR3 ligand), in the absence or presence of a COX-1/2 inhibitor, NF-kB- and TNFa inhibitors. mRNA assays, confocal microscopy, ELISA, chemotaxis assays and molecular biology assays were used to analyze the chemokine production and tumor-associated suppressive factors.

Results

We observed that poly-I:C induced activation of NF-kB- and COX-2 pathways leading to induction of COX2-dependent suppressive factors and Treg- and MDSC-attracting chemokines. These undesirable effects were blocked with inhibitors of both NF-kB- or COX-2 pathways. In contrast rintatolimod selectively induced the desirable chemokines, which were associated with lack of direct activation of NF-kB- or COX-2 pathways, and strongly suppressed attraction of Tregs and MDSCs, with elevated CTL attraction in ex vivo migration assays.

Conclusion

Our data implicates an important new role of helicases by the induction of tumor-promoting factors by dsRNA and points out to new targets to enhance the immunogenic and antitumor activities of adjuvants.
Th17 Cytokines Drive Liver Fibrosis Progression by Regulating TGF-β Signaling through Activation of MAPKs
Authors: Shoukry Naglaa H , Fabre Thomas , Molina Manuel Flores , Soucy Genevieve , Willems Bernard , Villeneuve Jean-Pierre , Bilodeau Marc
Published: January 2023

Activation of hepatic stellate cells (HSCs) is a key event in the initiation of liver fibrosis. We and others have demonstrated that IL-17A produced by Th17 cells promotes activation of HSCs. Th17 cells also produce IL-22, an enigmatic cytokine with pro-inflammatory and hepatoprotective properties. In addition, regulatory T cells (Treg) negatively modulate activation of HSCs. We hypothesized that liver fibrosis progression results from an alteration in the Th17/Treg ratio leading to an imbalance in pro-fibrotic Th17 cytokines in the liver. We examined ex vivo the frequency of Th17 and Treg populations and the cytokine profile of intrahepatic lymphocytes in liver biopsy samples (n=32). We observed increased Th17/Treg ratio in advanced as compared to moderate or no-fibrosis. Furthermore, we observed a bias towards Th17 cytokines in fibrotic livers with viral-hepatitis both in situ and ex vivo. All biopsies exhibited a 5-fold increase in IL-22 in fibrotic livers irrespective of aetiology. In vitro stimulation of HSCs with IL-22 sensitized them to suboptimal doses of TGF-β and activated p38 pathways. Chemical inhibition of p38 suppressed the pro-fibrogenic effect of IL-22. In vivo, lack of IL-22 signaling protected mice against liver fibrosis. IL-22RA1 Knockout mice exhibited reduced collagen deposition and expression of pro-fibrotic genes (ACTA2, LOXL2, TIMP-I, TGFb1, COL1A1) in comparison to wild-type littermates. Our results suggest a dysregulated Th17 response with increased IL-22 signaling in advanced fibrosis from all different aetiologies. Finally, we have identified IL-22 as a common factor in advanced liver fibrosis acting through sensitization of HSCs to TGF-β in a p38-dependent manner.
The use of tMUC1 highly specific chimeric antigen receptor-redirected T cells for the eradication of triple negative breast cancer
Authors: Zhou Ru , Roy Lopamudra Das , Yazdanifar Mahboubeh , Livasy Chad , Mukherjee Pinku
Published: January 2023

Antibody-derived Chimeric Antigen Receptor (CAR) T cells have great success in reduction of liquid form of tumor, but not solid tumor. Our novel antibody TAB 004 can specifically recognize the tumor form of MUC1 (tMUC1) while sparing the normal MUC1 in several subtypes of breast cancers including >95% of triple negative breast cancer (TNBC). In vivo delivery of TAB 004 specifically accumulates in the xenograft TNBC tumor only. Therefore, we hypothesize that TNBC can be specifically targeted with TAB 004-derived tMUC1-specific CAR T cells. A panel of 45 human breast cancer cell lines were tested for tMUC1 level, and 11 out of 13 TNBC cell lines showed higher frequency of tMUC1 expression compared to that on normal cells. We engineered a 2nd generation human CAR using TAB 004 that was coupled to the CD28-CD3zeta. CAR was well expressed on activated human primary CD8 and CD4 T cells. Compared to their mock control, CAR-T cells expressed higher CD25 and CD11c, particularly on CD8 T cells. Those CAR-T cells specifically recognized the tMUC1 antigen and formed tight synapse with tMUC1high tumor cells in vitro. The susceptibility of TNBC cells to CAR-T cell cytolysis was dramatic while still corresponding to their tMUC1 level. Only the tMUC1-specific CAR-T cells expressed and released large amount of IFN-gamma after antigen recognition on TNBC cells. However, IFN-gamma was not directly involved in the tumor killing by CAR-T cells. The in vivo treatment of TNBC tumor by CAR-T cells is ongoing. Taken together, our TAB 004-originated CAR-T cells are very potent at killing TNBC tumor cells in a tMUC1-highly specific manner, which is very promising to provide a safe, effective and tumor antigen-specific immunotherapeutic intervention.
Targeted deletion of liver myeloid derived suppressor cells by inhibition of STAT3 dependent survival pathways to rescue CAR-T function
Authors: Gardell Jillian , Guha Prajna , Cunetta Marissa , Lima Matthew , Darpolor Josephine , Espat N. Joseph , Junghans Richard P. , Katz Steven C
Published: January 2023

Myeloid derived suppressor cells (MDSC) subvert anti-tumor immunity. Previously we reported that the GM-CSF/ JAK2/STAT3 axis drives liver MDSC (L-MDSC) proliferation and CAR-T suppression. We hypothesized that STAT3 supports L-MDSC survival and suppressive function by inhibiting apoptosis. We treated liver metastasis (LM) in mice with STAT3 inhibitors (STATTIC or BBI) or with vehicle control. STAT3 inhibition caused a significant reduction in tumor burden (p<0.05) and L-MDSC frequency (DMSO 41±3% vs. STATTIC 29±3%/BBI 20±3%, p<0.0001, n=10) in association with lower pSTAT3 levels (DMSO 33±4% vs. STATTIC 11±3%/BBI 9±2%, p<0.0001, n=10). L-MDSC isolated from STATTIC or BBI treated mice were co-cultured with CAR-Ts and corresponding target tumor cells at 1:1:1 ratio. There was a significant decrease in tumor cell density (DMSO 100% vs. STATTIC 71±5%/BBI 20±3%, p<0.05, n=5) and enhancement of tumor cell killing (DMSO 28±4% vs. STATTIC 54±4%/BBI 52±7%, p<0.01, n=5). Rescue of CAR-T tumor killing function correlated with enhanced L-MDSC apoptosis signaling. We detected upregulation of pro-apoptotic proteins Bax, caspase 3, and Fas. Signaling molecules downstream of Fas, JNK and p38 MAPK (p<0.05), were also activated in L-MDSC. In contrast, L-MDSC pro-survival Bcl2, pErk, and pAkt (p<0.05) were downregulated in response to STAT3 inhibition. Microarray results confirmed the STAT3-induced changes in apoptotic and survival gene expression, which was validated by RT-PCR (p<0.05). Within LM, STAT3 inhibition drove L-MDSC apoptosis via the Fas/Fas- L pathway with downstream pro-apoptotic signaling through p38 MAPK. Blocking STAT3 may have clinical application for enhancing immunotherapy for LM.
Active Specific Immunotherapy plus Doxorubicin Chemotherapy Mediates Durable Remission of Lethal Canine Hemangiosarcoma
Authors: Konduri Vanaja , Halpert Matthew M , Chen Yunyu , Liang Dan , Levitt Jonathan M , Neal Brittany , Husdon Caleb , Hottinger Heidi , Piscoya Sindy , Wilson Nicola , DiBernardi Lisa , Stallings Shonda , Omarbekov Zarkhyn , Seelhoff Lori , Ramirez Oscar , Yao Qizhi C , Bhardwaj Ratan D , Ravi Vinod , Douglass Laurel , Decker William K
Published: January 2023

Angiosarcoma is a deadly neoplasm of the vascular endothelium. Metastases are often present at diagnosis, and 5-year survival is only 10–30%. While the disease is uncommon in humans, angiosarcoma is a major killer of companion dogs, responsible for 120,000 deaths per year in the US. The canine disease (HSA) presents as acute hemoabdomen secondary to splenic rupture. Even if life-saving splenectomy is performed, median OS is only 48 days, and one-year survival is 0%. Here we report an interim analysis of a phase I multi-site, open-label trial of chemo-immunotherapy performed on consecutively-presenting splenectomized canines with verified HSA. Subjects received doxorubicin plus an autologous cell-therapy that generates durable CD8+ memory. Disease was monitored monthly by abdominal ultrasound, chest x-ray, and echocardiogram. At the time of this submission, median OS had not yet been reached in the per protocol population, with 75% of animals alive and healthy at up to one year post-splenectomy (p<0.0002). Abdominal ultrasound documented resolution of local mesenteric disease in one animal, sternal lymphadenopathy in another, and hepatic metastases in a third. No significant adverse events were reported. Administration of this therapy appears feasible, safe, and highly efficacious in dogs. If subjects survived long enough to begin treatment, prognosis became highly favorable. The results suggest additional clinical studies in both canines and humans are warranted.
Cyclophosphamide preconditioning facilitates autoimmune vitiligo and enhanced antitumor efficacy following vaccination with activated dendritic cells.
Authors: Mullins David W , Alexander Matthew P
Published: January 2023

Cyclophosphamide (CTX) has been used as a lymphoablative conditioning agent to deplete regulatory T cells (Tregs) and enhance the efficacy of adoptive immune therapies. We initially investigated whether CTX conditioning could enhance the immunogenicity of melanoma Ag-pulsed dendritic cells (DC) in a murine model. CTX enhanced Ag-specific CD8+ T cell responses and control of SC-growing OVA+ melanoma following immunization with activated OVA257–265-pulsed DC; immunogenicity and tumor control were abrogated by reconstitution of Treg compartments. Unexpectedly, CTX-conditioned, DC-immunized animals developed significant vitiligo, despite the absence of exogenous melanocyte-specific Ag in the vaccine. CTX treatment or DC immunization alone failed to induce vitiligo. Injection of CTX-conditioned mice with MHC class II−/− DC, but not β2 microglobulin−/− DC, induced vitiligo regardless of exogenous Ag, suggesting that the induction of vitiligo may result from cross-presentation of endogenous melanocyte Ag in an environment of relaxed tolerance. Ab-mediated depletion of CD8 cells abrogated the vitiligo. These data suggest that CTX conditioning liberates class I-restricted melanocyte Ag for cross-presentation by activated DC to cognate CD8 cells, which in turn, achieve full activation and effector function in the absence of a normal regulatory environment. CTX conditioning may also open dermal compartments for increased effector T cell infiltration. Thus, CTX mediates multiple immunomodulatory mechanisms that may impact the future design of immunotherapy for melanoma.
Low-dose chemotherapy induces immunogenic tumor dormancy in mouse mammary carcinoma cells
Authors: Smith Timothy M , Butler Savannah E. , Wang Xiang-Yang Shawn , Manjili Masoud H
Published: January 2023

Patients with early stage cancer do not usually die from primary cancer, which tends to be responsive to therapy, but rather as a result of distant recurrence of the tumor in the form of advanced stage diseases. This phenomenon can be explained by cancer dormancy. However, many patients that harbor dormant tumor cells survive without experiencing recurrence. It has been suggested that certain chemotherapeutics that induce immunogenic tumor cell death (ICD) could prolong survival of patients because of inducing an anti-tumor immune response. However, tumor-reactive immune cells might fade away due to toxic doses of chemotherapy and/or tolerogenic properties of dormant tumor cells. Here, we hypothesize that some chemotherapeutic drugs at a low dose could also induce immunogenic tumor dormancy (ITD) for constant stimulation of the immune system. To test our hypothesis, we used neu overexpressing mouse mammary carcinoma (MMC) cells, and different doses of Adriamycin, Cyclophosphamide, or a combination therapy of 5-flurouracil, Adriamycin, and Cyclophosphamide (FAC). We demonstrated that low dose FAC was more effective in inducing ICD and ITD compared with other drugs or more cytotoxic doses. These findings warrant further examination into using a conditioning regimen of low-dose chemotherapeutics as a means of establishing ICD and ITD, thereby inducing and maintaining an immune response for the prevention of tumor recurrence.
Antibody-dependent Tumor Phagocytosis Enhances PD-L1 and IDOs Expression on Macrophages, Which Mediate Trastuzumab Resistance in Her2+ Breast Cancer
Authors: Song Erwei , Su Shicheng , Zhao Jinghua
Published: January 2023

The immune response has been linked to the therapeutic effect of trastuzumab, the monoclonal antibody against Her2. However, the underlying mechanism remains unclear. Here, we reported that the infiltration of tumor-associated macrophages is associated with the reduction of CD8+ T cells and NK cells in tumors and poor response of trastuzumab-containing neo-adjuvant chemotherapy in Her2+ breast cancer patients. In vitro, the macrophages suppress proliferation and tumoricidal effect of CD8+ T cells and NK cells after phagocytosis of Her2+ tumor cells mediated by trastuzumab in a CD16 and CD32a-dependent manner. By using global expression screening, we identified the immunosuppression effect is due to the upregulation of PD-L1 and IDOs on macrophages after tumor phagocytosis. Accordingly, optimal response of 4D5, the mouse anti-human Her2 antibody as trastuzumab parental drug, require macrophage depletion or anti-PD-L and IDO inhibition in tumor-bearing mice. In consistent with results form mice, the expression of PD-L1 and IDOs on tumor-associated macrophages increase in clinical samples of patients who received trastuzumab-containing neo-adjuvant chemotherapy, but not in those of patients who received neo-adjuvant chemotherapy without trastuzumab. Collectively, our data suggest that trastuzumab and double immune checkpoint blockade exert non-redundant clinical effects on Her2+ breast cancer patients with dense tumor-associated macrophages infiltration.
The antitumor function of anti-4-1BB therapy involves the suppression of CD73-A2BR axis
Authors: Chen Siqi , Fan Jie , Zhang Minghui , Qin Lei , Dominguez Donye , Long Alan W , Zhang Bin
Published: January 2023

Agonist antibodies (Ab) directed against costimulatory molecule CD137 (4-1BB) on the surface of antigen-primed T-lymphocytes are currently in various stages of pre-clinical and clinical trials, albeit with limited therapeutic benefit as single agents. Here, we examined the inhibitory role of ecto-enzyme CD73 (generating adenosine) on the antitumor efficacy of agonistic anti-4-1BB Ab therapy, as anti-4-1BB treatment can downregulate the expression of CD73 on T cells within tumor microenvironment. CD73-deificient mice treated with anti-4-1BB Ab completely rejected CD73-nonexpressing B16-SIY melanoma when compared to wild-type mice. To explore the therapeutic relevance of these observations, we combined anti-4-1BB and anti-CD73 Ab to treat mice with established B16-SIY melanoma. This combination therapy induced tumor regression associated with enhanced antitumor CD8+ T cell responses and reduced CD4+Foxp3+ regulatory T cell number and function. In contrast, neither anti-4-1BB nor anti-CD73 monotherapy is effective to control the growth of established melanoma. Notably, anti-4-1BB treatment was also able to downregulate specifically the expression of adenosine receptor A2BR. Moreover, anti-4-1BB treatment elicited potent antitumor effect in A2BR-deficient mice as that in CD-73-deficient hosts. Our study establishes a novel mechanistic link between the CD73-A2BR axis and 4-1BB engagement. Thus, the blockade of CD73-A2BR axis has potential implications for more effective clinical targeting of 4-1BB and possibly other costimulatory molecules.
Identification of inhibitors of myeloid-derived suppressor cells activity through phenotypic chemical Screening
Authors: Schröder Matthias , Loos Simone , Naumann Svenja Kerstin , Bachran Christopher , Krötschel Marit , Umansky Viktor , Helming Laura , Swee Lee Kim
Published: January 2023

Tumors are infiltrated by cells of the immune system that interact through complex regulatory networks. Although tumor-specific CD8 T cells can be found in peripheral blood and tumor samples from cancer patients, their function is inhibited by immunosuppressive cells such as regulatory T cells, tumor-associated macrophages and myeloid-derived suppressor cells (MDSC). Recent clinical successes have demonstrated that alleviating immunosuppression and T cell exhaustion translates into long-term clinical benefits. Although tremendous progress has been achieved, tools that afford unbiased approaches and screenings to uncover new potential inhibitors or gene targets are lacking. In the present study, we describe a system based on immortalized progenitors that allows straightforward investigation of myeloid cells. We show that bone marrow progenitors immortalized through the transduction of NUP98-HOXB4 transgene can be differentiated into CD11b+Gr-1+ MDSC that express arginase-1 and PD-L1, produce reactive oxygen and nitrogen species and suppress T cell function in vitro. To uncover chemical probes that interfere with MDSC biology, we performed a chemical phenotypic screening and identified 3-deazaneplanocin A as novel modulator of MDSC functions. We characterized and compared the effect of 3-deazaneplanocin-A and all-trans retinoic acid, a well-known modulator of MDSC activity, on the expression of effector molecules and immunosuppressive functions of MDSC. Altogether, this proof-of-principle opens new possibilities for the identification of drugs targeting myeloid cells with immunosuppressive activities.
The Ron receptor tyrosine kinase plays a protective role in the development of CNS inflammation during health and disease states
Authors: Dey Adwitia , Allen Joselyn N , Fraser James W , Snyder Lindsay M , Cantorna Margherita T , Paulson Robert F , Hankey-Giblin Pamela
Published: January 2023

Neurodegeneration is characterized by severe central nervous system (CNS) inflammation. Macrophages are immune cells that closely regulate inflammation by taking on different activation states. The Ron receptor tyrosine kinase is expressed on tissue resident macrophages including microglia. An in vivo deletion of Ron (Ron KO) promotes inflammatory (M1) and limits reparative (M2) macrophage activation. Whether this response influences CNS tissue inflammation is not characterized. The objective of this study was to elucidate whether Ron expression plays a critical role in CNS inflammation in homeostasis and subsequently in disease models. During homeostasis with no interventions, Ron KO mice developed an inflammatory CNS niche with increased tissue expression of M1-associated markers TNFα, Cox-2 and iNOS when compared to WT-mice (P<0.05). In a diet induced obesity(DIO) model of chronic inflammation, Ron KO mice exhibit exacerbated CNS inflammation with decreased expression of M2(Arg-1) and a robust increase in M1 markers compared to WT-mice following 27 weeks of high-fat-diet intervention(P<0.05). Lastly, we evaluated the role of Ron in a disease model of Multiple Sclerosis, experimental autoimmune encephalitis (EAE). Ron KO-EAE mice exhibited higher disease severity from peak disease state and onward when compared to WT-EAE mice (P<0.05). Ron KO mice had greater CNS expression of M1 markers relative to higher peripheral inflammation with increased IFNγ secretion from lymphoid organs during peak disease state(P<0.05). Collectively these results illustrate how maintenance of Ron expression in the CNS could then be a potential therapeutic approach to treating various grades of inflammation underlying neurodegeneration.
BMP-2 Increases Articular Chondrocyte Maturation and Hypertrophy through a COX-2 Dependent Mechanism Associated with Inflammation.
Authors: Cottrell Jessica , Michaels Sarah
Published: January 2023

Rheumatoid arthritis is an autoimmune disease where the body perceives joint tissue as a foreign object and attacks it through the immune response. During this process the immune response destroys the native joint tissue and later replaces it with nonfunctional scar tissue. The joints and surrounding tissues continue to have uncontrolled chronic inflammation which produces perpetual pain, swelling, and tissue destruction. Cyclooxygenase-2 (COX-2) metabolizes arachidonic acid into eicosanoids which can regulate the inflammatory response and affect bone metabolism. When articular cartilage is damaged, bone morphogenetic protein-2 (BMP-2) is released from cells and can upregulate COX-2 expression. It is hypothesized that this BMP-2 induced COX-2 expression results in cartilage producing cells known as chondrocytes to advance to hypertrophy. In this study, articular chondrocytes harvested from wild type mice were treated with BMP-2 to determine if COX-2 expression could increase chondrocyte maturation and hypertrophy. Samples were collected at 0, 1, 2, 4, 7, and 14 days after treatment to analyze protein and gene expression. BMP-2 treated cells had statistically higher COX-2 mRNA and protein levels when compared to control cells. The expression of COX-2 on days 1 and 3 was followed by the expression of collagen 10a1 (a marker of chondrocyte hypertrophy) on days 7 and 10. These data suggest that BMP-2 directed COX-2 expression can affect chondrocyte maturation. In conclusion, this mechanism may contribute to the bone loss associated with chronic inflammation in diseases like rheumatoid arthritis.
Mer receptor tyrosine kinase signaling prevents TLR-mediated self-ligand sensing and aberrant selection in germinal centers
Authors: Schell Stephanie L , Soni Chetna , Fasnacht Melinda J , Cooper Timothy K , Rahman Ziaur S.M.
Published: January 2023

Mer receptor tyrosine kinase (Mer) signaling maintains immune tolerance by clearing apoptotic cells and inducing immunoregulatory signaling cascades. We previously showed that Mer-deficient mice on a mixed B6 and 129 background (B6129F2.Mer−/−) have increased germinal center (GC) responses, T cell activation, and apoptotic cell accumulation within GCs. Whereas B6129F2.Mer−/− mice accumulate apoptotic cells in GCs, B6.Mer−/− mice do not accumulate apoptotic cells, resembling B6 mice. Therefore, we used these two models to mechanistically separate the effects of debris released from secondary necrotic cells as a result of apoptotic cell accumulation from those driven by the loss of immune regulation by Mer. Through the study of global and cell-type intrinsic TLR and MyD88 deficiency in B6129F2.Mer−/− mice, we found that GC B cell-intrinsic sensing of self-RNA, but not self-DNA, released from secondary necrotic cells drives self-antigen induced GC responses. While self-ligands drive the magnitude of GC response, the loss of Mer immunoregulatory signaling bolsters antigen presentation and APC-intrinsic TLR activation drives pro-inflammatory cytokine production. Further, the loss of Mer immunoregulatory signaling accelerates the onset of autoimmunity when B6.Mer−/− mice are crossed to autoimmune-prone B6.Sle1b mice (Sle1b.Mer−/−) due to the retention of autoreactive B cells in GCs. Collectively, Mer prevents the cell-intrinsic activation of GC B cells and APCs to regulate the magnitude of GC response and T cell activation, in addition to regulating GC selection events to prevent the development of autoimmunity.
The role of renal infiltrating CD11c+ cells in lupus nephritis
Authors: Liao Xiaofeng , Ren Jingjing , Reihl Alec , Pirapakaran Tharshikha , Reilly Christopher M , Luo Xin M
Published: January 2023

Lupus nephritis, a leading cause of morbidity and mortality in patients with Systemic Lupus Erythematosus (SLE), manifests as inflammatory infiltration of the kidney. Many types of leukocytes infiltrate the kidney during the progression of lupus nephritis, but the specific contributions of each infiltrating population are still unclear. In this study, we have found that CD11c+cells accumulate in nephritic kidneys of lupus-prone mice. These renal CD11c+ cells, forming clusters with infiltrating T cells, are proinflammatory and secreting cytokines that promote autoantibody production and Th17 response. They also produce chemokines that attract other leukocytes into the kidney. The surface phenotype (CD11b+CD115highCD135lowCX3CR1highFcgRIVhigh)suggests that they may be derived from mature monocytes and be activated by pathogenic immune complexes. Ex vivo co-culture experiments also suggest that they can maintain the survival of renal infiltrating T cells. To further demonstrate the role of these CD11c+ cells in vivo, we are currently depleting them by targeting CX3CR1, as they are a unique population of cells predominantly expressing a high level of CX3CR1. The results obtained from this study will be important for the understanding of lupus pathogenesis and identification of new therapeutic targets against lupus nephritis.
Complement protein C1q modulates lipoprotein metabolism in macrophage foam cells
Authors: Fraser Deborah A , Curiel Lizette , Budin Ryan , Pulanco Marc C
Published: January 2023

In the atherosclerotic lesion, macrophages ingest high levels of damaged, modified, low density lipoproteins (LDL), which accumulate in lipid droplets or defective lysosomes, generating macrophage foam cells which contribute to plaque instability and rupture. C1q, the recognition component of the classical complement cascade, opsonizes modified forms of LDL such as oxidized LDL, and promotes ingestion by macrophages. C1q has been shown to be protective in an atherosclerosis model in vivo and shown to improve macrophage foam cell survival in vitro. Autophagy is a catabolic pathway that plays a role in cellular survival and is an important process in many phagocytic cells, such as macrophages. We tested the hypothesis that C1q modulates macrophage lysosomal maturation during ingestion of oxLDL and increases autophagy in an atheroprotective manner to improve macrophage survival. Levels of autophagy initiation and elongation proteins, Beclin 1 and Atg5, are not modulated by C1q, but levels of autophagosome-associated proteins, LC3B-II and p62, were increased in immunoblot. Quantitative PCR revealed that the increase in p62 protein levels was not due to an increase in transcription. Immunofluorescence microscopy verified that the increase in LC3B and p62 by C1q resulted in an increase in autophagosome formation during oxLDL clearance. TEM verified the formation of double membrane vesicles. C1q also increased lysosomal acidification in macrophages during ingestion of oxLDL. These atheroprotective alterations to lipoprotein metabolism may be important in slowing disease progression, and provide insight into the protective role of C1q in early atherosclerosis.
Macrophages attenuate survival of intravenously administered human mesenchymal stromal cells by phagocytosis
Authors: Hasgur Suheyla , Desbourdes Laura M. , Guess Adam J. , Otsuru Satoru , Yu Minjun , Overholt Kathleen M. , Relation Theresa , Horwitz Edwin M.
Published: January 2023

Human mesenchymal stromal cells (MSCs) are increasing used in clinical trials for a diverse array of applications. Whether allogenic or autologous cells are used, unqualified efficacy has not been shown, suggesting the innate immune system may be impairing MSC activity. For example, MSCs inherently traffic to osteosarcoma (OS) rendering them potentially effective delivery vehicles of therapeutics; however, such strategies have not emerged, possibly due to inadequate tumor localization. We sought to enhance MSC trafficking to OS studying xenograft models of human osteosarcoma in nude and NOD-scid-IL2rγnull (NSG) mice. After IV infusion, firefly luciferase-expressing MSCs trafficked to the tumor in both models. Interestingly, we observed 3.5-fold greater bioluminescent signal per unit volume from the OS in NSG compared to nude mice, suggesting macrophages in nude mice may be clearing the MSCs. To test our hypothesis, we infused MSCs into tumor-bearing nude mice after clodronate-mediated macrophage depletion and in non-depleted controls. The macrophage-depleted nude mice showed 3.7-fold more MSCs tumor localization on day 11 compared with controls. To evaluate a role of macrophages, 4 hours after claret-labeled MSCs were infused into nude mice. Flow cytometric analysis of a single cell suspension of lung and spleen revealed a claret+ human CD90+ fraction, consistent with human MSCs, and a claret+ F4/80+ human CD90− fraction suggestive of macrophages that have phagocytosed MSCs. Fluorescence microscopy confirmed claret+ cells within macrophages. Since clodronate depletes splenic, but not pulmonary, macrophages the splenic macrophages likely have the greatest impact which can be mitigated by transient macrophage depletion.
Soy isoflavones and their metabolites modulate cytokine-induced NK cell function.
Authors: Mace Thomas A , King Samantha , Loftus Shannon , Farren Matthew R , McMichael Elizabeth , Young Gregory , Carson William E , Clinton Steven K , Lesinski Gregory B
Published: January 2023

Soybeans and foods derived from them are a rich source of phytochemicals that have been linked with beneficial effects on obesity, cancer, and inflammation. It is postulated that these immunomodulatory properties in particular, may contribute to their health promoting effects. However, the impact of soy bioactives on the functional properties of immune cell subsets is not fully understood. Upon ingestion, the profile of bioactive compounds derived from soy becomes increasingly complex. For example, soy isoflavones (genistein and daidzein) can be differentially metabolized by distinct populations of gut microbiota which promote their metabolism into compounds as O-desmethylangolensin (O-DMA) and equol. We hypothesized that soy phytochemicals alter natural killer (NK) cell signaling and function. Soy isoflavones and their metabolites did not alter the viability of healthy donor PBMC, even at high (25μM) concentrations. However, pre-treatment of PBMC with genistein (p=0.0009) and equol (p=0.002) decreased IL-12/18 induced IFN-γ production compared to controls. Genistein (p=0.002) and equol (p=0.0006) also decreased IL-12/18-induced IFN-γ production by human NK cells but did not alter cytotoxicity. Furthermore, in vivo studies revealed that C57BL/6 mice fed a soy-enriched diet produced less IFN-γ in plasma following administration of IL-12/IL-18 as compared to control-fed animals (p<0.0001). This study provides insight into how dietary soy modulates NK cell functions and suggest the use of soy may interact with during chemopreventive or immune based therapies that act via NK cells.
Leukotriene (LT) B4 metabolites inhibit the LTB4-mediated responses of human neutrophils.
Authors: Archambault Anne-Sophie , Poirier Samuel , Lefebvre Julie S. , Larose Marie-Chantal , Turcotte Caroline , Martin Cyril , Provost Véronique , Boudreau Luc H. , Laviolette Michel , Surette Marc E. , Flamand Nicolas
Published: January 2023

CONTEXT

LTB4 is recognized as an excellent promoter of host defense. Indeed, it stimulates leukocyte functions mainly through the engagement of the BLT1 receptor. Of note, LTB4 induces phagocyte recruitment, the release of antimicrobial peptides, and potentiates the ingestion and the killing of pathogens. In humans, LTB4 has a short half-life and is rapidly metabolized into 12-oxo-LTB4 by monocytes/macrophages, or into 20-OH- and 20-COOH-LTB4 by neutrophils. Although LTB4 metabolites bind to the BLT1 receptor with high affinity, they poorly activate neutrophils.

HYPOTHESIS

We postulated that LTB4 metabolites might act as inhibitors of BLT1-mediated responses, therefore limiting the impact of LTB4 on human neutrophil functions and that inhibiting LTB4 degradation would enhance its effects on neutrophils.

RESULTS

20-OH-, 20-COOH-, and 12-oxo-LTB4 inhibited all LTB4-mediated responses of neutrophils we tested (migration, degranulation, leukotriene biosynthesis). 20-OH-LTB4 was as efficient as the BLT1 antagonist CP 105,696 and all LTB4 metabolites were more potent than the anti-inflammatory and pro-resolving lipids resolvin E1 and 5(S),6(R)-lipoxin A4. In contrast, the fMLP- and IL-8-mediated responses were not affected by the LTB4 metabolites. Inhibiting LTB4 degradation into 20-OH-LTB4 in neutrophils notably amplified the LTB4-induced kinase phosphorylation and diminished the chemokinetic effect of LTB4.

CONCLUSIONS

Our data indicate that LTB4 metabolites act as natural inhibitors of LTB4-mediated responses and that preventing LTB4 metabolism can modulate some functions of neutrophils. These results support the idea that preventing LTB4 degradation might enhance host defense by boosting innate immunity.
The severity of pulmonary congestion stemming from pseudomonal wound infection is influenced by IL-22 produced by ILC3 in burn mice after γ-irradiation
Authors: Ito Ichiaki , Kobayashi Makiko , Cox Robert A , Finnerty Celeste C , Herndon David N , Suzuki Fujio
Published: January 2023

Sepsis is severely developed after pseudomonal wound infection in mice with combined injuries (burn plus γ-irradiation). In the present study, we investigated the severity of pulmonary congestion caused by pseudomonal wound infection in combined injured mice. Combined injured mice were prepared after exposure to 15% TBSA burn and 5 Gy whole body γ-irradiation, as previously described. Mice exposed to burn injury or γ-irradiation alone served as controls. P. aeruginosa (10 CFU/mouse) was applied to burn wound area i.d. Bacterial growth in various organs of these mice was determined by colony counting. Also, the infection was pathologically examined by histochemical staining of tissue sections. Various cytokines in the alveolar lavage fluids of combined injured mice were measured by ELISA, and ILC3 in the lung tissue homogenates were analyzed by flow cytometry. In the results, P. aeruginosa grew violently in the lung of combined injured mice, as compared with both groups of control mice. The lung of combined injured mice histologically exhibited an alveolar thickening, abnormal structure, and pulmonary congestion. Although the number of ILC3 with IL-22 producing capacities was not increased in the lung of both groups of control mice, the large number of ILC3 was isolated from the lung of combined injured mice. Elevated levels of IL-22 were detected in the lung of combined injured mice. These results suggest that the severity of sepsis stemming from pseudomonal burn wound infection is influenced by ILC3 increased in the lung and their IL-22 products in combined injured mice.
Memory B cells are poised for robust antigen presentation by HLA-DO
Authors: Mellins Elizabeth D , Jiang Wei , Adler Lital , Macmillan Henriette
Published: January 2023

The B cell is a uniquely efficient APC due to expression of BCR for antigen uptake and delivery to MHCII-enriched compartments (MIIC), and key regulatory molecules, invariant chain (Ii), HLA-DM and HLA-DO. All professional APCs express Ii and DM, whereas expression of DO is limited to B cells, certain DCs and thymic medullary epithelia. DO levels change dramatically in B cells entering or exiting the germinal center (GC). To determine effects of DO on other components of the MHCII pathway, we delineated the intracellular distribution of DO, DM, MHCII, and class II-associated CLIP in T2 cells with or without DO or DM expression using structured illumination microscopy. The distribution at steady state shows a very restricted lysosomal destination (LAMP1+) for DO and DM, and dual destinations (surface and LAMP1+) for CLIP and MHCII. DO expression increased overall CLIP compared to DM+DO-cells, but almost doubled the percent of CLIP co-localized with LAMP1, establishing peptide exchange potential (PEP) within MIICs, but not any other compartments or the cell surface. PEP enhancement associated with DO expression is likely due to its regulation of DM activity, as neither the overall CLIP level nor the percent of DR in MIICs increased. We then evaluated the extent of PEP in distinct primary B cell populations isolated from human tonsils, including memory (CD38−/IgD−), naïve (CD38−/IgD+), and GC (CD38+/IgD−) B cells. The measurement shows PEPMemory (significantly) > PEPNaïve > PEPGC−, although the percent of CLIP among all class II peptides is naïve > memory >> GC. These results argue that DO expression in memory B cells equips them for more efficient APC function upon re-encounter with antigen.
T-cell antigens in pediatric-onset multiple sclerosis: A unique window into early disease mechanisms
Authors: Mexhitaj Ina , Nyirenda Mukanthu , Arnold Douglas L , Marrie Ruth Ann , Banwell Brenda , Bar-Or Amit
Published: January 2023

Multiple sclerosis (MS) involves immune attacks on the CNS, leading to demyelination, axonal injury and increasing neurological dysfunction. Though T cells are implicated, the particular subsets and their antigenic targets remain unknown. In adult-onset MS, distinguishing immune responses that are consequences of, rather than cause of, injury, is difficult. In contrast, pediatric-onset MS offers an early window into disease mechanisms given the narrower gap from biological onset. We aim to identify and characterize disease-relevant antigen-specific effector T cell responses to traditional and novel antigenic targets involved early in the MS disease process.

Our group has implicated target antigens and T cell subsets in pediatric-onset MS, by following patients from time of an initial presentation with acquired demyelinating syndrome and comparing those confirmed to have MS with those who remain monophasic. A CSF proteomic study implicated novel axo-glial apparatus molecules as early injury targets, rather than traditional compact myelin antigens. A series of multiparameter flow-cytometry panels applied to pediatric peripheral blood mononuclear cells (PBMC) revealed that MS children harbor abnormally increased frequencies and pro-inflammatory cytokine responses of particular effector T cell subsets compared to controls.

We will develop assays, initially in fresh PBMC samples from adult MS and controls, then miniaturize the approach and validate it for use in the small numbers of available cryopreserved pediatric PBMC samples to quantify antigen-specific responses including proliferation and cytokine profiles of distinct disease-implicated T cell subsets to both traditionally and newly implicated antigens.
Enhanced susceptibility to alcohol-induced bacterial translocation, immune activation, and persistent inflammation in patients with alcoholic hepatitis: a prospective observational study
Authors: Li Wei , Amet Tohti , Xing Yanyan , Lan Jie , Liangpunsakul Suthat , Puri Puneet , Kamath Patrick , Sanyal Arun , Shah Vijay , Radaeva Svetlana , Crabb David , Chalasani Naga , Yu Andy
Published: January 2023

Up to 30% chronic heavy drinkers develop a spectrum of severe alcoholic liver diseases, including alcoholic hepatitis (AH). Alcohol-induced impairment of intestinal integrity and microbial translocation into the portal vein lead to immune activation and play a major role in the pathogenesis of AH. To better understand why AH develops only in some heavy drinkers and the effect of alcohol abstinence on immunity, we conducted a prospective study to compare plasma LPS levels and peripheral blood immunoprofiles between 68 AH patients and 65 heavy drinkers without liver disease (HDC) who were followed up to 12 months. Plasma samples were also obtained from 31 health donors (HD). Plasma LPS was detected in a higher portion of AH patients than HDC (80% vs 49%) at baseline, and in only 8% HD. However, LPS levels were not different between AH an HDC subjects with detectable LPS. Compared with HDC, AH patients had higher baseline plasma levels of sCD14, sCD163, sICAM-1, sVCAM-1, IL-22, TNF-α, IL-8, IP10, IL-4, IL-6, IL-9, IL-10, FGF2, IL-7, IL-15, and TGF-α, but lower levels of the anti-inflammatory chemokine MDC. AH patients also had more activated, yet dysfunctional monocytes, T cells, and B cells as they expressed higher levels of CD38 and CD69, but lower levels of HLA-DR, CD80, and CD86. In spite of having significant bacterial translocation, HDC showed little evidence of immune activation. With alcohol abstinence, levels of many dysregulated immune markers normalized in AH patients. However, TNF-α, IL-8, IL-10, sCD163, and sVCAM-1 levels remained higher.

Conclusion

AH patients were more susceptible to alcohol-induced bacterial translocation and immune activation. Alcohol abstinence reduced but did not fully reverse immunological abnormalities.
Lack of central memory cell generation by PD-1-deficient T cells under helper-deficient conditions
Authors: Kim Woojin , Ju Ji-Min , Choi Eun Young
Published: January 2023

An immune check point molecule PD-1 is associated with CD8 T cell exhaustion in tumor-infiltrating and chronic infection situations. Previously, we have demonstrated that CD8 T cells specific for a transplantation antigen, minor histocompatibility antigen H60, are exhausted when primed under CD4 helper-deficient conditions, showing PD-1hi phenotype, and the exhaustion is ascribed to inefficient antigen-clearance. We examined whether the helpless CD8 T cells could respond to re-challenge with H60 in absence of PD-1-mediated signaling using PD-1-deficient mice. Upon re-challenge with H60 of the PD-1-deficient mice, which had been primed against H60 under helper-deficient conditions, the H60-specific CD8 T cells expanded vigorously, exhibiting a memory-like response. Despite the restored proliferative potential to the antigen-re-encounter, the H60-specific PD-1-deficient CD8 T cells did not generate CD44hiCD6Lhicentral memory T cells under helper-deficient priming condition. They exhibited an exhausted status, with an increased CXCR3+KLRG1+ Tint cell fraction. When transferred to naïve hosts, PD-1-deficient helpless CD8 T cells showed reduced generation of central memory cells compared to their wild type counterparts. These results suggest that PD-1 signaling influences fate of effector CD8 T cells and provide information regarding CD8 T cell memory generation.
CD46 is a potent co-stimulatory receptor on CD8+ T cells
Authors: Hansen Aida Solhoej , Slater Josefine , Biltoft Mette , Höllsberg Per
Published: January 2023

Upon activation CD8+ T cells may proliferate extensively and become specialized effector cells with an important role in clearing viral infections. Similar to CD4+ T cells, precursor CD8+ T cells are thought to be dependent on a co-stimulatory signal through CD28. CD46 is another co-stimulatory receptor on CD4+ T cells that promotes proliferation and secretion of IFNγ followed by secretion of high amounts of IL-10, thereby inducing a regulatory phenotype. To our knowledge, the co-stimulatory function of CD46 on CD8+ T cells is yet unknown.

CD4+ and CD8+ T cells were isolated from peripheral blood mononuclear cells from healthy donors and activated in vitro with plate-bound αCD3/αCD28 or αCD3/αCD46 for 72 hrs. The proliferation was examined by CFSE staining and the secretion of IFNγ and IL-10 by ELISA. As expected, we observed increased proliferation as well as secretion of IFNγ and high amounts of IL-10 in the CD4+ T cells upon CD46 co-stimulation compared to CD28 co-stimulation. Intriguingly, CD46 co-stimulation also promoted proliferation of CD8+ T cells and secretion of high amounts of IFNγ compared to CD28 co-stimulation. In contrast to the regulatory phenotype of CD4+ T cells, it appears that CD46-costimulation promotes the effector response of CD8+ T cells, as we observed elevated IFNγ and only limited induction of IL-10 secretion in CD8+ T cells when compared with CD4+ T cells.

In conclusion, these results suggest that CD46 co-stimulation has a functional significance on both proliferation and effector functions of CD8+ T cells.
TET2 and TET3 regulate stability and function of regulatory T cells.
Authors: Nakatsukasa Hiroko , Yoshimura Akihiko
Published: January 2023

Regulatory T cells (Tregs) play a pivotal role in regulating immune response and maintaining immunological tolerance, and are characterized by the expression of Foxp3. Thymus-derived Tregs have been shown to be relatively stable compared with iTregs generated from naive T cells with TGFβ in vitro. Treg stability is maintained by the epigenetic modification, such as DNA demethylation of Treg-specific demethylation regions (TSDRs) during Treg development in the thymus. In fact, the TSDRs of iTreg are hypermethylated. Although DNA demethylase, ten-eleven translocation (TET) family proteins have been proposed to be involved in the DNA demethylation of TSDRs, actual contribution of TET family proteins in Treg stability and function remains to be elucidated. To investigate the role of TET proteins in Tregs, we generated Treg-specific Tet-deficient (Foxp3YFPCre+Tet2f/fTet3f/f; FDKO) mice. FDKO mice died at an average of 20 weeks of age with splenomegaly and lymph adenopathy accompanied by uncontrolled activation of T cells and B cells. These FDKO mice developed inflammation in the gut, and both Foxp3+ and Foxp3− T cells were expanded and produced high levels of IL-17 especially in gut mucosa and mesenteric lymph nodes. Transfer experiment of FDKO Tregs into lymphopenic mice revealed that Foxp3 expression in FDKO Tregs was unstable and Treg cells which have lost their Foxp3 expression were accumulated. TSDRs of FDKO Tregs were hyper-, but not completely, methylated compared with WT Tregs, indicating that there are TET2/3 dependent and independent mechanisms for TSDR demethylation. These data indicate that TET2 and TET3 are redundantly involved in TSDR demethylation and are important for the stability and homeostasis of Tregs.
Regulation of CD11c+ T-bet+ IgM+ Memory B-cells by TNFα
Authors: Popescu Maria , Cabrera-Martinez Berenice , Winslow Gary M.
Published: January 2023

Our studies of ehrlichial infection have identified a population of CD11c+ T-bet+ IgM memory B-cells that arise within 30 days post-infection. We and others have shown that CD11c+ T-bet+B cells require for their development or function a number of factors, including T-bet, IFNγ, CD4 T-cells, IL-21, and TLRs. Here, we addressed a possible role for TNFα in the generation of CD11c+ T-bet+IgM memory B-cells, because aside from being a major inflammatory cytokine, it can regulate germinal center development, and mediate apoptosis. We observed an 8-fold increase in the number of CD11c+IgM memory B-cells (also CD73+) in TNFα-deficient mice, relative to wild-type mice, on day 16 post-infection. The B cell population persisted at much higher frequency and number on day 30 post-infection. The changes in the IgM memory cell population were associated with a concomitant 10-fold relative increase in the frequency of GL7+ CD38lo CD95+germinal center B cells in TNFα-deficient mice. These latter data contrast with published studies that have reported that TNFα is required for GC development, suggesting that the requirement we have observed for TNFα is context-dependent. These data suggest that there may be a positive relationship between GC development and generation of CD11c+ IgM memory cells, and that TNFα may act to mediate IgM memory cell homeostasis by regulating apoptosis. Ongoing studies will address mechanisms whereby TNFα contributes to the genesis and maintenance of CD11c+ T-bet+IgM memory B-cells.