Expression Levels Of MHC-II Research Articles

CpG Oligodeoxynucleotide-Coated Chitosan Nanoparticles Enhance Macrophage Proinflammatory Phenotype In Vitro.

This study aimed to investigate the effects of CpG Oligodeoxynucleotide (CpG ODNs)-Coated Chitosan Nanoparticles (CNP) on the phenotype of murine macrophages and their pro-inflammatory cytokine profile in vitro. CNP-CpG ODNs loaded with FITC-scrambled siRNA were prepared using the ionotropic gelation method. Peritoneal macrophages were isolated and exposed to CNP-CpG ODNs. Treated macrophages were assessed for uptake capacity. Flow cytometry was used to evaluate the expression levels of MHC-II, CD40, and CD86 costimulatory molecules in treated macrophages. Furthermore, the secretion levels of proinflammatory cytokines (TNF-α and IL-6) and the release of nitric oxide (NO) were measured in the culture supernatant of treated macrophages using sandwich ELISA and the Griess reaction, respectively. These in vitro studies showed that CNP-CpG ODNs had no cytotoxic effect on macrophages and were efficiently taken up by them. Additionally, CNP-CpG ODNs significantly increased the production of TNF-α, IL-6, and NO in the culture supernatant compared to CNP alone. Moreover, CNP-CpG ODNs enhanced the expression of MHC-II, CD40, and CD86 costimulatory molecules on macrophages. These findings indicate that incorporating CpG ODNs into CNPs promotes macrophage maturation and a proinflammatory phenotype. Therefore, CNP-CpG ODNs may serve as an effective system for targeted gene delivery to macrophages, enhancing immune responses.

Anti- and pro-inflammatory milieu differentially regulate differentiation and immune functions of oligodendrocyte progenitor cells.

Oligodendrocyte progenitor cells (OPCs) were regarded for years solely for their regenerative role; however, their immune-modulatory roles have gained much attention recently, particularly in the context of multiple sclerosis (MS). Despite extensive studies on OPCs, there are limited data elucidating the interactions between their intrinsic regenerative and immune functions, as well as their relationship with the inflamed central nervous system (CNS) environment, a key factor in MS pathology. We examined the effects of pro-inflammatory cytokines, represented by interferon (IFN)-γ and tumour necrosis factor (TNF)-α, as well as anti-inflammatory cytokines, represented by interleukin (IL)-4 and IL-10, on OPC differentiation and immune characteristics. Using primary cultures, enzyme-linked immunosorbent assay and immunofluorescence stainings, we assessed differentiation capacity, phagocytic activity, major histocompatibility complex (MHC)-II expression, and cytokine secretion. We observed that the anti-inflammatory milieu (IL4 and IL10) reduced both OPC differentiation and immune functions. Conversely, exposure to TNF-α led to intact differentiation, increased phagocytic activity, high levels of MHC-II expression, and cytokines secretion. Those effects were attributed to signalling via TNF-receptor-2 and counteracted the detrimental effects of IFN-γ on OPC differentiation. Our findings suggest that a pro-regenerative, permissive inflammatory environment is needed for OPCs to execute both regenerative and immune-modulatory functions.

IMMU-04. LOW-INTENSITY PULSED ULTRASOUND ENHANCES CAR T CELL THERAPY BY INDUCING TWO-WAY IMMUNE CELL TRAFFICKING – T CELL INTO TUMOR AND MYELOID CELL INTO PERIPHERY IN A SYNGENEIC MOUSE GLIOBLASTOMA MODEL

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy has been successful against hematological malignancies. However, it has not established efficacy in glioblastoma (GBM) patients. Significant challenges currently limiting the effectiveness of CAR T-cell therapy for patients with GBM include poor CAR-T cell trafficking to GBM due to the blood-brain barrier, and the presence of highly immunosuppressive and pro-tumoral tumor-associated macrophages (TAMs) in the tumor microenvironment. Furthermore, the heterogeneous expression of target antigens in GBM can lead to antigen escape and therapeutic resistance. Low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has been shown to enhance CAR T-cell trafficking and improve survival in immunocompromised xenograft mice GBM models. In this study, we investigated whether multiple rounds of LIPU/MB can improve not only CAR T-cell but also endogenous T-cell infiltration into the tumors and induce myeloid cell trafficking into lymph nodes (LNs), thus enhancing CAR T-cell therapy against GBM in the immunocompetent syngeneic mice model. We recently developed a novel immunocompetent transgenic (Tg) mouse that expresses an anti-EGFRvIII CAR on T-cells and enables us to investigate the interaction between CAR T-cells and the endogenous immune environment. C57BL6/J mice bearing intracerebral EGFRvIII+ GBM received an intravenous administration of anti-EGFRvIII CAR T-cells derived from Tg-mice. The mice were stratified to receive single-LIPU (1 min before CAR T-cells administration) or triple-LIPU/MB (1 min before CAR T-cells administration, then two and four days later). Triple-LIPU/MB significantly suppressed tumor growth and improved survival and was associated with increased CAR T-cells and endogenous T-cells infiltration into the tumors compared with single-LIPU/MB or without LIPU. Furthermore, draining cervical LNs showed increased macrophages trafficking after triple-LIPU/MB. Moreover, TAMs isolated from triple-LIPU/MB mice brains showed higher levels of MHC-II expression. These results suggest that LIPU/MB may promote the tumor antigen presentation to endogenous T-cells and enhance the efficacy of CAR T-cell therapy for GBM.

GENIPOSIDE IMPROVES CLP-INDUCED SEPSIS MODEL PROGNOSIS BY UPREGULATING PPARγ TO MODULATE MONOCYTE PHENOTYPE AND CYTOKINE NETWORK.

Background : We explored the efficacy and main biological mechanism of geniposide intervention in sepsis. Methods : A sepsis model was established in male BALB/c mice through cecal ligation and puncture (CLP). Different doses of geniposide (20 or 40 mg/kg) were administered intravenously at 0 and/or 24 h after CLP surgery. The survival rate of different groups was observed. In addition, the expression levels of CD16 and major histocompatibility complex class II in monocytes were assessed using flow cytometry. The concentrations of TNF-α, IL-1β, IL-6, and IL-10 in the serum were measured by ELISA. We also observed the biological effects of geniposide on CD16 and MHC-II expression levels in RAW264.7 cells, as well as the secretion of TNF-α, IL-1β, IL-6, and IL-10 in the LPS-induced RAW264.7 cell model. The PPARγ levels were determined using western blot analysis. Results : Intravenous administration of 40 mg/kg of geniposide at 0 h after CLP significantly improved the survival outcomes in the septic mouse model, with no significant benefits from low dosing (20 mg/kg) or delayed administration (24 h). The effective dose of geniposide significantly decreased the serum cytokine TNF-α, IL-1β, IL-6, and IL-10 concentrations in septic mice ( P < 0.05). Notably, in vitro assays showed that geniposide specifically increased the IL-10 level. Geniposide significantly reduced the CD16 expression ( P < 0.05) and increased MHC-II expression in monocytes ( P < 0.05). In addition, geniposide elevated the PPARγ level in monocytes ( P < 0.05). Conclusions : High-dose early-stage geniposide administration significantly improved the survival rate in a CLP mouse sepsis model by modulating the monocyte phenotype and regulating the cytokine network (IL-6/IL-10 levels). The pharmacological mechanism of geniposide action might be exerted primarily through PPARγ upregulation.

PAD4 controls the anti-tumor response by fine-tuning IFNγ signaling in macrophages

Abstract MHC-II–mediated antigen presentation is essential for the function of the immune system. Recent evidence indicates that MHC-II expression shapes tumor immunity and response to immunotherapy. MHC-II expressing antigen presenting cells (APCs) mediate CD4 +T-cell priming and activation and license them to support the CD8 +T-cell response. Citrullination – catalyzed by peptidyl arginine deiminases (PADs) – is a post-translational modification at arginine residues. Here, we show that PAD4 negatively regulates anti-tumor immunity by tuning IFNγ-signaling gene expression, including that of MHC-II, in tumor associated macrophages (TAMs) in tumor bearing mouse models. Mechanistically, PAD4 citrullinates STAT1, thereby promoting the interaction between STAT1 and PIAS1 (protein inhibitor of activated STAT1) and resulting in reduced MHC-II expression in TAMs. Moreover, low levels of MHC-II expression in TAMs correlate with a reduced response to immunotherapy and are associated with poor cancer patient survival. These data suggest that targeting PAD4-mediated citrullination in macrophages may serve as a potential anti-cancer therapeutic approach. Supported by grants from the NCI (R01CA217510, PA-21-071)

MiR-148a regulation interferes in inflammatory cytokine and parasitic load in canine leishmaniasis.

Canine leishmaniasis (CanL) is a severe public health threat. Infected animals mediate transmission of the Leishmania protozoan to humans via the sandfly's bite during a blood meal. CanL progression depends on the degree of suppression of the immune response, possibly associated with microRNAs (miR), which can modulate mRNA translation into proteins and (consequently) regulate cell function. Increased miR-148a in splenic leukocytes (SL) of dogs with CanL was observed in previous studies, and in silico analysis, identified possible pathways involved in immune response regulation that are affected by this miR. Therefore, we evaluated the involvement of miR-148a in the regulation of TNF-α, IL-6, IL-12, IL-1β, iNOS, MHCII, CD80, CD3, T-bet, and GATA-3 transcription factors and their relationship with parasite load in SL of dogs with CanL. Splenic leukocytes obtained from healthy and diseased dogs were transfected with miR-148a mimic and inhibitor oligonucleotides. After 48 hours, expression levels of MHCII, CD80, iNOS, CD3, T-bet, and GATA-3 were evaluated by flow cytometry, and concentrations of TNF-α, IL-12, IL-6, and IL-1β were measured in culture supernatants by capture enzyme-linked immunosorbent assays. Transfection of SL with miR-148a mimics decreased iNOS levels in cells and TNF-α, IL-6, and IL-12 in the supernatants of cultured SL from CanL dogs. Interestingly, transfection with miR-148a inhibitor decreased parasite load in SL cells. These results suggest a direct or not regulatory role of this miR in the immune response to Leishmania infantum infection. We conclude that miR-148a can modulate immune responses by regulating inflammatory cytokines during CanL. Our results contribute to understanding the complex host/parasite interaction in CanL and could assist the development of treatments.

Chicken CSF2 and IL-4-, and CSF2-dependent bone marrow cultures differentiate into macrophages over time.

Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are utilized as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1) to differentiate BMMΦ and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMΦ and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMΦ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMΦ. All cultures were efficiently able to uptake 0.5 µm beads, but poorly phagocytosed 1 µm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC.

LAMTOR1 degrades MHC-II via the endocytic in hepatocellular carcinoma.

Tumor cell surface antigen recognition is a major hallmark of cancer therapy, and loss of major histocompatibility complex class I (MHC-I) is the most common mechanism that impairs tumor cell surface antigen processing and expression. In addition to this, MHC-II regulates antigen presentation in CD4+ T cell immune responses involved in tumor killing by CD8+ T cells, whereas the regulation of endocytosis regulating MHC-II antigen presentation has not been reported. Therefore, the regulation of the endocytosis pathway on the expression of MHC-II surface level and antitumor T cell response remains to be explored. In this experiment, we found that LAMTOR1 regulates the endocytic pathway through the GTPase domain of DNM2 and triggers the formation of autophagosomes. We performed flow cytometry and western blotting analyses, which revealed that the expression of MHC-II molecules on the surface of cells is influenced by LAMTOR1 through the endocytic pathway. We showed that the expression of MHC-II molecules, which recognize CD4+ T cells on the surface of cells, was regulated by LAMTOR1 through an endocytic pathway. By coculture experiments, we showed that CD8+/CD4+ T cells exhibit substantially higher levels of tumor cell apoptosis than those observed when hepatocellular carcinoma (HCC) cells were cocultured with CD8+ T cells alone. This study revealed that LAMTOR1 decreases the expression levels of MHC-II on cell surfaces in order to reduce antigen expression, leading to a decrease in antitumor T cell responses.

Class Switching is Required for Antigen-Dependent B cell Anti-Tumor Activity

Abstract Recent clinical studies demonstrated a positive correlation between B cell parameters and response to immune checkpoint blockade therapy. However, the potential mechanisms underlying B cell anti-tumor activities are still unclear. A better understanding of B cell-mediated immune responses in cancer will highlight novel vantage points to exploit therapeutically. To this end, we engineered tumor cells to express the B cell model antigen hen egg lysozyme (HEL). Adoptive transfer of CD45.1+ HEL-specific B cells (SWHEL B cells) into wild-type mice bearing HEL+ tumors significantly delayed tumor growth. SWHEL B cells from tumor-draining lymph nodes showed higher levels of MHCII expression, germinal center (GC) formation and class switch rate than non-tumor specific B cells. Of note, MD4 B cells, which recognize HEL but cannot class switch, were unable to control tumor growth, suggesting that immunoglobulin class M are not required for B cell anti-tumor activities. Accordingly, depletion of CD4 T cells (which are required for class switch) in mice bearing HEL+ tumors reverted the anti-tumor activity of SWHEL B cells. GC SWHEL B cells class switched to immunoglobulin G1 with a frequency between 20 and 80%, and resulted in a 10-fold increase of plasmablast (CD19+CD138+) and memory B cell (CD19+IgD-CD38+CCR6+) generation, as compared to non-tumor specific B cells. Collectively, these data point to a role for class-switched B cells and CD4 T cells in tumor immunosurveillance. Future studies will assess the ability of tumor antigen-specific B cells in priming naïve CD4 T cells and the role of other immunoglobulin classes in tumor control and escape. Supported by V Foundation for Cancer Research V2019-012

Estradiol inhibits differentiation of mouse macrophage into a pro-inflammatory phenotype by upregulating the IRE1α-XBP1 signaling axis

To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway. Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR. Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist. Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.

Reduced MHC class II expression in medullary thyroid cancer identifies patients with poor prognosis

Increasing body of recent studies determining the expression of tumor-specific major histocompatibility complex (MHC) class II protein supports its potential role in several malignancies, but little is known in human medullary thyroid cancer (MTC). Here, we report the expression of MHC-II and its clinicopathologic and prognostic relevance in MTC patients. Immunohistochemistry staining revealed a significant reduction in tumor cell-specific MHC-II expression in a higher AJCC stage and its poor prognostic correlation with human MTC development. Further statistical analysis identified the low MHC-II expression as a significant and independent risk factor for MTC recurrence and patient survival. Moreover, in vitro studies showed that the MHC-II expression was remarkably increased by RET inhibitors, which were prescribed to treat advanced MTC. Similarly, inhibitors blocking the MAPK/ERK and AKT/mTOR pathways also augmented MHC-II expression, suggesting their implications in RET-MHC-II signaling axis. Importantly, in vitro assays manifested enhanced peripheral blood leukocytes-mediated cytotoxicity in MTC cells treated with RET inhibitors, which were partially alleviated by HLA knock-down. Together, our study demonstrates that low MHC-II expression levels may serve as a prognostic biomarker for aggressive diseases in MTC patients and indicates that RET activation may promote MTC immune escape through downregulating MHC-II expression.

Anti-CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems.

Anti‐CD52 antibody (anti‐CD52‐Ab) leads to a rapid depletion of T and B cells, followed by reconstitution of immune cells with tolerogenic characteristics. However, very little is known about its effect on innate immune cells. In this study, experimental autoimmune encephalomyelitis mice were administered murine anti‐CD52‐Ab to investigate its effect on dendritic cells and monocytes/macrophages in the periphery lymphoid organs and the central nervous system (CNS). Our data show that blood and splenic innate immune cells exhibited significantly increased expression of MHC‐II and costimulatory molecules, which was associated with increased capacity of activating antigen‐specific T cells, at first day but not three weeks after five daily treatment with anti‐CD52‐Ab in comparison with controls. In contrast to the periphery, microglia and infiltrating macrophages in the CNS exhibited reduced expression levels of MHC‐II and costimulatory molecules after antibody treatment at both time‐points investigated when compared to controls. Furthermore, the transit response of peripheral innate immune cells to anti‐CD52‐Ab treatment was also observed in the lymphocyte‐deficient SCID mice, suggesting the changes are not a direct consequence of the mass depletion of lymphocytes in the periphery. Our study demonstrates a dynamic and tissue‐specific modulation of the innate immune cells in their phenotype and function following the antibody treatment. The findings of differential modulation of the microglia and infiltrating macrophages in the CNS in comparison with the innate immune cells in the peripheral organs support the CNS‐specific beneficial effect of alemtuzumab treatment on inhibiting neuroinflammation in multiple sclerosis patients.

Ridd Is Required for the Prevention of Chronic Gvhd By Targeting IRE-1a/Xbp-1s Signaling

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective therapeutic procedure to treat hematological malignancies. However, the benefit of allo-HCT is limited by a major complication, chronic graft-versus-host disease (cGVHD). Since transmembrane and secretory proteins are generated and modified in the endoplasmic reticulum (ER), the ER stress response is of great importance to secretory cells including B cells. By using conditional knock-out (KO) of XBP-1, IRE-1α or both specifically on B cells, we demonstrated that the IRE-1α/XBP-1s pathway, one of the major ER stress response mediators, plays a critical role in B cell pathogenicity on the induction of cGVHD in murine models of allo-HCT. Endoribonuclease activity of IRE-1α not only activates XBP-1s transcription factor by converting unspliced XBP-1 (XBP-1u) mRNA into spliced XBP-1 (XBP-1s) mRNA but also cleaves other ER-associated mRNAs through regulated IRE-1α-dependent decay (RIDD). Besides, it is known that ablation of XBP-1s production leads to unleashed activation of RIDD. Therefore, we hypothesized that RIDD plays an important role in B cells during cGVHD development. In this study, we found that B cells deficient for XBP-1s reduced ability to induce cGVHD, which however was reversed by inactivation of IRE-1α, highlighting the role of RIDD in controlling cGVHD (Fig. A). Activation of RIDD targets IgM mRNA of (Fig. B), a contributor to organ damage and fibrosis in cGVHD, which correlated with dysregulated expression of MHC II and costimulatory molecules such as CD86, CD40, and ICOSL in B cells (Fig. C). Alloreactive T cells need to be primed by APCs to initiate GVHD, and specifically, CD86 and CD40 mediated-costimulation from APCs has been demonstrated to play an essential role in eliciting cGVHD. We demonstrated that alloreactivity of T cells, especially CD4 T cells, can be recovered by suppressing RIDD in XBP-1s-deficient B cells (Fig. D). Since IRE-1α carrying a S729A mutation shows ablated RIDD activity without effect on splicing XBP-1 mRNA, we investigated the contribution of B cells from S729A knock-in mice to confirm the role of RIDD in B cells. We found that B cells from S729A mice increased GVHD severity (Fig. E). S729A B cells showed significant increases in IgM secretion (Fig. F), GC cell differentiation (Fig. G), and the expression levels of MHCII and co-stimulatory factors (Fig. H). In conclusion, these results provide a novel insight on how ER stress response regulates B cell activity after allo-HCT and suggest RIDD is an important mediator for reducing cGVHD pathogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pparα Ablation Suppresses T Cell Responses and Anti-Tumor Immunity By Compromising the Antigen-Presenting Properties of Tumor-Associated Macrophages

Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to nuclear hormone superfamily, with three distinct types identified: PPARapha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ). PPARs possess a critical role in the regulation of lipid metabolism, and thus play critical roles in the differentiation and fate of immune cells. PPARα is involved in lipid and carbohydrate metabolism and PPARα agonists, such as fibrates, have been used for the treatment of hypertriglyceridemia and cardiovascular diseases. PPARα has an anti-inflammatory role during infection, and similar to PPARγ, affects the polarization of macrophages. In acute myelogenous leukemia (AML), PPARα mutations correlate with chemoresistance, poor treatment outcomes and unfavorable prognosis. In experimental tumor models, it has been proposed that PPARα agonists might enhance anti-tumor T cell responses during PD-1 blocking immunotherapy. To dissect the mechanistic role of PPARα in tumor immunity, we used mice with global deletion of PPARα and examined tumor growth and profile of the immunological landscape, using various syngeneic tumor models. Significantly larger B16-F10 melanoma and MC-17 fibrosarcoma tumors were observed in PPARα KO mice compared with wild-type control, suggesting that PPARα deletion attenuated the immunological response against cancer. To dissect the role of PPARα in key populations of the innate and adaptive immune system involved in anti-tumor responses, we analyzed the immunological landscape of tumor, tumor draining lymph nodes (TDLN) and spleen, 14-16 days after tumor implantation. Assessment of CD4 + and CD8 + T cells, CD11b +F4/80 + tumor-associated macrophages (TAMs), CD11b +Ly6C hiLy6G - monocytic myeloid derived suppressor cells (M-MDSC), and CD11b +Ly6C loLy6G + polymorphonuclear myeloid derived suppressor cells (PMN-MDSC), by using flow cytometry, showed no quantitative differences between the two experimental groups. Functionally, MDSC from PPARα KO and WT mice showed comparable immunosuppressive properties as determined by suppression assay using splenocytes from OTI transgenic mice. However, PPARα KO TAMs demonstrated a less activated state, as determined by the lower expression levels of MHC-II that is critical for antigen presentation, and CD86 that is critical for T cell costimulation and prevention of T cell anergy and exhaustion. In agreement with these properties of TAMs, CD4 + T cells from TDLN of PPARα KO mice had diminished expression of activation markers, including PD-1, PD-L1 and ICOS, and numerically decreased central memory-like CD4 + T cells (T CM), compared to control tumor bearing mice. Furthermore, CD69, an emerging marker of T cell exhaustion, was significantly upregulated in CD4 + and CD8 + T cells from the TDLN of PPARα KO mice. To determine whether PPARα ablation altered the cell intrinsic properties of myeloid cells and/or T cells resulting in impaired anti-tumor function, we examined in vitro responses of isolated populations. In response to activation via TCR/CD3 and CD28, PPARα deficient T cells had no significant differences in expansion and cytokine production compared to control. In contrast, PPARα deficient Ly6C + monocytes isolated from the bone marrow displayed diminished responses to TLR-mediated signaling as determined by production of IL-6 and TNFα. Our in vitro and in vivo findings reveal a dominant role of PPARα in regulating the fate of innate immune cells thereby altering T cell responses and anti-tumor function. Our findings have implications for the development of new therapeutic approaches to enhance innate immune cell function for the improvement of cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1778: Bacterial regulation of innate and adaptive tumor immunity in metastatic breast cancer

Abstract In the tumor microenvironment (TME), innate and adaptive immune populations produce inflammatory cytokines that, while often critical for anti-tumor immunity, also mediate tumor-promoting functions. This paradoxical role of inflammation highlights the importance of defining the factors that control inflammatory signaling during tumor progression to optimize treatment of inflammation driven tumors and enhance our understanding of the role of inflammation in cancer immunology. Similar to inflammation, recent exploration of bacteria-mediated immune regulation reveals the microbiome mediates both anti-tumor and protumorigenic functions. In breast cancer for example, Helicobacter infection-induced inflammation augments primary tumor growth in a neutrophil-dependent manner. Similarly, an exogenously induced shift toward a more inflammatory microbiome in breast tumor-bearing mice leads to systemic inflammation and myeloid cell infiltration into the tumor that enhances dissemination into the lungs. Considering the lack of surgical options for patients with highly metastatic breast cancer (MBC), we seek to define how microbiome-mediated inflammation affects tumor progression in metastatic forms of the disease. Using a microbiome depletion model, our labs found bacterial depletion inhibited primary tumor growth of metastatic 4T1 breast cancer tumors specifically, while having no effect on non-metastatic 67NR tumors. When we compared immune populations in metastatic and non-metastatic tumors, we found that following microbiome depletion, MHCII expression was significantly reduced on macrophages in metastatic 4T1 tumors specifically. We also found CD4 Th17 populations were decreased both in terms of percentage and number, and that reduced Th17 numbers robustly correlated with reduced tumor volume. We then determined if the decrease in MHCII expression was due to M2 polarization. Analysis of CD206+ cells (M2 macrophages) revealed no differences between macrophages from control and microbiome-depleted animals, and when we analyzed MHCII expression in accordance with CD206, we found that reduced MHCII expression was associated specifically with CD206- cells, showing that down regulation occurs on the M1 population primarily responsible for activating Th17 cells. Additionally, rank correlation analyses revealed that reduced MHCII expression levels robustly correlated reduced tumor volumes in 4T1 tumors. These data suggest the microbiome induces tumor-promoting inflammation in MBC, and suggest a model whereby the microbiome regulates protumorigenic macrophage function by maintaining MHCII expression in MBC, and that bacterial-dependent macrophage activity augments tumor-promoting inflammation from Th17 cells to drive disease progression. Further analysis of this axis will identify pathways that can be targeted during treatment of inflammation-driven tumors. Citation Format: Zachary Gerbec, Shoukat Dedhar, Brett Finlay. Bacterial regulation of innate and adaptive tumor immunity in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1778.

High-Oxygen Submersion Fetal Thymus Organ Cultures Enable FOXN1-Dependent and -Independent Support of T Lymphopoiesis.

T cell development is effectively supported in fetal thymus organ cultures (FTOCs), which places thymus lobes atop an air-liquid interface (ALI) culture system. The direct exposure to air is critical for its success, as fetal thymus lobes placed in low oxygen submersion (LOS)-FTOCs fail to support thymocyte development. However, submersion cultures performed in the presence of high concentration of ambient oxygen (60~80%) allow for normal thymocyte development, but the underlying mechanism for this rescue has remained elusive. Here, we show that FOXN1 expression in thymic epithelial cells (TECs) from LOS-FTOCs was greatly reduced compared to conventional ALI-FTOCs. Consequently, the expression of important FOXN1 target genes, including Dll4 and Ccl25, in TECs was extinguished. The loss of DLL4 and CCL25 interrupted thymocyte differentiation and led to CD4+CD8+ cells exiting the lobes, respectively. High oxygen submersion (HOS)-FTOCs restored the expression of FOXN1 and its target genes, as well as maintained high levels of MHCII expression in TECs. In addition, HOS-FTOCs promoted the self-renewal of CD4−CD8−CD44−CD25+ cells, allowing for the continuous generation of later stage thymocytes. Forced FOXN1 expression in TECs rescued thymocyte developmental progression, but not cellularity, in LOS-FTOCs. Given that oxidative stress has been reported to accelerate the onset of age-associated thymic involution, we postulate that regulation of FOXN1 by oxygen and antioxidants may underpin this biological process.

The MHC class II transactivator modulates seeded alpha-synuclein pathology and dopaminergic neurodegeneration in an in vivo rat model of Parkinson's disease

BackgroundAbnormal folding, aggregation and spreading of alpha-synuclein (αsyn) is a mechanistic hypothesis for the progressive neuropathology in Parkinson's disease (PD). Spread of αsyn between cells is supported by clinical, neuropathological and experimental evidence. It has been proposed that a pro-inflammatory micro-environment in response to αsyn can promote its aggregation. We have previously shown that allelic differences in the major histocompatibility complex class two transactivator (Mhc2ta) gene, located in the VRA4 locus, alter MHCII expression levels, microglial activation and antigen presentation capacity in rats upon human αsyn over-expression. In addition, Mhc2ta regulated dopaminergic neurodegeneration and the extent of motor impairment. The purpose of this study was to determine whether Mhc2ta regulates αsyn aggregation, propagation and dopaminergic pathology in an αsyn pre-formed fibril (PFF)-seeded in vivo model of PD. MethodsThe DA and DA.VRA4 congenic rat strains share background genome but display differential microglial antigen presenting capacity due to different Mhc2ta alleles in the VRA4 locus. PFFs of human αsyn or BSA solution were injected unilaterally to the striatum of DA and DA.VRA4 rats two weeks after ipsilateral administration of recombinant adeno-associated virus (rAAV) vectors carrying human αsyn or GFP to the substantia nigra pars compacta. Behavioural assessment was performed at 2, 5 and 8 weeks while histological evaluation of αsyn pathology, inflammation and neurodegeneration as well as determination of serum cytokine profiles were performed at 8 weeks. ResultsrAAV-mediated expression of human αsyn in nigral dopaminergic neurons combined with striatal PFF administration induced enhanced αsyn pathology in DA.VRA4 compared to DA rats. Mhc2ta thus significantly regulated the seeding, propagation and toxicity of αsyn in vivo. This was reflected in terms of wider extent and anatomical distribution of αsyn inclusions, ranging from striatum to the forebrain, midbrain, hindbrain and cerebellum in DA.VRA4. Compared to DA rats, DA.VRA4 also displayed enhanced motor impairment and dopaminergic neurodegeneration as well as higher levels of the proinflammatory cytokines IL-2 and TNFα in serum. ConclusionsWe conclude that the key regulator of MHCII expression, Mhc2ta, modulates neuroinflammation, αsyn-seeded Lewy-like pathology, dopaminergic neurodegeneration and motor impairment. This makes Mhc2ta and microglial antigen presentation promising therapeutic targets for reducing the progressive neuropathology and clinical manifestations in PD.

Silencing EPB41 Gene Expression Leads to Cell Cycle Arrest, Migration Inhibition, and Upregulation of Cell Surface Antigen in DC2.4 Cells.

BackgroundProtein 4.1R (EPB41) is the main cytoskeleton component of the erythrocyte membrane and may be involved in cell migration and adhesion. Previous research discovered overexpression of 4.1R in the thymus of patients with myasthenia gravis (MG). The protein 4.1R on dendritic cells may play a pivotal role in MG pathogenesis.This research investigated the effects of small interfering RNA 4.1R-siRNA on cell migration, cell cycle, and surface antigen expression of DC2.4 mouse dendritic cells, thus providing a new direction for the study of MG pathogenesis.Material/MethodsThree 4.1R-specific siRNAs were designed, and the expression of 4.1R was detected by real-time PCR at the mRNA level and Western blot analysis at the protein level to select out the most efficient siRNAs. Changes in cell morphology were observed and cell migration ability was analyzed by Transwell assay. Cell cycle and surface antigen were both analyzed by flow cytometry.ResultsThe cell bodies of DC2.4 diminished, the synapses were increased, and protuberance became more obvious after being transfected with 4.1R-siRNA. After knockdown of 4.1R, cell migration ability decreased and the proportion of cells in S phase significantly increased (both P<0.05). The expression levels of MHCII, CD80, and CD86 were all increased in DC2.4 cells (all <0.05).ConclusionsSilencing the expression of 4.1R in dendritic cells resulted in inhibition of migration ability, cell cycle arrest, and increase in surface antigens, which suggest that 4.1R participates in MG autoimmunity.

Heterogeneity of porcine bone marrow-derived dendritic cells induced by GM-CSF

In vitro generation of dendritic cells (DCs) is advantageous for overcoming the low frequency of primary DCs and the difficulty of applying isolation techniques for studying DC immunobiology. The culture of bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used extensively to generate bone marrow-derived dendritic cells (BMDCs). Studies have reported the heterogeneity of cells grown in murine GM-CSF culture based on the levels of MHCII expression. Although porcine DCs are generated by this classical method, the exact characteristics of the BMDC population have not yet been defined. In this study, we discriminated GM-CSF-grown BMDCs from gnotobiotic miniature pigs according to several criteria including morphology, phenotype, gene expression pattern and function. We showed that porcine BMDCs were heterogeneous cells that differentially expressed MHCII. MHCIIhigh cells displayed more representative of DC-like morphology and phenotype, including costimulatory molecules, as well as they showed a superior T cell priming capacity as compared to MHCIIlow cell. Our data showed that the difference in MHCIIhigh and MHCIIlow cell populations involved distinct maturation states rather than the presence of different cell types. Overall, characterization of porcine BMDC cultures provides important information about this widely used cellular model.

Comparative analysis of mesenchymal stromal cells derived from rabbit bone marrow and Wharton’s jelly for adipose tissue engineering

ABSTRACT Purpose: To investigate the proliferative, adipogenic, and immunological properties of rabbit Mesenchymal stromal cells (MSCs) derived from bone marrow and umbilical cord Wharton’s jelly.Materials and Methods: We extracted rabbit MSCs from bone marrow (BMSCs) and umbilical cord Wharton’s jelly (WJ-MSCs). Both BMSCs and WJ-MSCs underwent adipogenic differentiation for 2 weeks, and then were transferred to non-inductive complete medium. Their adipogenic capacities were examined by histomorphometry and quantitative RT-PCR (qRT-PCR). The immunological markers were determined by mRNA expression of MHC-Ia, MHC-II, and RLA-DRA by qRT-PCR and protein expression of MHC-II by immunofluorescent staining. The proliferative capacities of adipogenic MSCs were also examined by counting kit-8 experiment and cell population doubling time.Results: We found that adipogenic differentiation increased the mRNA expression levels of adipogenic and immunological markers. The protein expression levels of MHC-II also increased after adipogenic differentiation in both groups. The adipogenic BMSCs showed higher mRNA expression levels of adipogenic and immunological markers. Removal of adipogenic agents after 2 weeks of adipo-differentiation inversely decreased the expression of immunological and adipogenic markers. The adipo-differentiation could decreased the proliferative capacities of both MSCs, but the adipogenic WJ-MSCs showed significantly higher proliferative capacities than BMSCs.Conclusions: Adipogenic differentiation increased the immunogenicity of both BMSCs and WJ-MSCs, and dedifferentiation inversely decreased their immunogenicity. Adipogenic WJ-MSCs showed significantly higher proliferative and immunoprivileged capacities than BMSCs, and the dedifferentiated BMSCs showed almost the same adipogenic capacity as WJ-MSCs. WJ-MSCs were more suitable than BMSCs for adipose tissue engineering.