Development of a Protocol for Obtaining a Homologous Cell Product of Animal Origin Intended for Preclinical Studies of Antitumor Vaccine CaTeVac.

When developing a program of preclinical studies of human cell-based drugs intended for adoptive immunotherapy of cancer patients, the biological effect should be substantiated by data describing their immunological action. Administration and study of human autologous dendritic cell vaccine to immunocompetent animals are not adequate in terms of immunological compatibility. It is possible to use immunocompromised, knockout, or transgenic animals or to obtain a homologous cellular product, namely, a preparation based on animal cells using a technology similar to obtaining the original preparation for clinical practice in humans. Within the framework of this study, we have developed a protocol for obtaining a homologous cell product based on animal dendritic cells (mice, rats) according to a similar technology for obtaining human vaccine dendritic cells, and demonstrated the comparability of morphological characteristics and expression of differentiation antigens of dendritic cells (CD11c, CD80, CD86, and CD83) of animals (mice) and humans.

PIKfyve, expressed by CD11c-positive cells, controls tumor immunity.

Cancer treatment continues to shift from utilizing traditional therapies to targeted ones, such as protein kinase inhibitors and immunotherapy. Mobilizing dendritic cells (DC) and other myeloid cells with antigen presenting and cancer cell killing capacities is an attractive but not fully exploited approach. Here, we show that PIKFYVE is a shared gene target of clinically relevant protein kinase inhibitors and high expression of this gene in DCs is associated with poor patient response to immune checkpoint blockade (ICB) therapy. Genetic and pharmacological studies demonstrate that PIKfyve ablation enhances the function of CD11c+ cells (predominantly dendritic cells) via selectively altering the non-canonical NF-κB pathway. Both loss of Pikfyve in CD11c+ cells and treatment with apilimod, a potent and specific PIKfyve inhibitor, restrained tumor growth, enhanced DC-dependent T cell immunity, and potentiated ICB efficacy in tumor-bearing mouse models. Furthermore, the combination of a vaccine adjuvant and apilimod reduced tumor progression in vivo. Thus, PIKfyve negatively regulates the function of CD11c+ cells, and PIKfyve inhibition has promise for cancer immunotherapy and vaccine treatment strategies.

Regulation of B-cell function and expression of CD11c, T-bet, and FcRL5 in response to different activation signals.

CD11c, FcRL5, or T-bet are commonly expressed by B cells expanding during inflammation, where they can make up >30% of mature B cells. However, the association between the proteins and differentiation and function in the host response remains largely unclear. We have assessed the co-expression of CD11c, T-bet, and FcRL5 in an in vitro B-cell culture system to determine how stimulation via the BCR, toll-like receptor 9 (TLR9), and different cytokines influence CD11c, T-bet, and FcRL5 expression. We observed different expression dynamics for all markers, but a largely overlapping regulation of CD11c and FcRL5 in response to BCR and TLR9 activation, while T-bet was strongly dependent on IFN-γ signaling. Investigating plasma cell differentiation and APC functions, there was no association between marker expression and antibody secretion or T-cell help. Rather the functions were associated with TLR9-signalling and B-cell-derived IL-6 production, respectively. These results suggest that the expression of CD11c, FcRL5, and T-bet and plasma cell differentiation and improved APC functions occur in parallel and are regulated by similar activation signals, but they are not interdependent.

A fluorescent reporter model for the visualization and characterization of TDC.

TDC are hematopoietic cells that combine dendritic cell (DC) and conventional T-cell markers and functional properties. They were identified in secondary lymphoid organs (SLOs) of naïve mice as cells expressing CD11c, major histocompatibility molecules (MHC)-II, and the T-cell receptor (TCR). Despite thorough characterization, a physiological role for TDC remains to be determined. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells, including T cells, upon activation. Here, we took advantage of Zbtb46-GFP reporter mice to explore the frequency and localization of TDC in different tissues at steady state and upon viral infection. RNA sequencing analysis confirmed that TDC sorted from Zbtb46-GFP mice have a gene signature that is distinct from conventional T cells and DC. In addition, this reporter model allowed for identification of TDC in situ not only in SLOs but also in the liver and lung of naïve mice. Interestingly, we found that TDC numbers in the SLOs increased upon viral infection, suggesting that TDC might play a role during viral infections. In conclusion, we propose a visualization strategy that might shed light on the physiological role of TDC in several pathological contexts, including infection and cancer.

CD11c+ and IRF8+ cell densities in rectal cancer biopsies predict outcomes of neoadjuvant chemoradiotherapy.

Approximately 20% of locally advanced rectal cancer (LARC) patients treated preoperatively with chemoradiotherapy (CRT) achieve pathologically confirmed complete regression. However, there are no clinically implemented biomarkers measurable in biopsies that are predictive of tumor regression. Here, we conducted multiplexed immunophenotyping of rectal cancer diagnostic biopsies from 16 LARC patients treated preoperatively with CRT. We identified that patients with greater tumor regression had higher tumor infiltration of pan-T cells and IRF8+HLA-DR+ cells prior to CRT. High IRF8+HLA-DR+ cell density was further associated with prolonged disease-specific survival with 83% survival at 5 y compared to 28% in patients with low infiltration. Contrastingly, low CD11c+ myeloid cell infiltration prior to CRT was a putative biomarker associated with longer 3- and 5-y disease-free survival. The results demonstrate the potential use of rectal cancer diagnostic biopsies to measure IRF8+ HLA-DR+ cells as predictors of CRT-induced tumor regression and CD11c+ myeloid cells as predictors of LARC patient survival.

CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours.

Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4-6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7-10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.

Integrated Bioinformatics Analysis to Screen Hub Gene Signatures for Fetal Growth Restriction.

Background: Fetal growth restriction (FGR) is the impairment of the biological growth potential of the fetus and often leads to adverse pregnancy outcomes. The molecular mechanisms for the development of FGR, however, are still unclear. The purpose of this study is to identify critical genes associated with FGR through an integrated bioinformatics approach and explore the potential pathogenesis of FGR. Methods: We downloaded FGR-related gene microarray data, used weighted gene co-expression network analysis (WGCNA), differentially expressed genes (DEGs), and protein-protein interaction (PPI) networks to screen hub genes. The GSE24129 gene set was used for validation of critical gene expression levels and diagnostic capabilities. Results: A weighted gene co-expression network was constructed, and 5000 genes were divided into 12 modules. Of these modules, the blue module showed the closest relationship with FGR. Taking the intersection of the DEGs and genes in the blue module as pivotal genes, 277 genes were identified, and 20 crucial genes were screened from the PPI network. The GSE24129 gene set verified the expression of 20 genes, and CXCL9, CXCR3, and ITGAX genes were identified as actual pivotal genes. The expression levels of CXCL9, CXCR3, and ITGAX were increased in both the training and validation sets, and ROC curve validation revealed that these three pivotal genes had a significant diagnostic ability for FGR. Single-gene GSEA results showed that all three core genes activated "hematopoietic cell lineage" and "cell adhesion molecules" and inhibited the "cGMP-PKG signaling pathway" in the development of FGR. CXCL9, CXCR3, and ITGAX may therefore be closely associated with the development of FGR and may serve as potential biomarkers for the diagnosis and treatment of FGR.

The prognostic signature based on glycolysis-immune related genes for acute myeloid leukemia patients.

Acute myeloid leukemia (AML) is widely considered an immunoresponsive malignancy. However, potential association between glycolysis-immune related genes and AML patients' prognosis has been seldom studied. AML-related data was downloaded from TCGA and GEO databases. We grouped patients according to Glycolysis status, Immune Score and combination analysis, basing on which overlapped differentially expressed genes (DEGs) were identified. The Risk Score model was then established. The results showed that totally 142 overlapped genes were probably correlated with glycolysis-immunity in AML patients, among which 6 optimal genes were screened to construct Risk Score. High Risk Score was an independent poor prognostic factor for AML. In conclusion, we established a relatively reliable prognostic signature of AML based on glycolysis-immunity related genes, including METTL7B, HTR7, ITGAX, TNNI2, SIX3 and PURG.

CD11c+ macrophages are proangiogenic and necessary for experimental choroidal neovascularization.

Patients with neovascular AMD (nAMD) suffer vision loss from destructive angiogenesis, termed choroidal neovascularization (CNV). Macrophages are found in CNV lesions from patients with nAMD. Additionally, Ccr2-/- mice, which lack classical monocyte-derived macrophages, show reduced CNV size. However, macrophages are highly diverse cells that can perform multiple functions. We performed single-cell RNA-Seq on immune cells from WT and Ccr2-/- eyes to uncover macrophage heterogeneity during the laser-induced CNV mouse model of nAMD. We identified 12 macrophage clusters, including Spp1+ macrophages. Spp1+ macrophages were enriched from WT lasered eyes and expressed a proangiogenic transcriptome via multiple pathways, including vascular endothelial growth factor signaling, endothelial cell sprouting, cytokine signaling, and fibrosis. Additionally, Spp1+ macrophages expressed the marker CD11c, and CD11c+ macrophages were increased by laser and present in CNV lesions. Finally, CD11c+ macrophage depletion reduced CNV size by 40%. These findings broaden our understanding of ocular macrophage heterogeneity and implicate CD11c+ macrophages as potential therapeutic targets for treatment-resistant patients with nAMD.

SERPINB8 and furin regulate ITGAX expression and affect the proliferation and invasion of melanoma cells.

In the past 10 years, the systemic treatment of advanced melanoma has undergone tremendous changes through the development of targeted therapy. However, there is still a long way to go. This study aims to characterize the function and interaction of ITGAX, SERPINB8 and furin in BRAF V600E mutant melanoma. Differentially expressed genes related to BRAF V600E mutation and BRAFi treatment were obtained by analysing GSE141484 and GSE22838. two kinds of BRAFi (Vemurafenib, 10 µM; Dabrafenib, 1 µM) were used to treat A375 and 1205Lu cells, respectively. The expression of ITGAX, SERPINB8 and Furin in A375 and 1205Lu cells was down-regulated by specific siRNAs, and cell proliferation, clone formation and invasion were detected by CCK-8, colony formation and transwell assays. The physical binding of furin and SERPINB8 was detected by immunoprecipitation. BRAFi treatment down-regulated the ITGAX and SERPINB8 expression and did not change furin expression. Knockdown of ITGAX and SERPINB8 both inhibited the proliferation and invasion of A375 and 1205Lu cells. Knocking down SERPINB8 down-regulated the expression of ITGAX. Furin knockdown and inhibitors all up-regulated the protein level of ITGAX. SERPINB8 can physically bind to furin. In summary, SERPINB8 and furin regulate the expression of ITGAX in melanoma cells, and ITGAX significantly promotes the proliferation and invasion of melanoma cells.

The epidermal immune microenvironment plays a dominant role in psoriasis development, as revealed by mass cytometry.

Psoriasis is a common chronic inflammatory skin disease. The diversity and heterogeneity of immune cells in human skin have been studied in recent years, but the spatial distribution of immune cells at the single-cell level in the human psoriatic epidermis and dermis remains unclear. In this study, we mapped psoriatic skin immune cells from paired lesional, perilesional, and nonlesional skin samples using mass cytometry. Phenotypic dendritic cells (DCs) were found in the psoriatic epidermis and dermis. Psoriatic dermal CD1c+CD11b+ cDC2s migrated to the epidermis in the perilesional skin during the preinitiation stage. CD1c+CD11b+ cDC2s rapidly replaced EpCAM+CD11clow LC cells and initiated inflammation. Simultaneously, CD207+CD11chi LC and CD5+ T cells accumulated in the psoriatic epidermis and orchestrated epidermal inflammation in psoriasis. The immune cell pool in the psoriatic dermis primarily included APCs and T cells. However, unlike that in the dermis, the epidermal immune environment was more significant and coincided with the inflammation occurring during psoriasis.The epidermal immune microenvironment plays a dominant role in psoriasis. Langerhans cells, epidermis-resident memory T cells and macrophages together contribute to healthy epidermal immune homeostasis. However, psoriatic CD1c+CD11b+ epidermal cDC2s are positioned in the perilesional area, replacing EpCAM+CD11clow LCs rapidly and initiating inflammation. Epidermal CD141+ cDC1s, CD1c+ cDC2s, CD14+ moDCs, and BDCA2+ pDCs orchestrate psoriatic inflammation. Meanwhile, CD11chi LCs and CD5+ T cells accumulate in the psoriatic epidermis.

Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis.

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C- cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c-/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.

Post-endotoxin exposure-induced lung inflammation and resolution consequences beneficially impacted by lung-delivered IL-10 therapy.

Although lung diseases typically result from long-term exposures, even a robust, one-time exposure can result in long-lasting consequences. Endotoxin is a ubiquitous environmental/occupational inflammatory agent often used to model airway inflammation. Using a murine model, the return to lung homeostasis following high dose inhalant lipopolysaccharide (LPS, 10-100 µg) exposure were delineated over 2 weeks. LPS-induced rapid weight loss, release of proinflammatory mediators, and inflammatory cell influx with prolonged persistence of activated macrophages CD11c+CD11b+ and recruited/transitioning CD11cintCD11b+ monocyte-macrophages out to 2 weeks. Next, lung-delivered recombinant (r) interleukin (IL)-10 was intratracheally administered for 3 doses initiated 5 h following LPS (10 µg) exposure for 2 days. IL-10 therapy reduced LPS-induced weight loss and increased blood glucose levels. Whereas there was no difference in LPS-induced bronchoalveolar lavage airway fluid cellular influx, total lung cell infiltrates were reduced (37%) with rIL-10 treatment. Post-LPS exposure treatment with rIL-10 strikingly reduced lavage fluid and lung homogenate levels of tumor necrosis factor-α (88% and 93% reduction, respectively), IL-6 (98% and 94% reduction), CXCL1 (66% and 75% reduction), and CXCL2 (47% and 67% reduction). LPS-induced recruited monocyte-macrophages (CD11cintCD11b+) were reduced (68%) with rIL-10. Correspondingly, LPS-induced lung tissue CCR2+ inflammatory monocyte-macrophage were reduced with rIL-10. There were also reductions in LPS-induced lung neutrophils, lymphocyte subpopulations, collagen content, and vimentin expression. These findings support the importance of studying resolution processes for the development of treatment after unintended environmental/occupational biohazard exposures. Short-term, lung-delivered rIL-10 favorably hastened inflammatory recovery processes following acute, high dose inhalant LPS exposure.

Protocol to isolate and analyze mouse bone marrow derived dendritic cells (BMDC).

Different types of immune cells are involved in atherogenesis and may act atheroprotective or atheroprogressive. Here, we describe an in vitro approach to analyze CD11c+ cells and CD11c+-derived ApoE in atherosclerosis. The major steps include harvesting mouse bone marrow, plating cells in culture dishes, treating them with differentiation factors, and collecting cells after removal of undesirable populations. This protocol can be adapted for CD11c+ cells in different contexts, thus, serving as models for different diseases and to analyze cell-specific molecules. For complete details on the use and execution of this protocol, please refer to Sauter et al. (2021).

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (>10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells (DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

The Effect of Age and Cell Culture Parameters on the Quantity and Function of Bone Marrow-derived Dendritic Cells.

Dendritic cells (DCs) are a group of bone marrow-derived cells that play a crucial role in innate and acquired immune responses. Bone marrow-derived dendritic cells (BMDC) are used in many studies, so the efficiency and purity of the differentiated cells are essential. This study aimed to investigate the effect of several parameters, including the age of mice, cell culture medium, and swirling of the culture plate, to increase the efficiency of the induced cells, considering the standard protocols. Bone marrow-derived dendritic cells were induced from both juvenile and adult mice bone marrow cells. Then, the purity of CD11c+ cells was compared between juvenile mice BMDCs and adult mice BMDCs. Cells were cultured in an enriched and non-enriched medium, and some wells were swirled when changing the medium on the 3rd day. Then the effect of enriched medium and swirling before medium replacement were evaluated based on the expression of the CD11c marker. The efficiency of DCs differentiation (CD11c+ cells) was higher when juvenile mouse bone marrow precursors were used compared to adult mice; using the enriched media with supplements and swirling the well before media replacement significantly affected the purity of immature CD11c+ cells. Due to our results, using juvenile mice, an enriched culture medium, and physical removal of granulocyte cells could significantly improve the purity and efficiency of CD11c+ cells. Therefore, considering these three items in the production protocol of these cells can probably reduce the use of lymphocyte-removing antibodies and purification methods.

Conditional knockout of oestrogen receptor alpha in CD11c+ cells impacts female survival and inflammatory cytokine profile in murine lupus.

Oestrogen and oestrogen receptor alpha (ERα) have been implicated in systemic lupus erythematosus pathogenesis. ERα signalling influences dendritic cell (DC) development and function, as well as inflammation and downstream immune responses. We previously reported that ERα modulates multiple Toll-like receptor-stimulated pathways in both conventional and plasmacytoid DCs in lupus-prone mice. For example, CD11chi MHCII+ cell numbers are reduced in mice with global ERα deficiency or when expressing a short variant of ERα. Herein, RNA-seq analysis of CD11chi cells from bone marrow of NZM2410 mice expressing WT ERα versus ERα short versus ERα null revealed differentially expressed complement genes, interferon-related genes and cytokine signalling (e.g., IL-17 and Th17 pathways). To better understand the role of ERα in CD11c+ cells, lupus prone NZM2410 mice with selective deletion of the Esr1 gene in CD11c+ cells were generated. Phenotype and survival of these mice were similar with the exception of Cre positive (CrePos) female mice. CrePos females, but not males, all died unexpectedly prior to 35 weeks. DC subsets were not significantly different between groups. Since ERα is necessary for robust development of DCs, this result suggests that DC fate was determined prior to CD11c expression and subsequent ERα deletion (i.e., proximally in DC ontogeny). Overall, findings point to a clear functional role for ERα in regulating cytokine signalling and inflammation, suggesting that further study into ERα-mediated regulatory mechanisms in DCs and other immune cell types is warranted.

CD11c+ T-bet+ CD21hi B Cells Are Negatively Associated With Renal Impairment in Systemic Lupus Erythematosus and Act as a Marker for Nephritis Remission.

Lupus nephritis (LN) is one of the most common manifestations of systemic lupus erythematosus (SLE), characterized by abnormal B cell activation and differentiation to memory or plasma effector cells. However, the role of these cells in the pathogenesis of LN is not fully understood, as well as the effect of induction therapy on B cell subsets, possibly associated with this manifestation, like aged-associated B cells (ABCs). Consequently, we analyzed the molecules defining the ABCs subpopulation (CD11c, T-bet, and CD21) through flow cytometry of blood samples from patients with lupus presenting or not LN, following up a small sub-cohort after six months of induction therapy. The frequency of ABCs resulted higher in LN patients compared to healthy subjects. Unexpectedly, we identified a robust reduction of a CD21hi subset that was almost specific to LN patients. Moreover, several clinical and laboratory lupus features showed strong and significant correlations with this undefined B cell subpopulation. Finally, it was observed that the induction therapy affected not only the frequencies of ABCs and CD21hi subsets but also the phenotype of the CD21hi subset that expressed a higher density of CXCR5. Collectively, our results suggest that ABCs, and more importantly the CD21hi subset, may work to assess therapeutic response since the reduced frequency of CD21hi cells could be associated with the onset of LN.