Cellular immunology最新文献

Mast cells initiate allergic inflammatory immune responses and play a role in disease by releasing various inflammatory and immunomodulatory mediators. Several mast cell-lines and primary cultured cells have been used as mast cell models with inconsistent results among research groups. Bone marrow-derived mast cells (BMMC) cultured from mouse bone marrow progenitor cells are often used as a representative model of mucosal mast cell behaviour, however their reported phenotype is variable due to inconsistent culture protocols. RBL-2H3 is a rat basophilic histamine-releasing cell line that has some characteristics of both mast cells and basophils but is not a true representation of either cell type. The murine mast cell line MC/9 is an IL-3-dependent mucosal mast cell model but has limited mast cell characteristics. In this study, we have compared the response of BMMC (derived from C57BL/6 male or female mice), two sources of RBL-2H3 (purchased directly from ATCC and a lab curated culture), and MC/9 (ATCC) at several critical stages to some common stimuli (IgE/Ag, A23187) and analyzed mast cell morphology, expression level of common mast cell surface markers (CD117 and FcεRI), protease expression, and function (growth kinetics, viability, ROS production, degranulation, cytokine release and FcεRI signaling). The objective of this study was to provide insight into the effects of culture conditions, biological sex, and age of the cells on variability among reported phenotypes and, to determine optimal conditions for activation of these cells. Our data show that factors that are often overlooked such as source, age and biological sex of mast cells play an integral role in phenotypic outcomes and may account for the reported variability in their function.

Oral immunotherapies are being developed for various autoimmune diseases and allergies to suppress immune responses in an antigen-specific manner. Previous studies have shown that anti-drug antibody (inhibitor) formation in protein replacement therapy for the inherited bleeding disorder hemophilia can be prevented by repeated oral delivery of coagulation factor antigens bioencapsulated in transplastomic lettuce cells. Here, we find that this approach substantially reduces antibody development against factor VIII in hemophilia A mice treated with adeno-associated viral gene transfer. We propose that the concept of oral tolerance can be applied to prevent immune responses against therapeutic transgene products expressed in gene therapy.

SIRPα is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on monocytes, dendritic cells, and macrophages. Studies recently showed that SIRPα is essential for priming of CD4 + T cells by DCs and for development of Th17 cell-mediated autoimmune diseases. We have now further evaluated the importance of SIRPα and that of its ligand CD47 in primary immune thrombocytopenia (ITP). In this study, we show that there was a low expression state of SIRPα on the surface of monocytes. Treatment of cells culture from ITP patients with a mAb to SIRPα that blocks the binding of SIRPα to CD47 downregulated the ITP response. The abilities of monocytes from ITP patients to stimulate an allogenic MLR were reduced. The proliferation of, and production of IL-2, by CD4 + T cells from ITP patients were inhibited, the Treg cell numbers and the production of IL-10 pairs were upregulated, and the production of TGF-β not was inhibited, by a mAb to SIRPα. Moreover, a mAb to SIRPα, the expression of HLA-DR and CD86 were markedly inhibited and the expression of CD80 was slightly upregulated, on the surface of CD14 + monocytes from ITP patients as compared with healthy subjects. However, blockade of SIRPα increased the secretion of TLR-dependent cytokines TNF-α, IL-6 and IL-1β by PBMCs, which may be considered as a reserve in response to danger signals. These results suggest that SIRPα on monocytes is essential for the priming of naive T cells and the development of ITP. Therefore, SIRPα is a potential therapeutic target for ITP and other autoimmune diseases.

Ulcerative colitis is a type of inflammatory bowel disease which in long run can lead to colorectal cancer (CRC). Chronic inflammation can be a key factor for the occurrence of CRC thus mitigating an inflammation can be a preventive strategy for the occurrence of CRC. In this study we have explored the anti-inflammatory potential of phloretin, in in vitro gut inflammation model, developed by co-culture of Caco2 (intestinal epithelial) cells and RAW264.7 macrophages (immune cells). Phloretin is a dihydrochalcone present in apple, pear and strawberries. An anti-inflammatory effect of phloretin in reducing LPS induced inflammation and maintenance of transepithelial electric resistance (TEER) in Caco2 cells was examined. Paracellular permeability assay was performed using Lucifer yellow dye to evaluate the effect of phloretin in inhibiting gut leakiness caused by inflammatory mediators secreted by activated macrophages. Phloretin attenuated LPS induced nitric oxide levels, oxidative stress, depolarization of mitochondrial membrane potential in Caco2 cells as evidenced by reduction in reactive oxygen species (ROS), and enhancement of MMP, and decrease in inflammatory cytokines IL8, TNFα, IL1β and IL6. It exhibited anti-inflammatory activity by inhibiting the expression of NFκB, iNOS and Cox2. Phloretin maintained the epithelial integrity by regulating the expression of tight junction proteins ZO1, occludin, Claudin1 and JAM. Phloretin reduced LPS induced levels of Cox2 along with the reduction in Src expression which further regulated an expression of tight junction protein occludin. Phloretin in combination to sodium pyruvate exhibited potential anti-inflammatory activity via targeting NFkB signaling. Our findings paved a way to position phloretin as nutraceutical in preventing the occurrence of colitis and culmination of disease into colitis associated colorectal cancer.

Chimeric antigen receptor (CAR)-T cells encounter many issues when treating solid tumors, including tumor antigen heterogeneity and immunosuppression. United targeting of two tumor-associated antigens (TAAs) and blocking of PD-1 may solve this problem and enhance the function of CAR-T. Mucin 1 (MUC1) and prostate stem cell antigen (PSCA) are overexpressed in non-small cell lung cancer (NSCLC). Here, we constructed a bivalent tandem CAR-T (Tan CAR-T), which can simultaneously target MUC1 and PSCA and evaluated its effects of inhibiting non-small cell lung cancer (NSCLC) in vitro and in vivo. Results indicated that the tumor killing effect of these Tan CAR-T was more effective than that of single-target CAR-T, its antitumor efficacy could be further strengthened by anti-PD-1 antibody. Our study reported a previously unstudied therapeutic effect of a Tan CAR-T in NSCLC, providing a preclinical rationale for anti-PD-1 antibody combined with Tan CAR-T targeting MUC1 and PSCA in the treatment of NSCLC.

CD5, a T-cell receptor (TCR) negative regulator, is reduced on the surface of CD8+ lymphocytes in the tumor microenvironment (TME). Reduced surface CD5 expression (sCD5) occurs due to the preferential transcription of HERV-E derived exon E1B, i.e., a non-conventional form of the cd5 gene instead of its conventional exon E1A. A tumor employs several mechanisms to evade anti-tumor response, and hypoxia is one such mechanism that prevails in the TME and modulates the infiltrated T lymphocytes. We identified hypoxia response elements (HREs) upstream of E1B. We showed binding of HIF-1α onto these HREs and increased E1B mRNA expression in hypoxic T cells. This results in decreased sCD5 expression and increased cytoplasmic accumulation in T cells. We also validated our study in a solid tumor, i.e., colorectal cancer (CRC) patient samples. This hypoxia-driven mechanism reduces the surface CD5 expression on infiltrated T-cells in solid tumors.

Rheumatoid arthritis (RA), multiple sclerosis (MS), type 1 diabetes (T1D), and celiac disease (CD), are strongly associated with susceptible HLA class II haplotypes. The peptide-binding pockets of these molecules are polymorphic, thus each HLA class II protein presents a distinct set of peptides to CD4⁺ T cells. Peptide diversity is increased through post-translational modifications, generating non-templated sequences that enhance HLA binding and/or T cell recognition. The high-risk HLA-DR alleles that confer susceptibility to RA are notable for their ability to accommodate citrulline, promoting responses to citrullinated self-antigens. Likewise, HLA-DQ alleles associated with T1D and CD favor the binding of deamidated peptides. In this review, we discuss structural features that promote modified self-epitope presentation, provide evidence supporting the relevance of T cell recognition of such antigens in disease processes, and make a case that interrupting the pathways that generate such epitopes and reprogramming neoepitope-specific T cells are key strategies for effective therapeutic intervention.

Although clinically effective, the actions of IFNα, either produced endogenously or by therapeutic delivery, remain poorly understood. Emblematic of this research gap is the disparate array of notable side effects that occur in susceptible individuals, such as neuropsychiatric consequences, autoimmune phenomena, and infectious complications. We hypothesised that these complications are driven at least in part by dysregulated cellular metabolism. Male Wistar rats were treated with either 170,000 IU/kg human recombinant IFNα-2a or BSA/saline (0.9% NaCl) three times per week for three weeks. Bone marrow (BM) immune cells were isolated from the excised femurs for glycolytic rate and mitochondrial function assessment using Agilent Seahorse Technology. Frequencies of immune cell populations were assessed by flow cytometry to determine whether leukopoietic changes had occurred in both blood and BM. Plasma levels of lactate and succinate were also determined. BMDMs were metabolically assessed as above, as well as their metabolic response to an antigenic stimulus (iH37Rv). We observed that BM immune cells from IFN-treated rats exhibit a hypermetabolic state (increased basal OCR/GlycoPER) with decreased mitochondrial metabolic respiration and increased non-mitochondrial OCR. Flow cytometry results indicated an increase in immature granulocytes (RP1- SSChi CD45lo) only in the blood, together with increased succinate levels in the plasma. BMDMs from IFN-treated rats retained the hypermetabolic phenotype after differentiation and failed to induce a step-up in glycolysis and mitochondrial respiration after bacterial stimulation. This work provides the first evidence of the effects of IFNα treatment in inducing hypermetabolic immune features that are associated with markers of inflammation, leukopoiesis, and defective responses to bacterial stimulation.

Elimination of apoptotic neutrophils by macrophages is as a major step for the resolution of inflammation. However, the fate and the cellular functionality of neutrophils aged in the absence of macrophages are not well documented. Herein, freshly isolated human neutrophils were aged for several days in vitro and then stimulated with agonists for determining their cell responsiveness. In vitro-aged neutrophils were still able to generate reactive oxygen species after 48 h, exert phagocytosis after 72 h, and increase their adhesion onto a cell substratum after 48 h. These data demonstrate that a portion of neutrophils cultivated for several days in vitro are still able to exert biological functions. This opens the possibility that, during inflammation, neutrophils may still respond to agonists, a condition that is likely to occur in vivo when they are not efficiently eliminated by efferocytosis.

The CD300 glycoproteins are a family of related leucocyte surface molecules that regulate the immune response via their paired triggering and inhibitory receptors. Here we studied CD300f, an apoptotic cell receptor, and how it modulates the function of human monocytes and macrophages. We showed that CD300f signalling by crosslinking with anti-CD300f mAb (DCR-2) suppressed monocytes causing upregulation of the inhibitory molecule, CD274 (PD-L1) and their inhibition of T cell proliferation. Furthermore, CD300f signalling drove macrophages preferentially towards M2-type with upregulation of CD274, which was further enhanced by IL-4. CD300f signalling activates the PI3K/Akt pathway in monocytes. Inhibition of PI3K/Akt signalling resulting from CD300f crosslinking leads to downregulation of CD274 expression on monocytes. These findings highlight the potential use of CD300f blockade in cancer immune therapy to target immune suppressive macrophages in the tumour microenvironment, a known resistance mechanism to PD-1/PD-L1 checkpoint inhibitors.