Upregulation Of HLA-DR Expression Research Articles

Mitochondrial damage-associated molecular patterns stimulate reactive oxygen species production in human microglia

Microglia are the resident innate immune cells of the central nervous system and exert functions of host defense and maintenance of normal tissue homeostasis, along with support of neuronal processes in the healthy brain. Chronic and dysregulated microglial cell activation has increasingly been linked to the status of neuroinflammation underlying many neurodegenerative diseases, including multiple sclerosis (MS). However, the stimulus (or stimuli) and mechanisms by which microglial activation is initiated and maintained MS are still debated. The purpose of our research was to investigate whether the endogenous mitochondrial (mt)-derived damage-associated molecular patterns (MTDs) mtDNA, N-formyl peptides and cardiolipin (CL) contribute to these phenomena. We characterized the effects of the abovementioned MTDs on microglia activation in vitro (i.e. using HMC3 cells) by evaluating the expression of gene coding for proteins involved in their binding and coupled to downstream signaling pathways, the up-regulation of markers of activation on the cell surface and the production of pro-inflammatory cytokines and reactive oxygen species. At the transcriptional level, significant variations in the mRNA relative expression of five of eleven selected genes were observed in response to stimulation. No changes in activation of antigenic profile or functional properties of HMC3 cells were observed; there was no up-regulation of HLA-DR expression or increased secretion of tumor necrosis factor-α and interleukin-6. However, after stimulation with mtDNA and CL, an increase in cellular oxidative stress, but not in the mt ROS O2−, compared to control cells, were observed. There were no effects on cell viability. Overall, our data suggest that MTDs could cause a failure in microglial activation toward a pro-inflammatory phenotype, possibly triggering an endogenous regulatory mechanism for the resolution of neuroinflammation. This could open a door for the development of drugs selectively targeting microglia and modulating its functionality to treat MS and/or other neurodegenerative conditions in which MTDs have a pathogenic relevance.

Xenotransplantation of interferon-gamma-pretreated clumps of a human mesenchymal stem cell/extracellular matrix complex induces mouse calvarial bone regeneration

BackgroundThree-dimensional cultured clumps of a mesenchymal stem cell (MSC)/extracellular matrix (ECM) complex (C-MSC) consists of cells and self-produced ECM. C-MSC can regulate the cellular function in vitro and induce successful bone regeneration using ECM as a cell scaffold. Potentiating the immunomodulatory capacity of C-MSCs, which can ameliorate the allo-specific immune response, may be helpful in developing beneficial “off-the-shelf” cell therapy for tissue regeneration. It is well reported that interferon (IFN)-γ stimulates the immunosuppressive properties of MSC via upregulation of the immunomodulatory enzyme IDO. Therefore, the aim of this study was to investigate the effect of IFN-γ on the immunomodulatory capacity of C-MSC in vitro and to test the bone regenerative activity of C-MSC or IFN-γ-pretreated C-MSC (C-MSCγ) xenografts in a mice calvarial defect model.MethodsHuman bone marrow-derived MSCs were seeded at a density of 2.0 × 105 cells/well into 24-well plates and cultured with growth medium supplemented with 50 μg/mL L-ascorbic acid for 4 days. To obtain C-MSC, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The cellular sheet was rolled to make a round clump of cells. C-MSC was stimulated with IFN-γ and IDO expression, immunosuppressive capacity, and immunophenotype were evaluated in vitro. Moreover, C-MSC or C-MSCγ was xenotransplanted into immunocompetent or immunodeficient mice calvarial defect models without artificial scaffold, respectively.ResultsIFN-γ stimulated IDO expression in C-MSC. C-MSCγ, but not C-MSC, attenuated CD3/CD28-induced T cell proliferation and its suppressive effect was reversed by an IDO inhibitor. C-MSCγ showed upregulation of HLA-DR expression, but its co-stimulatory molecule, CD86, was not detected. Xenotransplantation of C-MSCγ into immunocompetent mice calvarial defect induced bone regeneration, whereas C-MSC xenograft failed and induced T cell infiltration in the grafted area. On the other hand, both C-MSC and C-MSCγ xenotransplantation into immunodeficient mice caused bone regeneration.ConclusionsXenotransplantation of C-MSCγ, which exerts immunomodulatory properties via the upregulation of IDO activity in vitro, may attenuate xenoreactive host immune response, and thereby induce bone regeneration in mice. Accordingly, C-MSCγ may constitute a promising novel allograft cell therapy for bone regeneration.

Immune correlates of varlilumab (CDX-1127) treated cancer patients are consistent with CD27 costimulatory activity

Varlilumab is a human IgG1 agonist anti-CD27 antibody designed to activate T cells through CD27 costimulation. Preclinical studies have shown that Varlilumab efficiently activates human T cells when combined with T cell receptor stimulation, enhances antigen specific CD8 T cell responses in human CD27 transgenic mice when combined with vaccination, and mediates anti-tumor activity in these mice when challenged with syngeneic tumors. A multi-dose, dose-escalation/expansion trial of Varlilumab in patients with advanced solid tumors (n = 56) or lymphoma (n = 24) has demonstrated a good safety profile with no MTD reached through the 10 mg/kg dose level. Evidence of clinical activity includes a complete response in a treatment-refractory Stage IV Hodgkin’s patient (remains in remission at 12.9 months); a renal cell carcinoma (RCC) patient with a partial response (ongoing at 3.8 months); an additional RCC patient with extended stable disease (ongoing at 22.4+ months); and 12 additional patients with stable disease (range: 2.7+ to 14 months). We performed extensive correlative immune monitoring from serum and peripheral blood cells, particularly in melanoma and RCC patients. Differential gene expression analysis was assessed preand post-treatment by hybridizing RNA isolated from whole blood PAXgene RNA tubes onto Illumina HT-12 microarrays. Transcriptomic analysis showed significant impact of Varlilumab on immunological pathways, including T cell receptor signaling that was observed at early time points (day 2 and day 8 post treatment). Interestingly, these changes were most significant at low dose levels (0.1 and 0.3 mg/kg), however, a larger sample size would be required to confirm these results. Varlilumab administration was associated with enhanced T cell activity by evaluation of activation markers and functional analysis using IFN-g Elispot. In particular, evaluation of response to peptides derived from melanoma antigens in selected patients showed evidence that Varlilumab promoted CD8 T cell function against melanoma antigens. Specifically, expanded responses were detected to epitopes from gp100 and MART-1, while de novo responses to epitopes MAGE-A1, NY-ESO-1 and gp100 were evident. These responses were confirmed by MHC-multimer staining. These data, and our previous analyses demonstrating a transient increase in pro-inflammatory cytokines (IP-10, IL-6, MCP-1), up-regulation of HLA-DR expression on T cells, increase in NK cell numbers, and decrease in regulatory T cells in response to Varlilumab treatment, show a pattern consistent with CD27 costimulation. These results, together with our preclinical data, provide the rationale for initiating clinical studies of Varlilumab in combination with therapies such as vaccines, checkpoint inhibitors and targeted therapies.

Reactive microglia in aging and dementia: an immunohistochemical study of postmortem human brain tissue.

Significantly increased up-regulation of HLA DR (major histocompatibility complex class II antigen) was seen using immunohistochemistry in postmortem brain tissue from demented patients with Alzheimer's disease (AD) (73 cases, 61 females/12 males, mean age 84 +/- 9 years) compared to controls (22 cases, 10 females/12 males, mean age 78 +/- 9 years). The counts of HLA DR-expressing activated microglia were significantly higher in female AD patients compared to males, significantly higher in AD patients with the age at death greater than 75 years compared to those dying younger and higher, although not statistically significantly, in AD patients with the apolipoprotein E (ApoE) epsilon4 allele compared to those patients not carrying this allele. In contrast to the situation in AD patients, in the control cases the HLA DR expression was higher in males compared to females. Furthermore, in the very old non-demented patients (age at death > 80 years), a decrease in the up-regulation of HLA DR expression was observed. A significant correlation between activated microglia and neurofibrillary tangles was seen in female AD patients compared to males, in AD cases without ApoE epsilon4 allele compared to those with this allele, in sporadic cases compared to familial and in cases with senile rather than presenile onset of the disease. Our results indicate that there is an age- and/or sex-related variability in up-regulation of HLA DR expression of microglia and that the linkage between this up-regulation and AD lesions is significantly influenced by the ApoE epsilon4 allele, gender of subjects, age at onset and familiality of the disease.

Increased Intestinal Permeability and Altered Mucosal Immunity in Cholestatic Jaundice

To examine the effects of cholestatic jaundice on gut barrier function. Gut barrier failure occurs in animal models of jaundice. In humans, the presence of endotoxemia indirectly implicates failure of this host defense, but this has not previously been investigated in jaundiced patients. Twenty-seven patients with extrahepatic obstructive jaundice and 27 nonicteric subjects were studied. Intestinal permeability was measured using the lactulose-mannitol test. Small intestinal morphology and the presence of mucosal immunologic activation were examined in endoscopic biopsies of the second part of the duodenum. Systemic antiendotoxin core IgG antibodies and serum interleukin-6 and C-reactive protein were also quantified. Intestinal permeability was remeasured in 9 patients 5 weeks after internal biliary drainage. The median lactulose-mannitol ratio was significantly increased in the jaundiced patients. This was accompanied by upregulation of HLA-DR expression on enterocytes and gut-associated lymphoid tissue, suggesting immune activation. A significant increase in the acute phase response and circulating antiendotoxin core antibodies was also observed in the jaundiced patients. After internal biliary drainage, intestinal permeability returned toward normal levels. A reversible impairment in gut barrier function occurs in patients with cholestatic jaundice. Increased intestinal permeability is associated with local immune cell and enterocyte activation. In view of the role of gut defenses in the modern paradigm of sepsis, these data may directly identify an important underlying mechanism contributing to the high risk of sepsis in jaundiced patients.

The Inhibition of Interferon-γ-Induced Upregulation of HLA-DR Expression on Cultured Human Gingival Fibroblasts by Interleukin-1β or Tumor Necrosis Factor-α.

The purpose of this study was to examine the effect of inflammatory cytokines on IFN-γ-induced HLA-DR expression on cultured human gingival fibroblasts by flow cytometry. Natural human IFN-γ, recombinant human interleukin-1β (rhIL-1β), and rh tumor necrosis factor-α (rhTNF-α) were used. IFN-γ-induced upregulation of HLA-DR expression was inhibited by simultaneously adding rhIL-1β or rhTNF-α (65.9% and 31.4% inhibition, respectively). Both rhIL-1β and rhTNF-α induced endogenous Prostaglandin E2 (PGE2 ) from gingival fibroblasts, while IFN-γ did not. The inhibitory effect of rhIL-1β or rhTNF-α on IFN-γ-induced upregulation of HLA-DR expression was partially abated in the presence of indomethacin (reductions of 65.9% and 41.7%, respectively). Both rhIL-1β- and rhTNF-α-induced endogenous PGE2 synthesis were completely inhibited by adding indomethacin (P < 0.001). The addition of exogenous PGE2 inhibited the IFN-γ-induced HLA-DR expression (P < 0.001). These observations suggest that the MCH class II expression on human gingival fibroblasts are influenced by the cytokine network and indirectly by the cytokine-mediated fibroblast PGE2 . J Periodontol 1994;65:336-341.

Renal Allograft Rejection: Protection of Renal Tubular Epithelial Cells from Lymphokine Activated Killer Cell Mediated Lysis by Pretreatment with Cytokines

Human renal tissue was disaggregated by passage through a stainless-steel mesh and the resultant material was separated on the basis of particle size. The tubular fraction recovered from a 45 microns mesh was cultured and after a period of between 5 and 7 days outgrowing adherent epithelial cells were identified. All of these cells contained cytokeratin and after culture for at least 14 days fewer than 5% showed surface HLA-DR antigens. Cultured renal epithelial cells were rapidly lysed by lymphokine activated killer (LAK) cells which were produced from peripheral blood lymphocytes of normal subjects by incubation for 5 days in the presence of either recombinant interleukin-2 (IL-2) or mixed lymphocyte reaction (MLR) supernatant. Stimulation of the cultured epithelial cells for 5 days with either gamma-interferon or MLR supernatant resulted in 67% +/- 9% (mean +/- SD; n = 8) of the cells showing an upregulation of HLA-DR expression. These treated cells were markedly less sensitive to lysis by LAK cells than the untreated cells. Thus, if renal cell damaging LAK cells are generated from lymphocytes in vivo by the action of IL-2 produced during rejection, then gamma-interferon, which is also produced during rejection, may partially protect the renal cells from such LAK cell mediated damage.