Mabood Qureshi

Membrane-bound HSP70-engineered myeloma cell-derived exosomes stimulate more efficient CD8 CTL- and NK-mediated antitumour immunity than exosomes released from heat-shocked tumour cells expressing cytoplasmic HSP70

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour‐derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T‐cell immune responses. To enhance EXO‐based antitumour immunity, we generated an engineered myeloma cell line J558HSP expressing endogenous P1A tumour antigen and transgenic form of membrane‐bound HSP70 and heat‐shocked J558HS expressing cytoplasmic HSP70, and purified EXOHSP and EXOHS from J558HSP and J558HS tumour cell culture supernatants by ultracentrifugation. We found that EXOHSP were able to more efficiently stimulate maturation of DCs with up‐regulation of Iab, CD40, CD80 and inflammatory cytokines than EXOHS after overnight incubation of immature bone‐marrow‐derived DCs (5 × 106 cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A‐specific T‐cell responses after immunization. We demonstrate that EXOHSP are able to stimulate type 1 CD4+ helper T (Th1) cell responses, and more efficient P1A‐specific CD8+ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXOHS. In addition, we further elucidate that EXOHSP‐stimulated antitumour immunity is mediated by both P1A‐specific CD8+ CTL and non‐P1A‐specific natural killer (NK) responses. Therefore, membrane‐bound HSP70‐expressing tumour cell‐released EXO may represent a more effective EXO‐based vaccine in induction of antitumour immunity.

Dendritic Cells Recruit T Cell Exosomes via Exosomal LFA-1 Leading to Inhibition of CD8+ CTL Responses through Downregulation of Peptide/MHC Class I and Fas Ligand-Mediated Cytotoxicity

Active T cells release bioactive exosomes (EXOs). However, its potential modulation in immune responses is elusive. In this study, we in vitro generated active OVA-specific CD8+ T cells by cultivation of OVA-pulsed dendritic cells (DCOVA) with naive CD8+ T cells derived from OVA-specific TCR transgenic OTI mice and purified EXOs from CD8+ T cell culture supernatant by differential ultracentrifugation. We then investigated the suppressive effect of T cell EXOs on DCOVA-mediated CD8+ CTL responses and antitumor immunity. We found that DCOVA uptake OTI T cell EXOs expressing OVA-specific TCRs and Fas ligand via peptide/MHC Ag I–TCR and CD54–LFA-1 interactions leading to downregulation of peptide/MHC Ag I expression and induction of apoptosis of DCOVA via Fas/Fas ligand pathway. We demonstrated that OVA-specific OTI T cell EXOs, but not lymphocytic choriomeningitis virus-specific TCR transgenic mouse CD8+ T cell EXOs, can inhibit DCOVA-stimulated CD8+ CTL responses and antitumor immunity against OVA-expressing B16 melanoma. In addition, these T cell EXOs can also inhibit DCOVA-mediated CD8+ CTL-induced diabetes in transgenic rat insulin promoter-mOVA mice. Interestingly, the anti–LFA-1 Ab treatment significantly reduces T cell EXO-induced inhibition of CD8+ CTL responses in both antitumor immunity and autoimmunity. EXOs released from T cell hybridoma RF3370 cells expressing OTI CD8+ TCRs have a similar inhibitory effect as T cell EXOs in DCOVA-stimulated CTL responses and antitumor immunity. Therefore, our data indicate that Ag-specific CD8+ T cells can modulate immune responses via T cell-released EXOs, and T cell EXOs may be useful for treatment of autoimmune diseases.

A novel T cell-based vaccine capable of stimulating long-term functional CTL memory against B16 melanoma via CD40L signaling

The ultimate goal of antitumor vaccines is to develop memory CD8+ cytotoxic T lymphocytes (CTLs), which are critical mediators of antitumor immunity. We previously demonstrated that the ovalbumin (OVA)-specific CD4+ T cell-based (OVA-TEXO) vaccine generated using OVA-pulsed dendritic cell (DCOVA)-released exosomes (EXOOVA) stimulate CTL responses via IL-2 and costimulatory CD80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type (WT) C57BL/6 and gene-knockout mice with WT CD4+ OVA-TEXO cells or OVA-TEXO cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2Kb/OVA257–264 tetramer and FITC-anti-CD8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-10OVA. We demonstrated that CD4+ OVA-TEXO cells stimulated more efficient CTL responses compared to DCOVA. By assessing primary and recall CTL responses in mice immunized with OVA-TEXO or with OVA-TEXO lacking the costimulatory molecules CD40L, 4-1BBL or OX40L, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-TEXO. Interestingly, CD40L, but not 4-1BBL or OX40L, plays a crucial role in the development of functional memory CTLs against BL6-10OVA tumors. Overall, this work suggests that a novel CD4+ T cell-based vaccine that is capable of stimulating long-term functional CTL memory via CD40L signaling may represent a novel, efficient approach to antitumor vaccination.

Antitumor Immune Responses Derived from Transgenic Expression of CD40 Ligand in Myeloma Cells

Tumor cells engineered to express immunogenes have been used for cancer vaccines to induce the antitumor immunity and study the antitumor immune mechanisms derived from the immunogene expression. In the present study, we engineered a mouse myeloma cell line J558 with a cloned CD40 ligand (CD40L) gene. We demonstrated that (i) the engineered J558/CD40L tumor cells expressing the CD40 ligand molecule lost their tumorigenicity in syngeneic mice, and (ii) the inoculation of J558/CD40L tumor cells further lead to the protective immunity against wild-type J558 tumors. In animal studies using T-cell subset depleted mice, we further showed that the primary rejection of J558/CD40L tumors did not require T cells, but was mainly mediated by NK cells, whereas the effector phase of the protective immunity is mediated by CD8+ T cells. In addition, our data, for the first time, showed that the inoculation of engineered J558/CD40L tumor cells is able to stimulate stronger activation of dendritic cells with enhanced expression of B7-1 and ICAM-1 molecules than the wild-type J558 tumor cells Taken together, we demonstrated the antitumor effect of engineered J558/CD40L tumor cells that is mediated by the activation of the host dendritic cells in vivo. Our data indicate that the introduction of co-stimulatory CD40 ligand molecule will be useful as a new strategy of immunogene therapy against tumors.

Elevated Levels of Plasma Homocysteine in Hypertensive Patients with Diabetes Mellitus

Background: Homocysteine generates oxygen radicals (superoxide anion and hydrogen peroxide) that are known to produce vasoconstriction. Hypertension is a common problem in individuals with diabetes mellitus. It is possible that hypertension in diabetic patients may be due to increased levels of plasma homocysteine. We investigated the plasma levels of homocysteine, factors involved in homocysteine metabolism (serum folic acid and vitamin B12) and lipid peroxidation product in the serum of diabetic patients with hypertension. Methods and Results: The studies were conducted in three groups: 1) healthy controls, and diabetic patients who were 2) normotensive and 3) hypertensive. Plasma homocysteine, serum malondialdehyde (a lipid peroxidation product), vitamin B12, and folic acid were measured in these patients. Plasma homocysteine and serum malondialdehyde levels were elevated in diabetic patients compared to the control group. Plasma levels of homocysteine and serum levels of malondialdehyde were higher in the hypertensive diabetic patients than in those who were normotensive. Levels of serum folate were lower in hypertensive diabetic patients compared to the normotensive group. Levels of serum vitamin B12 were similar in both the normotensive and hypertensive diabetic patients. Conclusions: Levels of plasma homocysteine and serum malondialdehyde are elevated in hypertensive diabetic patients. Hyperhomocysteinemia may be involved in the induction and sustaining of hypertension in diabetic patients.

Transgene IL-6 enhances DC-stimulated CTL responses by counteracting CD4+25+Foxp3+ regulatory T cell suppression via IL-6-induced Foxp3 downregulation.

Dendritic cells (DCs), the most potent antigen-presenting cells have been extensively applied in clinical trials for evaluation of antitumor immunity. However, the efficacy of DC-mediated cancer vaccines is still limited as they are unable to sufficiently break the immune tolerance. In this study, we constructed a recombinant adenoviral vector (AdVIL-6) expressing IL-6, and generated IL-6 transgene-engineered DC vaccine (DCOVA/IL-6) by transfection of murine bone marrow-derived ovalbumin (OVA)-pulsed DCs (DCOVA) with AdVIL-6. We then assessed DCOVA/IL-6-stimulated cytotoxic T-lymphocyte (CTL) responses and antitumor immunity in OVA-specific animal tumor model. We demonstrate that DCOVA/IL-6 vaccine up-regulates expression of DC maturation markers, secretes transgene-encoded IL-6, and more efficiently stimulates OVA-specific CTL responses and therapeutic immunity against OVA-expressing B16 melanoma BL6-10OVA in vivo than the control DCOVA/Null vaccine. Moreover, DCOVA/IL-6-stimulated CTL responses were relatively maintained in mice with transfer of CD4+25+Foxp3+ Tr-cells, but significantly reduced when treated with anti-IL-6 antibody. In addition, we demonstrate that IL-6 down-regulates Foxp3-expression of CD4+25+Foxp3+ Tr-cells in vitro. Taken together, our results demonstrate that AdV-mediated IL-6 transgene-engineered DC vaccine stimulates potent CTL responses and antitumor immunity by counteracting CD4+25+ Tr immunosuppression via IL-6-induced Foxp3 down-regulation. Thus, IL-6 may be a good candidate for engineering DCs for cancer immunotherapy.

Atherosclerosis and the Hypercholesterolemic AGE-RAGE Axis.

Background Interaction of advanced glycation end products (AGE) with the receptor for advanced glycation end products (RAGE) has been implicated in the pathogenesis of atherosclerosis. Soluble receptors for advanced glycation end products (sRAGE) act as a decoy for AGE by competing with RAGE and suppressing developing atherosclerosis. Hypercholesterolemia and the oxidative stress are known factors involved in atherosclerosis. High-density lipoprotein cholesterol (HDL-C) is known to exert a protective effect against the development of atherosclerosis. We hypothesize that hypercholesterolemia-induced atherosclerosis may be mediated through the AGE-RAGE axis. Objectives Two objectives to be determined are: (1) if hypercholesterolemia is positively correlated with serum AGE, AGE/sRAGE, and malondialdehyde (MDA: a marker for oxidative stress) and (2) if the protective effect of HDL-C is positively associated with serum sRAGE and negatively correlated with the levels of AGE and AGE/sRAGE. Methods Measurement of serum lipid levels from 100 patients allowed the separation into two groups (hypercholesterolemic and normocholesterolemic). Measurements of serum levels of AGE, sRAGE, and MDA were performed. Results Serum levels of sRAGE were lower, while the levels of AGE and AGE/sRAGE were higher in hypercholesterolemic subjects as compared with normocholesterolemic subjects. sRAGE levels are positively correlated with HDL, while they are negatively correlated with low-density lipoprotein, triglycerides, total cholesterol, and MDA in hypercholesterolemic subjects. Conclusions Hypercholesterolemia is positively correlated with serum AGE, AGE/sRAGE, and MDA. The effect of HDL-C may be due to increases in sRAGE and decreases in the levels of AGE and AGE/sRAGE. Hypercholesterolemia-induced atherosclerosis may be mediated through the AGE-RAGE axis; however, more research must be conducted.

Oxidative stress in renal transplant patients who develop cardiovascular disease.

Cardiovascular disease limits life expectancy of successful renal transplant patients. Reactive oxygen species have been implicated in the development of atherosclerosis, and high levels could be due to increased production or a decrease in antioxidant reserve. Cardiovascular disease in renal transplant recipients could be due to elevated levels of malondialdehyde (an index of levels of reactive oxygen species) and homocysteine and reduced levels of glutathione. Renal transplant recipients with and without cardiovascular disease were studied along with healthy controls. Serum malondialdehyde, plasma homocysteine, and red blood cell glutathione were measured. The results suggest that levels of serum malondialdehyde and plasma homocysteine were higher in patients with or without cardiovascular disease compared with controls; however, the values were similar in both groups of transplant patients. Glutathione levels in red blood cells were similar in all 3 groups. Renal transplant recipients without cardiovascular disease have high levels of oxidative stress and may develop cardiovascular disease with time.

Candidate recommendations for protein electrophoresis reporting from the Canadian Society of Clinical Chemists Monoclonal Gammopathy Working Group

Protein electrophoresis is commonly used as an aid in the diagnosis of monoclonal gammopathies and is performed in many laboratories in Canada and throughout the world. However, unlike many other diagnostic tests, there is limited guidance for standardization and neither guidance nor specific recommendations for clinical reporting of serum (SPE) or urine (UPE) protein electrophoresis and immunotyping available in the literature. Therefore, a Canadian effort was undertaken to recommend standards that cover all aspects of clinical reporting with an ultimate goal towards reporting standardization. The Canadian Society of Clinical Chemists (CSCC) Monoclonal Gammopathy Interest Group (MGIG), which is composed of CSCC members with an interest in protein electrophoresis, has formed a Monoclonal Gammopathy Working Group (MGWG) to take initial steps towards standardization of SPE, UPE and immunotyping. Candidate standardization recommendations were developed, discussed and voted upon by the MGWG. Candidate recommendations that achieved 90% agreement are presented as consensus recommendations. Recommendations that did not achieve 90% consensus remain candidate recommendations and are presented with accompanying MGWG discussion. Eleven consensus recommendations along with candidate recommendations for nomenclature, protein fraction reporting, test utilization, interference handling and interpretive reporting options are presented.