Immunization of hypercholesterolemic mice with selected apoB-100 peptide antigens reduces atherosclerosis but the precise immune mediators of athero-protection remain unclear. In this study we show that immunization of apoE (-/-) mice with p210, a 20 amino acid apoB-100 related peptide, reduced aortic atherosclerosis compared with PBS or adjuvant/carrier controls. Immunization with p210 activated CD8+ T cells, reduced dendritic cells (DC) at the site of immunization and within the plaque with an associated reduction in plaque macrophage immunoreactivity. Adoptive transfer of CD8+ T cells from p210 immunized mice recapitulated the athero-protective effect of p210 immunization in naïve, non-immunized mice. CD8+ T cells from p210 immunized mice developed a preferentially higher cytolytic response against p210-loaded dendritic cells in vitro. Although p210 immunization profoundly modulated DCs and cellular immune responses, it did not alter the efficacy of subsequent T cell dependent or independent immune response to other irrelevant antigens. Our data define, for the first time, a role for CD8+ T cells in mediating the athero-protective effects of apoB-100 related peptide immunization in apoE (-/-) mice.

T cells modulate neointima formation after arterial injury but the specific T cell population that is activated in response to arterial injury remains unknown. The objective of the study was to identify the T cell populations that are activated and modulate neointimal thickening after arterial injury in mice. Arterial injury in wild type C57Bl6 mice resulted in T cell activation characterized by increased CD4+CD44hi and CD8+CD44hi T cells in the lymph nodes and spleens. Splenic CD8+CD25+ T cells and CD8+CD28+ T cells, but not CD4+CD25+ and CD4+CD28+ T cells, were also significantly increased. Adoptive cell transfer of CD4+ or CD8+ T cells from donor CD8−/− or CD4−/− mice, respectively, to immune-deficient Rag-1−/− mice was performed to determine the T cell subtype that inhibits neointima formation after arterial injury. Rag-1−/− mice that received CD8+ T cells had significantly reduced neointima formation compared with Rag-1−/− mice without cell transfer. CD4+ T cell transfer did not reduce neointima formation. CD8+ T cells from CD4−/− mice had cytotoxic activity against syngeneic smooth muscle cells in vitro. The study shows that although both CD8+ T cells and CD4+ T cells are activated in response to arterial injury, adoptive cell transfer identifies CD8+ T cells as the specific and selective cell type involved in inhibiting neointima formation.

Poly-IgM treatment reduced aortic and accelerated carotid atherosclerosis in apoE-/- mice in association with increased anti-oxLDL IgG titers, and reduced number and proliferative function of splenic CD4(+) T cells. Our study identifies a novel athero-protective and immunomodulatory role for non-immune polyclonal IgM.

Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis.

Glucocorticoid (GC)-evoked apoptosis of T-lymphoid cells is preceded by increases in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which may contribute to apoptosis. This report demonstrates that GC-mediated upregulation of the bZIP transcriptional repressor gene, E4BP4, is dependent on [Ca 2+ ] i levels, and correlates with GC-evoked apoptosis of GC-sensitive CEM-C7-14 cells. Calcium chelators EGTA and BAPTA reduced [Ca 2+ ] i levels and protected CEM-C7-14 cells from Dex-evoked E4BP4 upregulation as well as apoptosis. In the GC-resistant sister clone, CEM-C1-15, Dex treatment did not induce [Ca 2+ ] i levels, E4BP4 expression or apoptosis, however, the calcium ionophore A23187 restored Dex-evoked E4BP4 upregulation and apoptosis. CEM-C7-14 cells were more sensitive to GC-independent increases in [Ca 2+ ] i levels by thapsigargin, and a corresponding increase in E4BP4 expression and cell death, compared to CEM-C1-15 cells, suggesting a direct correlation between [Ca 2+ ] i levels, E4BP4 expression, and apoptosis.

Precursor B-lineage acute lymphoblastic leukemia (pre-B ALL) affects hematopoietic development and therefore is associated with immune deficiencies that can be further exacerbated by chemotherapy. It is unclear if and when monoclonal antibodies (mAbs) that stimulate antibody-mediated cellular cytotoxicity (ADCC) can be used for treatment because this depends on the presence of functional effector cells. Here, we used flow cytometry to determine that patient samples at diagnosis, post-induction and relapse contain detectable numbers of CD56+ cells. We were able to selectively expand CD56+ immune effector cells from bone marrow and peripheral blood samples at diagnosis and at various stages of treatment by co-culture with artificial antigen-presenting K562 clone 9.mbIL-21 cells. Amplified CD56+CD3- cells had spontaneous and anti-BAFF-R mAb-stimulated ADCC activity against autologous ALL cells, which could be further enhanced by IL15. Importantly, matched CD56+ effector cells also killed autologous ALL cells grown out from leukemia samples of the same patient, through both spontaneous as well as antibody-dependent cellular cytotoxicity. Since autologous cell therapy will not be complicated by graft-versus-host disease, our results show that expanded CD56+ cells could be applied for treatment of pre-B-ALL without transplantation, or for purging of bone marrow in the setting of autologous bone marrow transplants.

. Exogenous heat shock protein-70 inhibits cigarette smoke-induced intimal thickening. Am J Physiol Regul Integr Comp Physiol 295: R1320 -R1327, 2008. First published August 13, 2008; doi:10.1152/ajpregu.00624.2007.-Cigarette smoke is associated with increased carotid intimal thickening or stroke. Preliminary work showed that exposure to smoke resulted in a 4.5-fold reduction of heat shock protein-70 (HSP70) expression in spleens of mice using gene microarray analysis. In the current study, we investigated the role of extracellular HSP70 in carotid intimal thickening of mice exposed to cigarette smoke. Intimal thickening was induced by placement of a cuff around the right carotid artery of mice. Cuff injury resulted in increased HSP70 mRNA expression in carotid arteries that persisted for 21 days. Cigarette smoke exposure decreased arterial HSP70 expression and significantly increased intimal thickening compared with mice exposed to air. Treatment of mice exposed to cigarette smoke with intravenous recombinant HSP70 attenuated intimal thickening through reduced phosphorylated extracellular signal-regulated kinase (pERK) expression in the arterial wall. In vitro experiments with rat aortic smooth muscle cells confirmed that recombinant HSP70 decreases pERK and proliferating cell nuclear antigen (PCNA) expression in cells exposed to cigarette smoke extract and H 2O2. Our study suggests that decreased expression of arterial HSP70 is an important mechanism by which exposure to cigarette smoke augments intimal thickening. The effects of recombinant HSP70 suggest a role for extracellular HSP70. arterial injury; oxidative stress; extracellular signal-regulated kinase CIGARETTE SMOKING IS ASSOCIATED with increased carotid intimal thickening or incidence of stroke (4,10,17,20,29). We have previously reported that exposure to cigarette smoke accelerates the development of arterial intimal thickening in mice after injury (1, 35). However, the underlying mechanisms of these deleterious effects are unclear. We performed preliminary studies with gene microarray analysis of spleens from mice exposed to cigarette smoke and found a 4.5-fold decreased expression of stress-response heat shock protein-70 (HSP70) compared with mice exposed to air, identifying HSP70 as a potential target for further mechanistic studies.HSPs are part of the host stress-response mechanism. Arterial injury results in oxidative stress (1,6,14), and evidence suggests that the oxidative stress response in atherosclerosis involves HSPs (31,36). HSP70 is overexpressed in atherosclerotic lesions (16). Higher serum levels of HSP70 are associated with reduced atherosclerotic intimal thickening and lower risk of coronary artery disease (38). Furthermore, thermal treatment of rats increased arterial wall HSP70 expression and attenuated intimal thickening after injury (27).HSP70 modulation of cell signaling is, in part, mediated by sequestration of the Raf-1-activator, Bag-1, and decreased phosphorylated extracellular signal regulated kinase (pERK) activation (33...

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