Levels Of Granzyme Research Articles

Study of serum Granzyme B in heavy cigarette smokers with and without chronic obstructive pulmonary disease

BackgroundInflammation of the airways is present in COPD with increased number of inflammatory cells including killer cells that lyse their target cells by two mechanisms; membranolysis in which secreted molecules such as granzymes form pores in the membrane of target cells; and apoptosis. Granzyme B has the strongest apoptotic activity of all granzymes. Aim of this workAim of this work was to study the relation between Granzyme B, tobacco smoking and chronic obstructive pulmonary disease. MethodsThe study included 40 clinically stable COPD patients classified according to GOLD (2013) criteria into two groups; moderate (GOLD II) and severe (GOLD III) plus 40 apparently healthy control subjects (20 smokers and 20 nonsmokers). Pulmonary function results and serum levels of Granzyme B (measured by ELISA) were recorded. ResultsGranzyme B levels are elevated in COPD. Cigarette smoking appears to be a direct stimulus to Granzyme B production. Granzyme B could play a role in the pathogenesis of COPD. Aging seems to be a risk factor for Granzyme B production and pathogenesis of COPD. ConclusionGranzyme B levels are elevated in COPD. Cigarette smoking appears to be a direct stimulus to Granzyme B production. Granzyme B could play a role in the pathogenesis of COPD. Aging seems to be a risk factor for Granzyme B production and pathogenesis of COPD.

Cytotoxic effector CD4 T cells express NKG2C/E and are defined by a unique program dependent on Blimp-1. (IRC8P.482)

Abstract CD4 T cells with cytotoxic activity can be generated during many immune responses to acute viral infections and they can contribute to CD4-mediated viral control. During the peak of the CD4 T cell response to an acute infection of influenza A/PR8 [IAV], cytotoxic CD4 T cells [ThCTL] are found in the lung. The cytotoxicity they mediate is MHC II-restricted and perforin-dependent. We find the lung ThCTL express NKG2C and E that are members of the killer cell lectin-like receptor family [KLR]. Although the ThCTL cytotoxicity is strictly antigen dependent, we show that NKG2C/E can costimulate the cytotoxic activity of ThCTL in vivo, since target cells lacking the ability to present peptide on Qa-1b, which binds to NKG2C/E, are significantly protected from lysis by ThCTL during IAV infection. The ThCTL express a unique pattern of molecules including high levels of granzyme B, PSGL-1, PD-1 and CD27 as well as transcription factors Eomes and Blimp-1. We find that Blimp-1 is critical for the generation and differentiation of this subset, since a CD4 T cell intrinsic defect in Blimp-1 leads to a broad reduction of most of the ThCTL program including cytotoxic activity and migration to the lungs as well as expression of NKG2C/E, granzyme B, PSGL-1 and CD27. These features help to define a distinct ThCTL cell subset generated under distinct conditions during an acute viral infection that has novel target specificity and thus likely occupies a unique functional niche.

Plasmacytoid Dendritic Cells Contribute to Doxorubicin-induced Tumor Arrest in a Mouse Model of Pulmonary Metastasis

The biology of plasmacytoid dendritic cells (pDCs) in tumors is an emerging area of investigation. pDCs populate many human solid tumors, including lung cancer. The aim of our study was to understand the role of pDCs in pulmonary metastases during the treatment with conventional antitumor agents. For this purpose, C57Bl/6 mice were inoculated with the metastatic cell line B16-F10 or B16-Flt3L cells. The administration of doxorubicin significantly reduced the amount of pulmonary metastases in both experimental models. It is interesting to note that, 5 hours after injection, doxorubicin-induced tumor cell death was associated with higher influx of pDCs to the lung, which at 24 hours populated the mediastinal lymph nodes. In this context, lung tumor-derived pDCs obtained from mice treated with doxorubicin had higher levels of MHC I and MHC II that well correlated with the higher proliferation rate of CD4 and CD8 T cells, compared with PBS mice. Siglec-H and PD-L1 levels were not altered on lung tumor-associated pDCs derived from doxorubicin-treated and PBS-treated mice. In addition, lung tumor-associated pDCs obtained from mice treated with doxorubicin released higher levels of granzyme B. The administration of 2 consecutive doses of doxorubicin in lung tumor-bearing mice showed that B16-Flt3L-implanted mice had lower tumor burden than B16-F10-implanted mice. In conclusion, our data highlight the crucial role of the proinflammatory pDCs for the adaptive antitumor immunity. Strategies aiming at modulating pDC phenotype and activity in the tumor site might prove to be novel and effective, enhancing the conventional/actual antitumor strategies.

Immunomodulatory effects of Newcastle disease virus AF2240 strain on human peripheral blood mononuclear cells.

Immunotherapy has raised the attention of many scientists because it hold promise to be an attractive therapeutic strategy to treat a number of disorders. In this study, the immunomodulatory effects of low titers of Newcastle disease virus (NDV) AF2240 on human peripheral blood mononuclear cells (PBMC) were analyzed. We evaluated cytokine secretion and PBMC activation by cell proliferation assay, immunophenotyping and enzyme linked immunosorbent assay. The proliferation of the human PBMC was measured to be 28.5% and 36.5% upon treatment with 8 hemaglutinin unit (HAU) and 2 HAU of NDV respectively. Interestingly, the percentage of cells with activating markers CD16 and CD56 were increased significantly. Furthermore, the intracellular perforin and granzyme levels were also increased upon virus infection. Human PBMC treated with NDV titer 8 HAU was found to stimulate the highest level of cytokine production including interferon-γ, interleukin-2 and interleukin-12. The release of these proteins contributes to the antitumor effect of PBMC against MCF-7 breast cancer cells. Based on the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, activated human PBMC showed high cytolytic efficiency towards human breast tumor cells. In summary, NDV was able to stimulate PBMC proliferation, cytokine secretion and cytolytic activity.

Common Gamma Chain Cytokines Contribute To Acute GvHD Via Perforin/Granzyme B-Mediated Cytotoxicity Of CD8 T Cells

Allogeneic hematopoietic cell transplantation (allo-HCT) is an important treatment option for different hematological malignancies, but also for some nonmalignant hematological disorders such as sickle cell anemia, aplastic anemia or thalassemia(1). In the ladder group the graft-versus-leukemia (GvL) effect mediated by donor T cells is less important and prevention of graft-versus-host disease (GvHD), which occurs in 40-50% of allo-HCT patients, is a major priority. The common gamma chain (CD132) is a cytokine receptor sub-unit that is common to the interleukin (IL) receptors of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Since several of these cytokines were shown to be increased in the serum of patients developing acute GvHD, we reasoned that inhibition of CD132 could have a profound effect on acute GvHD by inhibiting the bioactivity of multiple proinflammatory cytokines.We observed that anti-CD132 treatment reduced GvHD potently with respect to survival, production of TNF, IFN-γ, IL-6, MCP-1 and GvHD histopathology. Protection was only seen when anti-CD132 was applied in a CD8 T cell-dependent GvHD model while no protection was seen when only CD4 T cells were given. Mechanistically, we could show that CD8 T cells isolated from mice treated with anti-CD132 had reduced levels of Granzyme B and that GvHD induced by Perforin-deficient T cells was resistant towards blockade by anti-CD132 treatment. These data indicated a role of the common gamma chain cytokines for the induction of Perforin/Granzyme B in CD8 T cells during GvHD. Compatible with this notion, exposure of CD8 T cells towards IL-2, IL-7, IL-15 and IL-21 alone or in combination induced increased levels of Granzyme B.Based on these findings, we concluded that CD8 T cells that are activated by common gamma chain cytokines during GvHD produce then Granzyme B which can be blocked by anti-CD132 treatment. This therapeutic approach has particular clinical potential for patients undergoing allogeneic transplantation for nonmalignant indications, since graft-versus-tumor activity is not required. Disclosures:No relevant conflicts of interest to declare.

Transcription factor Bach2 regulates naïve T-cell homeostasis and functional maturation of CD8 T-cell memory (P1309)

Abstract The transcriptional repressor Bach2 is considered as a B cell-specific transcription factor that maintains B cell identity by opposing terminal differentiation to plasma cells. Here, we show that Bach2 is expressed in CD8 T cells and the level of expression depends upon the cellular differentiation state. Bach2 is highly expressed in naïve and central memory T cells (TCM), but the more terminally differentiated effector cells and effector memory T cells (TEM) expressed low levels of Bach2. A larger fraction of CD8 T cells from Bach2-deficient (Bach2-/-) mice displayed the activated CD44HI phenotype and produced cytokines such as IFN-γ. Following an acute viral infection, Bach2-/- CD8 T cells showed normal clonal expansion and effector differentiation. However, without Bach2, effector to memory transition was markedly impaired. A substantially larger fraction of Bach2-/- memory CD8 T cells displayed the senescent KLRG1HI/CD127LO/CD27LO phenotype, with a marked reduction in the numbers of TCM cells. Functionally, Bach2-/- memory CD8 T cells produced lower levels of IFN-γ, TNFα and IL-2 but expressed higher levels of granzyme B, compared to WT. Thus, we have identified novel roles for Bach2 in maintaining naïve CD8 T-cell quiescence, promoting effector to memory transition and steering functional maturation of memory CD8 T cells, and development of TCM cells. These findings have improved our understanding of the transcriptional regulation of naïve and memory T-cell homeostasis.

CD4+ but not CD8+ memory T cells escape DN Tregs-mediated regulation via expression of Serpin Protease Inhibitor 6 (P2160)

Abstract Memory T cell (Tm) homeostasis is poorly understood and is a critical challenge to prevent transplant rejection. At present there are no effective clinical strategies to control Tm and contradictory reports exist as to their control by regulatory T cell (Treg). We previously found that TCRab+CD3+CD4-CD8-NK1.1- (double-negative, DN) Treg could effectively suppress CD4+ and CD8+ effector T cells and thus prevent transplant rejection. We utilized this model to test whether DN Tregs could similarly suppress Tm. Interestingly DN Tregs suppressed CD8+ Tm but had no effect on CD4+ Tm cells. As well DN Tregs express very high levels of granzyme B (GrB), suggesting a potential control mechanism and indeed no suppression was observed using perforin null DN Tregs. In BALB/c (H-2d) to B6-Rag1-/- mice (H-2b) skin allograft transplantation, DN Tregs significantly reduced total CD44high CD8+ Tm in recipients and significantly prolonged graft survival. In contrast, co-transfer of DN Tregs had no effect on CD4+CD44high Tm mediated rejection. There was no effect with co-transferred CD4+ Tm. CD4+ Tm cells express higher levels of GrB inhibitory Serpin Protease Inhibitor 6 (SPI-6) mRNA and protein than CD8+ Tm. Furthermore, SPI-6 null CD4+ Tm is not resistant to DN Treg-mediated regulation. We show for the first time, that DN Treg can control CD8+ Tm and suggest that DN Treg along with targeting SPI-6 may be useful to limit the expansion of both CD4+ and CD8+ Tm cells in transplantation.

A genetic variant in granzyme B is associated with progression of joint destruction in rheumatoid arthritis

Genetic factors account for an estimated 45-58% of the variance in joint destruction in rheumatoid arthritis (RA). The serine proteinase granzyme B induces target cell apoptosis, and several in vitro studies suggest that granzyme B is involved in apoptosis of chondrocytes. Serum levels of granzyme B are increased in RA and are also associated with radiographic erosions. The aim of this study was to investigate GZMB as a candidate gene accounting for the severity of joint destruction in RA. A total of 1,418 patients with 4,885 radiograph sets of the hands and feet from 4 independent cohorts were studied. First, explorative analyses were performed in 600 RA patients in the Leiden Early Arthritis Clinic cohort. Fifteen single-nucleotide polymorphisms (SNPs) tagging GZMB were tested. Significantly associated SNPs were genotyped in data sets representing patients from the Groningen, Sheffield, and Lund cohorts. In each data set, the relative increase in the annual rate of progression in the presence of a genotype was assessed. Data were summarized in a meta-analysis. The association of GZMB with the RNA expression level of the GZMB genomic region was tested by mapping expression quantitative trait loci (QTLs) on 1,469 whole blood samples. SNP rs8192916 was significantly associated with the rate of joint destruction in the first cohort and in the meta-analysis of all data sets. Patients homozygous for the minor allele of rs8192916 had a higher rate of joint destruction per year compared with other patients (P = 7.8 × 10(-4)). Expression QTL of GZMB identified higher expression in the presence of the minor allele of rs8192916 (P = 2.27 × 10(-5)). SNP rs8192916 located in GZMB is associated with the progression of joint destruction in RA as well as with RNA expression in whole blood.

Comparative Study of Serum levels of Granzyme H & Estrogen In Patients Suffering From Breast Cancer

GranymeH is a functional cytotoxic serine protease of NK cell granules,which expands the cell death-inducing repertoire of innate immune system.The purpose of this study was to determine GranymeH(GZMH)level in breast cancer(BC) and healty women.This study was performed on 30 patients with BC and 30 healty woman.GZMH and Estrogen levels were measured in cancer patients and healty women subsequently using ELISA and Radioimmunoassay(RIA) methods. Mean GZMH value was lower in BC than healty women(p<0.0001) and mean Estrogen level was higher in BC patients in comparison to healty women (p<0.003).Our finding indicates probability of existance of suppressor or a problem in production of GZMH in cancer patients

Granzyme A Produced by γ9δ2 T Cells Induces Human Macrophages to Inhibit Growth of an Intracellular Pathogen

Human γ9δ2 T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ9δ2 T cells produced soluble factors that could pass through 0.45 µm membranes and inhibit intracellular mycobacteria in human monocytes cultured below transwell inserts. Neutralization of TNF-α in co-cultures of infected monocytes and γ9δ2 T cells prevented inhibition, suggesting that TNF-α was the critical inhibitory factor produced by γ9δ2 T cells. However, only siRNA- mediated knockdown of TNF-α in infected monocytes, but not in γ9δ2 T cells, prevented mycobacterial growth inhibition. Investigations of other soluble factors produced by γ9δ2 T cells identified a highly significant correlation between the levels of granzyme A produced and intracellular mycobacterial growth inhibition. Furthermore, purified granzyme A alone induced inhibition of intracellular mycobacteria, while knockdown of granzyme A in γ9δ2 T cell clones blocked their inhibitory effects. The inhibitory mechanism was independent of autophagy, apoptosis, nitric oxide production, type I interferons, Fas/FasL and perforin. These results demonstrate a novel microbial defense mechanism involving granzyme A-mediated triggering of TNF-α production by monocytes leading to intracellular mycobacterial growth suppression. This pathway may provide a protective mechanism relevant for the development of new vaccines and/or immunotherapies for macrophage-resident chronic microbial infections.

Characterization of CD4+CD28null T cells in patients with coronary artery disease and individuals with risk factors for atherosclerosis

Risk factors for atherosclerosis may contribute to chronic low-grade inflammation. A highly cytotoxic and inflammatory CD4+ cell subset (CD4+CD28null cells) has been associated with inflammatory diseases, including acute coronary syndromes (ACS). The aim of this study was to quantify and characterize CD4+CD28null cells in individuals with risk factors for atherosclerosis and patients with coronary artery disease (CAD). In order to achieve this goal, peripheral blood mononuclear cells (PBMCs) from individuals with risk factors for atherosclerosis and patients with CAD were analyzed using flow cytometry to detect cytotoxic molecules and evaluate the expression of homing receptors and inflammatory cytokines in CD4+ cell subsets. The cells were evaluated ex vivo and after stimulation in culture. We found no differences in the proportions of CD4+CD28null cells among the groups. Compared with the CD4+CD28+ population, the ex vivo CD4+CD28null subset from all groups expressed higher levels of granzymes A and B, perforin, granulysin and interferon-γ (IFN-γ). Individuals with risk factors and patients with ACS showed the highest levels of cytotoxic molecules. After stimulation, tumor necrosis factor-α (TNF-α) expression in the CD4+CD28null subset from these groups increased more than in the other groups. Stimulation with LPS decreased the expression of cytotoxic molecules by CD4+CD28null cells in all groups. In conclusion, our results show that risk factors for atherosclerosis may alter the CD4+CD28null cells phenotype, increasing their cytotoxic potential. Our findings also suggest that CD4+CD28null cells may participate in the early phases of atherosclerosis.

Human conventional CD1c+ Dendritic Cells produce IL-12 and induce potent CD8+ T cell priming

Event Abstract Back to Event Human conventional CD1c+ Dendritic Cells produce IL-12 and induce potent CD8+ T cell priming Giulia Nizzoli1, Anja Weick2, Jana Krietsch2, Svenja Steinfelder2, Federica Facciotti1, Sergio Abrignani1 and Jens Geginat1* 1 INGM, Italy 2 DRFZ, Germany BDCA-3+ dendritic cells are thought to be the human equivalents of murine CD8α+ DC, which cross-prime CD8+ T-cell responses and secrete high amounts of IL-12. We compared human myeloid DC expressing CD1c or BDCA-3 and plasmacytoid DC for cytokine production and surface receptor expression in peripheral blood and lymphoid tissues, and assessed their capacities to induce cytotoxic T cell responses. We found that mDC1, but not other DC or monocyte subsets could secrete high amounts of IL-12p70. IL-12 production by mDC1 was tightly controlled, as it required combinational Toll-like receptor (TLR) stimulation and was fine-tuned by lymphocyte-derived stimuli. Conversely, mDC2 produced high levels of IFNlambda in response to TLR-3 stimulation alone. pDC secreted IFNalpha  expressed lower levels of MHC class-I and -II, CD40 and CD86 than myeloid DC. Moreover, they poorly cross-presented soluble antigens to CD8+ T cells. In contrast, both mDC1 and mDC2 could cross-present efficiently, but required different combinations of TLR ligands. Nevertheless, all DC subsets induced secondary expansions of CD8+ memory T-cells upon licensing by CD4+ T-cells. Furthermore, following appropriate TLR stimulation and help by CD4+ T cells, all DC primed naive CD8+ T-cells. However, while mDC1 induced high levels of Granzyme B via IL-12, mDC2 and pDC induced preferentially Granzyme A. Our results show that CD1c+ mDC1 are a relevant source of IL-12 for naïve T cell priming, and that specific pathogen-derived stimuli and CD4 help can instruct different DC subsets to induce cytotoxic T-cell responses. Keywords: human DC subsets, IL-12p70, cross-presentation, CD8+ priming, MDC1 Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Nizzoli G, Weick A, Krietsch J, Steinfelder S, Facciotti F, Abrignani S and Geginat J (2013). Human conventional CD1c+ Dendritic Cells produce IL-12 and induce potent CD8+ T cell priming. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00876 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Jens Geginat, INGM, Milano, Italy, geginat@ingm.org Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Giulia Nizzoli Anja Weick Jana Krietsch Svenja Steinfelder Federica Facciotti Sergio Abrignani Jens Geginat Google Giulia Nizzoli Anja Weick Jana Krietsch Svenja Steinfelder Federica Facciotti Sergio Abrignani Jens Geginat Google Scholar Giulia Nizzoli Anja Weick Jana Krietsch Svenja Steinfelder Federica Facciotti Sergio Abrignani Jens Geginat PubMed Giulia Nizzoli Anja Weick Jana Krietsch Svenja Steinfelder Federica Facciotti Sergio Abrignani Jens Geginat Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

CVID patients with autoimmunity have elevated T cell expression of granzyme B and HLA-DR and reduced levels of Treg cells

AimsCommon variable immunodeficiency (CVID) is a primary antibody immunodeficiency with approximately 20% of patients reporting additional autoimmune symptoms. The primary aim of this study was to compare the levels of...

Abstract 19582: Deletion of miRNA-146a Alters Host Immune Response to Coxsackievirus B3 with Activation of NK cells

Coxsackievirus B3 (CVB3) is a positive single-stranded RNA virus that causes a range of diseases, including myocarditis, hepatitis, and pancreatitis. Understanding complex interplay between the virus and host immune system will provide insight for development of future therapeutic interventions. miRNA-146a plays a role in immune system through regulation of NF-kB signaling. It is expressed at relatively high levels in hematopoetic cells, and upregulated with various immune stimuli. We hypothesized that miRNA-146a is involved in mediating immune response against CVB3 infection. Genetically modified C57BL/6 mice with global deletion of miRNA-146a, and wild type littermate controls were intraperitoneally injected with 10 3 -10 4 PFU of CVB3. miRNA-146a null mice had significantly higher mortality rate, reaching 45% by day 4 after infection (n=16), compared to zero mortality observed in wild type mice (n=13, p=0.006). Evans blue dye uptake was higher in the hearts of knock out animals 4 days post-infection, indicating disruption of cardiomyocyte membrane integrity. The degree of hepatocyte damage and disruption of the lobular architecture in liver was more pronounced in the miRNA-146a null mice 4 days post-infection. In normal uninfected state the majority of NK cells are localized in liver and spleen, and they are one of the first to get activated in CVB3 infection. We assessed the degree of NK cell activation 24 hours post infection with FACS analyses of mononuclear cells isolated from liver by Percoll gradient. The total percentage of NK cell population in liver was the same between both groups. On average 10+/-0.8% of total NK population in the livers of knock out mice were represented by the cells with high level of TNF-related apoptosis-inducing ligand (TRAIL) expression, whereas only 4.7+/-2% of the hepatic NK cells in the wild type controls had the same level of TRAIL expression (n=3, p=0.03). Liver mRNA levels of Granzymes A and B, and perforin were 4- to 8-fold higher in miRNA-146a null mice compared to wild type controls (n=6 for wild type, n=5 for knock out, p0.05). Taken together, these data indicate that deletion of miRNA-146a alters host response to CVB3 by increasing NK cell activation.

Assessment of different biomarkers provides valuable diagnostic standards in the evaluation of the risk of acute rejection

Acute rejection (AR) is a strong risk factor for chronic rejection in renal transplant recipients. Accurate and timely diagnosis of AR episodes is very important for disease control and prognosis. Therefore, objectively evaluated the immune status of patients is essential in the field of post-transplantation treatment. This longitudinal study investigated the usefulness of five biomarkers, human leukocyte antigen (HLA)-G5 and sCD30 level in sera, intracellular adenosine triphosphate (iATP) release level of CD4(+) T cells, and granzyme B/perforin expression in peripheral blood mononuclear cells (PBMCs) and biopsies, to detect AR and the resolution of biomarkers in a total of 84 cases of renal transplantation. The data demonstrated that recipients with clinical or biopsy proven rejection significantly increased iATP release level of CD4(+) T cells, and elevated sCD30 but lowered HLA-G5 level in sera compared with individuals with stable graft function. Expression levels of granzyme B and perforin were also elevated in PBMCs and graft biopsies of AR patients. Taken together, we identified that upregulation of sCD30, iATP, granzyme B, perforin, and downregulation of HLA-G5 could provide valuable diagnostic standards to identify those recipients in the risk of AR. And iATP may be a better biomarker than others for predicting the graft rejection episode.

Abstract 138: Deficiency of CD11a Reduces CD8+ T-Cell Activation and Proliferation in Adipose Tissue of Obese Mice

T cells, particularly CD8 + T cells, are major participants in obesity-linked adipose tissue (AT) inflammation. CD11a, a β 2 -integrin, is crucial for T cell adhesion and interactions with antigen-presenting cells. It has been shown that stimulation of CD11a lowered the threshold of T cell activation, yet the role of CD11a in T cell activation in AT remains unknown. We used CD11a -/- mice to test the hypothesis that CD11a deficiency reduces CD8 + T cell activation in AT. Mice fed high-fat diet for 3 months were used as an obesity model, with mice on normal diet as lean controls. CD8 + T cells and related cytokines were examined in AT by flow cytometry or quantitative RT-PCR. Compared to lean wild-type (WT) mice, obese WT mice had increased numbers of activated CD8 + T cells, with higher proportions of CD11a high CD8 + memory T cells, CD44 + CD62L - effector memory CD8 + T cells and CD44 + CD69 + effector CD8 + T cells; and increased mRNA levels of granzyme B and IFN-γ in AT. Compared to obese WT, obese CD11a -/- mice had lower proportions of CD44 + CD62L - effector memory CD8 + T cells and CD44 + CD69 + effector CD8 + T cells, and decreased mRNA levels of granzyme B and IFN-γ (P&lt;0.01), implying decreased activation of CD8 + T cells. IL-2, IL-12 and IL-18 levels were increased in AT of obese WT compared to that of leans, and were lower in AT of obese CD11a -/- mice than that of obese WT. In vitro treatment with IL-2, IL-12 and IL-18 induced proliferation and expression of IFN-γ and CD69 in CD8 + cells isolated from AT of WT mice. Compared to those from WT mice, CD8 + T cells from AT of CD11a -/- mice showed decreased response to cytokine stimulation with fewer cells expressing IFN-γ (7.0±1.1% in CD11a -/- vs. 11.3±1.1% in WT, n=3-5/group, P&lt;0.05). In addition, using Edu labeling techniques, we studied proliferation of CD8 + T cells in AT in vivo. At 3 hours after intraperitoneal injection of Edu, no Edu + T cells appeared in blood but we found CD8 + /Edu + T cells in AT of WT mice, indicating CD8 + T cells proliferated in AT. Compared to obese WT mice, obese CD11a -/- mice had decreased proportion of proliferating CD8 + T cells in AT (1.3±0.4% in CD11a -/- vs. 2.4±0.1% in WT, n=3/group, P&lt;0.01). In conclusion, we demonstrated that CD11a played a crucial role in CD8 + T cell activation and proliferation in AT associated with obesity.

Comparable Polyfunctionality of Ectromelia Virus- and Vaccinia Virus-Specific Murine T Cells despite Markedly DifferentIn VivoReplication and Pathogenicity

Vaccinia virus (VACV) stimulates long-term immunity against highly pathogenic orthopoxvirus infection of humans (smallpox) and mice (mousepox [ectromelia virus {ECTV}]) despite the lack of a natural host-pathogen relationship with either of these species. Previous research revealed that VACV is able to induce polyfunctional CD8(+) T-cell responses after immunization of humans. However, the degree to which the functional profile of T cells induced by VACV is similar to that generated during natural poxvirus infection remains unknown. In this study, we monitored virus-specific T-cell responses following the dermal infection of C57BL/6 mice with ECTV or VACV. Using polychromatic flow cytometry, we measured levels of degranulation, cytokine expression (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]), and the cytolytic mediator granzyme B. We observed that the functional capacities of T cells induced by VACV and ECTV were of a similar quality in spite of the markedly different replication abilities and pathogenic outcomes of these viruses. In general, a significant fraction (≥50%) of all T-cell responses were positive for at least three functions both during acute infection and into the memory phase. In vivo killing assays revealed that CD8(+) T cells specific for both viruses were equally cytolytic (∼80% target cell lysis after 4 h), consistent with the similar levels of granzyme B and degranulation detected among these cells. Collectively, these data provide a mechanism to explain the ability of VACV to induce protective T-cell responses against pathogenic poxviruses in their natural hosts and provide further support for the use of VACV as a vaccine platform able to induce polyfunctional T cells.

Increased Granzyme Levels in Cytotoxic T Lymphocytes Are Associated With Disease Severity in Emergency Department Patients With Severe Sepsis

Exocytosis of granules containing the cytolytic effector (CE) molecules granzyme A (GzmA), granzyme B (GzmB), and perforin is one major pathway of lymphocyte-mediated cytotoxicity. Studies in murine models and the finding of elevated granzyme levels in the plasma of septic patients have implicated cytotoxic lymphocytes in the pathogenesis of sepsis. We sought to evaluate the role of cytotoxic cells and CE in sepsis and determine if intracellular levels of CE in cytotoxic cells correlate with disease severity. We conducted a prospective cohort study of 40 patients enrolled into one of three groups: controls (C), acutely ill nonseptic illnesses, or patients with severe sepsis (SS) (lactate, >4 mmol/L; systolic blood pressure, <90 mmHg after 2 L normal saline). Peripheral blood mononuclear cells were isolated and stained for extracellular markers for defined subpopulations and for intracellular expression of GzmA and GzmB and perforin. Levels of CE were quantified by geometric mean fluorescent intensity (GMFI) via flow cytometry. Cytotoxic T lymphocyte (CTL) expression was higher in SS (P = 0.04). The GMFI of GzmB was significantly higher in CTLs of SS patients versus acutely ill nonseptic illnesses or C. The GMFI of each GzmA and GzmB in CTLs were associated with the Acute Physiology and Chronic Health Evaluation II score (P = 0.01). A significant increase in the number of granulocytes in the peripheral blood mononuclear cells of SS patients consisted primarily of low-density neutrophils, which expressed increased levels of GzmA (P < 0.01). The results suggest that CTLs are activated in SS and express significantly higher intracellular levels of GzmB and that GzmA and B levels correlate with disease severity.

What’s New in Shock, March 2012?

What’s New in Shock, March 2012?

Interruption of the IFNγR/CXCR3 Axis Results in Altered T Cell Trafficking in Vivo and Abrogation of GVHD While Maintaining a Robust GVL Response

The goal of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is to minimize graft-versus-host disease (GvHD) while maintaining a beneficial graft-versus-leukemia (GvL) effect. This can be achieved by infusing regulatory T cells (Tregs) which suppress GvHD but have only limited effects on GvL. Unfortunately, Tregs exist in low frequency in the peripheral blood, are costly to purify and expand, and after expansion are difficult to isolate due to the lack of cell surface markers. Thus, alternative therapeutic approaches are needed. Using an MHC-mismatched GvHD model, B6 (H-2b) → Balb/c (H-2d), we demonstrated that infusion of IFNγR deficient (IFNγR-/-) allogeneic donor T cells induced significantly less GvHD, compared to WT T cells, as determined by survival (74% vs. 0%; p = 0.0004), weight loss and percentages of B220+ B cells (12.4% vs. 3.8%; p = 0.0205), CD3+ T cells (14.3% vs. 4.3%; p = 0.0025) in blood. Of note was that the IFNγR-/- donor T cells induced a robust GvL effect in both systemic leukemia and solid tumor models using luciferase-expressing A20 cells derived from Balb/c. We found that IFNγR-/- T cells responded normally to allogeneic antigens in vitro, based on mixed lymphocyte reactions, and expressed similar levels of granzyme B, compared to WT T cells. Using bioluminescence imaging (BLI) and luciferase-transduced WT and IFNγR-/- T cells, we observed that IFNγR-/- T cells trafficked primarily to the spleen while WT T cells trafficked to gastrointestinal tract and lymph nodes, which are major GvHD target organs. These findings suggest that the reduced GvHD was not due to reduced function, altered subsets or relative deficiency of allogeneic donor T cells but from an alteration of in vivo trafficking of IFNγR-/- T cells compared to WT T cells. We further demonstrated that the IFNγR-mediated signaling in alloreactive donor T cells was required for expression of CXCR3 which has been implicated in trafficking of T cells to areas of inflammation and target organs, commonly known to be the sites of GvHD. In addition, overexpression of CXCR3 in IFNγR-/- T cells rescued the GvHD-inducing function of IFNγR-/- T cells. CXCR3-/- T cells and pharmacological approaches using inhibitors of IFNγR signaling recapitulated the anti-GvHD (and pro-GvL) effects seen in IFNγR-/- T cells after allo-HSCT. Thus, the IFNγR-CXCR3 axis represents a promising therapeutic target for future efforts to mitigate GvHD while maintaining GvL after allo-HSCT.