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Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Anti-viral ADCC requires three components: (1) antibody; (2) effector lymphocytes with the Fc-IgG receptor CD16A; and (3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity to CD16A than fucosylated antibodies; individuals' variation in afucosylation contributes to differences in ADCC. Current assays for afucosylated antibodies involve expensive methods. We report an improved bioassay for antibodies that supports ADCC, which encompasses afucosylation. This assay utilizes the externalization of CD107a by NK-92-CD16A cells after antibody recognition. We used anti-CD20 monoclonal antibodies, GA101 WT or glycoengineered (GE), 10% or ~50% afucosylated, and CD20-positive Raji target cells. CD107a increased detection 7-fold compared to flow cytometry to detect Raji-bound antibodies. WT and GE antibody effective concentrations (EC50s) for CD107a externalization differed by 20-fold, with afucosylated GA101-GE more detectable. The EC50s for CD107a externalization vs. 51Cr cell death were similar for NK-92-CD16A and blood NK cells. Notably, the % CD107a-positive cells were negatively correlated with dead Raji cells and were nearly undetectable at high NK:Raji ratios required for cytotoxicity. This bioassay is very sensitive and adaptable to assess anti-viral antibodies but unsuitable as a surrogate assay to monitor cell death after ADCC. [ABSTRACT FROM AUTHOR]

The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of studies and endpoints. While the functional assessment of CD8+ cytotoxic T-lymphocytes (CTL) is well-characterized in the clinic, the lack of a robust macaque CTL functional assay has been an important hurdle in evaluating and accurately quantifying cell-mediated CD8+ T-cell effector responses in the nonclinical setting. This paper describes the development of an assay to measure CTL activity in peripheral blood mononuclear cells (PBMC) isolated from Cynomolgus macaques. A human EGFR/CD3 Bispecific T-cell Engager (BiTE®) was used to mount a robust CD8+ T-cell response in the presence of target-expressing cells. Upon target engagement, degranulation of CD107a and production of interferon (IFN)-γ both reliably indicated a robust functional response in CD8+ T-cells. The BiTE®-mediated stimulation method proved to be favorable when compared to other methods of stimulation in the absence of target cells. These studies demonstrated acceptable longitudinal variability of the functional assay and sensitivity to dexamethasone-mediated immunosuppression. Taken together, the results indicated an assay leveraging CD3-bispecific antibodies and target-expressing cells can provide a robust approach to the in vitro or ex vivo assessment of CTL function in Cynomolgus macaques. Because the impairment of CTL activity by immunomodulators is recognized to be an important contributor to decreased antiviral defense and increased carcinogenicity risk, we believe that this novel assay to be a valuable addition to the immunotoxicology assessment of therapeutic drug candidates. [ABSTRACT FROM AUTHOR]

The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of studies and endpoints. While the functional assessment of CD8+ cytotoxic T-lymphocytes (CTL) is well-characterized in the clinic, the lack of a robust macaque CTL functional assay has been an important hurdle in evaluating and accurately quantifying cell-mediated CD8+ T-cell effector responses in the nonclinical setting. This paper describes the development of an assay to measure CTL activity in peripheral blood mononuclear cells (PBMC) isolated from Cynomolgus macaques. A human EGFR/CD3 Bispecific T-cell Engager (BiTE®) was used to mount a robust CD8+ T-cell response in the presence of target-expressing cells. Upon target engagement, degranulation of CD107a and production of interferon (IFN)-γ both reliably indicated a robust functional response in CD8+ T-cells. The BiTE®-mediated stimulation method proved to be favorable when compared to other methods of stimulation in the absence of target cells. These studies demonstrated acceptable longitudinal variability of the functional assay and sensitivity to dexamethasone-mediated immunosuppression. Taken together, the results indicated an assay leveraging CD3-bispecific antibodies and target-expressing cells can provide a robust approach to the in vitro or ex vivo assessment of CTL function in Cynomolgus macaques. Because the impairment of CTL activity by immunomodulators is recognized to be an important contributor to decreased antiviral defense and increased carcinogenicity risk, we believe that this novel assay to be a valuable addition to the immunotoxicology assessment of therapeutic drug candidates. [ABSTRACT FROM AUTHOR]

Acute‐phase neutralizing antibody (NAb) passive immunization in simian immunodeficiency virus (SIV)‐infected rhesus macaques (Macaca mulatta) can confer stringent viremia control with T‐cell augmentation. In one NAb‐infused SIV partial controller, we identify chronic‐phase Nef‐specific CD107a+ CD4+ T‐cell response maintenance, implicating that NAb infusion modulates long‐term T‐cell responses even within viremic control. [ABSTRACT FROM AUTHOR]

We hypothesised that CD8 SP + sp T cells have a greater cytotoxic potential in ITP patients who do not have detectable autoantibodies, compared to patients with autoantibodies. Autoantibody-negative ITP patients were not more likely to have high CD107a expression compared to autoantibody-positive patients (OR: 0-6, 95% CI: 0-1-3-5). Since CD107a expression in ITP patients was comparable with non-ITP, it is unclear whether the enhanced cytotoxic potential of CD8 SP + sp T cells in ITP is disease-specific. Whether or not ITP patients have CD8 SP + sp T cells specific to platelet autoantigens remains to be determined. [Extracted from the article]

The oncogenic role of human cytomegalovirus (HCMV) has been recently shown in different cancers like colorectal cancer (CRC). According to the recent immunotherapy approach to target the CMV-expressing tumor cells, we investigated the CMV peptidestimulated CD8+T cells functions in CRC patients compared to healthy individuals. All sixteen patients and seven controls were CMV seropositive. Blood samples were obtained from patients without chemotherapy and radiotherapy before surgery. Cytotoxic CD8+ T cells were generated using 14-day culture of PBMCs in the presences of CMV peptide epitopes and rhIL-2. In addition to the supernatant evaluations for TNF-α and IFN-γ, the functionality of CD8+ T cells was examined by detecting CD107a and intracellular IFN-γ using flow cytometry. CMV DNA was detected in tissues by Real Time PCR. CMV DNA was found in 31% of tumor tissues, while it was not seen in the adjacent non-tumor tissues. There was a close association between CMV in tumor tissue and tumor grade. Surface expression of CD107a and intracellular IFN-γ in CMV-stimulated CD8+T cells and the level of IFN-γ production in patient and control groups increased significantly after culture. The number of functions increased in patients (p<0.05) and healthy individuals after culture. Followingstimulation, expressions of CD107a and intracellular IFN-γ were elevated in tumor CMV positive patients while the TNF-α secretion was decreased. In vitro stimulation of PBMC in the presence of CMV peptide epitopes and IL-2 can be an applicable method to generate cytotoxic CD8+ T cells in CRC patients for future T cell therapy. [ABSTRACT FROM AUTHOR]

Peritoneal ascites are a distinguishable feature of patients with advanced epithelial ovarian cancer (EOC). The presence of different lymphocyte subsets has been reported in EOC-associated ascites, which also can or not contain malignant cells. The goal of this study was to analyze the functional characteristics of natural killer (NK) cells from EOC-associated ascites in terms of their expression of activating receptors and ascites’ contents of lymphocyte subtypes, cytokine profile and presence of EOC cells. NK cell function was evaluated by the expression of the degranulation marker CD107a in resting and interleukin (IL)-2 stimulated NK cells from ascites and blood. Degranulation of NK cells from EOC cell-free ascites was significantly (p < 0.05) higher than all the other groups, either in their resting state or after IL-2 stimulation, suggesting a previous local stimulation. In contrast, treatment with IL-2 had no effect on NK cells from ascites with EOC cells. The amount of regulatory T cells was significantly higher in ascites with EOC cells compared to EOC cell-free ascites. Ascites with EOC cells also had higher levels of tumor necrosis factor (TNF)-α, suggesting inflammation related to the malignancy. In conclusion, the functional performance of NK cells was distinct between EOC cell-free ascites and ascites with EOC cells. The impairment of NK cell response to IL-2 in ascites with EOC cells was consistent with an immunosuppressive tumor microenvironment. [ABSTRACT FROM AUTHOR]

Measuring degranulation through CD107a expression has become an advantageous tool for testing the functional capacity of cytotoxic cells. Such functional studies have been hampered in the rat by the lack of a suitable anti-rat CD107a antibody. In this study, we report a novel hybridoma generated by immunizing Armenian inbred hamsters with transfected Chinese hamster ovary cells expressing CD107a. The SIM1 clone exhibited specific reactivity with CD107a and measured degranulation from natural killer (NK) cells stimulated with target cells or mAb crosslinking of their activating receptors. Degranulation in IL-2-activated NK cells could also be measured, when using low effector to target ratios. SIM1 also stained activated CD8, but not CD4 T cells. This report characterizes the degranulation response in cytotoxic rat cells with a new antibody against rat CD107a. [ABSTRACT FROM AUTHOR]

The purpose of this study was to monitor abundance and activation of local CD8β-expressing T-cell populations during Eimeria tenella infections of naïve chickens and chickens immune by previous infections. Chickens were infected with E. tenella up to three times. Caecal T-cell receptor ( TCR) γ/δ- CD8β+ cells (cytotoxic T lymphocytes; CTL) and TCRγ/δ+ CD8β+ cells were characterized with respect to activation markers (blast transformation, CD25 and cell surface CD107a). Cells were also induced to degranulate in vitro as a measure of activation potential. Major findings included a prominent long-lasting, up to 6 weeks, increase in the proportion of CTL among caecal CD45+ cells in the later stages after primary E. tenella infection. These CTL also showed clear signs of activation, that is blast transformation and increased in vitro induced degranulation. At second and third E. tenella infection, chickens showed strong protective immunity but discrete signs of cellular activation were observed, for example increased in vitro induced degranulation of CTL. Thus, primary E. tenella infection induced clear recruitment and activation of local CTL. Upon subsequent infections of strongly immune chickens cellular changes were less prominent, possibly due to lower overall numbers of cells being activated because of the severe restriction of parasite replication. [ABSTRACT FROM AUTHOR]