The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo

Hermans, Ian F.; Silk, Jonathan D.; Yang, Jianping; Palmowski, Michael J.; Gileadi, Uzi; McCarthy, Corinna; Salio, Mariolina; Ronchese, Franca; Cerundolo, Vincenzo

doi:10.1016/j.jim.2003.10.017

Cited by 164 publications

(161 citation statements)

References 44 publications

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“…Vital assay was performed as described elsewhere (Hermans et al, 2004). In brief, PBMCs were pre-sensitized as described with ILMNDQEVGV, HIV and CEF peptides.…”

Section: Isolation Of Peripheral Blood Mononuclear Cells (Pbmcs)mentioning

confidence: 99%

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Identification of HLA-A01- and HLA-A02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

Weinzierl

Rudolf

Maurer

et al. 2008

Journal of General Virology

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Acute enteroviral infections ranging from meningitis, pancreatitis to myocarditis are common and normally well controlled by the host immune system comprising virus-specific CD8 + cytotoxic T lymphocytes (CTL). However, in some patients enteroviruses and especially coxsackieviruses of group B are capable of inducing severe chronic forms of diseases such as chronic myocarditis. Currently, it is not known whether divergences in the CTL-related immune response may contribute to the different outcome and course of enterovirus myocarditis. A pre-requisite for the study of CTL reactions in patients with acute and chronic myocarditis is the identification of CTL epitopes. In order to define dominant enterovirus CTL epitopes, we have screened, by using gamma interferon (IFN-c) ELISPOT, 62 HLA-A*01-and 59 HLA-A*02-positive healthy blood donors for pre-existing CTL reactions against 12 HLA-A*01 and 20 HLA-A*02 predicted CTL epitopes derived from coxsackieviruses of group B. Positive CTL reactions were verified by FACS analysis in a combined major histocompatibility complex-tetramer IFN-c staining. A total of 14.8 % of all donors reacted against one of the three identified epitopes MLDGHLIAFDY, YGDDVIASY or GIIYIIYKL. The HLA-A*02-restricted epitope ILMNDQEVGV was recognized by 25 % of all tested blood donors. For this peptide, we could demonstrate specific granzyme B secretion, a strong cytolytic potential and endogenous processing. All four epitopes were homologous in 36-92 % of group B enteroviruses, providing a strong basis for monitoring the divergence of T-cellbased immune responses in enterovirus-induced acute and chronic diseases.

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“…Vital assay was performed as described elsewhere (Hermans et al, 2004). In brief, PBMCs were pre-sensitized as described with ILMNDQEVGV, HIV and CEF peptides.…”

Section: Isolation Of Peripheral Blood Mononuclear Cells (Pbmcs)mentioning

confidence: 99%

“…Each peptide (2610 3 )-loaded T2 cell group was incubated for 24 h with varying amounts of pre-sensitized PBMCs (300-300 000) and subsequently analysed by FACS analysis on a FACSCantoII. The specific lysis was calculated according to Hermans et al (2004).…”

Section: Human Cd8mentioning

confidence: 99%

Identification of HLA-A01- and HLA-A02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

Weinzierl

Rudolf

Maurer

et al. 2008

Journal of General Virology

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“…Unfortunately, it is not able to simulate the biological environment of the body during in vitro CTL experiments due to numerous factors. Therefore, some researchers have suggested using in vivo CTL experiments (26)(27)(28), in which isogeneic target cells and antigen peptides are injected into animal bodies to compare the in vivo CTL activity of these 'treated' animals with that of the controls. However, these experiments are limited by the detection of reactions between specific CTLs and simple antigens.…”

Section: Discussionmentioning

confidence: 99%

High-dose immunosuppressant alters the immunological status of New Zealand white rabbits following skin transplantation

Cheng

Zhong

Jiang

et al. 2015

Experimental and Therapeutic Medicine

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Abstract. The aim of this study was to investigate the effect of an immunosuppressant on the immunological status of New Zealand white rabbits after skin grafting, and to evaluate a method for monitoring the immunological status of subjects with skin transplants. The rabbits were randomly divided into allograft rejection, autograft tolerance, nontransplant, allograft low-dose immunosuppressant and allograft high-dose immunosuppressant groups. The rabbits in the low-and high-dose immunosuppressant groups were treated with cyclosporine A intravenously 8 h prior to skin transplantation and once daily following transplantation at doses of 2 and 25 mg/kg, respectively. At 12 days after skin transplantation, the spleens of donor (female) rabbits and recipient (male) rabbits were harvested for the preparation of single-cell suspensions. The splenocytes from recipient and donor rabbits were labeled with 0.3 or 6 µM carboxy fluorescein diacetate succinimidyl ester, respectively, and a mixed cell suspension was prepared. The final preparation was intravenously injected into recipient New Zealand white rabbits. The ratio of the two fluorescently labeled cell populations in the peripheral blood was measured using flow cytometry at 1, 2, 4 and 8 h after the injection, and the cell death rate was calculated. Histological analysis was also performed on samples collected at the time of splenectomy. The cell death rates of the allograft rejection and low-dose immunosuppressant groups reached their highest levels 8 h after the injection of spleen cell suspension. Allogeneic spleen cells from donor male rabbits were almost completely removed within 8 h of injection. The cell death rate increased slowly in the nontransplant, autograft and high-dose immunosuppressant groups without specificity. This study provides a specific method for the in vivo monitoring of the immunological status of patients after skin grafting. This method can quickly and accurately detect the immunological status of recipients following the injection of a mixed splenocyte suspension, thereby indicating the strength of immune rejection by the immune systems of the recipients.

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“…These target cells were then combined in round-bottomed 96-well plate assay wells with 2-wk in vitro stimulated PBMC effector cells at effector-to-target ratio of 30:1, incubated at 37 C in 5% CO 2 for 6 and 16 h and analysed using a flow cytometry. Percent specific lysis was calculated as 100 -(number of pulsed / number of unpulsed surviving cells with effectors divided by number of pulsed / number of unpulsed surviving cells without effectors Â 100) [44].…”

Section: Ifn-c Elispot Assaymentioning

confidence: 99%

Induction of long‐lasting multi‐specific CD8⁺T cells by a four‐component DNA‐MVA/HIVA‐RENTA candidate HIV‐1 vaccine in rhesus macaques

Nkolola

Gléria

et al. 2006

Eur J Immunol

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As a part of a long‐term effort to develop vaccine against HIV‐1 clade A inducing protective T cell responses in humans, we run mutually complementing studies in humans and non‐human primates (NHP) with the aim to maximize vaccine immunogenicity. The candidate vaccine under development has four components, pTHr.HIVA and pTH.RENTA DNA, and modified vaccinia virus Ankara (MVA).HIVA and MVA.RENTA, delivered in a heterologous DNA prime‐MVA boost regimen. While the HIVA (Gag/epitopes) components have been tested in NHP and over 300 human subjects, we plan to test in humans the RENTA (reverse transcriptase, gp41, Nef, Tat) vaccines designed to broaden HIVA‐induced responses in year 2007. Here, we investigated the four‐component vaccine long‐term immunogenicity in Mamu‐A*01‐positive rhesus macaques and demonstrated that the vaccine‐induced T cells were multi‐specific, multi‐functional, readily proliferated to recall peptides and were circulating in the peripheral blood of vaccine recipients over 1 year after vaccine administration. The consensus clade A‐elicited T cells recognized 50% of tested epitope variants from other HIV‐1 clades. Thus, the DNA‐MVA/HIVA‐RENTA vaccine induced memory T cells of desirable characteristics and similarities to those induced in humans by HIVA vaccines alone; however, single‐clade vaccines may not elicit sufficiently cross‐reactive responses.

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The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo

Cited by 164 publications

References 44 publications

Identification of HLA-A01- and HLA-A02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

Identification of HLA-A01- and HLA-A02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

High-dose immunosuppressant alters the immunological status of New Zealand white rabbits following skin transplantation

Induction of long‐lasting multi‐specific CD8⁺T cells by a four‐component DNA‐MVA/HIVA‐RENTA candidate HIV‐1 vaccine in rhesus macaques

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The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo

Cited by 164 publications

References 44 publications

Identification of HLA-A*01- and HLA-A*02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

Identification of HLA-A*01- and HLA-A*02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

High-dose immunosuppressant alters the immunological status of New Zealand white rabbits following skin transplantation

Induction of long‐lasting multi‐specific CD8+ T cells by a four‐component DNA‐MVA/HIVA‐RENTA candidate HIV‐1 vaccine in rhesus macaques

Contact Info

Product

Resources

About

Identification of HLA-A01- and HLA-A02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

Identification of HLA-A01- and HLA-A02-restricted CD8+ T-cell epitopes shared among group B enteroviruses

Induction of long‐lasting multi‐specific CD8⁺T cells by a four‐component DNA‐MVA/HIVA‐RENTA candidate HIV‐1 vaccine in rhesus macaques