Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells

Mbitikon-Kobo, Florentin-Martial; Bonneville, Marc; Sékaly, Rafick‐Pierre; Trautmann, Lydie

doi:10.1016/j.jim.2011.09.008

Cited by 18 publications

(17 citation statements)

References 10 publications

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“…To determine the effectiveness of HIV-specific CD8 T cells from acute/early infection to lyse infected target cells, we measured the intrinsic killing capacity of HIV-specific CD8 T cells in a new assay to quantify killing in lytic units ( Figure 5G). 24 We observed that HIV-specific CD8 T cells in primary infection exhibited a significantly higher cytotoxic capacity than HIV-specific CD8 T cells in chronic infection (mean lytic units of 237 and 72, respectively) and BLOOD, 25 OCTOBER 2012 ⅐ VOLUME 120, NUMBER 17 For personal use only. on May 10, 2018. by guest www.bloodjournal.org From comparable with the cytotoxic capacity of CMV-specific CD8 T cells (mean lytic unit of 227; Figure 5H) with comparable frequencies of tetramer ϩ cells (supplemental Figure 1D).…”

Section: Discordant Cytokine Production and Cytolytic Capacity Of Hivmentioning

confidence: 78%

“…The cytotoxic capacity of HIV-and CMV-specific CD8 T cells was measured in lytic units using the protocol detailed in Mbitikon-Kobo et al 24 Briefly, autologous B cells were positively isolated and stained with low and high concentrations of CFSE. Cells stained with the low concentration of CFSE were loaded with the cognate peptide for 1 hour at 37°C, washed twice, and mixed with cells stained with the high concentration of CFSE.…”

Section: Analysis Of Antigen-specific Cd8 T-cell Cytotoxic Capacitymentioning

confidence: 99%

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Profound metabolic, functional, and cytolytic differences characterize HIV-specific CD8 T cells in primary and chronic HIV infection

Trautmann

Mbitikon-Kobo

Goulet

et al. 2012

Blood

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Immediate-early host-virus interactions that occur during the first weeks after HIV infection have a major impact on disease progression. The mechanisms underlying the failure of HIV-specific CD8 T-cell response to persist and control viral replication early in infection are yet to be characterized. In this study, we performed a thorough phenotypic, gene expression and functional analysis to compare HIV-specific CD8 T cells in acutely and chronically infected subjects.We showed that HIV-specific CD8 T cells in primary infection can be distinguished by their metabolic state, rate of proliferation, and susceptibility to apoptosis. HIV-specific CD8 T cells in acute/early HIV infection secreted less IFN-␥ but were more cytotoxic than their counterparts in chronic infection. Importantly, we showed that the levels of IL-7R expression and the capacity of HIV-specific CD8 T cells to secrete IL-2 on antigenic restimulation during primary infection were inversely correlated with the viral set-point. Altogether, these data suggest an altered metabolic state of HIV-specific CD8 T cells in primary infection resulting from hyperproliferation and stress induced signals, demonstrate the discordant function of HIV-specific CD8 T cells during early/acute infection, and highlight the importance of T-cell maintenance for viral control. (Blood. 2012;120(17):3466-3477)

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Section: Discordant Cytokine Production and Cytolytic Capacity Of Hivmentioning

confidence: 78%

Section: Analysis Of Antigen-specific Cd8 T-cell Cytotoxic Capacitymentioning

confidence: 99%

Profound metabolic, functional, and cytolytic differences characterize HIV-specific CD8 T cells in primary and chronic HIV infection

Trautmann

Mbitikon-Kobo

Goulet

et al. 2012

Blood

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“…Cytotoxic T lymphocyte (CTL) assays were done as previously described with a little modification (17). In briefly, splenocytes were stimulated in vitro with 60 Co-irradiated HPD-1NR tumor cells infected with dl312 at a ratio of 5:1 in RPMI1640 medium for 4 days.…”

Section: Ctl Assaysmentioning

confidence: 99%

The Efficacy of Oncolytic Adenovirus Is Mediated by T-cell Responses against Virus and Tumor in Syrian Hamster Model

Wang

et al. 2017

Clinical Cancer Research

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Purpose: Oncolytic adenoviruses (Ad) represent an innovative approach to cancer therapy. Its efficacy depends on multiple actions, including direct tumor lysis and stimulation of antiviral and antitumor immune responses. In this study, we investigated the roles of T-cell responses in oncolytic adenoviral therapy.Experimental Design: An immunocompetent and viral replication-permissive Syrian hamster tumor model was used. The therapeutic mechanisms of oncolytic Ad were investigated by Tcell deletion, immunohistochemical staining, and CTL assay.Results: Deletion of T cells with an anti-CD3 antibody completely demolished the antitumor efficacy of oncolytic Ad. Intratumoral injection of Ad induced strong virus-and tumorspecific T-cell responses, as well as antiviral antibody response.Both antiviral and antitumor T-cell responses contributed to the efficacy of oncolytic Ad. Deletion of T cells increased viral replication and extended the persistence of infectious virus within tumors but almost abrogated the antitumor efficacy. Preexisting antiviral immunity promoted the clearance of injected oncolytic Ad from tumors but had no effect on antitumor efficacy. Strikingly, the repeated treatment with oncolytic Ad has strong therapeutic effect on relapsed tumors or tumors insensitive to the primary viral therapy.Conclusions: These results demonstrate that T cell-mediated immune responses outweigh the direct oncolysis in mediating antitumor efficacy of oncolytic Ad. Our data have a high impact on redesigning the regimen of oncolytic Ad for cancer treatment.

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“…The percentage of specific lysis for each well was calculated as follows: % specific lysis = 100 -((CFSE low/CFSE high) in the presence of effector cells / mean of the three wells containing target cells alone in the absence of effector cells) x100. To determine the intrinsic cytolytic capacity of antigen-specific CD8+ T cells and compare results between donors, the cytolytic activity was expressed in lytic units as previously reported 8 . The effector versus target ratio (E/T) was measured by calculating the ratio between total tetramer+ CD8+ T cells and total CFSE-low CD4+ T cells for each well.…”

Section: For Each Well the Ratio Between Pulsed And Un-pulsed Live Cmentioning

confidence: 99%

“…We have previously shown that this method was reproducible, sensitive, specific and did not depend on the number of effector cells within the total CD8+ T cell population 8 . By enumerating both the number of effector and target cells in the coculture assay, the intrinsic capacity of CD8+ T cells to kill target cells can be calculated and expressed in lytic units 9 .…”

Section: Introductionmentioning

confidence: 95%

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Noto

Ngauv

Trautmann

2013

JoVE

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Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU₃₀/10(6) cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

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Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells

Cited by 18 publications

References 10 publications

Profound metabolic, functional, and cytolytic differences characterize HIV-specific CD8 T cells in primary and chronic HIV infection

Profound metabolic, functional, and cytolytic differences characterize HIV-specific CD8 T cells in primary and chronic HIV infection

The Efficacy of Oncolytic Adenovirus Is Mediated by T-cell Responses against Virus and Tumor in Syrian Hamster Model

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

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