Elsevier

Methods

Volume 61, Issue 2, 1 June 2013, Pages 105-109
Methods

In vivo assessment of specific cytotoxic T lymphocyte killing

https://doi.org/10.1016/j.ymeth.2013.02.007Get rights and content

Abstract

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18–24 h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells.

Introduction

Cellular cytotoxicity is the main effector mechanism of CD8+ T cells (also known as CTLs-cytotoxic T lymphocytes) constituting an important weapon against cancer and pathogen-infected cells. The scientific and technological advances in the last couple of decades have improved the analysis of CTL activation, differentiation and effector function. The classical test to assess cellular cytotoxicity is the radioactive chromium (51Cr) release assay, which was developed in 1968 by Brunner and colleagues based on binding and internalization of radioisotopes by target cells [1]. The development of the enzyme-linked immunosorbent spot assays (ELISPOT) and the construct of multimeric MHC molecules plus particular peptides improved the detection and characterization of antigen-specific T cells. However, in vitro and ex vivo analysis are less informative than in vivo assays since they do not take into account the complexity of the microenvironment where the immune response occurs and, therefore, do not always reproduce what occurs in vivo. For instance, it was observed that even after 300 days of exposure to viral agent, 84% of target cells transferred were eliminated in vivo, while negative results were observed in vitro or ex vivo [2]. In addition, several paradigms based on in vitro studies were dismissed or at least reformulated after in vivo experiments. In this case, the results showed that absence of granzymes A and B impair in vitro CTL killing [3]. The same authors showed later that mice deficient in both granzymes A and B are able to eliminate target cells in vivo as quickly and in a similar manner than wild-type mice without using any compensatory mechanisms, such as increased expression of orphan granzymes or FAS–FASL interactions [3]. Thus, in vivo assays have been gaining more and more attention as important tools to comprehensively evaluate CTL-mediated immune responses.

The in vivo CTL assay we describe here was first proposed by Oehen and colleagues [4] and is based on inoculation of immunized or infected mice with a 1:1 proportion of labeled syngeneic spleen cells, where one population is pulsed with a peptide for which the specific CTLs response will be analyzed. The presence of labeled cells in the spleen of the recipient mice is evaluated after different periods of time by flow cytometry. We detail the procedure and analysis of the in vivo CTL assay using a model of genetic immunization using as vector human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes and discuss the use and limitations of this test.

Section snippets

Immunization with recombinant adenoviruses

Adenoviruses have several characteristics that make them excellent candidates for use as gene delivery system (or vectors) to induce specific immune responses [5]. Adenoviruses exert an adjuvant-like effect by stimulating the innate immune system via both Toll-like receptors (TLR)-dependent and -independent mechanisms [6]. Inoculation of adenoviruses triggers a characteristic immune response based on the development of neutralizing antibodies and CTLs, aiming to neutralize the extracellular

Processing of target cells

Once a CTL bearing a specific TCR finds a cognate peptide associated with a MHC class I molecule on the surface of a target cell, the CTL become activated and can kill the target cell. Normally, cytosolic proteins are processed by the proteasome, generating peptides that are transported to the endoplasmic reticulum (ER) via a “pore” formed by TAP-1 and TAP-2 (TAP: transporter associated with antigen processing) proteins. There, the peptides associate with nascent MHC class I molecules and the

Labeling control and target cells with CFSE

To prepare the populations of target and control cells, splenocytes obtained as described above were divided into two tubes containing 5 × 107 cells/ml and labeled with pre-warmed (10 min, 37 °C) CFSE (carboxyfluorescein diacetate succinimidyl diester – Molecular Probes, Eugene, Oregon, USA) at either 1 μM (CFSElow) or 10 μM (CFSEhigh) of the fluorescent dye in PBS. Splenocyte suspensions should be homogenized well, incubated at 37 °C for 15 min and centrifuged at 300g for and 10 min at 4 °C. Supernatants

Pulse target cells with specific peptides

As mentioned above, CTL will only kill target cells bearing the specific peptide associated with self-MHC class I molecules. In the study described here, the target CFSEhigh cells were pulsed with 1 μg/ml of the H2-Kb restricted ICPMYARV peptide, a representative β-Gal epitope (β-Gal 497–504) [11], for 40 min at 37 °C (protected from light), while the control CFSElow cells remained unpulsed or were pulsed with an irrelevant peptide (PA8-VNHRFTLV). Alternatively, in the case of A/Sn mice, cells

Injection of labeled cells in mice

In procedures requiring the delivery of agents intravascularly in mice, it is common to use one of the lateral tail veins. However, this can be technically tricky and there is a high risk of failure. Retro-orbital injections are considered by many a more humane alternative and easier to perform [12]. Thus, we suggest the retro-orbital route to inoculate the CFSE-labeled cells. Briefly, each mouse received a mixture of 2 × 107 CFSElow and 2 × 107 CFSEhigh cells in a total volume of 200 μL of

Flow cytometry analysis/specificity of the immune response

Initially, we illustrated the specificity of this assay and its dependence on CD8 T cells by evaluating in vivo specific CTL response after immunization with HAd5 wild type and CD8 KO C57Bl/6 mice. To obtain back the injected CFSElow and CFSEhigh cells, splenocytes were prepared from the injected mice accordingly to the same procedure described above. Single-cell suspensions were washed three times in RPMI-10 and fixed with 1.0% paraformaldehyde. At least 100,000 events were acquired on a

Combined use of other dyes

Other dyes may be used as alternative to or in parallel with CFSE. Among the various commercially available vital markers we show here the results with Cell Trace Violet dye (Molecular Probes, Eugene, Oregon, USA). The labeling consists in a protocol similar to that of CFSE. Non target cells were labeled with 1 μM of CFSE and the target cell population labeled with 5 μM of Cell Trace Violet. As expected, we observed the disappearance of the cell trace violet-positive cells only when they were

Calculating the cytotoxicity

The percentage of specific lysis was determined using the formula:1-%CFSEhighinfected/%CFSElowinfected%CFSEhighnaive/%CFSElownaive×100%

Statistical analysis

All statistical analysis described here were performed with Graphpad Prism (Graphpad Software Company Incorporation). Values were expressed as means ± SD. These values were compared using two-way ANOVA followed by BonFerroni tests. The differences were considered significant when the P value was <0.05.

Tips

The in vivo CTL assay is relatively complex if taken into account the wide-range of manipulation that can be applied to the system, both during the cell processing and labelling and the data acquisition and analysis. However, the reproducibility can be very high if some considerations were followed:

  • 1.

    To make sure that the CFSE staining occurs with success without major loss in cell viability, the PBS used to prepare the CFSEhigh and CFSElow solutions should be pre-warmed at 37 °C and labeling

Conclusion

The protocol described in this study allows a meticulous in vivo evaluation of cytotoxic T cell activity.

Acknowledgments

We thank Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Instituto Nacional de Ciência e Tecnologia (INCT) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their ongoing support of the work carried out in our laboratories.

References (12)

  • D.S. Bangari et al.

    Vaccine

    (2006)
  • S.K. Mittal et al.

    Virus Res.

    (1993)
  • K.T. Brunner et al.

    Immunology

    (1968)
  • W. Barchet et al.

    Eur. J. Immunol.

    (2000)
  • M. Regner et al.

    J. Immunol.

    (2009)
  • S. Oehen et al.

    Immunology

    (1998)
There are more references available in the full text version of this article.

Cited by (19)

  • A versatile photothermal vaccine based on acid-responsive glyco-nanoplatform for synergistic therapy of cancer

    2021, Biomaterials
    Citation Excerpt :

    Furthermore, CTL activation by I-R-Ap-AcDEX2 NPs in vivo was evaluated next. Mice were vaccinated with I-R-Ap-AcDEX2 NPs by one injection, and the resulting CTL activities of immunized mice were determined by the in vivo CFSE assay [54]. As shown in Fig. 5b, I-R-Ap-AcDEX2 NPs vaccination by subcutaneous route activated higher levels of Ap-specific CTLs than other groups, leading to significant death of Ap containing target cells.

  • DNA hypomethylating agents increase activation and cytolytic activity of CD8<sup>+</sup> T cells

    2021, Molecular Cell
    Citation Excerpt :

    Consistent with human CD8+ T cell co-culture experiments, we also observed an increased killing ability of target cells by DAC-treated murine CD8+ T cells (Figure 3H). Next, we assessed the CD8+ T cell effector function in vivo by performing an OVA antigen-specific in vivo killing assay using CFSE-labeled target cells as previously described (Clemente et al., 2013). Mice were immunized with OVA antigen delivered using an adenoviral vector and randomized into two groups.

  • The Mevalonate Pathway Is a Druggable Target for Vaccine Adjuvant Discovery

    2018, Cell
    Citation Excerpt :

    Moreover, OVA re-stimulation strongly induced Th1 cytokine production in lymphocytes from DLNs of mice immunized with simvastatin-OVA (Figure 6B), as well as enhancing lymphocyte cell proliferation (Figure 6C). An in vivo cytotoxicity analysis (Clemente et al., 2013) conducted 28 days after OVA vaccination showed that simvastatin-treated mice had a stronger ability than did alum in killing OVA-bearing cells (Figure 6D). Tetramer staining showed that 1% of CD8+ T cells were positive for the OVA-tetramer in simvastatin-immunized mice, higher than in the alum or CpG adjuvant controls (Figure 6E).

  • Combining different types of multifunctional liposomes loaded with ammonium bicarbonate to fabricate microneedle arrays as a vaginal mucosal vaccine adjuvant-dual delivery system (VADDS)

    2017, Journal of Controlled Release
    Citation Excerpt :

    The anti-OVA Ab titer was defined as the smallest dilution of sera at which the 450 nm OA reading is 0.5 and presented as a logarithm value estimated from the curve plotted by OA vs dilution. Analysis of in vivo Ag-specific cytolysis of induced CTLs in the vaginal MA vaccine-treated mice was conducted using the CFSE-labeled cells bearing Ag epitopes as previously described [46]. Briefly, the syngeneic splenocytes isolated from naive mice were washed with PBS supplemented with 3% FCS and 5 mM EDTA and resuspended at 2 × 107 cells/mL in PBS.

View all citing articles on Scopus
View full text