利用<sup>3</sup>[H]-胸苷掺入分析和Luminex多重细胞因子分析技术检测免疫小鼠脾脏中抗原特异性T细胞

Maria  Agallou; Evdokia  Karagouni

doi:10.21769/BioProtoc.3252

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Peer-reviewed

Detection of Antigen-specific T cells in Spleens of Vaccinated Mice Applying ³[H]-Thymidine Incorporation Assay and Luminex Multiple Cytokine Analysis Technology

利用³[H]-胸苷掺入分析和Luminex多重细胞因子分析技术检测免疫小鼠脾脏中抗原特异性T细胞

Maria Agallou

Evdokia Karagouni email

发布: 2019年06月05日第9卷第11期 DOI: 10.21769/BioProtoc.3252 浏览次数: 11180

评审: Alexandros AlexandratosWoojong LeeMichela Perego

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Abstract

For many infectious diseases T cells are an important part of naturally acquired protective immune responses, and inducing these by vaccination has been the aim of much research. Here, we describe a protocol for the analysis of vaccine-induced antigen-specific immune responses. For this purpose, cells of whole spleens obtained from vaccinated BALB/c mice were ex vivo stimulated with the antigen incorporated in the vaccine. Evaluation and characterization of vaccine-induced adaptive T cell responses was performed by assaying spleen cell proliferation through radioactive ³[H]-thymidine incorporation and multiplex cytokine analysis of IL-2, IFN-γ and TNFα in supernatants from spleen cell suspensions. This protocol can be very useful as a starting point for assessing vaccine-induced memory T cell populations in pre-clinical studies.

Keywords: Splenocyte cultures (脾细胞培养)

Immunization (免疫接种)

Antigen-specific proliferation (抗原特异性增殖)

Multiplex cytokine detection (多重细胞因子检测)

Background

T cells have an essential role in protection against a variety of infections by controlling or delaying the onset of disease. Thus, vaccine development for these infections is focused on generating protective T-cell responses (Seder and Hill, 2000). Therefore, selecting T-cell readouts to measure in pre-clinical vaccines studies to assess immunogenicity would aid progress on T cell vaccines. Ideally, a T cell–vaccine would induce long-lived memory T cells, characterized as central memory (T_CM) within lymphoid tissues, i.e., lymph nodes and spleen, and effector memory (T_EM) within peripheral tissues. Memory T cells when encounter the previously given antigen via vaccination they are clonally expanded in order to differentiate into effector cells. A key mechanism by which T cells mediate their effector function is through the simultaneous production of IFN-γ, TNFα and IL-2 (Darrah et al., 2007). IFN-γ synergize with TNFα to promote killing of pathogens through activation of macrophages, whereas IL-2 strongly enhances the expansion of T cells leading to more efficient effector responses. Therefore, among the most common ways to assess the existence of vaccine-induced cell effector populations is to measure spleen cell proliferation and IL-2, TNFα and IFN-γ production upon ex vivo stimulation with the antigens that have been incorporated in the vaccine (Seder et al., 2008). Cell proliferation encompasses DNA synthesis, mitosis and an increase in cell number. The ³[H]-thymidine incorporation assay allows the measurement of ³[H]-thymidine into DNA during the S phase of the cell cycle as a quantitative measure of new DNA synthesis and yields statistically significant data even in the presence of low number of divided cells. This is achieved due to the linear relationship between the count rates and the rate of DNA synthesis (Ahern et al., 1976). Thus, this method has been a gold standard for measuring cell proliferation in mixed lymphocyte cultures. If the goal of the study is not only to determine DNA synthesis but also to characterize the fraction of cells in the S phase, then CFSE labeling stands out as a versatile alternative. Despite the fact that the ³[H]-thymidine incorporation assay is a radioactive method, it is still considered one of the best and most reliable methods providing statistically significant data. On the other hand, there are various established methods for assessing cytokine production. Typically, ELISA and ELISpot assays are the gold standard methods for quantifying antigen-specific cellular responses after vaccination but they are limited only to a single parameter readout (Ranieri et al., 2014). Intracellular cytokine staining and analysis by flow cytometry is another method of choice as it allows both multi-parameter cytokine analysis and phenotyping of cells (Freer and Rindi, 2013). However, it is characterized by many disadvantages. Among them are that it is labor intensive, it costs more per sample and requires a large number of cells (Freer and Rindi, 2013). On the other hand, multiplex arrays allow multiple cytokine analysis using different fluorescent beads that depending on the kit used a maximum of 80 cytokines can be investigated. The cytometric bead array system and the Luminex multi-analyte profiling (xMAP) technology both employ beads sets which are distinguishable under flow cytometry in the absence of Luminex machine. Moreover, they have been used in various pre-clinical studies to explore cytokines profiles induced by vaccination, since different samples can be used, i.e., serum or cell-supernatant (Flaxman and Ewer, 2018). Based on the above, we describe a detailed protocol for assessing activation and effector functions of antigen-specific spleen cell populations using ³[H]-thymidine incorporation and Luminex assays.

Materials and Reagents

Pipettes tips: 0.5-10 μl, 10-200 μl, 200-1,000 μl (Greiner Bio-One, catalog numbers: 771291, 739290, 740290)
50 ml sterile conical tube (Thermo Fischer Scientific, catalog number: 339652)
15 ml sterile conical tube (Thermo Fischer Scientific, catalog number: 339650)
Disposable sterile pipettes: 2 ml, 5 ml, 10 ml (Greiner Bio-One, catalog numbers: 710180, 606180, 607180)
96-well sterile cell culture U-bottom plate (Greiner Bio-One, catalog number: 650180)
24-well sterile cell culture plate (Greiner Bio-One, catalog number: 662160)
5 ml syringe (Terumo, catalog number: SS+05S21381)
70 μm Nylon Cell strainer (Falcon, catalog number: 352350)
Petri dishes (Greiner Bio-One, catalog number: 664102)
Eppendorf tubes (Greiner Bio-One, catalog number: 616201)
Glass fiber filtermat (PerkinElmer, catalog number:1205-401)
Sample Bags for Microbeta (PerkinElmer, catalog number: 1405-432)
0.22 μm filter (Merck Millipore, catalog number: SLGV004SL)
BALB/c female mice, 6-8 weeks old (Department of Animal Models for Biomedical Research, Hellenic Pasteur Institute)
Antigens of interest. For the purposes of the present protocol re-stimulation of spleen cells was conducted with multi-epitope peptides (i.e., CPA_160-198 and EF-2_1-137) contained in experimental vaccines against parasite Leishmania developed in our laboratory
RPMI-1640 (Biowest, catalog number: L0501-500)
HEPES (Biowest, catalog number: L0180-100)
Penicillin-streptomycin (100x) (Biowest, catalog number: L0022-100)
L-glutamine (100x) (Biowest, catalog number: X0550-100)
Fetal bovine serum Premium (FBS) (catalog number: LSB-015S)
Trypan blue for vital staining (B.D.H., catalog number: 34078)
Ammonium chloride (NH₄Cl), ACS reagent, ≥ 99.5% (Sigma-Aldrich, catalog number: 213330)
Potassium bicarbonate (KHCO₃), ACS reagent, ≥ 99.7% (Sigma-Aldrich, catalog number: 237205)
Hydrochloric acid (HCl) solution, 1 M (Sigma-Aldrich, catalog number 150696)
Ethylenediaminetetraacetic acid disodium salt dehydrate (Na₂EDTA), ACS reagent, 99.0-101.0% (Sigma-Aldrich, catalog number: E4884)
Sodium chloride 99.9% (NaCl) (Applichem, catalog number: 381659)
Potassium chloride (KCl), ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: 746336)
Sodium phosphate dibasic (Na₂HPO₄), ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: 795410)
Potassium phosphate monobasic (KH₂PO₄), ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: 795488)
Sodium hydroxide (NaOH) solution, 1 M (Sigma-Aldrich, catalog number 79724)
Concanavalin A from Canavalia ensiformis (Sigma-Aldrich, catalog number C5275)
Thymidine, [methyl-³H] (PerkinElmer, catalog number: NET27X001MC)
Scintillation fluid (PerkinElmer, catalog number: 1200-439)
Milliplex^® MAP Kit (MerckMillipore Corporation, catalog number: MCYTOMAG-70K)
(Optional) Array Mouse Th1/Th2 Cytokine Kit (BD Biosciences, catalog number: 551287); LEGENDplex Mouse Th1/Th2 Panel (8-plex) (Biolegend, catalog number: 740750); FlowCytomix mouse Th1/Th2 10plex (Bender MedSystems, catalog number: BMS720FF)
Ammonium chloride lysis solution (ACK, pH 7.2) (see Recipes, page 10)
Phosphate-buffered saline, 10x (PBS, pH 7.2) (see Recipes, page 10)
Complete RPMI medium (see Recipes, page 10)
Concanavalin A stock solution (see Recipes, page 10)
0.4% (w/v) Trypan blue exclusion dye (see Recipes, page 10)

Equipment

Dressing scissor straight sharp with blunt forceps, skylar dressing forcep and forcep full curved, all kept sterile with 70% ethanol
Gilson Pipettes (P-10, P-20, P-200, P-1000)
Multichannel pipette (Nichiryo, Nichipet 7000)
Neubauer counting chamber (Paul Marienfeld GmbH & Co.)
CO₂ incubator (New Brunswick, Galaxy 170S)
Laminar flow hood (Telstar, Bio-II-A)
Refrigerated centrifuge (Centurion Scientific Ltd, PrO-Research)
Light microscope (Olympus, BH)
Water bath (LabTech, LSB-015S)
Combi cell harvester (Skatron Instruments, 11025/11028)
Microplate scintillation and luminescence counter (Wallac, Microbeta 1450 Trilux)
Luminex^® 200^TM System with xPONENT^® Software (Life Technologies, Novex^®)

Software

Cytokine production data were analyzed using the xPONENT^® software (Luminex)
Statistical tests were performed using GraphPad Prism v6 software
Note: The different groups were compared using One-way ANOVA with the Tukey’s multiple comparison test, comparing all groups with the non-vaccinated mice group.

Procedure

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Agallou, M. and Karagouni, E. (2019). Detection of Antigen-specific T cells in Spleens of Vaccinated Mice Applying ³[H]-Thymidine Incorporation Assay and Luminex Multiple Cytokine Analysis Technology. Bio-protocol 9(11): e3252. DOI: 10.21769/BioProtoc.3252.