T淋巴细胞毒性试验Induction and Measurement of Cytotoxic T Lymphocyte Activity

SECTION IIIASSAYS FOR T CELL FUNCTION UNIT 3.11Induction and Measurement of Cytotoxic T Lymphocyte Activity Cytotoxic T lymphocytes (CTL) kill target cells on the basis of cell-surface antigen recognition and are important in the host response to tumors, transplants, and viruses. CTL react with a wide variety of antigens including minor and major histocompatibility alloantigens and antigens expressed by syngeneic cells, such as from haptens, tumor- associated products, and viruses. CTL are generated by precursor T lymphocytes (CTLp) following stimulation by specific antigens carried on “stimulator” cells in the presence of products of accessory and helper T cells (UNIT 3.12; Singer et al., 1984). In principle, any cell type can be used as targets for measuring CTL activity, although activated cells such as lymphoblasts, tissue culture cells, or tumor cells have proven to be best. This unit presents several protocols for generating and measuring CTL activity (see Fig. 3.11.1). The first (see Basic Protocol 1) describes generating CTL against some of the most commonly used target antigens. There are two methods for the quantita- tion of CTL activity, described in Basic Protocol 2 and Alternate Protocol 1. CTL use two pathways to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. In the chromium-release assay (see Basic Protocol 2), labeled antigenic targets are recognized and lysed, releasing ra- dioactivity into the supernatant. In the JAM test (see Alternate Protocol 1), CTL activity is determined by measuring degradation of radioactively labeled DNA in target cells that have undergone apoptotic cell death. Rather than measuring release of radioactivity, the JAM test measures the amount of DNA retained in target cells that are not killed by CTL. Support Protocols 1 and 2 detail the generation of CTL precursors (CTLp) against antigens that require priming in vivo. A second set of support protocols de- scribe the preparation of both stimulator and target cells for these responses using two representative antigens, trinitrophenyl (TNP; see Support Protocol 3) and viruses (see Support Protocol 4). Finally, Alternate Protocols 2 and 3 illustrate how to determine total CTLp activity in a population that might express cytolytic activity. These proto- cols bypass MHC restriction and the original antigen specificity of CTLp by polyclonal stimulation of CTLp with mitogens followed by attachment of CTL to target cells and subsequent cytolysis. BASIC PROTOCOL 1 INDUCTION OF CYTOLYTIC ACTIVITY IN CTL PRECURSORS Cytolytic activity can be induced in CTLp by a large number of antigens including major and minor alloantigens, haptens, and viruses. Generation of CTL involves culturing the responder cells, which include CTLp, with a population of stimulator cells that express the desired antigen. The population of responder cells may or may not have to be primed in vivo, depending upon the antigen type. In vivo priming is not required for studies employing allogeneic MHC antigens, because the frequency of T cells that recognize these antigens in most responder-stimulator combinations is sufficiently high to generate a primary response in vitro against stimulator lymphocytes; this is known as the mixed lymphocyte reaction (MLR; see also UNIT 3.12). Use of antigens not encoded by the MHC, on the other hand, requires that the responder cell population be derived from an in vivo Contributed by John Wonderlich, Gene Shearer, Alexandra Livingstone, and Andrew Brooks Current Protocols in Immunology (2006) 3.11.1-3.11.23 Copyright C© 2006 by John Wiley & Sons, Inc. In Vitro Assays for Mouse Lymphocyte Function 3.11.1 Supplement 72 Induction and Measurement of Cytotoxic T Lymphocyte Activity 3.11.2 Supplement 72 Current Protocols in Immunology In vivo priming of mice none minor histocompatibility antigens influenza viral antigens In vitro stimulation of splenocytes minor histocompatibility antigens influenza viral antigens major histocompatibility antigens (alloantigens) polyclonal induction and detection TNP-modified self antigens In vitro detection of CTL activity 51Cr-release assay (Basic Protocol 2) or JAM test none (Support Protocol 1) (Support Protocol 2) (Basic Protocol 1) (Basic Protocol 1) (Support Protocol 3) (Support Protocol 4) (Alternate Protocol 2) (Alternate Protocol 1) Figure 3.11.1 Induction and detection of cytotoxic T lymphocyte (CTL) activity using chromium- release assay. Relationships of protocols presented in this unit are indicated. primed source, in order to generate a significant CTL response in vitro (see Support Protocol 1 and Support Protocol 2). An exception to this rule is the response of CTLp to trinitrophenyl (TNP)-modified syngeneic stimulator cells (see Support Protocol 3). Finally, Basic Protocol 3 describes a flow cytometric method for measuring CTL activity in vivo. CTL effector cells are generated in virally infected or peptide-primed mice, which are then injected with peptide-pulsed target cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). CTL activity is then quantified by flow cytometry, using spleen or lymph node cells from these mice, by measuring the loss of the CFSE- bright cells. In this protocol, single-cell suspensions are prepared with responder mouse spleen cells (primed or unprimed) and stimulator cells (virus- or hapten-modified or unmodified, also from mouse spleen). Stimulator cell division is blocked by addition of mitomycin C (or by exposure to γ irradiation), then the cells are washed extensively to remove all traces of the compound. The two cell populations are then mixed at optimal densities that have been predetermined by titration. After a 5-day incubation, the nonadherent CTL are harvested and used as effector cells in the chromium-release assay (see Basic Protocol 2) or JAM test (see Alternate Protocol 1). Materials Source of responder cells: unprimed or in vivo primed mouse spleen cells (see Support Protocols 1 and 2) Sensitization medium (see recipe) Source of stimulator cells: unmodified or hapten-modified (see Support Protocol 3) mouse spleen cells Complete RPMI-10 medium (APPENDIX 2) In Vitro Assays for Mouse Lymphocyte Function 3.11.3 Current Protocols in Immunology Supplement 72 0.5 mg/ml mitomycin C prepared in HBSS (stored in dark) Con A or IL-2 (optional) 15-ml disposable polystyrene conical tubes with screw caps (e.g., Falcon) Sorvall H-1000B rotor (or equivalent) 24-well flat-bottom microtiter plate, 2-ml capacity, with lids (e.g., Costar) Additional materials for preparing single-cell suspensions (UNIT 3.1) and cell viability test using trypan blue exclusion (APPENDIX 3B) NOTE: The mice should be free of infectious agents that can affect results, such as mycoplasma and immunosuppressive viruses (e.g., Sendai). 1. In a 15-ml conical tube, prepare a single-cell suspension (UNIT 3.1) of −1 × 107 responder cells/ml in sensitization medium. For initial titration experiments (see step 5), prepare a total of 10 × 107 responder cells in 10 ml. For responses to MHC antigens or to hapten (TNP)-modified stimulator cells, responder cells can be prepared from unprimed mice. Responder cells from mice primed in vivo are prepared as in Support Protocols 1 or 2. 2. In a 15-ml conical tube, prepare a single-cell suspension of −1 × 107 stimulator cells/ml in complete RPMI-10 (UNIT 3.1). For initial titration experiments (see step 5), prepare a total of 5 × 107 responder cells in 5 ml. Red blood cells may be removed (UNIT 3.1), but this is not strictly necessary. For responses against major and minor histocompatibility antigens (e.g., H-Y antigens), use as stimulator cells normal spleen cells from strains that differ from the responding strain by the antigen(s) of interest. For responses against other antigens, use spleen cells syngeneic to the responder cells as stimulator cells following appropriate antigen exposure (see Support Protocols 3 or 4). 3. Add 0.5 mg/ml mitomycin C to the stimulator cell suspension to 25 µg/ml final. Incubate 20 min in a humidified 37◦C, 5% CO2 incubator (or at 37◦C in tightly capped tube protected from light). Mitomycin C treatment blocks cell division in the stimulating population. This is partic- ularly relevant for an MLR because the stimulator cells can also recognize alloantigens on the responder cells. Although syngeneic stimulator cells (such as those used for anti- viral and anti-TNP responses) will not recognize responder cells as foreign, blocking the division of stimulator cells is recommended for providing a clear distinction between responder and stimulator cells. Recovery of cells after mitomycin C treatment may be as low as 50%. An alternative treatment for blocking cell division is γ irradiation using 2000 rad (tumor cells may require very high doses of >10,000 rad; see UNIT 3.12). Irradiation with dose >2000 rad inhibits antigen-presenting activity of B cells but not that of macrophages or dendritic cells. 4. Fill tube containing stimulator cells with complete RPMI-10 and centrifuge 5 min at 200 × g (1000 rpm in Sorvall H-1000B rotor), room temperature. Discard supernatant and wash three times with −14 ml medium using these centrifuge conditions, ending the wash regime with stimulator cell pellets. Extensive washing is important, as carry over of mitomycin C could block function of responder cells. Aspirating the supernatant down to the pellet will improve the efficiency of the washing process. If stimulator cells are irradiated rather than treated with mitocyin C, a single subsequent wash is adequate. Adequacy of irradiation or mitomycin C treatment in blocking cell division is assessed by measuring radiolabeled thymidine incorporation (UNIT 3.12). Induction and Measurement of Cytotoxic T Lymphocyte Activity 3.11.4 Supplement 72 Current Protocols in Immunology 5. Using sensitization medium, adjust responder cells to 2–10 × 106 cells/ml and stim- ulator cells to 1–6 × 106 cells/ml. Optimal concentrations of responder and stimulator cells will vary somewhat with the nature of the antigenic differences and with prior activation of cells in situ. Titration of both cell types will be necessary to identify the optimal concentrations; a typical pilot experiment may include a cross-hatch of three concentrations of responder cells (8, 4, and 2 × 106/ml), and three concentrations of stimulator cells (1, 2, and 4 × 106 cells/ml). 6. Add 1 ml each of responder and stimulator cells to the wells of a 24-well microtiter plate. Final cell concentration (i.e., sum of responder and stimulator cells) should not exceed 12 × 106/well in a volume of 2 ml. Prepare replicates of wells sufficient to generate the required number of effector cells. For initial titration experiments (see step 5), it will be sufficient to prepare six wells for each concentration of responder and stimulator cells. Cell recovery after 5 days is generally 50% to 100% of the responder cells initially plated. Cultures can be scaled down to 0.2 ml in microtiter wells, or up to 10 ml in 25-cm2 upright tissue culture flasks, maintaining the same cell concentrations and ratios of responder to stimulator cells. Adding Con A supernatant (∼10% v/v) or human recombinant IL-2 (∼10 U/ml) when cultures are initiated may enhance the generation of CTL activity. However, the Con A must be neutralized with α-methyl-D-mannoside before adding supernatant to the cultures, and the supernatant preparation must be titrated to determine the best concentration for maximal generation of CTL activity and minimum generation of natural killer (NK)-like activity. If IL-2 is added, be aware that it can bypass functions of ancillary cells, such as helper T cells, required for generating CTL (see UNIT 6.3). 7. Culture cells 5 days in a humidified 37◦C, 5% CO2 incubator. Leave the lids attached to the plates but allow air exchange. 8. Harvest nonadherent effector cells as follows. Using a 5- or 10-ml disposable pipet, transfer cells to a 15-ml conical tube, centrifuge 5 min at 200 × g, room temperature, and resuspend the cell pellet in complete RPMI-10 at −1–5 × 106 cells/ml in a tightly capped tube. Maintain at room temperature or on ice until CTL activity is assayed by 51Cr-release (see Basic Protocol 2) or DNA degradation (see Alternate Protocol 1). Provided that cells are gently resuspended from time to time, they can remain at room temperature for at least 4 hr without any diminution of cytolytic activity. BASIC PROTOCOL 2 CHROMIUM-RELEASE ASSAY FOR MEASURING CTL ACTIVITY In this procedure, target cells are briefly labeled with 51Cr, washed, then mixed with effector CTL (prepared in Basic Protocol 1) at appropriate effector-to-target (E:T) ratios for varying periods of time. The amount of 51Cr released into the supernatant by killed target cells is quantitated. By comparison with 51Cr release of controls, the corrected percent lysis is calculated for each concentration of effector cells. Materials Target cells (e.g., single-cell suspension of splenic lymphoblasts, tissue culture cells, or tumor cells) Control target cells (matched to test target cells except for differences in antigen expression) Complete RPMI-10 medium (APPENDIX 2; antibiotics not necessary in medium used for this assay) Sensitization medium (see recipe) In Vitro Assays for Mouse Lymphocyte Function 3.11.5 Current Protocols in Immunology Supplement 72 Mitogen solution (optional; for preparing splenic lymphoblasts): 1 mg/ml Con A in PBS or 1 mg/ml LPS in H2O (to stimulate splenic T and B cells, respectively) ∼1 mCi/ml Na251CrO4 in isotonic medium, sterile and pyrogen-free (200 to 500 µCi/µg; DuPont NEN or Amersham) Fetal bovine serum (FBS), heat-inactivated 1 hr at 56◦C Effector cells (including CTL; see Basic Protocol 1) Control effector cells (nonsensitized spleen cells or spleen cells sensitized against irrelevant antigen) 2% (v/v) Triton X-100 in H2O 25-cm2 tissue culture flasks (e.g., Corning) 24-well flat-bottom microtiter plates, 2-ml capacity, with lids (e.g., Costar) Nylon filtration fabric, 112-µm mesh (optional; Tetco) 15-ml disposable polystyrene conical tubes with screw caps (e.g., Falcon) Sorvall H-1000B rotor (or equivalent) and microtiter plate carrier Multiwick supernatant harvesting system (Skatron) 96-well round-bottom microtiter plates with lids to fit supernatant harvesting system (Costar) Multichannel pipettor (50- to 200-µl) with disposable tips 51Cr counting tubes (Skatron) Additional materials for preparing single-cell suspension (UNIT 3.1) and cell viability test using trypan blue exclusion (APPENDIX 3B) CAUTION: Follow standard radiation-safety procedures when working with 51Cr solution and 51Cr-labeled target cells. Prepare target and effector cells 1. Prepare a single-cell suspension (UNIT 3.1) of target cells in complete RPMI-10 medium. If splenic lymphoblasts are to be used, preactivate (in vitro) ∼5 × 106 spleen cells/ml in ∼2 µg/ml Con A or −10 µg/ml LPS (both stocks diluted in sensitization medium) for stimulating T cells or B cells, respectively. Incubate spleen cells 2 to 3 days in a 25-cm2 upright tissue culture flask (containing ≤10 ml cells) or 2-ml flat-bottom microtiter plate (2 ml cells/well) in a humidified 37◦C, 5% CO2 incubator. Lymphoblasts, tissue culture cells, or tumor cells are recommended as sources of target cells because they take up more 51Cr with less subsequent spontaneous leakage than nonactivated, freshly explanted normal cells. If lymphoblasts are used, they must first be activated as described above. If tissue culture cell lines or tumor cells are used, they must be of the appropriate MHC phenotype; often-used tumor cells are P815 (H-2d), EL4 (H-2b), and LK (H-2k), all available from ATCC. An alternative to collecting spleens for stimulator cells (see Basic Protocol 1, step 2) on day 0 and again 2 to 3 days later for target cells is as follows. Collect spleens for both stimulator and target cells on day 0. Place tightly capped culture flasks for target cells at 4◦C; 2 to 3 days later add mitogen as noted in step 1, loosen cap, and transfer to a CO2 incubator for 2 to 3 days. Control target cells should not express the antigens used for sensitization and should be matched as closely as possible to the test cells, except for differences in antigen expression. Control cells are also prepared in complete RPMI-10. 2. Transfer cells to 15-ml conical tube and wash cells once in 14 ml complete RPMI- 10 by centrifuging 5 min in ∼200 × g (1000 rpm in Sorvall H-1000B rotor), room temperature. Discard supernatant. 3. Resuspend cells in 5 ml complete RPMI-10. Allow cell aggregates to settle by gravity for several minutes or pass cell suspension through a single layer of 112-µm-mesh Induction and Measurement of Cytotoxic T Lymphocyte Activity 3.11.6 Supplement 72 Current Protocols in Immunology nylon filtration fabric resting on the open top of a 15-ml conical tube (press a pipet tip into the nylon fabric to form a small funnel). If a funnel is difficult to form with a 15-ml conical tube, a 50-ml tube can be used. 4. Determine viable cell count of unsettled or filtered cells by trypan blue exclusion (APPENDIX 3B). Target cells should have a high viability (>80%). Ficoll-Hypaque gradient centrifugation (UNIT 3.1) can enrich the population of live cells, but live cells isolated from a starting pop- ulation with poor viability generally leak 51Cr more than cells from a starting population with high viability. 5. Centrifuge ≤5 × 107 cells 5 min at 200 × g, room temperature, in a 15-ml conical tube. Discard most of supernatant, but leave ∼0.1 ml medium on the pellet. 6. Gently resuspend cells in remaining complete RPMI-10. Add 0.2 ml of −1 mCi/ml 51Cr solution and 20 µl FBS. Mix gently and incubate in a loosely capped 15-ml conical tube ∼45 min for lymphocytes or 1 to 2 hr for tumor cells at 37◦C, 5% CO2. Proceed to step 7 during incubation. The half-life of 51Cr is 28 days. Smaller volumes (e.g., 0.05 to 0.1 ml) of 51Cr solution may be adequate for labeling cells. Tumor cells and cultured cell lines may label adequately with a specific activity of <200 µCi/µg. The volumes of all materials can be expanded if larger numbers of 51Cr-labeled target cells are required. Gently resuspending cells once during incubation may enhance labeling. 7. Prepare effector cells by repeating steps 2 to 4 and resuspend in complete RPMI- 10 at 107 cells/ml. Prepare controls that differ from the test effector cells only in antigen specificity (nonsensitized cells or cells sensitized against irrelevant antigens). Prepare a series of tubes with 3-fold serial dilutions in complete RPMI-10 for each source of effector cells. 8. Dispense 0.1 ml effector cells (or controls) to the wells of 96-well microtiter plates, with replicates of three or four wells for each effector cell concentration. “Effector cells” commonly refers to all cells in a suspension consisting of CTL and nonlytic cells. E:T refers to the ratio of the total number of effector cells (not just CTL) to target cells. A ratio of 106 effector cells to 104 target cells per well is represented by E:T = 100. Use the suspected level of CTL activity to calculate the range of effector cell concentrations. A small dose-response assay in which four different E:T ratios are used is a good starting point; this assay commonly includes replicates in which the E:T ratios are 100, 30, 10, and 3. Unusually active CTL can result in detectable lysis at effector:target ratios <1:1. Concentrations >2 × 106 cells/well commonly suppress activity. 9. Wash 51Cr-labeled target cells 2 to 3 times with 14 ml complete RPMI-10 as in step 2, aspirating down to the cell pellet between washes and collec