Glycoprotein methods protocols - biotechnology
Trang 138
Analysis of the Frequency of MHC-Unrestricted
MUC1-Specific Cytotoxic T-Cells in Peripheral Blood
by Limiting Dilution Assay
John R McKolanis and Olivera J Finn
1 Introduction
Mucins are highly glycosylated proteins present on the lumenal side of ductal epi-thelial cells MUC1 is the only mucin with a transmembrane region anchoring it to the cell surface The extracellular domain of MUC1 is composed of numerous tandem
repeats of a 20-amino acid sequence (1) Normal cells produce a highly glycosylated
and sialylated form of MUC1 The O-linked carbohydrate side chains of MUC1 on
tumor cells are shorter and less abundant, exposing previously unrecognized antigenic
sites on the polypeptide core of the molecule (2) This underglycosylated MUC1
mol-ecule on tumor cells can be recognized as a tumor-specific antigen by T-cells The major cytotoxic T-lymphocyte (CTL) response to tumor-specific MUC1 is T-cell receptor (TCR) mediated but major histocompatibility complex (MHC)-unrestricted Owing to the high density of repeating antigenic epitopes extending along each MUC1 molecule, it is postulated that a large number of TCRs can be triggered simultaneously
to activate the CTLs to kill tumor cells or proliferate (3–5) Antibodies specific for the
TCR, or for the defined MUC1 epitope recognized by the TCR, inhibit CTL recogni-tion Antibodies against MHC molecules have no effect.
In addition to the MHC-unrestricted CTLs, some MUC1-specific MHC-restricted CTLs have also been identified Patients who are HLA-A11 and HLA-A3 have CTLs that recognize a nine amino acid peptide from the tandem repeat region bound to these
alleles (6) Another peptide has been reported that binds HLA-A2.1 allele (7).
Although it is not yet clear whether these CTLs, MHC unrestricted or MHC restricted, have a therapeutic function, it has been considered important to measure their fre-quency pre- and post vaccination with MUC1-based immunogens used in various
clini-cal trials (7–9) in order to be able to correlate this specific component of the antitumor
response with the outcome of the trial.
From: Methods in Molecular Biology, Vol 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A Corfield © Humana Press Inc., Totowa, NJ
Trang 2We have established a limiting dilution assay (LDA) to evaluate the frequency of MHC-unrestricted MUC1-specific CTL This assay is convenient because the same conditions and reagents can be applied to all patients regardless of their HLA type We believe that these CTLs will be an important corollary of a tumor-specific immune response because they recognize an epitope that is expressed only on tumor cells and not on normal epithelial cells The same assay may be used to determine the frequency
of MHC-restricted CTL However, the assay must be custom tailored to each patient,
or at least to several specific HLA alleles.
2 Materials
1 RPMI-1640 tissue culture medium supplemented with 10% fetal calf serum (FCS) or
human AB serum, 2 mM L-glutamine, 100 Units/mL penicillin, 100 µg/mL streptomycin
2 Human male AB serum purchased from Gemini Bio Products, Calabasas, CA
3 FCS purchased from Life Technologies, Grand Island, NY
4 Glutamine, purchased as 100X stock from Life Technologies, Grand Island, NY
5 Penicillin-streptomycin, purchased as 100x stock from Life Technologies
6 Hepes buffer, purchased as 100X stock from Life Technologies
7 Recombinant interleukin-2 (rIL-2), obtained from several sources
8 Phytohemagglutinin (PHA-P), purchased from Sigma, St Louis, MO
9 Trypan blue, 0.4% stock solution, purchased from Life Technologies
10 Phosphate buffered physiologic saline (1.9 mM NaH2PO4, 8.1 mM NaHPO4, 154 mM
NaCl, pH 7.2); may also be purchased as a 10X stock solution
11 51Sodium chromate and 3H thymidine, purchased from Amersham, Arlington Heights, IL
12 Ficoll density gradient, purchased as LSM from Organon Technica Corp, Durham, NC
13 DNAase, purchased from Calbiochem, La Jolla, CA Filter-sterilized stock solution at
10 mg/mL in RPMI-1640 containing HEPES buffer should be kept frozen at –80°C
14 Dimethyl sulfoxide (DMSO) purchased from Sigma
15 Tumor cells that express MUC1 can be obtained from American Type Culture Collection (ATCC), Rockville, MD, or as gifts from various investigators Epstein-Barr virus (EBV) immortalized B-lymphoblastoid cell lines or other cells transfected with the MUC1 gene can be obtained from various investigators Natural killer and lymphokine-activated killer (LAK) cell target K562 can be purchased from ATCC
16 Styrofoam boxes for slow freezing of cells, purchased from Sarstadt, Newton, NC
17 96-well, U-bottomed microtiter plates, purchased from Becton Dickinson, Franklin Lakes, NJ
18 Cytotoxicity assay harvesting materials, purchased from Scatron Instruments, Sterling, VA
3 Methods
3.1 Isolation of Mononuclear Cells from Peripheral Blood
Blood samples are obtained from patients at specific time intervals, depending on the goals of the study Peripheral blood mononuclear cells are separated from the rest
of the blood using cenrifugation through Ficoll density gradient (LSM) This involves layering 10 mL of blood diluted 1:2 in RPMI medium without serum, over 3 mL of
LSM in a 15-mL conical centrifuge tube Following centrifugation at 400g for 20 min,
mononuclear cells can be removed from the interface between the two layers The cells are washed three times in phosphate-buffered saline (PBS) to remove contami-nating LSM From 1 mL of blood we obtain 1–2 × 106cells.
Trang 33.2 Cryopreservation of Cells
Mononuclear cells are frozen in 90% FCS and 10% DMSO at a concentration of 5–10 × 106 cells/mL The cells are first resuspended in cold FCS at one-half of the final volume The remaining volume of cold FCS with 20% DMSO is then added dropwise while swirling the tube A controlled rate of freezing can be simulated by simply placing the vials in a styrofoam box in a –80 °C freezer overnight The vials are then transferred to liquid nitrogen for long-term storage.
At the time of the assay, the vials are thawed rapidly in a 37 °C water bath The contents are transferred to 10 mL of RPMI-1640 containing 30 µg/mL of DNAase Following a 30-min incubation at 37 °C, the cells are washed three times in PBS Viable cells are counted by diluting the sample 1:2 in trypan blue and counting in a hemocy-tometer the cells that have not taken up the color blue.
3.3 Setting up the LDA Cultures
In the LDA culture assay, hundreds of peripheral blood lymphocyte microcultures are stimulated once in vitro with the appropriate antigen The cells are grown in 96-well
U bottomed plates.
1 At each dilution, seed peripheral blood lymphocytes in a 100-µL volume into 24 wells of
a round-bottomed 96-well microtiter plate (see Fig 1) We routinely use twofold
dilu-tions of cells starting with 2 × 105cells/well and ending with 6.25 × 103cells/well (see
Note 1) Culture medium is RPMI-1640 supplemented with 10% human AB serum,
peni-cillin, and streptomycin
2 Add irradiated (6000 RADS) stimulator cells expressing MUC-1 in a 50-µL volume at a concentration of 2 × 104/well
3 Use irradiated (3000 rad) autologous peripheral blood lymphocytes (PBLs) as “feeder” cells, and add at a concentration of 2 × 104/well in a 50-µL volume Suspend the “feeder”
cells in culture medium containing one half of the optimal concentration of IL-2 (see
Notes 1–4).
4 Following 7 d, in culture, remove 100 µL of culture medium from each well and replace with fresh medium containing IL-2 Perform the CTL assay on d 10
3.4 Setting up the CTL Assay
1 If adherent, trypsinize target cells and wash 1 × 106cells three times in PBS After the last wash, carefully aspirate the supernatant so that the smallest possible volume remains
2 Resuspend the cell pellet in 100 µL of fresh 51Cr, specific activity 1 mCi/mL, and incu-bate at 37°C for 1–2 h Shake the tube periodically to keep the cells in suspension
3 Following three washes, place 1 × 103 cells in a gamma counter to determine the uptake
of radioactivity and thus ensure proper labeling If less than 500 counts are obtained with tumor cells or 300 counts with EBV-transformed B-cells, a new batch of targets should be prepared The new targets should be labeled for a longer period of time or newer chro-mium should be used
4 While the targets are being prepared, the rest of the assay can be set up Each well of the original plates should be carefully mixed with a micropipettor and transferred into two wells of a 96-well microtiter V-bottomed plate (“split-well” assay) Thus, row A of the
original 96-well plate becomes rows A and B of the assay plate (see Fig 1) Each new
well contains 100 µL of the original 200-µL cell cuture
Trang 45 To each new well, add 2.5 × 104unlabeled LAK target cells in a volume of 50 µL For CTL assays with tumor cells as labeled target cells, we use unlabeled K562 cells When MUC1 transfected EBV B-cells are used as targets, we use unlabeled parental EBV cells
to compete out the LAK lysis
6 At each dilution, add 1 × 103of the labeled specific targets to one of the split wells and to the other well 1 × 103labeled control targets, in 50-µL volumes Include several wells containing only target cells for spontaneous and maximal release values
7 Centrifuge the plates at 150g for 5 min and incubate at 37°C for 4 h
8 Harvest supernatant from the wells and count the radioactive chromium released from lysed cells Obtain maximal release values following lysis of the cells in designated wells with acid or hypertonic saline
9 Calculate specific release using the following formula: 100 × (sample release –
spontane-ous release/maximal release – spontanespontane-ous release).
10 Calculate percentage of specific release for each well containing the specific target and compare this with the corresponding well containing the control target Wells are scored
positive if specific target cell lysis is 15% greater than the control (see Note 7).
Fig 1 An example of an experiment utilizing limiting dilution culture, showing the process
of setting up the original culture plates and the final assay plates In the culture plates, each cell concentration is represented by two rows (24 wells) Each culture well is split into two assay wells The black wells in the assay plates were scored positive for CTL activity Rows A con-tain the MUC1+ target, and rows B contain the negative control target
Trang 53.5 Calculation of the CTL Frequency
Inasmuch as positive wells may contain more than one responding T-cell, the fre-quency of antigen-specific cells is determined by the number of negative wells in each dilution The number of negative wells is divided by the total number of wells at each dilution This fraction is converted to its base 10 log and is plotted on a log scale against the number of cells added to each well The Maximal Likelihood method is one of the best statistical estimators used to find the best fitting straight line going through the
origin (10) Linear regression can also be utilized It is assumed in these assays that all
parameters for T-cell growth are optimal and that any well containing antigen-specific cells will be positive in the assay The single hit model of Poisson distribution predicts that when 37% of the test cultures are negative, there is an average of one precursor cell
per well (11) Several statistical computer programs are available to simply evaluate the
data Figure 2 shows graphic data from the assay represented by Fig 1 on an individual
with slightly elevated frequency of MUC1-specific cells.
3.6 LDA Special Considerations
3.6.1 Time of Assay
LDA assays can be used to quantitate specific CTL precursors that need in vitro reactiva-tion, or recently activated specific effector CTL Precursor CTL are determined following 10
or 11 d in culture with antigen Activated CTL could be assayed after 3–5 d of culture.
Fig 2 MUC1-specific CTL frequency derived from the results depicted in Fig 1 The solid
line represents CTL frequency derived in the absence, and the broken line represents CTL frequency derived in the presence of anti-HLA class I antibody W632, illustrating the MHC-unrestricted nature of the CTL The log of the fraction of negative cultures is plotted on a log scale against the number of cells per well Linear regression analysis was used to calculate the slope The wells containing 200,000 cells had no negative wells; thus, the value is 0 and is not plotted A frequency of 1/65,949 for untreated cultures and 1/57,542 for antibody-treated cul-tures is determined when 37% of the culcul-tures are negative This is shown at the intersection of the two plotted lines with the horizontal dashed line
Trang 63.6.2 Number of Cells
The frequency of MUC1-specific precursor cells in most healthy individuals is about 1 in 106cells This frequency is increased to varying degrees in cancer patients and is in the range of 1 in 150,000 to 1 in 300,000 Following immunization of cancer patients in phase I trials, we have observed that the frequency in some patients may
increase between two- to 11-fold (8) These are, nevertheless, relatively low
frequen-cies that require cell concentrations for the assays to start at 2 × 105cells/well We have found, however, that greater than 4 × 105cells/well can result in overcrowding of the well and inconsistent results Optimal statistical analysis requires at least 24 repli-cates of each dilution Thus, the minimum number of cells required is 1.3 × 107for each patient to be tested.
3.6.3 LAK vs Specific CTL Activity
Owing to the presence of IL-2 in the culture medium and additional IL-2 secreted following T-cell activation, some nonspecific cytokine-activated cell lysis can occur, which increases the background noise We use unlabeled K562 cells as cold-target competitors with labeled targets to minimize the background Other unlabeled (cold) targets could be used to compete out possible alloreactive responses or EBV-specific
responses (see Subheading 3.6.4.).
3.6.4 Target Cells
EBV-transformed MUC1-transfected B-cells are excellent stimulator cells because they also express costimulation molecules necessary for optimal T-cell activation However, unless these cell lines are derived from each patient and only autologous cells are used in the assay, it is possible to generate allospecific CTL during the culture period If autolo-gous cells are used, often EBV-specific CTL are activated together with the MUC1-spe-cific CTLs One way to overcome this problem is to use the parental untransfected B-cell line as the cold-target inhibitor Appropriate control target cell in the split-well assays would be the same untransfected EBV-transformed cell, or a tumor cell target that expresses MUC1 but does not share MHC alleles with the stimulator cells.
The MHC-unrestricted nature of the MUC1-specific CTL allows us to use a more standardized and less labor-intensive approach We stimulate the PBL in vitro with one MUC1+tumor cell line and test CTL function against another MUC1+tumor cell line that shares no MHC alleles with the stimulator tumor Allospecific cytotoxicity, if any generated in the cultures, would be ineffective against the second target.
4 Notes
1 It is most convenient to prepare dilutions of cells directly in the microtiter plates The first two rows receive 4 × 105cells in 200 µL Using a 12 -well multichannel pipettor, 12 serial dilutions can then be performed simultaneously by transferring 100-µL volumes into lower wells containing 100 µL of medium alone
2 Sufficient IL-2 is added to the culture to initiate the growth of antigen stimulated T-cells but not the growth of nonspecific lymphokine activated T-cells Thus, if 20 U/mL is the optimal concentration of IL-2, the stimulator cells are resuspended in 10 U/mL of IL-2 Thus, in this example, each well receives 0.5 U of the cytokine
Trang 73 It is also possible to culture the cells for 3 d before adding IL-2 This may reduce nonspe-cific background from cytokine-activated T-cells
4 Depending on the source, IL-2 is purchased in units/milliliter or micrograms/milliliter quantities Each new batch should be evaluated to determine the concentration that pro-motes optimal growth Dilutions of IL-2 from 0.1 to 100 U/mL or 0.1–100 ng/mL are prepared in RPMI-1640 containing 10% human AB serum, penicillin/streptomycin, glutamine, and HEPES Using 96-well microtiter U-bottomed plates, triplicate 100-µL aliquots of each dilution are added to 100 µL of human PHA blasts at a concentration of 1
× 105/mL PHA blasts are prepared by culturing 5 × 105Ficoll-separated peripheral blood mononuclear cells in 5 mL of 10% human AB serum with 10 µg/mL PHA for 3 d at 37°C
in a humidified CO2incubator The cells can be frozen in 10% DMSO and 90% serum, and stored in small aliquots indefinitely in liquid nitrogen Before use, the PHA blasts are thawed and rested by overnight incubation in 10% FCS at 37°C to obtain an accurate viable count Following incubation in different IL-2 dilutions for 3 d at 37°, 1 µCi of 3H thymidine is added After 18–20 h at 37°C the cells are harvested and counted The dilu-tion of IL-2 that gives 50% of maximal growth is considered the optimal concentradilu-tion
5 Successful culture of T-cells requires enriched media such as RPMI-1640 or AIM-5, human male AB serum, and IL-2 Owing to lot-to-lot variation, AB serum should also be tested for supporting optimal cell growth Testing of human serum is done with PHA blasts prepared as described above Vendors will supply small samples of several serum lots on request Cells (1 × 105) are thawed and cultured for 4 d in RPMI-1640 with 10%
test serum, and the optimal concentration of IL-2 determined as described in Note 4 A
viable cell count is a reliable way to determine proliferation A second method is to assess incorporation of 3H thymidine by mitotic cells This is done in triplicate using 96-well microtiter plates Viable cells (3 × 104) are cultured overnight with 1 µCi of 3H thymidine The cells are then harvested and counted for uptake of radionuclide by dividing cells
6 It may be possible to assay simultaneously more than two target cells by splitting the original well into a number of test wells, depending on how vigorously the T-cells have proliferated during the 10-d culture period
7 The arbitrary discrimination between positive and negative cultures is usually a 15% dif-ference in specific release Some patients have low levels of specific release and little or
no observed background LAK activity It may be possible in these patients to consider lower specific lysis as positive
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