Scad
J. lmmunol., Vol.
5 , Suppl. 5 , 1976.
Human Leucocyte Migration Inhibition G . BENDIXEN, K. BENDTZEN, J. E. CLAUSEN, M. KJER & M. Laboratory of Clinical Immunology, Medical Department TA, Rigshospitalet, Copenhagen University, Denmark
SOBORG
Abstract. Within the last decade a variety of techniques have been developed and used for the detection of cell-mediated immunity in man by means of leucocyte migration inhibition in vitro. A detailed description of the leucocyte migration capillary tube technique (LMCT) and the leucocyte migration agarose technique (LMAT) is given. The procedure for selecting and using the proper antigen concentration is described. A description is also given of the indirect LMAT and the technique for determination of concanavalin A-induced lymphocyte release of leucocyte migration inhibition factor. Applications of these techniques are mainly intended for the exploration of the immunobiology of lymphocytes and cellular interactions associated with the immune response and the investigation of clinical conditions in man, i.e. infectious diseases, autoimmune diseases, transplantation states, tumour diseases, contact hypersensitivity and immunological deficiency states. Selection and adaptation to suit the experimental aim is necessary to obtain optimal results with these techniques. Their usefulness may be increased through more extensive use of purified antigens and indirect assays.
Antigen-induced migration inhibition of peripheral blood leucocytes is the principle behind several modifications of in vitro methods for detection of cell-mediated immunity in man, developed on the basis of original observations by Rich & Lewis (45), George & Vaughan (32) and David and coworkers (26). In 1967 Serborg & Bendixen reported (56) a leucocyte migration capillary tube technique (LMCT) which gave a significantly good specific performance in brucella hypersensitivity in man, and developed and described a standard procedure for it (8). The biological interactions of the system were studied by Sraborg (52, 54), Thor et d. (57), Dumonde and coworkers (28, 29), Clausen (18, 19, 20) and several others (1 5). Some laboratories met with difficulties when trying to reproduce the LMCT ( 9 , 33), but the overall development clearly showed that the LMCI' was a useful tool for the detection of cell-mediated hypet-
sensitivity states, which had previously been difficult or impossible to demonstrate. The earliest observations of this type were the demonstration of organ-specific cell-mediated hypersensitivity in ulcerative colitis (4), glomerulonephritis ( 5 ) , idiopathic Addison's disease (44) and chronic liver diseases (49, 5 l), and of tumour-specific, cell-mediated hypersensitivity in mammary carcinoma ( I ) , and graft-specific, cell-mediated hypersensitivity after kidney transplantation (27, 30, 50). Organspecific, tumour-specific and graft-specific hypersensitivity have since then been main fields of application of the LMCT, and the results are partially reviewed elsewhere (2, 7, 46). Another important advantage of the LMCT was that it provided a possibility of performing sequential studies on a specific cell-mediated state of hypersensitivity without antigen exposure of the organism examined, an advantage utilized for instance by Andersen et
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G. Bendixen, K . Bendtzen, J . E. Clausen, M. K j m C. M.Ssborg
al. (3), S ~ b o r get al. (55) and KjiEr et al. (38, 41). In 1971 Clausen (17) described a technique in which leucocytes migrate from small cylindrical wells under an agarose gel in a plastic petri dish. This leucocyte migration agarose test (LMAT) had some advantages over the LMCT. It gave more significant results with some antigens, e.g. tuberculin ( 2 3 ) , whereas the LMCT was still preferable when other types of antigen, e.g. tissue extracts, were used. In two-step systems the LMAT immediately proved highly valuable as a tool for detection of cellular interactions and cell-released reaction products (18, 2 0 , 2 2 ) . Thc present report will give a detailed description of standardized, contemporary modifications of the LMCT and the LMAT, comprising methods for the selection of antigen dose, direct and indirect LMAT, semiquantitative detection of concanavalin A (con-A)induced Ieucocyte migration inhibition factor (LIF) and comments on the statistical value of the results. The various fields of application will be briefly discussed.
METHODS 1 . Leucocyte migration capillary tube technique (LMCT) The LMCT as described by Ssborg & Bendixen (56) and Bendixen & Saborg ( 8 ) , partly micromodified according to Federlin et al. (31) and Maini et al. ( 4 3 ) , is performed as follows: 40 ml venous blood is collected with moderate haemostasis in 10 ml Nundon@ polysterol tubes (Nunc, Roskilde, Denmark), each containing 250 i.u. of heparin (Heparin Leo, Ballerup, Denmark) and 2.0 m l of 5% dextran 2 5 0 (Pharmacia, Uppsala, Sweden) in NaCl 9.0 mg per ml. The tubes are slowly inverted 10 times and placed for 1 h at 37°C. The leucocyte-containing supernatants are transferred to soft polyethylene tubes (Thrombotest tubes, Dansk Laboratorieudstyr, Copenhagen, Denmark) and centrifuged at 225 g for 5 min. The cell pellets are resuspended at the original
volume with Hank’s balanced salt solution (HBSS) and washed (the routine three times) by similar centrifugations. Care must be taken at this stage to ensure monocellular suspension during all washing procedures and in the final suspension. This is accomplished by injecting HBSS through a needle-mounted syringe upon the pellets in the tubes followed by manual or machine vibration of the tubes. After the final washing in HBSS the cell pellets are resuspended in T C 199 with penicillin 67 i.u. and streptomycin 67 pg per ml (Difco Laboratories, Mich., USA) containing 10% horse serum without preservatives (Stzte Serum Institute, Copenhagen, Denmark, batch number important, since not all batches have been found to be equally good). Cell suspensions from all tubes are at this stage pooled to obtain a definite and final concentration of 2.2 x 108 cells per ml. Aliquots of cell suspension containing 3 x 106 leucocytes are transferred to a suitable number of 20 PI glass capillary tubes, internal diameter 0.6 mm (Drummond Hemocaps@, Drummond Scientific Supplies, Broomall, Pa., USA). The capillary tubes are sealed by melting one end in a flame, centrifuged at 900 g at room temperature for 10 min, and cut with a glass file just below the cellfluid interface to avoid clotting of the tube opening by thrombocytes. Immediately after cutting, each tube is placed in a 0.5 ml culture chamber (disposable polystyrene chamber for cell migration, Sterilin Ltd., Middlesex, England) containing the same TC 199 medium with 10% horse serum as described above. The capillary tubes are fixed onto the floor of the chambers by a small lump of silicone wax (Dow Corning Corp.) on the melted end of the tube. Care must be taken that the whole length of each tube rests on the chamber floor. Antigen is added to the culture chambers according to experimental schedule, and the chambers are filled to the brim with culture medium and sealed with a microscope coverslip, retained by silicone wax. All cultures in all concentrations of antigen employed and all control cultures are made in quadruplicate. Control cultures contain only culture medium and/or material comparable to the antigen but
Human Leurocyte Migration Inhibition
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Fig., 1, Plate with culture chainbers containing LMCT cultures after 24 h.
without the antigen investigated. After 24 h at 37OC the migration areas of leucocytes around the capillary tube openings are studied in a projection microscope, drawn on paper, cut and weighed. Within one set of identical quadruplicates the variation from one culture area to another should not be permitted to exceed 5%. The average migration areas of a set of antigen-containing cultures (Mx) and a set of control cultures (Mo) from the same blood sample are used to calculate the migration index (MI): Mx MI=-.
Mo
An MI below 1.0 indicates antigen-induced inhibition, and an MI above 1.0 antigen-induced stimulation of the leucocyte migration. A set of culture chambers is shown in Fig. 1. The antigen concentrations to be used in a particular investigation are determined by titrations as described by Kjaer ( 3 6 ) . At least two comparable preparations with and without the antigen studied are employed in serial dilution, as exemplified in Fig. 2. The titrations are performed in normal control persons and in patients and the aim is to explore and define the antigen concentrations at which the method clearly and consistently discriminates between an immunologically specific inhibition and a 'toxic' inhibition of the migration.
12
The direct leucocyte migration agarose tesi (LMAT) ( 1 7 ) a. Preparation of tissue culture media Liquid medium. Hepes-buffered TC medium is prepared from 100 ml lo-fold concentrated TC 199 (TC-medium 199, 10 X , Difco Laboratories, Detroit, Mich., USA), 6.2 g Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, Sigma Chemical Company, St. Louis, Mo., USA), 500,000 i.u. penicillin, 500 mg streptomycin, and distilled water added to 1000 ml. The p H of the medium is adjusted to 7.40 with 5 N NaOH. The medium is sterilized by filtration through Millipore@ filters, 0.22 pm pore size. Agarose medium. A 2% agarose (Litex, Glostrup, Denmark) solution in distilled water is prepared every two weeks and stored at 5°C. Fresh agarose culture medium is prepared every day. The 2% agarose gel is heated in boiling water. After sol formation, the agarose is cooled to 47OC in a water bath. For preparing 100 ml agarose culture medium, 50 ml 2% agarose solution is added to a mixture (prewarmed to 47°C) of 31 ml sterile distilled water, 9 ml 10-fold concentrated TC 199, 10 ml horse serum, 200 p1 penicillin-streptomycin in sterilized water (containing 6600 i.u. penicillin and 6.6 mg streptomycin), and 540 pI 10% sodium bicarbonate (TC sodium bicar-
2.
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G. Bendixen, K. Bendtzen, J. E. Clausen, M.Kjer & M.Soborg
MIXNON TOXIC DOSE
PROTEIN CONC
Fig. 2. Example of dose-related effect in the LMCT. LMCT reactivity in a patient with hypernephroma compared to a normal person (blood donor), using allogeneic hypernephroma extract at concentrations of 10-600 p g protein per ml. The specifically altered reactivity of the hypernephroma patient is evident at 300 and 400 pg per ml, whereas 600 p g per ml induces a migration inhibition of both normal and patient leucocytes. The use of allogeneic hypernephroma tissue extract in prospective studies has confirmed a dose-related specific response at 100, 300, and 400 p g per ml in renal carcinoma patients as compared to normal controls. A clearly toxic effect is evident in all persons at 600 pg per ml (36, 49).
bonate solution lo%, Difco Laboratories). Thus, the final agarose medium contains 1% agarose, single strength TC 199, 10% horse serum, 66 i.u./ml penicillin, and 66 pg/ml streptomycin. The 47" agarose culture medium is sucked into a prewarmed 25-ml burette, and 5-ml volumes are transferred to 48 x 8.5 mm plastic Petri dishes (Millipore Filter Corp., Bedford, Mass., USA). After the gel medium has solidified, the dishes are incubated at 37°C in a humidified atmosphere of 2% C02 in air. When the leucocyte cultures are to be made, six to eight wells of 2.3 mm diameter are punched in the gel. The bicarbonate/COz buffer, which results in a pH between 7.20 and 7.40, can be substituted by Hepes 0.62 g per 100 ml agarose medium (21). b. Procedure Venous blood is collected in 115 X 13 mm polystyrene tubes (8 ml blood/tube), each containing 250 i.u. heparin and 2 ml 5% dextran
Fig. 3. Petri dish with LMAT cultures after 20 h of migration.
250 in saline (0.9%) solution. After sedimentation at 37°C for 3/4-1 h, the leucocyte-rich plasma is withdrawn and the cells are washed three times in HBSS. Then the cells are resuspended in Hepes-buffered TC 199 with 10% horse serum to give a cell concentration of 2.4 X 1081rnl. Half of the leucocyte suspension is mixed with antigen-containing medium in the proportion 9:1; and, as control, the other half with medium without antigen. After incubation at 37°C for 30 min, 7 - 4 portions from each of the cell suspensions are placed in wells in agarose medium by means of micropipettes. The same number (three to four) of control and stimulated cultures is made on each of two agarose plates. The agarose cultures are incubated at 37'C in a humidified atmosphere of 2% C02 in air. During the following hours, the cells will migrate away from the well in the capillary cleft beneath the gel. A plate of agarose cultures is shown in Fig. 3. After 20 h of culture, the migration areas around the wells are studied under a projection microscope and measured by planimetry. The area of the well is not included in the migration area. Since the migration inhibition is
Human Leucocyte Migration Inhibition
often more pronounced early in the culture period, it can be an advantage to measure the areas after 4 h as well as after 20 h (19). The migration inhibition is expressed as a migration index which indicates the ratio between the average area of antigen-stimulated cultures and that of control cultures. In a previous study (17), the coefficient of variation for the eight control cultures in the individual migration investigation ranged from 1.5 to 9.491, averaging 5.1%. Correspondingly, the coefficient of variation for PPD-stimulated cultures ranged from 1.8 to 13.2% with an average of (5.2%. In 37 double determinations (17), the difference between the two corresponding MI varied between 0 and 0.11. The difference was independent of the mean MI of the double determination. Calculated from these double determinations, the 95% confidence limit for MI is & 0.06 In 12 cases, MI was determined twice at an interval of one to four days. The difference between the two determinations varied between 0.01 and 0.13 with an average of 0.05. 3 . The indirect (two-step) MIF agarose assay (18, 20) a. First step: Preparation of culture supernatants Venous blood with 250 i.u. heparin per 10 ml blood is diluted with an equal volume of HBSS. Seven millilitres of the diluted blood are carefully layered upon 3 ml of a mixture of 9.G% sodium metrizoate and 5.6% Ficoll (Lymphoprep, Nyegaard & Co., Oslo, Norway) in 110 x 17 mm polystyrene tubes. After centrifugation at 400 g for 40 min at room temperature, the mononuclear leucocytes (lymphocytes plus monocytes) in the interface between the plasma and the lymphoprep are pipetted off with a Pasteur pipette, washed three times in HBSS and resuspended in Hepes-buffered TC 199 containing 20% horse serum. Medium containing antigen is added to one half of the cell suspension, and, as control, the same volume of medium without antigen to the other half. The cell concentration is adjusted to 2 X 106/ml. The cultures
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can then be prepared by two different procedures. Cells cultured in the presence of antigen. The cell suspension is divided into three to four 500-pl aliquots and cultured for 24 h at 37°C in 38 x 12.5 mm polypropylene tubes. At the end of the culture period, the supernatants within each group are pooled and centrifuged at 2,000 g for 1 5 min. Antigen is added to the control supernatant to reconstitute it to the concentration present in the supernatant from the antigen-stimulated cultures. Cells preincubated with antigen for 2% h and thereafter cultured ivz antigen-free medium. After incubation of control as well as antigencontaining cell suspension at 37°C for 295 h, the cells are sedimented by centrifugation at 220 g for 5 min, washed three times in Hepesbuffered TC 199 and resuspended in Hepesbuffered TC 199 with :20% horse serum to a final cell concentration of 2 x 106/ml. Both cell suspensions are divided into three to four 5OO-pl aliquots and cultured for two days. At the end of the culture period, the supernatants within each group are pooled and centrifuged at 2,000 g for 15 min. b. Second step: Migration inhibition of indicator leucocytes by supernatants The migration inhibitory effect of culture supernatants containing antigen has to be tested on leucocytes from donors without immunity to the particular antigen, whereas supernatants not containing antigen c:an be tested on leucocytes without regard to the specific immune state of the donor. Leucocyte-rich plasma is prepared as described above and divided between a number of 80 x 16 mm polypropylene tubes corresponding to the number of cell-free culture supernatants which are to be tested for migration inhibitory activity. The indicator leucocytes are washed three times in HBSS. After the last centrifugation, the salt solution is tipped off and the cells resuspended in the different culture supernatants to give a leucocyte concentration of 2.1 x 108/ml. The migration inhibitory activity of culture supernatants can also be tested on purified polymorphs (18).
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G. Bendi.uen, K . Betidtzen,
I . E.
Clausen, M. Kjcer & M . Ssborg
After incubation at 37°C for h, six to eight 7-pl portions of each cell suspension are placed in wells in agarose medium, and the migration areas are measured after 20 h as described above. The migration inhibitory effect of a supernatant from an antigen-stirnulated culture is expressed as a migration index which indicates the ratio between the average area of indicator cells resuspended in supernatant from antigen-stimulated cultures and that of cells resuspended in supernatant from corresponding control cultures.
4. Concnnnvnlin A (con-A)-induced release o f leurocyte migrntion inhibition factor ( L I F ) a. First step Lymphokine pvodtlction. Ten millilitres of venous blood is collected in two 10-ml polystyrene tubes each containing 250 i.u. heparin and 5 ml HBSS. The diluted blood is carefully layered onto 3 ml Isopaque-Ficoll solution in 10 ml polystyrene tubes and centrifuged for 20 min at 400 g. The 95-99% pure mononuclear cell population in the interface between the plasma and the Isopaque-Ficoll solution is pipetted off and washed three times in HBSS. The cells are then suspended at a cell concentration of 2.5 X 106/ml, in serum-free medium TC-199 with penicillin 67 i.u./ml and streptomycin 67 pglml. Two aliquots of the cell suspension are incubated in the presence or absence (control supernatant) of con-A (Pharmacia, Uppsala, Sweden) 80 pg/ml. After 22 h of culture at 37°C in water-saturated atmospheric air with 2 % COz, the supernatants are harvested by centrifugation at 670 g for 10 min, and the control supernatant is reconstituted with con-A 80 pglml. Removal of con-A and salt from culture supernatants. Two identical Sephadex G-100 columns (Pharmacia) are equilibrated at 22°C with volatile buffer (0.05 M ammonium bicarbonate; acetic acid, pH 7.2, 0.1 mM CaC12, 0.1 mM MgC12 and 0.1 mM MnC12). The bed volumes are chosen five times the size of the supernatant volumes. The supernatants are carefully applied on the columns and eluted with volatile buffer at a flow rate of approximately 1
mllmin. The void volumes, initially determined by the elution profile of Blue dextran ( M W 2 x 106) (Pharmacia) (7), are discarded, and eluates twice the size of the sample volumes are collected. The eluates are then passed through Millipore filters (0.45 pm pore site), lyophilized and stored at 4°C until assay for LIF activity. The Sephadex G-100 columns can be reconstituted by washing with one bed volume of 0.1 M glucose folowed by three bed volumes of volatile buffer. b. Second step Assay f o r LIF activity. The second step of the indirect (two-step) migration inhibition factor agarose assay described above is e m ployed, using peripheral blood leucocytes from healthy, unrelated human donors as migratory cells. Semiquantitative results can be obtained by testing stepwise diluted supernatants for LIF activity. The mitogen-free, lyophilized supernatant material from con-A preincubated and control lymphocytes is redissolved in Tc-199 with penicillin, streptomycin and 10% horse serum as indicated above, to give the following concentrationsldilutions of the original supernatants: concentrated x +-concentrated x 3unconcentrated-diluted %-diluted *k (diluted The washed indicator leucocytes are suspended in the supernatants at a concentration of 2.1 x 108/ml. After incubation at 37OC for h, 7-pl aliquots of each cell suspension (1.5 x 106 cells) are placed in wells in agarose medium. Migration areas and the migration inhibitory effects of the active supernatants are measured and calculated as described previously. The greatest dilution which significantly inhibits the migration of cells is thereby determined. Comments. Since con-A at a concentration of more than 10 pgjml (13) inhibits the migration of human leucocytes under agarose, it is essential to remove the con-A activity from the supernatants before assay. This is effectively accomplished by passage through Sephadex G100 columns. Neither migration inhibitory activity nor mitogenic activity, as measured by
Human Leucocyte Migration Inhibition
the incorporation of %-thymidine into DNA, are detectable in the eluates (13, 14). Statistical methods. Statistically significant migration inhibition can be calculated by several statistical methods. The most commonly used is Student’s t test for paired comparisons, which is generally permissible. However, with less than six to eight simultaneous determinations, a non-parametric test (e.g. Wilcoxon’s test) is to be preferred. Reproducibility. The variation between areas within an individual LMAT corresponds to that described above. In repeated experiments the variation between migration inhibition using three different indicator cell populations incubated in the same supernatant was less than 5% (F test for analysis of variance) (14)* In the same study three consecutive LIF determinations with several months’ interval in a male donor varied between MI = 0.85 and 0.84 at a standard dilution of y3 of the supernatants. However, in a female donor five consecutive LIF determinations at a dilution of y3 of supernatants varied between MI = 0.79 and 1.16,and semi-quantitative estimations of LIF activity varied by a factor of 72. These variations are discussed in detail elsewhere (14).
APPLICATIONS 1. Direct
LMCT
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vivo tests for detection of cell-mediated immunity opens up the possibility for numerous applications in clinical research. c. The LMCT can detect cell-mediated immunity against antigens that cannot without objection be used for in vivo testing in man, such as tissue extracts, tumour extracts, etc. It can also give significant results when only crude, unpurified antigen-containing preparations are available. d. In titration experiments the LMCT can give semiquantitative information on the antigenic strength of comparable preparations (40). e. Using repeated cell washings, the LMCT
can demonstrate that tumour-specific cell-mediated immunity can be concealed by components that are gradually removed by cell washings and can be detected in the washing medium (37). In serum from tumour patients, the LMCT can detect components with inhibiting effect on the tumour-specific in vitro reactivity (3 5).
2. Direct LMAT
a. In general the LMA’C has the same fields of application and the same advantages over in vivo methods as the LMCT. Experience shows, however, that the LMAT is preferable when pure or comparatively pure antigen products are available. With less well defined antigen-containing preparations, the LMAT should not be the technique of choice, since it cannot replace the LMCT in a11 cases (42). b. The LMAT can be used for detection of leucocyte migration inhibitory activity released in mixed lymphocyte cultures (18).
a. The LMCT can be used for detection of cell-mediated immunity to for instance microbial antigens (23, 25, 47, 52, 53, 56, 58), to organ-specific tissue components in autoimmune diseases (4, 5, 6, 7, 16, 44,46,49, 51), to graft antigens after transplantation (27, 30, 50) and to tumour-specific antigens in neo3. Indirect LMAT plastic disorders (1, 2, 34, 36, 38, 39, 42). a. The indirect LMAT has similar applib. Since the LMCT permits registration of cell-mediated immunity without introduction cations to the LMAT. ’The indirect LMAT of antigenic material into the organism, it makes it possible to examine the leucocyte permits longitudinal studies with repeated ex- migration inhibitory activity on a neutral, nonaminations of the same person, for instance related migrating indicator cell population, during a clinical course or a controlled experi- which is an obvious advantage from several ment (3, 38, 41, 55). This advantage over in points of view.
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G. Bendixen, K . Bendtzen, J. E. Clausen, M. K i m & M.Snborg
b. The indirect LMAT is well suited for monitoring leucocyte migration inhibitory activity during purification procedures (1 1, 12). The major advantage is the small amount of lymphokine-containing supernatant needed for assay (90 $). c. The indirect LMAT can be used for studies that attempt biochemical or physical modulation of the biological activity of supernatants from antigen-stimulated lymphocytes. d. The indirect LMAT can be used to investigate whether leucocyte migration inhibitory activity is antigen-dependent or antigen-independent.
4. Con-A-LIF assay Determination of the con-A-induced release of leucocyte migration inhibition factor in a two-step technique can be used for: a. Assessment of lymphokine synthesis capacity of lymphocytes responding to a well-defined, standardized, non-specific stimulus (14). b. Production of large amounts of LIF for purification (1 1). c. Production of standardized, lyophilized LIF material (13). These preparations can be used to standardize migration inhibition assays. They have also been applied in the investigations of various pathological conditions, e.g. testing the ability of leucocytes from patients suffering from immunodeficiency disorders to respond to LIF. d. Assessment of effects of chemicals and drugs on leucocyte migration, lymphokine (LIF) production and lymphokine (L1F)-leucocyte interaction (10, 24). DISCUSSION
I . Direct assays Experience so far shows that direct assays utilizing the same peripheral blood cell population as the source of specifically reacting lymphocytes and as polymorphonuclear granulocyte indicator cells in a mixture have given significant results in the LMCT as well as the LMAT in a great number of studies of clinical hypersensitivity states. Direct LMAT has main-
ly proved useful with purified antigen products, such as PPD, virus, bacterial cultures, and KLH, and less useful with impure antigen-containing preparations such as tissue homogenates for the detection of organ-specific antigens and tumour-specific antigens. One exception is the use of spleen extracts as antigen in the LMAT for detection of transplantation hypersensitivity. In general, however, the LMAT seems to be preferable for purified or comparatively pure antigen, and the LMCT for cruder antigen preparations. The cause of this qualitative difference has not yet been analysed in depth, but one probable explanation is that the antigen in the LMAT is preincubated with the cell mixture in a small volume for a certain limited period before the cells are transferred to the milieu intended for migration. With a crude antigen mixture containing for instance tissue elements in the direct LMAT, lymphocytes as well as the granulocytes and monocytes of the cell mixture examined are heavily exposed to a considerable number of non-specific, potentially biologically active compounds, which may interfere with the migration inhibition reaction at several points. A purified antigen product would be less apt to cause this type of non-specific blocking of the system. I I . Indirect assays Clearly, the direct LMCT or LMAT are less time-consuming than two-step assays, so that if results of comparable significance can be obtained, they are preferable in for instance controlled clinical trials. The direct systems may even be considered more representative ad hoc, since they register the combined reactivity of the very cell population of the patient at the time of examination. However, with our limited knowledge of the complexity of the system, comparison in all analyses of one-step and two-step reactivity with own and unrelated indicator cells would improve the chances of significant conclusions. With our present knowledge it is impossible to say whether two-step methods could be developed to achieve higher sensitivity than one-step assays, and at present the best method is to purify the antigen and use the LMAT.
Human Leuroryte Migration Inhibition REFERENCES 1.Anderse1-1, V., Bendixen, G. & Schiedt, T. An in vitro demonstration of cellular immunity against autologous mammary carcinoma in man. Arta med. srand. 186, 101, 1969. 2. Andersen, V., Kjaer, M. & Bendixen, G. In vitrodemonstration of cellular hypersensitivity to tumour antigens in man. Ser. Haemat. S, 3, 1972. 3. Andersen, V., Seborg, M. & Serensen, S. F. Antigen-induced lymphocyte transformation in vitro during primary immunization in man. I. Development and course. Arta path. mirrobiol. srand. Section B, 79, 489, 1971. 4. Bendixen, G. Specific inhibition of the in vitro migration of leucocytes in ulcerative colitis and Crohn’s disease. Srand. J. Gastroent. 2, 214, 1967. 5. Bendixen, G. Organ-specific inhibition of the in vitro migration of leucocytes in human glomerulonephritis. Arta med. srand. 184, 99, 1968. 6. Bendixen, G. Cellular hypersensitivity to components of intestinal mucosa in ulcerative colitis and Gohn’s disease. Gut 10, 631, 1969. 7. Bendixen, G. Organ-specific cellular hypersensitivity in autoimmune diseases. Ann. lmmunol. 4, 3, 1972. 8. Bendixen, G. & Seborg, M. A leucocyte migration technique for in vitro detection of cellular (delayed type) hypersensitivity in man. Danish med. Bull. 16, 1, 1969. 9.Bendixen, G. & Seborg, M. Comments on the leucocyte migration technique as an in vitro method for demonstrating cellular hypersensitivity in man. J. Immunol. 104, 1551, 1970. 10. Bendtzen, K. Drug effects on human leucocyte migration and migra:ion inhibitory activity from lymphocytes stimulated with concanavalin A. Arta path. mirrobiol. srand. Section C, 83, 447, 1975. 11. Bendken, K. Inhibition of human leukocyte migration inhibitory factor (LIF) by a-L-fucose. Arta allerg. (Kbh.) 30, 327, 1975. 12. Bendtzen, K. A new technique for partial purification of human leucocyte migration inhibitory factor. J. lmmunol. Methods. (Submitted for publication). 1976. 13. Bendtzen, K. Unpublished data. 14. Bendtzen, K., Andersen, V. & Bendixen, G. An in vitro assay of leukocyte migration inhibitory activity from human lymphocytes stimulated with concanavalin A. Arta allerg. (Kbh.) 30, 133, 1975. 15. Bloom, B. R. & Glade, P. R. (eds.) In Vitro Methods in Cell-Mediated Immunity. Academic Press, New York and London, 1971. 16. Brostoff, J., Roitt, I. M. & Doniach, D. Leucocyte-migration inhibition in autoimmune diseases. Lancet 1969, 1, 1969. 17. Clausen, J. E. Tuberculin-induced migration inhibition of human peripheral leucocytes in agarose medium. Arta allerg. (Kbh.) 26, 56, 1971.
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18. Clausen, J. E. Migration inhibitory effect of cell-
free supernatants from mixed human lymphocyte cultures. J. lmmunol. 108, 453, 1972. 19. Clausen, J. E. Tuberculin-induced migration inhibition of human peripheral blood leukocytes after 2, 4, and 20 hours in agarose culture. Arta allerg. (Kbh.) 28, 28, 1973. 20. Clausen, J. E. Migration inhibitory effect of cellfree supernatants from tuberculin-stimulated cultures of human mononuclear leukocytes demonstrated by two-step MIF agarose assay. J. Immunol. 110, 146, 1973. 21. Clausen, J. E. Leukocyte migration agarose technique: some technical details. Arta allerg. (Kbh.) 28, 351, 1973. 22. Clausen, J. E. The Agarose Migration lnhibition
Technique f o r In Vitro Demonstration of CellMediated Immunity in Man. Thesis, N. Olaf Meller, Copenhagen, 1974. 23. Clausen, J. E. & Seborg, M. In vitro detection of tuberculin hypersensitivity in man. Arta med. srand 186, 227, 1969. 24. Coeugniet, E., Bendtzen, K. & Bendixen, G. Leucocyte migration inhibitory activity of concanavalin A-stimulated human lymphocytes. Modification by dipyridamole, lysine-acetylsalicylate and heparin. Arta med. srand. 199, 99, 1976. 25. Dardenne, M., Zabriskie, J. & Bach, J. F. Streptococcal sensitivity in chronic glomerulonephritis. Lancet 1, 126, 1972. 26. David, J. R., Al-Askari, S., Lawrence, H. S. & Thomas, L. Delayed hypersensitivity in vitro I. The specificity of inhibition of cell migration by antigens. J. Immunol. 93, 264, 1964. 27. Dormont, J., Sobel, A., Galaneaud, P., Gevon, M. C. & Colombine, J. Leucocyte migration inhibition with spleen extracts and other antigens in patients with renal allografts. Transplant. Pror. 4, 265, 1972. 28. Dumonde, D. ‘Lymphokines’: Molecular mediators of cellular immune responses in animals and man. Proc. Roy. Sor. Med. 63, 899, 1970. 29. Dumonde, D. C., Howson, W. T. & Wolstencroft, R. A. The role of macrophages and lymphocytes in reactions of delayed hypersensitivity. In Miescher, P. A. & Grabar, P. (eds.) Immunopathology, Schwabe & Co., Basel, 1968, p. 263. 30. Falk, R. E., Guttmann, R. D., Beaudoin, J. G., Morehouse, D. D. & Oh, J. H. Leucocyte migration in vitro and its relationship to human renal allograft rejection and enhancement. Trunsplantation 13, 461, 1972. 31. Federlin, K., Maini,
R. N., Russell, A. S. & Dumonde, D. C. A micro-method for peripheral leucocyte migration in tuberculin sensitivity. J. din. Path. 24, 533, 1971. 32.George, M. & Vaughan, J. H. In vitro cell migration as a model for delayed hypersensitivity. Proc. Sor. exp. Biol. Med 111, 514, 1962.
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G . Bendixen, K . Bendtzen, J . E. Clauseti, M . Kjier & Af. S o b o r ~
33. Kaltreider, H. B., Soghor, D., Taylor, J. B. & Decker, J. L. Capillary tube migration for detection of human delayed hypersmsitivity: Difficulties encountered with ‘buffy coat’ cells and tuberculin antigen. J. Immunol. 103, 179, 1969. 34. Kjaer, M. In vitro-demonstration of cellular hypersensitivity to tumour antigens by means of thz leucocyte migration technique in patients with renal carcinoma. Europ. J . Cancer, 10, 523, 1974. 35. Kjaer, M. The capillary tube leucocyte migration technique (LMT) as a method for detection of serum inhibiting activity of tumour-directed, cellular hypersensitivity in patients with renal carcinomas. Arta path. microbiol. srand. Section B. 82, 294, 1974. 36. Kjaer, M. The dose-related effect of tumour extract on the in vitro migration of leucocytes from patients with renal carcinoma. Euvop. J. Cancer. 11, 281, 1975. 37. Kjaer, M. The effect of leucocyte washings on the in vitro detection of tumour-directed, cellmediated hypersensitivity in patients with renal carcinoma. Submitted for publication. 38. Kjaer, M. & Bendixen, G . Tumour-directed, cellular hypersensitivity detected by means of the leucocyte migration technique in patients with renal carcinoma. Ann. N.Y. Arad. Sci. 1976 (in press). 39. Kjaer, M. & Bendixen, G. Clinical detection and monitoring of tumour specific cell-mediated immunity in man: Examination of hypernephroma with the leucocyte migration technique. In Bloom, B. & David, J. R. (eds.) Z n Vitro Methods in Cell-Mediated Immunity, 11. Academic Press, 1976 (in press). 40. Kjaer, M. & Christensen, N. Ability of renal carcinoma tissue extracts to induce leucocyte migration inhibition in patients with non-metastatic renal carcinoma: Correlation with clinical and histopathological findings. Submitted for publication. 41. Kjaer, M. & Christensen, N. Prognostic value of tumour directed, cellular hypersensitivity detected by means of the leucocyte migration technique in patients with renal carcinoma. 1976 (in preparation). 42.Kjaer, M. & Snrensen, T. B. Comparison between the direct capillary tube leucocyte migration technique (LMCT) and the direct agarose leucocyte migration technique (LMAT) as indicators of tumour-directed, cell-mediated hypersensitivity (TCMH) in patients with renal carcinoma. Arta allerg., (Kbh.) 31, 141, 1976. 43.Main4 R. N., Roffe, L. M., Magrath, J. T. & Dumonde, D. C. Standardization of the leucocyte migration test. lnt. Arch. Allergy 45, 308, 1973. 44. Nerup, J., Andersen, V. & Bendixen, G. Anti-
Received 3 March 1976
adrenal cellular hypersensitivity in Addison’s disease. Clin. exp. Immunol. 4, 355, 1969. 45. Rich, A. R. & Lewis, M. R. The nature of allergy in tuberculosis as revealed by tissue culture studies. Bull. Juhns Hupkins Hosp. 50, 115, 1932. 46. Rocklin, R. 1:. Clinical applications of in vitro lymphocyte test. In Schwartz, R. S. (ed.) Progress in Clinirnl Immunology, Grune & Stratton, New York, 1974, p. 21. 47.Rosenberg S. A. & David, J. R. Inhibition of leucocyte migration: An evaluation of this in vitro assay of delayed hypersensitivity in man to a soluble antigen. J . Immunol. 103, 1447, 1970. 48. Sephadex. Gel filtration in theory and practice. Pharmacia Fine Chemicals AB, IJppsala, Sweden. 49. Smith, M. G. M., Eddleston, A. L. W. F. & Williams, R. Leucocyte migration in active chronic hepatitis and primary biliary cirrhosis. International mecting on immunological aspects of liver disease. In Smith, M. & Williams, R. (eds.) Z m m u n o l u ~uf~ the L k e r . Heineman, London, 1971, p. 135. 50. Smith, M. G. M., Eddleston, A. L. W. F., Dominguez, J. A., Evans, D . B., Bewick, M. & Williams, R. Changes in leucocyte migration after renal transplantation. Brit. med. J . 4, 275, 1969. 51. Smith, M. G. M., Golding, P. L., Eddleston, A. L. W . F., Mitchell, C. G., Kemp, A. & Williams, R. Cell-mediated immune responses in chronic liver diseases. Brit. med. J. 1, 527, 1972. 52. Soborg, M. Bestemmelse uf cellulcer hypersensibilitet in vitro med migrution.rhiemnitigsreaktionen. Thesis. FADL, Copenhagen, 1971. 53. Soborg, M. In vitro detection of cellular hypersensitivity in man. Specific migration inhibition of white blood cells from brucella positive persons. Acta med. srand. 182, 167, 1967. 54. Soborg, M. Interaction of human peripheral lymphocytes and granulocytes in the migration inhibition reaction. Arta med. srand. 185, 221, 1969. 5 5 . Soborg, M., Andersen, V. & Serrensen, S. F. Antigen-induced lymphocyte transformation in vitro during primary immunization in man. 11. Correlation with development of cellular and humoral hypersensitivity. Arta path. mirrobiol. srand. Section B, 79, 495, 1971. 56.Soborg, M. & Bendixen, G. Human lymphocyte migration as a parameter of hypersensitivity. Acta med. srand. 181, 247, 1967. 57. Thor, D. E., Jureziz, R. E., Veach, S. R., Miller, E. & Dray, S. Cell migration inhibition factor released by antigen from human peripheral lymphocytes. Nature (Load.), 219, 755, 1968. 58. Zabriskie, J. B., Read, S. E., Ellis, R. J., Markowitz, S. & Rappaport, F. T. Cellular reactivity studies in human and experimental renal transplantation. Tramp. Pror. 4, 259, 1972.
J. lmmunol., Vol.
5 , Suppl. 5 , 1976.
Human Leucocyte Migration Inhibition G . BENDIXEN, K. BENDTZEN, J. E. CLAUSEN, M. KJER & M. Laboratory of Clinical Immunology, Medical Department TA, Rigshospitalet, Copenhagen University, Denmark
SOBORG
Abstract. Within the last decade a variety of techniques have been developed and used for the detection of cell-mediated immunity in man by means of leucocyte migration inhibition in vitro. A detailed description of the leucocyte migration capillary tube technique (LMCT) and the leucocyte migration agarose technique (LMAT) is given. The procedure for selecting and using the proper antigen concentration is described. A description is also given of the indirect LMAT and the technique for determination of concanavalin A-induced lymphocyte release of leucocyte migration inhibition factor. Applications of these techniques are mainly intended for the exploration of the immunobiology of lymphocytes and cellular interactions associated with the immune response and the investigation of clinical conditions in man, i.e. infectious diseases, autoimmune diseases, transplantation states, tumour diseases, contact hypersensitivity and immunological deficiency states. Selection and adaptation to suit the experimental aim is necessary to obtain optimal results with these techniques. Their usefulness may be increased through more extensive use of purified antigens and indirect assays.
Antigen-induced migration inhibition of peripheral blood leucocytes is the principle behind several modifications of in vitro methods for detection of cell-mediated immunity in man, developed on the basis of original observations by Rich & Lewis (45), George & Vaughan (32) and David and coworkers (26). In 1967 Serborg & Bendixen reported (56) a leucocyte migration capillary tube technique (LMCT) which gave a significantly good specific performance in brucella hypersensitivity in man, and developed and described a standard procedure for it (8). The biological interactions of the system were studied by Sraborg (52, 54), Thor et d. (57), Dumonde and coworkers (28, 29), Clausen (18, 19, 20) and several others (1 5). Some laboratories met with difficulties when trying to reproduce the LMCT ( 9 , 33), but the overall development clearly showed that the LMCI' was a useful tool for the detection of cell-mediated hypet-
sensitivity states, which had previously been difficult or impossible to demonstrate. The earliest observations of this type were the demonstration of organ-specific cell-mediated hypersensitivity in ulcerative colitis (4), glomerulonephritis ( 5 ) , idiopathic Addison's disease (44) and chronic liver diseases (49, 5 l), and of tumour-specific, cell-mediated hypersensitivity in mammary carcinoma ( I ) , and graft-specific, cell-mediated hypersensitivity after kidney transplantation (27, 30, 50). Organspecific, tumour-specific and graft-specific hypersensitivity have since then been main fields of application of the LMCT, and the results are partially reviewed elsewhere (2, 7, 46). Another important advantage of the LMCT was that it provided a possibility of performing sequential studies on a specific cell-mediated state of hypersensitivity without antigen exposure of the organism examined, an advantage utilized for instance by Andersen et
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al. (3), S ~ b o r get al. (55) and KjiEr et al. (38, 41). In 1971 Clausen (17) described a technique in which leucocytes migrate from small cylindrical wells under an agarose gel in a plastic petri dish. This leucocyte migration agarose test (LMAT) had some advantages over the LMCT. It gave more significant results with some antigens, e.g. tuberculin ( 2 3 ) , whereas the LMCT was still preferable when other types of antigen, e.g. tissue extracts, were used. In two-step systems the LMAT immediately proved highly valuable as a tool for detection of cellular interactions and cell-released reaction products (18, 2 0 , 2 2 ) . Thc present report will give a detailed description of standardized, contemporary modifications of the LMCT and the LMAT, comprising methods for the selection of antigen dose, direct and indirect LMAT, semiquantitative detection of concanavalin A (con-A)induced Ieucocyte migration inhibition factor (LIF) and comments on the statistical value of the results. The various fields of application will be briefly discussed.
METHODS 1 . Leucocyte migration capillary tube technique (LMCT) The LMCT as described by Ssborg & Bendixen (56) and Bendixen & Saborg ( 8 ) , partly micromodified according to Federlin et al. (31) and Maini et al. ( 4 3 ) , is performed as follows: 40 ml venous blood is collected with moderate haemostasis in 10 ml Nundon@ polysterol tubes (Nunc, Roskilde, Denmark), each containing 250 i.u. of heparin (Heparin Leo, Ballerup, Denmark) and 2.0 m l of 5% dextran 2 5 0 (Pharmacia, Uppsala, Sweden) in NaCl 9.0 mg per ml. The tubes are slowly inverted 10 times and placed for 1 h at 37°C. The leucocyte-containing supernatants are transferred to soft polyethylene tubes (Thrombotest tubes, Dansk Laboratorieudstyr, Copenhagen, Denmark) and centrifuged at 225 g for 5 min. The cell pellets are resuspended at the original
volume with Hank’s balanced salt solution (HBSS) and washed (the routine three times) by similar centrifugations. Care must be taken at this stage to ensure monocellular suspension during all washing procedures and in the final suspension. This is accomplished by injecting HBSS through a needle-mounted syringe upon the pellets in the tubes followed by manual or machine vibration of the tubes. After the final washing in HBSS the cell pellets are resuspended in T C 199 with penicillin 67 i.u. and streptomycin 67 pg per ml (Difco Laboratories, Mich., USA) containing 10% horse serum without preservatives (Stzte Serum Institute, Copenhagen, Denmark, batch number important, since not all batches have been found to be equally good). Cell suspensions from all tubes are at this stage pooled to obtain a definite and final concentration of 2.2 x 108 cells per ml. Aliquots of cell suspension containing 3 x 106 leucocytes are transferred to a suitable number of 20 PI glass capillary tubes, internal diameter 0.6 mm (Drummond Hemocaps@, Drummond Scientific Supplies, Broomall, Pa., USA). The capillary tubes are sealed by melting one end in a flame, centrifuged at 900 g at room temperature for 10 min, and cut with a glass file just below the cellfluid interface to avoid clotting of the tube opening by thrombocytes. Immediately after cutting, each tube is placed in a 0.5 ml culture chamber (disposable polystyrene chamber for cell migration, Sterilin Ltd., Middlesex, England) containing the same TC 199 medium with 10% horse serum as described above. The capillary tubes are fixed onto the floor of the chambers by a small lump of silicone wax (Dow Corning Corp.) on the melted end of the tube. Care must be taken that the whole length of each tube rests on the chamber floor. Antigen is added to the culture chambers according to experimental schedule, and the chambers are filled to the brim with culture medium and sealed with a microscope coverslip, retained by silicone wax. All cultures in all concentrations of antigen employed and all control cultures are made in quadruplicate. Control cultures contain only culture medium and/or material comparable to the antigen but
Human Leurocyte Migration Inhibition
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Fig., 1, Plate with culture chainbers containing LMCT cultures after 24 h.
without the antigen investigated. After 24 h at 37OC the migration areas of leucocytes around the capillary tube openings are studied in a projection microscope, drawn on paper, cut and weighed. Within one set of identical quadruplicates the variation from one culture area to another should not be permitted to exceed 5%. The average migration areas of a set of antigen-containing cultures (Mx) and a set of control cultures (Mo) from the same blood sample are used to calculate the migration index (MI): Mx MI=-.
Mo
An MI below 1.0 indicates antigen-induced inhibition, and an MI above 1.0 antigen-induced stimulation of the leucocyte migration. A set of culture chambers is shown in Fig. 1. The antigen concentrations to be used in a particular investigation are determined by titrations as described by Kjaer ( 3 6 ) . At least two comparable preparations with and without the antigen studied are employed in serial dilution, as exemplified in Fig. 2. The titrations are performed in normal control persons and in patients and the aim is to explore and define the antigen concentrations at which the method clearly and consistently discriminates between an immunologically specific inhibition and a 'toxic' inhibition of the migration.
12
The direct leucocyte migration agarose tesi (LMAT) ( 1 7 ) a. Preparation of tissue culture media Liquid medium. Hepes-buffered TC medium is prepared from 100 ml lo-fold concentrated TC 199 (TC-medium 199, 10 X , Difco Laboratories, Detroit, Mich., USA), 6.2 g Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, Sigma Chemical Company, St. Louis, Mo., USA), 500,000 i.u. penicillin, 500 mg streptomycin, and distilled water added to 1000 ml. The p H of the medium is adjusted to 7.40 with 5 N NaOH. The medium is sterilized by filtration through Millipore@ filters, 0.22 pm pore size. Agarose medium. A 2% agarose (Litex, Glostrup, Denmark) solution in distilled water is prepared every two weeks and stored at 5°C. Fresh agarose culture medium is prepared every day. The 2% agarose gel is heated in boiling water. After sol formation, the agarose is cooled to 47OC in a water bath. For preparing 100 ml agarose culture medium, 50 ml 2% agarose solution is added to a mixture (prewarmed to 47°C) of 31 ml sterile distilled water, 9 ml 10-fold concentrated TC 199, 10 ml horse serum, 200 p1 penicillin-streptomycin in sterilized water (containing 6600 i.u. penicillin and 6.6 mg streptomycin), and 540 pI 10% sodium bicarbonate (TC sodium bicar-
2.
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G. Bendixen, K. Bendtzen, J. E. Clausen, M.Kjer & M.Soborg
MIXNON TOXIC DOSE
PROTEIN CONC
Fig. 2. Example of dose-related effect in the LMCT. LMCT reactivity in a patient with hypernephroma compared to a normal person (blood donor), using allogeneic hypernephroma extract at concentrations of 10-600 p g protein per ml. The specifically altered reactivity of the hypernephroma patient is evident at 300 and 400 pg per ml, whereas 600 p g per ml induces a migration inhibition of both normal and patient leucocytes. The use of allogeneic hypernephroma tissue extract in prospective studies has confirmed a dose-related specific response at 100, 300, and 400 p g per ml in renal carcinoma patients as compared to normal controls. A clearly toxic effect is evident in all persons at 600 pg per ml (36, 49).
bonate solution lo%, Difco Laboratories). Thus, the final agarose medium contains 1% agarose, single strength TC 199, 10% horse serum, 66 i.u./ml penicillin, and 66 pg/ml streptomycin. The 47" agarose culture medium is sucked into a prewarmed 25-ml burette, and 5-ml volumes are transferred to 48 x 8.5 mm plastic Petri dishes (Millipore Filter Corp., Bedford, Mass., USA). After the gel medium has solidified, the dishes are incubated at 37°C in a humidified atmosphere of 2% C02 in air. When the leucocyte cultures are to be made, six to eight wells of 2.3 mm diameter are punched in the gel. The bicarbonate/COz buffer, which results in a pH between 7.20 and 7.40, can be substituted by Hepes 0.62 g per 100 ml agarose medium (21). b. Procedure Venous blood is collected in 115 X 13 mm polystyrene tubes (8 ml blood/tube), each containing 250 i.u. heparin and 2 ml 5% dextran
Fig. 3. Petri dish with LMAT cultures after 20 h of migration.
250 in saline (0.9%) solution. After sedimentation at 37°C for 3/4-1 h, the leucocyte-rich plasma is withdrawn and the cells are washed three times in HBSS. Then the cells are resuspended in Hepes-buffered TC 199 with 10% horse serum to give a cell concentration of 2.4 X 1081rnl. Half of the leucocyte suspension is mixed with antigen-containing medium in the proportion 9:1; and, as control, the other half with medium without antigen. After incubation at 37°C for 30 min, 7 - 4 portions from each of the cell suspensions are placed in wells in agarose medium by means of micropipettes. The same number (three to four) of control and stimulated cultures is made on each of two agarose plates. The agarose cultures are incubated at 37'C in a humidified atmosphere of 2% C02 in air. During the following hours, the cells will migrate away from the well in the capillary cleft beneath the gel. A plate of agarose cultures is shown in Fig. 3. After 20 h of culture, the migration areas around the wells are studied under a projection microscope and measured by planimetry. The area of the well is not included in the migration area. Since the migration inhibition is
Human Leucocyte Migration Inhibition
often more pronounced early in the culture period, it can be an advantage to measure the areas after 4 h as well as after 20 h (19). The migration inhibition is expressed as a migration index which indicates the ratio between the average area of antigen-stimulated cultures and that of control cultures. In a previous study (17), the coefficient of variation for the eight control cultures in the individual migration investigation ranged from 1.5 to 9.491, averaging 5.1%. Correspondingly, the coefficient of variation for PPD-stimulated cultures ranged from 1.8 to 13.2% with an average of (5.2%. In 37 double determinations (17), the difference between the two corresponding MI varied between 0 and 0.11. The difference was independent of the mean MI of the double determination. Calculated from these double determinations, the 95% confidence limit for MI is & 0.06 In 12 cases, MI was determined twice at an interval of one to four days. The difference between the two determinations varied between 0.01 and 0.13 with an average of 0.05. 3 . The indirect (two-step) MIF agarose assay (18, 20) a. First step: Preparation of culture supernatants Venous blood with 250 i.u. heparin per 10 ml blood is diluted with an equal volume of HBSS. Seven millilitres of the diluted blood are carefully layered upon 3 ml of a mixture of 9.G% sodium metrizoate and 5.6% Ficoll (Lymphoprep, Nyegaard & Co., Oslo, Norway) in 110 x 17 mm polystyrene tubes. After centrifugation at 400 g for 40 min at room temperature, the mononuclear leucocytes (lymphocytes plus monocytes) in the interface between the plasma and the lymphoprep are pipetted off with a Pasteur pipette, washed three times in HBSS and resuspended in Hepes-buffered TC 199 containing 20% horse serum. Medium containing antigen is added to one half of the cell suspension, and, as control, the same volume of medium without antigen to the other half. The cell concentration is adjusted to 2 X 106/ml. The cultures
179
can then be prepared by two different procedures. Cells cultured in the presence of antigen. The cell suspension is divided into three to four 500-pl aliquots and cultured for 24 h at 37°C in 38 x 12.5 mm polypropylene tubes. At the end of the culture period, the supernatants within each group are pooled and centrifuged at 2,000 g for 1 5 min. Antigen is added to the control supernatant to reconstitute it to the concentration present in the supernatant from the antigen-stimulated cultures. Cells preincubated with antigen for 2% h and thereafter cultured ivz antigen-free medium. After incubation of control as well as antigencontaining cell suspension at 37°C for 295 h, the cells are sedimented by centrifugation at 220 g for 5 min, washed three times in Hepesbuffered TC 199 and resuspended in Hepesbuffered TC 199 with :20% horse serum to a final cell concentration of 2 x 106/ml. Both cell suspensions are divided into three to four 5OO-pl aliquots and cultured for two days. At the end of the culture period, the supernatants within each group are pooled and centrifuged at 2,000 g for 15 min. b. Second step: Migration inhibition of indicator leucocytes by supernatants The migration inhibitory effect of culture supernatants containing antigen has to be tested on leucocytes from donors without immunity to the particular antigen, whereas supernatants not containing antigen c:an be tested on leucocytes without regard to the specific immune state of the donor. Leucocyte-rich plasma is prepared as described above and divided between a number of 80 x 16 mm polypropylene tubes corresponding to the number of cell-free culture supernatants which are to be tested for migration inhibitory activity. The indicator leucocytes are washed three times in HBSS. After the last centrifugation, the salt solution is tipped off and the cells resuspended in the different culture supernatants to give a leucocyte concentration of 2.1 x 108/ml. The migration inhibitory activity of culture supernatants can also be tested on purified polymorphs (18).
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I . E.
Clausen, M. Kjcer & M . Ssborg
After incubation at 37°C for h, six to eight 7-pl portions of each cell suspension are placed in wells in agarose medium, and the migration areas are measured after 20 h as described above. The migration inhibitory effect of a supernatant from an antigen-stirnulated culture is expressed as a migration index which indicates the ratio between the average area of indicator cells resuspended in supernatant from antigen-stimulated cultures and that of cells resuspended in supernatant from corresponding control cultures.
4. Concnnnvnlin A (con-A)-induced release o f leurocyte migrntion inhibition factor ( L I F ) a. First step Lymphokine pvodtlction. Ten millilitres of venous blood is collected in two 10-ml polystyrene tubes each containing 250 i.u. heparin and 5 ml HBSS. The diluted blood is carefully layered onto 3 ml Isopaque-Ficoll solution in 10 ml polystyrene tubes and centrifuged for 20 min at 400 g. The 95-99% pure mononuclear cell population in the interface between the plasma and the Isopaque-Ficoll solution is pipetted off and washed three times in HBSS. The cells are then suspended at a cell concentration of 2.5 X 106/ml, in serum-free medium TC-199 with penicillin 67 i.u./ml and streptomycin 67 pglml. Two aliquots of the cell suspension are incubated in the presence or absence (control supernatant) of con-A (Pharmacia, Uppsala, Sweden) 80 pg/ml. After 22 h of culture at 37°C in water-saturated atmospheric air with 2 % COz, the supernatants are harvested by centrifugation at 670 g for 10 min, and the control supernatant is reconstituted with con-A 80 pglml. Removal of con-A and salt from culture supernatants. Two identical Sephadex G-100 columns (Pharmacia) are equilibrated at 22°C with volatile buffer (0.05 M ammonium bicarbonate; acetic acid, pH 7.2, 0.1 mM CaC12, 0.1 mM MgC12 and 0.1 mM MnC12). The bed volumes are chosen five times the size of the supernatant volumes. The supernatants are carefully applied on the columns and eluted with volatile buffer at a flow rate of approximately 1
mllmin. The void volumes, initially determined by the elution profile of Blue dextran ( M W 2 x 106) (Pharmacia) (7), are discarded, and eluates twice the size of the sample volumes are collected. The eluates are then passed through Millipore filters (0.45 pm pore site), lyophilized and stored at 4°C until assay for LIF activity. The Sephadex G-100 columns can be reconstituted by washing with one bed volume of 0.1 M glucose folowed by three bed volumes of volatile buffer. b. Second step Assay f o r LIF activity. The second step of the indirect (two-step) migration inhibition factor agarose assay described above is e m ployed, using peripheral blood leucocytes from healthy, unrelated human donors as migratory cells. Semiquantitative results can be obtained by testing stepwise diluted supernatants for LIF activity. The mitogen-free, lyophilized supernatant material from con-A preincubated and control lymphocytes is redissolved in Tc-199 with penicillin, streptomycin and 10% horse serum as indicated above, to give the following concentrationsldilutions of the original supernatants: concentrated x +-concentrated x 3unconcentrated-diluted %-diluted *k (diluted The washed indicator leucocytes are suspended in the supernatants at a concentration of 2.1 x 108/ml. After incubation at 37OC for h, 7-pl aliquots of each cell suspension (1.5 x 106 cells) are placed in wells in agarose medium. Migration areas and the migration inhibitory effects of the active supernatants are measured and calculated as described previously. The greatest dilution which significantly inhibits the migration of cells is thereby determined. Comments. Since con-A at a concentration of more than 10 pgjml (13) inhibits the migration of human leucocytes under agarose, it is essential to remove the con-A activity from the supernatants before assay. This is effectively accomplished by passage through Sephadex G100 columns. Neither migration inhibitory activity nor mitogenic activity, as measured by
Human Leucocyte Migration Inhibition
the incorporation of %-thymidine into DNA, are detectable in the eluates (13, 14). Statistical methods. Statistically significant migration inhibition can be calculated by several statistical methods. The most commonly used is Student’s t test for paired comparisons, which is generally permissible. However, with less than six to eight simultaneous determinations, a non-parametric test (e.g. Wilcoxon’s test) is to be preferred. Reproducibility. The variation between areas within an individual LMAT corresponds to that described above. In repeated experiments the variation between migration inhibition using three different indicator cell populations incubated in the same supernatant was less than 5% (F test for analysis of variance) (14)* In the same study three consecutive LIF determinations with several months’ interval in a male donor varied between MI = 0.85 and 0.84 at a standard dilution of y3 of the supernatants. However, in a female donor five consecutive LIF determinations at a dilution of y3 of supernatants varied between MI = 0.79 and 1.16,and semi-quantitative estimations of LIF activity varied by a factor of 72. These variations are discussed in detail elsewhere (14).
APPLICATIONS 1. Direct
LMCT
181
vivo tests for detection of cell-mediated immunity opens up the possibility for numerous applications in clinical research. c. The LMCT can detect cell-mediated immunity against antigens that cannot without objection be used for in vivo testing in man, such as tissue extracts, tumour extracts, etc. It can also give significant results when only crude, unpurified antigen-containing preparations are available. d. In titration experiments the LMCT can give semiquantitative information on the antigenic strength of comparable preparations (40). e. Using repeated cell washings, the LMCT
can demonstrate that tumour-specific cell-mediated immunity can be concealed by components that are gradually removed by cell washings and can be detected in the washing medium (37). In serum from tumour patients, the LMCT can detect components with inhibiting effect on the tumour-specific in vitro reactivity (3 5).
2. Direct LMAT
a. In general the LMA’C has the same fields of application and the same advantages over in vivo methods as the LMCT. Experience shows, however, that the LMAT is preferable when pure or comparatively pure antigen products are available. With less well defined antigen-containing preparations, the LMAT should not be the technique of choice, since it cannot replace the LMCT in a11 cases (42). b. The LMAT can be used for detection of leucocyte migration inhibitory activity released in mixed lymphocyte cultures (18).
a. The LMCT can be used for detection of cell-mediated immunity to for instance microbial antigens (23, 25, 47, 52, 53, 56, 58), to organ-specific tissue components in autoimmune diseases (4, 5, 6, 7, 16, 44,46,49, 51), to graft antigens after transplantation (27, 30, 50) and to tumour-specific antigens in neo3. Indirect LMAT plastic disorders (1, 2, 34, 36, 38, 39, 42). a. The indirect LMAT has similar applib. Since the LMCT permits registration of cell-mediated immunity without introduction cations to the LMAT. ’The indirect LMAT of antigenic material into the organism, it makes it possible to examine the leucocyte permits longitudinal studies with repeated ex- migration inhibitory activity on a neutral, nonaminations of the same person, for instance related migrating indicator cell population, during a clinical course or a controlled experi- which is an obvious advantage from several ment (3, 38, 41, 55). This advantage over in points of view.
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G. Bendixen, K . Bendtzen, J. E. Clausen, M. K i m & M.Snborg
b. The indirect LMAT is well suited for monitoring leucocyte migration inhibitory activity during purification procedures (1 1, 12). The major advantage is the small amount of lymphokine-containing supernatant needed for assay (90 $). c. The indirect LMAT can be used for studies that attempt biochemical or physical modulation of the biological activity of supernatants from antigen-stimulated lymphocytes. d. The indirect LMAT can be used to investigate whether leucocyte migration inhibitory activity is antigen-dependent or antigen-independent.
4. Con-A-LIF assay Determination of the con-A-induced release of leucocyte migration inhibition factor in a two-step technique can be used for: a. Assessment of lymphokine synthesis capacity of lymphocytes responding to a well-defined, standardized, non-specific stimulus (14). b. Production of large amounts of LIF for purification (1 1). c. Production of standardized, lyophilized LIF material (13). These preparations can be used to standardize migration inhibition assays. They have also been applied in the investigations of various pathological conditions, e.g. testing the ability of leucocytes from patients suffering from immunodeficiency disorders to respond to LIF. d. Assessment of effects of chemicals and drugs on leucocyte migration, lymphokine (LIF) production and lymphokine (L1F)-leucocyte interaction (10, 24). DISCUSSION
I . Direct assays Experience so far shows that direct assays utilizing the same peripheral blood cell population as the source of specifically reacting lymphocytes and as polymorphonuclear granulocyte indicator cells in a mixture have given significant results in the LMCT as well as the LMAT in a great number of studies of clinical hypersensitivity states. Direct LMAT has main-
ly proved useful with purified antigen products, such as PPD, virus, bacterial cultures, and KLH, and less useful with impure antigen-containing preparations such as tissue homogenates for the detection of organ-specific antigens and tumour-specific antigens. One exception is the use of spleen extracts as antigen in the LMAT for detection of transplantation hypersensitivity. In general, however, the LMAT seems to be preferable for purified or comparatively pure antigen, and the LMCT for cruder antigen preparations. The cause of this qualitative difference has not yet been analysed in depth, but one probable explanation is that the antigen in the LMAT is preincubated with the cell mixture in a small volume for a certain limited period before the cells are transferred to the milieu intended for migration. With a crude antigen mixture containing for instance tissue elements in the direct LMAT, lymphocytes as well as the granulocytes and monocytes of the cell mixture examined are heavily exposed to a considerable number of non-specific, potentially biologically active compounds, which may interfere with the migration inhibition reaction at several points. A purified antigen product would be less apt to cause this type of non-specific blocking of the system. I I . Indirect assays Clearly, the direct LMCT or LMAT are less time-consuming than two-step assays, so that if results of comparable significance can be obtained, they are preferable in for instance controlled clinical trials. The direct systems may even be considered more representative ad hoc, since they register the combined reactivity of the very cell population of the patient at the time of examination. However, with our limited knowledge of the complexity of the system, comparison in all analyses of one-step and two-step reactivity with own and unrelated indicator cells would improve the chances of significant conclusions. With our present knowledge it is impossible to say whether two-step methods could be developed to achieve higher sensitivity than one-step assays, and at present the best method is to purify the antigen and use the LMAT.
Human Leuroryte Migration Inhibition REFERENCES 1.Anderse1-1, V., Bendixen, G. & Schiedt, T. An in vitro demonstration of cellular immunity against autologous mammary carcinoma in man. Arta med. srand. 186, 101, 1969. 2. Andersen, V., Kjaer, M. & Bendixen, G. In vitrodemonstration of cellular hypersensitivity to tumour antigens in man. Ser. Haemat. S, 3, 1972. 3. Andersen, V., Seborg, M. & Serensen, S. F. Antigen-induced lymphocyte transformation in vitro during primary immunization in man. I. Development and course. Arta path. mirrobiol. srand. Section B, 79, 489, 1971. 4. Bendixen, G. Specific inhibition of the in vitro migration of leucocytes in ulcerative colitis and Crohn’s disease. Srand. J. Gastroent. 2, 214, 1967. 5. Bendixen, G. Organ-specific inhibition of the in vitro migration of leucocytes in human glomerulonephritis. Arta med. srand. 184, 99, 1968. 6. Bendixen, G. Cellular hypersensitivity to components of intestinal mucosa in ulcerative colitis and Gohn’s disease. Gut 10, 631, 1969. 7. Bendixen, G. Organ-specific cellular hypersensitivity in autoimmune diseases. Ann. lmmunol. 4, 3, 1972. 8. Bendixen, G. & Seborg, M. A leucocyte migration technique for in vitro detection of cellular (delayed type) hypersensitivity in man. Danish med. Bull. 16, 1, 1969. 9.Bendixen, G. & Seborg, M. Comments on the leucocyte migration technique as an in vitro method for demonstrating cellular hypersensitivity in man. J. Immunol. 104, 1551, 1970. 10. Bendtzen, K. Drug effects on human leucocyte migration and migra:ion inhibitory activity from lymphocytes stimulated with concanavalin A. Arta path. mirrobiol. srand. Section C, 83, 447, 1975. 11. Bendken, K. Inhibition of human leukocyte migration inhibitory factor (LIF) by a-L-fucose. Arta allerg. (Kbh.) 30, 327, 1975. 12. Bendtzen, K. A new technique for partial purification of human leucocyte migration inhibitory factor. J. lmmunol. Methods. (Submitted for publication). 1976. 13. Bendtzen, K. Unpublished data. 14. Bendtzen, K., Andersen, V. & Bendixen, G. An in vitro assay of leukocyte migration inhibitory activity from human lymphocytes stimulated with concanavalin A. Arta allerg. (Kbh.) 30, 133, 1975. 15. Bloom, B. R. & Glade, P. R. (eds.) In Vitro Methods in Cell-Mediated Immunity. Academic Press, New York and London, 1971. 16. Brostoff, J., Roitt, I. M. & Doniach, D. Leucocyte-migration inhibition in autoimmune diseases. Lancet 1969, 1, 1969. 17. Clausen, J. E. Tuberculin-induced migration inhibition of human peripheral leucocytes in agarose medium. Arta allerg. (Kbh.) 26, 56, 1971.
183
18. Clausen, J. E. Migration inhibitory effect of cell-
free supernatants from mixed human lymphocyte cultures. J. lmmunol. 108, 453, 1972. 19. Clausen, J. E. Tuberculin-induced migration inhibition of human peripheral blood leukocytes after 2, 4, and 20 hours in agarose culture. Arta allerg. (Kbh.) 28, 28, 1973. 20. Clausen, J. E. Migration inhibitory effect of cellfree supernatants from tuberculin-stimulated cultures of human mononuclear leukocytes demonstrated by two-step MIF agarose assay. J. Immunol. 110, 146, 1973. 21. Clausen, J. E. Leukocyte migration agarose technique: some technical details. Arta allerg. (Kbh.) 28, 351, 1973. 22. Clausen, J. E. The Agarose Migration lnhibition
Technique f o r In Vitro Demonstration of CellMediated Immunity in Man. Thesis, N. Olaf Meller, Copenhagen, 1974. 23. Clausen, J. E. & Seborg, M. In vitro detection of tuberculin hypersensitivity in man. Arta med. srand 186, 227, 1969. 24. Coeugniet, E., Bendtzen, K. & Bendixen, G. Leucocyte migration inhibitory activity of concanavalin A-stimulated human lymphocytes. Modification by dipyridamole, lysine-acetylsalicylate and heparin. Arta med. srand. 199, 99, 1976. 25. Dardenne, M., Zabriskie, J. & Bach, J. F. Streptococcal sensitivity in chronic glomerulonephritis. Lancet 1, 126, 1972. 26. David, J. R., Al-Askari, S., Lawrence, H. S. & Thomas, L. Delayed hypersensitivity in vitro I. The specificity of inhibition of cell migration by antigens. J. Immunol. 93, 264, 1964. 27. Dormont, J., Sobel, A., Galaneaud, P., Gevon, M. C. & Colombine, J. Leucocyte migration inhibition with spleen extracts and other antigens in patients with renal allografts. Transplant. Pror. 4, 265, 1972. 28. Dumonde, D. ‘Lymphokines’: Molecular mediators of cellular immune responses in animals and man. Proc. Roy. Sor. Med. 63, 899, 1970. 29. Dumonde, D. C., Howson, W. T. & Wolstencroft, R. A. The role of macrophages and lymphocytes in reactions of delayed hypersensitivity. In Miescher, P. A. & Grabar, P. (eds.) Immunopathology, Schwabe & Co., Basel, 1968, p. 263. 30. Falk, R. E., Guttmann, R. D., Beaudoin, J. G., Morehouse, D. D. & Oh, J. H. Leucocyte migration in vitro and its relationship to human renal allograft rejection and enhancement. Trunsplantation 13, 461, 1972. 31. Federlin, K., Maini,
R. N., Russell, A. S. & Dumonde, D. C. A micro-method for peripheral leucocyte migration in tuberculin sensitivity. J. din. Path. 24, 533, 1971. 32.George, M. & Vaughan, J. H. In vitro cell migration as a model for delayed hypersensitivity. Proc. Sor. exp. Biol. Med 111, 514, 1962.
184
G . Bendixen, K . Bendtzen, J . E. Clauseti, M . Kjier & Af. S o b o r ~
33. Kaltreider, H. B., Soghor, D., Taylor, J. B. & Decker, J. L. Capillary tube migration for detection of human delayed hypersmsitivity: Difficulties encountered with ‘buffy coat’ cells and tuberculin antigen. J. Immunol. 103, 179, 1969. 34. Kjaer, M. In vitro-demonstration of cellular hypersensitivity to tumour antigens by means of thz leucocyte migration technique in patients with renal carcinoma. Europ. J . Cancer, 10, 523, 1974. 35. Kjaer, M. The capillary tube leucocyte migration technique (LMT) as a method for detection of serum inhibiting activity of tumour-directed, cellular hypersensitivity in patients with renal carcinomas. Arta path. microbiol. srand. Section B. 82, 294, 1974. 36. Kjaer, M. The dose-related effect of tumour extract on the in vitro migration of leucocytes from patients with renal carcinoma. Euvop. J. Cancer. 11, 281, 1975. 37. Kjaer, M. The effect of leucocyte washings on the in vitro detection of tumour-directed, cellmediated hypersensitivity in patients with renal carcinoma. Submitted for publication. 38. Kjaer, M. & Bendixen, G . Tumour-directed, cellular hypersensitivity detected by means of the leucocyte migration technique in patients with renal carcinoma. Ann. N.Y. Arad. Sci. 1976 (in press). 39. Kjaer, M. & Bendixen, G. Clinical detection and monitoring of tumour specific cell-mediated immunity in man: Examination of hypernephroma with the leucocyte migration technique. In Bloom, B. & David, J. R. (eds.) Z n Vitro Methods in Cell-Mediated Immunity, 11. Academic Press, 1976 (in press). 40. Kjaer, M. & Christensen, N. Ability of renal carcinoma tissue extracts to induce leucocyte migration inhibition in patients with non-metastatic renal carcinoma: Correlation with clinical and histopathological findings. Submitted for publication. 41. Kjaer, M. & Christensen, N. Prognostic value of tumour directed, cellular hypersensitivity detected by means of the leucocyte migration technique in patients with renal carcinoma. 1976 (in preparation). 42.Kjaer, M. & Snrensen, T. B. Comparison between the direct capillary tube leucocyte migration technique (LMCT) and the direct agarose leucocyte migration technique (LMAT) as indicators of tumour-directed, cell-mediated hypersensitivity (TCMH) in patients with renal carcinoma. Arta allerg., (Kbh.) 31, 141, 1976. 43.Main4 R. N., Roffe, L. M., Magrath, J. T. & Dumonde, D. C. Standardization of the leucocyte migration test. lnt. Arch. Allergy 45, 308, 1973. 44. Nerup, J., Andersen, V. & Bendixen, G. Anti-
Received 3 March 1976
adrenal cellular hypersensitivity in Addison’s disease. Clin. exp. Immunol. 4, 355, 1969. 45. Rich, A. R. & Lewis, M. R. The nature of allergy in tuberculosis as revealed by tissue culture studies. Bull. Juhns Hupkins Hosp. 50, 115, 1932. 46. Rocklin, R. 1:. Clinical applications of in vitro lymphocyte test. In Schwartz, R. S. (ed.) Progress in Clinirnl Immunology, Grune & Stratton, New York, 1974, p. 21. 47.Rosenberg S. A. & David, J. R. Inhibition of leucocyte migration: An evaluation of this in vitro assay of delayed hypersensitivity in man to a soluble antigen. J . Immunol. 103, 1447, 1970. 48. Sephadex. Gel filtration in theory and practice. Pharmacia Fine Chemicals AB, IJppsala, Sweden. 49. Smith, M. G. M., Eddleston, A. L. W. F. & Williams, R. Leucocyte migration in active chronic hepatitis and primary biliary cirrhosis. International mecting on immunological aspects of liver disease. In Smith, M. & Williams, R. (eds.) Z m m u n o l u ~uf~ the L k e r . Heineman, London, 1971, p. 135. 50. Smith, M. G. M., Eddleston, A. L. W. F., Dominguez, J. A., Evans, D . B., Bewick, M. & Williams, R. Changes in leucocyte migration after renal transplantation. Brit. med. J . 4, 275, 1969. 51. Smith, M. G. M., Golding, P. L., Eddleston, A. L. W . F., Mitchell, C. G., Kemp, A. & Williams, R. Cell-mediated immune responses in chronic liver diseases. Brit. med. J. 1, 527, 1972. 52. Soborg, M. Bestemmelse uf cellulcer hypersensibilitet in vitro med migrution.rhiemnitigsreaktionen. Thesis. FADL, Copenhagen, 1971. 53. Soborg, M. In vitro detection of cellular hypersensitivity in man. Specific migration inhibition of white blood cells from brucella positive persons. Acta med. srand. 182, 167, 1967. 54. Soborg, M. Interaction of human peripheral lymphocytes and granulocytes in the migration inhibition reaction. Arta med. srand. 185, 221, 1969. 5 5 . Soborg, M., Andersen, V. & Serrensen, S. F. Antigen-induced lymphocyte transformation in vitro during primary immunization in man. 11. Correlation with development of cellular and humoral hypersensitivity. Arta path. mirrobiol. srand. Section B, 79, 495, 1971. 56.Soborg, M. & Bendixen, G. Human lymphocyte migration as a parameter of hypersensitivity. Acta med. srand. 181, 247, 1967. 57. Thor, D. E., Jureziz, R. E., Veach, S. R., Miller, E. & Dray, S. Cell migration inhibition factor released by antigen from human peripheral lymphocytes. Nature (Load.), 219, 755, 1968. 58. Zabriskie, J. B., Read, S. E., Ellis, R. J., Markowitz, S. & Rappaport, F. T. Cellular reactivity studies in human and experimental renal transplantation. Tramp. Pror. 4, 259, 1972.
