Clin. exp. Immunol. (1976) 23, 481-490.

Specific blastogenesis and lymphokine production in DNCB-sensitive human leucocyte cultures stimulated with soluble and particulate DNP-containiing antigens W. R. L E V I S, J. J. W H A L E N & J. A. P O W E L L Dermatology Branch, National Cancer Institute, Bethesda, Maryland, U.S.A.

(Received 24 June 1975)

SUMMARY

A soluble hapten (dinitrobenzene sulphonic acid (DNBSO3)) and particulate antigens consisting of DNCB, DNFB or DNBSO3 complexed with erythrocytes, all induce a specific blastogenic response and lymphokine production in leucocyte cultures from human subjects topically sensitized to dinitrochlorobenzene (DNCB). While low concentrations (50-100 ug/ml) of DNBSO3 could be left in human leucocyte cultures the entire 4 or 5 days of culture and result in reasonable levels of blastogenesis, it was found that consistently higher degrees of blast transformation resulted when DNCBsensitive leucocytes were exposed to high concentrations of DNBSO3 (500 ug/ml) for a short period (2 hr). Cell-free supernatants from DNCB-sensitive leucocyte cultures harvested after 48 hr induced blastogenesis and DNA synthesis in secondary target leucocyte cultures from subjects not sensitized to DNCB. Such a blastogenic factor or lymphokine appeared to stimulate even in the absence of any residual antigen, since DNCB complexed to erythrocytes was removed by simple filtration through a 0 45 um Millipore filter. In contrast to DNCB complexes, the antigenic activity of DNBSO3 complexed with erythrocytes was not removed by such filtration. Thus, several DNP-containing haptens (DNCB, DNFB, DNBSO3) induce specific lymphocyte transformation and lymphokine production when exposed in several different manners to leucocytes from humans sensitized to DNCB. The ability to use either a particulate or soluble stimulant in vitro offers a versatile system for studying cell-mediated immunity in humans with a broad range of potential applicability in both investigative and clinical medicine.

INTRODUCTION Sensitivity to dinitrochlorobenzene (DNCB) is widely used as a model for evaluating cellmediated immunity in humans and guinea-pigs (Chase, 1967; Catalona et al., 1972; Kligman & Epstein, 1959). In recent years several approaches for in vitro evaluation of DNCB or dinitrofluorobenzene (DNFB) sensitivity have been reported in humans (Miller & Levis, 1973a, b) guinea-pigs, (Geczy & Baumgarten, 1970; Milner, 1970) and mice (Phanuphak, Moorhead & Claman, 1974). These studies utilized specific lymphocyte transformation to DNCB (Miller & Levis, 1973a, b), DNFB (Geczy & Baumgarten, 1970) or dinitrobenzene sulphonic acid (DNBSO3) (Phanuphak et al., 1974) as a means of in vitro stimulation. The animal and human studies have involved various means of sensitization and various manCorrespondence: Dr W. R. Levis, Dermatology Branch, National Cancer Institute, Bethesda, Maryland 20014, U.S.A.

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ners of presenting the antigen, either free or complexed, to the responding lymphocytes in culture. The advent of such in vitro studies with simple chemicals offers potential insight into the mechanism of contact sensitivity including the role of various carriers and allows a systematic approach for the study of cell-mediated immunity in humans. In this report we extend some previous in vitro animal studies of DNCB or DNFB contact sensitivity using human subjects topically sensitized with DNCB in acetone. We employ both soluble DNBSO3 and particulate antigens (DNCB, DNFB and DNBSO3 coupled to erythrocytes) to induce specific blastogenesis and lymphokine production. The ability to utilize both soluble and particulate antigens in vitro suggests that several alternative mechanisms may be involved in contact sensitivity and adds versatility to the in vitro method for evaluating cell-mediated immunity in humans.

MATERIALS AND METHODS Sensitization and patch testing with DNCB. A sensitizing dose of 2000 pg DNCB (1-chloro-2,4-dinitrobenzene) (Eastman Kodak Company, Rochester, New York) in 0 1 ml acetone was applied to the skin of the medial aspect of both upper arms within a 2-cm diameter polyethylene ring, allowed to evaporate, and covered by an adhesive bandaid for 2 days (Catalona, Taylor & Chretien, 1972). All sensitized subjects used in this study had been rechallenged with DNCB in a cream base (Acid Mantle Creme (Aluminum Acetate) Dome Laboratories, West Haven, Connecticut) on three or more occasions within the past year and were known to have maintained high levels of lymphocyte transformation to DNCB antigen (Miller & Levis, 1973b; Levis, Whalen & Powell, 1975) and all showed at least erythema and induration to 1-pg DNCB patch tests. Preparation ofleucocyte cultures andincubation with DNBSO3. 'Mononuclearleucocyte culture fluid' was prepared by diluting the 'mononuclear leucocyte-rich plasma' obtained by centrifugation (Levis & Robbins, 1972) with four parts of tissue culture medium (RPMI 1640 Grand Island Biological, number 187G) containing penicillin and streptomycin. This method yields a final concentration of 04-2 x 106 cells/ml of which 70-90%Y are lymphocytes. In this study, to assure a higher cell concentration, the top half of the plasma following the initial 5 min centrifugation was removed and the lower mononuclear leucocyte-rich plasma was collected separately. Following this manoeuvre all cultures were adjusted to a final concentration of 2 x 106 leucocytes/ml of culture. Before being placed in culture, leucocytes were incubated with varying concentrations of DNBSO3 in RPMI 1640 both with and without 20 per cent plasma. For any given experiment, leucocytes with and without DNBSO3 and with and without plasma were treated with identical washing procedures. Plasma was removed by washing the cells twice with RPMI 1640, centrifuging at 150 g for 6 min with each wash. Following the desired length of incubation with DNBSO3 leucocytes were again washed and final culture concentration was readjusted to 2 x 106 leucocytes/ml with 20%Y autologous plasma in RPMI 1640. Cultures were initially prepared in 1-ml volumes and quadruplicate 0-2 ml cultures were transferred to microtitre plates (Microtest II Tissue Culture Plate, catalog number 3040, Falcon Plastics, Oxnard, California) with Finnpipettes (Volumetrics, Incorporated, Wilmington, Massachusetts). Cultures were incubated for 4 or 5 days in a 5 % CO2 humidified atmosphere at 37°C. Preparation ofparticulate antigen with DNCB, DNFB and DNBS03. Details of the preparation of a particulate complex with DNCB (DNCB-antigen) have been previously reported (Miller & Levis, 1973b). Briefly, for DNCB-antigen, 1-ml of packed erythrocytes are incubated in 10 ml of undiluted DMSO (dimethyl sulphoxide) (Aldrich Chemical Company, Milwaukee, Wisconsin) containing 1 g %Y DNCB for 1 hr at 38'C. The resultant cell pellet was washed once with DMSO, twice with tissue culture medium and then lyophilized, pulverized and stored at 4°C. When DNFB (l-fluoro-2,4-dinitrobenzene, Eastman Kodak Company) or DNBSO3 (2,4-dinitrobenzenesulphonic acid sodium salt) (Eastman Kodak Company) were used to prepare a particulate antigen, the same procedure as outlined above was used. However, it was found that incubation of erythrocytes with DNBSO3 formed a more active antigen when performed at a concentration of 5 g % rather than the 1 g Y. used for DNCB and DNFB. These lyophilized particulate antigens were added to leucocyte cultures at varying concentrations as indicated in the results. Lymphokine supernatant harvest andassay. Details of this procedure are published elsewhere (Levis, Whalen & Miller, 1974). In brief, cultures for the production of lymphokine(s) were prepared by culturing leucocytes alone (unstimulated control cultures) or in the presence of DNBSO3 or particulate DNCB-antigen (DNCBantigen-stimulated cultures). These primary cultures were cultured in 5-ml volumes at 37°C in a 5% CO2 humidified atmosphere in 2-oz prescription flasks. In studies presented in this report, supernatants from these cultures were harvested by centrifugation between 46 and 51 hr and were filtered through 0 45 pm Millipore

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filter (Millipore Corporation, Bedford, Massachusetts); 0-2-ml aliquots of the primary cultures were removed at the time of supernate harvest, placed in microtitre plates and assayed for DNA synthesis 2 days later. Assay of lymphokine activity involved the addition of supernates of stimulated and unstimulated primary cultures to secondary leucocyte cultures prepared from subjects not sensitized to DNCB. The concentration of leucocytes was maintained constant at 2 x 106 leucocytes/ml. Secondary target leucocyte cultures were cultured in 0-2-ml volumes in microtitre plates. Addition of [3H]TdR to microcultures. Thymidine-methyl-3H ([3H]TdR) (17-22 Ci/mmole; I mCi/ml; catalogue number TRK 120), (Amersham/Searle, Chicago, Illinois) was added to obtain a final concentration of 10 uCi/ml of culture by dispensing 5 lambda aliquots into each 0-2 ml culture from a Hamilton repeating syringe (Hamilton Company, Reno, Nevada). Harvesting of [3H]TdR-labelledmicrocultures. After incubation with [3H]TdR for 3 hr, the microtitre plates were harvested with the Multiple Automated Sample Harvestor (MASH II) (Microbiological Associates, Bethesda, Maryland). The MASH II simultaneously aspirates cultures and passes the culture fluid through a glass fiber filter (Reeve Angel, Clifton, New Jersey) which retains the cells (Hartzman et al., 1972; Thurman et al., 1973). Culture wells were automatically rinsed with 0-85 M, pH 7-2, phosphate buffer. The filter paper was removed, the apparatus rinsed by aspirating distilled water through the system, a new filter paper was placed on the collecting tray, ready to harvest the next twenty-four cultures. When the filter paper is removed from the MASH II, circular areas corresponding to each of the twenty-four wells have already been partially punched out. The circular pieces of filter paper are then easily tapped into clean half-dram vials in preparation for liquid scintillation spectroscopy. Scintillation counting. Filters were counted by a double-vial technique reported previously (Robbins, Burk & Levis, 1970; Robbins et al., 1972). The half-dram vials containing the filters are placed in a drying oven in vacuo at 80'C for 5 min, removed, and allowed to cool to room temperature. The dried filter is wetted by adding to the vial 1 ml of a scintillation fluid composed of 20 ml of 'Liquifluor' (New England Nuclear Corporation, Boston, Massachusetts) in 1 pint of toluene. The vial is then screwed into its cap which has previously been cemented to the inner lining of the cap of a standard scintillation counting vial. Radioactivity on the filter within this 'double-vial' apparatus is then determined in a Model 4322 Packard Tri-Carb Scintillation Spectrometer (Packard Instrument Company, Downers Grove, Illinois). [3H]TdR incorporation of DNA synthesis is expressed as the mean of quadruplicate cultures ± S.E.

RESULTS

Fig. 1 illustrates two experiments in which DNBSO3-induced lymphocyte transformation in leucocyte cultures prepared from DNCB-sensitive subjects. Even when initial exposure time (a)

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FIG. 1. Effect of concentration and incubation time with DNBSO3 on lymphocyte transformation in DNCB-sensitive subjects. Time in hours represents the initial incubation time of leucocytes with DNBSO3. All leucocytes were washed after the designated incubation time and cultered for a total of 4 days at which time [3H]TDR incorporation was measured. (a) Experiment 1. (b) Experiment 2.

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was as short as 15 min, high levels of DNA synthesis were induced with concentrations of DNBSO3 of 500 jig/ml or more (e.g. 2000 jig for 15 min gave over 50,000 ct/min). However, when the initial incubation period with DNBS03 was prolonged, a marked diminution in DNA synthesis was noted at concentrations of 500pg/ml or greater (e.g. 2000 jg for 4 hr in experiment 1, Fig. 1, is already less than 3000 ct/min). At lower concentrations of DNBSO3 prolonged incubation resulted in increased DNA synthesis (e.g. 50,jg for 15 min showed only 1300 ct/min, whereas after 4 hr gave 6000 ct/min). In fact, concentrations of 50-100 jig could be left in culture the entire 96 hr and result in significant DNA synthesis as shown in Fig. 2 and as seen in other experiments (unpublished data). However, the degree of DNA synthesis achieved with such low concentrations was usually not as great as with high concentrations for shorter periods. We therefore settled on 500 jg DNBSO3/ml for a period of 2 hr (unless other times are indicated) for all subsequent experiments despite the disadvantage of the requisite washing procedure. (a)

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