Journal of Immunological Methods, 11 (1976} 107--116

107

© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

IN VITRO INDUCTION AND MEASUREMENT OF HEMOLYTIC P L A Q U E F O R M I N G C E L L S IN M A N

HANS-MICHAEL DOSCH and ERWIN W. GELFAND Department of Immunology, The Hospital For Sick Children, Toronto, Canada

(Received 1 October 1975, accepted 29 October 1975)

Following co-cultivation with sheep red ceils or ovalbumin, Hypaque--Ficoll-separated human tonsillar lymphocytes were demonstrated to generate specific hemolytic PFC with maximum numbers at day 5--7. PFC were enumerated on poly-L-lysine coupled red cell monolayers in Microtest-II-plates. Plaque formation appeared to be puromycin-sensitive, complement-dependent and showed clear specificity for the antigen present during the inductive culture. Treatment of PFC with p-chain specific antisera and complement resulted in complete inactivation of PFC; 3'-chain antisera had no effect. The development of such a simple and sensitive assay system permits the analysis of cellular interactions required for the induction of PFC responses in man. T h e i n d u c t i o n o f a p r i m a r y h u m o r a l i m m u n e r e s p o n s e as well as t h e d e t e c t i o n o f a n t i b o d y secreting cells, have b e e n s t u d i e d b y a v a r i e t y o f m e t h o d s p r o v i d i n g i n f o r m a t i o n c o n c e r n i n g t h e cellular a n d antigenic' r e q u i r e m e n t s as well as t h e n a t u r e o f b o t h t h e d i f f e r e n t i a t i v e a n d p r o l i f e r a t i v e e v e n t s w h i c h o c c u r d u r i n g i n d u c t i o n a n d d e v e l o p m e n t o f a h u m o r a l i m m u n e res p o n s e in v i t r o (Mishell a n d D u t t o n , 1 9 6 7 ; M a r b r o o k , 1 9 6 7 , 1 9 6 8 ; Cunningham and Szenberg, 1968). Whereas much of these data have been accumul a t e d f r o m a n i m a l m o d e l s , little d i r e c t k n o w l e d g e is available f o r h u m a n cell s y s t e m s (Platts-Mills a n d I s h i z a k a , 1 9 7 5 ; S l o y e r et al., 1 9 7 3 ; W a t a n a b e et al., 1 9 7 4 ) . T h e d e v e l o p m e n t o f such a m o d e l s y s t e m in m a n w o u l d a p p e a r t o h a v e several a d v a n t a g e s ; these include well d o c u m e n t e d m a r k e r s y s t e m s f o r h u m a n B a n d T l y m p h o c y t e s ( C o o m b s , 1 9 6 9 ; S e l i g m a n n e t al., 1 9 7 3 ; E d e n e t al., 1 9 7 3 ) a n d availability o f l y m p h o i d ceils f r o m t h e w i d e v a r i e t y o f n a t u r a l l y o c c u r r i n g i m m u n e d e f i c i e n c y disease states. As suggested r e c e n t l y b y P y k e et al. ( 1 9 7 5 ) d e v e l o p m e n t o f such m e t h o d s m a y also be h e l p f u l in t h e d e l i n e a t i o n o f t h e basic d e f e c t in several o f t h e s e c o n d i t i o n s . In this p a p e r , a s i m p l e p r o c e d u r e f o r s t u d y i n g t h e i n d u c t i o n a n d evaluat i o n o f h e m o l y t i c p l a q u e f o r m i n g cells ( P F C ) in m a n is d e s c r i b e d . O u r results Abbreviations used in this paper: PFC -- hemolytic plaque forming cells; HcPFC -- hu-

man culture PFC; HTC -- human tonsillar lymphocytes after Ficoll--Hypaque; HRC -human red cells, blood group O ; S R C - sheep red cells; O A - Ovalbumin; SRC-OA/HRCOA -- Ovalbumin coupled to SRC or HRC by means of chromic chloride; PLL -- poly-Llysine; PWM -- pokeweed mitogen.

108 indicate in this reproducible procedure the PFC response is specific for the antigen present during the incubation phase, that the response is mediated by cells carrying surface IgM and that the response is puromycin-sensitive. The micromodification of the hemolytic PFC assay developed is sensitive, and requires low numbers of effector cells. MATERIALS AND METHODS

Sera and media RPMI-1640 was used, supplemented with Penicillin (100 U/ml), Streptomycin (100 pg/ml), Fungizone (25 pg/ml), L-glutamine (2 mM) and 1% Trypticase-Soy broth solution (0.3 g/100 ml distilled water) (Gibco, Grand Island~ N.Y.). For culture, the medium was supplemented with 10% heatinactivated AB-serum from a pooled stock of 4 or more donors. These serum batches exhibited the usual (low) titers of Forssman-like antibody activity and were used unabsorbed.

Cell preparation Tonsils obtained following tonsillectomy were placed in medium and teased with dull scissors using sterile precautions. After filtration through glass wool, single cell suspensions were pelleted and separated on Ficoll-H y p a q u e gradients (Gelfand et al., 1972). The mononuclear cell layer (HTC) was washed three times in medium and usually contained 60--75% lymphocytes carrying membrane immunoglobulin and 1--6% macrophages as judged by staining for non-specific esterase (Yam and Crosby, 1971). Viability evaluated by Trypan Blue dye exclusion always exceeded 98%. The cell yields per one tonsil ranged from 8--15 × 10 s viable lymphocytes.

Antigens Commercially available sheep red cells (SRC) were washed 2--3 times in PBS. Varying numbers of SRC were added to the cultures in volumes of 20 or 50 pl. A stock solution of ovalbumin (45 mg/ml) (Sigma Chemicals Co., St. Louis., Fract. VI) was prepared in PBS, Millipore-filtered and stored at --20 ° C.

Complement Guinea pig serum was obtained by cardiac puncture of male outbred animals and absorbed three times for one hour with 100 #1 of washed, packed SRC/ml at 4°C. Absorbed serum was stored in 0.5 ml samples at --70°C, thawed prior to use at 37°C and diluted with medium to give a final dilution of 1 : 2 0 . Some of these samples were heat inactivated (30 min/

109 56°C) for complement-free controls. Following incubation of SRC with the inactivated serum (2 h/37°C) and three washings in PBS, reincubation (2 h/ 37°C) with the complement preserved serum failed to result in lysis suggesting the absence of anti-SRC activity in the complement preparations.

Induction o f PFC Cultures were performed in 15 ml plastic tubes (Falcon Plastics # 2 0 5 7 ) containing 10 ml culture medium and 5--10 X 106 viable HTC with or without antigen. Following varying incubation periods in a humidified atmosphere of 5.0% CO2 in air at 37°C, cultures were pelleted, washed twice and kept on ice in 1 ml serum-free medium. Cell counts and assessments of cell viability were performed using a h e m o c y t o m e t e r and the cell suspensions were adjusted to 2.5 X 10s--2.5 X 106 viable cells/ml. In other cultures, cell preparations were suspended in the original volume (following washing) w i t h o u t regard for cell recovery and aliquots of 20--50 pl were used for evaluation of PFC w i t h o u t further processing.

PFC assay Evaluation of PFC in Microtest II plates (Falcon Plastics # 3 0 4 0 ) was performed following a modification of the m e t h o d described by Kennedy and Axelrad (1971). Plates containing 50 ttl of poly-L-lysine (PLL, 25 lag~ ml distilled water) per well were incubated for 30 min at 37°C. The PLL was aspirated, the plates washed 3 times with PBS and 100 /~1 of washed indicator red cells (2--4%) were added immediately. Following an incubation period of 30--60 min at 37°C, u n b o u n d red cells were rinsed off.with PBS under continuous suction. The monolayers were overlayed with 25 ttl serum-free medium to prevent drying and were used the same day. For assessment of PFC, dilutions of cultured HTC were added in a volume of 20 #l/well in quadruplicates. In general, 5 X 103 to 5 X 104 viable HTC were added. Complement (20 pl) was then added and the plates were incubated for 40--60 min at 37°C. Hemolytic plaques were counted using an inverted microscope. In experiments using ovalbumin (OA) as antigen, red cells used for indicator monolayers were coated with OA b y means of chromic chloride (Sweet and Wellborne, 1971) prior to preparing the monolayers. Since binding of these cells to the PLL-treated wells was n o t as stable as that of the untreated cells, these monolayers were checked microscopically before addition of PFC and only intact monolayers were used. In specificity control experiments human red blood cells, blood group O (HRC), were also used as indicator cells.

Mitogenic stimulation Cultures with and w i t h o u t SRC were incubated in the presence of various concentrations of pokeweed mitogen (PWM). Mitogenic responses were determined on day 6. Cells were harvested and 5 X 104 viable cells reincu-

110

bated in fresh medium containing 0.2 pCi [3H]thymidine. After a further 4 h incubation at 37°C, cells were harvested using a Skatron multiple cell harvester (Skatron, Lierbyen/Norway) and radioactivity was counted by liquid scintillation. The stimulation index was calculated as the ratio of cpm of stimulated to unstimulated cultures.

Treatment o f PFC Puromycin (Sigma Chemicals Co., St. Louis, Mo.) was added to cultures 2--14 h before harvesting in a concentration of 10 pg/ml. The media used for washing and for the PFC assay contained the same concentration of the drug. In the presence of puromycin, the cell viability was f o u n d to be essentially unchanged when compared with puromycin-free controls for up to 8h. Monospecific goat antisera to h u m a n p or 7 chains * (50 pl) and 100 pl absorbed complement were incubated with 5--10 × 106 cultured HTC in a final volume of 500 pl in a shaking waterbath at 37°C. After 1 h cells were washed twice in the cold and twice pelleted through 1 ml AB-serum. RESULTS

Time course o f PFC generation In the first series of experiments, cultures containing 107 tonsil cells and %RECOVERY

CULTUREOF VIABLE

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DAYSOFCULTURE Fig. 1. T i m e c o u r s e o f P F C g e n e r a t i o n in cultures o f 1 × 107 HTC and an o p t i m a l conc e n t r a t i o n o f SRC (1 × 106). Viability o f r e c o v e r e d cells was assessed b y t r y p a n blue dye exclusion. • e : n o r m a l i z e d d a t a f r o m t h r e e e x p e r i m e n t s e x p r e s s e d as PFC/106 viable, recovered cells. A A: m e a n r e c o v e r y o f viable cells f r o m t h r e e e x p e r i m e n t s expressed as a p e r c e n t a g e o f the initial i n o c u l u m o f 107 HTC.

* Kindly provided by Dr. C. Alper, Boston, Mass.

111 104 SRC per tube were incubated in parallel and harvested at various times. As indicated in fig. 1, PFC's could first be detected after 3 days in culture and represented less than 1% of the maximal PFC numbers f o u n d on day 5-7. At the time of maximal PFC response, cell recovery had declined from its m a x i m u m f o u n d at day 4. PFC levels as well as recoverable cell numbers slowly declined beyond day 7. The decrease in PFC numbers could not be reduced or delayed by feeding the cultures with nutritional cocktails and/or renewing 20, 40 or 80% of culture medium 1--3 times despite an improvem e n t in cell survival. Handling of cells during culture, accompanied by resuspension, decreased the PFC response. Calculation of PFC numbers generated was performed in two ways. One was based on the number of viable cells assayed, and in the second, PFC were measured in a defined aliquot regardless of cell numbers or cell viability. As both calculations revealed similar results, this suggested t h a t plaque formation was a f u n c t i o n - o f living cells.

Effect of complement PFC were enumerated in the presence of complement-preserved guinea pig serum. Control-wells contained complement only or cultured cells together with heat inactivated guinea pig serum. Fewer than 1--3 plaques/ well were observed in these controls, suggesting a strict complement-dependance of the plaque formation observed. This was confirmed when varying numbers of cells were assessed (see further) where the number of plaques increased with increasing numbers of cells only in the presence of complement.

Analysis of plaque formation Fig. 2 illustrates a limiting assay with normalized data obtained from four

/ caLs/wm 10 10s Fig. 2. Demonstration of the linear relationship between the numbers of recovered cells after culture and PFC observed. Results of 4 experiments have been normalized and the means +-1 S.D. are shown. The slope of the line approached 1.

112

experiments. A linear relationship between the number of cells assayed and the number o f plaques observed was established. When plotted on a log-log scale, the slope of the resulting line approximated 1. This suggested a onecell rather than a pluricellular p h e n o m e n o n (Quintans and Lefcovits, 1974a). Plaque formation was assessed after 10 min to 180 min incubation at 37°C. It was f o u n d t h a t the plaque size increased with time, and at 90--180 min plaques became confluent. PFC numbers did not increase beyond 20 min.

Antigen requirement and specificity Varying the antigen concentration from 104--109 SRC per 107 tonsil cells resulted in establishment of a distinct dose response (table 1) with the maximal PFC response at a ratio of about one red cell per 10 lymphocytes (~ 1.3 + 0.4, N-12). Specificity of the PFC response was studied using the unrelated antigen OA, where a similar dose response curve was found. Following culture in the presence of either SRC or OA, PFC were evaluated on monolayers of indicator red cells which included SRC, SRC-OA or HRCOA. Anti-OA-PFC were maximal on day 5--6 and were of the same magnitude as anti-SRC-PFC (table 1). Furthermore the PFC generated showed clear specificity for the antigen present during the induction phase (table 1). PWM has been described to induce proliferation of both B and T lymphocytes and to increase immunoglobulin synthesis in man (Geha et al., 1973; TABLE 1 /

A n t i g e n d o s e r e s p o n s e and s p e c i f i c i t y o f PFC. PFC w e r e assayed o n poly-L-lysine c o u p l e d red cell m o n o l a y e r s a f t e r 6 days o f c u l t u r e o f 107 HTC in t h e p r e s e n c e o f varying a m o u n t s o f a n t i g e n and are e x p r e s s e d as P F C ± 1 S.D./106 viable, cells r e c o v e r e d a f t e r culture. Antigen

SRC SRC SRC SRC

Concentration]culture

10 s 106 107 10 s

P F C / 1 0 e cells SRC *

SRC-OA **

161 2922 899 241

119± 2782+_ 707 -+ 209-+

-+ 43 ± 122 +- 103 -+ 74

HRC-OA

56 92 91 62

In vitro induction and measurement of hemolytic plaque forming cells in man.

Journal of Immunological Methods, 11 (1976} 107--116 107 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands IN VITRO INDUC...
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