Antibody-forming foci in soft-agar cultures of human peripheral blood cells

Journal of hnmunological Methods, 45 ( 1981 ) 129--136 Elsevier/North-Holland Biomedical Press

ANTIBODY-FORMING F O C I IN S O F T - A G A R PERIPHERAL BLOOD CELLS

129

CULTURES

OF HUMAN

MARIA LUISA VILLA I and ENRICO CLERICI

Department of Immunology, Unieersity of Milan. and Istituto Nazionale Tumori, 20133 Milan, Italy (Received 8 January 1981, accepted 28 April 1981)

Human peripheral blood lymphocytes (PBL) from healthy blood donors were grown in soft-agar gel with sheep red blood cells (SRBC) and autologous plasma as a source of complement. After 4--6 days incubation, foci of proliferating hemolysin-forming cells, surrounded by a lytic area of 0.2--0.5 mm, were detected on the surface of the plates. The response was antigen specific, since new hemolytic areas were observed on pouring a fresh agar-SRBC mixture over the surface of primary cultures, but not on pouring a mixture containing rat or rabbit erythrocytes. The antibody response was significantly increased by addition to the cultures of polyethylene glycol 6000 (PEG), 8% final concentration. The mean number of foci was 8.4 + 2.2 in cultures without PEG and 36.2 + 2.3 in PEG+ cultures, both containing 9 , 106 lymphocytes. This finding is in agreement with observations on the frequency of precursors of antibody-forming cells among lymphoid populations, The explanation of the mechanism by which PEG 6000 modified the immune reactivity of PBL is not clear. However, we think that this technique provides a reliable methodology for PBL antigenic stimulation in vitro.

INTRODUCTION

M e t h o d s so far d e v e l o p e d f o r i n d u c i n g an i m m u n e r e s p o n s e in h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s ( P B L ) in v i t r o a r e v a l u a b l e b u t c a n n o t y e t b e used for purposes of diagnostic medicine because they yield results which are s e l d o m r e p r o d u c i b l e . T h i s is s u r p r i s i n g s i n c e t h e r e is n o d o u b t t h a t h u m a n b l o o d c o n t a i n s a n t i b o d y - p r e c u r s o r s cells, as d e m o n s t r a t e d b y t h e f a c t t h a t P B L c u l t u r e s s t i m u l a t e d b y p o l y c l o n a l a c t i v a t o r s s u c h as p o k e w e e d m i t o g e n or endotoxic lipopolysaccharide produce sheep erythrocyte (SRBC) hemolysing ceils w h i c h m a y b e d e t e c t e d b y s u i t a b l e a s s a y s ( F a u c i a n d P r a t t , 1 9 7 6 ) . However, when PBL cultures are stimulated by the antigen itself, rather than b y p o l y c l o n a l a c t i v a t o r s , t h e a n t i b o d y r e s p o n s e is less s a t i s f a c t o r y . E v e n t h e b e s t r e s u l t s , w h i c h h a v e b e e n o b t a i n e d b y c h a l l e n g i n g P B L w i t h S R B C in t h e p r e s e n c e o f E p s t e i n - B a r r virus, a r e c h a r a c t e r i z e d b y a s u b s t a n t i a l a m o u n t o f

I Correspondence

to: Maria Luisa Villa, M.D., Department of Immunology, via Venezian

1, 20133 Milan, Italy.

0022-1759/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press

130

neoative data, i.e., cultures devoid o f any a n t i b o d y p r o d u c t i o n ILuzzati et al., 19761. Since Stlch studies have usually been cm'ried OLIt with c o m p l e x antigens, it is hard to helieve that the i m m u n e lel'tne,~ is due to genetically d e t e r m i n e d unresponsiveness, like that observed in certain mouse and guinea pig strains challenged with very simple antigens such as s y n t h e t i c p o l y p e p tides. It is more likely that the i m m u n e inel'tness is caused by (ai the inadeq u a c y o f tile t e c h n i q u e s for culturing and sensitizing PBL in vitro, and or ~b) tile presence, a m o n g PBL, o f suppressor cells which seem to be very active in the blood o f certain individuals I H o f f m a n , 19801. F u r t h e r m o r e . peripheral blood is not a main site o f a n t i b o d y p r o d u c t i o n , the f u n c t i o n o f its l y m p h o cytes being to c o n n e c t the peril)heral l y m p h o i d organs and c o n t r o l i m m u n e reactivity. It might t h e r e f o r e be e x p e c t e d that antigen-reactive cells represent a tiny m i n o r i t y o f PBL and that critical culture conditiom, are required to elicit their a n t i b o d y - f o r m i n g capacity and nmke the i m m u n e response detectable by r o u t i n e techniques. In the present s t u d y we have c o n c e n t r a t e d on improving the survival and stimulation o f PBL in vitro by utilizing t e c h n i q u e s previously applied to the p r o d u c t i o n o f myeloid and l y m p h o i d cell colonies in agar, and by supplemeriting the culture media with p o l y e t h y l e n e glycol (PEG) 6000. By means o f these devices we o b t a i n e d an a n t i b o d y response against SRBC r e m a r k a b l y good, specific and r e p r o d u c i b l e . MATERIALS AND METHODS

Blood donors and l y m p h o o ' t e separation Heparinized blood o b t a i n e d from healthy blood d o n o r s was centrifuged on a F i c o l l - H y p a q u e ~ a d i e n t {BOyum. 19681. The plasma oll the top of the gradient and the cells at the interface were harvested. The cells were washed twice in D u l b e e c o ' s modified Eagle's m e d i u m ( D M E M ) a n d resuspended in a u t o l o g o u s fresh plasma (2 ml o f plasma for the cells from every 10 ml o f Mood L TJSSLIe cLtltltres Cultures were set up ill 60 nlm plastic Petri dishes ~Sterilin, cod. 123 S~. B o t t o m layers were prepared by mixing equal volumes o f 1¢7 agar (Nobleagar, Difco Lab. Inc., Detroit, MI), previously boiled and c o o l e d to 40~C. and d o u b l e strength DMEM with fetal calf serum at a final c o n c e n t r a t i o n o f 10% and 0.2 mgG D E A E d e x t r a n . Aliquots o f 6 ml o f this m i x t u r e were p o u r e d into Petri dishes az~cl allowed to gel at r o o m t e m p e r a t u r e . Plates were used oil the day they were prepared. The t o p layer m i x t u r e was prepared in such a way that each ml c o n t a i n e d 0.33% agar in DMEM + 6.6"~ fetal calf serum, 3 :,' 10 ~ PBL in a u t o l o g o u s fresh plasnm I final c o n c e n t r a t i o n 30%), 10'7 SRBC and 0.2 rag% D E A E d e x t r a n . Aliquots o f 3 ml o f this mixture, which was kept warm at 40~C. were t h e n p i p e t t e d o n t o previously prepared b o t t o m layers, taking cm'e to avoid bubbles. After cooling at r o o m

131 temperature, the plates were incubated 4--6 days at 37°C, in a humidified box with an atmosphere of 10~ CO: + 5.% O: + 85% N.,. In a second series of plates, PEG 6000 (Sigma Chemical Co.I was added to the t op layer, at a final concentration of 8%. In all experiments control plates containing SRBC but not PBL were set up to detect spurious hemolytic foci.

Hemolytic foci and cell colony Ibrmation Hemolytic foci and cell colony counting and size measurements were done with a dissecting microscope at ×10 magnification using an ocular micrometer. Counting was further facilitated by staining the entire plate with benzidine (Jerne et al., 1963). For microscopic examination of cell colonies, the center of the plaques areas was removed with a finely drawn Pasteur pipette and placed on a prestained glass slide (Testsimplet, Boehringer, Mannheim), or processed for immunofluorescence by routine techniques.

Double-layer technique for specificity control of hemolytic loci After 3 days incubation, some plates were removed from the incubator. The hemolytic foci were counted without using benzidine and their position marked with a dot on the b o t t o m of the plate. These plates were overlaid with 3 ml of 0.5% agar in DMEM, containing either SRBC or rabbit or rat e r y t h r o c y t e s at 10c~ final concentration. Following reincul)ation for 2 days, the plates were stained with benzidine, hemolytic areas in the overlayer were counted, and their coincidence with or divergence from previously detected foci was verified. In order to demonstrate that rat and rabbit RBCs cm~ be lysed under these experimental conditions, controls were included in which they were cultured together with PBL in agar plates for 3 days, and the cultures were then overlaid with 0.5% agar in DMEM containing rat or rabbit or sheep erythrocytes.

IgM detection in hemolytic foci by enzyme immunoassay This test was performed on some plates following the technique of Sharon et al. (1979} and Miggiano (1979), with some modifications. Filters (Millipore, pore size 0.45 pro} coated with SRBC ghosts, were gently dropped on the surface of some plates after 3 days incubation. The plates were reincubated overnight, and the filters were removed, treated with rabbit anti-lgM for 24 h, then incubated with peroxidase conjugated sheep anti-rabbit immunoglobulins for 30 min at 37°C and 16 h at 4 ° C. Filters were finally treated with diaminobenzidine and H:O.,. In about 20 min brown spots appeared which corresponded to IgM deposition areas. RESULTS

Hemolytic foci in cultures with and without PEG 6000 Fig. 1 shows the results obtained with PBL of 35 adult healthy blood donors cultured with or without PEG 6000. In the plates without PEG the

132 n of f o o per g, ~06 cells

70 65 60 55 50 45 40

°:o

35 30:

oee

25

0g0

20

m I

15

10

el •

ee

g•

o

D 0o egD m j

OQD

Q

D D Qm

PEG-

PEG +

Fig. 1. H e m o l y t i c foei in PBL c u l t u r e s p r e p a r e d with and w i t h o u t PEG 6 0 0 0 . Each point r e p r e s e n t s the n u m b e r o f t'oei scored in a single plate c o n t a i n i n g 9 ' 106 PBL.

n u m e r i c a l d i s t r i b u t i o n of h e m o l y t i c foci shows wide scatter, seven o f the d o n o r s p r o d u c i n g no h e m o l y t i c loci at all. T h e average n u m b e r o f h e m o l y t i c foci was 8.4 _+ 2.2 S.E.M. In tile cultures c o n t a i n i n g P E G the numl)er o f h e m o l y t i c foci is significantly increased and t h e r e are no longer negative results. Tile average n u m b e r o f foci was 36.2 -+ 2.3 S.E.M. Results ol)tained on r e p e a t i n g these tests a f t e r a o n e m o n t h interval satisfactorily replicated t h o s e o b s e r v e d in the first series. Fig. 2 s h o w s the h e m o l y t i c foci as t h e y a p p e a r in plates. C o n t r o l plates p r e p a r e d with S R B C but w i t h o u t a d d i t i o n o f PBL never s h o w e d the so-called s p u r i o u s h e m o l y t i c areas.

133

Fig. 2. Hemolytic loci, before benzidine staining. The lytic areas contain scattered unlysed ceils. Lymphoid cell colonies are present in the center of each focus (:z50 I. T h e n u m b e r and the d i a m e t e r o f the h e m o l y t i c loci increase f r o m d a y 1 to d a y 3, reaching a p e a k o n d a y 5. T h e c e n t e r o f each area c o n t a i n s a l y m p h o c y t i c c o l o n y c o m p o s e d o f a p p r o x i m a t e l y 5 - - 2 0 cells w h o s e m o r p h o l o g y is t h a t o f p l a s m a b l a s t s or l y m p h o b l a s t s , with large a m o u n t o f c y t o p l a s m , r o u n d nucleus with loosely p a c k e d c h r o m a t i n and large nucleolus. After r e m o v a l f r o m the agar, these cells d e m o n s t r a t e d a positive bright fluoresc e n c e for i n t r a c y t o p l a s m i c p and ~ or ~ chains.

t34 TABLE

1

Hemolytie loci (d)~erved in the pritl'lary eLllttlres and in the overlaye,s containing ,d~eel) o r rat o r rabbit erythrocytes. V i , - t u a l l y n o hernolytic foci developed in the overlaym',, c o n t a i n i n g red I)lood c e i l s d i f f e r e n t from that u~ed in p r i m a r y c u l t u r e s .

Experiment

P r i m a r y c ill t tll'e~,

Overlaym's

I10.

Red b l o o d cell.,

No. of hemolytic

Rod I ) l o o d cells

loci 1 2 3 4 5 6 7 .'~ 9 10 11 12 13 14

.qleep *heep ~heep .qlee p sheel) sheep sheep sheep .-heep sheep ;heep sheep rat ,'at

15

rat

16 17 1%

rabbit rabbit rabbit

2() 16 10 12 20 2:3 l 1 19 21 15 19 21 12 x 15 11 5 7

No. of hemolytic

foci sheep *heep ~heep -hePI) ~heep ,,heep rat ,a I cat rabbit .a b b i t ,abbit ral .heep

15 ] i) 10 ,~ 12 1~, I~ 1 1 2 II fl lO ,1

rabbit

1

ral)bit sheep rat

', 1 I~

Hcmoly tic loci in the oeerlayers The table shows the results of the double layer technique. 2 4 - - 4 8 h after reincubation, hemolytic areas could be observed in the SRBC-agar overlay, coincident with foci already counted in the first SRBC-agar layer after 3 days of incubation, the positions of which had been nmrkecl with dots on the bottoms of the plates. A l y m p h o c y t i c c o l o n y was invariably seen in the center of each hemolytic area. On the contrary, virtually no hemolytic areas developed in the overlayers prepared by mixing agar with rat or rabbit erythrocytes, i.e., with antigens different from those (SRBC) used in the first layer of the original cultures to sensitize the human lymphocytes. Comparable results were obtained in control cultures of rat or rabbit RBC, overlaid with rat or rabl)it or sheep erythroeytes.

lgM detection in hemolytic loci by enzyme immunoassay Brown spots of 0.2--0.8 mm developed on SRBC ghost-coated membranes delineating the sites on which hemolysin-forming areas were detected before the filters were placed on the surface of the plates, The observation of snmller spots was facilitated by use of a dissecting microscope.

135 DISCUSSION

These results show that PBL cultured in agar together with SRBC and autologous plasma as source of complement produce colonies of hemolysinforming cells. The antibody response is evident in the lytic areas which surround the cell colonies growing on the surface of the plates; such areas had, on average, a diameter of 0 . 2 - 0 . 5 ram. Tile antibody response is significantly increased (number of foci) by addition to the cultures of PEG 6000, 8% final concentration. Some success ill growing human B-cell colonies has recently been obtained by Muraguchi et al. (1980), by using polyclonal activators such as PHA or protein A; in the present work colonies were obtained without addition of mitogens to the culture media. A technique for triggering the immune response, somewhat similar to ours, has been used by other authors (Robinson et al., 1976; Cla6sson et al., 1978) working with m o u s e spleen cells. However, in order to obtain an optimal antibody response with h u m a n PBL, we had to modify the culture conditions, since those for mouse spleen lymphoeytes proved to be quite unsatisfacto~5" with human cells. Two of the main points emerging from our experience are the advantage of decreasing the O: concentration in the incubation chamber, as suggested by other authors (Mizrahi et al., 1972; Bradley et al., 1978), and the conspicuous enhancement of antibody production by addition of PEG. As shown in Fig. 1, the PBL response to challenge with SRBC in the absence of PEG 6000 is extremely variable. Seven donors produced no antibody at all and only one gave more than 60 loci, while in the majority the number of hemolytic foei was between 1 and 10. When PEG 6000 was added, the yield of hemolytic foci was increased. No negatives were observed and the average total number of areas was 36.2 ± 2.3. Since 9 X 1 0 6 PBL were sown in each plate, it may be calculated that in a million lymphocytes there are at least 3 or 4 precursor cells. It may also be deduced that the frequency of activated B-lymphocytes is 2 per 10 s, because only 20% of PBL are B-cells. The frequency of precursors of anti-SRBC hemolysin-producing cells in the mouse spleen has been calculated as 1--3 per 10 s lymphocytes (Quintans and Lefkovitz, 1973). However, comparison between peripheral blood and spleen cells is inappropriate since blood is not the main site of antibody production. To rule out the possibility that microaggregation of antibody-coated erythrocytes may generate false plaques following addition of complement (Muchmore et al., 1976), we always prepared control plates by sowing SRBC without PBL; pseudoplaques were never detected in these controls. We also confirmed the reproducibility of hemolytic loci by pouring a fresh agarSRBC mixture over the surface of the primary cultures after 3 days incubation. New hemolytic loci appeared in this overlayer after 2 days further reincubation, coinciding in position with those observed already. This indicates that preformed antibody was not involved and that the hemolytic areas

136 of the overlayer were p r o d u c e d by a diffusible factor, i.e., tie n o l o synthesized h e m o l y s i n c o m i n g from the top layer. By the overlayer techniqtte it was also possible to c h e c k the specificity o f the a n t i b o d y response and e x c l u d e i m m u n o g l o b u l i n synthesis by PBL polyclonal stimulation..,,race in the overlayers c o n t a i n i n g antigen different from that used for the p r i m a r y cultures, h e m o l y t i c foei were virtually never detected. To verify that the h e m o l y t i c areas were p r o d u c e d by IgM antibodies, we used a m o d i f i c a t i o n of the 'replica i m m u n o a d s o r p t i o n " t e c h n i q u e (Miggiano, 1 9 7 9 ; Sharon et al., 1979). The a p p e a r a n c e on the filters used for e n z y m e i m m u n o a s s a y o f b r o w n spots c o r r e s p o n d i n g to the h e m o l y t i c plaques further c o n f i r m e d the presence of IgM a n t i b o d i e s in those areas. The e x p l a n a t i o n o f the m e c h a n i s m by which PEG 6 0 0 0 modifies the i m m u n e reactivity o f PBL is not k n o w n . It is possible that it m a y intensify i m m u n o l o g i c a l interactions by "squeezing" antigens and cells to interact with each o t h e r , as suggested 1)3' Ben-Sasson and Henkart ~19771 and Ponzio (1980h ACKNOWLEDGEMENT This w o r k was s u p p o r t e d by Grant 8 0 . 0 1 5 1 7 . 9 6 . 1 1 5 . 4 7 5 0 f r o m CNR, Rome. REFERENCES Ben-Sasson, S.A. and P.A. Henkart, 1977, J. Immunol. 119, 2o7. B6yum, A., 1968, Seand. d. Clin. Lab. Invest. 21 (Suppl. 97 I, 1. Bradley, T.R., G.S. Hodgson and M. Rosendaal, 1978, J. Cell. Physiol. 97,517. Cla~;sson, M,H., J.E. Layton and G.A. Luehenbach, 1978, Immunology 35, 397. Fauci, A.S. and K.R. Pratt, 1976, J. Exp. Med. 144,674. Hoffman, S., 1980, Proc. Natl. Acad. Sci. U.S.A. 77, 1139. Jerne, N.K., A.A. Nordin and C. Henry, 1963, in: Cell-bound Antibodies IWislar Inst. Press, Philadelphia, PAIl p. 109. Luzzati, A.L., M.J. Taussig, T. Meo and B. Pernis, 1976, J. Exp. Med. 144, 573. Miggiano, V., 1979, in: Hybridoma Technology with Special Reference to Parasitic Diseases (WHO, Geneva) p. 97. Mizrahi, A., G.V. Vosselle¢, Y. Yagi and G.E. Moore, 1972, Proc. Soe. Exp. Biol. bled. 139, 118, Muehmore, A.V., I. Koski, N. Dooley and R.M. Blease, 1976, ,J, hnmunol. 116, 1016. Muraguchi, A., T. Kishimoto, T, Kuritani, T. Watanabe and Y. Yamamura, 1980, J. hnmunol. 125,564. Ponzio, N.M., 1980, Cell. Immunol. 49, 266. Quintans, S.J. and I. Lefkovitz, 1973, Eur. J. Immunol. 3, 39'-) . Robinson, W.A., d. Marbrook and E. Diener, 1976, J. Exp. Med. 126, 347. Sharon, J., S.L. Morrison and E.A. Kabat, 1979, Proe. Natl. Aead. Sci. U.S.A. 76, 420.