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Saturday 23 December 1967

EFFECT OF ANTI-LYMPHOCYTE SERUM ON

RESPONSES OF HUMAN PERIPHERAL-BLOODLYMPHOCYTES TO SPECIFIC AND

NON-SPECIFIC STIMULANTS IN VITRO

M. F. GREAVESB.Sc. Lond.

RESEARCH FELLOW

I. M. ROITTD.Phil. Oxon.

READER IN IMMUNOPATHOLOGY

R. ZAMIRM.Sc. Jerusalem

RESEARCH ASSISTANT

RHEUMATOLOGY RESEARCH DEPARTMENT,MIDDLESEX HOSPITAL MEDICAL SCHOOL, LONDON W.I

R. B. A. CARNAGHANM.R.C.V.S., M.C.Path.

SENIOR RESEARCH OFFICER II,MINISTRY OF AGRICULTURE, FISHERIES AND FOOD,

CENTRAL VETERINARY LABORATORY, WEYBRIDGE, SURREY

Summary Anti-lymphocyte sera (A.L.S.) againsthuman splenic and tonsillar cells were

raised in goats. These antisera when tested in vitro withhuman peripheral-blood lymphocytes had cytotoxic andagglutinating properties and could stimulate D.N.A. synthe-sis and transformation to blast cells. Exposure of humanperipheral-blood lymphocytes from Mantoux-positivedonors for 16 hours to a concentration of A.L.S. slightlygreater than the optimum concentration for inducingtransformation, inhibits a subsequent response to purifiedprotein derivative (P.P.D.); suboptimal concentrations ofA.L.S. may enhance the subsequent P.P.D. response. Pre-liminary findings suggest that the mixed-lymphocytereaction may also be suppressed by high doses of A.L.S.These responses may provide a basis for the in-vitroassessment of the immunosuppressive activity of A.L.S.

Introduction

THE immunosuppressive capacity of anti-lymphocytesera (A.L.S.) in vivo has been well established (Waksmanet al. 1961, Woodruff and Anderson 1964, Levey andMedawar 1966). In vitro, these antisera agglutinate andare cytotoxic to lymphocytes (Woodruff et al. 1967), andhave been shown to induce transformation of lymphocytesin vitro in a similar way to phytohsemagglutinin (P.H.A.)(Grasbeck et al. 1964, Holt et al. 1966, Woodruff et al.1967). Levey and Medawar (1966) suggested that immuni-suppression in vivo may be mediated by a

" sterile activa-tion " through lymphocyte transformation. Treatment ofhuman peripheral-blood lymphocytes with staphylococcalfiltrate or P.H.A., however, does not suppress their capacityto respond to a subsequent specific antigenic stimulus invitro (Ling and Holt 1967); hence, non-specifically stimu-lated lymphocytes may not necessarily be immunologicallyinactivated. A.L.S., although inducing a similar morpho-

logical response to that induced by non-specific mitogens,may still render the stimulated population immunologicallyinactive by covering specific receptor sites on the cellsurface (i.e., " blindfolding ") as also suggested by Leveyand Medawar (1966). The present report concerns theability of human peripheral-blood lymphocytes treated invitro with A.L.S. in the absence of complement to respondto subsequent specific and non-specific stimuli.

Materials and Methods

Antiserum PreparationHuman lymphoid cells were prepared by sterile techniques

from spleen and tonsils obtained at operation which were slicedinto small sections and teased through sieves (Endecote Ltd.).Any remaining gross clumps of cells were discarded by centri-fugation at low speed and the remaining cell suspension waswashed thoroughly in medium 199 to remove debris. Twogoats were given an initial intravenous injection of 0-5 X 1010mononuclear spleen cells, and 21 days later were re-injectedwith 1-2 x 109 tonsil mononuclear cells. Serum samples werecollected 1, 2, and 7 weeks after the second injection. Theexperiments reported here concern the activity of a serumsample taken 1 week after the second injection. Normal goatserum (N.G.S.) was used as a control. Sera were inactivated byheating to 56°C for 40 minutes.

Lymphocyte Cultures60-100 ml. of venous blood from healthy volunteers was

added to heparinised plastic containers and the erythrocytessedimented with 0-5 volumes of 6% dextran (Fisons Ltd.)in saline solution for 45 minutes at 37°C. The upper layer wasremoved and centrifuged in plastic tubes at 150 g for 10 min.The cell button was suspended in medium 199 and centrifugedat 125 g for 6 minutes. The cells were resuspended in a totalvolume of 3 ml. 20% foetal calf serum in medium 199 andadded to a 1 x 5 cm. column of styrene divinylbenzene copoly-beads (Thompson et al. 1966). Lymphocyte recovery was inthe range of 85-98 % and the cell suspension contained 95-99 %lymphocytes. In all restimulation experiments, 0-3 ml. of the3 ml. pre-column suspension was added to the post-columnmaterial to give an overall suspension of 85-90% lymphocytes,the purpose of this manceuvre being to avoid removing all theadhesive cells ( ? macrophages) that might play some role in theresponse of lymphocytes to antigenic stimuli (Oppenheim et al.1966). Cell numbers were adjusted to 1,0-1,75 x 106 per ml.in 20% inactivated foetal calf serum in medium 199, andtriplicate cultures were set up in McCartney bottles (1-2 ml.per bottle) and flushed with 5% CO2/02 before incubation at37°C. In restimulation experiments, lymphocytes were cul-tured in the presence of inactivated A.L.S. or normal goat serumfor 16 hours, washed once in medium 199 and recultured in20% inactivated foetal calf serum in 199 without further stimu-lation for 2 days. Addition ofP.H.A., dialysed tuberculin (P.P.D.)or mixing of lymphocytes was then carried out. Preliminaryexperiments on lymphocytes from Mantoux-positive donorsindicated that the optimal dose of P.P.D. for D.N.A. stimulationwas 50-250 units per ml., depending on the degree of intra-dermal reaction. The transformation was in the range of 8-15 %of surviving cells 5-6 days after addition of antigen. Lympho-cytes from Mantoux-negative volunteers gave no response.

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Mixed lymphocyte cultures were set up by preparing separatesuspensions from individual donors as described above, andpooling equal volumes and cell numbers from paired donors.The time course and degree of the response were more variablein this system, but usually 5-15% of surviving cells weretransformed 4-7 days after mixing. P.H.A. (Burroughs Well-come) was pooled from several vials. The optimal dose wasfound to be 0-015 ml. of the reconstituted P.H.A. per ml. ofculture medium. The proliferative response of the stimulatedlymphocytes was estimated by measuring the uptake of 2-[14C]-thymidine (0-0125 µC per ml. culture medium; specific activity55-8 mC/mM; Radiochemical Centre, Amersham) over 16 hoursusing liquid scintillation counting as described by Ling andHolt (1967).

Results

Initial experiments showed that the antiserum was

cytotoxic for lymphocytes in the presence of complementto a titre of 1/640 and agglutinated red cells and lympho-cytes at a dilution of 1 in 320. Antibodies to a serum

Fig. I-Effect of anti-lymphocyte serum on human peripheral-bloodlymphocytes in vitro.

< blast cells. 0 (14C]thymidine uptake.

protein of fast beta mobility were demonstrable byimmunoelectrophoresis. In the absence of complement,A.L.S. induced blastoid transformation of lymphocytes andD.N.A. synthesis (fig. 1). The same antiserum agglutinatedrhesus monkey peripheral-blood lymphocytes to a titre of1/32, but did not stimulate D.N.A. synthesis. Fractionationof the antiserum on Sephadex, G200 ’ and diethylamino-ethyl (D.E.A.E.)-cellulose showed thatthe mitogenic activity was almost

exclusively in the IgG fraction as previ-ously reported (Woodruff et al. 1967).Lymphoagglutinins were present almostequally in the IgG and IgM fractions,whereas cytotoxic antibodies were

mostly IgM.Incubation of lymphocytes with

A.L.S. at the optimal transforming dosefor 16 hours followed by resuspensionin fresh medium lacking A.L.s. resultedin a wave of D.N.A. synthesis (fig. 2).Hence, in order to detect the specificmitogenic effect due to subsequenttreatment with antigen, the washedcells had to be left in culture for 2 daysuntil the rate of D.N.A. synthesis hadfallen sufficiently before addition of

antigen and [14C]thymidine.The result of pretreatment of lym-

phocytes from a Mantoux-positive

Fig. 2-Effect of 16-hour A.L.S. stimulus on incorporation of[1‘C]thymidine into lymphocytes in vitro.

0 A.L.S. 0 normal goat serum.

donor with normal goat serum (N.G.S.) or an optimallystimulating dose of A.L.S., and restimulation with P.P.D.or P.H.A. is shown in figs. 3 and 4. N.G.s.-treated

lymphocytes gave a good response to P.P.D., D.N.A.

synthesis reaching a peak 4-6 days after addition of antigen.In contrast, A.L.s.-treated cells showed no response toP.P.D. at this time, although an early minor peak ofsynthesis was evident. Restimulation with P.H.A. showedthat A.L.s.-treated cells could none the less still proliferateand in fact responded sooner and to a greater extent thancontrol cells treated with normal goat serum (fig. 4).Partial-to-complete suppression of the P.P.D. response wasobtained on two further occasions, but in a subsequentexperiment, paradoxically, D.N.A. synthesis was consider-ably enhanced. These differences were shown to dependupon the concentration of A.L.S. used. As shown in fig. 5,a sub-optimal transforming dose of A.L.S. enhanced thesubsequent P.P.D. response. With an optimal concentra-tion, D.N.A. synthesis gave only a minor early peak (cf.fig. 3) whereas with a supra-optimal dose of A.L.S., thestimulation by P.P.D. was completely abolished. Lympho-cytes treated with these different concentrations of A.L.s.did not differ greatly in the intensity of their response toP.H.A. (fig. 6).

Fig. 3-Suppression of P.P.D. response byA.L.S. pretreatment.

0 A.L.s. alone. 8 A.L.S.+P.P.D.0 N.G.S. alone.. N.G.S.+P.P.D.

In this and subsequent figures the pointsrepresent the mean of duplicate or triplicatecultures.

Fig. 4-Effect of A.L.S. pretreatment onresponse to phytohsemaggtutlmia (samedonor as in fig. 3).0 A.L.S. alone. * A.L.S.+P.H.A.

0 N.G.S. alone. NN.G.s.+p.H.A.

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Fig. 5-Effect of dose of A.L.S. on subse-quent response to P.P.D.0 A.L.S.: a, 0’06ml./ml. medium; b,

0’04 ml./ml., c, 0-02 ml./ml. a N.G.S.

control. P.P.D. added to each culture.

Fig. 6-Effect of dose of A.L.S. on subse-quent response to phytohaEmagtutinin(same donor as in fig. 5).. A.L.S. : a, 0-06 ml./ml. medium, b,

0-04 ml./ml., c, 0-02 ml./ml. 0 N.G.S.

control. P.H.A. added to each culture.

Fig. 7-Suppression of mixed lymphocytereaction by A.L.S. pretreatment.0 A.L.S. only (sum of unmixed cultures);

. A.L.s.+mixing. N.G.S. only (sum ofunmixed cultures). 0 N.G.s.-(-mixing.

In a preliminary experiment with mixed lymphocytecultures, pretreatment of cells from both donors with anoptimally transforming dose of A.L.S. suppressed the sub-sequent mixed lymphocyte reaction over the period studied(fig. 7).

Discussion

The results of these experiments show that when

lymphocytes from Mantoux-positive donors are pretreatedwith anti-lymphocyte serum, they fail to respond signifi-cantly to a subsequent stimulus with P.P.D., although 4-8days after contact with antigen control cells treated withnormal goat serum give a typical peak of D.N.A. synthesis.Complete inhibition was obtained with doses of A.L.S.which were supra-optimal for the transformation of

lymphocytes. Sub-optimal concentrations of A.L.S., how-ever, enhanced the subsequent response to P.P.D. andhastened its onset, recalling the observations made byLing and Holt (1967) on the effect of pretreatment withstaphylococcal filtrate on specific antigenic stimulation.In the absence of complement, the cells in cultures

treated with a supra-optimal concentration of A.L.S.

showed only slightly decreased viability relative to theN.G.S. controls, whereas their ability to proliferate as

evidenced by the response to P.H.A. was, if anything,enhanced. But, since the number of cells capable ofresponding initially to P.P.D. may represent a very smallproportion of the total lymphocyte population in the

culture, the possibility cannot be excluded that cells

specifically primed to respond to antigen did not survivetreatment with high doses of A.L.s. Reversal of inhibition

by trypsin or by use of high concentrations of antigenwould establish that these cells were inactivated but not

destroyed by A.L.S.If selective cell death proves not to be the explanation

for the inhibition, one interpretation may be that A.L.S.alters the lymphocyte cell membrane in such a way as toprevent the interaction of antigen and specific receptor sitenecessary for the induction of the proliferative response.Other experiments are consistent with the view that A.L.s.may coat lymphocytes and thereby inhibit immunologicalreactivity. Thus, treating lymphocytes with A.L.S. preventssubsequent histocompatibility typing with isoantisera(Balner 1967) and lymphocytes coated with isoantisera

lose their ability to attack allogeneic monolayers in vitro(Moller 1967). Inhibition of the mixed lymphocytereaction by A.L.S. in the present experiments could beascribed to masking of the surface antigens or to a directeffect on the ability of the lymphocyte to recognise andrespond to homologous antigens-a response which is

particularly relevant to homograft rejection.Two factors make it difficult to compare the immuno-

suppressive action of A.L.s. in vivo and its effect on lympho-cyte cultures. First, since complement was not present inthe cultures, the lymphocytes were not exposed to cyto-toxic effects of A.L.S. Secondly, A.L.S. had to be removedfrom the cultures after 16 hours to prevent a second waveof thymidine uptake due to A.L.s. itself which would haveobscured the specific proliferative response to antigen;in vivo, the lymphocytes would be exposed to antibodiesover a long period.

Further investigation of the relation between the capacityof A.L.S. to block immunological responses in vivo andin vitro, and its ability to initiate transformation and D.N.A.synthesis in lymphocyte cultures may provide a basis forthe assessment in vitro of the immunosuppressive potencyof A.L.S. before it is used in therapy.

This work was supported by grants from the Medical ResearchCouncil, the Nuffield Foundation, the Arthritis and RheumatismCouncil, and the World Health Organisation. We are grateful toMrs. G. Stead for help in preparation of the manuscript and toMr. H. S. Drury for preparing the figures.

Requests for reprints should be addressed to 1. M. R.

REFERENCES

Balner, H. (1967) in Ciba Foundation Study Group no. 29: AntilymphocyteSerum (edited by G. E. W. Wolstenholme and M. O’Connor); p. 52.London.

Grasbeck, R., Nordman, C. T., De La Chapelle, A. (1964) Acta med. scand.suppl. 412, 39.

Holt, L. P. J., Ling, N. R., Stanworth, D. R. (1966) Immunochemistry, 3,359.

Levey, R. H., Medawar, P. B. (1966) Ann. N.Y. Acad. Sci. 129, 164.Ling, N. R., Holt, L. P. J. (1967) J. cell Sci. 2, 57.Moller, E. (1967) J. exp. Med. 126, 395.Oppenheim, J. J., Hersh, E. M., Block, J. B. (1966) in Proceedings of the

Symposium: The Biological Effects of Phytoh&aelig;magglutinin (edited byM. W. Elves); p. 183. Oswestry.

Thompson, A. E. R., Bull, J. M., Robinson, M. A. (1966) Br. J. H&oelig;matol.12, 433.

Waksman, B. H., Arbouys, S., Arnason, B. G. (1961) J. exp. Med. 114, 997.Woodruff, M. F. A., Anderson, N. F. (1964) Ann. N.Y. Acad. Sci. 120, 119.

&mdash; James, K., Anderson, N. F., Reid, B. L. (1967) in Ciba FoundationStudy Group no. 29: Antilymphocyte Serum (edited by G. E. W.Wolstenholme and M. O’Connor); p. 57. London.