IN VITRO DEMONSTRATION OF THE SUPPRESSIVE

T H E RABBIT AND THE MOUSE ON EFFECT BY ANTI-LEUKOCYTE SERA OF

ALLOGENEIC ANTIBODY-PRODUCING CELLS*

Susanna Harris and T. N . Harris The Children 6 Hospital of Philadelphia and the Department of Pediatrics, School of Medicine, U n iwrsity of Pennsylvania, Philadelphia, Pa.

The serum of rabbits injected with rabbit leukocytes has been found to contain antibody which suppresses the activity of antibody-forming rabbit lymph node cells. This has been indicated in lymph node cell transfer experi- ments by lower maximal titers of recipients of cells so treated, in comparison with titers in recipients of untreated cells.' The suppressive effect of such rabbit anti-leukocyte serum could be demonstrated, not only by injection of the serum into recipient animals, but also by in vitro incubation of such anti- serum with the lymph node cells before they were transferred.' Recent descriptions by Jerne and Nordin3 and by Ingraham and Bussard4 of a procedure which makes possible the detection and enumeration of antibody- producing cells in vitro suggested an attempt to apply this technique to our studies of the suppression of allogeneic lymph node cells, especially since the suppressive antibodies had been found, as indicated above, to be effective on in vitro incubation with lymph node cells prior to transfer. This paper will present data on the suppression of rabbit lymph node cells by rabbit anti- rabbit leukocyte serum, and of mouse spleen cells by mouse anti-mouse spleen cell sera, the test system being the formation of antibody plaques by anti- erythrocyte lymph node or spleen cells in vitro, rather than, as in the previous studies referred to, the maximal level of antibody produced by transferred lymph node cells in recipient animals.

MATERIALS AND METHODS

The Hemolytic-Antibody Plaque Test. Popliteal lymph nodes were ob- tained from rabbits four days after the injection of 0.2 ml of a 50 percent suspension of sheep erythrocytes in each hind foot pad. Cells obtained by teasing these lymph nodes were washed once and suspended in Hanksy5 solu- tion containing 0.5 per cent rabbit serum albumin, at a standard concentra- tion, usually 2 x 10' cells /ml. The plaque tests were carried out in shallow plastic plates 5 cm in diameter, in which a 3-ml layer of 1.1 percent agar in Earle's solution6 had been allowed to harden. A suspension of lymph node cells, containing 0.1 ml of the prepared suspension referred to above, 0.4 ml of reagents to be described below and 0.1 ml of normal guinea pig serum 1:2, was

*This study was supported by Research Grant HE-04598 of the National Institutes of Health, United States Public Health Service.

178

Harris & Harris: Ant i -Leukocyte Sera 179

mixed with 1 ml of 1.1 per cent agar containing 3 per cent sheep erythrocytes. Of this mixture, 1 ml was placed on the agar layer in the plate and agitated until i t covered the area of the plate. A 0.5-mm layer of agar containing lymph node cells and erythrocytes was thus produced. When the agar had hardened, normal guinea pig serum (1 ml of 1:4) was added to the plate, which was then incubated a t 37OC for 1 hour. The number of plaques in the plate was then determined.

Mouse splenic cells were obtained similarly from Balb/cJ and CBA/J mice which had received an IP injection of 1 ml of 50 per cent sheep RBC four days earlier, and tested in the same way.

Anti-Rabbit Leukocyte Serum. Anti-leukocyte sera were obtained by pool- ing blood leucocytes from 40 to 60 rabbits and injecting these into 12 or more rabbits, in doses of 150 million cells. After one or two such injections, sera were obtained from the injected rabbits on the 8th, 10th and 11th day, and pooled. Mouse anti-spleen cell sera were obtained by injecting Balb spleen cells into CBA mice or vice versa, one, two or more times a t various intervals. Orbital bleedings were obtained from the injected mice on five successive days, be- ginning one, two and three weeks after the injection of the spleen cells. The serum obtained from the blood specimens of each week was pooled.

Preparation of Soluble Rejection-Associated Antigen. The solubilized re- jection-associated antigen was prepared as described elsewhere. The release of lipoprotein from cell fragments by the use of Triton yields some water- soluble material, with some activity, referred to here as Triton-solubilized, and largely, water-insoluble lipoprotein. From the latter, active material can be solubilized by butanol.' The association of this antigen with cell rejection has been indicated by the fact that, when bound to Ecteola-cellulose, it can absorb the suppressive antibody from anti-leukocyte sera.

I

RESULTS

Reduction in Plaque Count by Suppressive Antisera

In early experiments, the incubation of anti-erythrocyte lymph node cells with anti-leukocyte serum pools, which were known from cell transfer tests to have suppressive activity, was not found to cause any reduction in the num- ber of plaques on subsequent plating. I t was then found that, when comple- ment was added to the incubation mixture of suppressive serum and lymph node cells, there was a reduction in the number of plaques produced, in com- parison with that produced by the control portion of the suspension of lymph node cells. This reduction was found to be almost complete within three or four two-fold steps of dilution of a given suppressive serum from the first dilution which caused any reduction in plaque number. As an estimate of relative concentration of suppressive antibody in various preparations of anti- leucocyte serum, a 50 percent endpoint was arrived at by converting all

180

No. of %of plaques control

82 20 215 51 368 84

436 388

6.0

Annals New York Academy of Sciences

No. of %of No. of %of No. of %of plaques control plaques control plarples control

30 15 100 50 11 11 140 I 0 45 45 4 8

76 76 19 42 35 78

206 94 47 192 102 42

1.0 8.1 9.2

plaque counts to percentages of the control level, and interpolating on a linear scale between the two successive concentrations of serum which gave percent- ages above and below 50. A number was thus arrived a t which gave the fraction of the log2 step which corresponded to 50 percent of the control level of plaque count. This procedure is illustrated in each of the five pairs of col- umns in TABLE 1.

In order to compare the suppressive activity of anti-leukocyte sera that might be tested under different conditions, it was necessary to examine the degree of plaque reduction in relation to the number of cells with which the suppressive serum was incubated. TABLE 1 shows the data obtained in an experiment in which appropriate dilutions of a standard rabbit anti-rabbit- leukocyte serum pool were incubated with a given suspension of lymph node cells, the latter being used in a range of concentrations progressing in two-fold steps from 16 to 1 x lo7. For each concentration a t which the lymph node cells were used, the lowest two numbers show the control levels, that is, the plaque count of cells untreated by the suppressive antiserum. In addition, the plaque counts are shown for each concentration of cells used after the incuba- tion with the antiserum a t appropriate concentrations. I t can be seen that the endpoint of the serum rises with increasing dilution of cells in the incubation mixture, and that for each of five successive two-fold dilutions of the cells the apparent endpoint of this serum changes by approximately 1 power of 2.

In the mouse, it was also found possible to reduce the numbers of hemo- lytic plaques of a given suspension of antibody-producing allogeneic spleen cells by incubation with anti-spleen-cell sera produced in mice of another strain. Some studies were carried out on the effect of such factors as the dose

TABLE 1 EFFECT OF CONCENTRATION OF LYMPH NODE CELLS ON

PLAQUE-REDUCTION ENDPOINT OF A SUPRESSIVE ANTISERUM

Dilution of standard

serum

1024

50%plaque reduction endpolnt, log2

610

5.05 I

Number of lymph node cells, X 106

8 1 4 1 2 1 '

Harris & Harris: Anti-Leukocyte Sera 181

of spleen cells and the interval between injections. Typical data are shown in TABLE 2 as log2 of 50 percent plaque-reduction endpoints.

In the upper part of the Table, it can be seen that, in sera of Balb mice given a single injection of CBA spleen cells, 50 percent plague reduction end- points a t or above the convenient threshold level of 22 were not obtained, if the injection dose was lo7 cells, but were obtained if 10' cells were injected. However, in the case of secondary injections, it can be seen that a dose of lo7 cells yielded sera with endpoints above 22, if the interval between the in- jections was sufficient. The secondary response to 10' cells was corre- spondingly higher, and still higher values were obtained a t this dose with substantially longer intervals following the previous injections.

The lower part of TABLE 2 shows typical data obtained in CBA mice injected with Balb spleen cells. A single injection of 10' Balb cells caused the production of antiserum with an endpoint of log2 1.8. Second injections, a t somewhat higher dosage levels, yielded sera with considerably higher end- points. An effect of the interval between primary and secondary injection is again seen within each of the groups of CBA mice divided into sub-groups for second injections after four and seven weeks, respectively. It can be seen that, a t each dose, the longer interval yielded a serum pool of about twice the activity of the shorter one. The specificity of the sera was examined by test- ing each serum against spleen cells of the opposite strain. No plaque-reducing effect was encountered, even when the sera were used a t concentrations 20 or more times their 50 percent endpoints.

Plaque-Reduction Endpoints of Anti-Leukocyte Sera in Relation to the Suppressive Effect of These Sera in Cell Transfer

Experiments.

A comparison of the measure of effectiveness of suppressive antisera in the reduction of plaque counts and in the suppression of transferred lymph node cells was made in a number of rabbit anti-rabbit leucocyte serum pools. Of these pools, the suppressive endpoint had been determined in cell transfer experiments, this endpoint being the reduction by 3 powers of 2 in the mean maximal antibody titer in sera of recipient animals from the level attained in recipients of cells incubated with pooled normal rabbit serum. Such pools, obtained after one or two injections of leucocytes, and a t an injection dose of 50 million or 150 million cells, were available in an 80-fold range of activity. Fifteen pools extending over this range of suppressive activity were examined by plaque-count reduction, and the endpoint of 50 percent reduction in plaque count so obtained was plotted against the suppressive endpoints of these sera as determined in cell transfer experiments. The resulting plot in- dicated a high degree of correlation between the suppressive endpoints ob- tained by plaque reduction in vitro and those in lymph node cell transfer experiments.

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Harris & Harris: Anti-Leukocyte Sera 183

That plaque reduction probably measures the same antibody in anti- leucocyte sera as that which causes the suppression of transferred lymph node cells was also indicated in another series of experiments. Rabbits were injected twice with the butanol-solubilized antigen associated with the rejection of transferred rabbit lymph node cells, described elsewhere,' and the resulting antisera were found to cause reduction in plaque number. In groups of rabbits so injected, the serum of each animal was examined for its plaque-reduction endpoint. A pool was prepared of the sera of rabbits showing the highest plaque-reduction titers, and another of those with the lowest titers of the group. In each of three such experiments, it was found that the grouping of the sera on the basis of their plaque-reducing endpoint produced pools of sera that were markedly different in their suppression of transferred lymph node cells, conforming with the average plaque-reducing titers of the constituent sera.

Relation of Plaque-Reducing Titer o f Serum to the Degree of Rejection o f Transferred Lymph Node Cells, in Rabbits

Actively Immunized with the Rejection- Associated Antigen

The relation between the plaque-reduction endpoint of serum and the degree of actively induced suppression of transferred lymph node cells was examined, again in rabbits given two injections of the butanol-solubilized, cell-rejection antigen. In these experiments, the rabbits thus immunized were bled, and the 50 percent plaque-reduction endpoint for each serum deter- mined just before the irradiation preceding cell transfer. The rabbits were then given lymph node cells from ferritin-injected donor rabbits and bled as usual after transfer. (The maximum anti-ferritin titer of recipients of such cells oc- curs two days after transfer, when the titer of plaque-reducing antibody resulting from the injection of the rejection-associated antigen is still a t its maximal level.) For each recipient of transferred cells the peak anti-ferritin titer was determined, and the difference between this level and the mean value of the control group was used as the measure of the degree of suppression of the transferred lymph node cells in that animal. This estimate of the suppres- sion of the transferred lymph node cells was plotted against the 50 percent plaque-reduction endpoint of its serum. Because of the ranges of sensitivity of the methods, a number of the plaque reduction endpoints were below a lower limit of measurement, and a number of the endpoints of suppression in cell transfer were above an upper limit. In each case, the opposite parameter was correspondingly of relatively low or high value. The points in the central part of the graph, where the measure of each parameter could be indicated, showed progressive increase in degree of rejection of transferred cells with increase in plaque-reduction endpoint of the recipient's serum.

184 Annals New York Academy of Sciences

Inhibition of Plaque Reduction by Soluble Cell-Antigen Preparations

I t was possible in this system to demonstrate inhibition of the cell- suppressive effect of anti-leukocyte Sera by using soluble preparations of the rejection-associated antigen without resorting to binding the antigen to cellu- lose for absorption of the suppressive antibody, as indicated above. Serial dilutions of the soluble antigen were incubated a t 37°C for 30 minutes with equal volumes of a dilution of the standard antiserum. The mixture was then added to a standard suspension of antisheep erythrocyte lymph node cells for theusual incubation and plating. The serum was titrated as used, in the same experiment, for a determination of the number of units of antibody neutral- ized. TABLE 3 shows data obtained in a typical experiment on the relations between relative amounts of suppressive antibody and of cell-antigen a t neutralization to a 50 per cent endpoint. I n this experiment, a standard pool of suppressive rabbit anti-rabbit-leukocyte serum was used at dilutions vary- ing in two-fold steps from 4 to 64. The solution of cell-antigen, a butanol- solubilized preparation from tissues representing nearly 100 rabbits, was incubated with each of these dilutions of serum in a range of concentrations which was expected, on the basis of previous experiments, to include tha t which would neutralize the suppressive antibody present. Because of the relation of the suppressive effect of such a n antiserum to the concentration of lymph node cells, it was necessary to use corresponding numbers of lymph,node cells in testing the mixtures involving each of the serum concentrations, so tha t each of these would constitute the same number of units of suppressive anti- body as used. In TABLE 3 are shown the plaque counts and their percentages of the control value for inhibition tests carried out against five successive two- fold dilutions of a standard rabbit suppressive serum. On interpolation to the 50 per cent endpoint, it can be seen that the neutralizing endpoints of the solubilized cell antigen are directly proportional t o the dilution of the serum against which they were tested.

With standardization of the conditions for estimating inhibition of the suppressive antibody, it was possible to determine the relative activity of preparations of the rejection-associated antigen. I n TABLE 4 are shown typical examples of these comparisons in the mouse system. Among prepara- tions of Balb antigen, it can be seen that the spleen yielded preparations of higher specific activity than the liver, and tha t , in each case, the activity of the butanol-solubilized preparation was greater than tha t of the material solubilized by Triton alone. The CBA liver butanol antigen was without inhibiting effect on the anti-Balb Serum at the highest concentration feasible for testing. The lower part of the Table shows tha t the CBA liver butanol preparation was approximately as effective in inhibiting anti-CBA serum as was the analogous Balb preparation in its own system. A specificity control is again shown for comparison.

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DISCUSSION

The data presented above indicate clearly tha t exposure of antibody- forming rabbit lymph node cells t o rabbit anti-rabbit-leukocyte sera can cause a substantial decrease in the number of cells which produce antibody, upon incubation in vitro, and that the same effect can be demonstrated on active mouse spleen cells between inbred strains of mice. T h a t this injury of al- logeneic cells is, in each case, caused by an antibody is indicated by the following: (1) Serum pools obtained after two injections of pooled cells are much more effective than those obtained after one; (2) The reaction requires complement; and (3) The suppression can be inhibited by the solubilized rejection-associated antigen prepared from tissues of tha t species or strain. The antigenicity of this solubilized preparation has been demonstrated by its ability to absorb the suppressive antibody from antisera used in cell-transfer experiments, and to induce actively, on injection into rabbits, the rejection of transferred allogeneic lymph node cells.77

The suppression of function of allogeneic cells by antibody has thus been demonstrated in one genetically heterogeneous species, the rabbit, and be-

Antigen preparation Concentration of CBA anti- Balb Balb anti-CBA antigen neutralizing

solubil- 1 unit of suppressive serum serum source ized by antibody

units units mg/ml Balb Butanol 0.16

Balb Triton 0.5 4

4 Balb Butanol 0.5

4

spleen

spleen

liver

liver

liver

4

Balb Triton 5

CBA Butanol > 8

CBA Butanol 0.8

Balb Butanol > 6

4

liver 3

liver 3

Harris & Harris: Anti-Leukocyte Sera 187

tween two inbred strains of mice. It is of interest that , in the titration of antisera to determine the 50 per cent plaque-reduction endpoints, the rate of change of plaque count with successive two-fold dilutions of the antiserum was much more rapid in the mouse system than in the rabbit. In the mouse, with its single major antigenic difference, the plaque count usually changed from nearly 100 per cent of the control level t o near zero within two two-fold steps of serum dilution, whereas, in the rabbit, with multiple antigens in the pooled lymph node cells and the corresponding antibodies in the serum pool, the rate of change of plaque count with serum dilution varied among individual assays, but was always slower than in the mouse.

The requirement for complement in this reaction was suggested after early failures of suppressive antisera to cause reduction in the number of plaques, because of concurrent cell transfer experiments with papain fragments of the suppressive antibody, in which the fragments failed to cause suppres- sion. The failure of the papain fragments suggested that complement might be required for the suppression of function of allogeneic cells whether in vitro or in recipient animals. In the cell transfer experiments, i t was not necessary to supply this requirement, since the complexes of transferred cells and suppres- sive antibody could fix complement in the recipient's serum. It should be pointed out that the requirement of complement for both the suppressive reaction described here and the cytotoxic effects indicated by changes in permeability does not mean that the antibodies involved in the two reactions are the same.

The relation of this plaque test to the mechanism of suppression of trans- ferred allogeneic lymph node cells in vivo was approached in two ways. The probable identity of the plaque-reducing antibody to the antibody which suppresses the function of transferred lymph node cells was indicated by the high degree of linear correlation between endpoints of given serum pools by the two tests, with a 45' slope of the line of regression, and by the fact that individual antisera, pooled on the basis of plaque tests, showed differences in degree of suppression of transferred lymph node cells that were marked and conformed with the results of the earlier plaque tests.

The second approach, which had special interest regarding the ques- tion of the mechanism of rejection of allogeneic lymph node cells in the recipient animal, involved rabbits which were immunized with the solubilized rejection-associated antigen and then themselves used as recipients of antigen- stimulated lymph node cells. In these rabbits, a clear relation could be shown between the serum plaque-reduction titer and the degree of suppression of the transferred lymph node cells. In the range where both quantities could be given a numerical value, these showed a reasonable degree of correlation, again about a 4 5 O line. While this relationship in no way precludes the possibility that other immunologic reactions may be involved in the suppres- sion of these cells, it is possible, on the basis of quantitative considerations which have been presented elsewhereg to account for the degree of suppres-

188 Annals New York Academy of Sciences

sion of the transferred lymph node cells in these actively immunized recipients by the concentration of suppressive antibody in their serum.

The in vitro plaque test has a considerable number of advantages over lymph node cell transfer for the estimation of the suppressive antibody or of the rejection-associated antigen. Technically, only a few hours are required (in comparison with almost a week), far less antibody is required, the agree- ment among replicates is much better, and adjustment can be made for the substantial differences in degree of immunologic activity of suspensions of donors’ cells from experiment t o experiment (by the preliminary plating). Perhaps - most important in this category of advantages is tha t the plaque test produces data distributed on a continuous scale-per cent reduction in num- ber of plaques-whereas cell transfer produces discrete-step data-geometric means of antibody concentrations which are determined by two-fold dilutions of serum.

Other advantages of the plaque technique are immunologic in nature, arising from the simpler in vitro system and the shorter time of the experi- ment. First, in the genetically heterogeneous rabbit, the transfer of lymph node cells carrying bound antibody into tissues that must share some antigens with the transferred cells may well lead, in this dynamic system, to the re- distribution of some of the antibodies from transferred cells to the recipients’ cells. This would be especially important a t limiting concentrations of the suppressive antibodies. This consideration would not, of course, apply in vitro. Secondly, in cell transfer experiments to demonstrate the activity of solubilized preparations of the rejection-associated antigens by inhibition of suppressive antibody, the high dilution of the incubation mixture on intra- venous injection, and the longer period of time involved presumably caused a significant degree of dissociation of antigen-antibody complexes which inter- fered with the demonstration of inhibition of the suppressive antibody. In the plaque test, this problem did not arise.

Of the in vitro tests devised for the estimation of antibodies present in animals given allogeneic skin grafts or cell-injections (cytotoxicity, cytoly- sis, leucoagglutination, complement-fixation, hemagglutinin, reverse passive anaphylaxis, mixed agglutination in tissue culture, antiglubulin consumption tests2’-”), all involve an antibody measured by a serologic test, or by an injurious effect on the donor’s cells as indicated by change in permeability. The test described here would appear to be of particu- lar relevance to the immunologic reaction involved in the rejection of allo- geneic cells, because, in this experimental situation, it is a synthetic function of the cell that is being measured, and the activity of the antibody is indicated by reduction in the ability of the cell to perform the function which it would have continued to perform in the donor’s tissues.

SUMMARY The production of hemolytic antibody plaques by lymph node cells of

rabbits injected with sheep erythrocytes can be inhibited by prior incubation

10

1 1 12 13 14-16

17 16-20

Harris & Harris: Anti-Leukocyte Sera 189

of the cells with rabbit anti-rabbit-leukocyte serum and complement. A similar effect can be demonstrated with spleen cells of inbred mice and antisera to like spleen cells produced in another inbred strain. The 50 per cent plaque- reduction endpoint of such sera is, over a wide range, inversely proportional to the concentration of the lymph node cell suspension used.

The identity of the antibody causing reduction in number of plaques by allogeneic lymph node cells with the antibody causing reduction of antibody production by these cells when transferred to recipient rabbits has been indicated by a high degree of correlation between endpoints of respective sera when tested in the two systems, and by tests in cell transfer of pools of individ- ual rabbit sera selected on the basis of their plaque-reducing titer.

In rabbits actively immunized with the rejection-associated antigen and then used as recipients in cell transfer, a relationship could be observed be- tween the degree of suppression of transferred lymph node cells and the plaque-reduction titer in the serum of the individual rabbit obtained shortly before cell transfer.

The reduction of plaque number by suppressive antisera could be inhibited by prior incubation of the serum with solubilized preparations of the cell antigen associated with the rejection of transferred allogeneic cells.

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