augmentation of immune activity by elimination of antibody and its implications in cancer

Augmentation of Immune Activity by Elimination of Antibody and i t s Implications in Cancer ................................................................................. .................................................................................

SAM ROSE, M.B.

A “procedure” consisting of thoracic duct fistula, intravenous return of cells, wasting lymph fluid, and administration of replacement therapy was performed on rats immunized with SRBC or nucleated cells from histo-incompatible donors. The “procedure” resulted in a large augmentation of the homoral initnune response described by the following parameters. The number of 19s antibody-producing cells in spleen and lumph nodes increased up to twenty-fold. The daily antibody content in the lymph increased exponentially, doubling every 10 to 18 hours, and reached peak values of 100 times the antibody content in the whole animal prior to fistula. The peak antibody production rate of experimental animals was 1000 times the pre-fistula level-this latter rate being the antibody production level in animals with a stable immunity. The immune augmentation which was achieved in these experimentds does not represent the therorectical or practical limit attainable. When the “procedure” was performed in nonsplenectomized animals without raised venous pressure, the blood titer fell and then rose to pre-fistula levels or higher, and the daily antibody content of the lymph samples reached a platueau. When the “procedure” was performed in splenectomized animals with raised venous pressure the blood titer fell and remained low and there was a continuing exponential increase in antibody content of progressive lymph samples throughout the duration of the fistula procedure. The “procedure” and results are discussed in relation to aspects of immune regulation and practical advantages for antibody production. The implications of the “procedure” as a means of manipulating components important in tumor-host immune relationship are discussed with respect to possible immunotherapy of cancer. The “procedure” caused regression of M. S . V. induced tumors growing in syngeneic rats. I t is suggested that the effect on the tumors was due to the mechanisms discussed.

............................................................................... ...............................................................................

From M. D. Anderson Hospital and Tumor Institute, Houston, Texas, and The Salk Institute for Biological Studies, La Jolla, California.

The author’s present address is Roswell Park Memorial Institute, 666 Elm Street, Buffalo, New York 14203.

Journal of Surgical Oncology @Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y. 1001 1 137

138 Rose

The immune system, like many other control systems in biology, has features of adaptation and self-regulatory homeostasis. These two features, acting concurrently, ensure a controlled response with respect to the relevant external and internal prevailing conditions. The minimal organizational requirement to achieve adaptation and homeostasis is that the element under control be affected by two antagonistic processes or regulatory factors: one which activates and one which inhibits the mechanism generating the element.

In order to obtain an understanding of the dynamics of any controlled system, it is necessary to study the relationship, over a wide range, between the levels of the regulatory factors and the element which is controlled. The levels of the regulatory factors can be altered by addition or subtraction and the response of the element can be measured.

Classical studies in endocrinology have involved subtraction and addition of regulatory factors. A retrospective analysis of these studies emphasizes the importance of complete removal of a gland in order to demonstrate its functional role, the organism’ maximum potential for correction, and the physiological and biochemical effects of graded additions of the hormone(s) extracted from the gland. By analogy with these endocrine studies one can predict that it will be difficult to demonstrate and study the possible endocrine role of tissues such as fat, skeletal or smooth muscle, because of the inherent difficulty of completely removing any of these tissues.

of antibodies, as regulatory products, have been used to study the control of the immune system.

Specific antibodies have been administered to animals prior to, or after, immunization. In both of these cases the addition can be considered as “extra” antibody because the animals were able to, OF had already, generated a certain level of circulting antibody. Moreover, in many of these studies the “extra” antibody was usually given at a stage of immunization when qualitatively similar antibodies had not been generated (1). Such experiments demonstrate that “extra” antibody can have an inhibitory effect on the immune system, but by themselves do not prove that antibody generated during the immune response has a physiologically important regulatory role.

antibody production, with consequent rise in blood level, reaching a peak titer 5 to 8 days after antibody removal. The peak titer in every case reached levels above those present before antibody removal (2). These experiments support the notion that specific antibody has a physiological regulatory feedback role. How- ever, removal of antibody by plasniaphoresis has limitations because only a proportion of the total antibody contained in the body is removed, the antibody is not removed continuously, and is removed only after the antibody has had a chance to circulate in the blood stream, and therefore after it had probably ex-

It is instructive to examine how the principle of addition and subtraction

Removal of specific antibody from plasma resulted in an increased rate of

1 39 Immune Augmentation and Cancer Therapy

erted some feedback action. It is not possible to predict, by extrapolation fro’m experiments involving partial removal, the response parameters that could be achieved following complete removal of feedback antibody.

siderable proliferation. A. Splenic cells from mice, immunized with sheep red blood cells (SRBC), were transferred into irradiated syngeneic recipients. After one week the transferred cells in these recipients were again transferred to other irradiated recipients. This process was repeated a number of times, each transfer of splenic cells was ac- companied by the administration of SRBC. It is possible to interpret the results from these experiments as showing that each antigen-sensitive cell is capable of dividing 40 times to produce 10l2 antibody-producing cells. It was suggested that this phenomenon was due to a lack of feedback inhibition (3).

However, repeated cell transfer into irradiated recipients introduces the complications of the milieu of the irradiated animals and its possible consequence on the biology of the transferred cells. In addition, repeated cell transfer does not result in the multiplication of antigen-sensitive cells in such a manner that large numbers of lymphocytes or their products can actually be collected and used. This limitation is evident because the immune cells which were generated were distributed in all the recipients. Moreover, even though calculation showed that each antigen-sensitive cell could generate 10l2 immune cells, no such number of cells were actually produced. The figure was derived from assays and was corrected for aliquots, dilutions, and proportions of splenic cells which “home” to the spleen when they were administered intravenously. In addition, because antigen-sensitive cells have a limited lifetime and capacity for multiplication (3), and because new antigen-sensitive cells are not generated in irradiated animals, repeated cell transfers into irradiated syngeneic recipients only provides data as to the maximum proliferative capacity of antigen-sensitive cells and not to the full potential for immune augmentation. B. Spleen cells from mice, immunized with SRBC, were cultured with SRBC. The number of plaque-forming cells increased exponentially with time; and after 5 to 6 days in culture the proportion of plaque-forming cells in the cultures was 50 times the maximum which normally develop in the intact spleen of immunized animals. It is thought that the continued proliferation (for 5 days) of the antigen- sensivitive cells in the tissue culture was due to the absence of regulatory specific antibodies (4, 5 ) . Biological studies carried out in tissue culture have inherent limitations, and these, rather than an inherent property in the cells, may explain the limited proliferation of the antigen-sensitive cells in these studies.

The present experiments were designed to study the proliferation of antigen-sensitive cells and the augmentation of the immune response by a procedure which can almost completely eliminate feedback antibody and which does not have the limitations inherent in the studies described above. The ex-

Two procedures demonstrate that antigen-sensitive cells are capable of con-

140 Rose

tent of immune augmentation will depend on the ability to eliminate the specific antibody quantitatively and prior to its exerting a negative feedback effect. The discussion which follows shows that this elimination can be achieved by a thoracic duct fistula in animals with splenectomy and raised venous pressure. The lymph fluid containing the specific feedback antibody is wasted, and the cells contained in the lymph are returned to the animal.

RATIONALE OF FISTULA PROCEDURE AS A METHOD FOR MAXIMUM ELIMINATION OF SPECIFIC ANTIBODY PRIOR TO ITS EXERTING FEEDBACK EFFECT

The rationale of the fistula procedure, as a method of eliminating nearly all specific antibody, immediately after it has been synthesized, and prior to its having exerted feedback effect, depends on the anatomical and physiological relations between antibody production, flow and feedback sites.

A. tion of antibody immediately produced by and surrounding the responding cells. Spleen cells from immunized mice were transferred into irradiated syngeneic recipients, which were themselves given antigen. The transferred cells were thus placed in an environment lacking antibody. In the presence of antigen, and because of the lack of anti-body to act as a feedback, the transferred antigen- sensitive cells proliferated. Repeated transfers into successive recipients resulted in some spleens made up of 80 to 90% specific antibody producing cells (3). If the immediately produced microenvironmental concentration of antibody had acted directly as a feedback, spleens with such a high proportion of antibody- producing cells could not have been produced. The data from this experiment suggest that the immediately produced microenvironmental concentration of antibody is ineffective in regulating the immune response.

tain a homeostatic systemic antibody concentration. The following experimental data support the hypothesis that the second type of control is operative. Re- peated antigenic administration produces a relatively constant antibody concentration in the systemic blood. Antibody subtraction from the systemic blood (2), or addition to the systemic blood (1). is effective in transiently alter- ing immune function. B. Most antibodies are synthesized in the lymph nodes and lymphoid masses of the body. From these sites of synthesis the antibodies leave by efferent lymphatic channels. Little, if any, antibody diffuses into, and leaves by the blood circulation (6-10).

Peripheral efferent lymphatics from one node usually enter other more proximal lymph nodes along the lymphatic chain (1 1). Antibodies and other

Theoretically, two types of control of the immune system could be operative. One type of control would depend on the microenvironmental concentra-

The second type of control depends on the organism attempting to main-

141 Immune Augmentation and Cancer Therapy

high molecular weight compounds, contained in peripheral efferent lymph ducts, traverse the more proximal lymph nodes and leave by their efferent ducts (12-14). Nearly all the efferent ducts finally join to form central large lymph trunks which open into veins at the base of the neck.

The left thoracic duct is the major pathway from the lymphatics to the venous system. However, alternate lymphatico-venous channels exist. These latter pathways are variable in their occurrence, anatomical positions, and contributions they make in returning, intravenously, a proportion of the total lymph produced. Moreover, their importance will be different under various physiological and pathological conditions (1 5-2 1).

In addition, communicating collateral channels are present between various lymph ducts. Like the alternate lymphatico-venous channels, these collateral channels vary in their occurrence, anatomical position and flow pattern (22-24).

Because of the existence of alternative lymphatico-venous and collateral lymph channels, a cannula inserted into the main thoracic duct will not collect all the lymph generated in the body.

However, the lymphatic circulation is a low pressure system made up of thin vessels which respond markedly and quickly to pressure and flow within them (25-29). A differential pressure between the venous and lymph circulation can be established by elevating the central venous pressure and inserting into the main thoracic duct a catheter which ensures free flow of lymph. Under these conditions, the flow in the lymphaticovenous channels ceases or reverses, the collateral lymph channels enlarge, and all the lymph with its contained antibodies flows out through the thoracic duct. C. Anatomical and histological studies of the spleen have generated conflicting data with respect to the lymphatic supply of the spleen. Recent anatomical data suggest that the lymph drainage system from the spleen does not drain the parenchyma, but is limited to the capsule and connective tissue traveculae (30). Physiological studies, in which ovaries were transplanted in the spleen of rats, support the view that the parenchyma of the spleen lacks a lymphatic drainage. The pituitary, vagina, and uterus of these animals were identical to the tissues of spayed animals, because all estrogen which was synthesized in the transplanted ovaries was absorbed directly into the portal circulation to reach and be inacti- vated in its first passage through the liver (31,32). These anatomical and physiological studies strongly suggest that antibodies synthesized by the spleen are absorbed directly into the vascular system. From this, it follows that splenectomy is required to achieve maximum elimination of antibody via the lymphatics, and prior to its circulation in the vascular system. D. The great majority of antibody-producing cells are resident in lymph nodes, lymph masses, and spleen. A very small number may be in the circulation, the actual number probably being determined by the antigen and physiological state of the animal (33-36). It is possible that many previous estimates of the

142 Rose

number of antibody-producing cells in the circulation have been erroneously high. For example, it has been reported thatthe Jerne Plaque-positive cells, found in the circulating blood of rats, immunized with SRBC, were false positives in that the cells did not synthesize antibody, but caused local hemolysis in the assay by virtue of antibody adsorbed on their surface (36). Mature antibody- producing plasma cells are rarely seen in lymph or blood (37-40). They remain in lymphoid tissue and appear to have a short intranodal life span (41 . 44). However, immature plasma cells do circulate in lymph and blood, and it is likely that they continue their development to maturity when they settle in lymph nodes (45-48). In these situations they produce antibody and possibly transfer information to other effector cells (8). Experimentally, circulating immature plasma cells may develop to maturity in tissue culture or in diffusion chambers (45,46). These types of experiments may introduce an erroneous interpretation, namely, that the cells under study would be antibody-producing while circulating in vivo. The importance of the number of circulating antibody-producing cells, with respect to the rationale of the present experiments is, that they determine how much antibody is secreted directly into the blood stream. E. Despite fistula, splenectomy, and raised venous pressure, it is likely that some antibody will enter the blood circulation by one mode or another. Antibody in the blood, particularly the smaller 7 s type, will quickly be eliminated because there is a daily turnover of approximately 50 to 100% of protein from plasma to thoracic duct lymph (49-52).

MATERIALS AND METHODS

With Respect to Humoral Immunity.

gm, were employed for experiments using sheep red blood cells (SRBC) or goat red blood cells (GRBC) as antigen. Parental rats of BN, Fischer, and Lewis strain (Microbiological Associates, Bethesda, Md.) were employed for experiments using transplantation antigens. BN rats differ from both, Fischer and Lewis, at the AgB locus (53).

times and then resuspended in saline at a concentration of lo9 cells/ml. Animals were injected intravenously every 3 days prior to and during fistula. The details will be presented with the results.

and Lewis rats, by a skin transplant and, after rejection of the transplant, by injections of 50 X lo6 nucleated cells subcutaneously every 2 to 4 weeks for 1 to 3 months. Three weeks prior to and during fistula the injections were given every thrid day. The aim of these immunizing regimes was to establish a stable immune state prior to fistula. Antigen administration was the same during

Animals. Outbred Holtzmann or Sprague Dawley rats, weighing 250-320

Antigens and immunizing regime. SRBC and GRBC were washed three

BN animals were immunized against transplantation antigens of Fischer


fistula as during the 3 weeks immediately prior t o fistula. This regime was adoped in order to minimize the number of variables at the time of fistula which might influence the results of fistula.

blood was allowed to clot, after which the serum was separated by centrifugation, heated at 56°C for 30 minutes, and then stored at -20°C. The bleeding schedule varied with the different immunizing repimes. In general, animals on fistula were bled daily or every second day.

Serological assays. Serum antibodies to SRBC were assayed by micro- agglutination technique, and to GRBC by hemolysin technique. Antibodies against transplantationantigens were measured by cytotoxicity using trypan blue dye exlusion and 51Cr release. The target cells were lymphocytes obtained by thoracic duct drainage.

and lumbar lymph nodes were assayed by the technique of Jerne et al., (54), which measures 19s antibody-forming cells. Triplicate assays were made at each of several dilutions of cell suspensions to ensure that plates were obtained with 20-100 plaques per plate.

General operative procedure on animals. The operations were carried out with clean instruments, but no attempt at complete asepsis was made. However, all catheters, tubing, and solutions were sterilized by appropriate methods. Animals were anesthetized with chloral hydrate injected intraperitoneally. Immediately on completion of the operation the animals received 1 t o 2 ml of citrated rat blood, depending on the amount of bleeding. The animals were operated on a warm pad and heat was applied to the animals in the immediate post-operative phase. The impression was gained that the blood transufsions and the warmth improved the post-operative recovery.

Thoracic duct cannula. The catheter was made to have a double lumen. The larger lumen tube, through which the lymph flowed, had a polyethylene tip (Clay Adams PE 50) which was attached to a silastic tube. The tip had a bevel for ease of entry, and a bump to ensure that the ties behind the bump kept the catheter in position. The silastic tube which formed the main part of the catheter provided flexibility, so that small errors of alignment were corrected without tension on the duct. The smaller lumen tube was made from polyethylene (Pe 50) and was “drawn out” in a stream of hot nitrogen to a diameter of 100 microns. The nitrogen prevented oxidation of the surface of the polyethylene during the “drawing;out” procedure. The unaltered polyethylene surface re- tained its excellent anticlotting property ( 5 5 ) . The smaller tube whch was inside the main tube carried heparin in a saline solution to the very tip of the catheter. The saline solution contained lOOU of heparin per ml and was infused up the catheter at a rate of 0.5 ml/hr. This prevented the lymph from clotting, and reduced the chance of blocking the catheter by cellular aggregates.

Antisera. The animals were bled from tail vein or retro-orbital sinus. The

Hemolytic plaque assays. The number of plaque-forming cells in spleen

144 Rose

Thoracic duct fistula. Insertion of the cannula was performed under a dis- secting microscope by the standard technique, with the modification that the exposure of the duct was achieved by holding the animal in the left hand with the fingers spread in an appropriate manner. The modification has the disad- vantage that it requires an assistant for ties, etc., but has the advantage that the operation can be performed more quickly and with less trauma and exposure of the viscera, compared to using standard retracting devices.

Gastric tube and placement. A silastic tube was inserted into the stomach and sealed in position by a double row of purse string nylon atraumatic sutures.

Intravenous catheters. The design of the catheter took into account the fact that cells as well as fluid would be returned through it. Thus, it seemed de- sirable that the catheter should be fine where it was inserted into the vein and be large at the end where the connection was made to a drip chamber. Polye- thylene tube, a PE 50, drawn down to 0.25 mm diameter for the intravenous section, or a PE 10, expanded for the connecting section, were used. Fine silicone rubber catheters were generally found to be unsatisfactory because pressure could not be applied to them if they became blocked.

Restraint of animals and externalizing of catheters. The 4 tubes (thoracic duct cannula, heparin line to cannula, intravenous catheter, and gastric tube) were made to pass (from their site of insertion) subcutaneously to emerge from beneath the skin of the tail. Consequently, when the animal was placed in a restraining cage the catheters were accessible to the investigator. The animals were partially immobilized by an abdominal harness held in position by tapes. The harness was attached by a bar to the restraining cage. The whole arrangement (levers, wheels, cage, etc.) was made so that the catheters were inaccessible to the rat, while giving maximum mobility to the animal. In addition, the apparatus was suitable for and was used as a metabolism cage.

carbonate centrifuge tubes, kept at 5°C. Prior to lymph collection, 5 ml of Ringers solution, containing 100 units of heparin, 0.5 mg of streptomycin, and 500 units of penicillin, were added to each centrifuge tube. The lymph was collected in 12-hourly batches and centrifuged at IOOOG for 10 minutes. In some experiments the lymph was evenly suspended prior to centrifugation and a sample taken for a lymphocyte count. The lymph fluid (mixed with the saline infused up the catheter, and the 5 ml of Ringers solution originally placed in the centrifuge tube) was withdrawn and kept in separate containers at - 20°C for subsequent assays. The dilution of the lymph (by the added saline and Ringers solution) was calculated to correct for subsequent serological assays.

The cells contained in the lymph were resuspended in 2 ml of citrated rat plasma and incubated for 10 minutes at 37°C. This was done as a prccautionary measure because it has been shown that citrated rat plasma can inactivate endotoxins effectively (56 , 57).

Lymph collection and cell return. The lymph was collected in 50 ml poly-


Preliminary experiments had shown that there was a variable but large (up to 50%) loss of lymphocytes if they were transferred by syringe and needles. Therefore, all procedures involving cell transfers were carried out using Pasteur pipettes. Preliminary experiments had shown that less than 2% of lymphocytes reached the vascular circulation if they were returned via the peritoneal cavity in animals with mild peritonitis. This latter state is usual after abdominal operations and particularly if catheters traverse the peritoneal cavity.

connected with the intravenous line. The following sequential steps were carried out at the time of cell transfer:

The cells were transferred by Pasteur pipettes into a small drip chamber

(1). The continuous intravenous infusion was stopped. (2) . The drip chamber was opened and the 1.5 ml of fluid contained in it

(3). The cells and plasma were pipetted into the near empty drip chamber. (4). Controlled air pressure, by means of a syringe, was applied to the drip

was removed by pipette.

chamber so that most of the plasma and cells were returned intravenously over a 10-minute period.

(5). 1.5 ml of saline was added to the drip chamber, which was then closed and the intravenous infusion recommenced.

(6). The remaining cells were slowly returned by the continuous intravenous infusion.

The details of the above procedure ensured minimum loss of cells, quick return for most cells, slow return for the remaining cells, and a minimum acute overloading of the vascular system.

Gastric feeding. Various feeding regimes were used. The most successful was to adapt the animals gradually by forced feeding to an increased volume of a high protein fluid diet for two weeks prior to fistula. Two to three feeds were given each day, each day increasing in volue from 3 ml to 10 ml.

The first day post-operatively, 1 ml of this diet was given three times per day. This was increased to 8-10 ml/feed after 2 to 3 days. Between gastric feeds, the animals were allowed to eat their usual pellets and drink 0.5 N saline ad libitum.

Intravenous fluid and electrolyte therapy. In general, this was controlled by measurement of input and output of fluid sodium and potassium. It was found advisable to err on the side of too little fluid for the first post-operative day, or until normal renal function returned.

Intravenous protein replacement therapy. This was determined by two types of experimental design and will be discussed in that section.

Measurement of venous pressure. This was achieved by either a continuous or discontinuous method. In the former, a continuous administration of saline at 0.5 ml/hr was given through one leg of a Y-piece, the second leg was a catheter placed in a central vein, the third leg consisting of tubing the same size as the catheter, made up the vertical arm of a manometer. In the second method the

146 Rose

intravenous line was disconnected and used as a temporary manometer. Design of experiments. Two types of experimental design were used.

Type A studied the augmentation of the immune response when the amount of antibody lost through the fistula was dictated by the natural anatomy of the thoracic duct, lymphatico-venous channels and the proportion of antibody produced by spleen and lymph nodes.

Type B studied the augmentation phenomenon consequent on maximum antibody elimination. In the early section of fhis paper, it was argued that maximum elimination would occur in splenectomized animals with raised venous pressure. The animals were splenectomized 2 to 4 weeks prior t o fistula, so that the animal and its immune system could recover from the operation. The venous pressure was raised to 12-15 cm H20 by the continuous administration of serum protein. The venous pressure was estimated 4 to 5 times per day and the rate of protein administration altered accordingly.

series received on average 30-35 ml of serum each day, as dictated by the venous pressure.

Calculation of results. Antibody (AB) content of fluids was calculated by multiplying the reciprocal of the titer by the volume of fluid. This is an approxi- mater calculation because it does not take into account binding affinity, etc., of the AB.

AB content of the rat was calculated on the basis that at equilibrium IgC is equally distributed between intra- and extra-vascular compartment (58), and that the volume of blood is 12% of the body weight.

AB production rate in animals with stable immunity was calculated from data showing that IgG in the rat has a half-life of 4.8 days (59).

AB production rate in animals on fistula could theoretically be calculated from formula: Production rate = Content of AB in daily lymph sample minus change in content of AB in the animal. AB loss early in the fistula period is due to combined effect of loss of prior formed AB plus newly synthesized AB. The proportion that each of these components contributed to the loss via the lymph was not calculated.

The above formula could be used only late in the fistula period and when the AB production was very high. Under these circumstances, errors due to biological half-life, state of hydration, and distribution between intra- and extra-vascular compartment could be ignored. In addition, late in the fistula, the AB content in daily lymph samples was high compared to the total, and even higher compared to the daily changes of the total AB content of the animal. Therefore, late in the fistula period changes in the total AB content of the animal were ignored and the production rate of AB was calculated as the content of AB in the daily or 12-hourly lymph samples.

Animals in type A series received 10- 12 ml and animals in the type B


With Respect to Tumor Growth and Regression

200-250 gm were used.

(MSV) into neonatally thymectomized rats (60). [The tumor was kindly supplied by Dr. R. Ting of the N.C.I., N.I.H.]

antigen as demonstrated by transplantation resistance (60).

of tumor cells. (0.5 X 106 animal passaged cells or 0.1 X 106 cultured tumor cells.)

fers when the tumor reached a diameter of approximately 1% to 2 cm.

inoculation of 0.5 X lo6 viable tumor cells as determined by trypan blue dye exclusion.

Experiments with cultured tumor cells were carried out after subcutaneous inoculation ofO.1 x 106 tumor cells.

Procedure. Operative procedure, care of animals, and cell return was the same as the described technique for studies on augmentation of humoral immunity. The animals were not splenectomized and the venous pressure was not elevated during the fistula. The control animals, for studies with animal passaged cells, received the same operation and treatment as experimental animals except cells and lymph were returned intravenously.

The control animals for studies with cultured tumor cells were unoperated controls.

Animals. Adult male BN rats (Microbiological Associates) weighing

Tumor. The tumor was induced by inoculation of Murine Sarcoma Virus

The tumor is transplantable and possesses a tumor-specific transplantation

The tumor regularly kills recipients if they are given an appropriate dose

The tumor was carried in tissue culture and in BN animals by serial trans-

Experiments with animal passaged cells were carried out after subcutaneous

RESULTS

With Respect to Humoral Immunity General. The results have been generated from 27 experimental (SRBC

were used as antigen in 12 animals, GRBC in 4 animals, and transplantation antigens in 11 animals) and 30 control rats. With respect to the experiemental group, 18 animals were studied using design type A (i. e., augmentation of immune response consequent on “natural” elimination of antibody through fistual) and 9 rats were studied using design type B (i.e., augmentation of immune response consequent on maximum elimination of antibody). Design type B required that the experiments be carried out in splenectomized animals with raised venous pressure. As described in the section on Methods, the venous pressure was elevated by serum infusions. The rate of serum infusions was re- gulated by periodic “manual” measurements of the venous pressure, thus making

148 Rose

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it possible that there were periods during the night in which the venous pressure was not elevated.

Antibody-producing cells. The number of 19s antibody-producing cells in the spleen and lymph nodes of control and experimental animals (design A) is is shown in Fig. 1 and Fig. 2 . There were 4 to 20 times as many 19s antibody- producing cells in the experimental animals as compared to controls at the same stage of immunization. No attempt was made to determine how much of this


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increase was due to recruitment or proliferation. Antibody content in lymph samples obtained early in fistula period. In

animals with a stable immunity the antibody content of daily lymph samples obtained during the first 2 days of fistula varied between 20 and 60% of the total antibody contained in the rat prior to fistula. These relatively low values are consistent with the low rate of antibody synthesis (equal to biological decay) in animals with stable immunity. The carly lymph sample values are also consistent

150 Rose

with data showing that in the rat there is a daily transfer of 75% of intravascular protein into the lymph (46). The results show that during the first few days of fistula the quantity of antibody eliminated in the lymph was of the same order as that contained in the rat. In addition, the animals were out of equilibrium with respect to fluid balance and intra- and extravascular compartment volumes. For these reasons, it was not possible, from serological data obtained during the first few days of fistula, to determine the rate of antibody production, or if there was a lag period prior to augmented antibody production.

lymph of fistula animals increased exponentially, usually doubling every 10 to 18 hours (Fig. 3-7).

The peak daily output of antibody in the lymph was 20 to 100 times the total content of antibody in the rat prior to the fistula. The daily output of antibody can be considered the production rate when these values are high compared to the total antibody content, or daily changes in antibody content in the rat. The relative contribution of increased numbers of antibody-producing cells and increased antibody synthesis per cell toward the augmented antibody production was not determined.

Two other methods of expressing the magnitude of the augmentation are of interest.

(1). The total amount of antibody eliminated in the lymph of any one experimental animal, over 7-9 days of fistula, was 100-500 times the amount which could have been obtained by complete exsanguination of that animal prior to fistula.

times the rate of pre-fistula synthesis; this latter rate is equal to the production rate in animals with stable immunity.

antibody titer of all animals in experimental groups fell to 10% of the pre-fistula level (Figs. 3-7).

Following the initial fall, the serum antibody titer in the experimental group A rose, often reaching peak values of 200% above the pre-fistula level on the 7th to 9th day of fistula (Fig. 5). The rise in serum antibody titer in the later stages of fistula occurred despite continued loss of large amounts of antibody in the lymph.

In 4 animals of group B the serum titer fell and remained low during the entire period of the fistula. These results are illustrated for SRBC in Fib. 4, and for transplantation antigens in Fig. 6.

Calculation shows that 0.5-2.0% of the antibody produced per day was present in the blood. The results from animals in group B, and data concerning the daily turnover of proteins from blood to lymph (46), suggest that loss of antibody from the blood circulation to lymph could not account for the low

Augmented antibody production. The daily output of antibody in the

(2). Peak antibody production rate in experimental animals was 200-1000

Serum antibody titer. During the first 2 t o 3 days of fistula, the serum

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serum titers. Direct loss of antibody in the lymph must be the major cause for the low serum antibody titers i n the face of high antibody production rates by these animals. Direct loss was presumably due to the combined effect of splenectomy and raised venous pressure, diverting nearly all of the newly synthesized antibody into the lymph prior to reaching the blood stream.

In 2 animals of experimental group B the serum titer fell and rose again.

152 Rose 10,000

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Days after fistula operation Fig. 4. Average results from Holtzniann rats 5 5 and 56. Serum antibody titer prior to and during fistula procedure and daily antibody loss in lymph. Rats received lo9 SRBC intravenously 14 days prior to fistula and every 3 days thereafter. Experimental design type B was employed. Rats were splenectomized 21 days prior to first antigen administration and venous pressure was elevated during fistula procedure.

The result from one of these animals (Fig. 7), shows that the pattern of serum titer recovery and overshoot follows a similar trend to animals in group A. The failure to maintain a low blood titer during the course of the fistula was probably due to the venous pressure not remaining continuously elevated throughout the fistula period.

-Relations between serum antibody titer and antibody production. In ex-

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Days after fistula operation Fig. 5. Average results from BN rats 882 and 883. Serum antibody titer prior to and during fistula procedure and daily antibody loss in lymph. BN rats received skin transplant from Fischer rats 2 months prior to fistula. After rejection of skin transplant the BN rats received 5 x lo7 nucleated Fischer cells subcutaneously every 2 weeks until 3 weeks prior to fistula at which time the animals received 5 x lo7 Lewis cells every third day. Design type A was employed. Rats were not splenectomized and the venous pressure was not elevated during fistula procedure.

perimental animals in group A (and in 2 animals of group B), the daily antibody production reached a plateau after 5 to 6 days of fistula, and the serum titer returned to, or increased beyond, the pre-fistula levels (Figs. 3 , 5 , and 7). In the later stage of the fistula period, these animals were in a new and relatively stable immune state. During this “stable” state the serum titer remained fairly constant, at a level the same or higher than the pre-fistula level, and the rate of

154 Rose

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Fig. 6. BN rat 893. Serum antibody titer prior to and during fistula procedure and daily antibody loss in lymph. BN rat received skin transplant from Lewis animal 3 months prior to fistula. After rejection of skin transplant the BN rat received 5 X lo7 nucleated Lewis cclls subcutaneously every 2 to 4 weeks prior to fistual, at which time the animal received 5 X lo7 cells every third day. Experimental design type B was employed. The rat was splenectomized 2 months prior to fistula and venous pressure was elevated during fistual procedure.

antibody production and loss was balanced. The antibody production rate in these lattcr animals was several hundred times the pre-fistula production rate, or the production rate in control animals with stable immunity. These results add further support to the hypothesis, discussed in the introduction, that the organism acts to regulate the concentration of circulating antibody rather than the antibody production rate of the “immediately produced local micro-

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Days after fistula operation Fig. 7. BN rat 891. Serum antibody titer prior to and during fistula procedure and daily antibody loss in lymph. BN rat received skin transplant from Lewis animal 3 months prior to fistula. After rejection of skin transplant the BN rat received 5 X 10’ nucleated Lewis cells subcutaneously every 2 to 4 weeks until 3 weeks prior to fistula procedure at which time the animal received 5 X lO’cells every third day. Experimental design type B was employed. The rat was splenectomized 2 months prior to fistula. The rise in serum titer to pre-fistula level may be due to the venous pressure not having been elevated continuously during the fistula period.

environmental concentration of antibody.” There was a continuing exponential increase in antibody production in

experimental animals of group B, in which the serum titer remained low during fistula (Figs. 4 and 6). It is hypothesized that the continuing exponential increase in antibody production was a result of antigen stimulation in the near absence of feedback control due to a low serum antibody titer.

156

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Fig. 8. Tumor growth after subcutaneous inoculation of 0.5 X lo6 “animal passaged murine sarcoma virus induced tumor” cells in control (curve shows average growth in 14 animals) and individual experimental BN rats. The 14 control rats were subjected to the thoracic duct fistual procedure and the lymph fluid and cells were returned intravenously. The cells in the lymph, but not the lymph fluid, were returned to the experimental rats.

With Respect to Tumor Growth and Regression

the tumor and the growth rate of the tumor are shown in Fig. 8. In 4 animals (H-12, H-17, H-23, and H-25) the entire procedure was technically successful. On the second day of fistula procedure the tumors became less firm and then completely regressed on days 4-7 (Fig. 8).

Tumor from cultured cells. The lag period and growth rate of these tumors are shown in Fig. 9. The tumors of all experimental animals undergoing a technically “successful” procedure showed marked tumor regression (Fig. 9). At autopsy the tumors appeared haemorrhagic and necrotic.

After inoculation of “animal Passaged tumor cells” into control animals, the tumor growth was slower than the tumors derived from cultured cells. The “fistula procedure” produced a more dramatic regression of tumors derived from animal passaged cells as compared to tumors derived from cultured cells. The explanation for these differences is not known. However, when animal passaged cells were transferred, a considerable number of “immune” cells of various kinds may also have been transferred and thus account for the above phenomenon.

Tumors from animal passaged cells. The lag period before appearance of

157

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Immune Augmentation and Cancer Therapy

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Fig. 9. Tumor growth after subcutaneous inoculation of 0.5 X lo6 “tissue cultured murine sarcoma virus induced tumor” cells in control (curve shows average growth in up to 81 animals) and individual experimental BN rats. The experimental animals were subjected to the fistula procedure. the cells contained in the lymph fluid were returned intravenously and the lymph fluid was wasted.

158 Rose

DISCUSSION Biological Effects of Fistula Procedure

Rats with thoracic duct fistula lose lymph fluid equal to approximately 25% of theri body weight per day. The animals must be given replacement therapy, which can be of various kinds and combinations. In order to use the “fistula procedure” to study immune augmentation, the replacement therapy must lack the specific antibody. The combination of fistula, and replacement therapy lacking specific antibody produces a makred reduction in the level of specific antibody in the circulating blood and extra-vascular tissue fluid. It is likely that the augmentation of the specific antibody production is a result of the uninhibited synthesis of antibody and uninhibited recruitment and proliferation of immune cells. The limit of augmented antibody production as a consequence of antibody elimination is not known. The theroretical limit may be determined by the proliferative ability and antibody production capacity of the relevant cells of the immune system. The practical limit may be reached when the antibody production is so high that enough antibody “sneaks” into the blood circulation to generate an antibody titer in the serum equal to or higher that the pre-fistula state.

lymph and receiving replacement therapy could produce depletions of various components which are normally present in the fluids of the body. For each component, the extent of depletion will be determined by the differential loss (rate of 100s through lymph minus rate of gain through replacement therapy) and the rate and capacity of the organism to compensate. The net effect of the “procedure” will vary with each component and will depend on many factors, such as the type of replacement therapy and properties of the component, for example, its molecular weight, permeability through capillary and lymphatic vessels, and its site(s) of synthesis and capillary or lymphatic exist from site(s) of synthesis.

It is interesting to consider whether the composition of the experimental animal could be maintained unaltered, apart from a depletion of the specific antibody. This may be achieved if the experimental animal received, at a rate equal to its lymph loss, fresh cell-free lymph from a pool of syngeneic donors. Even using this “ideal” replacement therapy, it might be argued that labile compounds could be lost during transfer of lymph from donors to experimental animals. However, this latter possibility is unlikely to produce a critical deficiency because there is generally a rapid turnover and homeostatic adjust- ment of labilc compounds in vivo.

Consequently, interpretations of the results of fistula could be complicated by alterations, other than antibody depletion, in the composition of body fluids and the function of certain tissues such as endocrine glands.

In addition to eliminating specific antibody, the “procedure” of losing

Experimentally, it may be difficult to achieve “ideal” replacement therapy.


It would be possible to increase the precision of the interpretation of results if the experimental animals were immunized against two unrelated antigens, and were given lymph replacement therapy from syngeneic donors immunized against only one of these antigens. Lymph from the donor animals should only be removed intermittently in order to avoid alterations in the donor’s general physiological or specific immune status which would be reflected in the composition of their lymph. The above design with a “built in” control may be particularly important in evaluating studies concerning the possibility of tumor regression by eliminating “blocking” factors. Thus, the animal could be in- oculated with two antigenically different tumors. The experimental animals on fistula would receive lumph replacement therapy from syngeneic donors bearing only one of these tumors.

Implications with Respect to Antibody Production

various antibodies by manipulating the immune system as described in this paper. The antibody may be obtained in increased total quantity, increased quantity per available antigen, increased specific activity, and decreased vari- ability by virtue of being generated in less animals than with usual methods of production.

There are likely to be practical and economical advantages of generating

Evolutionary Implications

existence of the enormous potential for immune augmentation.

and feedback inhibitor factor are synthesized by the organism. Both factors have probably evolved in a balanced way in the same species as the organism. Further, under natural environmental conditions the physiological level of these factors only fluctuate over a small range. These features may help to explain the organism’s relatively moderate and limited capacity to respond even if the system is severely distrubed. The immune system differs in many important re- spects from other systems in the organism. The activator is antigen which is generally not synthesized by the organism whereas the feedback inhibitory factor is specific antibody synthesized by the immune tissues of the organism. The major function of specific antibody is to hasten the elimination of antigen. This latter function is achieved (in part) by complexing the antigen, a process which eliminates antigen and antibody. In addition, the immune system evolved under conditions of enormous fluctuations of antigen load.

The above features may help to explain the evolutionary selection of organisms having an immune system which has the capacity for enormous augmentation and antibody synthesis.

The following is a speculative discussion concerning explanations for the

In most controlled systems of multicellular organisms the activator factor

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Implications with Respect to Immunotherapy of Cancer It is generally accepted that most neoplasms have tumor-specific trans-

plantation antigens. The immune system of the host responds to the presence of the tumor by generating cells and antibodies, some of which can kill the tumor cells. However, under usual conditions, the immune system does not act vigorously enough to cope with the growing cancer.

The nature of the interaction between the immune system and the tumor can be described as a relationship between a highly controlled immune system which has the inherent capacity for large augmentation, and an uncontrolled proliferation of tumor cells. Except over a limited range it is not possible for a servocontrolled system to deal with an uncontrolled system. With increasing time, the disparity between the two systems increases. For cancer the critical level of disparity for failed immune control of the tumor appears to occur when the tumor is small, that is of a size which is not clinically recognizable. Immunotherapy of cancer may require the nature of this relationship to be reversed. This may be achieved by a (relatively) unrestrained proliferation and activity of the appropriate components of the immune system and by elimination of excess immunologically active products such as tumor antigen, which may be continuously generated and released by the cancer cells.

has the capacity to manipulate, continuously and over a prolonged period, the critical components involved in the tumor-immune system interaction. The following discussion suggests that such manipulations could be achieved by variations of the “thoracic duct fistula procedure.”

In splenectomized tumor-bearing animals with raised venous pressure. the following components flow preferentially and directly into the thoracic duct fistula prior to entering the blood stream:

(1). The (augmented) capillary filtrate (less components removed by tissues and regional lymph nodes) from the entire body.

(2). Almost all newly synthesized antibody (prior to exerting a feedback effect).

(3). All lymphocytes which have traversed tissues (normal, tumor, and lymph node) and which have been recently generated in, and released from, lymphoid tissue. These cells will include recirculating lymphocytes, recently generated lymphocytes, and lymphocytes recently inhibited (possibly by tumor antibody-antigen complexes).

weight greater than 150,000, a high proportion of this antigen, not phagocytosed by regional lymph nodes, is likely to be present in the thoracic duct lymph.

In order to achieve these ends, it is necessary to devise a system which

(4). If tumor antigen is released from tumor cells and has a molecular

( 5 ) . Tumor antigen-antibody complexes. The above components are some of the critical factors influencing the


immunological relationship between tumor and host. For example, the balance of these components is involved in the operative mechanisms of immune enhancement, immune tolerance and possibly immune activity controlled to function at a low and therefore ineffective level. Data of various kinds suggest that specific antibody against tumor antigen, or tumor antigen complexes, limit the activity and the effectiveness of the immune system to reject tumor cells. The phenomenon has been called enhancement and has been the subject of recent reviews (61,62).

sulting in the failure of the immune system to reject tumors is not known. This is largely due to the fact thant enhancement has been studied by measuring the level of blocking factor(s) in the serum, or “adding extra blocking factor(s)” to animals bearing a tumor. However, only by removing blocking factor(s) can data be obtained as to their degree of interference and the full potential of the immune system in their absence to reject tumors.

Many possible mechanisms of enhancement have been suggested. The relative contributions of each of the suggested mechanisms to the in vivo problem is not known. Nonetheless, it is instructive (even though speculative) to analyze these mechanisms from the operational point of view, as to which of them could, or could not be reduced or eliminated by the “fistula procedure.”

Afferent inhibition. Antibody may decrease the availability of tumor antigen which is necessary for stimulation of the immune system (63,64).

Efferent inhibition. Effector cells are blocked from reacting with target cells because their antigenic sites are covered (65-69).

Antigenic modulation. There are data from one system [the TL antigen of mice (70,711 and suggestive data from another [Burkitt Lymphoma (72)] which is consistent with the notion that cells reversibly lose their tumor antigen in the presence of specific antibody. If a tumor loses its tumor antigen, it may not be susceptible to the otherwise cytotoxic action of lymphocytes which are immune-committed against the tumor antigen.

Inhibition of proliferation of sensitized lymphocytes in lymph nodes and tumor tissue. By analogy with antibody-producing cells (1,73,74), specific antibody may limit the number of sensitized lymphocytes which are generated.

Inhibition of macrophage function. Data from various experiments have been interpreted to show that specific antibody can inhibit the “antigen pro- cessing’’ ability of macrophates (75-79).

is produced by antigen-antibody complexes acting to inhibit lymphoid function (61,80). The phenomenon has recently been studied and recognized as an immune limiting mechanism, possibly different to those described above. The recognition of this form of enhancement arose from data showing that enhancement of grafts or host can be produced by very small amounts of antibody (81),

The contributions made by enhancement as the operative mechanism(s) re-

Low dose enhancement. It has been suggested that low dose enhancement

162 Rose

and by the findings that antibody does not remain on the cell surface for a long period (61).

The present experiments (using SRBC and transplantation antigens) have shown that a low blood titer can be achieved despite enormous (1000-fold) augmented antibody production, provided the fistula procedure is carried out in splenectomized animals with raised venous pressure. By analogy, it is reasonable to assume that a low blood titer of tumor-specific antibody could be achieved by the fistula procedure. By extrapolation, it is predicted that there will be an even greater depression in the tumor antigen titer in the blood. This latter pre- diciton is likely to be true because it is unlikely that there would be an increased tumor antigen production and release, consequent on tumor antigen elimination. Elimination of tumor antibody and tumor antigen must lead to low levels of tumor antigen-antibody complexes. Lowering the blood level of these factors could reduce enhancement and augment the generation and effectiveness of cell-mediated immune cells.

As a tentative working hypothesis, it is postulated that, in the experiments reported in this paper, thoracic duct fistula caused tumor regression by the above mechanisms. Elimination of “blocking factor” as a cause of tumor regression is not ruled out as a possible explanation in the present experiments even though the animals were not splenectomized and their venous pressure not raised. This statement is derived from data on studies of humoral immune augmentation (Figs. 3 and 5) . These results show that after fistula procedure the serum antibody titer was depressed and remained low for 3 to 4 days, even though these animals were not splenectomized and their venous pressure not elevated.

ACKNOWLEDGEMENTS

A portion of this work was supported by a grant from Dr. Armand

The author thanks Mrs. Nola Duffy, Mr. Peter Bina, Mr. James Lewis, Hammer to the Laboratory of Dr. Jonas Salk.

Mrs. Elsie Ward, Mrs. Ann Hobson, and Mr. Don Wegemer for their indispen- able assistance.

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