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The induction of long-term immunological tolerance
The Induction of Long-Term Immunological Tolerance By RAPHAELH. LEVEY
L
ONG-TERM DURARLE CELLULAR CHIMERISM is difficult to achieve in adult mice, and previously well-tolerated skin allografts will be slowly rejected after immunosuppression is terminated.’ The greater the genetic disparity between donor and recipient strains, the more difficult it is to induce lasting immunological tolerance. These obstacles are most severe when attempts are made to cross species barriers. The breakdown in tolerance will occur despite repeated injections of allogeneic cells,” suggesting that the regenerant host cells slowly crowd out the donor inoculum. which is at a selective numerical and immunological disadvantage. Experiments were undertaken, therefore, to determine whether the allogeneic or xenogeneic cellular inoculum could be placed on a more equal footing with the host lymphoid cell population by providini the donor cells with their usual microcellular environment; namely, that supplied by the stromal elements of lymph nodes. It was reasoned that allogeneic and xenogeneic cells would “home” to established lymph node grafts of the donor strain and survive to proliferate in this environment, and thus would be a continuing source of donor antigen. The cellular chimerism would then be reflected in a higher degree of tolerance than that achieved by injecting cells alone. MATERIALS ANDMETHODS
Animals CBA male and female mice 8-10 wk of age obtained from Jackson Laboratories, Bar Harbor, Maine, were used as recipient animals throughout. In experiments in which rat tissues were grafted to mice, the recipients were thymectomized at 6 wk of age. Thymectomy was performed under general pentobarbital anesthesia using a transcervical aspiration technique. Donor mice were of the C57BL/6 strain and were matched with the recipients for sex. Weanling
Lewis rats served as the xknogeneic cell and skin donors.
Antilymphocyte
Serum
Antilymphocyte serum (ALS) was raised in New Zealand white rabbits against outbred mouse thymocytes by a standard two-pulse intravenous technique, which has been reported elsewhere.3 All sera were absorbed with scrupulously washed, packed, mouse red blood cells on two occasions in a serum-to-cell ratio of 5 to 1.
From the Department of Surgery, Children’s Hospital Medical Center, and Harvard Medical School, Boston, Mass. Prevznted before the American Pediatric Surgical Associatiotl. Hamilton, Bermuda, April 22-24,197l. Supported by VSPHS Grant AI-09230. RAPHAEL H. LEVEY, M.D.: Department of Surgery, Children’s Hospital Medical Center, and Haroard Medical School, Boston, Mass. Dr. Leoey received a Faculty Research Fellowship of the American Cancer Society and is a Markel Scholar in Medicine.
@JHXALOF
PEDIATRIC SUHCEIIY,VOL. 6, No.5
( OCTOIIEH). 1971
533
544
RAPHAELH.LEVEY
Fig. I.-Illustration of nonimmunological cellular cooperation in immunological tolerance showing the experimental protocol of the induction of tolerance across strong intraspecies and interspecies barriers, The ALS volumes are in milliliters. The skin grafts, lymph node grafts, and s leen cells are all from thpe same donor strain of mouse or rat.
Cellular Inocula Monodispersed cellular suspensions were prepared by gentle sieving of donor spleens through a nylon mesh in medium 199 with 15% fetal calf serum added and then buffered with bicarbonate to a pH of 7.4. The suspensions were filtered through surgical cotton wool to remove any clumps, centrifuged at 1000 rpm, and washed once in fresh medium. The cells were resuspended and injected intravenously into the tail vein of the recipient mice in a volume of 0.5 ml.
Skin and Lymph Node Grafting Tail-skin allografts and xenografts were applied to the trunk of the recipient mice by a technique that has been described elsewhere. 4 Two lymph nodes of the appropriate donor strain were positioned under each kidney capsule of the recipient mice. Each kidney was exposed through a separate flank incision.
Experimental Protocol Figure 1 summarizes the protocol used in all experiments. On day 0 all mice were grafted with either allogeneic mouse or xenogeneic rat skin. Thereafter, 2.5 ml of antilymphocyte serum was administered subcutaneously in divided doses over a 21-day period commencing on day +2 following grafting, followed by biweekly injections thereafter. On day i-25 lymph node grafts of the appropriate donor strain were placed under both kidney capsules, using two axillary or inguinal nodes on each side. The antilymphocyte serum was restarted 2 days following the lymph node grafting and 1.0ml administered in the ensuing 10 days. Three days following the last dose of ALS, either allogeneic spleen cells or xenogeneic spleen cells were injected intravenously. The dose of allogeneic cells was 5 X 107 and of xenogeneic cells, 1 x 10.8 No treatment was given to the test animal thereafter. The skin grafts were observed starting on day +I2 and inspected daily. In both the allogeneic and xenogeneic experiments, the mice were divided into two groups: those that received ALS and cells only and those that received ALS, lymph node transplants, and cells. RESULTS The results are translated into survival curves that show, day by day, the number of mice still bearing intact grafts, and are expressed numerically as the median survival time (MST). In the experiments illustrated in Fig. 2, 36 CBA mice were grafted with C57BL/6 tail skin on day 0. The mice were
INDUCTION OF IMMUNOLOGICAL
TOLERANCE
545 Fig. 2-Allogeneic tolerance. (A) Survival on CBA mice of C57BL/6 strain skin grafts transplanted under the protective effect of ALS. (B) Survival on CBA mice receiving, in addition to ALS and C57BL/ 6 spleen cells, C57BL/6 lymph node grafts on the 25th day after grafting.
Fig. 3.-Xenogeneic tolerance. Recording the dayto-dav survival of Lewisrat t&l-skin xenograft on (B) thvmectomized CBA mice that had received ALS and 100 million rat spleen cells; and on (A) thvmectomized CBA mice that received, in addition, rat lymph node grafts under both kidney capsules.
then subdivided into two equal groups, one of which (Group B) was engrafted with C57BL/6 lymph node on day +25. The illustration shows that skin grafts in this group enjoyed a median survival time of greater than 210 days, with 15-18 grafts still surviving at this time. The grafts on the control mice (those that received ALS and cells only, Group A) had broken down more quickly. The MST of these grafts was 105 days. This experiment is still in progress. Figure 3 shows the survival pattern of Lewis rat-tail skin grafts on thymectomized CBA mice. In these experiments, two equal groups of 18 mice were treated according to the protocol of Fig. 1. Group A mice received, in addition to ALS and xenogeneic spleen cells, subcapsular kidney grafts on day +25. In this group, 15 of 18 grafts are surviving at 180 days whereas the MST of the control grafts is 65 days (maximum survival 120 days). Graft-vs.-host reactions were not seen in any recipient mice. DISCUSSION
The results presented here suggest that a greater degree of tolerance can be achieved in mice that are provided with lymph node grafts from the same strain or species as the donor of the skin graft and subsequent cellular lymphoid inocula. The mechanism by which this is achieved is not clear and experiments are in progress to evaluate the actual migration or “homing” of the injected cells to the lymph node graft and to study their survival patterns in these locations
546
RAPHAEL H. LEVEY
It is possible that the environment that is syngeneic with the injected cells provides the appropriate biochemical or structural milieu in which the injected cells can survive to proliferate at less of a selective disadvantage than in that of the allogeneic environment of the recipient animal. At the time of lymph node grafting, the recipient animals were depleted of circulating small lymphocytes by the prolonged course of antilymphocyte serum. Therefore, the lymph node graft is not rejected but repopulation of the graft by the host cells is precluded by this depletion. A Z-wk interval was allowed following the lymph node grafting during which the graft could become established, although the stromal elements could remain viable and acquire a new blood supply. The histologic appearance of these grafted lymph nodes is under study. It is suggested that repopulation of the node occurs by virtue of the large number of injected allogeneic or xenogeneic cells. An interesting and important sidelight of these experiments is that in no animals was graft-vs.-host disease noted. The recipient animals were clearly immunologically incompetent at the time of the injection of the cells; however, it would appear that they were protected by having high circulating levels of antilymphocyte serum that was able to clear those cells capable of initiating graft-V.-host reactions while allowing the putative stem cells to take up residence in the lymph nodes and spleen without giving rise to daughter cells that would subsequently cause secondary disease.
SUMMARY CBA mice were rendered tolerant by a prolonged course of antilymphocyte serum and the subsequent injection of allogeneic or xenogeneic lymphoid cells. Control mice received no other therapy. Experimental mice were grafted with allogeneic or xenogeneic lymph nodes prior to the administration of the cellular inocula. The survival pattern of tail-skin grafts was assessed in all mice. Those animals that were grafted with lymph node transplants failed to reject the skin grafts with the same rapidity or pattern as mice that were treated with ALS and cells alone. Furthermore, graft-V.-host disease was not noted in either the allogeneic or xenogeneic chimeras. The possible mechanisms of this durable chimerism are discussed. The experiments are still preliminary and others are in progress to evaluate the survival patterns of lymphoid cells in their heterotopic but syngeneic environment.
REFERENCES 1. Monaco, A. l’.: Use of antilymphocyte serm~i on the induction of immunological tolerance to tissue allografts. Fed. Proc. 29: 153, 1970. 2. Lance, E. M., and hledawar, P. B.: Quantitative studies on tissue transplantation immunity. IX. Induction of tolerance with antilymphocyte serum. Prnc. Roy. SW. Brit. 103:447, 1969.
3. Levey, It. H., and Medawar, P. B.: Nature and mode of action of antilymphocytic antiserum. Proc. Nat. Acad. Sci. U.S.A. 56: 1130, 1966. 4 Billingham, R. E., and Medawar, P. B.: The technique of free skin grafting in mammals. J. Exp. Biol. 28:385, 1951.
L
ONG-TERM DURARLE CELLULAR CHIMERISM is difficult to achieve in adult mice, and previously well-tolerated skin allografts will be slowly rejected after immunosuppression is terminated.’ The greater the genetic disparity between donor and recipient strains, the more difficult it is to induce lasting immunological tolerance. These obstacles are most severe when attempts are made to cross species barriers. The breakdown in tolerance will occur despite repeated injections of allogeneic cells,” suggesting that the regenerant host cells slowly crowd out the donor inoculum. which is at a selective numerical and immunological disadvantage. Experiments were undertaken, therefore, to determine whether the allogeneic or xenogeneic cellular inoculum could be placed on a more equal footing with the host lymphoid cell population by providini the donor cells with their usual microcellular environment; namely, that supplied by the stromal elements of lymph nodes. It was reasoned that allogeneic and xenogeneic cells would “home” to established lymph node grafts of the donor strain and survive to proliferate in this environment, and thus would be a continuing source of donor antigen. The cellular chimerism would then be reflected in a higher degree of tolerance than that achieved by injecting cells alone. MATERIALS ANDMETHODS
Animals CBA male and female mice 8-10 wk of age obtained from Jackson Laboratories, Bar Harbor, Maine, were used as recipient animals throughout. In experiments in which rat tissues were grafted to mice, the recipients were thymectomized at 6 wk of age. Thymectomy was performed under general pentobarbital anesthesia using a transcervical aspiration technique. Donor mice were of the C57BL/6 strain and were matched with the recipients for sex. Weanling
Lewis rats served as the xknogeneic cell and skin donors.
Antilymphocyte
Serum
Antilymphocyte serum (ALS) was raised in New Zealand white rabbits against outbred mouse thymocytes by a standard two-pulse intravenous technique, which has been reported elsewhere.3 All sera were absorbed with scrupulously washed, packed, mouse red blood cells on two occasions in a serum-to-cell ratio of 5 to 1.
From the Department of Surgery, Children’s Hospital Medical Center, and Harvard Medical School, Boston, Mass. Prevznted before the American Pediatric Surgical Associatiotl. Hamilton, Bermuda, April 22-24,197l. Supported by VSPHS Grant AI-09230. RAPHAEL H. LEVEY, M.D.: Department of Surgery, Children’s Hospital Medical Center, and Haroard Medical School, Boston, Mass. Dr. Leoey received a Faculty Research Fellowship of the American Cancer Society and is a Markel Scholar in Medicine.
@JHXALOF
PEDIATRIC SUHCEIIY,VOL. 6, No.5
( OCTOIIEH). 1971
533
544
RAPHAELH.LEVEY
Fig. I.-Illustration of nonimmunological cellular cooperation in immunological tolerance showing the experimental protocol of the induction of tolerance across strong intraspecies and interspecies barriers, The ALS volumes are in milliliters. The skin grafts, lymph node grafts, and s leen cells are all from thpe same donor strain of mouse or rat.
Cellular Inocula Monodispersed cellular suspensions were prepared by gentle sieving of donor spleens through a nylon mesh in medium 199 with 15% fetal calf serum added and then buffered with bicarbonate to a pH of 7.4. The suspensions were filtered through surgical cotton wool to remove any clumps, centrifuged at 1000 rpm, and washed once in fresh medium. The cells were resuspended and injected intravenously into the tail vein of the recipient mice in a volume of 0.5 ml.
Skin and Lymph Node Grafting Tail-skin allografts and xenografts were applied to the trunk of the recipient mice by a technique that has been described elsewhere. 4 Two lymph nodes of the appropriate donor strain were positioned under each kidney capsule of the recipient mice. Each kidney was exposed through a separate flank incision.
Experimental Protocol Figure 1 summarizes the protocol used in all experiments. On day 0 all mice were grafted with either allogeneic mouse or xenogeneic rat skin. Thereafter, 2.5 ml of antilymphocyte serum was administered subcutaneously in divided doses over a 21-day period commencing on day +2 following grafting, followed by biweekly injections thereafter. On day i-25 lymph node grafts of the appropriate donor strain were placed under both kidney capsules, using two axillary or inguinal nodes on each side. The antilymphocyte serum was restarted 2 days following the lymph node grafting and 1.0ml administered in the ensuing 10 days. Three days following the last dose of ALS, either allogeneic spleen cells or xenogeneic spleen cells were injected intravenously. The dose of allogeneic cells was 5 X 107 and of xenogeneic cells, 1 x 10.8 No treatment was given to the test animal thereafter. The skin grafts were observed starting on day +I2 and inspected daily. In both the allogeneic and xenogeneic experiments, the mice were divided into two groups: those that received ALS and cells only and those that received ALS, lymph node transplants, and cells. RESULTS The results are translated into survival curves that show, day by day, the number of mice still bearing intact grafts, and are expressed numerically as the median survival time (MST). In the experiments illustrated in Fig. 2, 36 CBA mice were grafted with C57BL/6 tail skin on day 0. The mice were
INDUCTION OF IMMUNOLOGICAL
TOLERANCE
545 Fig. 2-Allogeneic tolerance. (A) Survival on CBA mice of C57BL/6 strain skin grafts transplanted under the protective effect of ALS. (B) Survival on CBA mice receiving, in addition to ALS and C57BL/ 6 spleen cells, C57BL/6 lymph node grafts on the 25th day after grafting.
Fig. 3.-Xenogeneic tolerance. Recording the dayto-dav survival of Lewisrat t&l-skin xenograft on (B) thvmectomized CBA mice that had received ALS and 100 million rat spleen cells; and on (A) thvmectomized CBA mice that received, in addition, rat lymph node grafts under both kidney capsules.
then subdivided into two equal groups, one of which (Group B) was engrafted with C57BL/6 lymph node on day +25. The illustration shows that skin grafts in this group enjoyed a median survival time of greater than 210 days, with 15-18 grafts still surviving at this time. The grafts on the control mice (those that received ALS and cells only, Group A) had broken down more quickly. The MST of these grafts was 105 days. This experiment is still in progress. Figure 3 shows the survival pattern of Lewis rat-tail skin grafts on thymectomized CBA mice. In these experiments, two equal groups of 18 mice were treated according to the protocol of Fig. 1. Group A mice received, in addition to ALS and xenogeneic spleen cells, subcapsular kidney grafts on day +25. In this group, 15 of 18 grafts are surviving at 180 days whereas the MST of the control grafts is 65 days (maximum survival 120 days). Graft-vs.-host reactions were not seen in any recipient mice. DISCUSSION
The results presented here suggest that a greater degree of tolerance can be achieved in mice that are provided with lymph node grafts from the same strain or species as the donor of the skin graft and subsequent cellular lymphoid inocula. The mechanism by which this is achieved is not clear and experiments are in progress to evaluate the actual migration or “homing” of the injected cells to the lymph node graft and to study their survival patterns in these locations
546
RAPHAEL H. LEVEY
It is possible that the environment that is syngeneic with the injected cells provides the appropriate biochemical or structural milieu in which the injected cells can survive to proliferate at less of a selective disadvantage than in that of the allogeneic environment of the recipient animal. At the time of lymph node grafting, the recipient animals were depleted of circulating small lymphocytes by the prolonged course of antilymphocyte serum. Therefore, the lymph node graft is not rejected but repopulation of the graft by the host cells is precluded by this depletion. A Z-wk interval was allowed following the lymph node grafting during which the graft could become established, although the stromal elements could remain viable and acquire a new blood supply. The histologic appearance of these grafted lymph nodes is under study. It is suggested that repopulation of the node occurs by virtue of the large number of injected allogeneic or xenogeneic cells. An interesting and important sidelight of these experiments is that in no animals was graft-vs.-host disease noted. The recipient animals were clearly immunologically incompetent at the time of the injection of the cells; however, it would appear that they were protected by having high circulating levels of antilymphocyte serum that was able to clear those cells capable of initiating graft-V.-host reactions while allowing the putative stem cells to take up residence in the lymph nodes and spleen without giving rise to daughter cells that would subsequently cause secondary disease.
SUMMARY CBA mice were rendered tolerant by a prolonged course of antilymphocyte serum and the subsequent injection of allogeneic or xenogeneic lymphoid cells. Control mice received no other therapy. Experimental mice were grafted with allogeneic or xenogeneic lymph nodes prior to the administration of the cellular inocula. The survival pattern of tail-skin grafts was assessed in all mice. Those animals that were grafted with lymph node transplants failed to reject the skin grafts with the same rapidity or pattern as mice that were treated with ALS and cells alone. Furthermore, graft-V.-host disease was not noted in either the allogeneic or xenogeneic chimeras. The possible mechanisms of this durable chimerism are discussed. The experiments are still preliminary and others are in progress to evaluate the survival patterns of lymphoid cells in their heterotopic but syngeneic environment.
REFERENCES 1. Monaco, A. l’.: Use of antilymphocyte serm~i on the induction of immunological tolerance to tissue allografts. Fed. Proc. 29: 153, 1970. 2. Lance, E. M., and hledawar, P. B.: Quantitative studies on tissue transplantation immunity. IX. Induction of tolerance with antilymphocyte serum. Prnc. Roy. SW. Brit. 103:447, 1969.
3. Levey, It. H., and Medawar, P. B.: Nature and mode of action of antilymphocytic antiserum. Proc. Nat. Acad. Sci. U.S.A. 56: 1130, 1966. 4 Billingham, R. E., and Medawar, P. B.: The technique of free skin grafting in mammals. J. Exp. Biol. 28:385, 1951.