Mary L. Wood

STUDIES ON HETEROLOGOUS ANTI-LYMPHOCYTE SERUM IN MICE. III. IMMUNOLOGIC TOLERANCE AND CHIMERISM PRODUCED ACROSS THE H-2 LOCUS WITH ADULT THYMECTOMY AND ANTI-LYMPHOCYTE SERUM*

A number of studies in several species have clearly established that heterologous anti-lymphocyte serum is a remarkably potent immunosuppressive agent. Guinea pigs treated with anti-lymphocyte serum show reduced numbers of circulating lymphocytes and suppression of delayed sensitivity reactions.’” It has been noted, however, t h a t administration of sufficient quantities of anti-lymphocyte serum to guinea pigs to suppress delayed sensitivity reactions fails to modify the allograft rejection r e a ~ t i o n . ~ Rats given heterologous anti-lymphocyte serum show peripheral lymphopenia and lymphocyte depletion of lymphatic tissues, as well as an associated prolongation of allograft s ~ r v i v a l . ~ Woodruff and Anderson5 easily prolonged allografts in rats with anti-lymphocyte serum and showed that the effects of the serum and a thoracic duct fistula were additive. The properties of rabbit-anti-mouse lymphocyte serum (RAMLS) have been previously studied in detail in this l a b ~ r a t o r y . ~ * Rabbits appropriately immunized with purified suspensions of mouse lymph node cells produce antibodies to mouse lymphocytes demonstrable as lymphoagglutinins and cytotoxins. Such serum, when given to mice, causes profound and rapid lymphopenia and, if chronically administered, produces a remarkable lymphocyte depletion of the lymph nodes and other lymphatic tissues. I t was further shown tha t the duration of peripheral blood and tissue lymphopenia depended on the amount of serum administered, with all mice recovering normal blood and tissue lymphocyte levels after a given amount of time following cessation of a finite course of serum treatment. During the period of lymphopenia, the mice showed a generalized suppression of the immune response in tha t they were unable to make normal titers of humoral antibody to primary or secondary challenge with several antigens, or to reject skin allografts and xenografts. With restoration of normal lymphocyte levels following recovery from serum treatment, immunologic competence was completely restored in serum-treated mice.

Adult Thymectomy: Effect on Recovery from Immunologic Depression in Mice

A/Jax mice treated for I week with rabbit antiserum to A/Jax lymphocytes showed peripheral lymphopenia and tissue lymphocyte depletion which persisted for 4 weeks after initiation of serum treatment. During this time, such animals showed depression of the humoral antibody response and inability to reject skin allografts. After 4 weeks, mice recovered from effects of serum treatment. Normal blood and tissue lymphocyte levels returned, and immunologic competence was restored. Thymectomy of A/Jax mice (8 to 10 weeks old) prior to serum treatment resulted in peripheral and tissue lymphopenia and depression of antibody formation, which persisted twice as long as that shown by animals given serum alone. Similarly, skin allografts survive two to four times longer in serum-treated thymectomized mice, and, in some cases persisted over 100 days in perfect condition.

Antiserum to Lymphocytes: Prolonged Survival of Canine Renal Allografts

A horse immunized with dog lynmphocytes produced an antiserum which agglutinated canine lymphocytes in vitro and caused prolonged lymphopenia in dogs in vivo. Renal transplants in dogs treated with this antiserum survived for long periods, two of the grafts surviving beyond 350 days with normal function and histologic appearance.

SOME EFFECTS OF PURIFIED HETEROLOGOUS ANTIHUMAN LYMPHOCYTE SERUM IN MAN

A purified 7S-gamma globulin fraction of a rabbit anti-human lymphocyte serum (RAHLS) has been prepared. This material agglutinates and kills human lymph node cells and peripheral blood lymphocytes in vitro. Human subjects given 7S-RAHLS show transient lymphopenia associated with moderately prolonged abolition of positive delayed skin reactions and modest prolongation of skin allograft survival.

Leukemia Virus Activation during Homograft Rejection

Activation of murine leukemia viruses, as detected by the mixed culture cytopathogenicity (XC) assay, followed the transplantation of A/J skin onto immunosuppressed BALB/c mice. Virus was found in most of the mice receiving both skin grafts and antilymphocyte serum, but not in animals receiving either the serum alone, skin graft alone, or no treatment.

Suppressor cells in specific unresponsiveness to skin allografts in ALS-treated, marrow-injected mice.

Lymphoid cells from ALS-treated (C57BL/6 x A/J)F1 (B6AF1) mice bearing enhanced C3H/He grafts were assayed for their ability to suppress the response to C3H/He grafts after transfer to syngeneic B6AF1 recipients. Cells were transferred from ALS-treated B6AF1 mice that had received either a C3H/He graft alone, C3H/He marrow alone, or both a graft and marrow. Suppressor cells appeared in the spleens of ALS-treated B6AF1 mice that had received either a graft alone or both graft and marrow as early as day +13. They persisted only in the spleens of mice that had received both a graft and marrow, i.e., mice whose grafts showed significant prolongation. Suppressor cells did not appear in the lymph nodes of mice bearing enhanced grafts until day +42. Thymocytes and bone marrow cells were unable to transfer unresponsiveness. The cells which transferred unresponsiveness were specific for the graft donor strain as they did not transfer unresponsiveness to third-party grafts. The ability of cells to transfer suppression was abrogated by treatment with anti-theta serum.

The effect of injection of donor bone marrow on the frequency of donor-reactive CTL in antilymphocyte serum-treated, grafted mice.

The survival of C3H/He skin grafts can be prolonged on B6AF1 mice immunosuppressed with ALS by the injection of C3H/He marrow 1 week postgrafting. The precursor frequencies of donor-reactive CTL in the spleen and lymph nodes of ALS-treated, grafted mice given donor marrow were compared with CTL frequencies observed in ALS-treated, grafted controls. Spleens and nodes were removed from experimental and control mice on days +8, +14, +21, +58, and 1 year postgrafting, and used as effectors in the LDA. Donor-reactive CTL in the marrow-injected group remained suppressed as long as the recipients maintained their grafts. The frequency of CTL to third-party antigens was normal in mice bearing long-term C3H/He grafts. When marrow-injected mice rejected their grafts, the total donor-reactive CTL frequency returned to normal. In contrast, in ALS-treated controls that did not receive marrow, the total number of donor-reactive CTL returned to normal levels with recovery from the immunosuppressive effects of ALS. These results suggest that donor marrow suppresses the regeneration of donor-reactive CTL in the lymphoid tissues of ALS-treated mice, possibly by veto cell activity.

EFFECT OF BLOOD TRANSFUSION ON IL-2 PRODUCTION

Spleen cells from B10.A mice transfused with B10.D2 blood suppress the immune responses of normal B10. A to B10.D2 in coculture as early as 2 days posttransfusion. In addition, the ability of B10.A mice to respond in cell-mediated lymphocytotoxicity (CML) is significantly impaired as early as 2 days after B10.D2 transfusion. Experiments were performed to characterize the cells mediating the suppressive effect and to determine whether the inability of transfused mice to generate a cytotoxic response is due to an inhibition of IL-2 production. To characterize the suppressor cells, spleen cells from B10.A mice were assayed 2 or 16 days after B10.D2 transfusion for the ability to suppress mixed lymphocyte culture (MLC) and CML responses of normal B10.A mice in coculture. The putative suppressor cells were either passed over a Sephadex G-10 or nylon wool column, treated with anti-Thy antibody or left untreated before addition to the coculture. Untreated cells from transfused mice suppressed the CML response of normal B10.A both 2 and 16 days posttransfusion, while the effect on the MLC response was inconsistent. Passage of the cells over Sephadex G-10 or nylon wool before assaying abrogated the suppressive effect, while treatment with anti-Thy antibody had no effect. These results suggest that the suppressor cells appearing shortly after blood transfusion have the characteristics of macrophages and not T lymphocytes. To determine the effect of transfusion on IL-2 production, cells from transfused mice were assayed for their ability to produce IL-1 and IL-2 and for the formation of IL-2 receptors. In addition, the effect of exogenous IL-1 and IL-2 on restoring the CML response of transfused mice to normal was assayed. The production of IL-1 by transfused mice was normal, while the production of IL-2 was significantly suppressed both 2 and 16 days posttransfusion. Activated cells from normal and transfused mice showed equal ability to absorb IL-2, indicating that IL-2 receptor formation is normal after transfusion. The addition of exogenous IL-2, but not IL-1, to CML cultures containing cells from transfused mice as responders restored the response to normal. These results indicate that the inability of transfused mice to respond in CML is due, at least in part, to an inability to produce IL-2. This could be mediated by prostaglandins released by activated macrophages.

Superiority of sirolimus (rapamycin) over cyclosporine in augmenting allograft and xenograft survival in mice treated with antilymphocyte serum and donor-specific bone marrow.

BACKGROUND Sirolimus is a potent immunosuppressive agent with great therapeutic potential. The objective of our study was to evaluate the efficacy of sirolimus versus cyclosporine in augmenting the unresponsiveness induced by an antilymphocyte serum (ALS)/donor-specific bone marrow (BM)-based regimen across three levels of histoincompatibility: class I and II disparate (DBA/2 to B6AF1), complete mismatch (AKR to C57BL/6), and xenograft (ACI rat to B6AF1). METHODS Full-thickness skin grafts were taken from donors and placed on recipients in standard fashion. Seven groups of recipient mice (n=10-28) received various combinations of the following treatment protocols: sirolimus, 1.5 mg/kg (3.0 mg/kg for xenografts) every other day from day 0 to day 12; cyclosporine, 50 mg/kg every other day from day 10 through 22; ALS, 0.5 ml on days -1 and 2 for allografts and days -1, 2, and 4 for xenografts; and BM, 25 million donor-specific cells IV on day 7. RESULTS The administration of ALS or ALS/BM resulted in modest but significant prolongation of skin graft survival in all combinations tested. Cyclosporine combined with ALS or ALS/BM significantly extended allograft survival compared with ALS or ALS/BM alone (P<0.05) but had no effect on xenograft survival. In contrast, the combination of sirolimus with ALS or ALS/BM resulted in a two- to threefold increase in allograft survival and over a fourfold increase in xenograft survival when compared with the comparable cyclosporine-based regimen. Additionally, lymphocytes isolated from class I and II incompatible mice with skin grafts surviving >100 days demonstrated markedly reduced interleukin 2 and interferon-gamma secretion in response to irradiated donor-specific lymphocytes in culture. CONCLUSIONS In the regimens tested, sirolimus was superior to cyclosporine in augmenting donor BM-induced skin graft prolongation in ALS-treated mice across all levels of histoincompatibility.

Suppressor Cells In Mice Bearing Intact Skin Allografts After Blood Transfusions

Evidence for the presence of suppressor cells in B6AF1 mice bearing long-term C3H skin grafts after multiple blood transfusions and antilymphocyte serum (ALS) treatment is described. Transfer of spleen cells from these mice into secondary B6AF1 recipients given ALS and C3H skin grafts resulted in marked prolongation of graft survival. The spleen cells were also capable of specifically inhibiting the proliferative response of normal B6AF1 responders to C3H stimulator cells in coculture mixed lymphocyte culture (MLC) experiments.