Long-lasting skin allograft tolerance in adult mice induced across fully allogeneic (multimajor H-2 plus multiminor histocompatibility) antigen barriers by a tolerance-inducing method using cyclophosphamide.

A new method of cyclophosphamide (CP)-induced skin allograft tolerance in mice that can regularly overcome fully allogeneic (major H-2 plus non-H-2) antigen barriers in mice has been established. The components of the method are intravenous or intraperitoneal administration of 50-100 micrograms of anti-Thy-1.2 mAb on day -1, intravenous injection of 90 x 10(6) allogeneic spleen cells mixed with 30 x 10(6) allogeneic bone marrow cells from the same donor on day 0, and intraperitoneal injection of 200 mg/kg CP on day 2. In each of four fully allogeneic donor----recipient combinations, including C3H/HeJ (C3H; H-2k)----C57BL/6J(B6; H-2b), B6----C3H, BALB/cByJ (BALB; H-2d)----B6, and BALB----C3H, long-lasting survival of skin allografts was induced in most of the recipient mice. The specific tolerant state induced was dependent on the doses of the antibody and bone marrow cells used. The optimal timing of CP treatment to induce tolerance was found to be 1-3 d after the stimulating cell injection. Treatment with the anti-Thy-1.2 antibody together with CP on day 2 after the cell injection on day 0 also induced profound tolerance. In the B6 mice made tolerant of C3H with antibody, C3H spleen cells plus C3H bone marrow cells, and then CP, a minimal degree of stable mixed chimerism was established and the antitolerogen (C3H) immune responses examined here, including delayed footpad reaction (DFR), CTL activity, and capacity for antibody production against donor-strain antigens were abrogated in a tolerogen-specific manner. From cell transfer experiments, the mechanism of tolerance could be largely attributed to reduction of effector T cells reactive against the tolerogen, and strong suppressive influences that might prolong skin allograft survival directly were not detected in the tolerant mice. Moreover, pretreatment with anti-Thy-1.2 antibody or anti-L3T4 (CD4) antibody was more effective than pretreatment with anti-Lyt-1 (CD5) antibody or anti-Lyt-2 (CD8) antibody as an initial step in tolerance induction. These results suggest that permanent tolerance to fully allogeneic skin grafts may be induced because antibody given before the stimulating cell injection reduces the number of reactive T cells in the recipient mice. This antibody treatment may facilitate an antigen-stimulated destruction of responding and thus proliferating cells with CP by preventing a possibly less proliferative, more rapid maturation of reactive T cells or by destroying residual effector T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Intrathymic clonal deletion of V beta 6+ T cells in cyclophosphamide-induced tolerance to H-2-compatible, Mls-disparate antigens.

When C3H (H-2k, Mls-1b) mice were primed intravenously with 10(8) viable spleen cells from AKR (H-2k, Mls-1a) and treated intraperitoneally with 200 mg/kg of cyclophosphamide (CP) 2 d later, not only a long-lasting skin allograft tolerance but also a tolerance in mixed lymphocyte reaction to Mls-1a-encoded antigens was established. The cellular mechanisms of CP-induced tolerance were examined by assessing the V beta 6-bearing T cells that are strongly correlated with reactivity to Mls-1a-encoded antigens bound to MHC class II molecules. At the relatively early stage (2 or 5 wk) after the CP treatment, CD4+-V beta 6+ T cells of C3H origin were preferentially eliminated in the lymph nodes of the tolerant mice, whereas CD8+-V beta 6+ T cells remained. On the other hand, neither CD4+CD8- nor CD4-CD8+ thymocytes bearing a high density of V beta 6 was detected in the chimeric thymus. Namely, in the thymus of the tolerant C3H mice, neither mixed chimerism nor the clonal deletion of the V beta 6-bearing T cells was observed on day 14, whereas both of them were observed on day 35. The clonal deletion and mixed chimerism in the thymus were lasting for greater than 10 wk after the CP treatment. Expression of V beta 6 on the peripheral T cells in the tolerant C3H mice gradually reduced in the process of time. These results strongly suggested that the clonal deletion in the thymus was one of the essential mechanisms in the CP-induced tolerance system.

Drug-induced tolerance to allografts in mice. IV. Mechanisms and kinetics of cyclophosphamide-induced tolerance.

Mechanisms and kinetics of tolerance in AKR mice induced using i.v. priming with viable C57BL/6 spleen cells and treatment with cyclophosphamide 2 days later were analyzed. In this tolerance induction system, some lymphocyte populations mediating delayed foot-pad reaction and cytotoxic activity were resistant to tolerance induction and remained in a sensitized state after cyclophosphamide treatment. These populations were considered to be qualitatively distinct from populations sensitive to cyclophosphamide, because delayed foot-pad reaction and cytotoxic lymphocyte activity were stronger in tolerant mice than in control mice in the early stages of tolerance induction, but were not augmented after immunization with C57BL/6 spleen cells, C57BL/6 skin grafts, or EL4 tumor grafts in the absence of suppressor T cells. One of the important differences in these two lymphocyte populations may be the capacity for clonal expansion.

Drug-induced tolerance to allografts in mice. VI. Tolerance induction in H-2-haplotype-identical strain combinations in mice.

When C3H/HeN (C3H) mice were primed with viable AKR/J (AKR) spleen cells and treated with cyclophosphamide (CP) two days later, a profound tolerance to AKR skin grafts was induced. This tolerance was induced also in other combinations disparate only at minor histocompatibility (H) antigens (AKR-C3H and BALB/c[BALB]-DBA/2[DBA]). In C3H mice made tolerant to AKR, delayed foot-pad reaction (DFR), cytotoxic lymphocytes (CTL), and cytotoxic antibodies (CTAb) against AKR spleen cells were abrogated completely. Tolerance to AKR mice was also observed in C3H mice primed with viable AKR and C57BL/6 (B6) spleen cells and treated with CP, but tolerance to B6 was not induced because a cell population responsible for DFR and CTL against B6 H-2 antigens remained after tolerance induction. These results suggest that there is a lymphocyte population responsible for DFR and CTL against antigens allogeneic at both major and minor H that is less proliferative than the population responsible for DFR and CTL against minor H antigens.

The nature of tolerance in adult recipient mice made tolerant of alloantigens with supralethal irradiation followed by syngeneic bone marrow cell transplantation plus injection of F1 spleen cells.

The length of time after syngeneic bone marrow reconstitution when tolerance to alloantigens can be induced in adult mice during T cell differentiation from bone marrow cells was studied by exposing those T cells to (recipient x donor)F1 spleen cells. Supralethally irradiated C3H/He Slc(C3H; H-2k) mice were reconstituted with 1 x 10(7) syngeneic T cell-depleted bone marrow cells and then injected intravenously with 5 x 10(7) (C3H x C57BL/6[B6])F1 (B6C3F1; H-2bxk) or (C3H x AKR/J[AKR])F1 (AKC3F1; H-2kxk) spleen cells at various intervals. In the fully allogeneic combination of B6C3F1----C3H, EL-4 tumor originating from B6 was accepted, and survival of grafted B6 skin was significantly prolonged in the tolerant C3H mice treated with irradiation on day -1 followed by injection of syngeneic bone marrow cells on day 0 plus B6C3F1 spleen cells on days 0, 5, or 10, in a tolerogen-specific manner. In the multiminor histocompatibility antigen-disparate combination of AKC3F1----C3H, AKR skin grafts were permanently accepted in the tolerant C3H mice treated with AKC3F1 spleen cells on days 0, 5, 10, or 15. Immunological parameters, including cytotoxic T lymphocyte activity and delayed foot-pad reaction (DFR), were almost completely suppressed in C3H mice made tolerant of B6 or AKR antigens. A chimeric assay using a direct immunofluorescence method revealed that the tolerant C3H mice given B6C3F1 spleen cells on day 0 were mixed-chimeric for at least 8 weeks after syngeneic bone marrow reconstitution, but not definitely chimeric thereafter. The C3H mice given AKC3F1 spleen cells on day 0 were chimeric even 43 weeks after syngeneic bone marrow reconstitution, but the C3H mice given AKC3F1 spleen cells on day 15 showed temporal chimerism that disappeared within 43 weeks. The untolerant mice were never detectably chimeric. These data suggest that the earlier the timing of the injection of F1 spleen cells after syngeneic bone marrow reconstitution was, the more profound tolerance was induced. Moreover, the stronger the antigenic disparity between donor and recipient, the earlier the injection of F1 spleen cells was required to induce tolerance.

Drug-induced tolerance to allografts in mice. I. Difference between tumor and skin grafts.

When AKR mice were primed with viable C57BL/6(B6) spleen cells and treated with cyclophosphamide 1-3 days later, a profound tolerance to B6 tumor allografts was induced. The tolerant state was maintained completely for as long as 8 weeks. Although tumor allografts grew progressively even when inoculated after complete rejection of skin grafts, B6 skin grafts were rejected by tolerant mice. In mice tolerant of B6 tumors, production of cytotoxic antibody and cytotoxic activity was reduced profoundly, but the delayed-type hypersensitivity level decreased only slightly. We therefore presume that the decrease in cytotoxic activity may allow progressive growth of tumor allografts, but the maintenance of delayed-type hypersensitivity or a low level of cytotoxicity, or both, precludes acceptance of skin allografts.

Drug-induced in vitro tolerance to allogeneic antigens. II. Further analysis of in vitro-tolerized spleen cells in a fully allogeneic murine combination.

C3H/HeSlc (C3H, H-2k) spleen cells were made tolerant in vitro to C57BL/6CrSlc (B6, H-2b) at the cell-mediated cytotoxicity (CMC) level by in vitro stimulation for 48 hr with mitomycin C (MMC)-treated B6 spleen cells, and treatment with 5 micrograms/ml of 5-fluorouracil for a further 9 hr. These cells were given intraperitoneally to neonate (C3HxB6) F1 mice to examine whether these tolerized spleen cells would cause lethal graft-versus-host disease (GVHD). Despite the lack of CMC, the tolerized C3H spleen cells caused lethal GVHD in most of the neonate F1 mice. Evaluating from various immune parameters, it was evident that T cell populations responsible for IL-2 production, cytostasis, and delayed footpad reaction (DFR) were retained intact after in vitro tolerance induction, probably because of their less-proliferative characteristics in response to fully allogeneic antigen stimulation, and were considered to be responsible for lethal GVHD. Contribution of natural killer (NK) cells to lethal GVHD was not ruled out.

Drug-induced in vitro tolerance to allogeneic antigens. I. Establishment of a tolerance induction system in a fully allogeneic murine combination.

C3H/HeSlc (H-2k) spleen cells were cultured with mitomycin C (MMC)-treated C57BL/6CrSlc (H-2b) spleen cells for 2 days and incubated with 5-fluorouracil (5-FU) for a further 9 hr. Thereafter, those C3H/He spleen cells were recultured with the same allogeneic cells for 5 days. Cell-mediated cytotoxicity (CMC) and mixed lymphocyte reaction (MLR) were profoundly suppressed, antigen-specifically, in such C3H/He spleen cells. In contrast, interleukin 2(IL-2) production was not impaired in the restimulating mixed lymphocyte culture (MLC) with C57BL/6. Moreover, an adequate amount of exogenous IL-2 added to the restimulating MLC did not lead to a restoration of the depressed CMC. Suppressor cell activity in the CMC assay was not detected in the C3H/He spleen cells exposed to such a tolerance induction. These results suggest that the unresponsiveness to alloantigens in CMC and MLR was induced through a clonal deletion mechanism, and there may exist a 5-FU-resistant--thus less-proliferative--cell population that can produce IL-2 even after the tolerance induction.

Allograft rejection and immune responses against allogeneic antigens in neonatally thymectomized mice.

Skin allograft survival and immune responses against allogeneic antigens homologous to skin grafts were observed in BALB/c Cr Slc (BALB) mice (H-2d) thymectomized at 1 day after birth and grafted with skin from major histocompatibility complex (MHC)-incompatible, fully allogeneic C3H/HeN (C3H) (H-2k) or MHC-compatible allogeneic DBA/2 Cr Slc (DBA) mice (H-2d), at 14 weeks of age. In neonatally thymectomized (NTx) BALB mice, survival of C3H skin grafts was not prolonged at all, but survival of DBA skin grafts was prolonged significantly, although the survival periods of DBA skin grafts were very different among individual recipients. In NTx recipients grafted with C3H skin, delayed foot-pad reaction (DFR) was not reduced, but cytotoxic lymphocyte (CTL) activity and cytotoxic antibody (CTAb) production were appreciably depressed. CTL and CTAb were reduced profoundly and consistently in all NTx mice grafted with DBA skin, while DFR was reduced to various degrees in each. The degrees of depression of DFR in these NTx mice correlated well with the prolongation of DBA skin survival, although the sample number was small. The rejection of skin allografts appears to be attributable largely to a T cell subset, the function of which can be expressed as DFR. Thymus dependency in the ontogenic development is low as compared with other T cell subsets.

Drug-induced tolerance to allografts in mice. IX. Establishment of complete chimerism by allogeneic spleen cell transplantation from donors made tolerant to H-2-identical recipients.

Graft-versus-host reaction (GVH) after allogeneic spleen cell transplantation was completely suppressed in an H-2-matched murine combination (AKR/J Sea [H-2k]----lethally irradiated C3H/He Slc [H-2k]) by pretreatment of the donors with recipient spleen cell antigen plus cyclophosphamide (CP). Irradiated recipients receiving cells became chimeric. In contrast to the H-2 matched combination, lethal GVH reaction could not be prevented in an H-2-mismatched fully allogeneic combination (C57BL/6 Cr Slc [H-2b]----lethally irradiated C3H/He Slc [H-2k]) by pretreatment of the donors. The results suggest that the effectors responsible for the GVH reaction were abrogated by pretreatment of the donors with allogeneic recipient spleen cells plus CP in the H-2-matched combination, but donor pretreatment failed to abrogate GVH reaction in the H-2-mismatched combination.

Drug-induced tolerance to allografts in mice. XII. The relationships between tolerance, chimerism, and graft-versus-host disease.

When AKR/J Sea (AKR, H-2k) mice were primed i.v. with 1 X 10(8) viable spleen cells from naive C3H/He Slc (C3H, H-2k) mice and treated i.p. with 200 mg/kg cyclophosphamide (CP) 2 days later, a minimal degree of mixed chimerism associated with tolerance to C3H skin was established without graft-versus-host disease (GVHD) and maintained for at least one month. When AKR mice were primed i.v. with 1 X 10(8) viable spleen cells from C3H mice preimmunized i.v. 7 days earlier with 5 X 10(7) viable AKR spleen cells, and treated with 200 mg/kg CP, chimerism became exclusive, but lethal GVHD occurred in the AKR mice. Moreover, most of normal AKR mice primed with the preimmunized C3H spleen cells without CP died of GVHD. In contrast, in a major histocompatibility complex (MHC)-incompatible combination of AKR (H-2k)-C57BL/6 Cr Slc (B6, H-2b), mixed chimerism, tolerance to skin allografts, and GVHD were not observed, whether or not the mice had been treated with naive or preimmunized B6 spleen cells with or without CP.

Fractionated dosing of cyclophosphamide for establishing long-lasting skin allograft survival, stable mixed chimerism, and intrathymic clonal deletion in mice primed with allogeneic spleen cells.

BACKGROUND: Injection of allo-spleen cells (SC) followed by a single dose of cyclophosphamide (CP) can induce tolerance of tumor and/or skin allografts in mice. To minimize the damage caused by CP, fractionation of CP that can establish long-lasting skin graft survival, stable mixed chimerism, and intrathymic clonal deletion in the host was investigated in the present study. METHODS: Allo-SC (10(8)) were given intravenously on day 0. CP at 200 mg/kg was given intraperitoneally on day 2 in a single dose (CP 200x1 group). CP at 100, 66, 50, 40, and 33 mg/kg was given daily from day 1 through days 2, 3, 4, 5 and 6, respectively, in the fractionated doses (CP 100x2, 66x3, 50x4, 40x5, and 33x6 groups; total dose=200 mg/kg). Allografting was performed on day 14. RESULTS: In a fully allogeneic combination of C57BL/6 (H2b)-->AKR (H2k, Mls-1a), an EL-4 tumor (H2b) was specifically accepted to kill the AKR mice in all of the SC+CP 200x1, 100x2, 66x3, 50x4, 40x5, and 33x6 groups (n=6), but C57BL/6 skin graft survival was not prolonged in any of the tumor-tolerant groups. In an H2-identical combination of AKR-->C3H (H2k, Mls-1b), AKR skin graft survival was prolonged remarkably (80-90 days) in the SC+CP 200x1, 100x2, and 66x3 groups (n=5-11), but was prolonged moderately (20-60 days) in the SC+CP 50x4 and 40x5 groups. In both of the SC+CP 200x1 and 66x3 groups in the AKR-->C3H combination, mixed chimerism was maintained for as long as 100 days after tolerance induction in both the spleen and thymus, associated with intrathymic clonal deletion of Vbeta6+ T cells. The decreases in leukocyte count, hemoglobin level, spleen weight, SC count, and body weight were significantly smaller in the SC+CP 66x3 group than in the SC+CP 200x1 group. CONCLUSIONS: Fractionated CP is effective in ameliorating the compromised state induced by a single dose of CP. To induce a long-lasting skin allograft survival associated with stable mixed chimerism and intrathymic clonal deletion in an H2-identical combination, 200 mg/kg of CP can be divided into three or fewer fractions.

Donor-specific prolongation of rat skin graft survival induced by rat-donor cells and cyclophosphamide under coadministration of monoclonal antibodies against T cell receptor alpha beta and natural killer cells in mice.

Because of the recent interest in human xenotransplantation, we investigated the possibility of inducing tolerance in a xenogeneic combination using cyclophosphamide (CP). Donor-specific prolongation of xenogeneic Fisher 344 (F344) rat skin graft survival for up to 60 days was induced in C57BL/6 (B6) mice by giving F344 bone marrow cells and spleen cells on day 0, CP on day 2, and monoclonal antibodies against murine TCR-alpha beta and NK1.1 on days--1 and 3. The inoculation of the xenogeneic cells brought accelerated repopulation of TCR-alpha beta+ T cells, even under the administration of anti-TCR-alpha beta mAb. The quick increase of the host TCR-alpha beta+ T cells caused by the xenogeneic cell injection was deeply suppressed by CP. Mixed lymphocyte reaction, CTL activity, and antibody production against donor F344 were profoundly suppressed for 50 days. Mixed xenogeneic chimerism was observed for 1 month after the inoculation of donor cells in the spleen and peripheral blood of the recipient B6 mice, but was never observed in the thymus. Moreover, when irradiated F344 cells were used in place of viable cells, chimerism was never detected and graft survival was only slightly prolonged. Clonal deletion of V beta 5- or V beta 11-bearing murine T cells was not observed on day 50 in the thymus or spleen of the recipient B6 mice. These results suggest that treatment with viable xenogeneic donor cells, CP, and mAbs against T and NK cells can induce a temporary peripheral mixed chimerism and donor-specific prolongation of xenogeneic skin graft survival. The destruction with CP of T and B cells that are xenoreactive and thus proliferating after antigen stimulation, followed by mechanism other than intrathymic clonal deletion, may be the mechanism of the hyporesponsiveness in the present system.

Induction of tolerance across major barriers using a two-step method with genetic analysis of tolerance induction.

Using a murine skin allograft tolerance induction system that consists of intravenous injection of 1 x 10(8) allogeneic spleen cells followed by intraperitoneal (i.p.) injection of 200 mg/kg cyclophosphamide (CP) 2 days later, sensitivity to tolerance induction was examined across various histocompatibility (H) barriers. Although each group of class I, class II or multiminor H antigens was not by itself a prohibitively strong barrier, resistance to tolerance induction increased when the three types of barriers were combined in various ways. When the donor-recipient combinations were disparate at the entire spectrum of both H-2 plus non H-2 antigens (fully allogeneic), profound tolerance to skin allografts was not induced by this method in any of the combinations examined. Based on these results, induction of tolerance across fully allogeneic barriers was attempted in C57BL/10SnJ (B10; H-2b) mice against C3H/HeSnJ (C3H; H-2k) strain by addressing the 11 barriers as two separate challenges. B10 mice were first given B10.BR/SgSnJ (B10.BR; H-2k) spleen cells plus CP to make them tolerant to the H-2k component represented among C3H antigens, and then later were given C3H spleen cells plus CP to establish a tolerant state to the remainder of the disparate antigens of the C3H donors. After these two separate manipulations, C3H skin was accepted in the B10 mice, and normal hair growth was observed in the grafted C3H skin. By contrast, B10 mice given C3H spleen cells plus CP and then again another injection of C3H spleen cells plus CP were not rendered tolerant to C3H skin. In B10 mice, tolerance to C3H induced with B10.BR spleen cells plus CP and then C3H spleen cells plus CP was specific to C3H, and the tolerant B10 mice rejected third-party skin from DBA/2J (DBA; H-2d) strain in a normal fashion. In transfer experiments, the mechanism of tolerance was found to be based largely on reduction of the effector cells rather than on a mechanism involving active suppression. Assays for chimerism revealed that maintaining the tolerant state required persistence of cells of donor origin. These data indicate that in a primary immune response to a certain dose of allogeneic cells (tolerogen), the existence of a relatively large proportion of potentially reactive clones in the host may trigger proliferation of only a part of the population and some of the potentially reactive cells may differentiate rapidly without a prolonged period of proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)

The necessity of both allogeneic antigens and stem cells for cyclophosphamide-induced skin allograft tolerance in mice.

We have reported that in an H-2 identical murine combination of AKR/J (AKR, H-2k, Thy1.1) and C3H/HeJ (C3H, H-2k, Thy 1.2), specific tolerance to C3H skin in AKR mice is induced only when both intravenously (i.v.) 1 x 10(8) viable C3H spleen cells and, two days later, intraperitoneally (i.p.) 200 mg/kg cyclophosphamide (CP) have been given. To further examine this mechanism of tolerance, we used 2000R-irradiated C3H spleen cells as an antigen source and bone marrow cells depleted of Thy1.2+ cells and Ia+ cells as a stem cell source. When a mixture of 1 x 10(8) irradiated spleen cells and 3 x 10(7) bone marrow cells was used as tolerogen and 200 mg/kg CP was administered two days later, a profound and specific long-lasting tolerance was induced. This tolerant state, however, was less profound than that induced with spleen cells plus CP. When the number of irradiated spleen cells was fixed at 1 x 10(8), the tolerant state was dose-dependent on the quantity of bone marrow cells. On the other hand, when the number of bone marrow cells was fixed at 1 x 10(6), tolerance induction depended on the dosage of irradiated spleen cells. Tolerance induced with irradiated spleen cells plus bone marrow cells and CP was tolerogen specific. Tolerance was never induced when the bone marrow cells had been irradiated with 2000R prior to injection. Transfer experiments showed that the tolerant state, in its acute phase, appeared to be predominantly based on reduction of functionally reactive cells. The prolongation of skin allograft survival in tolerant mice could not be attributed directly to suppressor cells, nor was any evidence of suppressive factor induction observed. In the chronic phase, however, the importance of the suppressive mechanisms appeared to be relatively increased. EPICS analysis of the thymocytes using fluorescein-conjugated anti-Thy1.1 and anti-Thy1.2 antibodies showed that a minimal degree of mixed chimerism had been established in the tolerant mice. Moreover, both T cells and Ia+ cells had beneficial effects on the induction of tolerance. We conclude that in the tolerance induced by spleen cells plus CP, histocompatibility antigens expressed on the surface of the spleen cells were essential to the antigen-stimulated cell destruction mechanism. Stem cells contained in the spleen cells also appeared to be crucial for maintaining tolerance by establishing a minimal degree of mixed chimerism.

Cyclophosphamide-induced immunological tolerance: an overview.

A cyclophosphamide (CP)-induced tolerance system in mice that primarily consists of donor cell injection followed by CP-treatment was found useful for inducing a long-lasting allo- or xeno-tolerance to various solid organs. In the cells-followed-by-CP system, the sequential mechanisms of tolerance were clarified using the specific correlation between superantigens and certain T cell receptor (TCR) V beta segments. Those include the clonal destruction of antigen-stimulated mature T cells, the peripheral clonal deletion associated with peripheral chimerism, the intrathymic clonal deletion associated with intrathymic chimerism, and the clonal anergy. The generation of suppressor T cells was another important mechanism of tolerance in the late stage. Special care was taken to overcome the " hard" barriers of allo- or xeno-combinations by reducing the "split tolerance" produced through the clonal destruction mechanism. For this purpose, the tolerogen, antimitotic drugs, their doses, timing, route of administration, combined immunosuppressants, and supportive treatment were all crucial for successful induction of a long-lasting skin tolerance. This system may be applicable to human transplantation.

Drug-induced tolerance to allografts in mice II. Tolerance to tumor allografts of large doses associated with rejection of skin allografts and tumor allografts of small doses.

Antigen-specific tolerance to tumor allografts in mice was obtained by stimulation with allogeneic spleen cell antigens and treatment with cyclophosphamide (CY) 2 days later in various strain combinations between donor mice of spleen cell antigens and recipients. In such tolerant mice, skin allografts were rejected in various combinations of the donor-recipient. Furthermore, some of the tolerant mice showed accelerated rejection of tumor allografts of small doses, even though they allowed progressive growth of the same tumor allografts of large doses. Our results suggest that the rejection of tumor allografts of large doses may depend upon a mechanism that is distinct, qualitatively or quantitatively, from that one responsible for the rejection of tumor allografts of small doses and skin allografts. One of the most important properties of lymphocytes to separate these two different mechanisms may be a capacity of clonal expansion, namely, sensitivity to CY-induced tolerance of the lymphocytes mediating allograft rejection.

Drug-induced tolerance to allografts in mice. X. Augmentation of split tolerance in murine combinations disparate at both H-2 and non-H-2 antigens by the use of spleen cells from donors preimmunized with recipient antigens.

In a fully allogeneic murine combination of C3H/HeSlc (C3H) (H-2k) and C57BL/6CrSlc (B6) (H-2b), C3H mice were primed i.v. with 1 X 10(8) spleen cells from B6 mice preimmunized i.v. with 5 X 10(7) C3H spleen cells and then were given i.p. 200 mg/kg cyclophosphamide (CP) 2 days later (Im-B6-Sc plus CP group). The tolerant state in those recipient mice was compared with that in mice made tolerant conventionally with 1 X 10(8) naive B6 spleen cells plus 200 mg/kg CP (naive-B6-Sc plus CP group). B6 skin was rejected in an almost normal fashion in both the naive-B6-Sc plus CP group and the Im-B6-Sc plus CP group. However, EL4 tumor allografts (B6 origin) inoculated after complete rejection of B6 skin grafts were specifically accepted in both groups. Moreover, the tumor growth in the Im-B6-Sc plus CP group was faster than that in the naive-B6-Sc plus CP group. Mixed lymphocyte reaction, cytotoxic T lymphocyte activity, and antibody production against the tolerogen were depressed more profoundly in the Im-B6-Sc plus CP group than in the naive-B6-Sc plus CP group. These observations were consistent with the results from tumor allografting. The other immunological parameters examined in the present study, including helper T cell activity and delayed foot-pad reaction, were retained in the Im-B6-Sc plus CP group at the same levels as in the naive-B6-Sc plus CP group. These observations were consistent with the results from skin allografting. In conclusion, tumor allograft tolerance was made more profound by the use of spleen cells from donors preimmunized with recipient antigens as the tolerogen than by the use of naive spleen cells. However, skin allograft tolerance was not achieved at all by these same treatments. The contribution of graft-versus-host disease to this phenomenon was excluded by the chimeric analysis in AKR/JSea (H-2k) mice given the preimmunized (with AKR antigens) B6 spleen cells plus CP. These results strongly support the existence of a less proliferative lymphocyte population which does not evoke cell divisions to mature even after the strong stimulation with the preimmunized spleen cells and is resistant to tolerance induction.

Establishment of a novel method to induce tolerance in adult mice across fully allogeneic (entire H-2 plus multiminor histocompatibility) antigen barriers, using supralethal irradiation followed by injection of syngeneic bone marrow cells plus (donor X recipient) F1 spleen cells.

A novel method was established which can regularly induce profound tolerance in mice across entire H-2 plus multiminor histocompatibility (H) antigen (fully allogeneic) barriers. When recipient AKR/J Sea (AKR; H-2k) or C3H/He Slc (C3H; H-2k) mice were irradiated with 900 rad followed 1 day later by injection of 1 X 10(7) T cell-depleted syngeneic bone marrow cells plus 5 X 10(7) viable, but not mitomycin C-treated, [C57BL/6 Slc(B6) X AKR (or C3H)] F1 spleen cells via intravenous (i.v.) route, a specific tolerant state was induced against B6 (H-2b) antigens. In the tolerant C3H mice, the EL-4 tumor, which originates from B6, was accepted in a tolerogen-specific manner. Moreover, B6 skin grafts were permanently accepted in most of the tolerized AKR and C3H mice. Immunological parameters, including cytotoxic T lymphocyte (CTL) activity and the mixed lymphocyte reaction (MLR), were almost completely suppressed in the tolerant mice. An assay for chimerism using a direct immunofluorescence method revealed that the tolerant AKR mice were chimeric for the first 5 weeks after tolerance induction but not definitely chimeric thereafter. In the tolerant AKR mice, strong suppressor cells were not detected. This method could be used in order to investigate mechanisms of tolerance to allogeneic antigens in future experiments.

The effect of T cell depletion from spleen cell allografts on graft-versus-host disease and long-term immune reconstitution in H-2 haplotype-identical murine combinations.

Graft-versus-host disease (GVHD) and states of immune reconstitution in allogeneic chimera mice across minor histocompatibility antigens were analyzed in excess of 9 months after injecting AKR/JSea (AKR) spleen cells into irradiated C3H/HeSlc (C3H) mice. When T cell-depleted AKR spleen cells were used as inoculum cells, neither graft failure nor GVHD was seen for 9 months postgrafting in the C3H mice irradiated with 660 rad or more. In an AKR - C3H (850 rad) model, Thy1.1+ or L3T4+ T cell depletion from donor AKR spleen cells abolished both acute and chronic GVHD in lethally irradiated C3H mice. Lyt2+ T cell depletion, however, resulted in acute and chronic GVHD in more than half of the recipient C3H mice. Moreover, actual existence of donor (AKR)-type T cells with L3T4 phenotype, but not Lyt2 phenotype, was always observed in the spleen of the C3H mice suffering from acute GVHD. In addition, the C3H mice that were irradiated with 850 rad, grafted with AKR spleen cells depleted of Lyt2.1+ T cells, escaped from acute GVHD and survived for more than 10 mo postgrafting, showed impaired activities of immune responses such as delayed footpad reaction to sheep red blood cells, antibody production tested by IgM plaque forming cells and reactivity to an intracellular bacterium. Listeria monocytogenes as compared with the C3H mice reconstituted with syngeneic C3H spleen cells or Thy1.1+ or L3T4+ T cell-depleted AKR spleen cells. These results suggest that L3T4+ T cells, rather than Lyt2+ T cells, contained in the grafted cells not only cause acute GVHD but also a long-term immunodeficient state (chronic GVHD) in recipient mice in the H-2-identical murine combinations examined here.