Denise A. Pearson

Donor-derived interferon gamma is required for inhibition of acute graft-versus-host disease by interleukin 12.

We have demonstrated that a single injection of interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits acute graft-versus-host disease (GVHD) in mice. This effect of IL-12 can be diminished by anti-interferon (IFN)-gamma mAb. To determine the mechanism by which IFN-gamma affects IL-12-mediated GVHD protection, we have compared the effect of IL-12 on GVHD in C57BL/6 wild-type (WT) or IFN-gamma gene knockout (GKO) recipients of fully major histocompatibility complex plus minor antigen-mismatched allogeneic BMT from WT or GKO BALB/c mice. Lethal acute GVHD was readily induced in the absence of IFN-gamma. IL-12 inhibited GVHD mortality to a similar extent in WT and GKO recipients of WT allogeneic BMT. However, neither WT nor GKO recipients were protected by IL-12 from GVHD induced by GKO allogeneic BMT. Moreover, the effective inhibition of host-reactive donor T cell activation and expansion that is associated with IL-12-mediated GVHD protection was dependent on the ability of BALB/c donors to produce IFN-gamma. These results demonstrate that (a) acute GVHD can be induced in the absence of IFN-gamma, (b) host IFN-gamma does not play a critical role in IL-12-induced GVHD protection, and (c) the protective effect of IL-12 against GVHD is dependent on the ability of the donor to produce IFN-gamma.

Intrathymic deletion of alloreactive T cells in mixed bone marrow chimeras prepared with a nonmyeloablative conditioning regimen

BACKGROUND Mixed hematopoietic chimerism induced with a nonmyeloablative conditioning regimen leads to donor-specific transplantation tolerance. Analyses of specific Vbeta-bearing T-cell families that recognize endogenous superantigens demonstrated that donor-specific tolerance is due mainly to an intrathymic deletional mechanism in these mixed chimeras. However, superantigens are not known to behave as classical transplantation antigens. We therefore used T-cell receptor (TCR) transgenic (Tg) recipients expressing a clonotypic TCR specific for an allogeneic major histocompatibility complex antigen to further assess deletional tolerance. METHODS 2C TCR Tg mice (H2b), whose Tg TCR recognizes major histocompatibility complex class I Ld, were used as recipients of Ld+ bone marrow cells after conditioning with depleting anti-CD4 and CD8 monoclonal antibodies, 3 Gy whole-body irradiation, and 7 Gy thymic irradiation. Chimerism and deletion of CD8+ 2C recipient T cells was evaluated by flow cytometry and by immunohistochemical staining. Tolerance was tested with in vitro cell-mediated lympholysis assays and in vivo by grafting with donor skin. RESULTS Intrathymic and peripheral deletion of 2C+ CD8-single-positive T cells was evident in mixed chimeras, and deletion correlated with the presence of donor-type cells with dendritic morphology in the thymus, and with chimerism in lymphohematopoietic tissues. Chimeras showed tolerance to the donor in cell-mediated lympholysis assays and specifically accepted donor skin grafts. CONCLUSIONS Tolerance to transplantation antigens is achieved through intrathymic deletion of donor-reactive T cells in mixed chimeras prepared with a nonmyeloablative conditioning regimen and allogeneic bone marrow transplantation.

Anti-CD154 or CTLA4Ig obviates the need for thymic irradiation in a non-myeloablative conditioning regimen for the induction of mixed hematopoietic chimerism and tolerance.

BACKGROUND Thymic irradiation (TI) or repeated administration of T cell-depleting monoclonal antibodies (TCD mAbs) is required in a previously described non-myeloablative regimen allowing allogeneic marrow engraftment with stable mixed chimerism and tolerance. As both treatments might be associated with toxicity in the clinical setting, we evaluated whether T-cell costimulatory blockade could be used to replace them. METHODS C57BL/6 mice received depleting anti-CD4 and anti-CD8 mAbs on day -5, 3 Gy whole body irradiation (day 0), and 15x10(6) fully MHC-mismatched, B10.A bone marrow cells. In addition, hosts were injected with an anti-CD154 mAb (day 0) and/or CTLA4Ig (day +2). Chimerism in peripheral blood was followed by flow cytometric (FACS) analysis, and tolerance was assessed by skin grafting, and also by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. The frequency of certain Vbeta families was determined by FACS to assess deletion of donor-reactive T cells. RESULTS Chimerism was transient and tolerance was not present in animals receiving TCD mAbs on day -5 without costimulatory blockade. The addition of anti-CD154 and CTLA4Ig, alone or in combination, reliably permitted induction of high levels of stable (>6 months) multi-lineage chimerism, with specific tolerance to skin grafts and donor antigens by MLR and CML assays. Long-term chimeras showed deletion of donor-reactive CD4+ peripheral blood lymphocytes, splenocytes, and mature thymocytes. Administration of TCD mAbs only 1 day before bone marrow transplantation plus anti-CD154 also allowed induction of permanent chimerism and tolerance. CONCLUSIONS One injection of anti-CD154 or CTLA4Ig overcomes the need for TI or prolonged host TCD in a preclinical model for the induction of mixed chimerism and deletional tolerance and thus further decreases the toxicity of this protocol. Achievement of tolerance with conditioning given over 24 hr suggests applicability to cadaveric organ transplantation.

Specific prolongation of skin graft survival following retroviral transduction of bone marrow with an allogeneic major histocompatibility complex gene

Engrafted allogeneic hematopoietic cells have a unique capacity to induce a state of donor-specific transplantation tolerance across major histocompatibility complex barriers. This state allows permanent acceptance of donor-type organ grafts, with otherwise normal immunocompetence. We hypothesized that introduction of allogeneic MHC genes into autologous bone marrow which is then returned to recipient mice might similarly induce specific tolerance to products of the introduced MHC genes, without the risk of graft-vs-host disease. We demonstrate here that the introduction of MHC class I Kb cDNA by retrovirus-mediated gene transfer into B10.AKM (Kk) hematopoietic cells confers specific hyporesponsiveness to allogeneic skin grafts expressing Kb.

Dose and timing of interleukin(IL)-12 and timing and type of total-body irradiation : effects on graft-vs. -host disease inhibition and toxicity of exogenous IL-12 in murine bone marrow transplant recipients

Paradoxically, a single injection of recombinant murine interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits graft-vs.-host disease (GVHD) while preserving graft-vs.-leukemia (GVL) effects in lethally irradiated mice receiving fully MHC-mismatched bone marrow and spleen cells. These protective effects are mediated by interferon (IFN)-gamma, whose early secretion is induced by IL-12 treatment. We investigated the relationship of IL-12 dose and timing of administration, as well as timing and type of total-body irradiation (TBI), with the ability of IL-12 to inhibit GVHD or mediate toxicity. The results show that a relatively low dose of IL-12 (as little as 50 U in a single injection) can mediate significant GVHD protection. The timing of IL-12 administration, however, is a critical factor. IL-12 administered 1 hour before BMT was most protective, but protection was still observed when it was administered 1-12 hours after BMT. Delaying IL-12 administration to 36 hours post-BMT completely obviated its protective effect. Administration of a second IL-12 injection 6 days after BMT negated the protective effect of an initial injection at the time of BMT. While IL-12 protection was evident when TBI was administered by 137Cs-irradiator in one or two fractions on day -1 or day 0, the use of an X-irradiator to deliver TBI on day -1 was associated with marked IL-12 toxicity. Whereas the protective effect of IL-12 against GVHD depended on donor-derived IFN-gamma, toxicity depended on the ability of host cells to produce IFN-gamma. Careful studies are warranted to test the effects of IL-12 in the context of BMT with various conditioning regimens in large animal preclinical models before this novel approach to GVHD protection can be applied clinically.

Role of intrathymic rat class II+ cells in maintaining deletional tolerance in xenogeneic rat-->mouse bone marrow chimeras.

BACKGROUND Mixed xenogeneic bone marrow chimerism and tolerance can be induced in mice conditioned with a nonmyeloablative regimen followed by injection of T cell-depleted rat bone marrow cells. We hypothesized that, despite a gradual decline in rat hematopoiesis observed in these chimeras, as long as rat class II+ antigen-presenting cells remain in their thymi, tolerance will persist as a result of deletion of donor-reactive thymocytes. METHODS The level of chimerism and of mouse Vbeta5 and Vbeta11 T-cell deletion was followed over time. These results were correlated with the presence of rat class II+ cells in the thymus by immunohistochemistry and the presence of tolerance in long-term chimeras by in vivo and in vitro assays. RESULTS (1) Proliferation and cytotoxicity assays, as well as skin graft survival, demonstrated the presence of specific tolerance to host and to donor rat, with normal reactivity to third-party rat and mouse stimulators, even as late as 85 weeks after bone marrow transplantation. (2) The absence of mature Vbeta5+ and Vbeta11+ host T cells in the thymus and periphery was always associated with the presence of rat class II+ cells in the thymus, and incomplete deletion of T cells expressing these Vbeta families was observed in thymi in which rat class II+ cells were not detectable. CONCLUSIONS Donor-specific T-cell tolerance is maintained during the period when donor-type reconstitution declines, and is most likely mediated by intrathymic clonal deletion of T cells that recognize antigens expressed on class II+ rat cells.

Inhibition of graft-versus-host disease by interleukin-2 treatment is associated with altered cytokine production by expanded graft-versus-host-reactive CD4+ helper cells

In a fully MHC plus multiple minor antigen-mismatched murine bone marrow transplantation (BMT) model, we have demonstrated that a short course of high dose IL-2, begun on the day of BMT, protects against graft-versus-host disease (GVHD). This inhibitory effect is directed against donor CD4+ cells. To determine whether the mechanism of IL-2-induced GVHD protection involves clonal deletion or anergy of host-reactive donor T helper cells (Th), we performed limiting dilution analyses to measure the frequency of activated Th that reacted to donor, host, and thirdparty antigens in GVHD control and IL-2-protected mice. Marked and specific expansion of host-reactive Th was observed to a similar extent in GVHD control and IL-2-protected mice by day 5 after BMT, and the number of these cells in the spleen increased by several orders of magnitude between days 3 and 5 after BMT, which suggests that recirculation from other tissues occurred in this period. A high proportion (approximately 80%) of donor T cells expressed CD25 in both GVHD control and IL-2-protected mice on day 4 after BMT, which suggests a high level of bystander T cell activation. Since marked quantitative differences in the GVH response were not observed between GVHD control and IL-2-protected mice, we assessed both groups for qualitative differences in the Th response. Spleen cells isolated in the first 8 days after BMT were cultured with host-type, donor-type, or third-party stimulators or without stimulators, and cytokines were measured in supernatants harvested at 24 hr. GVHD was associated with marked increases in supernatant IFN- levels from day 3 to day 6 after BMT, and with increases in IL-2 levels compared with naive A/J controls or syngeneic BMT controls stimulated with host antigens. Production of these cytokines was specifically induced by host-type antigens. Supernatants from spleens of IL-2-treated mice showed delayed kinetics of IFN-y production, and tended to contain higher levels of IL-4 in response to host antigen compared with GVHD controls on days 2 and 4 after BMT. Both IL-4 and IFN- were produced almost exclusively by CD4+ cells in spleens of GVHD control and IL-2-protected mice on day 4. However, no consistent difference was observed between the groups in supernatant IL-2 or IL-10 levels, ruling out a simple Th1 to Th2 switch. Neutralizing antibody to IL-4 did not inhibit the protective effect of IL-2 against GVHD, and previous studies have indicated that changes in IFN- kinetics do not explain IL-2-induced GVHD protection. Thus, in vivo high dose exogenous IL-2 treatment does not inhibit the marked Th expansion and T cell activation that occurs in the first week of GVHD, but significantly perturbs the pattern of GVHD-associated CD4 cytokine production.

Discordant xenogeneic neonatal thymic transplantation can induce donor-specific tolerance.

The limited supply of human organs for transplantation necessitates the development of methods leading to acceptance of xenografts. To avoid the hazards of the high-dose chronic immunosuppressive pharmacotherapy which would otherwise be required for successful xenografting, it would be desirable to induce permanent tolerance to xenogeneic donors. We have recently demonstrated that xenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymic and hematopoietic tissue into thymectomized, T cell-depleted, and natural killer-cell-depleted mice, or into natural killer cell-depleted nude mice. We have now extended these studies by replacing fetal tissue with neonatal pig thymic and hematopoietic tissue, and by examining the in vivo responses of reconstituted mice to pig skin grafts. Neonatal tissue was studied because it might be more practicable than fetal tissue for the purpose of transplantation to primates. BALB/c nu/nu mice transplanted with neonatal (<24-hr-old) pig thymus and spleen fragments developed circulating mouse CD4+ cells. The pig thymus grafts were necessary for mouse T-cell development, as CD4 recovery did not occur in recipients of neonatal pig splenic tissue alone. The CD4+ cells that developed included Vbeta8.1/2+ T cells in similar proportions as in BALB/c mice, and Vbeta11+ and Vbeta5+ CD4 T cells were deleted almost as completely as in normal BALB/c mice. This deletion was detected among CD4 single-positive graft thymocytes. In 9 of 12 evaluable animals, mixed lymphocyte responses demonstrated tolerance to donor-type pig SLA antigens, with responsiveness to alloantigens and/or third-party pig xenoantigens. Furthermore, grafting of neonatal pig thymus conferred the ability to reject allogeneic mouse skin in 7 of 10 animals. In addition, 7 of 10 animals accepted paternal (donor SLA-matched) skin (median survival time [MST] > 100 days), whereas 4 of 4 animals rejected third-party SLA-mismatched pig skin (MST=40.5 days). We conclude that neonatal pig thymi transplanted to BALB/c nu/nu mice can support the development of mouse CD4+ cells that are functional and specifically tolerant to donor-type pig antigens.

Strain dependence of interleukin-2-induced graft-versus-host disease protection: evidence that interleukin-2 inhibits selected CD4 functions.

Summary: Treatment of lethally irradiated mice with a short course of highdose interleukin (IL)-2 markedly inhibits acute and chronic graft-versus-host disease (GVHD), while preserving a graft-versus-leukemia (GVL) effect of allogeneic T-Cells. We recently demonstrated that this GVL effect, observed with the EL4 leukemia/lymphoma in the A/JB10 strain combination, was mediated by CD8+ A/J T-cells in a CD4-independent fashion. IL-2 inhibited only the activity of CD4+ cells, and not that of CD4-independent CD8+ T-cells in A/J spleen cell inocula. This inhibition of CD4 function was sufficient to markedly inhibit GVHD, thus explaining the dissociation of GVHD and GVL in IL-2-treated mice. We have now performed studies to determine the capacity of IL-2 to inhibit GVHD induced across a variety of different histocompatibility bariers. IL-2 signifiacantly delayed GVHD morality in three of four additional fully major histocompatibility comples (MHC) plus minoe-disparated strain combinatins when CD4+ T.cells were given. Numbers of CD8+ T-cells were given Numbers of CD8+ T.cells comarable to those that might contaminate human marrow demonstrated a relatively poor capacity to produce acute GVHD when given without CD4+ cells in all of three additional strain combinations evaluated. In one of these strain combinations (B10BALB/c), IL-2 protected against acute but not chronic GVHD mortality when CD4+ cells were given with or without CD8+ cells. IL-2 was unable to inhibit CD8-mediated GVHD in strain combinations differing at isolated class I MHC loci. In a strain combination differing only at multiple minor histoincompatibility antigen (HA) loci, B10C3H.SW, GVHD was largely CD8-dependent, but IL-2 did not inhibit the small CD4-mediated component of GVHD. Together, these results suggest that IL-2 inhibits a restricted subset of CD4 cells or functions, and that the type of CD4 activities mediating GVHD is determined by the particular histoincompatibilities between donor and host.

Strain restricted typing sera (srts) for use in monitoring the genetic integrity of congenic strains.

A relatively simple procedure for serologic monitoring of the genetic integrity of congenic strains housed in a conventional colony is presented. Using a combination of 3 or 4 F1 immunizing cells, sera can be produced in each strain housed in the colony which will react in a complement-mediated cytotoxicity assay with peripheral lymphocytes from most if not all other strains in the colony. Routine screening of breeding stock with these strain restricted typing sera (SRTS) permits the sensitive detection of genetic contamination between the stocks maintained. These sera detect H-2, minor histocompatibility differences, and other cell surface differentiation antigens, and can also be used to identify the nature of a contaminant when isolated. In addition, when used within appropriate strain combinations, the sera can be useful in detecting antigenic determinants otherwise difficult to identify.