H Sefrioui

Transforming growth factor-beta inhibits lymphokine activated killer cytotoxicity of bone marrow cells: implications for the graft-versus-leukemia effect in irradiation allogeneic bone marrow chimeras.

BACKGROUND We have previously shown that allogeneic bone marrow (BM) chimeras preconditioned with total lymphoid irradiation and low-dose total body irradiation (TLI/TBI) develop a stronger graft-versus-leukemia (GVL) effect than chimeras preconditioned with high-dose total body irradiation only (TBI). Here, we report on the possible role of cytokines in the mechanism underlying this GVL effect. METHODS Splenic mRNA levels of the cytokines interleukin (IL)-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta (TGF-beta), and of inducible nitric oxide synthetase were determined by reverse transcription-polymerase chain reaction in TLI/TBI- or TBI-conditioned C3H/AKR BM chimeras challenged with AKR-type BW5147.3 leukemia cells. Ex vivo TGF-beta protein production by splenocytes was determined using ELISA. The possibility that cytokines influence the GVL effect by modulating the activity of IL-2-activated lymphocytes (LAK cells) was investigated by in vitro assays on donor-type BM cells. RESULTS Of all cytokine mRNA levels studied, those of TGF-beta and IL-7 were different between groups; both were significantly more elevated in TBI- than in TLI/ TBI-conditioned or normal mice. Differences were apparent after conditioning and were not influenced by additionally injected BM or leukemia cells. Cultured splenocytes of TBI-conditioned animals produced significantly more TGF-beta protein than those of TLI/TBI-conditioned ones or normal controls. r-TGF-beta but not r-IL-7 suppressed in vitro LAK activity of donor-type BM cells against BW5147.3 cells in a dose-dependent way. CONCLUSIONS High-dose TBI-induced, host-derived splenic TGF-beta may inhibit generation of LAK cells from subsequently transplanted donor BM cells, suppressing their capacity to generate cytotoxicity upon injection of leukemia cells. The cytokine profile, induced by irradiation in host hematopoietic organs, can significantly modify posttransplant immunological processes such as the GVL effect and graft-versus-host disease (GVHD).

Graft-versus-leukemia effect in minor antigen mismatched chimeras given delayed donor leucocyte infusion: immunoregulatory aspects and role of donor T and ASGM1-positive cells.

BACKGROUND Previous studies have demonstrated that delayed donor leukocyte infusion (DLI) can increase graft-versus-leukemia (GVL) without increasing graft-versus-host-disease (GVHD) in MHC mismatched bone marrow (BM) chimeras. In our report, the immune status of minor antigen mismatched BM chimeras given DLI was studied. Particularly the role of donor ASGM1 positive or T cells in the graft-versus-leukemia effect (GVL) was investigated. METHODS AKR mice (H2k, Mls1a, Thy1.1) received TBI (9,5 Gy) and T cell-depleted (TCD) C3H (H2k, Mls2a, Thy1.2) BM alone (BM chimeras), or TCD BM together with immunocompetent C3H spleen cells at the time of BM transplantation (BM+SP chimeras), or TCD BM and 3 weeks later C3H spleen cells (DLI chimeras). Chimerism and T lymphocyte subsets were scored using FACS and anti-Thy, anti-Vbeta6, anti-IL2-beta receptor, anti-CD4, anti-CD3, and anti-CD8 mAbs. Leukemia challenge consisted of 5 x 10(6) AKR T cell lymphoma (BW4157) cells injected i.v. ASGM1 positive (ASGM1+) cells and T cells were depleted using anti-ASGM1 or anti-Thy1.2 antibodies, respectively. Immune tolerance was studied using MLR and CML tests. RESULTS BM + SP chimeras developed acute and lethal GVHD, whereas DLI chimeras were totally free from GVHD. In DLI chimeras, host-reactive cytotoxic T cells (CTL) could not be induced and host-reactive CD8Vbeta6 cells were deleted whereas CD4Vbeta6 cells and MLR reactivity persisted temporarily. In contrast, in BM+SP chimeras, anti-host CTL were easily generated and an expansion of both host-reactive CD8Vbeta6 and CD4Vbeta6 T cells was found as well as high anti-host MLR reactivity. Depletion of either ASGM1 + cells or T cells from the DLI inoculum resulted in an abrogation of GVL reactivity, suggesting that both cell populations were involved in the protection against BW4157 leukemia. Three weeks after DLI, the GVL effect waned which correlated with the disappearance of host-reactive CD4 cells and MLR reactivity. CONCLUSION In minor antigen mismatched BM chimeras, anti-host reactivity after DLI is characterized by (1) an absence of clinical GVHD, (2) clonal deletion of host-reactive CD8 cells, (3) an absence of anti-host CTL induction, and ( 4) a temporary persistence of host-reactive CD4 T cells and of MLR reactivity. In addition, either donor ASGM1+ cells or an interaction between these cells and T cells contribute to the GVL effect.

Synthesis and in vitro evaluation of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)-pteridines as dual immunosuppressive and anti-inflammatory agents

Screening of a pteridine-based compound library led to the identification of compounds exhibiting immunosuppressive as well as anti-inflammatory activity. Optimization afforded a series of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)pteridine analogues. The most potent congeners in this series displayed low nM IC(50) values in the Mixed Lymphocyte Reaction (MLR) assay. In addition, these compounds also have potent anti-inflammatory activity as measured in the Tumor Necrosis Factor (TNF) assay.

Influence of the additional injection of host-type bone marrow on the immune tolerance of minor antigen-mismatched chimeras: possible involvement of double-negative (natural killer) T cells.

BACKGROUND It has previously been demonstrated that adding T cell-depleted (TCD) host bone marrow (BM) to an MHC-mismatched BM inoculum allows for induction of long-term stable chimeras without graft-versus-host disease (GVHD) even when non-TCD allogeneic BM was used. AIMS The present study was undertaken to investigate immune tolerance mechanisms in minor antigen-mismatched allogeneic BM chimeras when host-type BM was added to the BM inoculum. METHODS C3H (H2k, Thy 1.2, Mls 2a) recipients were conditioned with 9.5 gray (Gy) of total body irradiation. To exclude any interference with possible subclinical GVHD, 5x10(6) TCD AKR (H2k, Thy 1.1, Mls 1a) BM cells were injected with (syn + allo) or without (allo) 5x 10(6) TCD C3H BM cells. Chimerism, clonal deletion, and T lymphocyte subsets were scored using FACS and anti-mouse Thy, Vbeta6, Vbeta3, CD3, CD4, or CD8 monoclonal antibodies. The stability of tolerance was studied by investigating mixed lymphocyte reaction and cytotoxic T cell induction in chimeras after immunization with host, donor, or third-party (BALB/c) splenocytes. Breaking of chimerism was attempted by injecting nontolerant 40x10(6) host-type splenocytes 2 months after BM transplantation. Cytokines and Valpha14 mRNA were assayed using real time quantitative reverse transcriptase-polymerase chain reaction at 4 and 48 hr, respectively, after injection of nontolerant host-type splenocytes. RESULTS Both groups of mice became long-term stable mixed chimeras without any clinical sign of GVHD. Neither group was able to produce antihost nor antidonor cytotoxic T cells, even after immunization. The addition of syngeneic BM to the allogeneic inoculum reduced the overall level of allogeneic chimerism (from approximately 70% or approximately 85% in peripheral blood lymphocytes and spleen, respectively, in allo chimeras versus approximately 35% and approximately 60% in syn + allo chimeras). Moreover, it resulted in complete clonal deletion of both host-reactive (Vbeta3) and donor-reactive (Vbeta6) lymphocytes in syn + allo chimeras in contrast to in allo chimeras, in which only donor-reactive lymphocytes were completely deleted. After nontolerant C3H splenocyte injection, high levels of interleukin 2 mRNA were produced and chimerism decreased in syn + allo chimeras. In contrast, in allo chimeras, this maneuver was followed by the production of higher levels of interleukin 4 and interferon-gamma, and of Valpha14 mRNA, as well as by the proliferation of CD3+CD4-CD8- (double-negative) T cells and by an increase of donor chimerism. CONCLUSION The addition of host-type BM to the allogeneic inoculum has an influence on the level of chimerism, the extent of clonal deletion, and the reaction of chimeras after the injection of nontolerant host-type splenocytes. In the latter phenomenon, cytokine production and proliferation of Valpha14+ CD3+CD4-CD8- (double-negative, natural killer T) lymphocytes may be involved.

Stable mixed chimerism induced by total lymphoid irradiation or by total body irradiation is maintained by different mechanisms and leads to different graft versus leukemia or graft rejection reactions

Abstract ALLOGENEIC bone marrow (BM) transplantation is an effective procedure for the treatment of various malignant disorders of hematologic origin. 1,2 Several reports indicate that the fatality rate in leukemic patients can be significantly decreased after allogeneic bone marrow transplantation. 1,2 Previously, we demonstrated that mice conditioned with TLI or TBI were capable of developing stable bone marrow chimerism without signs of graft-versus-host-disease (GVHD) or rejection. 3 In the present work, we examined the mechanisms that maintained the state of the chimerism and which determined graft-versus-leukemia (GVL) or breaking of the tolerance in these stable chimeras.

Tolerance in Mlsa mismatched chimeras prepared with allogeneic bone marrow alone or in combination with syngeneic bone marrow.

ALLOGENEIC bone marrow (BM) transplantation remains a viable option for the treatment of both malignant and nonmalignant diseases of the immune system. However, certain serious disadvantages like graft rejection or graft-vs-host disease (GVHD) may limit its efficacy. When host-type BM and allogeneic BM were infused together, MHC-mismatshed chimeras showed specific tolerance to donor and host antigens as well as protection against GVHD. Nevertheless, the mechanism involved in this protection of host BM remains unclear. The present study was undertaken to investigate further the influence of additional injection of host BM together with allogeneic BM on the immune tolerance of Mlsa, minor histocompatibility antigen mismatshed chimeras. In addition, the behavior of those stable chimeras after challenging them with host-type splenocytes was also investigated.