Recipients Of Spleen Cells Research Articles

The contribution of cytotoxic and noncytotoxic function by donor T-cells that support engraftment after allogeneic bone marrow transplantation

The present studies were designed for investigation of the requirements for cytotoxic function in donor T-cells transplanted to support engraftment after infusion of allogeneic bone marrow. The experiments examined the capacity of donor CD8 T-cells lacking Fas ligand and/or perforin function to facilitate donor B6 congenic (B6-Ly5.1) BM engraftment across major histocompatibility complex class I/II barriers after transplantation. T-cell-depleted BM cells from B6-Ly5.1 donors were transplanted into sublethally irradiated (5.5 Gy) BALB/c recipients together with different lymphocyte populations from wild-type B6 (B6-wt) donors or donors lacking functional cytotoxic pathways. Early presence of lineage-committed donor progenitor cells was assessed by the presence of day 5 splenic colony-forming units-granulocyte-macrophage (CFU-GM). Recipients of BMT without donor T-cells did not demonstrate significant CFU-GM activity 5 days post-BMT. Lineage-committed progenitor cells in recipient spleens could be supported by addition to the BM of wild-type (B6-wt) and cytotoxically single- (perforin, B6-pko or FasL, B6-gld) or double-deficient (B6-cdd) CD8 T-cells. However, B220+-enriched B-cells could not support the presence of day 5 donor CFU-GM. For further assessment of the capacity of cytotoxically impaired T-cells to participate in the engraftment process, the ability of these and normal CD8 cells to support the homing of donor cells to the BM was examined after infusion of carboxyfluorescein diacetete succinimidyl ester-labeled progenitors. In a syngeneic model lacking resistance, cytotoxically impaired donor T-cells supported increased numbers of progenitor cells in the marrow equivalent to the support provided by wild-type donor T-cells. Examination of peripheral chimerism indicated that during the first month after B6-->BALB/c BMT, donor chimerism was detected in BMT recipients receiving unfractionated T-cells or CD8+ T-cells from B6-wt donors, and chimerism was maintained at least 80 days after BMT. In contrast, B6-cdd unfractionated or CD8+ T-cells failed to maintain long-term B6 donor chimerism in the host. Experiments with highly enriched populations of positively selected CD8+ T-cells from B6-pko, B6-gld, or B6-cdd donors demonstrated that although each of these T-cell populations could promote the initial presence of donor CFU-GM early post-BMT, B6-pko and B6-cdd CD8+ T-cell populations were not able to support long-term peripheral chimerism. These results demonstrate that donor T-cells lacking major cytotoxic effector pathways have functions that support initial donor progenitor cell presence in the host hematopoietic compartment after BMT. They also demonstrate that support of long-term donor BM engraftment requires CD8+ T-cells with intact cytotoxic, that is, perforin, function. Finally, syngeneic B6-->B6 BMT suggests activation of CD8+ T-cells posttransplantation apparently is required to support enhanced progenitor cell activity. This study provides new findings concerning the role of cytotoxic function in the process of facilitating allogeneic donor BM engraftment.

Liver-derived dendritic cells induce donor-specific hyporesponsiveness: use of sponge implant as a cell transplant model.

Spontaneously accepted mouse liver allografts are capable of protecting subsequently transplanted donor organs from rejection; however, the underlying mechanisms are unclear. Dendritic cells (DC) residing in liver grafts are likely important in tolerance induction. DC propagated from mouse liver with GM-CSF are phenotypically and functionally immature. They are poor allostimulators in MLR and prolong survival of pancreatic islet allografts. It has been problematic to perform mechanistic studies in an islet transplant model because of difficulties in obtaining sufficient graft infiltrating cells. In this study, we used a sponge allograft model [i.e.. a subcutaneously implanted sponge matrix loaded with B10 (H2b) spleen cells]. To investigate the influence of administration of donor (B10) liver-derived DC on alloimmune reactivity of C3H (H2k) hosts, sponge graft infiltrating cells (SGIC) and recipient spleen cells were isolated, and their immunophenotype and donor-specific cytotoxic T lymphocyte (CTL) activity were examined. The results illustrate that donor-specific CTL activity of T cells are lower in recipients that had received systemic treatment with liver-derived immature DC, associated with a decrease in CD8+ cell population and an increase in Gr-1+ cells in SGIC, compared with recipients treated with mature bone marrow (BM)-derived DC. Interestingly, administration of liver DC directly into the sponge did not inhibit T cell responses. These data suggest that systemic administration of donor liver DC induces donor-specific hyporesponsiveness, probably not by direct inhibition of graft infiltrating T cells. The increased Gr-1+ cells may play immune regulatory roles in induction of host donor-specific hyporesponsiveness.

Induction of specific transplantation immunity by oral immunization with allogeneic cells.

Oral administration of antigen has been shown to be effective for both positive and negative modulation of immune responses. In the present study we characterized changes in the reactivity of the immune system after oral immunization with allogeneic spleen cells. Mice were orally immunized for 10 consecutive days with fresh allogeneic spleen cells, and the phenotype, proliferative response, cytotoxic activity and cytokine production profile of recipient spleen cells were assessed 1 or 7 days after the last immunization dose. Although no significant changes in the proportion of CD4+, CD8+ or CD25+ cells were observed in the spleen of orally immunized mice, significant activation of alloreactivity in spleen cells was found. Cells from orally immunized mice exhibited enhanced proliferation and cytotoxic activity after stimulation with specific allogeneic cells in vitro, and produced considerably higher concentrations of interferon-gamma (IFN-gamma) and significantly less interleukin (IL)-4 than did cells from control mice. The production of IL-2 was essentially unchanged and that of IL-10 was only slightly increased. The systemic allosensitization induced by oral immunization was demonstrated in vivo by increased resistance to the growth of allogeneic tumours induced by subcutaneous inoculation of high doses of tumour cells. In addition, orthotopic corneal allografts in orally immunized recipients were rejected more rapidly (in a second-set manner) than in control, untreated recipients. These data show that oral immunization with allogeneic cells modulates individual components of the immune response and that specific transplantation immunity, rather than tolerance, is induced in the treated recipients.

GRAFT INFILTRATING T CELLS CAN RESPOND TO INDIRECTLY PRESENTED ANTIGEN DURING REJECTION OF FULLY MHC DISPARATE SKIN ALLOGRAFTS.

49 T cell alloreactivity during graft rejection has generally been defined through the study of immune responses in peripheral lymphoid organs. It has largely been assumed that such detected immune responses are representative of T cell reactivity in the graft itself, although frequencies and cytokine profiles of alloantigen specific T cells in the graft have not been well characterized. Furthermore, indirect presentation of donor derived determinants expressed in the context of recipient MHC (indirect presentation) occurs during rejection, but whether graft infiltrating T lymphocytes recognize indirectly presented antigens in the graft itself remains unknown. We used a cytokine ELISA spot assay to define the allorecognition pathways and cytokine profiles of skin graft infiltrating T lymphocytes during rejection of fully MHC disparate skin in mice. Purified T cells were isolated from B6 skin grafts on days 8-12 after placed on BALB/c recipient mice using collagenase digestion and negative selection. The cells were tested in IFNγ, IL-2 and IL-4 ELISA spot assays for their ability to response to intact donor spleen cells (direct pathway) or to syngeneic (recipient) spleen cells plus donor sonicate (indirect pathway) as published by our laboratory. The findings were correlated with analogously performed experiments using draining lymph node T cells. Graft infiltrating T cells were detectable by d 10-12 (but not on d 8) after skin graft placement. They produced predominantly IFNγ in response to directly presented alloantigens at a frequency of > 2000 per million T cells, and were ∼70% CD8+. IL-2-secreting, but not IL-4 secreting cells were found in lower frequenies. In addition, CD4+, IFNγ-secreting T cells responding to antigens presented by the indirect pathway were found in the graft, and comprised ∼5-10% of the total intragraft alloresponse. Interestingly, a kinetic analysis revealed that the intragraft responses tended to lag behind those of the draining lymph nodes by 1-2 days, although the frequencies and cytokine profiles were similar at the two sites. These studies reveal that indirect alloreactivity occurs in the graft itself as well as in the draining lymph nodes, providing further evidence that T cells responding to the indirect pathway are relevant participants of the rejection response. Our data further suggest that studies of immune responses in draining lymph nodes during acute skin graft rejection are reasonable reflections of the intragraft, alloreactive T cell repertoire.

Assessment of peripheral tolerance in anti-CD4 treated C57BL/6 mouse heart transplants recipients

The study was designed to compare second heart and skin grafts and in vitro assays as a means of assessing peripheral tolerance in C57BL/6 mice. Vascularized heterotopic BALB/c hearts were placed in C57BL/6 recipients treated with anti-CD4, GK1.5 (1 mg total per 20 g mouse i.p. on days 0, 1, 2, 3). Those mice in which hearts survived for >60 days were challenged with donor and third-party (CBA) skin grafts or with second heart grafts, of donor or third-party origin, attached to the carotid artery and jugular vein. In vitro alloreactivity was assessed by mixed lymphocyte reactions (MLR) and cell mediated lympholysis (CML) using recipient spleen cells. Parenchymal damage, cellular infiltration and vascular disease were assessed from the histology of long-term allografts and isografts. Allografts in untreated recipients were rapidly rejected while isografts survived >10 days. Primary allografts grafts in anti-CD4 treated recipients also survived >100 days, as did donor strain secondary heart transplants given at >60 days after the first graft. Third-party hearts were rapidly rejected, as were donor and third-party skin grafts placed on recipients with long-term allografts. These recipients showed low MLR response to both donor and third-party stimulators and donor-specific suppression of CML at 60 days post graft. Long-surviving heart allografts all showed evidence of parenchymal damage and vascular intimal thickening. Thus in the BALB/c to C57BL/6 donor-recipient strain combination, hearts, but not skin grafts, could be used to demonstrate peripheral tolerance, which seemed to be both organ and major histocompatibility complex (MHC) specific. Despite long survival, BALB/c hearts all showed evidence of parenchymal damage and vascular intimal thickening, a sign of chronic rejection.

CD40L is Necessary for the Priming of Effector Cells for Lymphocytic and Granulomatous Experimental Autoimmune Thyroiditis

The interaction of CD40 on antigen presenting cells (APC) with CD40L on mouse thyroglobulin (MTg)-specific T cells may deliver an essential signal for the development of CD4+experimental autoimmune thyroiditis (EAT) effector cells and anti-MTg producing B cells. To determine the requirement for CD40–CD40L interactions in G-EAT, donor mice were injected with an anti-CD40L monoclonal antibody (mAb) on days −1, 0, and +1 relative to immunization with MTg and adjuvant. Recipients of spleen cells from MTg-primed donor mice injected with anti-CD40L did not develop EAT, while spleen cells from similarly immunized hamster Ig-treated donors transferred severe G-EAT. Although the decreased EAT severity was accompanied by increased IL-4 mRNA expression by CD4+T cells from anti-CD40L-treated donors, the increased IL-4 wasnotnecessary for suppression of EAT, since anti-CD40L treatment prevented EAT in IL-4-deficient mice. Addition of MTg-primed B cells duringin vitroactivation of spleen cells from anti-CD40L-treated donors did not induce EAT in recipients, suggesting that anti-CD40L suppresses EAT by preventing the sensitization of EAT effector cells. Addition of anti-CD40L duringin vitroactivation of MTg-primed spleen cells or treatment of recipients with anti-CD40L had no effect on EAT severity, indicating that CD40–CD40L interactions are not required after EAT effector cells are primed to MTg.

MECHANISMS OF TOLERANCE INDUCED BY TOLEROGENIC ALLOCHIMERIC DONOR/RECIPIENT CLASS I MHC PROTEIN

42 Introduction: We previously reported that portal vein (p.v.) or oral administration of a donor/recipient tolerogenic protein (α1hu62-69-RT1.Aa) bearing four donor amino acids (Arg62, Glu63, Gln65, Gly69) in combination with a short course of cyclosporine (CsA) to ACI (RT1a) rats induced donor-specific transplantation tolerance to Wistar Furth (WF; RT1u) heart allografts (>100 days). The present study examines the mechanisms of tolerance using T cell adoptive transfer experiments and analysis of cytokine mRNA expression. Methods: Tolerance to WF hearts was induced by p.v. injection to ACI rats of 10 µg α1hu62-69-RT1.Aa (day 0) or oral gavage with 5 mg α1hu62-69-RT1.Aa (days 0-3) in combination with a 7-day CsA course (4 mg/kg/day; days 0-6). For adoptive transfer experiments, 40×106 T cells purified from spleen or lymph node cells of tolerant ACI recipients were injected intravenously into irradiated (500 rads) syngeneic ACI recipients of donor-type WF or third-party Brown Norway (BN; RT1n) heart allografts. Expression of cytokine mRNAs was analyzed in: WF heart allografts from tolerant ACI recipients (>100 days); WF hearts from untreated WF recipients (day 7; isograft controls); and WF heart allografts from untreated ACI recipients (day 7; allograft controls). Normal WF hearts (normal controls) were also used for analysis. Total RNA was purified by homogenizing hearts in guanidine isothiocyanate solution, followed by CsCl2 gradient with ultracentrifugation. A semi-quantitative RT-PCR method was used to measure the levels of cytokine mRNAs expression using the pairs of gene-specific primers, namely for IL-2, IFN-γ (T helper 1; Th1), IL-4, and IL-10 (Th2). Results: Irradiated ACI recipients rejected WF heart allografts at a mean survival time (MST) of 33.2±1.1 days. Adoptive transfer of 40×106 tolerant T cells, obtained from p.v.-induced (n=5) or orally induced (n=4) tolerant recipients, rendered irradiated ACI recipients tolerant to WF heart allografts (> 100 days, p<0.01). In contrast, the same tolerant 40×106 T cells did not prolong the survival of the third-party BN heart allografts; MSTs were 13.3±5.5 days in a p.v. tolerance model (n=3, NS) and 12.5±1.0 days (n=3, NS) in an oral tolerance model. Non-transferred ACI recipients rejected BN heart allografts at 11.6±0.9 days. WF heart allograft from untreated recipients had increased levels of mRNAs for IL-2 (++++), IFN-γ (++++), IL-4 (+), and IL-10 (++++) when compared with normal or isograft WF controls [IL-2 (-), IFN-γ (+), IL-4 (-), and IL-10 (+)]. In comparison to rejectors, tolerant WF heart allografts (from p.v.- or orally induced tolerant hosts) displayed reduced levels of IL-2 (+) and IFN-γ (+++) mRNAs, but not IL-4 (+) and IL-10 (++++) mRNAs. Conclusion: These results suggest that allochimeric protein-induced tolerance is mediated by regulatory T cells, possibly with selective activation of Th2-like cells.

DONOR PRETREATMENT WITH Flt-3 LIGAND AUGMENTS ANTIDONOR CYTOTOXIC T LYMPHOCYTE, NATURAL KILLER, AND LYMPHOKINE-ACTIVATED KILLER CELL ACTIVITIES WITHIN LIVER ALLOGRAFTS AND ALTERS THE PATTERN OF INTRAGRAFT APOPTOTIC ACTIVITY1,2

Liver allografts are accepted across major histocompatibility complex (MHC) barriers in mice and induce donor-specific tolerance without requirement for immunosuppressive therapy. There is evidence that passenger leukocytes may play a key role in tolerance induction. Flt-3 ligand (FL) is a recently cloned hematopoietic cytokine that strikingly augments functional dendritic cells (DCs) within lymphoid and nonlymphoid tissue. The expression of costimulatory molecules and MHC class II antigen on DCs isolated from livers of FL-treated B10 (H2b) mice (10 microg/day; 10 days) was examined by flow cytometric analysis, and their allostimulatory activity assessed in primary mixed leukocyte cultures. B10 livers from FL-treated donors were transplanted orthotopically into naive C3H (H2k) recipients. Donor cells (MHC class II+) in recipient spleens were identified by immunohistochemistry. Antidonor cytotoxic T lymphocyte activity, and both natural killer and lymphokine-activated killer cell activities of graft nonparenchymal cells and host splenocytes were determined using isotope release assays. Apoptotic activity within liver grafts was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. DCs isolated from livers of FL-treated donor mice exhibited increased cell surface expression of CD40, CD80, CD86, and IAb, and augmented T cell allostimulatory activity compared with controls. Within 24 hr of organ transplantation, the numbers of donor IAb+ cells within recipient spleens was augmented substantially compared with normal liver recipients. Livers from FL-treated donors were rejected acutely (median survival time, 5 days), whereas control B10 liver allografts survived >100 days. Nonparenchymal cells from rejecting grafts 4 days after transplantation exhibited increased antidonor cytotoxic T lymphocyte, natural killer, and lymphokine-activated killer cell activities compared with cells from spontaneously accepted grafts. This augmented cytotoxic reactivity was associated with histologic evidence of injury to bile duct epithelium and vascular endothelium that was not readily evident in controls. Thus, although normal livers provide allostimulatory signals sufficient to elicit an antidonor immune response, regulatory mechanisms that may include apoptosis of graft-infiltrating T cells, and that are overcome by augmenting the number of functional donor DCs, may account for inherent liver tolerogenicity.

PROLONGATION OF ORGAN ALLOGRAFT SURVIVAL BY SYSTEMIC ANTI-IL-10 ANTIBODY ADMINISTRATION IS MEDIATED BY B CELL SUPPRESSION

394 IL-10 has been regarded as an immunosuppressive cytokine because of its capacity to inhibit the function of antigen presenting cells (APC) and downregulate the synthesis of several proinflammatory cytokines It has been demonstrated previously however, that systemic post-transplant administration of recombinant mouse IL-10 can accelerate B10 (H-2b) cardiac allograft rejection in C3H (H-2k) recipients. This is associated with enhancement of donor-specific cytotoxic T lymphocyte (CTL) and alloantibody responses. There is also evidence that systemic post-transplant administration of a neutralizing anti-IL-10 monoclonal antibody (mAb) can significantly prolong B10 mouse organ allograft survival in normal and presensitized C3H recipients(the latter animals are exposed to donor alloantigens by skin grafting 2 weeks before organ transplantation). The inhibition by anti-IL-10 of secondary allograft rejection is of particular significance and potential clinical importance, as donor presensitization remains a difficult problem due to its resistance to conventional immunosuppressive therapy. In presensitized recipients, anti-IL-10 mAb (rat IgM; Schering-Plough Research Institute, Kenilworth, NJ; purity 85-95%) at 0.5 mg/d for 6 consecutive days after organ transplantation significantly prolonged survival of both vascularized cardiac and orthotopic liver grafts. Graft median survival times were extended from 5(n = 5) to 10 days (n = 6) p<0.05, and from 4 (n = 5) to 11 days (n = 7) p<0.05, respectively. To understand the underlying mechanisms, we examined the influence of administration of anti-IL-10 mAb on anti-donor CTL activity, circulating complement-dependent cytotoxic (CDC) antibody titers and the phenotype of graft-infiltrating and splenic immunocytes in the presensitized liver allograft recipients. Prolongation of liver allograft survival in presensitized recipients was not associated with a suppressive effect on CTL activity against donor alloantigens mediated by either graft-infiltrating cells or host spleen cells. Inhibition of rejection was however, associated with suppression of circulating CDC antibody titers, and with significant reductions in the incidence of B220+ B cells in both grafts and recipient spleens. In conclusion, the data provide evidence for a potential therapeutic role of anti-IL-10 mAb in allograft transplantation in both normal and presensitized recipients. The suppressive effect of anti-IL-10 mAb may be mediated by inhibition of B cell development and cytotoxic alloantibody production.

Active suppression induced by cutaneous exposure to bacterial superantigen is prevented by interleukin-12 treatment in vivo.

Exposure to the bacterial superantigen staphylococcal enterotoxin B (SEB) leads to inhibition of several immune responses and the induction of regulatory cells. The aim of this study was to characterize these regulatory cells further and to investigate the effect of interleukin-12 (IL-12) on superantigen-induced suppression. For this purpose BALB/c mice were injected subcutaneously with low doses of SEB that did not deplete the SEB-reactive V beta T cells. Intravenous transfer of unseparated local-draining lymph node cells from these SEB-treated animals suppressed the proliferative response of mononuclear spleen cells of naive syngeneic recipients for at least 3 weeks. The regulatory cells did not produce the type 2 cytokines, interleukin-4 (IL-4) or interleukin-10 (IL-10), or increased amounts of transforming growth factor-beta (TGF-beta). Depletion of CD8+ or SEB-reactive V beta 7+ and V beta 8+ T cells, prior to transfer, abrogated the suppressive effect. Intraperitoneal injections of IL-12 into donors, prior to SEB treatment, prevented the induction of functional regulatory cells, and treatment of recipients with IL-12, prior to receipt of cells from SEB-treated donors, prevented the suppressive effect of regulatory cells that were already induced. The data indicate that exposure to minute amounts of superantigens directly induces superantigen-reactive and CD8+ regulatory T cells and that superantigen-induced suppression can be prevented and reversed by IL-12 treatment in vivo.

Inhibition of murine chronic graft-versus-host disease by the chloroform extract of Tripterygium wilfordii Hook f

The effects of chloroform extract of Tripterygium Wilfordii Hook f (TWH extract) on chronic graft-versus-host disease (GVHD) were examined in a murine experimental model. Chronic GVHD was induced by intravenous transfer of parental DBA/2 spleen cells into unirradiated (C57BL/6 × DBA/2)F1 recipient mice. The effects of TWH extract on GVHD were assessed by measuring both the degree of splenomegaly and the total serum IgE levels 3 weeks after the cell transfer. Subcutaneous administration of TWH extract once a day for 3 weeks suppressed chronic GVHD in a dose-dependent manner. Significant suppression of splenomegaly was first noted in mice treated with 7.5 μg/kg of the agent. The maximum inhibition was observed when mice were treated with more than 10.0 μg/kg (but not 5.0 μg/kg) caused complete suppression of serum IgE hyperproduction. The ability of donor T cells purified from recipient spleen cells to produce interleukin 4 in response to stimulation with anti-CD3 monoclonal antibody was significantly abrogated when recipient mice were treated with 10.0 μg/kg of the agent. These results strongly suggest that TWH extract will be an addition to the cohort of immunosuppressive therapies used in solid organ and bone marrow transplantation.

Evaluation of microchimerism after orthotopic liver transplantation between allogeneic mice

In the orthotopic mouse liver transplantation model, allografts are accepted without immunosuppression, and donor-specific tolerance is induced upto 40 days. Although FK 506 is a well-known immunosuppressive agent, its influence on tolerance induction is not known. In this study, we examined the influence of FK 506 on tolerance induction in a mouse liver transplant model.Orthotopic liver transplantation was performed from B10.BR (H-2K) to B10.D2 (H-2D mice). In the experimental group, FK 506 (1 mg/kg/d) was given subcutaneously to the recipients from day 0 to day 21, whereas the control group received a placebo (1 mg/kg/d). On day 40, donor skin grafts were transplanted to the recipients to examine the survival times of the recipients and the skin grafts. On day 14, donor-type cells in recipient's blood, spleen, kidney, thymus, and lymph nodes were examined by RT-PCR using specific donor-type MHC class I and II primers.All recipients survived for more than 100 days. The mean survival time of skin grafts in the experimental group was significantly reduced compared to that of controls. On day 14, either donor-type MHC class I- or class II-positive cells were detected in the control group, whereas donor-derived MHC class II-positive cells disappeared in the experimental group.In the early period after mouse liver transplantation, FK 506 inhibits tolerance induction paradoxically. Some donor-derived MHC class II-positive cells might play an important role in tolerance induction.

Recipient Humoral Immunity Against Leukoreduced Allogeneic Platelets Is Suppressed by Aminoguanidine, a Selective Inhibitor of Inducible Nitric Oxide Synthase

Leukoreduced allogeneic platelet transfusions have been previously shown to initially stimulate an in vitro cellular cytotoxicity and subsequently Induce the formation of immunoglobulin G (IgG) antidonor alloantibodies. To further characterize these responses and determine if they are related, recipient BALB/c H-2d mice were treated with aminoguanidine (AMG), a selective inhibitor of inducible nitric oxide synthase (iNOS), and transfused weekly with 2 x 10(8) C57BL/6 H2b platelets. In control, non-AMG-treated mice, transfusion significantly (P < .01) increased serum levels of interferon-gamma (IFN-gamma) by day 1 posttransfusion (PT). IFN-gamma returned to pretransfusion levels by day 3 PT, and its production was not affected by AMG treatment. Serum interleukin-4 (IL-4), on the other hand, was undetectable before and during the transfusion protocol. By day 3 PT, recipient spleen cells could mediate in vitro anti-P815 (auto), anti-EL4 (allo), and anti-R1.1 (third-party MHC) cytotoxicity, and these responses were maximal by day 7 PT. Concurrently, a significant reduction in the vitro ability of recipient splenocytes to respond to Concanavalin A (ConA) was observed; this was not seen with lipopolysaccharide (LPS) stimulation. Elevated levels of NO2- were found in the ConA culture supernatants from transfused mice at day 3 PT. Serum antidonor alloantibodies were detected by the fifth platelet transfusion. AMG treatment of recipient mice significantly inhibited the transfusion. Induced cytotoxicity and ConA-stimulated NO2- production, and restored ConA-induced proliferation to normal levels. AMG appeared to selectively inhibit platelet-induced alloantibody production in that it did not affect antibody production induced by transfusions with 10(5) allogeneic leukocytes or by immunization with a foreign protein antigen, human gamma globulin, in adjuvant therapy. These results indicate that an in vivo AMG-sensitive mechanism is essential for recipients to initiate a humoral IgG immune response against allogeneic platelets.

Induction of graft versus leukemia effects by cell-mediated lymphokine-activated immunotherapy after syngeneic bone marrow transplantation in murine B cell leukemia.

The feasibility of inducing graft versus leukemia (GVL) effects with allogeneic T cells in recipients of autologous bone marrow transplantation (BMT) was studied in a murine model (BCL 1) of human B cell leukemia/ lymphoma. Allogeneic cell therapy, induced by infusion with peripheral blood lymphocytes, a mixture of allogeneic spleen and lymph node cells and allogeneic activated cell therapy, induced by in vitro recombinant-interleukin-2(rIL-2)-activated allogeneic bone marrow cells in tumor-bearing mice, prevented disease development in adoptive BALB/c recipients. Concomitant in vivo activation of allogeneic lymphocytes with rIL-2 suppressed even more effectively the development of leukemia in secondary adoptive recipients of spleen cells obtained from treated mice. In contrast, in vivo administration of rIL-2 after syngeneic BMT, with or without equal numbers of syngeneic lymphocytes, led to disease development in secondary recipients. Our data suggest that effective cell therapy can be achieved after SBMT by allogeneic but not syngeneic lymphocytes and that anti-leukemic effects induced by allogeneic lymphocytes can be further enhanced by in vitro or in vivo activation of allogeneic effector cells with rIL-2. Therefore, cell therapy by allogeneic lymphocytes following autologous BMT could become an effective method for inducing GVL-like effects on minimal residual disease provided that graft versus host disease can be prevented or adequately controlled.

Antigen-Specific IL-4- and IL-10-Secreting CD4+Lymphocytes Increasein VivoSusceptibility toTrypanosoma cruziInfection

Control of macrophage parasiticidal function by treatment with recombinant cytokines or their neutralizing antibodies modifies the severity of experimentalTrypanosoma cruziinfections. However, so far, no directin vivoevidence has demonstrated changes in disease outcome after altering the initial ratios of parasite-specific IFN-γ and IL-10/IL-4-secretor cells in secondary lymphoid organs. To this end, a population of predominantly CD4+parasite-Ag-reactive, IL-4- and IL-10-secreting T lymphocytes derived fromT. cruzi-immunized mice was adoptively transferred to naive recipients. Compared with cell responses from normal mice, spleen cells of uninfected recipients proliferated significantly toT. cruziAg and produced much greater amounts of IL-4 and IL-10; lower IFN-γ levels and increased IL-4/IL-10 levels were induced by Con A stimulation. Recipient mice challenged withT. cruzipresented overwhelming tissue and blood parasitemia and early death, contrasting with typically resistant controls. Uninfected recipients did not exhibit tissue damage following cell transfer. No disease exacerbation occurred in recipients of OVA-reactive CD4+, IL-4/IL-10-secreting T lymphocytes stimulated with OVA at the start of infection. On Day 6 postinfection, not only spleen cells but also LN cells from infected recipients showed decreased production of IFN-γ and augmented secretion of IL-4/IL-10 compared to cells from untransferred infected mice. The results indicate that an imbalance of Th cell populations leading to the predominance of secreted IL-4 and IL-10 at the start of infection and the concomitant down-regulation of IFN-γ secretion reversed the host's resistance toT. cruzi.Moreover, transfer of anti-T. cruziTh2-type cells most likely favored thein vivoexpansion of parasite-specific host cells toward a Th2 phenotype.

Suppression of the antibody response to a polymorphic peptide from the platelet alloantigen integrin beta3 with low molecular weight antigen arrays.

The allelic human platelet alloantigens PIA1/PIA2 are determined by a 33 Leu-Pro substitution in the second disulfide loop of the integrin beta3 subunit of the fibrinogen receptor, alphaIIb beta3 (GPIIb-IIIa). Alloantibodies to PIA1 cause neonatal alloimmune thrombocytopenia. We studied the suppression of specific Ab production to a disulfide-looped peptide spanning the polymorphic region of integrin beta3, 24AWCSDEALPLGSPRCD39 (LPL). Mice immunized with LPL coupled to OVA (LPL-OVA) produced Abs specific for LPL. When immunized animals were injected with low m.w. dextran heavily derivitized with LPL (Dex(low)LPL(high)), levels of Ab to LPL fell immediately and remained low 1 mo later. Both high affinity and total Abs were affected. Arrays with lower peptide density or a high m.w. backbone did not induce well sustained suppression. Abs to OVA were unaffected by the arrays. Naive mice given Dex(low)LPL(high) were tolerant to subsequent immunization with LPL-OVA. In transfer experiments, irradiated recipients of spleen cells or purified 8 cells from animals suppressed with Dex(low)LPL(high) did not respond to LPL-OVA. Spleen cells from suppressed animals did not suppress the response to LPL-OVA in recipients of immune B cells. These results demonstrate that peptide arrays, by a mechanism sensitive to molecular configuration, induce tolerance to peptide immunization and suppress an ongoing, high affinity Ab response. Peptide arrays induce the elimination or irreversible anergy of specific memory B cells and do not require a non-B spleen cell population to maintain suppression.

Indirect Allorecognition of Platelets by T Helper Cells During Platelet Transfusions Correlates With Anti-Major Histocompatibility Complex Antibody and Cytotoxic T Lymphocyte Formation

To study the cellular immunology of platelet-induced alloimmunization, a murine transfusion model was developed. BALB/c (H-2d) recipient mice were transfused weekly with 2 x 10(8) platelets or 10(3) leukocytes from C57BL/6 (H-2b) donor mice. Recipient antidonor major histocompatibility complex (MHC) class I alloantibodies could be detected in flow cytometric assays by the fifth platelet transfusion. In contrast, when leukocytes only were transfused, alloantibodies were not detected. In vitro assays demonstrated that murine H-2b platelets were positive for MHC class I expression but lacked MHC class II molecules on their membranes and were unable to stimulate proliferation or cytokine production when incubated with naive H-2d spleen cells. In vivo, however, platelet transfusions induced two distinct patterns of cell-mediated reactivity. First, during the initial transfusions and before alloantibody formation, there was induction of T-cell anergy, characterized by the inability of recipient T cells to respond to Concanavalin A (ConA) or to proliferate in an antidonor mixed lymphocyte reaction (MLR), together with suppressed natural killer (NK) cell activity. This unresponsiveness was associated with a transient increase in nitric oxide (NO)-dependent cytotoxicity and interleukin-1 (IL-1) production. Second, once alloantibodies developed, significantly increased antidonor CD8+ cytotoxic T lymphocyte (CTL) and NK cell responses were observed. At this time, when recipient spleen cells were depleted of CD8+ T cells and incubated with only donor platelets in 7-day antigen-presenting cell (APC) assays, enhanced proliferation and IL-2 production occurred. These cellular responses were not seen when 10(3) allogeneic leukocytes were transfused. Thus, the results suggest that leukoreduced platelet transfusions induce antidonor MHC antibodies and CD8+ CTL responses in recipient mice. At the same time, the transfusions induced recipient CD4+ T-cell activation when incubated with donor platelets in the presence of syngeneic APCs, an indirect recognition pathway that correlates with the time of alloantibody production.

SYSTEMIC LIPOPOLYSACCHARIDE RECRUITS DENDRITIC CELL PROGENITORS TO NONLYMPHOID TISSUES

Dendritic cells (DC) are thought to be the "passenger leukocytes" that sensitize the recipients of organ transplants against graft antigens and trigger allograft rejection. DC originate from MHC class II-negative (Ia-) progenitors in the bone marrow, which enter the tissues and develop into migratory cells with the specialized capacity to initiate primary immune responses. There is little information on which stimuli recruit DC progenitors to the tissues. Systemic administration of LPS to mice depletes Ia+ leukocytes from heart and kidney but recruits Ia- leukocytes (Roake JA, et al., see footnote 6). When these leukocytes were isolated and cultured overnight, Ia+ low density leukocytes developed that could stimulate primary T cell responses in vitro. Hearts from LPS-treated mice were transplanted to allogeneic recipients. One to 4 days after grafting, Ia+ donor cells were present in recipient spleens, localized to peripheral white pulp, and associated with CD4+, but not CD8+, T cells. Cells with the migratory characteristics of DC, therefore, originated from Ia- progenitors in the transplanted hearts. We conclude that LPS recruits Ia- DC precursors to the heart and kidneys. Hearts from LPS-treated donors were rejected by allogeneic recipients at the same tempo as normal hearts, implying that Ia- DC progenitors might ultimately contribute to heart graft rejection (direct sensitization). However, since hearts from cyclophosphamide-treated donors, which do not give rise to Ia+ cells in recipient spleens, were also rejected at a similar tempo, indirect sensitization could also play a role in heart graft rejection in this model.

Pathogenesis of late airway changes in long-term surviving lung allografts.

Late airway changes are frequently observed in long-term surviving rat lung allografts. In this present study, we investigate this problem using the mixed lymphocyte reaction, graft-versus-host assay (popliteal lymph node assay), skin grafting from the donor strain to the lung allograft recipient, and histology to evaluate this problem. The results show that spleen cells from recipient rats with long-term surviving lung allografts possessed alloreactivity in mixed lymphocyte reaction against both donor and third-party ACI antigens. Suppressor cell activity was not detected in spleen cells from these recipient rats. The popliteal lymph node assay showed that the response to recipient spleen cells was detectable but weaker than that of naive spleen cells from the same strain. Mean survival time of skin grafts from the donor strain to long-term allograft survivors was significantly longer than that between donor and recipient strains. Chest roentgenograms revealed opacification of long-term surviving lung allografts at 2 to 3 wk after skin transplantation that was coincident with the rejection of the skin grafts, and histologic examination of the lung allografts revealed changes compatible with acute rejection. We conclude that alloreactivity can be demonstrated in rats with long-term surviving lung allografts and suggest that late airway changes in these lungs are immunologically mediated.

INDUCTION OF NONSPECIFIC X-IRRADIATION-RESISTANT SUPPRESSOR CELL ACTIVITY IN VIVO AND PROLONGATION OF VASCULARIZED ALLOGRAFT SURVIVAL BY SK&amp;F 105685, A NOVEL IMMUNOMODULATORY AZASPIRANE

SK&F 105685 is a novel azaspirane with immunosuppressive activity in animal models of autoimmune disease. This study evaluates the efficacy and mechanism of action of the compound in rat recipients of cardiac allografts. Short-term SK&F 105685 therapy (20 mg/kg/day by gavage) proved effective both in the pretreatment (days -14 to -8 or -7 to -1; allograft at day 0) and treatment (days 0 to 6) protocols, with cardiac allograft survival prolonged to 14-17 days (acute rejection = 7 days; P < 0.001). SK&F 105685 pretreatment exerted at least additive effects with subtherapeutic CsA (1.5 mg/kg/day x 7 days i.m.) given after transplantation, with 50% of allografts surviving > 50 days. SK&F 105685 therapy diminished the immunohistological features of acute rejection, with the cellular infiltrate suppressed and the induction of IL-2/transferrin receptors, and elaboration of IL-2/IFN-gamma essentially abolished, as compared with the grafts in untreated hosts. These correlated with normal frequency of CD4, CD5, CD8 phenotype subsets and B cells in recipient lymphoid organs, as shown by flow microfluorimetry. Adoptive transfer of untreated or x-irradiated (2000 rads) spleen cells from SK&F 105685-modulated hosts significantly prolonged the survival of donor-specific or third-party test cardiac allografts to 10-15 days, suggesting the presence of nonspecific x-irradiation-resistant suppressor cells in the transferred inoculum. Their activity could be enriched by Percoll density centrifugation and screened by the ability to inhibit Con A-driven proliferation of normal cells in the coculture assay. The light-density x-irradiation-resistant spleen cell fraction (1.07 g/ml) was consistently and significantly more suppressive than the heavy-density (1.09 g/ml) interface, or the corresponding unseparated cells. Thus SK&F 105685 therapy abrogates rejection response and significantly prolongs the survival of vascularized cardiac allografts in rats. This effect is associated with selective depression of host alloreactivity/immune activation at the graft site, and simultaneous induction of suppressor cells in recipient spleen, comparable to natural or nonspecific suppressor cells generated by TLI. This unique activity profile is consistent with the concept that SK&F 105685 should be considered as a critical chemical adjunct in novel therapeutic strategies representing TLI-equivalent.