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A W Thomson

Publications and source records attributed to A W Thomson.

At least 73 records · Page 4Linked to original sources
PubMed1999

Hepatic dendritic cells: immunobiology and role in liver transplantation.

Traditional investigations of hepatic dendritic cells (DC) have focused on immunohistochemical studies of these cells within normal and pathological liver tissue. The recent availability of reagents for the improved characterization of DC, together with cytokine-based methods for the expansion of liver DC both in vivo and in vitro have begun to provide new insight into the immunobiology of these important antigen-presenting cells. Hepatic DC probably play a key role in the host response to blood-borne pathogens, and in the pathogenesis of infectious and autoimmune liver diseases. They appear to be important in determining the balance between liver transplant tolerance and rejection. Their possible role in oral and portal venous tolerance remains to be defined. In this article, we focus on emerging aspects of hepatic DC immunobiology, with particular reference to liver transplantation.

Animals↗
PubMed1999

Trafficking of APC from liver allografts of Flt3L-treated donors: augmentation of potent allostimulatory cells in recipient lymphoid tissue is associated with a switch from tolerance to rejection.

Livers transplanted across major histocompatibility complex (MHC) barriers in mice are normally accepted without recipient immune suppression, and induce a state of functional tolerance. However, markedly increasing functional dendritic cells (DC) in the 'passenger leucocyte' population by donor pretreatment with the hematopoietic growth factor Flt3-ligand (Flt3L; 10 microg/day for 10 days) results in acute allograft rejection. In this study, molecular, immunohistochemical and flow cytometric analysis of donor cell traffick into recipient lymphoid tissue 24 h after liver transplantation (C57BL/10 [H2b]-->C3H [H2k]) was performed. In addition, the capacity of donor-derived cells in these tissues to stimulate host T cell proliferation was examined. Reverse transcriptase polymerase chain reaction analysis revealed increases in donor genomic DNA in both thymi and spleens of mice given livers from Flt3L-treated donors compared to controls. Donor MHC class II+ (IAb+) cells in spleens were strikingly elevated (10-fold) in the former group. Two-colour flow cytometry revealed a similar increase in donor-derived H-2Kb+/I-Ab+ cells, and in the incidence of donor leucocytes expressing CD40, CD80, and CD86. CD11c+ DC comprised approximately 40% of the I-Ab+ cells in spleens of mice given livers from Flt3L-treated donors. These changes were associated with the presence, in spleens, of potent allostimulatory activity for naive recipient strain T cells, that was not observed in normal liver recipients. Elicitation of allograft rejection, associated with enhanced trafficking of stimulatory donor antigen-presenting cells (APC), in particular DC, suggests that normal liver graft survival and tolerance induction may be linked to failure/counter-regulation of APC-driven stimulation of effective anti-donor T cell responses.

Animals↗
PubMed1999

Adenoviral delivery of CTLA4Ig into myeloid dendritic cells promotes their in vitro tolerogenicity and survival in allogeneic recipients.

Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) that initiate and modulate immune responses. They are essential for naive T cell activation, but may also play roles both in central and peripheral tolerance. Blockade of costimulatory pathways that provide the crucial second signal for lymphocyte activation is one strategy to augment the potential tolerogenicity of DC. Here, in vitro propagated DC were transduced using an adenoviral (Ad) vector to express the gene encoding cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4lg), which blocks interaction of CD80 and CD86 on DC with CD28 on T cells. Supernatants of AdCTLA4lg-transduced DC strikingly inhibited mixed leukocyte reactions (MLR) induced by non-transduced DC. Whereas transduction of marker genes (LacZ or enhanced green fluorescence protein (EGFP)) did not alter their potent allostimulatory activity, DC transduced with CTLA4lg exhibited striking reductions in cell surface staining for CD86, but not MHC class II, and were poor stimulators of T cell proliferation and cytotoxic T lymphocyte (CTL) responses. In addition, they induced alloantigen-specific T cell hyporesponsiveness. They were detected, following local injection, in significantly increased numbers in the lymphoid tissue of unmodified allogeneic recipients. This is the first report of the functional properties of DC genetically engineered to express CTLA4lg.

Abatacept↗
PubMed1998

Retroviral delivery of viral interleukin-10 into myeloid dendritic cells markedly inhibits their allostimulatory activity and promotes the induction of T-cell hyporesponsiveness.

BACKGROUND: Dendritic cells (DC) play critical roles in the initiation and modulation of immune responses and may determine the balance between tolerance and immunity. Viral interleukin-10 (vIL-10), encoded by the Epstein-Barr virus, is highly homologous to the "immunosuppressive" cytokine, mammalian IL-10. It impairs antigen-presenting cell function but lacks certain immunostimulatory properties of mammalian IL-10. We accomplished the following: (1) evaluated the effects of vIL-10 protein on DC phenotype and function, (2) transduced mouse bone marrow-derived DC to express vIL-10, and (3) assessed the impact of transgene expression on DC allostimulatory activity. METHODS: DC progenitors propagated from bone marrow of B10 (H2b) mice in granulocyte-macrophage colony-stimulating factor plus IL-4 were repeatedly transduced by centrifugation, using retroviral supernatant obtained from the BOSC 23 ecotropic packaging cell line. To evaluate transduction efficiency, DC were transduced with the retroviral vector MFG-enhanced green fluorescence protein as a marker gene. Transgene and key cell surface molecule expression were examined by flow cytometry. The level of vIL-10 gene product in the culture supernatant was quantitated by ELISA. DC function was assessed by evaluation of the ability of DC to induce allogeneic (C3H;H2k) T-cell proliferation and cytotoxic T lymphocytes in primary mixed leukocyte reactions. Secondary mixed leukocyte reactions were used to test for T-cell hyporesponsiveness. RESULTS: The early addition of vIL-10 protein to cultures inhibited DC maturation and function. vIL-10 gene transfer was achieved with an approximate transduction efficiency of 35 to 40%. Transduced DC expressed vIL-10 at a level of 40 ng/10(6) cells/48 hr. In comparison with controls, vIL-10-transduced cells showed decreased surface expression of major histocompatibility complex class II and costimulatory molecules, reduced ability to stimulate T-cell proliferation and cytotoxic T lymphocyte generation, and potential to induce alloantigen-specific hyporesponsiveness. CONCLUSIONS: DC can be effectively transduced to express vIL-10 and limit their ability to stimulate in vitro. These genetically engineered antigen-presenting cells may have therapeutic potential to inhibit undesired immune responses to allo- or autoantigens.

Animals↗
PubMed1998

Systemic administration of anti-interleukin-10 antibody prolongs organ allograft survival in normal and presensitized recipients.

BACKGROUND: Systemic administration of cellular interleukin (IL)-10 at a dose of 100 microg/day for 1 week after transplantation accelerates mouse cardiac allograft rejection across MHC barriers. This effect is associated with enhancement of donor-specific cytotoxic T lymphocyte and alloantibody (alloAb) titers. To further evaluate the in vivo role of IL-10, we tested the influence of a neutralizing anti-IL-10 monoclonal antibody (mAb) in both normal and donor (skin) presensitized mouse organ allograft recipients. METHODS: Heart or liver transplants were performed from B10 (H2b) donors to C3H (H2k) recipients. Anti-IL-10 mAb (SXC.I) was administered intravenously in a single injection or repeated once daily injections. Cytotoxic activity of graft-infiltrating cells was determined by 51Cr-release assay. Circulating alloAb levels were quantified by complement-dependent cytotoxicity and flow cytometry. RESULTS: Survival of vascularized B10 cardiac allografts in normal recipients was prolonged significantly in the mAb-treated groups. A single injection of 1 mg of anti-IL-10 mAb immediately after heart transplantation gave a similar graft median survival time to repeated injections of lower dose mAb (0.5 mg/day for 6 days after transplantation) (Ig isotype control 11 days; single mAb injection 18 days; multiple injection 20 days). In presensitized recipients, anti-IL-10 mAb from days 0 to 6 significantly prolonged survival of both cardiac and orthotopic liver grafts. Graft median survival time was extended from 5 to 10 days and from 4 to 11 days, respectively. Prolongation of liver allograft survival in presensitized recipients was associated with suppression of circulating alloAb levels and with significant reductions in the incidence of B220+ cells in both grafts and recipient spleens. CONCLUSIONS: The data support an adverse role of anti-IL-10 in allograft rejection; it seems that by reducing alloAb responses, anti-IL-10 mAb may have potential for use as a therapeutic immunosuppressant, particularly in presensitized organ allograft recipients.

Animals↗
PubMed1998

Phenotype, function, and in vivo migration and survival of allogeneic dendritic cell progenitors genetically engineered to express TGF-beta.

BACKGROUND: Administration of donor bone marrow (BM)-derived dendritic cell (DC) progenitors (DCp) that are major histocompatibility complex (MHC) class II+ but costimulatory molecule (CD40, CD80, CD86)-deficient can prolong mouse heart allograft survival This is associated with microchimerism and inhibition of antidonor cytotoxic T lymphocyte (CTL) activity. Genetic modification of these donor antigen-presenting cells to express an immunosuppressive molecule(s) may enhance their in vivo survival and potential tolerogenicity. METHODS: The surface phenotype of B10(H-2b) DCp before and after gene transfer using replication-deficient adenoviral (Ad) vectors was determined by monoclonal antibody (mAb) staining and flow cytometry. Transforming growth factor-beta (TGF-beta) production was quantitated by enzyme-linked immunosorbent assay. Allostimulatory activity of the gene-transduced DCp was ascertained by mixed leukocyte reaction (MLR) and CTL induction. To assess their in vivo migratory activity and survival, the transduced cells were injected subcutaneously into one hind footpad of C3H (H-2k) mice. Tissues (draining popliteal lymph nodes [LN], spleens, and thymi) were removed 1, 2, 7, and 14 days later and stained for donor MHC class II using anti-LA(b) mAb in an immunohistochemical procedure. The mean number of IAb+ cells per unit area was determined. RESULTS: Transduction with a control Ad vector (Ad-LacZ) at 50 multiplicity of infection slightly increased CD40 and CD86 expression and up-regulated the poor allostimulatory activity of the DCp assessed by MLR and CTL responses. These effects on function were negated in Ad-TGF-beta1-transduced cells. After their injection into mouse footpads, the gene-transduced IAb+ cells were observed in maximal numbers in the popliteal LN at day 1 and in marginal zones and T-dependent areas of spleens (peak at day 7) but were rare in thymi. Transduction with Ad-LacZ reduced the numbers of IAb+ cells identified in both LN and spleens at all time points postinjection, suggesting that the vector alone affected DC life span in allogeneic recipients. TGF-beta1 transgene expression not only fully prevented the reduction in DC induced by Ad transduction alone, but also increased numbers and prolonged the survival of donor cells in the spleen, as shown by a two-to fivefold increase in IAb+ cells at days 2-14 compared with control (Ad-LacZ-transduced) DC. CONCLUSION: BM-derived DCp can be transduced efficiently to express TGF-beta1 using an Ad vector. They exhibit very poor allostimulatory activity and similar migration characteristics in vivo to unmodified DCp. Survival of TGF-beta gene-transduced DC, however, is enhanced significantly compared with unmodified and (especially) control Ad-LacZ gene-transduced DC. Genetic engineering of donor DC to express the immunosuppressive molecule TGF-beta promotes their survival in allogeneic hosts and may potentiate their previously reported tolerogenicity.

Adenoviridae↗
PubMed1998

Expansion of functional NK cells in multiple tissue compartments of mice treated with Flt3-ligand: implications for anti-cancer and anti-viral therapy.

The generation and activity of NK cells appear to be regulated by a particular set of cytokines. We examined the in vivo effects of recombinant human Flt3 ligand (Flt3-L), a recently cloned potent hemopoietic cytokine, on NK cell development in mice. Daily i.p. administration of Flt3-L consistently induced striking increases in both the absolute number and the total cytotoxic activity of mature nonactivated NK cells within various tissues. Dose- and time-dependent increases were observed in the bone marrow (approximately 2- and approximately 11-fold, respectively), thymus (approximately 2.8- and approximately 2.0-fold), blood (approximately 11- and approximately 15-fold), spleen (approximately 10- and approximately 9-fold), and liver (approximately 15- and approximately 39-fold). In addition, IL-2 induced a rapid increase in NK activity, NK cell proliferative responses, generation of lymphokine-activated killer activity, and development of activated adherent NK cells, which were all significantly increased by Flt3-L treatment. Thus, in addition to its recently reported capacity to stimulate dendritic cell production, Flt3-L has a prominent biologic role in NK cell generation in vivo. This is probably a result of selectively induced expansion of NK cell progenitors (pro-NK cells), because Flt3-L stimulates in vitro proliferation of pro-NK cells without affecting the cytotoxicity of mature NK cells. The results also indicate that either alone or in combination with a potent activator of NK cells, such as IL-2, Flt3-L could be used to markedly augment the number and activity of NK cells, especially in the liver. Flt3-L appears to have considerable potential for therapy of both cancer and viral infection.

Adjuvants, Immunologic↗
PubMed1998

Lymphoid/nonlymphoid compartmentalization of donor leukocyte chimerism in rat recipients of heart allografts, with or without adjunct bone marrow.

BACKGROUND: The role of leukocyte migration and chimerism in organ allograft acceptance has been obscured by the lack of information about the late localization of the donor cells. METHODS: Male Lewis rat-->female Brown Norway abdominal heart transplantation was performed under tacrolimus immunosuppression (days 0-13, 20, and 27) with or without donor bone marrow and (in bone marrow subgroups) a 1-week postoperative course of a possibly chimerism-enhancing drug. Using rat sex-determining region-Y-specific oligonucleotide primers, we determined the donor DNA concentration by polymerase chain reaction in serial venous blood samples for 100 days and in tissue specimens when animals were killed. RESULTS: Chimerism was detected out to 56 days in 89% of the blood samples but in none of the samples at 100 days. However, donor DNA was detected when animals were killed in 95% of the native hearts, 80% of the skin biopsy specimens, and 23% of the spleens. The presence and quantity of early and late chimerism were strongly correlated the administration of adjunct bone marrow and with a reduction in the vasculopathy and inflammation index in the cardiac allografts. Marginally significant further increases in chimerism and/or reductions in chronic heart rejection beyond those achieved with adjunct bone marrow alone were associated with additional treatment with the growth factors Flt-3 ligand, granulocyte colony-stimulating factor, and a recombinant molecular variant of interleukin-6 (interleukin-6 mutein) but not with hepatocyte growth factor or lisofylline. CONCLUSIONS: The previously suspected shift of early chimerism in the blood and lymphoid organs to dominance in host nonlymphoid tissues is consistent with the dual mechanisms of clonal exhaustion and immune indifference, governed by antigen migration and localization, that have been postulated elsewhere to account for organ allograft acceptance.

Adjuvants, Immunologic↗
PubMed1998

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 activity.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Animals↗
PubMed1998

Flt-3 ligand increases microchimerism but can prevent the therapeutic effect of donor bone marrow in transiently immunosuppressed cardiac allograft recipients.

C3H (H2k) mice received 50 x 10(6) B10 (H2b) bone marrow (BM) cells either alone or with flt-3 ligand (FL) (10 microg/day), tacrolimus (2 mg/kg/day), or both agents for 7 days. Donor MHC class II+ (IAb+) cells were quantitated in spleens by immunohistochemical analysis, and donor class II DNA detected in BM by PCR. Donor cells were rare in the BM alone and BM + FL groups, whereas there was a substantial increase in chimerism in the BM + tacrolimus group. Addition of FL to BM + tacrolimus led to a further eightfold increase in donor cells and enhanced donor DNA compared with the BM + tacrolimus group. This increase in donor cells was almost 500-fold compared with BM alone. C3H recipients of B10 heart allografts given perioperative B10 BM and tacrolimus (days 0-13) exhibited a markedly extended median graft survival time (MST, 42 days) compared with those given tacrolimus alone (MST, 22 days). Addition of FL (10 microg/day; 7 days) to BM + tacrolimus prevented the beneficial effect of donor BM (MST, 18 days). BM alone or BM + FL resulted in uniform early heart graft failure (MST < 8 days). Functional studies revealed maximal antidonor MLR and CTL activities in the BM- and BM + FL-treated groups, with minimal activity in the tacrolimus-treated groups. Thus, dramatic growth factor-induced increases in chimerism achieved under cover of immunosuppression may result in augmented antidonor T cell reactivity and reduced graft survival after immunosuppressive drug withdrawal. With FL, this may reflect striking augmentation of immunostimulatory dendritic cells.

Animals↗
PubMed1998

Donor bone marrow potentiates the effect of tacrolimus on nonvascularized heart allograft survival: association with microchimerism and growth of donor dendritic cell progenitors from recipient bone marrow.

BACKGROUND: The influence of donor hematopoietic cell microchimerism on organ allograft survival has been studied largely in vascularized transplant models. Here, we examine the impact of donor bone marrow (BM) cells administered intravenously together with transient systemic tacrolimus therapy on microchimerism, the survival of nonvascularized cardiac allografts, and growth of donor antigen-presenting cells [dendritic cells (DCs)] from recipient BM. METHODS: Adult male C3H (H2k) mice received heterotopic heart transplants from B10 (H2b) donors in the dorsal ear pinna. They were given no further treatment, or either a short course of tacrolimus (FK506; 2 mg/kg i.p. from day 0 to day 13), unmodified donor BM cells (50x10(6) i.v. on day 0) or both treatments. Grafts were examined daily for contractile activity. Anti-donor cytotoxic T lymphocyte responses were determined in recipients' spleens. Microchimerism (IAb+ cells) was demonstrated by immunocytochemical staining of spleens, and of cells expanded from recipient BM using cytokines and culture conditions that promote the growth of DCs. RESULTS: Tacrolimus alone significantly prolonged median heart graft survival time from 10 to 22 days (P<0.001). BM alone failed to prolong graft survival. By contrast, tacrolimus + donor BM resulted in a mean survival time of 42 days (P<0.01 compared with tacrolimus treatment alone). This marked increase in heart allograft survival was associated with reduced anti-donor cytotoxic T lymphocyte responses attributable to a nonspecific effect of tacrolimus. In addition, however, a link was observed between the beneficial effect of donor BM and comparatively large numbers of donor major histocompatibility complex class II (IAb+)-positive cells in recipients' spleens, and in cultures of granulocyte-macrophage colony-stimulating factor + interleukin-4-stimulated DCs from recipients' BM. No donor-derived cells were propagated from heart graft recipients given either tacrolimus or donor BM alone. CONCLUSIONS: This nonvascularized organ transplant model demonstrates the positive effect on allograft survival of donor BM given at the time of transplant to transiently immunosuppressed recipients. The findings also reveal links between hematopoietic cell chimerism, the presence of donor DC progenitors in recipient BM, and organ allograft survival.

Animals↗
PubMed1998

Impact of Flt-3 ligand on donor-derived antigen presenting cells and alloimmune reactivity in heart graft recipients given adjuvant donor bone marrow.

The influence of the haematopoietic growth factor Flt-3 ligand (FL) on the incidence and function of donor major histocompatibility complex (MHC) class II+ cells in the lymphoid tissues of noncytoablated recipients of heart allografts and donor bone marrow (BM) cells was investigated. C3H (H2k) mice received a nonvascularized B10 (H2b) heart allograft in the dorsal ear pinna, followed by an i.v. infusion of 50 x 10(6) donor BM cells. They were given FL (10 microg/day i.p., x7 days), tacrolimus (2mg/kg/day i.p., x13 days) or both agents immediately following heart transplantation (HTx) and were killed 10 or 21 days later. Their BM cells were propagated in vitro in granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 for 5 days to promote the growth of dendritic cells (DC). Donor DC were identified by immunocytochemical staining. Spleens were harvested, and donor (IAb+) cells enumerated by immunohistochemical analysis. Donor MHC class II DNA was detected in spleens and cultured BM-derived cells by reverse transcriptase-polymerase chain reaction (RT-PCR). A striking increase in donor MHC class II+ cells was noted in both the spleen and BM of the BM + tacrolimus-treated group compared to either the BM alone, or BM + FL-treated groups. Addition of FL treatment to BM + tacrolimus led to a further increase in donor cells in spleen (three-fold at 10 days, and two-fold at 21 days). The increase in donor cells at 10 days was almost 140-fold compared to that with donor BM alone. PCR analysis at this time revealed enhanced donor DNA in the BM + FL + tacrolimus group compared to that in the BM + tacrolimus group. FL treatment augmented mixed leucocyte reactions (MLR) and cytotoxic T lymphocyte (CTL) activity of host spleen cells against donor alloantigens. These effects were reversed by tacrolimus administration. Histopathology of heart grafts from tacrolimus-treated animals at 10 and 21 days showed absence or substantial reduction in cellular infiltration, and the preservation of viable myocardium. By contrast, in untreated mice, or animals given BM or BM + FL alone, there was marked cellular infiltration, and features of accelerated rejection. Donor-derived DC could be propagated in vitro from the BM of heart transplant recipients given donor BM, especially from mice that also received tacrolimus +/- FL. At day 21, donor-derived cells could only be propagated from the BM + FL + tacrolimus-treated group. These findings show that numbers of donor antigen presenting cells (APC) or their progenitors can be markedly increased in conventionally immunosuppressed organ allograft recipients given donor BM + a potent haematopoietic and DC-growth promoting cytokine. Although withdrawal of systemic immunosuppression appears to allow exhibition of the potential allostimulatory activity of these donor APC leading to rejection, the model provides a useful basis for further evaluation of the persistence and manipulation of donor haematopoietic cells and in particular, donor-derived APC, on the outcome of organ transplantation.

Adjuvants, Immunologic↗
PubMed1997

Blockade of the CD40-CD40 ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival.

BACKGROUND: Failure of costimulatory molecule-deficient donor dendritic cells (DCs) to induce indefinite allograft acceptance may be a result of the 'late" up-regulation of these molecules on the DCs after interaction with host T cells. Ligation of CD40 on antigen-presenting cells by its cognate ligand CD40L is thought to induce expression of CD80 (B7-1) and CD86 (B7-2). We examined the influence of anti-CD40L monoclonal antibody (mAb) on the capacity of donor-derived DC progenitors to induce long-term allograft survival. METHODS: High purity DC progenitors were grown from B10 (H2b) mouse bone marrow in granulocyte-macrophage colony-stimulating factor and transforming growth factor beta1 (TGFbeta1). Mature DC were propagated in granulocyte-macrophage colony-stimulating factor and interleukin-4. Their phenotype was characterized by flow cytometric analysis and their function by mixed leukocyte reactivity. Anti-donor cytotoxic T lymphocyte activity in grafts and spleens of vascularized heart allograft recipients was also assessed. RESULTS: The TGFbeta3-cultured cells were (1) DEC 205-positive, MHC class II-positive, CD80dim, CD86dim, and CD40dim, (2) poor stimulators of naive allogeneic T-cell proliferation, and (3) able to prolong significantly B10 cardiac allograft survival in C3H (H2k) recipients when given (2 x 10[6] i.v.) 7 days before organ transplantation (median survival time [MST] 26 days vs. 12 days in controls, and 5 days in interleukin-4 DC-treated animals). Their allostimulatory activity was further diminished by addition of anti-CD40L mAb at the start of the mixed leukocyte cultures. Anti-CD40L mAb alone (250 microg/mouse, i.p.; day -7) did not prolong cardiac graft survival (MST 12 days). In contrast, TGFbeta-cultured DCs + anti-CD40L mAb extended graft survival to a MST of 77 days, and inhibited substantially the anti-donor cytotoxic T lymphocyte activity of graft-infiltrating cells and host spleen cells assessed 8 days after transplant. CONCLUSIONS: The CD40-CD40L pathway appears important in regulation of allogeneic DC-T-cell functional interaction in vivo; its blockade increases markedly the potential of costimulatory molecule-deficient DCs of donor origin to induce long-lasting allograft survival.

Animals↗
PubMed1997

Augmentation of dendritic cells in murine organ donors by Flt3 ligand alters the balance between transplant tolerance and immunity.

Treatment of mice with the recently cloned hemopoietic growth factor Flt3 ligand (FL; 10 microg/day for 10 days) resulted in a large increase in myeloid lineage cells within the liver. While the number of nonparenchymal cells (NPC) harvested from liver increased about 9-fold, a 90-fold increase was observed in the proportion of CD11c+ dendritic cells (DC) recovered from NPC following overnight (18-h) culture in granulocyte-macrophage CSF. In contrast, only a 50% increase was seen in CD11c+ cells within heart single cell suspensions and in the number of DC obtained from hearts after 18-h culture. Liver NPC and heart cell suspensions freshly isolated from 10-day FL-treated animals exhibited increased T cell allostimulatory capacity compared with controls. Overnight cultured DC from livers of FL-treated animals expressed both higher levels of costimulatory molecules (CD80 and CD86) and allostimulatory activity than those from controls. Heart-derived DC also displayed enhanced stimulatory capacity. Pretreatment of organ donors with FL for either 5 or 10 days before transplant of organs to normal recipients abrogated the spontaneous liver allograft acceptance normally observed and resulted in delayed or acute graft rejection (median survival times, 40 and 12 days, respectively). Heart rejection was significantly accelerated by pretreatment of donors with FL for 5 or 10 days (median survival times, 8 and 7 days, respectively, vs 12 days in controls). These novel findings reveal the potent immunologic adjuvant properties of FL in vivo. They also show that substantial augmentation of the number of potential allostimulatory cells in donor organs before transplantation favors rejection rather than tolerance induction.

Animals↗