Biomedical subjects
Publications and source records attributed to A W Thomson.
Many new insights have been gained over the past 5 years into the mechanisms that regulate immune reactivity to cell and organ transplants. This new knowledge is being applied to the development of innovative experimental strategies that may soon be evaluated in the clinic.
The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.
Liver allografts are accepted spontaneously in all mouse strain combinations without immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we examined the effect of CD4+ CD25+ T regulatory cells (Treg) on the induction of mouse liver transplant tolerance. Orthotopic liver transplantation was performed from B10 (H2b) to C3H (H2k) mice. Depleting rat anti-mouse CD25 mAb (PC61) was given to the donors or recipients (250 microg/d IP) pretransplant or to the recipients postoperatively. At day 5 posttransplantation, both effector T cells (mainly CD8) and CD4+ CD25+ Treg were increased in the liver allografts and host spleens compared to naïve mice. Anti-CD25 mAb administration, either pretransplantation or posttransplantation, reduced the ratio of CD4+ CD25+ Treg to the CD3 T cells of liver grafts and recipient spleens and induced liver allograft acute rejection compared to IgG treatment. Anti-CD25 mAb administration elevated anti-donor T-cell proliferative responses and CTL and NK activities of graft infiltrates and host splenocytes; reduced CTLA4, Foxp3, and IDO mRNA levels; increased IL-10 and IFN-gamma; and decreased IL-4 mRNA levels in the livers or host spleens. The number of apoptotic T cells was reduced significantly in the liver grafts and treated host spleens. Therefore, anti-CD25 mAb administration changed the balance of CD4+ CD25+ Treg to activated T cells of liver graft recipients, preventing liver transplant tolerance. This was associated with enhanced anti-donor immune reactivity, downregulated Treg gene expression, and reduced T cell apoptosis in the grafts and host spleens.
Tolerance induction against donor allo-antigens (allo-Ag) remains one of the most challenging aspects of transplant immunology. The ability of dendritic cells (DC) to participate in immunity and tolerance makes them an excellent tool for tolerance induction. Here, we employed the immunosuppressive properties of apoptotic cells to deliver simultaneously an inhibitory signal and donor allo-Ag to recipient DC for treatment of allograft rejection. DC that captured apoptotic cells remained immature and activated deficiently anti-donor CD4(+) T cells that were unable to upregulate T-cell activation markers, to secrete IL-2 and IFN-gamma and to survive under homeostatic conditions due to low expression of Bcl-X(L), IL-7R and IL-15R. Administration of donor apoptotic cells decreased the systemic anti-donor T- and B-cell response and prolonged cardiac allograft survival in mice. The effect was donor specific and required the interaction of donor apoptotic cells with recipient quiescent CD8alpha(+) DC. When combined with CD40-CD154-blockade, administration of donor apoptotic cells resulted in indefinite graft survival mediated by generation of regulatory T cells. The use of the inhibitory effects of apoptotic cells on the anti-donor response provides a new approach to treat transplant rejection.
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Infusion of donor immature dendritic cells (DC) can significantly prolong survival of organ allografts, and this is believed to be due to antigen recognition by T cells in the absence of co-stimulation. In this study we report that a single pre-operative infusion of donor-mobilized immature plasmacytoid dendritic cells (pDCs) is superior to that of other DC sub-sets in suppressing allograft rejection. The combination of pDC infusion with injection of anti-CD154 monoclonal antibody further inhibited graft rejection and, in 50% of the mice, led to indefinite graft survival. This finding suggests a role for the plasmacytoid DC sub-set in facilitating organ transplant survival and also in the treatment of autoimmune disorders.
Dendritic cells (DC) exhibit remarkable plasticity in terms of their ability to induce and regulate innate and adaptive immune responses. Human and mouse interferon alpha-producing plasmacytoid (p) DC have been found to regulate allogeneic T cell responses in vitro. Evidence is emerging that pDC may also regulate immune reactivity in vivo, including the responses that underlie graft-versus-host disease and organ transplant rejection. These cells may also offer potential for therapy of adverse immune responses and the promotion of tolerance induction.
We examined the recovery of circulating monocytoid (Lin- CD11+ HLA-DR+) and plasmacytoid (Lin- CD123+ HLA-DR+) precursor (pre) dendritic cell (DC) subsets after allogeneic stem cell transplantation (SCT) in 39 children, using age-matched healthy children as controls. The frequencies of DCs in peripheral blood samples were determined by flow cytometry. The initial recovery of DC occurred simultaneously with myeloid engraftment. However, with time, DC subset values declined, being very low 40-50 days after SCT. Low monocytoid and plasmacytoid DC values were associated significantly with the development of severe acute graft-versus-host disease (aGVHD) (P=0.042 and 0.017, respectively). Plasmacytoid DC values were lower than in the age-matched controls for the entire follow-up period (range 102-2569 days), although, with time, values approached normal levels. Normal monocytoid DC numbers were observed within 300-400 days post SCT. The severity of chronic GVHD did not correlate with quantitative recovery of DC. We conclude that in pediatric SCT, initial recovery of DC production is concurrent with that of myelopoiesis, yet with time, DC subset values decline and low counts are associated with severe aGVHD. Monocytoid DC numbers approach normal levels within a year of SCT, but plasmacytoid DC counts recover very slowly.
Our knowledge of the role of dendritic cells (DC) in the generation and maintenance of T-cell tolerance has expanded rapidly and is now a key area of research in basic and applied DC biology. This minireview highlights recent developments in the field that are leading to new avenues for exploiting DC in the promotion of transplant tolerance.
Dendritic cells (DC) are a heterogeneous group of uniquely well-equipped bone marrow-derived antigen-presenting cells. They circulate in blood as precursor cells (preDC). In humans, two blood-borne subtypes of preDC can be distinguished by their differential expression of CD11c (CD11c+ preDC; monocytoid DC) and CD123 (CD123+ preDC; plasmacytoid DC). We studied the incidence of monocytoid and plasmacytoid DC in peripheral blood samples from 39 children of various ages (0.4-16.8 years) by flow cytometry, and found a significant negative correlation between the number of plasmacytoid DC and age (r = 0.421, P = 0.012). Monocytoid DC counts did not change significantly with age. Similarly, we analysed DC subsets in 19 children with cancer at the time of diagnosis prior to initiation of any myelosuppressive or antiproliferative treatment and compared the results with those obtained from gender- and age-matched control children. Patients with cancer had significantly less circulating monocytoid DC than controls (medians 13.2 versus 21.4 cells/ micro l, respectively, P = 0.042) at diagnosis, whereas absolute plasmacytoid DC counts did not differ significantly between the study groups. However, clinical outcome of the children with cancer (2.9-5 years follow-up after diagnosis) correlated with plasmacytoid DC count. Children with high plasmacytoid DC counts at diagnosis (above median) survived significantly worse (6/10 deceased) than those with low counts (1/9 deceased) (P = 0.034). Thus, circulating plasmacytoid DC counts are related to age during childhood, and development of cancer is associated with low number of monocytoid DC. A low circulating plasmacytoid DC count at diagnosis was a good prognostic sign.
C57BL/10 (B10; H2(b)) bone marrow-derived myeloid dendritic cells (DC) propagated in GM-CSF + IL-4 were transduced with r adenoviral (Ad) vectors encoding either control neomycin-resistance gene (Ad-Neo) or murine IL-4 (Ad-IL-4) on day 5 of culture following CD11c immunomagnetic bead purification. Both Ad-Neo- and Ad-IL-4-transduced DC displayed upregulated surface MHC class II and costimulatory molecules (CD40, CD80, CD86). Ad-IL-4 DC secreted higher levels of bioactive IL-12p70 after CD40 ligation or LPS stimulation than either Ad-Neo or unmodified DC. Only Ad-IL-4 DC produced IL-12p70 in primary MLR, in which they induced augmented proliferative responses of naïve allogeneic C3H/HeJ (C3H; H2(k)) T-cells. Compared with Ad-Neo DC, Ad-IL-4 DC were also more effective in priming naïve allogeneic recipients to exhibit specifically enhanced anti-donor T-cell proliferative and CTL responses. T-cells primed in vivo 7 days previously with Ad-IL-4 DC displayed enhanced secretion of Th2 (IL-4, IL-10) but also higher Th1 cytokine (IFNgamma) production following ex vivo challenge with donor alloAg. Moreover, pretreatment of vascularized heart graft recipients with i.v. Ad-IL-4 DC, 1 week before transplant, significantly accelerated rejection and antagonized the therapeutic effect of anti-CD40L (CD154) mAb. These data contrast markedly with recently reported inhibitory effects of autologous Ad-IL-4 DC on autoimmune inflammatory disease.
Dendritic cells (DC) are uniquely able to either induce immune responses or to maintain the state of self tolerance. Recent evidence has shown that the ability of DC to induce tolerance in the steady state is critical to the prevention of the autoimmune response. Likewise, DC have been shown to induce several type of regulatory T cells including Th2, Tr1, Ts and NKT cells, depending on the maturation state of the DC and the local microenvironment. DC have been shown to have therapeutic value in models of allograft rejection and autoimmunity, although no success has been reported in allergy. Several strategies, including the use of specific DC subsets, genetic modification of DC and the use of DC at various maturation stages for the treatment of allograft rejection and autoimmune disease are discussed. The challenge for the future use of DC therapy in human disease is to identify the appropriate DC for the proposed therapy; a task made more daunting by the extreme plasticity of DC that has recently been demonstrated. However, the progress achieved to date suggests that these are not insurmountable obstacles and that DC may become a useful therapeutic tool in transplantation and autoimmune disease.
Hepatic dendritic cells (DC) unquestionably play important roles in the induction and regulation of immune responses. Due to their paucity, functional characterisation of these important antigen presenting cells has been slow but use of DC growth factors (in particular GM-CSF and Flt3L) that markedly enhance their numbers has proved helpful in furnishing adequate study material. While there is growing evidence that DC function is affected in the pathogenesis of liver disease, most work to date has been performed on non-hepatic DC. Increasing knowledge of hepatic DC biology is likely to improve our understanding of disease pathogenesis and resistance to and therapy of liver disease.
Possible mechanisms by which liver antigen presenting cells (APC) may facilitate tolerance induction are discussed. Tolerance may be facilitated by a distinctive combination of factors, linked to the unique anatomical and microenvironmental features of the liver.
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BACKGROUND: Considerable interest has focused recently on murine CD8alpha- and CD8alpha+ dendritic cell (DC) subsets, because of their roles in initiating and regulating immune responses. Attention has also centered on their presumed human counterparts, DC1 and DC2, respectively, and their precursors. Identification and quantification of these subsets in the blood may be crucial to understanding and monitoring of their immunologic significance, particularly in humans, where blood may be the only tissue readily or routinely available. METHODS: Leukocytes were isolated from anticoagulated human or mouse (C57BL/10J) blood using conventional procedures. Four-color, rare-event, flow cytometric analysis was used to identify DC1 precursors (pDC1; lineage [lin]- CD4+ CD11c+ HLA-DR+) or DC2 precursors (pDC2; lin- CD4+ CD11c- CD123(hi) [IL-3Ralpha(hi)] HLA-DR+) in normal humans. In mice, CD8alpha+ (CD11b(lo), CD11c+) and CD8alpha- (CD11b(hi), CD11c+) DC subsets were identified both in normal animals and after administration of the potent DC growth factor, fms-like tyrosine kinase 3 ligand (Flt3L). RESULTS: All human subjects examined had discrete populations of pDC1 and pDC2 comprising approximately 0.6% and 0.1% of blood mononuclear cells. CD8alpha- and CD8alpha+ DC constituted approximately 0.75% and 0.2%, respectively, of blood mononuclear cells in normal mice, and 12% and 0.5%, respectively, in Flt3L-treated animals. Flt3L administration substantially increased the absolute numbers of circulating CD11c+ DC by approximately 200-fold. CONCLUSIONS: In addition to pDC1 and CD8alpha- DC, pDC2 and CD8alpha+ DC can be identified in normal human or mouse blood, respectively. Monitoring and isolation or characterization of these cells may provide novel insights into their functional significance in transplantation and other clinical conditions.
BACKGROUND: Mouse liver grafts are accepted across major histocompatibility complex (MHC) barriers and induce donor-specific tolerance without immunosuppressive therapy. By contrast, hepatic allografts from donors treated with the hematopoietic growth factor fms-like tyrosine kinase 3 ligand (FL), which dramatically increases hepatic interstitial dendritic cells (DC), are rejected acutely (median survival time 5 days). This switch from tolerance to rejection is associated with a marked reduction in apoptotic activity of graft-infiltrating T cells. We hypothesized that T-cell costimulation, provided by markedly enhanced numbers of donor antigen presenting cells (APCs), might inhibit apoptosis, promote expansion of T helper 1 cells and play a key role in acute liver rejection. METHODS: C3H (H2k) recipients of orthotopic liver grafts from FL-treated B10 (H2b) donors were given cytotoxic T-lymphocyte antigen 4: immunoglobulin (CTLA4Ig), a chimeric fusion protein that blocks the B7-CD28 costimulatory pathway, or control human immunoglobulin (200 microg) on the day of transplantation (day 0). Livers and spleens were removed on day 4. Cryostat sections were stained for interleukin (IL)-12 or by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL). Expression of mRNA encoding interferon (IFN)-gamma and IL-10 was determined by RNase protection assay. Suspensions of graft-infiltrating cells (GICs) and spleen cells were analyzed for apoptotic (TUNEL+) T-cell subsets by flow cytometry. CTL activity of GICs and circulating alloantibody levels were determined by cytotoxicity assays. RESULTS: Survival of liver grafts from FL donors was markedly prolonged by CTLA4Ig administration. This effect was associated with reductions in IFN-gamma and IL-10 gene transcripts within the GIC population, and with decreases in donor-specific CTL and NK cell activities and circulating anti-donor alloantibody levels. At the same time, there were marked increases in TUNEL+ CD4+ and especially CD8+ cells, both within the grafts and in the spleens of CTLA4Ig-treated mice. CONCLUSIONS: Signaling via the B7-CD28 pathway appears to play a key role in the switch from tolerance to rejection that is precipitated by markedly enhanced numbers of donor DCs. Inhibition of acute liver allograft rejection by CTLA4Ig, linked to restoration of apoptotic activity of graft-infiltrating T cells, further suggests that deletion of these cells may be critical for promotion of long-term allograft survival.
The mission of the recently established Immune Tolerance Network includes the development of protocols for the induction of transplant tolerance in organ allograft recipients and the development of assays that correlate with and may be predictive of the tolerant state. The state of clinical organ transplant tolerance seems to already exist in a small minority of conventionally immunosuppressed liver and, more rarely, kidney transplant patients. Immunosuppressive drug therapy has been withdrawn from these patients for a variety of reasons, including protocolized weaning for a uniquely large group of liver patients at the University of Pittsburgh. In this study, we propose to evaluate the validity of a variety of in vitro immunologic and molecular biologic tests that may correlate with, and be predictive of, the state of organ transplant tolerance in stable liver patients off immunosuppression. Only peripheral blood will be available for the execution of these tests. Both adult and pediatric liver graft recipients will be studied, in comparison to appropriate controls. We shall examine circulating dendritic cell (DC) subsets [precursor (p) DC1 and p DC2] including cells of donor origin, and assess both the frequency and function of donor-reactive T cells by ELISPOT and by trans-vivo delayed-type hypersensitivity analysis in a surrogate murine model. Cytokine gene polymorphism and alloantibody titers will also be investigated. It is anticipated that the results obtained may provide physicians with a tolerance assay "profile" that may determine those patients from whom immunosuppressive therapy may be safely withdrawn.