anti-CD40 Ligand mAb Research Articles

Costimulation Blockade of Both Inducible Costimulator and CD40 Ligand Induces Dominant Tolerance to Islet Allografts and Prevents Spontaneous Autoimmune Diabetes in the NOD Mouse

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4+CD25+ T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into naïve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti–CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.

Costimulation Blockade of Both Inducible Costimulator and CD40 Ligand Induces Dominant Tolerance to Islet Allografts and Prevents Spontaneous Autoimmune Diabetes in the NOD Mouse

Costimulation blockade is a promising strategy for preventing allograft rejection and inducing tolerance. Using a fully allogeneic mouse model, we tested the effectiveness of the combined blockade of the CD40 ligand and the inducible costimulator (ICOS) on islet allograft survival and in the prevention of autoimmune diabetes in the NOD mouse. Recipients treated with blocking monoclonal antibodies (mAbs) to ICOS and the CD40 ligand had significant prolongation of graft survival, with 26 of 28 functioning for >200 days. Long-term engrafted mice maintained antidonor proliferative and cytotoxic responses, but donor-specific immunization did not induce graft rejection, and challenge with second, same donor but not third-party grafts resulted in long-term acceptance. The immunohistology of tolerant grafts demonstrated the presence of CD4(+)CD25(+) T-cells expressing Foxp3, and islet/kidney composite grafts from tolerant mice, but not from mice lacking lymphocytes, were accepted indefinitely when transplanted into naïve B6 mice, suggesting that recipient T-cells were necessary to generate dominant tolerance. Combined anti-ICOS and anti-CD40 ligand mAb therapy also prevented diabetes in NOD mice, with only 11% of treated recipients developing diabetes compared with 75% of controls. These data demonstrate that the blockade of CD40 ligand and ICOS signaling induces islet allograft tolerance involving a dominant mechanism associated with intragraft regulatory cells and prevents autoimmune diabetes in NOD mice.

38. AAV-Mediated Gene Therapy for Systemic Lupus Erythematosus (SLE)

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by polyclonal B-cell activation, autoantibody production and immune complex-mediated glomerulonephritis(GN). NZB/W F1 mice spontaneously develop SLE-like syndromes and have been widely used as an animal model for SLE. Even though the etiologic cause of autoimmunity in both human and murine lupus is not clearly understood, mounting evidence indicates the involvement of autoreactive B cells and T cells. Blockade of costimulatory pathways using CTLA4Ig fusion protein and anti-CD40 ligand mAb was able to suppress autoantibody production and inhibit neprhitis in NZB/W F1 mice. However, for such a chronic disease long term intervention is necessary and gene therapy may be advantageous.

IFN-gamma production is specifically regulated by IL-10 in mice made tolerant with anti-CD40 ligand antibody and intact active bone.

We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.

B-Cell activation and allosensitization after left ventricular assist device implantation is due to T-Cell activation and CD40 ligand expression

Left ventricular assist device (LVAD) implantation is frequently complicated by B-cell activation and allosensitization, posing a significant risk to successful transplant outcome. This study investigated whether B-cell hyperreactivity and alloantibody production in LVAD recipients involves T-cell dependent pathways. T-cell calcium flux and nuclear translocation of NFATc were used to determine states of T-cell activation. Flow cytometry was used to assess human T- and B-cell activation after culture with LVAD-derived biomaterial particles. Sera from LVAD recipients and controls were tested for the presence of anti-HLA antibodies, and for soluble CD40 ligand. LVAD-derived biomaterial induced rapid and sustained calcium flux into normal T cells, resulting in calcineurin-dependent nuclear translocation of NFATc. This resulted in increased T-cell expression of CD40 ligand and subsequent B-cell activation, which was reduced by inhibitors of T-cell activation (CsA or anti-CD25 mAb) or by anti-CD40 ligand mAb. LVAD recipients demonstrated higher frequencies of anti-HLA antibodies and serum levels of soluble CD40 ligand compared with heart failure controls. The results indicate that exposure of human mononuclear cells to LVAD-derived biomaterial leads to T-cell dependent B-cell activation via CD40–CD40 ligand interaction, and suggest that treatment with calcineurin inhibitors or monoclonal antibodies against either CD25 or CD40 ligand could be effective at preventing B-cell hyperreactivity and allosensitization after LVAD implantation.

CD4 T cell-mediated alloresistance to fully MHC-mismatched allogeneic bone marrow engraftment is dependent on CD40-CD40 ligand interactions, and lasting T cell tolerance is induced by bone marrow transplantation with initial blockade of this pathway.

Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.

Allogeneic chimerism with low-dose irradiation, antigen presensitization, and costimulator blockade in H-2 mismatched mice

AbstractWe have previously shown that the keys to high-level nontoxic chimerism in syngeneic models are stem cell toxic, nonmyelotoxic host treatment as provided by 100-cGy whole-body irradiation and relatively high levels of marrow stem cells. This approach was unsuccessful in H-2 mismatched B6.SJL to BALB/c marrow transplants, but with tolerization, stable multilineage chimerism was obtained. Ten million B6.SJL spleen cells were infused intravenously into BALB/c hosts on day −10 and (MR-1) anti-CD40 ligand monoclonal antibody (mAb) injected intraperitoneally at varying levels on days −10, −7, −3, 0, and +3 and the BALB/c mice irradiated (100 cGy) and infused with 40 million B6.SJL/H-2 mismatched marrow cells on day 0. Stable multilineage chimerism at levels between 30% to 40% was achieved in the great majority of mice at 1.6 mg anti-CD40 ligand mAb per injection out to 64 weeks after transplantation, without graft-versus-host disease. The transplanted mice were also tolerant of donor B6.SJL, but not third-party CBA/J skin grafts at 8 to 9 and 39 to 43 weeks after marrow transplantation. These data provide a unique model for obtaining stable partial chimerism in H-2 mismatched mice, which can be applied to various clinical diseases of man such as sickle cell anemia, thalassemia, and autoimmune disorders.

Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor.

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients' malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-gamma, TNF-alpha, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.

NOD mice have a generalized defect in their response to transplantation tolerance induction.

A protocol consisting of a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (anti-CD40 ligand mAb) induces permanent islet allograft survival in chemically diabetic mice, but its efficacy in mice with autoimmune diabetes is unknown. Confirming a previous report, we first observed that treatment of young female NOD mice with anti-CD154 mAb reduced the frequency of diabetes through 1 year of age to 43%, compared with 73% in untreated controls. We also confirmed that spontaneously diabetic NOD mice transplanted with syngeneic (NOD-Prkdc(scid)/Prkdc(scid)) or allogeneic (BALB/c) islets rapidly reject their grafts. Graft survival was not prolonged, however, by pretreatment with either anti-CD154 mAb alone or anti-CD154 mAb plus DST. In addition, allograft rejection in NOD mice was not restricted to islet grafts. Anti-CD154 mAb plus DST treatment failed to prolong skin allograft survival in nondiabetic male NOD mice. The inability to induce transplantation tolerance in NOD (H2g7) mice was associated with non-major histocompatibility complex (MHC) genes. Treatment with DST and anti-CD154 mAb prolonged skin allograft survival in both C57BL/6 (H2b) and C57BL/6.NOD-H2g7 mice, but it was ineffective in NOD, NOD.SWR-H2q, and NOR (H2g7) mice. Mitogen-stimulated interleukin-1beta production by antigen-presenting cells was greater in strains susceptible to tolerance induction than in the strains resistant to tolerance induction. The results suggest the existence of a general defect in tolerance mechanisms in NOD mice. This genetic defect involves defective antigen-presenting cell maturation, leads to spontaneous autoimmune diabetes in the presence of the H2g7 MHC, and precludes the induction of transplantation tolerance irrespective of MHC haplotype. Promising islet transplantation methods based on overcoming the alloimmune response by interference with costimulation may require modification or amplification for use in the setting of autoimmune diabetes.

Normal B cells fail to secrete interleukin-12.

Interleukin-12 is a key regulatory cytokine produced by antigen-presenting cells (APC) which drives the development of interferon-gamma (IFN-gamma)-producing cells and promotes cell-mediated immunity. Following subcutaneous immunization with protein antigen in adjuvant, dendritic cells (DC) but not small nor large B cells in immune lymph nodes express antigenic complexes and secrete substantial amounts of bioactive IL-12 p75 upon antigen-specific interaction with T cells. We have analyzed secretion of IL-12 p40 and p75 by cell populations enriched in DC, macrophages or B cells in response to nonspecific stimulation or to interaction with antigen-specific CD4+ cells. These APC populations do not produce IL-12 constitutively but, upon stimulation with heat-fixed Staphylococcus aureus and IFN-gamma, IL-12 p40 and p75 are secreted by DC and macrophages, whereas B cells fail to produce IL-12. B cells also fail to secrete IL-12 in response to stimulation with LPS and IFN-gamma. Co-culture with CD4+ T hybridoma cells and antigen induces IL-12 secretion by DC. Up-regulation of IL-12 secretion by interaction with antigen-specific CD4+ T cells is abrogated by anti-class II monoclonal antibodies (mAb), by soluble CD40 molecules and by anti-CD40 ligand mAb, demonstrating a positive feedback between T cells and DC mediated by TCR-peptide/class II and by CD40-CD40 ligand interactions. Expression of class II and CD40 molecules is comparable in B cells and DC, and both APC types activate CD4+ T cells. Yet, even upon interaction with antigen-specific T cells, B cells fail to secrete IL-12. The capacity of B cells to present antigen but not to secrete IL-12 may explain their propensity to selectively drive T helper type 2 cell development.

T cell-derived IL-4 and dendritic cell-derived IL-12 regulate the lymphokine-producing phenotype of alloantigen-primed naive human CD4 T cells.

Previous studies on human Th subset development were restricted to the analysis of naive T cells activated with anti-CD3 mAb in the absence of physiologic APC. In this study, we have analyzed the role of cytokines and physiologic APC on T cell maturation in an Ag-specific system, in which naive neonatal CD4 T cells were primed with allogeneic dendritic cells (DC). We found that the cytokine profile of primed cells was dependent upon 1) the ratio between T cells and allogeneic DC and 2) the endogenous production of IL-4 and IL-12. Neutralization of IL-4 during primary MLR increased IFN-gamma production at priming and shifted the phenotype of primed cells from Th0 to Th1. These effects were IL-12 dependent, in that they were suppressed by anti-IL-12 Abs. The production of IL-12 in primary MLR was further evidenced by the presence of IL-12 p40 in the culture supernatant fluids. IL-12 production was suppressed by exogenous IL-4 and increased by anti-IL-4 blocking mAbs, indicating that endogenous IL-4 down-regulated IL-12 production by DC. Finally, IL-12 was produced as a result of T cell/DC interaction involving the CD40/CD40 ligand and CD28/B7 costimulation pathways, as revealed by the inhibitory effect of anti-CD40 ligand mAb and CTLA-4Ig. These observations suggest that in neutral conditions, Ag presentation by DC results in the coordinate production of naive T cell-derived IL-4 and DC-derived IL-12 that in concert shape the cytokine profile of Th cells.

High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10.

We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.

T and B cell collaboration is essential for the autoantibody response to DNA topoisomerase I in systemic sclerosis.

To elucidate the mechanisms controlling anti-DNA topoisomerase I (topo I) antibody production in patients with systemic sclerosis (SSc), in particular the role of interactions between topo I-specific Th cells and B cells, we established an in vitro system for the analysis of anti-topo I antibody production. In vitro anti-topo I antibody synthesis in PBMC cultures was induced by recombinant topo I and PWM, and was measured by a topo I-specific ELISA. Anti-topo I antibody was detected in PBMC culture supernatants from 11 (61%) of 18 anti-topo I-positive SSc patients. In contrast, anti-topo I antibody was not detected in the PBMC culture supernatants from 4 anti-topo I-negative SSc patients or 10 healthy donors. Characterization of in vitro anti-topo I antibody production showed that 1) the anti-topo I antibody isotype produced was IgG; 2) the anti-topo I antibody levels in culture supernatants correlated with those in patients' sera; 3) CD4+ T cells were necessary for antibody synthesis; and 4) antibody synthesis was restricted by HLA-DR, but not by HLA-DQ or DP. In addition, separation of cultured T and B cells by a semipermeable membrane or culture with anti-CD40 ligand mAb blocked in vitro anti-topo I antibody production. These results indicate that a contact-mediated and HLA-DR-restricted collaboration between topo I-specific T and B cells is essential for in vitro anti-topo I antibody production in a subset of SSc patients.

IL-4-producing CD8+ T cell clones can provide B cell help.

Interactions between CD4+ T cells and B cells are mediated by both soluble factors and cell surface molecules. The Ag-independent interaction between the CD40 ligand, expressed on activated T cells, and its CD40 receptor, expressed on B cells, enhances B cell proliferation in response to IL-4 stimulation. The expression of the CD40 ligand is induced on CD4+ T cells by stimulation with Ag-pulsed APC or mitogens. Here, we show that at least some IL-4-producing murine CD8+ T cell clones can be induced to express the CD40 ligand when stimulated with anti-CD3 mAb. Additionally, such activated CD8+ IL-4-producing clones potentiate the proliferative response of small resting B cells to IL-4 and induce Ig secretion by small resting B cells to IL-4 and IL-5. Proliferation of small resting B cells cultured with IL-4 in the presence of activated IL-4-producing CD8+ murine T cell clones appeared to be mediated by the expression of the CD40 ligand on the T cell because an anti-CD40 ligand mAb inhibited this proliferative response. A conventional murine CD8+ CTL clone, which did not produce IL-4 or express CD40 ligand upon activation, did not potentiate proliferation of small resting B cells exposed to IL-4. Thus, under some circumstances, CD8+ T cells that are able to express CD40 ligand may be able to provide B cell help.