CD40-CD154 Interaction Research Articles

P40 Homodimers Indirectly Induce Endogenous Donor-Reactive Memory CD8 T Cell Proliferation within High-Risk Allografts via IL-15 signaling

Abstract Increasing cold ischemic storage (CIS) time from 0.5 to 8 hrs prior to transplant promotes the increased proliferation and effector functions of endogenous donor-reactive memory CD4 and CD8 T cells infiltrating mouse cardiac allografts 24–48 hrs after transplant. This increased mCD8 T cell proliferation within high-risk allografts requires mCD4 T cell help via CD40-CD154 interactions with graft dendritic cells to induce IL-12p40 homodimer (p40 HD) production required for this mCD8 T cell proliferation. This study tested if p40HD directly or indirectly stimulate the mCD8 T cell proliferation within high risk allografts. While endogenous mCD8 T cells within highly ischemic allografts expressed both IL12Rβ1 and β2, most proliferating CD8 T cells did not express the IL12Rβ1 needed for p40 HD mediated signaling. Moreover, endogenous mCD8 T cells isolated from allografts did not proliferate ex vivo when cultured with p40 HD. qPCR analysis indicated increased mRNA expression of IL2Rα (CD25), β(CD122) and IL15 Rain infiltrating mCD8 T cells purified from allografts subjected to prolonged vs. minimal CIS on day 2 post-transplant. ELISA indicated longer CIS time markedly increased IL-12p40 and IL-15 protein in the allografts and these increases were dependent on recipient CD4 T cells. Blocking of p40 or IL-15 signaling with anti-p40 or anti-CD122 mAb inhibited endogenous mCD8 T cell proliferation within high-risk cardiac allografts and extended the survival of the high-risk allografts from day 18 to day 54 in CTLA-4Ig conditioned recipients. These results suggest that the p40 HD stimulate proliferative cytokines such as IL-15, which directly stimulate endogenous donor-reactive mCD8 T cell proliferation within the higher-risk allografts.

PTPN22 1858C>T polymorphism is associated with increased CD154 expression and higher CD4+ T cells percentage in rheumatoid arthritis patients.

CD40 is a costimulatory molecule for B cells, and CD154 is a marker of CD4+ T cells activation. CD40-CD154 interaction promotes pro-inflammatory cytokines secretion and autoantibodies production. PTPN22 gene encodes LYP protein, an inhibitor of T- and B-cell activation. PTPN22 1858C>T polymorphism confers rheumatoid arthritis (RA) susceptibility. Hence, we evaluate the relationship between 1858C>T polymorphism with CD40 and CD154 expression and IFN-γ secretion in RA patients. PTPN22 1858C>T polymorphism was genotyped in 315 RA patients and 315 control subjects (CS) using PCR-RFLP method. Later, we selected only ten anti-CCP-positive RA patients, naïve to disease-modifying antirheumatic drugs and ten CS, all with known 1858C>T PTPN22 genotype. The CD40 and CD154 membrane expressions were determined by flow cytometry in peripheral B and T cells, correspondingly. The B cells percentage and mCD40 expression were similar between RA and CS (P>0.05) and we did not find an association between these variables and the 1858C>T polymorphism. The CD4+ T cells percentage was higher in RA patients than CS (P=0.003), and in the RA group, the CD4+ T cells percentage and mCD154 expression were higher in the 1858T allele carriers (P=0.008 and P=0.032, respectively). The IFN-γ levels were lower in RA patients carrying the PTPN22 risk allele (P=0.032). The PTPN22 1858T risk allele is associated with increased CD4+ T cells percentage and high mCD154 expression in RA patients, which could favor the pro-inflammatory cytokine release and the establishment of the inflammatory response at the seropositive RA.

Inherited Immunodeficiencies With High Predisposition to Epstein-Barr Virus-Driven Lymphoproliferative Diseases.

Epstein–Barr Virus (EBV) is a gamma-herpes virus that infects 90% of humans without any symptoms in most cases, but has an oncogenic potential, especially in immunocompromised individuals. In the past 30 years, several primary immunodeficiencies (PIDs) associated with a high risk to develop EBV-associated lymphoproliferative disorders (LPDs), essentially consisting of virus-associated hemophagocytic syndrome, non-malignant and malignant B-cell LPDs including non-Hodgkin and Hodgkin’s types of B lymphomas have been characterized. Among them are SH2D1A (SAP), XIAP, ITK, MAGT1, CD27, CD70, CTPS1, RASGRP1, and CORO1A deficiencies. Penetrance of EBV infection ranges from 50 to 100% in those PIDs. Description of large cohorts and case reports has refined the specific phenotypes associated with these PIDs helping to the diagnosis. Specific pathways required for protective immunity to EBV have emerged from studies of these PIDs. SLAM-associated protein-dependent SLAM receptors and MAGT1-dependent NKG2D pathways are important for T and NK-cell cytotoxicity toward EBV-infected B-cells, while CD27–CD70 interactions are critical to drive the expansion of EBV-specific T-cells. CTPS1 and RASGRP1 deficiencies further strengthen that T-lymphocyte expansion is a key step in the immune response to EBV. These pathways appear to be also important for the anti-tumoral immune surveillance of abnormal B cells. Monogenic PIDs should be thus considered in case of any EBV-associated LPDs.

Graft dendritic cell p40 homodimers activate donor-reactive endogenous memory CD8 T cells within higher risk allografts

Abstract Memory T cells with donor-reactivity pose a major barrier to successful transplantation and tolerance induction. Longer cold ischemic storage (CIS) prior to transplantation promotes endogenous donor-reactive memory CD8 T cell infiltration and activation within cardiac allografts to directly mediate CTLA-4Ig-resistant rejection between days 15–22 post-transplant vs. > 60 days for allografts subjected to minimal CIS. Using BrdU labeling, graftinfiltrating memory CD4 and CD8 T cells proliferated much more strongly within allografts subjected to 8 vs. 0.5 hr CIS and proliferation of endogenous memory CD8 T cells within the higher risk allografts required recipient CD4 T cells, graft dendritic cells (DC) and graft expression of class II MHC, CD40 and IL-12p40, but not p35. IL-12p40 but not IL-12 p35 or IL-23p19 mRNA was elevated at 48 hr post-transplant in allografts subjected to 8 vs. 0.5 hr CIS and the increase in p40 mRNA was reduced to the levels seen in 0.5 CIS allografts by recipient CD4 T cell depletion, but was restored by treating with agonist anti-CD40 mAb. p40 homodimers, but not p70 heterodimers, were increased in allografts subjected to 8 hr CIS and were dependent on recipient CD4 T cells. Peri-transplant anti-p40 mAb reversed CTLA-4Ig-resistant rejection of higher risk allografts and peri-transplant p40 homodimers induced endogenous memory CD8 T cell proliferation within allografts subjected to 0.5 hr CIS to the levels observed in allografts subjected to 8 hr CIS. These data indicate that endogenous memory CD8 T cell activation within higher risk cardiac allografts requires CD4 T cell help via CD40-CD154 interactions with graft DC to induce their production of p40 homodimers.

Defective innate immune responses lead to hepatic damage in CD154-deficient mice following infection with attenuated Salmonella typhimurium

Abstract In addition to its established role in immunoglobulin isotype switching, CD40–CD154 interaction plays a role in the regulation of multiple innate immune system cell types. Of note, we have shown that CD154-deficient (CD154−/−) mice are hyper-susceptible to intraperitoneal infection by attenuated strains of Salmonella enterica serovar Typhimurium. Compared to their normal counterparts, CD154−/− mice exhibited progressive morbidity during the early phases of disease and succumbed to infection 3–4 weeks after inoculation. Since the liver is a key target organ in Salmonella infections, we carried out a comparative analysis of infection-associated hepatic changes at different time-points. Bacterial loads in the liver were significantly higher in CD154−/− compared to WT mice, and this correlated with progressively worsening hepatic damage and rising levels of liver enzymes. Liver histomorphology revealed focal necrosis early in the infection that was self-limiting in WT mice. In contrast, infected CD154−/− mice developed large and generalized necrosis coupled with the appearance of large venous thrombi. Immunohistochemical analysis showed a retarded inflammatory response affecting recruitment of myeloid cells in the liver of CD154−/− animals. Moreover, neutrophil recruitment into the peritoneal cavity, as well as the activation of the respiratory burst, was defective in CD154−/− mice. Taken together, our findings demonstrate that CD154-deficiency results in a failure to mount an appropriate innate immune response to contain bacterial proliferation during the acute phase of disease. This renders the animals incapable of limiting the extent of histopathological damage in the liver resulting, ultimately, in their demise.

RD-05, a novel anti-CD154 antibody, efficiently inhibits generation of anti-drug antibody without the risk of thrombus formation in non-human primates

Antibody formation against therapeutic agents, such as tumor necrosis factor inhibitors and Factor VIII, that leads to treatment failure has become a major challenge in the treatment of rheumatoid arthritis and hemophilia. It is well known that anti-CD154 antibodies have the highest potential to inhibit these types of adverse immune responses. Nevertheless, the formation of thromboemboli is the major hurdle in the clinical application of these anti-CD154 blocking antibodies. For this, we attempted to derive an idea as to how this major complication can be eliminated. Consequently, we developed a novel anti-CD154 chimeric antibody, which was made by genetic modification of a portion of human IgG4 Fc. This antibody has an almost comparable antigen binding affinity to a previously developed 5C8 clone and near completely inhibited CD40-CD154 interaction and T cell-dependent B cell activation in vitro. Even under the condition, where we injected immune complexes comprised of RD-05 and CD154 antigen, the formation of thromboembolism was not seen in human FcγRIIA-transgenic mice, whereas the converse was exactly true in the case of 5C8 antibody. Notably, just two injections of RD-05 antibody was sufficient to inhibit the antibody formation against adalimumab during 3–4 months in cynomolgus macaques, in which adalimumab was repeatedly injected for 12 weeks. Based on these findings, we suggest that this RD-05 antibody can be applied to antibody-mediated autoimmune diseases, including systemic lupus erythematosus and immune thrombocytopenic purpura.

Therapeutic effects of anti-CD154 antibody in cynomolgus monkeys with advanced rheumatoid arthritis

Rheumatoid arthritis is one major chronic inflammatory systemic autoimmune disease. The CD154-CD40 interactions play a critical role in the regulation of immune responses and the maintenance of autoimmunity. Therefore, we aimed to determine whether anti-CD154 antibody treatment show positive effects on immunomodulation and clinical improvement of sustained severe rheumatoid arthritis in cynomolgus monkeys. Arthritis was induced using chicken type II collagen (CII) and arthritic monkey were divided into control and anti-CD154 treatment groups based on their concentrations of anti-CII antibodies on week 7 post-immunization. Blood and tissue samples were collected on week 16 post-immunization. Anti-CD154 antibody treatment improved arthritis and movement, and significantly decreased the numbers of proliferating B cells and the serum levels of anti-type II collagen antibody and sCD154 compared with non-treatment group. Further anti-CD154 antibody treatment significantly decreased the percentage of CD4+ cells and the ratio of CD4+ to CD8+ T cells and significantly increased the percentage of CD8+ cells and effector memory CD8+ cells in peripheral blood. We have shown for the first time in a nonhuman primate model of RA that CD154 blockade has beneficial effects. This study might be valuable as preclinical data of CD154 blockade in nonhuman primate models of severe rheumatoid arthritis.

CD27 co-stimulation increases regulatory T cell survival and limits progression of atherosclerosis.

Abstract Co-stimulatory signals are important shaping adaptive immunity which is pivotal to all stages of atherosclerosis. The interaction of the co-stimulatory molecules CD27 and CD70 modulates Treg development. Furthermore, T cell activation, proliferation, and also differentiation are affected by CD27-CD70 interactions. Thus, we hypothesized that the deficiency of CD27 will lead to exacerbated atherosclerosis. Cd27−/− mice were crossed with Apoe−/− mice. Cd27−/−Apoe−/− and littermate controls (Cd27+/+Apoe−/−) were sacrificed at the age of 18 and 28 weeks. Bone marrow of Cd27−/−Apoe−/− and littermate controls was transplanted into Apoe−/− recipient mice. Lesion size, histology and cellular composition were analyzed in the ascending aorta. Human and murine T cells co-localize with CD27 in atherosclerotic lesions. At early stages of atherosclerosis, Cd27−/−Apoe−/− mice displayed bigger and more complex atherosclerotic lesions. Flow cytometry revealed a significant decrease in abundance of splenic and aortic Tregs and increased apoptosis of Treg in the thymus of Cd27−/−Apoe−/− mice. In contrast, at later stages of atherosclerosis (28 weeks), splenic and lesional Treg content and atherosclerotic burden were similar. Transplantation of Cd27−/−Apoe−/− bone marrow, in contrast to control, induced increased atherosclerotic plaque size and a profound pro-inflammatory plaque phenotype accompanied by reduced frequency of aortic and splenic Tregs. Furthermore, the number of circulating leukocytes was increased. Taken together, CD27 deficiency impairs thymic Treg development which exacerbates early atherogenesis. However, the role of CD27–CD70 interactions does not affect later stages of atherosclerosis.

Effect of ZNT7 on the CD40 signaling pathway in B lymphocytes: a possible mechanism for a regulatory role of zinc in immune function

Zinc deficiency impairs the immune system leading to frequent infections. Although it is known that zinc plays critical roles in maintaining healthy immune function, the underlying molecular targets are largely unknown. In this study, we demonstrate that zinc is important for the CD154‐CD40‐mediated activation of downstream signaling pathways in human B lymphocytes. CD40 is a receptor localized on the cell surface of many immune cells, including B lymphocytes. When the T helper lymphocyte (Th) is activated by antigens, it expresses a membrane protein, CD154 that allows Th to bind to B cells via CD154‐CD40 interaction leading to B cell activation. We show that cellular zinc deficiency impaired the CD154‐CD40‐mediated p38 MAPK phosphorylation. Most importantly, we demonstrate that ZNT7 interacted with CD40. ZNT7 knockdown in B lymphocytes had a negative effect on the cell surface expression of CD40. Consequently, the CD40‐mediated p38 MAPK signaling transduction was down‐regulated in ZNT7KD B lymphocytes. Conversely, this p38 MAPK signaling activity was up‐regulated by over‐expression of ZNT7 in B lymphocytes. We conclude that prevention of zinc deficiency is a key to maintain an optimal function of immune system and CD40 is the molecule target for ZNT7 in regulation of immune function of B lymphocytes.Support or Funding InformationThis work was supported by the United States Department of Agriculture, ARS CRIS project 2032‐51000‐004‐00D.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Th40 cells (CD4+CD40+ Tcells) drive a more severe form of Experimental Autoimmune Encephalomyelitis than conventional CD4 T cells.

CD40-CD154 interaction is critically involved in autoimmune diseases, and CD4 T cells play a dominant role in the Experimental Autoimmune Encephalomyelitis (EAE) model of Multiple Sclerosis (MS). CD4 T cells expressing CD40 (Th40) are pathogenic in type I diabetes but have not been evaluated in EAE. We demonstrate here that Th40 cells drive a rapid, more severe EAE disease course than conventional CD4 T cells. Adoptively transferred Th40 cells are present in lesions in the CNS and are associated with wide spread demyelination. Primary Th40 cells from EAE-induced donors adoptively transfer EAE without further in-vitro expansion and without requiring the administration of the EAE induction regimen to the recipient animals. This has not been accomplished with primary, non-TCR-transgenic donor cells previously. If co-injection of Th40 donor cells with Freund’s adjuvant (CFA) in the recipient animals is done, the disease course is more severe. The CFA component of the EAE induction regimen causes generalized inflammation, promoting expansion of Th40 cells and infiltration of the CNS, while MOG-antigen shapes the antigen-specific TCR repertoire. Those events are both necessary to precipitate disease. In MS, viral infections or trauma may induce generalized inflammation in susceptible individuals with subsequent disease onset. It will be important to further understand the events leading up to disease onset and to elucidate the contributions of the Th40 T cell subset. Also, evaluating Th40 levels as predictors of disease onset would be highly useful because if either the generalized inflammation event or the TCR-honing can be interrupted, disease onset may be prevented.

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency.

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)-related diseases. Three patients presented with EBV-associated Hodgkin's lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro-generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70-CD27 interactions therefore play a nonredundant role in T and B cell-mediated immunity, especially for protection against EBV and humoral immunity.

Viral Persistence Induces Antibody Inflation without Altering Antibody Avidity.

Antibodies are implicated in long-term immunity against numerous pathogens, and because of this property, antibody induction is the basis for many vaccines. Little is known about the influence of viral persistence on the evolving antibody response. Here, we examined the characteristics of antibody responses to persistent infection by employing the prototypic betaherpesvirus family member cytomegalovirus (CMV) in experimental mouse models. During the course of infection, mouse CMV (MCMV)-specific IgM and IgG responses are elicited; however, IgG levels gradually inflate in the persistent phase of infection while IgM levels are stably maintained. Whereas CD27-CD70 interactions are dispensable, the CD28/B7 costimulatory pathway is critical for the class switching of MCMV-specific IgM-to-IgG B cell responses, which corresponds to the CD28/B7-dependent formation of CD4(+)T follicular helper cells (TFH) and germinal center (GC) B cells. Furthermore, the initial viral inoculum dose dictates the height of the antibody levels during IgG antibody inflation and relates to the induction of long-lived plasma cells and memory B cells. Antibody avidity nonetheless is not altered after the establishment of viral persistence and occurs independently of the inoculum doses. However, repetitive challenge with intact viral particles, accompanied by increased GC reactivity, promotes the development of high-avidity IgG responses with neutralizing capacity. These insights can be used for the rational design of CMV-based vaccines aimed at inducing antibody responses. Antibodies provide long-term protection to different pathogens. However, how antibody responses develop during persistent virus infection is not entirely clear. Here, we characterize factors that influence the virus-specific antibody response to persistent CMV. This study describes that during persistent infection, CMV-specific IgM antibody levels are stably maintained while IgG2b and IgG2c levels gradually inflate over time. In contrast, the IgG avidity remains similar after the establishment of viral persistence. The induction of T follicular helper cells and GC B cells requires CD4(+)T cell help and CD28/B7 costimulation signals and is essential for the development of CMV-specific IgG antibody responses. Furthermore, neutralizing CMV-specific antibodies appear to develop late after infection, yet the neutralizing capacity can be improved upon repetitive viral challenge that is associated with increased GC reactivity. The results described here could inform the use of CMV-based vaccines and may help to understand how our immune system copes with this persistent virus.

Exploratory computational assessment of possible binding modes for small molecule inhibitors of the CD40-CD154 co-stimulatory interaction.

Protein-protein interactions (PPI) tend to involve extensive, flat, and featureless interfaces that are difficult to disrupt by small molecule binding. However, recently, PPIs are being recognized as increasingly valuable 'druggable' targets. We have identified several small molecule inhibitors of the immunologically relevant CD40-CD154 co-stimulatory interaction that bind to the homotrimeric CD154, a member of the tumor necrosis factor superfamily (TNFSF). Recently, on the basis of the co-crystal structure of CD154 with another small molecule (BIO8898), it has been suggested that these PPIs could be particularly susceptible to small molecule blockade due to a subunit fracture mechanism resulting in a distortion of the trimeric structure. To investigate whether this mechanism can occur with our organic dye-related inhibitors, we performed exploratory computational docking experiments. Possible druggable pockets that can serve as binding sites for small molecule inhibitors were identified with the FFT map algorithm both along the CD154-CD40 binding interface (competitive, orthosteric model) and in the interior core of the CD154 trimer corresponding to the BIO8898 binding site (allosteric model). Docking experiments (using Glide) were performed at these sites using the PDB ID: 3QD6 (CD40-CD154) and 3LKJ (BIO8898-CD154) co-crystal structures, respectively. The docking algorithm was able to better discriminate binders from non-binders at the deeper allosteric site than at the competitive site. Accordingly, an allosteric inhibitory mechanism that involves intercalation between monomeric subunits seems feasible for our small molecules making the constitutively trimeric CD154 a likely druggable target.

Expression of the immune regulation antigen CD70 in osteosarcoma.

Osteosarcoma is the most frequent bone cancer in children and young adults. The outcome of patients with advanced disease is dismal. Exploitation of tumor-immune cell interactions may provide novel therapeutic approaches. CD70-CD27 interactions are important for the regulation of adaptive immunity. CD70 expression has been reported in some solid cancers and implicated in tumor escape from immunosurveillance. In this study, expression of CD70 and CD27 was analyzed in osteosarcoma cell lines and tumor specimens.CD70 protein was expressed on most osteosarcoma cell lines (5/7) and patient-derived primary osteosarcoma cultures (4/6) as measured by flow cytometry. In contrast, CD70 was detected on few Ewing sarcoma cell lines (5/15) and was virtually absent from neuroblastoma (1/7) and rhabdomyosarcoma cell lines (0/5). CD70+ primary cultures were derived from CD70+ osteosarcoma lesions. CD70 expression in osteosarcoma cryosections was heterogeneous, restricted to tumor cells and not attributed to infiltrating CD3+ T cells as assessed by immunohistochemistry/immunofluorescence. CD70 was detected in primary (1/5) but also recurrent (2/4) and metastatic (1/3) tumors. CD27, the receptor for CD70, was neither detected on tumor cells nor on T cells in CD70+ or CD70− tumors, suggesting that CD70 on tumor cells is not involved in CD27-dependent tumor-immune cell interactions in osteosarcoma. CD70 gene expression in diagnostic biopsies of osteosarcoma patients did not correlate with the occurrence of metastasis and survival (n = 70).Our data illustrate that CD70 is expressed in a subset of osteosarcoma patients. In patients with CD70+ tumors, CD70 may represent a novel candidate for antibody-based targeted immunotherapy.

IFN-γ production by memory helper T cells is required for CD40-independent alloantibody responses.

Cognate T-B cell interactions and CD40-CD154 costimulation are essential for productive humoral immunity against T-dependent Ags. We reported that memory CD4 T cells can deliver help to B cells and induce pathogenic IgG alloantibodies in the absence of CD40-CD154 interactions. To determine cytokine requirements for CD40-independent help, we used CD40(-/-) mice containing differentiated subsets of donor-reactive memory Th cells as heart allograft recipients. Th1 and Th17, but not Th2, memory CD4 T cells elicited high titers of anti-donor Ab. Abs induced by Th17 memory CD4 T cells had decreased reactivity against donor MHC class I molecules and inferior ability to cause complement deposition in heart allografts compared with Abs induced by Th1 cells, suggesting a requirement for IFN-γ during CD40-independent help. IFN-γ neutralization inhibited helper functions of memory CD4 T cells in both CD40(-/-) recipients and wild type recipients treated with anti-CD154 mAb. Our results suggest that IFN-γ secreted by pre-existing memory helper cells determines both isotype and specificity of donor-reactive alloantibodies and can thus affect allograft pathology. This information may be valuable for identifying transplant patients at risk for de novo development of pathogenic alloantibodies and for preventing alloantibody production in T cell-sensitized recipients.

A CD40-targeted peptide controls and reverses type 1 diabetes in NOD mice.

The CD40-CD154 interaction directs autoimmune inflammation. Therefore, a long-standing goal in the treatment of autoimmune disease has been to control the formation of that interaction and thereby prevent destructive inflammation. Antibodies blocking CD154 are successful in mouse models of autoimmune disease but, while promising when used in humans, unfortunate thrombotic events have occurred, forcing the termination of those studies. To address the clinical problem of thrombotic events caused by anti-CD154 antibody treatment, we created a series of small peptides based on the CD154 domain that interacts with CD40 and tested the ability of these peptides to target CD40 and prevent type 1 diabetes in NOD mice. We identified a lead candidate, the 15-mer KGYY15 peptide, which specifically targets CD40-positive cells in a size- and sequence-dependent manner. It is highly efficient in preventing hyperglycaemia in NOD mice that spontaneously develop type 1 diabetes. Importantly, KGYY15 can also reverse new-onset hyperglycaemia. KGYY15 is well tolerated and functions to control the cytokine profile of culprit Th40 effector T cells. The KGYY15 peptide is 87% homologous to the human sequence, suggesting that it is an important candidate for translational studies. Peptide KGYY15 constitutes a viable therapeutic option to antibody therapy in targeting the CD40-CD154 interaction in type 1 diabetes. Given the involvement of CD40 in autoimmunity in general, it will also be important to evaluate KGYY15 in the treatment of other autoimmune diseases. This alternative therapeutic approach opens new avenues of exploration in targeting receptor-ligand interactions.

Unexpected Efficacy of CTLA-4Ig in Controlling Ongoing Allospecific Responses and Treating Acute Rejection.

Once rejection is initiated, halting the immune response becomes much more difficult because of increased frequencies of activated effector and memory alloreactive T and B cells and of plasma cells. Costimulation blockade targeting CD28-B7 and CD40-CD154 interactions is reported to be particularly ineffective at controlling memory T cell and T-dependent B cell responses. Here we report on the unexpected efficacy of CTLA-4Ig in controlling ongoing antibody responses and treating acute rejection. To test the efficacy of CTLA-4Ig at halting ongoing alloimmune response, C57BL/6 mice were sensitized with a subcutaneous injection of BALB/c splenocytes (DST) and treated with biweekly CTLA-4Ig from d0, d7, or d14 post-DST. Without CTLA-4Ig, donor-specific Abs (DSA) were detected on d7 and peaked on d21 post-DST. Delayed treatment with CTLA-4Ig from d7 or d14 post-DST halted the DSA response, and DSA titers tended towards baseline by d42. To test whether delayed CTLA-4Ig prevented development of memory responses, CTLA4-Ig treatment was stopped on d42 and 2 weeks later, the mice were challenged with DST without CTLA4-Ig. Mice receiving d0-42 CTLA-4Ig exhibited no increase in alloantibody production, consistent with tolerance, whereas those receiving d14-42 CTLA-4Ig exhibited recall antibody responses indistinguishable from untreated. Notably, mice receiving d7-42 CTLA-4Ig exhibited a blunted antibody relative to recall as well as to primary response. Finally in a BALB/c into C57BL/6 heart transplantation model, where unmodified rejection occurs on d7-8 post-transplant, biweekly treatment with CTLA4-Ig from 6d post-transplant prevented rejection in 2 of 3 recipients (>42d). A second BALB/c heart transplanted on d21 was also accepted (>21d). These results demonstrate the unexpected efficacy of delayed CTLA4-Ig at halting ongoing alloreactive responses, inhibiting memory formation, facilitating the development of a hyporesponsive state and in treating acute rejection.

Characterization of ASKP1240, a Fully Human Antibody Targeting Human CD40 With Potent Immunosuppressive Effects

Blocking the CD40–CD154 interaction is reported to be effective for transplantation management and autoimmune disease models in rodents and nonhuman primates. However, clinical trials with anti-CD154 mAbs were halted because of high incidence of thromboembolic complications. Thus, we generated and characterized a fully human anti-CD40 mAb ASKP1240, as an alternative to anti-CD154 mAb. In vitro ASKP1240 concentration-dependently inhibited human peripheral blood mononuclear cell proliferation induced by soluble CD154. In addition, ASKP1240 did not destabilize platelet thrombi under physiological high shear conditions while mouse anti-human CD154 mAb (mu5C8) did. And ASKP1240 itself did not activate platelet and endothelial cells. In vivo administration of ASKP1240 (1 or 10 mg/kg, intravenously) to cynomolgus monkeys, weekly for 3 weeks, significantly attenuated both delayed-type hypersensitivity and specific antibody formation evoked by tetanus toxoid. The immunosuppressive effect was well correlated with the CD40 receptor saturation. Thus, these results suggest that ASKP1240 is immunosuppressive but not prothromboembolic, and as such appears to be a promising therapeutic candidate for the management of solid organ transplant rejection and autoimmune diseases therapy.

AB0126 CD70 Expressing CD4 T Cells Produce IFN-Γ and IL-17 in Rheumatoid Arthritis

BackgroundCD70 expressing CD4 T cells are enriched in rheumatoid arthritis (RA) and promote autoimmunity via costimulatory CD70-CD27 interaction. To date, it is unknown whether CD70+ CD4 T cells contribute to...