Agonist anti-ChemR23 mAb reduces tissue neutrophil accumulation and triggers chronic inflammation resolution.

Resolution of inflammation is elicited by proresolving lipids, which activate GPCRs to induce neutrophil apoptosis, reduce neutrophil tissue recruitment, and promote macrophage efferocytosis. Transcriptional analyses in up to 300 patients with Inflammatory Bowel Disease (IBD) identified potential therapeutic targets mediating chronic inflammation. We found that ChemR23, a GPCR targeted by resolvin E1, is overexpressed in inflamed colon tissues of severe IBD patients unresponsive to anti-TNFα or anti-α4ß7 therapies and associated with significant mucosal neutrophil accumulation. We also identified an anti-ChemR23 agonist antibody that induces receptor signaling, promotes macrophage efferocytosis, and reduces neutrophil apoptosis at the site of inflammation. This ChemR23 mAb accelerated acute inflammation resolution and triggered resolution in ongoing chronic colitis models, with a significant decrease in tissue lesions, fibrosis and inflammation-driven tumors. Our findings suggest that failure of current IBD therapies may be associated with neutrophil infiltration and that ChemR23 is a promising therapeutic target for chronic inflammation.

Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

Clinical efficacy of a new CD28-targeting antagonist of T cell co-stimulation in a non-human primate model of collagen-induced arthritis.

T cells have a central pathogenic role in the aetiopathogenesis of rheumatoid arthritis (RA), and are therefore a favoured target of immunotherapy aiming at physical or functional elimination. Here we report an efficacy test of FR104, a new co-stimulation inhibitor directly targeting CD28 on T cells, in a translationally relevant model, the rhesus monkey model of collagen-induced arthritis (CIA). As a relevant comparator we used abatacept [cytotoxic T lymphocyte antigen immunoglobulin (CTLA Ig)], an antagonist of CTLA-4 binding to CD80/86 clinically approved for treatment of RA. Treatment with either compound was started at the day of CIA induction. Although FR104 previously demonstrated a higher control of T cell responses in vitro than abatacept, both compounds were equally potent in the suppression of CIA symptoms and biomarkers, such as the production of C-reactive protein (CRP) and interleukin (IL)-6 and anti-collagen type II (CII) serum antibody (IgM/IgG). However, in contrast to abatacept, FR104 showed effective suppression of CII-induced peripheral blood mononuclear cell (PBMC) proliferation. The current study demonstrates a strong potential of the new selective CD28 antagonist FR104 for treatment of RA.

Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

FR104, an antagonist anti-CD28 monovalent fab' antibody, prevents alloimmunization and allows calcineurin inhibitor minimization in nonhuman primate renal allograft.

Selective targeting of CD28 might represent an effective immunomodulation strategy by preventing T cell costimulation, while favoring coinhibition since inhibitory signals transmitted through CTLA-4; PD-L1 and B7 would not be affected. We previously showed in vitro and in vivo that anti-CD28 antagonists suppress effector T cells while enhancing regulatory T cell (Treg) suppression and immune tolerance. Here, we evaluate FR104, a novel antagonist pegylated anti-CD28 Fab' antibody fragment, in nonhuman primate renal allotransplantation. FR104, in association with low doses of tacrolimus or with rapamycin in a steroid-free therapy, prevents acute rejection and alloantibody development and prolongs allograft survival. However, when FR104 was associated with mycophenolate mofetil and steroids, half of the recipients rejected their grafts prematurely. Finally, we observed an accumulation of Helios-negative Tregs in the blood and within the graft after FR104 therapy, confirmed by Treg-specific demethylated region DNA analysis. In conclusion, FR104 reinforces immunosuppression in calcineurin inhibitor (CNI)-low or CNI-free protocols, without the need of steroids. Accumulation of intragraft Tregs suggested the promotion of immunoregulatory mechanisms. Selective CD28 antagonists might become an alternative CNI-sparing strategy to B7 antagonists for kidney transplant recipients.

Preclinical efficacy and immunological safety of FR104, an antagonist anti-CD28 monovalent Fab' antibody.

Antagonist anti-CD28 antibodies prevent T cell costimulation and differentiate from CTLA4Ig since they cannot block CTLA-4 and PDL-1 coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function and immune tolerance. However, anti-CD28 antibodies devoid of immunotoxicity and with a good pharmacokinetic profile have not yet been developed. Here, we describe FR104, a novel humanized pegylated anti-CD28 Fab' antibody fragment presenting a long elimination half-life in monkeys. In vitro, FR104 failed to induce human T cell proliferation and cytokines secretion, even in the presence of anti-CD3 antibodies or when cross-linked with secondary antibodies. Furthermore, in humanized NOD/SCID mice adoptively transferred with human PBMC, whereas superagonist and divalent antibodies elicited rapid cytokines secretion and human T cell activation, FR104 did not. These humanized mice developed a florid graft-versus-host disease, which was prevented by administration of FR104 in a CTLA4-dependent manner. Interestingly, administration of high doses of CTLA4-Ig was ineffective to prevent GVHD, whereas administration of low doses was partially effective. In conclusion, we demonstrated that FR104 is devoid of agonist activity on human T cells and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA-4, as compared to CD80/CD86 antagonists.

CD28-specific immunomodulating antibodies: what can be learned from experimental models?

Tolerance induction to alloantigens remains a major challenge in transplant immunology. Progress in the last decade of our understanding of T-cell activation has led to the development of new immunotherapeutic strategies to replace conventional immunosuppression which inhibits the immune system in a nonspecific way. In particular, positive and negative costimulatory molecules of the CD28 family have been consistently demonstrated to be critical for the development of productive immune responses as well as the establishment and maintenance of peripheral tolerance. However, recent discoveries of novel costimulatory interactions confer a novel dimension to the immunoregulatory interactions within the B7:CD28 family and compels a revised view within a "quintet" of costimulatory molecules: CD28/B7/CTLA-4/PD-L1/ICOSL. Complexity introduced in this more detailed costimulatory pathway has important implications in therapeutic interventions against human immunological diseases and, especially, highlight the fundamental differences in selectively targeting CD28 molecules instead of B7 counterparts. In this review, we discuss these differences and emphasize different CD28-specific immunomodulating strategies evaluated in experimental models of transplantation and autoimmune diseases.

Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyl-transferase transgenic pig kidneys into baboons.

Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.

Antibody-mediated depletion of lymphocyte-activation gene-3 (LAG-3(+) )-activated T lymphocytes prevents delayed-type hypersensitivity in non-human primates.

Lymphocyte-activation gene-3 (LAG-3, CD223) is a marker for recently activated effector T cells. Activated T lymphocytes are of major importance in many autoimmune diseases and organ transplant rejection. Therefore, specifically depleting LAG-3(+) T cells might lead to targeted immunosuppression that would spare resting T cells while eliminating pathogenic activated T cells. We have shown previously that anti-LAG-3 antibodies sharing depleting as well as modulating activities inhibit heart allograft rejection in rats. Here, we have developed and characterized a cytotoxic LAG-3 chimeric antibody (chimeric A9H12), and evaluated its potential as a selective therapeutic depleting agent in a non-human primate model of delayed-type hypersensitivity (DTH). Chimeric A9H12 showed a high affinity to its antigen and depleted both cytomegalovirus (CMV)-activated CD4(+) and CD8(+) human T lymphocytes in vitro. In vivo, a single intravenous injection at either 1 or 0·1 mg/kg was sufficient to deplete LAG-3(+) -activated T cells in lymph nodes and to prevent the T helper type 1 (Th1)-driven skin inflammation in a tuberculin-induced DTH model in baboons. T lymphocyte and macrophage infiltration into the skin was also reduced. The in vivo effect was long-lasting, as several weeks to months were required after injection to restore a positive reaction after antigen challenge. Our data confirm that LAG-3 is a promising therapeutic target for depleting antibodies that might lead to higher therapeutic indexes compared to traditional immunosuppressive agents in autoimmune diseases and transplantation.

Immune regulation by non-lymphoid cells in transplantation.

Regulatory cells play a crucial role in the induction and maintenance of tolerance by controlling T cell as well as B and natural killer (NK) cell-mediated immunity. In transplantation, CD4+CD25+forkhead box P3+ T regulatory cells are instrumental in the maintenance of immunological tolerance, as are several other T cell subsets such as NK T cells, double negative CD3+ T cells, gammadelta T cells, interleukin-10-producing regulatory type 1 cells, transforming growth factor-beta-producing T helper type 3 cells and CD8+CD28(-) cells. However, not only T cells have immunosuppressive properties, as it is becoming increasingly clear that both T and non-T regulatory cells co-operate and form a network of cellular interactions controlling immune responses. Non-T regulatory cells include tolerogenic dendritic cells, plasmacytoid dendritic cells, mesenchymal stem cells, different types of stem cells, various types of alternatively activated macrophages and myeloid-derived suppressor cells. Here, we review the mechanism of action of these non-lymphoid regulatory cells as they relate to the induction or maintenance of tolerance in organ transplantation.

Physiomer reduces the chemokine interleukin-8 production by activated human respiratory epithelial cells.

The authors have recently shown that the transcription factor nuclear factor-kappaB (NF-kappaB) is a central mediator in the NaCl-mediated interleukin (IL)-8 production by human airway epithelial cells. In this study, it was investigated whether Physiomer, an isotonic sea water-derived solution commercialized for cleaning the nasal mucosa, impaired the chemokine IL-8 expression and secretion by human respiratory epithelial cells compared with that obtained with an isotonic 9% NaCl solution. Primary human bronchial gland (HBG) epithelial cells were incubated either in Physiomer or in a NaCl 9% solution and activated either with 20 ng x mL(-1) tumour necrosis factor-alpha, or IL-1beta, respectively. Physiomer significantly reduced the IL-8 protein release in basal and activated HBG cells in comparison with that obtained with the 9% NaCl solution. In contrast to the effects of Physiomer observed on resting HBG cells, Physiomer did not significantly reduce the level of phosphorylation of the NF-kappaB inhibitor protein IkappaBalpha or the steady-state IL-8 messenger ribonucleic acid levels in activated HBG cells, suggesting that Physiomer would have a post-transcriptional effect on IL-8 expression in activated HBG cells. The authors conclude that Physiomer is potentially useful in the reduction of airway mucosal inflammation.

Direct recognition of foreign MHC determinants by naive T cells mobilizes specific Vbeta families without skewing of the complementarity-determining region 3 length distribution.

The capacity of T cells to interact with nonself-APC, also referred to as direct allorecognition, is an essential feature of the cellular response involved in graft rejection. However, there is no study on TCR repertoire biases associated with direct restricted T cell activation. In this paper, we have addressed the impact of direct recognition on the whole naive T cell repertoire, using a new approach that provides, for the first time, an integrated depiction of the quantitative and qualitative alterations in the TCR Vbeta transcriptome. This method can differentiate resting patterns from polyclonally activated ones, as evidenced by superantigen usage. According to this new readout, we show that direct recognition of nonself-MHC molecules triggers mRNA accumulation of several TCR Vbeta families, specific to the combination studied. Moreover, in marked contrast to the situation that prevails in indirect allorecognition, T cell activation through the direct presentation pathway was not associated with skewing of the complementarity determining region (CDR) 3 length distribution. Altogether, these data argue for the significance of TCR contacts with the MHC framework in direct allorecognition. In addition, the TCR diversity mobilized by this interaction and the massive TCRbeta mRNA accumulation observed after a few days of culture suggest that a significant proportion of naive T cells receive a signal leading to TCRbeta transcriptional activation even though only a few of them engage in mitosis.

Mechanisms of tolerance induction: blockade of co-stimulation.

Induction of tolerance to transplantation antigens is believed to be a promising way to achieve long-term allograft survival without a deleterious immunosuppressive regimen. T-cell activation, which is an essential feature of graft rejection, requires a first signal provided by T-cell receptor (TCR) ligation and a second signal provided by engagement of co-stimulatory molecules with their respective ligands on antigen-presenting cells. The coordinated triggering of these two independent signalling systems ensures the full T-cell activation, including proliferation and acquisition of effector function. TCR occupancy in the absence of co-stimulatory signals leads to a sustained loss of antigen responsiveness called clonal anergy, which could be of major importance in transplantation. In vivo, co-stimulation blockade was indeed shown to allow for long-term allograft survival in several transplantation models. However, the current continuous identification of new co-stimulatory molecules suggests that a functional redundancy of the system exists and that tolerance to transplantation antigens might be achieved more easily through the combined blockade of two or several co-stimulatory signals. In this review, we analyse the biological effects of the disruption of some co-stimulation pathways in vitro and in vivo and discuss their potential interest for tolerance induction.

T-cell-mediated rejection of vascularized xenografts in the absence of induced anti-donor antibody response.

T cells are considered to play a major indirect role in the pathogenesis of xenograft vascular rejection, by promoting the induction of anti-donor antibodies that trigger complement- and antibody-dependent cell cytotoxicity. However, how vigorous the T cell xenoresponse is in vivo, and whether, besides their helper function, T cells are capable of directly affecting the graft is still unclear. We have previously shown that cyclosporine A (CsA) withdrawal in accommodated cardiac xenograft recipient allows for a rapid and dense T-cell infiltration, concomitant to an acute graft rejection. In this paper we further characterize the role of T cells in this rejection process and we demonstrate that adoptive transfer of CD4+ T cells in irradiated recipients of long-term cardiac xenografts is sufficient to trigger acute rejection, in the absence of any detectable induced anti-hamster antibody response. Therefore, our data suggest that unusually strong T-cell response will be another major barrier to xenotransplantation, even if antibody-mediated vascular rejection is controlled.

Induction of anti-Forssman antibodies in the hamster-to-rat xenotransplantation model.

BACKGROUND: In the hamster-to-rat heart xenotransplantation model, the serum response of the host contributes to determine whether the xenograft is accommodated or rejected. METHODS: To further characterize the serum response in this model, we compared anti-hamster antibodies found in naive LEW-1A rats, or in LEW-1A rats rejecting or accommodating a hamster heart, using a combination of cobra venom factor (CVF) and cyclosporin A (CsA) given for 10 days, and then CsA alone. RESULTS: Hamster hearts grafted into rat recipients contained IgG and IgA deposits to the same extent whether the xenograft was rejected or accommodated. Only immunoglobulins of the IgM isotype were found to be more abundant in recipients rejecting their graft. A significant part of this IgM response was directed toward the Forssman antigen, a sphingolipid present in the hamster but not in the rat. However, although anti-Forssman antibodies bind in situ to hamster tissues, this binding was not able to induce hyperacute rejection after antibody transfer. Furthermore, depletion of anti-Forssman antibodies from a rejecting serum did not modify its rejection properties. CONCLUSION: Unlike the pig-to-primate discordant xenotransplantation model, in which preexisting anti-carbohydrate antibodies are directly responsible for hyperacute rejection, in the concordant hamster-to-rat situation, the evoked IgM anti-Forssman carbohydrate antibodies do not appear to be the main cause of the vascular rejection.

T cell repertoire alterations of vascularized xenografts.

The role of T cells in the rejection of vascularized xenografts has been little explored. Because of the high potential diversity of xenoantigens, it has been suggested that xenotransplantation could induce a strong cellular response that could contribute to delayed rejection. Alternatively, alterations in molecular interactions could impair the T cell response. Because the analysis of TCR repertoire in vivo indirectly reflects the nature and the magnitude of T cell xenorecognition, we took advantage of the possibility of obtaining long term survival of hamster heart xenografts in rat recipients treated with a combination of cobra venom factor and cyclosporin A (CsA), to analyze T cell infiltration and, for the first time, V beta TCR usage, at the complementarity-determining region 3 level, in accommodated and rejected xenografts, compared with allografts. After withdrawal of CsA (on day 40), the analysis of V beta family expression and corresponding complementarity-determining region 3 lengths in rejected xenografts revealed a Gaussian pattern, in contrast to a much more restricted pattern in rejected allografts (p = 0.002), suggesting that, after withdrawal of CsA, all the underrepresented T cell clones are rapidly expanded in xenografts. These results correlate with the rapid kinetics of rejection (4 +/- 1 days), the high number of T cells, the rapid expression of markers of activation (IL-2 receptor alpha-chain and class II receptor), and the strong deposit of IgG Abs in rejected xenografts. Taken together, these results suggest that the intensity and diversity of the T cell response to xenografts could be stronger than the response to allografts in vivo.