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Background: Polyclonal rabbit antithymocyte globulins (ATGs) are commonly used in organ transplantation as induction. Anti- N -glycolylneuraminic acid carbohydrate antibodies which develop in response to rabbit carbohydrate antigens might lead to unwanted systemic inflammation. LIS1, the first new generation of antilymphocyte globulins (ALGs) derived from double knockout swine, lacking carbohydrate xenoantigens was already tested in nonhuman primates and rodent models., Methods: This open-label, single-site, dose escalation, first-in-human, phase 1 study evaluated the safety, T cell depletion, pharmacokinetics, and pharmacodynamics of LIS1. In an ascending dose cohort (n = 5), a primary kidney transplant recipient at low immunologic risk (panel reactive antibody [PRA] < 20%), received LIS1 for 5 d at either 0.6, 1, 3, 6, or 8 mg/kg. After each patient completed treatment, the data safety monitoring board approved respective dose escalation. In the therapeutic dose cohort (n = 5) in patients with PRA <50% without donor specific antibodies, 2 patients received 8 mg/kg and 3 patients 10 mg/kg., Results: CD3 + T cell depletion <100/mm 3 at day 2 was observed in all patients who received 6, 8, and 10 mg/kg of LIS1. The terminal half-life of LIS1 was 33.7 d with linearity in its disposition. Lymphocyte repopulation was fast and pretransplant lymphocyte subpopulation counts recovered within 2-4 wk. LIS1 was well tolerated, neither cytokine release syndrome nor severe thrombocytopenia or leukopenia were noticed. Antibodies to LIS1 were not detected., Conclusions: In this first-in-human trial, genome-edited swine-derived polyclonal LIS1 ALG was well tolerated, did not elicit antidrug antibodies, and caused time-limited T cell depletion in low- and medium-risk kidney transplant recipients., Competing Interests: J.R., P.-J.R., G.E., O.D., and B.V. are employees of Xenothera. O.D., J.-P.S., J.-M.B., and C.G. are cofounders of Xenothera. The other authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Humanized immune system immunodeficient mice have been extremely useful for the in vivo analyses of immune responses in a variety of models, including organ transplantation and graft versus host disease (GVHD) but they have limitations. Rat models are interesting complementary alternatives presenting advantages over mice, such as their size and their active complement compartment. Immunodeficient rats have been generated but human immune responses have not yet been described., Methods: We generated immunodeficient Rat Rag-/- Gamma chain-/- human signal regulatory protein alpha-positive (RRGS) rats combining Rag1 and Il2rg deficiency with the expression of human signal regulatory protein alpha, a negative regulator of macrophage phagocytosis allowing repression of rat macrophages by human CD47-positive cells. We then immune humanized RRGS animals with human peripheral blood mononuclear cells (hPBMCs) to set up a human acute GVHD model. Treatment of GVHD was done with a new porcine antihuman lymphocyte serum active through complement-dependent cytotoxicity. We also established a tumor xenograft rejection model in these hPBMCs immune system RRGS animals by subcutaneous implantation of a human tumor cell line., Results: RRGS animals receiving hPBMCs showed robust and reproducible reconstitution, mainly by T and B cells. A dose-dependent acute GVHD process was observed with progressive weight loss, tissue damage, and death censoring. Antihuman lymphocyte serum (L1S1) antibody completely prevented acute GVHD. In the human tumor xenograft model, detectable tumors were rejected upon hPBMCs injection., Conclusions: hPBMC can be implanted in RRGS animals and elicit acute GVHD or rejection of human tumor cells and these are useful models to test new immunotherapies.

Nephrotoxicity of calcineurin inhibitors and uncontrolled effector function of alloreactive T lymphocytes are main drivers of transplant dysfunctions. T lymphocytes either directly damage tissues or indirectly promote inflammation and antibody responses. Beside inhibitors of calcium-dependent pathways and antimetabolites, modulators of T-cell costimulation are elected pharmacological tools to enable interference with immune-mediated transplant dysfunctions. CD28 and CTLA-4 are major costimulatory and coinhibitory cell surface signaling molecules interacting with CD80/86, known to be critically important for immune response of committed T cells and regulation. Initial bench to beside translation, 2 decades ago, resulted in the development of belatacept CTLA-4 fused with an immunoglobulin Fc domain, a biologic inhibiting interaction of both CD28 and CTLA-4 with CD80/86. Despite proven effectiveness in inhibiting alloimmune responses, clinical use of belatacept in kidney transplantation revealed a substantially high incidence of acute, cell-mediated rejection. The cause of belatacept-resistant graft rejection was allocated to elevated pretransplant frequencies of CD28 memory T cells. Owing to different requirements in CD28 costimulatory and CTLA-4 coinhibitory signals to control naive and memory T cells, selective antagonists of CD28-CD80/86 interactions have been developed on the rationale that preservation of CTLA-4-mediated regulatory mechanisms would result in a better control of alloreactivity and would represent a regulatory T-cell-compatible immunosuppression. After the successful testing of selective CD28 antagonists in First In Human studies, this review delineates how this shift in paradigm performed in preclinical transplantation models and evaluates its clinical potential.

Background: Graft-versus-host disease (GVHD) is a severe complication of hematopoietic stem cell transplantation. Current therapies to prevent alloreactive T cell activation largely cause generalized immunosuppression and may result in adverse drug, antileukemia and antipathogen responses. Recently, several immunomodulatory therapeutics have been developed that show efficacy in maintaining antileukemia responses while inhibiting GVHD in murine models. To analyze efficacy and better understand immunological tolerance, escape mechanisms, and side effects of clinical reagents, testing of species cross-reactive human agents in large animal GVHD models is critical., Methods: We have previously developed and refined a nonhuman primate (NHP) large animal GVHD model. However, this model is not readily amenable to semi-high throughput screening of candidate clinical reagents., Results: Here, we report a novel, optimized NHP xenogeneic GVHD (xeno-GVHD) small animal model that recapitulates many aspects of NHP and human GVHD. This model was validated using a clinically available blocking, monovalent anti-CD28 antibody (FR104) whose effects in a human xeno-GVHD rodent model are known., Conclusions: Because human-reactive reagents may not be fully cross-reactive or effective in vivo on NHP immune cells, this NHP xeno-GVHD model provides immunological insights and direct testing on NHP-induced GVHD before committing to the intensive NHP studies that are being increasingly used for detailed evaluation of new immune therapeutic strategies before human trials.

Background: Lymphocyte-activated gene-3 (LAG-3, CD223) is upregulated during the early stages of T-cell activation and could be the target of cytotoxic antibodies for induction therapy in transplantation., Methods: Fully vascularized heterotopic allogeneic heart transplantation was performed in rats across a full major histocompatibility complex-mismatch barrier (LEW.1W into LEW.1A). Recipients received two injections (day 0 and 3) of cytotoxic antibodies directed to the extra-loop of LAG-3 immunoglobulin (Ig)-like N-terminal domain or control antibodies., Results: LAG-3 mRNA transcripts accumulated in cardiac allografts undergoing rejection, but not in peripheral lymphoid organs. Administration of anti-LAG-3 antibodies on the day of transplantation did not modify alloreactivity of T lymphocytes from the spleen and did not change the alloantibody response. However, it inhibited graft infiltration by effector mononuclear cells, reduced intragraft levels of interferon-gamma mRNA and prolonged allograft survival from 6 days in controls to a median of 27 days. Anti-LAG-3 antibodies were also active in prolonging survival when administered in a delayed manner, after rejection onset. LAG-3 being also expressed by activated regulatory T (Treg) cells, we tested the effect of anti-LAG-3 antibodies on graft acceptance after donor blood transfusions, a Treg-dependent tolerance induction model. We found that tolerance induction was prevented by anti-LAG-3 antibodies., Conclusions: Targeting LAG-3-positive cells with cytotoxic antibodies is immunosuppressive in transplantation by depleting effectors T cells and therefore may represent a treatment for rejection episodes focused only on pathogenic cells. However, it might not be compatible with tolerance-induction strategies.

Background: The innate immune system, including complement and natural killer (NK) cells, plays a critical role in activation and damage of endothelial cells (ECs) during xenograft rejection. The semisynthetic proteoglycan analog dextran sulfate (DXS, molecular weight 5,000) is known to inhibit the complement and coagulation cascades. We hypothesized that DXS may act as an "EC-protectant" preventing complement and NK lysis by functionally replacing heparan sulfate proteoglycans that are shed from the EC surface on activation of the endothelium., Methods: Binding of DXS to ECs, deposition of human complement, cytotoxicity, and heparan sulfate expression after exposure to normal human serum were analyzed by flow cytometry. The efficacy of DXS to protect ECs from xenogeneic NK cell-mediated cytotoxicity was tested in standard 51Cr-release assays., Results: DXS dose-dependently inhibited all three pathways of complement activation. Binding of DXS to porcine cells increased on treatment with human serum or heparinase I and correlated positively with the inhibition of human complement deposition. This cytoprotective effect of DXS was still present when the challenge with normal human serum was performed up to 48 hr after DXS treatment of the cells. DXS incubation of porcine ECs with and without prior tumor necrosis factor-alpha stimulation reduced xenogeneic cytotoxicity mediated by human NK cells by 47.3% and 25.3%, respectively., Conclusions: DXS binds to porcine cells and protects them from complement- and NK cell-mediated injury in vitro. It might therefore be used as a novel therapeutic strategy to prevent xenograft rejection and has potential for clinical application as an "EC protectant."

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.