Negative Vaccination Strategies for Promotion of Transplant Tolerance

Abstract

Organ transplantation requires the use of immunosuppressive medications that lack antigen specificity, have many adverse side effects, and fail to induce immunological tolerance to the graft. The safe induction of tolerance to allogeneic tissue without compromising host responses to infection or enhancing the risk of malignant disease is a major goal in transplantation. One promising approach to achieve this goal is based on the concept of “negative vaccination”. Vaccination (or actively-acquired immunity) involves the presentation of both a foreign antigen and immunostimulatory adjuvant to the immune system to induce antigen-specific immunity. By contrast, negative vaccination, in the context of transplantation, involves the delivery of donor antigen prior to or after transplantation, together with a “negative adjuvant” to selectively inhibit the allo-immune response. This review will explore established and emerging negative vaccination strategies for promotion of organ or pancreatic islet transplant tolerance. These include donor regulatory myeloid cell infusion, that has progressed to early phase clinical trials, apoptotic donor cell infusion that has advanced to nonhuman primate models, and novel nanoparticle antigen-delivery systems.

Introduction

Clinical transplantation provides life-saving therapy to patients with organ system failure. However, allogeneic transplantation requires the use of immunosuppressive (IS) medications that lack antigen (Ag) specificity, have a multitude of adverse side effects, and fail to induce tolerance to the transplanted organ. The safe induction of immunologic tolerance to allogeneic tissue, without compromising host responses to infection or enhancing the risk of malignant disease, is a major goal in transplantation. Observations of naturally-occurring hematopoietic cell chimerism and tolerance were first made in cattle by Owen in 1945.¹ Later experiments in mice by Medawar et al published in 1953, revealed that fetal introduction of donor hematopoietic cells led to tolerance of foreign skin grafts in adulthood.² Over the past 70 years, numerous experimental approaches, including induction of chimerism, or use of diversely-acting pharmacologic or biologic agents, have been shown to induce transplant tolerance in adult rodents. However, identification of a therapeutic regimen that can safely and predictably induce organ transplant tolerance in humans has proved elusive.

Multiple strategies to promote tolerance have been tested in humans. These include the induction of donor hematopoietic cell chimerism that, despite limited success,³ cannot currently be generalized due to risks associated with the conditioning regimens required to promote chimerism. They also include adoptive cell therapies such a T regulatory cells (T regs) and chimeric antigen receptor (CAR) T regs, for which unequivocal evidence of efficacy has yet to be documented in organ transplantation.⁴ An approach that holds considerable promise based on extensive preclinical testing in recent years is based on the concept of “negative vaccination”. On the one hand, vaccination (or actively-acquired immunity) involves the presentation of both foreign Ag and an immunostimulatory adjuvant to the immune system to induce Ag-specific immunity. On the other, negative vaccination to promote tolerance, involves the delivery of donor Ag together with a “negative adjuvant” that in combination, selectively and durably inhibit the alloimmune response. Instigation of either response is dependent on specialized Ag-presenting cells (APCs) of the innate immune system with the capacity to induce or regulate allo-immunity.^5,6

Antigen-presenting myeloid cells as key to “negative vaccination” – the roles of dendritic cells and macrophages

A potential tolerance vaccine must interact with endogenous Ag-presentation systems. The most proficient Ag-acquiring, processing, and presenting cell of the immune system is the dendritic cell (DC). DCs were first described by Steinman and Cohn in 1973⁷ and are a heterogeneous population of uniquely well-equipped “professional” APCs derived from bone marrow precursors, that play critical roles in both inducing and modulating innate and acquired immune responses.^8,9

Most DCs are ultimately derived from common myeloid/lymphoid bone-marrow progenitors. As immature DCs, they migrate via blood to tissues throughout the body where they act as sentinels. As such, they sample the local microenvironment and recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) that trigger their maturation. Consequently, they become well-equipped to present Ag to and activate immune effector cells in secondary lymphoid tissue. While all conventional (c) DCs in blood and tissue inherently express major histocompatibility complex (MHC) class II Ag and cluster of differentiation (CD) 11c to varying degrees, multiple distinct subsets of DCs with different ontogeny, cell surface phenotypes and function have been described. In addition to cDC, these include nonconventional plasmacytoid DCs (pDC), epidermal Langerhans cells (LC), and inflammatory monocyte-derived DCs (MoDC).^10,11

cDCs are distributed ubiquitously throughout all lymphoid and nonlymphoid tissues, including all commonly-transplanted organs. They present endogenous Ag through MHC class I and exogenous Ag through MHC class II molecules. Two principal subsets of cDC: cDC1 and cDC2, are distinguished in humans by the presence of cell-surface molecules CD141 and CD1c respectively. cDC1s are proficient in cross-presentation and priming of CD8⁺ T cells against extracellular Ags, while cDC2 are potent stimulators of naïve T cells, with ability to induce either T helper (Th) cell polarization or regulatory T cells (T-regs). pDC (CD11c⁺CD123⁺) are found in peripheral organs, or as circulating cells that express interferon (IFN) regulatory factor (IRF) 7 and secrete type-1 IFN upon viral infection.¹² They also exhibit tolerogenic properties, favoring a T-reg response.¹² MoDC are monocyte-derived inflammatory cells that infiltrate local tissues as a consequence of inflammation or infection. They release tumor necrosis factor (TNF) and inducible nitric oxide synthase (iNOS) in response to pathogens. Unlike the other DC populations, epidermal LCs have an embryonic origin in hematopoietic precursors and migrate following activation to skin-draining lymph nodes where they present Ags and exhibit immune regulatory functions.^13,14 Harnessing the immunoregulatory properties of these diverse DC populations offers a key approach to tolerance induction.

Macrophages are versatile phagocytic innate immune cells derived from circulating monocytes, with considerable phenotypic/functional plasticity and functional diversity in response to the local microenvironment factors and immune cell signaling. They can act as APCs and drive a spectrum of immune responses dependent on local environmental factors, with inflammatory phenotypes that drive Th1 and Th17 cell responses, to anti-inflammatory/tissue repair phenotypes.¹⁵

DC-regs and M-regs

DCs maintain self-tolerance in the healthy steady-state.¹⁶ Each DC subset appears to have the capacity to promote immune regulation or tolerance through several mechanisms, T cell anergy, apoptosis/deletion and the induction of T-regs. So-called “tolerogenic” or “regulatory” DCs (DC-regs) with capacity to inhibit naïve or memory T cell responses are not defined by a specific cell-surface marker, but in general exhibit characteristic upregulation of negative T cell coregulatory or suppressive molecules such as program death ligand (PD-L) 1 and Fas ligand, and release anti-inflammatory cytokines such as IL-10, indoleamine 2,3-dioxygenase (IDO), adenosine, and other factors. DC-reg are weak T cell stimulators and inhibit their responses by inducing anergy or apoptosis.¹⁷

A subset of regulatory macrophages (M-regs) with regulatory and suppressive activity is found in vivo, or can be induced in culture by a variety of signals including monocyte colony-stimulating factor (M-CSF) and interleukin (IL)-10.¹⁸ Thus, macrophages are also key agents/targets for promotion of tolerance. They can also spare or induce T-regs.¹⁹ M-regs appear to inhibit allo-Ag responses by suppressing T cell proliferation via induction of IL-10-producing T-regs mediated by transforming growth factor- β (TGF-β), retinoic acid and IDO, and by eliminating allo-reactive T cells.^20,21

Production and dosing of DC-regs and M-regs for therapeutic applications

Numerous protocols have been described for generation of DC-regs in animal models and humans.²² Processes for manufacturing DC-regs for early phase clinical testing in transplantation and autoimmunity have been described in detail,^18,23,24 without consensus regarding an optimal protocol. Usually, peripheral blood monocytes are isolated from leukapheresis products by elutriation and cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4, as well as biological agents such as rapamycin, dexamethasone, or Vitamin D3, or anti-inflammatory cytokines such as IL-10.^18,22 Upon generation, manufactured DC-regs have low expression of co-stimulatory molecules (CD80 and CD86) but comparatively high levels of PD-L1, and produce minimal levels of IL-12 but high levels of IL-10 in response to CD40 ligation.²⁵ In addition and importantly, unlike immature DC in vivo, they also resist potent maturation signals such as Toll-like receptor (TLR)-4 ligation, or pro-inflammatory cytokines.^18,22,25

Guidelines for manufacturing M-regs have also been developed.²⁶ Similarly to DC-reg generation, CD14⁺ monocytes are isolated from PBMC and cultured in growth factor (M-CSF). After this initial induction, M-regs are stimulated with IFN-γ and express high IDO activity.²⁷ Donor-derived M-regs have been tested safely in human renal transplantation.^27,28

In experimental transplantation and in patients, adoptively-transferred regulatory myeloid cells can be either donor- or recipient-derived. Donor cells can immediately present donor Ag to host T cells via the direct pathway of allo-recognition. However, it is unlikely that these allogeneic cells survive for significant periods following their infusion. Indeed, donor MHC Ags and other molecules are soon conveyed to recipient APCs via donor-derived small extracellular vesicles²⁹ that appear to mediate the immune-modifying effect of the donor cell infusion.^30,31 Manufactured recipient-derived regulatory cells can either be pulsed with donor allo-Ag or infused unpulsed a day before transplantation, whereupon they acquire and process allograft-derived Ag in vivo. Practically, generation of donor regulatory myeloid cells is not feasible for infusion before deceased donor transplantation due to the time required for their production, although this approach has been documented in nonhuman primate (NHP) kidney graft recipients^32–35 and is feasible in human live donor transplant recipients.^29,36 Pretransplant infusion of autologous DC-regs can extend cardiac allograft survival and induce donor-specific tolerance in rats.³⁷ Moreover, an early phase live-donor human kidney transplant trial of peritransplant autologous DC-reg infusion has been undertaken and shown to be feasible and safe, with some preliminary evidence of DC-reg function.^24,28 Mechanistically, it appears that the immunomodulatory effect of autologous DC-reg infusion can be shaped to induce donor-specific tolerance in rats when the cells are co-administered with suboptimal levels of IS agents. However, it is unclear whether this represents indirect Ag presentation in vivo, or an alternative mechanism.³⁷ An additional unanswered question is whether myeloid APCs generated ex vivo from patients with preexisting end-stage organ disease retain the same functional properties and potential as those from healthy individuals.

In terms of dosage, a single donor-derived DC-reg infusion of 2.5–10 ×10⁶ cells/kg has been administered pretransplant in NHP renal transplantation^33–35 with a similar dose range in phase 1/2a clinical trials in live donor liver transplantation.²⁹ Similar single doses have been used in early phase human trials of donor-derived M-regs (8 × 10⁶ cells/kg,²⁷) and autologous DC-regs (1 × 10⁶ cells/kg,²⁴) in kidney transplantation.

Efficacy of ex vivo-generated regulatory myeloid APCs in animal models

In mice, donor DC-reg infusion without IS therapy prior to islet allografting doubles graft survival time from 15 to 30 days.³⁸ Similarly, recipient DC-reg infusion prior to sex-mismatched skin grafting significantly prolonged graft survival time.³⁹ However, when directly compared in a heart allograft model, rapamycin-treated donor DC-reg infusion 1 week prior to transplant resulted in superior, although not indefinite graft survival.⁴⁰ Untreated, immature bone-marrow-derived donor DC-regs have been investigated for therapeutic effect, with a modest increase in median survival of murine cardiac transplants from 9.5 to 22 days when infused without IS therapy 1 week prior to transplantation.⁴¹ Lutz et al however showed that donor-derived DC-regs that induced T cell unresponsiveness in vitro and in vivo prolonged haplotype-specific cardiac allograft survival (from 8 days to >100 days median survival time) when they were administered 7 days (but not 3, 14, or 28 days) before transplantation.⁴² Similar results have been obtained in mice using a protocol to generate DC-regs from pluripotent stem cells,^30,43 or with monocyte-derived M-reg infusion.²⁰ Alternative approaches with donor-derived M-reg infusion alone prior to heterotopic heart transplantation in mice have also demonstrated significant extension of graft survival from <10 days to >30 days.²⁰

In a NHP model of kidney transplantation, DC-reg infusion has shown significant promise. Pretransplant (day −7) donor DC-reg infusion combined with a minimal IS regimen of co-stimulatory CD28 blockade using CTLA-4 Ig and tapered rapamycin therapy safely led to prolongation of median graft survival time from 39.5 to 113.5 days, with no adverse events or evidence of host sensitization.^32,33,35 Prolongation of renal graft survival was also observed when recipient-derived DC-regs pulsed with donor allo-Ag were administered 1 day before transplant.³⁴

In summary, regulatory myeloid APC infusions given preemptively before transplant can effectively prolong graft survival and promote donor-specific tolerance in rodents, principally through 1) induction of Foxp3⁺ T-regs, 2) induction of T cell anergy, 3) elimination of allo-reactive T cells.^44,45 Donor-derived DC-reg infusion also safely prolongs graft survival in NHP when combined with minimal IS drug therapy. A summary of regulatory myeloid cell infusion studies in preclinical models and early-phase clinical trials can be found in Table 1.

Table 1 –

Preclinical and clinical studies of regulatory myeloid cell infusion prior to transplantation

Reference	Model	Treatment	Outcomes
Rodent models
Rastellini et al 199538	Islet allograft Mouse	Treatment: Donor DCreg infusion 7 days prior to islet transplantation without other IS Methods: Hepatic DC progenitors cultured with GM-CSF Control: Syngeneic cell infusion	Mean allograft survival 15.3 days in control group vs 30.3 days in donor cell infusion group (p < 0.001)
Fu et al 199641	Heart allograft Mouse	Treatment: Donor DCreg infusion 7 days prior to heart transplantation without other IS Methods: Bone marrow-derived DC cultured with GM-CSF Control: GM-CSF + IL-4 stimulated donor DC infusion	Mean allograft survival 7 days in control group vs 22 days in donor cell infusion group (p < 0.001)
Lutz et al 200042	Heart allograft Mouse	Treatment: Donor DCreg infusion 7 days prior to transplantation without other IS Methods: Bone marrow-derived DC cultured with low concentration GM-CSF Control: Multiple groups, primary comparator no infusion	Mean allograft survival 8 days in control vs >100 days in donor cell infusion group (p < 0.01)
Taner et al 200540	Heart allograft Mouse	Treatment: Recipient (syngeneic) DCreg infusion 10, 3, and 0 days prior to transplantation without other IS Methods: Bone marrow-derived DC cultured with GM-CSF, IL-4, and rapamycin, pulsed with allogeneic donor antigen Control: Multiple groups, primary comparator no infusion	Mean allograft survival 9 days in control vs >59 days in recipient cell infusion group (p = 0.0014)
Turnquist et al 200784	Heart allograft Mouse	Treatment: Recipient (syngeneic) DCreg infusion 7 days prior to transplantation with concomitant rapamycin administration Methods: Bone marrow-derived DC cultured with GM-CSF and rapamycin, pulsed with allogeneic antigens Control: Multiple groups, primary comparator recipient DCreg infusion without rapamycin co-administration	Mean allograft survival 12 days in control vs >100 days in recipient cell infusion plus rapamycin group (p = 0.0013)
Li et al 2008107	Heart allograft Mouse	Treatment: Recipient (syngeneic) DCreg infusion 3 days prior and 1 day post transplantation with whole body irradiation Methods: Syngeneic DCregs isolated from previously tolerised syngeneic recipients using CD45RA Ab and a deoxyspergualin analog. Control: Control DCs isolated from syngeneic rejecting recipients	Mean allograft survival 13 days in control vs 39.4 days in DCreg infusion group (p < 0.001)
Divito et al 201030	Heart allograft Mouse	Treatment: Donor DCreg infusion 7 days prior to transplantation without other IS Methods: Bone marrow-derived DC cultured with GM-CSF, IL-4, and Vitamin D3. Control: Multiple groups, primary comparator no infusion	Mean allograft survival 11.1 days in control vs 52.2 days in DCreg infusion group (p < 0.001)
Riquelme et al 201320	Heart allograft Mouse	Treatment: Donor Mreg infusion 8 days prior to transplantation without other IS Methods: Bone marrow-derived monocytes cultured with M-CSF and pulsed with IFNγ Control: Multiple groups, primary comparator no infusion	Mean allograft survival 8.7 days in control vs 32.9 days in Mreg infusion group (p < 0.001)
Segovia et al 201439	Skin allograft Mouse	Treatment: Recipient (syngeneic) DCreg infusion 1 day prior to transplantation with 5 doses anti-CD3 antibody between day −1 to day +7 Methods: Bone marrow-derived DC cultured with low-dose GM-CSF Control: Multiple groups, primary comparator anti-CD3 alone	Median allograft survival approximately 50 days in control vs >75 days in DCreg infusion plus anti-CD3 group (exact values and p values not reported)
Cai et al 201743	Heart allograft Mouse	Treatment: Donor DCreg infusion 7 days prior to transplantation without other IS Methods: Induced pluripotent stem cells cultured with GM-CSF, TGF-β, IL-10, with late IFN-γ stimulation Control: Multiple groups, primary comparator no infusion	Mean allograft survival 8 days in control vs >100 days in DC-reg infusion group (p < 0.001)
Bériou et al 200537	Heart allograft Rat	Treatment: Recipient (syngeneic) DCreg infusion ± a deoxyspergualin analog as additional IS Methods: Bone marrow-derived DC cultured with GM-CSF and IL-4 Control: Multiple groups, primary comparator no treatment	Median allograft survival 6 days in control vs 21.5 days in DCreg infusion alone (p < 0.01) vs 100 days in DCreg infusion plus the deoxyspergualin analog (p < 0.01)
Pêche et al 2005108	Heart allograft Rat	Treatment: Recipient (syngeneic) DCreg infusion 1 day prior to transplantation without other IS Methods: Bone marrow-derived DC cultured with GM-CSF and IL-4 Control: Multiple groups, primary comparator no treatment, second comparator donor DCreg infusion 1 day prior to transplantation	Median allograft survival 6 days in no treatment group vs 16.5 days in donor DCreg infusion control (p < 0.05 vs no treatment) vs 22.5 days in recipient DCreg infusion group (p < 0.01 vs no treatment)
Zheng et al 201075	Heart allograft Rat	Treatment: Recipient (syngeneic) DCreg infusion 7 days prior to transplantation without other IS Methods: Recipient (syngeneic) bone marrow-derived DCs co-cultured with apoptotic donor splenocytes treated with UV-A irradiation Control: No treatment, or recipient DCs cultured with nonapoptotic donor splenocytes	Median allograft survival approximately <10 days in control groups vs >25 days in DC infusion group (p < 0.01)
Nonhuman primate models
Ezzelarab et al 201333,35	Renal allograft	Treatment: Donor DCreg infusion 7 days prior to transplantation with CTLA4Ig between days −7 and 10, and daily low dose rapamycin for 6 months Methods: Donor leukapheresis pretransplantation, DC culture with GM-CSF, IL-4, IL-10 and Vitamin D3 Control: No DC-reg infusion, but other IS (CTLA4 Ig and tapering rapamycin) identical	Median allograft survival 39.5 days in control vs 113.5 days in donor DCreg infusion (p = 0.0138)
Ezzelarab et al 201734	Renal allograft	Treatment: Recipient (autologous) DCreg infusion 1 day prior to transplantation with CTLA4 Ig between days −7 and 10, and daily low dose rapamycin for 6 months Methods: Recipient leukapheresis pretransplantation, DC culture with GM-CSF, IL-4, IL-10 and Vitamin D3. DCregs pulsed with donor Ag prior to infusion. Control: No DCreg infusion, but other IS (CTLA4Ig and tapering rapamycin) identical (shared control group with Ezzelarab et al 201335)	Median allograft survival 39.5 days in control vs 56 days in recipient DCreg infusion (p = 0.1133; NS)
Clinical trials
Hutchinson et al 2008104	Renal allograft (live donor)	Treatment: Donor Mreg infusion 5 days prior to transplantation with ATG, tacrolimus, mycophenolate, and weaning steroid Methods: Donor leukapheresis pretransplantation, Mreg culture with M-CSF, co-cultured with recipient PBMC Control: No control, single-arm study	Clinical safety, no immediate adverse outcomes (n = 5) 4/5 weaned to tacrolimus monotherapy 1/5 tolerated 8 months of no immunosuppression 2/5 acute rejection episode year 1
Hutchinson et al 2008105	Renal allograft (deceased donor)	Treatment: Donor Mreg infusion 5 days posttransplantation with tacrolimus, tapered sirolimus, and tapered steroid Methods: Donor leukapheresis pretransplantation, Mreg culture with M-CSF, co-cultured with recipient PBMC Control: No control, single-arm study	Clinical safety, no immediate adverse outcomes (n = 10) 7/10 acute rejection episodes year 1
Hutchinson et al 201127	Renal allograft (live donor)	Treatment: Donor Mreg infusion 6–7 days prior to transplantation with tacrolimus, azathioprine, and weaning steroids Methods: Donor leukapheresis pretransplantation, Mreg culture with M-CSF and IFNγ Control: No control, single-arm study	Clinical safety, no immediate adverse outcomes (n = 2)
Macedo et al 202129 Tran et al 202336	Liver allograft (live donor)	Treatment: Donor DCreg infusion 7 days prior to transplantation with tacrolimus, mycophenolate mofetil, and weaning steroids Methods: Donor leukapheresis pretransplantation, DCreg culture with GM-CSF, IL-4, IL-10 and Vitamin D3 Control: No control; single-arm study	Clinical safety, no immediate adverse outcomes (n = 14) Clinical observations and detailed mechanistic studies up to 1 year posttransplant (follow-up to Macedo et al, 2021)
Sawitzki et al 202028 Follow-up reported in Moreau et al 202324	Renal allograft (live donor)	Treatment: Recipient (autologous; no Ag) DCreg infusion OR donor Mreg infusion 1 day prior to transplantation plus tacrolimus, mycophenolate mofetil, and standard steroids Methods: Recipient leukapheresis pretransplantation, DCreg culture with GM-CSF Control: Basiliximab induction plus tacrolimus, mycophenolate mofetil, and standard steroids	Clinical safety, no immediate adverse outcomes (n = 8 in treatment group) 100% graft survival at 3 years in all groups with 5/8 DCreg infusion patients able to reduce immunosuppression

In vivo targeting of DCs: apoptotic donor cells as negative vaccines

The immune system removes billions of circulating apoptotic cell fragments daily while maintaining self-tolerance.⁴⁶ This is mediated through a process termed “efferocytosis”, involving phagocytosis of apoptotic fragments by immune cells. Apoptotic cells present pro-phagocytic signals to the immune system in a manner that necrotic cells do not.⁴⁷ Phagocytic tyrosine kinase receptors mediate this process in an immunologically silent manner with triggering of anti-inflammatory signaling pathways, the release of anti-inflammatory cytokines, and suppression of pro-inflammatory cytokines by inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway.^47,48

These host anti-inflammatory pathways can be harnessed to induce donor-specific tolerance. Thus, apoptotic donor cells generated using a variety of methods have been used in experimental models of transplant tolerance. To present the full complement of donor class I and II MHC Ags, donor splenocytes or ex vivo-expanded donor B cells have been used.⁴⁹ In mice, donor apoptotic cells localize to the recipient’s splenic marginal zone within hours⁵⁰ and are phagocytosed by recipient DCs and macrophages through host efferocytic receptor tyrosine kinase pathways.^51,52 Recipient DCs and macrophages that phagocytose apoptotic donor cells broadly cause an increase in anti-inflammatory cytokines,^50,53,54 a reduction in pro-inflammatory cytokines,⁵⁵ and subsequent induction of donor Ag-specific Foxp3⁺ T-regs.^55,56

Murine models show marked increases in anti-inflammatory cytokines such as IL-10, IL-13, and TGF-β following apoptotic donor leukocyte infusion.^53,54 The effect is rapid, with IL-10 production by splenic macrophages within 10 minutes of infusion rapidly altering the phenotype of F4/80⁺ macrophages to favor high PD-L1 expression.⁵⁰ In mouse heterotopic heart transplantation, expression of anti-inflammatory cytokines is also increased within allograft tissue.⁵⁴ Concomitantly, there is a reduction in circulating pro-inflammatory cytokines, including IFN-γ, TNF-α, IL-1β, IL-6, IL-17, and IL-23.⁵⁴ The direct and immediate effect of the apoptotic body response is observed in mixed leukocyte reactions, where co-culture with apoptotic donor cells results in a reduction in IFN-γ production by allogeneic T cells, and a corresponding increase in cardiac allograft survival.⁵⁷

Another mechanism by which donor apoptotic cells may induce tolerance is via upregulation of the negative coregulatory molecules PD-L1 and 2 by recipient CD11c⁺ DCs,⁵² – pathways that appear crucial for downregulation of T effector cells.⁵⁸ CD8α⁺ DC that capture apoptotic cells remain immature in a DC-reg-like state, activating anti-donor CD4⁺ T cells that are deficiently responsive to IL-7 and IL-15, and cannot upregulate the anti-apoptotic protein B-cell lymphoma-extras large (BCL-X_L) or secrete pro-inflammatory cytokines.⁴⁷ Simultaneously, CD4⁺ T cells with indirect allo-specificity undergo an initial expansion, followed by profound clonal contraction and sequestration in the spleen without trafficking to the allograft or draining lymphatic system.⁵² This inhibits CD8⁺ T cell responses as the remaining CD4⁺ T cell population suppresses CD40 signaling.⁵⁹

While allo-specific T cells are downregulated, there is an increase in CD4⁺Foxp3⁺ T-regs following apoptotic donor cell administration.^55,56 Splenic macrophages are polarized towards the alternatively-activated M2 phenotype, leading to expansion of T-regs in the spleen and allograft-draining lymph nodes.⁵³ Direct in vitro culture of apoptotic allogeneic APCs with naïve CD4⁺ and CD4⁺CD25⁻ T cells induces a Treg phenotype with upregulation of Foxp3, high expression of CD62L and CD2, and low expression of co-stimulatory signaling molecules that do not stimulate proliferation or inflammatory cytokine secretion by co-cultured CD4⁺ T cells.⁶⁰ Similarly, there appears to be an increase in circulating regulatory B cell phenotypes, with a simultaneous reduction in allo-specific effector B cells.⁶¹

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous suppressor cell population that express the myeloid marker glucocorticoid receptor 1 (Gr1) and appear to have an important role in immune tolerance, infiltrating the allograft and suppressing local inflammatory cell responses. In mice, apoptotic donor cell infusions increase CD11b⁺ MDSCs that maintain the ability to suppress T cell proliferation in vitro. In a mouse cardiac allograft model, these MDSCs migrate to the allograft,⁶² mediating a protective effect by local inhibition of CD8⁺ T effector cells and potential induction of Treg in an IFN-y dependent process.^62,63 Adoptive transfer of MDSCs in mice has also shown promise by prolonging cardiac and skin allograft survival.^64,65

In summary, apoptotic donor leukocytes package the entire spectrum of major and minor donor Ags in a fashion that induces donor-specific tolerance through (1) rapid Ag uptake by recipient APCs; (2) an increase in anti-inflammatory cytokines and concomitant decrease in inflammatory cytokines that drive the allo-immune response; (3) phenotypic alteration of DCs to promote a regulatory response; (4) downregulation of allo-specific CD4⁺ T cells, inhibition of CD8⁺ effector T cell responses; (5) induction of Foxp3⁺ T-regs; and (6) activation of MDSCs.

Production and dosing of apoptotic cells for therapeutic application

Apoptotic cells for Ag-specific tolerance have generally been manufactured using either radiation- or chemically-induced apoptosis. UV radiation combined with freezing/thawing cycles induces apoptosis^47,66 though generally, chemically-induced apoptosis has been preferred using the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (ECDI). ECDI was first synthesized in 1961,⁶⁷ later finding use as a coupling agent for Ag presentation⁶⁸ and as an apoptotic agent for Ag-specific tolerance.⁶⁹

Donor leukocytes are the most common cell source, and both splenocytes and PBMCs have been used. Donor spleens can be processed to a single cell suspension, providing a sufficient number of MHC class I and II positive cells for infusion. However, numbers may not be sufficient for the provision of multiple infusions. Donor B cells can also be isolated from PBMCs and expanded ex vivo to achieve similar cell doses for infusion.⁴⁹ An alternative approach is ECDI-coupling of recipient leukocytes with donor allo-Ag.^50,59 It is important to avoid use of necrotic or late apoptotic cells as they interact differently with the immune system and can actually lead to DC maturation and sensitization.⁶⁶

With regards to an appropriate dose of apoptotic cells, the aim is to induce tolerance, while avoiding theoretical saturation of efferocytic systems and causing paradoxical sensitization. Historical studies have shown significant variation in immune responsiveness to foreign HLA antigens with different Ag loads.⁷⁰ A total of 1×10⁸ ECDI-splenocytes can provide indefinite islet allograft survival in mice.⁵⁸ Further dose-finding studies showed doses as low as 5×10⁶ cells compromised graft protection, but 1×10⁷ cells provided similar protection and may be an appropriate target dose.⁷¹ In translational NHP studies, a similar threshold calculated to 0.25 ×10⁹ cells/kg demonstrated efficacy in islet transplantation.⁶¹

Efficacy of apoptotic donor cells in animal models

Transplant tolerance using apoptotic cells has been demonstrated in a variety of animal models (mouse, rat, NHP), and with a variety of allografts (islet, heart, skin).

In murine skin allograft models, donor ECDI-splenocyte infusions together with concurrent rapamycin administration significantly extended graft survival time for both full-thickness and vascularized grafts.^53,55 Long-term mouse cardiac allograft survival (>100 days) has also been achieved following donor ECDI-splenocyte infusion, including full MHC mismatch models and (together with OX40-OX40L blockade) in presensitized recipients.^47,57,62,72 Similarly, donor ECDI-splenocyte infusion alone can induce tolerance of islet allografts,^52,56,58 with co-administration of B cell blockade using anti-CD20 inducing tolerance in a rat-to-mouse islet xenograft model.⁷³

An alternative method of activating apoptotic mechanisms is through extracorporeal photopheresis (ECP) of donor leukocytes and pretransplant infusion that induces recipient DC-regs and T-regs for long-term graft survival in mouse models.⁷⁴ Also, in a rat cardiac allograft model, infusion of donor lymphocytes treated with 8-methoxypsoralen and UV A light leads to uptake by recipient DCs, inducing T-regs and suppression of heart allograft rejection.⁷⁵

Importantly, it does appear that apoptotic donor cell-induced tolerance can be disrupted in the experimental setting by specific immunological challenges. CMV infection breaks established tolerance in a model of murine islet allotransplantation,⁷⁶ a mechanism that appears dependent on conversion of anergic T cells to IFN-γ-producing effector T cells.⁷⁷ Other similar models of tolerance using donor-specific transfusion have shown that disruption of tolerance also occurs with TLR engagement following CpG administration.⁷⁸

NHPs are valuable preclinical models for transplant immunology research, with highly-conserved MHC proteins between NHP and humans.^79,80 Two major studies have investigated the use of apoptotic cells for tolerance induction following islet allo-transplantation in monkeys. One study in cynomolgus monkeys combined ECDI-donor lymphocyte infusion with induction IS comprising anti-thymocyte globulin (ATG), anti-IL-6 antibody (Ab), and 30 days rapamycin administration. The experimental group displayed prolonged graft survival (85.5 versus 13.5 days), with the longest graft survival 133 days.⁸¹ Another study in rhesus macaques combined ECDI-donor lymphocyte infusion with induction IS comprising ATG and 30 days co-stimulatory pathway (CD40/CD154) blockade, anti-IL-6 Ab, soluble TNF receptor, and rapamycin. This led to long-term graft tolerance >1 year with no further IS after 21 days posttransplant.⁶¹ A broad array of mechanisms were implicated in these studies, including increased circulating T-regs, B regulatory cells (B-regs), and MDSCs following apoptotic donor cell infusion.⁶¹ A summary of donor apoptotic cell infusion studies in preclinical models and clinical transplantation can be found in Table 2.

Table 2 –

Preclinical and clinical studies of donor apoptotic cell infusion prior to transplantation

Reference	Model	Treatment	Outcomes
Rodent models
Kaneko et al 200357	Heart allograft Mouse	Treatment: ECDI-treated donor DC infusion 7 days prior to transplantation without other IS Methods: Bone-marrow derived DC cultured with GM-CSF and IL-4, pulsed with donor cell lysate and ECDI Control: Control donor DCs or no treatment	Median survival approximately 10 days in control groups vs >25 days in ECDI-DC infusion group (exact values not reported; p < 0.01)
Wang et al 200647	Heart allograft Mouse	Treatment: Apoptotic donor splenocyte infusion 7 days prior to transplantation without other IS Methods: Donor splenocytes with apoptosis induced by UV-B irradiation Control: Necrotic donor splenocytes or no treatment	Median survival approximately 20 days in control groups vs 40 days in apoptotic donor splenocyte infusion group (exact values not reported, p = 0.0001)
Luo et al 200858	Islet allograft Mouse	Treatment: ECDI-treated donor splenocyte infusion 7 days prior and 1 day after transplantation without other IS Methods: Donor splenocytes with apoptosis induced by ECDI Control: Multiple groups, primary comparator no treatment	Median survival 20 days in control groups vs >100 days in ECDI-splenocyte infusion group (p < 0.0036)
Martin et al 201059	Sex-mismatched skin allograft Mouse	Treatment: ECDI-treated recipient splenocyte infusion 7 days prior and 0 days after transplantation without other IS Methods: Recipient splenocytes ECDI-cross-linked to Y chromosomal CD4 antigen Control: Alternative non-CD4 Ag ECDI-cross linked to recipient splenocytes, or no treatment	Median survival approximately 20 days in control groups vs >100 days in ECDI-splenocyte infusion group (p < 0.001)
Kheradmand et al 201156	Islet allograft Mouse	Treament: ECDI-donor splenocyte infusion 7 days prior and 1 day after transplantation without other IS Methods: Donor splenocytes with apoptosis induced by ECDI Control: No infusion	Median survival <20 days in control group vs >150 days in ECDI-splenocyte infusion group (p value not reported)
Kheradmand et al 201252	Islet allograft Mouse	Treatment: ECDI-donor splenocyte infusion 7 days prior and 1 day after transplantation ± anti-CD20 antibody or macrophage depletion or CD11c depletion Methods: Donor splenocytes with apoptosis induced by ECDI, macrophage depletion with lipo-clodronate Control: No infusions	Median survival <10 days in control group vs <20 days in ECDI-splenocyte infusion plus CD11c depletion group vs >100 days in ECDI-splenocyte (p = 0.008) ± anti-CD20 ± macrophage depletion groups
Chen et al 201262	Heart allograft Mouse	Treatment: ECDI-donor splenocyte infusions 7 days prior and 1 day after; ± 7 / 14 days after transplantation; ± rapamycin from 1 day prior to 8 days posttransplant Methods: Donor splenocytes with apoptosis induced by ECDI Control: No infusions	Mean survival 9 days in control vs 45 days in 2 infusion group (p = 0.0005) vs 85 days in 4 infusion group (p = 0.012) vs >150 days in 2 infusion group with rapamycin (p = 0.0018)
Wang et al 201373	Islet xenograft Rat >Mouse	Treatment: ECDI-donor splenocyte infusions 7 days prior and 1 day after transplantation plus depleting anti-CD20 antibody Methods: Donor splenocytes with apoptosis induced by ECDI Control: No infusions	Median survival approximately <20 days in control vs >40 days in ECDI-splenocyte infusion group (p = 0.0026; exact values not reported). Indefinite graft survival when combined with anti-CD20 Ab
Bryant et al 201463	Heart allograft Mouse	Treatment: ECDI-donor splenocytes infusions 7 days prior and 1 day after transplantation without other IS Methods: Donor splenocytes with apoptosis induced by ECDI Control: No infusion	Median survival approximately 10 days in control vs >20 days in ECDI-splenocyte infusion group (p < 0.05, exact values not reported)
Wang et al 201571	Islet allograft Mouse	Treatment: ECDI-donor splenocytes infusions 7 days prior and 1 day after transplantation without other IS Methods: Donor splenocytes with apoptosis induced by ECDI, or recipient splenocytes ECDI-coupled to donor splenocyte lysate Control: Multiple groups, primary comparator no infusion or recipient splenocytes infusions 7 days prior and 1 day after transplantation	Median survival approximately <20 days in control vs >100 days for either donor or antigen-pulsed recipient ECDI-splenocyte infusion (p = 0.0017)
Lai et al 201754	Heart allograft Mouse	Treatment: ECDI-donor splenocytes infusions 7 days prior and 1 day after transplantation ± α1-antitrypsin from 1 day prior to 8 days after transplantation Methods: Donor splenocytes with apoptosis induced by ECDI Control: No infusion	Mean survival 7 days in control vs 42 days in ECDI-splenocyte infusion group (p = 0.0006) vs >90 days in ECDI-splenocyte infusion plus α1-antitrypsin infusion group (p = 0.0005)
Ding et al 201855	Skin allograft Mouse	Treatment: ECDI-donor splenocytes 1–3 infusions pre- and posttransplantation ± low-dose rapamycin Methods: Donor splenocytes with apoptosis induced by ECDI Control: No infusion	Median survival approximately <20 days in control vs >20 days in 2x ECDI-splenocyte infusions plus rapamycin (p < 0.01, exact values not reported)
Zhang et al 201951	Islet OR heart allografts Mouse	Treatment: ECDI-donor splenocytes infusions 7 days prior and 1 day after transplantation without other IS Methods: Donor splenocytes with apoptosis induced by ECDI Control: No infusion	Islet allograft: Median survival approximately < 20 days in control vs > 100 days in ECDI-splenocyte infusion group (p < 0.001, exact values not reported) Heart allograft: Median survival approximately <10 days in control vs 15 days in ECDI-splenocyte infusion group (p < 0.01; exact values not reported)
Zhou et al 201953	Skin allograft Mouse	Treatment: ECDI-donor splenocytes infusion 7 days prior and 1 day after transplantation plus rapamycin Methods: Donor splenocytes with apoptosis induced by ECDI Control: ECDI-donor splenocytes infusions alone	Median survival 15 days in control vs 20 days in ECDI-splenocyte infusion plus rapamycin group (no p value reported)
Xingqiang et al 201988	Heart allograft Mouse	Treatment: ECDI-donor splenocyte infusions 7 days prior and 1 day after transplantation plus cordycepin daily from day 0 to day 7 Methods: Donor splenocytes with apoptosis induced by ECDI Control: Multiple groups, primary comparator ECDI-donor splenocytes infusions alone, and no infusion	Median survival approximately 10 days in no infusion group vs > 40 days in ECDI-donor splenocyte infusion alone (p = 0.0006) vs 80 days in ECDI-donor splenocyte infusion with cordycepin infusion (p = 0.0001; exact values not reported)
Dangi et al202085	Islet allograft Sensitized mouse	Treatment: ECDI-donor splenocyte infusion 7 days prior and 1 day after transplantation plus rapamycin daily from 7 days prior to 10 days after transplantation with 4 doses of anti-CD40 Ab. Methods: Donor splenocytes with apoptosis induced by ECDI Control: Multiple groups, primary comparator no anti-CD40 Ab, and no infusion	Median survival approximately 5 days in no infusion group vs 10 days in ECDI-donor splenocyte infusion no anti-CD40 group vs 30 days in anti-CD40 (p < 0.05, exact values not reported)
Lai et al 202072	Heart allograft Mouse	Treatment: ECDI-donor splenocytes + OX40L Ab Methods: Donor splenocytes with apoptosis induced by ECDI Control: Multiple groups, primary comparator no anti-OX40L antibody, and no infusion	Median survival approximately < 10 days in no infusion group vs > 20 days in ECDI-donor splenocyte infusion alone group vs >100 days in ECDI-donor splenocyte infusion plus anti-OX40L Ab group (p < 0.001; exact values not reported)
Schneiderman et al 202274	Heart, renal, or liver allograft Mouse	Treatment: Apoptotic donor leukocyte infusion 7 days prior to transplantation ± rapamycin ± tacrolimus Methods: Donor splenocyte extracorporeal photopheresis by incubating with methoxypsoralen and UV-A irradiation Control: Multiple groups, primary comparator infusion of untreated cells, and no infusion group	Heart allograft: Median survival < 10 days in no infusion group vs > 15 days in apoptotic cell infusion group (p < 0.0001) vs >50 days in apoptotic cell infusion plus rapamycin group (p < 0.05) Liver allograft: Median survival <20 days in no infusion group vs >200 days in apoptotic cell infusion group (p < 0.0001)
Chen et al 201587	Renal allograft Rat	Treatment: ECDI-donor splenocyte infusions 7 days prior and 1 day after transplantation, ± α1-antitrypsin 1 day prior and 1/3 days after transplantation Methods: Donor splenocytes with apoptosis induced by ECDI Control: No infusion	Median survival approximately <10 days in control group vs 50 days in ECDI-splenocyte infusion group vs >200 days in ECDI-splenocyte with α1-antitrypsin infusion (p < 0.05; exact values not reported)
Nonhuman primate models
Lei et al 201581	Islet allograft	Treatment: ECDI-donor lymphocyte infusion day 0 after transplantation plus pretransplant ATG 2 doses, anti-IL-6 Ab 4 doses, and rapamycin for 30 days posttransplant Methods: Lymphoid cells isolated from donor spleen and lymph nodes, incubated with ECDI prior to infusion Control: Identical immunosuppression without ECDI-donor lymphocyte infusion	Mean survival 13.5 days in control vs 85.5 days in ECDI-donor lymphocyte infusion group (p < 0.0177)
Singh et al 201961	Islet allograft (5/6 HLA mismatch model)	Treatment: ECDI-donor lymphocyte infusions 7 days prior and 1 day after transplantation plus anti-CD40 Ab 3 doses, soluble TNF receptor 4 doses, anti-IL-6 Ab 3 doses, and rapamycin for 14 days Methods: Donor blood draw with B cell isolation, B cell culture and expansion; ECDI treatment of donor B cells prior to infusion. Control: Identical immunosuppression without ECDI-donor lymphocyte infusion	Median survival approximately <180 days in control group vs >365 days in ECDI-donor lymphocyte infusion group (p = 0.021)
Clinical trials
Mevorach et al 2014106	Allogeneic hematopoietic stem cell transplant	Treatment: Single infusion of donor mononuclear apoptotic cells plus usual induction therapy with either busulfan or total body irradiation Methods: Donor leukapheresis with cellular apoptosis induced with freezing/thawing cycle followed by cell culture with methylprednisolone Control: No infusion, usual induction therapy	Clinical safety, no immediate adverse outcomes (n = 13) No episodes of GVHD in high dose group (0/6) compared to 43% in low dose group (3/7)

The “negative adjuvant” effect: leveraging concomitant immunosuppression strategies to enhance Ag-specific tolerance

Infusion of regulatory myeloid cells or apoptotic donor cells prolongs allograft survival and can induce donor-specific tolerance in rodent models. However, these strategies alone are usually insufficient to induce permanent graft tolerance. An adjuvant is a substance that helps and enhances the effect of a drug or biologic system. “Negative adjuvants” that promote a tolerogenic milieu have thus been used to enhance the immune-modifying effect of DC-reg or apoptotic cell infusions.

Mechanistic target of rapamycin (mTOR) inhibitors have immunoregulatory and tolerogenic properties,⁸² -they inhibit maturation of DCs, enhance Foxp3⁺ T-reg responses and activity, and inhibit alloreactive T effector cells.^60,61,83 Rapamycin co-administered during the peritransplant period has been used as an adjuvant to DC-reg or apoptotic cell infusions, with successful establishment of tolerance in murine models.^37,40,62,84

Co-stimulation blockade co-administered at the time of DC-reg infusion reduces the possibility of signal 2-dependent allo-stimulation. Thus DC-reg infusion with CD28 pathway blockade using cytotoxic T lymphocyte-associated antigen (CTLA)-4 immunoglobulin (Ig) in combination with rapamycin prolongs renal allograft survival in NHP models.^32,33,35

Co-stimulation blockade strategies, together with apoptotic cell infusion have focused on blockade of CD40/CD154⁴⁷ and late co-stimulatory molecule OX40-OX40L signaling,⁷² rather than on B7/CD28 co-stimulation. Apoptotic donor DC infusion 7 days after transplant, together with CD40/CD154 blockade, significantly prolonged murine heart allograft survival from <20 to >100 days.⁴⁷ Notably, while it appeared that ECDI-treated splenocyte infusion alone was insufficient for the induction of tolerance in a sensitized mouse islet allograft model, addition of CD40 blockade and anti-CD20 therapy facilitated tolerance induction and long-term allograft survival >180 days.⁸⁵ Similarly, administration of OX40/OX40L blockade with multiple infusions of ECDI-treated splenocytes from day −7 to day +14 posttransplant induced tolerance in a presensitized mouse cardiac allograft model when ECDI-treated cell infusion alone was insufficient.⁷²

Anti-inflammatory molecules have been used in combination with apoptotic donor leukocyte infusions to reduce potential activation of allo-immune responses. α1-antitrypsin is a serine protease inhibitor with immunoregulatory properties mediated via inhibition of inflammatory cytokine production and complement activation.⁸⁶ It has been used in combination with apoptotic (ECDI-fixed) cell infusion to induce mouse heart allograft tolerance⁵⁴ and to prolong rat renal allograft survival.⁸⁷ Cordycepin is a fungal compound with anti-inflammatory properties that promotes an M2 regulatory macrophage phenotype, reduces pro-inflammatory cytokine release and increases IL-10 production by PBMCs.⁸⁸ Combining cordycepin with donor apoptotic cell infusion significantly prolonged mouse heart allograft survival from approximately 21 days with apoptotic cell infusion alone, to > 40 days with combined treatment.⁸⁸

Lymphodepletion represents an opportunity to reshape a newly “naïve” immune system. This concept has been used in NHPs, where leukodepletion followed by apoptotic donor cell infusions and a regimen of rapamycin, co-stimulation blockade, and anti-inflammatory molecules (anti-IL-6 and soluble TNF receptor) prolonged islet allograft survival.^61,81 Thus overall, a variety of pharmacologic and biologic agents have proven effective as adjuvants to promote the tolerogenicity of donor apoptotic cells.

Other conventional immunosuppressive agents, including calcineurin inhibitors and mycophenolate do not potentiate or interfere with apoptotic cell infusion-induced tolerance in murine models.⁷¹ DC-reg infusions in human clinical trials have been combined with conventional immunosuppression using tacrolimus, mycophenolate, and steroids,^24,36 although there is concern that calcineurin inhibitors could potentially impair T-reg induction.⁸⁹ Clinical translation remains focused on the safe introduction of negative vaccination strategies to existing clinical practice, and it is likely that future studies will combine these approaches with triple immunosuppression as standard of care, while acknowledging that the most promising animal models have used combinations of co-stimulation blockade and mTOR inhibition.

Developing alternative methods of negative vaccination via targeting of APCs in vivo

Apoptotic cell infusions are a well-developed approach to target the in vivo Ag-presentation system. However, alternative methods of Ag delivery to inhibit anti-donor immune reactivity and promote tolerance are under investigation.

Exosomes are small extracellular membrane vesicles secreted/released by hematopoietic cells, including DCs that express MHC class II. Pretransplant infusion of exosomes secreted by immature donor DCs prolongs MHC-mismatched heart graft survival in rats.⁹⁰ Moreover, combined with IS or additional donor Ag-specific regulatory cell therapy (T-regs), exosome administration induces tolerance in a rat liver transplant model.⁹¹ Exosome-based methods avoid any potential risk of regulatory cell maturation in vivo and host sensitization.

Donor Ag coupling to specialized drug-delivery nanoparticles represents a promising technology for targeting APCs in situ that does not require isolation and culture of donor or recipient cells ex vivo. Delivery platforms such as poly(lactide-co-glycolide) (PLG) in ovalbumin (OVA)-sensitized mice,^92,93 and β sheets of proteinaceous aggregates in murine skin transplant models⁹⁴ have shown promise. These nanoparticles can also encapsulate immune-modifying drugs such as rapamycin,^93,95,96 and be used to target myeloid cells in vivo at the time of transplant to promote tolerance.^97,98

DCs sample their environment through an array of pattern recognition receptors (PRRs) including C-type lectin receptors (CLR). CLRs can act as adhesion molecules and as endocytic receptors, recognising a wide array of carbohydrate-containing molecules. The nature of the ligand and local microenvironment appears to determine whether the immune response triggered by receptor activation is inflammatory or tolerogenic. Approaches to activate these receptors use Ag fused to either anti-receptor antibodies or receptor-activating carbohydrate moieties. One molecule of interest expressed on cDC1s is the endocytic receptor DEC-205 (CD205).⁹⁹ Anti-CD205 fusion antibodies bound to specific Ag have shown promise in experimental models of autoimmune disease,¹⁰⁰ but their complex distribution across different cell types in humans may limit translation to clinical studies.⁹ CD-209 (the C-type lectin DC-SIGN) expressed on DCs has also been targeted using porous silicon nanoparticles loaded with rapamycin, and in vitro experiments have demonstrated phenotypic change towards a regulatory phenotype in DCs, with an increase in Ag-specific splenic T-regs when administered in OVA-sensitized mice.^95,96 Other preclinical models have used glycan-modified and sialylated Ags to target other CLRs and sialic-acid binding immunoglobulin-type lectins (Siglecs) with some success in murine models of experimental autoimmune encephalomyelitis and allergy.^101,102 It is unclear how applicable these methods will be in transplantation where tolerance is desired across a broad array of polymorphic MHC molecules. A summary of alternative antigen delivery approaches in preclinical models can be found in Table 3.

Table 3.

Preclinical studies of alternative forms of donor antigen delivery

Reference	Model	Treatment	Outcomes
Rodent models
Bryant et al 2014109	Islet allograft Mouse	Treatment: Donor antigen-coupled poly(d,l-lactide-co-glycolide) (PLG) particles infused 7 days prior and 1 day after transplantation without other IS Methods: PLG coupled by ECDI to donor spleen lysate to create donor antigen-couple PLG particles Control: Multiple dose groups, primary comparator sham injection of carrier molecule without donor antigen coupling	Median survival <20 days in control group vs >20 days in nanoparticle-treated group (p = 0.0099; exact values not reported)
Shah et al 201992	Skin allograft Mouse	Treatment: Donor antigen-coupled PLG infused 7 days prior and 1 day after transplantation without other IS Methods: PLG coupled by ECDI to donor protein to create donor antigen-coupled PLG particles Control: Multiple dose groups, primary comparator sham injection of carrier molecule without donor antigen coupling	Median survival <20 days in control group vs >20 days in nanoparticle-treated group (p < 0.001; exact values not reported)
Luo et al 2023110	Heart allograft Mouse	Treatment: Donor BMDC-derived exosomes injected daily from 0 days to 3 days after transplantation without other IS Methods: BM-derived donor DCs genetically altered to create high PD-L1 expression, with supernatant collected when cell fusion rate reached 80% Control: Multiple groups, primary comparator no injection of exosomes	Mean survival approximately 6 days in control group vs 12 days in exosome-treated group (p = 0.001; exact values not reported)
Pêche et al2003111	Heart allograft Rat	Treatment: Donor BMDC-derived exosomes 10μg injected 14 days and 7 days prior to transplantation without other IS Methods: BM-derived donor cells co-cultured with GM-CSF and IL-4 with supernatant collected at day 10 Control: Multiple dose groups, primary comparator no injection of exosomes	Median survival 6 days in control group vs 25 days in exosome-treated group (p < 0.01)
Pêche et al 200690	Heart allograft Rat	Treatment: Donor BMDC-derived exosomes 25μg injected 0 days and 6 days after transplantation without other IS Methods: BM-derived donor cells co-cultured with GM-CSF and IL-4 with supernatant collected at day 10 Control: Multiple dose groups, primary comparator no injection of exosomes	Median survival 6 days in control group vs 25.5 days in exosome-treated group (p < 0.05)
Ma et al 201691	Liver allograft Rat	Treatment: Donor BMDC-derived exosomes 25μg injected 7 days prior, 0 days and 7 days after transplantation without other IS Methods: BM-derived donor (syngeneic) cells co-cultured with GM-CSF and IL-4 with supernatant collected at days 6 and 11 Control: Multiple dose groups, primary comparator no injection of exosomes	Median survival 10 days in control group vs 37 days in exosome-treated group (p < 0.0001)

Clinical translation

The 2022 Fifth International Sam Strober Workshop on clinical transplant tolerance described ongoing efforts at clinical translation of tolerance-promoting strategies in organ transplantation.¹⁰³ Preclinical NHP studies have demonstrated the feasibility, safety, and efficacy of donor-derived DC-reg infusion prior to transplant in prolonging renal allograft survival,^35,79,81 and of 2-dose pre- and posttransplant apoptotic donor cell treatment in promoting islet allograft tolerance.⁶¹

Early phase clinical trials of DC-regs and M-regs have been undertaken internationally. The ONE study was a concerted evaluation of 7, small, single-center phase 1/2a clinical trials of regulatory cell-based therapy in live-donor kidney transplantation using autologous T-regs or DC-regs, or donor-derived M-regs.²⁸ Autologous DC-reg infusion 1 day prior to transplantation was compared to standard-of-care IS with basiliximab induction, tacrolimus, mycophenolate, and tapering steroid. DC-reg infusions were safe, with acute rejection in 2/8 patients in the treatment group and 1/9 in the reference group – all were treated with glucocorticoids and graft survival at 3 years was 100%. There were no differences in infectious complications. IS was reduced with mycophenolate tapering in 5/8, and tacrolimus monotherapy achieved in 2/8 patients.²⁴

Two earlier small phase 1/2a studies demonstrated the safety of either donor- or recipient-derived M-reg infusion prior to kidney transplantation. These cell infusions in combination with standard induction IS appeared to facilitate periods of reduced or no IS within the first year of transplant. However, rates of acute rejection were high at 4/10 and long-term IS was universally required.^104,105 An ongoing phase 1/2a trial at the University of Pittsburgh will report on 13 live donor liver transplant recipients given donor-derived DC-regs (within the dose range that proved efficacious in NHP) a week before transplant, and who received a protocol graft biopsy at 1 year. Patients with “permissive” biopsies at 1 year then began early withdrawal of IS therapy. In a second trial at the same center, 14 live donor liver transplant recipients with a permissive biopsy at 1–2 years posttransplant have received donor-derived DC-regs within the same dose range as in the initial trial, followed by weaning of IS therapy. In a further trial at the University of Pittsburgh dose escalation of donor-derived DC-reg infusion pretransplant in live donor kidney recipients is being conducted in a total of 14 donor-recipient pairs. These DC-reg trials incorporate detailed mechanistic studies to ascertain the fate of the infused regulatory cells, and their influence on allo-immune responses.^29,36

Apoptotic donor cell therapy has not yet been studied in human organ transplantation. However, apoptotic donor mononuclear cell infusion in 13 patients receiving HLA-matched allogeneic bone marrow transplants for the prevention of GVHD was successful in a small phase 1/2a trial.¹⁰⁶ Those in the high-dose group had no episodes of GVHD as compared to 23% in the low-dose group.¹⁰⁶ Trials in solid organ transplantation are likely to be undertaken once feasible and effective treatment protocols for live and deceased donor transplantation have been established in NHP models.

Conclusions

Induction of tolerance in humans to allogeneic tissue expressing the full complement of donor MHC and non-HLA Ags remains a challenging task. “Negative vaccination” approaches that deliver donor Ag together with a negative adjuvant have progressed significantly (Fig 1). Regulatory myeloid APC therapy has progressed to human trials, while the use of donor apoptotic cells shows significant promise in NHP models. It remains to be seen whether true tolerance will be achieved in clinical translation, or whether using the promising strategies described herein, a reduced burden of IS - which would in itself be of significant benefit – can be achieved, especially if accomplished early posttransplant.

Figure 1:

Diagrammatic representation of strategies for tolerance induction using negative vaccination in solid organ transplantation. Created using biorender.com.

Abbreviations

Ab

antibody

Ag

antigen

APC(s)

antigen-presenting cell(s)

ATG

anti-thymocyte globulin

BCL-X_L

B-cell lymphoma – extra large

CAR

chimeric antigen receptor

CD

cluster of differentiation

CTLA-4 Ig

cytotoxic T lymphocyte associated protein 4 immunoglobulin

DAMP

danger associated molecular pattern

DC(s)

dendritic cell(s)

DC-reg(s)

regulatory dendritic cells

ECDI

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

Foxp3

forkhead boxp3

GM-CSF

granulocyte macrophage-colony stimulating factor

Gr1

glucocorticoid receptor 1

GVHD

graft versus host disease

IL

interleukin

IS

immunosuppressive/immunosuppression

IFN

interferon

Inos

inducible nitric oxide synthase

M-CSF

macrophage-colony stimulating factor

MDSC(s)

myeloid-derived suppressor cells

MHC

major histocompatibility complex

M-reg(s)

regulatory macrophage(s)

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NHP

nonhuman primate

OVA

ovalbumin

PAMP

pathogen-associated molecular pattern

PBMC

peripheral blood mononuclear cell

PD-L1

programmed death ligand-1

PLG

polylactide-co-glycolide

Th cell

T helper cell

TGF-β

transforming growth factor β

TLR

Toll-like receptor

TNF

tumor necrosis factor

T-reg(s)

regulatory T cell(s)