Donor Macrophage Depletion Permits Post-Transplant Tolerance Induction in a Murine Islet Transplant Model

Abstract

Current strategies for experimental tolerance induction for allogeneic transplantation typically require recipient preparation days to weeks prior to transplantation, making them not applicable to deceased donor transplantation. Developing tolerance strategies feasible for deceased donor transplantation would greatly increase the pool of eligible patients for tolerance induction. Here, we aimed to induce tolerance with post-transplant only interventions in a murine pancreatic islet transplant model. We demonstrated that transplant tolerance induction by recipient infusions of ethylcarbodiimide-treated donor splenocytes (ECDI-SPs) could be reliably delayed to the post-transplant timeframe provided that donor islets were depleted of intra-islet macrophages prior to transplantation. Mechanistically, islet production of CCL3, CCL4, and CCL5 was significantly reduced by intra-islet macrophage depletion. On POD+1, islet allograft depleted of donor intra-islet macrophages exhibited significantly reduced infiltration of recipient innate immune cells, including monocytes, macrophages, and neutrophils. Interestingly, perioperative inhibition of CCR5, the receptor for CCL3, CCL4 and CCL5, also reduced POD+1 innate immune cell infiltration, and similarly permitted tolerance induction by post-transplant donor ECDI-SP infusions. This study thus demonstrates the efficacy of a strategy that would allow transplant tolerance induction by post-transplant-only interventions, thereby expanding the applicability of tolerance induction regimens to additional clinically relevant settings.

Introduction

Transplant tolerance induction permits survival of transplanted organs without indefinite global immunosuppression, thereby lowering medical and financial burdens to transplant recipients^1,2. For most clinical and preclinical models of tolerance induction, however, pre-transplant recipient conditioning is mandatory. These include induction of donor chimerism and donor-specific transfusions^3,4. As a result, these strategies are only applicable to living donor transplantation where the timing of donor availability is predictable. Yet, living donor transplantation made up only 22.8% of kidney transplants and 5.7% of liver transplants in the U.S. in 2023^5,6, and the overall trend has not changed in recent years.

For transplant tolerance induction, our lab has pioneered a strategy of recipient injections of donor splenocytes (SPs) treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs)⁷, and has demonstrated its robust efficacy in several murine and non-human primate (NHP) models of transplantation^8–11. This strategy was initially developed in models of autoimmunity, with splenocytes bound to proteolipid proteins, the main component of myelin, being able to reduce incidence of experimental autoimmune encephalomyelitis^12,13. A Phase I clinical trial found that treatment with autologous PBMCs coupled with myelin peptides was well-tolerated and resulted in reduced antigen-specific T cell activity, demonstrating clinical feasibility of this strategy¹⁴. Efficacy of ECDI-SPs relies on the tolerogenic nature of cells rendered apoptotic by ECDI. ECDI-SPs are rapidly taken up by APCs, which then release tolerogenic cytokines including TGF-β and IL-10⁸. Treatment with ECDI-SPs also results in increased antigen-specific regulatory T cells (Tregs)⁹ and myeloid-derived suppressor cells (MDSCs)¹¹. When applied to transplant, ECDI-SP injections can provide graft protection in murine islet, heart, and kidney transplant models^11,15,16. In humanized mice, donor ECDI-SPs also provide protection to porcine islet xenografts¹⁷. Some success using ECDI-treated donor lymphocytes has been demonstrated in NHP islet transplantation¹⁸. Ongoing research in our lab has also found benefit in NHP kidney transplant (manuscript in preparation). Donor ECDI-SPs are typically administered on days-7 and +1 (in reference to transplantation on day 0), with the dose on day-7 being crucial for efficacy of the treatment⁸, again limiting its utility to living donor transplantation. Therefore, an effective tolerance strategy that can be implemented entirely post-transplant is urgently needed for applications in deceased donor transplantation.

Previously, researchers have found that organ resident macrophages and dendritic cells contribute to graft rejection¹⁹. In mice, donor CCR2⁺ macrophages contribute to heart allograft rejection²⁰. However, total depletion of cardiac macrophages does not benefit graft survival, potentially due to the otherwise beneficial impact of CCR2⁻ macrophages²¹. In liver transplantation, some strain combinations result in spontaneous graft acceptance, a phenomenon associated with donor dendritic cell precursors later detected in recipient bone marrow²². Furthermore, donor treatment with IL-4 results in a larger M2-like macrophage population which enhances graft acceptance²³. In kidney transplantation, we have demonstrated that donor tissue-resident macrophages contribute to post-transplant graft recipient immune infiltration¹⁵. In non-transplant pathologies, macrophages have been found to contribute to tissue degeneration, suggesting a role in disease pathogenesis²⁴.

The role of donor macrophages in transplant tolerance induction, however, is unknown. Islets of Langerhans contain macrophages with a distinctly M1-like profile, expressing high levels of major histocompatibility class II (MHC II) and costimulatory molecules^25,26; therefore, are highly inflammatory^27,28. We hypothesized that their depletion prior to transplantation would reduce post-transplant inflammation and allow tolerance induction to be delayed to the post-transplant timeframe.

In this study, using a model of murine allogeneic islet transplantation, we showed that depletion of intra-islet donor macrophages prior to transplantation abrogated the immediate graft recipient immune infiltration. When combined with post-transplant infusions of donor ECDI-SPs, this strategy resulted in donor-specific tolerance. We additionally demonstrated that pancreatic macrophages promote the release of chemokines CCL3, CCL4 and CCL5; consequently, perioperative blockade of CCR5, their common receptor²⁹, also reduced graft recipient immune infiltration and permitted tolerance induction by post-transplant donor ECDI-SP infusions. This study thus demonstrates an effective strategy for post-transplant tolerance induction and expands the applicability of tolerance induction regimens to deceased donor transplantation.

Results

Donor Macrophage Depletion Combined with Post-Transplant Donor ECDI-SPs Results in Indefinite Immunosuppression-free Islet Allograft Survival

To investigate the impact of donor macrophage depletion on the efficacy of post-transplant tolerance induction, we used a murine allogeneic islet transplant model. As shown in Figure 1A, islet resident macrophages in donor BALB/c mice were depleted by two intraperitoneal injections of anti-CD115 antibody³⁰ on day-11 and day-7, followed by islet isolation and transplantation to diabetic C57BL/6 (B6) recipients on day0. Recipients were then treated with BALB/c ECDI-SP infusions on post-operative day +1 (POD+1) and POD+7. Flow cytometry³¹ verified a near complete (>95%) depletion of islet macrophages (Figure 1B). As shown in Figure 1C, combining donor macrophage depletion with post-transplant donor ECDI-SP infusions on POD+1 and POD+7 resulted in indefinite islet allograft survival in 8/9 recipients. Graft survival was superior to either post-transplant donor ECDI-SP infusions alone or donor macrophage depletion alone.

Figure 1: Donor macrophage depletion combined with post-transplant donor ECDI-SP infusions results in indefinite islet allograft survival.

(A) Diagram of treatment schedule for delayed tolerance protocol. BALB/c donors are treated with two doses of 500 μg anti-CD115 antibody intraperitoneally (i.p.) as described in Methods. Donor macrophage-depleted (DMac-Depleted) or non-depleted (Control) islets are transplanted on day 0 into diabetic C57BL/6 mice. Recipients are then treated with two doses of donor ECDI-SPs on POD+1 and POD+7. (B) Representative FACS plots depicting gating strategy of pancreatic islet macrophages. BALB/c donors received two doses of 500 μg intraperitoneal anti-CD115 antibody on days-11 and −7 prior to islet isolation on day0. Islets were harvested and immediately dissociated before staining for flow cytometry. Two donors were pooled for each data point, and the number of macrophages was normalized to the number of donors. The bar graph depicts the average number of donor islet macrophages (DMac) per donor, N=4 for each group. (C) Blood glucose was tracked to determine graft function, with two consecutive days of blood glucose >250 mg/dL defined as graft rejection. Survival of grafts with each treatment regimen is represented in the survival plot as days post-transplant, significance *p < 0.05.

Donor Macrophage Depletion Results in a Reduction of Early Graft Innate Immune Cell Infiltration

We hypothesized that the efficacy in promoting transplant tolerance by the POD+1 dose of donor ECDI-SPs would be influenced by the immune milieu of the graft at that time. Following allogeneic transplantation, grafts are quickly infiltrated by innate immune cells³², with T cells following thereafter³³. Therefore, we first investigated how early innate immune cell infiltration of the islet allograft was affected by donor macrophage depletion. Recipient and donor immune cells were differentiated by congenic markers CD45.1/CD45.2 and the gating strategy for recipient (CD45.1⁺) neutrophils, monocytes and macrophages is shown in Figure 2A. As expected, CD45.2⁺ donor intra-islet macrophages were demonstrably reduced in recipients of donor macrophage-depleted grafts (Supplemental Figure 1A, 1B). On POD+1, there was a significant decrease in graft-infiltrating recipient CD11b⁺ cells to islet grafts depleted of donor macrophages (Figure 2B). Among subsets of CD11b⁺ cells, Ly6G⁺ neutrophil and Ly6C⁺ monocyte infiltration were significantly reduced. F4/80⁺ macrophages trended strongly towards a reduced infiltration as well. Interestingly, infiltration of innate immune cell populations progressively increased over time in both groups, such that by POD+10 (Figure 2C) these populations reached similar numbers in either group.

Figure 2: Donor macrophages contribute to early innate graft infiltration.

(A) Representative FACS plots demonstrating gating strategy for innate immune cell infiltration post-transplantation. (B) Bar graphs show total infiltration of each cell type per graft, comparing donor macrophage-depleted (DMac-Depleted) and non-depleted (control) islet grafts at POD+1. (C) The same as (B) but on POD+10. Recipients received either donor DMac-Depleted or Control BALB/c islet grafts. Grafts were collected on POD+1 (prior to the first dose of donor ECDI-SPs) or on POD+10 (after receiving two doses of ECDI-SPs), followed by dissociation, staining, and analysis. N=8–9 on POD+1. N=5 on POD+10.

Donor Macrophage Depletion Combined with Post-Transplant Donor ECDI-SPs Results in a Reduction of Late T Cell Infiltration and Reduced Donor-Specific T Cell Activation

In murine pancreatic islet transplant, graft-infiltrating CD4 and CD8 T cells are independently capable of graft rejection. However, graft rejection is typically more robust when both subsets are present³⁴. Therefore, we next investigated how donor macrophage depletion impacted the kinetics of CD4 and CD8 T cell infiltration of the islet allograft. Diabetic B6 mice were transplanted as in Figure 1A and grafts were harvested on indicated days for evaluation. T cell gating strategy is shown in Figure 3A.

Figure 3: Donor macrophages contribute to T cell infiltration and donor-specific T cell activation.

(A) Representative FACS plots demonstrating gating strategy for T cell infiltration post-transplantation. (B) Grafts were collected on POD+2, +7, +14 and +21 from recipients transplanted either with DMac-Depleted or control (non-depleted) islet allografts and were analyzed by FACS to enumerate recipient T cell infiltration of the graft. All recipients were treated with BALB/c ECDI-SPs infusions on POD+1 and +7. Bar graphs show total CD4 or CD8 T cell infiltration per graft, comparing DMac-depleted and control grafts. POD+2, n=6–7 per group. POD+7, n=7–8 per group. POD+14, n=12–14 per group, POD+21, n=8–9 per group. (C) POD+21 recipient splenic T cells were isolated to perform mixed lymphocyte reactions. T cells were cultured with APCs from BALB/c or C3H spleens for three days. T cell proliferation was measured using eFluor 450 proliferation dye. Graphs represent the percentage of recipient T cells which proliferated in response to stimulation. n=5–6 for each group. (D) Recipient spleens were harvested at POD+21. Spleens were analyzed for CD4⁺FoxP3⁺ cells using FACS. Graph represents percent of CD4 T cells expressing FoxP3. n=9 for each group.

As shown in Figure 3B, on POD+2, there were a relatively small but significant number of both CD4 and CD8 T cells infiltrating the islet allografts in both groups and there was no significant difference between the two groups. Interestingly, at this time, mRNA expression of several inflammatory molecules indicating T cell activation already showed differences between donor macrophage-depleted versus control grafts (Supplemental Figure 2). On POD+7, CD4 and CD8 T cell infiltration was substantially increased in both groups, although there was still no significant difference in their numbers between groups. However, by POD+14, their numbers were now significantly lower in islets with donor macrophage depletion in comparison to those without, and these decreases were sustained on POD+21 (Figure 3B).

To determine how recipient T cells were functionally altered by donor intra-islet macrophage depletion, we conducted mixed lymphocyte reactions (MLRs) with recipient splenic T cells on POD+21. As shown in Figure 3C, T cells from mice receiving donor macrophage-depleted islet allografts showed a significant reduction in proliferation following BALB/c stimulation in comparison to T cells from mice receiving non-depleted islet allografts. Responses to third party C3H was not significantly different, indicating donor-specificity of the T cell hypo-responsiveness achieved by our treatment.

We have previously demonstrated that donor ECDI-SP infusions on day-7 and +1 is characterized by an increase in splenic FoxP3⁺ Tregs on POD+20^9,35. Therefore, we also investigated splenic FoxP3⁺ Tregs in our two experimental groups. As shown in Figure 3D, on POD+21, the spleen of recipients receiving donor macrophage-depleted islets contained a significantly higher percentage of FoxP3⁺ Tregs than that of recipients receiving non-depleted islets.

Collectively, these data support that donor macrophage depletion combined with post-transplant donor ECDI-SPs results in a donor-specific T cell hypo-responsiveness and enhanced splenic Tregs, concomitant with a substantial percentage of such recipients achieving indefinite islet allograft survival.

Intra-islet Macrophages Promote Chemokine Release

To determine potential molecular mechanisms by which depletion of intra-islet macrophages contributed to reduced early innate immune cell infiltration, we next investigated the release of chemokines by islets with or without intra-islet macrophage depletion. Following isolation, islets were placed into culture medium with the addition of 10ng/mL IFN-γ to mimic the inflammatory milieu following allogeneic transplantation. Chemokine release was measured in supernatant after 72 hours (Figure 4A)³⁶. We first performed a screen by a broad multiplex panel analysis of the supernatant. While secretion of several cytokines and chemokines was found to be reduced from macrophage-depleted islets, three highly reduced analytes were CCL3, CCL4, and CCL5 (Supplemental Figure 3), which are all ligands for CCR5 whose signaling has been previously strongly associated with islet allograft rejection^37–39. Therefore, we narrowed our subsequent investigations to CCL3, CCL4, and CCL5.

Figure 4: Islet macrophages contribute to release of CCR5 ligands by pancreatic islets.

(A) Schematic of islet culture system. DMac-depleted or control islets were harvested from BALB/c mice and placed into culture. Approximately 300 islets were placed in a single well of a 24-well plate with 0.5 mL of media. IFN-γ was added at a concentration of 10 ng/mL. Following 48 or 72 hours of culture, islets and supernatant were harvested for analysis. (B) Relative mRNA expression of CCL3, CCL4, and CCL5 by DMac-depleted and control islets in culture. Islets were collected at 48 hours and placed into Trizol for mRNA isolation. N=3 for each group. (C) Multiplex analysis of secreted chemokines from DMac-depleted and non-depleted islets. Supernatant was collected after 72 hours of culture for analysis. N=3 for each group. (D) Relative mRNA expression of CCL3, CCL4, and CCL5 in DMac-depleted and control islet grafts harvested on POD+1 and POD+2 after transplant. Grafts were harvested on POD+1 or +2, dissociated with collagenase, and placed in Trizol for mRNA isolation. n=4–5 per group. (E) Single-cell transcriptomic map of wildtype B6 murine pancreatic islet cell populations. Sequencing analysis was performed on a public NCBI data set (GEO accession no. GSE232474). Violin plots show Log2 expression of CCL3, CCL4, and CCL5 in islet cell populations.

We first confirmed the above by targeted chemokine examinations. Cultured islets depleted of macrophages showed a significant reduction in mRNA expression of CCL3, CCL4, and CCL5 compared to non-depleted control islets (Figure 4B); and their supernatant showed a significant reduction of CCL4 and CCL5 secretion, with a strong trend of reduction of CCL3, in comparison to control islets (Figure 4C). To determine whether this phenomenon was replicated in vivo following transplant, we harvested islet grafts at POD+1 and POD+2 for analysis. Using qPCR of whole islet grafts, we found that on POD+1, CCL5 was reduced in donor macrophage-depleted islet grafts compared to controls (Figure 4D). However, this difference was no longer observed on POD+2. Additionally, CCL3 and CCL4 expression was not different between groups at either timepoint. We postulate that the lack of differential expression between groups by whole graft qPCR of CCL5 on POD+2 and CCL3 and CCL4 on both days could be due in part to infiltrating recipient immune cells diluting the signal from the very few donor macrophages.

To pinpoint the cellular source of these chemokines, we analyzed a publicly available single cell transcriptomics dataset from freshly isolated islets of B6 mice (NCBI; Gene Expression Omnibus [GEO] Accession Number GSE232474)⁴⁰. As shown in Figure 4E, cell clustering revealed that the largest population in B6 islets was β cells, with further clusters of other endocrine cells (α/δ/PP cells), endothelium, resident macrophages, and B cells, in descending order of frequency. Interestingly, only macrophages showed strong expressions of CCL3, CCL4 and CCL5 (Figure 4E violin plots). This analysis supports our hypothesis that islet macrophages are the primary source of these chemokines.

Intra-islet Chemokines Contribute to Early Graft Innate Immune Cell Infiltration

We next examined whether chemokines released from the islet allograft contributed to the early post-transplant innate immune cell infiltration. We first evaluated the expression of CCR5, the receptor for CCL3, CCL4 and CCL5, on the infiltrating innate immune cells. As shown in Figure 5A, on POD+1, infiltrating recipient Ly6C⁺ monocytes and F4/80⁺ macrophages, but not Ly6G⁺ neutrophils, expressed CCR5. We next used maraviroc, a small molecule CCR5 inhibitor, to test the impact of CCR5 inhibition on early graft innate immune cell infiltration. As shown in Figure 5B, recipients were given maraviroc or vehicle daily on day-1 and day0, transplanted on day0 and analyzed on POD+1. Maraviroc treatment notably reduced CD11b⁺ cell graft infiltration. When breaking down to subpopulations of CD11b⁺ cells, monocyte and macrophage graft infiltration was significantly reduced, although no appreciable difference was seen with neutrophil infiltration. The lack of an effect on neutrophil infiltration by CCR5 blockade is not surprising, as we did not see CCR5 expression on recipient infiltrating neutrophils (Figure 5A), suggesting that the observed effect of donor macrophage depletion on early graft neutrophil infiltration (Figure 2B) was mediated via a CCR5-independent mechanism.

Figure 5: CCR5 inhibition reduces early graft innate immune cell infiltration and promotes transplant tolerance induction by post-transplant donor ECDI-SP infusions.

(A) Grafts of untreated recipients were harvested at POD+1. Grafts were analyzed by FACS for expression of CCR5, the receptor for CCL3, CCL4, and CCL5. Gating strategy is the same as in Figure 2A. (B) Schematic for treatment of recipient with maraviroc, a small molecule CCR5 inhibitor. Recipients were given 25 mg/kg/day maraviroc via i.p. injection on day-1 and 0 immediately following islet transplant. Grafts were harvested on POD+1 to enumerate graft infiltrating recipient cells using FACS. Graphs represent total number of each cell type per graft. Gating strategy is the same as in Figure 2A. N=10 for each group. (C) Schematic for two peritransplant maraviroc treatment regimens. Recipients were given daily injections of maraviroc from day-1 to POD+7 or to POD+1. Maraviroc-treated and vehicle-treated (control) recipients were all given POD+1 and +7 donor ECDI-SPs infusions. Graft survival for each group is plotted as days post-transplant. (D) Graft infiltration by T cells on POD+7 in DMac-depleted or maraviroc-treated grafts. n=4–7 for each group. (E) POD+7 recipient splenic T cells were isolated to perform mixed lymphocyte reactions. T cells were cultured with APCs from BALB/c or C3H spleens for three days. T cell proliferation was measured using eFluor 450 proliferation dye. Graphs represent the percentage of recipient T cells which proliferated in response to stimulation. n=4–5 per group.

Lastly, we tested whether CCR5 inhibition would also allow tolerance induction by post-transplant donor ECDI-SP infusions. B6 recipients were treated with daily injections of maraviroc from days-1 to +7, and additionally received donor ECDI-SPs infusions on POD+1 and +7. As shown in Figure 5C, (−1 to +7) maraviroc combined with post-transplant donor ECDI-SPs resulted in ~80% recipients achieving indefinite graft survival. We also tested a shorter course of maraviroc, treating recipients only from days-1 to +1. The rationale for days-1 to +1 maraviroc dosing stemmed from the observed transient nature (detected on POD+1 but not on POD+2, Figure 4D) of differential CCL5 expression from donor macrophage depleted versus non-depleted islet allografts. We found the (−1 to +1) maraviroc course to be equally effective as the (−1 to +7) maraviroc in prolonging graft survival when combined with post-transplant donor ECDI-SPs (Figure 5C). As a control, maraviroc alone (−1 to +7) did not improve graft survival (Figure 5C).

To rule out that this synergistic protective effect of maraviroc was due to it directly inhibiting T cell graft infiltration or activation rather than inhibiting early innate immune cell infiltration, we compared graft T cell infiltration between (−1 to +7) maraviroc-treated islet recipients versus DMac-depleted islet recipients. Both groups received donor ECDI-SPs infusions on POD+1 and +7. We found that on POD+7, after the last dose of maraviroc, graft T cell infiltration in maraviroc-treated recipients was not significantly different from that in DMac-depleted recipients (Figure 5D), which was not different from controls (Figure 3B). Furthermore, when recipient splenic T cells from POD+7 were stimulated with donor APCs in cultures, maraviroc treatment did not impact T cell proliferation (Figure 5E).

Donor Macrophage Depletion Combined with Post-Transplant Donor ECDI-SP Infusions Results in Donor-Specific Transplant Tolerance

To determine whether the observed indefinite islet allograft survival in Figure 1C was indeed donor-specific tolerance, recipient mice were nephrectomized to remove the first functioning islet allograft followed by retransplanting a second same-donor islet allograft without any further intervention (schematically shown in Figure 6A). Removing the first functioning islet allograft resulted in recipient hyperglycemia in the following 2–3 days as shown in Figure 6B. Following retransplantation with the same-donor (BALB/c) islets, grafts were accepted and functioned for >100 days without further treatment (Figure 6C). However, third-party (C3H) islets were promptly rejected in these recipients (Figure 6C) with the same tempo as in naïve recipients (data not shown). These data demonstrated that combining donor macrophage depletion with POD+1 and +7 donor ECDI-SP infusions resulted in donor-specific transplant tolerance.

Figure 6: Post-transplant donor ECDI-SP infusions induce donor-specific tolerance.

(A) Schematic of re-transplant experiment. Long-term stable recipients (>100d with functioning islet allografts) were nephrectomized to remove the original graft. New BALB/c (original donor) or C3H (third-party) grafts were placed on the contralateral kidney and blood glucose was observed to determine graft survival. (B) Recipient blood glucose before and after graft nephrectomy. Blood glucose was monitored to confirm reestablished diabetic blood glucose levels following nephrectomy and before re-transplant on day 0. (C) Survival curve of retransplanted grafts. New graft survival is represented as days post-retransplant. *p < 0.05.

Discussion

Current experimental transplant tolerance strategies target recipients of living donor transplantation. These strategies have relied on recipient preconditioning before transplant. More recently, several centers have investigated post-transplant tolerance strategies. Specifically, Stanford investigators have found success in post-transplant chimerism and tolerance induction in MHC-matched, but not MHC-mismatched, transplants⁴¹. In pediatric liver transplant, some case studies have demonstrated successful deceased-donor chimerism and tolerance induction^42,43. In heart and kidney transplant, investigators at Massachusetts General Hospital were able to induce chimerism-mediated tolerance post-transplant in non-human primates^44–46, but have not yet examined in clinical settings. To date, non-chimerism-based tolerance strategies have not been tested in the post-transplant timeframe.

Our lab has previously established that infusions with donor ECDI-SPs on days-7 and +1 induce donor-specific tolerance in several transplant models, including murine islets, heart, and kidney, and non-human primate islet transplant models^8,10,11. However, we have previously also demonstrated that eliminating the day-7 dose results in failure of tolerance induction⁸. The current study examined a strategy aiming to overcome this limitation, so that donor ECDI-SPs could be administered entirely in the post-transplant timeframe. Our results here suggest that this is achievable provided that donor graft is first depleted of tissue-resident macrophages prior to transplantation. The immune milieu at the initiation of tolerance induction may determine the strategy’s success. With the standard −7 and +1 ECDI-SP treatment schedule, the first dose is administered prior to inflammation induced by introduction of an allogeneic graft. The failure of post-transplant ECDI-SP administration to consistently induce tolerance without additional treatments may be due in part to increased inflammation at the time of treatment initiation. Donor macrophage depletion reduces this inflammation thereby allowing post-transplant ECDI-SP treatment to be effective in inducing tolerance.

Previous literature has established that donor passenger leukocytes can contribute to transplant rejection^10,15,47. While few in number, islet resident macrophages have a highly immunogenic phenotype which may contribute to their striking impact on tolerance induction^48,49. We have previously shown that chemokine release by kidney resident macrophages results in a greater graft infiltration of recipient immune cells and worse kidney allograft function¹⁵. Consistent with our previous study, here we showed that macrophage-depleted islets produced a significantly lower level of CCL3, CCL4, and CCL5. Furthermore, we showed that chemokine-CCR5 interaction contributed to early innate immune cell infiltration of the islet allograft; consequently, CCR5 inhibition reduced such graft infiltration on POD+1. We further demonstrated that peritransplant CCR5 inhibition combined with post-transplant donor ECDI-SPs resulted in donor-specific transplant tolerance.

It is important to note that previous studies combining chemokine blockade with tolerance induction have been quite limited and often contradictory. For instance, in a cardiac transplant model, tolerance by costimulation blockade effective in wildtype recipients was no longer effective in CCR7^−/− recipients and correlated with an increase of infiltrating effector T cells and a reduction in Tregs⁵⁰. Contrastingly, in a model antigen lung transplant model, it was shown that CXCR3^−/− antigen-specific T cells could become Tregs to promote graft tolerance⁵¹. The role of CCR5 in transplant rejection and tolerance is also complex. In one study, CCR5^−/− recipients experienced prolonged islet allograft survival³⁹. Similarly, CCR5^−/− recipients of fully MHC-mismatched renal allografts showed improved allograft function⁵². However, in a single MHC-mismatched cardiac transplant model where grafts in wildtype recipients survive >100 days, CCR5^−/− recipients universally rejected their grafts in less than 24 days. The authors attributed this phenomenon to dysregulation of Treg trafficking⁵³. Nonetheless, results of our current study support that chemokine-chemokine receptor inhibition may lower the threshold for tolerance induction, and when combined with a pro-tolerogenic approach such as donor ECDI-SP infusions permit delayed tolerance induction to the post-transplant timeframe.

Our study further demonstrates that macrophage depletion combined with post-transplant donor ECDI-SPs resulted in durable donor-specific T cell hyporesponsiveness. This hyporesponsiveness was characterized by a decrease of graft-infiltrating effector T cells, an increase in splenic Tregs, and a reduction of donor-specific T cell activation. The current study did not investigate how donor macrophage release of CCR5 ligands may contribute to T cell graft infiltration or activation. Since we did not observe differential T cell infiltration until after donor macrophages had disappeared from the graft (Supplemental Figure 1C), any influence they have on T cell infiltration is likely indirect. The effect on T cells may primarily arise from difference in efficacy of tolerance induction now enhanced by donor macrophage depletion and a reduction of early post-transplant inflammation. Future research will be needed to explore the mechanisms by which donor macrophages promote T cell infiltration and activation.

While the strategy tested here requires donor treatment prior to transplantation, future work based on the same principle should now operationalize this approach in a more clinically applicable fashion. For example, as islets are typically cultured for up to 72 hours prior to transplantation, islet macrophages may be depleted during this period⁵⁴. In addition, with rapid advancements in ex vivo organ perfusion circuits, resident macrophage depletion may be achieved post-organ harvest and further optimized through innovative designs of perfusion circuits and/or perfusion solutions. Similarly, the extension of organ viability via perfusion circuits may permit recipient treatment with agents such as maraviroc prior to transplant.

Besides releasing CCR5 ligands, donor macrophages likely play additional roles in impairing tolerance induction. Evidence for this complexity can be found in neutrophils’ reduced infiltration in response to donor macrophage depletion, but not to CCR5 inhibition (Figure 2B and 5B). In addition, donor macrophages may also release a wide range of other chemokines and cytokines to promote alloimmunity. Our initial chemokine screen indicated that IP-10 release was reduced by donor macrophage depletion (Supplemental Figure 3). Literature has found that IP-10-producing macrophages contribute to tissue inflammation in transplant rejection⁵⁵, possibly via IP-10-CXCR3 interaction. Consequently, concurrent inhibition of CCR5 and CXCR3 has been shown to produce greater benefit in murine heart transplant⁵⁶. Combining these therapies may prove to be further beneficial to tolerance induction. In addition, donor macrophages have been shown to traffic to graft draining lymph nodes where they directly stimulate recipient immune cells. Lastly, it has been demonstrated that donor cells can also distribute donor antigens to secondary lymphoid tissues by releasing extracellular vesicles (EVs)^57–59. Therefore, it is conceivable that donor islet macrophages also release donor antigen-laden EVs, engage recipient APCs, indirectly promote alloimmunity and increase tolerance threshold. EV release by donor macrophages as a parallel mechanism underlying our observations is being actively investigated in our lab.

In conclusion, we have demonstrated a strategy for post-transplant tolerance induction in an islet transplant model, making tolerance induction by infusions of donor ECDI-SPs more applicable to deceased donor transplantation. We have shown that donor macrophages, while few in number, have a strong impact on tolerance induction to islet allografts. These macrophages contribute to release of chemokines which interact with recipient CCR5 and promote early graft innate immune cell infiltration. Targeting donor macrophages and chemokines may provide an avenue to increase the effectiveness of tolerance induction strategies and make them applicable to deceased donor transplants. Future research of such a strategy in vascularized organ transplants would make these findings more generalizable to solid organ transplantation.

Methods

Sex as a biological variable

Our current study examined male mice only as per approval by our IACUC protocols. Future experiments will extend all of our experiments in this study to female mice. We expect our findings to be relevant to more than one sex.

Donor Islet Macrophage Depletion

Donor BALB/c mice were treated with anti-CD115 (anti-CSF1R, BioXCell #BE0213) antibody to deplete pancreatic islet resident macrophages. Donors were treated with two i.p. injections of 500 μg each, administered four days apart. After the second injection, donors were rested for a week prior to islet harvest.

Maraviroc Treatment

To inhibit CCR5 activity in vivo, maraviroc (MedChem Express) was administered to recipients at 25mg/kg/day. Maraviroc stock was prepared by suspending 100mg/mL in DMSO. The final injection vehicle consisted of 10% DMSO, 40% PEG300, 5% Tween-80, and 45% ddH₂O per manufacturer recommendation to ensure complete resuspension. Maraviroc was injected via i.p. during the treatment duration.

Statistical Analysis

Statistics were analyzed using GraphPad Prism v10.6.1. Descriptive statistics are presented as mean ± SD for parametric data. Graft survival was compared using Kaplan-Meier survival curves with log-rank test. Welch's t test or analysis of variance (ANOVA) was used to compare means of groups. P < .05 was considered statistically significant.

Additional Methods:

Please see “Supplemental Materials”.

Abbreviations:

ANOVA

Analysis of Variance

APC

antigen presenting cell

B6

C57BL/6

CCL

C-C chemokine ligand

CCR

C-C chemokine receptor

CD115

Colony Stimulating Factor 1 Receptor

DMac

donor macrophage

DMSO

dimethyl sulfoxide

ECDI-SPs

ethylcarbodiimide-treated splenocytes

ECDI

ethylcarbodiimide

EV

extracellular vesicle

i.p.

intraperitoneal

i.v.

intravenous

IFN-γ

interferon gamma

IL

interleukin

iNOS

inducible nitric oxide synthase

IS

immunosuppression

MHC

major histocompatibility class

MLR

mixed lymphocyte reaction

mRNA

messenger ribonucleic acid

PFA

paraformaldehyde

POD

post-operative day

rt-qPCR

reverse transcriptase quantitative polymerase chain reaction

Treg

T regulatory cell

UMAP

uniform manifold approximation and projection