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

196 31 Main text: 3991 32 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 3

33 Current strategies for experimental tolerance induction for allogeneic transplantation typically 34 require recipient preparation days to weeks prior to transplantation, making them not applicable to 35 deceased donor transplantation. Developing tolerance strategies feasible for deceased donor 36 transplantation would greatly increase the pool of eligible patients for tolerance induction. Here, we 37 aimed to induce tolerance with post-transplant only interventions in a murine pancreatic islet transplant 38 model. We demonstrated that transplant tolerance induction by recipient infusions of ethylcarbodiimide-39 treated donor splenocytes (ECDI-SPs) could be reliably delayed to the post-transplant timeframe provided 40 that donor islets were depleted of intra-islet macrophages prior to transplantation. Mechanistically, islet 41 production of CCL3, CCL4, and CCL5 (RANTES) was significantly reduced by intra- islet macrophage 42 depletion. On POD+1, islet allograft depleted of donor intra- islet macrophages exhibited significantly 43 reduced infiltration of recipient innate immune cells, including monocytes, macrophages, and neutrophils. 44 Interestingly, perioperative inhibition of CCR5, the receptor for CCL3, CCL4 and CCL5, also reduced POD+1 45 innate immune cell infiltration, and similarly permitted tolerance induction by post-transplant donor ECDI-46 SP infusions. This study thus demonstrates the efficacy of a strategy that would allow transplant tolerance 47 induction by post -transplant-only interventions, thereby expanding the applicability of tolerance 48 induction regimens to additional clinically relevant settings. 49 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 4

50 Transplant t olerance induction permit s survival of transplanted organs without the need for 51 indefinite global immunosuppression, thereby lowering medical and financial burdens to transplant 52 recipients1,2. For m ost clinical and preclinical models of tolerance induction, however, pre -transplant 53 recipient conditioning is mandatory . These include induction of donor chimerism and donor -specific 54 transfusions3,4. As a result, these strategies are only applicable in living donor transplantation where the 55 timing of donor availability is predictable. Yet, living donor transplantation made up only 22.8% of kidney 56 transplants and 5.7% of liver transplants in the U.S. in 20235,6, and the overall trend has not changed in 57 more recent years. 58 For transplant tolerance induction, our lab has pioneered a strategy of recipient injections of 59 donor splenocytes (SPs) treated with the chemical cross -linker ethylcarbodiimide (ECDI- SPs)7, and has 60 demonstrated its robust efficacy in several murine and non-human primate models of transplantation8-11. 61 Donor ECDI-SPs are typically administered on days-7 and +1 (in reference to transplantation on day 0), 62 with the dose on day-7 being crucial for efficacy of the treatment8, again limiting its utility in living donor 63 transplantation. Therefore, an effective tolerance strategy that can be implement ed entirely by post-64 transplant treatments is urgently needed for applications in deceased donor transplantation . In the 65 current study, we utilized a murine pancreatic islet transplant model to investigate such a strategy. 66 Donor passenger leukocytes are known to impact alloimmunity in transplantation12,13. We have 67 previously demonstrated that donor tissue-resident macrophages contribute to post-transplant recipient 68 immune infiltration in a mouse model of allogeneic kidney transplantation13. Their role in transplant 69 tolerance induction, however, is unknown. Islets of Langerhans contain macrophages with a distinctly 70 M1-like profile, expressing high levels of major histocompatibility class II (MHC II) and costimulatory 71 molecules14,15; t herefore are highly inflammatory 16,17. We hypothesized that their depletion prior to 72 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 5 transplantation would reduce post-transplant inflammation and allow tolerance induction to be delayed 73 to the post-transplant timeframe. 74 In this study, using a model of murine allogeneic islet transplantation, we showed that depletion 75 of intra- islet donor macrophages prior to transplantation abrogated the immediate influx of recipient 76 innate immune cells to the islet allograft. When combined with post-transplant infusions of donor ECDI-77 SPs, this strategy resulted in donor-specific tolerance and indefinite islet allograft survival. We further 78 demonstrated that pancreatic macrophages promote the release of chemokines CCL3, CCL4 and CCL5 ; 79 consequently, perioperative blockade of CCR5, their common receptor18, also reduced graft infiltration of 80 recipient innate immune cells and permitted tolerance induction by post -transplant donor ECDI -SP 81 infusions. The observed graft protection was further characterized by a reduction of late graft-infiltrating 82 T effector cells and an increase of systemic FoxP3+ T regulatory cells (Tregs). This study thus demonstrates 83 an effective strategy for post -transplant tolerance induction and expands the applicability of tolerance 84 induction regimens to deceased donor transplantation. 85 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 6

86 Donor Macrophage Depletion Combined with Post -Transplant Donor ECDI- SP Infusions Result in 87 Indefinite Immunosuppression-free Islet Allograft Survival 88 To investigate the impact of donor macrophage depletion on the efficacy of post-transplant 89 tolerance induction, we used a murine allogeneic islet transplant model. As shown in Figure 1A, islet 90 resident macrophages in donor BALB/c mice were depleted by t wo intraperitoneal injections of anti-91 CD115 antibody19 on day-11 and day-7, followed by islet isolation and transplantation to diabetic C57BL/6 92 (B6) recipients on day0 . Recipients were then treated with BALB/c ECDI-SP infusions on post-operative 93 day +1 (POD+1) and POD+7. Flow cytometry was used as previously published20 to verify a near complete 94 (>95%) depletion of islet macrophages (Figure 1B). As shown in Figure 1C, combining donor macrophage 95 depletion with post-transplant donor ECDI-SP infusions on POD+1 and POD+7 resulted in indefinite (>100 96 days) islet allo graft survival in the complete absence of immunosuppression in 8/9 recipients ( filled 97 triangle). Graft survival was superior to either post-transplant donor ECDI-SP infusions alone (filled circle) 98 or donor macrophage depletion alone (open square). 99 100 Donor Macrophage Depletion Results in a Reduction of Early Graft Innate Immune Cell Infiltration 101 We hypothesized that the efficacy in promoting transplant tolerance by the POD+1 dose of donor 102 ECDI-SPs would be influenced by the immune milieu of the graft at th at time. F ollowing allogeneic 103 transplantation, grafts are quickly infiltrated by innate immune cells21, with T cells following thereafter22. 104 Therefore, we first investigated how early post-transplant innate immune cell infiltration of the islet 105 allograft was affected by donor intra-islet macrophage depletion. 106 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 7 Donor islets were depleted of macrophages and subsequently transplanted as in Figure 1A. Grafts 107 were harvested on POD+1, prior to the first dose of ECDI -SPs, for analysis. Recipient and donor immune 108 cells were differentiated by congenic markers CD45.1 and CD45.2 respectively (Figure 2A). CD45.2+ donor 109 intra-islet macrophages were demonstrably reduced in recipients of donor macrophage -depleted grafts 110 (Supplemental Figure 1). Gating strategy for recipient (CD45.1 +) neutrophils, monocytes and 111 macrophages is also shown in Figure 2A. As shown in Figure 2 B top panels , on POD+1, there was a 112 significant decrease in graft -infiltrating recipient CD11b+ cells to islet grafts depleted of donor 113 macrophages. Among subsets of CD11b + infiltrating cells, Ly6G+ neutrophil infiltration of the graft was 114 reduced, as was Ly6C+ monocyte infiltration. F4/80 + macrophages trended strongly towards a reduced 115 infiltration as well. Interestingly, infiltration of innate immune cell populations progressively increased 116 over time in both groups (data not shown), such that by POD+10 ( Figure 2B lower panels ) these 117 populations reached similar numbers in either donor macrophage-depleted or non-depleted grafts. 118 119 Intra-islet Macrophages Promote Chemokine Release 120 To determine potential molecular mechanisms by which depletion of intra-islet macrophages 121 contributed to reduced early innate immune cell infiltration , we next investigated the release of 122 chemokines by islets with or without intra-islet macrophage depletion. BALB/c mice were treated either 123 with anti-CD115 or an isotype control. Following isolation, islets were placed into culture medium with 124 the addition of 10ng/mL IFN -γ t o mimic the inflammatory milieu following allogeneic transplantation. 125 Chemokine release was measured in supernatant after 72 hours (Figure 3A)23. We first performed a screen 126 by a broad multiplex panel analysis of the supernatant. While several cytokines and chemokines were 127 found to be reduced from macrophage-depleted islets, three of the f our most reduced analytes were 128 CCL3, CCL4, and CCL5 (Supplemental Figure 2), which are all ligands for CCR5. CCR5 signaling has been 129 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 8 previously strongly associated with islet allograft rejection24-26. Because of this association, we decided to 130 narrow our subsequent investigations to CCR5 ligands CCL3, CCL4, and CCL5. 131 We first confirmed the above initial screening findings by targeted chemokine examinations. 132 Cultured islets were harvested for analysis. Islets depleted of macrophages showed a significant reduction 133 in mRNA expression of CCL3, CCL4, and CCL5 compared to non -depleted control islets (Figure 3B); and 134 their supernatant showed a significant reduction of CCL4 and CCL5 levels, with a strong trend of reduction 135 of CCL3, in comparison to control islets (Figure 3C). 136 To determine the cellular source of the se chemokines, we utilized a publicly available single cell 137 transcriptomics dataset from freshly isolated islets from B6 mice (NCBI; Gene Expression Omnibus [GEO] 138 Accession Number GSE232474)27 and performed an independent analysis using Seurat in R. As shown in 139 Figure 3D, cell clustering revealed that the largest population in B6 mouse islets was β cells, with further 140 clusters of other endocrine cells ( α/δ/PP cells) , endothelium, B cells, and resident macrophages, in 141 descending order of frequency. Interestingly, when querying for expressions of CCL3, CCL4 and CCL5, we 142 found that only islet macrophages showed strong expression of each transcript (Figure 3D violin plots) 143 whereas other cell s showed negligible expressions. This analysis supports our hypothesis that islet 144 macrophages are the primary source of CCL3, CCL4 and CCL5. 145 146 Intra-islet Chemokines Contribute to Early Graft Innate Immune Cell Infiltration 147 We next examined whether chemokines released from the islet allograft contributed to the early 148 innate immune cell infiltration following transplantation. We first evaluated the expression of CCR5, the 149 receptor for CCL3, CCL4 and CCL5, on the infiltrating innate immune cells. We identified infiltrating innate 150 immune cell populations using the same gating strategy as in Figure 2 A. On POD+1, we found that 151 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 9 infiltrating recipient Ly6C+ monocytes and F4/80 + macrophages, but not Ly6G + neutrophils, expressed 152 CCR5 (Figure 4A). 153 We next used maraviroc, a small molecule CCR5 inhibitor, to test the impact of CCR5 inhibition on 154 early graft innate immune cell infiltration. Recipients were given maraviroc or vehicle daily on day-1 and 155 day0, transplanted on day0 and analyzed on POD+1 (Figure 4B) . As shown in Fi gure 4B, maraviroc 156 treatment notably reduced CD11b+ cell infiltration to the islet allograft . When breaking down to 157 subpopulations of CD11b + cells, monocyte and macrophage infiltration of the graft was significantly 158 reduced on POD+1, although no appreciable difference was seen with neutrophil infiltration. The lack of 159 an effect on neutrophil infiltration by CCR5 blockade is not surprising, as we did not see CCR5 expression 160 on recipient infiltrating neutrophils (Figure 4A); suggesting that the observed effect of donor macrophage 161 depletion on early graft neutrophil infiltration (Figure 2B) was mediated via a CCR5 -independent 162 mechanism. 163 Lastly, we tested whether CCR5 inhibition would also allow tolerance induction by post-transplant 164 donor ECDI -SP infusions. As shown in Figure 4C, B6 recipients were treated with daily injections of 165 maraviroc from days -1 to +7. During this period , recipients also received donor ECDI-SPs infusions on 166 POD+1 and +7. As shown in Figure 4C, peritransplant CCR5 inhibition by maraviroc combined with post-167 transplant donor ECDI-SPs resulted in ~80% recipients achieving indefinite graft survival, a graft survival 168 significantly more superior in comparison to vehicle treated recipients. 169 170 Donor Macrophage Depletion Combined with Post-Transplant Donor ECDI-SPs Results in a Reduction of 171 Late T Cell Infiltration and Reduced Donor-Specific T Cell Activation 172 In murine pancreatic islet transplant, graft-infiltrating CD4 and CD8 T cells are independently 173 capable of graft rejection. However, graft rejection is typically more robust when both subsets are 174 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 10 present28. Therefore, we next investigated how donor macrophage depletion impacted the kinetics of 175 CD4 and CD8 T cell infiltration of the islet allograft . We transplanted B6 mice with either macrophage -176 depleted or non-depleted BALB/c islets, followed by injection of BALB/c ECDI-SPs on POD+1 and +7. Grafts 177 were harvested at POD+2 or +14 for evaluation. T cell gating strategy is shown in Figure 5A. 178 While innate immune cell infiltration showed difference in macrophage-depleted versus control 179 non-depleted islet allografts at a very early timepoint (Figure 2B), T cell infiltration showed a different 180 kinetics. As shown in Figure 5B , o n POD+2, there w ere a small number of both CD4 and CD8 T cells 181 infiltrating the islet allografts in both groups and there was no significant difference in their numbers 182 between groups . Interestingly, at this time, mRNA expression of several inflammatory molecules 183 indicating T cell activation already showed difference s between donor macrophage -depleted versus 184 control grafts (Supplemental Figure 3). On POD+7, there was a market increase of islet -infiltrating CD4 185 and CD8 T cells in both groups, although there was still no significant difference in their numbers between 186 groups (Supplemental Figure 4). However , by POD+14, their numbers were now significantly lower in 187 islets with donor macrophage depletion in comparison to those without (Figure 5B). 188 To determine how recipient T cells were functionally altered by donor intra-islet macrophage 189 depletion, we conducted mixed lymphocyte reactions (MLRs) with recipient splenic T cells from these two 190 groups on POD+21. As shown in Figure 5C, T cells from mice receiving donor macrophage -depleted islet 191 allografts showed a significant reduction in proliferation following BALB/c stimulation in comparison to T 192 cells from mice receiving non- depleted islet allografts. However, T cell response to third party C3H 193 stimulation was not significantly different between the two groups, indicating that a donor-specific T cell 194 hypo-responsiveness was achieved by our treatment strategy. As a negative control, T cells had minimal 195 response to syngeneic B6 stimulation (data not shown). 196 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 11 We have previously demonstrated that donor ECDI-SP infusions on day-7 and +1 is characterized 197 by an increase in splenic FoxP3+ Tregs on POD+20.9,29 Therefore, we also investigated splenic FoxP3+ Tregs 198 in our two experimental groups. As shown in Figure 5D, on POD+21, the spleen of recipients receiving 199 donor macrophage-depleted islet allografts contained a significantly higher percentage of FoxP3 + CD4 T 200 cells than that of recipients receiving non-depleted islet allografts. 201 Collectively, these data support that donor macrophage depletion combined with post-transplant 202 donor ECDI -SPs results in a donor-specific T cell hyporesponsiveness and enhanced splenic Tregs, 203 concomitant with a substantial percentage of such recipients achieving indefinite immunosuppression -204 free islet allograft survival. 205 206 Donor Macrophage Depletion Combined with Post-Transplant Donor ECDI-SP Infusions Results in Donor-207 Specific Transplant Tolerance 208 To determine whether the observed indefinite islet allo graft survival was a result of systemic 209 tolerance or a local protective effect, recipient mice were nephrectomized to remove the first functioning 210 islet allograft followed by retransplanting a second same -donor islet allo graft without any further 211 intervention (schematically shown in Figure 6A). Removing the first functioning islet allograft resulted in 212 recipient hyperglycemia in the following 2-3 days as shown in Figure 6B. Following retransplantation with 213 the same- donor ( BALB/c) islets, grafts were accepted and functioned for > 100 days with out further 214 treatment (Figure 6C). However, third-party (C3H) islets were promptly rejected in these recipients (Figure 215 6C) with the same tempo as in naïve recipients (data not shown) . These data demonstrated that 216 combining donor macrophage depletion with POD+1 and +7 donor ECDI -SP infusions resulted in donor -217 specific transplant tolerance. 218 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 12

219 Current experimental transplant toler ance strategies primarily target recipients of living donor 220 transplantation. One such strategy is to induce mixed chimerism where recipients receive a donor bone 221 marrow transplant along with the same-donor solid organ transplant30. These strategies have relied on 222 recipient preconditioning before transplant and therefore have only been experimented in living donor 223 transplantation. More recently, several centers have begun to investigate post-transplant tolerance 224 strategies. For example, investigators at Stanford ha ve found success in post -transplant chimerism and 225 tolerance induction in MHC -matched, but not MHC -mismatched, transplants31. In pediatric liver 226 transplant, some case studies have demonstrated successful deceased-donor chimerism and tolerance 227 induction32,33. In heart and kidney transplant, investigators at Massachusetts General Hospital were able 228 to induce chimerism-mediated tolerance post-transplant in non- human primates34-36, but ha ve not yet 229 experimented these strategies in clinical settings . To date, non -chimerism-based tolerance strategies 230 have not been tested in the post-transplant timeframe. 231 Our lab has previously established that infusions with donor ECDI-SPs on days-7 and +1 (relative 232 to transplantation on day0) induce donor-specific tolerance in several transplant models, including murine 233 islets, heart, and kidney, and non-human primate islet transplant models8,10,11. In humanized mice, donor 234 ECDI-SPs also provide protection to islet xeno grafts37. However, we have previously demonstrated that 235 eliminating the day-7 dose results in failure of tolerance induction8, limiting the application of this strategy 236 to only recipients of living donor transplants. The current study aimed to overcome this limitation and 237 investigated the efficacy of a potential strategy for transplant tolerance induction by donor ECDI -SPs 238 administered entirely in the post-transplant timeframe. 239 Our results from the current study suggest that tolerance induction by post-transplant donor 240 ECDI-SP infusions can be achieved provided that donor graft is first depleted of tissue-resident 241 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 13 macrophages prior to transplantation. We have shown previously that POD+1-only donor ECDI-SP infusion 242 is insufficient to induce tolerance to murine islet allograft 8. However, we now show that with repeated 243 post-transplant dosing of donor ECDI -SP infusions (POD+1 and +7) , over a third of the recipients can in 244 fact be tolerized (Figure 1C); and the efficacy of tolerance induction can be further augmented by 245 depleting donor tissue-resident macrophages prior to transplantation. 246 Previous literature has established that donor passenger leukocytes can contribute to transplant 247 rejection10,12,13. While few in number, islet resident macrophages have a highly immunogenic phenotype 248 which may contribute to their striking impact on tolerance induction38,39. We have previously shown that 249 chemokine release by kidney resident macrophages results in a greater graft infiltration of recipient 250 immune cells and worse kidney allograft function13. Consistent with our previous study, here we showed 251 that macrophage-depleted islets produce d a significantly lower level of CCL3, CCL4, and CCL5 . We 252 hypothesized that the reduced chemokine production by depletion of donor tissue-resident macrophages 253 contributed to lowering the threshold for tolerance induction. To test this hypothesis, we investigated 254 maraviroc, an FDA-approved small molecule inhibitor of CCR5, receptor for CCL3, CCL4 and CCL5. 255 Pr evious studies combining chemokine blockade with tolerance induction have been quite limited 256 and often contradictory. For instance, in a cardiac transplant model, tolerance by costimulation blockade 257 effective in wildtype recipients was no longer effective in CCR 7-/- recipients and correlated with an 258 increase of infiltrating effector T cells and a reduction in Tregs in the draining lymph node40. Contrastingly, 259 in a model antigen lung transplant model, it was shown that CXCR3-/- antigen-specific CD8 T cells could 260 become Tregs to promote graft tolerance41. The role of CCR5 in transplant rejection and tolerance is also 261 complex. In one study, CCR5-/- recipients experienced prolonged islet allograft survival26. Similarly, CCR5-262 /- recipients of fully MHC-mismatched renal allografts showed improved allograft function42. However, in 263 a single MHC-mismatched cardiac transplant model where grafts in wildtype recipients survive >100 days, 264 CCR5-/- recipients universally rejected their grafts in less than 24 days . The authors attributed this 265 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 14 phenomenon to dysregulation of Treg trafficking43. This possibility led us to choose a short course of CCR5 266 inhibition in our model to minimize an effect on Treg trafficking (Figure 4C). 267 In our model, we showed that chemokine-CCR5 interaction contributed to early innate immune 268 cell infiltration of the islet allo graft; consequently, CCR5 inhibition reduc ed such graft infiltration on 269 POD+1. We further demonstrated that peritransplant CCR5 inhibition combined with post -transplant 270 donor ECDI-SPs resulted in donor-specific transplant tolerance. The same principles may be applied to 271 solid organ transplant models. Results of the current study support that chemokine-chemokine receptor 272 inhibition will likely lower the threshold for tolerance induction, and when combined with a pro -273 tolerogenic approach such as donor ECDI-SP infusions will permit delayed tolerance induction to the post-274 transplant timeframe. 275 Besides releasing CCR5 ligands, donor macrophages likely play additional roles in antagonizing 276 tolerance induction. Evidence for this complexity can be found in neutrophils’ reduced infiltration in 277 response to donor macrophage depletion, but not to CCR5 inhibition (Figure 2B and 4B) . In a complex 278 allograft setting, donor macrophages may also release a wide range of other chemokines and cytokines 279 to promote alloimmunity. Donor macrophages have been additionally shown to traffic to graft-draining 280 lymph nodes where they directly stimulate the maturation and activation of recipient immune cells . 281 Lastly, previous literature has demonstrated that donor cells can distribute donor antigens to secondary 282 lymphoid tissues by releasing extracellular vesicles (EVs)44-46. Therefore, it is conceivable that donor islet 283 macrophages also release donor antigen-laiden EVs, engage recipient antigen presenting cells, indirectly 284 promote alloimmunity and increase tolerance threshold . This hypothesis linking EV release to donor 285 macrophages as a parallel mechanism underlying our observations is being actively investigated in our 286 lab. 287 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 15 In conclusion, we have demonstrated a strategy for post-transplant tolerance induction in an islet 288 transplant model, making tolerance induction by infusions of donor ECDI-SPs more applicable to deceased 289 donor transplantation. We have shown that donor macrophages, while few in number, have a strong 290 impact on tolerance induction to islet allografts. These macrophages contribute to release of chemokines 291 which interact with recipient CCR5 and promote early graft innate immune cell infiltration. Targeting 292 donor macrophages and chemokines may provide an avenue to increase the effectiveness of tolerance 293 induction strategies and make them applicable to deceased donor transplants. Future research of such a 294 strategy in vascularized organ transplants would make these findings more generalizable to solid organ 295 transplantation. 296 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 16

297 Sex as a biological variable 298 Our current study examined male mice only as per approval by our current IACUC protocols. 299 Future experiments will extend all of our experiments in this study to female mice. We expect our findings 300 to be relevant to more than one sex. 301 302 Donor Islet Macrophage Depletion 303 Donor BALB/c mice were treated with anti -CD115 (anti-CSF1R, BioXCell # BE0213) antibody to 304 deplete pancreatic islet resident macrophages. Donors were treated with two i.p. injections of 500 µg 305 each, administered four days apart. After the second injection, donors were rested for a week prior to 306 islet harvest. Macrophage depletion was verified via flow cytometry. 307 308 Pancreatic Islet Culture 309 Pancreatic islets were harvested as described8. Islets were immediately placed into culture at 37°C 310 in RPMI (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin- streptomycin 311 (Gibco). IFN-γ (R&D Systems) was added at 10 ng/mL. Approximately 300 islets were placed into each 312 well of a 24 -well plate in 500 µ L of media. Either islets were harvested for qPCR at 48h, or supernatant 313 was collected at 72h for multiplex analysis. 314 315 Tolerization with Donor ECDI-SP infusions 316 BALB/c spleens were processed to single-cell suspension by mechanical disruption and red blood 317 cells were lysed with ACK lysing buffer (Lonza). BALB/c splenocytes (SP) were incubated with ECDI 318 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 17 (Calbiochem, 30 mg/mL) on ice for 1 hr with agitation followed by washing. The final product was passed 319 through a 70 µm filter to remove clumps. 5x107 BALB/c ECDI-SP were injected i.v. to recipients on day +1 320 and day +7. 321 322 Mixed Lymphocyte Reaction 323 For the mixed lymphocyte reactions (MLR), recipient spleens were harvested and processed to 324 single cell suspension. A small number of cells were set aside for flow cytometry analysis. The remainder 325 of the spleen was purified for CD3 T cells using the T Cell Isolation EasySep kit (StemCell Techonologies, 326 19851A). Following isolation, T cells were washed and then stained with eFluor 450 proliferation dye 327 (Invitrogen, #65-0842-90) according to manufacturer instructions. 328 Antigen presenting cells (APCs) were harvested as previously described 47. Briefly, spleens were 329 harvested from naïve B6, C3H, and BALB/c mice. Spleens were perfused using 3 mL of collagenase type IV 330 (2 mg/mL, Worthington Biochemical Corporat ion). Perfused spleens were incubated at 37°C for 30 331 minutes. Following incubation, spleens were processed to single cell suspension. Splenocytes were 332 resuspended in 3 mL of 30% bovine serum albumin (BSA). 1 mL of PBS was layered on top of the BSA. Cells 333 were centrifuged at 1000 relative centrifugal force (rcf) for 30 minutes with no brake. Cells at the interface 334 were collected and washed. These cells were used as enriched APCs for the MLR. 335 T cells and APCs were cultured at a ratio of 1:1 for three days in RPMI 1640 supplemented with 336 10% fetal bovine serum (Gibco), 1% penicillin -streptomycin (Gibco), 1% HEPES buffer (Corning), 1% 337 sodium pyruvate (Gibco), 1% minimum essential media (Gibco), 0.1% gentamicin (Gibco), and 0.05 mM 2-338 mercaptoethanol (Millipore). The MLR was performed in a 96 -well U-bottom plate. Cells were counted 339 and placed into U -bottom 96 well plates with 1x10 5 T cells and enriched APCs per well. Samples were 340 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 18 harvested after three days. T cell proliferation was quantified using flow cytometry and eFluor 450 341 proliferation dye intensity. 342 343 Maraviroc Treatment 344 To inhibit CCR5 activity in vivo, maraviroc (MedChem Express) was administered to recipients at 345 25 mg/kg/day. Maraviroc stock was prepared by suspending 100 mg/kg in DMSO. The final injection 346 vehicle consisted of 10% DMSO, 40% PEG300, 5% Tween -80, and 45% ddH 2O per manufacturer 347 recommendation to ensure complete resuspension. Maraviroc was injected via i.p. during the treatment 348 duration. 349 350 Statistical Analysis 351 Statistics were analyzed using GraphPad Prism v10. 6.1. Descriptive statistics are presented as 352 mean ± SD for parametric data. Graft survival was compared using Kaplan-Meier survival curves with log-353 rank test. Welch's t test or analysis of variance (ANOVA) was used to compare means of groups. P < .05 354 was considered statistically significant. 355 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 19 Author contributions: 356 MD and XL designed the research study. MD and XL analyzed the data and wrote the manuscript. MD, OF, 357 YY, and CZJ performed the experiments. YY performed RNA-Seq data analysis. XL supervised the overall 358 project. 359 360 Acknowledgments: 361 This work was supported by National Institutes of Health research grant R01 DK 132889. Multiplex assays 362 were performed in the Duke Cancer Institute Flow Cytometry Facility at Duke University, Durham, NC, 363 which is supported by the NCI Cancer Center Support Grant (CCSG) award number P30CA014236. 364 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 20 Figure Legend 365 Figure 1: Donor macrophage depletion combined with post-transplant donor ECDI-SP infusions results 366 in indefinite islet allograft survival. (A) Diagram of treatment schedule for delayed tolerance protocol. 367 BALB/c donors are treated with two doses of 500 μg anti-CD115 antibody intraperitoneally (i.p.) as 368 described in Methods. Donor macrophage-depleted (DMac-Depleted) or non-depleted (Control) islets are 369 transplanted on day 0 into diabetic C57BL/6 mice. Recipients are then treated with two doses of donor 370 ECDI-SPs on POD+1 and POD+7. (B) Representative FACS plots depicting gating strategy to count 371 pancreatic islet macrophages . BALB/c donors received two doses of 500 μg intraperitoneal anti-CD115 372 antibody on days-11 and - 7 prior to islet isolation on day0 . Islets were harvested and immediately 373 dissociated before staining for flow cytometry. Two donors were pooled for each data point, and the 374 number of macrophages was normalized to the number of donors. The bar graph depicts the average 375 number of donor islet macrophages (DMac) per donor, N=4 for each group. (C) Blood glucose was tracked 376 to determine graft function , with two consecutive days of blood glucose > 250 mg/dL defined as graft 377 rejection. Survival of grafts with each treatment regimen is represented in the survival plot as days post-378 transplant, significance *p < 0.05. ***p < .005. 379 380 Figure 2: Donor macrophages contribute to early innate graft infiltration. (A) Representative FACS plots 381 demonstrating gating strategy for innate immune cell infiltration post -transplantation. (B) Bar graphs 382 show total infiltration of each cell type per graft, comparing donor macrophage -depleted (DMac-383 Depleted) and non-depleted (control) islet grafts at POD+1 and POD+10. Recipients received either donor 384 DMac-Depleted or Control BALB/c islet grafts. Grafts were collected on POD+1 (prior to the first dose of 385 donor ECDI-SPs) or on POD+10 (after receiving two doses of ECDI-SPs), followed by dissociation, staining, 386 and analysis. N=8-9 on POD+1. N=5 on POD+10. 387 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 21 388 Figure 3: Islet macrophages contribute to release of CCR5 ligands by pancreatic islets. (A) Schematic of 389 islet culture system. DMac-depleted or control islets were harvested from BALB/c mice and placed into 390 culture. Approximately 300 islets were placed in a single well of a 24-well plate with 0.5 mL of media. IFN-391 γ was added at a concentration of 10 ng/mL. Following 48 or 72 hours of culture, islets and supernatant 392 were harvested for analysis. (B) Relative mRNA expression of CCL3, CCL4, and CCL5 by DMac-depleted and 393 control islets in culture. Islets were collected at 48 hours and placed into Trizol for mRNA isolation. N =3 394 for each group. (C) Multiplex analysis of secreted chemokines from macrophage depleted and non -395 depleted islets. Supernatant was collected after 72 hours of culture for analysis. N=3 for each group. (D) 396 Single-cell transcriptomic map of wildtype B6 murine pancreatic islet cell populations. Sequencing analysis 397 was performed on a public NCBI data set (GEO accession no. GSE232474 ). Violin plots show Log2 398 expression of CCL3, CCL4, and CCL5 in islet cell populations. Shaded areas represent the 25th to 75 th 399 percentiles. 400 401 Figure 4: CCR5 inhibition reduces early graft innate immune cell infiltration and promotes transplant 402 tolerance induction by post-transplant donor ECDI-SP infusions. (A) Grafts of untreated recipients were 403 harvested at POD+1. Grafts were analyzed by FACS for expression of CCR5, the receptor for CCL3, CCL4, 404 and CCL5. Gating strategy is the same as shown in Figure 2A. (B) Schematic for treatment of recipient with 405 maraviroc, a small molecule CCR5 inhibitor. Recipients were given 25 mg/kg /day maraviroc via i.p. 406 injection on day-1 and 0 immediately following islet transplant. Grafts were harvested on PO D+1 to 407 enumerate graft infiltrating recipient cells using FACS. Graphs represent total number of each cell type in 408 the graft. N=10 for each group. (C) Schematic for short -term peritransplant maraviroc treatment. 409 Recipients were given daily injections as described above from day-1 through POD +7. Maraviroc-treated 410 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 22 and vehicle-treated (control) recipients were both given POD+1 and +7 donor ECDI-SPs infusions. Graft 411 survival for each group is plotted as days post-transplant. *p < 0.05 412 413 Figure 5: Donor macrophages contribute to T cell infiltration and donor -specific T cell activation . (A) 414 Representative FACS plots demonstrating gating strategy for T cell infiltration post -transplantation. (B) 415 Grafts were collected on POD+2 and POD+14 from recipients transplanted either with DMac-Depleted or 416 control (non-depleted) islet allografts and were analyzed by FACS to enumerate recipient T cell infiltration 417 of the graft. All recipients were treated with BALB/c ECDI-SPs infusions on POD+1 and +7. Bar graphs show 418 total CD4 or CD8 T cell infiltration per graft, comparing DMac-depleted and control grafts. POD+2, N=6-7 419 per group. POD+14, N=12-14 per group. (C) POD+21 recipient splenic T cells were isolated to perform 420 mixed lymphocyte reactions. T cells were cultured with APCs from BALB/c or C3H spleens for three days. 421 T cell proliferation was measured using eFluor 450 proliferation dye. Graphs represent the percentage of 422 recipient T cells which proliferated in response to stimulation. N=5-6 for each group. (D) Recipient spleens 423 were harvested at POD+21. Spleens were analyzed for CD4 +FoxP3+ cells using FACS. Graph represents 424 percent of CD4 T cells expressing FoxP3. N=9 for each group. 425 426 Figure 6: Post-transplant donor ECDI-SP infusions induce donor-specific tolerance. (A) Schematic of re-427 transplant experiment. Long-term stabl e recipients (> 100d with functioning islet allografts ) were 428 nephrectomized to remove the original graft. New BALB/c (original donor) or C3H (third-party) grafts were 429 placed on the contralateral kidney and blood glucose was observed to determine graft survival. (B) 430 Recipient blood glucose before and after graft nephrectomy . Blood glucose was monitored to confirm 431 reestablished diabetic blood glucose levels following nephrectomy and before re-transplant on day 0. (C) 432 Survival curve of retransplanted grafts. New graft survival is represented as days post-retransplant. 433 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698403doi: bioRxiv preprint 23

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