Rapamycin containing nanoparticles (ImmTOR) were manufactured as described earlier (2, 7). Briefly, PLA, pegylated polylactic acid (PLA-PEG), and rapamycin were dissolved in dichloromethane to form the oil phase. An aqueous solution was then added to the oil phase and emulsified by sonication (Branson Digital Sonifier 250A). Following emulsification, a double emulsion was created by adding an aqueous solution of polyvinylalcohol and sonicating a second time. The double emulsion was added to a beaker containing phosphate buffer solution and stirred at room temperature for 2 h to allow the dichloromethane to evaporate. The resulting NPs were washed twice by centrifuging at 75,600 × g and 4°C followed by resuspension of the pellet in PBS. Fluorescent Cy5-containing NPs were manufactured as described above using PLA-Cy5 conjugate. PLA with a butyl amine end group was prepared from PLA-acid, which was then treated with Cy5-acid in the presence of a coupling agent (O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate) to afford the conjugates. ImmTOR doses were based on rapamycin content ranging from 200 to 400 µg. Empty nanoparticles were manufactured in an identical manner, but without rapamycin. Rapamycin (sirolimus) was manufactured by Concord Biotech (Ahmedabad, India).

Immunologically naïve, female C57BL/6 mice aged 36-52 days (or 17-18g) were purchased from Charles River Laboratories (Wilmington, MA). Similarly aged B6.Cg-Tg(TcraTcrb)425Cbn/J mice (also known as OT-II mice), expressing the T cell receptor (TCR), which is specific for chicken ovalbumin 323-339 peptide (OVA_323-339 or OP-II) in the context of I-A^b resulting in CD4⁺ T cells that primarily recognize OP-II when presented by the MHC-II molecule were purchased from Jackson Laboratories (Bar Harbor, ME). To minimize the potential effects of stress, mice were acclimated to the Animal Care Facility at Selecta for at least three days prior to injection. All the experiments were conducted in strict compliance with NIH Guide for the Care and Use of Laboratory Animals and other federal, state and local regulations and were approved by Selecta’s IACUC.

Mice were injected (i.v., tail vein or retro-orbital plexus) with ImmTOR or empty nanoparticles in the effective range of 200-400 µg. Molar equivalent of soluble rapamycin was administered i.p.

For given timepoints (most of the time, several overlapping time-points were tested using the same set of treatments, always including naïve and/or placebo or free rapamycin controls), mice were euthanized, livers and/or spleens harvested and rendered into single cell suspensions via collagenase 4 (Worthington Biochemical, Lakewood, NJ) enzymatic digest according to manufacturer’s recommended protocol. Next, a red blood cell lysis step was performed for 5 min at room temperature in 150 mM NH₄Cl, 10 mM KHCO₃, 10 μM Na₂-EDTA; washed in PBS, 2% bovine serum; then filtered on a 70 µm nylon mesh. LSECs were isolated via CD146 positive selection with immunomagnetic beads according to manufacturer’s instructions (Miltenyi Biotec, San Diego, CA). To prevent non-specific antibody binding, cells were incubated 20 min. on ice with anti-CD16/32 then stained with antibodies (all from BioLegend, San Diego, CA) for given cell surface phenotype. Analysis was performed via FACSCanto flow cytometer (BD Biosciences) with subsequent data analysis using FlowJo software (TreeStar, Ashland, OR).

Flow cytometry was used for the phenotyping of hepatocytes, LSEC, KC, hepatic stellate cells (HSC) and liver T cells from liver cell suspension and of LSEC post CD146 positive selection. Dead cells were always excluded from analysis. Phenotypic changes were assessed as percentage of parent population as shown; measuring of absolute mean fluorescent intensity (MFI) gave essentially the same results. Gating strategies for all the major hepatic cell populations assessed in the study are shown in Supplementary Figure S1 . All antibodies to cell surface markers were from BioLegend (San Diego, CA), with the exception of that against LRP-1 (A2MR-α2) being from Thermo Fischer (Waltham, MA). The following primary antibodies were used to identify liver parenchyma cells: anti-LRP-1 (conjugated with R-PE using SiteClick R-PE labeling kit (Thermo Fischer) according to manufacturer’s instructions), F4/80 (BV510), anti-CD68 (APC/Cy™7), anti-CD11b (PE/Cy7), anti-mannose receptor (MR) (BV-eFlour450), anti-CD146-FITC, anti-CD38 (APC/Cy7) and anti-GFAP (BV421). To confirm the purity of the LSEC population after CD146 positive selection anti-MR (BV eFlour450), anti-F4/80 (BV 510) and anti-CD68 (APC/Cy7) were used. For LSEC and KC phenotyping, the following antibodies were utilized: anti-MHCII (Alexa Flour^® 488), anti-CD80 (PE), anti-CD86 (PE/Cy7) and anti-PD-L1 (PerCP-Cy5.5), all from BioLegend. The following antibodies were used for T cell differentiation and characterization; anti-CD127 (PE), anti-PD-1 (PerCP-Cy5.5), anti-CD4 (PE/Cy7), anti-CD25 (APC/Cy7), anti-CD3 (BV421), anti-CD8α (BV510), anti-CD62L (Alexa Flour^® 488), anti-CD44 (PE) and anti-NK1.1 (APC-Fire). Annexin B was used to evaluate cell apoptosis. Additionally, anti-CD11c (BV510), anti-PDCA1 (Alexa Flour^® 488), anti-CD45 (APC/Cy7), anti-CD152(PE/Cy5) and anti-MHCI (PerCP-Cy5.5) were used to analyze dendritic cells (DC) and their activation status. Cells were incubated with anti-CD16/32 antibodies to prevent non-specific binding, then incubated with primary antibodies for 30 min at 4°C and washed. Analysis of cells were carried out on BD FACSCanto™ II flow cytometer (BD Biosciences) with data analysis using FlowJo software (TreeStar, Ashland, OR).

LSEC were purified as described above and KC were purified using positive selection F4/80 microbeads (Miltenyi Biotec) with >80% purity of either population confirmed by flow cytometry. For cell proliferation studies LSEC isolated from ImmTOR-treated or naïve mice were cultured in limiting dilutions (starting at 1.25 x 10⁵ cells/well) with a fixed number of OT-II splenocytes (2.5 x 10⁴ cells/well) stimulated with OVA_323-339 peptide (Anaspec, Fremont, CA) at 1 µg/ml. Cultures were carried out in triplicates in 96-well round-bottomed plates and cell proliferation was evaluated 72 hours after initiating the cultures using two separate methods, namely resazurin-based fluorescence (17) with PrestoBlue™ HS cell viability reagent (Thermo Fisher, Waltham, MA) according to manufacturer’s instructions and via intracellular flow cytometry using labeling with anti-Ki-67 (Alexa Fluor^® 647, BioLegend), a protein known to be absent in non-dividing cells (18) and to positively correlate with mouse T cell proliferation (19).

Serum cytokine levels were analyzed with Meso Scale Discovery (MSD) 96-Well MULTI-SPOT^® Ultra-Sensitive Human Immunoassay Kits, using electrochemiluminescence detection on an MESO^® QuickPlex SQ 120 with Discovery Workbench software (version 3.0.18) (MSD^®, Gaitherburg, MD). Cytokines were measured using the U-PLEX TH1/TH2 combo (ms) 10-plex kit and the U-PLEX TGF-β Combo (ms) 3-plex kit. Assays were performed according to manufacturer’s instructions, and without alterations to the recommended standard curve dilutions. OR).

Concanavalin A (Con A) induced liver toxicity model was employed essentially as earlier described (20, 21). Briefly, mice were injected (i.v., r.o.) Con A at 12 mg/kg and then terminally bled at 6, 8, 12 or 24 hours post-challenge with cytokine levels in serum determined by MSD as described above and liver tissues collected simultaneously for single-cell suspension analysis by flow cytometry as described above or for hematoxylin-eosin staining followed by microscopic evaluation.

Statistical analyses were performed using GraphPad Prism 8.0.2. To compare the mouse experimental groups pairwise either multiple t test (for several time-points) or Mann-Whitney two-tailed test (for a single time-point; individual comparison of two groups presented within the same graph) were used. Significance is shown for each figure legend (* – p < 0.05, ** – p < 0.01; *** – p < 0.001; **** – p < 0.0001; not significant – p > 0.05). All data for individual experimental groups is presented as mean ± SD (error bars).

ImmTOR has been previously shown to preferentially accumulate in the liver and spleen after intravenous delivery (7). In order to discern its hepatic trafficking in more detail, we analyzed individual liver cell populations by flow cytometry after injection of fluorescent-labeled ImmTOR ( Figure 1 ). Not surprisingly, there was a massive uptake of ImmTOR by Kupffer cells, especially those with a phagocytic phenotype ( Figures 1A, B ). Moreover, there was significant ImmTOR uptake by hepatocytes ( Figure 1C ) and LSECs ( Figure 1D ). Similar results were seen at earlier and later time-points, spanning from one day to two weeks post injection (not shown). Collectively, intravenous injection of ImmTOR led to its simultaneous uptake by all major resident liver cell populations tested.

Expression of cell surface molecules on phagocytic KCs that play key roles in antigen presentation and immune co-stimulation were affected as early as 1-3 days after ImmTOR treatment ( Figure 2 ). Specifically, expression of the immune checkpoint ligand, PD-L1, was already elevated at day 1 ( Figure 2A ), while expression of MHC class II and the co-stimulatory CD80 molecule were decreased by day 3 ( Figures 2B, C ). All of these effects peaked around days 5-7 post-ImmTOR administration and returned to baseline levels by day 10. Other populations of professional APC, such as myeloid DC, showed similar increases in PD-L1 and decreases in CD80 expression, while plasmacytoid DC and cytokine-producing KC showed modest but significant decreases in CD80 and CD86 expression ( Supplementary Figure S2 ).

A broad effect of ImmTOR was detected when analyzing the surface molecule expression profile of LSECs, which have been shown to play a major role in tolerogenic immune responses in the liver (16). Specifically, a consistent suppression of MHC class II, CD80 and especially, CD86 was detected as early as one day after ImmTOR treatment and then maintained throughout the first week after ImmTOR administration ( Figures 3A–C ). Of these, CD80 was downregulated early, but then gradually increased over the first week ( Figure 3B ), while MHC-II was modestly downregulated over days 1-10 post injection ( Figure 3A ). CD86 expression was profoundly suppressed for at least two weeks after ImmTOR treatment ( Figure 3C ). None of these effects were observed if placebo nanoparticles (NP-Empty) were used (not shown).

In contrast, expression of PD-L1 was markedly elevated on LSECs at 1 day after ImmTOR administration and peaking around days 3-5 post-treatment ( Figure 3D ). When combined with analysis of CD80/86 expression, a profound increase in LSEC with a tolerogenic phenotype (PD-L1⁺CD80^lowCD86^low) was apparent during the first week after ImmTOR administration, which was maintained through at least day 14 ( Figure 3E ). By this time CD80 and MHC class II ( Figures 3A, B ) expression was gradually restored to baseline levels, while CD86 expression remained suppressed ( Figure 3C ). A similar phenotype of LSEC was observed if a purified LSEC population selected for CD146 expression was used (not shown) and this tolerogenic LSEC surface phenotype was the same irrespective of whether MR or CD146 was used for LSEC identification ( Supplementary Figure S3 ). As with professional hepatic APC, no effect on LSEC surface expression of CD80, CD86 and MHC class II molecules was seen when NP-Empty was used (see Supplementary Figure S3 for representative images).

There was little or no difference in expression of PD-L1 or MHC class II when comparing total hepatocytes from mice treated with ImmTOR vs naïve controls ( Supplementary Figure S4 ). However, those hepatocytes that took up ImmTOR, as evidenced by use of fluorescent-labeled ImmTOR, showed a profound upregulation of PD-L1 and down-regulation of MHC class II expression. Specifically, such fluorescent ImmTOR-positive hepatocytes expressed two times less MHC class II ( Supplementary Figure S4A ) and two times more PD-L1 ( Supplementary Figure S4B ) than ImmTOR-negative hepatocytes or hepatocytes from naïve untreated mice. A similar pattern was observed in hepatic stellate cells ( Supplementary Figure S5 ), which by day 5 took up ImmTOR particles with similar efficiency as other parenchymal cells ( Supplementary Figure S5A ) and then upregulated PD-L1 and downregulated MHC class II molecules ( Supplementary Figures S5B, C ), but only in those cells that took up ImmTOR ( Supplementary Figures S5D, E ). The same effect was seen at 7 days post-injection (not shown) with MHC class II expression on HSC decreased from being nearly 100% in HSC that were ImmTOR-negative to less than 20% of cells that were positive for fluorescent ImmTOR.

ImmTOR administration led to a profound impact on hepatic T cells ( Figure 4 ). Specifically, a massive decrease in CD4⁺ T cells was observed as early as 3 days after ImmTOR treatment ( Figure 4A ), which was accompanied by a decrease of CD8⁺ cells as well ( Figure 4B ). There was no evidence of increased apoptosis among either CD4 or CD8 T cells whether 7-AAD ( Supplementary Figures S6A, B ) or Annexin B (not shown) were used. However, there was a substantial increase in double-negative (DN, CD3⁺CD4^–CD8^–) T cells that persisted for at least 14 days ( Figures 4C, D ). The increase in DN T cells was not reproduced by systemic injection of free rapamycin ( Figures 4B–D ), but was observed to a lesser degree with empty particles (NP-Empty) ( Figure 4D ). This effect was observed only in hepatic T cells, as a change in DN T cells was not seen in splenic T cells ( Figure 4E ).

Additionally, ImmTOR administration led to induction of PD-1 expression on remaining CD4⁺ and CD8⁺ T cell subpopulations within three days and maintained for at least 14 days post-administration and was not seen when equal doses of free rapamycin were used ( Figures 5A–C ). Empty nanoparticles showed a modest, non-significant trend to increasing PD-1 expression on T cells. The increase in PD-1 expressing T cells was more pronounced on CD4⁺CD25⁺ T cell cells ( Figures 5D, E ) and specifically on T cells with a regulatory phenotype (CD4⁺CD25⁺CD127^low (22, 23); ( Figure 5F ). However, there was no significant increase in intracellular FoxP3 expression within hepatic CD4⁺CD25⁺ cells ( Supplementary Figure S6C ). We then tested whether immune checkpoint-related pathways are involved in this process, especially taking into consideration ImmTOR-mediated PD-L1 elevation on hepatic professional and non-professional APC ( Figures 2 , 3 ). ImmTOR-mediated increase in PD-1^high hepatic T cells ( Figure 6A ) and CD4⁺CD25⁺CD127^low T cells ( Figure 6B ), but not the emergence of hepatic DN T cells ( Figure 6C ), was inhibited by anti-PD-1 or anti-CTLA-4 antibodies.

There was no significant impact of ImmTOR on hepatic NK T cells ( Supplementary Figure S6D ) and the key features of T cell impact such as CD4 and CD8 surface downregulation, DN T cell emergence and PD-1 surface induction were the same irrespective of whether female ( Figure 5 ) or male ( Supplementary Figure S7 ) mice were used to assess ImmTOR effects. ImmTOR treatment did not affect surface expression of CTLA-4 and CD28 on hepatic T cells (not shown).

The ability of ImmTOR to suppress immune activation was further assayed using several ex vivo and in vivo models. Firstly, LSEC from ImmTOR-treated or naïve mice were isolated and co-incubated with cognate-peptide stimulated splenocytes from OT-II mice. While LSEC from naïve mice exhibited minimal and non-specific inhibitory effect on OT-II cell proliferation, the effect of LSEC from ImmTOR-treated animals was dose-dependent and of much higher scale (by the factor of 3 to 10) and was confirmed by two independent methods of proliferation measurement ( Figures 7A, B ). When cytokine expression by Kupffer cells and LSEC isolated from ImmTOR-treated mice was assessed in vitro vs. that in naïve mice, there was a significant decrease in expression of KC/GRO ( Figure 7C ), a neutrophil chemokine with a known role in several models of inflammatory response (24, 25).

Since neutrophils along with several other immune cell types are known to play a key role in a model of immune-mediated hepatic cell damage inflicted by concanavalin A (Con A) administration (20, 21, 26), we have then tested the ability of ImmTOR to prevent Con A-mediated liver inflammation. When mice treated with ImmTOR at 7 days prior to Con A challenge were evaluated, there was massive decrease in systemic cytokine induction vs. that seen in untreated mice or those treated with NP-Empty placebo, with the difference in levels of IFN-γ, KC/GRO, TNFα, IL-2 and IL-10 being especially pronounced at several post-challenge time-points tested ( Figure 8 ). Similarly, intrahepatic appearance of activated neutrophils as well as activation of liver-resident macrophages and T cells was significantly suppressed by ImmTOR, but not by NP-Empty pre-treatment ( Figure 9 ) as was histological evidence of leukocyte infiltration of blood vessel-adjacent liver tissues and Con A-induced tissue necrosis ( Figure 10 ). Similar effect, but of lesser scale was seen if ImmTOR treatment was administered 3 days prior to Con A challenge (not shown).