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Oxidized-ATP Attenuates Kidney Allograft Rejection By Inhibiting T-Cell, B-Cell, And

Macrophage Activity

Xiang Ding1*, Nancy A. Wilson2*, Robert R. Redfield III3, Sarah E. Panzer2, Bret Verhoven3,

Shannon R. Reese2, Weixiong Zhong4, Lei Shi5, William J. Burlingham3, Loren C. Denlinger5,

Arjang Djamali2

*Contributed equally

1Department of Organ Transplantation, Xiangya Hospital, Central South University, China

2Department of Medicine, Division of Nephrology, University of Wisconsin, Madison

3Department of Surgery, Division of Transplant, University of Wisconsin, Madison

4Department of Pathology and Lab Medicine, University of Wisconsin, Madison

5Department of Medicine, Division of Pulmonary and Critical Care, University of Wisconsin,

Madison

Kidney360 Publish Ahead of Print, published on February 3, 2020 as doi:10.34067/KID.0000692019

Copyright 2020 by American Society of Nephrology.

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Keywords: allograft, kidney, oATP, P2X7R, purinergic receptor, ABMR

Abbreviations:

ATP adenosine triphosphate

oATP oxidized ATP

CsA cyclosporine A

Scr serum creatinine

BUN Blood urea nitrogen

TCMR T cell mediated rejection

ABMR Antibody mediated rejection

MLR Mixed lymphocyte reaction

P2X7R Receptor P2X7

BN Brown Norway rat

DSA Donor specific antibody

RT PCR Real Time Polymerase Chain Reaction

BzAPT 20-30-O-(4-benzoylbenzoyl) adenosine 5’ triphosphate

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Abstract

Background: Extracellular adenosine triphosphate (ATP) binds to purinergic receptors and

promotes inflammatory responses. We tested whether oxidized ATP (oATP), P2X7 receptor

antagonist can attenuate acute kidney allograft rejection.

Methods: Brown Norway kidney allografts were transplanted into Lewis recipients. Three groups

were defined: oATP (n=8), Cyclosporine A (n=6), and no treatment (n=8). On day seven, we

assessed kidney allograft survival, function, and rejection characteristics. We further determined

T-cell, B-cell, and macrophage response to oATP in vivo and in vitro and examined intragraft

inflammatory gene transcripts.

Results: Kaplan-Meier survival analyses demonstrated significantly better graft survival rates in

oATP and CsA groups compared to no treatment (p<0.05). Similarly, SCr and BUN levels were

significantly lower in oATP and CsA groups (p<0.05). oATP reduced both T cell mediated

rejection (TCMR) and antibody mediated rejection (ABMR), inhibited B cell and T cell activation,

and downregulated intragraft IL-6 mRNA levels (p<0.0001). In vitro, oATP prevented proliferation

in Mixed Lymphocyte Reaction assays, and inhibited macrophage P2X7R activity in a dose

dependent manner.

Conclusions: Our findings suggest that oATP mitigates kidney allograft rejection by inhibiting T

cell, B cell, and macrophage activity and indicate a potential role for the purinergic system and

oATP in solid organ transplantation.

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Introduction

Extracellular ATP may serve as a mediator of cell-to-cell communication by interacting with

specific cell surface molecules known as P2 purinergic receptors; it can also be a mediator of

cytotoxic cell-dependent lysis (1). Activation of ATP receptor P2X7 (P2X7R) leads to ion channel

activity, which can depolarize the cell and activate several signaling cascades including PKC and

MAPK pathways (2, 3). Oxidized ATP (oATP) acts as an antagonist of P2X7R (3, 4). oATP has

demonstrated anti-inflammatory effects in experimental models of asthma, neurodegenerative

disease, infections, and autoimmunity (3, 5-9).

Recent evidence supports a protective role for oATP in renal ischemia reperfusion injury

(10) and islet allograft rejection (11). Intraperitoneal delivery of oATP prior to ischemia-

reperfusion injury in mice decreased serum creatinine (Scr), blood urea nitrogen (BUN), tubular

injury scores, and tubular epithelial cell apoptosis after injury (10). The infiltration of dendritic cells,

neutrophils, macrophages, CD69+CD4+, and CD44+CD4+ T cells was attenuated, but renal

Foxp3+CD4+ Tregulatory cell (Treg) infiltration was increased by oATP. It was concluded that

oATP attenuated acute renal damage and facilitated renal recovery in ischemia-reperfusion injury

by expansion of Tregs (10). Similarly, in vivo short-term P2X7R targeting with oATP delayed islet

allograft rejection in mice, reduced the ratio of Th1/Th17 cells, and induced hyporesponsiveness

toward donor antigens (11). The combination of oATP and rapamycin synergized in inducing long-

term islet function in 80% of transplanted mice and resulted in reshaping of the recipient immune

system (11).

These observations led us to believe that oATP may be used in solid organ transplantation

to attenuate the alloimmune response. We tested this hypothesis in a robust model of acute

kidney allograft rejection using Brown Norway (BN) donor kidneys and Lewis recipients. We

observed that oATP therapy improved graft survival and function, reduced both T cell mediated

rejection (TCMR) and antibody mediated rejection (ABMR), inhibited B cell and T cell activation,

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and downregulated intragraft IL-6 mRNA levels. In vitro, oATP prevented macrophage and

splenocyte proliferation in Mixed Lymphocyte Reaction (MLR) assays, and inhibited macrophage

P2X7R activity in a dose dependent manner. Altogether, our findings suggest that oATP mitigates

kidney allograft rejection by inhibiting T cell, B cell, and macrophage activity; the anti-inflammatory

effects of oATP may be in part mediated via P2X7R.

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Materials and Methods

Animal Model: Adult (175-200g) male Lewis (RT1l) and Brown Norway (RT1n) rats were

purchased from Envigo Laboratories (Madison, WI) and were housed in the animal care facility at

the William S. Middleton VA Hospital in Madison, WI. All procedures were performed in

accordance with the Animal Care and Use Policies at the VA Hospital and University of Wisconsin.

Guidelines from “The Principles of Laboratory Animal Care” NIH publication Vol 25, No. 28 revised

1996: http://grants.nih.gov/grants/guide/noticefiles/not96-208.html) were followed. This model of

acute rejection including allogeneic kidney transplantation with simultaneous bilateral native

nephrectomy has been previously reported by our laboratory (12, 13). We defined three groups:

oATP 3 mg/kg/d intraperitoneal (i.p.) x 7 days (#A6779 Sigma Aldrich, diluted with normal saline

and filter sterilized), CsA 10 mg/kg/d i.p. x 7 days, and no treatment (Figure 1. A.). The dose of

oATP was selected based on previous studies in mice ranging from 150 microg x 1 to 5 mg/kg/d

(3, 8, 10). Blood and tissue samples were collected and analyzed on day 7 or earlier if animals

died sooner from rejection.

Kidney function: Blood urea nitrogen (BUN) and Serum Creatinine (Scr) were measured in

serum samples using VetTest Analyzer technology (IDEXX Laboratories, Westbrook, ME) as

previously described (12).

Histology: Formalin-fixed, paraffin embedded kidneys were cut into 5microm sections. Slides

were deparaffinized, rehydrated from xylene through a graded ethanol series to ddH2O and

subsequently treated as described below. Slides were viewed on an Olympus BX52 microscope

equipped with an Olympus DP70 camera and software. All Hematoxylin-eosin and C4d slides

were reviewed by Dr. Weixiong Zhong, MD, PhD, transplant pathologist and scored according to

Banff 2015 (14).

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Immunostaining: Immunoperoxidase studies were done as previously described (15-17). The

following antibodies with used for immunohistochemical studies: Rabbit anti-C4D (200-1,

American Research Products, cat 12-5000), anti-rat P2X7R (Alamone, cat APR-004).

Mixed lymphocyte Reaction (MLR): BN and Lewis splenocytes were purified using standard

protocols. Target BN cells were irradiated with 2,000 rad to prevent proliferation. 1 x 105 cells of

Lewis cells and irradiated BN cells were mixed in a 6 well plate and incubated for 3 days with or

without 50 microM oATP. At the end of the assay, cells were tested for proliferation using a cell

counting assay (Dojindo Molecular Technologies, Code: CK04). Cell death was assessed using

a Cytotoxicity LDH assay (Dojindo Molecular Technologies, Code: CK12).

Flow cytometry: Splenocytes, bone marrow cells, lymph node cells and peripheral blood

monocytes (PBMC) were isolated and aliquoted 500,000 cells per tube. Cells from the MLR were

washed off the plate, washed and aliquoted 500,000 cells per tube. All assays included a

live/dead discrimination GhostRed 780 (Tonbo), even though typically cells were >95% viable.

The B cell stain antibodies included IgD (MCA190B-biotinylated, BioRad) with PE-CF594

streptavidin (562284 BD Biosciences), FITC IgM clone G53-238, BD Biosciences, PerCP CD138

(clone B-A38, Abcam), PE CD38 (clone 14.27, BioLegend), PECy7 CD45R (B220) (clone HIS24

eBioscience), BV605 or BV510 CD27 (clone LG.3A10 BD Biosciences), APC CD24 (130-106-

217 Miltenyi Biotec). The Marginal Zone B cell stain antibodies included: FITC anti-rat Marginal

Zone (clone HIS57 BD Biosciences), PECy5 CD45RA (clone OXj-33, BD Biosciences). The T

cell stain antibodies included: PerCP CD8a (clone OX-8 BioLegend), PE CD11b (clone OX-42

BD Biosciences), BV605 or BV510 CD27 (clone LG.3A10 BD Biosciences), APC CD4 (clone OX-

35 BD Biosciences). The Tfh stain antibodies included: AlexaFluor 488 rCCR4 (FAB1567G R&D

Systems), PerCP/Cy5.5 CD278 (iCOS) (clone C398.4A BioLegend), PE CCR6 (FAB8320P R&D

systems), PECy7 CD4 (W3/25 BioLegend), rabbit anti-rat CXCR5 (ab133706 Abcam), BV421

polyclonal Goat anti-rabbit IgG (cat: 565017 BD Biosciences), AlexaFluor647 CD3 (clone 1F4

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BioLegend). All flow was gated first through the live gate (GhostRed 780 negative), then singlets

before gating for lymphocytes or plasma cells. Flow data were analyzed in FlowJo v 10.2. All flow

cytometry was perfomed at the UWCCC Flow cytometry facility either on the LSR II or LSR

Fortessa.

Donor Specific Antibody (DSA) analysis was performed essentially as previously described

(12, 13). Briefly, donor (RT1n) splenocytes were freshly isolated from spleen macerated though

a 40 microm sieve, washed, after RBC lysis, cells were re-suspended and 500,000 cells were

aliquoted into cluster tubes. Splenocytes were incubated for 30 minutes in a 37°C CO2 incubator

with 50 microl of 1:4 dilution of plasma, then washed and stained for rat antibody isotypes.

Antibodies used for the rat DSA included: AlexaFluor647 CD3 (clone 1F4 BioLegend), PECy7

CD45R (B220) (clone HIS24 eBioscience), FITC IgG1 (clone RG11/39.4 BD Biosciences), PE

IgG2a (clone RG7/1.30 BD Biosciences), FITC IgG2b (clone RG7/11.1 BD Biosciences),

biotinylated IgG2c (clone A92-1 BD Biosciences), Streptavidin Pacific Blue (S11222 Life

Technologies), PE IgM (clone G53-238 BD Biosciences). Cells were put through a singlet gate,

then gated through either CD3+ cells (for MHC class I allo antibodies) or CD45R+ cells (for MHC

class I and MHC class II allo antibodies), then MFI was determined for each isotype.

RT PCR: Real time PCR analyses were completed as described previously (12, 15). Briefly, total

RNA was extracted from snap frozen kidney tissue using Trizol (Life Technologies, Grand Island,

NY). RNA was further purified using the RNAeasy kit (Qiagen, the Netherlands). cDNA was

created using the SuperScript IV first strand synthesis system (18091050 Invitrogen). Taqman

gene expression assays were ordered from ThermoFisher. GAPDH was used to normalize

values (delta CT), all cytokine CTs were compared to cytokine CTs for the untreated group (delta-

delta CT). Delta-delta CTs were used to calculate fold changes.

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YoPro Pore Assay: The YOPRO dye uptake assay was described previously (6, 18). Briefly,

1x105 purified Lewis splenic macrophages per well were stimulated for 30 min with increasing

concentrations of oATP or A740003 in the presence of 250 microM 20-30-O-(4-benzoylbenzoyl)

adenosine 5’ triphosphate (BzATP, Sigma Chemical, St. Louis, MO) with 10 mM YO-PRO-1

(Invitrogen, Carlsbad, CA), then read on a plate reader at ODEX=491nm ODEM=509nm.

Statistical analysis: Student t test and Mann-Whitney rank sum test were used to compare

parametric and nonparametric continuous data, as appropriate. One way ANOVA was used when

comparing between multiple groups simultaneously. Chi square or Fisher’s exact tests were used

to compare categorical data between groups. A Kaplan Meier analysis was used for the survival

data. P values of 0.05 or less were considered significant.

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Results

oATP was associated with improved graft survival and function (Figure 1). To determine

the effect of oATP on kidney transplant survival and function, we compared animal survival and

Scr and BUN levels on day seven between three groups: oATP (n=8), CsA (n=6), and no

treatment (n=6) (Figure 1). Kaplan-Meier survival analyses demonstrated significantly better graft

survival rates in oATP and CsA groups compared to no treatment (Figure 1B, p<0.05). Similarly,

Scr and BUN levels were significantly lower in oATP and CsA groups (Figure 1C, p<0.05).

oATP attenuated T cell mediated rejection (TCMR) and antibody mediated rejection (ABMR)

(Table 1, Figure 2, Figure 3). To define the specific effect effect of oATP on kidney allograft

rejection, we compared the histopathological findings between the three groups according to

Banff (14). All six surviving animals from the untreated group demonstrated severe mixed

rejection (TCMR and ABMR) with large areas of interstitial hemorrhage and cortical necrosis on

day 7 (Table 1, Figure 2 panels a,b). No recipient in the oATP or CsA group had evidence of

cortical necrosis/interstitial hemorrhage (p<0.001). Neither oATP nor CsA were completely

effective in preventing rejection; however, oATP reduced the severity of tubulitis (t score, p<0.001)

and endarteritis (v score, p=0.04) lesions while CsA was associated with a more robust

improvement in i, t, v, g, ptc scores compared to no treatment (p < 0.05) (Figure 3). C4d scores

were not different between the three groups, but the intensity of C4d staining was much weaker

in the CsA group.

oATP treatment inhibited the proliferation of splenocytes and macrophages in vitro (Figure

4). We performed Mixed Lymphocyte Reactions (MLR) to test the effect of oATP on cell

proliferatation upon stimulation with allogeneic cells. Splenocytes were isolated from BN and

Lewis spleens. Lewis splenocytes were incubated with irradiated Lewis* cells (negative control,

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Syn) or irradiated BN* cells (positive control, Allo). In a third group, Lewis splenocytes were co-

incubated with irradiated BN* splenocytes and 50 microM oATP (Allo + oATP).

As noted in Figure 4A, Lewis splenocytes proliferated significantly in the presence of

irradiated BN* splenocytes. However, splenocyte proliferation was significantly inhibited by oATP

(p<0.05). A similar result was obtained looking only at macrophages (Figure 4B).

oATP therapy was associated with a decline in B cells and T cell activation after renal

transplantation (Figure 5). We next examined the effect of oATP on B cell and T cell phenotypes

in vivo following kidney transplantation. We directly tested the status of the cell populations in the

treated versus non-treated allograft recipients on day 7 post-transplant for activation status; the

cell populations were isolated from the recipient spleen, bone marrow, lymph nodes, and PBMC

and tested for activation markers. Gating strategies used were demonstrated in Supplemental

Figure 1. These studies showed that oATP treatment was associated with a significant decline in

CD45R+ B cells in the spleen and BM (Figure 5A, p<0.05). Furthermore, transitional B cell

(CD45R+CD24+CD38+) were reduced in the spleen, LN, and PBMC (Figure 5B, p<0.05), and

activated B cells (CD45R+CD27+) were reduced in the spleen (Figure 5C, p<0.05). The total

number of T cells did not change significantly (Figure 5D); however, both CD3+CD4+iCOS+ T

cells (Figure 5E) and CD3+CD4+CCR6+ T cells (Figure 5F) declined significantly in the LN,

PMBC ± spleen (p<0.05).

oATP treatment was associated with a decline in Donor Specific Antibodies (DSA) post-

transplant (Figure 6). To determine the effect of oATP treatment on DSA, we examined IgM,

IgG2b, and IgG2c allo-antibodies on day 7 (Figure 6, Panels A-C). Normal Lewis plasma alone

served as a negative control for the assay. These studies demonstrated that DSA were

significantly reduced in animals treated with oATP. This observation was particularly evident for

IgM DSA in the absence of previous exposure and memory response.

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oATP significantly reduced IL-6 mRNA in the kidney allograft (Figure 7). To determine the

effect of oATP on intrarenal pro-inflammatory gene expression, we compared IL1-beta, IL-2, IL-

6, IL-10, IL-17, and IFN-gamma mRNA levels using real-time PCR analyses, normalizing gene

expression in oATP treated kidneys to controls (Figure 7). These studies showed that oATP

therapy was associated with a drastic downregulation of IL-6 mRNA (~80 fold, p<0.0001) in kidney

allografts.

oATP inhibited macrophage P2X7R in Lewis rats (Figure 8). Because macrophages are an

important source of IL-6, we sought to examine whether oATP inhibited Lewis (recipient)

macrophages through their P2X7 receptor (P2X7R). Activation of the P2X7R leads to the

formation of a membrane pore that allows molecules up to ~900 Da to permeate upon ATP

binding (19, 20). We first used increasing concentrations of agonistic BzATP to open P2X7R

pores on macrophages isolated from the spleen (Figure 8A). We observed that BzATP opened

P2X7R pores in a dose-dependent manner until a concentration of approximately 500 micromolar;

thereafter, there was a decline in BzATP activity. Next, we compared the effects of oATP and

A740003 (selective inhibitor of P2X7R), on closing the P2XR pores in the presence of a constant

concentration of BzATP (250 microM, Figures 8B and 8C). These studies determined that while

all concentrations of A740003 were effective in inhibiting BzATP, only higher concentrations of

oATP (500 microM) inhibted P2X7R (Figures 8B and 8C).

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Discussion

Our studies determined that oATP improves graft survival and function, attenuates TCMR and

ABMR in a robust model of acute rejection, inhibits B-cell and T-cell activation in vivo and in vitro,

and downregulates intragraft IL-6 mRNA levels. In vitro, oATP prevents splenic macrophage and

lymphocytes proliferation while inhibiting macrophage P2X7R activity. In aggregate, these

findings suggest that oATP may attenuate kidney allograft rejection by inhibiting T-cell, B-cell, and

macrophage activity. The inhibition of macrophage P2X7R indicates a potential molecular

pathway regulating the anti-inflammatory properties of oATP in kidney transplantation by

interfering with downstream signaling.

The anti-inflammatory effects of oATP, including the downregulation of IL-6 in an

experimental model of renal injury are well established (10). oATP is a Schiff-base-forming

reagent that has been used for some years as an antagonist at the P2X7 receptor (P2X7R) (4,

21). In a mouse macrophage-like cell line, low oATP concentrations (100 microM) completely

blocked early permeabilization of plasma membrane, cell swelling, vacuolization, and lysis

mediated by extracellular ATP (4). Antagonism developed slowly, as an incubation at 37 degrees

C for at least 2 h in the presence of oATP was needed and was irreversible, thus suggesting that

the inhibitory action was due to covalent modification of the P2X7R receptor (4). In our study,

although oATP inhibited macrophage P2X7R in vitro, we did not delineate whether IL-6 inhibition

was mediated through P2X7R antagonism. Furthermore, while all concentrations of A740003

(the selective inhibitor of P2X7R) were effective in inhibiting BzATP, only higher concentrations

of oATP (500 microM) inhibited the receptor, suggesting that some anti-inflammatory effects of

oATP may not be due to blockade of the P2X7R. Consistent with this observation, oATP can

reduce NFkappaB activation and IL-8 release in cells lacking P2X7R (21). Similarly, other

investigators have reported inhibitory effects of oATP on proinflammatory responses in three

human cell types lacking the expression of P2X7R: umbilical vein endothelial cells (HUVEC),

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HEK293 cells, and 1321N1 astrocytes (22). In these studies, oATP decreased by 40-70% the

secretion of interleukin IL-8 stimulated by TNF-alpha in all three cell types, by IL-1beta in HUVEC

and 1321N1 cells, and by endotoxin in HUVEC (22). Attenuation of TNF-alpha-stimulated IL-8

secretion by oATP was similar in wild-type HEK cells or HEK cells stably expressing recombinant

P2X7R (22).

We further demonstrated a significant inhibition of T cell, B cell, and macrophage

activation in vitro and in vivo. The total number of T cells did not change significantly; however,

both CD3+CD4+iCOS+ T cells and CD3+CD4+CCR6+ T cells declined significantly in secondary

lymphoid organs. These in vivo phenotypes define memory and effector T cells with high

inflammatory potential (23, 24). Our findings are consistent with previous studies demonstrating

the inhibition of T cell activation and Th1/Th17 phenotype by oATP in autoimmune disease, islet

transplantation, and infection (3, 9, 11, 24). In a mouse model of islet transplantation, P2X1R and

P2X7R were induced in islet allograft-infiltrating cells, but only P2X7R was increasingly expressed

during alloimmune response (11). In vivo short-term P2X7R targeting with oATP delayed islet

allograft rejection, reduced the frequency of Th1/Th17 cells, and induced hyporesponsiveness

toward donor antigens (11). oATP-treated mice displayed preserved islet grafts with reduced Th1

transcripts and in vitro P2X7R targeting using oATP reduced T-cell activation and diminished

Th1/Th17 cytokine production. The investigators concluded that beneficial effects of oATP

treatment revealed a role for the purinergic system in islet allograft rejection, and the targeting of

P2X7R could be a novel strategy to induce long-term islet allograft function (11). We add new

data to these observations, by demonstrating a downregulation of transitional B cells

(CD45R+CD24+CD38+), activated B cells (CD45R+CD27+), and donor specific antibodies in a

solid organ transplant model.

Our studies did not examine the potential effects of oATP on non-immune cells, as P2XR

expression can be upregulated in glomerular and endothelial cells during acute injury (25).

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However, taken together, our findings suggest that oATP can attenuate proinflammatory signaling

by mechanisms that are partially mediated via P2 receptor subtypes (1, 4, 21, 22). This

observation was associated with improvement in short-term kidney allograft survival and function,

attenuation of acute TCMR and ABMR, inhibition of B-cell, T-cell, and macrophage activation and

proliferation, and downregulation of intragraft IL-6 mRNA levels. Further studies are needed to

determine the role of the purinergic system and oATP in kidney transplantation.

Disclosures:

Conflict of interest: L Denlinger reports grants from NIH-NHLBI during the conduct of the study;

personal fees from Sanofi-Regeneron and grants and personal fees from AstraZeneca outside

the submitted work; in addition, L Denlinger has a patent to US Patent #7,560,243 B2 issued and

a patent to US Patent #7,960,131 B2 issued. None of the remaining authors have any relevant

conflict of interest to disclose.

Funding:

Support: HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI): R01 HL115118

Author Contrbutions:

Xiang Ding: Conceptualization; Data curation; Formal analysis; Methodology; Writing - original draft

Nancy Wilson: Conceptualization; Data curation; Formal analysis; Methodology; Writing - original draft

Robert Redfield: Resources; Supervision; Writing - review and editing

Sarah Panzer: Resources; Supervision; Writing - review and editing

Bret Verhoven: Methodology; Writing - review and editing

Shannon Reese: Methodology; Supervision; Writing - review and editing

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Weixiong Zhong: Methodology; Writing - review and editing

Lei Shi: Methodology; Writing - review and editing

William Burlingham: Methodology; Resources; Supervision; Writing - review and editing

Loren Denlinger: Methodology; Resources; Supervision; Writing - review and editing

Arjang Djamali: Conceptualization; Data curation; Funding acquisition; Methodology; Resources; Supervision; Writing - original draft; Writing - review and editing

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Figure Legends:

Figure 1. oATP was associated with improved graft survival and function. We compared

animal survival and Scr/BUN levels on day seven between three groups: oATP (n=8), CsA (n=6),

and no treatment (n=6). Kaplan-Meier survival analyses demonstrated significantly better graft

survival rates in oATP and CsA groups compared to no treatment (Figure 1B, p<0.05). Similarly,

Scr and BUN levels were significantly lower in oATP and CsA groups (Figure 1C, p<0.05).

Figure 2. oATP attenuated T cell mediated rejection (TCMR) and antibody mediated

rejection (ABMR). All six surviving animals from the untreated group demonstrated severe mixed

rejection (TCMR and ABMR) with large areas of interstitial hemorrhage and cortical necrosis on

day 7 (Figure 2 panels a,b). No recipient in the oATP or CsA group had evidence of cortical

necrosis/interstitial hemorrhage (p<0.001). Neither oATP nor CsA were completely effective in

preventing rejection; however, oATP reduced the severity of tubulitis (t score, p<0.001) and

endarteritis (v score, p=0.04) while CsA was associated with a more robust improvement in i, t, v,

g, ptc scores compared to no treatment (p < 0.05). C4d scores were not different between the

three groups, but the intensity of C4d staining was much weaker in the CsA group.

Figure 3. oATP was associated with a reduction in BANFF scores. Banff scores were

analyzed using a grouped analysis and presented as interleaved bar graphs, with error bars

representing mean and SEM. Animals that received no treatment are represented by open bars,

those that received cyclosporine A (CsA) are in grey and animals that received oATP are in black.

* Several of the animals in the no treatment group displayed such extensive necrosis that BANFF

scores could not be determined (scores reported as NA in Table 1). For these animals, an

aribitrary score of 4 was assigned for the purpose of making a representative graph.

21

Figure 4. oATP treatment inhibited the proliferation of splenocytes and macrophages in

vitro. Splenocytes were isolated from BN and Lewis spleens. Lewis splenocytes were incubated

with irradiated Lewis* cells (negative control, Syn) or irradiated BN* cells (positive control, Allo).

Irradiated cells are indicated with a *. In a third group, Lewis splenocytes were co-incubated with

irradiated BN* splenocytes and 50 microM oATP (Allo + oATP). As noted in Figure 4A, Lewis

splenocytes proliferated significantly in the presence of irradiated BN* splenocytes. However,

splenocyte proliferation was significantly inhibited by oATP (p<0.05). A similar result was

obtained when macrophages analyzed separately from total lymphocytes in these cultures (Figure

4B).

Figure 5. oATP therapy was associated with a decline in B cells and T cell activation in

vivo. Seven days post transplant, oATP treatment was associated with a significant decline in

CD45R+ B cells in the spleen and BM (Figure 5A, p<0.05). Transitional B cell

(CD45R+CD24+CD38+) were reduced in the spleen, LN, and PBMC (Figure 5B, p<0.05), and

activated B cells (CD45R+CD27+) were reduced in the spleen (Figure 5C, p<0.05). The total

number of T cells did not change significantly (Figure 5D); however, both CD3+CD4+iCOS+ T

cells (Figure 5E) and CD3+CD4+CCR6+ T cells (Figure 5F) declined significantly in the LN,

PMBC and spleen (p<0.05).

Figure 6. oATP treatment was associated with a decline in Donor Specific Antibodies (DSA)

post-transplant. To determine the effect of oATP treatment on DSA, we examined IgM, IgG2b,

and IgG2c allo-antibodies on day 7 (Figure 6, Panels A-C). Plasma alone served as a negative

control for the assay. These studies demonstrated that DSA were significantly reduced in animals

treated with oATP. This observation was particularly evident for IgM DSA in the absence of

previous exposure and memory response.

22

Figure 7. oATP significantly reduced IL-6 mRNA in the kidney allograft. To determine the

effect of oATP on intrarenal pro-inflammatory gene expression, we compared IL1-beta, IL-2, IL-

6, IL-10, IL-17, and IFN-gamma mRNA levels using real-time PCR analyses, normalizing gene

expression in oATP treated kidneys to controls. These studies showed that oATP therapy was

associated with a drastic downregulation of IL-6 mRNA (~80 fold, p<0.0001) in kidney allografts.

Figure 8. oATP inhibited macrophage P2X7R in Lewis rats. Activation of the P2X7 receptor

leads to the formation of a membrane pore that allows molecules up to ~900 Da to permeate upon

ATP binding. To test this activity, we first used increasing concentrations of agonistic BzATP to

open P2X7R pores on Lewis macrophages isolated from the spleen (Figure 8A). BzATP (filled

circles) opened P2X7R pores in a concentration-dependent manner until approximately 500

micromolar (microM); thereafter, there was a decline in BzATP activity. As a control, the ability

of YOPRO to enter cells with no agonist, YOPRO alone, is shown as a straight line. Next, we

compared the effects of oATP (filled squares) and A740003 (filled circles, selective inhibitor of

P2X7R), on closing the P2XR pores in the presence of a constant concentration of BzATP (250

microM Figures 8B and 8C). Again, as a control, we indicated the ability of YOPRO to enter cells

by using YOPRO alone (line). These studies determined that while all concentrations of A740003

were effective in inhibiting BzATP, only higher concentrations of oATP (500 microM) inhibited

P2X7R (Figures 8B and 8C).

Pathology Score According to Banff Classification of Kidney Allograft Pathology 2015

animal ID Treatment i t v g ptc C4d Cortical necrosis/Interstitial hemorrhage Diagnosis

1 oATP 2 2 2 2 3 3 - IIb TCMR and ABMR2 oATP 3 2 1 3 3 3 - IIa TCMR and ABMR3 oATP 3 2 3 3 3 3 - III TCMR and ABMR4 oATP 3 2 1 3 2 3 - IIa TCMR and ABMR5 oATP 3 2 1 3 3 3 - IIa TCMR and ABMR6 oATP 3 2 1 3 3 3 - IIa TCMR and ABMR7 oATP 3 2 2 3 3 3 - IIb TCMR and ABMR8 oATP 3 1 1 3 3 3 - IIa TCMR and ABMR9 Control 3 3 2 3 2 3 + IIb TCMR and ABMR

10 Control NA NA NA NA NA 3 + ABMR11 Control NA NA NA NA NA 3 + ABMR12 Control NA NA 3 NA NA 3 + ABMR13 Control NA NA NA NA NA 3 + ABMR14 Control 3 3 3 3 3 3 + III TCMR and ABMR15 CsA 1 0 0 2 1 3 - ABMR16 CsA 1 0 2 2 1 3 - ABMR17 CsA 1 0 0 2 1 3 - ABMR18 CsA 0 0 0 2 3 3 - ABMR19 CsA 1 0 0 1 1 3 - ABMR20 CsA 2 1 0 2 1 3 - Ia TCMR and ABMR

NA: Not Assessed due to diffuse kidney necrosis

Table 1. oATP attenuated TCMR and ABMR

Day 0 1 2 3 4 5 6 7

EuthanizeA

no treatment

oATP 3mg/kg/day

CsA 10mg/kg/day

or

or

Allogeneic Allo + CsA Allo + oATP

Mg/

dL

C

* * * *P<0.05 P<0.05 P<0.05 P<0.05

Allogeneic Allo + CsA Allo + oATP

Figure 1. oATP treatment associated with improved graft function and survival

Serum creatinine BUN

Allo + CsA

Allo + oATP

Allogeneic

vi.

bleeding

ptc

ptc

gt

Diffuse C4d

ptc

minimal C4d

Diffuse C4d

Figure 2. oATP attenuated TCMR and ABMRC

4d

H&E

a

h

d

g

cb

fe

A. Allogeneic

necrotic area non-necrotic area

B. Allogeneic + CsA C. Allogeneic + oATP

Figure 3. oATP therapy was associated with attenuated Banff scores

A. Splenocyte Proliferation B. Macrophage Proliferation

oATP - - 50µMLew* + - -BN* - + + Lew + + +

Figure 4. oATP inhibited splenocyte and macrophage proliferation in vitro

Spleen BM LN PBMC

Cel

ls p

er 1

x105

lym

phoc

ytes

Spleen BM LN PBMCSpleen BM LN PBMC

Spleen BM LN PBMCSpleen BM LN PBMCSpleen BM LN PBMC

A. Total B Cell (CD45R+) B. Transitional B cell (CD45R+CD24+CD38+) C. Activated B cells (CD45R+CD27+)

D. T Cells (CD3+CD4+) E. CD3+CD4+iCOS+ F. CD3+CD4+CCR6+

**

*

*p<0.05

**

*p<0.05

*p<0.05

*p<0.05 *p<0.05

*

*

** *

*

AlloAllo + oATP

AlloAllo + oATP

AlloAllo + oATP

AlloAllo + oATP

AlloAllo + oATP

AlloAllo + oATP

Cel

ls p

er 1

x105

lym

phoc

ytes

Figure 5. oATP treatment associated with reduced B and T cell activation in vivo

Figure 6. oATP treatment was associated with reduced DSAM

ean

Fluo

resc

ence

Inte

nsity

(MFI

)

Control Allo Allo + oATP

Mea

n Fl

uore

scen

ce In

tens

ity (M

FI)

Mea

n Fl

uore

scen

ce In

tens

ity (M

FI)

C. IgG2c

P<0.04

P<0.0001

Control Allo Allo + oATP Control Allo Allo + oATP

P<0.04

A. IgM B. IgG2b

Figure 7. oATP significantly reduced intragraft IL-6 gene expression

Figure 8. oATP inhibited macrophage P2X7R in Lewis rats

A. Agonist BzATP opened P2X7R pores B. Both oATP and A740003 inhibited the effect of BzATP

C. The inhibitory effect of oATP was dose dependent

Inhibitor (oATP or A749993) concentration (uM)