Anti-donor MHC Class II Alloantibody Induces Glomerular Injury in Mouse Renal Allografts Subjected to Prolonged Cold Ischemia

Significance Statement

Prolonged cold-ischemia storage of donor organs is a major risk factor for acute and chronic graft injury, but the immunologic mechanisms underlying deleterious effects of cold-ischemia time and subsequent ischemia-reperfusion injury are poorly defined. In a mouse model of allogeneic kidney transplantation, the authors found that subjecting donor kidneys to prolonged cold ischemia enhanced early humoral and cellular anti-donor immune responses. In particular, recipients generated donor-specific antibodies against MHC class II (but not class I) antigens and exhibited extensive transplant glomerulopathy. The results indicate that antibodies against donor MHC class II antigens are critical mediators of glomerular injury caused by prolonged cold ischemia. Establishing mechanistic links between cold-ischemia storage and late transplant tissue injury may guide future therapies to ameliorate manifestations of chronic kidney allograft rejection.

Visual Abstract

Abstract

Background

The mechanisms underlying the effects of prolonged cold-ischemia storage on kidney allografts are poorly understood.

Methods

To investigate effects of cold ischemia on donor-reactive immune responses and graft pathology, we used a mouse kidney transplantation model that subjected MHC-mismatched BALB/c kidney allografts to cold-ischemia storage for 0.5 or 6 hours before transplant into C57BL/6 mice.

Results

At day 14 post-transplant, recipients of allografts subjected to 6 versus 0.5 hours of cold-ischemia storage had increased levels of anti–MHC class II (but not class I) donor-specific antibodies, increased donor-reactive T cells, and a significantly higher proportion of transplant glomeruli infiltrated with macrophages. By day 60 post-transplant, allografts with a 6 hour cold-ischemia time developed extensive glomerular injury compared with moderate pathology in allografts with 0.5 hour of cold-ischemia time. Pathology was associated with increased serum levels of anti–class 2 but not anti–class 1 donor-specific antibodies. Recipient B cell depletion abrogated early macrophage recruitment, suggesting augmented donor-specific antibodies, rather than T cells, increase glomerular pathology after prolonged cold ischemia. Lymphocyte sequestration with sphingosine-1-phosphate receptor 1 antagonist FTY720 specifically inhibited anti–MHC class II antibody production and abrogated macrophage infiltration into glomeruli. Adoptive transfer of sera containing anti-donor MHC class II antibodies or mAbs against donor MHC class II restored early glomerular macrophage infiltration in FTY720-treated recipients.

Conclusions

Post-transplant inflammation augments generation of donor-specific antibodies against MHC class II antigens. Resulting MHC class II–reactive donor-specific antibodies are essential mediators of kidney allograft glomerular injury caused by prolonged cold ischemia.

Prolonged cold-ischemia storage (CIS) of donor organs is detrimental to the immediate graft function and long-term transplant survival.^1–10 Recent analysis of a large cohort of recipients of renal transplants demonstrated that each additional hour of cold-ischemia time significantly increases the risk of graft failure and mortality.¹¹ Hypoxia and nutrient deprivation during ischemia lead to vasoconstriction, small vessel occlusion, local edema, activation of the coagulation system, and cell death.¹² After the restoration of blood supply, the injury is further exacerbated by the generation of reactive oxygen species, release of danger-associated molecular patterns by necrotic cells, upregulation of proinflammatory cytokines and adhesion molecules, and leukocyte infiltration.¹³

Innate and adaptive immunity are recognized as important players in ischemia-reperfusion injury (IRI). All innate immune cells (including neutrophils, macrophages, dendritic cells, natural killer cells, and platelets) as well as the complement system, promote IRI-induced inflammation.¹³ The importance of T lymphocytes, in particular the CD4⁺ subset, has been demonstrated in several animal models of IRI.^14–17 The effect of B cells is more controversial and is often related to the presence of natural antibodies against neoantigens exposed during ischemia.^16,18–20

Beyond initial tissue injury, IRI promotes ensuing adaptive immune responses by increasing the maturation of antigen-presenting cells (APCs), the production of proinflammatory cytokines and chemokines, and upregulating MHC and costimulatory molecule expression on endothelial and epithelial cells.^14,21–26 IRI also generates endogenous ligands for pathogen recognition receptors that enhance APC function and T and B lymphocyte activation.²⁷ This may have critical consequences for the activation of donor-reactive immune responses and allograft tissue injury. However, the influence of IRI on the magnitude and duration of alloimmune responses has not been investigated in detail.

The goal of our study was to assess the effects of prolonged CIS on anti-donor immune responses and graft outcome in a mouse model of fully MHC-mismatched kidney transplantation. We found that extending donor kidney CIS time from 0.5 to 6 hours significantly elevated the generation of donor-specific antibody (DSA) against MHC class II (but not class I) and donor-reactive, IFNγ-producing T cells. Compared with 0.5-hour CIS controls, 6-hour CIS allografts had a significantly higher proportion of glomeruli infiltrated with macrophages by day 14 post-transplant and revealed extensive glomerular injury at 60 days post-transplant. Recipient B cell depletion prevented early macrophage glomerular recruitment, indicating a key role of DSA in this process. Notably, lymphocyte sequestration with sphingosine-1-phosphate receptor 1 antagonist FTY720 specifically inhibited anti–MHC class II but not class I production and abrogated macrophage infiltration into glomeruli. Furthermore, adoptive transfer of anti-donor MHC class II–containing sera or mAb restored early glomerular macrophage infiltration in FTY720-treated recipients but had little effect on the development of interstitial fibrosis. These results indicate that, whereas IRI augments both humoral and cellular alloimmunity, MHC class II–reactive DSA is the essential mediator of transplant glomerular injury after prolonged CIS.

Methods

Animals

Male C57BL/6J (B6, H-2^b), BALB/c (H-2^d), and SJL/J-Pde6b^rd1 (SJL, H2^s) mice, aged 6–8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal procedures were approved by the Institutional Animal Care and Use Committee at Cleveland Clinic.

Kidney Transplantation

Murine kidney transplantation was performed as previously described.²⁸ Briefly, the donor kidney with vascular supply and ureter were harvested en bloc and the donor artery and vein were anastomosed to the recipient abdominal aorta and inferior vena cava. The collected kidneys were perfused with University of Wisconsin (UW) solution (320 mOsm; Preservation Solutions, Elkhorn, WI) and stored on ice for either 0.5 or 6 hours before transplantation. The remaining native kidney was removed at the time of transplant so that recipient survival was dependent on the kidney graft. Kidney graft survival was assessed by daily examination of overall animal health and measurement of BUN levels using the BUN Colorimetric Detection Kit (Thermo Fisher Scientific, Waltham, MA). No immunosuppression was administered to the recipients.

Recipient Treatment

To deplete B cells, recipients were treated with anti-mouse CD20 mAb (mouse IgG2a, clone 18B12; Biogen Idec, San Diego, CA) with 250 µg intravenously on day −3 and 100 µg intraperitoneally on day 8 post-transplant. FTY720 was administered at 3 µg/ml in drinking water throughout the duration of the experiment starting 3 days before transplantation. For the generation of anti-donor serum, BALB/c kidney allografts were subjected to 6 hours of CIS and transplanted into B6 recipients. Recipients were euthanized on day 14 post-transplant and sera were collected, analyzed for the presence of MHC class I and II reactive DSA by ELISA, pooled, and stored at −20°C. A volume of 200 µl or resulting serum was injected intravenously into a new group of B6 recipients of 6-hour CIS BALB/c allografts treated with FTY720 on days 3, 7, and 11 post-transplant. For adoptive transfer of mAb, B6 recipients of 6-hour CIS BALB/c kidney allografts were treated with FTY720 and intravenously injected with 100 µg of either control mouse IgG2a (clone C1.18.4; BioXCell, West Lebanon, NH), anti-K^d (clone 34-5-3; SouthernBiotech, Birmingham, AL), or anti–I-A^d (clone SF1.1.10; BioXCell) on days 3, 7, and 11 post-transplant.

Histologic Graft Evaluation

Harvested grafts were embedded in paraffin and stained as previously described.²⁸ Stained sections were scored in a blinded fashion. The numbers of glomeruli containing zero to five, five to ten, or more than ten Mac-2⁺ cells were counted in ten fields per graft section at 100× magnification. Glomerular pathology was evaluated on complete cross-sections of kidney allografts by capturing four to six digital images at 100× magnification. An average of 25 glomeruli were scored from each allograft without knowledge of the treatment group. The extent of capillary injury was graded on a scale of zero to three for each glomerulus. The number of glomeruli with lobular and total sclerosis was also counted in these images. Periarterial, periglomerular, glomerular, and interstitial T cell infiltrates were quantified in serial sections stained for CD4 and CD8. Each compartment was scored as illustrated in Supplemental Figure 1. The scoring was performed by a pathologist (W.M.B.) who had no knowledge of experimental groups.

IFNγ Enzyme-Linked Immunospot Assay

The frequencies of IFNγ-secreting spleen cells were measured as previously described using capture and detecting anti-mouse IFNγ antibody from BD Pharmingen.^29–31 Recipient spleen cells were stimulated with mitomycin C–treated donor BALB/c or third party SJL spleen cells for 24 hours.

IgG Enzyme-Linked Immunospot Assay

The frequencies of antibody-secreting cells (ASC) producing IgG against donor MHC class I or II molecules were determined using ELISpot^PLUS kit for mouse IgG (MABTECH AB, Nacka Strand, Sweden) as previously published.^32,33

Evaluation of Serum Alloantibody against Donor MHC Class I and Class II Molecules

Recipient serum samples were collected by tail-vein bleeding at 7, 14, and 60 days after transplantation and stored at −20°C. Flat-bottomed 96-well Nunc plates (Thermo Fisher Scientific) were coated overnight at 4°C with biotinylated donor class 1 D^d (folded with RGPGRAFVTI peptide), donor class 2 I-A^d (folded with PVSKMRMATPLLMQA class 2–associated invariant chain peptide), control D^b (folded with HGIRNASFI peptide), or I-A^k (folded with PVSKMRMATPLLMQA class 2–associated invariant chain peptide) MHC monomers at 1 µg/ml in PBS. The peptide/MHC monomers were provided by the National Institutes of Health (NIH) Tetramer Core Facility at Emory University (Atlanta, GA). Plates were washed with PBS and blocked with 1% BSA in PBS solution for 1 hour at room temperature. A total of 100 µl of serum samples diluted 1:500 were added to the plates and incubated overnight at 4°C. After washes with PBS and PBS-0.25% Tween, plates were incubated with goat anti-mouse IgG–horse radish peroxidase (HRP) conjugate (1:20,000 dilution; Thermo Fisher Scientific) for 1 hour at room temperature, followed by washes and development with ABTS Peroxidase Substrate (KPL, Gaithersburgh, MD). The absorbance at 415 nm was measured using the Bio-Rad iMark Microplate Reader (Bio-Rad Laboratories, Hercules, CA). For IgG isotype evaluation, 1:100 dilutions of pooled sera samples were tested by ELISA as outlined above, with goat anti-mouse IgG₁-HRP, goat anti-mouse IgG_2a-HRP, and goat anti-mouse IgG_2b-HRP conjugates used as detecting antibody at 1:5000 dilutions (all from SouthernBiotech).

Statistical Analysis

The data were analyzed using a nonparametric equivalent of one-way ANOVA, the Kruskal–Wallis test. When the overall P value was <0.05, pairwise comparisons were carried out using the Mann–Whitney test. A value of P<0.05 was considered statistically significant.

Results

Prolonged CIS of Renal Allografts Augments Both Humoral and Cellular Anti-donor Immune Responses

To evaluate the effects of prolonged CIS on alloimmunity, we used a fully MHC-mismatched model of mouse renal transplantation. BALB/c (H-2^d) kidneys were harvested and stored on ice in UW solution for either 0.5 or 6 hours followed by transplantation into B6 (H-2^b) recipients (0.5- and 6-hour CIS groups, respectively). The remaining native kidney was removed at the time of transplant so that recipient survival was dependent on the kidney graft, and no immunosuppression was administered. By day 14 after transplantation, 6-hour CIS recipients had significantly higher serum levels of IgG Ab against donor MHC class II molecule I-A^d (reciprocal serum titers of 200 versus 150 in 6- versus 0.5-hour CIS recipients, respectively) whereas Ab against donor class 1 molecule D^d were comparable in both groups with reciprocal serum titers of 200 (Figure 1, A and B, and not shown). Both anti-donor class 1 and class 2 DSA were predominantly of IgG₁ and IgG_2b isotypes, with minimal levels of IgG_2c (Supplemental Figure 2). Transplantation of B6 isografts subjected to 6-hour CIS did not induce detectable H-2^d–reactive DSA (Figure 1, A and B). The numbers of spleen cells secreting anti-donor MHC class I and class II antibodies were elevated in some allograft recipients (Figure 1C). Prolonged donor allograft CIS also significantly enhanced priming of donor-reactive IFNγ T cells in the spleen (Figure 1D).

Figure 1.

Prolonged CIS of renal allografts augments humoral and cellular anti-donor immune responses. B6 mice were transplanted with BALB/c renal allografts subjected to 0.5 or 6 hours of CIS in UW solution. Control B6 recipients were transplanted with 6-hour CIS B6 isografts. (A) Serum IgG antibody against donor MHC class I and (B) MHC class II were measured by ELISA at days 7, 14, and 60 post-transplant. The levels of antibodies against self or third party MHC molecules were ≤0.1 OD415 units for all groups. (C) The frequencies of cells secreting antibodies against donor MHC class I or class II and (D) the frequencies of donor-reactive IFNγ-producing T cells were determined in the spleen by enzyme-linked immunospot assays at day 14 and 60 post-transplant. For IFNγ enzyme-linked immunospot, recipient spleen cells were restimulated with donor BALB/c or third party SJL splenocytes. The frequency of cells secreting IFNγ in response to SJL stimulator cells was <50 per 1×10⁶ spleen cells in all groups. Graph symbols represent individual animals. n=9 per group for naive mice, 4 per group for isograft recipients, and 20–25 per group for allograft recipients.

Prolonged CIS Increases Early Glomerular Macrophage Infiltration and Exacerbates Renal Allograft Pathology at Day 60 Post-transplant

Prior to transplantation, 6-hour CIS grafts did not reveal significant tissue injury when compared with native kidney, with largely intact tubular brush border, minimal edema, and no epithelial or endothelial cells positive for caspase-3 staining (data not shown). At day 14 post-transplant, kidney allografts in both groups were heavily infiltrated with mononuclear cells including CD3⁺ lymphocytes and macrophages (Figure 2, A and B). Consistent with the presence of serum IgG DSA, grafts in both groups had prominent vascular and glomerular deposition of C4d (Figure 2A). Periodic acid–Schiff stains demonstrated focal expansion of the mesangial matrix with interposition of mesangium into the glomerular capillary walls, generating duplication of the basement membrane consistent with the possibility of antibody-mediated injury by 14 days (Figure 2D). However, these findings were not limited to the 6-hour CIS group and were also present in some grafts from the control group. Such an early manifestation of glomerular basement membrane duplication compared with human transplant recipients could be due to the absence of ongoing immunosuppression. Notably, distribution of Mac-2⁺ cells within the graft tissue was altered by prolonged CIS. In particular, allografts from the 6-hour CIS group had a significantly higher proportion of glomeruli infiltrated with macrophages compared with 0.5-hour CIS controls (Figure 2C). Despite intense T cell infiltration, histologic analysis of CD4 and CD8 staining revealed only modest tubulitis that was comparable in 0.5- and 6-hour CIS allografts (Supplemental Figure 3, Supplemental Table 1). Importantly, isografts subjected to 6 hours of CIS revealed minimal signs of tubular atrophy, tubulitis, cellular infiltrate, or complement deposition (Supplemental Figure 4), underscoring that these pathologic features depend on alloimmune responses.

Figure 2.

Prolonged CIS increases early glomerular macrophage infiltration. (A) Hematoxylin and eosin and immunoperoxidase staining for CD3 and C4d performed on day 14 post-transplant. The photographs are representative of six to eight animals in each group. (B) Immunoperoxidase staining for Mac-2 on day 14 post-transplant. (C) The numbers of glomeruli containing zero to five, five to ten, or more than ten Mac-2⁺ cells were counted in ten fields per graft section at 200× magnification and presented as percentage of total glomeruli in the section. Graph symbols represent individual grafts. n=7–8 allografts per group. (D) Periodic acid–Schiff (PAS) staining on day 14 post-transplant. n=12–19 allografts or 5 isografts per group.

Regardless of allograft CIS duration, all recipients survived for >60 days post-transplant. Histologic evaluation at this time point revealed the 0.5-hour CIS grafts had moderate segmental sclerosis in a limited number of glomeruli. In contrast, 6-hour CIS grafts had extensive glomerular injury including thickened capillary loops, segmental or global sclerosis, and thrombotic microangiopathy with red cell congestion. The most prominent glomerular pathology was mesangial cell lysis with aneurysmal capillary ballooning and intracapillary fibrinogen deposition (Figure 3, A–D). The proportion of glomeruli with aneurysms or sclerosis was increased in the 6- versus 0.5-hour CIS group (Figure 3E). Consistent with exacerbated manifestations of renal transplant injury, BUN levels were elevated in 6-hour CIS allograft recipients compared with 6-hour CIS isograft or 0.5-hour CIS allograft recipients (Figure 3F).

Figure 3.

Prolonged CIS exacerbates renal pathology at day 60 post-transplant. (A) Trichrome staining of 0.5-hour CIS and 6-hour CIS allografts. The images are representative of at least ten grafts per group. Original magnification, ×100. (B) Representative trichrome stains demonstrating severe glomerular pathology of 6-hour CIS allografts at 60 days after transplantation. (B–D) Varying degrees of glomerular sclerosis. (B) Severe mesangiolysis in the glomerulus on the right. (C) Large capillary aneurysm with fibrin deposition. Original magnification, ×400. (E) Proportion of glomeruli showing evidence of aneurysm (left), sclerosis (middle), and aneurysm or sclerosis (right) in 0.5- versus 6-hour CIS allografts, calculated as described in the Methods section. (F) BUN levels were measured at day 60 post-transplant by ELISA.

Chronic Glomerular Injury Induced by Prolonged CIS Is Associated with Increased DSA against Donor MHC Class II Molecules

To begin addressing immunologic mechanisms underlying exacerbated pathology of renal allografts after 6 hours of CIS, we evaluated the kinetics of DSA development in allograft recipients. Consistent with our findings at day 14 post-transplant, prolonged allograft CIS did not alter levels of donor class 1 (D^d) reactive DSA either early (day 7) or late (day 60) after transplantation (Figure 1A). In contrast, 6-hour CIS recipients had increased serum levels of anti-donor class 2 (I-A^d), both at day 14 and at day 60 post-transplant (Figure 1B). Despite the disparate CIS effects on class 1 versus class 2 serum DSA, by day 60 post-transplant, recipients of 6-hour CIS renal allografts had significantly elevated numbers of spleen cells secreting DSA against both MHC class I and class II molecules (Figure 1C). Regardless of CIS time, the numbers of IFNγ-producing donor-reactive T cells in the spleen were decreased to the levels observed in naive, nontransplanted mice (Figure 1D). These results suggested DSA rather than T cells are responsible for the exacerbated glomerular pathology in 6-hour CIS allografts.

B Cell Depletion Inhibits Early Macrophage Infiltration into Allograft Glomeruli

To test the role of DSA in early macrophage infiltration into kidney transplant glomeruli, we depleted recipient B cells before transplantation. Treatment with anti-mouse CD20 mAb markedly reduced the numbers of B220⁺ cells in peripheral blood and the spleen, while sparing B220^loCD138⁺ plasma cells (Figure 4B, Supplemental Figure 5). As anticipated, B cell depletion inhibited MHC class I– and class II–reactive DSA generation (Figure 4C). Notably, 6-hour CIS recipients depleted of B cells had reduced intragraft C4d deposition and macrophage glomerular infiltration at day 14 post-transplant (Figure 4, D and E), consistent with the hypothesis that the initial glomerular injury after prolonged CIS is alloantibody mediated. Anti-CD20 mAb treatment decreased the frequency of donor-reactive T cells, suggesting B cells act as major APCs in this model. In addition, recipients depleted of B cells had diminished T cell infiltration into the graft (Supplemental Figure 5). Therefore, the findings in B cell–depleted recipients did not entirely rule out the possibility that T cells mediate early macrophage recruitment into glomeruli of 6-hour CIS allografts.

Figure 4.

B cell depletion inhibits DSA development, intragraft complement deposition, and macrophage glomerular infiltration in recipients of 6-hour CIS renal allografts. (A) Experimental design. (B) Percentage of B220⁺ cells among live cells in peripheral blood. (C) Alloantibody reactive to donor MHC class I (D^d) and class II (I-A^d) molecules detected in recipient sera at day 14 by ELISA. Symbols represent individual animals. (D) Hematoxylin and eosin (H&E), C4d, and Mac-2 graft staining performed on day 14 post-transplant. The photographs are representative of seven to eight animals per group. (E) The numbers of glomeruli containing zero to five, five to ten, or more than ten Mac-2⁺ cells were counted in ten fields per graft section at 200× magnification and presented as percentage of total glomeruli in the section. Graph symbols represent individual grafts. n=6–8 allografts per group. i.p., intraperitoneal; i.v., intravenous.

MHC Class II–Reactive DSA Induce Glomerular Macrophage Infiltration in 6-Hour CIS Kidney Allografts

To test the role of donor-reactive T cells in glomerular injury, we continually treated 6-hour CIS recipients with the sphingosine-1-phosphate receptor 1 agonist, FTY720.^30,34,35 As anticipated, the treatment severely reduced numbers of circulating T and B lymphocytes (Supplemental Figure 6A). The numbers of spleen CD4⁺ and CD8⁺ T cells were diminished by FTY720, whereas spleen B cell populations remained largely unaffected (Supplemental Figure 6B). FTY720 treatment markedly inhibited priming of donor-reactive T cells in the spleen, consistent with previously reported effects of this reagent on donor APCs (Figure 5B).³⁶

Figure 5.

FTY720 inhibits T cell priming and donor MHC class II–reactive DSA in recipients of 6-hour CIS renal allografts. (A) Experimental design. B6 mice were transplanted with BALB/c kidney allografts subjected to 6 hours of CIS. Recipients were treated with FTY720 (3 µg/ml in drinking water) throughout the duration of the experiment starting 3 days before transplantation. (B) The frequencies of donor-reactive IFNγ-producing T cells were determined by enzyme-linked immunospot assay on day 14 post-transplant. (C) DSA against MHC class I (D^d) and class II (I-A^d) molecules were detected by ELISA on day 14 post-transplant. Symbols represent individual recipients.

Despite lymphocyte sequestration and decreased activation of donor-reactive IFNγ-producing T cells, FTY720 treatment did not alter the production of anti-donor MHC class I IgG antibodies compared with control 6-hour CIS recipients. In contrast, the levels of MHC class II–reactive DSA were markedly reduced in FTY720-treated recipients (Figure 5C). Despite the presence of anti–class 1 DSA, 6-hour CIS renal allografts from FTY720-treated recipients had variable vascular complement deposition and no macrophage infiltrate within glomeruli at day 14 post-transplant (Figure 6, A–C). To confirm the pathogenic role for anti–class 2 DSA, FTY720-treated recipients were injected with sera from untreated 6-hour CIS recipients with high levels of MHC class II–reactive DSA (Figure 6B). Sera transfer partially restored complement deposition and macrophage glomeruli infiltrate in the absence of T cell activation (Figure 6, A–C). To specifically test the role of class 1 versus class 2 DSA in this model, FTY720-treated recipients were intravenously injected with mAb against donor MHC class II molecule (I-A^d) or anti-donor MHC class I (K^d) (Figure 6B). In the absence of mAb transfer, such recipients develop endogenous anti–class 1 but not anti–class 2 DSA at levels observed in nontreated recipients and have variable C4d staining intensity (Figure 5C). Whereas mAb transfer including the isotype control elevated the intensity of peritubular C4d deposition, the changes in C4d deposition scores were not statistically significant (Supplemental Figure 7, A–C). T cell graft infiltrates were moderate and comparable between the groups (Supplemental Table 2). Importantly, however, the transfer of donor class 2–reactive, but not class 1–reactive or isotype control, mAb restored macrophage infiltration within glomeruli (Figure 6C). In addition, the C4d staining of glomeruli in recipients transferred with anti–class 2 mAb had a patchy pattern compared with uniform C4d deposition in anti–class 1 mAb–injected recipients, suggesting disruption of normal capillary structure (Supplemental Figure 7C). Taken together, these results indicate that augmented MHC class II–reactive DSA mediate macrophage recruitment into glomeruli of renal allografts subjected to prolonged CIS.

Figure 6.

MHC class II–reactive DSA induce glomerular macrophage infiltration in 6-hour CIS kidney allografts. (A) Histologic evaluation of BALB/c renal allografts subjected to 6 hours of CIS. Recipients were either untreated (top), treated with FTY720 on days −3 to 14 relative to transplantation (middle), or treated with FTY720 and injected with anti-donor sera collected from B6 recipients of 6-hour CIS BALB/c kidney allografts at day 14 post-transplant (bottom, see also panel 6B). Hematoxylin and eosin (H&E) and immunoperoxidase staining for CD3 C4d and Mac-2 was performed on day 14 post-transplant. The photographs are representative of four to eight animals in each group. (B) Experimental design. B6 recipients of BALB/c kidney allografts subjected to 6 hours of CIS were treated with FTY720 on days −3 to 14 relative to transplantation and additionally injected with anti-donor sera collected from B6 recipients of 6-hour CIS BALB/c kidney allografts at day 14 post-transplant, control mouse IgG2a (mIgG), anti-K^d (aK^d), or anti–I-A^d (aI-A^d) on days 3, 7, and 11 after transplantation. (C) The numbers of glomeruli containing zero to five, five to ten, or more than ten Mac-2⁺ cells were counted in ten fields per graft section at 200× magnification. Graph symbols represent individual grafts. n=4–8 allografts for all groups except IgG2a (two per group). NT, not treated.

Discussion

Although the average CIS time for clinical cadaveric donor renal grafts is about 18 hours,³⁷ most animal transplantation studies use minimal graft processing time (30–45 minutes) and thus minimize the effects of post-transplant inflammation in alloimmune responses. Using a mouse kidney transplantation model, we provide novel insights into the effects of prolonged donor organ CIS on cellular and humoral anti-donor immunity and graft outcome.

We found that both effector T cell priming and DSA generation were increased in response to 6- versus 0.5-hour CIS allografts early after transplantation (Figure 1). However, the effects of CIS time on long-lasting immunity were distinct for T and B cell responses. Donor-reactive T cell responses at day 60 post-transplant were low and comparable in both groups (Figure 1D), indicating that prolonged CIS did not sustain the development of T cell memory. In contrast, the increased serum DSA levels and higher frequencies of donor-reactive ASC were observed in the 6-hour CIS group throughout the duration of the study (Figure 1, A–C).

The changes in anti-donor immunity caused by prolonged CIS had little effect on tubular injury (Supplemental Figure 3, Supplemental Table 1) but increased early macrophage accumulation within transplant glomeruli. By day 60 after transplantation, recipients of 6-hour CIS allografts developed various manifestations of chronic glomerular injury and had elevated BUN levels (Figure 3). Although observed glomerular aneurysms representing severe end stage microangiopathy are not typically present in human transplant patients due to ongoing immunosuppression and careful monitoring of renal function, these results nevertheless suggest that glomerular macrophage infiltration in our model translates into chronic injury. The role of graft-infiltrating macrophages in tissue injury and repair is a subject of active investigation.³⁸ Clinical studies showed that accumulation of intragraft macrophages is associated with increased risk of chronic rejection and worse transplant outcomes.^39,40 Furthermore, early glomerulitis has been strongly associated with the later development of transplant glomerulopathy in a study by Bagnasco et al.⁴¹ Using a mouse model of heterotopic heart transplantation, we recently reported that macrophage infiltration within different graft compartments may determine acute versus chronic tissue injury.⁴² The factors driving macrophage recruitment into glomeruli as well as the effector molecules mediating ensuing glomerulopathy (such as proinflammatory cytokines) in our model remain to be determined. In addition, the mechanisms underlying enhanced humoral alloimmunity after 6-hour CIS donor organ storage, such as increased antigen release and acute response cytokines, are the focus of ongoing investigations in our group.

Prolonged CIS and subsequent IRI can influence both innate and adaptive immune responses.^13–20 Importantly, isografts subjected to 6 hours of CIS revealed little apparent tissue injury, indicating adaptive alloimmune responses are the chief cause of the observed pathology (Supplemental Figure 4). As both cellular and humoral alloimmunity were affected by prolonged CIS, either graft-infiltrating T cells or DSA could mediate early macrophage infiltration within glomeruli. The numbers of IFNγ-secreting, donor-reactive T cells were increased in the 6-hour CIS group, and CD3⁺ cells were prominent in graft infiltrates at day 14 post-transplant. Pretransplant B cell depletion completely abrogated both anti–MHC class I and class II DSA generation and early glomerular macrophage accumulation, suggesting the critical role of DSA in ensuing graft tissue injury. However, T cell priming was also affected by B cell depletion and the contribution of T cells to the observed glomerular pathology could not be entirely ruled out by this approach. Interestingly, B cell depletion had no significant effect on T cell responses in recipients of 0.5-hour CIS allografts, suggesting enhanced post-transplant inflammation enables B cells to function as essential APCs.

We initially used FTY720 to test the role of T lymphocytes in early macrophage recruitment. Unexpectedly, we observed distinct effects of lymphocyte sequestration on the production of anti-donor MHC class II versus class I antibodies. One explanation for these findings is that, due to the lower antigen availability, class 2–reactive B cells are more dependent on T cell helper signals that are limited by FTY720 treatment. Even more likely, FTY720 inhibits T cell activation and recruitment into the graft thus inhibiting donor MHC class II upregulation by T cell–derived cytokines. The mechanisms linking initial IRI and specific elevation of anti–MHC class II DSA are being further investigated in our laboratory. Regardless of these mechanisms, adoptive transfer of anti-donor MHC class II antibody made possible glomerular macrophage accumulation in FTY720-treated recipients. These data indicate that, after high levels of anti–class 2 DSA are generated, they are sufficient to induce glomerular injury independent of T cells.

Transplantation of 6-hour CIS renal allografts significantly increased the frequencies of plasma cells secreting antibodies against both donor MHC class I and class II molecules. At the same time, we observed elevated serum levels of donor MHC class II– but not class I–reactive DSA (Figures 1 and 5). This discrepancy could be due to the deposition of MHC class I–reactive DSA within the graft subjected to 6 hours of CIS. Alternatively, MHC class II–reactive antibody may have higher affinity for target antigens. Regardless of the mechanisms, prolonged CIS favors the generation of donor MHC class II–reactive DSA that in turn can mediate chronic transplant glomerulopathy. Studies are ongoing in our laboratory to directly address the role of MHC class II–reactive DSA in the chronic glomerular injury caused by prolonged CIS.

The relevance of our findings in the mouse model to clinical transplantation is underscored by recent studies revealing correlations between anti–class 2 DSA and late antibody-mediated rejection in patients who received renal transplants.^43–47 For example, studies of unsensitized recipients of renal transplants demonstrated association between longer cold ischemic time and higher risk of de novo DSA which in turn correlated with significantly reduced graft survival at 10 years post-transplant. Notably, donor class 2 DSA were the predominant type in recipients with de novo alloantibody (96% recipients developed class 2 DSA, and 68% had only class 2 DSA).⁴⁷ In another large-scale study, the presence of class 2 DSA was strongly associated with early transplant glomerulopathy that translated into increased graft failure by 5 years post-transplant.⁴³

The experimental model of mouse kidney transplantation is a powerful tool to study alloimmune responses against fully vascularized and functional organ transplants. That said, there is a great variability in mouse renal transplant recipient survival reported by different groups depending on donor/recipient strain combinations, strain genetic drifts, details of the surgical procedure (such as the time of second native kidney removal), the duration of warm-ischemia time, and the potential exposure to environmental immune stimuli.^48–53 Importantly, our model allows studying relationship between IRI, alloimmune responses, and late transplant tissue injury in the absence of acute allograft rejection and confounding effects of immunosuppression. The results indicate that post-transplant inflammation not only increases the magnitude of anti-donor adaptive immune responses, but specifically enhances the development of anti–MHC class II DSA that in turn mediate renal allograft pathology. This information may guide future therapies to ameliorate manifestations of chronic allograft rejection in recipients of renal transplants from deceased donors.

Disclosures

Dr. Baldwin reports other support from Mallinckrodt Pharmaceuticals and from University of Washington, outside the submitted work.

Funding

This work was supported by a NIH grant 1P01 AI087586 (Dr. Valujskikh, Dr. Baldwin, and Dr. Fairchild).

Supplemental Material

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018111169/-/DCSupplemental.

Supplemental Table 1. Quantification of T cell infiltrate in 0.5 hour versus 6 hour CIS allografts on day 14 posttransplant.

Supplemental Table 2. Quantification of T cell graft infiltrate after adoptive transfer of donor class I- versus class II-reactive monoclonal antibodies.

Supplemental Figure 1. Examples of CD4 and CD8 graft infiltrates.

Supplemental Figure 2. Prolonged CIS of renal allografts augments generation of anti-class II DSA.

Supplemental Figure 3. Scores of tubular T cell infiltrate at day 14 posttransplant.

Supplemental Figure 4. Histological evaluation of isografts subjected to 6 hour CIS.

Supplemental Figure 5. Effect of anti-mCD20 mAb treatment on spleen B cell subsets and anti-donor T cell responses.

Supplemental Figure 6. The effect of FTY720 treatment on peripheral lymphocyte subsets.

Supplemental Figure 7. Histological evaluation of Collagen IV and C4d deposition after mAb transfer.