Enhanced De Novo Alloantibody and Antibody-Mediated Injury in Rhesus Macaques

Animal selection and transplantation

Male rhesus macaques were obtained from Alpha Genesis (Yemassee, SC, USA) and Yerkes National Primate Research Center Field Station (Lawrenceville, GA, USA). All animals weighed 4–7 kg, were tested specific pathogen free, and expressed the FN18 epitope, the immunotoxin binding site. Donor–recipient pairs were selected based on avoidance of MHC class I matches (6 alleles tested), and class II maximal mismatch among available animals. Donor reactivity was confirmed in vitro with carboxyfluorescein succinimidyl ester-labeled (CFSE, Molecular Probes, Eugene, OR, USA) mixed lymphocyte reaction (MLR). Each animal underwent a donor nephrectomy followed by recipient transplantation, with at least 3 weeks between the two operations. This domino approach was used for all animals in the study. All medications and procedures were approved by the Emory University Institutional Animal Care and Use Committee, and were conducted in accordance with Yerkes National Primate Research Center and the National Institutes of Health guidelines.

Immunosuppression agents

Six animals were designated as untreated controls. All animals given immunosuppression received anti-CD3 immunotoxin (A-dmDT390-scfbDb (C207), Massachusetts General Hospital, Dana Farber-Harvard Cancer Center Recombinant Protein Expression and Purification Core Facility, Boston, MA, USA). Four animals were given immunotoxin (IT) plus tacrolimus (Astellas Pharma US, Inc., Deerfield, IL, USA), and another four received IT, tacrolimus, and alefacept (LFA3-Ig, Astellas Pharma US, Inc). IT was administered at 0.025 mg/kg intravenously twice daily from postoperative day (POD) 0–3. To alleviate potential symptoms of cytokine storm, methylprednisolone 125 mg was injected intravenously on days the animals received IT. They also received methylprednisolone on days 4–6 when they were administered bromodeoxyuridine. Tacrolimus (Tac) was started at 0.05 mg/kg intramuscular injection twice daily on the day of transplantation, and titrated throughout the life of the allograft to maintain a trough of 8–12 ng/mL. Alefacept was administered at 0.3 mg/kg once weekly for 8 weeks, starting on POD 3, 0, then 7, 14 etc. Rescue immunosuppression with methylprednisolone 125 mg IV × 3 days was used when the animal exhibited signs of acute rejection.

Allograft and immune monitoring

Peripheral blood was obtained weekly by femoral venipuncture for allograft and immune monitoring. Three milliliter were designated for serum chemistries and preserving serum, 0.5 mL for complete blood count, 1 mL for immune surveillance by polychromatic flow cytometry and 0.5 mL for monitoring tacrolimus trough levels. For flow cytometric analysis, peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll method using 5 mL of lymphocyte separation medium (Mediatech, Inc, Manassas, VA, USA) per sample. Washed PBMCs were surfaced stained with the following antibodies: Alexa-Fluor 700 conjugated anti-CD3, PerCP-Cy5.5 conjugated anti-CD4, APC-Cy7 conjugated anti-CD8 (BD Pharmingen, San Diego, CA, USA), PE-Cy7 conjugated anti-CD28, eFluor450 conjugated anti-CD95 (Ebioscience, San Diego, CA, USA) and PE conjugated anti-CD25 (Myltenyi Biotec, Auburn, CA, USA). Cells were then processed to detect FoxP3 and Ki-67 using intracellular staining with a Fix/Perm solution (Ebioscience), Pacific Blue conjugated anti-FoxP3 (BioLegend, San Diego, CA, USA) and PE conjugated anti-Ki-67 (BD Pharmingen).

Upon necropsy, rejected renal allograft tissues were incubated for 30 min at 37°C in Clostridium histolyticum collagenase (C8051 Blend Type H, Sigma-Aldrich, St. Louis, MO, USA). Graft and lymph node tissues were crushed through a 100 μm strainer, washed and stained with the above antibodies.

Samples were collected with an LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software 9.2. (Tree Star, Ashland, OR, USA)

Detection of donor-specific antibodies

Alloantibody production was retrospectively assessed by flow cytometric crossmatch of donor PBMCs with serially collected recipient serum samples. Donor PBMCs were coated with ChromPure goat IgG (Jackson ImmunoResearch, West Grove, PA, USA) and incubated with recipient serum. Cells were stained with FITC-labeled antimonkey IgG (KPL, Inc. Gaithersburg, MD, USA), PE CD20 (BD Pharmingen) and PerCP CD3 (BD Pharmingen, San Diego, CA). The threshold for alloantibody positivity varies widely across the literature, ranging from a 10 to a 100 shift in mean fluorescence intensity for both CD3+ and CD20+ cells (15,40–43); thus, in this study, a twofold increase in mean fluorescence intensity from pretransplant values was used to determine alloantibody positivity, with all positive shifts being greater than 100 shift in mean fluorescence intensity.

Immunohistochemistry and electron microscopy

Renal allograft tissues were obtained at time of rejection and fixed in 10% neutral-buffered formalin and embedded in paraffin. Embedded tissue blocks were sliced into 5 μm-thick sections, deparaffinized in xylene, and rehydrated through graded ethanol to water for H&E and PAS stains as well as immunohistochemical analysis. For immunohistochemical assessment, the slides were subjected to heat-induced epitope recover in a 10 mM citrate buffer and then rinsed with Tris-buffered saline. The nonspecific sites were blocked by sequential treatment with 3% hydrogen peroxide, avidin-biotin blocking and protein blocking solutions. Tissue sections were labeled with antigen-specific primary antibodies CD3 (DAKO; dilution, 1:200) and C4d (ARP; dilution, 1:40) for T cell and C4d recognition and subsequently visualized using the Dako LSAB + labeled Streptavidin-Biotin kit (Dako). The positive staining were detected by 3,3-diaminobenzidine peroxidation and counterstained with hematoxylin. The slides stained with hematoxylin and eosin, PAS and antihuman CD3 and C4d antibodies were prepared by the Emory Transplant Center immunohistochemist. Formalin-preserved graft tissues were submitted to the Apkarian Integrated Electron Microscopy Core (Emory University, Atlanta, GA, USA). Renal pathologists reviewed in a blinded fashion all histology, immunohistochemistry and electron microscopy images.

In vitro assays

PBMCs from nine MHC-disparate macaque donor–recipient pairs were isolated and prepared into CFSE MLR. Each MLR was cocultured with three dosings of alefacept: 0 μg/mL, 100 μg/mL and 500 μg/mL; recipient lymphocytes without irradiated cell stimulation were used as controls. Upon incubation at 37°C, 5% CO2 for 5 days, the cells were recovered, washed and stained with the following antibodies: Alexa-Fluor 700 conjugated anti-CD3, PerCP-Cy5.5 conjugated anti-CD4, APC-Cy7 conjugated anti-CD8 (BD Pharmingen), PE-Cy7 conjugated anti-CD28, eFluor450 conjugated anti-CD95, PE conjugated anti-CD2 (Ebioscience, San Diego, CA, USA) and APC-Cy7 conjugated anti-CD20 (BioLegend).

Statistics

All statistical analyses were conducted using Statistical Package for the Social Sciences 19.0 (IBM SPSS, Chicago, IL, USA). Continuous variables were ranked and analyzed with the Mann–Whitney U or Kruskall–Wallis tests. Survival graphs were obtained with the Kaplan–Meier analysis. A p-value of less than 0.05 was considered significant.

IT induces profound peripheral depletion with minimal improvement in graft survival

Three male rhesus macaques treated with IT alone experienced renal allograft survival of 8, 10 and 13 days, which was marginally increased (p = 0.048) compared to controls without immunosuppression (Figure 1A). All three animals experienced maximal peripheral T cell depletion (99%+) by day 3 after transplantation (Figure 1B). Despite profound peripheral depletion, IT did not induce T cell depletion uniformly across lymphoid compartments, particularly in lymph nodes (Figure 1C). Consistent with that, in peripheral blood, a dramatic increase in central memory T cells (CD28+CD95+) was observed by day 7 in IT-treated animals (Figure 1D). Histological analysis of rejected grafts revealed acute cellular rejection.

The addition of tacrolimus and alefacept prolongs graft survival

In 2005, Pearl et al. described a relative sparing of CD4+ effector memory T cells in human kidney recipients receiving T cell depletion with alemtuzumab or thymoglobulin. Calcineurin inhibitors, especially tacrolimus, best inhibited the function of these CD4+ memory cells in vitro (44). We had also observed a significant decrease in acute rejection when adding tacrolimus to human kidney transplant recipients undergoing alemtuzumab-based depletion with sirolimus maintenance therapy. To target the rapidly repopulating CD4+ memory population in our immunotoxin-treated animals, we added tacrolimus as maintenance immunosuppression and observed a prolongation in graft survival (Figure 2A, p = 0.034). Three of four animals rejected with acute cellular rejection and without donor-specific antibodies by an average of 31 days (Figure 1B). The fourth animal survived to 106 days, with C4d deposition observed in his rejected graft. In contrast to the IT-treated animals, the IT + tacrolimus treated animals showed delayed repopulation of memory T cells, with the most pronounced difference in CD4+ central memory T cells (Figure 2C). All IT + tacrolimus treated animals experienced a significant decrease in CD8 effector memory cells one week after induction T cell depletion (mean 12% of CD8+ cells, p = 0.02); however, by the time of graft rejection, this population increased to 58% of CD8+ cells (p = 0.043).

Improved inhibition of effector memory T cells was necessary to prevent early acute rejection in our model. As alloreactive T cells express higher amounts of CD2, they have been shown to be susceptible to and preferentially inhibited by the CD2-specific fusion protein alefacept/LFA3-Ig (45,46). Four transplanted animals received IT, tacrolimus and alefacept (Figure 3A), and experienced graft survival of 38, 63, 73 and 87 days. Although no statistical difference was found in comparison with the IT + tacrolimus group (p = 0.248), Figure 3B illustrates an incremental increase in graft survival with the addition of each immunosuppressive agent. Alefacept-treated animals still experienced a rapid repopulation of CD4+ central memory T cells, with a 285% increase from POD 7 (time of maximal depletion) to POD 21 (p = 0.02). Clinically, these animals demonstrated evidence of rejection at an average of 43 postoperative days, with increasing BUN and creatinine, and decrease in appetite and activity. One week prior to clinical manifestations of rejection, a significant increase from baseline (p = 0.02) in CD8 effector memory T cells could be detected by flow cytometry. Serum creatinine trends are displayed in Figure 3C.

All IT-treated animals sustained low levels of peripheral blood T cells (average 19.8% at 4 weeks, 22% at 8 weeks) throughout the life of their grafts, as depicted in Figure 3D. B cell counts experienced an initial decrease, likely due to methylprednisolone (47,48), but were disproportionately increased in animals surviving past 4 weeks. No difference in T- and B cell absolute counts was observed between IT + tacrolimus and IT + tacrolimus + alefacept treated animals within the 4 weeks (Figure 3D, right). Despite low numbers of T cells, differential proportions of CD4+ and CD8+ were observed, with a relative sparing of CD4+ lymphocytes seen after IT induction: this difference was greatest by 1–2 weeks (p = 0.001) (Figure 3E). While all animals had a skewing toward a CD4+ dominant population, animals treated with alefacept experienced greater CD4+ proliferation (measured by intracellular Ki67+ staining) by weeks 3–4 compared to those without; no difference was observed in CD8+ proliferation. No differences in peripheral blood regulatory T cells (CD4+CD25+FoxP3+) were observed across the treatment groups (data not shown).

Alefacept-treated animals have increased alloantibody production and morphologic evidence of antibody-mediated injury

Alloantibody was detected in all animals treated with alefacept (Figure 4A). Flow crossmatch showed at least a —two- to sixfold mean channel shift by 2–4 weeks for both T and B cells in 100% of alefacept-treated animals. The kinetics of alloantibody production for each of the alefacept-treated animals is shown in Figure 4B.

Graft histology was evaluated in a blinded fashion and graded using Banff criteria (Table 1). Among the diagnoses listed in Table 1, glomerulitis, allograft glomerulopathy, mesangial matrix increase, peritubular capillaritis (PTC) and C4d deposition are commonly seen with antibody-mediated injury (4,24,49). These scores were combined to represent an antibody-mediated injury score: the IT alone group had an average combined score of 3.33, IT + tacrolimus 6.25 and IT + tacrolimus + alefacept 6.75. These data suggest that depletion by IT results in antibody-mediated injury that is detectable if the animal and graft survive to at least 4 weeks. The finding that all alefacept-treated animals developed de novo alloantibody makes it the preferred regimen for an AMR model. It should be noted that all animals exhibited varying degrees of PTC; although PTC is indicative of antibody-mediated rejection, it is also related to inflammation due to acute tubular necrosis, pyelonephritis, necrosis and infarction (50), which could confound the scoring system.

Figures 4C and D depict morphologic changes observed in the IT + tacrolimus + alefacept group. These include transplant glomerulopathy, subendothelial accumulation of ground substance (arteriolar endothelial injury) and C4d deposition (Figure 4C). Glomerular basement membrane thickening and duplication were noted on electron microscopy (Figure 4D). While donor-specific antibodies were present in all four animals in this treatment group, their graft immunohistochemistry reflected weak and varying intensities of C4d staining. This could be due to the time course of antibody-mediated injury. The average graft survival in this group was 65 days, which is shorter than the onset of C4d deposition detected by Smith et al.(51); furthermore, they report alloantibodies as the first to appear in the progression of chronic humoral rejection. Einecke et al. also underscored the importance of microcirculation changes and HLA antibodies in defining antibody-mediated rejection, as they preceded late kidney failures regardless of C4d+ status (52).

Alefacept increases CD4+ effector memory T cell proliferationin vitro

As de novo alloantibodies were consistently detected in the animals treated with IT + tacrolimus + alefacept, we investigated the effects of alefacept on lymphocyte proliferation and memory phenotypes in vitro. Alefacept was titrated at 0, 100 and 500 μg/mL in CFSE-mixed lymphocyte culture. Increased doses of alefacept corresponded to significantly increased CD3+ lymphocyte proliferation (Figure 5A); no difference was seen in CD20+ proliferation (Figure 5B). Specifically, CD4+ effector memory T cells exhibited the highest increase in proliferation with increased alefacept doses (Figures 5C and D). This phenomenon was not identified in vivo, as CD4+ effector memory cells are minimally detected in peripheral blood. They were, however, much more prevalent in rejected grafts (Figure 5E), suggesting that alefacept-driven proliferation of CD4+ effector memory cells occurs locally at the site of the alloantigen presentation.