Objectives: Antibody-mediated injury in chronic active antibody-mediated rejection, possibly with other effector T cells, may play a role in graft injury. The role of inflammatory cells in the inflammation and fibrosis and tubular atrophy region has been recently advocated in the progression of injury. Cytotoxic T cells play a prominent role in T-cell-mediated rejection; however, the possible role of cytotoxic T cells in circulation and the intragraft compartment in chronic active antibody-mediated rejection, a common immunological cause of long-term graft failure, has not been well-studied.

Materials and Methods: We measured the frequency of circulating cytotoxic T cells with flow cytometry, serum granzyme B level by enzyme-linked immunosorbent assay and intragraft granzyme B+ cell, and mRNA by immunohistochemistry and real-time polymerase chain reaction in biopsy tissue from living donor renal allograft recipients with stable graft function and chronic active antibody-mediated rejection.

Results: The frequency of CD3+ and CD3+CD8+ T cells was similar in both stable graft function patients and chronic active antibody-mediated rejection patients. The frequency of CD3+CD8+granzyme B+ cytotoxic T cells was significantly lower in peripheral blood. Serum granzyme B level and intragraft number of granzyme B+ cells (counts/mm2) were also significantly higher in the chronic active antibody-mediated rejection group compared with that of patients with stable graft function. The intragraft granzyme B+ T cell count was positively correlated with serum creatinine and 24-hour urine proteinuria but negatively correlated with estimated glomerular filtration rate.

Conclusions: Granzyme B mediates covert graft injury in patients with chronic active antibody-mediated rejection in addition to antibody-mediated injury.

Key words : Cytotoxic T cells, Intragraft granzyme B, mRNA expression, Stable graft function

Introduction

Chronic active antibody-mediated rejection (CABMR) is one of the important immunological causes of graft loss in the late posttransplant period. Chronic active antibody-mediated rejection is clinically characterized by proteinuria, hypertension, a decline in glomerular filtration rate, and the presence of circulating donor-specific antibodies. The common histological features are multilayering in peritubular capillary basement membrane along with complement fragment C4d deposition, glomerulitis, along with the varying degree of interstitial inflammation and fibrosis, and tubular atrophy.¹ Patients with chronic antibody-mediated rejection respond poorly to conventional immunosuppressive regimens and rapidly progress to end-stage graft failure.²

A short-term interaction of naive CD8+ T cells with allograft peptide results in CD8+ T-cell activation.³ The persistent allogeneic stimulation activates CD8+ T cells to synthesize and secrete granzyme B, perforin, and expression of other armamentaria, such as Fas ligand (FasL), granulysin, lymphotoxin, tumor necrosis factor α, and interferon γ, which may lead to allograft injury.⁴ A few studies showed that inhibition of the FasL-dependent pathway could not inhibit apoptosis in the peritubular epithelial cell. In contrast, inhibition of the perforin/granzyme B pathway with concanamycin A and higher endogenous protease inhibitor 9 inhibited apoptosis and cell lysis, suggesting perforin/granzyme B as an indispensable pathway for allograft cell death and rejection.^5,6 Granzyme B, a serine protease of broad substrate specificity, mediates cytotoxicity and induces apoptosis in target cells.⁷ Few studies have reported urinary, intragraft, and circulating granzyme B as a reliable marker of acute rejection.8 In addition to antibody-mediated injury, T-cell-mediated injury also occurs in the allograft. In studies of the role of inflammatory cells within interstitial and tubular atrophy region, inflammation and fibrosis and tubular atrophy were associated with poor prognosis and rapid graft failure.^9,10 T-cell infiltration and tubulitis are often observed in for-cause biopsies with a diagnosis of antibody-mediated rejection, and such mixed T-cell antibody-mediated pathology may occur in 10% to 90% of graft biopsies.^11-13 There are relatively few studies published on the changes in the cytotoxic T lymphocyte (CTL) in peripheral circulation and intragraft tissue in CABMR. In this study, we investigated the circulating and intragraft profiles of CTL in patients with CABMR in comparison with those of patients with stable graft function (SGF).

Materials and Methods

Patient recruitment
A total of 42 living donor first renal allograft recipients (10 SGF and 32 CABMR), without a prior history of rejections, were included in the study. Based on histopathology reports (per the 2017 Banff criteria), independently evaluated by the 2 histopathologists (Manoj Jain, Vinita Agarwal), patients were categorized into 2 groups: SGF and CABMR groups.1 All CABMR cases were defined per the criteria in the Banff classification for fulfillment of morphological, immunohistological, and serological tests.1 Patients with SGF were those who had stable serum creatinine levels for 6 months without significant proteinuria and less than 10% cortical surface area showing evidence of interstitial fibrosis and tubular atrophy on histology. These patients had an absence of C4d staining in peritubular capillaries and donor-specific antibodies in blood.

This study was approved by the institutional ethics committee (ethics approval code-IEC.2012-117-PhD-63), and signed written consent was obtained from patients and/or relatives.

Sample collection
Renal allograft biopsy was performed with the standard graft biopsy technique, and 3 cores of biopsy were obtained; 1 core was for histopathological diagnosis, and a second core was divided for immunofluorescence and electron microscopy examination. The third core was used for RNA isolation for gene transcript expression analysis by real-time polymerase chain reaction (PCR). Simultaneously, venous blood was collected in heparinized and plain vials for cellular flow cytometry and granzyme B level analysis. Granzyme B immunostaining was performed with the paraffin block of a routine biopsy core. All SGF patients included in this study agreed and consented for the graft biopsies.

Immunostaining for granzyme B in intragraft tissue
Granzyme B immunostaining was performed with formalin-fixed, paraffin-embedded tissue sections using an anti-human mouse monoclonal antibody against granzyme B (1:100 dilution, Santa Cruz Biotechnology) in steps as mentioned in our previous study.¹⁴

Cytotoxic T-cell staining and analysis
Whole blood cells were stimulated following the protocol mentioned in our previous study.¹⁵ We stained 100 μL of stimulated blood cells for analysis. We used 10 μL of peridinin-chlorophyll-cyanine 5.5-conjugated mouse monoclonal anti-human CD3 and 10 μL of allophycocyanin-conjugated mouse monoclonal anti-human CD8 antibody for surface staining following a brief incubation for 30 minutes in the dark at room temperature. After red blood cell lysis with red blood cell lysis buffer for 13 minutes, cells were fixed and permeabilized with BD Cytofix/Cytoperm fixation/permeabilization solution, followed by intracellular staining with 5 μL of phycoerythrin-conjugated mouse monoclonal anti-human granzyme B antibody for 30 minutes. An appropriate fluorochrome-conjugated isotype-matched antibody against CD8 and granzyme B was used to nullify any false-positive signal and setting quadrate for differentiating positive population. All reagents and antibodies were purchased from BD Bioscience (BD Pharmingen).

We used a BD FACSCalibur machine to acquire 10 000 lymphocyte gated cells, and we analyzed the cells with FCS Express 6 software. The gating strategy was as follows: CD3+ T cells were calculated in the lymphocyte gate (gate 1); then in the CD3+ T cell gate (gate 2), CD8+granzyme B+ (double-positive) T cells were considered to be CTL.

Serum granzyme B level detection
Blood in plain vials were centrifuged at 1500 revolutions per minute for 5 minutes, and serum was separated and stored at -80°C until analysis. Enzyme-linked immunosorbent assay was performed with BioLegend LEGEND MAX human granzyme B ELISA kit. The minimum detection limit for the kit was 2.4 ± 1.2 pg/mL.

Granzyme B gene expression analysis by TaqMan real-time polymerase chain reaction
We used a Polytron rotor/stator homogenizer (Kinematica) to homogenize the biopsy specimens, and we used a Qiagen RNA extraction mini kit to extract the RNA, according to the manufacturer’s protocol. The purity and concentration of RNA were determined on a NanoDrop spectrophotometer (Thermo Scientific). We checked the integrity of the RNA on a 1% agarose gel. To prepare copy DNA, we used SuperScript II RT reverse transcriptase (Invitrogen) to process 200 ng of RNA and random hexamer primer in a 20-μL reaction volume, following the manufacturer’s protocol. We used an Applied Biosystems GenAmp 7700 sequence detection system with a predesigned primer and probe for granzyme B (HS01554355_m1; Applied Biosystems) to perform TaqMan real-time PCR with 2 μL cDNA in a 20-μL reaction volume. We used glucose-6 phosphate dehydrogenase (Hs99999905_m1) as an endogenous housekeeping control to normalize the RNA starting quantity. We used the 2-∆∆Ct method to calculate relative expression.¹⁶

Statistical analyses
We analyzed the data with SPSS software (version 20), and continuous variables were analyzed with an independent t test. Data are expressed as mean values ± SD. We used the chi-square test and the Fischer exact test, whenever applicable, to analyze the categorical variables. We used the Pearson correlation matrix to analyze the correlations among the different variables. P < .05 with the 2-tailed test was considered to be significant.

Results

Baseline characteristics of patients
The clinical characteristics of patients with CABMR and SGF are shown in Table 1. All patients were living donor organ recipients and related renal donors were spouse, parent, or sibling in both of the groups. Patients in the CABMR group had significantly reduced estimated glomerular filtration rate and hemoglobin and an increased level of serum creatinine and proteinuria. The histological grading of allograft injury in both groups is shown in Table 2. The histological scoring for peritubular capillaritis, glomerulitis, tubulitis, tubular atrophy, and inter­stitial fibrosis was higher in CABMR compared with SGF patients.

Circulating cytotoxic T-cell profiles of the patients
The frequency of CD3+ T cells and CD3+CD8+ T cells was similar in both groups. However, the frequency of CD3+CD8+granzyme B+ CTL was significantly lower in the CABMR group than in the SGF group ( Table 3, Figure 1).

Intragraft granzyme B+ cell count and mRNA expression
Intragraft granzyme B+ cell count (per mm2) and granzyme B mRNA expression were significantly higher in the CABMR group than in the SGF group ( Figure 2).

Intragraft granzyme B+ cell count was positively correlated with graft function parameters
Intragraft granzyme B+ cell count (per mm2) was positively correlated with the 24-hour proteinuria level and the serum creatinine level but negatively correlated with the estimated glomerular filtration rate ( Figure 3).

Serum granzyme B level
Serum granzyme B level was significantly higher in the CABMR group than in the SGF group ( Figure 3D).

Discussion

In this study, the most crucial observation was a lower frequency of circulating CTL and higher serum granzyme B level and intragraft granzyme B+ cell count in CABMR patients compared with results shown in SGF patients. These findings suggested a pathogenic association of CTL with allograft injury in cases of CABMR. Ashton-Chess and colleagues have also observed that granzyme B mRNA transcript expression was significantly increased in the graft and significantly decreased in the peripheral blood of patients with CABMR.¹⁷ They have also emphasized that the quantification of peripheral blood granzyme mRNA may have the potential to aid in the noninvasive diagnosis of CABMR.

The persistent allo-stimulating signal leads to CTL, activation, and increased synthesis of granzyme
B mRNA and protein.^18,19 The low frequency of circulating cell CD3+CD8+granzyme B+ T cells compared with that of SGF patients may be caused by the sequestration of granzyme B+ T cells into the intragraft tissue in cases of CABMR. CD8+ T cells in peripheral circulation also released granzyme B into the serum, which may have subsequently sequestered into the graft and thereby mediated allograft injury. The activated CTL acquire different homing transient surface molecules such as the HML-1, human mucosal lymphocyte, the VLA-4 very late activation antigen, the LFA-1 leukocyte function-associated antigen, CD103, and the CXCR3 chemokine receptor, which helps in intragraft sequestration and adherence of CTL via the cognate receptor expressed on activated endothelium and epithelial cells of allograft tissues, for example, the ICAM-1 intercellular adhesion molecule, the MCP-1 monocyte chemoattractant protein, the MIP-1α macrophage inflammatory protein, and integrin αE(CD103)β7.^20,21 Higher granzyme B+ cell count on immunohistochemistry in our findings also suggests activation and infiltration from circulating blood into the graft tissue, similar to Ashton-Chess and colleagues.17 Although we did not stain T-cell-specific markers, there may be possible involvement of natural killer (NK) cells in granzyme B release in graft tissue.

Protease granzyme B has a pleiotropic function
Granzyme B may have systemic and local effects leading to graft injury and activation of inflammatory cells in circulating blood.²² Granzyme B is a serine protease of broad substrate specificity, cleaves cytoskeleton proteins, matrix metalloproteinase, nuclear lamina of a cell, and activates proinf­lammatory cytokines interleukin 1β linked with many inflammatory diseases.^7,22 Granzyme B cleaves extracellular matrix protein and releases active transforming growth factor β from cells into the interstitial space, which may induce fibrosis and de novo differentiation of regulatory T cells.²³ Simultaneously, increased regulatory T cells in the local milieu at the site of injury helps in suppressing the inflammatory cell and sustaining the inflam­mation for longer duration and perpetuating the allograft injury.^17,24

Cytotoxic T cells also mediate target cell death by activating executor caspase 3 and reactive oxygen species formation by cleavages of NDUFV1, NDUFS1, and NDUFS2 subunits of the NADH:ubiquinone oxidoreductase complex I inside mitochondria,^23,25 which may lead to the death of graft cells and graft dysfunction. Our findings also suggest an association between graft dysfunction and granzyme B-mediated injury. There was a significant positive correlation between tissue granzyme B-positive cell count and degree of proteinuria and serum creatinine level and a negative correlation with the decline in estimated glomerular filtration rate in our study.

To circumvent the devastating effects of granzyme B, peritubular epithelial cells of allografts also express a higher level of granzyme inhibitor protease, serpin proteinase inhibitor 6, which binds to granzyme B covalently and inhibits granzyme B-mediated tubular cell damage; however, one of the studies has already shown that serpin proteinase inhibitor 6 could not prevent subclinical injury in acute rejection.²⁶ An inhibition of a caspase 3/reactive oxygen species inhibitor like isatin sulfonamide, ascorbate, or tocopherol may have beneficial effects in retarding granzyme B-driven CABMR. However, this requires further study before use in practice. Recently, inflammatory cells with inflammation and fibrosis and tubular atrophy region of the microscopy showed association with rapid progression and graft failure. In our previous study, we have demonstrated that T-bet-positive cells in allografts were associated with poor prognosis.¹⁴

The major limitation of our study was that we had not investigated perforin expression in CTL required for granzyme B delivery and coexpression of CD8+granzyme B+ (double-positive) T cells in intragraft tissue. The exact mechanisms of granzyme B-induced injury, whether by caspase or reactive oxygen species dependent in the allograft, remain unclear. Therefore, a more comprehensive study is required to fully understand the role of CTL and NK cells, which release granzyme B in cases of CABMR.

Conclusions

Higher granzyme B+ cell count in renal allograft tissue in patients of CABMR compared with that of SGF patients indicates granzyme-mediated covert injury in patients with CABMR. Graft tissue granzyme level was also associated with graft dysfunction in CABMR. Persistent allogeneic stimulation to the CTL leads to increased synthesis and release of granzyme B. Activated CTL infiltrates into the different compartments of the allograft and mediates granzyme B-dependent injury, resulting in the development of chronic allograft dysfunction and, ultimately, graft loss. Inhibition of granzyme B synthesis/release and prevention of CTL sequestration in intragraft tissue may be useful strategies for the prolongation of graft survival in the future.

References:

1. De Serres SA, Noel R, Cote I, et al. 2013 Banff criteria for chronic active antibody-mediated rejection: assessment in a real-life setting. Am J Transplant. 2016;16(5):1516-1525. doi:10.1111/ajt.13624
   CrossRef - PubMed
   
2. Gheith O, Al-Otaibi T, Halim MA, et al. Early versus late acute antibody-mediated rejection among renal transplant recipients in terms of response to rituximab therapy: a single center experience. Exp Clin Transplant. 2017;15(Suppl 1):150-155. doi:10.6002/ect.mesot2016.P32
   CrossRef - PubMed
   
3. Parish IA, Kaech SM. Diversity in CD8(+) T cell differentiation. Curr Opin Immunol. 2009;21(3):291-297. doi:10.1016/j.coi.2009.05.008
   CrossRef - PubMed
   
4. Sharma VK, Bologa RM, Li B, et al. Molecular executors of cell death: differential intrarenal expression of Fas ligand, Fas, granzyme B, and perforin during acute and/or chronic rejection of human renal allografts. Transplantation. 1996;62(12):1860-1866. doi:10.1097/00007890-199612270-00031
   CrossRef - PubMed
   
5. Wever PC, Boonstra JG, Laterveer JC, et al. Mechanisms of lymphocyte-mediated cytotoxicity in acute renal allograft rejection. Transplantation. 1998;66(2):259-264. doi:10.1097/00007890-199807270-00021
   CrossRef - PubMed
   
6. Rowshani AT, Florquin S, Bemelman F, Kummer JA, Hack CE, Ten Berge IJ. Hyperexpression of the granzyme B inhibitor PI-9 in human renal allografts: a potential mechanism for stable renal function in patients with subclinical rejection. Kidney Int. 2004;66(4):1417-1422. doi:10.1111/j.1523-1755.2004.00903.x
   CrossRef - PubMed
   
7. Wowk ME, Trapani JA. Cytotoxic activity of the lymphocyte toxin granzyme B. Microbes Infect. 2004;6(8):752-758. doi:10.1016/j.micinf.2004.03.008
   CrossRef - PubMed
   
8. Li B, Hartono C, Ding R, et al. Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. N Engl J Med. 2001;344(13):947-954. doi:10.1056/NEJM200103293441301
   CrossRef - PubMed
   
9. Nankivell BJ, Shingde M, Keung KL, et al. The causes, significance and consequences of inflammatory fibrosis in kidney transplantation: the Banff i-IFTA lesion. Am J Transplant. 2018;18(2):364-376. doi:10.1111/ajt.14609
   CrossRef - PubMed
   
10. Roufosse C, Simmonds N, Clahsen-van Groningen M, et al. A 2018 Reference Guide to the Banff Classification of Renal Allograft Pathology. Transplantation. 2018;102(11):1795-1814. doi:10.1097/TP.0000000000002366
    CrossRef - PubMed
    
11. Nickeleit V, Andreoni K. The classification and treatment of antibody-mediated renal allograft injury: where do we stand? Kidney Int. 2007;71(1):7-11. doi:10.1038/sj.ki.5002003
    CrossRef - PubMed
    
12. Randhawa P. T-cell-mediated rejection of the kidney in the era of donor-specific antibodies: diagnostic challenges and clinical significance. Curr Opin Organ Transplant. 2015;20(3):325-332. doi:10.1097/MOT.0000000000000189
    CrossRef - PubMed
    
13. Sun Q, Liu ZH, Cheng Z, et al. Treatment of early mixed cellular and humoral renal allograft rejection with tacrolimus and mycophenolate mofetil. Kidney Int. 2007;71(1):24-30. doi:10.1038/sj.ki.5001870
    CrossRef - PubMed
    
14. Yadav B, Prasad N, Agrawal V, et al. T-bet-positive mononuclear cell infiltration is associated with transplant glomerulopathy and interstitial fibrosis and tubular atrophy in renal allograft recipients. Exp Clin Transplant. 2015;13(2):145-151.
    CrossRef - PubMed
    
15. Prasad N, Jaiswal AK, Agarwal V, et al. Differential alteration in peripheral T-regulatory and T-effector cells with change in P-glycoprotein expression in childhood nephrotic syndrome: a longitudinal study. Cytokine. 2015;72(2):190-196. doi:10.1016/j.cyto.2014.12.028
    CrossRef - PubMed
    
16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods. 2001;25(4):402-408. doi:10.1006/meth.2001.1262
    CrossRef - PubMed
    
17. Ashton-Chess J, Dugast E, Colvin RB, et al. Regulatory, effector, and cytotoxic T cell profiles in long-term kidney transplant patients. J Am Soc Nephrol. 2009;20(5):1113-1122. doi:10.1681/ASN.2008050450
    CrossRef - PubMed
    
18. Soni C, Karande AA. Glycodelin A suppresses the cytolytic activity of CD8+ T lymphocytes. Mol Immunol. 2010;47(15):2458-2466. doi:10.1016/j.molimm.2010.06.008
    CrossRef - PubMed
    
19. Simon T, Opelz G, Wiesel M, Ott RC, Susal C. Serial peripheral blood perforin and granzyme B gene expression measurements for prediction of acute rejection in kidney graft recipients. Am J Transplant. 2003;3(9):1121-1127. doi:10.1034/j.1600-6143.2003.00187.x
    CrossRef - PubMed
    
20. Kim CH, Broxmeyer HE. Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol. 1999;65(1):6-15. doi:10.1002/jlb.65.1.6
    CrossRef - PubMed
    
21. Smyth LJ, Kirby JA, Cunningham AC. Role of the mucosal integrin alpha(E)(CD103)beta(7) in tissue-restricted cytotoxicity. Clin Exp Immunol. 2007;149(1):162-170. doi:10.1111/j.1365-2249.2007.03385.x
    CrossRef - PubMed
    
22. Goldbach-Mansky R, Suson S, Wesley R, Hack CE, El-Gabalawy HS, Tak PP. Raised granzyme B levels are associated with erosions in patients with early rheumatoid factor positive rheumatoid arthritis. Ann Rheum Dis. 2005;64(5):715-721. doi:10.1136/ard.2003.007039
    CrossRef - PubMed
    
23. Jacquemin G, Margiotta D, Kasahara A, et al. Granzyme B-induced mitochondrial ROS are required for apoptosis. Cell Death Differ. 2015;22(5):862-874. doi:10.1038/cdd.2014.180
    CrossRef - PubMed
    
24. Boivin WA, Shackleford M, Vanden Hoek A, et al. Granzyme B cleaves decorin, biglycan and soluble betaglycan, releasing active transforming growth factor-beta1. PLoS One. 2012;7(3):e33163. doi:10.1371/journal.pone.0033163
    CrossRef - PubMed
    
25. Afonina IS, Cullen SP, Martin SJ. Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B. Immunol Rev. 2010;235(1):105-116. doi:10.1111/j.0105-2896.2010.00908.x
    CrossRef - PubMed
    
26. Lau A, Khan K, Pavlosky A, et al. Serine protease inhibitor-6 inhibits granzyme B-mediated injury of renal tubular cells and promotes renal allograft survival. Transplantation. 2014;98(4):402-410. doi:10.1097/TP.0000000000000237
    CrossRef - PubMed