Objectives: We aimed to study the role of T-bet-positive mononuclear cell infiltration in different compartments of kidney graft tissues in patients with chronic transplant glomerulopathy, interstitial fibrosis and tubular atrophy, and stable graft function.

Materials and Methods: There were 80 living-related renal transplant recipients included (chronic transplant glomerulopathy, n = 28; interstitial fibrosis and tubular atrophy, n = 28; stable graft function, n = 24). Histologic characteristics and scoring for peritubular capillaritis, glomerulitis, interstitial fibrosis and tubular atrophy, and intimal arteritis were performed according to Banff 2007 classification and compared between the groups. Immunohistologic staining was performed for transcription factor T-bet, T-bet mononuclear cells were counted, and T-bet infiltration score was compared between groups.

Results: Patients in different groups had similar clinical profiles and human leukocyte antigen mismatches, except the groups differed in serum creatinine and proteinuria. The prevalence and scoring of peritubular capillaritis and glomerulitis were significantly higher in chronic transplant glomerulopathy than interstitial fibrosis and tubular atrophy (P = .001) and stable graft function (P < .001). Tubulitis was observed in 6 patients (21.4%) with chronic transplant glomerulopathy but no patients with interstitial fibrosis and tubular atrophy. The C4d/donor-specific antibody was positive in 100% patients with chronic transplant glomerulopathy, 0% patients with interstitial fibrosis and tubular atrophy, and 4.1 % patients with stable graft function. Interstitial fibrosis and tubular atrophy was seen in 100% patients who had interstitial fibrosis and tubular atrophy; in patients who had chronic transplant glomerulopathy, 24 patients (85.7%) had interstitial fibrosis and 78.5% patients had tubular atrophy. The degree and severity of T-bet-positive cell infiltration were significantly higher in chronic transplant glomerulo-pathy than interstitial fibrosis and tubular atrophy or stable graft function; however, 85% patients with interstitial fibrosis and tubular atrophy also had T-bet-positive infiltration, suggesting a role of T-bet-positive cells in interstitial fibrosis and tubular atrophy.

Conclusions: Chronic transplant glomerulopathy is a consequence of chronic active immune-mediated injury. Interstitial fibrosis and tubular atrophy may be associated with T-bet-positive mononuclear cell infiltration in the peritubular region. The T-bet infiltration should be evaluated in patients with chronic allograft injury.

Key words : Chronic allograft dysfunction, Immunology, Inflammation

Introduction

Renal transplant is the preferred modality of treatment for end-stage renal failure patients. Tacrolimus-based immunosuppressions has reduced the incidence of acute rejection, but chronic allograft injury due to chronic transplant glomerulopathy (CTG), interstitial fibrosis and tubular atrophy (IF/TA), calcineurin inhibitor toxicity, and recurrence of native kidney diseases are major causes of allograft loss in the long term.^1-4 The reported incidence of CTG is 1% to 4% at 1 year and 20% at 5 years after transplant.⁵ Recent evidence suggests that CTG may be associated with chronic antibody-mediated injury and may be manifested by either C4d-positivity or presence of donor-specific antibody (DSA) against either class of donor human leukocyte antigen (HLA).⁶ However, idiopathic CTG may be negative for both C4d and DSA.⁷ The IF/TA, which is characterized by presence of marked interstitial fibrosis and tubular atrophy (without changes of chronic hypertension, calcineurin inhibitor toxicity, chronic obstruction, bacterial pyelonephritis, or viral cytopathologic change) is a controversial issue in renal transplant because precise pathogenesis is unknown.⁸

The extent of immune-mediated injury and the contribution of T-cell-lineage-related injury in renal allograft in patients with CTG and IF/TA are not well studied. The transcription factor T-bet of the T box transcription family is essential for Th1 cell lineage commitment from the precursor T cell. The T-bet is encoded by a TBX21 gene in humans.⁹ Other immune cells such as natural killer, dendritic, B, and CD8-positive-T cells also express T bet.^10-14 The T-bet has a pleiotropic role, directs the expression of interleukin 1α, macrophage inflammatory protein-1α in dendritic cells,¹⁵ interferon γ in Th1 cells,¹⁶ and class switching in B cells.¹⁷ A recent study revealed that intraglomerular T-bet expression is associated with antibody-mediated rejection in Chinese people.¹⁸ In this study, we aimed to analyze T-bet-positive lymphocyte infiltration in renal allografts and compare the prevalence between patients who had CTG, IF/TA, or stable graft function (SGF).

Materials and Methods

Patients
A total of 56 living-donor renal transplant recipients with chronic allograft injury (CTG, 28 patients; IF/TA, 28 patients), and 24 patients with SGF as control, were retrospectively selected from our transplant database and included in the study. The demographic and clinical profiles of all the patients and donors were noted from the patient history and computerized hospital information system of the institute. Patients were categorized into CTG, IF/TA, and SGF groups based on clinical profile and histopathology according to the Banff 2007 classification of allograft injury.¹⁹ The SGF was defined by < 25% increase in serum creatinine level from baseline on follow-up without proteinuria and < 10% cortical surface area showing any evidence of tubular atrophy, interstitial fibrosis, or glomerular changes on light microscopy. Patients with evidence of calcineurin inhibitor toxicity, hypertensive changes, pyelo-nephritis, recurrence of native kidney disease, and viral-associated cytopathologic changes were excluded from the study. Patients with second or multiple transplant, previous positive crossmatch, panel reactive anti-body (PRA) positivity > 20%, multiple pregnancies, and history of > 2 blood transfusions before transplant also were excluded from the study.

Clinically, CTG was defined by the presence of proteinuria, hypertension, progressive decline in creatinine clearance, and graft loss.20 Histologically, CTG was characterized by peritubular capillary membrane duplication, glomerulitis, and C4d deposition along capillary walls.²⁰ All patients who had CTG were either C4d-positive or DSA-positive. The study was approved by the ethics committee of the institute and performed according to the ethical guidelines of the Helsinki declaration.

Light microscopy and immunohistologic staining
In each case, 2 cores of ultrasonography-guided needle biopsy were obtained (1 for formalin fixation and 1 in normal saline for cryosectioning and immunofluorescence study). Hematoxylin-eosin, periodic acid Schiff, and Masson trichrome staining were performed for routine testing with formalin-fixed tissue. Indirect immunofluorescence staining was performed for C4d, C1q, C3, and immuno-globulin (Ig) M, A, and G using antihuman mouse monoclonal antibody and fluorescein isothiocyanate-conjugated secondary antimouse IgG rabbit antibody with normal saline core. Immunohistochemistry was performed with formalin-fixed, paraffin-embedded tissue sections. Heat induced antigen retrieval in Tris ethylenediaminetetraacetic acid buffer (pH 8.6) was performed. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 30 minutes. Nonspecific binding sites were blocked with 5% nonfat skimmed milk. An antihuman mouse monoclonal antibody against T-bet was used in 1:100 dilution (Santa Cruz Biotechnology, Santa Cruz CA, USA). Horseradish peroxidase-conjugated antimouse rabbit polyclonal secondary antibody in 1:250 dilution (Dako, Carpinteria, CA, USA) and color substrate 3,3’-diaminobenzidine (Dako, Glostrup, Denmark) were used to develop color, and hematoxylin was used as counter stain to distinguish the nucleus. Immunohistochemical staining of each section was performed in duplicate. The result was analyzed by 2 independent, blinded, research-experienced pathologists; ≥ 10 high power fields per slide (original magnification × 200; mean, 10.5 ± 3.5 high power fields per slide) were chosen to count the T-bet-positive cells. The result was expressed in terms of number of only T-bet-positive mononuclear cell infiltration in peritubular capillaries, glomeruli, tubules, intimal layer of arteries, and interstitial regions. Interobserver variability was > 10% in none of the cases. A photographic image of each section was made (Olympus photomicroscope, Tokyo, Japan).

The scoring of peritubular capillaritis, C4d positivity, glomerulitis, interstitial infiltrate, and arteritis was performed according to Banff 2007 classification and the scores were compared between patients who had CTG, IF/TA, and SGF. Furthermore, we compared T-bet-positive mononuclear cell infiltration in patients who had CTG, IF/TA, and SGF.

Statistical analyses
The data were analyzed using software (IBM SPSS Statistics for Windows, Version 20.0, IBM Corp., Armonk, NY, USA). The data were expressed as mean ± standard deviation (SD). Categorical variables were compared using chi-square or Fischer exact test. The continuous variables between groups were compared using 1-way analysis of variance, and post hoc analysis with Bonferroni adjustment was applied to compare the significance between the different groups. The death-censored graft survival was analyzed using Kaplan-Meier method, and log-rank test was used to compare the significance of differences between the 2 groups. Death-censored graft survival was calculated from the date of transplant to the stage of irreversible graft failure when the patient was determined to have end-stage renal disease and advised to return to long-term dialysis or retransplant, or the date of last follow-up when the transplant was still functioning. In the event of death with a functioning graft, follow-up was censored at the date of death. A value of P < .05 was considered significant.

Results

The demographic and clinical characteristics showed that patients in all groups were similar in recipient age, sex ratio, donor age, and time of biopsy after transplant (Table 1). Mean serum creatinine level was significantly greater in patients with CTG and IF/TA than SGF (P < .001). Proteinuria was significantly higher in CTG than IF/TA or SGF patients. The proportion of patients with HLA mismatch was significantly higher in patients with CTG and IF/TA than SGF. The proportion of patients receiving basiliximab and induction therapy was similar between all groups of patients. The mean trough level of tacrolimus at graft biopsy was similar between all groups of patients (Table 1).

Table 2 shows the prevalence of peritubular capillaritis, glomerulitis, interstitial fibrosis and tubular atrophy, intimal arteritis, and C4d-positive status in the different groups of patients according to Banff 2007 classification. The prevalence of peritubular capillaritis, glomerulitis, tubulitis, and intimal arteritis in patients with CTG was 92.85%, 85.7%, 64.2%, and 64.2%; in patients with IF/TA was 10.7%, 42.8%, 0%,and 17.85%; and in patients with SGF was 25%,12.5%, 0%, and 3.57% respectively. Interstitial fibrosis was observed in 85.7% and tubular atrophy in 78.5% of CTG patients. In the 28 patients with CTG, all 28 patients (100%) were either C4d-positive (20 patients [71.4%]) or DSA-positive (8 patients [28.6%]); none of the patients who had IF/TA were C4d-positive, and only 1 patient (4.1%) who had SGF was C4d-positive.

The histologic scoring of peritubular capillaritis, glomerulitis, interstitial fibrosis and tubular atrophy, and intimal arteritis based on Banff 2007 classification is shown in Table 3. The degrees of capillaritis, glomerulitis, tubulitis, and arteritis were significantly higher in patients who had CTG than IF/TA or SGF (P < .001). The severity and degree of interstitial fibrosis and tubular atrophy were similar in IF/TA and CTG patients and significantly higher than in SGF patients (P < .001).

T-bet-positive mononuclear cell infiltration
Table 4 shows the degree and severity of T-bet-positive mononuclear cell infiltration in different regions of renal allograft histology. The percentage of patients showing T-bet-positive mononuclear cell infiltration in different compartments of renal allografts in different categories is shown in Figure 1. The T-bet-positive mononuclear infiltration in the peritubular capillary region was observed in 25 of 28 patients who had CTG and 24 of 28 patients who had IF/TA, but the degree of cell infiltration was less in patients who had IF/TA than CTG. Representative photomicrographs are shown in Figure 2. The degree and severity of T-bet-positive mononuclear cell infiltration in glomeruli were significantly higher in patients who had CTG than IF/TA or SGF (P < .001) (Figure 2). The T-bet-positive infiltration in tubules was observed in 18 patients (64.2%) who had CTG but none of the patients who had IF/TA or SGF. The T-bet-positive interstitial infiltrate was significantly higher in patients who had CTG than IF/TA. We observed intimal arteritis in 15 patients (53.5%) who had CTG but only 3 patients (10.7%) who had IF/TA and 1 patient (4.1%) who had SGF (P < .001). The C4d/DSA-positivity was observed in 28 patients (100%) who had CTG, none of the patients who had IF/TA, and 1 patient who had SGF.

Death censored graft survival
The mean death-censored graft survival in patients who had CTG was 108.1 months (95% confidence interval [CI]: 89.46 to 126.7 mo); IF/TA was 113.65 months (95% CI: 94.1 to 133.2 mo); and SGF 149.4 months (95% CI: 131.4 to 167.3 mo). The death-censored graft survival after renal transplant at 3, 5, and 10 years in the CTG group was 96.3%, 92.4%, and 43.2%; in the IF/TA group was 88.9%,76.9%, and 53.8%; and in the SGF group was 100%, 100%, and 77.1% (P = .013) (Figure 3).

Discussion

The present study showed that long-term death-censored renal allograft survival was less in patients who had CTG than IF/TA. We also observed that the prevalence and severity scores of peritubular capillaritis, glomerulitis, and intimal arteritis and tubulitis were significantly higher in patients who had CTG than IF/TA or SGF. All IF/TA patients had interstitial fibrosis and tubular atrophy, but patients who had CTG also had high frequency of interstitial fibrosis (85.7% patients) and tubular atrophy (78.5% patients). Based on light microscopy and immuno-fluorescence study, peritubular capillaritis was observed in only 3 of 28 patients and was of low severity (p1) in patients who had IF/TA. However, focusing only on T-bet-positive mononuclear cell infiltration, we observed that 71.4% of IF/TA had 1 to 2 cells and 14.2% of IF/TA had 3 to 4 cells of T-bet-positive cell infiltration in peritubular capillaries; moreover, T-bet-positive mononuclear cell infiltration in the glomerulus, tubules, and interstitium was not significantly different between patients who had IF/TA and CTG. The findings suggest that T-bet-positive mononuclear cells may be associated with IF/TA in renal transplant patients in which no other specific etiology is identified.

In a recent study, Sun and associates showed that T-bet was associated with the development of CTG, and T-bet expression also distinguished CTG from IF/TA and stable grafts.²¹ Their study showed that inflammatory cell infiltration was limited primarily to the peritubular capillary region and intra-glomerular capillary vessels in cases with CTG. Similarly, we also observed that the severity of infiltration of T-bet-positive mononuclear cells was higher in patients who had CTG than IF/TA, and a low grade of T-bet-positive mononuclear cell infiltration was observed in the peritubular capillary region in 85% patients who had IF/TA. The T-bet-positive cells secrete effector cytotoxic molecules that cause local injury. Peritubular capillaritis and glomerulitis are forms of micro-circulating inflammasomes.²¹ These microcirculating inflammasomes are considered a consequence of active immune-mediated injury. Antibody-mediated activation of classical complement deposition in patches leads to renal injury in such cases.²² We have observed that all CTG patients were positive for C4d or DSA against 1 of the classes of HLA antigens, which also highlights the role of B-cell-mediated injury.²³ It appears that injury in CTG and IF/TA which may progress via a hierarchical phenomenon that starts just after transplant or after several months, due to multiple factors, and may progress via capillaritis, glomerulitis, interstitial infiltration, and tubulitis, resulting in IF/TA and graft loss.

It is possible that a severe degree of injury leads to CTG and less severe, slow injury progresses to IF/TA in cases for which the etiology of IF/TA may not be specified. The role of T-bet cells in fibrotic mechanisms and alloimmune injury has been reported previously in cases of kidney and pulmonary fibrosis.^24,25 The possible role of early ischemia/reperfusion injury on subsequent development of IF/TA has been hypothesized.²⁶ Ischemia reperfusion injury might selectively express cell adhesion molecules such as intracellular, vascular, or platelet cell adhesion molecules that may modulate the homing and adherence of T-bet-positive mononuclear cells in peritubular capillary, glomerular, or interstitial regions, leading to renal fibrosis and peritubular capillary membrane duplication, and later, leading to CTG and IF/TA.

In conclusion, peritubular and intraglomerular T-bet-positive cell infiltration is significantly higher in patients who had CTG than IF/TA or SGF. The evidence suggests that T-bet testing should be performed routinely in patients with chronic allograft injury. However, robust supportive data are lacking, and further investigation is warranted with a prospective study to evaluate such infiltration on protocol biopsies.

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