Unravelling the Multifactorial Pathogenesis and Immune Cell Infiltrates in Transplant Glomerulopathy

Introduction

Kidney transplantation remains the treatment of choice for end-stage kidney disease, as it significantly enhances both quality of life and patient survival. In India, ∼3,500 renal transplants are performed annually.¹ Advances in pre-transplant donor selection and immunosuppressive therapies have reduced acute rejection rates.² However, chronic graft outcomes remain a significant concern.³

Chronic allograft injury (CAI) occurs due to persistent immune injury to the graft, recurrent or de novo glomerulonephritis, chronic thrombotic microangiopathy, and infections such as polyomavirus nephropathy.⁴ Chronic antibody-mediated rejection (cABMR) results primarily from repeated endothelial injury caused by donor-specific HLA antibodies,⁵ and less frequently, non-HLA antibodies.^6,7 This leads to glomerular basement membrane reduplication, resulting in the characteristic TG lesion observed under light microscopy.⁸

Chronic active T-cell-mediated rejection (c-aTCMR) is characterized histologically by moderate or severe tubulitis, interstitial inflammation, and interstitial fibrosis and tubular atrophy.⁹ While TG is well-recognized in cases of chronic ABMR, it has also been documented in T-cell-mediated rejection (TCMR). Other causes of TG include hepatitis C infection and thrombotic microangiopathy.

Several studies have explored inflammatory cell types involved in acute renal graft rejection, suggesting potential roles for cytotoxic T-cells, natural killer (NK) cells, and macrophages.^10-12 However, data on the types, spatial distribution, and clinical relevance of immune cell infiltrates in renal allograft biopsies with TG lesions from various etiologies remain limited.

We investigated the causes of TG and analyzed the types of immune cell infiltrates in renal allograft biopsies.

Materials and Methods

All allograft biopsies performed between 2016 and 2022 with TG were retrieved from the Department of Histopathology. Hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), C4d immunostaining, and direct immunofluorescence findings for all cases were reviewed and re-evaluated according to the Banff 2019 guidelines.

Of the 9,338 renal biopsies received during the 6-year period (2016–2022), 3,600 (38.5%) were allograft kidney biopsies. Of these, 212 showed features of TG, accounting for 2.3% and 5.9% of all renal and allograft biopsies, respectively. The study involved 80 cases with sufficient material in the paraffin blocks.

Clinical details, follow-up information, and other laboratory parameters were obtained from the records of the Department of Renal Transplant Surgery and the Department of Nephrology. There was no extra sample taken for this study. A biopsy taken for diagnostic purposes was utilised for the study. Consent was obtained at the time of biopsy. And the study was ethically approved by our institute’s ethics committee.

Sections of 4-5 μm thickness were cut from paraffin blocks and mounted on freshly prepared slides coated with 0.01% poly-L-lysine. Slides were dried overnight at 37°C, dewaxed in xylene, and rehydrated through graded alcohols. This was followed by three washes in phosphate-buffered saline (PBS) for 5 minutes each.

Antigen retrieval was performed by steaming slides in 0.01 M sodium citrate buffer (pH 6.0) at 99-100°C for 20 minutes, after which they were left in the buffer to cool for 20 minutes at room temperature. Slides were then rinsed in PBS. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 20 minutes, followed by three PBS washes.

Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 20 minutes, followed by PBS washes. Immunohistochemistry was performed using the Ventana Benchmark XT auto-stainer. After initial processing, sections were incubated with primary antibodies for CD68, CD163, and CD56. Secondary antibody incubation was performed, followed by counterstaining with hematoxylin. Diaminobenzidine was used as the chromogen in all reactions. The slides were then mounted with DPX mountant.

Slides were incubated with the primary antibody for 60 minutes at room temperature (RT), washed in PBS, and then incubated with Biocare’s MACH 2 Double Stain 1 for 30 minutes at RT. After washing with PBS, chromogen (diaminobenzidine, DAB) was applied, and sections were incubated for 5 minutes at RT, followed by another PBS wash. Vulcan Fast Red (Biocare) was then applied for 10–20 minutes at RT. The sections were counterstained with hematoxylin, rinsed with deionized water, and mounted.

Dual-color immunohistochemistry was performed for CD3/CD20 (BioCare, RTU) and CD4/CD8 (BioCare, RTU). Single-color immunohistochemistry was conducted for CD68 (BioCare, RTU), CD163 (BioCare, RTU), and CD56 (Sigma Aldrich) to determine the type and distribution of immune cell infiltrates.

The glomeruli, tubules, peritubular capillaries, and interstitium with maximum involvement were graded for immune cell infiltration. Immune cell infiltrates were graded as 0: No expression for immunomarkers, 1+: 1-4 cells, 2+: 5-9 cells and 3+: >10 cells per glomerulus, tubule, peritubular capillary, or high-power field (HPF) of interstitium

Results

Based on current and prior biopsy findings, along with C4d status, TG was attributed to various causes. ABMR was the most common, in 39 cases (49%). Among these, 29 exhibited active injury characterized by microvascular injury and/or C4d positivity and were classified as chronic active ABMR. Within this group, 17 cases (58.6%) demonstrated C4d positivity; the remaining 10 had a history of ABMR but lacked active microvascular injury or C4d positivity in the current biopsy, leading to chronic ABMR classification.

A subset (10 or 12.5%) displayed a combination of ABMR and T-cell-mediated rejection (TCMR) features. Another 10 cases (12.5%) were attributed solely to TCMR, with no microvascular injury, C4d positivity, or prior ABMR documentation.

Hepatitis C infection was associated with TG in 12 cases (15%), with five and two also exhibiting ABMR features and immune complex deposits, respectively. Direct immunofluorescence showed immune complex deposition in eight cases (10%); four of them had concurrent ABMR features. Four cases indicated chronic microangiopathy changes, such as intimal thickening with myxoid changes, with associated ABMR features.

No definitive etiology could be determined for 10 cases (12.5). Renal biopsy findings of cases with TG and associated concurrent features have been presented in Figure 1.

Close

Figure 1:

Transplant glomerulopathy with varied histological findings: (a-c) Renal biopsy showing glomerulitis (single blue arrow) in a glomerulus (H&E, 200x) with thickening and double contouring of the glomerular capillary walls, best visualized with Periodic Acid-Schiff (PAS) stain (H&E, 400x) and C4d positivity in peritubular capillaries (400x), indicating chronic active antibody-mediated rejection (ABMR). (d) Transplant glomerulopathy with no evidence of microvascular injury in the glomerulus or peritubular capillaries (PAS, 200x). (e) Transplant glomerulopathy with chronic thrombotic microangiopathy changes in the arterioles (double blue arrow) (PAS, 200x). (f) Transplant glomerulopathy with concurrent features of acute T-cell–mediated rejection (PAS, 100x). H&E: Hematoxylin and Eosin.

Export to PPT

The clinical details of the cases included in this study with TG have been presented in Table 1. The age range was 18-65 years, with a mean age of 40.9±11.13 years. The cohort comprised 18 females and 62 males. Of the 80 patients, 62 received allografts from live donors, and eight from deceased donors. The donor status was unknown for 10 patients. There was no statistically significant difference in the time to develop TG between live and deceased donor transplant recipients.

The mean duration from transplantation to the development of TG was 6.8 years. Mean serum creatinine at the time of biopsy was 3.4 ± 2.9 mg/dL, while 24-hour proteinuria ranged from 0.1 mg/24hr to 13.5 mg/24hr.

Biopsy records from prior indications were available for 26 cases. Of these,11 showed acute ABMR; two demonstrated acute TCMR. The remaining cases had no morphological evidence of rejection and were documented as acute tubular injury or calcineurin inhibitor (CNI) toxicity features.

The mean follow-up period was 19.4 months. Follow-up biopsies were available in 22 patients. Interstitial fibrosis and tubular atrophy (IFTA) percentages at diagnosis and follow-up were compared. The mean IFTA at diagnosis was 21.36%, which increased to 30.36% during follow-up. IFTA progressed in all TG cases irrespective of causes.

The grading and distribution of immune cell infiltrates in allograft biopsies, assessed using immunohistochemistry, have been summarized in Table 2 and Figure 2. In the glomeruli, CD68⁺ and CD163⁺ cells were observed in 43 (53.8%) and 31 cases (38.7%), respectively, making macrophages the predominant immune cells in this compartment. Four (5%) patients showed CD20⁺ B-cells, 14 (17.5%) showed CD3⁺ T-cells, four (5%) showed CD4⁺ helper T-cells, and 10 (12.5%) showed CD8⁺ cytotoxic T-cells.

Close

Figure 2:

Distribution of immune cell infiltrates in chronic active antibody-mediated rejection (CABMR). (a–c) CD20/CD3 dual-color immunostaining in a case of chronic active ABMR, demonstrating CD3+ T cells (single blue arrow) in the glomerulus and peritubular capillaries. The tubulointerstitial compartment (blue star) shows a mixed population of CD20+ B cells (brown, Diaminobenzidine chromogen) and CD3+ T cells (red, Vulcan Fast Red chromogen). (a: 200x; b, c: 400x). (d–f) CD4/CD8 dual-color immunostaining in a chronic active ABMR case, highlighting predominant CD8+ cytotoxic T-cell infiltration in the tubulointerstitial compartment and arterial walls (double blue stars) (CD4: brown, Diaminobenzidine chromogen; CD8: red, Vulcan Fast Red chromogen). (d: 200x; e, f: 400x). (g–i) Immunostaining for CD163+ macrophages highlights glomerular infiltration by CD163+ cells (red arrow) (g, Diaminobenzidine chromogen, 400x). Abundant CD163+ macrophages in the tubulointerstitial compartment (h) and periarterial region (i) (h, i: Diaminobenzidine chromogen, 100x).

Export to PPT

In TG cases with ABMR features, glomeruli showed CD68⁺ and CD163⁺ cells in 25 (64.1%) and 19 (48.7%) cases, respectively, reinforcing the dominant role of macrophages, particularly M2, in chronic ABMR. Other immune cell markers observed in ABMR included CD20⁺ B-cells in four cases (10.2%), CD3⁺ T-cells in 11 (28.2%), CD4⁺ helper T-cells in three (7.6%), and CD8⁺ cytotoxic T-cells in nine (23%). CD56, a marker for NK cells, was faintly expressed in only two chronic active ABMR cases.

Macrophages were also the dominant immune cells in the tubulointerstitial compartment, particularly in fibrotic regions. CD68⁺ macrophages were noted in 71 cases (88.7%), while CD163⁺ M2 were seen in 78 (97.5%). T- (CD3⁺, CD4⁺, CD8⁺) and B-lymphocytes (CD20⁺) were also widely distributed in this compartment, with their presence as follows: CD3⁺ T-cells in 78 cases (97.5%), CD4⁺ helper T-cells in 63 cases (78.7%), CD8⁺ cytotoxic T-cells in 76 cases (95%), and CD20⁺ B-cells in 70 cases (87.5%).

Statistical analysis revealed a significant association between CD3⁺ and CD8⁺ T-cell infiltration in the tubules and TG cases with TCMR (p = 0.004). Similarly, TG cases were strongly associated with mixed TCMR and ABMR (p = 0.001).

Discussion

TG is a common morphological lesion in CAI and a significant cause of post-transplant proteinuria.¹³ It independently impacts graft survival and arises from chronic endothelial damage through various pathophysiological processes. TG is reported in 5%-20% of post-transplant biopsies, with mean of 33 months post-transplant, as seen in Aubert et al.’s study (6.7% prevalence in 8207 biopsies).¹⁴ Similarly, our study found a 5.9% prevalence, with TG appearing as early as 2 months post-transplant and three cases developing within the first year. Proteinuria, a hallmark of TG, ranged from 1+ to 3+ on dipstick tests and 0.1-13.5 mg/24 hrs in our cohort.

ABMR is the primary cause of TG, along with thrombotic microangiopathy, hepatitis C infection, and T-cell mediated rejection (TCMR), all linked to recurrent endothelial injury.⁸ Donor-specific antibodies (DSA) are crucial in TG development, as highlighted in the Banff 2019 criteria for chronic active ABMR (CAAMR). Loupy et al. reported TG in 43% of DSA-positive patients with subclinical ABMR within a year.¹⁵ In our study, 13.7% of TG cases had prior ABMR, though subclinical ABMR may have been underdiagnosed due to limited protocol biopsies.

Non-antibody mechanisms also play a role in TG.¹⁶ Lesage et al.¹⁷ and Sis et al.¹⁸ found TG cases lacking both DSA and C4d staining, implicating other pathways. Studies by Gloor et al.¹⁹ reported that TCMR often precedes TG, with the tubuloglomerular feedback mechanism hypothesized to mediate ischemic glomerular injury. Repeated non-obstructive ischemic insults to the glomerulus could lead to TG development in patients with only morphological TCMR evidence in both current and prior biopsies. In our cohort, 10.2% of TG cases exhibited only TCMR features, without ABMR or C4d positivity.

Hepatitis C virus (HCV) is another potential contributor to TG, as suggested by Gallay et al.,²⁰ who linked it to membranoproliferative glomerulonephritis (MPGN). HCV may exacerbate TG through immune dysregulation, alloimmune response upregulation, or antiphospholipid antibody production. In our study, 12 patients had prior HCV diagnoses, with 6 showing C4d positivity and 2 having immune complex deposits.

Chronic thrombotic microangiopathies can contribute to TG. In renal allograft recipients, thrombotic microangiopathy (TMA) may occur de novo or as a recurrent disease.²¹ TMA can have primary causes, including hereditary or acquired factors, but it is most commonly associated with ABMR. In our institutional study, 55% of 59 patients with de novo TMA were C4d positive, indicating that ABMR was the most frequent underlying cause.²² Other potential contributors to TMA include calcineurin inhibitor toxicity and viral infections such as cytomegalovirus (CMV), parvovirus B19, hepatitis C, and influenza. Four cases showed chronic microangiopathic changes with concurrent antibody mediated rejection features.

The predominance of CD68⁺ (53.8%) and CD163⁺ M2 (38.7%) macrophages in glomeruli suggests their critical role in TG, and graft injury. In chronic ABMR, M2 macrophages (48.7%) and T-lymphocytes (28.2%) dominated the immune infiltrates, consistent with their role in antibody-mediated endothelial damage. CD20⁺ B-cells were sparse (10.2%), reflecting their secondary role in TG compared to macrophages and T cells. These results align with prior studies, including Sablik et al.,²³ who highlighted the importance of macrophages and CD8⁺ T-cells in mediating glomerular and tubular injury in chronic ABMR. The presence of M2-polarized macrophages, marked by CD163 expression, indicates an environment conducive to tissue remodeling and contributes to glomerular basement membrane reduplication, a hallmark feature of classical TG lesions.

Hidalgo et al.²⁴ demonstrated through transcriptomic studies that NK cells might contribute to endothelial injury in ABMR. However, in our study, only two cases showed faint CD56 positivity in chronic ABMR glomeruli, indicating minimal NK cell presence documented through immunohistochemistry. Similarly, Sablik et al.,²³ using multiplex immunofluorescence staining, found only a few NK cells in CAAMR, unable to confirm their significant involvement. Parkes et al.²⁵ highlighted that >50% the transcripts increased in CD16a-activated NK cells were also elevated in activated CD8⁺ T-cells, suggesting functional overlap, including shared effector cytokines, chemokines, and molecules critical for immune function.

Macrophages were the dominant immune cells in the tubulointerstitial compartment, with CD68⁺ and CD163⁺ cells identified in 88.7% and 97.5% of cases, respectively. The admixture of T- and B-lymphocytes in this compartment underscores the complex immune interplay contributing to graft injury. CD3⁺ and CD8⁺ T-cells showed significant associations with TCMR (p = 0.004) and mixed rejection (p = 0.001).

Persistent macrophage-mediated inflammation in the tubulointerstitium likely exacerbates chronic injury through profibrotic cytokine production and promotion of fibrosis.²⁶ This aligns with prior reports indicating that M2 macrophages are key mediators of chronic graft dysfunction. Their presence in fibrotic regions highlights their role in the transition from inflammation to fibrosis, further reinforcing their central role in TG pathogenesis.^27,28

This study investigates the diverse causes of TG and characterizes inflammatory cell infiltrates in the glomeruli and tubulointerstitial (TI) compartments of affected patients. Although the sample size is limited, the findings offer valuable insights into the potential pathogenesis of TG.

The macrophage predominance, particularly M2 macrophages, and CD8⁺ T-cells in TG suggests potential therapeutic targets. Therapeutic intervention by depleting M2 macrophages may delay TG and IFTA progression, help mitigate chronic injury, and improve graft survival.