Hyperacute Rejection of a Kidney Allograft Associated with Anti-Histidyl-tRNA Synthetase and Anti-Signal Transducer and Activator of Transcription 6 Antibodies

Introduction

Hyperacute rejection represents 1 of the most catastrophic forms of kidney allograft failure, typically driven by preformed donor-specific antibodies targeting human leukocyte antigen (HLA) or ABO blood group antigens.1, 2, 3 With routine pretransplant anti-HLA antibody screening and lymphocyte crossmatch testing, the incidence of hyperacute rejection has markedly declined.4, 5, 6 Nevertheless, its occurrence in ABO-compatible and lymphocyte crossmatch negative recipients remains an alarming clinical scenario.^7,8

Recent insights into transplant immunology have expanded the scope of alloimmune injury beyond the traditional HLA paradigm.^8,9 Non-HLA antibodies have been increasingly implicated in antibody-mediated rejection (ABMR).^9,S1,S2 However, the contribution of non-HLA antibodies to hyperacute rejection was rarely reported.S1

In this report, we describe a case of hyperacute rejection in a kidney transplant recipient with no detectable HLA sensitization. Retrospective investigations revealed the presence of non-HLA antibodies targeting histidyl-tRNA synthetase (HARS) and signal transducer and activator of transcription 6 (STAT6).

Key teaching points of this case are presented in Table 1.

Table 1.

Teaching points

The occurrence of hyperacute rejection in ABO-compatible and lymphocyte crossmatch negative kidney transplantation remains a clinically alarming scenario.

Retrospective investigations identified the preformed non-HLA antibodies targeting HARS and STAT6 in a patient who developed hyperacute rejection following kidney transplant, despite the absence of ABO incompatibility and HLA sensitization.

Adding non-HLA antibody screening alongside conventional immunological assessment may further reduce the risk of hyperacute rejection in kidney transplantation.

HARS, histidyl-tRNA synthetase; HLA, human leukocyte antigen; STAT6, signal transducer and activator of transcription 6.

Case Presentation

A 45-year-old male with end-stage renal disease of unknown etiology had been on hemodialysis for the past 3 years and had no previous history of transfusions, autoimmune diseases, or organ transplants. The patient underwent ABO-compatible deceased donor kidney transplantation in June 2024. Pretransplant immunological screening showed no detectable anti-HLA antibodies using LABScreen Single Antigen Kits (One Lambda, Thermo Fisher Scientific, Los Angeles, CA) and negative complement-dependent cytotoxicity crossmatch assays with both donor T and B cells. The warm ischemia time was 4 minutes and the cold ischemia time was approximately 6 hours. Induction immunosuppression consisted of i.v. basiliximab (20 mg) and methylprednisolone (500 mg), followed by maintenance therapy with tacrolimus (3 mg twice daily) and enteric-coated mycophenolate sodium (720 mg twice daily). Intraoperative urine output was approximately 200 ml, and there were no other immediate complications.

Within the first hour post-transplant, urine output declined to less than 50 ml/h and continued to decrease over time. The patient began to report discomfort at the allograft site, which progressed to severe pain. Bedside Doppler ultrasonography demonstrated patent arterial and venous anastomoses but showed markedly reduced intrarenal perfusion. Then a noncontrast computed tomography scan showed perinephric fluid accumulation and hemorrhage. Emergent surgical exploration was conducted approximately 13 hours post transplantation, revealing allograft cyanosis, venous thrombosis but patent arterial anastomosis. A graft nephrectomy was performed, along with iliac vein thrombectomy and vascular repair. The patient subsequently achieved hemodynamic stability and resumed maintenance hemodialysis.

Kidney Allograft Biopsy

Histopathological examination of the excised allograft revealed characteristics of hyperacute rejection (Figure 1). Macroscopically, the graft was congested with cortical discoloration. Microscopically, extensive thrombi were identified in both glomerular and peritubular capillaries, and there were thrombi in focal small arteries. The glomerular and peritubular capillaries exhibited neutrophilic infiltration. There was marked fibrinoid necrosis of interlobular arteries, widespread intimal thickening with luminal narrowing of arteries, and prominent interstitial hemorrhage. Immunohistochemical studies revealed the widespread deposition of IgG on endothelial cells of glomerular capillaries, peritubular capillaries, and focal small arteries. C4d staining was detected in focal segmental glomerular capillary loops and endothelial cells of small arteries; however, peritubular capillaries were C4d-negative. Additionally, direct immunofluorescence studies demonstrated positive C1q staining in focal interlobular arteries.

Figure 1.

Histopathological examination of the excised kidney allograft. Kidney allograft biopsy showed: (a) congestion with cortical discoloration, (b) diffuse thrombi in both glomerular and peritubular capillaries (arrows), (c) thrombi in interlobular arteries, (d) neutrophil infiltration in glomerular capillaries (arrow), (e) neutrophilic peritubular capillaries (arrows), (f) fibrinoid necrosis of interlobular arteries superimposed on intimal thickening, (g) intimal thickening with luminal narrowing in interlobular arteries (b–g: hematoxylin and eosin (H&E), original magnification ×400), (h) diffuse interstitial hemorrhage (H&E, original magnification ×200), (i) deposition of IgG within glomerular capillaries, (j) IgG deposited on endothelial cells of peritubular capillaries and afferent arterioles (arrows), (k) IgG accumulation on endothelial cells of interlobular arteries (i–k: IgG immunoperoxidase stain, original magnification ×400), (l) C4d staining detected in segmental glomerular capillary loops, (m) C4d deposited on endothelial cells of afferent or efferent arteriole but not peritubular capillaries, (n) C4d staining observed on the endothelial cells of interlobular artery (l–n: C4d immunoperoxidase stain, original magnification ×400), (o) C1q staining on endothelial cells of interlobular arteries (immunofluorescence, original magnification ×400).

Non-HLA Antibodies Detection

Post-transplant anti-HLA antibody testing remained negative. Archived pre- and post-transplant serum samples were evaluated for non-HLA antibodies. Testing for antimajor histocompatibility complex class I-related chain A and anti-angiotensin II type 1 receptor antibodies was negative. However, antibodies against HARS and STAT6 showed strong signal. The median fluorescence intensity of anti-HARS antibodies in the pre- and post-transplant serum samples were 9710 and 8133, respectively, and those of anti-STAT6 antibodies were 4300 and 2646, respectively (LIFECODES non-HLA Antibody kit, Immucor, Norcross, GA; cutoffs for anti-HARS and anti-STAT6 antibodies were set at 1941 and 957, respectively, based on 3-times the average of nontransfused males). The flow cytometry crossmatch demonstrated patient serum IgG bound to human umbilical vein endothelial cells but not to donor-derived peripheral blood mononuclear cells, suggesting that the antibodies targeted endothelial cell rather than lymphocytic antigens (Supplementary Figure S1).

Immunohistochemical staining for HARS and STAT6 revealed widespread HARS expression in endothelial cells of glomerular capillaries, peritubular capillaries and small arteries, and diffuse STAT6 expression in endothelial cells of afferent and efferent arterioles (Supplementary Figure S2). Furthermore, no pathogenic mutations were identified in genes related to immune dysregulation, thrombophilia, or complement regulation through whole exome sequencing.

Discussion

This patient developed hyperacute rejection following kidney transplant, despite the lack of HLA sensitization and ABO incompatibility. After extended testing, non-HLA antibodies against HARS and STAT6 were identified in pre- and post-transplant serum samples.

Hyperacute rejection manifested with negative C4d staining for peritubular capillaries in this patient. Evidence indicated C4d-negative ABMR in many cases and C4d staining in peritubular capillaries is no longer a mandatory feature for ABMR diagnosis in kidney allografts.^2,S3 Furthermore, antibody-dependent cell-mediated cytotoxicity is another important mechanism for ABMR.S4–S6 We speculate that hyperacute rejection in this patient was primarily driven by antibody-dependent cell-mediated cytotoxicity.

HARS (Jo-1) is a homodimer enzyme responsible for charging tRNA with histidine, mainly located in the cytoplasm.S7 However, it is best known as a frequent target of autoantibodies in idiopathic inflammatory myopathies, particularly antisynthetase syndrome.S8 The exposure of HARS is thought to arise because of injury or infections.S7,S9 The presence of anti-HARS antibodies within 6 months post-transplant was found to be significantly associated with the development of chronic kidney allograft injury.S2 STAT6 is a transcription factor well studied in the context of autoimmune diseases and allergic inflammation.S10,S11 More recently, pretransplant anti-STAT6 antibodies were identified as an independent risk factor for the histology of ABMR in kidney allograft.S12

Given the high level of concordance between the HARS and STAT6 expression areas and the IgG deposition sites, we hypothesize that intracellular HARS and STAT6 in endothelial cells were exposed because of ischemia-reperfusion injury and recognized by preexisting antibodies, initiating ABMR process. Non-HLA antibodies against intracellular proteins are believed to recognize cryptic antigens that become exposed following tissue damage prompted by ischemia-reperfusion injury, alloimmunity, or chronic inflammation.S6,S13 Intracellular antigen exposure induced by ischemia-reperfusion injury has been verified in mouse models and in vitro experiments.S14,S15

Non-HLA antibody cutoffs were established by the manufacturer as 3 times the average median fluorescence intensity of nontransfused males, in the absence of clinically relevant thresholds anchored to a clinical end point. Senev et al.S12 found that pretransplant anti-STAT6 antibodies were independently associated with ABMR histology using vendor-defined cutoff in 774 kidney transplant patients. In an analysis of 74 patients on the kidney transplant waiting list, the positive frequencies for anti-STAT6 and anti-HARS antibodies were 18.9% and 6.8% respectively according to the manufacturer’s instructions.S16 Notably, when a calculated cutoff for anti-HARS antibodies was set as mean plus 2 times SD for median fluorescence intensity of healthy individuals (calculated cutoff = 2234; vendor-recommended cutoff = 1326) was applied, it was negative in all 30 ABMR negative kidney transplant recipients on the day of transplant, but positive in 7.0% ABMR ones.⁹ Therefore, the markedly elevated levels of anti-HARS antibodies in this patient were highly likely to have contributed to allograft injury.

The rapid progression to thrombosis, ischemia, and graft loss in our patient—despite negative traditional immunologic screening—underscores a critical limitation of standard pretransplant risk assessment protocol. Traditional panel-reactive antibody testing and complement-dependent cytotoxicity crossmatch assays focus on lymphocyte-expressed HLA antigens and fail to detect antibodies that bind exclusively to endothelial non-HLA targets. This case highlights the need for non-HLA antibody screening before transplant.

There are several limitations in this case study. First, there is no direct evidence that anti-HARS and anti-STAT6 antibodies were directly triggered or involved in hyperacute rejection. Other undetected non-HLA antibodies might have been involved in the acute rejection process. Secondly, the positive flow cytometry crossmatch with human umbilical vein endothelial cells could also result from other unknown non-HLA antibodies.

In summary, we present a case of hyperacute rejection in kidney transplant associated with non-HLA antibodies against HARS and STAT6. This case illustrates the potential detrimental effect of non-HLA antibodies in the development of hyperacute rejection and underscores the need for more comprehensive immunologic assessment before transplant. Future researches are needed to define the clinically meaningful cutoffs and pathogenic mechanisms of non-HLA antibodies in kidney transplantation.

Disclosure

All the authors declared no competing interests.

Patient Consent

The authors declare that they have obtained consent from the patients discussed in this report.

Supplementary Material

Supplementary File (PDF)

Supplementary References. Figure S1. The seroreactivity against human umbilical vein endothelial cells and donor-derived peripheral blood mononuclear cells in the pre- and post-transplant sera from the patient was examined by flow cytometry crossmatch. Figure S2. Immunohistochemical staining of HARS (rabbit monoclonal primary antibody (Abcam, Waltham, MA)) and STAT6 (rabbit monoclonal primary antibody (LBP SYSTEM, Guangzhou, China)) in the excised kidney allograft.