REGULAR ARTICLE

Allogeneic T cells cause acute renal injury after hematopoietic celltransplantation
Masahiro Miyata,1 Eri Matsuki,1 Kazunobu Ichikawa,1 Tomohiro Takehara,1 Yuka Hosokawa,2 Erika Sekiguchi,3 Daniel Peltier,4Pavan Reddy,5 Kenichi Ishizawa,2 Masafumi Watanabe,1 and Tomomi Toubai2

Key Points• Donor-derived T cells

are increased andactivated in the kidney
after allo-HCT.
• Allogeneic immune

responses induced bydonor T cells
contribute to renal
endothelial and tubularepithelial cell damage
after allo-HCT.

Acute kidney injury (AKI) is a frequent complication of allogeneic hematopoietic celltransplantation (allo-HCT). There are many causes of AKI after allo-HCT, but it is unknown
whether renal acute graft-versus-host disease (aGVHD) caused by direct allogeneic donor
T-cell–mediated renal damage contributes. Here, we tested whether allogeneic donor T cells
attack kidneys in murine models of aGVHD. To avoid confounding effects of nephrotoxic
agents, we did not administer immunosuppressants for GVHD prophylaxis. We found thaturinary N-acetyl-β-D-glucosaminidase, a marker of tubular injury, was elevated inallogeneic recipients on day 14 after allogeneic bone marrow transplantation. Donor major
histocompatibility complex–positive cells were present and CD3+ T cells were increased in
the glomerulus, peritubular capillaries, interstitium, and perivascular areas in the kidneysof allo-HCT recipient mice. These T cells included both CD4+ and CD8+ cells with elevatedactivation markers, increased exhaustion markers, and greater secretion ofproinﬂammatory cytokines and cytotoxic proteins. Consistent with allo-T-cell–mediatedrenal damage, expression of neutrophil gelatinase-binding lipocalin, a marker of AKI, andelaﬁn, a marker of aGVHD, were increased in renal tissue of allogeneic recipients. Because
apoptosis of target cells is observed on histopathology of aGVHD target tissues, we
conﬁrmed that alloreactive T cells increased apoptosis of renal endothelial and tubularepithelial cells in cytotoxic T-lymphocyte assays. These data suggest that immune responses
induced by donor T cells contribute to renal endothelial and tubular epithelial cell injury inallo-HCT recipients and that aGVHD may contribute to AKI after allo-HCT.

Introduction
Acute graft-versus-host disease (aGVHD) is a life-threatening complication of allogeneic hematopoietic
cell transplantation (allo-HCT). The skin, liver, and gastrointestinal tract are the organs most often
targeted and damaged by aGVHD.1 Although chronic GVHD can cause nephrotic syndrome andglomerulonephritis,2,3 the kidney is not generally recognized as a target of aGVHD.4

Submitted 9 January 2023; accepted 21 August 2023; prepublished online on Blood
Advances First Edition 25 September 2023. https://doi.org/10.1182/
bloodadvances.2023009721.
Data are available on request from the corresponding author, Tomomi Toubai(toubai@med.id.yamagata-u.ac.jp).

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The full-text version of this article contains a data supplement.
© 2023 by The American Society of Hematology. Licensed under Creative Commons
Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0),
permitting only noncommercial, nonderivative use with attribution. All other rights
reserved.

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1
Department of Cardiology, Pulmonology, and Nephrology and 2 Division of Hematology and Cell Therapy, Department of Internal Medicine III, Yamagata University School of
Medicine, Yamagata, Japan; 3 Keio University Hospital, Tokyo, Japan; 4 Division of Pediatric Hematology and Oncology, Department of Pediatrics, Herman B. Wells Center for
Pediatric Research, Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN; and 5 Dan L. Duncan Comprehensive Cancer Center, Baylor College ofMedicine, Houston, TX

Acute kidney injury (AKI) is a frequent complication of allo-HCT
occurring in >50% of patients.5 The differential diagnosis for AKI
after allo-HCT is broad, ranging from drug-induced nephropathy tothrombotic microangiopathy (TMA).6,7 Clinical studies have foundan association between aGVHD and the development of AKI8;
however, it is unknown whether aGVHD directly causes AKI.

In this report, we tested whether donor allogeneic T cells cause
kidney damage by using murine models of allo-HCT. We found that
donor-derived T cells inﬁltrate the kidney of allo-HCT recipients anddirectly attack endothelial and epithelial cells, resulting in renal
aGVHD. Thus, renal aGVHD may contribute to AKI after allo-HCT.

Methods
Mice
BALB/c (H-2d) and C57BL/6 (B6, H-2b) mice were purchasedfrom CLEA Japan, Inc (Tokyo, Japan). B6D2F1 (H-2b/d) mice werepurchased from Japan SLC, Inc (Hamamatsu, Japan). C3H.sw
mice were bred in our facility. Age-matched female mice (1014 weeks old) were used for all experiments. Animals were cared
for according to the animal welfare regulations of Yamagata University, and the study protocol was approved by the Animal Subjects Committee of Yamagata University. The investigation was
conducted in accordance with the Guide for the Care and Use ofLaboratory Animals published by the US National Institutes of
Health.

BMT
BMTs were performed as previously described.14-16 Brieﬂy, splenic
T cells from donor animals were isolated by magnetic cell sorting
using CD90.2 microbeads (Miltenyi Biotec, Bergisch Gladbach,
Germany). We used well-established HCT models. In a major histocompatibility complex (MHC)-mismatched model, BALB/c animals were used as recipients and received 7 or 4 Gy total body
irradiation (TBI; TITAN-225S, Shimadzu Industrial Systems, Kyoto,
Japan) on day −1 and were injected IV with 0.5 × 106 CD90.2+

28 NOVEMBER 2023 • VOLUME 7, NUMBER 22

Systemic analysis of GVHD and urinalysis
We monitored survival after HCT daily and assessed the degree ofclinical GVHD weekly, as described previously.17 We performed
weekly qualitative urinalysis with urine test strips that react toalbumin (URIACE-KC, Terumo, Tokyo, Japan). Concentrations ofN-acetyl-β-D-glucosaminidase (NAG) in the urine were measured
14 and 42 days after HCT by enzyme-linked immunosorbent assay
using QuantiChrom β-N-acetylglucosaminidase Assay Kit(BioAssay Systems, Hayward, CA). Assays were performed
according to the manufacturer’s protocol and read at 405 nm using
a microplate reader (xMark; Bio-Rad Laboratories, Hercules, CA).

Histopathological analysis
Kidney tissue was ﬁxed in 4% paraformaldehyde (FUJIFILM Wako
Pure Chemical, Osaka, Japan) and embedded in parafﬁn. Tissues
were sectioned (5 μm thickness) and periodic acid-Schiff (PAS)stained for light microscopic examination. To detect donor-derived
cells, immunoﬂuorescence staining for H-2b was performed using
a ﬂuorescein isothiocyanate–conjugated mouse monoclonal antimouse MHC class I H-2Kb antibody (clone AF6-88.5, LSC188568, LSBio, Seattle, WA) diluted 1:100 overnight at 4◦ C.
Vector TrueVIEW Autoﬂuorescence Quenching Kit (Vector Laboratories, Newark, CA) was used to diminish autoﬂuorescence.
Staining with 4′ ,6-diamidino-2-phenylindole (Bio-Rad Laboratories)
was also performed on each section. To detect T cells and elaﬁnpositive cells, parafﬁn-embedded kidney specimens were stainedusing an avidin-biotin-peroxidase complex technique. Primary antiCD3 rabbit polyclonal antibody (17617-1-AP, proteintech, Rosemont, IL; 1:300),and anti-elaﬁn rabbit polyclonal antibody(ab272906, Abcam, Cambridge, United Kingdom, 1:500) wereincubated with specimens, overnight at 4◦ C. Secondary horseradish peroxidase–conjugated anti-rabbit immunoglobulin G(W4018, PROMEGA, Madison, WI) was diluted 1:200 and incubated for 1 hour at room temperature. Bound antibody was
detected with 3,3’Diaminobenzidine (Takara Bio, Kusatsu, Japan).
Slides were then counterstained with hematoxylin, dehydrated, andcovered. Sections were observed using a microscope (OLYMPUS,
Tokyo, Japan). For each sample, CD3+ cells in the glomerular,interstitial, and periarterial regions were counted from 10 randomly
selected 200× magniﬁcation ﬁelds.

Cell isolation
Kidneys were minced and dissociated with collagenase D (Roche,
Basel, Switzerland) and DNase I (Roche) at 37◦ C for 50 minutes toobtain single-cell suspensions. Cells were then strained through a

DONOR T CELLS MEDIATE RENAL INJURY AFTER ALLO-HCT

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aGVHD is initiated by the activation of antigen-presenting cells(APCs) after conditioning-related tissue damage. Donor-derived T
cells are then activated and help coordinate target organ injury.9
Strategies to prevent GVHD typically use immunosuppressive
agents such as calcineurin inhibitors (CNIs), mycophenolate
mofetil, and posttransplant cyclophosphamide.1 Many of these
immunosuppressive agents are also nephrotoxic,10 which complicates efforts to determine whether aGVHD directly targets thekidneys. Determining the etiology of AKI after allo-HCT is further
complicated by the frequent inability to obtain diagnostic kidney
biopsies because of the increased risk of bleeding after HCT.10
Despite these challenges, several studies suggest that the kidneys may be targeted by aGVHD. For example, urine markers of
AKI were increased, expression of inﬂammatory cytokines in thekidneys were increased, and cellular inﬁltration of renal tubules andinterstitium were observed in allo-HCT recipient mice.11 In a rat
model of bone marrow transplant (BMT), donor-derived leukocytes
inﬁltrated the kidneys of recipient animals12; however, the classiﬁcation and function of these cells were not determined. These priorstudies indicate that alloimmune responses may contribute to kidney damage, but deﬁnitive evidence and mechanisms of direct
alloimmune-mediated kidney damage have not been established.13

T cells together with 5 × 106 bone marrow (BM) cells from either
syngeneic or allogeneic C57BL/6 (B6) donors on day 0. In aparent-to-F1 model, B6D2F1 animals received 11 Gy TBI on
day −1 and were injected with 3.5 × 106 CD90.2+ splenic T cells
together with 5 × 106 BM cells from either syngeneic or allogeneic
B6 donors on day 0. In an MHC-matched multiple minor antigen–mismatched model, C3H.sw animals received 10.5 Gy TBI on
day −1 and were injected with 1 × 106 CD90.2+ splenic T cells
together with 5 × 106 BM cells from either syngeneic or allogeneic
B6 donors on day 0. The mice were randomly assigned to syngeneic or allogeneic transplant groups in each experiment. The
investigators were not blinded to allocation during the experimentsor outcome assessment.

BALB/c (H-2d)

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D

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A

100-μm cell ﬁlter followed by incubation with red blood cell lysis
buffer (BioLegend, San Diego, CA) at room temperature for3 minutes.

Flow cytometry

Western blot analysis
Kidneys were lysed in radio-immunoprecipitation assay buffer, andtotal proteins were extracted as previously reported.20 Equal
amounts of protein extracts were subjected to 12% sodium

Cell culture
BALB/c mouse primary kidney endothelial cells (BALB-5014; Cell
Biologics, Chicago, IL) were cultured in complete mouse endothelial cell medium (M1168; Cell Biologics). BALB/c mouse primary proximal tubular epithelial cells (BALB-5015; Cell Biologics)
were cultured in complete epithelial cell medium (M6621; Cell
Biologics).

In vitro allogeneic cytotoxic T-cell assay
Effector splenic T cells from naïve B6 and BALB/c animals wereactivated by coculture with irradiated (20 Gy) red blood cell–lysed
splenocytes from BALB/c mice at an effector-to-target ratio of 5:2
for 6 days. Target cells consisted of 4 × 104 BALB/c-derived renal
endothelial cells or proximal tubular epithelial cells that were
cultured overnight on collagen-coated 24-well plates (AGC
Techno Glass, Yoshida, Japan). After aspirating the medium, 2 ×
106 effector cells suspended in RPMI supplemented with 10%
fetal bovine serum were added to each well and incubated for 4
hours. After removing the ﬂoating cells and medium, the wells were
washed with phosphate-buffered saline, followed by terminal
deoxynucleotidyl transferase–mediated deoxyuridine triphosphatenick end labeling (TUNEL) using the CF 594 TUNEL Assay
Apoptosis Detection Kit (Biotium, Fremont, CA). Staining with
4′ ,6-diamidino-2-phenylindole (Bio-Rad Laboratories) and wheat
germ agglutinin 488 (Thermo Fisher Scientiﬁc, Waltham, MA) was
also performed on each well. Cells were observed using a microscope (BZ-X700, KEYENCE, Osaka, Japan).

Figure 1. Renal injury markers are elevated in allo-HCT mice. (A-E) BALB/c mice received 7 Gy of TBI on day −1, and received transplantation with 0.5 × 106 CD90.2+
splenic T cells together with 5 × 106 BM cells from either syngeneic (syn) BALB/c or allogeneic (allo) MHC-mismatched B6 donors. (A-C) n = 6 to 10 mice per group, pooled
from 2 experiments. (D-E) n = 3 to 5 mice per group; representative data from 3 experiments are shown. (F-J) B6D2F1 mice received 11 Gy of TBI on day −1 and received
transplantation with 3.5 × 106 CD90.2+ splenic T cells together with 5 × 106 BM cells from either syngeneic B6D2F1 or allogeneic parent-to-F1 B6 donors. (F-H) n = 6 to 10
mice per group, pooled from 2 experiments. (I) n = 4 to 5 mice per group, (J) n = 3 to 5 mice per group; representative data from 2 experiments are shown. (K-O) C3H.sw animals
received 10.5 Gy of TBI on day −1 and received transplantation with 1 × 106 CD90.2+ splenic T cells together with 5 × 106 TCD-BM cells from either syn C3H.sw or
MHC-matched multiple minor antigen–mismatched B6 donors. (K-M) n = 6 to 10 mice per group, pooled from 2 experiments. (N-O) n = 3 to 5 mice per group; representative datafrom 2 experiments are shown. Survival (A,F,K), clinical GVHD score (B,G,L), body weight change (C,H,M), and urinalysis (E,J,O) are shown. Urinary NAG levels of recipient miceon day 14 after BMT was signiﬁcantly higher in the allogeneic group in the MHC-mismatched (D) and parent-to-F1 (I) models than in other models. (N) In the MHC-matched
multiple minor antigen–mismatched model, urinary NAG levels tended to be higher in the allo group than in the syn group. Results summarize at least 2 independent experiments.
Data represent the mean ± standard error of the mean (SEM). Student t test was used for statistical analysis; *P < .05; **P < .01; ***P < .001. UOB, urinary occult blood; UP,urinary protein.

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Flow cytometry was performed as previously described.16,18,19
Brieﬂy, to analyze donor T-cell expansion and activation status,
on days 14 and 42 after HCT, kidney cells from animals thatreceived transplantation were resuspended in 2% fetal bovine
serum (Sigma-Aldrich) in phosphate-buffered saline and stainedwith conjugated monoclonal antibodies (mAbs). Zombie Aqua dye(BioLegend) was used to selectively stain dead cells according to
the manufacturer’s protocol before incubation with primary antibodies. The following antibodies were used: ﬂuorescein
isothiocyanate–conjugated mAbs against mouse H-2Kd (clone
SF1-1.1, 116606; 1:200) and H-2Kb (clone AF-6-88.5, 116506;
1:200); phycoerythrin (PE)-conjugated mAbs against mouse CD69
(clone H1.2F3, 104508; 1:200), programmed cell death protein 1(PD-1) (clone 29F.1A12, 135206; 1:200), and CD62L (clone
MEL-14, 104408; 1:200); APC-conjugated mAbs against mouse
CD25 (clone PC61, 102012; 1:200), CD366 (Tim-3, clone RMT323, 119706; 1:200), and CD44 (clone IM7, 103012; 1:200);
APC/cyanine7-conjugated mAb against mouse CD3 (clone GK1.5,
100428; 1:200); and paciﬁc blue–conjugated mAbs againstmouse CD4 (clone 1A8, 127614; 1:200) and CD8a (clone 53-6.7,
100725; 1:200) (BioLegend). After staining, the cells were washedtwice and ﬁxed with BD ﬂuorescence-activated cell sorting lysing
solution (Becton, Dickinson and Company, Franklin Lakes, NJ). For
intracellular cytokine staining, cells were permeabilized after ﬁxationand stained with PE-conjugated mAbs against mouse interferon
gamma (IFN-γ; clone XMG1.2, 505808; 1:200) and granzyme B(clone QA16A02, 372208; 1:200) as well as APC-conjugatedmAbs against mouse tumor necrosis factor α (TNF-α; clone
MP6-XT22, 506308; 1:200) and perforin (clone S16009B,
154404; 1:200) (BioLegend). To detect regulatory T cells, cells
were stained with PE-conjugated mAbs against mouse FOXP3(clone MF-14, 126404; 1:200; BioLegend) after permeabilization
with Perm/Wash Buffer (Becton, Dickinson and Company). Cells
were run on a FACSMelody cell sorter (Becton, Dickinson andCompany) and analyzed using FlowJo version 10.8.1.

dodecyl sulfate–polyacrylamide gel electrophoresis and transferred
to polyvinylidene diﬂuoride membranes. Membranes were blockedwith 20 mM tris-HCl, pH 7.4, containing 150 mM NaCl, 0.1%
Tween (tris-buffered saline with Tween 20), and 5% milk or 5%
bovine serum albumin. The membranes were probed overnight at
4◦ C with the following primary antibodies: neutrophil gelatinaseassociated lipocalin (NGAL; clone H-7, sc-515876, Santa Cruz,
Dallas, TX; 1:500), elaﬁn (ab272906, Abcam; 1:1000), andβ-tubulin (no. 2146, Cell Signaling Technology, Danvers, MA;
1:3000). After incubation with horseradish peroxidase, conjugatedsecondary antibodies diluted in tris-buffered saline with Tween 20
containing 5% milk or 5% bovine serum albumin, immunoreactive
bands were detected using Clarity Max Western ECL Substrate(Bio-Rad Laboratories). The bands were recorded using the Light
Capture II system using CS Analyzer version 3.0 software (ATTO,
Tokyo, Japan). Protein expression levels were normalized toβ-tubulin.

B

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× 200

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Mononuclear cells/fieldsynCD3+ cells/cross-section

AStatistical analysis
Bars and error bars represent the mean and standard error of themean, respectively. We used unpaired t tests for statistical comparisons, except for survival analyses. P values < .05 wereconsidered signiﬁcant. Statistical signiﬁcance of survival was
determined using the log-rank test. All statistical analyses were
performed using EZR version 1.5321 and GraphPad Prism 9. All
sample sizes are detailed in each ﬁgure legend.

Renal injury marker levels are elevated in allo-HCT
mice
To test whether allogeneic immune responses cause renal injury,
we used murine models of HCT. To avoid confounding nephrotoxic
effects, GVHD prophylaxis was not used. As expected, survival was
lower, clinical signs of aGVHD were more severe, and weight loss
was more pronounced in BALB/c allogeneic recipients of MHCmismatched C57BL/6 cells relative to that in syngeneic recipients (Figure 1A-C). Levels of rinary protein and NAG, a marker oftubular damage, which were measured 14 days after HCT, weresigniﬁcantly elevated in allo-HCT recipients compared with those insyngeneic mice (Figure 1D-E). To rule out strain-dependent effects,
we used a parent-to-F1 (B6 into B6D2F1) model and observed
similar results (Figure 1F-J). In addition, we observed a similar but
nonsigniﬁcant increase in urine NAG in an MHC-matched multiple
minor antigen–mismatched B6 into C3H.sw model (Figures 1K-O).
These data collectively showed that urine markers of renal injury are
increased in allo-HCT recipient mice, suggesting that alloimmune
responses may directly cause renal damage.

Donor T cells inﬁltrate the kidneys of allogeneic
recipients and are associated with histopathologicalsigns of kidney damage.
Next, we determined whether the increased urine markers of renal
damage corresponded with renal histopathological changes. Renal
tissue was harvested 14 days after allogeneic (C57BL/6 into
BALB/c) or syngeneic HCT. We observed increased peritubular
capillaritis (PTCitis) and tubulitis accompanied by mononuclear cellinﬁltration on PAS staining in the kidneys of allogeneic micecompared with in those of syngeneic mice (Figure 2A-B). The
glomeruli appeared unaffected in both groups upon PAS staining,
and no evidence of TMA was found (Figure 2A). We used immunostaining to characterize the inﬂammatory cellular inﬁltrate.
H-2Kb–positive (donor-type) cells were found in the interstitium ofKidney-inﬁltrating donor T cells are activated andproduce inﬂammatory cytokines
To determine whether the donor T cells inﬁltrating the kidneys ofallo-HCT recipients expressed markers consistent with activationand inﬂammation, we performed ﬂow cytometry on day 14 after
HCT. We analyzed H-2Kd+ cells isolated from kidneys from syngeneic recipients and H-2Kb+ cells from kidneys of allogeneic
recipients. The amount of kidney-inﬁltrating CD4+ and CD8+ T
cells was increased in allogeneic mice (Figure 3A-B). CD4+ andCD8+antigen–experienced
effector
memoryT
cells(CD44+CD62L−) were increased in kidneys of allo-HCT recipients.
Consistent with this, activation markers (CD69 and CD25) andexhaustion markers (PD-1 and Tim-3) were also increased in T cells
isolated from the kidneys of allo-HCT recipients (Figure 3C-H;
supplemental Figure 1A-D). However, CD4+CD25+Foxp3+ regulatory T cells were not increased in the kidneys, even in the allogeneic recipients (supplemental Figure 2).
Production of the proinﬂammatory cytokines, TNF-α and IFN-γ,
were also increased in T cells isolated from kidneys of allo-HCT
recipients (Figure 4A-B; supplemental Figure 1E-F). Furthermore,
CD8+ T cells from the kidneys of allo-HCT recipients secreted
more cytotoxic markers, including granzyme B and perforin(Figure 4C-E; supplemental Figure 1G-H). Collectively, these data
demonstrated that allogeneic donor T cells in the kidney wereactivated and expressed cytotoxic proteins and inﬂammatory
cytokines that might have induced tissue damage.

Tissue injury markers were increased in the kidneysof allogeneic recipients
To determine whether alloimmune responses could produce kidneydamage, we measured the expression of protein markers of kidneydamage and alloimmune-mediated injury in the kidneys of recipient
mice on day 14 after HCT. NGAL, an indicator of AKI, wassigniﬁcantly higher 14 days after HCT in kidney lysates of allogeneic compared with that in kidney lysates of syngeneic recipients(Figure 5A-B). Similarly, elaﬁn, a tissue marker of acute GVHD,22
was higher in the allogeneic group (Figure 5C-D). Immunohistochemical staining to evaluate the localization of elaﬁn revealed that

Figure 2. Renal damage and donor T-cell inﬁltration are increased in allo-HCT recipients. BALB/c (H-2d) animals were lethally irradiated and received transplantation
with CD90.2+ splenic T cells together with BM cells from either syn BALB/c or allo MHC-mismatched B6 (H-2b) donors. The kidneys were collected from recipient mice on day 14
after HCT. (A) Representative images of PAS-stained kidneys after HCT are shown. Compared with the syn group, there was increased mononuclear cell inﬁltration in theperitubular capillaries (PTCs; see arrows in center bottom row image) and tubules (see arrows in right bottom row image) of allogeneic recipients. (B) Quantiﬁcation of inﬁltratingmononuclear cells (MNCs) in kidneys is shown. The number of inﬁltrating MNCs per 200× original magniﬁcation ﬁeld is signiﬁcantly higher in allo mice than in syn mice. (C)Representative H-2Kb (green) and 4′ ,6-diamidino-2-phenylindole (DAPI; blue) immunoﬂuorescence images are shown. H-2Kb–positive donor-derived cells were found in thekidneys of allogeneic recipients. (D) Representative images of immunohistological staining for CD3 are shown. Allogeneic recipients demonstrated increased CD3+ T-cell
inﬁltration in the peritubular, perivascular, and glomerular areas relative to syngeneic recipients. (E-G) Quantiﬁcation of T cells inﬁltrating the kidneys is shown. The absolute
number of CD3+ T cells inﬁltrating the glomeruli (E), interstitium (F), and perivascular area (G) was signiﬁcantly higher in allogeneic mice than in syngeneic mice; n = 3 to 5
mice per group. Data shown represent 2 experiments. Magniﬁcations are shown in the ﬁgures. The bars represent the mean ± SEM. Student t test was used for statistical analysis;*P < .05; **P < .01; ***P < .001 for panels B,E-G.

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Results

allogeneic mice (Figure 2C), and the number of CD3+ T cells was
signiﬁcantly increased in the glomeruli, peritubular capillaries/
interstitium, or periarterial areas of allo-HCT recipients comparedwith that of syngeneic recipients (Figures 2D-G). Thus, the kidneysof allo-HCT recipients had greater inﬂammation on histopathology
than kidneys of syngeneic recipients and were inﬁltrated withgreater amounts of donor and CD3+ cells.

synin H-2Kd+ cells
105 1.11

allo
in H-2Kb+ cells
105 0.12

10.9

36.0

105 91.8

10

103

103

102

102

101 81.9

6.13

101 2.03

5

1

allo
in CD4+ cells

6.12

105 99.3

3

4

10 10 10 10 10

104

103

103

102

102

3

4

11.1

101 1.57

5

3

4

69.2

10

5

5

1

30.8

2

3

4

10

10

3

103

10

***

0.18
0.18
101
1
2
3
4
10 10 10 10 105

CD62L

syn

CD62L

100%

allo

80%
2

other

60%

Naïve

40%

CM
EM

1

20%

****

D
8

D
4

CD25

syn

Cell count (×106)

allo

3

al
lo
_C

0.3

**

F

CD69
**

sy

E

sy

n_

C

D

4

CD8

8

0%
CD4

2
1
0

ns
syn
allo

0.2

0.1

0.0
CD4

G

CD8

CD4

H

PD-1
**

Cell count (×106)

syn
allo

3

CD8

Tim-3
1.5

****

2
1
0

**

**

syn
allo

1.0

0.5

0.0
CD4

CD8

CD4

CD8

Figure 3.
6942

MIYATA et al

0.92

102
0

101
101 102 103 104 105

3

4

98.7

10

4

0

5

5

D
***

4

4

0
0
101
1
2
3
4
10 10 10 10 105

in CD8+ cells

1020Cell count (×106)

2

10 10 10 10 10

39.9

CD3

Cell count (×106)

1

4

10 10 10 10 10

Cell count (×106)

2.04

0

101

57.7

CD3

5

102

5

10 10 10 10 10

B

102

D

2

103

in CD8+ cells

105 0.90

104

1

4

10

10 10 10 10 102.27

101 84.9

3

10

3

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105 1.65

61.8
2

10

al
lo
_C

2

0.67

4

4

C

1

CD44

10

CD44

CD4

syn
in CD4+ cells

4

4

CD8

C

n_

A

elaﬁn-positive cells were predominantly present in the distal tubularepithelium of allogeneic recipients (Figure 5E-F). These datasuggested that the kidneys in allogeneic animals were damaged byalloimmune responses.

Renal damage caused by alloreactive T cells waspresent in the late phase of allo-HCT and persisted
independent of the radiation dose used for
conditioning

In addition, we performed BMT with a reduced radiation dose (4Gy) to conﬁrm that the events observed in the kidneys of HCT
recipients were not due to radiation toxicity. Compared with myeloablative conditioning, recipients of reduced intensity conditioning
had less severe GVHD and lower urinary protein and NAG. However, the trend toward more renal damage in allogeneic recipients
than in syngeneic recipients persisted, and the number of T cells
inﬁltrating the kidney remained signiﬁcantly higher in renal
glomeruli, interstitium, and perivascular regions of allogeneic
recipients (supplemental Figure 5).

Allogeneic donor T cells directly attack renal cells
To conﬁrm whether allogeneic donor T cells could directly attack
renal cells, TUNEL staining was performed on histopathological
specimens of the kidneys of recipient animals 14 days after HCT. In
this in vivo experiment, few TUNEL-positive cells were present, evenin the allogeneic group (supplemental Figure 6). Therefore, we performed an in vitro cytotoxic T-lymphocyte killing assay. We activated
BALB/c (syngeneic) or B6 (allogeneic) effector T cells by culturing
each with irradiated BALB/c splenocytes for 6 days. These effector
T cells were then cocultured with target BALB/c-derived renal
endothelial or tubular epithelial cells. Apoptotic target cells detected
by TUNEL staining were signiﬁcantly higher when cocultured with

Discussion
AKI is a frequent complication of allo-HCT. The leading causes arethought to be nephrotoxic drugs or transplant complications.7
However, the etiology of renal injury is often not clear10 because
renal biopsies early after allo-HCT are frequently contraindicated.23
One potentially underrecognized cause of AKI after allo-HCT isrenal aGVHD. Previous studies reported an association between
aGVHD and AKI,8,24-26 but it was not known whether this association was direct or indirect.
In this report, we tested whether alloimmune responses directly
cause renal damage using murine models of HCT. To avoid theconfounding effects of nephrotoxic drugs, we did not use GVHD
prophylaxis. Compared with syngeneic recipients, allogeneic
recipients developed increased renal glomerular and tubular injury–
associated markers in the urine as well as greater donor T-cell
inﬁltration of the renal glomeruli, tubulointerstitium, and perivasculature. These inﬁltrating donor T cells expressed higher levelsof activation and exhaustion markers and secreted more inﬂammatory and cytotoxic cytokines than those from syngeneic controls.
Furthermore, activated allogeneic T cells directly caused apoptosisof cocultured renal cells, suggesting that allogeneic donor T cells
may directly mediate renal injury after allo-HCT.
Several prior studies have suggested that alloimmune responses
cause renal damage after HCT. Similar to our data, these studies
demonstrated kidney dysfunction indicated by increased urinary
albumin11 and NAG11,12 in preclinical models of allo-HCT. Thesestudies also showed renal inﬁltration of CD4+ T cells, CD8+ T cells,
FoxP3+ regulatory T cells, and macrophages, primarily in the
tubules and interstitium. However, these prior studies did not
determine whether these cells were of donor or recipient origin.
Here, we clearly showed that donor T cells inﬁltrated the kidneys ofallo-HCT recipients. We further demonstrated that these donor
T cells produced increased amounts of TNF-α and IFN-γ relative to
T cells isolated from syngeneic recipients. This is consistent withthe cytotoxic potential of these cytokines and prior reports showing
their upregulation in the kidneys of animal models of allo-HCT.11,12
Moreover, we showed that these donor T cells expressed
increased markers of activation and produced increased amountsof the cytotoxins granzyme B and perforin. These data collectively
suggest that donor T cells may directly contribute to kidney
dysfunction in allo-HCT recipients.

Figure 3. Activation and exhaustion markers were increased on kidney-inﬁltrating donor T cells from allogeneic recipients. BALB/c (H-2d) animals were lethally
irradiated and received transplantation with CD90.2+ splenic T cells together with BM cells from either syn BALB/c or allo MHC-mismatched B6 (H-2b) donors. On day 14 after
HCT, T cells inﬁltrating the kidneys (H-2Kd+ cells in syn recipients or H-2Kb+ cells in allo recipients) were analyzed by ﬂow cytometry. (A) Representative dot plots of donor
T-cell (CD3+) expansion in the kidneys are shown. (B) Absolute numbers of total CD3+CD4+ and CD3+CD8+ cells among gated live donor cells inﬁltrating the kidneys are shown.
(C-D) Subsets of donor-derived CD4+ T cells (top) and CD8+ T cells (bottom) in the kidney of the allo-HCT recipients are shown. Representative dot plots (C) and percentages ofeach subset (D) are shown. (E-H) T-cell activation (CD69 and CD25) and exhaustion (PD-1 and Tim-3) markers were analyzed in CD3+CD4+ and CD3+CD8+ populations. CD69(E), CD25 (F), PD-1 (G), and Tim-3 (H) were higher in the allo group; n = 3 to 5 mice per group. Data representing 3 experiments are shown. The bars depict the mean ± SEM.
Student t test was used for statistical analysis for panels B,E-H. Naïve, naïve T cell (CD44−CD62L+); CM, central memory T cell (CD44+CD62L+); EM, effector memory T cell
(CD44+CD62L−).

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To determine whether the renal damage observed in recipient
kidneys in the early phase of HCT (on day 14 after HCT) remained
present at later time points, we also examined the kidneys on day
42 after HCT. In this later phase of the disease, the elevation ofurinary NAG, mononuclear cell inﬁltration in histopathologicalsamples, and increased NGAL expression were observed in allogeneic recipients (supplemental Figure 3). These data suggested
that renal damage in the early phase continued in the late phase.
Analysis of donor-derived T cells by ﬂow cytometry showed that
CD4+ T-cell inﬁltration was reduced to the same extent as insyngeneic recipients, but CD8+ T-cell inﬁltration and expression ofactivation (CD69) and exhaustion markers (PD-1 and Tim-3) wereincreased in allogeneic recipients similar to that observed in theacute phase (supplemental Figure 4). These data suggested that
donor-derived allogeneic T cells remained activated and likely
mediated kidney damage late in the disease course.

allogeneic vs syngeneic effector T cells. The increase in apoptosis
after coculture with allogeneic T-cell effectors was similar between
renal endothelial and epithelial target cells (Figure 6A-C). These datasuggested that allogeneic donor T cells could directly attack renal
cells and may mediate aGVHD in the kidney.

Figure 4. Production of cytotoxic proteins and

A

B

TNF-D
ns

cytokines was increased in kidney-inﬁltrating donor
T cells of allo-HCT recipients. BALB/c (H-2d) animals

0.20

**

0.06

*

syn

Cell count (×106)

Cell count (×106)

IFN-J

allo

0.04

0.02

***

0.15

syn

were lethally irradiated and received transplantation withallo

CD90.2+ splenic T cells together with BM cells from either
syn BALB/c or allo MHC-mismatched B6 (H-2b) donors. On
day 14 after HCT, kidneys were harvested, and disrupted,

0.10

and incubated for 5 hours with phorbol 12-myristate
13-acetate, ionomycin, brefeldin A, and monensin, followed

0.05

by intracellular staining. (A-B) Donor TNF-α– (A) and IFNγ–producing (B) T cells in the kidney at day 14 after allo-HCT

0.00
CD4

CD8

CD4

CD8

are shown. (C-E) Donor granzyme B– (C-D) and perforin–
producing (E) CD8+ T cells in the kidney on day 14 after allo-C

synin CD8+ cells

allo
in CD8+ cells
100

80

80

% of max

100

60

60

40

40

20

20
0
101

0
102

103

104

105

*P < .05; **P < .01; ***P < .001.

102

1.0
0.5
0.0

103

104

syn

**

allo

0.2

0.1

CD8

The histopathological ﬁndings we observed in the kidneys of alloHCT recipients resembled those seen in the rejection of kidneyallografts. For example, acute T-cell–mediated rejection often
exhibits tubulitis, interstitial nephritis, intimal and transmural arteritis,
and mild glomerulitis on histopathology, with ﬁndings of PTCitis insome cases.27 PTCitis, tubulitis, and mild glomerulitis were present
in the kidneys of our aGVHD mouse model. Immunostaining suggested these inﬁltrating cells were most likely T cells. Thus, thepathology of T-cell–mediated allograft rejection and renal aGVHD
are likely similar. However, we acknowledge that additional mechanisms of immunological attack, such as those associated with
antibody-mediated rejection of kidney allograft,28 may also
contribute to the renal damage we observed. These possibilities
will need to be systematically examined in future studies.
Transplantation-associated TMA (TA-TMA) is a known cause of AKI
after allo-HCT. TA-TMA is thought to be due to endothelial damage

MIYATA et al

syn

0.0
CD8

6944

105

Perforin
0.3

allo

1.5

representative of 3 experiments. The bars show the mean ±Granzyme B

Cell count (×106)

Cell count (×106)***

in allogeneic recipients; n = 3 to 5 mice per group. Data are
SEM. Student t test was used for statistical analysis;

E

Granzyme B
2.0

control. Production of granzyme B (D) and perforin (E) by

41.5

Granzyme BDgranzyme B (C) are shown. Blue histograms indicate isotype
renal-inﬁltrating cytotoxic T cells was signiﬁcantly increased

22.2

101

HCT are shown. Representative ﬂow cytometry images offrom TBI, chemotherapy, and CNIs.10 Recent studies indicate thataGVHD of the vascular endothelium also contributes to TATMA.29,30 Although we found no evidence of pathological TA-TMA
in the kidneys of allo-HCT recipients, Ma et al observed ﬁndings
suggestive of TA-TMA, including mesangiolysis, mesangial proliferation, mesangial edema, subendothelial thickening, ﬁbrinoid
necrosis of afferent arterioles, microthrombi, and complement
deposition.31 Therefore, the histopathological changes of renal
aGVHD may more closely resemble that of TA-TMA rather than thecharacteristic aGVHD histopathological ﬁndings of epithelial cell
apoptosis and lymphocyte inﬁltration seen in the main aGVHD
target organs (ie, the skin, liver, and gut).32 Consistent with this, we
conﬁrmed that donor-derived cytotoxic T cells directly attack andinduce apoptosis in renal endothelial and tubular cells in vitro. We
did not observe signiﬁcant renal or endothelial cell apoptosis
in vivo. Apoptosis of target tissue cells is not always observed on
histopathologic analysis, particularly for liver aGVHD.33 However,

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0.00

A

B
*

NGAL

E-tubulin
syn

allo

8
6
4
2
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NGAL/E-tubulin (fold increase)

10

0
syn

C

allo

D

ElafinE-tubulin
syn

allo

Elafin/E-tubulin (fold increase)

2.5
**
2.0
1.5
1.0
0.5
0.0
syn

E

syn

allo

F

allo

**

50

× 200

100 µm× 200

100 µmElafin+ cells/field

40
30
20
10
0
syn

× 400

50 µm

× 400

allo

50 µm

Figure 5. Alloimmune-mediated renal tissue damage was increased in allo recipients. BALB/c animals were lethally irradiated and received transplantation with CD90.2+
splenic T cells together with BM cells from either syn BALB/c or allo MHC-mismatched B6 donors. Kidneys were harvested on day 14 after HCT to investigate the expression of
NGAL and elaﬁn in whole-kidney lysates, and immunohistochemically stained for elaﬁn. (A) Representative western blot image of NGAL is shown. (B) Quantiﬁcation of NGAL
protein level is shown. (C) Representative western blot image of elaﬁn is shown. (D) Quantiﬁcation of elaﬁn protein level is shown. (E-F) Representative images (E) andquantiﬁcation (F) of immunohistochemical staining for elaﬁn are shown. Arrows highlight elaﬁn-positive cells. Elaﬁn staining was positive mainly in distal tubular epithelial cells.
Magniﬁcations are shown in the ﬁgures in panel E. The number of elaﬁn-positive cells per 200× original magniﬁcation ﬁeld was higher in the kidneys of allo mice than in those of
syn mice in panel F. n = 3 to 5 mice per group. The data are representative of 2 experiments. The bars show the mean ± SEM. Student t test was used for statistical analysis forpanels B,D,F.

28 NOVEMBER 2023 • VOLUME 7, NUMBER 22

DONOR T CELLS MEDIATE RENAL INJURY AFTER ALLO-HCT

6945

A

B

end
50

allo

syn

TUNEL+ cells (%)

40

end

****

30
20
10

syn

C

allo

epi
50

**

TUNEL+ cells (%)

40
epi

30
20
10

TUNEL, DAPI, WGA
0
syn

allo

Figure 6. Allo donor T cells directly attack renal cells. Splenic effector T cells from either syn BALB/c or allo B6 mice were activated by coculturing with irradiated whole
splenocytes from BALB/c mice for 6 days. Target BALB/c-derived renal endothelial cells (end) or proximal tubular epithelial cells (epi) were cocultured with these activatedeffector T cells for 4 hours. Target cell apoptosis was detected by TUNEL assay. (A) Representative TUNEL-stained images of renal endothelial cells (top) and proximal tubularepithelial cells (bottom) are shown. Arrows highlight TUNEL-positive cells. TUNEL, red; DAPI, blue; wheat germ agglutinin (WGA), green. (B-C) The proportion of TUNEL-positive
cells among renal endothelial cells (B) and proximal tubular epithelial cells (C) is shown; n = 3 to 4 mice per group. The bars show the mean ± SEM. Student t test was used forstatistical analysis in panels B-C.

the lack of apoptotic renal cells in vivo may also be because of theearly examination time points. Future studies will need to explore
the evolution of renal aGVHD histopathology over time. In addition,comparing the renal histopathological changes we observed withthat observed in other potential nonclassical target organs ofaGVHD (ie, the lungs, central nervous system, and BM34) may
reveal new insights into the pathogenesis and spectrum of allogeneic immune–mediated tissue injury.
Elaﬁn is a speciﬁc marker for cutaneous GVHD.22 It is a small
protein abundantly expressed in epithelial tissues and inhibits
neutrophil elastase and proteinase-3.35 Elaﬁn increased in the urineof patients after HCT,36 suggesting that it is involved in the pathogenesis of kidney disease in HCT recipients. Interestingly, we
found that elaﬁn expression was elevated in the kidneys of allogeneic mice after HCT. These results suggest that alloimmune
responses induce markers of alloimmune-mediated tissue injury in
the kidneys of allo-HCT recipient mice. Whether urinary elaﬁnwould be useful for identifying renal aGVHD as a cause of AKI after
allo-HCT will need further study in prospective trials.
There are several limitations of our studies. First, because wecannot speciﬁcally label donor-derived cells in syngeneic

6946

MIYATA et alrecipients in our BMT models, the origin of T cells in the kidneysof syngeneic mice cannot be strictly determined. Second, we did
not use any GVHD prophylaxis, which differs from clinicalpractice. We omitted GVHD prophylaxis to avoid confounding
nephrotoxic effects of these agents. By doing so, we might haveunmasked renal aGVHD that is not clinically signiﬁcant in thesetting of GVHD prophylaxis. Although there are reports ofinﬂammatory cell inﬁltration in the renal pathology of patients
who receive allo-HCT who are on immunosuppressive drugs,6
the origin and type of cells have not been studied in detail.
Indeed, the kidney is not typically recognized as a major aGVHD
target organ despite being susceptible to other autoimmunemediated diseases such as systemic lupus erythematosus andantineutrophil cytoplasmic antibody–associated vasculitis thatcan result in AKI.37 One hypothesis for why the intensity ofalloimmune responses varies among organs is that organs differ
in their capacity to withstand immune attack.38 Indeed, renal
tissue repair after an AKI follows a robust pathway involving
dedifferentiation, proliferation, and redifferentiation of surrounding epithelial cells.39 Differences in the microbiome amongorgans may also inﬂuence alloimmune responses and contributeto differences in alloimmune-mediated damage among organs.38

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0

Notably, the kidneys are typically sterile, unlike the main aGVHD
target organs. Hence, the kidneys may have a relatively hightolerance for alloimmune responses because of a sterile environment that impedes the establishment of a severe alloimmune
reaction and the presence of robust intrinsic repair pathways.
Future studies will need to conﬁrm that renal aGVHD develops inthe setting of aGVHD prophylaxis.

Our data indicate that the kidney is targeted by aGVHD andopen several avenues of future study that may affect renal
function outcomes after allo-HCT. One future avenue is thedevelopment of a noninvasive biomarker for renal aGVHD.
Establishing such a biomarker would help clinicians tailor thetreatment of AKI to the underlying etiology in each patient. For
instance, the treatment of CNI nephropathy includes CNI withdrawal,40 which is the exact opposite treatment of aGVHD.41,42
In addition to identifying renal aGVHD biomarkers, further
research is needed to elucidate the unique aspects of renal
aGVHD pathogenesis that may lead to the development ofspeciﬁc therapies.
Collectively, our data demonstrated that donor T-cell–mediatedimmune responses (aGVHD) exacerbate renal endothelial andtubular epithelial injury in allo-HCT recipients. Uncovering kidneyspeciﬁc mechanisms of aGVHD may lead to improved renal outcomes after allo-HCT.

The authors thank Emiko Nishidate, Hiroko Sasaki, and Tomomi
Seino for their excellent technical assistance.
This work was supported by JSPS KAKENHI grants
JP20K08704 (T. Toubai) and JP21K08410 (K. Ichikawa), aJapanese Society of Hematology Research grant (T. Toubai),
Takeda Science Foundation Research grant (T. Toubai), the Amy
Strelzer Manasevit Research Program, which is funded through the
Be The Match Foundation (D.P.), and National Heart, Lung, andBlood Institute of the National Institutes of Health grant K08
HL157619 (D.P.).

Authorship
Contribution: M.M. designed and performed experiments, analyzed
the data, and wrote the manuscript; E.M., T. Takehara, Y.H., andE.S. performed experiments; D.P. critically reviewed and edited themanuscript; P.R. provided advice on the experimental design andcritically reviewed the manuscript; M.W. and K. Ishizawa provided
advice on the experimental design; and T. Toubai designed andsupervised the research and wrote the manuscript.
Conﬂict-of-interest disclosure:competing ﬁnancial interests.

The

authors

declareno

ORCID proﬁle: T.T., 0000-0001-6658-7543.
Correspondence: Tomomi Toubai, Division of Hematology andCell Therapy, Department of Internal Medicine III, Yamagata University School of Medicine, 2-2-2, Iida-Nishi, Yamagata 990-9585,
Japan; email: toubai@med.id.yamagata-u.ac.jp; and Kazunobu
Ichikawa, Department of Cardiology, Pulmonology, andNephrology, Yamagata University School of Medicine, 2-2-2, IidaNishi, Yamagata 990-9585, Japan; email: ichikawa-k@med.id.
yamagata-u.ac.jp.

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Additional limitations of our study include that TBI conditioning
might have contributed to the renal injury we observed; however,
we controlled for this by including a syngeneic group also
conditioned with TBI. Prerenal AKI due to dehydration fromexpected greater diarrhea in the allo-HCT recipients also might
have contributed to the renal damage we observed but would notaccount for the activated donor T-cell inﬁltration seen in allo-HCT
recipient mice.

Acknowledgments

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