Kamatani et al. Inflammation and Regeneration
https://doi.org/10.1186/s41232-021-00190-7(2022) 42:4

RESEARCH ARTICLE

Inflammation and Regeneration

Open Access

Evaluation of immunosuppression
protocols for MHC-matched allogeneic iPS
cell-based transplantation using a mouseskin transplantation model
Tomoki Kamatani1, Ryo Otsuka1, Tomoki Murata1, Haruka Wada1, Takeshi Takahashi2, Akihiro Mori3,
Soichiro Murata3, Hideki Taniguchi3,4 and Ken-ichiro Seino1*

Abstract
Background: Off-the-shelf major histocompatibility complex (MHC)-matched iPS cells (iPSC) can potentially initiate
host immune responses because of the existence of numerous minor antigens. To suppress allo-immune responses,
combination of immunosuppressants is usually used, but its efficacy to the allogeneic iPSC-based transplantation
has not been precisely evaluated.
Methods: Three transplantation models were used in this study; MHC-matched, minor antigen-mismatched mouseskin or iPSC-graft transplantation, and fully allogeneic human iPSC-derived liver organoid transplantation in immunehumanized mice. The recipients were treated with triple drugs combination (TDC; tacrolimus, methylprednisolone, andmycophenolate mofetil) or co-stimulatory molecule blockade (CB) therapy with some modifications. Graft survival aswell as anti-donor T and B cell responses was analyzed.
Results: In the mouse skin transplantation model, immunological rejection caused by the minor antigen-mismatch
ranged from mild to severe according to the donor-recipient combination. The TDC treatment could apparently
control the mild skin graft rejection when combined with a transient T cell depletion, but unexpected anti-donor T or
B cell response was observed. On the other hand, CB therapy, particularly when combined with rapamycin treatment,
was capable of attenuating both mild and severe skin graft rejection and allowing them to survive long-term without
any unfavorable anti-donor immune responses. The efficacy of the CB therapy was confirmed in both mouse andhuman iPSC-derived graft transplantation.
Conclusions: The findings suggest that the CB-based treatment seems suitable to well manage the MHC-matchedallogeneic iPSC-based transplantation. The TDC-based treatment may be also used to suppress the rejection, but
screening of its severity prior to the transplantation seems to be needed.
Keywords: iPS cell transplantation, Transplantation immunology, MHC-matched, Minor antigen, Immunosuppressive
agents, Costimulatory molecule blockade, Humanized mouse* Correspondence: seino@igm.hokudai.ac.jp
1
Division of Immunobiology, Institute for Genetic Medicine, Hokkaido
University, Kita-15, Nishi-7, Sapporo, Hokkaido 060-0815, Japan
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which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
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Kamatani et al. Inflammation and Regeneration(2022) 42:4

Introduction
In the field of transplantation medicine, promoting graft
acceptance and prolonging graft survival are important
issues because of the limited number of donors [1]. Rejection is a major cause of graft loss, and many studies
have focused on preventing rejection for decades [2].
Major histocompatibility complex (MHC), or humanleukocyte antigen (HLA) in humans, plays an importantrole in the immune response after allogeneic transplantation as they are expressed on the cell surface andundergo surveillance by recipient immune cells, especially T cells [3]. Therefore, in order to reduce immunological barriers, efforts have been devoted to matching
HLA gene haplotypes between donors and recipients ofsolid organ or bone marrow transplantation [4–6]. Although the main cause of graft loss is HLA mismatch,other antigens, such as minor antigens, can also cause
rejection in human [7]. In clinical practice, two large
multicenter studies using independent registry data
showed decreased long-term survival of kidney transplants performed between HLA haplotype-matchedsiblings, emphasizing the importance of non-HLA immunity to allogeneic grafts [8, 9]. More recently, genetic mismatch of a kidney-expressed antigen has
shown its contribution to a higher risk of rejection inhuman [10].
The establishment of iPS cells (iPSC) by Takahashi
and Yamanaka in 2006 has ushered in a new era for thefields of regenerative medicine, stem cell biology, anddrug discovery [11]. In regenerative medicine, cell therapy using iPSC is expected to be a promising solution todonor organ shortage [12]. Practically speaking, however,clinical-grade autotransplantation that meets quality assurance is unlikely to become standard therapy due toits high cost and long preparation time per patient [13,14]. Therefore, the project has launched to establish wellqualified “off-the-shelf” HLA-homozygous iPSC stock,which is considered to be effective at the point of reducing or avoiding immunological rejection [15, 16]. However, rejection caused by minor antigens mismatch
remains to be resolved. Our previous study has shownthat it was difficult to control rejection of MHCmatched mouse skin grafts even with high-dose tacrolimus (Tac) immunosuppression [17]. In addition, there isa report that MHC-matched iPSC-derived cardiomyocytes transplanted into non-human primates survived
for 12 weeks but needed immunosuppressive therapy
with methylprednisolone (MP) and Tac [18]. As indicated by these observations, our understanding of theimmunological response to MHC-matched iPSC-based
allogeneic transplantation and the optimal immunosuppression strategy are still incomplete.
In the present study, we examined the efficacy of traditional combinatory immunosuppressants treatment andPage 2 of 13

of targeted therapy to T cell co-stimulatory molecules
on MHC-matched transplantation, measured by thestringent transplantation model of skin grafting. We
found that susceptibility to rejection and response totreatment varied among MHC-matched donor-recipient
pairs. Conventional immunosuppressants exerted theireffect on mild rejection combination while costimulatory pathway-directed therapy could well-manage
rejection regardless of its severity. We further evaluatedthese treatment efficacies in mouse and immune humanized mouse transplantation model of iPSC grafts, which
presented consistent results to our observation in theskin transplantation model.

Methods
Mice

Male BALB/c (haplotype: H2d/d), C57BL/6 J (B6) (H-2b/b), CBA/N (CBA) (H-2 k/k), 129X1/SvJ (H-2b/b), and female C3H/He (H-2 k/k) were purchased from Japan
SLC, Inc. (Shizuoka, Japan). Then, 129X1/SvJ males werecrossed with C3H/He females to generate male C3129F1(F1) mice. Nonobese diabetic/Shi-scid, IL-2RγKO Jic(NOG) mice were purchased from In-Vivo Science Inc.
(Tokyo, Japan), and immune humanized mice were generated as follows. Male and female NOG mice were irradiated with 160 cGy of X-rays (MBR-1520R-4, Hitachi,
Hitachi, Japan), and the umbilical cord blood CD34+
cells (5 × 104 cells, StemExpress, Folsom, CA, USA) weretransplanted intravenously on the next day. To analyze
the hematopoietic chimerism in mice reconstituted withthe human immune system, a multicolor flow cytometricanalysis was performed using BD LSRFortessa (BD
Biosciences, Franklin Lakes, New Jersey, USA). The
peripheral blood was periodically collected from theretro-orbital venous plexus using capillary pipettes
with sodium heparinization (Paul Marienfeld GmbH &Co.KG, Lauda-Königshofen, Germany) under anesthesia
with isoflurane every 4 weeks. Red blood cells were lysedusing an ammonium-chloride-potassium solution (150mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA-Na2),and the mononuclear cells were stained with antibodies
for flow cytometry. Human CD45 chimerism was calculated using human CD45+ cells relative to the total CD45+
cells, which included the human and mouse CD45+ cell
populations.
All animal procedures were approved by the Hokkaido
University Animal Care Committee (approval number:
18-0004) and the Central Institute for Experiment andYokohama City University’s animal experiment committee (approval number: F-A-17-025, F-A-20-021). All animal experiments were performed in accordance with the
ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

Kamatani et al. Inflammation and Regeneration(2022) 42:4

Cell culture

Mouse iPSC were maintained on laminin-coated culture
dish with maintenance medium consisted of advancedDMEM/F-12 (1:1 mixed, Thermo, SIGMA) supplemented with 0.5 × Neuro Brew-21 (Miltenyi), 0.5 × N2
supplement (Wako), and 10 U/ml penicillin, 100 μg/ml
streptomycin, 0.1 mM 2-mercaptoethanol (Nacalai),
0.03% L-glutamine (Gibco), 3 μM CHIR99021 (Adooq),
1 μM PD0325901 (Tocris), and recombinant humanleukemia inhibitory factor (produced in our laboratory).
Fibroblasts were maintained on gelatin-coated culture
dish with DMEM (Wako) supplemented with 10% fetal
bovine serum (Sigma), 0.1 mM non-essential aminoacids, 1 mM sodium pyruvate, and 10 U/ml penicillin,
100 μg/ml streptomycin. Cells were maintained in a 5%
CO2/air environment at 37 °C.
Luc-iPSC generation

CBA and B6-derived iPSC were generated from fibroblasts isolated from fetal CBA and B6 mice and validated
by previously described methods [19]. Luciferase (Luc)
lentiviral particles were generated by transfecting LentiX 293 T cells with psPAX2 (Addgene, #12260), pMD2.5(Addgene, #12259), and pHIV-Luc-ZsGreen (Addgene,#39196) using polyethylenimine max. Supernatants containing lentiviral particles were collected and used to infect mouse iPSC, which were then continuously selectedby ZsGreen expression.
Human iPSC-derived liver organoids generation

The generation of human iPSC-derived liver organoids
was carried out as described previously [20]. We collected hepatic endoderm, endothelial, and mesenchymal
cells and seeded them on Elplasia six-well plates (Corning, Corning, NY, USA). The seeded cell number forhepatic endoderm was 2.5 × 106 cells, for endothelial, 1
× 106 cells, and, for mesenchymal cells, 1 × 106 cells per
well. The culture medium used was the same as reported
previously [21]. Y-27632 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was added on day one.
On day two, small liver organoids were collected andreseeded on a cell culture insert (Corning, Corning, NY,
USA). The medium was changed every other day.
Transplantation

For mouse skin transplants, recipient mice were anesthetized by intraperitoneal injection of a three-drug mixtureof medetomidine (Domitor Nippon Zenyaku Kogyo Co.,
Ltd.), midazolam (midazolam injection, TEVA, Takeda
Pharmaceutical Co., Ltd.), and butorphanol (Vetorphale,
Meiji Seika), and the dorsal side of the auricle skins fromdonor mice were transplanted into dorsal thoracic of therecipients. Recipient mice were wrapped with bandagesfor 7 days to protect skin grafts and warmed up until

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they moved freely as previously described [22]. To assess
rejection, the graft diameter was measured. The day ofgraft rejection was determined as the day on which thegraft diameter reached less than 30% of the initial diameter or the recipients died. Graft survival was calculatedusing the following formula: (total number of transplanted grafts − number of rejected grafts)/total numberof transplanted grafts × 100. All the mice were euthanized by cervical dislocation at the end of experiments.
For mouse iPSC-derived Luc-iPSC-graft transplants,
recipient mice were anesthetized by intraperitoneal injection of a three-drug mixture of medetomidine, midazolam, and butorphanol, and 2.5 × 106 CBA or B6derived Luc iPSC were injected into the gastrocnemius
muscle of recipient mice. Transplanted cell survival was
longitudinally monitored via in vivo bioluminescent imaging (BLI) by using IVIS Spectrum Imaging Systems(Spectrum-FL-TKHD; Caliper Life Sciences Ltd.) (POD0,2, 4, 6, 8, 11, 13, 17, 21, 25). D-Luciferin (Cayman) was administered intraperitoneally at a dose of 120 mg/kg ofbody weight. After 20 min from the administration of DLuciferin, recipient mice were placed in a light-tightchamber, and photons emitted from Luc-expressing cells
were collected with integration times of 20 s. BLI signal
was quantified in maximum photons per second per centimeter square per steradian (p/s/cm2/sr) and presented aslog10 [photons per second]. All the mice were euthanizedby cervical dislocation at the end of experiments.
For liver organoids transplants, the mice were anesthetized by isoflurane and the opened peritoneum. The
mesothelium of the left liver lobe was peeled off, and
iPSC-derived liver organoids were transplanted onto thepeeled-off portion. The transplanted cell number was 2
× 106 cells in hepatic endoderm equivalents. After thetransplantation, the transplant was covered with the
middle lobe of the liver and the closed peritoneum. All
the mice were euthanized by cervical dislocation at theend of experiments [23].
Immunosuppressants

For the conventional triple drugs combination (TDC)
treatment, tacrolimus (Astellas, Japan), methylprednisolone (Cayman, #15013), and mycophenolate mofetil(CHUGAI PHARMACEUTICAL CO., Ltd., Japan) wereadministered once daily by intraperitoneal injections,
0.5 mg/kg/day for tacrolimus, 5 mg/kg/day for methylprednisolone, and 100 mg/kg/day for mycophenolate
mofetil. For T cell depletion, anti-CD4 (GK1.5) and CD8(53-6.72) monoclonal antibodies (mAb) (purified in ourlaboratory) were administered by intraperitoneal injections on days -6, and -1 post-transplantation days. For
co-stimulatory molecule blockade (CB) therapy, antiCD40 ligand (CD40L) (MR-1 for mouse and #BE0292
for human, BioXcell) and CTLA4Ig (Orencia®, Bristol

Kamatani et al. Inflammation and Regeneration(2022) 42:4

Myers Squibb for mouse and #BE0099, BioXcell for human) were administered at a dose of 500 μg for antiCD40L, 400 μg for CTLA4Ig on days 0, 2, 4, and 6 after
transplantation. Rapamycin (LC Laboratories, R-5000)
were administered 2.0 mg/kg by intraperitoneal injections once every 3 days starting the day before transplantation. We have determined the dosage of
immunosuppressants referring to previous reports [24–27]. Especially for TDC, we made the dosage of themmore than those used in human (per kilogram). We havemeasured bodyweight of the treated mice and found that
there was no abnormal weight-loss in each treatmentgroup (Additional file 1: Supplementary Fig. 4E-H).

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Measurement of alloantibody production

In vivo antibody production was analyzed as previously
described [22]. Briefly, 100 days after skin graft transplantation, the sera were harvested from F1 recipient
mice by blood collection. To detect antibodies against
the donor antigen in the sera, thymocytes from BALB/c,
B6, CBA, or F1 mice Fc receptor were blocked and thenincubated with the sera. After washout of unbound antibodies, the cells were further incubated with Alexa
fluor-488-conjugated goat anti-mouse IgG antibody andPE anti-mouse CD19 and then were analyzed with an
FACSCelesta and FlowJo software.
Human albumin quantification

HE and immunohistochemistry

The skin grafts were fixed in 4% paraformaldehyde andembedded in paraffin. The blocks were sliced into 5-μm
sections and stained with anti-mouse CD3ε (A0452,
Dako, Glostrup, Denmark) as the primary antibody andthen with anti-rabbit IgG-horse radish peroxidase(K4003, Dako) as a secondary antibody. Antibody binding was detected with a chromogenic substrate forhorseradish peroxidase. The samples were counterstained with hematoxylin and eosin (HE) or Kernechtrot(Merck Millipore, Billerica, MA, USA).

Quantification of human albumin was carried out as described previously [21]. All blood samples of the mice
were collected before transplantation and then were collected every week after transplantation. Blood samples
were centrifuged by 4000 rpm, 20 min at 4 °C, and theplasma was collected. The collected plasma was diluted,
and the human albumin concentrations were assessed byenzyme-linked immunosorbent assay (Bethyl Laboratories, Montgomery, TX, USA), according to the manufacturer’s instructions.
Statistical analyses

Flow cytometry and antibodies

Flow cytometry was performed using FACSCelesta (BD
Biosciences), SH800S (SONY), or FACSAria II (BD
Biosciences), and the data were analyzed by the
FlowJo software V10.7.1 (Tree Star, Ashland, OR,
USA). The antibodies used in FACS were listed in
Additional file 2: Supplementary Table 1. For analysis,
live cells were gated based on forward and side
scatter as well as a lack of propidium iodide (PI) or
4′,6-diamidino-2-phenylindole (DAPI) uptake.
Mixed lymphocyte reaction

To assess the recipients’ T cell response, we performed amixed lymphocyte reaction as follows. Recipient mice
fresh whole splenocytes were used as responder cells. As
stimulator cells, 30 Gy-irradiated fresh whole splenocytes
from BALB/c, B6, CBA, or F1 were used. Carboxyfluorescein diacetate succinimidyl ester (CFSE) (Dojindo Laboratories, Kumamoto, Japan)-labeled responder cells (2× 105) were co-cultured with stimulator cells (8 × 105) in
96-well round-bottomed culture plates in RPMI-1640
high-glucose supplemented with 10% fetal bovine serum,
0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 100 U/mL penicillin,and 100 μg/mL of streptomycin (all from Life Technologies, Carlsbad, CA, USA). On day 4 of culture, T cellproliferation was assessed by measuring the reduction in
CFSE fluorescence by flow cytometry.

Statistical data were analyzed with the JMP software(JMP Pro Version 15.2.0, SAS Institute, Inc., Cary, NC,
USA; JMP license was distributed by Hokkaido University Graduate School of Medicine.). Kaplan-Meier survival curves were analyzed using the log-rank test or
Tukey’s honestly significant difference (HSD) test. P
values less than 0.05 were considered to indicate statistically significant results.

Results
Application of standard immunosuppressive therapy inhuman solid organ transplantation to MHC-matchedmouse skin transplantation

We first attempted to control rejection in an MHCmatched mouse skin graft model, which imitates an
iPSC-based tissue transplantation, by conventional immunosuppressive therapy commonly used in clinicalsolid organ transplantation. MHC homo-to-hetero skintransplantation was performed using a method based on
our previous report [17] where F1 (H-2b/k) mice weregrafted with skin from B6 (H-2b/b), CBA (H-2 k/k), or
BALB/c (H-2d/d) mice (Fig. 1A). Without any treatment,
the grafts from every donor were completely rejected
within 27 days [17]. Recipients were treated with a calcineurin inhibitor (tacrolimus, Tac), inosine monophosphate dehydrogenase inhibitor (mycophenolate mofetil,
MMF), and corticosteroid (methylprednisolone, MP),that have long been combinatory used to control

Kamatani et al. Inflammation and Regeneration(2022) 42:4

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Fig. 1 Standard immunosuppressive therapy in solid organ transplantation has limited efficacy in mouse skin transplantation, even in MHCmatched situations. (A) Schematic overview of auto or allogeneic skin transplantation. (B) TDC-based immunoregulation protocol.
Immunosuppression was performed by using a protocol of administrating a combination of methylprednisolone, tacrolimus, and mycophenolate
mofetil daily (TDC) with or without anti-CD4 mAb and anti-CD8mAb on day 6 and 1 (TDC mAb). (C) Skin graft survival under immunosuppression
with TDC (n = 9, C3129F1; n = 9, BALB/c; n = 9, C57BL/6; n = 9, CBA/N). (D) Skin graft survival under immunosuppression with TDC + mAb (n =10, C3129F1; n = 11, BALB/c; n = 10, C57BL/6; n = 9, CBA/N). (E) Macroscopic observation (upper panels) and hematoxylin and eosin stained
section (lower panels) of autologous (C3129F1) and CBA/N skin grafts on day 100. Scale bars: 100 μm. (F) Immunohistochemical staining for CD3of CBA/N skin grafts harvested from TDC or TDC + mAb treated groups on 100 days post-transplantation. Upper and lower panels represent CD3
specific staining and isotype control stained section, respectively. Scale bars: 100 μm. (G) Recipient T cell response in MHC-matched but minor
antigen-mismatched skin transplantation. The T cell proliferation rates in each treatment group were normalized to that of C3129F1 micestimulated with autologous irradiated splenocytes. Error bars indicate standard error of technical triplicates. Similar results were obtained in two
independent experiments. *p < 0.05, **p < 0.01 (Tukey’s HSD test). (H) De novo anti-donor antibody production in the recipients. Error bars
indicate standard error of biological replicates (n = 8, naive; n = 4-5, non-treatment; n = 9, TDC; n = 7, TDC + mAb). tx, transplantation; mAb,
monoclonal antibody; TDC, three drug combination; POD, post-operative day

rejection in human MHC-mismatched transplantation(Fig. 1B) [28]. In the triple drugs combination (TDC)
group, recipient F1 mice rejected MHC-mismatched
BALB/c skin grafts within 21 days with a median survival
time (MST) of 17 days (Fig. 1C). Similarly, MHCmatched but minor antigen-mismatched B6 skin grafts
were rejected within 25 days (MST = 21 days) (Fig. 1C).
On the other hand, more than 40% (4/9) of skin graftingfrom MHC-matched but minor antigen-mismatched CBA
to F1 remained accepted on 100 days post-transplantation(Fig. 1C). Although we have previously demonstrated thathigh dose treatment (2 mg/kg/day) of Tac monotherapy
was not effective enough to control rejection against B6
and CBA skin grafts in this model [17], combinatory administration resulted in prolonged CBA skin engraftment.
However, macroscopic observation of survived CBA skins

Kamatani et al. Inflammation and Regeneration(2022) 42:4

on day 100 exhibited epidermal atrophy compared to autologous grafts, and HE staining analysis of paraffinembedded sections revealed skin graft hyperplasia and immune cell infiltration. We further analyzed graft infiltrated
cells by immunostaining, revealing CD3+ cell infiltrationin the TDC-treated CBA skin grafts (Fig. 1E and F). In linewith our previous observation, these results suggest thatthe severity of graft rejection in MHC homo-to-heterotransplantation under the TDC treatment varies frommild (e.g., CBA) to severe (e.g., B6) depending on the
combination of donor and recipient.
Transient T cell depletion significantly improved theefficacy of TDC treatment, particularly for the mild
rejection

Usually, tissue or cellular grafts are suffered from severe
rejection than solid organ grafts in mouse allogeneictransplantation [29]. In pancreatic islet transplantationin human, addition of anti-thymocyte globulin to conventional immunosuppression significantly improved theoutcome [30]. Therefore, we next investigated mouseskin graft survival with a transient T cell depletion byanti-CD4 and CD8 monoclonal antibodies (mAb) as preconditioning prior to TDC therapy (TDC + mAb) (Fig.
1B). As a result, rejection of CBA skin graft was not observed, and all grafts survived more than 100 days (Fig.
1D). In addition, the MST of BALB/c and B6 skinshowed improvement to 35 and 49 days, respectively,
and 10% (1/10) of B6 grafts reached 100-day survival(Fig. 1D). Visual observation of survived CBA skin grafts
on day 100 showed no epidermal atrophy, and HE staining showed the similar observation image as autologous
grafts. In addition, immunostaining showed no significant infiltration of CD3+ cells into the TDC + mAbtreated CBA skin grafts (Fig. 1E and F).
Evaluation of anti-donor T and B cell responses in TDCbased treatment

We performed mixed lymphocyte reaction (MLR) using
splenocytes from the recipient mice of each treatmentgroup collected later than 100 days post-transplantation.
Recipient splenocytes were labeled with CFSE and cocultured with irradiated donor splenocytes. As a result,the proliferation rate of CD8+ T cells against BALB/c
was prominent which was dramatically decreased in thetreated groups, while the proliferation rate against B6
and CBA in non-treatment group was relatively low, andsuppression effect by TDC-based treatment was hardly
observed (Fig. 1G). On the other hand, significant CD4+
T cell proliferation was observed against every donor in
the TDC + mAb group, while no change was observedin the TDC alone group compared to the non-treatmentgroup (Fig. 1G). The drastic proliferation of CD4+ T
cells in the TDC + mAb group would reflect the

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emergence of in vivo formation and activation of allospecific memory T cells, which were suggested to occur
following transient T cell depletion in both mouse andhuman [31–33]. Although memory T cells can conduct
graft rejection, continuous tacrolimus administration
may have effectively suppressed their activation as indicated previously in clinical cases [34], and consequently
mild-combination (CBA) grafts survived long-term.
We also investigated the production of donor-reactive
antibodies, which was reported to be one of the causesof chronic rejection [35–37]. As a result, sera collectedfrom the non-treatment group did not contain anti-B6
or anti-CBA antibodies consistent with our previous observation [17] (Fig. 1H). Interestingly, analysis of serafrom TDC-treated recipients indicated the presence of
CBA reactive antibody while it was abolished by transient T cell depletion (Fig. 1H). MHC-mismatched BALB/
c provoked substantial antibody production in the nontreatment group, but TDC treatment decreased thatlevel, and transient T cell depletion minimized antiBALB/c antibody production (Fig. 1H). Considering thedetection of antibodies against CBA but not B6 in TDCtreated group, our findings suggest that prolonged graft
survival might elicit antibody production even in MHCmatched transplantation, and the T cell presence at theperi-transplantation period may have a significant role inanti-minor antigen antibody production.
The above results suggest that transient T cell depletion
enables TDC therapy to regulate MHC homo-to-heterotransplantation in the mild-combination setting in termsof no significant infiltration of immune cells into the grafts
and inhibition of antibody production. However, robust
CD4+ T cell proliferation in MLR would be reminiscent ofmemory T cell formation triggered by the transient T cell
depletion, and it could not significantly suppress the CD8+
T cell proliferation compared to the non-treatment group.
In addition, in severe-combination settings like B6 to F1,
the impact of T cell depletion was marginal. Therefore,
TDC-based immune suppression seemingly achieved
long-term graft survival but may remain to be improved,
particularly evaluating their combinatory usage with othertherapeutic reagents.
Blockade of leukocyte co-stimulatory molecules permits
long-term engraftment of mouse skin grafts from MHCmatched allogeneic donors

Co-stimulatory molecules such as CD28 or CD40L on T
cell surface conduct activation signals through binding totheir counterpart on antigen-presenting cell (APC), andtheir inhibition has been experimentally shown to be effective in controlling allograft rejection. We next attempted tocontrol rejection by combinatory administering costimulatory molecule blocking (CB) reagents (Fig. 2A). In
the treatment group with CB, recipient F1 mice rejectedKamatani et al. Inflammation and Regeneration(2022) 42:4

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Fig. 2 Blockade of leukocyte costimulatory molecules permits long-term engraftment of mouse skin grafts from MHC-matched allogeneic donors.
(A) CB-based immunoregulation protocol. Immunosuppression was performed by using a protocol of administrating a combination of anti-CD40L
mAb and CTLA4-Ig daily (CB) with or without rapamycin on every 3 days from POD 1 (CB + rapa). (B) Skin graft survival under
immunosuppression with CB (n = 9, C3129F1; n = 9, BALB/c; n = 9, C57BL/6; n = 9, CBA/N). (C) Skin graft survival under immunosuppression with
CB + rapa (n = 9, C3129F1; n = 8, BALB/c; n = 9, C57BL/6; n = 9, CBA/N). (D) Macroscopic observation (upper panels) and hematoxylin and eosin
stained section (lower panels) of autologous (C3129F1), CBA/N, and C57BL/6 skin grafts on day 100. Scale bars: 100 μm. (E) Immunohistochemical
staining for CD3 of C57BL/6 skin grafts harvested from CB or CB + rapa treated groups on 100 days post-transplantation. Upper and lower panels
represent CD3 specific staining and isotype control stained section, respectively. Scale bars: 100 μm. (F) Recipient T cell response in MHC-matchedbut minor antigen-mismatched skin transplantation. The T cell proliferation rates in each treatment group were normalized to that of C3129F1
mice stimulated with autologous irradiated splenocytes. Error bars indicate standard error of technical triplicates. Similar results were obtained in
two independent experiments. *p < 0.05, **p < 0.01 (Tukey’s HSD test). (G) De novo anti-donor antibody production in the recipients. Error bars
indicate standard error of biological replicates (n = 8, naive; n = 4, non-treatment; n = 9, CB; n = 9, CB + rapa). tx, transplantation; mAb,
monoclonal antibody; CB, co-stimulatory molecule blocking; rapa, rapamycin; POD, post-operative day

MHC-mismatched BALB/c skin grafts within 79 days
(MST = 20) (Fig. 2B). On the other hand, both CBA andB6 grafts were mostly viable at 100 days posttransplantation (Fig. 2B). Additionally, we attempted tocontrol the rejection of skin grafts by supplementing rapamycin (mTOR inhibitor) maintenance therapy in addition
to CB (CB + rapa) (Fig. 2A). We have previously reported
that rapamycin monotherapy could not induce long-term
graft acceptance in this skin transplantation model [17].
The results showed that 100% of the grafts were viable at
100 days post-transplantation in both CBA and B6 to F1

transplantation (Fig. 2C). In this setting, 75% of BALB/c
grafts were viable at 100 days post-transplantation (Fig. 2C).
We have directly compared the effect of TDC- andCB-based treatments in the mouse skin transplantation
model. The graft survival data with respect to eachdonor strain were shown in Additional file 1: Supplemental Fig. 4A-D. Both TDC + mAb and CB + rapa
treatments induced long-term graft acceptance (> 100days) in CBA donor, while only CB + rapa but not TDC+ mAb induced it in B6 donor (MST for TDC + mAb in
B6 was 49 days). Similarly, in the group pf BALB/c

Kamatani et al. Inflammation and Regeneration(2022) 42:4

donor which was MHC-mismatched, all the grafts werecompletely rejected within 70 days with TDC + mAb
treatment, while 75% (6/8) grafts were accepted morethan 100 days with CB + rapa treatment. Therefore, we
concluded that CB + rapa was more effective to induce
long-term graft acceptance than TDC + mAb in themodel of mouse skin transplantation.
Macroscopic observation of the CB-treated skin grafts
on day 100 indicated that the CBA skin grafts did notshow epidermal atrophy as in the autografts, while theB6 skin grafts showed partial tissue damage (Fig. 2D). In
addition, HE staining revealed CB-treated B6 skin grafts
underwent hyperplasia accompanying CD3+ cell infiltration, whereas CB + rapa treatment inhibited T cell infiltration and showed normal skin histology (Fig. 2D and
E). The MLR results showed that the proliferation ratesof CD8+ T cells in both treatment groups were significantly decreased compared with non-treatment group(Fig. 2F). CD4+ T cell proliferation levels against B6 and
CBA were mainly comparable to those of the nontreatment group regardless of long-term engraftment.
Interestingly, CB-based treatment significantly suppressed CD4+ and CD8+ T cell proliferation even against
MHC-mismatched BALB/c splenocytes, which was notobserved in TDC-based treatment, particularly for CD4+
T cells. We then tested for the presence of donorspecific antibody production in the serum of recipient
mice treated with CB or CB + rapa, but no donorspecific antibodies were detected except for anti-BALB/cin the non-treatment group (Fig. 2G). Therefore, immunomodulation targeting the co-stimulation pathway in MHCmatched tissue allografting, particularly when reinforced
with rapamycin maintenance treatment, can control graft
rejection without any immunological signs such as robust
T cell proliferation in MLR and antibody production.
Co-stimulatory blockade effectively prevents rejection to
iPSC-grafts

We next attempted to control the immune response in
the transplantation of iPSC in MHC-matched/minor
antigen-mismatched situation. The iPSC used for the experiments were generated from embryonic fibroblasts of
B6 or CBA mice and then were transduced with firefly
luciferase (Luc) gene to monitor iPSC survival by detecting bioluminescence signals. The Luc activity was validated in vitro by titrating the cell number, resulting incomparable signal intensity between B6 and CBA LuciPSC (Additional file 1: Supplementary Fig. 1). We
injected Luc-iPSC into the gastrocnemius muscle of recipient mice, and growing Luc-expressing iPSC-derived
teratomas was sequentially monitored for up to 25 days
after injection (Fig. 3A). When the iPSCs were injectedin syngeneic mice, B6-derived one grew faster to form
teratoma than CBA-derived one did (Additional file 1:

Page 8 of 13

Supplementary Fig. 5). For controlling immune responsein the allogeneic transplantation, TDC + mAb or CB +rapa therapies were conducted similar to Figs. 1B or 2A,
respectively. Luminescence signals of iPSC-grafts in
non-treatment control groups were significantly lower
than that of TDC + mAb or CB + rapa groups on 25
days post-injection, while both immunosuppression regimens allowed Luc-iPSC to gradually grow until the endpoint (Fig. 3B-D). Although we did not observe asignificant difference in Luc signal intensity between thetwo immunosuppressive therapies, teratomas in the
TDC + mAb group contained infiltrated immune cells inthe vicinity of differentiated tissues, whereas this was not
the case in the CB + rapa group (Fig. 3E). We performed
MLR using splenocytes from the recipient mice of eachtreatment group collected later than 25 days posttransplantation. As a result, significant CD4+ T cell proliferation was observed against both donors in the TDC+ mAb group, as in the skin transplantation (Fig. 3F).
We also tested for the presence of donor-specific antibody in the serum of recipient mice treated with TDC +mAb or CB + rapa, but no donor-specific antibodies weredetected in both treatment groups (Fig. 3G). Interestingly,
we found that frequency of naive (CD44loCD62Lhi) T cells
in the spleen of CB + rapa recipients was relatively higher
than that of TDC + mAb ones (Additional file 1: Supplementary Fig. 2). These findings support the robust efficacy
of CB-based immunosuppressive therapy in the case of
MHC matched/minor antigen mismatched iPSC-basedtransplantation.
Co-stimulatory blockade alleviates immune response
against human iPSC-derived liver organoids

In order to confirm the efficacy of CB-based treatment
on human immune cell reaction to iPSC-grafts, we generated immune humanized mice and transplanted themwith human iPSC-derived liver organoids of allogeneic
donor origin (Fig. 4A). We have beforehand observedthat tacrolimus substantially prolonged the liver organoid survival in this model [38], indicating that T cellmediated immune reaction is at least partially responsible for the loss of iPSC-derived allogeneic grafts. As tacrolimus could solely regulate the rejection as describedabove, TDC-based treatment could not be evaluatedwith this model. Thus, in this study, we focused on thetherapeutic potential of CB-based treatment. Humanized
mice generation and liver organoid transplantation werecarried out following procedures we have previously described [38]. Given that one of the reported limitationsof the humanized mice is an insufficient lymphoid immune response due to poor lymph node development[39, 40], we used HLA haplotype A mismatched combination in this experiment. In brief, highly immunodeficient NOG mice were injected with human HLA-A2+

Kamatani et al. Inflammation and Regeneration(2022) 42:4

Page 9 of 13

Fig. 3 Co-stimulatory blockade effectively prevents rejection to iPSC-grafts. (A) Schematic overview of CBA/N or C57BL/6 Luc-iPSC transplantation. (B)Bioluminescence images of C57BL/6 (upper panels) or CBA/N (lower panels) Luc-iPSC transplanted mice that received non-treatment, TDC + mAb, or CB+ rapa. n = 3-5 per group. (C, D) Quantitative bioluminescence intensity of C57BL/6 (C) or CBA/N (D) Luc-iPSC-transplanted mice that received nontreatment, TDC + mAb, or CB + rapa. n = 3-5 per group. *p < 0.05, **p < 0.01 (Tukey’s HSD test). (E) Hematoxylin and eosin stained sections of C57BL/6(left panel) or CBA/N (right panel) Luc-iPSC-derived teratomas under immunosuppression with TDC + mAb or CB + rapa on day 35. Lower panels show
enlargement of insets in the upper panels. Scale bars: 100 μm. (F) Recipient T cell response in MHC-matched but minor antigen-mismatched iPSC
transplantation. The T cell proliferation rates in each treatment group were normalized to that of C3129F1 mice stimulated with autologous irradiatedsplenocytes. Error bars indicate standard error of technical triplicates. Similar results were obtained in two independent experiments. *p < 0.05, **p < 0.01
(Tukey’s HSD test). (G) De novo anti-donor antibody production in the recipients. Error bars indicate standard error of biological replicates (n = 3, nontreatment; n = 5, TDC + mAb; n = 5, CB + rapa). Luc, luciferase; TDC, three drug combination; mAb, monoclonal antibody; CB, co-stimulatory molecule
blocking; rapa, rapamycin; POD, post-operative day

hematopoietic stem cells, and the reconstitution of human immune cells was confirmed by FACS analysis of
the peripheral blood (Additional file 1: Supplementary
Fig. 3). Then, allogeneic human liver organoids derived
from HLA-A24+ homozygous iPSC were transplanted
onto the liver surface of the immune humanized mice.

Consistent with our previous report, survival of the liverorganoids was gradually decreased in the non-treatmentgroup, which was traced by detecting human albumin
concentration in the recipient mouse serum, comparedwith the control group which was not reconstituted withhuman hematopoietic stem cells (Fig. 4B). On the otherKamatani et al. Inflammation and Regeneration(2022) 42:4

Page 10 of 13

Fig. 4 Co-stimulatory blockade alleviates immune response against human iPSC-derived liver organoids. (A) Schematic overview of human iPSCderived liver organoid transplantation. (B) Human albumin concentration in the serum of liver organoid recipient mice (n = 12, control; n = 8-9,
non-treatment; n = 7, CB; n = 5, CB + rapa). *p < 0.05, **p < 0.01 (two-way repeated measure ANOVA Tukey’s multiple comparisons test). hiPSC,
human iPSC; tx, transplantation; CB, co-stimulatory molecule blocking; rapa, rapamycin; POD, post-operative day; hALB, human albumin

hand, the level of human albumin was continuously
comparable between CB-treated and control groups
until the study endpoint (Fig. 4B). We found that, unexpectedly, maintenance rapamycin treatment worsened
the organoid survival. The adverse effect by rapamycinin this setting may be explained by a liver-damaging effect of mTOR1 inhibition and may discourage using
rapamycin for the liver organoid transplantation [41].

Discussion
Our skin transplantation model revealed diverse susceptibility to immunological rejection among minor
antigen-mismatched donors from mild to severe,
showing distinct responses to immunosuppression
regimens with TDC or TDC + mAb, regardless of
their general use in clinical settings. In the case of severe combination, represented by B6 in this study,
TDC-based therapy could not efficiently regulate theimmune response against the skin and iPSC-derived
grafts (Figs. 1 and 3E). These observations may giverise to an important argument in the process ofdeveloping an immune regulatory strategy for allogeneic iPSC-based transplantation. Particularly considering that graft rejection in cellular or tissuetransplantation, which is generally thought to be performed in iPSC-based transplantation, is relatively difficult to regulate, an alternative approach other thanTDC-based treatment would be required. In thisstudy, we have shown that CB treatment, particularly
combined with rapamycin, is effective to regulate theimmune response to not only mild but also severerisk donor-derived skin (Fig. 2) or iPSC (Fig. 3) grafts
with only a few doses of CB in the early stages oftransplantation. In the experiment of allogeneic human iPSC-derived liver organoid transplantation (Fig.
4), the efficacy of CB treatment was basically confirmed. Similar indications were provided previouslythat CB treatment enhanced MHC-mismatched mouse
iPSC-graft survival through the downregulation of T
cell activation gene signatures [27]. Thus, our present
study strongly suggests a limited effect of TDC but apotent inhibitory effect of CB on iPSC-derived

Kamatani et al. Inflammation and Regeneration(2022) 42:4

allograft rejection, even in MHC-matched but minor
antigen-mismatched situations.
A major impediment of clinical translation of CB therapy
is that conventional anti-CD40L mAb is currently not available due to prothrombotic toxicities [42]. Recently, a collaborative work by Viela Bio, AstraZeneca, and MedImmune
demonstrated that novel CD40L-targeting non-antibody
scaffold protein did not induce platelet aggregation in vitro
and improved disease activity of patients with autoimmunity without any complications [43]. Such technical advances
will support the future application of CB therapy in clinicalpractice.
It is worth establishing that a method to examine
whether the donor iPSC and recipient combination is at
risk of severe rejection. If this was possible, mild combination iPSC could be selected, and conventional TDCbased therapy would be a choice to control rejection.
Furthermore, the type of cells or tissues derived fromiPSC would determine the levels of immune responses,
as transplanted grafts from different origin exert various
levels of immunological rejection, e.g., rodent skin, small
bowel, and lung transplantation are suffered from muchseverer rejection, whereas heart, kidney, and liver areprogressively more easily accepted [44–48]. Therefore,
when cells or tissues were generated from iPSC fortransplantation, immunological characteristics of themshould be carefully estimated before the transplantation.
In this regard, evaluating the response of recipient immune cells mixed with cellular grafts may be a tool,
which demonstrated to reflect the underlying potentialof rejection [49]. However, further methodological development is required to make this reliable because wefound that simply co-culturing donor and recipient cells
was not sensitive enough to distinguish mild and severe
combinations in MHC-matched pairs [17]. Even in thiscase, attention should be paid to signatures of rejection
onset as CD4+ T cell proliferation and antibody production were detected in vitro analysis of TDC therapy
recipients.
The findings reported in this article provide clear evidence of various rejection severity and response to immunosuppression in MHC-matched and minor antigen
mismatched transplantation. To date, the modality of
immunosuppression is not limited to traditional chemical compounds or antibody-based strategies, but cellulartreatment is becoming an alternative option [50]. Since
this study did not evaluate the efficacy of the cellular immunosuppressive intervention, the degree of its effect onsevere and mild rejection caused by minor antigen mismatch remains to be elucidated. A better understandingof immunological issues and further development of
conventional and state-of-the-art technologies of regulating the immune system are of great importance to expedite the clinical application of iPSC-based therapy.

Page 11 of 13

Conclusions
Minor histoincompatibility is one of the limitations ofsuccessful transplantation, which may hinder the clinicalapplication of MHC-homozygous iPSC. The immunological rejection caused by minor antigen-mismatch
ranged from mild to severe. Conventional drug-based
immunosuppression showed a limited effect as it could
control only mild rejection when combined with T cell
depletion. On the other hand, CB therapy successfully
prevented rejection regardless of the rejection severity.
These results suggest the importance of carefully evaluating rejection and immunosuppressive therapy in
MHC-matched iPSC transplantation.
Abbreviations
iPSC: iPS cells; CB: Co-stimulatory molecule blockade; MHC: Major
histocompatibility complex; HLA: Human leukocyte antigen; NOG: Nonobese
diabetic/Shi-scid; BLI: Bioluminescent imaging; PI: Propidium iodide;
DAPI: 4′,6-diamidino-2-phenylindole; HSD: Honestly significant difference;
F1: C3129F1; B6: C57BL/6 J; CBA: CBA/N; Tac: Tacrolimus;
MMF: Mycophenolate mofetil; MP: Methylprednisolone; TDC: Triple drugs
combination; MST: Median survival time; HE: Hematoxylin and eosin;
mAb: Monoclonal antibodies; MLR: Mixed lymphocyte reaction;
CFSE: Carboxyfluorescein diacetate succinimidyl ester; CD40L: CD40 ligand;
APC: Antigen-presenting cell; rapa: Rapamycin; Luc: Luciferase

Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s41232-021-00190-7.
Additional file 1: Supplementary Fig. 1. Comparison of CBA/N or
C57BL/6 iPSC number with light emission signal. Luciferase expression
level in each cell line was measured using SYNERGY4 luminescentdetection reader with Gen5 software. Luc, luciferase. Supplementary
Fig. 2. Naive and memory T cell frequencies in the spleen of iPSC-graft
recipients. Flow cytometry analysis (POD 32) of memory (CD44hiCD62Llo/hi) and naive (CD44loCD62Lhi) T cell frequency in the spleen of recipient(C3129F1) mice transplanted with CBA/N or C57BL/6 Luc-iPSC. Luc, luciferase; TDC, three drug combination; mAb, monoclonal antibody; CB, costimulatory molecule blocking; rapa, rapamycin. Supplementary Fig. 3.
Human hematopoietic cell chimerism in the humanized NOG mice peripheral blood. hCD45 chimerism (left-top) (n = 16), hCD33+ cells (middletop) (n = 16), hCD19+ cells (right-top) (n = 16), hCD3+ cells (left-bottom)(n = 16), hCD4+ cells (middle-bottom) (n = 21), and hCD8+ cells (rightbottom) (n = 21). hALB, human Albumin. Supplementary Fig. 4. Mouseskin graft survival and body weight changes of recipient mice over time
under each immunosuppressive regimen. (A-D) were rearranged from thedata shown in Fig. 1C, D, and 2 B, and C. (A-D) Skin graft survival undervarious immunosuppression (n > 8). *p < 0.05 (Two-way repeatedmeasurement analysis of variance (ANOVA) followed by Holm–Sidak’s
multiple comparison test). (E-H) Body weight changes of recipient miceover time under various immunosuppression. n = 9 per group. Normalized to POD-1 weight. Supplementary Fig. 5. Bioluminescence imagesof C57BL/6 or CBA/N Luc-iPSC transplanted into syngeneic mice. The
mice were untreated. n = 3 per group.
Additional file 2: Supplementary Table 1. Antibody list for flow
cytometryAcknowledgements
Not applicable.
Authors’ contributions
T.K. conducted experiments, analyzed data, and wrote the manuscript. R.O.
analyzed data and wrote the manuscript. T.M. conducted experiments andacquiring data. H.W. analyzed data. T.T., A.M., S.M., and H.T. conducted theKamatani et al. Inflammation and Regeneration(2022) 42:4

humanized mouse experiments. K.S. supervised and designed all of thisstudy and wrote the manuscript. The authors read and approved the final
manuscript.
Funding
This study was supported in part by research grants from Japan Agency for
Medical Research and Development (AMED), the project of junior scientist
promotion in Hokkaido University, and the Photo-excitonix Project in Hokkaido University to K.S.
Availability of data and materials
All data generated or analyzed during this study are included in thispublished article [and its supplementary information files].

Declarations
Ethics approval and consent to participate
All animal procedures were approved by the Hokkaido University Animal
Care Committee (approval number: 18-0004) and the Central Institute for Experiment and Yokohama City University’s animal experiment committee (approval number: F-A-17-025, F-A-20-021). All animal experiments were
performed in accordance with the ARRIVE (Animal Research: Reporting of In
Vivo Experiments) guidelines.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Division of Immunobiology, Institute for Genetic Medicine, Hokkaido
University, Kita-15, Nishi-7, Sapporo, Hokkaido 060-0815, Japan. 2Central
Institute for Experimental Animals (CIEA), Kawasaki 210-0821, Japan.
3
Department of Regenerative Medicine, Yokohama City University Graduate
School of Medicine, 3-9, Fuku-ura, Kanazawa-ku, Yokohama, Kanagawa
236-0004, Japan. 4Department of Regenerative Medicine, Center for Stem
Cell Biology and Regenerative Medicine, The Institute of Medical Science,
The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639,
Japan.
Received: 21 September 2021 Accepted: 22 December 2021

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