Safe Administration of Immunosuppression in Japanese Monkeys: Relevance to Preclinical Xenotransplantation Studies

Abstract

The Japanese monkey has been used in several animal studies; however, its potential as a recipient model for xenotransplantation is unclear. The potential of the Japanese monkey as a recipient for xenotransplantation was assessed using two experimental models. The first model evaluated the optimal dose of tacrolimus without severe adverse events. The plasma tacrolimus levels, blood counts, and hepatic and renal function tests were evaluated. The second model assessed the immunosuppressive effects of thymoglobulin and tacrolimus. Immunosuppression was evaluated using blood counts and flow cytometry to measure lymphocytes in peripheral blood mononuclear cells (PBMCs). In the first model, the target trough level (10–15 ng/ml) was achieved and maintained with tacrolimus administration at 1.6 mg/kg/day in all monkeys. There were no adverse events related to the blood count or to liver, kidney, or nutrient parameters at this dose, except for hemoglobin. In the second model, a decrease in white blood cells was observed. Flow cytometry revealed a temporary decrease in T- and B-cell numbers among PBMCs on day 4. We consider that the Japanese monkey is acceptable to be used as a recipient model for preclinical xenotransplantation. The safe administration of tacrolimus and thymoglobulin is clarified for this model.

Graphical Abstract.

Introduction

Porcine xenotransplantation is one of the therapeutic choices for organ failures. It has long been considered experimental because of challenges in regulating immunity and zoonotic risks arising from the species differences between donor and recipient^1–7. Nevertheless, these hurdles might be overcome with the recent progress of gene-editing technology. Xenotransplantation has recently gained widespread attention following clinical trials of heart and kidney xenotransplantation using gene-edited porcine organs in the United States^8,9 (https://nyulangone.org/news/node/34872#:~:text=Surgeons%20at%20NYU%20Langone%20Health,and%20hope%20of%20modern%20medicine).

For the promotion of clinical porcine xenotransplantation, preclinical studies using suitable non-human primates (NHPs) are essential. The chimpanzee is the closest among the numerous NHPs ¹⁰ ; however, its experimental use has decreased following recommendations from the National Institutes of Health (https://www.nih.gov/about-nih/who-we-are/nih-director/statements/timeline-nihs-decision-end-use-chimpanzees-research). Furthermore, the cynomolgus monkey (Macaca fascicularis), an NHP widely used in xenotransplantation studies^11,12, is challenging to use because of rising costs. Therefore, other NHP candidates for preclinical xenotransplantation are needed.

The Japanese monkey (Macaca fuscata), an approximately 10-kg NHP belonging to the Macaca genus and endemic to Japan, is classified as an Old World monkey, like the cynomolgus monkey and rhesus monkey (Macaca mulatta) (Fig. 1A). Japanese monkey harbors some original characteristics which distinguish from other Macaca. For example, Japanese monkey has short tail reaching no more than 10 cm, unlike cynomolgus and rhesus monkeys. The body weight of Japanese monkey is heavier than cynomolgus monkey ¹³ . Furthermore, Japanese monkey is susceptible to malaria, while cynomolgus monkey shows comparative resistance against this infection ¹⁴ . The Japanese monkey is considered a preferable animal model and is used in various studies, including those focusing on neuroscience ¹⁵ , radiation ¹⁶ , and infection ¹⁷ . However, few studies involving the Japanese monkey have focused on transplantation ¹⁸ . In this study, we assessed the potential of the Japanese monkey as a recipient animal model for xenotransplantation. This potential was evaluated using two experimental models: one to determine the precise dose of tacrolimus, a calcineurin inhibitor, without severe adverse events; and the other to elucidate the immunosuppressive effects of thymoglobulin, an anti-human thymocyte immunoglobulin, in combination with tacrolimus. Both tacrolimus and thymoglobulin are widely used as immunosuppressants in clinical transplantations and in preclinical xenotransplantation studies^19–21.

Figure 1.

Study design: (A) Image of Japanese monkey (#24001, inserted central venous access port and gastrostomy tube). The general body length and weight of this monkey are 50–70 cm and 6–18 kg, respectively. The body color is brown and gray. The tail length is no more than 10 cm. (B) Study design model 1. Three Japanese monkeys (#21004, #21005, and #23002) received enteric administration of tacrolimus. During the first week, the monkeys were administered 0.2 mg/kg/day via gastrostomy tube for 4 days (days 0–3), followed by a 3-day rest period (days 4–6). The tacrolimus dose was doubled weekly (0.4 mg/kg/day in the second week, 0.8 mg/kg/day in the third week, and 1.6 mg/kg/day in the last week). In the final week, tacrolimus administration continued from days 0 to 10. Blood samples for a blood count, biochemical tests (liver and kidney function parameters), the BG level, and the plasma tacrolimus level were collected on day 0 (prior to tacrolimus administration) and day 4. In the last week, samples were collected on days 0, 4, 7, and 11, except for the plasma tacrolimus level, which was sampled daily from days 0 to 11. (C) Study design model 2. The time course of thymoglobulin and tacrolimus administration is shown. Two Japanese monkeys (#24001 and #24002) received intravenous thymoglobulin at 1.5 mg/kg/day for 4 days (days 0–3) via a central venous access port for immunosuppression induction. Intravenous hydrocortisone (0.5 mg/kg) was administered before and after thymoglobulin to prevent cytokine release syndrome. Tacrolimus was administered daily at 1.6 mg/kg/day for maintenance. Blood samples for a blood count, biochemical tests, the BG level, and the plasma tacrolimus level, as well as for acquiring peripheral blood mononuclear cells, were collected on days 0, 4, 11, 18, and 25 in #24001 and days 0, 4, 7, and 11 in #24002. Flow cytometry using PBMCs from #24001 was performed on days 0, 4, 11, 18, and 25 and #24002 on days 0, 4, 7, and 11 during immunosuppression (thymoglobulin: days 0–3; tacrolimus: days 0–).

Materials and Methods

Animals and Ethics

Japanese monkeys were provided by the National BioResource Project “Japanese Macaques.” Animals were housed under specific pathogen-free conditions with free access to food and water. The care of the animals and the experimental procedures complied with the Principles of Laboratory Animal Care [Guide for the Care and Use of Laboratory Animals, 8th edition (National Research Council, 2011)]. The experimental protocol was approved by the Animal Care and Use Committee of Fukuoka University (Approval number: 2308040).

Insertion of Gastrostomy Tube

Prior to tacrolimus administration, we inserted a gastrostomy tube percutaneously using a SOPH-A-PORT (#SP20410; TOKIBO Co., Ltd., Tokyo, Japan) (Fig. 1). In brief, the monkeys were positioned in the supine position under general anesthesia with inhaled isoflurane (#095-06573; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). After performing a laparotomy in the upper median abdomen, a small incision was made in the anterior wall of the gastric body. The gastrostomy tube was inserted through the incision, and its tip was moved to the duodenum. The anterior wall was then closed, and the tube was fixed in position. The tube was tunneled by Witzel’s method using the gastric serosa and extended outside the body. Finally, the tube was connected to the port, which was embedded in the subcutaneous region. Tacrolimus dissolved in water was injected into the port.

Insertion of Central Venous Access Port

A central venous access port was also inserted prior to tacrolimus injection to monitor the plasma tacrolimus level and other laboratory parameters (Fig. 1). A 6Fr central venous access port (Bard X-port isp, #0607530; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was inserted via the femoral vein, and the tip of the tube was fixed in the vena cava.

Schedule for Determination of Precise Dose of Tacrolimus (Model 1)

Three Japanese monkeys (#21004, #21005, and #23002) received intragastric administration of tacrolimus (Graceptor capsules, #3999014N1028, 2024, 3020; Astellas Pharma Inc., Tokyo, Japan). Fig. 1B shows the administration schedule for tacrolimus. During the first week, the monkeys were administered 0.2 mg/kg/day via the gastrostomy tube for 4 days (days 1–4), followed by a 3-day rest period (days 5–7). The tacrolimus dose was doubled weekly (0.4 mg/kg/day in the second week, 0.8 mg/kg/day in the third week, and 1.6 mg/kg/day in the last week). In the final week, tacrolimus was administered continuously from days 0 to 11. Blood samples for a blood count, biochemical testing (liver and kidney function parameters), the blood glucose (BG) level, and the plasma tacrolimus level were collected on day 0 (prior to tacrolimus administration) and day 4. During the last week, samples were collected on days 0, 4, 7, and 11.

Schedule for Determination of Immunosuppressive Effects of Thymoglobulin/Tacrolimus (Model 2)

The time course for administering thymoglobulin and tacrolimus is shown in Fig. 1C. Two Japanese monkeys (#24001 and #24002) received intravenous administration of thymoglobulin at 1.5 mg/kg/day (#876399; Sanofi S.A., Paris, France) for 4 days (days 0–3) via a central venous access port for the induction of immunosuppression. Intravenous hydrocortisone (0.5 mg/kg) was administered before and after thymoglobulin to prevent cytokine release syndrome. Tacrolimus was administered daily at 1.6 mg/kg/day for maintenance until the schedule was completed. This protocol is based on the immunosuppression protocol for clinical islet transplantation in Japan. Blood samples for a blood count, biochemical tests, the BG level, the plasma tacrolimus level, and the peripheral blood mononuclear cell (PMBC) count were collected on days 0, 4, 11, 18, and 25 in #24001 and days 0, 4, 7, and 11 in #24002, respectively.

Measurement of Laboratory Parameters

The blood count was performed using the Celltac α analyzer (Nihon Kohden Europe, Rosbach, Germany) to measure the white blood cell (WBC) count, hemoglobin (Hb) level, and platelet (Plt) count. Plasma samples for biochemical examination were isolated from heparinized blood by centrifugation at 6,000 rpm for 5 min. Liver function parameters [total bilirubin (T-Bil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), γ-glutamyltransferase (γGTP), and alkaline phosphatase 2 (ALP2)], kidney function parameters [blood urea nitrogen (BUN), uric acid (UA), and creatinine (Cre)], and nutritional status indicators [total protein (TP) and albumin (Alb)] were measured using the Spotchem™ EZ SP4430 (ARKRAY, Inc., Kyoto, Japan). BG was measured using the Glutestmint II (PHC Co., Tokyo, Japan). The plasma tacrolimus level was measured by SRL, Inc. (Tokyo, Japan).

Assessment of Adverse Events Due to Administration of Tacrolimus

The grade of adverse events due to tacrolimus administration was assessed using the criteria described in the Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0. The severity of adverse events was classified by grading as follows: grade 1 (mild), grade 2 (moderate), grade 3 (severe or medically significant but not immediately life-threatening), grade 4 (life-threatening consequences), and grade 5 (death related to adverse event). The grading of adverse events in this study is summarized in Table 1.

Table 1.

Grading of Adverse Events Due to Tacrolimus Based on CTCAE Version 5.0.

	Grade 1	Grade 2	Grade 3	Grade 4	Grade 5
WBC (/mm3)	3,300–3,000	3,000–2,000	2,000–1,000	<1,000
Hb (g/dl)	13.7–10.0	10.0–8.0	<8.0
Plt (/mm3)	158,000–75,000	75,000–50,000	50,000–25,000	<25,000
T-Bil (mg/dl)	1.5–2.25	2.25–4.5	4.5–15.0	>15.0
AST (IU/L)	30–90	90–150	150–600	>600
ALT (IU/L)	42–126	126–210	210–840	>840
LDH (IU/L)	>222
γGTP (IU/L)	64–160	160–320	320–1,280	>1,280
ALP2 (IU/L)	113.0–282.5	282.5–565	565–2,260	>2,260
Cre (mg/dl)	1.07–1.6	1.6–3.2	3.2–6.4	>6.4
Alb (g/dl)	3.0–4.0	2.0–3.0	~2.0
BG (mg/dl)	~109
Vomiting	Intervention not indicated	Outpatient IV hydration; medical intervention indicated	Tube feeding, TPN, or hospitalization indicated	Life-threatening consequences
Diarrhea	Increase of <4 stools per day over baseline; mild increase in ostomy output compared to baseline	Increase of 4–6 stools per day over baseline; moderate increase in ostomy output compared to baseline; limiting instrumental ADL	Increase of ≥7 stools per day over baseline; hospitalization indicated; severe increase in ostomy output compared to baseline; limiting self-care ADL	Life-threatening consequences; urgent intervention indicated

Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

PBMCs were isolated from blood samples following the manual for the BD Vacutainer® CPT™ Mononuclear Cell Preparation Tube—Sodium Heparin (#362753; Becton, Dickinson and Company). In brief, 8-ml blood samples were centrifuged at 1,500 × g for 15 min. After plasma removal, PBMCs that had centrifuged above the gel barrier were collected.

Flow Cytometry

Flow cytometry using PBMCs from Monkey #24001 was performed on days 0, 4, 11, 18, and 25 and from #24002 on days 0, 4, 7, and 11 during immunosuppression (thymoglobulin: days 0–3; tacrolimus: days 0–). PBMCs were first incubated with Human BD Fc Block™ (#564219; Becton, Dickinson and Company) for 10 min on ice and then incubated with fluorochrome-conjugated specific or isotype-matched nonspecific antibodies for 30 min on ice. Alexa Fluor® 488 Mouse Anti-Human CD3 [SP34-2] (#557705; Becton, Dickinson and Company), PE Mouse IgG2b, κ Isotype Ctrl Antibody [MPC-11] (#400311; BioLegend, San Diego, CA, USA), PE Mouse Anti-Human CXCR5 [MU5UBEE] (#12-9185-41; Thermo Fisher Scientific, Waltham, MA, USA), and APC Mouse Anti-Human CD4 [L200] (#551980; Becton, Dickinson and Company) were used for staining. Flow cytometry was performed using the BD Accuri™ C6 Plus flow cytometer (Becton, Dickinson and Company). Dead cells were gated out using 7-amino-actinomycin D (Becton, Dickinson and Company), and lymphocytes were identified by forward and side scatter. Doublets, determined by forward scatter height and width, were excluded from analysis. Singlets were then gated as CD3⁺CD4⁺ (CD4⁺ T cells), CD3⁺CD4⁻ (CD8⁺ T cells), CD3⁺CD4⁺CXCR5⁺ [CD4⁺ T follicular helper (Tfh) cells], and CD3⁺CD4⁻CXCR5⁺ (CD8⁺ Tfh cells).

Statistical Analysis

The unpaired t-test or Dunnett’s test was used for paired and multiple comparisons, respectively. Data are presented as mean ± standard error of the mean. A P-value of <0.05 was used to define statistical significance. Statistical analyses were conducted using JMP® 18.0.0 (SAS Institute Inc., Cary, NC, USA).

Results

Administration of Tacrolimus at 1.6 mg/kg/day Maintained Plasma Tacrolimus Level Within Trough Range

Table 2 shows the characteristics of the three Japanese monkeys, each weighing approximately 10 kg. In this study, the trough level of tacrolimus was defined as 10–15 ng/ml, based on guidelines for each indication. For example, the initial trough level in kidney transplantation is 8.0–15.0 ng/ml (https://jsn.or.jp/journal/document/58_4/562-567.pdf). For islet transplantation, the trough level was set at 10.0–12.0 ng/ml (http://www.ptccc.jp/pdf/implementation_guideline_04.pdf). In addition, the trough level for ulcerative colitis, as stated in the drug information sheet, is 10.0–15.0 ng/ml (https://pins.japic.or.jp/pdf/newPINS/00065389.pdf). The maximum trough level was kept below 20 ng/ml because tacrolimus may cause nephrotoxicity if plasma levels exceed 20 ng/ml due to reduced renal arterial blood flow and the development of tubulopathy, which leads to a decreased glomerular filtration rate^22,23.

Table 2.

Characteristics of Japanese Monkeys Used in This Study.

	#21004	#21005	#23002	#24001	#24002
Age (years)	5	5	12	4	16
Body weight at admission (kg)	8.0	9.4	10.6	7	10.2
WBC (/mm3) at day 0	61	78	93	79	99
Hb (g/dl) at day 0	13.7	12.7	16.1	17.3	14.1
Plt (/mm3) at day 0	4.7	22.5	30.2	14.6	31.3
T-Bil (mg/dl) at day 0	0.2	0.2	0.2	0.2	0.2
AST (IU/L) at day 0	10	10	10	10	11
ALT (IU/L) at day 0	13	35	93	10	22
LDH (IU/L) at day 0	346	318	257	314	305
γGTP (IU/L) at day 0	80	81	392	76	71
ALP2 (IU/L) at day 0	233	226	207	321	95
BUN (mg/dl) at day 0	16	22	16	13	14
UA (mg/dl) at day 0	1	1	1	1	1
Cre (mg/dl) at day 0	0.8	1	0.9	0.8	0.9
TP (g/dl) at day 0	6.6	6.5	7.2	6.6	6.6
Alb (g/dl) at day 0	4.2	4.1	4.5	3.6	3.3
BG (mg/dl) at day 0	85	51	83	76	74

Fig. 2 shows the plasma tacrolimus levels before and after administration of tacrolimus in model 1. The plasma tacrolimus levels increased on day 4 following administration of 0.2, 0.4, and 0.8 mg/kg/day. Significant differences were observed at 0.4 and 0.8 mg/kg/day, but levels did not exceed the trough range (1.08 ± 0.24 ng/ml, P = 0.12 at 0.2 mg/kg/day; 2.95 ± 0.06 ng/ml, P < 0.001 at 0.4 mg/kg/day; and 5.40 ± 0.70 ng/ml, P < 0.01 at 0.8 mg/kg/day) (Fig. 2A–C). The trough level was achieved with administration at 1.6 mg/kg/day in all three monkeys and was maintained during the dosing period (Fig. 2D). The maximum concentration of plasma tacrolimus was 13.0 ng/ml in Monkey #21004, 11.5 ng/ml in #21005, and 17.3 ng/ml in #23002 (Fig. 2D). Therefore, we consider the preferable dose of tacrolimus for Japanese monkeys to be 1.6 mg/kg/day. This dose is significantly higher than the dose used in clinical transplantation in Japan (approximately 0.3 mg/kg/day).

Figure 2.

Plasma tacrolimus level: Plasma tacrolimus levels before (day 0) and after (day 4 and onward) administration of tacrolimus at (A) 0.2, (B) 0.4, (C) 0.8, and (D) 1.6 mg/kg/day. The yellow-shaded area represents the target trough range for the plasma tacrolimus levels.

Severe Pancytopenia Did Not Occur Under Administration of Tacrolimus at 1.6 mg/kg/day

Fig. 3 and Supplemental Figure 1 show the changes in each blood count parameter in model 1. The WBC count in each monkey remained above the lower limit of normal (3,300/mm³) throughout the observation period, regardless of the tacrolimus dose (Fig. 3A and Supplemental Figure 1A). The Hb levels were also maintained above the lower limit of normal (13.7 g/dl) in two monkeys (#21004 and #23002). No significant changes were observed between pre-administration (day 0) and post-administration (day 4) of tacrolimus at doses of 0.2, 0.4, and 0.8 mg/kg/day (Supplemental Figure 1B). A mild decrease in the Hb levels was observed with tacrolimus at 1.6 mg/kg/day (from 13.5 to 12.1 g/dl), considered a grade 1 adverse event (Fig. 3B and Supplemental Figure 1B). For the Plt count, it was difficult to assess adverse event severity because Plt counts lower than the normal limit (158,000/mm³) were observed before tacrolimus administration in two monkeys (#21004 and #21005) (Supplemental Figure 1C). We concluded that there were no adverse events with respect to the Plt count because the counts remained consistent between pre- and post-tacrolimus administration (Fig. 3C and Supplemental Figure 1C). A significant decrease in the Plt count was observed in Monkey #21005 at a tacrolimus dose of 0.2 mg/kg/day (from 225,000/mm³ to 15,000/mm³, defined as grade 4); however, such a decrease was not observed at doses of 0.4, 0.8, and 1.6 mg/kg/day (Fig. 3C and Supplemental Figure 1C). There were no significant differences in the WBC count, Hb level, or Plt count between pre- and post-tacrolimus administration (Fig. 3).

Figure 3.

Changes in blood count parameters with tacrolimus administration at 1.6 mg/kg/day: (A) WBC count, (B) Hb level, and (C) Plt count before (day 0) and after (day 4 and onward) administration of tacrolimus.

Other Severe Adverse Events Did Not Occur Under Administration of Tacrolimus at 1.6 mg/kg/day

Fig. 4 and Supplemental Figure 2 show the changes in each liver parameter before and after tacrolimus administration. The T-Bil and AST levels were within normal limits, with no changes observed between pre- and post-administration of tacrolimus (Fig. 4A, B and Supplemental Figure 2A, B). Monkey #23002 showed an increase in the ALT level after administration of tacrolimus at 1.6 mg/kg/day; however, the ALT levels remained within normal limits in all three monkeys (<42 IU/L) (Fig. 4C and Supplemental Figure 2C). The LDH levels were slightly above the upper limit of normal (222 IU/L) in two monkeys (#21004 and #21005) but remained stable throughout the observation period in all three monkeys (Fig. 4D and Supplemental Figure 2D). Both the γGTP and ALP2 levels were also above the upper limit of normal, but no changes were observed between pre- and post-administration of tacrolimus, similar to the LDH levels (Fig. 4E, F and Supplemental Figure 2E, F). These data indicate that tacrolimus administration did not affect liver function at a dose of 1.6 mg/kg/day.

Figure 4.

Changes in liver function parameters with tacrolimus administration at 1.6 mg/kg/day: (A) T-Bil, (B) AST, (C) ALT, (D) LDH, (E) γGTP, and (F) ALP2 levels before (day 0) and after (day 4 and onward) administration of tacrolimus.

Renal dysfunction is a frequent adverse event of tacrolimus ²⁴ . Therefore, assessment of kidney parameters is essential when evaluating recipient animals administered tacrolimus. Our results showed no elevation in any kidney parameters (BUN, UA, and Cre) despite tacrolimus administration (Fig. 5A–C and Supplemental Figure 3A–C). In particular, the Cre level was maintained within normal limits (<1.07 mg/dl) (Fig. 5C and Supplemental Figure 3C). Similar to liver parameters, kidney parameters were not affected by tacrolimus administration at doses below 1.6 mg/kg/day.

Figure 5.

Changes in kidney function parameters with tacrolimus administration at 1.6 mg/kg/day: (A) BUN, (B) UA, and (C) Cre levels before (day 0) and after (day 4 and onward) administration of tacrolimus.

Next, we assessed the influence of tacrolimus on TP, Alb, and BG as indicators of the nutritional condition and islet injury. No significant decreases in TP and Alb were observed despite tacrolimus administration, and levels remained above 6.0 and 3.5 g/dl, respectively. Furthermore, no significant increases in BG were observed in any of the three monkeys (Fig. 6A, B and Supplemental Figure 4A, B). Regarding physical findings related to the gastrointestinal tract, including appetite loss, vomiting, and diarrhea, no adverse events were detected in any of the three monkeys. There were no symptoms related to the nutritional condition following tacrolimus administration at 1.6 mg/kg/day.

Figure 6.

Changes in nutrient parameters with tacrolimus administration at 1.6 mg/kg/day: (A) TP, (B) Alb, and (C) BG levels before (day 0) and after (day 4 and onward) administration of tacrolimus.

Immunosuppression Was Achieved by Administration of Thymoglobulin and Tacrolimus

Finally, we assessed the immunosuppressive effects of both thymoglobulin and tacrolimus in Japanese monkeys (Model 2). As mentioned in the Materials and Methods, Monkey #24001 and #24002 received thymoglobulin at 1.5 mg/kg/day for 4 days and tacrolimus at 1.6 mg/kg/day throughout the observation period. Figs. 7A and 8A show the plasma tacrolimus level of these monkeys, which reached the target trough level on day 4 and was maintained during the observation period. The tacrolimus dose was reduced to 0.8 mg/kg/day on day 20 in #24001 because of an elevated plasma tacrolimus level and appetite loss. The WBC level gradually decreased following thymoglobulin administration until day 8 (from 7,900/mm³ and 9,900/mm³ on day 0 to 5,200/mm³ and 7,900/mm³ in #24001 and on day 8; former #24001, later #24002) and then increased after day 11 (Figs. 7B and 8B). Flow cytometry revealed that the T-cell counts and non-T-cell counts (CD3⁻, CD3⁺CD4⁺, and CD3⁺CD4⁻) temporally decreased on day 4 in both monkeys (Figs. 7C and 8C). In terms of cell frequency, a temporary decrease in CD3⁺CD4⁻ cells (considered CD8⁺ killer T cells) was observed on day 4 in #24001 (Fig. 7C). In addition, a temporary decrease in CD3⁺CD4⁺CXCR5⁺ cells (Tfh cells) was also observed on day 4 in both monkeys (Supplemental Figure 5A and B). We consider that these decreases in T cells were due to thymoglobulin administration.

Figure 7.

Assessment of immunosuppression with thymoglobulin and tacrolimus administration in Monkey #24001: (A) Plasma tacrolimus levels during tacrolimus administration. (B) WBC counts during administration of thymoglobulin (1.5 mg/kg/day for 4 days) and tacrolimus (1.6 mg/kg/day throughout the observation period). (C) Flow cytometry of PBMCs collected on days 0, 4, 11, 18, and 25, showing distributions of CD3⁺, CD3⁺CD4⁺, and CD3⁺CD4⁻ cells. Quantitative data (frequencies and cell numbers) are shown in the lower panel.

Figure 8.

Assessment of immunosuppression with thymoglobulin and tacrolimus administration in Monkey #24002: (A) Plasma tacrolimus levels during tacrolimus administration. (B) WBC counts during administration of thymoglobulin (1.5 mg/kg/day for 4 days) and tacrolimus (1.6 mg/kg/day throughout the observation period). (C) Flow cytometry of PBMCs collected on days 0, 4, 7, and 11, showing distributions of CD3⁺, CD3⁺CD4⁺, and CD3⁺CD4⁻ cells. Quantitative data (frequencies and cell numbers) are shown in the lower panel.

Regarding adverse events from thymoglobulin and tacrolimus, no severe events were observed in terms of pancytopenia, liver and kidney dysfunction, nutritional impairment, or cytokine release syndrome from thymoglobulin (Supplemental Figure 6). Only appetite loss due to tacrolimus, which was reversible, was noted in #24001.

Discussion

Recent clinical trials using gene-edited pigs for xenotransplantation have shown the potential for this treatment to be feasible across various organs and tissues^8,9. Preclinical studies using suitable recipient animals are essential for advancing clinical xenotransplantation. Regarding this issue, cynomolgus monkey is the most popular and reliable recipient animal model used for preclinical transplantation studies including allogeneic organ transplantation ²⁵ , porcine organ xenotransplantation ²⁶ , pluripotent stem cell-derived cell transplantation ²⁷ , or CAR T-cell therapy ²⁸ . Nevertheless, cynomolgus monkey plays an important role for fighting human infectious diseases such as HIV ²⁹ and COVID-19 ³⁰ . On the other hand, recent inflated price of the macaque after COVID-19 outbreak becomes a hurdle for promoting preclinical studies using this animal model. Indeed, the price of individual cynomolgus monkey in 2023 is $20,000–24,000 USD, while the price before COVID-19 outbreak was from $2,800 to $5,000 ³¹ . Due to the limitation of using cynomolgus monkeys, we focused on Japanese monkey as a promising recipient animal model for preclinical transplantation studies.

Japanese monkey has several advantages. One is its size: as previously mentioned, the adult Japanese monkey weighs approximately 10 kg, allowing for numerous procedures, such as drug administration and blood sampling, to be performed by one or two experimenters. Another advantage is its reliability as a laboratory animal. As noted in the Introduction, Japanese monkeys have historically been used in many medical studies. Furthermore, similarity of anatomy to human is an advantage of this animal as a recipient model for preclinical transplantation study, which means the surgical technique performed to Japanese monkey can be directly applied to human. We consider the characteristics of Japanese monkeys in size and similarity in anatomy might contribute to training for pediatric surgeon. For example, pediatric surgeons can train pediatric liver transplantation using Japanese monkeys because the size of monkey is similar to 1 to 2 years old child. Indeed, a half of children in congenital bile duct atresia receive liver transplantation before 2 years of age ³² . Therefore, we consider the potential of Japanese monkey as a recipient model is comparable to other macaques, while the preclinical transplantation studies using these animals are few^33,34.

In this study, we aimed to demonstrate the potential of Japanese monkeys as a recipient model for xenotransplantation. First, we determined the optimal dose of tacrolimus for maintaining trough levels in Japanese monkeys. Tacrolimus is an important immunosuppressant used for maintenance in clinical transplantation^35–39. It functions as an immunosuppressant by blocking nuclear factor of activated T cells (NFAT) signaling, which plays a role in the development and differentiation of Tfh cells^40,41. We found that the optimal tacrolimus dose for Japanese monkeys was 1.6 mg/kg/day, which is notably higher than the dose used in humans. According to drug information, the initial dosage in humans ranges from 0.30 mg/kg/day for kidney, liver, pancreas, and intestine transplants; 0.06–0.30 mg/kg/day for heart transplantation; 0.10–0.30 mg/kg/day for lung transplantation; 0.12 mg/kg/day for bone marrow transplantation; and 0.05 mg/kg/day for ulcerative colitis (https://pins.japic.or.jp/pdf/newPINS/00065389.pdf). Our data also suggest that Japanese monkeys appeared to tolerate the use of immunosuppressants better. Importantly, symptoms of renal dysfunction (elevation of BUN and Cre levels), a major complication of tacrolimus, were not observed. The safe dose of tacrolimus for Japanese monkeys was revealed through these findings. The preferable dose of tacrolimus in other macaques is similar to that of Japanese monkeys, which were clarified in this study. Regarding cynomolgus monkey, previous studies indicated that the dose of tacrolimus is 1.0–2.0 mg/kg^25,42,43.

While tacrolimus is a major maintenance immunosuppressant, thymoglobulin is recognized as a pivotal induction agent that suppresses immunity by depleting lymphocytes ⁴⁴ . In this study, we administered a combination of thymoglobulin and tacrolimus to one Japanese monkey. The thymoglobulin dose was based on that used for clinical islet transplantation, and we confirmed a temporary decrease in the WBC count, including the lymphocyte count, on day 4 in this model without severe adverse events such as pancytopenia, liver or kidney dysfunction, or nutritional impairment. We consider thymoglobulin to be safe and effective in achieving temporary immunosuppression, which may help prevent early graft loss at this dose. However, it is necessary to determine the optimal thymoglobulin dose for Japanese monkeys undergoing porcine xenotransplantation in future studies.

This study had some limitations. First, the sample size was small. Second, the immunosuppression protocol consisting of tacrolimus and thymoglobulin might be insufficient to achieve xenotransplantation in Japanese monkey. Regarding this, we will perform porcine islet xenotransplantation under this protocol and assess the graft survival. Then, try to prolong the graft survival by improvement of this protocol by adding other important immunosuppressants such as mycophenolate mofetil, belatacept, and anti-CD154 antibody^45,46. Third, the validity of the dose of tacrolimus which was established in this study needs to be discussed. There is no evidence whether trough level for monkey is similar to that for human. Fourth, we lacked precise criteria for assessing adverse events related to immunosuppression in recipient monkeys. The CTCAE is the standard for evaluating adverse events in humans, not monkeys, and normal reference levels for laboratory data in monkeys are not well defined. In addition, our study assessed the suitability of Japanese monkeys as a recipient model over a short period (approximately 1 week). Future studies are needed to clear these limitations.

In conclusion, we demonstrated the potential of the Japanese monkey as a recipient model for preclinical xenotransplantation. The safe administration of tacrolimus and thymoglobulin for this model is clarified in this study.