CD4⁺/CD8⁺ T cell ratio correlates with the graft fate in pig-to-nonhuman primate islet xenotransplantation

Abstract

Background.

Xenogeneic islet transplantation using porcine pancreata has been a promising option for substituting human islet transplantation. Moreover, recent advances in pre-clinical results have put islet xenotransplantation closer to the possibility of clinical application. While preparing for the era of clinical xenotransplantation, developing non-invasive immune monitoring method which could predict the graft fate could benefit the patient. However, there are few reports showing predictive immune parameters associated with the fate of the graft in islet xenotransplantation.

Methods.

The absolute number and ratio of T cell subsets have been measured via flow cytometry from the peripheral blood of 16 rhesus monkeys before and after porcine islet xenotransplantation. The correlation between the graft survival and the absolute number or ratio of T cells were retrospectively analyzed.

Results.

The ratio of CD4⁺ versus CD8⁺ T cells were significantly reduced due to CD8⁺ effector memory cells’ increase. Correlation analyses revealed that CD4⁺/CD8⁺, CD4⁺/CD8⁺naïve, CD4⁺naïve/CD8⁺naïve, and CD4⁺central memory/CD8⁺naïve cell ratios negatively correlated with the duration of graft survival. Conversely, further analyses discovered strong, positive correlation of CD4⁺/CD8⁺ cell ratios within the early graft-rejected monkeys (≤ 60 days).

Conclusions.

This retrospective study demonstrated that CD4⁺/CD8⁺ ratios correlated with graft survival, especially in recipients which rejected the graft in early post-transplantation periods. CD4⁺/CD8⁺ ratios could be used as a surrogate marker to predict the graft fate in pig-to-NHP islet xenotransplantation.

1. Introduction

Ever since the success of pancreatic islet transplantation as a therapy for type 1 diabetes mellitus patients,¹ islet transplantation has been applied in the clinics with desirable safety and efficacy.² Undoubtedly, the shortage of donor organs has always been, and always will be the bottleneck in the application of islet transplantation to the patients in need.³ In these contexts, many researchers have turned their attention to the xenogeneic islet transplantation using the relatively unlimited porcine pancreas which also was suitable scientifically, socially, and ethically.⁴ Fortunately, recent advances in pre-clinical study of pig-to-nonhuman primate (NHP) islet xenotransplantation⁵ have opened the possibility of clinical trial initiation. Currently, however, except for some islet encapsulation strategies,⁶ immunosuppressive regimens are inevitable to control the immune responses to xeno-antigens.⁴

Among the diverse immune responses in xenotransplantation, T cell immune responses have been thought to be the most important barrier to be overcome.^4,7 To control the T cell immune responses to the xenografts, we and other groups have tested various immunosuppressants such as anti-CD154 mAb, anti-CD40 mAb, tacrolimus, and anti-IL-6R in pre-clinical porcine islet xenotransplantation.^5,8–11 Our efforts to establish the clinically applicable immunosuppressive regimen with wild type, naked porcine islets were followed by comprehensive monitoring of peripheral blood T lymphocyte subsets, because we postulated that monitoring the number of peripheral blood T cell subsets would render a possibility to predict the graft fate and enable us to intervene before the advent of immunological harm process.

Hence, we analyzed the peripheral blood T cell subsets’ number and ratio before and after pig-to-NHP islet xenotransplantation with various combinations of immunosuppressants in hopes of discovering the putative biomarkers to predict the graft fate. In this retrospective analysis, noticeable repopulation of particular T cell subsets were observed. On one hand, there was no significant difference seen in graft survival neither in the occurrence of rejection or the ratio of T cell recovery. On the other hand, while the ratio of functional subsets within CD4⁺ or CD8⁺ cells did not correlate well with graft survival, CD4⁺/CD8⁺ cell ratios in the recipients showed significant correlation with the duration of graft survival. Thus, the CD4⁺/CD8⁺ ratios in the early post-transplantation period might be a potential biomarker for graft fate prediction.

2. Materials and methods

2.1. Animals and grouping

A total of 16 rhesus monkeys (Macaca mulatta) were used in this study. All procedures were carried out in compliance with the guidelines set forth in the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86–23, revised 2011), and approved by the Seoul National University (SNU) Institutional Animal Care and Use Committee (IACUC no. 15–0297-S1A0). SNU miniature pigs were bred and maintained in a barrier-sustained and specific pathogen-free facility. Monkeys were divided into 3 groups depending on the use of specific maintenance immunosuppressant: αCD154 (anti-CD154 mAb), αCD40 (anti-CD40 mAb), or Tacro (anti-CD40 mAb plus tacrolimus).

2.2. Porcine islet isolation

Porcine islets were isolated as previously described.⁵ Briefly, islet isolation was performed using the modified Ricordi method after pancreatectomy. The procured pancreas was distended intraductally using a preservation solution containing CIzyme™ collagenase and CIzyme™ BP protease (VitaCyte, Indianapolis, IN).

2.3. Induction of diabetes and islet transplantation in NHPs

Diabetes induction and islet transplantation were conducted as previously described.⁵ Briefly, streptozotocin (110 mg/kg; Sigma-Aldrich, St. Louis, MO) was administrated to the monkeys via intravenous (i.v.) route. Diabetes induction was confirmed by persistent hyperglycemia and the absence of porcine C-peptide. The porcine islets were infused into the jejunal vein after the laparotomy.

2.4. Immunosuppression

The three groups of monkeys were universally treated with rabbit anti-thymocyte globulin (rATG; Thymoglobulin1; Genzyme, Cambridge, MA), sirolimus (Rapamune®; Pfizer, New York, NY), methylprednisolone (M p/d; Predisol, Reyon Pharmaceutical, Korea), Cobra venom factor (CVF; Quidel, San Diego, CA), and adalimumab (Humira®; Abbott Laboratories Ltd., Queenborough, UK). rATG (5 mg/kg) was administered i.v. on days −3, −1 or days −6, −4, −1 or days −6, −4, −1, 0 until sufficient T cell depletions were noticed. Sirolimus was administered p.o. daily from day −9, −5, or −3 to reach the trough level of 3–8 ng/mL. M p/d (5 mg/kg) was administered i.v. on days −4 and −2 or −3 and −1. CVF (100 U/kg) was administered on day −1 of the transplantation to prevent complement activation. TNF-α neutralizing mAb, adalimumab (5 mg/kg) was administered subcutaneously at 2–3 hours before islet infusion.

αCD154 group was treated with anti-human CD154 mAb (5C8; National Institutes of Health NHP Reagent Resource, Bethesda, MD). Anti-CD154 mAb (20 mg/kg) was infused i.v. on days −10, −7, −4, 0, 3, 7, and 12, weekly for 3 months and then biweekly thereafter of transplantation. R080 was administered with human factor H (i.v., 18 mg/kg) kindly provided by Prof. Hee Jung Kang at Hallym University College of Medicine. αCD40 group was treated with anti-CD40 mAb (2C10R4; National Institutes of Health NHP Reagent Resource). Anti-CD40 mAb (30–50 mg/kg) was infused i.v. on days −4, 0, 4, 7, 10, and 14, weekly for three months and then biweekly thereafter of transplantation. R091 and R095 were treated with belatacept (i.v., 20 mg/kg; Nulojix; Bristol-Myers Squibb, New York, NY) on days −2, 0, 3, 7 and weekly after that. R091 was also treated with basiliximab (Simulect; Novartis, Basel, Switzerland; i.v., 0.3 mg/kg) on days 27 and 29. For Tacro group, in addition to anti-CD40 mAb, tacrolimus (Advagraf®; Astellas Pharma Korea, Korea) was administered p.o. daily from day −3 to 30 to achieve stable trough levels (3–6 ng/mL). R008, R087, and R131 were treated with belatacept (i.v., 20 mg/kg) on day 16, 20, or 26 and weekly after that. In R143, R144, and R147, tocilizumab (Actemra®; Joongwae pharma, Korea) was infused i.v. at 10 mg/kg at 1 hour before the islet infusion. The descriptions of recipient monkeys and the immunosuppressive regimen are organized into Table 1.

Table 1.

Immunosuppressive regimen and islet graft survival of recipient monkeys (n = 16)

Group	Immunosuppresive regimen	Subject number	Graft survival (days)	Reported year
αCD154†	Anti-CD154 mAb, human factor H (R080)	5	168, 180, 303, 513, 784	Shin et al., 2015║
αCD40‡	Anti-CD40 mAb, belatacept (R091, R095), basiliximab (R091)	4	6, 7, 9, 25	Shin et al., 2018¶
Tacro§	Anti-CD40 mAb, tacrolimus, belatacept (R008, R087, R131), tocilizumab (R143, R144, R147)	7	3, 11, 14, 54, 60, 180, 193	Shin et al., 2018¶ and Min et al., 2018††

Note. All groups were treated with rATG (i.v., 5mg/kg), sirolimus (p.o., trough level of 3–8 ng/mL), M p/d (i.v., 5 mg/kg), CVF (i.v., 100 U/kg), and adalimumab (subcut, 5 mg/kg, day 0).

^†αCD154 group was treated with anti-human CD154 mAb (i.v., 20 mg/kg), and R080 was additionally treated with human factor H (i.v., 18 mg/kg).

^‡αCD40 group was treated with anti-CD40 mAb (i.v., 30–50 mg/kg). R091 and R095 were treated with belatacept (i.v., 20 mg/kg) and R091 was treated additionally with basiliximab (0.3 mg/kg).

^§Tacro group was treated with anti-CD40 mAb (i.v., 30–50 mg/kg) and tacrolimus (p.o., trough level of 3–6 ng/mL). R008, R087, and R131 were treated with belatacept (i.v., 20 mg/kg). R143, R144, R147 was treated with tocilizumab (i.v., 10 mg/kg).

^║Shin, J. S., et al. “Long‐term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets.” American Journal of Transplantation 15.11 (2015): 2837–2850.

^¶Shin, Jun‐Seop, et al. “Pre‐clinical results in pig‐to‐non‐human primate islet xenotransplantation using anti‐CD 40 antibody (2C10R4)‐based immunosuppression.” Xenotransplantation 25.1 (2018): e12356.

^††Min, Byoung‐Hoon, et al. “Delayed revascularization of islets after transplantation by IL‐6 blockade in pig to non‐human primate islet xenotransplantation model.” Xenotransplantation 25.1 (2018): e12374.

2.5. Flow cytometry

Flow cytometric analyses of peripheral blood leukocytes were performed using the following mAbs: FITC-anti-monkey CD3 mAb (FN-18; U-CyTech biosciences, Utrecht, The Netherlands), APC-Cy7-anti-human CD4 mAb (OKT; BioLegend, San Diego, CA), PE-Cy7-anti-human CD8 mAb (SK1; eBioscience, San Diego, CA), PE-anti-human CD95 mAb (DX2; eBioscience), and PerCP-Cy5.5-anti-human CD28 mAb (CD28.2; eBioscience). The absolute number of lymphocytes were determined with 123count eBeads™ (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. FACSCanto II flow cytometer (BD Biosciences; San Jose, CA) was used for flow cytometry and the data were analyzed using FACSDiva software (BD Biosciences). The subsets of lymphocytes in peripheral blood leukocytes were monitored once or twice per week until the incidence of definite graft failure or euthanasia for animal welfare.

2.6. Statistical analysis

The statistical software Graphdpad Prism 6 (GraphPad Software, Inc., La Jolla, CA) was used to perform correlation analysis and compute the t-test with two-tailed p-value. Data are presented as mean ± SD from monkey blood samples, unless otherwise stated. In correlation analysis, Pearson correlation value was used. P-values less than 0.05 were considered to be statistically significant.

3. Results

3.1. Homeostatic repopulation of CD4⁺ and CD8⁺ T cells after various combinations of immunosuppression

Previously, few studies reported rather brief, longitudinal monitoring of T lymphocytes in clinical and pre-clinical islet transplantation (Table 2), and all of them except for the one from our group⁵ were on allo-transplantation models.^12–14 Thus, as the first step of this retrospective study we presented the absolute counts of T cells in the peripheral blood of the porcine islet recipients in the early post-transplantation periods. Simultaneously, we gathered the pre-ATG peripheral blood T cell count data in monkeys to set the reference ranges (Supplementary Fig. 1) since there are individual size variations within the lymphocyte compartment in both human and nonhuman primates.^15,16 In our experiments, the CD3⁺ T cell count after the induction rATG treatment was aimed for less than 500 cells/μL in peripheral blood according to a previous report.⁸ Two to five times of rATG administration (5 mg/kg, i.v.) successfully achieved the target number except for one monkey (R086) at DPT 0. Depletion of CD3⁺ T cells were also observed in the secondary lymphoid organs (Supplementary Fig. 2). MFI (mean fluorescent intensity) of CD3 after the depletion was significantly lower than that before the depletion (Supplementary Fig. 3). Recovery kinetics of CD3⁺ T cell numbers in peripheral blood varied among the monkeys: Five out of sixteen monkeys failed to reach 10^th percentile value of the reference range during the follow-up period (R089, R092, R131, R144, R147). It took 19.1±10.3 days (Fig. 1A–C) to reach pre-ATG level or 10^th percentile of the reference range in eleven monkeys. To evaluate the effect of immunosuppression regimen on T cell repopulation patterns, the monkeys were divided into 3 groups according to the maintenance immunosuppression protocol and analyzed. There were no differences in recovery times between the groups, suggesting little effect of various immunosuppression on the homeostatic repopulation of CD3⁺ T cells (Fig. 1D).

Table 2.

Previous immune monitoring studies in pre-clinical and clinical islet transplantation models

Model	Induction	Maintenance	Markers	Reference
Human (n=13)	Daclizumab	Sirolimus & tacrolimus (n=10)	CD3, CD4, CD8, CD19	Monti et al., 2008†
		Sirolimus only (n=3)
Human (n=42)	Daclizumab (n=16)	Sirolimus & tacrolimus	CD4, CD8, CD20, CD25, CD45RA, CD45RO, CD62L, FoxP3, HLA-DR, NKT2	Toso et al., 2009‡
	rATG & etanercept (n=12)	Tacrolimus & MMF
	Alemtuzumab (n=14)	Tacrolimus & MMF, sirolimus & low-dose tacrolimus
Rhesus monkey (n=18)	Anti-LFA-1 antibody & basiliximab (n=5)	Sirolimus	CD3, CD4, CD8, CD11a, CD20, CD95, CD28	Badell et al., 2010§
	Anti-LFA-1 antibody (n=5)	Belatacept
	Anti-LFA-1 antibody (n=2)	None
	Basiliximab (n=3)	Sirolimus
	None (n=3)	Belatacept
Rhesus monkey (n=5)	rATG	Anti-CD154 mAb & sirolimus	CD4, CD8, CD28,	Shin et al., 2015║

Note. All of the above studies implemented allo-transplantation model except the one by Shin et al., which is pig-to-NHP islet xenotransplantation.

^†Monti, Paolo, et al. “Islet transplantation in patients with autoimmune diabetes induces homeostatic cytokines that expand autoreactive memory T cells.” The Journal of Clinical Investigation 118.5 (2008): 1806–1814.

^‡Toso, Christian, et al. “Effect of different induction strategies on effector, regulatory and memory lymphocyte sub‐populations in clinical islet transplantation.” Transplant International 22.2 (2009): 182–191.

^§Badell, Idelberto R., et al. “LFA-1–specific therapy prolongs allograft survival in rhesus macaques.” The Journal of Clinical Investigation 120.12 (2010): 4520–4531.

^║Shin, J. S., et al. “Long‐term control of diabetes in immunosuppressed nonhuman primates (NHP) by the transplantation of adult porcine islets.” American Journal of Transplantation 15.11 (2015): 2837–2850.

Figure 1. Depletion of T lymphocytes with rATG and time-series kinetics of CD3⁺ T cell count.

Time-series CD3⁺ T cell numbers after rATG treatment in (A) αCD154 group, (B) αCD40 group, and (C) Tacro group. Data were displayed before the rATG treatment until DPT 30–37 or when CD3⁺ T cell counts reached more than 500 cells/μL. (D) First days when CD3⁺ T cell count was above pre-ATG level or 10^th percentile of the reference range in each group. αCD154, anti-CD154 mAb; αCD40, anti-CD40 mAb; Tacro, tacrolimus.

Next, the repopulation patterns of CD4⁺ and CD8⁺ T cells were then analyzed. Before the depletion of T cells, almost all of the monkeys’ CD4⁺ T cell counts were more than or tantamount to CD8⁺ T cell count (Fig. 2A–D; c.f. Fig. 2A is processed from a previously published data by Shin et al.⁵). However, after the rATG treatment, fourteen out of sixteen monkeys exhibited CD8⁺ T cell count dominance over CD4⁺ T cell count (Fig. 2D–E). These results suggested that after the depletion of T cells, homeostatic repopulation of CD8⁺ T cells happened more dynamically than that of CD4⁺ T cells.

Figure 2. Homeostatic repopulation of CD4⁺ and CD8⁺ T cells with various combinations of immunosuppression.

Time-series CD4⁺ (red) and CD8⁺ (blue) T cell numbers after rATG treatment in (A) αCD154 group, (B) αCD40 group, and (C) Tacro group. (D) CD4⁺ T cell percentage among total CD3⁺ T cells before and after the rATG treatment. (E) CD8⁺ T cell percentage among total CD3⁺ T cells before and after the rATG treatment. Fig. 2A was quoted from our previously published result ⁵. **, P < 0.01 and ****, P < 0.0001 by the two-tailed Student’s t-test. αCD154, anti-CD154 mAb; αCD40, anti-CD40 mAb; Tacro, tacrolimus.

3.2. Homeostatic repopulation of subsets of CD4⁺ and CD8⁺ T cells

We further examined the kinetics of CD4⁺ and CD8⁺ T cell subsets. Rhesus monkey T cells can be classified as three functional T cell subsets including naïve T cells (CD28⁺CD95^-), central memory T cells (T_CM; CD28⁺CD95⁺) and effector memory T cells (T_EM; CD28^-CD95⁺).^17,18 Hence, we classified CD4⁺ and CD8⁺ T cells with these surface markers and investigated their kinetics (Fig. 3A–C). We observed that the naïve subset showed significant change in proportion among three CD4⁺ subsets (P=0.0098; Fig. 3D) after the transplantation. Meanwhile in CD8⁺ T cells, significant changes were observed both in naïve (P=0.0009) and T_EM (P<0.0001) subsets after repopulation (Fig. 3E).

Figure 3. Homeostatic repopulation of CD4⁺ and CD8⁺ functional subsets with various combinations of immunosuppression.

Time-series absolute cell counts of functional subpopulations of CD4⁺ and CD8⁺ T cells after rATG treatment: (A) αCD154 group, (B) αCD40 group, and (C) Tacro group. Naïve (black), central memory cells (red), and effector memory cells (blue) represent CD95^-CD28⁺, CD95⁺CD28⁺, and CD95⁺CD28^- T cells, respectively. (D) The relative ratios of three CD4⁺ cell subsets before ATG treatment and at DPT30–37. (E) The relative ratios of three CD8⁺ cell subsets before ATG treatment and at DPT30–37. αCD154, anti-CD154 mAb; αCD40, anti-CD40 mAb; Tacro, tacrolimus.

3.3. Correlation between T cell subset recovery and the graft survival.

It has been recognized that the depletion of T cells prior to islet transplantation improved the clinical outcomes of islet transplantation,¹⁹ so we postulated that the recovery of T cell populations in peripheral blood would naturally increase the risk of T cell-mediated graft failure. The first step was to discover the T cell populations which had correlations with the early graft rejection or in other words putative surrogate markers that could predict the graft survival. To connect the T cell monitoring data (Fig. 2–3) and graft survival, the islet graft survival was first defined with the measured serum porcine c-peptide level (> 0.15 ng/mL) within the recipient monkeys.¹⁰ Then, the relationship of the graft survival and the recovery of T cell subsets to the pre-ATG level after rATG treatment (on DPT 30–37) was checked. Whether T cells or their specific subsets recover or not within the early post-transplantation period did not result in significant difference of graft survival (Supplementary Fig. 4A). Since the pre-ATG level of T cell subsets varied among the recipient monkeys (Supplementary Fig. 1A), the recovery of T cell subsets was also defined based on its absolute numbers being over the 10^th percentile value of each subset (Supplementary Fig. 1B), the reference value of which had been used previously.²⁰ Likewise, it was shown that the T cell recovery after depletion indeed did not result in the significant difference of the duration of graft survival (Supplementary Fig. 4B).

Nevertheless, the CD4⁺ and CD8⁺ cells had shown distinct repopulation kinetics, so the correlation of graft survival and the pre-ATG to DPT 30–37 ratio of cells within each T cell subset was investigated (Supplementary Fig. 5). As it was postulated that T cell recovery would negatively correlate with graft survival, it was expected that the higher cell ratio in the early post-transplantation period would be positively correlated with graft survival. However, pre-ATG to DPT 30–37 ratio of CD4⁺ subsets (red) or CD8⁺ subsets (blue) did not significantly correlate with the graft survival. Altogether, it was discovered that the recovery rate of blood T lymphocytes in the early post-transplantation period did not correlate with the graft survival.

3.4. Correlation between the graft survival and the ratios of functional subsets within CD4⁺ or CD8⁺ cells

Since T_EM cells seemed to recover more proficiently than other subsets, the ratio of cell numbers (on the same day of examination) between each subset in both CD4⁺ and CD8⁺ cells after rATG treatment was plotted against graft survival to search for correlations. As illustrated in Supplementary Fig. 6, it was observed that the ratios within CD4⁺ (Supplementary Fig. 6A) or CD8⁺ (Supplementary Fig. 6B) cell subsets did not correlate significantly with graft survival despite the distinct shift in T cell subset compositions.

Regardless of the results in Supplementary Fig. 6, the reversed dominance of CD4⁺ and CD8⁺ cells before and after rATG treatment and the differential repopulation kinetics of the functional subsets required deeper analysis. Thus, it was investigated whether CD4⁺/CD8⁺ cell ratio within the early post-transplantation periods would correlate with graft fate in our experimental settings. Notably, negative correlation was found between CD4⁺/CD8⁺ cell ratio and graft survival in the recipient monkeys (Supplementary Fig. 7A). Subsequently, the same CD4⁺/CD8⁺ ratios were calculated for the naïve, T_CM, and T_EM subsets (Supplementary Fig. 7B–P), and it was discovered that CD4⁺/CD8⁺naïve (Supplementary Fig. 7E), CD4⁺naïve/CD8⁺naïve (Supplementary Fig. 7F), and CD4⁺T_CM/CD8⁺naïve (Supplementary Fig. 7G) ratios also correlated significantly with graft survival. In sum, our results indicated that higher CD4⁺/CD8⁺ cell ratio significantly correlated with shorter graft survival in the porcine islet-transplanted monkeys regardless of immunosuppressive regimen (Table 3).

Table 3.

Pearson r between the ratio of cells and the duration of graft survival in all recipients (n=16)

	CD4+ Total	CD4+ Naïve	CD4+ TCM	CD4+ TEM	CD8+ Total	CD8+ Naïve	CD8+ TCM	CD8+ TEM
CD4+ Total		n.s.	n.s.	n.s.
CD4+ Naïve			n.s.	n.s.
CD4+ TCM				n.s.
CD4+ TEM
CD8+ Total	−0.1637*	n.s.	n.s.	n.s.		n.s.	n.s.	n.s.
CD8+ Naïve	−0.1497*	−0.1891*	−0.2071**	n.s.			n.s.	n.s.
CD8+ TCM	n.s.	n.s.	n.s.	n.s.				n.s.
CD8+ TEM	n.s.	n.s.	n.s.	n.s.

Note. Ratios in each cell are calculated as: the absolute number of subsets indicated by column headers as the numerator and the absolute number of subsets indicated in the row headers as the denominator. The correlations presented with r are statistically significant.

n.s., not significant;

^*P<0.05

^**P<0.01

3.5. Correlation between CD4⁺/CD8⁺ ratio and graft survival in recipient monkey subgroups

Consequently, the same correlation analysis was performed separately on the αCD154, αCD40, and Tacro group of monkeys to uncover the existence of a particular pattern for each group (Table 4). Notably, highly significant, positive correlations between CD4⁺/CD8⁺ cell ratios and graft survival were found in αCD40 group of monkeys (bottom panel). On the other hand, only 1 ratio each in αCD154 group and Tacro group showed significant, negative correlation (top and middle panel). Since the graft survival durations in αCD40 group of monkeys were relatively low (Table 1), it was hypothesized that a distinct correlation might exist for the xeno-islet recipients with short- or long-term graft survival. To define the category for short-term survival, the mean of CD4⁺/CD8⁺ ratios were plotted against each monkey with the fixed duration of graft survival (Supplementary Fig. 8). In this way, the collective CD4⁺/CD8⁺ ratios (Supplementary Fig. 7) could be re-organized temporally. Through this analysis, graft survival for less than or equal to 60 days was chosen arbitrarily to define short-term survival, and further correlation analyses were performed. Remarkably, CD4⁺/CD8⁺ cell ratios in the short-term survival group showed highly significant, positive correlations with the graft survival (Table 5, left panel), while only some ratios in the long-term survival group showed significant, negative correlations (Table 5, right panel). All in all, the CD4⁺/CD8⁺ cell ratios demonstrated unique correlations with the graft survival depending on the subgroup of monkeys chosen, especially in recipient monkeys with short-term graft survival (≤ 60 days).

Table 4.

Pearson r between the ratio of T cells and the duration of graft survival in three monkey subgroups of different immunosuppressive regimen

αCD154	CD4+ Total	CD4+ Naïve	CD4+ TCM	CD4+ TEM
CD8+ Total	n.s.	n.s.	n.s.	n.s.
CD8+ Naïve	n.s.	n.s.	n.s.	n.s.
CD8+ TCM	n.s.	n.s.	n.s.	n.s.
CD8+ TEM	n.s.	n.s.	−0.3511*	n.s.

Tacro	CD4+ Total	CD4+ Naïve	CD4+ TCM	CD4+ TEM
CD8+ Total	n.s.	n.s.	n.s.	n.s.
CD8+ Naïve	n.s.	n.s.	n.s.	n.s.
CD8+ TCM	n.s.	−0.2127*	n.s.	n.s.
CD8+ TEM	n.s.	n.s.	n.s.	n.s.

αCD40	CD4+ Total	CD4+ Naïve	CD4+ TCM	CD4+ TEM
CD8+ Total	n.s.	0.3341*	0.5476***	n.s.
CD8+ Naïve	0.7059****	0,9078****	0.7464****	n.s.
CD8+ TCM	n.s.	n.s.	0.4701**	n.s.
CD8+ TEM	0.4294**	0.4240**	0.4834**	n.s.

Note. Ratios in each cell are calculated as: the absolute number of subsets indicated by column headers as the numerator and the absolute number of subsets indicated in the row headers as the denominator. The correlations presented with r are statistically significant.

n.s., not significant;

^*P<0.05

^**P<0.01

^***P<0.001

^****P<0.0001

Table 5.

Pearson r between the ratio of cells and the duration of graft survival in two monkey subgroups

≤ 60 days	CD4+ Total	CD4+ Naïve	CD4+ TCM	CD4+ TEM
CD8+ Total	0.4135****	0.2826**	0.3626***	0.3424***
CD8+ Naïve	0.5257****	0.6151****	0.3159**	0.4156§
CD8+ TCM	n.s.	n.s.	0.2724**	n.s.
CD8+ TEM	0.4879****	0.3312**	0.3600***	0.4251****

> 60 days	CD4+ Total	CD4+ Naïve	CD4+ TCM	CD4+ TEM
CD8+ Total	−0.2171*	n.s.	−0.2710*	n.s.
CD8+ Naïve	n.s.	n.s.	−0.2245*	n.s.
CD8+ TCM	n.s.	n.s.	−0.3414**	n.s.
CD8+ TEM	−0.2375*	n.s.	−0.3329**	n.s.

Note. Ratios in each cell are calculated as: the absolute number of subsets indicated by column headers as the numerator and the absolute number of subsets indicated in the row headers as the denominator. The correlations presented with r are statistically significant.

n.s., not significant;

^*P<0.05

^**P<0.01

^***P<0.001

^****P<0.0001

4. Discussion

Currently, reliable biomarkers in the early post-transplantation period to predict the outcome has been lacking in islet xenotransplantation field.²¹ Enzyme-linked immunospot assay has been utilized to monitor the porcine antigen-specific T cell immune responses in NHPs.^7,22,23 However, standardization of the protocol has been a big challenge. Recently, Kang et al. reported that the time-weighted average levels of D-dimer and anti-Gal IgG negatively correlated with the survival of porcine islet grafts in NHPs.²⁴ Also, though in pig-to-NHP corneal xenotransplantation model, a study by Yoon et al. showed potentials of CD8⁺IFNγ⁺ cells and aqueous humor C3a as useful biomarkers in graft rejection prediction.²⁵ Even though these studies have their own limitations or the lack of relevance, respectively, more promising biomarkers are being discovered in islet xenotransplantation.

We believed that the T cell immune monitoring which included enumerating CD4⁺ and CD8⁺ T cells and further classifying differential functional T cell subsets such as naïve (CD28⁺CD95^-), central memory cells (CD28⁺CD95⁺) and effector memory cells (CD28^-CD95⁺) from peripheral blood had a potential to discover reliable biomarkers because of the well-known fact that the T cells are the key player in organ rejection. Since comprehensive study on time-series profiling of T cell subsets from peripheral blood in islet transplantation recipients has been lacking (Table 2), this study was designed. It was intriguing that we observed decreased values of MFI of CD3 molecules after the depletion (Supplementary Fig. 3). It could be explained by two hypotheses: First, CD3^low populations’ resistance to rATG treatment. Second, CD3 epitope hiding by polyclonal rATG-binding. Considering the restoration of MFI at 30 DPT, the latter explanation seems more feasible but it may need further investigation.

In the recipient monkeys, rATG treatments depleted CD3⁺ T cell counts to < 500 cells/μL except R086 (Fig. 1). After then, CD8⁺ cells started to increase more rapidly than CD4⁺ cells (Fig. 2). Since we confirmed that our rATG protocol depleted T cells in not only peripheral blood but secondary lymphoid organ (Supplementary Fig. 2), rapid CD8⁺ cell expansion might have been due to its quicker proliferation than CD4⁺ T cells in the secondary lymphoid organs.^26,27 Moreover, it’s been known that CD8⁺ T cells have higher proliferation capacity than CD4⁺ T cells,²⁸ and there have been previous reports of CD8⁺ T cells dominating the T cell repertoire after T cell depletion therapies.²⁹ Furthermore, we noticed that the major populations of expanded CD8⁺ T cells were CD95⁺CD28^- T_EMs (Fig. 3). This observation was concordant with previous reports in clinical solid organ transplantation.^30,31

Initially, we started analyzing whether the recovery of T lymphocytes to pre-ATG level had impact on the graft survival. Through comparison and correlation analysis, it was revealed that T cell recovery in the early period of transplantation, even the graft-unfavorable memory subsets,³² did not correlate with the islet xenograft survival (Supplementary Fig. 4 & 5). A previous study by Nadazdin et al. also showed that there was no significant difference between the expansion of memory T cells of graft-rejected and graft-accepted monkeys.³³ This observation suggested that rather than the absolute number of T cell subsets in the peripheral blood, antigen-specific T cells or the balance of the immune cell composition would have affected the graft fate. Since we haven’t characterized antigen-specific T cells in our context, we decided to inspect T cell subsets’ ratio as the next step.

The differential repopulation of T cell functional subsets after ATG treatment has been previously recognized.^34,35 Considering the various role of lymphocyte subpopulations in islet graft rejection,³² it was presumed that the repopulation kinetics of T cell subsets observed in our xenotransplantation settings would affect the graft fate. Although the shifts in subpopulation composition within CD4⁺ or CD8⁺ cells were not correlated with the graft survival, the ratios of CD4⁺/CD8⁺ produced meaningful results: lower the ratios of CD4⁺/CD8⁺, longer the survival of the islet graft (Table 3). It is not clear why CD4⁺/CD8⁺ ratios in the early post-transplantation period were negatively correlated with the graft survival considering the well-known role of CD8⁺ cells in islet xenograft rejection.³⁶ However, as our group previously suggested the graft protective roles of CD8⁺CD28^- cells in islet xenotransplantation,⁵ rapid expansion of these graft protective CD8⁺CD28^- cell populations after the rATG-mediated T lymphocyte depletion might have promoted better survival of the grafts. Even though there have been several reports which claimed the regulatory functions of CD8⁺CD28^- cells in various conditions,^37–39 there were few studies that dealt with this population of cells after homeostatic expansion condition following lympho-depletion. Thus, our results warrant further in-depth characterizations of the CD4⁺ and CD8⁺ subsets’ immune-modulating activities in the post-transplantation period.

It was noteworthy in our observations that the correlation between CD4⁺/CD8⁺ ratios and the graft survival demonstrated completely opposite patterns within short-term graft-survived monkeys (Table 5). In contrast to what we have observed, CD4⁺/CD8⁺ ratios correlated positively with graft survival in the monkeys with graft survival of ≤ 60 days. Though this correlation was highly significant statistically and this ratio could serve as a negative marker, the explanation for this results remains obscure. However, our results might have touched on the potential role in CD4⁺ regulatory T cell’s role in CD40-CD154 axis blockade regimen. Our group recently observed in mice model that if CD4⁺CD25⁺ regulatory T cells were depleted in the early phase of porcine islet xenotransplation with CD40-CD154 axis blockade, grafts were not protected by the T cells.⁴⁰ It also implies that CD4⁺/CD8⁺ ratios as a biomarker should be interpreted in this context.

There are several limitations of our study. First, because it is a retrospective analysis, the immunosuppressant protocols and the measuring dates of blood T lymphocytes are highly variant for each recipient monkey to reach a stable conclusion. Second, the number of recipient monkeys are fairly small to produce robust statistics. Last but not least, the relatively high P-values of the 4 ratios which were found to be correlated with graft survival (CD4⁺/CD8⁺, CD4⁺/CD8⁺naïve, CD4⁺naïve/CD8⁺naïve, and CD4⁺T_CM/CD8⁺naïve) indicate that these markers need to be further investigated in a larger study. Indeed, the kinetics of CD4⁺/CD8⁺ ratios have been explored in each monkey or as a whole to find any pattern or unearth a specific time point which these ratios could act strongly as biomarkers, but such features could not be found (data not shown). Most of all, presumptions on the roles of such T lymphocytes in xeno-islet graft rejection should be made very cautiously from our observations because there would be discrepancies in lymphocytes detected in the peripheral blood and the actual events occurring in the graft. Linking our observations with the studies on antigen-specific T lymphocytes proximal to the grafts⁴⁰ could be our next step to understand such disparities.

Herein, we report a retrospective analysis based on the T cell functional subsets’ time-series kinetics after rATG treatment in NHPs after porcine islet xenotransplantation. CD4⁺/CD8⁺ cell ratios were negatively correlated with the graft survival, indicating that they might have the potential to be used as biomarkers for the graft fate prediction. We hope this study ultimately leads to the development of a clinically applicable biomarker which is less time-consuming and minimally invasive to the transplant recipients.

Abbreviations:

(rATG)

rabbit anti-thymocyte globulin

(NHP)

nonhuman primate

(DPT)

days post-transplantation