Low regulatory T-cells frequency is associated with graft rejection after small bowel transplantation: Clinical and experimental evidence

Abstract

Background: Intestinal transplantation (ITx) represents the only curative option for patients with irreversible intestinal failure. Nevertheless, its rejection rate surpasses that of other solid organ transplants due to the heightened immunological load of the gut. Regulatory T-cells (Tregs) are key players in the induction and maintenance of peripheral tolerance, suggesting their potential involvement in modulating host vs. graft responses after ITx. Thus, we investigated the association of Tregs with allograft outcomes in pediatric patients and in an experimental model of small bowel transplantation.

Methods: Treg frequency in human samples was analyzed by Flow cytometry (CD4+CD25highCD127-, blood samples) and immunohistochemistry (FoxP3, graft samples). Experimental allogenic-heterotopic small bowel transplantation was performed in rats and animals divided into 3 groups: non-immunosuppressant treatment, rapamycin (2 mg/kg), and tacrolimus (0.6 mg/kg) treatment. Acute cellular rejection (ACR) was diagnosed based on clinical and histological findings, graft gene expression of pro- and anti-inflammatory mediators assessed by RT-qPCR, serum IL-6 and IL-10 levels by Luminex, and Treg frequency analyzed by flow cytometry (CD4+CD25highFoxP3+).

Results: Blood samples from patients undergoing ACR exhibited a significant reduction in the Treg number compared to those with normo-functional grafts. Similarly, a diminished number of FoxP3+ cells was observed in mucosa samples with ACR. In the experimental model, rapamycin-treated animals displayed clinical and histological findings resembling those not receiving immunosuppression treatment. Notably, ACR correlated with a high CD8/CD4 ratio, loss of T-cell chimerism, mRNA upregulation of pro-inflammatory genes and diminished graft Treg frequency. In contrast, tacrolimus treatment prevented ACR and facilitate blood and graft Treg expansion. Remarkably, recipients who achieved Treg expansion within the graft remained free of ACR even after discontinuation of the immunosuppressant treatment and this phenomenon was associated with increased levels of serum IL-10.

Conclusion: Our clinical and experimental findings underscore the association between Treg frequency and graft rejection after ITx, advocating for strategies that promote their expansion within the gut mucosa to enhance long-term outcomes.

Fig 1. Regulatory T cell frequency is reduced in pediatric patients during intestinal cellular rejection.

(A) CD4⁺CD25^highCD127^- Treg frequency in blood of patients with and without graft rejection (n°/μL of blood; non-rejection n = 8, rejection n = 5). (B) Percentage of blood Tregs HLA-DR⁺, CD45RA⁺, CD45RA⁺HLA-DR⁺, and CD45RA^-HLA-DR⁺ (non-rejection n = 5, rejection n = 3). (C) Representative dot plots showing that CD4⁺CD25^highCD127^- blood lymphocytes express the FoxP3 intracellular marker (n = 3); CD25^highFoxP3⁺ T-cells are depicted with black dots (right plot). (D) Frequency of lymphocytes, T-cells, and CD4⁺T-cells in blood samples of patients with and without graft rejection (n°x10³/μL of blood). (E) Number of FoxP3 cells per field in the small bowel lamina propria (non-rejection n = 8, rejection n = 3). (F) Representative graft immunohistochemistry’s of patients without rejection (upper panel) and with active rejection (lower panel), black arrows indicate the FoxP3 positive cells. Results are presented as violin plots with mean and SEM (dotted lines). *p < .05.

Fig 2. Survival, clinical and histological signs of acute cellular rejection in intestinal graft recipients.

(A) Flow diagram of the experimental model. (B) Survival curves of intestinal graft recipients. (C) Percentage of body weight loss compared with day 0 after transplant. (D) Recipients clinical score from day 0 after transplant until de endpoint. (E) Histological score of ACR in control samples (before implanting the graft) and at day 3, 7, 14 or endpoint. (F) Microscopic graft appearance in representative cases of normal histology or indeterminate, mild moderate and severe graft ACR. Results are presented as mean ± SEM or as violin plots with mean and ESM (dotted lines). Lines/dots: Black, animals without immunosuppressive treatment (w/o IS); Blue, animals treated with 2mg/kg/day of rapamycin (Rapa); Red, animals treated with 0,6mg/kg/day of tacrolimus (Tac); Grey, graft samples taken before implantation (control). In all cases n = 5/group. *p < .05; ** p < .01; *** p < .001.

Fig 3. Frequency of CD4 and CD8 T-cell in blood and graft after transplant.

(A) CD4 T-cell frequency in blood samples of control animals and animals without immunosuppression treatment, rapamycin treatment, and tacrolimus treatment at days 3 and 7 after transplant. (B) CD8 T-cell frequency in blood samples of control animals and animals without immunosuppression treatment, rapamycin treatment, and tacrolimus treatment at days 3 and 7 after transplant. (C) Frequency of donor CD4 T-cells (BN MHC I⁺ CD4⁺) in the blood at days 3 and 7 after transplant. (D) Frequency of donor CD8 T-cells (BN MHC I⁺ CD8⁺) in the blood at days 3 and 7 after transplant. (E) CD4 T-cell frequency in graft samples of control animals and animals without immunosuppression treatment, rapamycin treatment, and tacrolimus treatment at days 3 and 7 after transplant. (F) CD8 T-cell frequency in graft samples of control animals and animals without immunosuppression treatment, rapamycin treatment, and tacrolimus treatment at days 3 and 7 after transplant. (G) Frequency of recipient CD4 T-cells (LEW MHC I⁺ CD4⁺) in the graft at days 3 and 7 after transplant. (H) Frequency of recipient CD8 T-cells (LEW MHC I⁺ CD4⁺) in the graft at days 3 and 7 after transplant. LEW: Lewis; BN: Brown Norway; w/o IS: Animals without immunosuppressant treatment (black dots); Rapa: Rapamycin treatment (blue dots); Tac: Tacrolimus treatment (red dots); grey dots: control samples. Results are presented as violin plots with mean and SEM (dotted lines). *p < .05; **p < .01; *** p < .001; **** p < .0001.

Fig 4. Frequency of regulatory T-cells in blood and graft.

(A) Representative dot plots depicting Treg frequency in blood samples from control animals and those without immunosuppressant treatment, rapamycin treatment and tacrolimus treatment at days 3 and 7 after transplant. (B) Treg frequency in blood samples from control animals and animals without immunosuppressant treatment, rapamycin treatment and tacrolimus treatment at days 3 and 7 after transplant. (C) Donor Treg frequency (BN MHC I⁺) in blood at days 3 and 7 after transplant. (D) Representative dot plots depicting Treg frequency in graft samples from control animals and samples from animals without immunosuppressant treatment, rapamycin treatment and tacrolimus treatment at days 3 and 7 after transplant. (E) Treg frequency in graft samples from control animals and animals without immunosuppressant treatment, rapamycin treatment and tacrolimus treatment at days 3 and 7 after transplant. (F) Recipient Treg frequency (LEW MHC I⁺) in graft samples at days 3 and 7 after transplant. LEW: Lewis; BN: Brown Norway; w/o IS: Animals without immunosuppressant treatment (black dots); Rapa: Rapamycin treatment (blue dots); Tac: Tacrolimus treatment (red dots). Grey dots: control samples. Results are presented as violin plots with mean and SEM (dotted lines). *p < .05; **p < .01; *** p < .001, ****p <, 0001.

Fig 5. Increase frequency of Treg is associated with graft protection against acute cellular rejection.

(A) Flow diagram of the experimental group. (B) Histological score of ACR at the endpoint in animals treated with tacrolimus during 14 consecutive days and those treated with tacrolimus for only 7 of the 14 days. (C) Frequency of CD4 and CD8 cells in blood of control animals and at day 14 after transplant in animals treated with tacrolimus for only 7 days. (D) Donor CD4 and CD8 T-cell frequency at day 14 post-transplant. (E). Heat map showing mRNA relative expression of MCP-1, IL-6, TNF, IFNγ, CCL-11, CXCL-10, IL-13, IL-17, IL-22, IL-10, IDO and TGFβ in small bowel control samples (before engraftment), samples from animals without immunosuppression treatment, and graft samples of animals receiving Tac for only 7 days. (F) Principal component analysis of mRNA gene expression. (G) Percentage of Tregs in blood and graft in control samples and at day 14 after transplant in animals that receive tacrolimus for only 7 days. (H) Representative dot plot of the Treg frequency in blood and graft at day 14 in animals that receive tacrolimus for only 7 days. (I) and (J) Serum IL-10 and IL-6 levels in control samples, and samples taken at the endpoint in animals without immunosuppression treatment and tacrolimus treatment for only 1 week. Red dots: Tacrolimus treated animals; grey dots: control samples; green dots: Tacrolimus for 7 days followed by 7 days without immunosuppressant treatment. Tac: Tacrolimus. w/o IS: without immunosuppression. EP: endpoint. Dotted line: IL-6 detection limit. Results are presented as violin plots with mean and SEM (dotted lines). *p < .05, **p < .01.