Trogocytosis-derived regulatory cluster of differentiation 8−positive T cells facilitate induction of immune tolerance after lung transplantation

Abstract

Objective

Donor-specific regulatory T cells are crucial for establishing immune tolerance in lung transplantation. We sought to elucidate the specific mechanisms by which major histocompatibility complex class II (MHC-II⁺) trogocytosis Tregs induce transient immune tolerance in lung transplantation.

Methods

A rat lung transplantation model with spontaneous tolerance was established. CD8⁺CD45RCl^ow Tregs and plasmacytoid dendritic cells (pDCs) were isolated by magnetic separation and characterized by flow cytometry, cytokine profiling, and confocal microscopy. Mixed lymphocyte reactions and targeted interventions were used to dissect the mechanisms governing MHC-II acquisition and the involvement of the interferon-gamma (IFN-γ)/indoleamine 2,3-dioxygenase (IDO) signaling axis. Adoptive transfer experiments were performed to evaluate the therapeutic potential of these cells.

Results

Lung grafts from spontaneously tolerant rats exhibited significantly increased frequencies of MHC-II compared with acutely rejected grafts (13.9 ± 0.9% vs 3.5 ± 0.5%, P < .001). Confocal microscopy assay enhanced presence of MHC-II⁺CD8⁺CD45RC^lowTregs may be attributed to a specialized trogocytosis interaction between CD8⁺CD45RC^lowTregs and pDCs, which facilitated the transfer and chimerism of MHC-II molecules on the surfaces of both CD8⁺CD45RC^lowTregs and pDCs as well. Intervention with IFN-γ resulted in an increased production of MHC-II⁺CD8⁺CD45RC^lowTregs during mixed lymphocyte reactions (P < .01). Conversely, blockade of IDO with anti−IFN-γ antibody or 1-MT significantly decreased the expression (P < .001). MHC-II⁺CD8⁺CD45RC^lowTregs secreted high levels of IL-10, and the adoptive transfer of MHC-II⁺ trogocytosis Tregs notably prolonged the survival of lung-transplanted rats (40.2 ± 8.1 days vs 9.05 ± 2.3 days).

Conclusions

MHC-II⁺CD8⁺CD45RC^lowTregs mediate lung transplantation transient immune tolerance through the IFN-γ/IDO signaling pathway, providing theoretical support for the development of donor-specific immune tolerance clinical strategies.

Trogocytosis-derived MHC-II⁺ CD8⁺ Tregs induce lung transplant tolerance via IFN-γ/IDO.

Central Message.

MHC-II⁺trogocytosisCD8⁺Tregs mediate transient LT immune tolerance through the IFN-γ/IDO signaling pathway, providing theoretical support for the development of immune tolerance strategies.

Perspective.

Donor-specific Tregs are crucial for establishing immune tolerance in lung transplantation. We sought to elucidate the specific mechanisms by which MHC-II + trogocytosis Tregs induce immune tolerance in lung transplantation. MHC-II + CD8+CD45RClowTregs mediate lung transplantation immune tolerance through the IFN-γ/IDO signaling pathway, providing support for the development of clinical strategies.

Lung transplantation (LT) represents the most efficacious intervention for patients with end-stage lung disease. Recent data from the International Society for Heart and Lung Transplantation indicate that the annual global volume of lung transplant procedures has consistently surpassed 4000 since 2013.^1,2 The immunosuppressive agents administered to lung transplant recipients function through nonspecific inhibition of T-cell immune responses, resulting in persistent immunosuppression. Consequently, lung transplant recipients, relative to recipients of other solid-organ transplants, exhibit heightened vulnerability to opportunistic infections, metabolic disorders, and malignancies. Furthermore, these immunosuppressive agents fail to mitigate or suppress the incidence and progression of chronic rejection, thereby posing substantial challenges to long-term survival.³ Inducing donor-specific immune tolerance emerges as the pivotal solution to these challenges. Achieving immune tolerance for the transplanted organ remains a paramount scientific inquiry within this domain. Empirical evidence underscores the pivotal role of regulatory T cells (Tregs) in maintaining immune homeostasis and promoting self-tolerance.4, 5, 6 Tregs are predominantly categorized into CD4⁺Tregs and CD8⁺Tregs on the basis of the expression of surface molecules. Within the milieu of organ transplant immune tolerance, CD4⁺Tregs are recognized for their significant contributions.⁷ Although extant research predominantly concentrates on the CD4⁺ Treg subset within the immune regulatory network, findings from murine models of solid organ transplantation and graft-versus-host disease reveal that CD8⁺Tregs, particularly CD8⁺CD45RC^low Tregs, exhibit superior suppressive efficacy compared with CD4⁺Tregs.^8,9 Consequently, the induction of donor-specific CD8⁺Tregs represents a promising frontier for overcoming current impediments in the field of immune tolerance research.¹⁰

Trogocytosis, the transfer of membrane fragments and cytoplasmic components between immune cells through direct contact, has recently gained attention in transplant immunology.¹¹ In recent years, our team collaborated with a French group and identified that trogocytosis creates a novel immune cell type with strong immune-tolerance potential (Figure 1). For example, Ménoret and colleagues¹² demonstrated the presence and significance of CD8+ Tregs in transplant models, showing that their suppressive function can occur through both cell-contact-dependent and independent mechanisms. Dendritic cells (DCs), which express high levels of major histocompatibility complex class II (MHC-II), can exchange surface molecules with T cells during immune synapses—T cells can acquire MHC-II from DCs whereas DCs gain T-cell markers.^13,14 This trogocytosis process has been observed across various T-cell subsets and DC interactions. Meanwhile, IDO (indoleamine 2,3-dioxygenase) is an immunoregulatory enzyme that catalyzes the degradation of tryptophan into kynurenine, contributing to an immunosuppressive microenvironment. 1-MT (1-methyl-tryptophan) is a well-established pharmacologic inhibitor of IDO, often used to assess the functional relevance of IDO signaling in immune modulation. In our previous work, we found that blocking the IDO pathway (eg, with the IDO inhibitor 1-MT) prevented the transfer of tolerance, suggesting that IDO-generated metabolites support CD8⁺ Treg-mediated allograft protection.¹⁵

Figure 1.

Illustrative diagram of immune trogocytosis mechanism. pDC, Plasmacytoid dendritic cell; IFN-γ, interferon-gamma; IDO, indoleamine 2,3-dioxygenase; MHC-II, major histocompatibility complex class II.

Our previous work involved the development of an extensive organ transplantation models and conducted in-depth studies on Tregs and DCs.¹⁶ Notably, in our spontaneous LT immune tolerance rat model, the trogocytosis between CD8+CD45RClow Tregs and plasmacytoid dendritic cells (pDCs), leading to the enhanced expression of MHC-II antigens on CD8+CD45RClow Tregs. We also confirmed that the expression of interferon-gamma (IFN-γ) + Treg cells in transplant recipients is intricately linked to the establishment of immune tolerance.¹⁷ We hypothesize that MHC-II + CD8+CD45RClow Tregs may effectively induce transient immune tolerance in LT and that the IFN-γ/IDO signaling pathway may play a pivotal role in regulating this process. However, the precise molecular mechanisms by which the IFN-γ/IDO axis modulates the tolerance-inducing capacity of MHC-II + CD8+CD45RClow Tregs remain to be elucidated. Therefore, this study aims to expand on our previous research to elucidate the detailed mechanisms by which MHC-II + trogocytosisTregs facilitate the induction of immune tolerance in LT.

Methods

Establishment and Grouping of the Rat LT Spontaneous Immune Tolerance Model

All experimental donor and recipient rats were inbred, male, and aged 6 to 8 weeks, including F344 and WKY rats purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd The study was performed under a project license (no. 2024-D.-42) granted by the Ethics Committee of Beijing Chaoyang Hospital in March 1, 2024, and complied with institutional guidelines for animals’ care and use. No human participants were included in this animal study. The rat LT model was performed using the “cuff” anastomosis technique: donor arteries, veins, and bronchi were managed with a cuff method. In the recipient, the bronchus was anastomosed first. The donor lung was then sequentially aligned with the pulmonary artery and vein using the bronchus as an axis, ensuring that all cuffs were inserted and anastomosed without tension to avoid vascular tearing during repeated anastomosis in the transplantation process. The survival of the rats was observed (Figure 2). Spontaneous immune tolerance was defined as the return to normal lung function 1 week posttransplantation, with the recipient rats surviving more than 100 days without any immunosuppressive drugs and maintaining stable vital signs. According to our previous modeling experience, approximately 70% of WKY→F344 rat transplantation models exhibited spontaneous immune tolerance.

Figure 2.

Establishment and grouping of the rat lung transplantation spontaneous immune tolerance model. A, Grouping of the rat lung transplantation model. B, Surgical procedure of the “cuff” technique for orthotopic left lung transplantation in rats. PA, Pulmonary artery; B, bronchus; PV, pulmonary vein.

The experimental groups (12 rats per group) were arranged as follows.

• (1)Acute rejection group: transplant donors were F344 rats, and recipients were WKY rats.
• (2)Spontaneous immune tolerance group: Transplant donors were WKY rats, and recipients were F344 rats.
• (3)Control group: transplant donors were F344 rats, and recipients were WKY rats. The day before surgery, an equivalent volume of saline was reinfused to match the dose used for cell infusion.
• (4)Reinfusion group: Donor DC cells and transplant donors were F344 rats, and recipients were WKY rats. The day before surgery, MHC-II + trogocytosis Tregs were reinfused.

In each group, 3 rats were used for early posttransplant tissue analysis (on postoperative day 7), and the remaining 9 rats were monitored for long-term survival and included in survival analyses.

Magnetic Bead Sorting and Flow Cytometry Analysis of MHC-II⁺ CD8⁺ CD45RC^low Tregs

Spleen tissues from WKY and F344 rats were excised, homogenized, and filtered to isolate lymphocytes. PBMCs were then isolated from WKY rat spleen lymphocytes using density gradient centrifugation at 800g for 30 minutes. The cells were subsequently labeled with fluorescent antibodies targeting T-cell receptor (TCR), CD4, CD8, CD25, CD45RC, and MHC-II. Using magnetic-activated cell sorting (MACS), TCR⁺CD4⁺CD25⁻ cells were sorted as CD4+ effector T cells, and TCR⁺CD8⁺CD45RC^low cells were sorted as CD8⁺CD45RC^lowTregs.

F344 rat spleen cells were subjected to immunomagnetic bead sorting to deplete T lymphocytes, B cells, and natural killer cells. The remaining cells were then stained with fluorescent antibodies against MHC-I, MHC-II, CD45R, and CD4. Using flow cytometry, MHCI^highMHCII^highTCR^-CD45RA^-CD4⁺CD45R⁺ cells were isolated as pDCs, serving as donor antigen-presenting cells (Table E1).

Observation of Trogocytosis via Confocal Microscopy

To visualize trogocytosis, CD8+CD45RC^lowTregs were labeled with the fluorescent dye CFSE, whereas pDCs were labeled with PKH26. The labeled CD8⁺Tregs and pDCs were then co-cultured at a density of 10⁶ cells/mL in a 4:1 ratio using RPMI 1640 medium. Under the guidance of confocal microscopy, the co-cultures were observed to monitor cell-to-cell interactions and the occurrence of trogocytosis. Images were captured and analyzed to evaluate the frequency and nature of these cellular interactions and the extent of trogocytosis events.

Blockade of Molecular Signaling Pathways

Mixed lymphocyte co-cultures (mixed lymphocyte reaction, MLR) of CD8⁺CD45RC^lowTregs and pDCs were set up to investigate the role of the IFN-γ/IDO pathway. The enzyme IDO was inhibited with 1-methyl-D-tryptophan (1-MT, 20 μM). Neutralizing antibodies against IFN-γ and interleukin-10 (IL-10; 10 μg/mL each) were added to some cultures to block these cytokines, whereas recombinant IFN-γ or IL-10 and an IDO pathway metabolic analog (3,4-DAA) were added to others to enhance signaling. Each experimental condition was performed in 6 replicates. Groups included (1) a control co-culture group (Tregs + pDCs without any intervention), and (2-6) 5 intervention groups: addition of exogenous IFN-γ, addition of anti-IFN-γ neutralizing antibody, addition of the IDO inhibitor 1-MT, pDCs pretreated with 1-MT, and CD8⁺ Tregs pretreated with 1-MT, respectively.

Western Blotting for Protein Expression Analysis

Protein expression of key molecules was analyzed by Western blotting. Cells from the co-cultures were lysed in RIPA buffer containing protease and phosphatase inhibitors, and total protein was extracted and quantified (BCA assay). Equal amounts of protein (20-50 μg per sample) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto PVDF membranes. Membranes were blocked with 5% bovine serum albumin and incubated overnight at 4 °C with primary antibodies against glyceraldehyde 3-phosphate dehydrogenase, IFN-γ, and IDO (each at 1:1000 dilution). After washing, membranes were incubated with IRDye-conjugated secondary antibodies (1:10,000). Bands were visualized using the Odyssey CLx infrared imaging system and quantified with ImageJ software; target protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase.

Enzyme-linked Immunosorbent Assay (ELISA) for Assessing CD8⁺CD45RC^lowTregs and pDCs MLRs System

Concentrations of cytokines in the CD8⁺Treg-pDC co-culture supernatants were measured by ELISA. Standard curves were prepared using serial dilutions from 2000 to 0 pg/mL. Then, 96-well plates were coated with capture antibodies and incubated overnight at 4 °C. The plates were then washed and blocked with assay buffer. Samples and standards were added to the wells and incubated at room temperature, followed by addition of detection antibodies and streptavidin-enzyme conjugate. After incubation with TMB substrate, the reaction was stopped and absorbance at 450 nm was measured with a microplate reader. Cytokine concentrations (eg, IL-10, IFN-γ) in the samples were calculated from the standard curve.

Ex Vivo Induction of Donor-specific MHC-II⁺CD8⁺CD45RC^lowTregs

Isolated CD8⁺CD45RC^lowTregs and pDCs were co-cultured at a 4:1 ratio for 1 week to induce the generation of trogocytotic MHC-II + CD8+CD45RClowTregs. After 1 week, the culture medium was replaced, and the mixed lymphocyte culture was continued for an additional 2 to 3 weeks. This ex vivo induction process resulted in a 50- to 100-fold proliferation of trogocytotic MHC-II + CD8+CD45RClowTregs. The cultured cells were then collected and subjected to lymphocyte sorting to isolate the trogocytotic MHC-II + CD8+CD45RClowTregs.

Adoptive Cell Transfer Technique

Recipient rats underwent myeloablative preconditioning with total body irradiation at a dose of 4.5 Gy 1 day before LT. CD8+CD45RClow Tregs were labeled with the CFSE bio-dye, and pDCs were labeled with PKH26 for mixed lymphocyte culture and proliferation. The ex vivo expanded and isolated trogocytotic MHC-II + CD8+CD45RClowTregs were then adoptively transferred into the lung transplant recipients after myeloablation, Approximately 1 × 10⁶ cells in 0.6 mL of phosphate-buffered saline were injected via the tail vein of each recipient rat one day before transplantation. The survival duration of the transplanted lungs in various experimental groups was observed to validate the induction of immune tolerance and donor-specificity. Specimens from the transplant recipients were collected to assess the occurrence of immune rejection.

Statistical Analysis

Data are presented as mean ± standard deviation or median (interquartile range) as appropriate. For comparisons between 2 groups, the Student t test was used for normally distributed data and the Mann-Whitney U test for nonparametric data. Categorical variables were compared using the χ² test or Fisher exact test (when expected counts were <5). For comparisons among 3 or more groups, one-way analysis of variance was performed, followed by least significant difference-t or Student-Newman-Keuls post hoc tests. All statistical analyses were conducted using SPSS version 24.0.

Results

Elevated Expression of CD8⁺CD45RC^lowTregs in a Rat Model of Spontaneous LT Tolerance

Our investigation demonstrated that, in comparison with the acute rejection group, rats exhibiting spontaneous immune tolerance after LT had a significantly greater proportion of CD8+CD45RClowTregs (35.9 ± 2.65% vs 16.45 ± 2.87%, P < .001). Moreover, a subset of these Tregs showed markedly increased expression of MHC-II antigens (13.9 ± 0.9% vs 3.5 ± 0.5%, P < .001) and IL-10 (16.8 ± 0.6% vs 4.5 ± 0.6%, P < .001) (Figure 3). These results underscore the critical role of MHC-II + CD8+CD45RClowTregs in the establishment and maintenance of immune tolerance in LT.

Figure 3.

High surface expression of MHC-II molecules on CD8+ CD45RClow Treg cells in the lung transplant tolerance (TOL) model. A-B, Immune tolerance model had a significantly greater proportion of CD8+ CD45RClow Tregs than acute rejection (AR) model, One-way ANOVA, n = 11, ns > .05, ∗P < .05, ∗∗∗P < .001. C, A subset of CD8+CD45RClow Tregs showed markedly increased expression of IL-10 in the group of immune tolerance model, one-way ANOVA, n = 11, ns > .05, ∗P < .05, ∗∗∗P < .001. D, A subset of CD8+ CD45RClow Tregs showed markedly increased expression of MHC-II antigens in the group of immune tolerance model, One-way ANOVA, n = 11, ns > .05, ∗P < .05, ∗∗∗P < .001. MHC-II, Major histocompatibility complex class II; ANOVA, analysis of variance.

Sorting Efficiency and Staining Viability of CD8⁺CD45RC^lowTregs and pDCs

Using a 2-step cell sorting strategy, we achieved >97% purity for both CD8⁺CD45RC^lowTregs (TCR⁺CD8⁺CD45RC^low) and pDCs (TCR^-CD45RA^-CD4⁺CD45R⁺). In addition, fluorescent labeling was highly efficient and did not affect cell viability: >99% of CD8⁺CD45RC^lowTregs were CFSE-positive with >99% viability, and >99% of pDCs were PKH26-positive with >99% viability. These results confirm that our cell isolation and labeling methods yielded highly pure, viable CD8⁺Tregs and pDCs for subsequent experiments.

Investigation of Trogocytosis Between CD8⁺CD45RC^low Tregs and pDCs

Flow cytometry analysis demonstrated that CD8⁺CD45RC^lowTregs initially exhibited low surface expression of MHC-II molecules, with a positivity rate of (3.7 ± 0.5)%. However, after a 6-day co-culture with syngeneic or allogeneic pDCs, the positivity rate of MHC-II molecules on CD8+CD45RClowTregs significantly increased to (9.5 ± 0.5)% and (10.7 ± 0.3)%, respectively (P < .001). Fluorescence microscopy revealed that CFSE-labeled CD8+CD45RClowTregs emitted green fluorescence, whereas PKH26-labeled pDCs exhibited red fluorescence. Post 6-day co-culture, confocal microscopy imaging identified punctate red fluorescent signals on the surface of green fluorescence-labeled CD8+CD45RClowTregs, suggesting the acquisition of surface molecules from pDCs by CD8+CD45RClowTregs. Furthermore, flow cytometry analysis identified a subset of cells double-positive for CFSE and PKH26. These double-positive CD8+CD45RClowTregs displayed significantly elevated surface expression of MHC-II molecules compared with CFSE single-positive CD8+CD45RClowTregs, with positivity rates of (3.1 ± 0.8)% and (15.1 ± 0.6)% respectively (P < .001) (Figure 4). These observations, corroborated by confocal microscopy results, indicate that the increased MHC-II expression on CD8+CD45RClowTregs is attributable to the transfer of MHC-II from pDCs.

Figure 4.

CD8+CD45RClow Tregs acquire MHC-II molecules from pDCs through trogocytosis. A, Representative flow cytometry plot showing MHC-II + CD8+CD45RClow Tregs after co-culture with CFSE and PKH26 dual staining and the expression level of MHC-II molecules on the surface of CFSE + PKH26 + CD8+CD45RClow Tregs is significantly greater than that on CFSE + PKH26-CD8+CD45RClow Tregs. Independent t-test, n = 5, ∗∗∗P < .001. B, Confocal microscopy imaging identified punctate red fluorescent signals on the surface of green fluorescence-labeled CD8+CD45RClow Tregs, suggesting the acquisition of surface MHC-II molecules from pDCs by CD8+CD45RClow Tregs. MHC-II, Major histocompatibility complex class II; pDCs, plasmacytoid dendritic cells.

Elevated Expression Levels of IL-10 and IFN-γ in MHC-II⁺ CD8⁺ CD45RC^lowTregs

To further elucidate the immunoregulatory mechanisms associated with MHC-II⁺CD8⁺CD45RC^lowTregs, we assessed the expression of immune stimulatory and inhibitory cytokines. Intracellular cytokine staining revealed that, after a 6-day co-culture, MHC-II + CD8+CD45RClowTregs demonstrated significantly elevated expression levels of IL-10 (25.7 ± 5.1 vs 10.7 ± 3.1, P < .001) and IFN-γ (17.9 ± 2.5 vs 6.0 ± 0.7, P < .01) compared with MHC-II-CD8+CD45RClowTregs (Figure 5, A and B). In addition, ELISA analysis of the supernatants from the co-culture of CD8+CD45RClowTregs and pDCs revealed a slight increase in IL-10 expression in the experimental group compared with the control group, although this difference was not statistically significant (Figure 5, C). In contrast, IFN-γ expression was markedly higher in the experimental group compared with the control group (Figure 5, D). These findings indicate that MHC-II + CD8+CD45RClowTregs generated in vitro exhibit potent immunoregulatory properties, likely mediated through the secretion of IL-10 and IFN-γ.

Figure 5.

Differential analysis of IL-10 and IFN-γ expression in MHC-II + CD8+CD45RClow Tregs and MLR supernatants. A and B, Comparative flow cytometry analysis of IL-10 and IFN-γ in MHC-II + CD8+CD45RClow Tregs, total CD8+CD45RClow Tregs, and MHC-II-CD8+CD45RClow Tregs. Results are expressed as the percentage of positive cells. One-way ANOVA, n = 5, ns > .05, ∗P < .05, ∗∗∗P < .001. C-D, ELISA detection of IL-10 and IFN-γ concentrations in MLR supernatants. One-way ANOVA, n = 5, ns > .05, ∗P < .05. ELISA, Enzyme-linked immunosorbent assay. IL-10, interleukin-10; IFN-γ, interferon-gamma; MHC-II, major histocompatibility complex class II; MLR, mixed lymphocyte reaction; ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay.

Investigation of CD8⁺Tregs Pathways in Inducing Immune Tolerance after LT

Our previous studies have established that adoptive transfer of CD8+CD45RClowTregs can induce and maintain long-term survival of cardiac allografts, with IDO and IFN-γ being pivotal in this process. To further investigate whether the generation of MHC-II + CD8+CD45RClowTregs is associated with the IFN-γ/IDO pathway, we conducted interventions in the co-culture system by adding IFN-γ, anti-IFN-γ antibody (anti-IFN-γAb), or IDO inhibitor 1-MT. Western blot analysis revealed significantly elevated protein expression levels of IFN-γ and IDO in the MLR group compared with the control group (Figure 6). Flow cytometry analysis showed that, under IFN-γ stimulation, the incidence of MHC-II + CD8+CD45RClowTregs was marginally increased compared with the control group, although this difference did not reach statistical significance. Conversely, under anti-IFN-γAb or 1-MT intervention, the surface expression of MHC-II molecules on CD8+CD45RClowTregs was significantly reduced compared with the control group. Similar outcomes were observed when either CD8+CD45RClowTregs (4.1 ± 0.5)% or pDCs (5.0 ± 0.3)% were pretreated with 1-MT. These findings suggest that the generation of MHC-II + CD8+CD45RClowTregs is contingent upon the IFN-γ/IDO signaling pathway. IFN-γ intervention augmented the production of MHC-II + CD8+CD45RClowTregs in MLRs (P < .01), whereas blockade of IDO with anti-IFN-γAb or 1-MT resulted in a marked decrease in the expression of MHC-II + CD8+CD45RClowTregs (P < .001).

Figure 6.

Correlation analysis of MHC-II + CD8+CD45RClow Treg Induction with the IFN-gamma/IDO pathway. A, Expression levels of MHC-II molecules on CD8+CD45RClow Tregs under various intervention conditions. Compared with the control group, IFN-γ treatment resulted in a modest increase in MHC-II expression, whereas interventions with anti-IFN-γ antibody and 1-MT did not significantly elevate MHC-II expression levels. One-way ANOVA, n = 3, ns > 0.05, ∗∗∗P < .001. B, Comparative analysis of IFN-γ and IDO protein expression in MLRs cells. MHC-II, Major histocompatibility complex class II; IFN-γ, interferon-gamma; IDO, indoleamine 2,3-dioxygenase; 1-MT, 1-methyl-tryptophan; ANOVA, analysis of variance.

Adoptive Transfer of Trogocytosis-Derived Tregs Prolongs Survival in Acute Rejection Rat Models

Our research has further elucidated and validated that the median survival duration of lung-transplanted rats in the adoptive transfer group was 40.2 ± 8.1 days, markedly exceeding the 9.05 ± 2.3 days observed in the acute rejection control group (P < .01). Histopathologic analysis revealed an absence of significant microscopic rejection signs in the graft lungs of the adoptive transfer group within the same time frame. These findings indicate that the adoptive transfer of induced Tregs not only prolonged the survival of rats undergoing acute rejection but also effectively suppressed the rejection-induced damage (Figure 7).

Figure 7.

Adoptive transfer of trogocytosis-derived Tregs prolongs survival in acute rejection rat models. A, Survival curve analysis of rats after lung transplantation in different groups. B, Hematoxylin and eosin−stained images of transplanted lungs in rats from the acute rejection group and adoptive transfer group after lung transplantation. IFN-γ, Interferon-gamma; 1-MT, 1-methyl-tryptophan; pDCs, plasmacytoid dendritic cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Discussion

Over the past 3 decades, the field of LT has not seen substantial improvements in long-term recipient survival. LT recipients continue to experience greater morbidity and mortality than those receiving other solid organs, largely due to early and late complications such as primary graft dysfunction and chronic lung allograft dysfunction.¹⁸ The prolonged administration of immunosuppressants results in chronic graft failure, metabolic disorders, graft injury, opportunistic infections, and malignancies, which collectively undermine long-term outcomes. Transplantation immune tolerance emerges as a viable solution to mitigate rejection and these drug-related complications. In recent years, adoptive cell therapy has shown promise in transplantation and autoimmune diseases.^19,20 However, the challenge of inducing donor-specific tolerance, whereby the recipient's immune system selectively tolerates the donor graft while preserving overall immune function, remains unresolved in transplant immunology. CD8⁺Tregs were the first suppressive T-cell subset to be identified,²¹ yet they have received less attention than CD4⁺Tregs. One reason is that CD8⁺T cells express markedly lower levels of the Treg lineage transcription factor Foxp3 compared with CD4⁺T cells in mammals,²² leading researchers to focus predominantly on CD4⁺Tregs and overlook the potential of CD8⁺Tregs. Nonetheless, CD8⁺Tregs can mediate immune tolerance through various mechanisms-including the secretion of inhibitory cytokines, cytotoxic effects, and metabolic disruption. Different subsets of CD8⁺Tregs may utilize distinct suppressive pathways, or a dominant subset may employ multiple mechanisms.²³ Beyond their direct effects on effector T cells, CD8⁺Tregs can modulate the maturation and function of DCs, thereby diminishing DC-mediated antigen presentation and reducing effector T-cell activation. In transplant immunology, both CD4⁺Tregs and CD8⁺Tregs belong to the inducible Treg subset. Although the mechanisms of action for CD4⁺Tregs are relatively well characterized, there remains significant scope for research on CD⁺Tregs. A critical distinction between CD8⁺ and CD4⁺Tregs lies in their antigen restriction: CD8⁺Tregs recognize antigens presented by MHC class I (expressed by nearly all nucleated cells), whereas CD4⁺Tregs recognize antigens on MHC-II (expressed mainly by professional antigen-presenting cells). Consequently, CD8⁺Tregs can exert their suppressive activity on a broader range of cell types-including all donor-derived cells and the endothelial cells of a graft's vasculature-potentially making them especially powerful in preventing transplant rejection and in graft-versus-host disease.²⁴ This broad MHC I−restricted activity also suggests the possibility of using allogeneic “off-the-shelf” CD8⁺Tregs; the host's anti-donor immune response could be controlled via MHC I recognition, whereas CD4⁺Tregs would require donor MHC II on host cells (which may necessitate high levels of IFN-γ to induce).²⁵ In the realm of LT, studies examining the role of CD8⁺Tregs are limited. Gauthier and colleagues²⁶ emphasized the importance of CD8⁺Tregs in mitigating rejection and promoting tolerance of lung allografts. Subsequently, Lin and colleagues²⁷ confirmed that expansion of CD8⁺Tregs was significantly associated with reduced incidence of acute and chronic rejection, as well as the maintenance of optimal graft function. Their findings also indicated that high-dose immunosuppressive therapy inhibits the generation of CD8⁺Tregs, and that acute rejection depletes a substantial number of CD8⁺Tregs, suggesting that strategies to preserve or enhance CD8⁺Tregs could help sustain long-term transplant tolerance. Our study identified a novel phenomenon in a rat lung transplant model: a subset of CD8⁺CD45RC^lowTregs expressing high levels of MHC-II. Typically, T cells do not express MHC-II, and to date there have been no reports of CD8⁺CD45RC^lowTregs with MHC-II expression. In our models, tolerant rats (those with long-term graft survival without immunosuppression) showed normal lung histology and elevated levels of CD8⁺CD45RC^lowTregs and IL-10, compared with acute rejection rats. This is consistent with reports that CD8⁺CD45RC^lowTregs predominantly produce Th2 cytokines (eg, IL-4, IL-10, IL-13), which are associated with promoting transplant tolerance.²⁸ We propose that these MHC-II^high CD8⁺Tregs contribute significantly to maintaining immune tolerance. However, the precise function of this subset and the mechanism by which they acquire MHC-II are unclear. Because chronic rejection remains a major obstacle to long-term graft survival, further investigation of Treg-based therapies (particularly involving this subset) is warranted. Trogocytosis may explain how CD8⁺Tregs acquired MHC-II in our model. This process, involving the exchange of membrane components between cells, generates chimeric cells that express markers from both origin cells.²⁹ A fundamental barrier in transplantation is the recipient's recognition of donor MHC molecules via direct and indirect pathways.³⁰ Given that DCs express abundant MHC-II and can transfer membrane fragments to T cells during immunologic synapses,^13,14 we hypothesized that pDCs could be the source of MHC-II on CD8⁺Tregs through trogocytosis. Our in vitro observations support this: when CD8⁺CD45RC^lowTregs were co-cultured with pDCs, the Tregs acquired PKH26-labeled membrane fragments from the pDCs, and these trogocytosis-positive Tregs exhibited substantially higher MHC-II levels than those that did not engage in trogocytosis. This finding confirms that pDCs can transfer MHC-II to CD8⁺Tregs via trogocytosis. Our data also shed light on the immunoregulatory pathways involved in generating these MHC-II⁺CD8⁺Tregs. We observed a correlation between IFN-γ–producing Tregs and transplant tolerance,¹⁷ implicating IFN-γ in the development of this Treg subset. It is well established that cytokines such as IFN-γ and IL-10 are pivotal for the induction and function of Tregs, influencing both immune cells and tissue cells.^8,31,32 IFN-γ, in particular, induces IDO expression in pDCs, an enzyme that creates a tolerogenic environment by depleting tryptophan and producing metabolites that favor Treg development. Indeed, in a previous study, blocking the IDO pathway (for example, using a metabolite analog) prevented the adoptive transfer of tolerance.⁹ Building on these insights, we propose that the IFN-γ/IDO signaling pathway is a key regulator of trogocytosis-mediated MHC-II acquisition by CD8+ Tregs. In our co-culture experiments, exogenous IFN-γ increased the generation of MHC-II⁺CD8⁺Tregs, whereas neutralizing IFN-γ or inhibiting IDO (with 1-MT) prevented CD8⁺Tregs from upregulating MHC-II. Likewise, pre-treating CD8⁺Tregs or pDCs with 1-MT sharply reduced trogocytosis and the emergence of MHC-II⁺ Tregs. These results, in line with previous reports that IDO activity in DCs promotes Treg differentiation and suppresses effector T cells.33, 34, 35 indicate that the trogocytosis of MHC-II from pDCs to CD8⁺Tregs is highly dependent on IFN-γ-driven IDO activity. We speculate that a positive feedback loop exists wherein IFN-γ produced by Tregs upregulates IDO in pDCs, which in turn facilitates further trogocytosis and expansion of MHC-II⁺CD8⁺Tregs, thereby reinforcing immune tolerance. In vivo, the adoptive transfer of these trogocytosis-induced MHC-II⁺CD8⁺Tregs significantly prolonged graft survival (Figure E1). However, permanent tolerance was not achieved, as eventually even the treated recipients developed rejection, underscoring that additional strategies are required to achieve indefinite tolerance. Optimizing this approach will likely require fine-tuning multiple factors, including the dose of transferred Tregs, their phenotypic composition, the timing of administration, and the frequency of cell therapy. Furthermore, the donor-specific nature of the tolerance induced by these MHC-II⁺CD8⁺Tregs needs to be confirmed. And in our previous studies involving liver transplantation models, we employed a dual-infusion protocol (pretransplant and on postoperative day 7). However, for the current LT model, we adopted a single pretransplant infusion strategy. We recognize that multiple infusions may further enhance the efficacy of tolerance induction. This will be systematically evaluated in future studies. Addressing these questions will be critical for refining this strategy and moving closer to achieving durable immune tolerance in LT.

Figure E1.

Mechanistic model illustrating how MHC-II⁺CD8⁺CD45RClow regulatory T cells (Tregs), generated through trogocytosis, mediate donor-specific immune tolerance in lung transplantation. In spontaneous lung transplant tolerance model (WKY→F344), CD8⁺CD45RClow Tregs acquire MHC-II molecules from donor-derived pDCs through trogocytosis, a direct membrane transfer process occurring at the T cell-DC synapse. This acquisition is enhanced by IFN-γ, which induces IDO expression in pDCs, promoting a tolerogenic microenvironment. The resulting MHC-II⁺CD8⁺CD45RClow Tregs secrete IL-10 and IFN-γ, reinforcing their regulatory phenotype and suppressive function. Adoptive transfer of these trogocytosis-derived MHC-II⁺ Tregs into recipient rats (F344→WKY) prolongs graft survival and attenuates acute rejection. Blockade of IFN-γ or IDO (eg, with anti-IFN-γ monoclonal antibody or 1-MT) disrupts this circuit, abolishing the generation of MHC-II⁺CD8⁺ Tregs and leading to graft rejection. MHC-II, Major histocompatibility complex class II; pDCs, plasmacytoid dendritic cells; DC, dendritic cell; IFN-γ, interferon-gamma; IDO, indoleamine 2,3-dioxygenase; 1-MT, 1-methyl-tryptophan.

Conclusions

MHC-II⁺CD8⁺CD45RC^lowTregs mediate LT transient immune tolerance through the IFN-γ/IDO signaling pathway, providing theoretical support for the development of donor-specific immune tolerance clinical strategies.

Conflict of Interest Statement

The authors reported no conflicts of interest.

The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest.

Appendix E1

Table E1.

Mouse anti-rat antibodies

Alternative name	Fluorescence channel	Clone number	Brand
CD4	FITC/V450	OX-35	BD Pharmingen
CD8a	FITC/PeCy7	OX-8	BD Pharmingen
CD25	APC	OX-39	BD Pharmingen
CD45RA	PE/V450	OX-33	BD Pharmingen
CD45RC	PE/APC	OX-22	BD Pharmingen
CD11b/c	PE	OX-42	BD Pharmingen
TCRαβ	PerCp/PE	R73	BD Pharmingen
CD45R	PE	HIS24	BD Pharmingen
MHC-II	FITC/APC	HIS24	Invitrogen
IL-10	Alexa Fluor 647	A5-4	BD bioscience
IFN-γ	APC	DB-1	Invitrogen

FITC, Fluorescein isothiocyanate; APC, antigen-presenting cell; PE, phycoerythrin; MHC-II, major histocompatibility complex class II; IL-10, interleukin-10; IFN-γ, interferon-gamma.