Regulatory T Cells and Kidney Transplantation

Abstract

The ability of the immune system to differentiate self from nonself is critical in determining the immune response to antigens expressed on transplanted tissue. Even with conventional immunosuppression, acceptance of the allograft is an active process often determined by the presence of regulatory T cells (Tregs). Tregs classically are CD4⁺ cells that constitutively express high levels of the IL-2 receptor α chain CD25, along with the transcription factor Foxp3. The use of Tregs in the field of solid organ transplantation is related specifically to the objective of achieving tolerance, with the goal of reducing or eliminating immunosuppressive drugs as well as maintaining tissue repair and managing acute rejection. A key issue in clinical use of Tregs is how to effectively expand the number of Tregs, either through increasing numbers of endogenous Tregs or by the direct infusion of exogenously expanded Tregs. In order to realize the benefits of Treg therapy in solid organ transplantation, a number of outstanding challenges need to be overcome, including assuring an effective expansion of Tregs, improving long-term Treg stability and reduction of risk-related to off-target, nonspecific, immunosuppressive effects related specially to cancer.

Introduction

The ability of the immune system to differentiate self from nonself is critical in determining the immune response to antigens expressed on transplanted tissue. The immune system responds to the antigens through the interaction of T cells with the MHC.

According to animal model studies (1), acceptance/tolerance of an allograft is an active process often determined by the presence of regulatory T cells (Tregs). Therefore, numerous studies have focused on ways of either expanding endogenously occurring Tregs or using exogenously expanded Tregs to achieve acceptance of the allograft while avoiding the complications of long-term immunosuppression (2).

Types of Tregs: Phenotyping

Regulatory T cells are broadly classified as thymus derived (natural) Tregs, or peripheral inducible Tregs. Inducible Tregs can be generated from natural Tregs or naïve CD4⁺CD25⁻ cells upon T cell receptor stimulation in the presence of cytokines such as TGF-β and IL-2 (3,4).

CD4⁺ Treg cells constitutively express high levels of the IL-2 receptor α chain CD25, together with the transcription factor Foxp3, whose methylation status is a reliable marker for identifying a stable population of these cells (5). The surface marker CD127 is inversely correlated with Foxp3 expression (6). Multiple other cellular markers have been shown to be expressed by Tregs (Table 1) (7,8).

Table 1.

Additional markers defining Treg subpopulations

Marker	Positive (+) /Negative (−)	Related to
CD45RA	+	Naïve cells
CD45RO	+	Memory cells
ICOS	+	Production of IL-10 or TGF-β
CD39/73	+	cAMP- or adenosine-mediated suppression
LAP	+	LAP/TGF-β complex in activated cells
CD103	+	Effector and memory cells
GITR	+	Costimulatory
CD31	+	Adhesion and transmigration
CD147	+	Activated Treg
CCR4	+	Migrate to the graft
CD49d	−	Part of lymphocyte homing receptor, useful for purification Tregs with CD127lo
CD62L	+/Low	Naïve cells/ effector and memory cells
CD161	+	Proinflammatory potential
CTLA-4	+	Coinhibitory molecule
CD71	+	Activated Treg
GARP	+	Binds to TGF-β in activated cells
CD137	+	Antigen-specific T cells

ICOS, inducible costimulator; LAP, latency-associated peptide; GITR, glucocorticoid-induced TNF receptor; CCR4, C-C chemokine receptor type 4; CTLA-4, cytotoxic T lymphocyte antigen-4; GARP, glycoprotein A repetitions predominant.

CD4⁺CD25⁺FOXP3⁺ Tregs constitute 5%–10% of all circulating CD4⁺ cells (9).

Mechanism of Actions, Interactions with Other Cell Types

Contact dependent and independent mechanisms have been shown to contribute to the suppressive activity of regulatory T cells (Figure 1) and may act simultaneously depending on the context (2,7).

Figure 1.

Mechanisms of Treg suppression. Contact-dependent mechanisms: Induction of apoptosis via engagement of cytotoxic T lymphocyte antigen-4 (CTLA-4) and programed cell death 1 (PD1), granzyme A/B, TNF-related apoptosis-inducing ligand (TRAIL), the Fas/Fas-ligand pathway, the galectin-9/T cell immunoglobulin and mucin domain-3 (TIM-3) pathway and through IL-2 deprivation. Interaction of CTLA-4 with its ligand CD80/86 on antigen presenting cells (APCs) delivers a negative signal that prevents T cell activation. Cell surface lymphocyte activation gene 3 (LAG-3) binds to MHC class II molecules preventing the maturation and the ability of APCs to activate effector T cells. Expression of membrane-bound active TGFβ-1. Disruption of metabolic pathways through the production of adenosine from the ectoenzymes CD39/CD73-mediated degradation of extracellular ATP to AMP. ATP-derived adenosine increases intracellular cAMP levels on Tregs that is transferred to T effector cells through gap junctions, leading to the upregulation of inducible cAMP early repressor (ICER) and in turn, the inhibition of nuclear factor of activated T-cells (NFAT) and IL-2 transcription. Contact-independent mechanisms: Anti-inflammatory cytokine production (IL-10, IL-35, and TGF-β) has been linked with inhibition of T cell activation in vivo. Transfer of micro ribonucleic acid (miRNA) through exosomes can silence specific genes in T effector cells, preventing proliferation and cytokine production.

The ability of Tregs to cause “linked” or “bystander” suppression of effector T cells with different antigen specificities, or confer tolerance to T cells that recognize a third-party antigen (“infectious tolerance”), are other ways of amplifying and extending their suppressor function (10,11). Finally, although Tregs can suppress the function of activated CD4⁺ and CD8⁺ T cells, B cells, macrophages, and dendritic cells, memory T cells are less responsive to their suppressive effect (12).

Expansion of Natural Tregs

Within a clinical context it is important to consider the effects of immunosuppressive drugs on Tregs. Treatment with calcineurin inhibitors decreases Treg viability and proliferation (13). The effects of mycophenolic acid on Tregs appear to be more variable (14–16) whereas glucocorticoids appear not to affect Tregs, although one study has described steroid-related IL-2–dependent expansion of Tregs (17). The mTOR inhibitors have been shown to promote the differentiation and expansion of Tregs (18) as well as increase Foxp3 expression (19), although this effect may decline over time (20). In certain scenarios, induction therapy with thymoglobulin has been shown to favorably shift the Treg to T effector ratio (21,22). Because of the high expression of CD25 on Tregs, it is thought that anti-CD25 (basiliximab) therapy may have a deleterious effect on Treg populations (23). The use of alemtuzumab has also been shown to lead to the generation/expansion of Tregs (20). Unsurprisingly, because of the importance of the B7:CD28 interaction for the maintenance and generation of Tregs (24), the use of costimulatory molecule blocker belatacept seems to reduce their number (25,26).

Interestingly, two nonimmunosuppressive drugs have been shown to increase Treg populations. Metformin promotes phosphorylation of p-STAT5 and FOXP3 (27). Erythropoietin, on the other hand, is reported to inhibit proliferation of conventional T cells, while simultaneously sparing Treg proliferation (28). Finally, the use of low-dose recombinant IL-2 has been considered as a potential means of expanding Tregs (29), although it appears to have a narrow therapeutic window because of the risk of stimulating natural killer cell activity (30).

Inducible Tregs: Types, Ways of Expansion, and Stability

Most protocols for Treg isolation from peripheral blood utilize leukapheresis followed by serial enrichment for a CD25⁺ population. The observations that ex vivo–expanded Tregs are more suppressive compared with freshly isolated Tregs (31) justifies the need of expansion.

This expansion can be done in an antigen-specific or -nonspecific way. Donor alloantigen-specific Tregs have been shown to be more effective than polyclonal cells; however, polyclonal cells are easier to produce (7). Protocols for nonspecific stimulation involve T cell receptor (CD3) stimulation with CD28 costimulation (32). Specific stimulation uses exposing Tregs to alloantigens or indirect presentation of donor peptide on recipient dendritic cells in the presence of IL-2 (33). Additional activation with antigen and IL-12 induces more potent, antigen-specific Th1-like Tregs that express T-bet and IFN-γ (34).

Finally, Tregs can be genetically engineered for alloantigen specificity (35). In order to avoid contamination with T effector cells during the expansion procedure, Treg friendly rapamycin has been used in the manufacturing process (36). An alternative approach to increase Treg specificity would be to find ways to optimize Treg homing to the allograft (37). Additionally, long-term stability of Tregs is also a concern, as some Tregs have shown the ability to switch to an effector phenotype (38).

Clinical Use of Tregs

The indications for the use of Tregs in the field of solid organ transplantation are related to the objective of achieving tolerance while maintaining tissue repair and managing acute rejection.

The first objective is described in this review, but with respect to tissue repair, Tregs may play an important role during repair in the kidney with ischemic–reperfusion injury. Kidney-infiltrating Tregs have been shown to play a direct role in promoting healing, probably by negative modulation of the proinflammatory cytokines produced by other T cells (39). In terms of acute rejection, it is conceivable that conventional recipient-derived T cells contribute to graft tolerance if they acquire a resting tissue-resident phenotype (40). Interestingly, a recent study has shown that inducible Treg treatment in a murine model decreased serum donor-specific antibody levels and deposition of IgG within allografts, indicating a potential use in the treatment of humoral rejection (41).

With regards to the number of Tregs necessary to achieve tolerance, more important than the total dose administered may be increase Tregs to at least 30% of T effectors (3). Tang and Lee (42) have calculated that if polyclonal Tregs were administered after lymphodepletion with thymoglobulin, a dose of 3–5×10⁹ could effectively increase the Treg percentage. The clinical trials related to Treg and kidney transplantation that are ongoing are presented in Table 2 according to the information obtained from Clinicaltrials.gov (43–54).

Table 2.

Clinical trials related to Treg and kidney transplantation that are ongoing

Study	Status	Location
Treg adoptive therapy for subclinical inflammation in kidney transplantation (43)	Active, not recruiting	University of California, San Francisco, California
Treg therapy in subclinical inflammation in kidney transplantation (44)	Recruiting	University of Alabama at Birmingham, Birmingham, Alabama
		Cedars-Sinai Medical Center, Los Angeles, California
		University of California, San Francisco, San Francisco, California
		(and three more...)
Rapamycin and regulatory T cells in kidney transplantation (45)	Completed	Policlinico Fondazione IRCCS “San Matteo,” Pavia, Italy
Infusion of T-Regulatory cells in kidney transplant recipients (The ONE Study) (46)	Active, not recruiting	Massachusetts General Hospital Boston, Massachusetts
A pilot study using autologous regulatory T cell infusion zortress (Everolimus) in renal transplant recipients (47)	Not yet recruiting	University of Kentucky Medical Center Lexington, Kentucky
Effect of different therapeutic strategies on regulatory T cells in kidney transplantation (48)	Completed	Policlinico Fondazione IRCCS “San Matteo” Pavia, Italy
Donor-alloantigen-reactive regulatory T cell (darTreg) therapy in renal transplantation (The ONE Study) (49)	Recruiting	University of California San Francisco - Transplant Department, San Francisco, California
Effects of sevoflurane and desflurane on Treg (50)	Completed	Ramathibodi Hospital, Ratchathewi, Bangkok, Thailand
Trial of adoptive immunotherapy with TRACT to prevent rejection in living donor kidney transplant recipients (51)	Active, not recruiting	Northwestern University Comprehensive Transplant Center, Chicago, Illinois
Role of T helper 17 and regulatory T cells in delayed graft function (52)	Active, not recruiting	McGill University Health Center, Montreal, Quebec, Canada
Ultra-low-dose subcutaneous IL-2 in renal transplantation (53)	Active, not recruiting	Brigham and Women’s Hospital, Boston
The differential effects of 3 different immunosuppressive (54)	Completed	Northwestern Memorial Hospital, Chicago, Illinois

TRACT, T-reg Adoptive Cell Transfer.

Safety: Need for Tumor Surveillance

Numerous studies have shown that patients with cancer with a poorer prognosis have an increased number of Tregs in the circulation, as well in the lymphoid tissues and tumor microenvironment. However, induction or infusion of Tregs in patients with a chronic inflammation process may restrict cancer initiation through reducing local expression of cancer-driving proinflammatory cytokines (55).

Accordingly, it may be best to develop a screening protocol in order to rule out an established tumor before Treg infusion, so as to expand Tregs that can express markers for migration only to inflammatory sites.

Monitoring the Effect of Tregs on the Immune Response

It cannot be assumed that tolerance induced by Tregs is totally stable and one of the real challenges will be to develop a means of monitoring the immune response. Some studies have labeled expanded Tregs with deuterium to monitor the adoptively transferred cells by flow cytometry (56). However, performing protocol kidney biopsies after Treg infusion to assess changes in inflammation (57) and monitoring of donor-specific antibodies may be necessary.

Finally, because metabolic alterations represent a state of immediate cellular responses to stresses, metabolomic measurements may be useful in this context (58).

In conclusion, a more realistic intermediate goal for Treg therapy in solid organ recipients is the reduction of immunosuppressive treatment and defining more individualized immunosuppressive protocols rather than tolerance. In order to achieve this goal, it will be necessary to assure effective expansion of Tregs, improve their long-term stability, and reduce the risk of nonspecific immunosuppression, related specially to cancer, by optimizing Treg homing to the allograft.

Disclosures

None.