Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation

Short‐term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen‐related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end‐stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3⁺CD4⁺CD25⁺CD127^lo surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg‐specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.     

Low‐Dose IL‐2 for In Vivo Expansion of CD4+ and CD8+ Regulatory T Cells in Nonhuman Primates

IL‐2 is a known potent T cell growth factor that amplifies lymphocyte responses in vivo. This capacity has led to the use of high‐dose IL‐2 to enhance T cell immunity in patients with AIDS or cancer. However, more recent studies have indicated that IL‐2 is also critical for the development and peripheral expansion of regulatory T cells (Tregs). In the current study, low‐dose IL‐2 (1 million IU/m² BSA/day) was administered to expand Tregs in vivo in naïve nonhuman primates. Our study demonstrated that low‐dose IL‐2 therapy significantly expanded peripheral blood CD4⁺ and CD8⁺ Tregs in vivo with limited expansion of non‐Treg cells. These expanded Tregs are mainly CD45RA^? Foxp3 ^high activated Tregs and demonstrated potent immunosuppressive function in vitro. The results of this preclinical study can serve as a basis to develop Treg immunotherapy, which has significant therapeutic potential in organ/cellular transplantation.     

The Effect of Costimulatory and Interleukin 2 Receptor Blockade on Regulatory T Cells in Renal Transplantation

Regulatory T cells (Treg) are critical regulators of immune tolerance. Both IL‐2 and CD28‐CD80/CD86 signaling are critical for CD4⁺CD25⁺FOXP3⁺ Treg survival in mice. Yet, both belatacept (a second‐generation CTLA‐4Ig) and basiliximab (an anti‐CD25 monoclonal antibody) are among the arsenal of current immunotherapies being used in kidney transplant patients. In this study, we explored the direct effect of basiliximab and belatacept on the Tregs in peripheral blood both in the short term and long term and in kidney biopsies of patients with acute rejection. We report that the combined belatacept/basiliximab therapy has no long‐term effect on circulating Tregs when compared to a calcineurin inhibitor (CNI)‐treated group. Moreover, belatacept‐treated patients had a significantly greater number of FOXP3⁺ T cells in graft biopsies during acute rejection as compared to CNI‐treated patients. Finally, it appears that the basiliximab caused a transient loss of both FOXP3⁺ and FOXP3^? CD25⁺ T cells in the circulation in both treatment groups raising important questions about the use of this therapy in tolerance promoting therapeutic protocols.     

Development and function of Foxp3+ regulatory T cells

Regulatory T cells (Tregs) have been recognized as having a major role in maintaining peripheral tolerance and preventing and limiting autoimmune and chronic inflammatory diseases. Tregs derive from the thymus and also develop peripherally. In this review, we discuss recent progress in our understanding of the basic mechanisms involved in Treg development and function in protecting against autoimmunity in the periphery, including thymic selection, peripheral induction and the many mechanisms of Treg suppression. Specifically in kidney disease, Tregs have been shown to play a role in limiting injury and may potentially have a therapeutic role.     

Clinical Grade Manufacturing of Human Alloantigen‐Reactive Regulatory T Cells for Use in Transplantation

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen‐reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short‐term cultures using good manufacturing practice‐compliant reagents. This process uses CD40L‐activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100‐ to 1600‐fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen‐expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.     

Migratory properties of ex vivo expanded regulatory T cells: Influence of all-trans retinoic acid and rapamycin

Adoptively transferred regulatory T-cells represent a promising therapeutic approach for tolerance induction in autoimmunity and transplantation medicine. However, a major hurdle for clinical application is the manufacturing of sufficient Treg cell numbers with respect to the low frequency of naturally occurring Tregs in the peripheral blood. Therefore, ex vivo large-scale expansion is mandatory for most of the clinical conditions. Besides the Treg cell number other parameters of the cell product are of high relevance for safe and efficient clinical Treg cell application like Treg cell purity, suppressive capacity and genetic stability of the Treg cell phenotype. Moreover, migratory properties of ex vivo expanded Tregs should be defined very clearly in order to predict their migration to secondary lymphoid organs as sites of antigen-specific activation, in vivo proliferation and subsequent trafficking to affected target organs. Therefore, we studied different cell culture conditions for Treg large-cell expansion using all-trans retinoic acid (ATRA) and/or rapamycin (Rapa) with focus on their migratory properties. The tested culture conditions revealed comparable chemokine receptor expression profiles (CXCR3, CCR4, CCR6, CCR7) and functional migration capabilities (IP10 and CCL19) with respect to Th1 and Th2 inflammatory conditions. However, the most striking difference was detected for the expansion capacity, suppressive potency and genetic stability likely predisposing large-scale expansion with ATRA and/or Rapa for therapeutic intervention in acute GvHD and without supplementation for chronic GvHD.     

Antigen‐specific regulatory T cells can induce tolerance to immunogenic grafts without the need for chronic immunosuppression

Regulatory CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) play an important role in the induction of allospecific tolerance. However tolerance in solid organ transplantation by mere transfer of Tregs has been difficult. Besides this the stability of the differentiation phenotype of Tregs has recently been questioned. We therefore aimed in generating large numbers of stable allospecific Tregs from naïve T cells by retroviral transduction with Foxp3. These were tested in an immunogenic skin transplantation model (C57BL/6→BALB/c). We established a system of transduction of mouse T cells with ecotropic retroviruses expressing Foxp3 and Thy1.1 as a surface marker to follow up transduced T cells. Alloantigen‐specific Tregs were generated by stimulating naïve recipient CD4⁺ T cells with irradiated donor splenocytes. CD25⁺ and/or CD69⁺ allospecific recipient CD4⁺ T cells were isolated and transduced with Foxp3. Alloantigen‐specific Foxp3 T cells (iTregs) showed high expression for the Treg markers Foxp3, CTLA4 and GITR. They could suppress a MLR in an alloantigen‐specific manner. Furthermore, they could be expanded up to 18 fold in vitro while maintaining their Treg phenotype and expression of lymph node homing markers like CCR7 and CD62L. iTregs prevented skin graft rejection without the need for chronic immunosuppression and recipients showed systemic allospecific allotolerance. Alloantigen‐specific Tregs were far more potent than polyspecific Tregs. Mechanisms of tolerance were graft specific homing, expansion and long‐term persistence of Tregs within the graft (>100 days, 90% of intragraft Tregs were alloantigen‐specific). In fact, tolerance could be transferred with re‐transplantation of the tolerant graft onto secondary recipients. Third party grafts were readily rejected demonstrating specificity of tolerance. Due to the Foxp3 transduction, iTregs did not lose their Treg phenotype. The results prove that large numbers of stable alloantigen‐specific Tregs can be generated from a polyclonal repertoire of naïve T cells. This is the first time that allotolerance was achieved in a non‐lymphopenic transplant model using skin grafts in an immunogenic strain combination. Therefore, antigen‐specific Tregs might have a huge therapeutic potential after solid organ transplantation.     

Applicability,safety, and biological activity of regulatory T cell therapy in liver transplantation

Regulatory T cells (Tregs) are a lymphocyte subset with intrinsic immunosuppressive properties that can be expanded in large numbers ex vivo and have been shown to prevent allograft rejection and promote tolerance in animal models. To investigate the safety, applicability, and biological activity of autologous Treg adoptive transfer in humans, we conducted an open‐label, dose‐escalation, Phase I clinical trial in liver transplantation. Patients were enrolled while awaiting liver transplantation or 6‐12 months posttransplant. Circulating Tregs were isolated from blood or leukapheresis, expanded under good manufacturing practices (GMP) conditions, and administered intravenously at either 0.5‐1 million Tregs/kg or 3‐4.5 million Tregs/kg. The primary endpoint was the rate of dose‐ limiting toxicities occurring within 4 weeks of infusion. The applicability of the clinical protocol was poor unless patient recruitment was deferred until 6‐12 months posttransplant. Thus, only 3 of the 17 patients who consented while awaiting liver transplantation were dosed. In contrast, all six patients who consented 6‐12 months posttransplant received the cell infusion. Treg transfer was safe, transiently increased the pool of circulating Tregs and reduced anti‐donor T cell responses. Our study opens the door to employing Treg immunotherapy to facilitate the reduction or complete discontinuation of immunosuppression following liver transplantation.     

Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection

Regulatory T cell (Treg) therapy using recipient‐derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor‐MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA‐A2‐specific CARs were engineered: one comprising a CD28‐CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA‐A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA‐A2‐expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune‐mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation.     

Frequency of regulatory T cells in peripheral blood and in tumour‐infiltrating lymphocytes correlates with poor prognosis in renal cell carcinoma

What’s known on the subject? and What does the study add? Treg overexpression has been demonstrated in several neoplasms, including liver, breast, pancreas and melanoma, while it has not been well evaluated in renal cancer. In renal cancer patients versus controls we found an increased expression of these cells, especially in tumour‐infiltrating lymphocytes. Moreover, Treg frequency significantly correlated with pathological stage, nuclear grade and prognostic models.

OBJECTIVE

• ? To compare the frequency of T regulatory cells (Tregs) in peripheral blood of patients (pPB) affected by renal cell carcinoma (RCC) both with the frequency of Tregs found in PB of healthy donors (hPB) and that of Tregs present in tumour infiltrating lymphocytes (TILs). To verify in vitro the inhibitory activity of tumour isolated Tregs on the effector T cells and, finally, to assess the prognostic role of Treg frequency determination.

PATIENTS AND METHODS

• ? Treg frequency in hPB, pPB and TILs was evaluated in 30 patients and 20 healthy controls by measuring both membrane‐CD25 and intracytoplasmic‐Foxp3 expression by flow cytometry.
• ? Treg inhibitory activity was evaluated by an in vitro proliferation assay performed on total, CD25‐depleted mononuclear cells (MNC) and CD25‐depleted MNC cultured in the presence of purified CD25⁺ Tregs.
• ? Finally, Treg frequency in pPB and TIL were correlated with conventional prognostic factors and scores of University of California Los Angeles and Kattan predictive models.

RESULTS

• ? Treg frequency was higher in TILs than in pPB (P= 0.002), whereas there were no important differences between hPB and pPB. CD25⁺ cells isolated either from PB and tumours showed the ability to significantly suppress in vitro both proliferation and interferon‐γ production by CD25‐depleted MNC, thus demonstrating that they are active Tregs.
• ? Treg frequency was found to significantly correlate both with pathological stage (pPB, P= 0.03; TIL, P= 0.04) and nuclear grade (TIL, P= 0.005), both for UCLA and Kattan models (all: P < 0.05 for both pPB and TIL).

CONCLUSION

• ? Treg frequency is significantly higher in TIL than in pPB of patients with RCC. Tregs showed in vitro an inhibitory activity on effector T cells isolated from kidney tumours. The increase in both peripheral and intratumoral Tregs in subjects affected with RCC were associated with worse prognosis.

     

Characterization,biology, and expansion of regulatory T cells in the Cynomolgus macaque for preclinical studies

Reliable in vitro expansion protocols of regulatory T cells (Tregs) are needed for clinical use. We studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed four in vitro Treg expansion protocols for translational studies. Tregs expanded 3000‐fold when artificial antigen presenting cells (aAPCs) expressing human CD80, CD58 and CD32 were used throughout the culture. When donor peripheral blood mononuclear cells (PBMCs) were used as the single source of APCs followed by aAPCs, Tregs expanded 2000‐fold. Tregs from all protocols suppressed the proliferation of anti‐CD2CD3CD28 bead‐stimulated autologous PBMCs albeit with different potencies, varying from 1:2‐1:4 Treg:PBMC ratios, up to >1:32. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed greater suppression of stimulation by anti‐CD2CD3CD28 beads of PBMCs from the same donor used for stimulation during the Treg cultures and of autologous cells than of third‐party PBMC responders. Similar to humans, the Treg–specific demethylated region (TSDR) within the Foxp3 locus correlated with suppressive activity and expression of Foxp3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression. In summary, we have characterized MCM Tregs and developed four Treg expansion protocols that can be used for preclinical applications.     

Two‐year follow‐up of a prospective study of circulating regulatory T cells in renal transplant patients

San Segundo D, Fernández‐Fresnedo G, Ruiz JC, Rodrigo E, Benito MJ, Arias M, López‐Hoyos M. Two‐year follow‐up of a prospective study of circulating regulatory T cells in renal transplant patients.
Clin Transplant 2010: 24: 386–393. © 2009 John Wiley & Sons A/S. Abstract: CD4⁺CD25^highFOXP3⁺ regulatory T cells (Tregs) are involved in alloreactivity and may be associated with protection from rejection. Their quantification in peripheral blood could guide clinicians in the management of renal transplant patients. Thus, we prospectively monitored the levels and in vitro suppression of circulating Tregs in 33 renal transplant patients from deceased donors within the first two yr of transplantation. Patients received maintenance immunosuppression with tacrolimus, mofetil mycophenolate and prednisolone. Results showed that peripheral blood Tregs were significantly lower six months after transplantation and recovered to almost basal levels at first post‐transplant year. The number of circulating Tregs increased significantly over basal levels afterwards. The decrease in circulating Tregs at six months may be explained by the high load of tacrolimus, as demonstrated by the inverse correlation between the blood concentration of Tregs and tacrolimus. Likewise, nine patients treated with anti‐CD25 antibodies showed higher numbers of Tregs at six months than those that did not, although differences were not observed later. In conclusion, circulating Tregs decrease in the first six months but recover thereafter up to two yr after kidney transplantation. Such a decrease is favored by high levels of tacrolimus but not by induction protocols with anti‐CD25.     

CD4+CD25+FOXP3+ Regulatory T Cells Increase De Novo in Kidney Transplant Patients After Immunodepletion with Campath-1H

Campath-1H (Alemtuzumab) is an effective immunodepletion agent used in renal transplantation. To evaluate its influence on T lymphocytes during repletion, we analyzed peripheral blood from Campath-1H-treated renal allograft recipients for the presence of FOXP3⁺ regulatory T (Treg) cells. Flow cytometry demonstrated that CD4⁺CD25⁺FOXP3⁺ lymphocytes increased significantly within the CD4⁺ T-cell population, skewing Treg/Teff (T effector) ratios for up to several years. In contrast, Treg levels in patients treated with anti-CD25 (Basiliximab) and maintained on CsA demonstrated a sustained decrease. The increase in Tregs in Campath-1H treated patients developed independent of maintenance immunosuppression. Importantly, the increase in Tregs was not fully explained by their homeostatic proliferation, increased thymic output, or Treg sparing, suggesting de novo generation/expansion. Consistent with this, in vitro stimulation of PBMCs with Campath-1H, with or without anti-CD3, activation led to an increase in CD4⁺CD25⁺FOXP3⁺ cells that had suppressive capabilities. Together, these data suggest that Campath-1H promotes an increase in peripheral Tregs and may act as an intrinsic generator of Tregs in vivo .     

Polyclonal Regulatory T Cell Therapy for Control of Inflammation in Kidney Transplants

Early subclinical inflammation in kidney transplants is associated with later graft fibrosis and dysfunction. Regulatory T cells (Tregs) can reverse established inflammation in animal models. We conducted a pilot safety and feasibility trial of autologous Treg cell therapy in three kidney transplant recipients with subclinical inflammation noted on 6‐month surveillance biopsies. Tregs were purified from peripheral blood and polyclonally expanded ex vivo using medium containing deuterated glucose to label the cells. All patients received a single infusion of ~320 × 10⁶ (319, 321, and 363.8 × 10⁶) expanded Tregs. Persistence of the infused Tregs was tracked. Graft inflammation was monitored with follow‐up biopsies and urinary biomarkers. Nearly 1 × 10⁹ (0.932, 0.956, 1.565 × 10⁹) Tregs were successfully manufactured for each patient. There were no infusion reactions or serious therapy‐related adverse events. The infused cells demonstrated patterns of persistence and stability similar to those observed in non‐immunosuppressed subjects receiving the same dose of Tregs. Isolation and expansion of Tregs is feasible in kidney transplant patients on immunosuppression. Infusion of these cells was safe and well tolerated. Future trials will test the efficacy of polyclonal and donor alloantigen‐reactive Tregs for the treatment of inflammation in kidney transplants.     

Differing Effects of Rapamycin or Calcineurin Inhibitor on T‐Regulatory Cells in Pediatric Liver and Kidney Transplant Recipients

In a cross‐sectional study, we assessed effects of calcineurin inhibitor (CNI) or rapamycin on T‐regulatory (Treg) cells from children with stable liver (n = 53) or kidney (n = 9) allografts several years posttransplant. We analyzed Treg number, phenotype, suppressive function, and methylation at the Treg‐specific demethylation region (TSDR) using Tregs and peripheral blood mononuclear cells. Forty‐eight patients received CNI (39 as monotherapy) and 12 patients received rapamycin (9 as monotherapy). Treg numbers diminished over time on either regimen, but reached significance only with CNI (r =?0.424, p = 0.017). CNI levels inversely correlated with Treg number (r =?0.371, p = 0.026), and positively correlated with CD127+ expression by Tregs (r = 0.437, p = 0.023). Patients with CNI levels >3.6 ng/mL had weaker Treg function than those with levels <3.6 ng/mL, whereas rapamycin therapy positively correlated with Treg numbers (r = 0.628, p = 0.029) and their expression of CTLA4 (r = 0.726, p = 0.041). Overall, CTLA4 expression, TSDR demethylation and an absence of CD127 were important for Treg suppressive function. We conclude that rapamycin has beneficial effects on Treg biology, whereas long‐term and high dose CNI use may impair Treg number, function and phenotype, potentially acting as a barrier to attaining host hyporesponsiveness to an allograft.     

Evaluation of the impact of conventional immunosuppressant on the establishment of murine transplantation tolerance – an experimental study

Regulatory T cells (Tregs) play a significant role in immune tolerance. Since Treg function deeply depends on Interleukin‐2 signaling, calcineurin inhibitors could affect their suppressive potentials, whereas mammalian target of rapamycin (mTOR) inhibitors may have less impact, as mTOR signaling is not fundamental to Treg proliferation. We previously reported a novel mixed hematopoietic chimerism induction regimen that promotes Treg proliferation by stimulating invariant natural killer T cells under CD40 blockade. Here, we use a mouse model to show the impact of tacrolimus (TAC) or everolimus (EVL) on the establishment of chimerism and Treg proliferation in the regimen. In the immunosuppressive drug‐dosing phase, peripheral blood chimerism was comparably enhanced by both TAC and EVL. After dosing was discontinued, TAC‐treated mice showed gradual graft rejection, whereas EVL‐treated mice sustained long‐term robust chimerism. Tregs of TAC‐treated mice showed lower expression of both Ki67 and cytotoxic T lymphocyte antigen‐4 (CTLA‐4), and lower suppressive activity in vitro than those of EVL‐treated mice, indicating that TAC negatively impacted the regimen by interfering with Treg proliferation and activation. Our results suggest that the usage of calcineurin inhibitors should be avoided if utilizing the regimen to induce Tregs in vivo for the establishment of mixed hematopoietic chimerism.     

Peripheral Blood Regulatory T Cells Are Diminished in Kidney Transplant Patients With Chronic Allograft Nephropathy

Background

Our aim in this study was to assess peripheral blood CD4⁺CD25⁺FOXP3⁺ regulatory T cell (Treg) levels in patients with chronic allograft nephropathy (CAN) 1 year after kidney transplantation.