A Phase I/IIa study of autologous tolerogenic dendritic cells immunotherapy in kidney transplant recipients.

Kidney transplant survival is shortened by chronic rejection and side effects of standard immunosuppressive drugs. Cell-based immunotherapy with tolerogenic dendritic cells has long been recognized as a promising approach to reduce general immunosuppression. Published trials report the safety and the absence of therapy-related adverse reactions in patients treated with tolerogenic dendritic cells suffering from several inflammatory diseases. Here, we present the first phase I clinical trial results using human autologous tolerogenic dendritic cells (ATDC) in kidney transplantation. Eight patients received ATDC the day before transplantation in conjunction with standard steroids, mycophenolate mofetil and tacrolimus immunosuppression with an option to taper mycophenolate mofetil. ATDC preparations were manufactured in a Good Manufacturing Practice-compliant facility and fulfilled cell count, viability, purity and identity criteria for release. A control group of nine patients received the same standard immunosuppression, except basiliximab induction replaced ATDC therapy and mycophenolate tapering was not allowed. During the three-year follow-up, no deaths occurred and there was 100% graft survival. No significant increase of adverse events was associated with ATDC infusion. Episodes of rejection were observed in two patients from the ATDC group and one patient from the control group. However, all rejections were successfully treated by glucocorticoids. Mycophenolate was successfully reduced/stopped in five patients from the ATDC group, allowing tacrolimus monotherapy for two of them. Regarding immune monitoring, reduced CD8 T cell activation markers and increased Foxp3 expression were observed in the ATDC group. Thus, our results demonstrate ATDC administration safety in kidney-transplant recipients.

Feasibility, long-term safety, and immune monitoring of regulatory T cell therapy in living donor kidney transplant recipients.

Short-term outcomes in kidney transplantation are marred by progressive transplant failure and mortality secondary to immunosuppression toxicity. Immune modulation with autologous polyclonal regulatory T cell (Treg) therapy may facilitate immunosuppression reduction promoting better long-term clinical outcomes. In a Phase I clinical trial, 12 kidney transplant recipients received 1-10 × 106 Treg per kg at Day +5 posttransplantation in lieu of induction immunosuppression (Treg Therapy cohort). Nineteen patients received standard immunosuppression (Reference cohort). Primary outcomes were rejection-free and patient survival. Patient and transplant survival was 100%; acute rejection-free survival was 100% in the Treg Therapy versus 78.9% in the reference cohort at 48 months posttransplant. Treg therapy revealed no excess safety concerns. Four patients in the Treg Therapy cohort had mycophenolate mofetil withdrawn successfully and remain on tacrolimus monotherapy. Treg infusion resulted in a long-lasting dose-dependent increase in peripheral blood Tregs together with an increase in marginal zone B cell numbers. We identified a pretransplantation immune phenotype suggesting a high risk of unsuccessful ex-vivo Treg expansion. Autologous Treg therapy is feasible, safe, and is potentially associated with a lower rejection rate than standard immunosuppression. Treg therapy may provide an exciting opportunity to minimize immunosuppression therapy and improve long-term outcomes.

Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.

BACKGROUND: Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. METHODS: The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. FINDINGS: The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2-18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. INTERPRETATION: Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression. FUNDING: The 7th EU Framework Programme.

CD8+ Immunosenescence Predicts Post-Transplant Cutaneous Squamous Cell Carcinoma in High-Risk Patients.

Most morbidity associated with malignancy in long-term renal transplant recipients is due to cutaneous squamous cell carcinoma (SCC). Previously identified measures to stratify SCC risk have limited use, however. We hypothesized that an increased proportion of senescent, terminally differentiated CD8(+) T cells would identify renal transplant recipients at elevated SCC risk. Peripheral blood lymphocytes were isolated from 117 stable transplant recipients at high risk of SCC and analyzed phenotypically by flow cytometry. Participants were followed up prospectively for SCC development. The predictive value of variables was assessed using Cox regression. Age at transplant and enrollment, dialysis duration, and previous disease were predictive of SCC development during follow-up. Previously published clinical phenotype-based risk scores lost predictive value with the removal of age as a covariate. The percentage of CD57-expressing CD8(+) T cells was the strongest immunologic predictor of future SCC and correlated with increasing CD8(+) T cell differentiation. We dichotomized participants into those with a majority (CD57hi) and a minority (CD57lo) of CD8(+) T cells expressing CD57; CD57hi participants were more likely to develop SCC during follow-up (hazard ratio, 2.9; 95% confidence interval, 1.0 to 8.0), independent of potential confounders, and tended to develop earlier recurrence. The CD57hi phenotype was stable with time and associated with increasing age and cytomegalovirus seropositivity. Our results show that the CD57hi phenotype is a strong predictor of SCC development and recurrence in this cohort of long-term, high-risk renal transplant recipients. This information may allow identification of recipients who may benefit from intensive dermatologic screening and immunosuppression reduction.

Regulatory T cells: first steps of clinical application in solid organ transplantation.

Solid organ transplantation is the treatment of choice for patients with end-stage organ failure. To prevent rejection of the transplanted organ continuous treatment with immunosuppressive medication is needed. Immunosuppression may be harmful to the transplant recipient, increasing the risk of cancer, infections and cardiovascular disease. To improve transplant and patient survival, there is a need for an immune-modulatory regimen that is not only potent in preventing rejection of the transplanted organ, but has less side effects compared to current immunosuppressive regimens. Increasingly, transplantation research focusses on regulatory T cell (Treg) therapy to achieve this aim, in which Treg are used as a strategy to allow reduction of immunosuppression. Currently, the first clinical trials are underway investigating the safety and feasibility of Treg therapy in renal transplantation. This review gives an overview of the rationale of using Treg therapy in transplantation, previous experience with Treg therapy in humans, and the expected safety, potential efficacy and cost-effectiveness of Treg therapy in solid organ transplantation.

Late Treatment With Autologous Expanded Regulatory T-cell Therapy After Alemtuzumab Induction Is Safe and Facilitates Immunosuppression Minimization in Living Donor Renal Transplantation.

BACKGROUND: The TWO Study (Transplantation Without Overimmunosuppression) aimed to investigate a novel approach to regulatory T-cell (Treg) therapy in renal transplant patients, using a delayed infusion protocol at 6 mo posttransplant to promote a Treg-skewed lymphocyte repopulation after alemtuzumab induction. We hypothesized that this would allow safe weaning of immunosuppression to tacrolimus alone. The COVID-19 pandemic led to the suspension of alemtuzumab use, and therefore, we report the unique cohort of 7 patients who underwent the original randomized controlled trial protocol. This study presents a unique insight into Treg therapy combined with alemtuzumab and is therefore an important proof of concept for studies in other diseases that are considering lymphodepletion. METHODS: Living donor kidney transplant recipients were randomized to receive autologous polyclonal Treg at week 26 posttransplantation, coupled with weaning doses of tacrolimus, (Treg therapy arm) or standard immunosuppression alone (tacrolimus and mycophenolate mofetil). Primary outcomes were patient survival and rejection-free survival. RESULTS: Successful cell manufacturing and cryopreservation until the 6-mo infusion were achieved. Patient and transplant survival was 100%. Acute rejection-free survival was 100% in the Treg-treated group at 18 mo after transplantation. Although alemtuzumab caused a profound depletion of all lymphocytes, including Treg, after cell therapy infusion, there was a transient increase in peripheral Treg numbers. CONCLUSIONS: The study establishes that delayed autologous Treg therapy is both feasible and safe, even 12 mo after cell production. The findings present a new treatment protocol for Treg therapy, potentially expanding its applications to other indications.

Update on the long-term complications of renal transplantation.

INTRODUCTION: Powerful immunosuppressive regimens have reduced rejection risk, leading to an expanding cohort of long-term kidney transplant recipients who are likely to encounter practitioners in other specialties. SOURCES OF DATA: Key review papers and primary literature identified through searches of PubMed, Google Scholar and Medline. AREAS OF AGREEMENT: Death from cardiovascular disease and malignancy remain the chief causes of transplant loss. Risk factors and phenotypes for these differ from the general population. AREAS OF CONTROVERSY: Many guidelines for renal transplant recipients are based on extrapolation from studies on non-transplant cohorts and may not be appropriate. Emerging studies demonstrate that established interventions in the general population are less efficacious in transplant recipients. GROWING POINTS: The influence of immunosuppression on the development of complications. AREAS TIMELY FOR DEVELOPING RESEARCH: Markers to guide individualized optimal immunosuppression and predict the development of complications would allow for targeted early intervention.

Conversion to sirolimus in kidney transplant recipients with squamous cell cancer and changes in immune phenotype.

BACKGROUND: Conversion to sirolimus from calcineurin inhibitor- (CNI), azathioprine- (AZA) and mycophenolate-based regimens reduces the risk of development of squamous cell carcinoma of the skin (SCC) in kidney transplant recipients (KTRs). Sirolimus conversion may also be protective by permitting beneficial changes in immune phenotype. It is not known how sirolimus will affect immune phenotype in KTRs with SCC. METHODS: Thirty-two KTRs with SCC were enrolled into this single-blinded randomized study and 13 KTRs randomized to sirolimus (4-10 ng/mL) and prednisolone 5 mg/day. RESULTS: Six-month post conversion to sirolimus FOXP3(+) CD127(low)CD25(high)CD69(-), the number of T cells (putative Treg) increased significantly (P = 0.008). Natural killer (NK) and CD56(bright) NK cells also increased significantly (P = 0.039 and 0.02). T-cell number only significantly increased in those KTRs where CNI was ceased as part of the conversion to mammalian target of rapamycin inhibitors (mTORi's) (P = 0.031) implying CNI cessation rather than mTORi initiation induced an increase in T-cell number. Increases in the NK cell number was only significant in those KTRs where AZA was ceased (P = 0.040), implying AZA cessation rather than mTORi initiation caused the NK cell number to increase. At 6 months, sirolimus conversion reduces new SCC/year, rate ratio 0.49 (95%CI: 0.15-1.63), P = 0.276. On therapy analysis and intention-to-treat analysis over 24 months, the rate ratios were 0.84 and 0.87, respectively, and did not reach significance. CONCLUSIONS: Conversion to mTORi from CNI may reveal a pre-existing high Treg phenotype by unmasking CNI inhibition of FOXP3 expression. Cessation of AZA leads to increased NK cell number. High FOXP3(+) T-cell number on conversion to mTORi may predict those KTRs who continue to accrue SCC.

Immune resilience despite inflammatory stress promotes longevity and favorable health outcomes including resistance to infection.

Some people remain healthier throughout life than others but the underlying reasons are poorly understood. Here we hypothesize this advantage is attributable in part to optimal immune resilience (IR), defined as the capacity to preserve and/or rapidly restore immune functions that promote disease resistance (immunocompetence) and control inflammation in infectious diseases as well as other causes of inflammatory stress. We gauge IR levels with two distinct peripheral blood metrics that quantify the balance between (i) CD8+ and CD4+ T-cell levels and (ii) gene expression signatures tracking longevity-associated immunocompetence and mortality-associated inflammation. Profiles of IR metrics in ~48,500 individuals collectively indicate that some persons resist degradation of IR both during aging and when challenged with varied inflammatory stressors. With this resistance, preservation of optimal IR tracked (i) a lower risk of HIV acquisition, AIDS development, symptomatic influenza infection, and recurrent skin cancer; (ii) survival during COVID-19 and sepsis; and (iii) longevity. IR degradation is potentially reversible by decreasing inflammatory stress. Overall, we show that optimal IR is a trait observed across the age spectrum, more common in females, and aligned with a specific immunocompetence-inflammation balance linked to favorable immunity-dependent health outcomes. IR metrics and mechanisms have utility both as biomarkers for measuring immune health and for improving health outcomes.

World Kidney Day 2012: the global role of kidney transplantation.

There remain major challenges to providing optimal treatment for ESRD worldwide and a need, particularly in low-income economies, to mandate more focus on community screening and implementation of simple measures to minimize progression of chronic kidney disease. The recent designation of renal disease as an important noncommunicable disease at the United Nations High Level Meeting on Noncommunicable Diseases is one step in this direction.(32) But early detection and prevention programs will never prevent ESRD in everyone with chronic kidney disease, and kidney transplantation is an essential, viable, cost-effective, and life-saving therapy which should be equally available to all people in need. It may be the only tenable long-term treatment option for ESRD in low-income countries since it is both cheaper and provides a better outcome for patients than other treatment for ESRD. However, the success of transplantation has not been delivered evenly across the world and substantial disparities still exist in access to transplantation.We remain troubled by commercialization of living donor transplantation and exploitation of vulnerable populations for profit. There are solutions available. These include demonstrably successful models of kidney transplant programs in many developing countries; growing availability of less-expensive generic immunosuppressive agents; improved clinical training opportunities; governmental and professional guidelines legislating prohibition of commercialization and defining professional standards of ethical practice; and a framework for each nation to develop self-sufficiency in organ transplantation through focus on both living donation and especially nationally managed deceased organ donation programs. The Transplantation Society and the ISN have pledged to work together in coordinated joint global outreach programs to help establish and grow appropriate kidney transplant programs in low- and middle-income countries utilizing their considerable joint expertise. World Kidney Day 2012 provides a focus to help spread this message to governments, all health authorities and communities across the world.

Dampened Inflammatory Signalling and Myeloid-Derived Suppressor-Like Cell Accumulation Reduces Circulating Monocytic HLA-DR Density and May Associate With Malignancy Risk in Long-Term Renal Transplant Recipients.

Background: Malignancy is a major cause of morbidity and mortality in transplant recipients. Identification of those at highest risk could facilitate pre-emptive intervention such as reduction of immunosuppression. Reduced circulating monocytic HLA-DR density is a marker of immune depression in the general population and associates with poorer outcome in critical illness. It has recently been used as a safety marker in adoptive cell therapy trials in renal transplantation. Despite its potential as a marker of dampened immune responses, factors that impact upon monocytic HLA-DR density and the long-term clinical sequelae of this have not been assessed in transplant recipients. Methods: A cohort study of stable long-term renal transplant recipients was undertaken. Serial circulating monocytic HLA-DR density and other leucocyte populations were quantified by flow cytometry. Gene expression of monocytes was performed using the Nanostring nCounter platform, and 13-plex cytokine bead array used to quantify serum concentrations. The primary outcome was malignancy development during one-year follow-up. Risk of malignancy was calculated by univariate and multivariate proportionate hazards modelling with and without adjustment for competing risks. Results: Monocytic HLA-DR density was stable in long-term renal transplant recipients (n=135) and similar to non-immunosuppressed controls (n=29), though was suppressed in recipients receiving prednisolone. Decreased mHLA-DRd was associated with accumulation of CD14+CD11b+CD33+HLA-DRlo monocytic myeloid-derived suppressor-like cells. Pathway analysis revealed downregulation of pathways relating to cytokine and chemokine signalling in monocytes with low HLA-DR density; however serum concentrations of major cytokines did not differ between these groups. There was an independent increase in malignancy risk during follow-up with decreased HLA-DR density. Conclusions: Dampened chemokine and cytokine signalling drives a stable reduction in monocytic HLA-DR density in long-term transplant recipients and associates with subsequent malignancy risk. This may function as a novel marker of excess immunosuppression. Further study is needed to understand the mechanism behind this association.

Transition from pediatric to adult renal services: a consensus statement by the International Society of Nephrology (ISN) and the International Pediatric Nephrology Association (IPNA).

The transfer of young patients from pediatric to adult renal care takes place after a transition process which involves both sides. It is important that it is individualized for each young person, focusing on self-management skills as well as assessing support structures. The consensus statement has been developed by the panel of adult and pediatric nephrologists and endorsed by the councils of both ISN and IPNA. It is hoped that the statement will provide a basis for the development of locally appropriate recommendations for clinical practice.

Immune phenotype predicts risk for posttransplantation squamous cell carcinoma.

Cutaneous squamous cell cancer (SCC) affects up to 30% of kidney transplant recipients (KTRs) within 10 years of transplantation. There are no reliable clinical tests that predict those who will develop multiple skin cancers. High numbers of regulatory T cells associate with poor prognosis for patients with cancer in the general population, suggesting their potential as a predictive marker of cutaneous SCC in KTRs. We matched KTRs with (n = 65) and without (n = 51) cutaneous SCC for gender, age, and duration of immunosuppression and assessed several risk factors for incident SCC during a median follow-up of 340 days. Greater than 35 peripheral FOXP3(+)CD4(+)CD127(low) regulatory T cells/microl, <100 natural killer cells/microl, and previous SCC each significantly associated with increased risk for new cutaneous SCC development (hazard ratio [HR] 2.48 [95% confidence interval (CI) 1.04 to 5.98], HR 5.6 [95% CI 1.31 to 24], and HR 1.33 [95% CI 1.15 to 1.53], respectively). In addition, the ratio of CD8/FOXP3 expression was significantly lower in cutaneous SCC excised from KTRs (n = 25) compared with matched SCC from non-KTRs (n = 25) and associated with development of new cutaneous SCCs. In summary, monitoring components of the immune system can predict development of cutaneous SCC among KTRs.

The role of kidney transplantation as a component of integrated care for chronic kidney disease.

Kidney transplant provides superior outcomes to dialysis as a treatment for end-stage kidney disease. Therefore, it is essential that kidney transplantation be part of an integrated treatment and management plan for chronic kidney disease (CKD). Developing an effective national program of transplantation is challenging because of the requirement for kidney donors and the need for a multidisciplinary team to provide expert care for both donors and recipients. This article outlines the steps necessary to establish a national kidney transplant program, starting with the requirement for effective legislation that provides the legal framework for transplantation whilst protecting organ donors, their families, recipients, and staff and is an essential requirement to combat organ trafficking. The next steps involve capacity building with the development of a multiskilled workforce, the credentialing of transplant centers, and the reporting of outcomes through national or regional registries. Although it is accepted that most transplant programs will begin with living related kidney donation, it is essential to aspire to and develop a deceased donor program. This requires engagement with multiple stakeholders, especially the patients, the general community, intensivists, and health departments. Development of transplant centers should be undertaken in concert with the development of a dialysis program. Both are essential components of integrated care for CKD and both should be viewed as part of the World Health Organization's initiative for universal health coverage. Provisions to cover the costs of treatment for patients need to be developed taking into account the state of development of the overall health framework in each country.

Development of a framework for minimum and optimal safety and quality standards for hemodialysis and peritoneal dialysis.

Substantial heterogeneity in practice patterns around the world has resulted in wide variations in the quality and type of dialysis care delivered. This is particularly so in countries without universal standards of care and governmental (or other organizational) oversight. Most high-income countries have developed such oversight based on documentation of adherence to standardized, evidence-based guidelines. Many low- and lower-middle-income countries have no or only limited organized oversight systems to ensure that care is safe and effective. The implementation and oversight of basic standards of care requires sufficient infrastructure and appropriate workforce and financial resources to support the basic levels of care and safety practices. It is important to understand how these standards then can be reasonably adapted and applied in low- and lower-middle-income countries.

Academic achievement and employment in young adults with end-stage kidney disease.

BACKGROUND: Young adults with end-stage kidney disease (ESKD) are at a pivotal stage of life: progressing through education, seeking employment and developing relationships. We set out to explore how ESKD impacts education and employment attainment in a matched UK and USA patient cohort. Moreover, we aimed to determine if there were significant differences in reported perceptions of impact. DESIGN: A mixed methods design combining previously validated quantitative questionnaire surveys and qualitative semi-structured interviews. PARTICIPANTS: Young people with ESKD aged 18-30 years (N = 27), attending single-centre follow-up in Oxford, UK were matched with 27 comparable young people aged 19-30 years, under follow-up in Denver, USA. Twelve of these patients from Denver were selected for interview. MEASUREMENTS: Self-report questionnaires surveyed patient demographics, educational and employment achievement and experiences. Questionnaire categorical data for matched pairs were analysed using Bowker's test of symmetry. Sequential flow analyses of interview content delineated perception patterns through thematic coding. RESULTS: Sixty percent of non-student Oxford participants were employed compared with 41% in Denver (p = 0.023). Forty-four percent of Oxford patients compared with 52% in Denver, reported illness had made it difficult to gain employment (p = 0.88). In Oxford, 32% completed high school as their highest educational achievement, versus 68% in Denver (p = 0.22). Qualitative themes included fatigue, self-esteem loss, social isolation and low mood. The impact of dialysis and poor understanding from educators/employers resulted in lost work time, and/or limited educational attainment. CONCLUSION: ESKD profoundly impacts on education and employment of young adults in the United States and United Kingdom, generating substantial barriers. Poor understanding appears prevalent amongst educators and employers. Healthcare providers must recognise these problems and invest resources towards tailored support in order to improve associated psychosocial and clinical outcomes.

Seven-year prospective study of nonmelanoma skin cancer incidence in U.K. renal transplant recipients.

Nonmelanoma skin cancer (NMSC) causes significant morbidity and mortality posttransplantation. We examined the annual incidence of NMSC in U.K. renal transplant recipients (RTRs). A total of 269 (95% of potential population) RTRs of skin type I-IV were recruited into a prospective study of NMSC incidence between 1998 and 2006. A total of 244 (91% enrolled) RTRs were screened on at least one occasion. The mean incidence per year of NMSC was 7.82% (SD: 1.84), comprising a mean (SD) incidence per year of squamous cell carcinoma 3.45% (1.36), basal cell carcinoma 3.58% (1.17), and Bowen's disease 2.52% (0.91). The risk of developing NMSC increased with duration posttransplantation: the mean incidence per year of NMSC was 3.27% (0.53) in RTRs <5 years posttransplantation, 5.86% (3.1) in RTRs 5-10 years posttransplant, and 11.1% (1.85) in those >10 years posttransplant. Relatively low NMSC incidence rates within the first 5 years posttransplantation suggests that duration posttransplantation may determine the optimum frequency of surveillance of RTRs in the United Kingdom.

CD45RA Distinguishes CD4+CD25+CD127-/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression.

BACKGROUND: Adoptive transfer of forkhead box protein (FOX)3 regulatory T (Treg) cells offers a promising strategy to reduce damage to an allograft by the recipient's immune system. Identification of cell surface markers sufficient to purify Treg cells expanded ex vivo to remove cellular contaminants requires optimization. Furthermore, the expanded Treg must be able to survive, expand, and suppress in allograft recipients exposed to immunosuppressants, such as tacrolimus (TAC). Reduced CD127 expression enhances identification of Treg in the human CD4CD25 population. CD45RA expression identifies naive CD4CD25 Treg with an enhanced stability of Treg phenotype. METHODS: We combine an analysis of CD45RA, CD25, and CD127 expression to identify subpopulations of CD4CD127CD25 cells. Regulatory T cells were sorted according to expression of CD25 and CD45RA and expanded in the presence of a physiological relevant concentration of TAC. Regulatory T cell-specific demethylation region (TSDR) demethylation, FOXP3 expression, and suppression were analyzed. RESULTS: CD4CD127CD25CD45RA Treg cells had a stable TSDR demethylated FOXP3 phenotype after expansion whereas CD4CD127CD25CD45RA Treg cell lost the TSDR demethylated phenotype. CD45RA Treg had a greater capacity to suppress after expansion with TAC. CONCLUSIONS: Although CD45RA Treg retained a greater suppressive capacity when expanded with TAC, the marked loss of the TSDR demethylated status highlights the potential for loss of stability of these cells in transplant recipients treated with TAC based immunosuppression. We show that a population of CD4CD127CD45RA Regulatory T cell may offer the best compromise between susceptibility to loss of suppression when exposed to TAC and maintenance of a TSDR demethylated phenotype following in vitro expansion.

Application of Operational Tolerance Signatures Are Limited by Variability and Type of Immunosuppression in Renal Transplant Recipients: A Cross-Sectional Study.

BACKGROUND: Renal transplant recipients (RTR) frequently develop complications relating to chronic immunosuppression. Identifying RTR who could safely reduce immunosuppression is therefore highly desirable. We hypothesized that "signatures" described in RTR who have stopped immunosuppression but maintained stable graft function ("operational tolerance") may enable identification of immunosuppressed RTR who are candidates for immunosuppression minimization. However, the effect of immunosuppression itself on these signatures and circulating B-cell populations is currently unknown. METHODS: We undertook a cross-sectional study of 117 RTR to assess the effect of immunosuppression upon circulating B cell populations, humoral alloresponse and 2 previously published "signatures" of operational tolerance. RESULTS: Immunosuppression associated with alterations in both published "signatures." Azathioprine associated with a decrease in transitional and naive B-cell numbers and calcineurin inhibition associated with an increase in the number of circulating plasmablasts. However, only azathioprine use associated with the presence of donor-specific anti-HLA IgG antibodies. Calcineurin inhibition associated with an increase in total serum IgM but not IgG. Data were corrected for age, time since last transplant, and other immunosuppression. CONCLUSIONS: Current signatures of operational tolerance may be significantly affected by immunosuppressive regimen, which may hinder use in their current form in clinical practice. Calcineurin inhibition may prevent the development of long-lasting humoral alloresponses, whereas azathioprine therapy may be associated with donor specific antibody development.