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Introduction: donor-derived modified immune cells (MIC) induced long-term specific immunosuppression against the allogeneic donor in preclinical models of transplantation. In a phase I clinical trial (TOL-1 Study), MIC treatment resulted in a cellular phenotype that was directly and indirectly suppressive to the recipient's immune system allowing for reduction of conventional immunosuppressive therapy. Here, we describe a protocol for a randomised controlled, multicentre phase-IIb clinical trial of individualised immunosuppression with intravenously administered donor MIC compared with standard-of-care (SoC) in living donor kidney transplantation (TOL-2 Study). Methods and analysis: sixty-three living donor kidney transplant recipients from six German transplant centres are randomised 2:1 to treatment with MIC (MIC group, N=42) or no treatment with MIC (control arm, N=21). MIC are manufactured from donor peripheral blood mononuclear cells under Good Manufacturing Practice conditions. The primary objective of this trial is to determine the efficacy of MIC treatment together with reduced conventional immunosuppressive therapy in terms of achieving an operational tolerance-like phenotype compared with SoC 12 months after MIC administration. Key secondary endpoints are the number of patient-relevant infections as well as a composite of biopsy-proven acute rejection, graft loss, graft dysfunction or death. Immunosuppressive therapy of MIC-treated patients is reduced during follow-up under an extended immunological monitoring including human leucocyte antigen-antibody testing, and determination of lymphocyte subsets, for example, regulatory B lymphocytes (Breg) and antidonor T cell response. A Data Safety Monitoring Board has been established to allow an independent assessment of safety and efficacy. Ethics and dissemination: ethical approval has been provided by the Ethics Committee of the Medical Faculty of the University of Heidelberg, Heidelberg, Germany (AFmu-580/2021, 17 March 2022) and from the Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Institute, Langen, Germany (Vorlage-Nr. 4586/02, 21 March 2022). Written informed consent will be obtained from all patients and respective donors prior to enrolment in the study. The results from the TOL-2 Study will be published in peer-reviewed medical journals and will be presented at symposia and scientific meetings. Trial registration number NCT05365672., Preparation of the study was funded by the Federal Ministry of Education and Research, Berlin, Germany (FKZ 161B0560A, 161B0560B, and FKZ 031B0560A, 031B0560B), and TolerogenixX GmbH, Heidelberg, Germany (N/A). Conduct of the study is funded by TolerogenixX GmbH (N/A).

An imbalance between CD4+-regulatory T-cells (Tregs) and CD4+-responder T-cells (Tresps) correlates with active disease flares in systemic lupus erythematosus (SLE) patients. Both cell subsets consist of highly proliferating Tregs/Tresps expressing inducible T-cell co-stimulatory molecule (ICOS) and less proliferating ICOS--Tregs/Tresps.Six-colour-flow-cytometric analysis was used to examine the effect of ICOS+- and ICOS--Treg/Tresp cell differentiation on the composition of the total CD4+-T-helper cell pool with ICOS+- and ICOS--Tregs/Tresps. Functionality of Tregs was examined using suppression assays.In 83 healthy volunteers, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age, while that of ICOS--Tregs/ICOS--Tresps did not change. In 86 SLE patients (SLEDAI7), disease activity was associated with an age-independently increased ratio of both ICOS+-Tregs/ICOS+-Tresps and ICOS--Tregs/ICOS--Tresps. In these patients, the functional activity of ICOS+-Tregs, but not of ICOS--Tregs, was preserved. In 13 markedly active disease patients (SLEDAI7), the percentage of both ICOS+-Tregs and ICOS+-Tresps, was strongly increased within total CD4+-T-helper cells. However, the increased ratio of ICOS+-Tregs/ICOS+-Tresps was not maintained in these patients, due to terminal differentiation and accumulation of naïve cells within total ICOS+-Tregs. Despite increased differentiation of both ICOS--Tregs and ICOS--Tresps, the percentage of ICOS--Tregs increased within CD4+-T-helper cells, while that of ICOS--Tresps decreased, resulting in a significantly increased ratio of ICOS--Tregs/ICOS--Tresps independent of age.Our data reveal a crucial role of Treg immune senescence for the occurrence of disease flares in SLE patients, with ICOS+-Treg cells being most affected.

Chronic kidney failure (KF) provokes the development of immune senescent CD8+ cytotoxic T cells, affecting the occurrence of graft rejection, viral infections, and malignancies after kidney transplantation. In this study, we analyzed the impact of KF, subsequent dialysis treatment, and kidney transplantation on the differentiation of CD8+CD31+CD45RA+CCR7+ recent thymic emigrant (CCR7+ RTE) Tregs/Tresps into CD8+CD31-CD45RA- memory (CD31- memory) Tregs/Tresps and its effect on the release of cytokines, Fas receptor, Fas ligand as well as cytotoxic mediators by naïve, central memory (CM), effector memory (EM), and terminally differentiated effector memory (TEMRA) Tresps. We found that normal age-dependent differentiation of CD8+ Tregs/Tresps generally differs in the way that TEMRA cells only arise in Tresps. Compared to healthy controls, KF patients revealed an age-independently decreased frequency of CCR7+ RTE Tregs/Tresps, but increased frequencies of CCR7+ MN Tregs/Tresps and CD31- memory Tregs/Tresps, suggesting an increased differentiation via CD31+CD45RA- memory (CD31+ memory) Tregs/Tresps into CD31- memory Tregs/Tresps. Intensified differentiation via CD31+ memory Tresps increased the emergence of apoptosis-resistant CM Tresps with strong Fas ligand-mediated cytotoxicity. CCR7+ RTE Tresp proliferation generated TEMRA Tresps, secreting high levels of cytotoxic mediators. In dialysis and transplant patients, CD31+ TEMRA Tregs/Tresps accumulated, proposing an impaired CCR7+ RTE Treg/Tresp differentiation via CD31+ memory Tregs/Tresps into CD31- memory Tregs/Tresps. Increased percentages of CD31- TEMRA Tresps, but not of CD31- TEMRA Tregs, were observed in all patient groups, indicating impaired proliferation of CCR7+ RTE Tresps, but not of CCR7+ RTE Tregs, into CD31- memory Tregs/Tresps. In transplant patients, CCR7+ RTE Tregs accumulated, while frequencies of CCR7+ RTE Tresps were decreased, suggesting that the immunosuppressive therapy only prevented excessive CCR7+ RTE Treg differentiation but not that of CCR7+ RTE Tresps. Presumably, this caused the accumulation of TEMRA Tresps with decreased release of cytotoxic mediators, such as perforin. In conclusion, we propose that chronic KF affects both the differentiation of CD8+ Tregs and CD8+ Tresps. However, the immunosuppressive therapy after transplantation may successfully prevent excessive Treg differentiation, but not as suffciently that of Tresps. Therefore, the risk for graft rejection may be reduced, while the susceptibility for infections and malignancies may be increased in these patients.

To study the impact of glucocorticoid maintenance dose and treatment duration on outcomes in patients with AAV (ANCA-associated vasculitis) with emphasis on infectious complications. A total of 130 AAV patients from two German vasculitis centers diagnosed between August 2004 and January 2019 treated with cyclophosphamide and glucocorticoids for induction therapy and glucocorticoids for maintenance therapy were retrospectively enrolled. We investigated the influence of glucocorticoid maintenance therapy on patient survival, time to relapse, kidney function, infectious complications and irreversible physical damage. The patients were divided into the following groups: patients treated according to the predefined reduction scheme ($$\ge $$ ≥ 7.5 mg had an increased rate of infectious episodes per patient (1.7 vs. 0.6; p p = 0.007), pneumonia (p = 0.003), opportunistic pneumonia (p = 0.022) and sepsis (p = 0.008). Especially pneumonia during the first 24 months after disease onset [hazard ratio, 3.0 (95% CI 1.5 − 6.1)] led to more deaths from infection (p = 0.034). Glucocorticoid maintenance therapy after 6 months had no impact on relapse rate or patient survival and decline in kidney function was comparable. Glucocorticoid maintenance therapy with $$\ge $$ ≥ 7.5 mg after 6 months is associated with more severe infectious complications leading to an increased frequency of deaths from infection. Glucocorticoid maintenance therapy has no effect on time to relapse or patient survival and should therefore be critically revised throughout the aftercare of AAV patients.

Objectives: humoral immunity wanes over time after two-dose BNT162b2 vaccination. Emerging variants of concern, such as the B.1.617.2 (delta) variant, are increasingly responsible for breakthrough infections owing to their higher transmissibility and partial immune escape. Longitudinal data on neutralization against the B.1.617.2 (delta) variant are urgently needed to guide vaccination strategies. Methods: in this prospective longitudinal observational study, anti-S1 IgG and surrogate neutralizing antibodies were measured in 234 collected samples from 60 health care workers after two-dose vaccination with BNT162b2 at five different time points over an 8-month period. In addition, antibodies against various severe acute respiratory syndrome coronavirus 2 epitopes, neutralization against wild-type, and cross-neutralization against the B.1.617.2 (delta) variant using a live virus assay were measured 6 weeks (second time point) and 8 months (last time point) after first vaccine dose. Results: median (interquartile range) anti-S1 IgG, surrogate neutralizing, and receptor-binding domain antibodies decreased significantly from a maximum level of 147 (102–298), 97 (96–98), and 20 159 (19 023–21 628) to 8 (4–13), 92 (80–96), and 15 324 (13 055–17 288) at the 8-month follow-up, respectively (p < 0.001 for all). Neutralization against the B.1.617.2 (delta) variant was detectable in all 36 (100%) participants at 6 weeks and in 50 of 53 (94%) participants 8 months after first vaccine dose. Median (interquartile) ID50 as determined by a live virus assay decreased from 160 (80–320) to 40 (20–40) (p < 0.001). Discussion: although humoral immunity wanes over time after two-dose BNT162b2 vaccination in healthy individuals, most individuals still had detectable neutralizing activity against the B.1.617.2 (delta) variant after 8 months., Dietmar Hopp Stiftung; Heidelberg Faculty of Medicine Rahel Goitein-Strauss Program; Heidelberg Faculty of Medicine Physician Scientist Program; German Federal Research Network Applied Surveillance and Testing (BFAST); Network University Medicine; DKFZ@fightCOVID; Helmholtz Association Initiative and Networking Fund Project Virological and Immunological Determinants of COVID-19 Pathogenesis

Hemodialysis patients are at high risk for severe COVID-19, and impaired seroconversion rates have been demonstrated after COVID-19 vaccination. Humoral immunity wanes over time and variants of concern with immune escape are posing an increasing threat. Little is known about protection against the B.1.617.2 (delta) variant of concern in hemodialysis patients before and after third vaccination. We determined anti-S1 IgG, surrogate neutralizing, and IgG antibodies against different SARS-CoV-2 epitopes in 84 hemodialysis patients directly before and three weeks after a third vaccine dose with BNT162b2. Third vaccination was performed after a median (IQR) of 119 (109-165) days after second vaccination. In addition, neutralizing activity against the B.1.617.2 (delta) variant was assessed in 31 seroconverted hemodialysis patients before and after third vaccination. Triple seropositivity for anti-S1 IgG, surrogate neutralizing, and anti-RBD antibodies increased from 31/84 (37%) dialysis patients after second to 80/84 (95%) after third vaccination. Neutralizing activity against the B.1.617.2 (delta) variant was significantly higher after third vaccination with a median (IQR) ID50 of 1:320 (1:160-1:1280) compared with 1:20 (0-1:40) before a third vaccine dose (P, Dietmar Hopp Stiftung; Heidelberg Faculty of Medicine Rahel Goitein-Strauss Program

Dysregulations in the differentiation of CD4+-regulatory-T-cells (Tregs) and CD4+-responder-T-cells (Tresps) are involved in the development of active systemic lupus erythematosus (SLE). Three differentiation pathways of highly proliferative inducible costimulatory molecule (ICOS)+- and less proliferative ICOS−-CD45RA+CD31+-recent-thymic-emigrant (RTE)-Tregs/Tresps via CD45RA−CD31+-memory-Tregs/Tresps (CD31+-memory-Tregs/Tresps), their direct proliferation via CD45RA+CD31−-mature naïve (MN)-Tregs/Tresps, and the production and differentiation of resting MN-Tregs/Tresp into CD45RA−CD31−-memory-Tregs/Tresps (CD31−-memory-Tregs/Tresps) were examined in 115 healthy controls, 96 SLE remission patients, and 20 active disease patients using six color flow cytometric analysis. In healthy controls an appropriate sequence of these pathways ensured regular age-dependent differentiation. In SLE patients, an age-independently exaggerated differentiation was observed for all Treg/Tresp subsets, where the increased conversion of resting MN-Tregs/Tresps particularly guaranteed the significantly increased ratios of ICOS+-Tregs/ICOS+-Tresps and ICOS−-Tregs/ICOS−-Tresps during remission. Changes in the differentiation of resting ICOS+-MN-Tresps and ICOS−-MN-Tregs from conversion to proliferation caused a significant shift in the ratio of ICOS+-Tregs/ICOS+-Tresps in favor of ICOS+-Tresps and a further increase in the ratio of ICOS−-Tregs/ICOS−-Tresps with active disease. The differentiation of ICOS+-RTE-Tregs/Tresps seems to be crucial for keeping patients in remission, where their limited production of proliferating resting MN-Tregs may be responsible for the occurrence of active disease flares.

Seroconversion rates following infection and vaccination are lower in dialysis patients compared to healthy controls. There is an urgent need for the characterization of humoral responses and success of a single-dose SARS-CoV-2 vaccination in previously infected dialysis patients. We performed a dual-center cohort study comparing three different groups: 25 unvaccinated hemodialysis patients after PCR-confirmed COVID-19 (Group 1), 43 hemodialysis patients after two-time BNT162b2 vaccination without prior SARS-CoV-2 infection (Group 2), and 13 single-dose vaccinated hemodialysis patients with prior SARS-CoV-2 infection (Group 3). Group 3 consists of seven patients from Group 1 and 6 additional patients with sera only available after single-dose vaccination. Anti-S1 IgG, neutralizing antibodies, and antibodies against various SARS-CoV-2 protein epitopes were measured 3 weeks after the first and 3 weeks after the second vaccination in patients without prior SARS-CoV-2 infection, 6 weeks after the onset of COVID-19 in unvaccinated patients, and 3 weeks after single-dose vaccination in patients with prior SARS-CoV-2 infection, respectively. Unvaccinated patients after COVID-19 showed a significantly higher neutralizing antibody capacity than two-time vaccinated patients without prior COVID-19 [median (IQR) percent inhibition 88.0 (71.5–95.5) vs. 50.7 (26.4–81.0); P = 0.018]. After one single vaccine dose, previously infected individuals generated 15- to 34-fold higher levels of anti-S1 IgG than age- and dialysis vintage-matched unvaccinated patients after infection or two-time vaccinated patients without prior SARS-CoV-2 infection with a median (IQR) index of 274 (151–791) compared to 18 (8–41) and 8 (1–21) (for both P < 0.001). With a median (IQR) percent inhibition of 97.6 (97.2–98.9), the neutralizing capacity of SARS-CoV-2 antibodies was significantly higher in single-dose vaccinated patients with prior SARS-CoV-2 infection compared to other groups (for both P < 0.01). Bead-based analysis showed high antibody reactivity against various SARS-CoV-2 spike protein epitopes after single-dose vaccination in previously infected patients. In conclusion, single-dose vaccination in previously infected dialysis patients induced a strong and broad antibody reactivity against various SARS-CoV-2 spike protein epitopes with high neutralizing capacity.

Background and Aims An imbalance between CD4+-regulatory T-cells (Tregs) and CD4+-responder T-cells (Tresps) correlates with active disease flares in Systemic Lupus Erythematosus (SLE) patients. Both cell subsets consist of highly proliferative inducible T-cell co-stimulatory molecule expressing (ICOS+-) and less proliferating ICOS--Treg/Tresp cells. Method In this study we aimed to examine the role of age-dependent differentiation for maintaining the ICOS+-Treg/ICOS+-Tresp or ICOS--Treg/ICOS--Tresp balance in SLE remission patients compared to active disease patients and to evaluate the influence of proliferation inhibitor or glucocorticoid therapy concerning the maintenance of these equilibria. Results In 83 healthy volunteers, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age, while that of ICOS--Tregs/ICOS--Tresps did not change. In 86 SLE remission patients, medication controlled disease activity was associated with an age-independently increased ratio of both ICOS+-Tregs/ICOS+-Tresps and ICOS--Tregs/ICOS--Tresps. In 13 active disease patients this increased ICOS+-Treg/ICOS+-Tresp ratio could not be maintained, due to an age-dependent earlier terminal differentiation of ICOS+-Tregs than of ICOS+-Tresps. The increased ratio of ICOS--Treg/ICOS--Tresp was maintained in active disease patients, but it decreased with age. This was attributable to an age-independent aberrant differentiation of ICOS--Tresps and a resulting loss of age-dependent differentiation of ICOS--Tregs. Especially the administration of proliferation inhibitors (azathioprine or mycophenolic acid) ensured prolonged differentiation and maintenance of functional ICOS+-Tregs with age. There was a similar but weaker effect of this medication on ICOS+-Tresps, causing a significant decrease of these cells with age. The proliferation capacity was neither affected in ICOS+-Tregs/Tresps nor in ICOS--Tregs/Tresps. As a result, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age and kept the patients in remission. In contrast, glucocorticoids increased the differentiation of ICOS--Tregs especially in younger individuals, but had an adverse effect on their proliferation capacity. Conclusion Our data reveal a crucial role of the ICOS+-Treg/ICOS+-Tresp balance for the maintenance of self-tolerance and avoidance of disease flares in SLE. The used immunosuppressive therapy, especially the administration of proliferation inhibitors, has a decisive effect on the preservation of regular differentiation of all Treg/Tresp subsets with age, and most importantly avoids the terminal differentiation and immune senescence of ICOS+-Tregs.

Premature aging of both CD4+ regulatory T (Treg) and CD4+ responder‐T (Tresp) cells in patients with end‐stage renal disease (ESRD) is expected to affect the success of later kidney transplantation. Both T‐cell populations are released from the thymus as inducible T‐cell co‐stimulator‐positive (ICOS +) and ICOS − recent thymic emigrant (RTE) Treg/Tresp cells, which differ primarily in their proliferative capacities. In this study, we analysed the effect of ESRD and subsequent renal replacement therapies on the differentiation of ICOS + and ICOS − RTE Treg/Tresp cells into ICOS + CD31− or ICOS − CD31− memory Treg/Tresp cells and examined whether diverging pathways affected the suppressive activity of ICOS + and ICOS − Treg cells in co‐culture with autologous Tresp cells. Compared with healthy controls, we found an increased differentiation of ICOS + RTE Treg/Tresp cells and ICOS − RTE Treg cells through CD31+ memory Treg/Tresp cells into CD31− memory Treg/Tresp cells in ESRD and dialysis patients. In contrast, ICOS − RTE Tresp cells showed an increased differentiation via ICOS − mature naive (MN) Tresp cells into CD31− memory Tresp cells. Thereby, the ratio of ICOS + Treg/ICOS + Tresp cells was not changed, whereas that of ICOS − Treg/ICOS − Tresp cells was significantly increased. This differentiation preserved the suppressive activity of both Treg populations in ESRD and partly in dialysis patients. After transplantation, the increased differentiation of ICOS + and ICOS − RTE Tresp cells proceeded, whereas that of ICOS + RTE Treg cells ceased and that of ICOS − RTE Treg cells switched to an increased differentiation via ICOS − MN Treg cells. Consequently, the ratios of ICOS + Treg/ICOS + Tresp cells and of ICOS − Treg/ICOS − Tresp cells decreased significantly, reducing the suppressive activity of Treg cells markedly. Our data reveal that an increased tolerance‐inducing differentiation of ICOS + and ICOS − Treg cells preserves the functional activity of Treg cells in ESRD patients, but this cannot be maintained during long‐term renal replacement therapy.

It has been demonstrated that patients on hemo- or peritoneal dialysis are particularly susceptible to SARS-CoV-2 infection and impaired seroconversion compared to healthy controls. Follow-up data on vaccination response in dialysis patients is limited but is greatly needed to individualize and guide (booster) vaccination strategies. In this prospective, multicenter study we measured anti-spike S1 and neutralizing antibodies in 124 hemodialysis patients, 41 peritoneal dialysis patients, and 20 age- and sex-matched healthy controls over 12 weeks after homologous BNT162b2 vaccination. Compared to healthy controls, both hemodialysis and peritoneal dialysis patients had lower anti-S1 IgG antibodies (median (IQR) 7.0 (2.8–24.3) and 21.8 (5.8–103.9) versus 134.9 (23.8–283.6), respectively, p &lt, 0.001 and p &lt, 0.05) and a reduced SARS-CoV-2 spike protein–ACE2 binding inhibition caused by vaccine-induced antibodies (median (IQR) 56% (40–81) and 77% (52–89) versus 96% (90–98), respectively, 0.01) three weeks after the second vaccination. Twelve weeks after the second vaccination, the spike protein–ACE2 binding inhibition significantly decreased to a median (IQR) of 45% (31–60) in hemodialysis patients and 55% (36–78) in peritoneal dialysis patients, respectively (p &lt, 0.05). Peritoneal dialysis patients mounted higher antibody levels compared with hemodialysis patients at all time points during the 12-week follow-up. Individual booster vaccinations in high-risk individuals without seroconversion or rapidly waning neutralizing antibody levels are required and further data on the neutralization of emerging variants of concern in these patients are urgently needed.

Despite limited data on safety and immunogenicity, heterologous prime-boost vaccination is currently recommended for individuals with ChAdOx1 nCoV-19 prime immunization in certain age groups. In this prospective, single-center study we included 166 health care workers from Heidelberg University Hospital who received either heterologous ChAdOx1 nCoV-19/BNT162b2, homologous BNT162b2 or homologous ChAdOx1 nCoV-19 vaccination between December 2020 and May 2021. We measured anti-S1 IgG, SARS-CoV-2 specific neutralizing antibodies, and antibodies against different SARS-CoV-2 fragments 0–3 days before and 19–21 days after boost vaccination. Before boost, 55/70 (79%) ChAdOx1 nCoV-19-primed compared with 44/45 (98%) BNT162b2-primed individuals showed positive anti-S1 IgG with a median (IQR) anti-S1 IgG index of 1.95 (1.05–2.99) compared to 9.38 (6.26–17.12). SARS-CoV-2 neutralizing antibodies exceeded the threshold in 24/70 (34%) of ChAdOx1 nCoV-19-primed and 43/45 (96%) of BNT162b2-primed individuals. After boosting dose, median (IQR) anti-S1 IgG index in heterologous ChAdOx1 nCoV-19/BNT162b2 vaccinees was 116.2 (61.84–170), compared to 13.09 (7.03–29.02) in homologous ChAdOx1 nCoV-19 and 145.5 (100–291.1) in homologous BNT162b2 vaccinees. All boosted vaccinees exceeded the threshold for neutralization, irrespective of their vaccination scheme. Vaccination was well-tolerated overall. We show that heterologous ChAdOx1 nCoV-19/BNT162b2 vaccination is safe and induces a strong and broad humoral response in healthy individuals.

Dialysis patients have deficiencies regarding the generation of immune responses and show an increased susceptibility for infections. Persisting uremic conditions are made responsible for the increased aging of their immune system. In this study, we analyzed whether age-related differences in the differentiation of both recent-thymic-emigrant-(RTE)-regulatory (Tregs) and RTE-responder T cells (Tresps) into CD31--memory Tregs/Tresps led to differences in the suppressive activity of naive and memory Tregs on autologous Tresps between healthy volunteers and dialysis patients. We found that regardless of age, the differentiation of RTE-Treg/Tresps into CD31--memory-Treg/Tresps was significantly increased in dialysis patients. By analyzing the age-related differences in the differentiation of Tregs/Tresps, we saw that in healthy volunteers RTE-Tregs differentiate via CD31+-memory Tregs into CD31--memory Tregs, which may strengthen the suppressive activity of the total Treg pool. In contrast RTE-Tresps of healthy volunteers differentiate via mature naive (MN)-Tresps into CD31--memory-Tresps, which may weaken the reactivity of the total Tresp pool. Our data revealed that this normal differentiation via MN-Tresps was lost in dialysis patients, suggesting that their Tresps are less sensitive to Treg-mediated immunosuppression. Functional analysis of Tregs on autologous Tresps showed an increasing suppressive activity with age in healthy individuals, who therefore may have a lower risk of developing autoimmune diseases but owing to decreased reactivity of their Tresps are more likely to suffer from infections. In contrast, dialysis patients exhibited a decreasing suppressive activity with age, owing to strengthened Tresp reactivity, which could explain the higher prevalence of chronic inflammatory conditions in these patients.

The introduction of calcineurin inhibitors (CNI) has greatly improved graft survival in the past three decades. However, long-term graft survival is still limited due to chronic allograft injury and side-effects of immunosuppressive medication. Areas covered: The present overview gives an update on pharmacotherapeutic strategies after kidney transplantation. The main focus is on CNI-sparing regimens using co-stimulatory blockade and on new substances on the horizone. Expert opinion: CNI sparing regimens are well-established. Complete CNI avoidance after kidney transplantation was often associated with impaired graft survival until the approval of the co-stimulation blocker belatacept for de novo immunosuppression after kidney transplantation. Concerns still exist with respect to severe T-cell-mediated rejection episodes in the early phase after transplantation. Thus, a triple drug regimen with CNI, mycophenolic acid and steroids still represents the gold-standard of immunosuppressive therapy. Alternative substances expand the possibilities of tailoring individual immunosuppression for different indications such as biopsy-proven CNI toxicity, polyoma virus BK nephropathy or CNI-triggered thrombotic microangiopathy. However, a change of the immunosuppressive therapy must always be balanced against each patient´s individual immunological risk in order to address the importance of chronic antibody-mediated rejection driven by donor specific antibodies (DSA).

Refinement of immunosuppressive strategies has led to further improvement of kidney graft survival in recent years. Currently, the main limitations to long-term graft survival are life-threatening side effects of immunosuppression and chronic allograft injury, emphasizing the need for innovative immunosuppressive regimens that resolve this therapeutic dilemma. Several cell therapeutic approaches to immunosuppression and donor-specific unresponsiveness have been tested in early phase I and phase II clinical trials in kidney transplantation. The aim of this overview is to summarize current cell therapeutic approaches to immunosuppression in clinical kidney transplantation with a focus on myeloid suppressor cell therapy by mitomycin C-induced cells (MICs). MICs show great promise as a therapeutic agent to achieve the rapid and durable establishment of donor-unresponsiveness in living-donor kidney transplantation. Cell-based therapeutic approaches may eventually revolutionize immunosuppression in kidney transplantation in the near future.

Problem Recent studies revealed appropriate differentiation of recent thymic emigrant (RTE)-regulatory T cells (Tregs) to be crucial for maintaining healthy pregnancy. Currently, the role of responder T cells (Tresps) is not known. Method of study Six-color flow cytometric analysis was used to detect differences in the differentiation of highly proliferative inducible co-stimulatory (ICOS)+-RTE-Tregs/Tresps and apoptosis-sensitive ICOS−-RTE-Tregs/Tresps into mature naive (MN)-, CD31+-memory and CD31−-memory Tregs/Tresps in women with spontaneous preterm labor (sPL) compared to healthy pregnancy. Results Healthy pregnancy was characterized by an increased differentiation of ICOS+- and ICOS−-RTE-Tregs, as well as ICOS+-RTE-Tresps via CD31+-memory Tregs/Tresps into CD31−-memory Tregs/Tresps. Women with sPL showed an early interruption of RTE-Treg/Tresp differentiation. Instead, ICOS+-MN-Tresps and partly ICOS−-MN-Tregs differentiated into CD31−-memory Tregs/Tresps, causing a significant reduction of both ICOS+-Tregs and ICOS+-Tresps, but an increase of ICOS−-Tresps within total CD4+-T-helper cells. Conclusion Aberrant differentiation of ICOS+-T cells is associated with sPL.

Background CD4+ T cells are of great importance in the pathogenesis of systemic lupus erythematosus (SLE), as an imbalance between CD4+ regulatory T cells (Tregs) and CD4+ responder T cells (Tresps) causes flares of active disease in SLE patients. In this study, we aimed to find the role of aberrant Treg/Tresp cell differentiation for maintaining Treg/Tresp cell balance and Treg functionality. Methods To determine differences in the differentiation of Tregs/Tresps we calculated the percentages of CD45RA+CD31+ recent thymic emigrant (RTE) Tregs/Tresps and CD45RA+CD31− mature naive (MN) Tregs/Tresps, as well as CD45RA−CD31+ and CD45RA−CD31− memory Tregs/Tresps (CD31+ and CD31− memory Tregs/Tresps) within the total Treg/Tresp pool of 78 SLE remission patients compared with 94 healthy controls of different ages. The proliferation capacity of each Treg/Tresp subset was determined by staining the cells with anti-Ki67 monoclonal antibodies. Differences in the autologous or allogeneic Treg function between SLE remission patients and healthy controls were determined using suppression assays. Results With age, we found an increased differentiation of RTE Tregs via CD31+ memory Tregs and of RTE Tresps via MN Tresps into CD31− memory Tregs/Tresp in healthy volunteers. This opposite differentiation of RTE Tregs and Tresps was associated with an age-dependent increase in the suppressive activity of both naive and memory Tregs. SLE patients showed similar age-dependent Treg cell differentiation. However, in these patients RTE Tresps differentiated increasingly via CD31+ memory Tresps, whereby CD31− memory Tresps arose that were much more difficult to inhibit for Tregs than those that emerged through differentiation via MN Tresps. Consequently, the increase in the suppressive activity of Tregs with age could not be maintained in SLE patients. Testing the Tregs of healthy volunteers and SLE patients with autologous and nonautologous Tresps revealed that the significantly decreased Treg function in SLE patients was not exclusively attributed to an age-dependent diminished sensitivity of the Tresps for Treg suppression. The immunosuppressive therapy reduced the accelerated age-dependent Tresp cell proliferation to normal levels, but simultaneously inhibited Treg cell proliferation below normal levels. Conclusions Our data reveal that the currently used immunosuppressive therapy has a favorable effect on the differentiation and proliferation of Tresps but has a rather unfavorable effect on the proliferation of Tregs. Newer substances with more specific effects on the immune system would be desirable. Electronic supplementary material The online version of this article (10.1186/s13075-018-1778-6) contains supplementary material, which is available to authorized users.

Regulatory T cells (Tregs) were shown to be involved into the pathogenesis of acute rejection after transplantation. The suppressive activity of the total regulatory T cell pool depends on its percentage of highly suppressive HLA-DR(+) -Treg cells. Therefore, both the suppressive activity of the total Treg pool and the extent of HLA-DR expression of HLA-DR(+) -Tregs (MFI HLA-DR) were estimated in non transplanted volunteers, patients with end-stage renal failure (ESRF), healthy renal transplant patients with suspicion on rejection, due to sole histological Bord-R or sole acute renal failure (ARF), and patients with clinically relevant borderline rejection (Bord-R and ARF). Compared to patients with only Bord-R or only ARF, the suppressive activity of the total Treg cell pool was exclusively reduced in patients with clinically relevant Bord-R. In parallel, the HLA-DR MFI of the DR(+) -Treg subset was significantly decreased in these patients, due to a significantly lower proportion of DR(high+) -Tregs, which were shown to have the highest suppressive capacity within the total Treg pool. Our findings clearly demonstrate that the determination of the HLA-DR MFI of the HLA-DR(+) -Treg subset allows a highly sensitive, specific and non-invasive discrimination between patients with clinically relevant Bord-R (Bord and ARF) and patients with subclinical rejection or other causes of transplant failure.

Methylprednisolone is widely used to improve immune suppression in transplanted patients threatened by acute rejection. Recently, we showed that the suppressive activity of a Treg cell population depends decisively on their percentage of highly suppressive HLA-DR(high+)-Treg cells, which are strongly reduced in rejecting transplant patients. In order to examine whether the composition of the total CD4(+)CD127(low+/-)FoxP3(+)-Treg cell pool with different Treg-subsets (DR(high+)CD45RA(-)-Tregs, DR(low+)CD45RA(-)-Tregs, DR(-)CD45RA(-)-Tregs, DR(-)CD45RA(+)-Tregs) is affected by methylprednisolone bolus therapy we compared the percentages of these four different Treg cell subsets in transplant patients with biopsy proven rejection before and after steroid bolus therapy (n=23). In patients treated with steroid bolus therapy, the percentage of the naïve DR(-)CD45RA(+)-Tregs was significantly decreased, whereas the percentage of the DR(+)CD45RA(-)-Tregs was significantly increased. By that, the strongest increase was detected for the most suppressive DR(high+)CD45RA(-)-Tregs. However, these effects were only temporarily and closely associated to the duration of the bolus therapy. Our results suggest that besides various anti-inflammatory effects on cells of the adaptive and innate immune system, methylprednisolone also has the capacity to enhance the suppressive activity of the total Treg cell pool by increasing its percentage of highly differentiated and highly suppressive DR(high+)CD45RA(-)-Tregs.

Dysregulations concerning the composition and function of regulatory T cells (T(regs)) are assumed to be involved in the pathophysiology of complicated pregnancies. We used six-colour flow cytometric analysis to demonstrate that the total CD4(+) CD127(low+/-) CD25(+) forkhead box protein 3 (FoxP3)(+) T(reg) cell pool contains four distinct T(reg) subsets: DR(high+) CD45RA(-), DR(low+) CD45RA(-), DR(-) CD45RA(-) T(regs) and naive DR(-) CD45RA(+) T(regs). During the normal course of pregnancy, the most prominent changes in the composition of the total T(reg) cell pool were observed between the 10th and 20th weeks of gestation, with a clear decrease in the percentage of DR(high+) CD45RA(-) and DR(low+) CD45RA(-) T(regs) and a clear increase in the percentage of naive DR(-) CD45RA(+) T(regs). After that time, the composition of the total T(reg) cell pool did not change significantly. Its suppressive activity remained stable during normally progressing pregnancy, but decreased significantly at term. Compared to healthy pregnancies the composition of the total T(reg) cell pool changed in the way that its percentage of naive DR(-) CD45RA(+) T(regs) was reduced significantly in the presence of pre-eclampsia and in the presence of preterm labour necessitating preterm delivery (PL). Interestingly, its percentage of DR(high+) CD45RA(-) and DR(low+) CD45RA(-) T(regs) was increased significantly in pregnancies affected by pre-eclampsia, while PL was accompanied by a significantly increased percentage of DR(-) CD45RA(-) and DR(low+) CD45RA(-) T(regs). The suppressive activity of the total T(reg) cell pool was diminished in both patient collectives. Hence, our findings propose that pre-eclampsia and PL are characterized by homeostatic changes in the composition of the total T(reg) pool with distinct T(reg) subsets that were accompanied by a significant decrease of its suppressive activity.

Background: After transplantation of solid organs like allogeneic kidneys, the administration of immunosuppressive drugs such as cyclosporine A (CSA) and steroids is mandatory. This regimen exerts toxicity to the graft and makes transplant recipients prone to opportunistic infections. Replacement of the immunosuppressive drugs by a transfusion of tolerogenic cells might overcome these noxious side effects. Mitomycin-induced cells (MICs) are donor-derived monocytes that gain immunosuppressive properties after incubation with the proliferation inhibitor mitomycin C and have a myeloid-derived suppressor cell (MDSC) character. Materials and methods: Peripheral blood mononuclear cells (PBMCs) were harvested from living kidney donors by leukapheresis and MIC cells were manufactured under Good Manufacturing Practice (GMP) conditions in the clean room of our University Hospital. Kidney transplant recipients received either 1.5x10E6 MIC cells per kg body weight on day -2 (N=3, group A) or 1.5x10E8 MIC cells per kg body weight on day -2 (N=3, group B) or on day -7 (N=4, group C) before living donor kidney transplantation. Patients received immunosuppressive therapy with cyclosporine a (CSA), enteric coated mycophenolate sodium (EC-MPS) and corticosteroids. The primary outcome was measured by the frequency of adverse events (AEs) on post-transplant day 30 with a follow-up until post-transplant day 360 for all patients. Results: Clinically, all kidney transplant recipients showed a median serum creatinine of 1.4 mg/dL at day 30 and remained stable with a median creatinine of 1.48 mg/dL at day 180 without significant proteinuria (median 10 g/mol creatinine at day 180) and without rejection episode. In total 72 AEs were observed including three severe AEs which were not associated with the MIC cell transfusion. Besides two infectious complications, no positive cross match results, no de novo donor-specific antibodies or rejection episodes were recorded. In group C, a reduction of immunosuppressive therapy was effective in the observational phase with low-dose CSA and low-dose EC-MPS. Immunologically, CD19+ B cells increased up to a median of 300/µL until day 30, followed by a decrease to a median of 35/µL at day 180 in group C. Notably, CD19+CD24highCD38high regulatory B cells were significantly increased from a median of 2% on day 30 to a median of 20% on day 180. The plasma IL-10/TNF-α ratio increased from a median of 0.05 before cell therapy to a median of 0.11 at day 180. Moreover, recipient lymphocytes showed no or only minimal reactivity against irradiated donor PBMCs, while reactivity against 3rd party healthy donor PBMCs in vitro was not impaired. Additionally, the quality assessment demonstrated that MIC cells have the capability to induce tolerogenic dendritic cells (tDCs) by down-regulating the costimulatory molecules CD80 and CD86, and the maturation molecule CD83, while up-regulating the immunosuppressive molecule CD103. MIC-induced tDCs showed the capacity to inhibit donor specific allo-reactive CD4 and CD8 T cell proliferation. Conclusion: A stable function was observed in all transplant recipients receiving the MIC cells product without any allograft injury or rejection episodes even under reduction of conventional therapy with immunosuppressive drugs. MIC cells constitute a novel tool for immunotherapy with a high potential in transplantation medicine. Disclosures No relevant conflicts of interest to declare.

Introduction: Quality assessment in terms of product specificity and product potency is obligatory and strictly required by EMA and FDA to provide a platform for analysis of product comparability, stability and compatibility, and for clinical development to help predict product clinical efficacy. Mitomycin-treated donor peripheral blood mononuclear cells (MICs) could induce donor-specific tolerance via generation of recipient-derived tolerogenic dendritic cells (tDCs). To evaluate the immunomodulating capacity of the MIC product, we assessed the phenotype of tDCs, the immunosuppressive capacity of tDCs on allo-reactive CD4+ and CD8+ T cells as well as on CMV-specific CD8+ T cells. Methods: For standardization of the potency assay, third-party PBMCs positive for HLA-A2 and anti-CMV IgG were used. Immature DCs (iDCs) were generated 3 days before MIC production. MICs were generated under Good Manufacturing Practice (GMP) conditions. Each batch of MICs was introduced to iDCs at different ratios (1:0, 1:1, 1:10, 1:20) for a two-hour interaction followed by adding a DC maturation cocktail for overnight co-culture. Thereafter tDCs were purified by magnetic negative separation. The morphology of tDCs was observed by microscopy. The expression of HLA-DR, CD80, CD83, CD86 and CD103 was analyzed by flow cytometry. The inhibitory capacity on allo-reactive T cells and virus specific T cells was determined by mixed lymphocyte reaction (MLR) assay, by ELISpot assay and by Tetramer staining assay after one week of expansion in mixed lymphocyte peptide culture (MLPC). Results: In light microscopy (magnification: x 40), tDCs showed a relative smooth membrane surface. While the conventional mDCs were significantly larger in size with rough surface, richer ruffles on the cell membrane, and bigger, longer protrusions or pseudopodia. MIC products could inhibit the expression of costimulatory molecules on tDCs with an inhibition of 41% of CD80, 27% of CD83 and 23% of CD86. In parallel, the inhibitory marker CD103 was up-regulated about 65% on tDCs. Functionally, both tDCs and MICs could inhibit the IFN-γ secretion by CMV-specific CD8+ T cells, respectively. Moreover, the proliferation of allo-reactive T cells and CMV-specific T cells could be inhibited by tDCs and MICs. Conclusions: In summary, the potency assays, including the measurement of physicochemical parameters, the morphology and marker expression of tDCs, as well as the biologic characterization, the functionally immunosuppressive capacity of tDCs, comprise valuable parameters for the evaluation of clinically used advanced therapeutic cellular products. Disclosures No relevant conflicts of interest to declare.

Background. Chronic loss of renal allograft function is associated with interstitial fibrosis and tubular atrophy (IF/TA). Independent of the underlying reason, one initial step in the development of fibrosis is chemokine-driven invasion of leukocytes from the blood vessels into the allograft. We studied the role of chemokines in kidney allografts with delayed graft function and the subsequent long-term outcome of renal function and fibrosis. Methods. We examined repetitive biopsies of 30 patients without signs of acute rejection but with initially delayed graft function for IF/TA. In addition, we examined the expression of chemokine receptor (CCR)-1 and CCR2 on invaded leukocytes and macrophages and the corresponding ligands regulated upon activation, normal t-cell expressed, and secreted (RANTES) and monocyte chemotactic protein-1 on residential kidney cells. Results. The initial expression of CCR1 positive invading cells and RANTES in glomerular cells correlated with the allograft function 12 months after transplantation and at last follow-up. The expression was independent of donor characteristics such as age, gender, infectious state, cause of death, or use of vasopressive agents. Furthermore, it did not correlate with the duration of cold ischemia time. Among the patients with the most progressive loss of allograft function follow-up biopsy specimen did not reveal any signs of rejection but showed increased CCR1 and RANTES expression in the interstitium suggesting ongoing inflammation and fibrosis. Conclusion. An early expression of RANTES in renal allografts with delayed graft function with consecutive invasion of CCR1 positive cells seems to promote ongoing IF/TA and to worse renal allograft outcome.

Two different subsets of naturally occurring regulatory T cells (nTregs), defined by their expression of the inducible co-stimulatory (ICOS) molecule, are produced by the human thymus. To examine the differentiation of ICOS(+) and ICOS(-) CD45RA(+) CD31(+) recent thymic emigrant (RTE) T regs during normal pregnancy and in the presence of pre-eclampsia or haemolysis elevated liver enzymes low platelet (HELLP)-syndrome, we used six-colour flow cytometric analysis to determine the changes in the composition of the ICOS(+) and ICOS(-) T reg pools with CD45RA(+) CD31(+) RTE T regs, CD45RA(+) CD31(-) mature naive (MN) T regs, CD45RA(-) CD31(+) and CD45RA(-) CD31(-) memory Tregs. With the beginning of pregnancy until term, we observed a strong differentiation of both ICOS(+) and ICOS(-) CD45RA(+) CD31(+) RTE, but not CD45RA(+) CD31(-) MN T regs, into CD45RA(-) CD31(-) memory T regs. At the end of pregnancy, the onset of spontaneous term labour was associated with a significant breakdown of ICOS(+) CD45RA(-) CD31(-) memory T regs. However, in the presence of pre-eclampsia, there was a significantly increased differentiation of ICOS(+) and ICOS(-) CD45RA(+) CD31(+) RTE T regs into CD45RA(-) CD31(+) memory T regs, wherein the lacking differentiation into CD45RA(-) CD31(-) memory T regs was partially replaced by the increased differentiation of ICOS(+) and ICOS(-) CD45RA(+) CD31(-) MN Tregs into CD45RA(-) CD31(-) memory T regs. In patients with HELLP syndrome, this alternatively increased differentiation of CD45RA(-) CD31(-) MN T regs seemed to be exaggerated, and presumably restored the suppressive activity of magnetically isolated ICOS(+) and ICOS(-) T regs, which were shown to be significantly less suppressive in pre-eclampsia patients, but not in HELLP syndrome patients. Hence, our findings propose that the regular differentiation of both ICOS(+) and ICOS(-) CD45RA(+) CD31(+) RTE T regs ensures a healthy pregnancy course, while their disturbed differentiation is associated with the occurrence of pre-eclampsia and HELLP syndrome.

To give an overview over cell therapeutic approaches to immunosuppression in clinical kidney transplantation. A focus is on myeloid suppressor cell therapy by mitomycin C-induced cells (MICs). Literature review with an emphasis on already existing therapies. Several cell therapeutic approaches to immunosuppression and donor-specific unresponsiveness are now being tested in early phase I and phase II trials in clinical kidney transplantation. Cell products such as regulatory T cells or regulatory macrophages, or other myeloid suppressor cell therapies, may either consist of donor-specific, third-party, or autologous cell preparations. Major problems are the identification of the suppressive cell populations and their expansion to have sufficient amount of cells to achieve donor unresponsiveness (e.g., with regulatory T cells). We show a simple and safe way to establish donor unresponsiveness in living-donor kidney transplantation by MIC therapy. A phase I clinical trial is now under way to test the safety and efficacy of this cell therapeutic approach. Cell therapeutic approaches to immunosuppression after kidney transplantation may revolutionize clinical transplantation in the future.

During pregnancy, regulatory T cells (Tregs) have a key role in maternal immune tolerance to the semi-allogeneic fetus. Our previous results showed that the naive CD45RA(+)-Treg pool is functionally improved in pregnant women compared with non-pregnant women. Therefore, we examined the thymic output and differentiation of CD45RA(+)CD31(+) recent thymic emigrant (RTE)-Tregs during normal pregnancy and in the presence of preeclampsia. With the onset of pregnancy, the composition of the total CD4(+)CD127(low+/-)FoxP3(+)-Treg pool changed in the way that its percentage of RTE- and CD45RA(-)CD31(+)-memory Tregs decreased strongly, whereas that of the CD45RA(+)CD31(-)-mature naive (MN)-Tregs did not change and that of the CD45RA(-)CD31(-)-memory Tregs increased complementary. Thereby, the ratio of RTE-/MN-Tregs decreased from 1.0 to 0.7 leading to a significant increase in the suppressive activity of the naive CD45RA(+)-Treg pool. This effect was confirmed by re-assembling separated RTE- and MN-Tregs from non-pregnant women in the ratio of pregnant women. The suppressive activity of both separated naive Treg subsets was equally high in non-pregnant and pregnant women, but considerably reduced in preeclampsia patients, who showed significantly increased percentages of CD45RA(-)CD31(+)-memory Tregs, but decreased percentages of RTE- and MN-Tregs. Our results suggest a reduced thymic output of RTE-Tregs during pregnancy, which causes a decrease in the ratio of RTE-/MN-Tregs and thus an increase in the differentiation of RTE-Tregs towards CD45RA(-)CD31(-)-memory Tregs. Presumably, this differentiation of RTE-Tregs, which was impaired in preeclampsia patients, ensures the improved suppressive activity of the CD45RA(+)-naive Treg pool and thus retains the maintenance of pregnancy.

STUDY QUESTION Are there differences in composition of the total regulatory T cell (Treg) pool and distinct Treg subsets (naive CD45RA(+)-Tregs, HLA-DR(-)- and HLA-DR(+)-memory Tregs) between successfully and non-successfully IVF/ICSI-treated women? SUMMARY ANSWER Non-successfully IVF/ICSI-treated women have a decreased percentage of naive CD45RA(+)-Tregs and an increased percentage of HLA-DR(-)-memory Tregs within the total Treg pool. WHAT IS KNOWN ALREADY Immunosuppressive Tregs play a significant role in human reproduction and studies have shown that their number and function are reduced in reproductive failure and complications of pregnancy such as pre-eclampsia and preterm labor. However, no data exist concerning the importance of Tregs for a successful outcome following assisted reproduction technologies. STUDY DESIGN, SIZE, DURATION Blood samples were obtained from 210 women undergoing IVF/ICSI treatment, where 14 patients were excluded due to biochemical pregnancy or missed abortion. Age control blood samples were collected from 20 neonates and 176 healthy female volunteers. The study was performed between October 2010 and March 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS In this study, we determined prospectively the quantity and composition of the total CD4(+)CD127(low+/-)CD25(+)FoxP3(+)-Treg pool and three different Treg subsets (naive CD45RA(+)-Tregs, HLA-DR(-)- and HLA-DR(+)-memory Tregs) in all women undergoing IVF/ICSI treatment. We examined whether there were differences between those who became pregnant (n = 36) and those who did not (n = 160). The blood samples were collected within 1 h before the embryo transfer and analyzed by six-color flow cytometry. In order to evaluate these results with regard to the normal age-related changes in composition of the total Treg pool, the same analysis was performed using samples of umbilical cord blood and from healthy female volunteers aged between 17 and 76 years. The composition of the total Treg pool was documented for successfully IVF/ICSI-treated women (n = 5) throughout their pregnancy and we assessed the suppressive activity of each Treg subset in pregnant (n = 10) compared with non-pregnant women (n = 10) using suppression assays. MAIN RESULTS AND ROLE OF CHANCE The percentage of CD4(+)CD127(low+/-)CD25(+)FoxP3(+)-Tregs within the total CD4(+)-T cell pool did not change with age and did not differ between IVF/ICSI-treated women who did or did not become pregnant. For the total Treg pool, the percentage of the naive CD45RA(+)-Tregs decreased continuously, while the percentage of HLA-DR(-)- and HLA-DR(+)-memory Tregs increased with aging. From the age of about 40 years, the increase in HLA-DR(+)-memory Tregs in particular became less pronounced, indicating that conversion of naive CD45RA(+)Tregs into HLA-DR(+)-memory Tregs decreases with age. Women who did not achieve a pregnancy with IVF/ICSI were older than those who did (P < 0.01). However, multiple logistic regression analysis revealed that irrespective of age, the percentage of naive CD45RA(+)-Tregs within the total Treg pool was decreased (P < 0.05), while the percentage of HLA-DR(-)-memory Tregs was increased (P < 0.01) in women who did not become pregnant compared with those who did. At the beginning of pregnancy, naive CD45RA(+)-Tregs showed a major decrease but increased again during pregnancy and these cells showed a higher suppressive activity (P < 0.0001) in pregnant compared with non-pregnant women. LIMITATIONS, REASONS FOR CAUTION There was a large variation in the percentages of the Treg subsets within the total Treg pool between successfully and non-successfully IVF/ICSI-treated women. Therefore, their determination would not allow us to predict the IVF/ICSI outcome with sufficient specificity and sensitivity. We did not examine the antigen specificity of the Treg subsets and therefore could not discern whether the naive CD45RA(+)-Tregs recognized maternal or paternal antigens. WIDER IMPLICATIONS OF THE FINDINGS Our findings suggest that Tregs, especially the naive CD45RA(+)-Treg subset, may play a role in determining the probability of both becoming pregnant and maintenance of the pregnancy. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the German Research Council (DFG) grant STE 885/3-2 (to A.S.). All authors declare to have no conflict of interest.

Recent studies show that regulatory T cells (Tregs) play an essential role in tolerance induction after organ transplantation. In order to examine whether there are differences in the composition of the total CD4(+)CD127(low+/-)FoxP3(+)- Treg cell pool between stable transplant patients and patients with biopsy proven rejection (BPR), we compared the percentages and the functional activity of the different Treg cell subsets (DR(high+)CD45RA(-)-Tregs, DR(low+)CD45RA(-)-Tregs, DR(-)CD45RA(-)-Tregs, DR(-)CD45RA(+)-Tregs). All parameters were determined during the three different periods of time after transplantation (0-30 days, 31-1,000 days, >1,000 days). Among 156 transplant patients, 37 patients suffered from BPR. The most prominent differences between rejecting and non-rejecting patients were observed regarding the DR(high+)CD45RA(-)-Treg cell subset. Our data demonstrate that the suppressive activity of the total Treg pool strongly depends on the presence of these Treg cells. Their percentage within the total Treg pool strongly decreased after transplantation and remained relatively low during the first year after transplantation in all patients. Subsequently, the proportion of this Treg subset increased again in patients who accepted the transplant and reached a value of healthy non-transplanted subjects. By contrast, in patients with acute kidney rejection, the DR(high+)CD45RA(-)-Treg subset disappeared excessively, causing a reduction in the suppressive activity of the total Treg pool. Therefore, both the monitoring of its percentage within the total Treg pool and the monitoring of the HLA-DR MFI of the DR(+)CD45RA(-)-Treg subset may be useful tools for the prediction of graft rejection.

Regulatory T cells (Tregs) are known to suppress alloimmune responses during pregnancy and post organ transplantation. We demonstrate that a distinct subset of FoxP3(+)DR(+)-Tregs among the total CD4(+)CD127(low+/-)CD25(+)-Treg cell pool is critically involved in preterm labor induction and kidney transplant rejection as well. Compared to healthy pregnancies and non-rejecting kidney recipients, we found that the percentage of the FoxP3(+)DR(+)-Treg subset was not reduced, but that the level of HLA-DR expression of such Tregs was strongly diminished in preterm laboring women and in patients with acute renal allograft rejection. In addition, both patient collectives showed a significantly reduced suppressive activity of their circulating CD4(+)CD127(low+/-)CD25(+)-Treg cell pool. Our findings propose that the FoxP3(+)DR(+)-Treg subset may be decisively responsible for the suppressive activity of the total CD4(+)CD127(low+/-)CD25(+)-Treg cell pool and that the immunologic mechanisms leading to preterm labor necessitating preterm delivery may be similar to those leading to allograft rejection after transplantation.