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Aims/hypothesis: There is increasing evidence for heterogeneity in type 1 diabetes mellitus (T1D): not only the age of onset and disease progression rate differ, but also the risk of complications varies markedly. Consequently, the presence of different disease endotypes has been suggested. Impaired T and B cell responses have been established in newly diagnosed diabetes patients. We hypothesized that deciphering the immune cell profile in peripheral blood of adults with longstanding T1D may help to understand disease heterogeneity., Methods: Adult patients with longstanding T1D and healthy controls (HC) were recruited, and their blood immune cell profile was determined using multicolour flow cytometry followed by a machine-learning based elastic-net (EN) classification model. Hierarchical clustering was performed to identify patient-specific immune cell profiles. Results were compared to those obtained in matched healthy control subjects., Results: Hierarchical clustering analysis of flow cytometry data revealed three immune cell composition-based distinct subgroups of individuals: HCs, T1D-group-A and T1D-group-B. In general, T1D patients, as compared to healthy controls, showed a more active immune profile as demonstrated by a higher percentage and absolute number of neutrophils, monocytes, total B cells and activated CD4+CD25+ T cells, while the abundance of regulatory T cells (Treg) was reduced. Patients belonging to T1D-group-A, as compared to T1D-group-B, revealed a more proinflammatory phenotype characterized by a lower percentage of FOXP3+ Treg, higher proportions of CCR4 expressing CD4 and CD8 T cell subsets, monocyte subsets, a lower Treg/conventional Tcell (Tconv) ratio, an increased proinflammatory cytokine (TNFα, IFNγ) and a decreased anti-inflammatory (IL-10) producing potential. Clinically, patients in T1D-group-A had more frequent diabetes-related macrovascular complications., Conclusions: Machine-learning based classification of multiparameter flow cytometry data revealed two distinct immunological profiles in adults with longstanding type 1 diabetes; T1D-group-A and T1D-group-B. T1D-group-A is characterized by a stronger pro-inflammatory profile and is associated with a higher rate of diabetes-related (macro)vascular complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 He, Wang, van Heck, van Cranenbroek, van Rijssen, Stienstra, Netea, Joosten, Tack and Koenen.)

Natural killer (NK) cells, with a unique NK cell receptor phenotype, are abundantly present in the non-pregnant (endometrium) and pregnant (decidua) humanuterine mucosa. It is hypothesized that NK cells in the endometrium are precursors for decidual NK cells present during pregnancy. Microenvironmental changes can alter the phenotype of NK cells, but it is unclear whether decidual NK cell precursors in the endometrium alter their NK cell receptor repertoire under the influence of pregnancy. To examine whether decidual NK cell precursors reveal phenotypic modifications upon pregnancy, we immunophenotyped the NK cell receptor repertoire of both endometrial and early-pregnancy decidual NK cells using flow cytometry. We showed that NK cells in pre-pregnancy endometrium have a different phenotypic composition compared to NK cells in early-pregnancy decidua. The frequency of killer-immunoglobulin-like receptor (KIR expressing NK cells, especially KIR2DS1, KIR2DL2L3S2, and KIR2DL2S2 was significantly lower in decidua, while the frequency of NK cells expressing activating receptors NKG2D, NKp30, NKp46, and CD244 was significantly higher compared to endometrium. Furthermore, co-expression patterns showed a lower frequency of NK cells co-expressing KIR3DL1S1 and KIR2DL2L3S2 in decidua. Our results provide new insights into the adaptations in NK cell receptor repertoire composition that NK cells in the uterine mucosa undergo upon pregnancy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Feyaerts, Benner, Comitini, Shadmanfar, van der Heijden, Joosten and van der Molen.)

Introduction: Thermal injury often leads to prolonged and excessive inflammation, which hinders the recovery of patients. There is a notable absence of suitable animal-free models for investigating the inflammatory processes following burn injuries, thereby impeding the development of more effective therapies to improve burn wound healing in patients., Methods: In this study, we established a human full skin equivalent (FSE) burn wound model and incorporated human peripheral blood-derived monocytes and T cells., Results: Upon infiltration into the FSEs, the monocytes differentiated into macrophages within a span of 7 days. Burn-injured FSEs exhibited macrophages with increased expression of HLA-DR + and elevated production of IL-8 (CXCL8), in comparison to uninjured FSEs. Among the T cells that actively migrated into the FSEs, the majority were CD4 + and CD25 + . These T cells demonstrated augmented expression of markers associated with regulatory T cell, Th1, or Th17 activity, which coincided with significant heightened cytokine production, including IFN-γ, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IP-10 (CXCL10), and TGF-β1. Burn injury did not impact the studied effector T cell subsets or cytokine levels., Discussion: Collectively, this study represents a significant advancement in the development of an immunocompetent human skin model, specifically tailored for investigating burn-induced innate or adaptive immune reactions at the site of burn injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mulder, Vlig, Elgersma, Rozemeijer, Mastenbroek, Middelkoop, Joosten, Koenen and Boekema.)

Inflammation is a physiological state where immune cells evoke a response against detrimental insults. Finding a safe and effective treatment for inflammation associated diseases has been a challenge. In this regard, human mesenchymal stem cells (hMSC), exert immunomodulatory effects and have regenerative capacity making it a promising therapeutic option for resolution of acute and chronic inflammation. T cells play a critical role in inflammation and depending on their phenotype, they can stimulate or suppress inflammatory responses. However, the regulatory effects of hMSC on T cells and the underlying mechanisms are not fully elucidated. Most studies focused on activation, proliferation, and differentiation of T cells. Here, we further investigated memory formation and responsiveness of CD4 + T cells and their dynamics by immune-profiling and cytokine secretion analysis. Umbilical cord mesenchymal stem cells (UC-MSC) were co-cultured with either αCD3/CD28 beads, activated peripheral blood mononuclear cells (PBMC) or magnetically sorted CD4 + T cells. The mechanism of immune modulation of UC-MSC were investigated by comparing different modes of action; transwell, direct cell-cell contact, addition of UC-MSC conditioned medium or blockade of paracrine factor production by UC-MSC. We observed a differential effect of UC-MSC on CD4 + T cell activation and proliferation using PBMC or purified CD4 + T cell co-cultures. UC-MSC skewed the effector memory T cells into a central memory phenotype in both co-culture conditions. This effect on central memory formation was reversible, since UC-MSC primed central memory cells were still responsive after a second encounter with the same stimuli. The presence of both cell-cell contact and paracrine factors were necessary for the most pronounced immunomodulatory effect of UC-MSC on T cells. We found suggestive evidence for a partial role of IL-6 and TGFβ in the UC-MSC derived immunomodulatory function. Collectively, our data show that UC-MSCs clearly affect T cell activation, proliferation and maturation, depending on co-culture conditions for which both cell-cell contact and paracrine factors are needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sengun, Wolfs, van Bruggen, van Cranenbroek, Simonetti, Ophelders, de Jonge, Joosten and van der Molen.)

The systemic and local immune response in burn patients is often extreme and derailed. As excessive inflammation can damage healthy tissues and slow down the healing process, modulation of inflammatory responses could limit complications and improve recovery. Due to its complexity, more detailed information on the immune effects of thermal injury is needed to improve patient outcomes. We therefore characterized and quantified subsets of immune cells and mediators present in human burn wound tissue (eschar), sampled at various time points. This study shows that after burn injury, the number of immune cells were persistently increased, unlike the normal wound healing process. There was an immediate, strong increase in neutrophils and a moderate increase in monocytes/macrophages and lymphocytes, especially in the second and third week post burn. The percentage of classical (CD14 high CD16 - ) monocytes/macrophages demonstrated a steady decrease over time, whereas the proportion of intermediate (CD14 high CD16 + ) monocytes/macrophages slowly increased. The absolute numbers of T cells, NK cells and B cells increased up to week 3, while the fraction of γδ T cells was increased only in week 1. Secretome profiling revealed high levels of chemokines and an overall pro-inflammatory cytokine milieu in burn tissue. The local burn immune response shows similarities to the systemic immune reaction, but differs in neutrophil maturity and lymphocyte composition. Altogether, the neutrophil surges, high levels of pro-inflammatory cytokines and limited immunosuppression might be key factors that prolong the inflammation phase and delay the wound healing process in burns., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mulder, Vlig, Fasse, Stoop, Pijpe, van Zuijlen, Joosten, Boekema and Koenen.)

CD4 + T-helper cells play an important role in alloimmune reactions following transplantation by stimulating humoral as well as cellular responses, which might lead to failure of the allograft. CD4 + memory T-helper cells from a previous immunizing event can potentially be reactivated by exposure to HLA mismatches that share T-cell epitopes with the initial immunizing HLA. Consequently, reactivity of CD4 + memory T-helper cells toward T-cell epitopes that are shared between immunizing HLA and donor HLA could increase the risk of alloimmunity following transplantation, thus affecting transplant outcome. In this study, the amount of T-cell epitopes shared between immunizing and donor HLA was used as a surrogate marker to evaluate the effect of donor-reactive CD4 + memory T-helper cells on the 10-year risk of death-censored kidney graft failure in 190 donor/recipient combinations using the PIRCHE-II algorithm. The T-cell epitopes of the initial theoretical immunizing HLA and the donor HLA were estimated and the number of shared PIRCHE-II epitopes was calculated. We show that the natural logarithm-transformed PIRCHE-II overlap score, or Shared T-cell EPitopes (STEP) score, significantly associates with the 10-year risk of death-censored kidney graft failure, suggesting that the presence of pre-transplant donor-reactive CD4 + memory T-helper cells might be a strong indicator for the risk of graft failure following kidney transplantation., Competing Interests: The authors of this manuscript have conflicts of interest to disclose. The UMC Utrecht has filed a patent application on the prediction of an alloimmune response against mismatched HLA. ES is listed as inventor on this patent. MN is employed by PIRCHE AG, which publishes the PIRCHE web-portal. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Peereboom, Matern, Tomosugi, Niemann, Drylewicz, Joosten, Allebes, van der Meer, Hilbrands, Baas, van Reekum, Verhaar, Kamburova, Seelen, Sanders, Hepkema, Lambeck, Bungener, Roozendaal, Tilanus, Voorter, Wieten, van Duijnhoven, Gelens, Christiaans, van Ittersum, Nurmohamed, Lardy, Swelsen, van der Pant, van der Weerd, ten Berge, Bemelman, de Vries, de Fijter, Betjes, Roelen, Claas, Otten, Heidt, van Zuilen, Kobayashi, Geneugelijk and Spierings.)

Pregnancy after renal transplantation is associated with an increased risk of complications. While a delicately balanced uterine immune system is essential for a successful pregnancy, little is known about the uterine immune environment of pregnant kidney transplant recipients. Moreover, children born to kidney transplant recipients are exposed in utero to immunosuppressive drugs, with possible consequences for neonatal outcomes. Here, we defined the effects of kidney transplantation on the immune cell composition during pregnancy with a cohort of kidney transplant recipients as well as healthy controls with uncomplicated pregnancies. Maternal immune cells from peripheral blood were collected during pregnancy as well as from decidua and cord blood obtained after delivery. Multiparameter flow cytometry was used to identify and characterize populations of cells. While systemic immune cell frequencies were altered in kidney transplant patients, immune cell dynamics over the course of pregnancy were largely similar to healthy women. In the decidua of women with a kidney transplant, we observed a decreased frequency of HLA-DR + Treg, particularly in those treated with tacrolimus versus those that were treated with azathioprine next to tacrolimus, or with azathioprine alone. In addition, both the innate and adaptive neonatal immune system of children born to kidney transplant recipients was significantly altered compared to neonates born from uncomplicated pregnancies. Overall, our findings indicate a significant and distinct impact on the maternal systemic, uterine, and neonatal immune cell composition in pregnant kidney transplant recipients, which could have important consequences for the incidence of pregnancy complications, treatment decisions, and the offspring's health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Feyaerts, Gillard, van Cranenbroek, Rigodanzo Marins, Baghdady, Comitini, Lely, van Hamersvelt, van der Heijden, Joosten and van der Molen.)

Background: Pregnancy is a portentous stage in life, during which countless events are precisely orchestrated to ensure a healthy offspring. Maternal microbial communities are thought to have a profound impact on development. Although antibiotic drugs may interfere in these processes, they constitute the most frequently prescribed medication during pregnancy to prohibit detrimental consequences of infections. Gestational antibiotic intervention is linked to preeclampsia and negative effects on neonatal immunity. Even though perturbations in the immune system of the mother can affect reproductive health, the impact of microbial manipulation on maternal immunity is still unknown., Aim: To assess whether antibiotic treatment influences maternal immunity during pregnancy., Methods: Pregnant mice were treated with broad-spectrum antibiotics. The maternal gut microbiome was assessed. Numerous immune parameters throughout the maternal body, including placenta and amniotic fluid were investigated and a novel machine-learning ensemble strategy was used to identify immunological parameters that allow distinction between the control and antibiotic-treated group., Results: Antibiotic treatment reduced diversity of maternal microbiota, but litter sizes remained unaffected. Effects of antibiotic treatment on immunity reached as far as the placenta. Four immunological features were identified by recursive feature selection to contribute to the most robust classification (splenic T helper 17 cells and CD5 + B cells, CD4 + T cells in mesenteric lymph nodes and RORγT mRNA expression in placenta)., Conclusion: In the present study, antibiotic treatment was able to affect the carefully coordinated immunity during pregnancy. These findings highlight the importance of inclusion of immunological parameters when studying the effects of medication used during gestation., Competing Interests: JG is a Director of Immunology at Nutricia Research, Netherlands. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Benner, Lopez-Rincon, Thijssen, Garssen, Ferwerda, Joosten, van der Molen and Hogenkamp.)

Long-term changes in the immune system of successfully treated people living with HIV (PLHIV) remain incompletely understood. In this study, we assessed 108 white blood cell (WBC) populations in a cohort of 211 PLHIV on stable antiretroviral therapy and in 56 HIV-uninfected controls using flow cytometry. We show that marked differences exist in T cell maturation and differentiation between PLHIV and HIV-uninfected controls: PLHIV had reduced percentages of CD4+ T cells and naïve T cells and increased percentages of CD8+ T cells, effector T cells, and T helper 17 (Th17) cells, together with increased Th17/regulatory T cell (Treg) ratios. PLHIV also exhibited altered B cell maturation with reduced percentages of memory B cells and increased numbers of plasmablasts. Determinants of the T and B cell composition in PLHIV included host factors (age, sex, and smoking), markers of the HIV reservoir, and CMV serostatus. Moreover, higher circulating Th17 percentages were associated with higher plasma concentrations of interleukin (IL) 6, soluble CD14, the gut homing chemokine CCL20, and intestinal fatty acid binding protein (IFABP). The changes in circulating lymphocytes translated into functional changes with reduced interferon (IFN)- γ responses of peripheral blood mononuclear cells to stimulation with  Candida albicans  and  Mycobacterium tuberculosis. In conclusion, this comprehensive analysis confirms the importance of persistent abnormalities in the number and function of circulating immune cells in PLHIV on stable treatment., Competing Interests: This study was supported by an AIDS-fonds (#P-29001) Netherlands and an ERC Advanced Grant (#833247). LV was supported by FWO (grant 1.8.020.09.N.00) and Collen-Francqui Research Professor Mandate. SR received a strategic basic research fund of the Research Foundation – Flanders (FWO, 1S32916N). TS and JL were employed by ViiV Healthcare. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Van de Wijer, van der Heijden, Horst, Jaeger, Trypsteen, Rutsaert, van Cranenbroek, van Rijssen, Joosten, Joosten, Vandekerckhove, Schoofs, van Lunzen, Netea, Koenen, van der Ven and de Mast.)

Severe burn injury causes local and systemic immune responses that can persist up to months, and can lead to systemic inflammatory response syndrome, organ damage and long-term sequalae such as hypertrophic scarring. To prevent these pathological conditions, a better understanding of the underlying mechanisms is essential. In this longitudinal study, we analyzed the temporal peripheral blood immune profile of 20 burn wound patients admitted to the intensive care by flow cytometry and secretome profiling, and compared this to data from 20 healthy subjects. The patient cohort showed signs of systemic inflammation and persistently high levels of pro-inflammatory soluble mediators, such as IL-6, IL-8, MCP-1, MIP-1β, and MIP-3α, were measured. Using both unsupervised and supervised flow cytometry techniques, we observed a continuous release of neutrophils and monocytes into the blood for at least 39 days. Increased numbers of immature neutrophils were present in peripheral blood in the first three weeks after injury (0.1-2.8 × 10 6 /ml after burn vs. 5 × 10 3 /ml in healthy controls). Total lymphocyte numbers did not increase, but numbers of effector T cells as well as regulatory T cells were increased from the second week onward. Within the CD4 + T cell population, elevated numbers of CCR4 + CCR6 - and CCR4 + CCR6 + cells were found. Altogether, these data reveal that severe burn injury induced a persistent innate inflammatory response, including a release of immature neutrophils, and shifts in the T cell composition toward an overall more pro-inflammatory phenotype, thereby continuing systemic inflammation and increasing the risk of secondary complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mulder, Vlig, Boekema, Stoop, Pijpe, van Zuijlen, de Jong, van Cranenbroek, Joosten, Koenen and Ulrich.)

Maintenance of regulatory T cells CD4 + CD25 high FOXP3 + (Treg) stability is vital for proper Treg function and controlling the immune equilibrium. Treg cells are heterogeneous and can reveal plasticity, exemplified by their potential to express IL-17A. TNFα-TNFR2 signaling controls IL-17A expression in conventional T cells via the anti-inflammatory ubiquitin-editing and kinase activity regulating enzyme TNFAIP3/ A20 (tumor necrosis factor-alpha-induced protein 3). To obtain a molecular understanding of TNFα signaling on IL-17 expression in the human effector ( eff Treg, CD25 high CD45RA - ) Treg subset, we here studied the kinome activity regulation by TNFα signaling. Using FACS-sorted naïve ( naïve Treg, CD25 high CD45RA + ) and eff Treg subsets, we demonstrated a reciprocal relationship between TNFα and IL-17A expression; eff Treg (TNFα low /IL-17A high ) and naïve Treg (TNFα high /IL-17A low ). In eff Treg, TNFα-TNFR2 signaling prevented IL-17A expression, whereas inhibition of TNFα signaling by clinically applied anti-TNF antibodies led to increased IL-17A expression. Inhibition of TNFα signaling led to reduced TNFAIP3 expression, which, by using siRNA inhibition of TNFAIP3 , appeared causally linked to increased IL-17A expression in eff Treg. Kinome activity screening of CD3/CD28-activated eff Treg revealed that anti-TNF-mediated neutralization led to increased kinase activity. STRING association analysis revealed that the TNF suppression eff Treg kinase activity network was strongly associated with kinases involved in TCR, JAK, MAPK, and PKC pathway signaling. Small-molecule-based inhibition of TCR and JAK pathways prevented the IL-17 expression in eff Treg. Together, these findings stress the importance of TNF-TNFR2 in regulating the kinase architecture of antigen-activated eff Treg and controlling IL-17 expression of the human Treg. These findings might be relevant for optimizing anti-TNF-based therapy and may aid in preventing Treg plasticity in case of Treg-based cell therapy., (Copyright © 2020 Urbano, He, Heeswijk, Filho, Tijssen, Smeets, Joosten and Koenen.)

Regulatory T cells (Treg) are well-known for their immune regulatory potential and are essential for maintaining immune homeostasis. The rationale of Treg-based immunotherapy for treating autoimmunity and transplant rejection is to tip the immune balance of effector T cell-mediated immune activation and Treg-mediated immune inhibition in favor of Treg cells, either through endogenous Treg expansion strategies or adoptive transfer of ex vivo expanded Treg. Compelling evidence indicates that Treg show properties of phenotypic heterogeneity and instability, which has caused considerable debate in the field regarding their correct use. Consequently, for further optimization of Treg-based immunotherapy, it is vital to further our understanding of Treg proliferative, migratory, and suppressive behavior. It is increasingly appreciated that the functional profile of immune cells is highly dependent on their metabolic state. Although the metabolic profiles of effector T cells are progressively understood, little is known on Treg in this respect. The objective of this review is to outline the current knowledge of human Treg metabolic profiles associated with the regulation of Treg functionality. As such information on human Treg is still limited, where information was lacking, we included insightful findings from mouse studies. To assess the available evidence on metabolic pathways involved in Treg functionality, PubMed, and Embase were searched for articles in English indexed before April 28th, 2019 using "regulatory T lymphocyte," "cell metabolism," "cell proliferation," "migration," "suppressor function," and related search terms. Removal of duplicates and search of the references was performed manually. We discerned that while glycolysis fuels the biosynthetic and bioenergetic needs necessary for proliferation and migration of human Treg, suppressive capacity is mainly maintained by oxidative metabolism. Based on the knowledge of metabolic differences between Treg and non-Treg cells, we additionally discuss and propose ways of how human Treg metabolism could be exploited for the betterment of tolerance-inducing therapies., (Copyright © 2019 Kempkes, Joosten, Koenen and He.)

The best treatment for patients with end-stage renal disease is kidney transplantation. Although graft survival rates have improved in the last decades, patients still may lose their grafts partly due to the detrimental effects of donor-specific antibodies (DSA) against human leukocyte antigens (HLA) and to a lesser extent also by antibodies directed against non-HLA antigens expressed on the donor endothelium. Assays to detect anti-HLA antibodies are already in use for many years and have been proven useful for transplant risk stratification. Currently, there is a need for assays to additionally detect multiple non-HLA antibodies simultaneously in order to study their clinical relevance in solid organ transplantation. This study describes the development, technical details and validation of a high-throughput multiplex assay for the detection of antibodies against 14 non-HLA antigens coupled directly to MagPlex microspheres or indirectly via a HaloTag. The non-HLA antigens have been selected based on a literature search in patients with kidney disease or following transplantation. Due to the flexibility of the assay, this approach can be used to include alternative antigens and can also be used for screening of other organ transplant recipients, such as heart and lung.

A crucial issue for Treg-based immunotherapy is to maintain a bona fide Treg phenotype as well as suppressive function during and after ex vivo expansion. Several strategies have been applied to harness Treg lineage stability. For instance, CD28 superagonist stimulation in vitro , in the absence of CD3 ligation, is more efficient in promoting Treg proliferation, and prevention of pro-inflammatory cytokine expression, such as IL-17, as compared to CD3/CD28-stimulated Treg. Addition of the mTOR inhibitor rapamycin to Treg cultures enhances FOXP3 expression and Treg stability, but does impair proliferative capacity. A tumor necrosis factor receptor 2 (TNFR2) agonist antibody was recently shown to favor homogenous expansion of Treg in vitro . Combined stimulation with rapamycin and TNFR2 agonist antibody enhanced hypo-methylation of the FOXP3 gene, and thus promoting Treg stability. To further explore the underlying mechanisms of rapamycin and TNFR2 agonist-mediated Treg stability, we here stimulated FACS-sorted human Treg with a CD28 superagonist, in the presence of rapamycin and a TNFR2 agonist. Phenotypic analysis of expanded Treg revealed an autocrine loop of TNFα-TNFR2 underlying the maintenance of Treg stability in vitro . Addition of rapamycin to CD28 superagonist-stimulated Treg led to a high expression of TNFR2, the main TNFR expressed on Treg, and additional stimulation with a TNFR2 agonist enhanced the production of soluble as well as membrane-bound TNFα. Moreover, our data showed that the expression of histone methyltransferase EZH2, a crucial epigenetic modulator for potent Treg suppressor function, was enhanced upon stimulation with CD28 superagonist. Interestingly, rapamycin seemed to downregulate CD28 superagonist-induced EZH2 expression, which could be rescued by the additional addition of TNFR2 agonist antibody. This process appeared TNFα-dependent manner, since depletion of TNFα using Etanercept inhibited EZH2 expression. To summarize, we propose that an autocrine TNFα-TNFR2 loop plays an important role in endorsing Treg stability.

Individual HLA mismatches may differentially impact graft survival after kidney transplantation. Therefore, there is a need for a reliable tool to define permissible HLA mismatches in kidney transplantation. We previously demonstrated that donor-derived Predicted Indirectly ReCognizable HLA Epitopes presented by recipient HLA class II (PIRCHE-II) play a role in de novo donor-specific HLA antibodies formation after kidney transplantation. In the present Dutch multi-center study, we evaluated the possible association between PIRCHE-II and kidney graft failure in 2,918 donor-recipient couples that were transplanted between 1995 and 2005. For these donors-recipients couples, PIRCHE-II numbers were related to graft survival in univariate and multivariable analyses. Adjusted for confounders, the natural logarithm of PIRCHE-II was associated with a higher risk for graft failure [hazard ratio (HR): 1.13, 95% CI: 1.04-1.23, p  = 0.003]. When analyzing a subgroup of patients who had their first transplantation, the HR of graft failure for ln(PIRCHE-II) was higher compared with the overall cohort (HR: 1.22, 95% CI: 1.10-1.34, p  < 0.001). PIRCHE-II demonstrated both early and late effects on graft failure in this subgroup. These data suggest that the PIRCHE-II may impact graft survival after kidney transplantation. Inclusion of PIRCHE-II in donor-selection criteria may eventually lead to an improved kidney graft survival.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

In order to combat chronic immune disorders (CIDs), it is an absolute necessity to understand the bigger picture, one that goes beyond insights at a one-disease, molecular, cellular, and static level. To unravel this bigger picture we advocate an integral, cross-disciplinary approach capable of embracing the complexity of the field. This paper discusses the current knowledge on common pathways in CIDs including general psychosocial and lifestyle factors associated with immune functioning. We demonstrate the lack of more in-depth psychosocial and lifestyle factors in current research cohorts and most importantly the need for an all-encompassing analysis of these factors. The second part of the paper discusses the challenges of understanding immune system dynamics and effectively integrating all key perspectives on immune functioning, including the patient's perspective itself. This paper suggests the use of techniques from complex systems science in describing and simulating healthy or deviating behavior of the immune system in its biopsychosocial surroundings. The patient's perspective data are suggested to be generated by using specific narrative techniques. We conclude that to gain more insight into the behavior of the whole system and to acquire new ways of combatting CIDs, we need to construct and apply new techniques in the field of computational and complexity science, to an even wider variety of dynamic data than used in today's systems medicine.

Clinical translation of tolerance-inducing cell therapies requires a novel approach focused on innovative networks, patient involvement, and, foremost, a fundamental paradigm shift in thinking from both Academia, and Industry and Regulatory Agencies. Tolerance-inducing cell products differ essentially from conventional drugs. They are personalized and target interactive immunological networks to shift the balance toward tolerance. The human cell products are often absent or fundamentally different in animals. This creates important limitations of pre-clinical animal testing for safety and efficacy of these products and calls for novel translational approaches, which require the combined efforts of the different parties involved. Dedicated international and multidisciplinary consortia that focus on clinical translation are of utmost importance. They can help in informing and educating regulatory policy makers on the unique requirements for these cell products, ranging from pre-clinical studies in animals to in vitro human studies. In addition, they can promote reliable immunomonitoring tools. The development of tolerance-inducing cell products requires not only bench-to-bedside but also reverse translation, from bedside back to the bench.