Timm Heinbokel

Obesity and its impact on transplantation and alloimmunity.

Obesity has become an increasing problem in healthcare worldwide with far-reaching consequences. More obese patients with irreversible end-stage organ failure undergo organ transplantation, and organs from obese donors are more frequently used. A growing body of evidence suggests more frequent postoperative complications and inferior patient and graft survival linked to obesity. More recently, adipose tissue has been linked to chronic inflammatory processes potentially impacting alloimmune responses and graft quality.

Immunosenescence and organ transplantation

Increasing numbers of elderly transplant recipients and a growing demand for organs from older donors impose pressing challenges on transplantation medicine. Continuous and complex modifications of the immune system in parallel to aging have a major impact on transplant outcome and organ quality. Both, altered alloimmune responses and increased immunogenicity of organs present risk factors for inferior patient and graft survival. Moreover, a growing body of knowledge on age-dependent modifications of allorecognition and alloimmune responses may require age-adapted immunosuppression and organ allocation. Here, we summarize relevant aspects of immunosenescence and their possible clinical impact on organ transplantation.

Impact of immunosenescence on transplant outcome.

Aging affects all compartments of the immune response and has a major impact on transplant outcome and organ quality. Although clinical trials in the aging transplant population remain rare, our current understanding of immunosenescence provides a basis for an age‐adapted immunosuppression and organ allocation with the goal to optimize utilization and to improve outcomes in older recipients. From a more general perspective, understanding the mechanisms and consequences of immunosenescence will have a broad impact on immune therapies in and beyond transplantation.

NAD(+) regulates Treg cell fate and promotes allograft survival via a systemic IL-10 production that is CD4(+) CD25(+) Foxp3(+) T cells independent.

CD4+ CD25+ Foxp3+ Tregs have been shown to play a central role in immune homeostasis while preventing from fatal inflammatory responses, while Th17 cells have traditionally been recognized as pro-inflammatory mediators implicated in a myriad of diseases. Studies have shown the potential of Tregs to convert into Th17 cells, and Th17 cells into Tregs. Increasing evidence have pointed out CD25 as a key molecule during this transdifferentiation process, however molecules that allow such development remain unknown. Here, we investigated the impact of NAD+ on the fate of CD4+ CD25+ Foxp3+ Tregs in-depth, dissected their transcriptional signature profile and explored mechanisms underlying their conversion into IL-17A producing cells. Our results demonstrate that NAD+ promotes Treg conversion into Th17 cells in vitro and in vivo via CD25 cell surface marker. Despite the reduced number of Tregs, known to promote homeostasis, and an increased number of pro-inflammatory Th17 cells, NAD+ was able to promote an impressive allograft survival through a robust systemic IL-10 production that was CD4+ CD25+ Foxp3+ independent. Collectively, our study unravels a novel immunoregulatory mechanism of NAD+ that regulates Tregs fate while promoting allograft survival that may have clinical applications in alloimmunity and in a wide spectrum of inflammatory conditions.

CD11c+ Dendritic Cells Accelerate the Rejection of Older Cardiac Transplants via Interleukin-17A

Background— Organ transplantation has seen an increased use of organs from older donors over the past decades in an attempt to meet the globally growing shortage of donor organs. However, inferior transplantation outcomes when older donor organs are used represent a growing challenge. Methods and Results— Here, we characterize the impact of donor age on solid-organ transplantation using a murine cardiac transplantation model. We found a compromised graft survival when older hearts were used. Shorter graft survival of older hearts was independent of organ age per se, because chimeric young or old organs repopulated with young passenger leukocytes showed comparable survival times. Transplantation of older organs triggered more potent alloimmune responses via intragraft CD11c+ dendritic cells augmenting CD4+ and CD8+ T-cell proliferation and proinflammatory cytokine production, particularly that of interleukin-17A. Of note, depletion of donor CD11c+ dendritic cells before engraftment, neutralization of interleukin-17A, or transplantation of older hearts into IL-17A−/− mice delayed rejection and reduced alloimmune responses to levels observed when young hearts were transplanted. Conclusions— These results demonstrate a critical role of old donor CD11c+ dendritic cells in mounting age-dependent alloimmune responses with an augmented interleukin-17A response in recipient animals. Targeting interleukin-17A may serve as a novel therapeutic approach when older organs are transplanted.

Age-Dependent Metabolic and Immunosuppressive Effects of Tacrolimus.

Immunosuppression in elderly recipients has been underappreciated in clinical trials. Here, we assessed age‐specific effects of the calcineurin inhibitor tacrolimus (TAC) in a murine transplant model and assessed its clinical relevance on human T cells. Old recipient mice exhibited prolonged skin graft survival compared with young animals after TAC administration. More important, half of the TAC dose was sufficient in old mice to achieve comparable systemic trough levels. TAC administration was able to reduce proinflammatory interferon‐γ cytokine production and promote interleukin‐10 production in old CD4+ T cells. In addition, TAC administration decreased interleukin‐2 secretion in old CD4+ T cells more effectively while inhibiting the proliferation of CD4+ T cells in old mice. Both TAC‐treated murine and human CD4+ T cells demonstrated an age‐specific suppression of intracellular calcineurin levels and Ca2+ influx, two critical pathways in T cell activation. Of note, depletion of CD8+ T cells did not alter allograft survival outcome in old TAC‐treated mice, suggesting that TAC age‐specific effects were mainly CD4+ T cell mediated. Collectively, our study demonstrates age‐specific immunosuppressive capacities of TAC that are CD4+ T cell mediated. The suppression of calcineurin levels and Ca2+ influx in both old murine and human T cells emphasizes the clinical relevance of age‐specific effects when using TAC.

Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation

Background: Given their unique capacity for antigen uptake, processing, and presentation, antigen‐presenting cells (APCs) are critical for initiating and regulating innate and adaptive immune responses. We have previously shown the role of nicotinamide adenine dinucleotide (NAD+) in T‐cell differentiation independently of the cytokine milieu, whereas the precise mechanisms remained unknown. Objective: The objective of this study is to further dissect the mechanism of actions of NAD+ and determine the effect of APCs on NAD+‐mediated T‐cell activation. Methods: Isolated dendritic cells and bone marrow–derived mast cells (MCs) were used to characterize the mechanisms of action of NAD+ on CD4+ T‐cell fate in vitro. Furthermore, NAD+‐mediated CD4+ T‐cell differentiation was investigated in vivo by using wild‐type C57BL/6, MC−/−, MHC class II−/−, Wiskott‐Aldrich syndrome protein (WASP)−/−, 5C.C7 recombination‐activating gene 2 (Rag2)−/−, and CD11b‐DTR transgenic mice. Finally, we tested the physiologic effect of NAD+ on the systemic immune response in the context of Listeria monocytogenes infection. Results: Our in vivo and in vitro findings indicate that after NAD+ administration, MCs exclusively promote CD4+ T‐cell differentiation, both in the absence of antigen and independently of major APCs. Moreover, we found that MCs mediated CD4+ T‐cell differentiation independently of MHC II and T‐cell receptor signaling machinery. More importantly, although treatment with NAD+ resulted in decreased MHC II expression on CD11c+ cells, MC‐mediated CD4+ T‐cell differentiation rendered mice resistant to administration of lethal doses of L monocytogenes. Conclusions: Collectively, our study unravels a novel cellular and molecular pathway that regulates innate and adaptive immunity through MCs exclusively and underscores the therapeutic potential of NAD+ in the context of primary immunodeficiencies and antimicrobial resistance.

Senolytic Treatment Attenuates mtDNA-Mediated Inflammatory injury in Old Donors and Improves Cardiac Allograft Survival

Introduction Solid organ transplantation has seen a significant increase in the utilization of older organs. Here, we investigate how aging-associated kinetics of damage associated molecular patterns (DAMPs) including mitochondrial DNA (mtDNA) are driving the augmented susceptibility of older organs to ischemia and reperfusion injury (IRI) through dendritic cells (DCs) with subsequent inferior graft survival. Methods Old and young mice underwent bilateral clamping of the renal pedicles (22 min ischemic time at controlled normothermia). mtDNA, cytokine and senescence marker levels were tested by qPCR; DC and T cell activation were characterized by FACS. Old and young DCs were adoptively transferred into young recipients that subsequently received young or old cardiac allografts. Results DCs of old naïve mice showed higher frequencies and levels of maturation in parallel to increased baseline levels of mtDNA. Importantly, renal IRI induced a prominent release of mtDNA into the circulation of old animals (n=5/group after 48h; p=0.019) and increased IFN-&ggr; expression in splenic CD8+ T cells (p=0.0001). Isolated DCs showed a dose-dependent up-regulation of CD40 with augmented amounts of IL-6 in the presence of mtDNA; the addition of a TLR9 antagonist attenuated this pro-inflammatory response of old DCs. In addition, old DCs promoted IFN-&ggr; and IL-17 responses of allogeneic T cells in vitro. Of particular relevance, adoptive transfer of old but not young DCs prior to transplantation shortened cardiac allograft survival (log-rank test: p<0.0001; n=8-10/group). Treatment with the senolytic agents Dasatinib (5mg/kg) and Quercetin (50mg/kg) not only reduced local expression of senescence marker p16 and p21 in kidneys, but also drastically reduced local and systemic mtDNA levels in old mice. In parallel, levels of IFN-&ggr; and IL-17 and systemic frequencies of CD8+IFN-&ggr;+ and CD4+IL-17+ T cells were reduced subsequent to the treatment with senolytics. These in-vitro effects translated into prolonged allograft survival of cardiac allografts procured from old donor mice that underwent pretreatment with senolytic agents, compared to allografts procured from old untreated donors (log-rank test: p<0.0280; n=6/group). Conclusion Our results suggest a pivotal role for mtDNA in driving allogeneic immune responses to old donor organs, critically affecting graft survival. Senolytic treatment reduced mtDNA release and associated inflammation with prolonged graft survival, thus representing a novel approach for increased utilization of marginal donor organs.

Costimulatory Blockade with CTLA4-Ig Abrogates Prolonged Graft Survival in Old Recipients

Introduction Although they represent a rapidly growing population, elderly organ transplant recipients are underrepresented in clinical trials. Age-specific aspects of established immunosuppressants are therefore poorly understood. Methods Here, we assessed the impact of immunosuppressive treatment with CTLA4-Ig, a fusion protein blocking costimulatory signaling between APCs and T cells through CD28, on alloimmune responses in old and young recipients (2-3 months vs. 16 months) in a fully MHC-mismatched murine transplantation models. Results While treatment with CTLA4-Ig prolonged skin graft survival in young recipients, the same treatment was unable to prolong graft survival in old recipients. Conversely, cardiac allografts in young mice treated with CTLA4-Ig survived indefinitely, while 80% of old recipients treated with CTLA4-Ig had lost their graft after 100 days (log-rank test, p<0.001; n=5/group; Fig. 1).{{AbstractFigure.1}} CTLA4-Ig reduced the in-vivo proliferation of CD4+ and CD8+ T cells (as assessed by BrdU incorporation) uniformly in both young and old recipients; in contrast, CTLA4-Ig reduced CD4+ central-memory and effector-memory T cells only in young but not old recipients. Moreover, systemic frequencies of CD4+IFN-&ggr;+ T cells and systemic levels of IFN-&ggr; cytokine production were reduced in young recipients, but remained unchanged in old. CTLA4-Ig caused significant perturbation in the Treg compartments with reduced frequencies and compromised proliferation of CD4+CD25+Foxp3+ cells. These differences correlated with a significant reduction in expression of CD28 on T cells in old mice, while levels of CTLA4 remained stable. Figure. No caption available. Conclusion Immunosuppressive effects of costimulatory blockade with CTLA4-Ig showed distinct age-dependent effects in both skin and cardiac transplantation. Reduced expression of CD28 with aging may represent an escape mechanism for old alloreactive T cells, with unique clinical consequences for immunosuppression in the growing population of elderly transplant recipients.

Tacrolimus Exerts Age-Specific Immunosuppressive Effects Linked to Modifications of CD4+ T cells Metabolism

Background Energy metabolism of immune cells has been identified as critical in driving alloimmunity. While metabolic capacities change with aging, aspects linking age, immunosuppression, and T-cell metabolism remain unclear. We submit that age-specific immunosuppressive capacities of Tacrolimus (TAC) are operative through specific changes in T-cell metabolism. Methods Full thickness skin grafts were transplanted from young (DBA/2 mice) to either young or old C57BL/6 recipients; animals were treated with TAC and graft survival was tested. To test the impact of T-cell metabolism, aging and TAC treatment, naïve CD4+ T cells were collected from recipient animals and activated with anti-CD3 and anti-CD28. Oxidative phosphorylation (OXPHOS) and aerobic glycolysis were assessed by oxygen consumption (OCR) and extracellular acidification rate (ECAR) using a XFe24 extracellular flux analyzer. Results Old and young recipients received weight-adapted doses of TAC. The majority of older recipients survived until the end of the observation period (day 30) while young animals rejected allografts by day 11 (mean survival time; n = 7; p < 0.0004;). Old but not young CD4+ T cells demonstrated compromised metabolic rates with significantly lower OCR and ECAR (p<0.0001). Next, we co-cultured naïve CD4+ T cells with TAC at different concentrations and measured OCR and ECAR by 24 hrs. Dose-response curves of ECAR to TAC revealed a significantly reduced IC50 of old CD4+ T cells (p<0.0001), suggesting an age-specific effect of TAC on glycolysis. Next, we tested if energy metabolism in CD4+ T cells impacts IL-2 cytokine production in an age-specific manner. Here, we used glyceraldehyde 3- phosphate (G3P) to induce aerobic glycolysis in-vitro. G3P is the sole substrate of GAPDH, the pivotal enzyme in aerobic glycolysis. Activation of aerobic glycolysis through G3P increased IL-2 significantly in old, but not young TAC treated CD4+ T cells. Moreover, IL-2 expression increased dramatically (by 82%) subsequent to the introduction of G3P in old TAC treated T cells; notably, only a minor (26%) increase of G3P was observed in old naïve T cells suggesting a compromised aerobic glycolysis in old CD4+ T cells, an effect that is exacerbated subsequent to the treatment with TAC. Conclusions TAC appears significantly effective in older recipients leading to specific prolongation of graft survival in old recipients. Those effects are linked to age-specific metabolic changes in CD-4 T-cell metabolism with a compromised glycolysis of old CD4+ T cells that is exacerbated age-specifically subsequent to TAC treatment. Moreover, IL-2 expression increased dramatically in old CD4+ T cells following the activation of glycolysis. Taken together, our results suggest that age-specific aspects of TAC are linked to a compromised glycolysis of CD4+ T cells Those findings provide a novel concept to refine immunosuppression in and beyond aging.