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American journal of transplantation 22(11), 2529-2547 (2022). doi:10.1111/ajt.17154, Published by Elsevier, [Amsterdam]

Long-term results following solid organ transplantation do not mirror the excellent short-term results achieved in recent decades. It is therefore clear that current immunosuppressive maintenance protocols primarily addressing the adaptive immune system no longer meet the required clinical need. Identification of novel targets addressing this shortcoming is urgently needed. There is a growing interest in better understanding the role of the innate immune system in this context. In this review, we focus on macrophages, which are known to prominently infiltrate allografts and, during allograft rejection, to be involved in the surge of the adaptive immune response by expression of pro-inflammatory cytokines and direct cytotoxicity. However, this active participation is janus-faced and unspecific targeting of macrophages may not consider the different subtypes involved. Under this premise, we give an overview on macrophages, including their origins, plasticity, and important markers. We then briefly describe their role in acute allograft rejection, which ranges from sustaining injury to promoting tolerance, as well as the impact of maintenance immunosuppressants on macrophages. Finally, we discuss the observed immunosuppressive role of the vitamin-like compound tetrahydrobiopterin and the recent findings that suggest the innate immune system, particularly macrophages, as its target.

Summary Background and Objectives We examined retrospectively whether the combination of standard dacarbazine (DTIC) and/or fotemustine chemotherapy and autologous peptide‐loaded dendritic cell (DC) vaccination may improve survival of stage IV melanoma patients. Furthermore, a small cohort of long‐term survivors was studied in more detail. Patients and methods Between 1998 and 2008, 41 patients were vaccinated at least three times with DCs while receiving chemotherapy and compared to all other 168 patients in our database who only received chemotherapy (1993–2008). Results Median life expectancy of patients receiving additional DC‐vaccination was 18 months, compared to eleven months for patients under standard chemotherapy alone. In contrast to patients with other haplotypes, the HLA‐A1/A1 subset of DC‐treated patients showed significantly lower median survival (12 vs. 25 months). Autoantibodies were frequently detected in serum of both vaccinated and non‐vaccinated patients, and there was no correlation between titers, loss or appearance of autoantibodies and survival. Additionally, phenotyping of DCs and PBMCs also did not reveal any conspicuous correlation with survival. Conclusions Combining standard chemotherapy and DC vaccination appears superior to chemotherapy alone. The impact of HLA haplotypes on survival emphasizes the importance of a careful selection of patients with specific, well‐defined HLA haplotypes for future vaccination trials using peptide‐pulsed DCs, possibly combined with checkpoint inhibitors.

Study question Within this prospective study, we aim to differentiate immune cell subpopulations in recurrent implantation failure (RIF) patients and fertile controls. Summary answer A misbalanced immune profile of NK cell subpopulations is present in RIF patients and might be a potential risk factor that requires further detailed analysis. What is known already So far, there is no conclusive opinion on the prognostic value of testing immune cell populations in women with RIF. Increased numbers of cytotoxic (CD56dimCD16bright) peripheral natural killer (pNK)-cells and CD56brightCD16dim mainly in the uterus occurring NK cells (uNK) seemed to be more prevalent in RIF patients. NK cell cytotoxicity is regulated by a complex interaction of activating and inhibiting receptors, such as the NKGD2 and natural cytotoxicity receptors including NKp46, NKp30 and NKp44. Dysregulated pNK cells could affect the adhesion and implantation of the embryo thereby contributing to RIF. Study design, size, duration Within this prospective study between March 2018 and August 2020 immune diagnostics of pNK cells and subpopulations as well as regulatory T-cells in RIF patients (defined as ≥ 3 failed fresh or frozen embryo transfers of good quality embryos (Istanbul criteria) and non-pregnant controls (nulli- and multipara) were performed using flow cytometry analysis. Participants/materials, setting, methods In total, n = 42 RIF and n = 85 controls were included. Absolute numbers and percentages of total lymphocytes of CD56dimCD16bright, CD56brightCD16dim NK cells, CD45+CD25+FoxP3+-regulatory T-cells and activation markers (CD57+, CD62L+, NKGD2+, NKp46+) were measured in patients and controls (n = 60 nulligravida, n = 25 para) in the mid-luteal phase. Statistical analysis was performed using SPSS Version 26 considering p Main results and the role of chance RIF patients showed significantly lower numbers and percentages of CD56dimCD16bright pNK cells (mean±SD per µl: 187,5±113,3 vs. 281,9±163,4 p = 0.001;%: 87,4±8,8 vs. 90,6±6,0 p = 0.017) and higher levels of CD56brightCD16dim pNK cells (mean±SD per%: 10,5±8,3 vs. 7,6±5,5 p = 0.021) compared to controls. Further, lower percentages of CD56dimCD16brightCD62L + (mean±SD per%: 23,5±11,1 vs. 32,0±14,0 p = 0.001), CD56dimCD16brightNKGD2 + (mean±SD per%: 94,0±6,8 vs. 96,4±4,2 p = 0.014) and CD56dimCD16brightNKp46 + (mean±SD per%: 65,8±19,5 vs. 76,1±14,0 p = 0.001) were observed in RIF patients (p Limitations, reasons for caution As controls composed out of not only nulli- but also multipara, higher levels of pNK cells in controls, could be induced by fetal microchimerism in multiparas, however, results remained significant after removing multipara from statistical analysis. Wider implications of the findings: These findings condense into the assumption of a non-linear association between NK cytotoxicity and successful pregnancy. A lower NK cytotoxicity in RIF patients could potentially lead to an altered immune environment impeding a successful implantation process. Trial registration number Drks00020803

Rigosertib, via reactive oxygen species (ROS), stimulates cJun N-terminal kinases 1/2 (JNK1/2), which inactivate RAS/RAF signaling and thereby inhibit growth and survival of tumor cells. JNK1/2 are not only regulated by ROS&mdash, they in turn can also control ROS production. The prooxidant and cell death function of p66Shc requires phosphorylation by JNK1/2. Here, we provide evidence that establishes p66Shc, an oxidoreductase, as a JNK1/2 effector downstream of Rigosertib-induced ROS production, DNA damage, and cell death. This may provide a common pathway for suppression of tumor cell growth by Rigosertib.

Mitochondria sense changes resulting from the ischemia and subsequent reperfusion of an organ and mitochondrial reactive oxygen species (ROS) production initiates a series of events, which over time result in the development of full-fledged ischemia-reperfusion injury (IRI), severely affecting graft function and survival after transplantation. ROS activate the innate immune system, regulate cell death, impair mitochondrial and cellular performance and hence organ function. Arresting the development of IRI before the onset of ROS production is currently not feasible and clinicians are faced with limiting the consequences. Ex vivo machine perfusion has opened the possibility to ameliorate or antagonize the development of IRI and may be particularly beneficial for extended criteria donor organs. The molecular events occurring during machine perfusion remain incompletely understood. Accumulation of succinate and depletion of adenosine triphosphate (ATP) have been considered key mechanisms in the initiation; however, a plethora of molecular events contribute to the final tissue damage. Here we discuss how understanding mitochondrial dysfunction linked to IRI may help to develop novel strategies for the prevention of ROS-initiated damage in the evolving era of machine perfusion.

Resistance to chemotherapy is one of the primary obstacles in acute myeloid leukemia (AML) therapy. Micro-RNA-23a (miR-23a) is frequently deregulated in AML and has been linked to chemoresistance in solid cancers. We, therefore, studied its role in chemoresistance to cytarabine (AraC), which forms the backbone of all cytostatic AML treatments. Initially, we assessed AraC sensitivity in three AML cell lines following miR-23a overexpression/knockdown using MTT-cell viability and soft-agar colony-formation assays. Overexpression of miR-23a decreased the sensitivity to AraC, whereas its knockdown had the opposite effect. Analysis of clinical data revealed that high miR-23a expression correlated with relapsed/refractory (R/R) AML disease stages, the leukemic stem cell compartment, as well as with inferior overall survival (OS) and event-free survival (EFS) in AraC-treated patients. Mechanistically, we demonstrate that miR-23a targets and downregulates topoisomerase-2-beta (TOP2B), and that TOP2B knockdown mediates AraC chemoresistance as well. Likewise, low TOP2B expression also correlated with R/R-AML disease stages and inferior EFS/OS. In conclusion, we show that increased expression of miR-23a mediates chemoresistance to AraC in AML and that it correlates with an inferior outcome in AraC-treated AML patients. We further demonstrate that miR-23a causes the downregulation of TOP2B, which is likely to mediate its effects on AraC sensitivity.

It has already been shown that neutralization of the activating NK cell receptor NKG2D in combination with co-stimulation blockade prolongs graft survival of vascularized transplants. In order to clarify the underlying cellular mechanisms, we transplanted complete MHC-disparate BALB/c-derived cardiac grafts into C57BL/6 wildtypes or mice deficient for NKG2D (Klrk1-/- ). Although median survival was 8 days for both recipient groups, we detected already at day 5 posttransplantation significantly greater intragraft frequencies of NKp46+ NK cells in Klrk1-/- recipients than in wildtypes. This was followed by a significantly greater infiltration of CD4+ , but a lesser infiltration of CD8+ T cell frequencies. Contrary to published observations, co-stimulation blockade with CTLA4-Ig resulted in a significant acceleration of cardiac rejection by Klrk1-/- recipients, and this result was confirmed by applying a neutralizing antibody against NKG2D to wildtypes. In both experimental setups, grafts derived from Klrk1-/- recipients were characterized by significantly higher levels of interferon-γ mRNA, and both CD4+ and CD8+ T cells displayed a greater capacity for degranulation and interferon-γ production. In summary, our results clearly illustrate that NKG2D expression in the recipient is important for cardiac allograft survival, thus supporting the hypothesis that impairment of NK cells prevents the establishment of graft acceptance.

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation. Electronic supplementary material The online version of this article (10.1007/s00018-017-2700-0) contains supplementary material, which is available to authorized users.

Background Clinical data suggest that iron disturbances deleteriously affect graft survival after heart transplantation (HTx), but immunological mechanisms underlying this phenomenon have not yet been elucidated. Methods To identify the mechanistic influence of iron in a murine model of HTx, fully allogeneic BALB/c donor organs were transplanted into iron-overloaded or iron-deficient C57BL/6 mice, and recipients were analyzed for functional and immunological parameters. Results After HTx, iron overload accelerated acute rejection as observed by shortened graft survival (HTx vs HTx + iron; p = 0.01), elevated rejection score ( p p + T cells ( p − NKp46 + natural killer cells ( p p p = 0.0051), which was rescued after treatment with the iron chelator deferoxamine. Iron deficiency (ID) also resulted in enhanced intragraft infiltration of inflammatory cells and accelerated rejection in the acute setting (HTx vs HTx + ID; p = 0.02) and after co-stimulation blockade ( p = 0.0059). Conclusions We provide novel insights into the understanding of disturbances in iron homeostasis and their consequences after HTX, allowing novel insights regarding improvements in personalized immunosuppression to prolong allograft survival.

The definition of biological donor organ age rather than chronological age seems obvious for the establishment of a valid pre-transplant risk assessment. Therefore, we studied gene expression for candidate markers in 60 zero-hour kidney biopsies. Compared with 29 younger donors under age 55, 31 elderly donors age 55 and older had significant mRNA expression for immunoproteasome subunits (PSMB8, PSMB9 and PSMB10), HLA-DRB, and transcripts of the activating cytotoxicity receptor NKG2D. Gene expression was validated in an independent donor cohort consisting of 37 kidneys from donors 30 years and under (Group I), 75 kidneys from donors age 31-54 years (Group II) and 75 kidneys from donors age 55 and older (Group III). Significant gene induction was confirmed in kidneys from Group III for PSMB9 and PSMB10. Strikingly, transcripts of NKG2D had the significantly highest gene induction in Group III versus Group II and Group I. Similar results were obtained for CDKN2A, but not for telomere length. Both NKG2D and CDKN2A mRNA expression were significantly correlated with creatinine levels at 24 months after transplantation. Univariate regression analysis showed significant predictive power regarding graft function at 6 and 12 months for NKG2D and CDKN2A. However, only NKG2D remained significantly predictive in the multivariate model at 12 months. Thus, our results reveal novel candidate markers in aged renal allografts, which could be helpful in the assessment of organ quality.

OBJECTIVES Ischaemia and subsequent reperfusion during heart transplantation inevitably result in donor organ injury. Toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viral and endogenous RNA released by injured cells. We hypothesized that ischaemia/reperfusion injury (IRI) leads to RNA release with subsequent TLR3 activation in transplanted hearts. METHODS Human endothelial cells were subjected to IRI and treated with TLR3 agonist polyinosinic–polycytidylic acid or a TLR3/double-stranded RNA complex inhibitor. TLR3 activation was analysed using reporter cells. Gene expression profiles were evaluated via next-generation sequencing. Neutrophil adhesion was assessed in vitro. Syngeneic heart transplantation of wild-type or Tlr3−/− mice was performed following 9 h of cold ischaemia. Hearts were analysed for inflammatory gene expression, cardiac damage, apoptosis and infiltrating leucocytes. RESULTS IRI resulted in RNA release with subsequent activation of TLR3. Treatment with a TLR3 inhibitor abrogated the inflammatory response upon IRI. In parallel, TLR3 stimulation caused activation of the inflammasome. Endothelial IRI resulted in TLR3-dependent adhesion of neutrophils. Tlr3−/− animals showed reduced intragraft and splenic messenger ribonucleic acid (mRNA) expression of proinflammatory cytokines, resulting in decreased myocardial damage, apoptosis and infiltrating cells. Tlr3 deficiency protected from cardiac damage, apoptosis and leucocyte infiltration after cardiac transplantation. CONCLUSIONS We uncover the release of RNA by injured cells with subsequent activation of TLR3 as a crucial pathomechanism of IRI. Our data indicate that TLR3 represents a novel target in the prevention of IRI in solid organ transplantation.

A comparative analysis of inflammation between solid organs following donor brain death (BD) is still lacking and the detailed influence of BD accelerating ischaemia-reperfusion injury (IRI) post-transplantation remains to be addressed. Applying a murine model of BD, we demonstrated that 4 h after BD organs were characterized by distinct inflammatory expression patterns. For instance, lipocalin 2 (LCN2), a marker of acute kidney injury, was selectively induced in BD livers but not in kidneys. BD further resulted in significantly reduced frequencies of CD3(+) CD4(+) , CD3(+) CD8(+) T cells and NKp46(+) NK cells in the liver, whereas BD kidneys and hearts were characterized by significantly lower frequencies of conventional dendritic cells (cDCs). Syngeneic models of kidney (KTx) and heart transplantation (HTx) illustrated stronger gene expression in engrafted BD hearts only, but 20 h post-transplantation both organs displayed comparable intragraft lymphocyte frequencies, except for NK cells and graft function. Moreover, the complement factor C3d deposit detected in small vessels and capillaries in cardiac syngrafts did not significantly differ between BD and sham-transplanted groups. Finally, no further influence of donor BD on graft survival was detected in an allogeneic heart transplantation setting (C57BL/6 grafts into BALB/c recipients). We show for the first time that BD organs are characterized by a varying inflammatory profile; however, BD does not accelerate IRI in syngeneic KTx and HTx. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Antibody-mediated rejection (AMR) has been identified among the most important factors limiting long-term outcome in cardiac and renal transplantation. Therapeutic management remains challenging and the development of effective treatment modalities is hampered by insufficient understanding of the underlying pathophysiology. However, recent findings indicate that in addition to AMR-triggered activation of the classical complement pathway, antibody-dependent cellular cytotoxicity by innate immune cell subsets also promotes vascular graft injury. This review summarizes the accumulating evidence for the contribution of natural killer cells, the key mediators of antibody-dependent cellular cytotoxicity, to human AMR in allotransplantation and xenotransplantation and illustrates the current mechanistic conceptions drawn from animal models.

A comparative analysis of inflammation between solid organs following donor brain death (BD) is still lacking and the detailed influence of BD accelerating ischaemia-reperfusion injury (IRI) post-transplantation remains to be addressed. Applying a murine model of BD, we demonstrated that 4 h after BD organs were characterized by distinct inflammatory expression patterns. For instance, lipocalin 2 (LCN2), a marker of acute kidney injury, was selectively induced in BD livers but not in kidneys. BD further resulted in significantly reduced frequencies of CD3(+) CD4(+) , CD3(+) CD8(+) T cells and NKp46(+) NK cells in the liver, whereas BD kidneys and hearts were characterized by significantly lower frequencies of conventional dendritic cells (cDCs). Syngeneic models of kidney (KTx) and heart transplantation (HTx) illustrated stronger gene expression in engrafted BD hearts only, but 20 h post-transplantation both organs displayed comparable intragraft lymphocyte frequencies, except for NK cells and graft function. Moreover, the complement factor C3d deposit detected in small vessels and capillaries in cardiac syngrafts did not significantly differ between BD and sham-transplanted groups. Finally, no further influence of donor BD on graft survival was detected in an allogeneic heart transplantation setting (C57BL/6 grafts into BALB/c recipients). We show for the first time that BD organs are characterized by a varying inflammatory profile; however, BD does not accelerate IRI in syngeneic KTx and HTx.

BACKGROUND A daily injection of low-molecular-weight heparin (LMWH) is often prescribed to women with unexplained recurrent pregnancy loss (RPL), although evidence suggesting a benefit is questionable. OBJECTIVE To determine whether LMWH increases ongoing pregnancy and live-birth rates in women with unexplained RPL. DESIGN Controlled, multicenter trial with randomization using minimization conducted from 2006 to 2013. (ClinicalTrials.gov: NCT00400387). SETTING 14 university hospitals and perinatal care centers in Germany and Austria. PATIENTS 449 women with at least 2 consecutive early miscarriages or 1 late miscarriage were included during 5 to 8 weeks' gestation after a viable pregnancy was confirmed by ultrasonography. INTERVENTION Women in the control group received multivitamin pills, and the intervention group received vitamins and 5000 IU of dalteparin-sodium for up to 24 weeks' gestation. MEASUREMENTS Primary outcome was ongoing pregnancy at 24 weeks' gestation. Secondary outcomes included the live-birth rate and late pregnancy complications. RESULTS At 24 weeks' gestation, 191 of 220 pregnancies (86.8%) and 188 of 214 pregnancies (87.9%) were intact in the intervention and control groups, respectively (absolute difference, -1.1 percentage points [95% CI, -7.4 to 5.3 percentage points]). The live-birth rates were 86.0% (185 of 215 women) and 86.7% (183 of 211 women) in the intervention and control groups, respectively (absolute difference, -0.7 percentage point [CI, -7.3 to 5.9 percentage points]). There were 3 intrauterine fetal deaths (1 woman had used LMWH); 9 cases of preeclampsia or the hemolysis, elevated liver enzyme level, and low platelet count (HELLP) syndrome (3 women had used LMWH); and 11 cases of intrauterine growth restriction or placental insufficiency (5 women had used LMWH). LIMITATION Placebo injections were not used, and neither trial staff nor patients were blinded. CONCLUSION Daily LMWH injections do not increase ongoing pregnancy or live-birth rates in women with unexplained RPL. Given the burden of the injections, they are not recommended for preventing miscarriage. PRIMARY FUNDING SOURCE Pfizer Pharma.