Advanced in vitro Research Models to Study the Role of Endothelial Cells in Solid Organ Transplantation

The endothelium plays a key role in acute and chronic rejection of solid organ transplants. During both processes the endothelium is damaged often with major consequences for organ function. Also, endothelial cells (EC) have antigen-presenting properties and can in this manner initiate and enhance alloreactive immune responses. For decades, knowledge about these roles of EC have been obtained by studying both in vitro and in vivo models. These experimental models poorly imitate the immune response in patients and might explain why the discovery and development of agents that control EC responses is hampered. In recent years, various innovative human 3D in vitro models mimicking in vivo organ structure and function have been developed. These models will extend the knowledge about the diverse roles of EC in allograft rejection and will hopefully lead to discoveries of new targets that are involved in the interactions between the donor organ EC and the recipient's immune system. Moreover, these models can be used to gain a better insight in the mode of action of the currently prescribed immunosuppression and will enhance the development of novel therapeutics aiming to reduce allograft rejection and prolong graft survival.</p

On the interactions between mesenchymal stem cells and regulatory T cells for immunomodulation in transplantation

Experimental studies have established the use of mesenchymal stem cells (MSC) as a candidate immunosuppressive therapy. MSC exert their immunomodulatory function through the inhibition of CD4+ and CD8+ T cell proliferation. It is unknown whether MSC impair the immunosuppressive function of regulatory T cells (Treg). In vitro and in vivo studies suggest that MSC mediate their immunomodulatory effects through the induction of Treg. In this review we will focus on the interactions between MSC and Treg, and evaluate the consequences of these cellular interplays for prospective MSC immunotherapy in organ transplantation

Tissue-resident Lymphocytes Are Released During Hypothermic and Normothermic Machine Perfusion of Human Donor Kidneys

BACKGROUND: Machine perfusion is the preferred preservation method for deceased donor kidneys. Perfusate fluid, which contains a complex mixture of components, offers potential insight into the organ's viability and function. This study explored immune cell release, particularly tissue-resident lymphocytes (TRLs), during donor kidney machine perfusion and its correlation with injury markers.METHODS: Perfusate samples from hypothermic machine perfusion (HMP; n = 26) and normothermic machine perfusion (NMP; n = 16) of human donor kidneys were analyzed for TRLs using flow cytometry. Residency was defined by expressions of CD69, CD103, and CD49as. TRL release was quantified exclusively in NMP. Additionally, levels of cell-free DNA, neutrophil gelatinase-associated lipocalin, and soluble E-cadherin (sE-cadherin) were measured in NMP supernatants with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.RESULTS: Both HMP and NMP samples contained a heterogeneous population of TRLs, including CD4+ tissue-resident memory T cells, CD8+ tissue-resident memory T cells, tissue-resident natural killer cells, tissue-resident natural killer T cells, and helper-like innate lymphoid cells. Median TRL proportions among total CD45+ lymphocytes were 0.89% (NMP) and 0.84% (HMP). TRL quantities in NMP did not correlate with donor characteristics, perfusion parameters, posttransplant outcomes, or cell-free DNA and neutrophil gelatinase-associated lipocalin concentrations. However, CD103+ TRL release positively correlated with the release of sE-cadherin, the ligand for the CD103 integrin.CONCLUSIONS: Human donor kidneys release TRLs during both HMP and NMP. The release of CD103+ TRLs was associated with the loss of their ligand sE-cadherin but not with general transplant injury biomarkers.</p

Membrane particles from mesenchymal stromal cells reduce the expression of fibrotic markers on pulmonary cells

Background: Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with limited treatment options in which the telomere shortening is a strong predictive factor of poor prognosis. Mesenchymal stromal cells (MSC) administration is probed in several experimental induced lung pathologies; however, MSC might stimulate fibrotic processes. A therapy that avoids MSC side effects of transformation would be an alternative to the use of living cells. Membranes particles (MP) are nanovesicles artificially generated from the membranes of MSC containing active enzymes involved in ECM regeneration. We aimed to investigate the anti-fibrotic role of MP derived from MSC in an in vitro model of pulmonary fibrosis. Methods: Epithelial cells (A549) and lung fibroblasts, from IPF patients with different telomere length, were co-cultured with MP and TGF-β for 48h and gene expression of major pro-fibrotic markers were analyzed. Results: About 90% of both types of cells effectively took up MP without cytotoxic effects. MP decreased the expression of profibrotic proteins such as Col1A1, Fibronectin and PAI-1, in A549 cells. In fibroblasts culture, there was a different response in the inhibitory effect of MP on some pro-fibrotic markers when comparing fibroblast from normal telomere length patients (FN) versus short telomere length (FS), but both types showed an inhibition of Col1A1, Tenascin-c, PAI-1 and MMP-1 gene expression after MP treatment

Tissue-resident memory T cells in human kidney transplants have alloreactive potential

The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. This study investigates the alloreactive potential of TRM cells in kidney explants from 4 patients who experienced severe acute rejection leading to graft loss. Alloreactive T cell receptor (TCR) clones were identified in pretransplant blood samples through mixed lymphocyte reactions, followed by single-cell RNA and TCR sequencing of the proliferated recipient T cells. Subsequently, these TCR clones were traced in the TRM cells of kidney explants, which were also subjected to single-cell RNA and TCR sequencing. The proportion of recipient-derived TRM cells expressing an alloreactive TCR in the 4 kidney explants varied from 0% to 9%. Notably, these alloreactive TCRs were predominantly found among CD4+ and CD8+ TRM cells with an effector phenotype. Intriguingly, these clones were present not only in recipient-derived TRM cells but also in donor-derived TRM cells, constituting up to 4% of the donor population, suggesting the presence of self-reactive TRM cells. Overall, our study demonstrates that T cells with alloreactive potential present in the peripheral blood prior to transplantation can infiltrate the kidney transplant and adopt a TRM phenotype.</p

Reparative effect of mesenchymal stromal cells on endothelial cells after hypoxic and inflammatory injury

Background: The renal endothelium is a prime target for ischemia-reperfusion injury (IRI) during donation and transplantation procedures. Mesenchymal stromal cells (MSC) have been shown to ameliorate kidney function after IRI. However, whether this involves repair of the endothelium is not clear. Therefore, our objective is to study potential regenerative effects of MSC on injured endothelial cells and to identify the molecular mechanisms involved. Methods: Human umbilical vein endothelial cells (HUVEC) were submitted to hypoxia and reoxygenation and TNF-α treatment. To determine whether physical interaction or soluble factors released by MSC were responsible for the potential regenerative effects of MSC on endothelial cells, dose-response experiments were performed in co-culture and transwell conditions and with secretome-deficient MSC. Results: MSC showed increased migration and adhesion to injured HUVEC, mediated by CD29 and CD44 on the MSC membrane. MSC decreased membrane injury marker expression, oxidative stress levels, and monolayer permeability of injured HUVEC, which was observed only when allowing both physical and paracrine interaction between MSC and HUVEC. Furthermore, viable MSC in direct contact with injured HUVEC improved wound healing capacity by 45% and completely restored their angiogenic capacity. In addition, MSC exhibited an increased ability to migrate through an injured HUVEC monolayer compared to non-injured HUVEC in vitro. Conclusions: These results show that MSC have regenerative effects on injured HUVEC via a mechanism which requires both physical and paracrine interaction. The identification of specific effector molecules involved in MSC-HUVEC interaction will allow targeted modification of MSC to apply and enhance the therapeutic effects of MSC in IRI. [Figure not available: see fulltext.

Tissue-resident memory T cells in human kidney transplants have alloreactive potential

The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. This study investigates the alloreactive potential of TRM cells in kidney explants from 4 patients who experienced severe acute rejection leading to graft loss. Alloreactive T cell receptor (TCR) clones were identified in pretransplant blood samples through mixed lymphocyte reactions, followed by single-cell RNA and TCR sequencing of the proliferated recipient T cells. Subsequently, these TCR clones were traced in the TRM cells of kidney explants, which were also subjected to single-cell RNA and TCR sequencing. The proportion of recipient-derived TRM cells expressing an alloreactive TCR in the 4 kidney explants varied from 0% to 9%. Notably, these alloreactive TCRs were predominantly found among CD4+ and CD8+ TRM cells with an effector phenotype. Intriguingly, these clones were present not only in recipient-derived TRM cells but also in donor-derived TRM cells, constituting up to 4% of the donor population, suggesting the presence of self-reactive TRM cells. Overall, our study demonstrates that T cells with alloreactive potential present in the peripheral blood prior to transplantation can infiltrate the kidney transplant and adopt a TRM phenotype.</p

Pretransplant identification of acute rejection risk following kidney transplantation

Lack of an accepted definition for 'high immunological risk' hampers individualization of immunosuppressive therapy after kidney transplantation. For recipient-related risk factors for acute rejection, the most compelling evidence points to younger age and African American ethnicity. Recipient gender, body mass, previous transplantation, and concomitant infection or disease do not appear to be influential. Deceased donation now has only a minor effect on rejection risk, but older donor age remains a significant predictor. Conventional immunological markers (human leukocyte antigen [HLA] mismatching, pretransplant anti-HLA alloantibodies, and panel reactive antibodies) are being reassessed in light of growing understanding about the role of donor-specific antibodies (DSA). At the time of transplant, delayed graft function is one of the most clear-cut risk factors for acute rejection. Extended cold ischemia time (≥24 h) may also play a contributory role. While it is not yet possible to establish conclusively the relative contribution of different risk factors for acute rejection after kidney transplantation, the available data point to variables that should be taken into account at the time of transplant. Together, these offer a realistic basis for planning an appropriate immunosuppression regimen in individual patients