CD14highCD16+ monocytes are the main producers of Interleukin-10 following clinical heart transplantation.

Introduction: Following heart transplantation, a cascade of immunological responses is initiated influencing the clinical outcome and long-term survival of the transplanted patients. The anti-inflammatory cytokine interleukin-10 (IL-10) was shown to be elevated in the blood of heart transplant recipients directly after transplantation but the releasing cell populations and the composition of lymphocyte subsets following transplantation have not been thoroughly studied. Methods: We identified immune cells by immunophenotyping and analyzed intracellular IL-10 production in peripheral blood mononuclear cells (PBMC) of heart transplanted patients (n= 17) before, directly after and 24h post heart transplantation. The cells were stimulated with lipopolysaccharide or PMA/Ionomycin to enhance cytokine production within leukocytes in vitro. Results and discussion: We demonstrate that intermediate monocytes (CD14highCD16+), but not CD8+ T cells, CD4+ T cells, CD56+ NK cells or CD20+ B cells appeared to be the major IL-10 producers within patients PBMC following heart transplantation. Consequently, the absolute monocyte count and the ratio of intermediate monocytes to classical monocytes (CD14+CD16-) were specifically increased in comparison to pre transplant levels. Hence, this population of monocytes, which has not been in the focus of heart transplantation so far, may be an important modulator of clinical outcome and long-term survival of heart transplant recipients. Alteration of blood-circulating monocytes towards a CD14highCD16+ phenotype could therefore shift the pro-inflammatory immune response towards induction of graft tolerance, and may pave the way for the optimization of immunosuppression.

Increased frequency of CD4+ CD25high CD127low T cells early after lung transplant is associated with improved graft survival - a retrospective study.

In this retrospective study, we analyzed the presence of any association of three CD4+ CD25high regulatory T-cell subpopulations at 3 weeks after lung transplantation with the later incidence of chronic lung allograft dysfunction and graft survival. Among lung-transplanted patients between January 2009 and April 2018, only patients with sufficient T-cell measurements at 3 weeks after transplantation were included into the study. Putative regulatory T cells were defined as CD4+ CD25high T cells, detected in peripheral blood and further analyzed for CD127low , FoxP3+ , and CD152+ using fluorescence-activated cell sorting (FACS) analysis. Associations of regulatory T cells with chronic lung allograft dysfunction (CLAD) and graft survival were evaluated using Cox analysis. During the study period, 724 (71%) patients were included into the study. Freedom from chronic lung allograft dysfunction (CLAD) and graft survival amounted to 66% and 68% at 5 years. At the multivariable analysis, increasing frequencies of CD127low were associated with better freedom from CLAD (hazard ratio for each 1% increase of %CD127low , HR = 0.989, 95% CI = 0.981-0.996, P = 0.003) and better graft survival (HR = 0.991, 95% CI = 0.984-0.999, P = 0.026). A higher frequency of CD127low regulatory T cells in peripheral blood early after lung transplantation estimated a protective effect against chronic lung allograft dysfunction, mortality, and re-transplantation.

Prediction of transplant outcome after 24-hour ex vivo lung perfusion using the Organ Care System in a porcine lung transplantation model.

Ex vivo lung perfusion (EVLP) has become routine practice in lung transplantation. Still, running periods exceeding 12 hours have not been undertaken clinically to date, and it remains unclear how the perfusion solution for extended running periods should be composed and which parameters may predict outcomes. Twenty-four porcine lungs underwent EVLP for 24 hours using the Organ Care System (OCS). Lungs were ventilated and perfused with STEEN's solution enriched with erythrocytes (n = 8), acellular STEEN's solution (n = 8), or low-potassium dextran (LPD) solution enriched with erythrocytes (n = 8). After 24 hours, the left lungs were transplanted into recipient pigs. After clamping of the contralateral lung, the recipients were observed for 6 hours. The most favorable outcome was observed in organs utilizing STEEN solution enriched with erythrocytes as perfusate, whereas the least favorable outcome was seen with LPD solution enriched with erythrocytes for perfusion. Animals surviving the observation period showed lower peak airway pressure (PAWP) and pulmonary vascular resistance (PVR) during OCS preservation. The results suggest that transplantation of lungs following 24 hours of EVLP is feasible but dependent on the composition of the perfusate. PAWP and PVR during EVLP are early and late predictors of transplant outcome, respectively.

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse.

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

Preemptive treatment of early donor-specific antibodies with IgA- and IgM-enriched intravenous human immunoglobulins in lung transplantation.

This retrospective study presents our 4-year experience of preemptive treatment of early anti-HLA donor specific antibodies with IgA- and IgM-enriched immunoglobulins. We compared outcomes between patients with antibodies and treatment (case patients) and patients without antibodies (control patients). Records of patients transplanted at our institution between March 2013 and November 2017 were reviewed. The treatment protocol included one single 2 g/kg immunoglobulin infusion followed by successive 0.5 g/kg infusions for a maximum of 6 months, usually combined with a single dose of anti-CD20 antibody and, in case of clinical rejection or positive crossmatch, with plasmapheresis or immunoabsorption. Among the 598 transplanted patients, 128 (21%) patients formed the case group and 452 (76%) the control group. In 116 (91%) patients who completed treatment, 106 (91%) showed no antibodies at treatment end. Fourteen (13%) patients showed antibody recurrence thereafter. In case versus control patients and at 4-year follow-up, respectively, graft survival (%) was 79 versus 81 (P = .59), freedom (%) from biopsy-confirmed rejection 57 versus 53 (P = .34), and from chronic lung allograft dysfunction 82 versus 78 (P = .83). After lung transplantation, patients with early donor-specific antibodies and treated with IgA- and IgM-enriched immunoglobulins had 4-year graft survival similar to patients without antibodies and showed high antibody clearance.

Isolation of human antigen-specific regulatory T cells with high suppressive function.

Adoptive transfer of regulatory T (Treg) cells could be an alternative to chronic immunosuppression for prevention of allogeneic graft rejection. While polyspecific Treg cells can prevent immune responses under lymphopenic conditions, Ag-specific Treg cells are needed to treat autoimmunity and graft rejection. Yet, reliable markers for Ag-specific Treg cells are missing. We report that latency-associated peptide (LAP) and glycoprotein A repetitions predominant (GARP) can identify human Ag-specific Treg cells. In addition, we show that the depletion of CD154(+) cells from LAP(+) or GARP(+) Treg cells increases the Treg-cell purity to over 90%, as assessed by epigenetic analysis. These Ag-specific Treg cells can be isolated magnetically and might contribute to the development of GMP-based protocols. In addition, Ag-specific Treg cells are functionally far superior to CD4(+) CD25(high) or CD4(+) CD25(high) CD127(low) Treg cells in vitro and in preventing strong alloreactions in humanized mice. They could, therefore, have a high therapeutic potential for the control of alloimmune, autoimmune, and allergic immune responses in patients.