Mutations in JAK/STAT and NOTCH1 Genes Are Enriched in Post-Transplant Lymphoproliferative Disorders

Post-transplant lymphoproliferative disorders (PTLD) are diseases occurring in immunocompromised patients after hematopoietic stem cell transplantation (HCT) or solid organ transplantation (SOT). Although PTLD occurs rarely, it may be associated with poor outcomes. In most cases, PTLD is driven by Epstein-Barr virus (EBV) infection. Few studies have investigated the mutational landscape and gene expression profile of PTLD. In our study, we performed targeted deep sequencing and RNA-sequencing (RNA-Seq) on 16 cases of florid follicular hyperplasia (FFH) type PTLD and 15 cases of other PTLD types that include: ten monomorphic (M-PTLD), three polymorphic (P-PTLD), and two classic Hodgkin lymphoma type PTLDs (CHL-PTLD). Our study identified recurrent mutations in JAK3 in five of 15 PTLD cases and one of 16 FFH-PTLD cases, as well as 16 other genes that were mutated in M-PTLD, P-PTLD, CHL-PTLD and FFH-PTLD. Digital image analysis demonstrated significant differences in single cell area, major axis, and diameter when comparing cases of M-PTLD and P-PTLD to FFH-PTLD. No morphometric relationship was identified with regards to a specific genetic mutation. Our findings suggest that immune regulatory pathways play an essential role in PTLD, with the JAK/STAT pathway affected in many PTLDs.

Induction of Donor-Specific Immune Hypo-Responsiveness Across Major Histocompatibility Complex Barriers in Murine Models of Solid Organ Transplant: Repurposing Extracorporeal Photochemotherapy

Recipients of solid organ transplantation (SOT) rely on life-long immunosuppression (IS), which is associated with significant side effects. Extracorporeal photochemotherapy (ECP) is a safe, existing cellular therapy used to treat transplant rejection by modulating the recipient’s own blood cells. We sought to induce donor-specific hypo-responsiveness of SOT recipients by infusing ECP-treated donor leukocytes prior to transplant. To this end, we utilized major histocompatibility complex (MHC) mismatched rodent models of allogeneic cardiac, liver, and kidney transplantation to test this novel strategy. Leukocytes isolated from donor-matched spleens for ECP treatment (ECP-DL) were infused into transplant recipients seven days prior to SOT. Pre-transplant infusion of ECP-DL without additional IS was associated with prolonged graft survival in all models. This innovative approach promoted the production of tolerogenic dendritic cells and regulatory T-cells with subsequent inhibition of T-cell priming and differentiation, along with a significant reduction of donor-specific T-cells in the spleen and grafts of treated animals. This new application of donor-type ECP-treated leukocytes provides insight into the mechanisms behind ECP-induced immunoregulation and holds significant promise in the prevention of graft rejection and reduction in need of global immune suppressive therapy in patients following SOT.

Antiviral T-Cell Frequencies in a Healthy Population: Reference Values for Evaluating Antiviral Immune Cell Profiles in Immunocompromised Patients

Viral infections and reactivations are major causes of morbidity and mortality after hematopoietic stem cell (HSCT) and solid organ transplantation (SOT) as well as in patients with immunodeficiencies. Latent herpesviruses (e.g., cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6), lytic viruses (e.g., adenovirus), and polyomaviruses (e.g., BK virus, JC virus) can cause severe complications. Antiviral drugs form the mainstay of treatment for viral infections and reactivations after transplantation, but they have side effects and cannot achieve complete viral clearance without prior reconstitution of functional antiviral T-cell immunity. The aim of this study was to establish normal ranges for virus-specific T-cell (VST) frequencies in healthy donors. Such data are needed for better interpretation of VST frequencies observed in immunocompromised patients. Therefore, we measured the frequencies of VSTs against 23 viral protein-derived peptide pools from 11 clinically relevant human viruses in blood from healthy donors (n = 151). Specifically, we determined the VST frequencies by interferon-gamma enzyme-linked immunospot assay and classified their distribution according to age and gender to allow for a more specific evaluation and prediction of antiviral immune responses. The reference values established here provide an invaluable tool for immune response evaluation, intensity of therapeutic drugs and treatment decision-making in immunosuppressed patients. This data should make an important contribution to improving the assessment of immune responses in immunocompromised patients.

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product

Background A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). Results The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. Conclusions These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

A Mortality Case Caused by Thrombotic Microangiopathy after Successful Bloodless Living Donor Liver Transplantation

Thrombotic microangiopathy (TMA) after solid organ transplantation is infrequent and its etiology remains still unclear. Nevertheless, if early diagnosis and early therapies are not performed, it can lead to severe life-threatening complications and even death. A 54-year-old female (a Jehovah’s Witness) was diagnosed with drug-induced toxic hepatitis and was decided to undergo bloodless living donor liver transplantation (LDLT). After the successful LDLT, the patient’s condition deteriorated, and she was diagnosed with TMA during further evaluation. We tried to proceed with plasma exchange-based treatment, but she and her family declined according to their religious beliefs. The patient expired 4 days after the diagnosis. Physicians should maintain a high level of suspicion for TMA after liver transplantation when clinical manifestations are observed.