Clonotype analysis is essential for complete characterization of antigen-specific T cells. Moreover, knowledge on clonal identity allows tracking of antigen-specific T cells in whole blood and tissue infiltrates and can provide information on antigenic specificity. Here, we developed a next generation sequencing (NGS)-based platform for the highly quantitative clonotype characterization of T cells and determined requirements for the unbiased characterization of the input material (DNA, RNA, ex vivo derived or cell culture expanded T cells). Thereafter we performed T cell receptor (TCR) repertoire analysis of various specimens in clinical settings including cytomegalovirus (CMV), polyomavirus BK (BKV) reactivation and acute cellular allograft rejection. Our results revealed dynamic nature of virusspecific T cell clonotypes; CMV reactivation was linked to appearance of new highly abundant antigenspecific clonalities. Moreover, analysis of clonotype overlap between BKV-, alloantigen-specific T cell-, kidney allograft-and urine-derived lymphocytes provided hints for the differential diagnosis of allograft dysfunction and enabled appropriate therapy adjustment. We believe that the established approach will provide insights into the regulation of virus-specific/ anti-tumor immunity and has high diagnostic potential in the clinical routine.

Sustained BK viruria is a reliable marker allowing an early identification of patients at high risk of BKVAN development and therefore assure precocious therapeutic interventions.

Rabbit antithymocyte globulin-GenzymeTM is used to prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Common disadvantages of treatment are infectious complications. The effects of rabbit antithymocyte globulin-Genzyme TM on thymic function have not been well-studied. Multicolor flow cytometry was used to analyze the kinetics of conventional and regulatory T cells in adult patients treated (n=12) or not treated (n=8) with rabbit antithymocyte globulin-Genzyme TM during the first 6 months after allogeneic hematopoietic stem cell transplantation. Patients treated with rabbit antithymocyte globulin-Genzyme TM had almost undetectable levels of recent thymic emigrants (CD45RA + CD31 + ) of both conventional and regulatory CD4 T cells throughout the 6 months after allogeneic hematopoietic stem cell transplantation whereas CD4 + CD45RA -memory T cells were less affected, but their levels were also significantly lower than in patients not treated with rabbit antithymocyte globulin-Genzyme TM . In vitro, rabbit antithymocyte globulin-Genzyme TM induced apoptosis and cytolysis of human thymocytes, and its cytotoxic effects were greater than those of rabbit antithymocyte globulin-Fresenius TM . Rabbit antithymocyte globulin-Genzyme TM in combination with a conditioning regimen strongly impairs thymic recovery of both conventional and regulatory CD4 + T cells. The sustained depletion of conventional and regulatory CD4 + T cells carries a high risk of both infections and graft-versus-host disease. Our data indicate that patients treated with rabbit antithymocyte globulin-Genzyme TM could benefit from thymus-protective therapies and that trials comparing this product with other rabbit antithymocyte globulin preparations or lymphocyte-depleting compounds would be informative.

No abstract

Rabbit antithymocyte globulin (Thymoglobulin®) is commonly used as graft-versus-host disease (GvHD) prophylaxis. Since we found similar total CD8 T cell numbers in patients with and without Thymoglobulin® therapy within the first six months after allogeneic hematopoietic stem cell transplantation, we have analyzed the reconstitution of the CD8 T cell compartment in detail. After T cell-depletion, higher and more sustained proliferative capacity of memory CD8 T cells resulted in their rapid expansion, whereas the fraction of naive CD8 T cells decreased. Importantly, this shift towards effector memory CD8 T cells did not accelerate the incidence of GvHD.

No abstract