In Vivo T Cell Depletion Using Rabbit Derived ATG Leads to An Increased EBV-PTLD Risk Due to An Induced Imbalance Between B and T Cell Recovery Which Is Not Seen After Horse Derived ATG or Alemtuzumab

Abstract

Abstract 1979Control of replication of endogenous viruses like CMV and EBV is fully dependent on CMV or EBV specific T cells after allogeneic stem cell transplantation (alloSCT). In the absence of specific CD8 T cell control, proliferation of EBV infected B cells can lead to post transplantation lymphoproliferative disease (PTLD). In an initial cohort of patients treated with horse derived anti thymocyte globulin (h-ATG), no early PTLD was observed. However, due to unavailability in Europe, h-ATG had to be replaced by rabbit derived ATG (r-ATG), leading to an unacceptable high incidence of EBV-PTLD (26% during first 3 months after alloSCT). Replacement of r-ATG by alemtuzumab (ALT) significantly reduced the incidence of EBV-PTLD (3 months incidence of EBV-PTLD 2%). To determine the immunological basis of these findings we performed a detailed analysis of immune reconstitution in these three cohorts of transplanted patients. The first cohort (41 patients) received h-ATG (Lymphoglobulin) 10 mg/kg/day for 4 days. The second cohort (19 patients) received r-ATG (Thymoglobulin) 2.0 or 3.5 mg/kg/day for 4 days and the third cohort (60 patients) received ALT, 15 mg/day for 2 days. All grafts consisted of PBSC to which 20 mg of ALT was added for in vitro T cell depletion. All patients received a fludarabin and busulphan based conditioning regimen. No standard post transplantation immunosuppressive treatment was given.In the r-ATG cohort, early EBV-PTLD occurred after a median of 7 weeks (range 4–12 weeks) post alloSCT. Three r-ATG treated patients died while high levels of circulating EBV-DNA were present (> log 4.0 copies/mL). Incidence of CMV disease was not significantly different in the three cohorts (5%, 6% and 0%, respectively). In contrast to the other 2 cohorts, immune reconstitution in the r-ATG cohort was characterized by an imbalance between recovery of B cells and CD8 T cells. Already 3 weeks after alloSCT, the majority (67%) of r-ATG patients showed a more rapid reconstitution of B cells than CD8 T cells, leading to B cells outnumbering CD8 T cells. This was seen in only a small minority of patients after h-ATG and ALT (17% and 6%, respectively, p<0.01 versus r-ATG).Because rapid recovery of T cells in the alemtuzumab patients was frequently found in the presence of circulating ALT (mean concentration 0.43 μg/mL and 0.12 μg/mL after 3 and 6 weeks, respectively), the phenotype of circulating CD4 and CD8 T cells at 6 weeks after ALT was analyzed. The majority of circulating CD8 and CD4 T cells lacked CD52 expression (56% (range 0–99%) and 81% (range 0–93%), respectively). Using tetramer staining, cytotoxicity assays and analysis of cytokine production, we demonstrated the presence of functional CD52 negative as well as CD52 positive CMV and EBV specific CD8 T cells. Based on FLAER negativity, it was demonstrated that the CD52 negative T cells are GPI anchor deficient, representing a PNH-like clone escaping ALT induced cell lysis. Because almost half of the circulating CD8 T cells were CD52 positive, we examined expression of CD52 and the in-vitro sensitivity to ALT-mediated complement-dependent cell lysis (CDC) of B cells, CD4 and CD8 T cells of healthy donors. The highest CD52 expression was observed on B cells (mean fluorescence intensity (MFI) 120), resulting in 95% lysis after incubation with ALT and complement. Differential expression of CD52 was observed on CD4 and CD8 T cells, MFI 120 and 101 respectively, resulting in relative protection of CD52 positive CD8 compared to CD4 T cells against ALT-mediated CDC (52% and 90% lysis).We conclude that the high incidence of EBV-PTLD after in-vivo T cell depletion with r-ATG is caused by an induced imbalance between B and T cell recovery, which is not seen after h-ATG or ALT. In-vivo T cell depletion with ALT is associated with a relatively low risk of EBV disease because of efficient B cell depletion and persistent EBV immunity due to the relative insusceptibility for ALT of CD8 T cells and the development of functional CD52 negative escape variants of CD4 and CD8 T cells. Disclosures:Off Label Use: Alemtuzumab and Anti Thymocyte Globulin used for in vivo T cell depletion prior to allogeneic stem cell transplantation.