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Abstract 1979 Control 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 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.

Allogeneic stem cell transplantation following reduced-intensity conditioning is being evaluated in patients with advanced B-cell chronic lymphocytic leukemia (B-CLL). The curative potential of this procedure is mediated by donor-derived alloreactive T cells, resulting in a graft-versus-leukemia effect. However, B-CLL may escape T-cell-mediated immune reactivity since these cells lack expression of costimulatory molecules. We examined the most optimal method to transform B-CLL cells into efficient antigen-presenting cells (APC) using activating cytokines, by triggering toll-like receptors (TLRs) using microbial pathogens and by CD40 stimulation with CD40L-transfected fibroblasts. CD40 activation in the presence of IL-4 induced strongest upregulation of costimulatory and adhesion molecules on B-CLL cells and induced the production of high amounts of IL-12 by the leukemic cells. In contrast to primary B-CLL cells as stimulator cells, these malignant APCs were capable of inducing the generation of B-CLL-reactive CD8(+) CTL lines and clones from HLA class I-matched donors. These CTL lines and clones recognized and killed primary B-CLL as well as patient-derived lymphoblasts, but not donor cells. These results show the feasibility of ex vivo generation of B-CLL-reactive CD8(+) CTLs. This opens new perspectives for adoptive immunotherapy, following allogeneic stem cell transplantation in patients with advanced B-CLL.

Treatment of patients with relapsed or persistent acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL) after allo-SCT with donor lymphocyte infusions (DLI) is frequently unsuccessful, probably due to the limited immunogenicity of these malignant B cells, and the lack of specificity of the T cells. In this study, we generated a broadly applicable stimulation and isolation procedure for the induction of leukemia-reactive T cells under good manufacturing practice (GMP) conditions. First, B-cell leukemias were modified into professional antigen presenting cells (APC). We previously demonstrated that CD40 crosslinking was required for the production of appropriate malignant B cell-APCs. CD40L expressing mouse fibroblasts were potent activators of CD40, but not GMP approved. CD40L trimers and CD40 antibodies crosslinked to plates or beads only minimally triggered CD40 on malignant B cells. As previously demonstrated, activated T cells briefly express CD40L. We first determined the dynamics and kinetics of CD40L expression on peripheral blood (PB) donor T cells after stimulation with CD3/28 Dynal T cell expansion-beads added in a ratio of 1 bead/10 PBMC. The kinetics were highly variable, with the optimal surface expression of CD40L on a median of 20% of the T cells (range 10–35%, n=6) being detectable between day 1 and 5. We next investigated the capacity of these activated T cells to stimulate malignant B cells in PB from patients with ALL or CLL containing 60–95% leukemic cells. We developed a two-step strategy using special CD3/28 Dynal isolation beads applicable not only for T cell activation, but also for T cell isolation. Using these beads, the T cells were depleted from the PB of the patient, directly irradiated and added back to the B cell cultures. After 3–7 days all B cells displayed a good APC phenotype with expression of CD80, CD86, and CD83 in the patient samples containing >5% T cells. If insufficient T cell numbers were present, donor T cells could be used as source of CD40L. Leukemia-reactive T cells could be reproducibly generated after two stimulations of fully HLA matched donor T cells with the leukemic APC under mild stimulatory conditions, followed by isolation of the responding T cells based on their production of interferon gamma (IFNg). Next, we investigated whether the leukemia-reactive T cells were the result of the successful induction of a primary immune response or that recognition of mimicry epitopes by previously activated T cells was underlying the activity. We separated unmodified donor T cells into CD45 RO+ memory cells, CD45RA+/CD27+ naive cells, and CD45RA+/CD27− effector cells by MACS CD45RO depletion, followed by FACS sorting on CD27, and stimulated these fractions with malignant APCs. Whereas high frequencies of IFNg producing T cells (5–15%) with cytotoxic activity against the primary leukemia (20–50% lysis) were induced from the naive T cell population, no leukemia-reactive T cells could be isolated from the memory or effector T cell populations (n=3). In conclusion, T cells stimulated with CD3/28 Dynal beads have a transient expression of CD40L and can be used as an alternative source of CD40L to generate good APC of malignant B cells under GMP conditions. These malignant B cell APCs were capable of inducing efficient priming of primary anti-leukemic immune responses by activating naive donor T cells.

Allogeneic SCT is being explored as treatment modality for patients with advanced MCL. Complete sustained remissions have been observed after allogeneic SCT illustrating susceptibility of MCL cells to graft-versus-lymphoma (GVL) effect.To potentiate this GVL effect and to reduce graft-versus-host disease (GVHD) reactivity, adoptive transfer of in vitro-selected cytotoxic T cells (CTLs) with specificity for MCL or for hematopoiesis-restricted minor histocompatibility antigens could be an attractive approach. The lack of expression of costimulatory molecules on MCL cells hampers the generation of MCL-reactive T cell-responses. To transform MCL cells into efficient antigen-presenting cells (APCs) we tested the B-lineage specific activating cytokines (IL-4), the unique MCL proliferating cytokine (IL-10) and the ligand of toll like receptor 9, CpG.Furthermore, CD40 triggering using irradiated CD40-L transfected murine fibroblasts (tCD40L) in combination with the cytokines and CpG was examined. The expression of the costimulatory and adhesion molecules CD80, CD86, CD83, CD54 and CD58 of MCL cells of 7 patients, all carrying the t(11;14) translocation, was analyzed by flowcytometry. No upregulation of any of these molecules was observed using the cytokines or CpG. Ligation of CD40 on MCL cells caused a significant upregulation of CD54,CD58, CD80 and CD86 (p