High Mutation Frequency of the PIGA Gene in T Cells Results in Reconstitution of GPI Anchor-/CD52- T Cells That Can Give Early Immune Protection after Alemtuzumab-Based T Cell-Depleted Allogeneic Stem Cell Transplantation.

Alemtuzumab (ALM) is used for T cell depletion in the context of allogeneic hematopoietic stem cell transplantation (alloSCT) to prevent acute graft-versus-host disease and graft rejection. Following ALM-based T cell-depleted alloSCT, relatively rapid recovery of circulating T cells has been described, including T cells that lack membrane expression of the GPI-anchored ALM target Ag CD52. We show, in a cohort of 89 human recipients of an ALM-based T cell-depleted alloSCT graft, that early lymphocyte reconstitution always coincided with the presence of large populations of T cells lacking CD52 membrane expression. In contrast, loss of CD52 expression was not overt within B cells or NK cells. We show that loss of CD52 expression from the T cell membrane resulted from loss of GPI anchor expression caused by a highly polyclonal mutational landscape in the PIGA gene. This polyclonal mutational landscape in the PIGA gene was also found in CD52- T cells present at a low frequency in peripheral blood of healthy donors. Finally, we demonstrate that the GPI-/CD52- T cell populations that arise after ALM-based T cell-depleted alloSCT contain functional T cells directed against multiple viral targets that can play an important role in immune protection early after ALM-based T cell-depleted transplantation.

Complement-dependent cytotoxicity induced by therapeutic antibodies in B-cell acute lymphoblastic leukemia is dictated by target antigen expression levels and augmented by loss of membrane-bound complement inhibitors.

To optimally utilize therapeutic monoclonal antibodies in the treatment of B-cell acute lymphoblastic leukemia (B-ALL) understanding their mechanisms of action and the factors influencing these mechanisms is required. We show strong correlations between target antigen expression levels and sensitivity to complement-dependent cytotoxicity (CDC) induced by rituximab, ofatumumab, or alemtuzumab in a panel of cell lines derived from primary B-ALL cells and in primary B-ALL samples. Simultaneous loss of expression of membrane-bound complement regulatory proteins (mCRP) CD55 and CD59 due to glycophosphatidylinositol-anchor deficiency, significantly increased sensitivity to CDC. Accordingly, induced increase in CD55 or CD59 expression protected cells against CDC. The extent of protection co-depended on antigen expression and antibody concentration. In contrast, natural variation in mCRP expression could not be used as a single factor to predict sensitivity to CDC. In conclusion, sensitivity of B-ALL cells to CDC was predominantly determined by antibody concentration and target antigen expression.

Effective treatment of refractory CMV reactivation after allogeneic stem cell transplantation with in vitro-generated CMV pp65-specific CD8+ T-cell lines.

To treat patients with refractory cytomegalovirus (CMV) reactivation after allogeneic stem cell transplantation, a phase I/II clinical study on adoptive transfer of in vitro-generated donor-derived or patient-derived CMV pp65-specific CD8* T-cell lines was performed. Peripheral blood mononuclear cells from CMV seropositive donors or patients were stimulated with HLA-A*0201-restricted and/or HLA-B*0702-restricted CMV pp65 peptides (NLV/TPR) and 1 day after stimulation interferon-γ)-producing cells were enriched using the CliniMACS Cytokine Capture System (interferon-γ), and cultured with autologous feeders and low-dose interluekin-2. After 7-14 days of culture, quality controls were performed and the CMV-specific T-cell lines were administered or cryopreserved. The T-cell lines generated contained 0.6-17 × 10(6) cells, comprising 54%-96% CMV pp65-specific CD8 T cells, and showed CMV-specific lysis of target cells. Fifteen CMV-specific T-cell lines were generated of which 8 were administered to patients with refractory CMV reactivation. After administration, no acute adverse events and no graft versus host disease were observed and CMV load disappeared. In several patients, a direct relation between administration of the T-cell line and the in vivo appearance of CMV pp65-specific T cells could be documented. In conclusion, administration of CMV pp65-specific CD8* T-cell lines was found to be feasible and safe, and enduring efficacy of administered CMV pp65-specific CD8* T-cell lines could be demonstrated.

Functional analysis of killer Ig-like receptor-expressing cytomegalovirus-specific CD8+ T cells.

Killer Ig-like receptors (KIR) are expressed by human NK cells and T cells. Although Ag-specific cytolytic activity and cytokine production of KIR(+) T cells can be inhibited by KIR ligation, the effect of KIR on proliferation is unclear. KIR(+) T cells have been reported to have a general proliferative defect. To investigate whether KIR(+) T cells represent end-stage dysfunctional T cells, we characterized KIR(+) CMV-specific T cells in allogeneic stem cell transplantation patients and healthy donors. In both patients and healthy donors, a significant percentage KIR(+) T cells was detected at various time points. All stem cell transplantation patients studied showed KIR expression on CMV-specific T cells, while not all donors had KIR-expressing CMV-specific T cells. From two of the patients and one donor KIR(+) CMV-specific T clones were isolated and analyzed functionally. T cells were detected that expressed KIR that could not encounter their corresponding KIR ligands in vivo, illustrating that KIR expression by these T cells was not based on functional selection but a random process. Our data demonstrate that KIR(+) T cells are fully functional T cells that are only restricted in effector functions and proliferation upon KIR ligation. The level of KIR-mediated inhibition of the effector functions and proliferation depended on the strength of TCR stimulation. We observed no diminished general proliferative capacity and therefore we conclude that these T cells do not represent end-stage dysfunctional T cells.

Genetic engineering of virus-specific T cells with T-cell receptors recognizing minor histocompatibility antigens for clinical application.

BACKGROUND: Donor lymphocyte infusion is an effective form of adoptive immunotherapy for hematologic malignancies after allogeneic stem cell transplantation. Graft-versus-host disease, however, often develops due to recognition of ubiquitously-expressed minor histocompatibility antigens. Transfer of T-cell receptors recognizing hematopoiesis-restricted minor histocompatibility antigens to virus-specific T cells may be a powerful anti-tumor therapy with a low risk of graft-versus-host disease. The purpose of this study was to develop an optimal T-cell receptors-encoding multi-cistronic retroviral vector and an efficient method for generating T-cell receptors-engineered virus-specific T cells. DESIGN AND METHODS: Retroviral vectors encoding the T-cell receptors for the hematopoiesis-restricted minor histocompatibility antigen HA-2 with and without selection markers were compared for T-cell receptors surface expression and HA-2-specific lysis. In addition, two different methods, i.e. peptide stimulation of CD8(+) cells and Pro5 MHC pentamer-based isolation of antigen-specific T cells, were investigated for their efficiency to generate T-cell receptors-transduced virus-specific T cells. RESULTS: Bi-cistronic vectors without selection markers most efficiently mediated T-cell receptors surface expression and HA-2-specific lysis. Furthermore, both methods were useful for generating gene-modified cells, but the purity of virus-specific T cells was higher after pentamer isolation. Finally, the capacity of gene-modified cells to express the transgenic T-cell receptors at the cell surface markedly differed between virus-specific T cells and was correlated with lysis of relevant target cells. CONCLUSIONS: Our data support T-cell receptors gene transfer to pentamer-isolated virus-specific T cells using bi-cistronic retroviral vectors and illustrate the relevance of selection of gene-modified T cells with appropriate transgenic T-cell receptors surface expression for clinical gene therapy.

Up-regulated expression in nonhematopoietic tissues of the BCL2A1-derived minor histocompatibility antigens in response to inflammatory cytokines: relevance for allogeneic immunotherapy of leukemia.

T cells directed against hematopoietic-restricted minor histocompatibility antigens (mHags) may mediate graft-versus-leukemia (GVL) reactivity without graft-versus-host disease (GVHD). Recently, the HLA-A24-restricted mHag ACC-1 and the HLA-B44-restricted mHag ACC-2 encoded by separate polymorphisms within the BCL2A1 gene were characterized. Hematopoietic-restricted expression was suggested for these mHags. We demonstrate BCL2-related protein A1 (BCL2A1) mRNA expression in mesenchymal stromal cells (MSCs) that was up-regulated by the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and/or interferon gamma (IFN-gamma). Analysis of cytotoxicity and IFN-gamma production illustrated that ACC-2-specific T cells did not recognize untreated MSCs or IFN-gamma-treated MSCs but showed specific recognition and killing of MSCs treated with TNF-alpha plus IFN-gamma. We hypothesize that under steady-state circumstances BCL2A1-specific T cells may exhibit relative specificity for hematopoietic tissue, but reactivity against nonhematopoietic cells may occur when inflammatory infiltrates are present. Thus, the role of BCL2A1-specific T cells in differential induction of GVL reactivity and GVHD may depend on the presence of inflammatory responses that may occur during GVHD.