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The Na+/H+ exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pHi). It is a ubiquitous, amiloride-sensitive, growth factor–activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pHi than normal hematopoietic tissue. We then show that a direct correlation exists between pHi and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na+/H+ exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pHiand induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pHi decreases, apoptosis—measured by annexin-V and TUNEL methodologies—rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents.

Summary:Refractory acute lymphoblastic leukemia (ALL) is often incurable, and relapse rates following allogeneic bone marrow transplantation (BMT) remain high. We have reported that patients who develop increased numbers of ␥␦ + T cells soon after BMT are significantly less likely to relapse. We now show in seven donor/recipient pairs that donor-derived V␦1 + CD4 − CD8 − ␥␦ + T cells are activated and proliferate in response to recipient primary ALL blasts. In addition, these cells have been shown to bind and lyse the recipient ALL blasts. Separately, ␥␦ + T cells proliferate poorly or not at all in mixed lymphocyte culture against HLA-mismatched unrelated stimulator cells. These observations suggest that allogeneic ␥␦ + T cells could be an effective immunotherapeutic strategy against refractory disease without the risk of graft-versus-host disease. Bone Marrow Transplantation (2001) 27, 601-606.

Cytomegalovirus (CMV) reactivation in immunocompromised recipients of allogeneic stem cell transplantation is a cause of morbidity and mortality from viral pneumonitis. Antiviral drugs given to reactivating patients have reduced the mortality from CMV but have toxic side effects and do not always prevent late CMV disease. Cellular immunotherapy to prevent CMV disease is less toxic and could provide prolonged protection. However, a practical approach to generating sufficient quantities of CMV-specific cytotoxic T cells (CTLs) is required. This study describes a system for generating sufficient CMV-specific CTLs for adoptive immuno-

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Use of a partially mismatched related donor (PMRD) is an option for patients who require allogeneic transplantation but do not have a matched sibling or unrelated donor. Epstein-Barr virus (EBV)-induced lymphoma is a major cause of mortality after PMRD transplantation. In this study, we present a clinical grade culture system for donor-derived EBV-specific cytotoxic T cells (CTLs) that do not recognize haplo-identical recipient cells. The EBV-specific CTLs were tested for cytolytic specificity and other functional properties, including ability to transgress into tissues, propensity for apoptosis, degree of clonality, stability of dominant T-cell clones, and Tc and Th phenotypes. The EBV-specific CTLs were routinely expanded to greater than 80 x 10(6) over a period of 5 weeks, which is sufficient for clinical application. A CD8+ phenotype predominated, and the CTLs were highly specific for donor lymphoblastoid cell lines (LCLs) without killing of recipient targets or K562. Vbeta spectratyping showed an oligoclonal population that was stable on prolonged culture. The EBV-specific CTLs were activated (D-related human leukocyte antigen [HLA-DR+], L-selectin+/-) and of memory phenotype (CD45RO+). Expression of the integrin VLA-4 suggested that these CTLs could adhere to endothelium and migrate into tissues. The Bcl-2 message was upregulated, which may protect the CTLs from the apoptosis. The first demonstration of overexpression of bcl-2 in human memory CTLs. In addition, we show that lymphoblastoid cell lines used to generate CTLs are readily genetically modified with recombinant lentivirus, indicating that genetically engineered antigen presentation is feasible.