The impact of stem cell therapy in hematology and oncology

The transplantation of hematopoietic stem cells (HSCT) is an established part of the therapy of hematologic neoplasia and certain solid tumors. In the allogeneic approach hematopoietic stem cells are harvested from healthy donors, while in the autologous setting preparations originating from the patient himself are being used. Both therapies use high dose cytotoxic medication for the induction of higher remission rates in malignant diseases. While autologous HSCT rescues hematopoiesis after high dose chemotherapy, in allogeneic HSCT donor immune cells exert an additional allo-reactivity towards recipient tissue and residual malignant cells. Autologous HSCT is mainly used in relapsed malignant high-grade lymphoma. Allogeneic HSCT results in cure from acute leukemia with unfavorable prognosis in a high percentage of patients. Recent developments target the expansion of the donor pool for allogeneic stem cells and want to reduce chemotherapeutic toxicity of allogeneic transplantation with sustained anti-leukemia efficacy.     

Die Bedeutung der Stammzelltherapie in der Hämatologie und Onkologie

The transplantation of hematopoietic stem cells (HSCT) is an established part of the therapy of hematologic neoplasia and certain solid tumors. In the allogeneic approach hematopoietic stem cells are harvested from healthy donors, while in the autologous setting preparations originating from the patient himself are being used. Both therapies use high dose cytotoxic medication for the induction of higher remission rates in malignant diseases. While autologous HSCT rescues hematopoiesis after high dose chemotherapy, in allogeneic HSCT donor immune cells exert an additional allo-reactivity towards recipient tissue and residual malignant cells. Autologous HSCT is mainly used in relapsed malignant high-grade lymphoma. Allogeneic HSCT results in cure from acute leukemia with unfavorable prognosis in a high percentage of patients. Recent developments target the expansion of the donor pool for allogeneic stem cells and want to reduce chemotherapeutic toxicity of allogeneic transplantation with sustained anti-leukemia efficacy.     

Rapid salvage treatment with virus-specific T cells for therapy-resistant disease

Background.?Viral infections are major complications after allogeneic hematopoietic stem cell transplantation (HSCT). During posttransplant immunosuppression the regular T-cell control is compromised. Even if treatment strategies against infections caused by herpes viruses such as cytomegalovirus, Epstein-Barr virus, and adenovirus have improved, the mortality rate is still considerable. If primary antiviral therapy fails or cannot be tolerated, adoptive therapy with virus-specific cytotoxic T cells (CTL) can be utilized. Methods.?In this study, we used virus-specific CTLs to treat 8 patients suffering from severe viral infections after allogeneic HSCT. Using positive selection with HLA multimers and magnetic beads, we isolated CTLs from both frozen donor material as well as third-party donors within hours. Results.?At 90 days after CTL infusions 7 out of 8 patients were still living. CTLs infused from third-party donors were detected in 5 of 6 patients up to 76 days after infusion. No graft-versus-host disease associated with CTL infusions was observed. Conclusions.?Our separation approach offers a rapid alternative for adoptive CTL therapy if primary antiviral treatment strategies fail. Because no prolonged expansion steps are needed, this method may be used for early treatment of patients suffering from life-threatening infectious complications.     

Generation of autologous cytotoxic and helper T-cell responses against the B-cell leukemia-associated antigen HB-1: relevance for precursor B-ALL-specific immunotherapy

Tumor relapses in patients with precursor B-cell acute lymphoblastic leukemia (BALL) occur frequently after primary treatment. Therefore, development of additional treatment modalities to eliminate residual tumor cells is needed. Active immunotherapy using dendritic cells (DCs) loaded with tumor-associated antigens is a promising approach to induce specific T-cell immunity in patients with cancer. In previous studies, we described HB-1 as a B-cell lineage-specific antigen that is recognized by donor-derived cytotoxic T lymphocytes (CTLs) on allogeneic B-ALL tumor cells. Here, we investigated the potential use of the HB-1 antigen as an autologous T-cell vaccine target. To determine whether HB-1-specific CTL precursors are present within the T-cell repertoire, we induced expansion of CD8+ T cells using mature monocyte-derived DCs pulsed with the previously identified HB-1.B44 antigenic peptide. In 6 of 8 donors, CD8+ CTL lines have been generated that exert cytotoxicity against target cells exogenously pulsed with peptide or endogenously expressing the HB-1 antigen. From one of these HB-1-specific T-cell lines, we isolated a CD8+ CTL that produces interferon-gamma on stimulation with B-ALL tumor cells. Interestingly, the HB-1 antigen also induced CD4+ T-helper responses on activation with protein-loaded mature monocyte-derived DCs. We identified 2 novel epitopes recognized in the context of HLA-DR4 and HLA-DR11 with the use of HB-1-specific CD4+ T-cell clones generated from different donors. These present data, that HB-1 induces both helper and cytotoxic T-cell responses, indicate that the HB-1 antigen is a candidate target to induce T-cell-mediated antitumor immunity in patients.     

Ex vivo priming for long-term maintenance of antileukemia human cytotoxic T cells suggests a general procedure for adoptive immunotherapy.

Adoptive cellular immunotherapy has proven to be a successful approach in preventing and curing cytomegalovirus infection and Epstein-Barr virus-associated lymphomas after bone marrow transplantation. Translation of this approach for preventing leukemia relapse after bone marrow transplantation might require ex vivo priming and long-term maintenance of leukemia blast-specific T cells. To accomplish this goal, procedures were optimized for the in vitro priming of naive CD8 using dendritic cells activated by CD40 ligation, interleukin-12 (IL-12), and IL-7. Using T lymphocytes and dendritic cells obtained from HLA-matched allogeneic bone marrow transplantation donors and leukemia blasts as a source of tumor antigens, anti-acute myeloid leukemia cytotoxic T lymphocytes (CTLs) were induced. In these experiments, it was found that though it is possible to induce CTLs using immature dendritic cells, IL-12, and IL-7, obtaining long-term CTLs requires the presence of CD4 T cells in the priming phase. Using this approach, long-term antileukemia CTL lines could be generated from 4 of 4 bone marrow donors. Because this procedure does not require definition of the target antigen and because it selects responding cells from a virgin T-cell repertoire, its general application is suggested in adoptive immunotherapy and in the definition of tumor rejection antigens.     

Targeted immunotherapy for acute myeloid leukemia

Allogeneic hematopoietic stem cell transplantation (HSCT) demonstrated convincingly the potential of allogeneic T cells in causing sustained remissions in high-risk hematologic malignancies. However toxicity of allogeneic HSCT limits its application to a broader group of patients. An improved understanding of NK biology along with mechanisms of natural killer (NK) cell and T-cell-mediated alloreactivity against leukemia has led to several clinical immunotherapeutic strategies that preserve graft versus leukemia (GVL) while minimizing the toxicity of HSCT. Here we review strategies being explored both in HSCT and non-HSCT settings that include an emphasis on two key aspects: (a) Maximizing cytotoxicity of alloreactive cells, ie, NK cells and T cells, and (b) Targeted manipulation of critical pathways in T and NK cells contributing to sustained anti-leukemia effects.     

Clinical grade expansion of CD45RA, CD45RO, and CD62L-positive T-cell lines from HLA-compatible donors: high cytotoxic potential against AML and ALL cells

OBJECTIVE: Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. MATERIALS AND METHODS: Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span, granulocyte macrophage colony-stimulating factor, interleukin-4, and calcium ionophore. All B-precursor ALL (N22) and AML (N13), but not T-cell ALL (N3), differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). RESULTS: Upon in vitro culture, donor-derived CTL acquired a memory T phenotype, showing concomitant high CD45RA, CD45RO, CD62L expression. CD8(+) cells, but not CD4(+) cells, were granzyme, perforine, and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%, 30:1 E:T ratio), but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%, 1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. CONCLUSIONS: Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation.     

Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia

Donor lymphocyte infusion (DLI) into patients with a relapse of their leukemia or multiple myeloma after allogeneic stem cell transplantation (alloSCT) has been shown to be a successful treatment approach. The hematopoiesis-restricted minor histocompatibility antigens (mHAgs) HA-1 or HA-2 expressed on malignant cells of the recipient may serve as target antigens for alloreactive donor T cells. Recently we treated three mHAg HA-1- and/or HA-2-positive patients with a relapse of their disease after alloSCT with DLI from their mHAg HA-1- and/or HA-2-negative donors. Using HLA-A2HA-1 and HA-2 peptide tetrameric complexes we showed the emergence of HA-1- and HA-2-specific CD8(+) T cells in the blood of the recipients 5-7 weeks after DLI. The appearance of these tetramer-positive cells was followed immediately by a complete remission of the disease and restoration of 100% donor chimerism in each of the patients. Furthermore, cloned tetramer-positive T cells isolated during the clinical response specifically recognized HA-1 and HA-2 expressing malignant progenitor cells of the recipient and inhibited the growth of leukemic precursor cells in vitro. Thus, HA-1- and HA-2-specific cytotoxic T lymphocytes emerging in the blood of patients after DLI demonstrate graft-versus-leukemia or myeloma reactivity resulting in a durable remission. This finding implies that in vitro generated HA-1- and HA-2-specific cytotoxic T lymphocytes could be used as adoptive immunotherapy to treat hematological malignancies relapsing after alloSCT.     

Generating CTLs against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies

The Epstein-Barr virus (EBV)-encoded LMP1 protein is expressed in EBV-positive Hodgkin disease and is a potential target for cytotoxic T-lymphocyte (CTL) therapy. However, the LMP1-specific CTL frequency is low, and so far the generation of LMP1-specific CTLs has required T-cell cloning. The toxicity of LMP1 has prevented the use of dendritic cells (DCs) for CTL stimulation, and we reasoned that an inactive, nontoxic LMP1 mutant (DeltaLMP1) could be expressed in DCs and would enable the activation and expansion of polyclonal LMP1-specific CTLs. Recombinant adenoviral vectors expressing LMP1 or DeltaLMP1 were tested for their ability to transduce DCs. LMP1 expression was toxic within 48 hours whereas high levels of DeltaLMP1 expression were achieved with minimal toxicity. DeltaLMP1-expressing DCs were able to reactivate and expand LMP1-specific CTLs from 3 healthy EBV-seropositive donors. LMP1-specific T cells were detected by interferon-gamma (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) assays using the HLA-A2-restricted LMP1 peptide, YLQQNWWTL (YLQ). YLQ-specific T cells were undetectable (less than 0.001%) in donor peripheral blood mononuclear cells (PBMCs); however, after stimulation the frequency increased to 0.5% to 3.8%. Lysis of autologous target cells by CTLs was dependent on the level of LMP1 expression. In contrast, the frequency of YLQ-specific CTLs in EBV-specific CTLs reactivated and expanded using lymphoblastoid cell lines was low and no LMP1-specific cytotoxic activity was observed. Thus, DeltaLMP1 expression in DCs is nontoxic and enables the generation of LMP1-specific CTLs for future adoptive immunotherapy protocols for patients with LMP1-positive malignancies such as EBV-positive Hodgkin disease. Targeting LMP1 in these malignancies may improve the efficacy of current adoptive immunotherapy approaches.     

High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic and leukemic-precursor cells

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies.     

Direct visualization of cytomegalovirus-specific T-cell reconstitution after allogeneic stem cell transplantation

Cytomegalovirus (CMV) remains an important cause of morbidity and mortality after allogeneic stem cell transplantation (SCT), but cytotoxic T lymphocytes (CTL) may play a critical role in controlling CMV reactivation. Fluorescent HLA-peptide tetramers containing immunodominant peptides from CMV were used to prospectively monitor the recovery of CMV CTL in recipients of allogeneic transplants from siblings (n = 13) or unrelated donors (n = 11). In patients given allografts from a sibling when both the patient and donor were seropositive for CMV before SCT, recovery of CMV-specific CTL was rapid and reached up to 21% of all CD8(+) T cells. Early reconstitution of CMV-specific immunity was not observed if either the donor or recipient was seronegative for CMV. In recipients of transplants from volunteer unrelated donors, recovery of CMV-specific CTL was delayed in comparison to that in recipients of transplants from siblings and no CTL were observed within the first 100 days after SCT. CTL numbers were increased after episodes of CMV reactivation but were suppressed by prednisolone therapy. Recovery of CMV-specific CTL to levels greater than 10 x 10(6)/L was associated with protection from CMV disease. It was concluded that use of HLA-peptide tetramers to quantify CMV CTL is valuable for studying T-cell responses after allogeneic SCT. It should allow prediction of CMV reactivation in individual patients and assist in the development of adoptive T-cell immunotherapy.     

Induction of allogeneic tumour- and lymphokine-activated lymphocytes against hepatocellular carcinoma

For clinical application of adoptive immunotherapy against hepatocellular carcinoma (HCC), it is not easy to prepare tumour specific effector cells such as cytotoxic T lymphocytes (CTL). To induce potent and broad-spectrum effectors, allogeneic cultured hepatoma cell lines (JHH-4 and HuH-6) were used as stimulators of peripheral blood lymphocytes (PBL) instead of autologous HCC cells. Allogeneic tumour- and lymphokine-activated killer cells (ATLAK) were generated by a mixed culture of lymphocytes and allogeneic cultured tumour cells with recombinant interleukin-2 (rIL-2). The tumour-killing activity of ATLAK induced by HuH-6 was confirmed against HuH-6 and other different HCC cell lines (JHH-2, HuH-7 and PLC). These activated lymphocytes were significantly more potent than lymphokine-activated killer cells (LAK) in [51Cr]-releasing assay. The JHH-4 stimulated ATLAK was reactive not only with JHH-4 but also with JHH-2. The lysis of allogeneic targets could be partially inhibited by anti-CD8 and anti-CD3 but not by anti-CD4. Anti-tumour cytotoxicity in these cultures might be mediated by CD3+CD56- and CD3+CD56+ effectors. These results imply that adoptive immunotherapy for HCC with ATLAK may be more feasible than that with LAK.     

Allorestricted cytotoxic T cells specific for human CD45 show potent antileukemic activity

Recent advances have made haploidentical transplantation for leukemia feasible, but the rigorous T-cell depletion used contributes to the high relapse rates observed. We have attempted to improve the graft-versus-leukemia (GVL) effect by generating allorestricted cytotoxic T lymphocytes (CTLs) directed against human CD45. Such CTLs should recognize patient hematopoietic cells including leukemia, enhancing donor cell engraftment and improving the GVL effect, but they should not recognize host nonhematopoietic tissues or donor cells from the graft. Using the T2 binding assay, 4 CD45-derived peptides were found to bind HLA-A2 molecules. These peptides were used to generate cytotoxic T-cell lines from HLA-A2(-) donors by sequential stimulation with peptide-pulsed HLA-A2(+) stimulators, and the lines obtained were screened for peptide-specific cytotoxicity. Using one of these peptides (P1218), it was possible to generate peptide-specific, allorestricted CTLs in 3 of 7 responders. P1218-specific CTL lines show potent cytotoxicity against hematopoietic cell lines coexpressing HLA-A2 and CD45 but not CD45 loss variants. Studies with stable transfectants of 293 cells demonstrated recognition by P1218-specific CTLs of endogenously expressed CD45. Likewise P1218-specific CTLs recognized peripheral blood mononuclear cells (PBMCs) from HLA-A2(+) patients with chronic myeloid leukemia (CML) and leukemic blasts in HLA-A2(+) patients with acute myeloid leukemia (AML), but they were unable to lyse HLA-A2(+) fibroblasts or HLA-A2(-) normal PBMCs. Coculture of CD34(+) PBMCs and bone marrow mononuclear cells (BMMCs) with P1218-specific CTL significantly inhibited colony-forming unit-granulocyte macrophage (CFU-GM) formation in HLA-A2(+) healthy controls and CML patients but resulted in no significant inhibition in HLA-A2(-) healthy controls. These studies demonstrate that P1218-specific cytotoxic T lymphocytes (CTLs) have potent activity against leukemic progenitors and suggest that adoptive immunotherapy with allorestricted CTLs directed against CD45 epitopes may be useful in restoring the GVL effect after HLA-A2-mismatched haploidentical transplantation. Further, because P1218-specific CTLs also recognize healthy HLA-A2(+) progenitors, such CTLs could also contribute to host myeloablation and enhance donor cell engraftment.     

Successful cell‐mediated cytokine‐activated immunotherapy for relapsed acute myeloid leukemia after hematopoietic stem cell transplantation

Acute myeloid leukemia (AML) is an extremely aggressive disease with a high relapse rate even after allogeneic hematopoietic stem cell transplantation (HSCT). We report the successful outcome of cell‐mediated cytokine‐activated immunotherapy in a high‐risk pediatric AML patient who relapsed shortly after allogeneic HSCT. Donor lymphocyte infusion along with interferon induced a graft‐versus‐leukemia effect, presenting as a reversible episode of graft‐versus‐host disease, which led to stable complete donor chimerism and total eradication of AML for over 24 months, at the time of this report. The curative potential of immunotherapy in hematological malignancies is discussed. Am. J. Hematol., 2009. © 2008 Wiley‐Liss, Inc.     

Allo-major histocompatibility complex-restricted cytotoxic T lymphocytes engraft in bone marrow transplant recipients without causing graft-versus-host disease.

Previous experiments in humans and mice have shown that allogeneic donors can serve as a source of cytotoxic T lymphocytes (CTL) specific for proteins, such as cyclin-D1 and mdm-2, expressed at elevated levels in tumor cells. In vitro, allo-major histocompatibility complex (MHC)-restricted CTL against these proteins selectively killed allogeneic tumor cells, including lymphoma, but not normal control cells. This suggested that these CTL may be useful for adoptive tumor immunotherapy, provided that they (1) survive in MHC-disparate hosts, (2) maintain their killing specificity, and (3) do not attack normal host tissues. Here, we used cloned allo-restricted CTL isolated from BALB/c mice (H-2(d)) that killed H-2(b)-derived tumor cells expressing elevated levels of the mdm-2 target protein. When these CTL were injected into bone marrow transplanted (BMT) C57BL/6 (H-2(b)) recipients, they consistently engrafted and were detectable in lymphoid tissues and in the bone marrow (BM). Long-term survival was most efficient in spleen and lymph nodes, where CTL were found up to 14 weeks after injection. The administration of CTL did not cause graft-versus-host disease (GVHD) normally associated with injection of allogeneic T cells. These data show that allo-restricted CTL clones are promising reagents for antigen-specific immunotherapy in BMT hosts, because they engraft and retain their specific killing activity without causing GVHD.     

Biologic activity of irradiated,autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic stem cell transplantation

Through an immune-mediated graft-versus-leukemia effect, allogeneic hematopoietic stem cell transplantation (HSCT) affords durable clinical benefits for many patients with hematologic malignancies. Nonetheless, subjects with high-risk acute myeloid leukemia or advanced myelodysplasia often relapse, underscoring the need to intensify tumor immunity within this cohort. In preclinical models, allogeneic HSCT followed by vaccination with irradiated tumor cells engineered to secrete GM-CSF generates a potent antitumor effect without exacerbating the toxicities of graft-versus-host disease (GVHD). To test whether this strategy might be similarly active in humans, we conducted a Phase I clinical trial in which high-risk acute myeloid leukemia or myelodysplasia patients were immunized with irradiated, autologous, GM-CSF-secreting tumor cells early after allogeneic, nonmyeloablative HSCT. Despite the administration of a calcineurin inhibitor as prophylaxis against GVHD, vaccination elicited local and systemic reactions that were qualitatively similar to those previously observed in nontransplanted, immunized solid-tumor patients. While the frequencies of acute and chronic GVHD were not increased, 9 of 10 subjects who completed vaccination achieved durable complete remissions, with a median follow-up of 26 months (range 12–43 months). Six long-term responders showed marked decreases in the levels of soluble NKG2D ligands, and 3 demonstrated normalization of cytotoxic lymphocyte NKG2D expression as a function of treatment. Together, these results establish the safety and immunogenicity of irradiated, autologous, GM-CSF-secreting leukemia cell vaccines early after allogeneic HSCT, and raise the possibility that this combinatorial immunotherapy might potentiate graft-versus-leukemia in patients.     

The immune system as a foundation for immunologic therapy and hematologic malignancies: a historical perspective

In this review we aim to provide a historical overview of the immunotherapeutic approaches which have been developed for the treatment of hematological malignancies. After briefly summarizing the development of the theory of cancer immune surveillance, we describe how initial studies discovering the efficacy of the immune-mediated graft-versus-tumor effects after allogeneic hematopoietic cell transplantation led to new transplantation approaches (termed non-myeloablative transplantation) relying almost exclusively on graft-versus-tumor effects for tumor eradication. We then summarize important steps in the development of tumor vaccines and autologous adoptive immunotherapy in patients with hematological malignancies. Finally, we describe historical discoveries leading to the recent success with monoclonal antibodies as treatment for lymphomas, chronic lymphocytic leukemia, and acute myeloid leukemia.     

Isolation and expansion of human cytomegalovirus- specific cytotoxic T lymphocytes using interferon-gamma secretion assay

OBJECTIVE: The aim of this study was to isolate and expand donor-derived human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs) for adoptive transfer of 10(7) cells per m(2) of body surface area to restore protective immunity after stem cell transplantation. MATERIALS AND METHODS: A new strategy to generate HCMV-specific CTLs using the interferon-gamma (IFN-gamma) secretion assay, followed by expansion to numbers sufficient for clinical application with interleukin-2 and feeder cell stimulation, is described. RESULTS: From 1 to 5 x 10(4) HCMV peptide-specific T lymphocytes (greater than 90% CD3(+)CD8(+)) were isolated from 1 to 2 x 10(8) peripheral blood mononuclear cells comparable to 50 to 100 mL of blood from HLA-A*0201 HCMV seropositive blood donors (n = 14) and expanded ex vivo after a median of 16 days (range 8-28 days; n = 13) to greater than 10(7)/m(2) HCMV peptide-specific CTLs using autologous (n = 2) or allogeneic (n = 11) feeder cell stimulation. In three experiments, expansion to 6 weeks was performed, achieving a median of 1.6 x 10(9) cells (range 6.1 x 10(8)-3.3 x 10(9)). Characterization of these HCMV-specific CTL lines revealed an average purity of 89.2% (range 66.2-99.3%) using HCMV pp65 peptide HLA-A*0201 tetramer staining (n = 14) and 89.4% (range 64.4-99.5%) by peptide-specific IFN-gamma secretion (n = 7). A median of 82.6% (range 76.0-88.0%) showed perforin secretion (n = 3) and 57.5% (range 22.2-80.7%) specific lysis of peptide-pulsed T2 cells (n = 5). A median of 52.2% (range 35.2-7.3%) revealed specific killing of HCMV-infected autologous, but not allogeneic, fibroblasts (n = 6). CONCLUSIONS: IFN-gamma secretion assay allows development of a simple and rapid protocol with short expansion times for generation of greater than 10(7)/m(2) HCMV-specific CTLs for adoptive immunotherapy.     

Adoptive T-cell therapy for EBV-associated post-transplant lymphoproliferative disease

Increased understanding of the mechanisms by which T lymphocytes recognize virus and tumor-specific antigens has fueled the use of adoptive immunotherapy for viral and malignant diseases. An ideal candidate for such treatment is Epstein-Barr virus (EBV). EBV-associated post-transplant lymphoproliferative disorder (PTLD) is a serious complication post-solid organ transplant (SOT) or hematopoietic stem cell transplant (HSCT). The disease is essentially the result of suppression of cytotoxic T-cell function and despite various treatment strategies the course may still be fulminant and lethal. Therefore, an adoptive immunotherapeutic approach using ex vivo derived EBV-specific CTL offers a promising solution not only for the treatment but also as prophylaxis for PTLD. The infusion of EBV-CTL has been demonstrated to be safe and effective in allogeneic HSCT recipients and their use post-SOT is being evaluated.