Effect of 5-[(125)I]iodo-2'-deoxyuridine uptake on the proliferation and pluripotency of human embryonic stem cells.

PURPOSE: Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because, in principle, they can differentiate into any cell type found in the human body. In addition, studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state, as well as the consequences of IR exposure on the development of organisms. However, the effect of IR, in particular radionuclide uptake, on the pluripotency, proliferation and survival of hESC has not been extensively studied. METHODS: In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU), a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC. RESULTS: We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However, treatment with 0.1 µCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 µCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls. CONCLUSIONS: Our results provide important initial insights into the sensitivity of hESC to IR, and especially that produced by the decay of an internalized radionuclide.

Lymphodepletion is permissive to the development of spontaneous T-cell responses to the self-antigen PR1 early after allogeneic stem cell transplantation and in patients with acute myeloid leukemia undergoing WT1 peptide vaccination following chemotherapy.

PR1, an HLA-A*0201 epitope shared by proteinase-3 (PR3) and elastase (ELA2) proteins, is expressed in normal neutrophils and overexpressed in myeloid leukemias. PR1-specific T cells have been linked to graft-versus-leukemia (GVL) effect. We hypothesized that lymphopenia induced by chemo-radiotherapy can enhance weak autoimmune responses to self-antigens such as PR1. We measured PR1-specific responses in 27 patients 30-120 days following allogeneic stem cell transplant (SCT) and correlated these with ELA2 and PR3 expression and minimal residual disease (MRD). Post-SCT 10/13 CML, 6/9 ALL, and 4/5 solid tumor patients had PR1 responses correlating with PR3 and ELA2 expression. At day 180 post-SCT, 8/8 CML patients with PR1 responses were BCR-ABL-negative compared with 2/5 BCR-ABL-positive patients (P = 0.025). In contrast, PR1 responses were detected in 2/4 MRD-negative compared with 4/5 MRD-positive ALL patients (P = 0.76). To assess whether the lymphopenic milieu also exaggerates weak T-cell responses in the autologous setting, we measured spontaneous induction of PR1 responses in 3 AML patients vaccinated with WT1-126 peptide following lymphodepletion. In addition to WT1-specific T cells, we detected PR1-specific T cells in 2 patients during hematopoietic recovery. Our findings suggest that lymphopenia induced by chemo-radiotherapy enhances weak autoimmune responses to self-antigens, which may result in GVL if the leukemia expresses the relevant self-antigen.

Repeated PR1 and WT1 peptide vaccination in Montanide-adjuvant fails to induce sustained high-avidity, epitope-specific CD8+ T cells in myeloid malignancies.

BACKGROUND: We previously showed that vaccination with one dose of PR1 and WT1 peptides induces transient anti-leukemia immunity. We hypothesized that maintenance of a sustained anti-leukemia response may require frequent boost injections. DESIGN AND METHODS: Eight patients with myeloid malignancies were enrolled in this phase II study, and 6 completed 6 injections of PR1 and WT1 peptides in Montanide-adjuvant with GM-CSF, every two weeks. RESULTS: Both high- and low-avidity PR1 or WT1-specific CD8(+) T cells were detected in all evaluable patients after the first vaccine dose. Repeated vaccination led to selective deletion of high avidity PR1- and WT1-specific CD8(+) T cells and was not associated with significant reduction in WT1-expression. Additional boosting failed to increase vaccine-induced CD8(+) T-cell frequencies further and in all patients the response was lost before the 6(th) dose. PR1- or WT1-specific CD8(+) T cells were not detected in bone marrow samples, excluding their preferential localization to this site. Following a booster injection three months after the 6(th) vaccine dose, no high-avidity PR1 or WT1-specific CD8(+) T cells could be detected, whereas low-avidity T cells were readily expanded. CONCLUSIONS: These data support the immunogenicity of PR1 and WT1 peptide vaccines. However, repeated delivery of peptides with Montanide-adjuvant and GM-CSF leads to rapid loss of high-avidity peptide-specific CD8(+) T cells. These results may offer an explanation for the lack of correlation between immune and clinical responses observed in a number of clinical trials of peptide vaccination. New approaches are needed to induce long-term high-avidity memory responses against leukemia antigens.

Donor KIR Genes 2DL5A, 2DS1 and 3DS1 are associated with a reduced rate of leukemia relapse after HLA-identical sibling stem cell transplantation for acute myeloid leukemia but not other hematologic malignancies.

Stem cell transplantation (SCT) from a healthy donor can be curative for patients with hematologic malignancies resistant to other treatments. Elimination of malignant cells through a graft-versus-leukemia (GVL) effect involves donor T and natural killer (NK) cells, but their relative contribution to this process is poorly defined. NK cell alloreactivity and GVL effects are controlled by the nature of the interaction of NK activation receptors and killer-immunoglobulin-like-receptors (KIR) with major histocompatibility locus class I antigens on the target cell. We performed KIR-genotyping of HLA-identical sibling donors in 246 T cell-depleted SCTs to identify genetic factors affecting transplant outcome (treatment-related mortality [TRM], leukemic relapse, and survival). Univariate and multivariate analysis of transplant-related risk factors and KIR genotyping was performed to identify independent variables predictive of outcome for different forms of leukemia. Further to confirming known predictive factors for TRM and survival (CD34 cell dose, patient age, disease stage), statistical analysis revealed that 3 donor B haplotype KIR genes, 2DL5A, 2DS1, and 3DS1, were associated with significantly less relapse in patients with acute myelogenous leukemia (AML) (13% versus 57%) but not in patients with other myelogenous or lymphoid malignancies. AML patients receiving SCT from donors with these KIR genes relapsed 4 times less frequently than patients transplanted from donors with other KIR genotypes. These findings suggest specific, genetically determined, interactions between NK cells and AML cells that facilitate the GVL effect, and have implications for donor selection for AML patients.

Ex vivo characterization of polyclonal memory CD8+ T-cell responses to PRAME-specific peptides in patients with acute lymphoblastic leukemia and acute and chronic myeloid leukemia.

Preferentially expressed antigen of melanoma (PRAME) is aberrantly expressed in hematologic malignancies and may be a useful target for immunotherapy in leukemia. To determine whether PRAME is naturally immunogenic, we studied CD8(+) T-cell responses to 4 HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and healthy donors. CD8(+) T cells recognizing PRAME peptides could be detected ex vivo in 4 of 10 ALL, 6 of 10 AML, 3 of 10 CML patients, and 3 of 10 donors by HLA-A2 tetramer analysis and flow cytometry for intracellular interferon-gamma. The frequency of PRAME-specific CD8(+) T cells was greater in patients with AML, CML, and ALL than healthy controls. All peptides were immunogenic in patients, while responses were only detected to PRA300 in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to greater than or equal to 2 PRAME epitopes compared with low PRAME expression levels (4/7 vs 0/23, P = .001), suggesting a PRAME-driven T-cell response. PRAME-specific T cells were readily expanded in short-term cultures in donors and patients. These results provide evidence for spontaneous T cell reactivity against multiple epitopes of PRAME in ALL, AML, and CML. The potential for developing PRAME as a target for immunotherapy in leukemia deserves further exploration.

Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted by in vitro expanded natural killer cells, which are functionally enhanced by bortezomib.

Primitive quiescent CD34(+) chronic myeloid leukemia (CML) cells are more biologically resistant to tyrosine kinase inhibitors than their cycling counterparts; however, graft-versus-leukemia (GVL) effects after allogeneic stem cell transplantation (SCT) probably eliminate even these quiescent cells in long-term surviving CML transplant recipients. We studied the progeny of CD34(+) cells from CML patients before SCT, which were cultured 4 days in serum-free media with hematopoietic growth factors. BCR-ABL expression was similar in both cycling and quiescent noncycling CD34(+) populations. Quiescent CD34(+) cells from CML patients were less susceptible than their cycling CD34(+) and CD34(-) counterparts to lysis by natural killer (NK) cells from their HLA-identical sibling donors. Compared with cycling populations, quiescent CD34(+) CML cells had higher surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. Bortezomib up-regulated TRAIL receptor expression on quiescent CD34(+) CML cells, and further enhanced their susceptibility to cytotoxicity by in vitro expanded donor NK cells. These results suggest that donor-derived NK cell-mediated GVL effects may be improved by sensitizing residual quiescent CML cells to NK-cell cytotoxicity after SCT. Such treatment, as an adjunct to donor lymphocyte infusions and pharmacologic therapy, may reduce the risk of relapse in CML patients who require treatment by SCT.

High PR3 or ELA2 expression by CD34+ cells in advanced-phase chronic myeloid leukemia is associated with improved outcome following allogeneic stem cell transplantation and may improve PR1 peptide-driven graft-versus-leukemia effects.

The primary granule proteins elastase (ELA2) and proteinase 3 (PR3) both contain the nonapeptide PR1, which can induce cytotoxic T lymphocyte (CTL) responses against chronic myeloid leukemia (CML) cells. To investigate whether eradication of CML after allogeneic stem cell transplantation (SCT) was influenced by PR3 and ELA2 gene expression or PR1-specific CTL responses, we studied cells from 87 CML patients and 27 HLA-A*0201(+) donors collected prior to T-cell-depleted HLA-identical sibling SCT. For patients in advanced phase (AdP), a higher expression of both PR3 and ELA2 in CD34(+) progenitors before SCT was associated with a lower incidence of relapse-related death, improved leukemia-free survival (LFS), and overall survival (OS); in chronic phase patients, no differences were observed. PR1-CTL responses were detected in 7 of 27 HLA-identical sibling donors, and associated with improved LFS and OS after SCT on follow-up. PR1-CTL responses detected in 7 of 28 CML patients before transplantation were not predictive of outcome and correlated inversely with PR3 and ELA2 expression. These findings suggest that assessment of PR3 and ELA2 expression in leukemic progenitors is useful for predicting posttransplantation outcome in AdP patients undergoing SCT. The presence of a donor immune response against PR1 may be advantageous and could be exploited therapeutically.

T-cell large granular lymphocyte leukemia is characterized by massive TCRBV-restricted clonal CD8 expansion and a generalized overexpression of the effector cell marker CD57.

INTRODUCTION: Large granular lymphocyte leukemia (LGL) is a clonal lymphoproliferative disease of CD8+ T cells expressing the CD57 activation marker. It is, however, unknown whether the CD57+ population represents the LGL clone or not. We previously demonstrated that the clone can be found in both CD8+CD57+ and CD8+CD57- cells, indicating that the LGL clone also resides in the CD57- fraction. MATERIALS AND METHODS: Here, we quantified the extent of the clonal CD8 expansion in LGL using T-cell receptor Vbeta (TCRBV)-specific monoclonal antibodies, and determined whether the CD4 population also contained skews. Furthermore, dominant TCRBV populations were assessed for clonal status using T-cell receptor-gamma (TCRG) PCR on genomic DNA. RESULTS: We show that the dominant TCRBV in LGL contains CD57+ and CD57- cells. Molecular analysis of CD8+CD57+ and CD8+CD57- subfractions of the dominant TCRBV by TCRG PCR demonstrates that indeed both fractions are clonal, and that the clone is absent from the dominant TCRBV-negative population. Furthermore, we show that CD57 overexpression is not restricted to the LGL clone, but a general phenomenon in CD8 cells of LGL patients. CONCLUSION: We therefore conclude that the primary characteristic of LGL is a clonal expansion of CD8 cells, with a concomitant upregulation of CD57 on this clone and uninvolved cells.

Molecular and flow cytometric characterization of the CD4 and CD8 T-cell repertoire in patients with myelodysplastic syndrome.

We studied 18 patients with myelodysplastic syndrome (MDS), measuring clonality and T-cell receptor Vbeta (TCRBV) expression of CD4 and CD8 T cells by polymerase chain reaction and by flow cytometric analysis of TCRBV families. The CD4 and CD8 T-cell repertoire in most MDS patients is characterized by an abnormal TCRBV-restricted expansion of T cells in CD4 and CD8 cells, and increased expression of the CD8 effector marker CD57 of multiple TCRBV in CD8 cells. Clonality analysis of CD4 and CD8 cells showed that seven of 10 patients analysed had a major clone in the CD8 cells but not in CD4 cells. Furthermore, in one patient we found that both the CD57- and CD57+ fraction contained the clone (which was absent from the TCRBV-negative fraction). These data suggest that, in MDS, multiple T-cell expansions can be found in both helper and cytotoxic T cells, and that, in the CD8 cells, T cells functionally differentiate in vivo from memory to effector T cells. Together, these data support the hypothesis of the involvement of T cells in the pathogenesis of MDS.