Rhoda Eniafe

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.

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 6th 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 6th 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. (ClinicalTrials.gov Identifier: NCT00499772)

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.

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.

Hematopoietic stem cells and progenitors of chronic myeloid leukemia express leukemia-associated antigens : implications for the graft-versus-leukemia effect and peptide vaccine-based immunotherapy

The cure of chronic myeloid leukemia (CML) patients following allogeneic stem cell transplantation (SCT) is attributed to graft-versus-leukemia (GVL) effects targeting alloantigens and/or leukemia-associated antigens (LAA) on leukemia cells. To assess the potential of LAA-peptide vaccines in eliminating leukemia in CML patients, we measured WT1, PR3, ELA2 and PRAME expression in CD34+ progenitor subpopulations in CML patients and compared them with minor histocompatibility antigens (mHAgs) HA1 and SMCY. All CD34+ subpopulations expressed similar levels of mHAgs irrespective of disease phase, suggesting that in the SCT setting, mHAgs are the best target for GVL. Furthermore, WT1 was consistently overexpressed in advanced phase (AdP) CML in all CD34+ subpopulations, and mature progenitors of chronic phase (CP) CML compared to healthy individuals. PRAME overexpression was limited to more mature AdP-CML progenitors only. Conversely, only CP-CML progenitors had PR3 overexpression, suggesting that PR1-peptide vaccines are only appropriate in CP-CML. Surface expression of WT1 protein in the most primitive hematopoietic stem cells in AdP-CML suggest that they could be targets for WT1 peptide-based vaccines, which in combination with PRAME, could additionally improve targeting differentiated progeny, and benefit patients responding suboptimally to tyrosine kinase inhibitors, or enhance GVL effects in SCT patients.

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

Summary. 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.

Flow cytometric quantitation and characterization of the T-lymphocyte memory response to CMV in healthy donors.

BACKGROUND Levels of circulating CMV Ag-specific lymphocytes determine CMV reactivation risk in immunocompromised individuals. METHODS Frequencies of T cells producing cytokines after stimulation by CMV Ag were measured in hematopoietic stem-cell donors using flow cytometry. RESULTS In seropositive individuals (n = 75) the mean number of CD8(+) (CD8(bright), CD8(dim)) and CD4(+) cells producing IFN-gamma was respectively 3.1% (12.6/microL) and 0.38% (3.2/microL), over 10-fold higher than in seronegative subjects (n = 22). CMV stimulation induced tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in both CD4(+) and CD8(+) cells (usually together), with a shift from memory- to effector-cell phenotype, while only a small proportion of CD4(+) cells produced IL-4. Although the normal range was wide, neither age, sex nor HLA type affected the frequency. DISCUSSION These quantitative studies and the recognition of CD4(+) cells as potential effectors of CMV immunity are of relevance for immunotherapeutic approaches to prevent CMV disease after stem-cell transplantation.

Improved outcome following allogeneic stem cell transplantation in chronic myeloid leukemia is associated with higher expression of BMI-1 and immune responses to BMI-1 protein

BMI-1 and EZH2 are polycomb group (PcG) proteins that maintain self-renewal of stem cells, and are overexpressed in leukemia. To investigate the potential of PcG proteins as leukemia-associated antigens, and as targets for graft-versus-leukemia (GVL) effects, we studied cells obtained from 86 patients with chronic myeloid leukemia (CML) and 25 human leukocyte antigen (HLA)-A*0201+ sibling donors collected before allogeneic stem cell transplantation (SCT). Although BMI-1 overexpression in CD34+ cells of CML patients treated with pharmacotherapy is associated with poor prognosis, we found, conversely, that in CML patients treated with SCT, a higher expression of BMI-1, and correspondingly a lower expression of its target for repression, CDKN2A, is associated with improved leukemia-free survival. Cytotoxic T-lymphocyte (CTL) responses to the BMI-1 peptide were detected in 5 of 25 (20%) donors, and in 8 of 19 (42%) HLA-A*0201+ CML patients. BMI-1 generated more total and high-avidity immune responses, and was more immunogenic than EZH2. PcG-specific CTLs had a memory phenotype, were readily expanded in short-term cultures and were detected after SCT in recipients of PcG-specific CTL-positive donors. A higher BMI-1 expression in CML CD34+ progenitors was associated with native BMI-1 immune responses. These immune responses to PcG proteins may target leukemia stem cells and have relevance for disease control by GVL.

Tissue-restricted T cell alloresponses across HLA barriers: selection and identification of leukemia-restricted CTL in HLA-mismatched stimulator-responder pairs.

Summary:Exploiting the graft-versus-leukemia (GVL) effect in mismatched transplants requires its separation from graft-versus-host disease (GVHD). We generated leukemia-specific cytotoxic T lymphocytes (CTL) in three haplotype-mismatched, two class I-mismatched and two single HLA-A locus-matched stimulator–responder pairs. Six patients with chronic myelogenous leukemia and one patient with acute myeloid leukemia transformed from MDS were studied. CTL generated after 10 days stimulation with unselected leukemic peripheral blood mononuclear cells inhibited leukemic CFU-GM colony growth (>85% at 10:1 effector:target ratio) with no third-party colony inhibition. In five pairs, responders were cultured separately with leukemia cells, PHA-B or LCL from the stimulator. After 2–4 restimulations, the T cell repertoire was examined by flow analysis using Vβ-specific antibodies. Test cultures (but not controls) showed preferential expansion of 1–4 Vβ families either common to two or more stimulators or unique to a particular stimulator. Notably, we elicited leukemia-specific TCR Vβ expansions on four out of five occasions. In two pairs, responder cells selected for the appropriate leukemia-specific Vβ family were shown to have leukemia-specific cytotoxicity. These leukemia-restricted T-cells were CD8+ or CD4+ and CD25+ or CD57+. The results support the development of strategies to selectively deplete GVHD and conserve GVL reactivity in mismatched transplants.

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

Abstract 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-[125I]iodo-2′-deoxyuridine (125IdU), 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 125IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However, treatment with 0.1 μCi/ml 125IdU 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 125IdU (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.