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.

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 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.

Measuring seasonal variations of moisture in a landfill with the partitioning gas tracer test.

Seven pilot-scale partitioning gas tracer tests (PGTTs) were conducted to assess the accuracy and reproducibility of this method for measuring water in municipal solid waste landfills. Tests were conducted in the same location over a 12-month period, and measured moisture conditions ranged from possible dry waste to refuse with a moisture content of 24.7%. The final moisture content of 24.7% was in reasonable agreement with gravimetric measurements of excavated refuse, where the moisture content was 26.5+/-6.0 CI%. Laboratory tests were used to assess the utility of the PGTT for measuring water in small pores, water sorbed to solid surfaces, and the influence of dry waste on PGTTs. These experiments indicated that when refuse surfaces are not completely solvated with water, PGTTs may produce misleading results (negative estimates) of water saturation and moisture content.

Quantification and modelling of 2,4-dinitrotoluene reduction with high-purity and cast iron.

Cast iron has been used as a reactive material in permeable reactive barriers (PRBs) for site remediation. While reactions are generally believed to occur on the iron (oxide) surface, a recent study by [Oh, S.Y., Cha, D.K., Chiu, P.C., 2002a. Graphite-mediated reduction of 2,4-dinitrotoluene with elemental iron. Environ. Sci. Technol. 36 (10), 2178-2184] showed that graphite inclusions in cast iron can also serve as reaction sites for 2,4-dinitrotoluene (DNT). These authors also found that graphite-mediated reduction of DNT has a regioselectivity that is different from that for iron surface. In this study, we quantified the observations reported by Oh et al. and examined the role of graphite in cast iron through numerical modelling. Models containing one and two reaction sites were developed to evaluate the mass transfer, sorption and reaction rates for DNT reduction in batch systems containing high-purity and cast iron. Our simulations showed that the regioselectivity, defined as the ratio of the ortho- and para-nitro reduction rate constants, was 0.37+/-0.04 S.E. (S.E.=one estimated standard error) for iron surface and 3.59+/-0.76 S.E. for graphite surface. In the cast iron-water system, we estimated that at least 66+/-2% S.E. of the DNT was reduced on graphite surface, despite the low graphite content and the lower DNT reduction rate with graphite than with iron. Graphite played such an important role because of the rapid adsorption of DNT to graphite. In the batch experiments conducted by Oh et al., external mass transfer was not rate limiting. Surface reaction was the rate-limiting step for DNT reduction on the graphite surface in cast iron, whereas internal mass transfer and/or adsorption and surface reaction were important for high-purity iron.

Leukemia-associated antigen-specific T-cell responses following combined PR1 and WT1 peptide vaccination in patients with myeloid malignancies.

We describe the safety and immunogenicity of a combined vaccine of 2 leukemia-associated antigenic peptides, PR1 and WT1. Eight patients with myeloid malignancies received one subcutaneous dose each of PR1 and WT1 vaccines in Montanide adjuvant, with granulocyte-macrophage colony-stimulating factor. Patients were reviewed weekly for 4 weeks to monitor toxicity and immunologic responses. Toxicity was limited to grades 1 to 2. Using peptide/HLA-A 0201 tetramers and intracellular interferon-gamma staining, CD8(+) T cells against PR1 or WT1 were detected in 8 of 8 patients after a single vaccination. To monitor the kinetics of vaccine-induced CD8(+) T-cell responses and disease regression after vaccination, absolute PR1 and WT1(+)CD8(+) T-cell numbers and WT1 expression were studied weekly after vaccination. Responses occurred as early as 1 week after vaccination. After vaccination, the emergence of PR1 or WT1(+)CD8(+) T cells was associated with a decrease in WT1 mRNA expression as a marker of minimal residual disease, suggesting a vaccine-driven antileukemia effect. Conversely, loss of response was associated with reappearance of WT1 transcripts (P < .01). This is the first demonstration that a combined PR1 and WT1 vaccine is immunogenic. These results support further studies of combination immunization strategies in leukemia patients.