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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-and͞or HA-2-positive patients with a relapse of their disease after alloSCT with DLI from their mHAg HA-1-and͞or HA-2-negative donors. Using HLA-A2͞HA-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 malignances relapsing after alloSCT.T reatment of patients with leukemia relapsing after allogeneic stem cell transplantation (alloSCT) by donor lymphocyte infusion (DLI) can induce long-lasting complete remissions through graft-versus-leukemia (GVL) reactivity (1-4). Complete molecular remissions (mCRs) of relapsed chronic myeloid leukemia (CML) in chronic phase have been obtained in 70-80% of treated patients (5-7). In contrast, patients with relapsed acute leukemia or CML in accelerated phase or blast crisis respond in only 20-35% of the cases (3,7,8). In a minority of patients with relapsed or persistent multiple myeloma, a graft-versus-myeloma effect after DLI has been demonstrated as well (9-11).Little is known about the nature and kinetics of antileukemic T cell responses involved in the GVL or graft-versus-myeloma effect after DLI. In patients with relapsed CML after alloSCT who have been treated with low-dose DLI, the time to achieve an mCR may vary from several weeks to 1 year (5, 12). Previously we showed that 5-15 weeks after DLI for relapsed CML significantly increased numbers of cytotoxic T lymphocytes (CTLs) recognizing malignant hematopoietic progenitor cells (HPCs) could be detected in peripheral blood of the recipients (13).In HLA genotypically identical donor-recipient pairs alloreactive donor T cells may recognize minor histocompatibility antigens (mHAgs) expressed on recipient cells (14). Ubiquitously expressed mHAgs such as HY (15-20), HA-3, HA-4, HA-6, HA-7 (14, 15), and HA-8 (21) may play a role in both graftversus-hos...

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Genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T-cell immunity toward viral infections and malignancies. We previously demonstrated redirected antileukemic reactivity of cytomegalovirus (CMV)-specific T cells by transfer of minor histocompatibility antigen HA-2-specific T-cell receptors (TCRs). HA-2-TCR-transferred CMV-specific T cells were potent effectors against HA-2-expressing leukemic cells, as well as CMV-expressing cells.Functional activity of these T cells correlated with TCR cell-surface expression. In the present study we analyzed which properties of transferred and endogenous TCRs are crucial for efficient cellsurface expression. We demonstrate that expression of the introduced TCR is not a random process but is determined by characteristics of both the introduced and the endogenously expressed TCR. The efficiency of TCR cell-surface expression is controlled by the intrinsic quality of the TCR complex. In addition, we demonstrate that chimeric TCRs can be formed and that efficiency of TCR expression is independent of whether TCRs are retrovirally introduced or naturally expressed. In conclusion, introduced, endogenous, and chimeric TCRs compete for cell-surface expression in favor of the TCR-CD3 complex with best-pairing properties. (Blood. 2007;109: [235][236][237][238][239][240][241][242][243]

We studied a dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human serum samples. A high degree of concordance was observed between the results of the dipstick assay and an IgM enzyme-linked immunosorbent assay (ELISA). Application of the dipstick assay for the detection of acute leptospirosis enabled the accurate identification, early in the disease, of a high proportion of the cases of leptospirosis. Analysis of a second serum sample is recommended, in order to determine seroconversion or increased staining intensity. All serum samples from the patients who were confirmed to be positive for leptospirosis by either a positive microscopic agglutination test or a positive culture but were found to be negative by the dipstick assay were also judged to be negative by the IgM ELISA or revealed borderline titers by the IgM ELISA. Some cross-reactivity was observed for sera from patients with diseases other than leptospirosis, and this should be taken into account in the interpretation of test results. The dipstick assay is easy to perform, can be performed quickly, and requires no electricity or special equipment, and the assay components, a dipstick and a staining reagent, can be stored for a prolonged period without a loss of reactivity, even at elevated temperatures.

Since HA-1-specific T cells have been shown to make a significant contribution to the clinical responses in patients with relapsed leukemia, we investigated the feasibility of adoptive transfer of in vitro induced HA-1-specific CD8 positive T cells to patients with relapsed leukemia after allogeneic stem cell transplantation. The in vitro generation of clinical grade HA-1-specific T-cell lines from HA-1 negative donors was seen to be feasible and 3 patients were treated with HA-1-specific T-cell lines. No toxicity after infusion was observed. Although in one patient, during a period of stable disease, HA-1-specific T cells could be detected in the peripheral blood and bone marrow, these patients had no clear clinical response.Key words: allogeneic stem cell transplantation, adoptive cellular immunotherapy, minor histocompatibility antigen, HA-1, CTL Citation: Meij P, Jedema I, van der Hoorn MAWG, Bongaerts R, Cox L, Wafelman AR, Marijt EWA, Willemze R, and Falkenburg JHF. Generation and administration of HA-1-specific T-cell lines for the treatment of patients with relapsed leukemia after allogeneic stem cell transplantation: a pilot study . Haematologica 2012;97(8):1205-1208. doi:10.3324/haematol.2011 This is an open-access paper. ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o nculturing CD14 + cells derived from donor peripheral blood with 100 ng/mL GM-CSF, 500 IU/ml IL-4 and 100 IU/mL IFNα-2a (Roferon-A, Roche, Basel, Switzerland) for five days. Immature CD34 or CD14 derived DC were maturated using DKTP (diphtheria, whooping cough, tetanus and polio) vaccine and diluted 1:1000 (RIVM, Bilthoven, The Netherlands). Generation of HA-1-specific T-cell linesAfter obtaining informed consent, HA-1-specific donor T-cell lines were generated by culturing donor peripheral blood mononuclear cells (PBMC) with donor-derived dendritic cells (DC) pulsed with the specific HA-1 peptide (VLHDDLLEA, synthesized at the Department of Immunohematology, LUMC, Leiden, The Netherlands) at a responder:stimulator (R/S) ratio of 10:1. DC (5-10x10 6 /mL) were pulsed by adding 1 μg/mL HA-1 peptide for 2 h at 37°C followed by extensive washing. Cells were cultured in IMDM containing penicillin and streptomycin, L-glutamin, 10% heat inactivated human serum and 6 IU/mL IL-2 (Proleukin (Aldesleukin) Novartis Pharmaceuticals, Horskam, UK). At Day 5 of the immune response, 120 IU/mL IL-2 was added. Until a maximum of five weeks, T-cell cultures were restimulated once a week with HA1 peptide pulsed-DC, at an R/S ratio of 10:1 in T-cell medium containing 120 U/mL IL-2. 8 Cultures were evaluated for the presence of HA-1-specific T cells by tetramer staining as previously described.1,9 HA-1-specific T-cell lines had to meet the following specifications before being released for administration: i) no microbiological contamination; ii) donor origin of the HA-1-specific CTL line established by chimerism analysis as previously described; 1 iii) more than 80% CD3 + T cells; iv) more than 5% HA-1-specific CD8 + T cells within the CD3 populatio...

Killer Ig-like receptors (KIR) are expressed by human NK cells and T cells. Although Ag-specific cytolytic activity and cytokine production of KIR+ T cells can be inhibited by KIR ligation, the effect of KIR on proliferation is unclear. KIR+ T cells have been reported to have a general proliferative defect. To investigate whether KIR+ T cells represent end-stage dysfunctional T cells, we characterized KIR+ CMV-specific T cells in allogeneic stem cell transplantation patients and healthy donors. In both patients and healthy donors, a significant percentage KIR+ T cells was detected at various time points. All stem cell transplantation patients studied showed KIR expression on CMV-specific T cells, while not all donors had KIR-expressing CMV-specific T cells. From two of the patients and one donor KIR+ CMV-specific T clones were isolated and analyzed functionally. T cells were detected that expressed KIR that could not encounter their corresponding KIR ligands in vivo, illustrating that KIR expression by these T cells was not based on functional selection but a random process. Our data demonstrate that KIR+ T cells are fully functional T cells that are only restricted in effector functions and proliferation upon KIR ligation. The level of KIR-mediated inhibition of the effector functions and proliferation depended on the strength of TCR stimulation. We observed no diminished general proliferative capacity and therefore we conclude that these T cells do not represent end-stage dysfunctional T cells.

BackgroundDonor lymphocyte infusion is an effective form of adoptive immunotherapy for hematologic malignancies after allogeneic stem cell transplantation. Graft-versus-host disease, however, often develops due to recognition of ubiquitously-expressed minor histocompatibility antigens. Transfer of T-cell receptors recognizing hematopoiesis-restricted minor histocompatibility antigens to virus-specific T cells may be a powerful anti-tumor therapy with a low risk of graft-versus-host disease. The purpose of this study was to develop an optimal T-cell receptors-encoding multi-cistronic retroviral vector and an efficient method for generating T-cell receptors-engineered virus-specific T cells.