IntroductionMultiple myeloma (MM) is a malignant neoplasm characterized by clonal proliferation of plasma cells in the bone marrow (BM). This disease accounts for approximately 2% of all cancer deaths and nearly 20% of deaths caused by hematologic malignancies. 1 Although allogeneic stem-cell transplantation 2-5 occasionally cures these patients, and drugs such as thalidomide, lenalidomide, and bortezomib have improved outcomes, 6 high-dose chemotherapy followed by autologous stem-cell transplantation (ASCT) 7 still appears to be the best treatment for patients up to 70 years of age. However, the great majority of patients with MM are incurable due to the persistence of minimal residual disease. 8,9 Thus, novel modalities complementing or improving current treatment options are needed.Natural killer (NK) cells are cytotoxic lymphocytes that lyse certain tumor-and virus-infected cells without any prior stimulation or immunization. 10 The cytotoxic activity of NK cells is tightly controlled by a balance between activating and inhibitory signals from receptors on the cell surface. A main group of receptors that inhibit NK-cell activation is the inhibitory killer immunoglobulinlike receptors (KIRs). Upon recognition of self-MHC class I molecules on target cells, these receptors deliver an inhibitory signal that stops the activating signaling cascade, sparing cells with normal MHC class I expression from NK cell-mediated lysis. Activating receptors include the natural cytotoxicity receptors (NCRs) and NKG2D, all of which push the balance toward cytolytic action through engagement with separate ligands on the target cell surface. 11,12 This role of NK cells indicates their possible use for adoptive immunotherapeutic strategies, in particular against malignancies that express low levels of MHC class I molecules. 13 The aim of this study was to investigate whether, and under what conditions, NK cells from patients with MM can be expanded numerically using good manufacturing practice (GMP)-compliant components. Furthermore, we aimed to investigate if NK cells expanded ex vivo could target autologous MM cells and thus prove to be favorable candidates for immunotherapeutic approaches against MM. Methods Patients and acquisition of patient materialPeripheral blood and BM samples from 7 newly diagnosed patients at different stages of MM were included in this study. The patients were admitted to the Department of Hematology, Karolinska University Hospital Huddinge, Stockholm, Sweden. The study was approved by the south Stockholm research ethics committee. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. Patients' characteristics at the time of blood and BM sampling are given in Table 1.Peripheral blood mononuclear cells (PBMCs) as well as BM mononuclear cells (BMMCs) were isolated by gradient centrifugation, using Lymphoprep (Axis-Shield, Oslo, Norway). PBMCs and BMMCs were washed twice with phosphate-buffered saline (PBS; Gibco, Grand Island, NY), and cell viability was assessed...

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Hantavirus infection was diagnosed serologically by mu-capture IgM and IgG ELISAs in hemorrhagic fever with renal syndrome (HFRS) patients admitted to Tuzla Hospital, Bosnia-Herzegovina. The results indicated that more than one hantavirus caused the outbreak. To address the question of which hantavirus serotypes were involved, sequentially drawn sera were analyzed by focus reduction neutralization test (FRNT) for antibodies against Puumala, Hantaan, Dobrava, and Seoul hantaviruses. The data revealed that acute- or early convalescent-phase sera, even when drawn as late as 3 weeks after the onset of disease, could not be used for typing of the causative hantavirus; a significant number of these samples showed similar reactivity of neutralizing antibodies to several different hantavirus serotypes. Moreover, although several acute-phase sera showed the highest FRNT titer to Hantaan virus, convalescent sera from these patients in all cases showed high specificity for Puumala or Dobrava viruses. This phenomenon, interpreted as a cross-neutralizing primary antibody response, makes several earlier reports concerning causative agents of HFRS questionable. Serological examination of small rodents trapped in the endemic area identified Puumala- and Dobrava-like virus infections. RT-PCR and sequencing of rodent lung samples identified Dobrava virus in one yellow-necked field mouse (Apodemus flavicollis). Cross-FRNT data, using polyclonal rabbit antibodies, clearly confirmed Dobrava virus as a unique hantavirus serotype. In conclusion, the results revealed that both Puumala- and Dobrava-like viruses caused HFRS in Bosnia-Herzegovina, whereas no signs of Hantaan or Seoul virus involvement were found.

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The chimeric state after allogeneic hematopoietic stem cell transplantation provides a platform for adoptive immunotherapy using donor-derived immune cells. The major risk with donor lymphocyte infusions (DLIs) is the development of graft-versus-host disease (GvHD). Development of new DLI products with antitumor reactivity and reduced GvHD risk represents a challenging task in cancer immunotherapy. Although natural killer (NK) and NK-like T cells are promising owing to their antitumor activity, their low concentrations in peripheral blood mononuclear cells reduces their utility in DLIs. We have recently developed a system that allows expansion of clinical-grade NK and NK-like T cells in large numbers. In this study, the safety of donor-derived long-term ex vivo-expanded human NK and NK-like T cells given as DLIs was investigated as immunotherapy for cancer in five patients following allogeneic stem cell infusion. Infusion of the cells was safe whether administered alone or with IL-2 subcutaneously. No signs of acute GvHD were observed. One patient with hepatocellular carcinoma showed markedly decreased serum alpha-fetoprotein levels following cell infusions. These findings suggest that the use of ex vivo-expanded NK and NK-like T cells is safe and appears an attractive approach for further clinical evaluation in cancer patients.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world. It is currently an incurable disease, making new treatment options such as immunotherapy desirable. Monoclonal antibodies (Mabs) to surface antigens of the tumor cell is one option. Administration of cytotoxic cells such as natural killer (NK) and natural killerlike T (NKT) cells expanded in vitro might be a useful treatment modality alone or in combination with MAbs. A limiting step in the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Here, we report the feasibility of expanding populations of the human killer cells, CD3ÀCD56 þ NK and CD3 þ CD56 þ NKT cells, from peripheral blood mononuclear cells (PBMCs) of B-CLL patients. The influence of tumor B cells on the in vitro expansion of killer cells was assessed by depleting B cells from PBMCs by microbead separation before culture. The 21-day cultures from both B-cell-and non-B-cell-depleted PBMC showed a marked expansion of NK cells, and also of T cells, among which almost half had the NKT phenotype. Depletion of B cells before culture did not change the expansion rates of NK and NKT cells significantly. In patients with progressive B-CLL, NK cell expansion capacity was improved after fludarabine treatment when compared to samples obtained before treatment. Repeated samples of PBMCs from individual untreated patients with both indolent and progressive disease cultured under identical conditions gave similar NK cell expansion rates. Expanded killer cell populations had cytotoxic function against the NK-sensitive target K562 cell line and expressed high levels of Granzyme B. From our studies, we conclude that NK cells as well as NKT cells from the peripheral blood of B-CLL patients can be expanded, and that these cells have cytotoxic capacity.