CD3+CD56−, CD4 and CD8 double negative T (DNT) cells comprise 1–3% of peripheral blood mononuclear cells. Their role in tumor immunity remains largely unknown due to their limited numbers and lack of effective methods to expand them. Here we developed a novel protocol by which DNT cells can be expanded ex vivo to therapeutic levels in 2 weeks from 13 of 16 acute myeloid leukemia (AML) patients during chemotherapy-induced complete remission. The expanded DNT cells expressed similar or higher levels of IFN-γ and TNF-α, and Granzyme B as that seen in bulk activated CD8 T cells from the same patient but significantly higher levels of perforin. The expanded DNT cells could effectively kill both allogeneic and autologous primary CD34+ leukemic blasts isolated from peripheral blood of AML patients in a perforin-dependant manner. These results demonstrate, for the first time, that DNT cells from AML patients can be expanded ex vivo even after intensive chemotherapy, and are effective at killing both allogeneic and autologous primary leukemic blasts. These findings warrant studies further exploring the potential of DNT cells as a novel adjuvant immunotherapy to decrease the risk of relapse in patients with AML and, perhaps other cancers.

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Soluble TRAIL (sTRAIL) can be produced by myeloid-derived cells to kill cancer cells. Whether this mechanism is used by T cells, and if so, how sTRAIL production is regulated, remains unclear. Our previous studies showed that ex vivo expanded human γδ T cells express TRAIL and NK receptor group 2 (R2), member D (NKG2D), and possess potent anticancer activities both in vitro and in vivo. Here, we investigated in greater detail the mechanisms by which γδ T cells utilize TRAIL and NKG2D to kill lung cancer cells. We demonstrate that human lung cancer cells express TRAIL R2 and NKG2D ligands. Blocking TRAIL or NKG2D during γδ T-cell-lung cancer cell co-cultures significantly reduced γδ T-cell-mediated cytotoxicity. Cross-linking NKG2D with anti-NKG2D antibody to mimic ligand binding promoted γδ T cells to produce sTRAIL, which induced apoptosis in lung cancer cells through TRAIL R2. Either neutralizing sTRAIL or blocking lung cancer cell TRAIL R2 significantly reduced γδ T-cell-mediated cytotoxicity to lung cancer cells. This study demonstrates that γδ T cells can mediate anticancer immunity via NKG2D-regulated production of sTRAIL. Additional supporting information may be found in the online version of this article at the publisher's web-site Introduction γδ T lymphocytes are innate immune cells that can prevent tumorigenesis by direct cytotoxicity against epithelial and hematopoietic cancers [1][2][3][4][5]. They can also augment the function of other immune cells including NK, B, and αβ T cells [6,7]. However, the precise mechanisms by which γδ T cells recognize and kill cancer cells remain poorly understood. Keywords: Lung cancerCorrespondence: Dr. Li Zhang e-mail: lzhang@uhnresearch.ca TRAIL is a member of the TNF superfamily that can induce apoptosis in transformed cells through two functional receptors (TRAIL R1/R2) [8,9]. TRAIL can be expressed in membranebound TRAIL (mTRAIL) and soluble TRAIL (sTRAIL) forms [10], each of which has been implicated in diverse biological functions, including cancer immunity [11][12][13][14][15][16][17]. mTRAIL is a well-established anticancer cytotoxic effector molecule [16][17][18], and sTRAIL has * These authors contributed equally to this work. [23], suggesting that production of sTRAIL by T cells may be involved in anticancer responses. However, to the best of our knowledge, direct evidence showing sTRAIL production as a γδ T-cell anticancer effector mechanism has not been reported previously. NK receptor group 2, member D (NKG2D) is a stimulatory receptor expressed on NK and T lymphocytes, including γδ T cells [5,24], that, in humans, recognizes stressed or malignant cells marked by expression of its ligands MHC class I-related inducible chains A and B (MICA/B) [25] and the UL16-binding proteins (ULBPs, reviewed in Champsaur [26]). Both NKG2D and TRAIL contribute to tumor surveillance and lysis [1,24,26,27], but their relationship in γδ T-cell-mediated cancer immunity is not known.We have developed a protocol that expands therapeutic numbers of γδ T cells from healthy do...

To evaluate the genetic damage induced by tuberculosis infection and also by a combination of therapeutic exposure to anti-tuberculosis (anti-TB) drugs (isoniazid + rifampicin + pyrazinamide + ethambutol or streptomycin) we have considered chromosome aberrations (CA) and micronuclei (MN) in binucleate peripheral blood lymphocytes from 36 patients diagnosed with pulmonary tuberculosis prior to receiving anti-TB drugs in the first phase and in the second phase after 6 months of therapy with anti-TB drugs. The same cytogenetic study has also been performed on 36 healthy individual controls. It was shown that the frequencies of both CA and micronucleated binucleate cells increased significantly after therapy with anti-TB drugs as compared with TB patients and controls. In addition, we observed that both cytogenetic markers (CA and MN) in patients before treatment with anti-TB drugs were significantly (P: < 0.05) higher than controls. No relationship was found between the frequency of MN and donor age. Thus, apart from drug effects, infection with tuberculosis is associated with increased CA and MN.

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Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.

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