IntroductionNatural killer (NK) cells contribute to innate immune responses against virally infected and neoplastic cells. 1 NK cells usually recognize and attack tumor cells that lack major histocompatibility complex (MHC) class I. 2 Our previous studies in murine tumor models clearly demonstrated that gamma delta (␥␦) T lymphocytes play an important role in the regulation of antitumor NK-cell function. 3 Specifically, we have shown that ␥␦ T lymphocytes are required for the antitumor activity of NK cells in vivo. More recently, we have demonstrated that culturing human peripheral blood mononuclear cells (PBMCs) with agents that activate ␥␦ T lymphocytes induce NK cell-mediated cytotoxicity against tumors that normally resist NK killing. 4 These findings are concordant with other studies that show that ␥␦ T lymphocytes regulate the early phase of NK cell-mediated antibacterial responses in mice. 5 Taken in concert these data strongly suggest that ␥␦ T lymphocytes are important in the regulation of NK-cell functions.␥␦ T cells are characterized by the expression of a T-cell receptor (TCR) consisting of both gamma and delta chains, 6 and account for 1% to 10% of CD3 ϩ cells in the peripheral blood of healthy adults. 7 Approximately 70% of ␥␦ T lymphocytes express the V␥2V␦2 TCR and can be expanded and activated by phosphoantigens such as the cholesterol biosynthesis intermediate, isopentenylpyrophosphate (IPP), or synthetic bisphosphonates (eg, pamidronate disodium and zoledronic acid). [8][9][10] Upon stimulation, ␥␦ T lymphocytes acquire the capacity to destroy solid tumors of diverse origins such as squamous cell carcinoma of the head and neck (SCCHN), melanoma, colon cancer, and breast carcinoma, 4,[11][12][13] suggesting that ␥␦ T lymphocytes are important antitumor effector cells. The validity of this antitumor function is further supported by mouse models demonstrating that mice deficient in ␥␦ T cells have increased sensitivity to the development of methylcholanthrene (MCA)-induced tumors. 14 In addition, a recent pilot clinical study showed that ␥␦ T lymphocyte adoptive therapy for patients with advanced renal cell carcinoma was well tolerated and induced antitumor immune responses. 15 The antitumor effects of ␥␦ T lymphocytes are recognized to result from both direct killing of tumor targets and trans-activation of adaptive immune responses. For example, recent data demonstrate that activated ␥␦ T lymphocytes cause the maturation of dendritic cells that promote development of acquired immunity. 16 In addition, ␥␦ T cells are known to cross-present tumor antigens (Ags) to CD8 ϩ cytolytic T lymphocytes. 17,18 Despite their wellcharacterized role in mediating adaptive immune responses, the mechanisms by which ␥␦ T cells regulate cells of the innate immune system, such as NK cells, are unclear.In this report we demonstrate that ␥␦ T lymphocytes provide a costimulatory function for NK cells stimulated with suboptimal doses of immobilized human immuglobulin G1 (hIgG1). Costimulated NK cells display up-regulat...

Purpose: The expression of CD56, a natural killer cell^associated molecule, on ah T lymphocytes correlates with their increased antitumor effector function. CD56 is also expressed on a subset of gy T cells. However, antitumor effector functions of CD56 + gy T cells are poorly characterized. Experimental Design: To investigate the potential effector role of CD56 + gy T cells in tumor killing, we used isopentenyl pyrophosphate and interleukin-2^expanded gyTcells from peripheral blood mononuclear cells of healthy donors. Results: Thirty to 70% of expanded gy T cells express CD56 on their surface. Interestingly, although both CD56+ and CD56 -gy Tcells express comparable levels of receptors involved in the regulation of gy T-cell cytotoxicity (e.g., NKG2D and CD94), only CD56 + gy T lymphocytes are capable of killing squamous cell carcinoma and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules because CD56 + gy T lymphocytes expressed higher levels of CD107a compared with CD56 -controls following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanamycin A and a combination of anti-gy T-cell receptor + anti-NKG2D monoclonal antibody, suggesting that the lytic activity of CD56 + gy T cells involves the perforin-granzyme pathway and is mainly gy T-cell receptor/NKG2D dependent. Importantly, CD56-expressing gy T lymphocytes are resistant to Fas ligand and chemically induced apoptosis. Conclusions: Our data indicate that CD56 + gy Tcells are potent antitumor effectors capable of killing squamous cell carcinoma and may play an important therapeutic role in patients with head and neck cancer and other malignancies.

CD137 (4-1BB)-mediated costimulation plays an important role in directing the fate of Ag-stimulated T cells and NK cells, yet the role of CD137 in mediating B cell function is unknown. We found that CD137 is expressed in vitro on anti-Ig–stimulated peripheral blood B cells and in vivo on tonsillar B cells with an activated phenotype. In vitro CD137 expression is enhanced by CD40 stimulation and IFN-γ and is inhibited by IL-4, -10, and -21. The expression of CD137 on activated human B cells is functionally relevant because engagement with its ligand at the time of activation stimulates B cell proliferation, enhances B cell survival, and induces secretion of TNF-α and -β. Our study suggests that CD137 costimulation may play a role in defining the fate of Ag-stimulated human B cells.

Macrophages and natural killer (NK) cells are important antitumor effectors by virtue of their ability to produce cytokines, chemokines and interferons (IFNs) and to mediate tumor cytotoxicity. Little is known about the impact of Toll-like receptor (TLR) and nucleotide binding and oligomerization domain (NOD)-like receptor (NLR) pathways on NK cell functions, and the role of TLRs and NLRs in macrophage activation is incompletely understood. In this study, we examined the capacities of expressed TLRs and NLRs to elicit cytokine production in human NK cells and THP1 macrophages, and to activate NK cytotoxicity against the squamous cell carcinoma of head and neck cell line Tu167 and erythroleukemia K562 cells. We found that NK cells express high levels of NOD2, NLRP3, TLR3, TLR7, and TLR9, while NOD1 was expressed at low levels. All tested NLR and TLR agonists potentiated NK cytotoxicity against Tu167 cells, whereas only poly (I:C) increased NK cytotoxicity against K562 cells. Poly (I:C) and Escherichia coli RNA markedly up-regulated TNF-α and IFN-γ expression in the NK92 cell line and human CD56(+)CD3(-) primary NK cells. High levels of NOD2, TLR7 and TLR9 proteins were observed in human THP1 cells, followed by TLR3, NOD1, and NLRP3. Stimulation of NLRP3 with E. coli RNA led to the highest induction of TNF-α, IL-6, IL-12p40, RANTES and IFN-β, whereas TLR7, TLR3, TLR9, NOD1 and NOD2 agonists had lower effects. Our data reveal involvement of TLRs and NLRs in potentiation of antitumor cytotoxicity and cytokine-producing activities of human NK cells and macrophages.

Thirty stool filtrates known to contain Clostridium difficile toxin based on previous testing on McCoy cells were tested for toxicity on primary African green monkey kidney (AGMK), McCoy, MRC-5, primary rhesus monkey kidney (RMK), and Vero cells. All 30 filtrates showed cytotoxic effect at-1:100 dilution on McCoy and Vero cells. A total of 22 filtrates were positive on MRC-5 monolayers, while only 16 and 10 filtrates showed positive cytotoxic effect on AGMK and RMK cells, respectively. Another 630 stool specimens were tested on McCoy and Vero cells only. Of these stool filtrates, 70 were positive and 560 were negative with both cell lines, which thus gave 100% agreement. Vero cells can be used interchangeably with McCoy cells for the detection of C. difficile toxin in stool filtrates. Detection of Clostridium difficile cytotoxin in stools from patients with diarrhea or pseudomembranous colitis must be rapid for optimal patient care. Testing for cell culture cytotoxicity is an accepted procedure (2, 3, 5, 6) for the detection of C. difficile cytotoxin within 24 h of inoculation. However, delays in testing may result if the specimen arrives when no suitable cell culture is available. This study was undertaken to determine which cell fines available in our laboratory could be used to detect C. difficile