Activating and expanding T cells using T-cell receptor (TCR) cross-linking antibodies and interleukin 2 (IL-2) results in potent cytotoxic effector cells capable of recognizing a broad range of malignant cell targets, including autologous leukemic cells. The mechanism of target cell recognition has previously been unknown. Recent studies show that ligation of NKG2D on natural killer (NK) cells directly induces cytotoxicity, whereas on T cells it costimulates TCR signaling. Here we demonstrate that NKG2D expression is up-regulated upon activation and expansion of human CD8+ T cells. Antibody blocking, redirected cytolysis, and small interfering RNA (siRNA) studies using purified CD8+ T cells demonstrate that cytotoxicity against malignant target cells occurs through NKG2D-mediated recognition and signaling and not through the TCR. Activated and expanded CD8+ T cells develop cytotoxicity after 10 to 14 days of culture, coincident with the expression of the adapter protein DAP10. T cells activated and expanded in low (30 U/mL) and high (300 U/mL) concentrations of IL-2 both up-regulated NKG2D expression equally, but only cells cultured in high-dose IL-2 expressed DAP10 and were cytotoxic. Collectively these results establish that NKG2D triggering accounts for the majority of major histocompatibility complex (MHC)-unrestricted cytotoxicity of activated and expanded CD8+ T cells, likely through DAP10-mediated signaling.

IntroductionT cells with natural killer (NK) cell activity have been identified in both murine and human tissues. [1][2][3][4] Murine NKT cells typically express phenotypic markers found on T cells such as CD3 and the ␣␤T-cell receptor (TCR), as well as the NK markers NK1.1 and DX5. 5 Two populations of murine NKT cells have been described. One population either does not express the co-receptors CD4 or CD8 or are CD4 ϩ . These NKT cells express the invariant V␣14-J␣281 TCR and have been found in the liver, thymus, bone marrow (BM), and spleen with approximately 0.5 to 1.5 million cells per organ. [6][7][8] Functionally, CD4 ϩ NK1.1 ϩ NKT cells produce large amounts of interleukin 4 (IL-4) on activation. 8,9 Therefore, this population of IL-4-producing NKT cells are thought to play an important role in the regulation of immune responses, especially those of the T H 2 type. 10 CD4 ϩ NKT cells are positively selected by the major histocompatibility complex (MHC) class I-like molecule CD1d and associated ␤-2 microglobulin since these cells are markedly reduced in mice deficient in these molecules. 9,[11][12][13] Recently, a second population of NKT cells has been described that expresses a variable TCR repertoire and is not dependent on CD1d for maturation and development. 14 In addition, these CD1d-independent splenic NKT cells expressed mainly CD8 or were double negative with respect to CD4 and CD8 expression. Approximately 1% to 2% of splenocytes from C57BL/6 mice are ␣␤TCR ϩ NK1.1 ϩ , and, of these cells, approximately 20% are CD8 ϩ . 14 Little is known about the function of this later population of CD8 ϩ NKT cells.T cells are readily expandable following mitogenic stimulation with monoclonal antibodies (MAbs) directed against the TCR complex. 15 The combination of anti-CD3 and IL-2 results in the expansion of murine and human T cells capable of lysing a variety of different tumor cell lines, some of which are resistant to NK cells. [16][17][18] The expanded cells also have in vivo antitumor cell activity against murine tumors 16,19 and human tumors transplanted into immunodeficient mice. 18,20 It has been demonstrated that T cells activated with anti-CD3 and IL-2 and cultured for 6 to 8 days have a decreased capacity for inducing graft-versus-host disease (GVHD). Both CD4 ϩ and CD8 ϩ cells were less likely to result in GVHD following activation with anti-CD3 and IL-2. 21 In this report we have used culture conditions that allow for the growth of large numbers of T cells that share functional and phenotypic properties with NK cells. Following activation and culture, ␣␤TCR ϩ , CD8 ϩ , and NK1.1 ϩ T cells with NK cell function are readily expandable. We characterize the phenotype, cytokine production, and in vitro and in vivo biological activity of this population of CD8 ϩ NKT cells that have antitumor activity yet limited capacity to cause GVHD. Materials and methods AnimalsBalb/c (H-2 d ), C57BL/6 (H-2 b ), B10.D2/nSnJ (H-2 d ), FVB/J (H-2 q ), and the following mice of C57BL/6 background, fas deficient, fas li...

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