Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24+/CD44+, CD133+, and aldehyde dehydrogenasebright) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies.

Bortezomib is a proteasome inhibitor that has direct antitumor effects. We and others have previously demonstrated that bortezomib could also sensitize tumor cells to killing via the death ligand, TRAIL. NK cells represent a potent antitumor effector cell. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing. Preincubation of tumor cells with bortezomib had no effect on short-term NK cell killing or purified granule killing assays. Using a 24-h lysis assay, increases in tumor killing was only observed using perforin-deficient NK cells, and this increased killing was found to be dependent on both TRAIL and FasL, correlating with an increase in tumor Fas and DR5 expression. Long-term tumor outgrowth assays allowed for the detection of this increased tumor killing by activated NK cells following bortezomib treatment of the tumor. In a tumor purging assay, in which tumor:bone marrow cell mixtures were placed into lethally irradiated mice, only treatment of these mixtures with a combination of NK cells with bortezomib resulted in significant tumor-free survival of the recipients. These results demonstrate that bortezomib treatment can sensitize tumor cells to cellular effector pathways. These results suggest that the combination of proteasome inhibition with immune therapy may result in increased antitumor efficacy.

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8 ؉ T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8 ؉ T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor ␣ chain), and led IntroductionMemory T cells represent an arm of the adaptive immune system that are long-lived, and capable of rapid antigen-specific responses. Memory T cells have been shown to have functional advantages more than naive T cells, as they develop more rapidly into cytolytic effector cells and produce greater amounts of cytokines after antigenic stimulation. 1 Although T cells classically require T-cell receptor (TCR) engagement and proper costimulation for complete activation and proliferation, memory T cells have also been observed to proliferate in response to various cytokines during viral infections. [2][3][4][5][6] These "bystander cells" proliferate and gain effector functions in response to the cytokine milieu produced during the course of viral and bacterial infections in mice and humans. [7][8][9][10] Cytokines alone can induce this as a single dose of recombinant type-I interferon (IFN) resulting in a transient increase in the proliferation of CD8 ϩ , CD62L ϩ CD44 high memory T cells, which was independent of coligation of the TCRs. 11 Such proliferation was not induced by the direct effects of type-I IFNs on CD8 ϩ T cells, but was because of type-I IFN-driven production of secondary cytokines such as 12 Effector and memory CD8 ϩ T cells express elevated levels of the receptors for IL-12 and IL-18, and secrete IFN-␥ in response to stimulation with both cytokines, 13 which suggests that other cytokine pathways can also induce their expansion. Similar to the secondary cytokine-driven proliferation observed after type-I IFN stimulation, IL-2, and toll-like receptor (TLR) agonists, that is, CpG and Poly:IC have also been described as having the capacity to induce bystander proliferation of CD8 ϩ CD44 high T cells. 12,14,15 The extent of antigen-specific proliferation versus bystander expansion has been the subject of considerable debate and may be contingent on the pathogen model and tissue examined. 9,13,16 Cancer therapies that target the stimulation of the immune system via agonist antibodies, cytokine-based modalities, or TLR agonists have been shown to result in potent CD8 ϩ T cell-mediated antitumor effects. 17,18 We have previously shown that a combination immunotherapy consisting of an agonist CD40 antibody and IL-2 results in synergistic antitumor effects. 19 Treatment of mice with other cytokine or TLR agonist combinations, such as CpGs and IL-15 or IL-2 and IL-12, also resulted in marked antitumor effects. 18 In all of these models, the antitumor effects were associated with rapid, extensive CD8 ϩ T-cell expansion. The antitumor effec...

IntroductionNatural killer (NK) cells represent a vital arm of innate immunity, mediating important antitumor and antiviral effects. 1 Although classically viewed as effectors that exhibit MHC-unrestricted cytotoxicity toward target cells, NK cells also produce numerous cytokines and mediate immunoregulatory functions. 2 The precise mechanisms by which NK cells recognize their targets have not been completely elucidated. NK cells use several receptor systems that recognize MHC for discrimination of abnormal cells from healthy cells. Both human and rodent NK cells exist as subsets bearing receptors that bind MHC class I molecules capable of inducing powerful inhibitory signals. Although the human killer-cell immunoglobulin-like receptors (KIRs) and rodent Ly49 receptors differ in structure, they are very similar in function. Both families are composed of members with either inhibitory or stimulatory activity that regulate the function of the NK cell after binding the appropriate MHC class I molecules. 3,4 It has been postulated that these inhibitory receptors exist as a means to prevent autoreactive attack by the NK cell; however, there are NK-cell subsets that do not appear to bear receptors binding "self" MHC molecules. This suggests that whereas the abilities of these molecules to bind MHC may indeed play critical roles in governing NK-cell development and activity, other means exist for their control.The concept of NK-cell education by "licensing" or "arming" the cells expressing the appropriate Ly49 inhibitory receptor has been proposed. 5,6 This concept was supported by data showing that NK cells bearing inhibitory Ly49 receptors for self-MHC demonstrated an increased ability to respond to stimulation in vitro. 7 This would provide the licensed NK cells with an advantage in the initial response to a stimulus and perhaps result in a more sustained response. However, this hypothesis has been brought into question recently with the demonstration that "unlicensed" Ly49 ϩ NK cells were the predominant subset that expanded after mouse cytomegalovirus (MCMV) infection and provided the greatest protection after adoptive transfer. 8 Therefore, the role of NK-cell licensing in vivo with regard to development and function remain unclear. Ly49 receptor expression occurs early in development and its distribution may or may not be driven by MHC. 9,10 NK cells have been under intense study for their potential use in HSCT and cancer therapy after reports demonstrating that the use of the appropriate KIR or KIR ligand mismatches in allogeneic HSCT improved clinical outcome. 11-13 However, very little is known regarding the role of mouse NK-cell subsets in reconstitution after HSCT or the response of the different subsets to activation stimuli induced by infection or administration of cytokines in vivo. In the present study, we show a preferential expansion of a Ly49 receptor in both reconstitution and activation models that is completely independent of the MHC haplotype. Methods MiceFemale C57BL/6 (B6, H-2 b ), Ly5.2 congen...

Summary Primary T cell activation involves the integration of three distinct signals delivered in sequence: 1)antigen recognition, 2)costimulation, and 3)cytokine-mediated differentiation and expansion. Strong immunostimulatory events such as immunotherapy or infection induce profound cytokine release causing “bystander” T cell activation, thereby increasing the potential for autoreactivity and need for control. We show that during strong stimulation, a profound suppression of primary CD4+ T cell-mediated immune responses ensued and was observed across preclinical models and patients undergoing high-dose interleukin-2 (IL-2) therapy. This suppression targeted naïve CD4+ but not CD8+ T cells and was mediated through transient suppressor of cytokine signaling-3 (SOCS3) inhibition of the STAT5b transcription factor signaling pathway. These events resulted in complete paralysis of primary CD4+ T cell activation affecting memory generation, induction of autoimmunity, as well as impaired viral clearance. These data highlight the critical regulation of naïve CD4+ T cells during inflammatory conditions.