Isabel Barao

Sensitization of Tumor Cells to NK Cell-Mediated Killing by Proteasome Inhibition

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.

Combination Therapy Using IL-2 and Anti-CD25 Results in Augmented Natural Killer Cell-Mediated Antitumor Responses

Interleukin (IL)-2 has been extensively examined to promote clinical T and natural killer (NK) cell responses. Regulatory T cells (Tregs) have been shown to regulate many aspects of the immune system, including NK cell-mediated responses. We have demonstrated that in vivo administration of IL-2 led to activation and expansion of both NK cells and immunosuppressive Tregs. Therefore, we attempted to augment NK cell antitumor effects by concurrently depleting Tregs using anti-CD25. Increased NK cell activation by IL-2 was found to be correlated with an increase in classical, short-term NK cell in vitro killing assays regardless of the depletion of Tregs. But when splenocytes of the treated mice were used in long-term tumor outgrowth experiments, we observed that prior depletion of Tregs from IL-2 administration led to improved antitumor effects compared with either treatment alone. Importantly, these in vitro data are correlated with subsequent in vivo survival of leukemia-bearing mice, in which co-treatment of IL-2 with anti-CD25 led to significantly improved survival compared with mice treated with either IL-2 alone or with Treg depletion. Prior depletion of NK1.1(+) cells, but not of CD8(+) cells, completely abrogated all antitumor effects mediated by IL-2 and anti-CD25 combination therapy. These findings demonstrate that superior NK cell-mediated antileukemic effects can be achieved with IL-2 administration and concurrent depletion of CD25(+) cells.

Hematopoietic progenitor cell regulation by CD4+CD25+ T cells

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) possess the capacity to modulate both adaptive and innate immune responses. We hypothesized that Tregs could regulate hematopoiesis based on cytokine effector molecules they can produce. The studies here demonstrate that Tregs can affect the differentiation of myeloid progenitor cells. In vitro findings demonstrated the ability of Tregs to inhibit the differentiation of interleukin-3 (IL-3)/stem cell factor (colony-forming unit [CFU]-IL3)-driven progenitor cells. Inhibitory effects were mediated by a pathway requiring cell-cell contact, major histocompatibility complex class II expression on marrow cells, and transforming growth factor-beta. Importantly, depletion of Tregs in situ resulted in enhanced CFU-IL3 levels after bone marrow transplantation. Cotransplantation of CD4(+)FoxP3(+)(gfp) Tregs together with bone marrow was found to diminish CFU-IL3 responses after transplantation. To address the consequence of transplanted Tregs on differentiated progeny from these CFU 2 weeks after hematopoietic stem cell transplantation, peripheral blood complete blood counts were performed and examined for polymorphonuclear leukocyte content. Recipients of cotransplanted Tregs exhibited diminished neutrophil counts. Together, these findings illustrate that both recipient and donor Tregs can influence hematopoietic progenitor cell activity after transplantation and that these cells can alter responses outside the adaptive and innate immune systems.

IL-15-mediated induction of LFA-1 is a late step required for cytotoxic differentiation of human NK cells from CD34+Lin- bone marrow cells

Optimal differentiation of cytotoxic NK cells is important to provide protective innate immunity to patients after bone marrow transplantation. In vitro differentiation of CD56+CD3− NK cells takes weeks and is supported by several cytokines, including IL-2, IL-7, and IL-15, and thus can be useful for immunotherapy. However, IL-2 therapy is problematic in vivo, and NK cells differentiated in vitro with only IL-7 lack cytotoxicity. We assessed whether human NK cells initially differentiated in vitro from CD34+Lin− bone marrow cells with IL-7 could acquire cytotoxicity after exposure to additional cytokines and what changes promoted cytotoxicity. The cells cultured with IL-7 already had granzyme B as well as perforin, as previously reported, the proteins of cytotoxic granules. The cells also lacked LFA-1. After 1 wk of secondary culture with either IL-2 or IL-15, but not with IL-12 or IL-18, the IL-7-cultured cells acquired cytotoxicity. IL-2 or IL-15 also induced LFA-1. Ab to the LFA-1 subunits CD11a and CD18 blocked lysis by the NK cells, indicating that the new LFA-1 correlated with, and was essential for, the cytotoxic function of the in vitro generated cells. The LFA-1 also participated in target cell binding by the in vitro differentiated cells. In this study, we demonstrated a new function for IL-15, the induction of LFA-1 in NK progenitor cells, and that IL-15 does more than merely support NK progenitor cell proliferation. The efficacy after only 1 wk of IL-15 administration is a positive practical feature that may apply to human therapy.

The TNF receptor-ligands 4-1BB-4-1BBL and GITR-GITRL in NK cell responses

Interactions between several tumor necrosis factor (TNF)-TNF receptor (TNFR) superfamily members that are expressed by T cells and natural killer (NK) cells and various other cell types modulate immune responses. This review summarizes the current understanding of how the TNF ligand-TNFR interactions 4-1BBL with 4-1BB, and GITRL with glucocorticoid-induced TNFR-related (GITR) regulate NK cell mediated antitumor responses and discuss its therapeutic implications.

The Interplay between Natural Killer Cells and Human Herpesvirus-6

Human Herpesvirus 6 (HHV-6) is a set of two closely related herpes viruses known as HHV-6A and HHV-6B. Both are lymphotropic viruses that establish latency in the host. The ability to evade the immune responses of effector cells is likely a major factor contributing to the development of a persistent HHV-6A/B (collectively termed HHV-6) infection. Natural killer (NK) cells are lymphocytes that, along with neutrophils and monocytes/macrophages, participate in the critical innate immune response during viral infections, but can also mediate the antigen-specific memory responses generally associated with adaptive immunity. NK cells compose the first barrier that viruses must break through to continue replication and dissemination, and a weak NK cell response may predispose an individual to chronic viral infections. Both HHV-6A and HHV-6B can interfere with NK cell-mediated anti-viral responses but the mechanisms by which each of these viruses affect NK cell activity differs. In this review, we will explore the nuanced relationships between the two viruses and NK cells, discussing, in addition, relevant disease associations.

Human natural killer cell development in a xenogeneic culture system

Summary. In vivo and in vitro xenogeneic models have shown the ability of a non‐human environment in supporting human haemopoiesis. In the present study, we evaluated the effect of fetal sheep thymic stroma in the invitro development of natural killer (NK) cells from humanhaemopoietic progenitors. CD34+HLA‐DR+ (CD34+ DR+)Lin– and CD34+DR–Lin– bone marrow (BM) progenitors were cultured for 3 weeks with or without interleukin 2 (IL‐2), in fetal sheep thymic stroma contact and transwell cultures. Both progenitors gave rise to NK cells, defined as CD45+CD56+ cells, in the presence or absence of IL‐2; however, the percentage of NK cells originated in cultures with IL‐2 was significantly higher. Direct contact with stroma seemed to be required for the most immature progenitors, CD34+DR–Lin–, to differentiate along the NK cell lineage. Functional assays revealed that only cells grown in the presence of IL‐2 were cytolytic against K562 targets and, curiously, NK cells derived from CD34+DR–Lin– progenitors were more cytotoxic that NK cells derived from CD34+DR+Lin– progenitors. These studies suggest that the ability of fetal sheep thymic stroma in promoting the generation of human NK cells from haemopoietic progenitors may have relevance in terms of NK cell ontogeny and induction of tolerance in transplantation.

HHV-6A Infection of Endometrial Epithelial Cells Induces Increased Endometrial NK Cell-Mediated Cytotoxicity

Background: We have recently reported the presence of Human herpesvirus-6A (HHV-6A) DNA in the 43% of endometrial epithelial cells from primary idiopathic infertile women, with no positivity in fertile women. To investigate the possible effect of HHV-6A infection in endometrial (e)NK cells functions, we examined activating/inhibitory receptors expressed by eNK cells and the corresponding ligands on endometrial cells during HHV-6A infection. Methods: Endometrial biopsies and uterine flushing samples during the secretory phase were obtained from 20 idiopathic infertile women and twenty fertile women. HHV-6A infection of endometrial epithelial cells was analyzed by Real-Time PCR, immunofluorescence and flow cytometry. eNKs receptors and endometrial ligands expression were evaluated by immunofluorescence and flow cytometry. Results: We observed the presence of HHV-6A infection (DNA, protein) of endometrial epithelial cells in the 40% of idiopathic infertile women. The eNK from all the subgroups expressed high levels of NKG2D and NKG2A receptors. Functional studies showed that NKG2D activating receptor and FasL are involved in the acquired cytotoxic function of eNK cells during HHV-6A infection of endometrial epithelial cells. In the presence of HHV-6A infection, eNK cells increased expression of CCR2, CXCR3 and CX3CR1 chemokine receptors (p = 0.01) and endometrial epithelial cells up-modulated the corresponding ligands: MCP1 (Monocyte chemotactic protein 1, CCL2), IP-10 (Interferon gamma-induced protein 10, CXCL10) and Eotaxin-3 (CCL26). Conclusion: Our results, for the first time, showed the implication of eNK cells in controlling HHV-6A endometrial infection and clarify the mechanisms that might be implicated in female idiopathic infertility.

Gene expression in INTERLEUKIN-7 differentiated natural killer cells derived from CD34+lin− bone marrow cells

Abstract Human NK cells can be generated in vitro from CD34+Lin− bone marrow (BM) cells in long term cultures supplemented with stem cell factor (SCF or c-kit ligand), Interleukin-1α (IL-1α), Interleukin-2 (IL-2) or Interleukin-7 (IL-7). However, only IL-2 differentiated cells show cytotoxicity against the NK-sensitive K562 cell line. The cytotoxic activity defect seen in IL-7 differentiated NK cells could be due in part to the: a) lack of expression of adhesion molecules essential for the formation of the effector:target conjugates or/and b) lack of perforin and granzymes mRNA and enzymatic activity. In previous studies our results shown that these cells express low levels of the adhesion molecules CD11a and CD18 when compared with IL-2 differentiated NK cells. The addition for one week of IL-2 or Interleukin-15 (IL-15) to IL-7 differentiated NK cells induced an increase in the expression of CD11a and CD18 to similar levels seen in IL-2 differentiated NK cells. This increase seems to be related with their increase inthe cytolytic activity suggesting that these cell surface markers are important for cell killing. NK cells have cytotoxic granules containing effector molecules like perforin and granzymes which are able to trigger pathways leading to DNA fragmentation and apoptosis on target cells. IL-7 differentiated NK cells express perforin and Granzyme A (GA) mRNA but not Hu-Met-1mRNA. We also detected by RT-PCR the expression of Granzyme B (GB) and Granzyme H (GH) mRNA in these cells. These results suggest that the defect seen in the cytotoxic activity in not due to lack of perforin or granzyme mRNA expression.