Janelle A. Olson

NK cells mediate reduction of GVHD by inhibiting activated, alloreactive T cells while retaining GVT effects

Natural killer (NK) cells suppress graft-versus-host disease (GVHD) without causing GVHD themselves. Our previous studies demonstrated that allogeneic T cells and NK cells traffic similarly after allogeneic bone marrow transplantation (BMT). We therefore investigated the impact of donor NK cells on donor alloreactive T cells in GVHD induction. Animals receiving donor NK and T cells showed improved survival and decreased GVHD score compared with controls receiving donor T cells alone. Donor T cells exhibited less proliferation, lower CD25 expression, and decreased interferon-gamma (IFN-gamma) production in the presence of NK cells. In vivo, we observed perforin- and Fas ligand (FasL)-mediated reduction of donor T cell proliferation and increased T cell apoptosis in the presence of NK cells. Further, activated NK cells mediated direct lysis of reisolated GVHD-inducing T cells in vitro. The graft-versus-tumor (GVT) effect was retained in the presence of donor NK cells. We demonstrate a novel mechanism of NK cell-mediated GVHD reduction whereby donor NK cells inhibit and lyse autologous donor T cells activated during the initiation of GVHD.

In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity.

Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. CIK cells are cytotoxic against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD). However, the precise mechanism of reduced GVHD is not fully understood. Therefore, we evaluated the trafficking and survival of luciferase-expressing CIK cells in an allogeneic bone marrow transplant model. The initial trafficking patterns of CIK cells were similar to conventional T cells that induced GVHD; however, CIK cells infiltrated GVHD target tissues much less and transiently. CIK cells accumulated and persisted in tumor sites, resulting in tumor eradication. We evaluated different properties of CIK cells compared with conventional T cells, demonstrating a slower division rate of CIK cells, higher susceptibility to apoptosis, persistent increased expression of interferon gamma (IFN-gamma), and reduced acquisition of homing molecules required for entry of cells into inflamed GVHD target organs that lack expression of NKG2D ligands recognized by CIK cells. Due to these properties, allogeneic CIK cells had reduced expansion and caused less tissue damage. We conclude that CIK cells have the potential to separate graft-versus-tumor effects from GVHD.

Low doses of natural killer T cells provide protection from acute graft-versus-host disease via an IL-4–dependent mechanism

CD4(+) natural killer T (NKT) cells, along with CD4(+)CD25(+) regulatory T cells (Tregs), are capable of controlling aberrant immune reactions. We explored the adoptive transfer of highly purified (> 95%) CD4(+)NKT cells in a murine model of allogeneic hematopoietic cell transplantation (HCT). NKT cells follow a migration and proliferation pattern similar to that of conventional T cells (Tcons), migrating initially to secondary lymphoid organs followed by infiltration of graft-versus-host disease (GVHD) target tissues. NKT cells persist for more than 100 days and do not cause significant morbidity or mortality. Doses of NKT cells as low as 1.0 × 10(4) cells suppress GVHD caused by 5.0 × 10(5) Tcons in an interleukin-4 (IL-4)-dependent mechanism. Protective doses of NKT cells minimally affect Tcon proliferation, but cause significant reductions in interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production by donor Tcons and in skin, spleen, and gastrointestinal pathology. In addition, NKT cells do not impact the graft-versus-tumor (GVT) effect of Tcons against B-cell lymphoma-1 (BCL-1) tumors. These studies elucidate the biologic function of donor-type CD4(+)NKT cells in suppressing GVHD in an allogeneic transplantation setting, demonstrating clinical potential in reducing GVHD in HCT.

Tissue-Specific Homing and Expansion of Donor NK Cells in Allogeneic Bone Marrow Transplantation

NK cells have potential therapeutic impact in suppressing graft-versus-host disease (GVHD) and enhancing antitumor effects as a cellular therapy for hematologic malignancies. However, few studies have addressed the trafficking and in vivo behavior of NK cells in murine models of bone marrow transplantation (BMT). We investigated NK cell trafficking and survival following allogeneic and syngeneic BMT using a novel bioluminescence-based imaging strategy. Transplantation of luciferase-expressing NK cells revealed CD62L-mediated trafficking to lymphoid organs and trafficking to GVHD target tissues, as evidenced by in vivo and ex vivo bioluminescence imaging. The NK cells persisted for ∼4 wk after transplantation in allogeneic recipients, but were not detectable in syngeneic recipients. CFSE-labeling studies showed extensive NK cell proliferation in vivo. Transplanted NK cells up-regulated molecules necessary for homing to the lymph nodes, gastrointestinal tract, and skin, yet did not cause clinical GVHD. This expansion and tissue-specific homing was not solely due to the conditioning regimen, as NK cells proliferated and reached lymphoid and GVHD target tissue in unconditioned allogeneic RAG2−/− γ-chain−/− recipients. IL-2 enhanced expansion and antitumor activity of NK cells. These results provide significant insight into the behavior and potential therapeutic impact of NK cells in BMT.

Single-cell analysis of siRNA-mediated gene silencing using multiparameter flow cytometry

Use of synthetic short interfering RNAs (siRNAs) to study gene function has been limited by an inability to selectively analyze subsets of cells in complex populations, low and variable transfection efficiencies, and semiquantitative assays for measuring protein down‐regulation. Intracellular flow cytometry can overcome these limitations by analyzing populations at the single‐cell level in a high‐throughput and quantitative fashion. Individual cells displaying a knockdown phenotype can be selectively interrogated for functional responses using multiparameter analysis.