Bone Marrow-derived Natural Killer Cells Research Articles

GARP-mediated active TGF-β1 induces bone marrow NK cell dysfunction in AML patients with early relapse post–allo-HSCT

AbstractRelapse is a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML). However, the underlying mechanisms remain poorly understood. Natural killer (NK) cells play a crucial role in tumor surveillance and cancer immunotherapy, and NK cell dysfunction has been observed in various tumors. Here, we performed ex vivo experiments to systematically characterize the mechanisms underlying the dysfunction of bone marrow−derived NK (BMNK) cells isolated from AML patients experiencing early relapse after allo-HSCT. We demonstrated that higher levels of active transforming growth factor β1 (TGF-β1) were associated with impaired effector function of BMNK cells in these AML patients. TGF-β1 activation was induced by the overexpression of glycoprotein A repetitions predominant on the surface of CD4+ T cells. Active TGF-β1 significantly suppressed mTORC1 activity, mitochondrial oxidative phosphorylation, the proliferation, and cytotoxicity of BMNK cells. Furthermore, pretreatment with the clinical stage TGF-β1 pathway inhibitor, galunisertib, significantly restored mTORC1 activity, mitochondrial homeostasis, and cytotoxicity. Importantly, the blockade of the TGF-β1 signaling improved the antitumor activity of NK cells in a leukemia xenograft mouse model. Thus, our findings reveal a mechanism explaining BMNK cell dysfunction and suggest that targeted inhibition of TGF-β1 signaling may represent a potential therapeutic intervention to improve outcomes in AML patients undergoing allo-HSCT or NK cell−based immunotherapy.

Methods for Isolating and Defining Single-Cell Transcriptomes of Tissue-Resident Human NK Cells.

Natural killer (NK) cells are innate lymphocytes that control tumors and microbial infections. Human NK cells are transcriptomically and phenotypically heterogeneous. The site where NK cells develop and reside determines their phenotype and effector functions. Our current knowledge about human NK cells is primarily from blood- and bone marrow-derived NK cells. The major limitation in formulating organ-specific clinical therapy is the knowledge gap on how tissue-resident NK cells develop, home, and function. Thus, it is crucial to define the transcriptomic profiles and the transcriptional regulation of tissue-resident NK cells. The major challenges in studying tissue-resident NK cells include their total number and the complexity of the tissue. Additionally, during isolation, keeping them viable and naïve without activation are challenging tasks. Here, we provide methods for isolating and performing transcriptomic analyses of NK cells at the individual cell level. Single-cell RNA sequencing provides a higher resolution of cellular heterogeneity and a better understanding of cell-cell interactions within the microenvironment. Using these methods, we can efficiently identify distinct populations of NK cells in tissues and define their unique transcriptomic profiles.

Irradiation and bone marrow reconstitution affect the functional Ly49 natural killer cell repertoire in rats.

Total body irradiation (TBI) is part of the preconditioning regimen for allogeneic bone marrow transplantation (alloBMT) and the procedure is associated with treatment-related toxicity and delayed immune reconstitution. Natural killer (NK) cells develop and acquire functional competence in close interaction with stromal bone marrow cells that are considered relatively radioresistant compared to the hematopoietic compartment. We thus undertook a study to assess the effect of TBI on the reconstitution of class I MHC-specific Ly49 NK cell receptors in a rat model of alloBMT. In rats subjected to TBI alone or followed by MHC-matched BMT, the irradiation conditioning induced a skewing of the Ly49 repertoire. Specifically, the activating Ly49s3bright subset exhibited increased frequency and receptor density which correlated with augmented alloreactivity relative to untreated control rats. Our results highlight the plasticity of NK cells and indicate that ionizing radiation (IR) affects the stromal compartment and as a consequence the maturation and functional properties of bone marrow-derived NK cells. These changes lasted throughout the 6 months observation period, showing that irradiation induces long term effects on the generation of the NK cell receptor repertoire.

Development of thymic NK cells from double negative 1 thymocyte precursors

AbstractThe differentiation of natural killer (NK) cells and a subpopulation of NK cells which requires an intact thymus, that is, thymic NK cells, is poorly understood. Previous in vitro studies indicate that double negative (CD4−CD8−, DN) thymocytes can develop into cells with NK cell markers, but these cells have not been well characterized. Herein, we generated and characterized NK cells differentiating from thymic DN precursors. Sorted DN1 (CD44+CD25−) CD122−NK1.1− thymocytes from Rag1−/− mice were adoptively transferred into Rag1−/−Ly5.1 congenic mice. After intrathymic injection, donor-derived cells phenotypically resembling thymic NK cells were found. To further study their differentiation, we seeded sorted DN1 CD122−NK1.1− thymocytes on irradiated OP9 bone marrow stromal cells with IL-15, IL-7, Flt3L, and stem cell factor. NK1.1+ cells emerged after 7 days. In vitro differentiated NK cells acquired markers associated with immature bone marrow–derived NK cells, but also expressed CD127, which is typically found on thymic NK cells. Furthermore, we found that in vitro cells generated from thymic precursors secreted cytokines when stimulated and degranulated on target exposure. Together, these data indicate that functional thymic NK cells can develop from a DN1 progenitor cell population.

Characterization of a subset of bone marrow-derived natural killer cells that regulates T cell activation in rats

We report that bone marrow-derived natural killer (BMNK) cells from DA or F344 rats inhibit PMA/ionomycin-induced T cell proliferation. These NK-regulatory cells are NKR-P1A(dim), whereas a minor subpopulation is NKR-P1A(bright). Only the NKR-P1A(dim) BMNK cells inhibit T cell proliferation. If activated with rat Con A supernatant, the NKR-P1A(dim) cells become NKR-P1A(bright) and lose the ability to inhibit T cell proliferation. In contrast to BMNK cells, all DA and F344 rat NK cells isolated from the blood, spleen, cervical, or mesenteric lymph nodes or Peyer's patches are NKR-P1A(bright) and lack the ability to inhibit T cell proliferation. Inhibition of T cell proliferation correlates with significant down-regulation of CD3, suggesting that this may be the mechanism through which the NKR-P1A(dim) cells mediate suppression. The nitric oxide synthase inhibitor N(G)-monomethyl-arginine acetate-abrogated NKR-P1A(dim) cell inhibition of T cell proliferation. We conclude that rat bone marrow NKR-P1A(dim) cells represent a unique population that may play a role in maintaining immune homeostasis by regulating the clonal expansion of activated T cells.

Extrinsic and intrinsic regulation of early natural killer cell development

Natural killer (NK) cells are lymphocytes that play a critical role in both adaptive and innate immune responses. These cells develop from multipotent progenitors in the embryonic thymus and neonatal or adult bone marrow and recent evidence suggests that a subset of these cells may develop in the thymus. Thymus- and bone marrow-derived NK cells have unique phenotypes and functional abilities supporting the hypothesis that the microenvironment dictates the outcome of NK cell development. A detailed understanding of the mechanisms controlling this developmental program will be required to determine how alterations in NK cell development lead to disease and to determine how to harness this developmental program for therapeutic purposes. In this review, we discuss some of the known extrinsic stromal-cell derived factors and cell intrinsic transcription factors that function in guiding NK cell development.

Chronic alcohol consumption perturbs the balance between thymus-derived and bone marrow-derived natural killer cells in the spleen

Alcohol consumption reduces peripheral NK cell numbers and compromises NK cell cytolytic activity; however, the underlying mechanism is not understood completely. It was found recently that the peripheral NK cell pool consists largely of bone marrow (BM)-derived and thymus-derived cells, which are phenotypically and functionally different. The effects of alcohol consumption on these subpopulations have not been studied previously. Using a well-established alcohol-feeding model, we found that chronic alcohol consumption decreases the percentage and number of peripheral NK cells, especially those expressing a mature phenotype. Alcohol consumption did not alter NK cells in the thymus. NK cells in the BM were increased significantly; however, proliferation rate was not altered by alcohol consumption, which increased CD127+ and decreased Ly49D+ NK cells in the spleen but not in the BM. Chronic alcohol consumption increased IFN-gamma-producing NK cells and GATA-3 expression in splenic NK cells. Collectively, these results indicate that chronic alcohol consumption perturbs the balance between thymus-derived and BM-derived NK cells. The increased proportion of thymus-derived NK cells in the spleen likely results from impaired NK cell release from the BM.

IL-15 and IL-2 oppositely regulate expression of the chemokine receptor CX3CR1

AbstractThe chemokine receptor CX3CR1 (CX3C chemokine receptor 1) is expressed in mouse blood on natural killer (NK) cells and on monocytes. Because interleukin-15 (IL-15) is an essential cytokine for NK cell development and maintenance, we hypothesized that it may induce CX3CR1 expression on this cell type. In contrast, we found that in primary mouse bone marrow-derived NK cells IL-15 specifically inhibited CX3CR1 protein and mRNA accumulation, whereas the related cytokine IL-2 did not inhibit but instead increased CX3CR1 expression. Consistent with this finding, intravenous injection of a single dose of recombinant IL-15 into C57BL/6 mice decreased steady-state CX3CR1 levels 24 hours after injection in freshly isolated peripheral blood mononuclear cells (PBMCs), splenocytes, and bone marrow cells, and treatment of mouse PBMCs with IL-15 in vitro inhibited CX3CL1 (ligand for CX3CR1)-induced chemotaxis. These data suggest that IL-15 may be a negative regulator of innate immunity by inhibiting CX3CR1 expression. These data also suggest that IL-15 inhibition of CX3CR1 may subvert potential cell immunotherapy strategies in which IL-15 is used to expand NK cell populations in vivo or ex vivo. Finally, our results provide additional evidence for differential signaling by IL-2 and IL-15, despite usage of common βγc receptor chains. (Blood. 2003;102:3494-3503)

Interleukin-12 and tumor necrosis factor alpha mediate innate production of gamma interferon by group B Streptococcus-treated splenocytes of severe combined immunodeficiency mice

The existence of interleukin-12-mediated innate immune responses to group B streptococci (GBS) was tested by examining T-lymphocyte-independent gamma interferon (IFN) production in cultured splenocytes from severe combined immunodeficiency mice. Splenocytes were cultured with killed or living GBS for 48 h, and then IFN was measured by enzyme-linked immunosorbent assay. Type III GBS as well as other extracellular bacterial agents of neonatal sepsis (staphylococci and enterococci) induced IFN production, which was enhanced by interleukin-2 and was inhibited by neutralizing antibodies to tumor necrosis factor alpha and to mouse interleukin-12. Interleukin-12 bioactivity was present in conditioned medium from GBS-treated adherent macrophages. Adherent peritoneal macrophages and bone marrow-derived natural killer cells from severe combined immunodeficiency mice cultured separately with GBS did not produce IFN, whereas cocultures did produce IFN. Functional macrophage activation was evident by nitric oxide production in GBS-treated splenocyte cultures. The results show that extracellular pathogens such as GBS, similarly to intracellular microbes, induce macrophage interleukin-12 and tumor necrosis factor alpha, which promote natural killer cell secretion of IFN, which then enhances innate phagocyte resistance mechanisms.

Hematopoiesis on Suspended Nylon Screen-Stromal Cell Microenvironments

A three-dimensional culture system for the growth of primate and rodent bone marrow was developed in our laboratory. This method involves the seeding of stromal cells onto a nylon screen and the inoculation of fresh or cryopreserved bone marrow hematopoietic cells after stromal cell processes had extended across 3 to 4 out of every 5 mesh openings. Stromal cells attach, grow, and secrete matrix proteins which contribute to an intricate microenvironment for the support of multilineage hematopoiesis, which was observed for greater than 270 days in the rat model and for greater than 12 weeks in the human system, as evidenced by flow cytometry analysis and in vitro clonogenic assays. The adherent zones of these suspended nylon screen cultures consisted primarily of immature cells. These cultures could also be used as substrates for cytotoxicity measurements; treatment of rat bone marrow cultures of various ages with cytosine beta-D arabinofuranoside, cyclophosphamide, 5-fluorouracil, or methotrexate resulted in a dose-dependent decrease in CFU-C numbers and altered the phenotypic distribution of hematologic cells in the adherent zone. The use of a modification of this method to generate large numbers of active cytolytic cells after greater than 75 days culture of rat bone marrow-derived natural killer cells is described also. Suspended nylon screen bone marrow culture also has potential uses in genetic insertion and graft vs. host disease studies, blood component therapy, the evaluation of ex vivo purging programs, and in marrow expansion for transplantation.