Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy-CD19, CD33, and CD123-to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

Dietary AhR Ligands Regulate AhRR Expression in Intestinal Immune Cells and Intestinal Microbiota Composition.

A diet rich in vegetables and fruit is generally considered healthy because of a high content of phytochemicals, vitamins, and fiber. The phytochemical indole-3-carbinol (I3C), a derivative of glucobrassicin, is sold as a dietary supplement promising diverse health benefits. I3C metabolites act as ligands of the aryl hydrocarbon receptor (AhR), an important sensor for environmental polyaromatic chemicals. Here, we investigated how dietary AhR ligand supplementation influences AhR target gene expression and intestinal microbiota composition. For this, we used AhR repressor (AhRR)-reporter mice as a tool to study AhR activation in the intestine following dietary I3C-supplementation in comparison with AhR ligand-deprived diets, including a high fat diet. AhRR expression in intestinal immune cells was mainly driven by dietary AhR ligands and was independent of microbial metabolites. A lack of dietary AhR ligands caused enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis and correlated with the expansion of Enterobacteriaceae, whereas Clostridiales, Muribaculaceae, and Rikenellaceae were strongly reduced. I3C supplementation largely reverted this effect. Comparison of I3C-induced changes in microbiota composition using wild-type (WT), AhRR-deficient, and AhR-deficient mice revealed both AhR-dependent and -independent alterations in the microbiome. Overall, our study demonstrates that dietary AhR ligand supplementation has a profound influence on Ahrr expression in intestinal immune cells as well as microbiota composition.

Indoleamine 2,3-Dioxygenase Activity During Acute Toxoplasmosis and the Suppressed T Cell Proliferation in Mice.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite and belongs to the phylum Apicomplexa. T. gondii is of medical and veterinary importance, because T. gondii causes the parasitic disease toxoplasmosis. In human cells, the interferon-gamma inducible indoleamine 2,3-dioxygenase 1 (IDO1) is an antimicrobial effector mechanism that degrades tryptophan to kynurenine and thus limits pathogen proliferation in vitro. Furthermore, IDO is described to have immunosuppressive properties, e.g., regulatory T cell differentiation and T cell suppression in humans and mice. However, there is only little known about the role of IDO1 in mice during acute toxoplasmosis. To shed further light on the role of mIDO1 in vivo, we have used a specifically adjusted experimental model. Therein, we infected mIDO1-deficient (IDO-/-) C57BL/6 mice and appropriate wild-type (WT) control mice with a high dose of T. gondii ME49 tachyozoites (type II strain) via the intraperitoneal route and compared the phenotype of IDO-/- and WT mice during acute toxoplasmosis. During murine T. gondii infection, we found mIDO1 mRNA and mIDO1 protein, as well as mIDO1-mediated tryptophan degradation in lungs of WT mice. IDO-/- mice show no tryptophan degradation in the lung during infection. Even though T. gondii is tryptophan auxotroph and rapidly replicates during acute infection, the parasite load was similar in IDO-/- mice compared to WT mice 7 days post-infection. IDO1 is described to have immunosuppressive properties, and since T cell suppression is observed during acute toxoplasmosis, we analyzed the possible involvement of mIDO1. Here, we did not find differences in the intensity of ex vivo mitogen stimulated T cell proliferation between WT and IDO-/- mice. Concomitant nitric oxide synthase inhibition and interleukin-2 supplementation increased the T cell proliferation from both genotypes drastically, but not completely. In sum, we analyzed the involvement of mIDO1 during acute murine toxoplasmosis in our specifically adjusted experimental model and found a definite mIDO1 induction. Nevertheless, mIDO1 seems to be functional redundant as an antiparasitic defense mechanism during acute toxoplasmosis in mice. Furthermore, we suggest that the systemic T cell suppression observed during acute toxoplasmosis is influenced by nitric oxide activity and IL-2 deprivation.