Innate lymphoid cell characterization in the rat and their correlation to gut commensal microbes.

Innate lymphoid cells (ILCs) are important for tissue immune homeostasis, and are thoroughly characterized in mice and humans. Here, we have performed in-depth characterization of rat ILCs. Rat ILCs were identified based on differential expression of transcription factors and lack of lineage markers. ILC3s represented the major ILC population of the small intestine, while ILC2s were infrequent but most prominent in liver and adipose tissue. Two major subsets of group 1 ILCs were defined. Lineage- T-bet+ Eomes+ cells were identified as conventional NK cells, while lineage- T-bet+ Eomes- cells were identified as the probable rat counterpart of ILC1s based on their selective expression of the ILC marker CD200R. Rat ILC1s were particularly abundant in liver and intestinal tissues, and were functionally similar to NK cells. Single-cell transcriptomics of spleen and liver cells confirmed the main division of NK cells and ILC1-like cells, and demonstrated Granzyme A as an additional ILC1 marker. We further report differential distributions of NK cells and ILCs along the small and large intestines, and the association of certain bacterial taxa to frequencies of ILCs. In conclusion, we provide a framework for future studies of ILCs in diverse rat experimental models, and novel data on the potential interplay between commensals and intestinal ILCs.

Functional characterization of a conserved pair of NKR-P1 receptors expressed by NK cells and T lymphocytes in liver and gut.

Natural killer cell receptor protein 1 (NKR-P1) molecules are C-type lectin-like receptors modulating cellular responses toward target cells expressing C-type lectin-like related (Clr) molecules. Although the function of the prototypic rat NKR-P1A receptor and its inhibitory counterpart NKR-P1B are known, little is known about NKR-P1F and NKR-P1G apart from their promiscuity for Clr ligands. Here we generated mAbs against both receptors for phenotypic and functional analyses in rat tissues. NKR-P1F induced redirected lysis and robust Ca(2+) signaling in NK cells, which were prevented by simultaneous engagement of NKR-P1G. NKR-P1G also inhibited NK-cell lysis of Clr transfectants. NKR-P1F was expressed by most NK cells and NKR-P1A(+) T cells in all tissues analyzed, and by many NKR-P1A(-) intestinal T cells, while NKR-P1G was expressed by subsets of these cells with highest prevalence in gut and liver. In the intraepithelial compartment, the proportion of NKR-P1A(+) and NKR-P1F(+) cells was high at birth and thereafter declined, while NKR-P1B(+) and NKR-P1G(+) cells increased with age. Expression levels were also modulated by cytokines, with an increase of NKR-P1B and NKR-P1G induced by inflammatory cytokines, and a reduction of NKR-P1A by TGF-ß. The physiological impact of NKR-P1 receptors might thus be dependent on age, tissue, and inflammatory status.

Frontline Science: A hyporesponsive subset of rat NK cells negative for Ly49s3 and NKR-P1B are precursors to the functionally mature NKR-P1B+ subset.

Rat NK cells are divided into major subsets expressing either Ly49 receptors or the inhibitory NKR-P1B receptor in conjunction with NKG2A/C/E receptors. A minor subset of NKp46+ cells lacking expression of both Ly49 receptors and NKR-P1B is present in blood and spleen and is associated with decreased functional competence. We hypothesized that this subset may represent precursors to Ly49+ and/or NKR-P1B+ NK cells. When cultured in vitro in IL-2 and IL-15 or adoptively transferred to syngeneic hosts, a portion of NKR-P1B-Ly49s3- cells transformed to express NKR-P1B, but very little Ly49s3. Acquisition of NKR-P1B by NKR-P1B-Ly49s3- cells coincided with increased degranulation. In addition, although NKR-P1B-Ly49s3- cells highly proliferate, proliferative activity was reduced upon acquisition of NKR-P1B at comparable levels to bona fide NKR-P1B+ NK cells. A fraction of NKR-P1B-Ly49s3- cells remained negative for NKR-P1B, both in vitro and after adoptive transfer in vivo. Most NKR-P1B-Ly49s3- cells expressed the transcription factor Eomesodermin and NK cell markers, indicating that these cells represent conventional NK cells. Our findings suggest that the NKR-P1B-Ly49s3- NK cells are precursors to NKR-P1B single-positive cells and that functional competence is acquired upon expression of NKR-P1B.

IL-12, IL-15, and IL-18 pre-activated NK cells target resistant T cell acute lymphoblastic leukemia and delay leukemia development in vivo.

NK cells have shown promise in therapy of hematological cancers, in particular against acute myeloid leukemia. In contrast, the more NK cell-resistant acute lymphoblastic leukemia (ALL) is difficult to treat with NK-cell-based therapies, and we hypothesized that pre-activation of NK cells could overcome this resistance. We show in pediatric and adult patients with T-cell ALL (T-ALL) perturbed NK cell effector functions at diagnosis. Using an in vivo rat model for T-ALL, Roser leukemia (RL), suppressed NK cell effector functions were observed. NK cells from T-ALL patients had reduced expression of the activating receptors NKp46 and DNAM-1, but not NKG2D. In contrast to T-ALL patients, NKG2D but not NKp46 was downregulated on NK cells during rat RL. Decreased frequencies of terminally differentiated NKG2A+CD57-CD56dim NK cells in human T-ALL was paralleled in the rat by reduced frequencies of bone marrow NK cells expressing the maturation marker CD11b, possibly indicating impairment of differentiation during leukemia. RL was highly resistant to autologous NK cells, but this resistance was overcome upon pre-activation of NK cells with IL-12, IL-15, and IL-18, with concomitant upregulation of activation markers and activating receptors. Importantly, adoptive transfers of IL-12, IL-15, and IL-18 pre-activated NK cells significantly slowed progression of RL in vivo. The data thus shows that T-ALL blasts normally resistant to NK cells may be targeted by cytokine pre-activated autologous NK cells, and this approach could have potential implications for immunotherapeutic protocols using NK cells to more efficiently target leukemia.

Increased levels of inflammatory mediators and proinflammatory monocytes in patients with type I diabetes mellitus and nephropathy.

AIMS: To investigate and describe the relationship between diabetic nephropathy and systemic inflammation in patients with type 1 diabetes mellitus (T1DM). METHODS: Patients with T1DM, with or without reduced renal function due to diabetic nephropathy, were included. Differences in inflammatory mediators, adhesion molecules, markers of endothelial dysfunction and subsets of monocytes were studied in patients with mean disease duration of 31years. RESULTS: Patients with T1DM with and without renal failure were compared. Patients with nephropathy had increased plasma levels of proinflammatory monocytes, as well as circulatory PAI-1, syndecan-1, VEGF, IL-1ß, IL-1Ra and CCL4. Peripheral blood mononuclear cells from patients with nephropathy numerically increased soluble ICAM and PAI-1 in co-culture with primary endothelial cells compared to cells from patients without nephropathy. CONCLUSIONS: T1DM patients with kidney failure have higher levels of proinflammatory monocytes and circulatory inflammatory mediators compared to patients with T1DM alone. The results highlight the importance of inflammation and endothelial dysfunction in diabetic nephropathy with reduced GFR.

Degranulation Response in Cytotoxic Rat Lymphocytes Measured with a Novel CD107a Antibody.

Measuring degranulation through CD107a expression has become an advantageous tool for testing the functional capacity of cytotoxic cells. Such functional studies have been hampered in the rat by the lack of a suitable anti-rat CD107a antibody. In this study, we report a novel hybridoma generated by immunizing Armenian inbred hamsters with transfected Chinese hamster ovary cells expressing CD107a. The SIM1 clone exhibited specific reactivity with CD107a and measured degranulation from natural killer (NK) cells stimulated with target cells or mAb crosslinking of their activating receptors. Degranulation in IL-2-activated NK cells could also be measured, when using low effector to target ratios. SIM1 also stained activated CD8, but not CD4 T cells. This report characterizes the degranulation response in cytotoxic rat cells with a new antibody against rat CD107a.

Syndecan-4 is a major syndecan in primary human endothelial cells in vitro, modulated by inflammatory stimuli and involved in wound healing.

Syndecans are important cell surface proteoglycans with many functions; yet, they have not been studied to a very large extent in primary human endothelial cells. The purpose of this study was to investigate syndecan-4 expression in cultured human umbilical vein endothelial cells (HUVECs) and assess its role in inflammatory reactions and experimental wound healing. qRT-PCR analysis revealed that syndecan-3 and syndecan-4 were highly expressed in HUVECs, whereas the expression of syndecan-1 and -2 was low. HUVECs were cultured with the inflammatory mediators lipopolysaccharide (LPS) and interleukin 1ß (IL-1ß). As a result, syndecan-4 expression showed a rapid and strong increase. Syndecan-1 and -2 expressions decreased, whereas syndecan-3 was unaffected. Knockdown of syndecan-4 using siRNA resulted in changes in cellular morphology and focal adhesion sites, delayed wound healing and tube formation, and increased secretion of the pro-inflammatory and angiogenic chemokine, CXCL8. These data suggest functions for syndecan-4 in inflammatory reactions, wound healing and angiogenesis in primary human endothelial cells.