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

Maternity services in multi-cultural Britain: using Q methodology to explore the views of first- and second-generation women of Pakistani origin

Objective to explore first- and second-generation Pakistani women’s experiences of maternity services and the inter generational differences/comparisons. Design a retrospective Q methodology study of Pakistani women following childbirth. Setting two Children's Centres in an inner city in the West Midlands. Participants women self-identified following distribution of information leaflets at Children’s Centres. Fifteen women took part in interviews (Stage one) using a semi-structured design and 16 women participated in the completion of the Q grid sorting (Stage four). Methods a standard five-stage Q methodology process took place: (1) initial data were gathered using a combination of individual face-to-face and focus group semi-structured community-based interviews (developing the concourse); (2) transcribed interviews were analysed for ‘themes’; (3) the themes were reduced to ‘statements’ that reflected the overall content of the concourse using an unstructured evolving approach (giving the Q set); (4) participants were asked to sort the statements (Q sorting) according to a pre-designed distribution grid providing individual participant response grids; and (5) the response grids were factor analysed using PQ Method (V2.11), which generates clusters of participants rather than clusters of variables. Factor loadings were calculated using factor analysis by principal components with varimax rotation. This produced a list of factors, each of which represents a ‘story’ of women’s experiences of maternity services. Throughout the process, an Urdu interpreter was involved. Findings six factors were identified: (1) confidence and empowerment of women who had attended higher education and had family support; (2) isolation of some women from both family and maternity services; (3) women who had poor experiences of maternity services but good family support, and wanted opportunities to be involved in service development; (4) women with positive experiences of maternity care and influenced by traditional cultural practices; (5) importance of information and support from health-care professionals; and (6) importance of midwifery care to women. Conclusion there were no clear inter generational differences identified, but a breadth of opinion and experience that seemed to be influenced by level of both education and social support was found. Whereas some women had few demands of maternity services, those who had less support and those with language barriers had additional needs. Implications for practice care given should be based on individual need but given within a wider collaborative context in order to support women effectively. Increased maternity service user involvement would also be welcomed for future planning of maternity services

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

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