preparation-single-cell-suspension-mouse-thymic-epithelial-cells

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the ...

National Institutes of Health

Invariant NKT (NKT) cells are thymus-generated innate-like T cells, comprised of three distinct subsets with divergent effector functions. The molecular mechanism that drives the lineage trifurcation of immature NKT cells into the NKT1, NKT2, and NKT17 subsets remains a controversial issue that remains to be resolved. Because cytokine receptor signaling is necessary for NKT cell generation, cytokines are proposed to contribute to NKT subset differentiation also. However, the precise roles and requirements of cytokines in these processes are not fully understood. Here, we show that IL-2Rβ, a nonredundant component of the IL-15 receptor complex, plays a critical role in both the development and differentiation of thymic NKT cells. While the induction of IL-2Rβ expression on postselection thymocytes is necessary to drive the generation of NKT cells, surprisingly, premature IL-2Rβ expression on immature NKT cells was detrimental to their development. Moreover, while IL-2Rβ is necessary for NKT1 generation, paradoxically, we found that the increased abundance of IL-2Rβ suppressed NKT1 generation without affecting NKT2 and NKT17 cell differentiation. Thus, the timing and abundance of IL-2Rβ expression control NKT lineage fate and development, thereby establishing cytokine receptor expression as a critical regulator of thymic NKT cell differentiation.

The Timing and Abundance of IL-2Rβ (CD122) Expression Control Thymic NKT Cell Generation and NKT1 Subset Differentiation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected  already on resting NK cells; moreover, the frequency of NKp44 NK cells was higher than that of NKp46 NK cells in most animals analyzed. Upon  stimulation with IL-2 or IL-15, the frequency of NKp44 NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.

The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) Is Expressed on the Majority of Porcine NK Cells  Without Stimulation.

Thymocytes bearing autoreactive T cell receptors (TCRs) are agonist-signaled by TCR/co-stimulatory molecules to either undergo clonal deletion or to differentiate into specialized regulatory T (T) or effector T (T) CD4 cells. How these different fates are achieved during development remains poorly understood. We now document that deletion and differentiation are agonist-signaled at different times during thymic selection and that T and T cells both arise after clonal deletion as alternative lineage fates of agonist-signaled CD4CD25 precursors. Disruption of agonist signaling induces CD4CD25 precursors to initiate Foxp3 expression and become T cells, whereas persistent agonist signaling induces CD4CD25 precursors to become IL-2 T cells. Notably, we discovered that transforming growth factor-β induces Foxp3 expression and promotes T cell development by disrupting weaker agonist signals and that Foxp3 expression is not induced by IL-2 except under non-physiological in vivo conditions. Thus, TCR signaling disruption versus persistence is a general mechanism of lineage fate determination in the thymus that directs development of agonist-signaled autoreactive thymocytes.

Assiatu Crossman

Flow Cytometry Facility,
Experimental Immunology Branch,
Center for Cancer Research,
National Cancer Institute,
Flow Cytometry Facility, Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute

National Institutes of Health

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

Assiatu Crossman

Flow Cytometry Facility,Experimental Immunology Branch,Center for Cancer Research,National Cancer Institute,Flow Cytometry Facility, Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute

National Institutes of Health

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

Flow Cytometry Facility,
Experimental Immunology Branch,
Center for Cancer Research,
National Cancer Institute,
Flow Cytometry Facility, Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute