The p110δ of PI3K plays a critical role in NK cell terminal maturation and cytokine/chemokine generation

Phosphatidylinositol 3-kinases (PI3Ks) play a critical role in regulating B cell receptor– and T cell receptor–mediated signaling. However, their role in natural killer (NK) cell development and functions is not well understood. Using mice expressing p110δD910A, a catalytically inactive p110δ, we show that these mice had reduced NK cellularity, defective Ly49C and Ly49I NK subset maturation, and decreased CD27High NK numbers. p110δ inactivation marginally impaired NK-mediated cytotoxicity against tumor cells in vitro and in vivo. However, NKG2D, Ly49D, and NK1.1 receptor–mediated cytokine and chemokine generation by NK cells was severely affected in these mice. Further, p110δD910A/D910A NK cell–mediated antiviral responses through natural cytotoxicity receptor 1 were reduced. Analysis of signaling events demonstrates that p110δD910A/D910A NK cells had a reduced c-Jun N-terminal kinase 1/2 phosphorylation in response to NKG2D-mediated activation. These results reveal a previously unrecognized role of PI3K-p110δ in NK cell development and effector functions

Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes

Rap1 GTPases control immune synapse formation and signaling in lymphocytes. However, the precise molecular mechanism by which Rap1 regulates natural killer (NK) cell activation is not known. Using Rap1a or Rap1b knockout mice, we identify Rap1b as the major isoform in NK cells. Its absence significantly impaired LFA1 polarization, spreading, and microtubule organizing center (MTOC) formation in NK cells. Neither Rap1 isoform was essential for NK cytotoxicity. However, absence of Rap1b impaired NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine production. Upon activation, Rap1b colocalized with the scaffolding protein IQGAP1. This interaction facilitated sequential phosphorylation of B-Raf, C-Raf, and ERK1/2 and helped IQGAP1 to form a large signalosome in the perinuclear region. These results reveal a previously unrecognized role for Rap1b in NK cell signaling and effector functions

Regulation of intestinal inflammation by CD45

Factors secreted by intestinal immune cells such as retinoic acid and cytokines are crucial in maintaining homeostasis in the gut. Dysregulation in the secretion of these factors can lead to inflammation and development of colitis. CD45 is a leukocyte specific tyrosine phosphatase important for T cell development and antigen receptor signaling. Here, I show that upon DSS-induced colitis, CD45-/- mice unexpectedly have significant numbers of T cells in their colon and exhibit more severe colitis. This was attributed to increased expression of the gut homing molecule, α4β7 and increased production of IFNγ and IL17A by the CD45-/- T cells. However, in the absence of adaptive immunity, CD45 is required for optimal intestinal innate immune responses. CD45-/- innate lymphoid cells had decreased IL-22 and GM-CSF production and CD45-/- myeloid cells have lower retinoic acid production. This led to less severe colitis when CD45 ⁺/⁺ T cells were transferred into CD45RAG-/- mice as it led to reduced expression of gut homing molecules and reduced homing of T cells to the colon. This defect was corrected by the addition of GM-CSF, which restored retinoic acid production. Induction of colitis by the transfer of naïve T cells into RAG-/- and CD45RAG-/- mice delayed the development of systemic disease in CD45RAG-/- mice, but led to comparable intestinal inflammation at the RAG-/- weight loss endpoint and significantly greater inflammation at the CD45RAG-/- endpoint, corresponding with increased CD45-/- myeloid cells in the colon. Since there was no difference in Foxp3+ regulatory T cells systemically, other options of inhibition of systemic inflammation in CD45RAG-/- mice were explored. CD45RAG-/- mice had increased CD71+TER119+ erythroid cells in the spleen prior to and post colitis induction and failed to downregulate erythroid progenitors upon T cell induced colitis. These suppressive erythroid cells may contribute to the delayed systemic inflammation in these mice. Overall, these results show novel roles for CD45 in the regulation of innate and adaptive immune cell cytokine production, as well as in erythrocyte maturation.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Transforming growth factor-beta-activated kinase 1 regulates natural killer cell-mediated cytotoxicity and cytokine production

Carma1, a caspase recruitment domain-containing membrane-associated guanylate kinase, initiates a unique signaling cascade via Bcl10 and Malt1 in NK cells. Carma1 deficiency results in reduced phosphorylation of JNK1/2 and activation of NF-κB that lead to impaired NK cell-mediated cytotoxicity and cytokine production. However, the precise identities of the downstream signaling molecules that link Carma1 to these effector functions were not defined. Here we show that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is abundantly present in NK cells, and activation via NKG2D results in its phosphorylation. Lack of Carma1 considerably reduced TAK1 phosphorylation, demonstrating the dependence of TAK1 on Carma1 in NKG2D-mediated NK cell activations. Pharmacological inhibitor to TAK1 significantly reduced NK-mediated cytotoxicity and its potential to generate IFN-γ, GM-CSF, MIP-1α, MIP-1β, and RANTES. Conditional in vivo knockdown of TAK1 in NK cells from Mx1Cre(+)TAK1(fx/fx) mice resulted in impaired NKG2D-mediated cytotoxicity and cytokine/chemokine production. Inhibition or conditional knockdown of TAK1 severely impaired the NKG2D-mediated phosphorylation of ERK1/2 and JNK1/2 and activation of NF-κB and AP1. Our results show that TAK1 links Carma1 to NK cell-mediated effector functions