SLAM-family receptors promote resolution of ILC2-mediated inflammation.

Type 2 innate lymphoid cells (ILC2) initiate early allergic inflammation in the lung, but the factors that promote subsequent resolution of type 2 inflammation and prevent prolonged ILC2 activation are not fully known. Here we show that SLAM-family receptors (SFR) play essential roles in this process. We demonstrate dynamic expression of several SFRs on ILC2s during papain-induced type 2 immunity in mice. SFR deficiency exacerbates ILC2-driven eosinophil infiltration in the lung, and results in a significant increase in IL-13 production by ILC2s exclusively in mediastinal lymph nodes (MLN), leading to increased dendritic cell (DC) and TH2 cell numbers. In MLNs, we observe more frequent interaction between ILC2s and bystander T cells, with T cell-expressed SFRs (especially SLAMF3 and SLAMF5) acting as self-ligands to suppress IL-13 production by ILC2s. Mechanistically, homotypic engagement of SFRs at the interface between ILC2s and T cells delivers inhibitory signaling primarily mediated by SHIP-1. This prevents activation of NF-κB, driven by IL-7 and IL-33, two major drivers of ILC2-mediated type 2 immunity. Thus, our study shows that an ILC2-DC-TH2 regulatory axis may promote the resolution of pulmonary type 2 immune responses, and highlights SLAMF3/SLAMF5 as potential therapeutic targets for ameliorating type 2 immunity.

SLAMF3 and SLAMF4 are immune checkpoints that constrain macrophage phagocytosis of hematopoietic tumors.

The interaction of SIRPα with CD47 represents a major mechanism for preventing macrophage phagocytosis. However, CD47-independent mechanisms are poorly defined. Here, we report a critical role of SLAM family receptors (SFRs), ubiquitously expressed on hematopoietic cells and forming homotypic interactions, in constraining macrophage phagocytosis. We found that SFR deficiency triggered macrophage phagocytosis of hematopoietic cells, leading to severe rejection of donor hematopoietic graft in recipient mice. Specific SFR members, mainly SLAMF3 and SLAMF4, were identified as "don't eat me" receptors on macrophages. These receptors inhibited "eat me" signals, such as LRP1-mediated activation of mTOR and Syk, through SH2 domain-containing phosphatases. SFRs combined with, but were independent of, CD47 to mitigate macrophage phagocytosis, and the combined deletion of SFRs and CD47 resulted in hematopoietic cytopenia in mice. This SFR-mediated tolerance was compromised in patients with hemophagocytic lymphohistiocytosis, a syndrome characterized by inappropriate phagocytosis toward hematopoietic cells. Loss of SFRs potently elicited macrophage rejection of hematopoietic tumors. Deletion of SFRs also significantly enhanced the phagocytosis of CD19-positive hematopoietic targets by the macrophages expressing the chimeric CD19 antigen receptor. Therefore, SFR-mediated inhibition of macrophage phagocytosis is critical to hematopoietic homeostasis, and SFRs may represent previously unknown targets for tumor immunotherapy.

mTORC1 and mTORC2 coordinate early NK cell development by differentially inducing E4BP4 and T-bet.

Natural killer (NK) cell development is a multistep process that requires a variety of signals and transcription factors. The lack of mammalian target of rapamycin (mTOR) kinase severely impairs NK cell development in mice. mTOR binds to Raptor and Rictor to form two complexes, mTORC1 and mTORC2, respectively. How mTOR and its two complexes regulate NK cell development is not fully understood. Here, we developed two methods to inactivate mTOR, Raptor, or Rictor in early stage NK cells (using CD122-Cre) or in late-stage NK cells (using Ncr1-CreTg). First, we found that when mTOR was deleted by CD122-Cre during and after NK cell commitment, NK cell development was severely impaired, while Ncr1-CreTg mediated mTOR deletion slightly affected NK cell terminal differentiation, suggesting that mTOR is essential for early NK cell differentiation. Second, we found that CD122-mediated deletion of Raptor significantly limited the differentiation of CD27+CD11b- immature NK (iNK) cell into mature NK cells. In contrast, the absence of Rictor significantly interfered with the differentiation of CD27-CD11b- early iNK cells. Third, Ncr1-mediated deletion of Raptor, rather than Rictor, moderately affected NK cell terminal differentiation. In terms of mechanism, mTORC1 mainly promotes the expression of NK cell-specific transcription factor E4 promoter-binding protein 4 (E4BP4), while both mTORC1 and mTORC2 can enhance the expression of T-bet. Therefore, mTORC1 and mTORC2 subtly coordinate NK cell development by differentially inducing E4BP4 and T-bet.

Regulation of MHC class I-independent NK cell education by SLAM family receptors.

Seven members of signaling lymphocytic activation molecule (SLAM) family receptors (SFRs) are ubiquitously expressed on hematopoietic cells and they play critical roles in immune cell differentiation and activation. The engagement of these receptors transmits intracellular signaling mainly by recruiting SLAM-associated protein (SAP) and its related adaptors, EWS-FLI1-activated transcript-2 (EAT-2) and EAT-2-related transducer (ERT). The critical roles of SFRs and SAP-family adaptors are highlighted by the discovery that SAP is mutated in human X-linked lymphoproliferative (XLP1) disease in which the contact between T and B cells in germinal center and cytotoxic lymphocytes (NK cells and CD8+ T cells) function are severely compromised. These immune defects are closely associated with the defective antibody production and the high incidence of lymphoma in the patients with XLP1. In addition to these well-known functions, SLAM-SAP family is involved in NK cell education, a process describing NK cell functional competence. In this chapter, we will mainly discuss these unappreciated roles of SAP-dependent and SAP-independent SFR signaling in regulating MHC-I-independent NK cell education.

Stage-specific requirement of kinase PDK1 for NK cells development and activation.

Phosphoinositide-dependent kinase-1 (PDK1) is an important enzyme for immune cell development by connecting PI3K to downstream mTOR signaling. It is needed to investigate how PDK1 spatiotemporally orchestrates NK cells development and whether this kinase is required for NK cells effector function. In this study, we used three genetic models to delete pdk1 at respective developmental stages, including hematopoietic stem cells (Vav1-Cre used), NK cell progenitor (NKp, CD122-Cre used) and terminal NK cells (Ncr1-Cre used). We found that CD122-Cre mediated deletion of pdk1 caused a severe loss of NK cells to an extent comparable to that of deletion by Vav1-Cre, and further revealed that PDK1 was necessary for NK cells master transcription factor E4BP4 expression at the NKp stage. Moreover, Ncr1-Cre-mediated inactivation of pdk1 delayed NK cells terminal differentiation. These PDK1-deficient NK cells secreted decreased amounts of the cytokine IFN-γ, likely due to impaired downstream mTOR activation. They also exhibited reduced degranulation in response to tumor cells. Mechanistically, PDK1 was critical for the formation of NK-target conjugates and lytic synapses. Therefore, we clarify the stage-specific roles of the metabolic regulator PDK1 in NK cells biology.

Dissection of SAP-dependent and SAP-independent SLAM family signaling in NKT cell development and humoral immunity.

Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) mutations in X-linked lymphoproliferative disease (XLP) lead to defective NKT cell development and impaired humoral immunity. Because of the redundancy of SLAM family receptors (SFRs) and the complexity of SAP actions, how SFRs and SAP mediate these processes remains elusive. Here, we examined NKT cell development and humoral immunity in mice completely deficient in SFR. We found that SFR deficiency severely impaired NKT cell development. In contrast to SAP deficiency, SFR deficiency caused no apparent defect in follicular helper T (TFH) cell differentiation. Intriguingly, the deletion of SFRs completely rescued the severe defect in TFH cell generation caused by SAP deficiency, whereas SFR deletion had a minimal effect on the defective NKT cell development in SAP-deficient mice. These findings suggest that SAP-dependent activating SFR signaling is essential for NKT cell selection; however, SFR signaling is inhibitory in SAP-deficient TFH cells. Thus, our current study revises our understanding of the mechanisms underlying T cell defects in patients with XLP.

NK cell development requires Tsc1-dependent negative regulation of IL-15-triggered mTORC1 activation.

Activation of metabolic signalling by IL-15 is required for natural killer (NK) cell development. Here we show that Tsc1, a repressor of mTOR, is dispensable for the terminal maturation, survival and function of NK cells but is critical to restrict exhaustive proliferation of immature NK cells and activation downstream of IL-15 during NK cell development. Tsc1 is expressed in immature NK cells and is upregulated by IL-15. Haematopoietic-specific deletion of Tsc1 causes a marked decrease in the number of NK cells and compromises rejection of 'missing-self' haematopoietic tumours and allogeneic bone marrow. The residual Tsc1-null NK cells display activated, pro-apoptotic phenotype and elevated mTORC1 activity. Deletion of Raptor, a component of mTORC1, largely reverses these defects. Tsc1-deficient NK cells express increased levels of T-bet and downregulate Eomes and CD122, a subunit of IL-15 receptor. These results reveal a role for Tsc1-dependent inhibition of mTORC1 activation during immature NK cell development.