Homeostatic MyD88-dependent signals cause lethal inflamMation in the absence of A20

Toll-like receptors (TLRs) on host cells are chronically engaged by microbial ligands during homeostatic conditions. These signals do not cause inflammatory immune responses in unperturbed mice, even though they drive innate and adaptive immune responses when combating microbial infections. A20 is a ubiquitin-modifying enzyme that restricts exogenous TLR-induced signals. We show that MyD88-dependent TLR signals drive the spontaneous T cell and myeloid cell activation, cachexia, and premature lethality seen in A20-deficient mice. We have used broad spectrum antibiotics to demonstrate that these constitutive TLR signals are driven by commensal intestinal flora. A20 restricts TLR signals by restricting ubiquitylation of the E3 ligase tumor necrosis factor receptor–associated factor 6. These results reveal both the severe proinflammatory pathophysiology that can arise from homeostatic TLR signals as well as the critical role of A20 in restricting these signals in vivo. In addition, A20 restricts MyD88-independent TLR signals by inhibiting Toll/interleukin 1 receptor domain–containing adaptor inducing interferon (IFN) β–dependent nuclear factor κB signals but not IFN response factor 3 signaling. These findings provide novel insights into how physiological TLR signals are regulated

Mature natural killer cells reset their responsiveness when exposed to an altered MHC environment

Some mature natural killer (NK) cells cannot be inhibited by major histocompatibility complex (MHC) I molecules, either because they lack corresponding inhibitory receptors or because the host lacks the corresponding MHC I ligands for the receptors. Such NK cells nevertheless remain self-tolerant and exhibit a generalized hyporesponsiveness to stimulation through activating receptors. To address whether NK cell responsiveness is set only during the NK cell differentiation process, we transferred mature NK cells from wild-type (WT) to MHC I–deficient hosts or vice versa. Remarkably, mature responsive NK cells from WT mice became hyporesponsive after transfer to MHC I–deficient mice, whereas mature hyporesponsive NK cells from MHC I–deficient mice became responsive after transfer to WT mice. Altered responsiveness was evident among mature NK cells that had not divided in the recipient animals, indicating that the cells were mature before transfer and that alterations in activity did not require cell division. Furthermore, the percentages of NK cells expressing KLRG1, CD11b, CD27, and Ly49 receptors specific for H-2b were not markedly altered after transfer. Thus, the functional activity of mature NK cells can be reset when the cells are exposed to a changed MHC environment. These findings have important implications for how NK cell functions may be curtailed or enhanced in the context of disease

Responsiveness and Tolerance of Natural Killer Cells

A major role of natural killer cells is distinguishing between “self” and “non–self”. This is accomplished through their ability to recognize MHC class I molecules and eliminate cells that have lost MHC I expression, a common feature of cancers and virus–infected cells. The interaction between NK receptors and MHC I on another cell is usually inhibitory and when MHC I is absent from target cells inhibition is relieved, leading to killing by NK cells (in a context of activating ligand expression). A subset of NK cells in wild–type mice does not express any MHC I specific inhibitory receptors and NK cells in MHC I–deficient (e.g. β2–microglobulin knockout) animals are never exposed to inhibition through MHC I. In both cases the NK cells do not attack MHC I deficient cells that are otherwise normal and exhibit a hyporesponsive phenotype when stimulated through their activating receptors, as evidenced by lack of inflammatory cytokine production. Work presented in this dissertation addresses the relationship of hyporesponsiveness and self–tolerance and demonstrates, for the first time, that the two phenomena are distinguishable. Additionally, the plasticity of NK cell education, the roles of hematopoietic vs. non–hematopoietic cells in this process, and finally the effects of inflammation on NK cell tolerance are also investigated. In order to investigate whether responsiveness of NK cells is set once during their development or can be changed in mature NK cells based on their environment we employed an adoptive transfer model. We transferred splenocytes containing mature NK cells from WT mice into β2m–deficient mice, or vice–versa. 8 to 10 days after transfer we assessed the responsiveness of the transferred NK cells to activating receptor crosslinking, as well as the capacity of the cells to reject grafts of β2m−/−spleen cells. We found that upon transfer into β2m knockout hosts WT NK cells reset their responsiveness downward and lost the capacity to reject MHC I — negative grafts. Conversely, when β2m–deficient NK cells were transferred to MHC I — expressing animals their responsiveness was reset upwards. Interestingly — despite increased responsiveness — these NK cells did not acquire the ability to reject β2m — deficient grafts. To address the roles or hematopoietic vs. non–hematopoietic cells in NK cell education we generated fetal liver chimeras by reconstituting WT or β2m−/−hosts with WT, β2m−/−, or a 1:1 mixture of WT and β2m−/−fetal liver cells. We found that NK cells that developed in an MHC I–expressing host acquired normal responsiveness whereas those that developed in an MHC I — negative host were hyporesponsive. Interestingly, however, NK cells that developed in the presence of β2m — deficient cells, of either non–hematopoietic or hematopoietic origin, acquired tolerance to β2m−/−grafts. Therefore, our data show that responsiveness of natural killer cells is mainly set by non–hematopoietic cells, while tolerance can be imposed either by hematopoietic or non–hematopoietic cells. Thus our data show for the first time that responsiveness of NK cells can be (partially) dissociated from tolerance of NK cells. These findings, taken together, lead to the proposal that two mechanisms operate to ensure NK cell self–tolerance, which are imposed by different cell types in vivo. Finally, we investigated whether inflammation can affect the ability of NK cells to respond to “missing self”. We infected mixed fetal liver chimeras with mouse cytomegalovirus and monitored the maintenance of β2m−/−donor cells. We found that β2m−/−donor cells were selectively rejected when the infected host was MHC I — sufficient, whereas no rejection was observed when the host was MHC I–deficient. These data indicate that different mechanisms of self tolerance are differentially regulated by inflammation, and that the form associated with hyporesponsiveness is more stable in the face of inflammation associated with infections.Altogether, the data presented in this dissertation demonstrate that different interacting cell types play different roles in the process of NK cell education, with non–hematopoietic cells being especially critical for regulating the sensitivity of activating NK receptors. The data also show, however, that under steady state conditions (no infection) responsiveness of mature NK cells can be re–set upward or downward depending on corresponding changes in their MHC environment. This dissertation provides the first evidence that tolerance to MHC I — deficient cells can be decoupled from hyporesponsiveness. Finally, we show that infection can break tolerance of NK cells towards MHC I — negative grafts only a setting where it is accompanied by high responsiveness, whereas tolerance that is accompanied by low responsiveness is more stable and cannon be broken as a result of infection. These findings represent a substantial revision of current notions of NK cell self tolerance and hyporesponsiveness

Responsiveness and Tolerance of Natural Killer Cells

A major role of natural killer cells is distinguishing between “self” and “non–self”. This is accomplished through their ability to recognize MHC class I molecules and eliminate cells that have lost MHC I expression, a common feature of cancers and virus–infected cells. The interaction between NK receptors and MHC I on another cell is usually inhibitory and when MHC I is absent from target cells inhibition is relieved, leading to killing by NK cells (in a context of activating ligand expression). A subset of NK cells in wild–type mice does not express any MHC I specific inhibitory receptors and NK cells in MHC I–deficient (e.g. β2–microglobulin knockout) animals are never exposed to inhibition through MHC I. In both cases the NK cells do not attack MHC I deficient cells that are otherwise normal and exhibit a hyporesponsive phenotype when stimulated through their activating receptors, as evidenced by lack of inflammatory cytokine production. Work presented in this dissertation addresses the relationship of hyporesponsiveness and self–tolerance and demonstrates, for the first time, that the two phenomena are distinguishable. Additionally, the plasticity of NK cell education, the roles of hematopoietic vs. non–hematopoietic cells in this process, and finally the effects of inflammation on NK cell tolerance are also investigated. In order to investigate whether responsiveness of NK cells is set once during their development or can be changed in mature NK cells based on their environment we employed an adoptive transfer model. We transferred splenocytes containing mature NK cells from WT mice into β2m–deficient mice, or vice–versa. 8 to 10 days after transfer we assessed the responsiveness of the transferred NK cells to activating receptor crosslinking, as well as the capacity of the cells to reject grafts of β2m−/−spleen cells. We found that upon transfer into β2m knockout hosts WT NK cells reset their responsiveness downward and lost the capacity to reject MHC I — negative grafts. Conversely, when β2m–deficient NK cells were transferred to MHC I — expressing animals their responsiveness was reset upwards. Interestingly — despite increased responsiveness — these NK cells did not acquire the ability to reject β2m — deficient grafts. To address the roles or hematopoietic vs. non–hematopoietic cells in NK cell education we generated fetal liver chimeras by reconstituting WT or β2m−/−hosts with WT, β2m−/−, or a 1:1 mixture of WT and β2m−/−fetal liver cells. We found that NK cells that developed in an MHC I–expressing host acquired normal responsiveness whereas those that developed in an MHC I — negative host were hyporesponsive. Interestingly, however, NK cells that developed in the presence of β2m — deficient cells, of either non–hematopoietic or hematopoietic origin, acquired tolerance to β2m−/−grafts. Therefore, our data show that responsiveness of natural killer cells is mainly set by non–hematopoietic cells, while tolerance can be imposed either by hematopoietic or non–hematopoietic cells. Thus our data show for the first time that responsiveness of NK cells can be (partially) dissociated from tolerance of NK cells. These findings, taken together, lead to the proposal that two mechanisms operate to ensure NK cell self–tolerance, which are imposed by different cell types in vivo. Finally, we investigated whether inflammation can affect the ability of NK cells to respond to “missing self”. We infected mixed fetal liver chimeras with mouse cytomegalovirus and monitored the maintenance of β2m−/−donor cells. We found that β2m−/−donor cells were selectively rejected when the infected host was MHC I — sufficient, whereas no rejection was observed when the host was MHC I–deficient. These data indicate that different mechanisms of self tolerance are differentially regulated by inflammation, and that the form associated with hyporesponsiveness is more stable in the face of inflammation associated with infections.Altogether, the data presented in this dissertation demonstrate that different interacting cell types play different roles in the process of NK cell education, with non–hematopoietic cells being especially critical for regulating the sensitivity of activating NK receptors. The data also show, however, that under steady state conditions (no infection) responsiveness of mature NK cells can be re–set upward or downward depending on corresponding changes in their MHC environment. This dissertation provides the first evidence that tolerance to MHC I — deficient cells can be decoupled from hyporesponsiveness. Finally, we show that infection can break tolerance of NK cells towards MHC I — negative grafts only a setting where it is accompanied by high responsiveness, whereas tolerance that is accompanied by low responsiveness is more stable and cannon be broken as a result of infection. These findings represent a substantial revision of current notions of NK cell self tolerance and hyporesponsiveness

NK cell self tolerance, responsiveness and missing self recognition.

Natural killer (NK) cells represent a first line of defense against pathogens and tumor cells. The activation of NK cells is regulated by the integration of signals deriving from activating and inhibitory receptors expressed on their surface. However, different NK cells respond differently to the same stimulus, be it target cells or agents that crosslink activating receptors. The processes that determine the level of NK cell responsiveness have been referred to collectively as NK cell education. NK cell education plays an important role in steady state conditions, where potentially auto-reactive NK cells are rendered tolerant to the surrounding environment. According to the "tuning" concept, the responsiveness of each NK cell is quantitatively adjusted to ensure self tolerance while at the same time ensuring useful reactivity against potential threats. MHC-specific inhibitory receptors displayed by NK cells play a major role in tuning NK cell responsiveness, but recent studies indicate that signaling from activating receptors is also important, suggesting that the critical determinant is an integrated signal from both types of receptors. An important and still unresolved question is whether NK cell education involves interactions with a specific cell population in the environment. Whether hematopoietic and/or non-hematopoietic cells play a role is still under debate. Recent results demonstrated that NK cell tuning exhibits plasticity in steady state conditions, meaning that it can be re-set if the MHC environment changes. Other evidence suggests, however, that inflammatory conditions accompanying infections may favor high responsiveness, indicating that inflammatory agents can over-ride the natural tendency of NK cells to adjust to the steady state environment. These findings raise many questions such as whether viruses and tumor cells manipulate NK cell responsiveness to evade immune-recognition. As knowledge of the underlying processes grows, the possibility of modulating NK cell responsiveness for therapeutic purposes is becoming increasingly attractive, and is now under serious investigation in clinical studies

Mature natural killer cells reset their responsiveness when exposed to an altered MHC environment.

Some mature natural killer (NK) cells cannot be inhibited by major histocompatibility complex (MHC) I molecules, either because they lack corresponding inhibitory receptors or because the host lacks the corresponding MHC I ligands for the receptors. Such NK cells nevertheless remain self-tolerant and exhibit a generalized hyporesponsiveness to stimulation through activating receptors. To address whether NK cell responsiveness is set only during the NK cell differentiation process, we transferred mature NK cells from wild-type (WT) to MHC I-deficient hosts or vice versa. Remarkably, mature responsive NK cells from WT mice became hyporesponsive after transfer to MHC I-deficient mice, whereas mature hyporesponsive NK cells from MHC I-deficient mice became responsive after transfer to WT mice. Altered responsiveness was evident among mature NK cells that had not divided in the recipient animals, indicating that the cells were mature before transfer and that alterations in activity did not require cell division. Furthermore, the percentages of NK cells expressing KLRG1, CD11b, CD27, and Ly49 receptors specific for H-2(b) were not markedly altered after transfer. Thus, the functional activity of mature NK cells can be reset when the cells are exposed to a changed MHC environment. These findings have important implications for how NK cell functions may be curtailed or enhanced in the context of disease

Differential Role of Hematopoietic and Nonhematopoietic Cell Types in the Regulation of NK Cell Tolerance and Responsiveness.

Many NK cells express inhibitory receptors that bind self-MHC class I (MHC I) molecules and prevent killing of self-cells, while enabling killing of MHC I-deficient cells. But tolerance also occurs for NK cells that lack inhibitory receptors for self-MHC I, and for all NK cells in MHC I-deficient animals. In both cases, NK cells are unresponsive to MHC I-deficient cells and hyporesponsive when stimulated through activating receptors, suggesting that hyporesponsiveness is responsible for self-tolerance. We generated irradiation chimeras, or carried out adoptive transfers, with wild-type (WT) and/or MHC I-deficient hematopoietic cells in WT or MHC I-deficient C57BL/6 host mice. Unexpectedly, in WT hosts, donor MHC I-deficient hematopoietic cells failed to induce hyporesponsiveness to activating receptor stimulation, but did induce tolerance to MHC I-deficient grafts. Therefore, these two properties of NK cells are separable. Both tolerance and hyporesponsiveness occurred when the host was MHC I deficient. Interestingly, infections of mice or exposure to inflammatory cytokines reversed the tolerance of NK cells that was induced by MHC I-deficient hematopoietic cells, but not the tolerance induced by MHC I-deficient nonhematopoietic cells. These data have implications for successful bone marrow transplantation, and suggest that tolerance induced by hematopoietic cells versus nonhematopoietic cells may be imposed by distinct mechanisms