Kannan Natarajan

Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand.

Natural killer (NK) cell function is regulated by NK receptors that interact with MHC class I (MHC-I) molecules on target cells. The murine NK receptor Ly49A inhibits NK cell activity by interacting with H-2Dd through its C-type-lectin-like NK receptor domain. Here we report the crystal structure of the complex between the Ly49A NK receptor domain and unglycosylated H-2Dd. The Ly49A dimer interacts extensively with two H-2Dd molecules at distinct sites. At one interface, a single Ly49A subunit contacts one side of the MHC-I peptide-binding platform, presenting an open cavity towards the conserved glycosylation site on the H-2Dd α2 domain. At a second, larger interface, the Ly49A dimer binds in a region overlapping the CD8-binding site. The smaller interface probably represents the interaction between Ly49A on the NK cell and MHC-I on the target cell, whereas the larger one suggests an interaction between Ly49A and MHC-I on the NK cell itself. Both Ly49A binding sites on MHC-I are spatially distinct from that of the T-cell receptor.

The Adaptor MAVS Promotes NLRP3 Mitochondrial Localization and Inflammasome Activation

NLRP3 is a key component of the macromolecular signaling complex called the inflammasome that promotes caspase 1-dependent production of IL-1β. The adaptor ASC is necessary for NLRP3-dependent inflammasome function, but it is not known whether ASC is a sufficient partner and whether inflammasome formation occurs in the cytosol or in association with mitochondria is controversial. Here, we show that the mitochondria-associated adaptor molecule, MAVS, is required for optimal NLRP3 inflammasome activity. MAVS mediates recruitment of NLRP3 to mitochondria, promoting production of IL-1β and the pathophysiologic activity of the NLRP3 inflammasome in vivo. Our data support a more complex model of NLRP3 inflammasome activation than previously appreciated, with at least two adapters required for maximal function. Because MAVS is a mitochondria-associated molecule previously considered to be uniquely involved in type 1 interferon production, these findings also reveal unexpected polygamous involvement of PYD/CARD-domain-containing adapters in innate immune signaling events.

Post‐thymectomy autoimmune gastritis: fine specificity and pathogenicity of anti‐H/K ATPase‐ reactive T cells

Thymectomy at day 3 of life (d3Tx) results in the development of organ‐specific autoimmunity. We have recently shown that d3Tx BALB/c mice which develop autoimmune gastritis contain CD4+ T cells specific for the gastric parietal cell proton pump, H/K ATPase. Here, we demonstrate that freshly explanted gastric lymph node (LN) cells from d3Tx mice react significantly to the H/K ATPase α chain, but only marginally to the β chain. Two H/K ATPase‐reactive T cell lines were derived from the gastric LN of d3Tx mice. Both are CD4+ , TCR α / β+ , and I‐Ad restricted, and recognize distinct peptides from the H/K ATPase α chain. One cell line secretes Th1 and the other Th2 cytokines, but both are equally potent in inducing gastritis with distinct profiles of cellular infiltration in nu/nu recipient animals. Neither of the cell lines induced disease in normal BALB/c recipients and transfer of disease to nu/nu recipients was blocked by co‐transfer of normal BALB/c spleen cells containing CD4+ CD25+ cells. Although CD4+ CD25+ T cells are thought to emigrate from the thymus after day 3 of life, they could be identified in LN of 2‐day‐old animals. The capacity of CD4+ CD25+ T cells to abrogate the pathogenic activity in vivo of both activated Th1/Th2 lines strongly suggests that this suppressor T cell population may have a therapeutic role in other models of established autoimmunity. The availability of well‐characterized lines of autoantigen‐specific T cells should greatly facilitate the analysis of the mechanism of action and target of the CD4+ CD25+ immunoregulatory cells.

Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b).

The Ly49 family of natural killer (NK) receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I. Ly49 receptors show complex patterns of MHC class I cross-reactivity and, in certain cases, peptide selectivity. To investigate whether specificity differences result from topological differences in MHC class I engagement, we determined the structure of the peptide-selective receptor Ly49C in complex with H-2Kb. The Ly49C homodimer binds two MHC class I molecules in symmetrical way, a mode distinct from that of Ly49A, which binds MHC class I asymmetrically. Ly49C does not directly contact the MHC-bound peptide. In addition, MHC crosslinking by Ly49C was demonstrated in solution. We propose a dynamic model for Ly49–MHC class I interactions involving conformational changes in the receptor, whereby variations in Ly49 dimerization mediate different MHC-binding modes.

A T cell receptor transgenic model of severe, spontaneous organ-specific autoimmunity

The development of mouse models of human organ‐specific autoimmune diseases has been hampered by the need to immunize mice with autoantigens in potent adjuvants. Even autoantigen‐specific T cell receptor transgenic models of autoimmunity have proven to be complex as the transgenic mice frequently fail to develop disease spontaneously. We have isolated a CD4+ T cell clone (TxA23)that recognizes the gastric parietal cell antigen, H/K ATPase α‐chain630–641, from a mouse with autoimmune gastritis that developed after thymectomy on day 3 of life. The T cell receptor α and β genes from this clone were used to generate A23 transgenic mice. All A23 transgenic animals spontaneously developed severe autoimmune gastritis, and evidence of disease was detected as early as day 10 of life. Gastritis could be transferred to immunocompromised mice with a limited number of transgenic thymocytes (103), but as many as 107 induced only mild disease in wild‐type animals. Due to the complete penetrance of spontaneous disease, identity of the auto‐antigen, susceptibility to immunoregulation, and close relation to autoimmune gastritis in man, A23 transgenic mice represent a unique CD4+ T cell‐mediated disease model for understanding the multiple factors regulating organ‐specific autoimmunity.

CONTROL OF AUTOIMMUNITY BY REGULATORY T CELLS

The development of autoimmune disease involves a breakdown in the mechanisms that control self vs non-self discrimimation. The primary mechanism that leads to self tolerance is thymic deletion of autoreactive T cells, but thymic deletion is not perfect and autoreactive T cells do escape to the periphery. Cells that escape thymic deletion are then subject to mechanisms of peripheral tolerance including the induction of anergy t as well as T cell ignorance/indifference of the recognition of autoantigens. However, anergy can be reversed and ignorant T cell populations have the potential to be activated when their target self-antigens are released into the lymphoid system during the course of an infectious insult or when activated by cross-reactive antigens present on infectious agents. Passive mechanisms for the induction of self tolerance may therefore be insufficient to control the activation of autoreactive T cells. Evidence has recently been obtained for an active mechanism of immune suppression in which a distinct subset of T cells suppresses the activation of autoreactive T cells that have escaped the other mechanisms of tolerance induction. Two experimental models have been developed which have allowed the definition of unique populations of regulatory T cells. In one model, autoimmunity is induced by depletion of regulatory T cells from adult animals, while in the second model, the development of regulatory T cells is abolished in neonatal animals.

Structural basis of MHC class I recognition by natural killer cell receptors

Summary: Natural killer (NK)‐cell function is regulated by NK receptors that recognize MHC class I (MHC‐I) molecules on target cells. Two structurally distinct families of NK receptors have been identified, the immunoglobulin‐like family (killer cell immunoglobulin‐like receptors (KIRs), leukocyte immunoglobulin‐like receptors (LIRs)) and the C‐type lectin‐like family (Ly49, CD94/NKG2A, NKG2D, CD69). Recently, the three‐dimensional structures of several NK receptors were determined, in free form or bound to MHC‐I. These include those of unbound KIRs, NKG2D, CD69, LIR‐1 and the CD94 subunit of the CD94/NKG2A heterodimer. Together, these structures define the basic molecular architecture of both the immunoglobulin‐like and C‐type lectin‐like families of NK receptors. In addition, crystal structures have been reported for the complex between Ly49A and H‐2Dd, and for KIR2DL2 bound to HLA‐Cw3. The complex structures provide a framework for understanding MHC‐I recognition by NK receptors from both families and reveal striking differences in the nature of this recognition, despite the receptors’ functional similarity.

Structural Basis of the CD8αβ/MHC Class I Interaction: Focused Recognition Orients CD8β to a T Cell Proximal Position

In the immune system, B cells, dendritic cells, NK cells, and T lymphocytes all respond to signals received via ligand binding to receptors and coreceptors. Although the specificity of T cell recognition is determined by the interaction of T cell receptors with MHC/peptide complexes, the development of T cells in the thymus and their sensitivity to Ag are also dependent on coreceptor molecules CD8 (for MHC class I (MHCI)) and CD4 (for MHCII). The CD8αβ heterodimer is a potent coreceptor for T cell activation, but efforts to understand its function fully have been hampered by ignorance of the structural details of its interactions with MHCI. In this study we describe the structure of CD8αβ in complex with the murine MHCI molecule H-2Dd at 2.6 Å resolution. The focus of the CD8αβ interaction is the acidic loop (residues 222–228) of the α3 domain of H-2Dd. The β subunit occupies a T cell membrane proximal position, defining the relative positions of the CD8α and CD8β subunits. Unlike the CD8αα homodimer, CD8αβ does not contact the MHCI α2- or β2-microglobulin domains. Movements of the CD8α CDR2 and CD8β CDR1 and CDR2 loops as well as the flexibility of the H-2Dd CD loop facilitate the monovalent interaction. The structure resolves inconclusive data on the topology of the CD8αβ/MHCI interaction, indicates that CD8β is crucial in orienting the CD8αβ heterodimer, provides a framework for understanding the mechanistic role of CD8αβ in lymphoid cell signaling, and offers a tangible context for design of structurally altered coreceptors for tumor and viral immunotherapy.

Interaction of the NK Cell Inhibitory Receptor Ly49A with H-2Dd:Identification of a Site Distinct from the TCR Site

Natural killer cell function is controlled by interaction of NK receptors with MHC I molecules expressed on target cells. We describe the binding of bacterially expressed Ly49A, the prototype murine NK inhibitory receptor, to similarly engineered H-2Dd. Despite its homology to C-type lectins, Ly49A binds independently of carbohydrate and Ca2+ and shows specificity for MHC I but not bound peptide. The affinity of the Ly49A/H-2Dd interaction as determined by surface plasmon resonance is from 6 to 26 microM at 25 degrees C and is greater by ultracentrifugation at 4 degrees C. Biotinylated Ly49A stains H-2Dd-expressing cells. Competition experiments indicate that the Ly49A and T cell receptor (TCR) binding sites on MHC I are distinct, suggesting complex regulation of cells that bear both TCR and NK cell receptors.

Structural Basis of the CD8[alpha beta]/MHC Class I Interaction: Focused Recognition Orients CD8[beta] to a T Cell Proximal Position[superscript 1,2]

In the immune system, B cells, dendritic cells, NK cells, and T lymphocytes all respond to signals received via ligand binding to receptors and coreceptors. Although the specificity of T cell recognition is determined by the interaction of T cell receptors with MHC/peptide complexes, the development of T cells in the thymus and their sensitivity to Ag are also dependent on coreceptor molecules CD8 (for MHC class I (MHCI)) and CD4 (for MHCII). The CD8αβ heterodimer is a potent coreceptor for T cell activation, but efforts to understand its function fully have been hampered by ignorance of the structural details of its interactions with MHCI. In this study we describe the structure of CD8αβ in complex with the murine MHCI molecule H-2Dd at 2.6 Å resolution. The focus of the CD8αβ interaction is the acidic loop (residues 222–228) of the α3 domain of H-2Dd. The β subunit occupies a T cell membrane proximal position, defining the relative positions of the CD8α and CD8β subunits. Unlike the CD8αα homodimer, CD8αβ does not contact the MHCI α2- or β2-microglobulin domains. Movements of the CD8α CDR2 and CD8β CDR1 and CDR2 loops as well as the flexibility of the H-2Dd CD loop facilitate the monovalent interaction. The structure resolves inconclusive data on the topology of the CD8αβ/MHCI interaction, indicates that CD8β is crucial in orienting the CD8αβ heterodimer, provides a framework for understanding the mechanistic role of CD8αβ in lymphoid cell signaling, and offers a tangible context for design of structurally altered coreceptors for tumor and viral immunotherapy.