Llewellyn H. Mason

LGL-1: a non-polymorphic antigen expressed on a major population of mouse natural killer cells.

Rat mAb have been raised to mouse liver-derived large granular lymphocytes (LGL). One of these mAb (4D11) binds specifically to mouse LGL and appears to recognize a non-allelic determinant on NK-active cell populations. The Ag recognized by 4D11 is expressed on LGL of all mouse strains tested, including C57BL/6 (B6), BALB/c, C3H/HeJ, and SJL/J; thus, we have provisionally called this Ag LGL-1. Analysis of various lymphoid and hemopoietic tissues has indicated that only normal tissues known to contain NK activity have 4D11+ cells. With B6 and B6 congenic strains, a positive correlation exists between the number of LGL in a sample and the percentage of 4D11 immunofluorescence-positive cells detected by flow cytometric analysis. Dual color immunofluorescence analyses indicate that some LGL-1+ cells are also stained for Ly-1 and Thy-1. A very small subset exists that is weakly positive for CD3 and LGL-1. However, virtually no cells are seen which co-express LGL-1 and Ly-2. LU activity against YAC-1 targets was increased 7- to 700-fold in LGL-1+ spleen cells obtained by cell sorting from several different strains of mice (B6, BALB/c, C3H/HeJ, SJL/J, and athymic/nude). Sorted, LGL-1- spleen cells contained little or no NK activity. Cells positively selected for LGL-1 also contained between 50 and 60% LGL by morphology. By using facilitated in vitro antibody plus C treatments, the majority of NK activity can be depleted from both B6 spleen and liver-derived leukocyte populations enriched for NK cells. mAb 4D11 was also shown to precipitate a protein of approximately 87 kDa from the surface of enriched murine NK cells. This mAb should prove valuable for understanding the role of NK cells in the immune response.

Induction of DAP12 phosphorylation, calcium mobilization, and cytokine secretion by Ly49H.

The ability of several Ly49 family members to inhibit natural killer (NK) cell functions through recruitment of SHP‐1 phosphatase has been reported. In contrast, the mechanisms underlying the activating signal generated by Ly49D are poorly understood. A homodimeric phosphoprotein (pp16) that physically and functionally associates with Ly49D has been described. In this study, a rabbit anti‐mouse pp16 antiserum was generated and used to demonstrate that pp16 corresponds to the recently described DAP12 molecule. In addition, we show that a second Ly49 family member that lacks an immunoreceptor tyrosine‐based inhibitory motif and contains a charged residue in the transmembrane domain, Ly49H, also associates with DAP12. Furthermore, we show that engagement of the Ly49H/DAP12 complex results in phosphorylation of DAP12, intracellular calcium mobilization, and tumor necrosis factor secretion in transfected cells. These results thus provide evidence that Ly49H is an activating receptor that associates with DAP12, previously described as a pp16 component of the Ly49D receptor complex. J. Leukoc. Biol. 66: 165–171; 1999.

Interaction of Ly-49D+ NK Cells with H-2Dd Target Cells Leads to Dap-12 Phosphorylation and IFN-γ Secretion

Murine Ly-49D augments NK cell function upon recognition of target cells expressing H-2Dd. Ly-49D activation is mediated by the immunoreceptor tyrosine-based activation motif-containing signaling moiety Dap-12. In this report we demonstrate that Ly-49D receptor ligation can lead to the rapid and potent secretion of IFN-γ. Cytokine secretion can be induced from Ly-49D+ NK cells after receptor ligation with Ab or after interaction with target cells expressing their H-2Dd ligand. Consistent with the dominant inhibitory function of Ly-49G, NK cells coexpressing Ly-49D and Ly-49G show a profound reduction in IFN-γ secretion after interaction with targets expressing their common ligand, H-2Dd. Importantly, we are able to demonstrate for the first time that effector/target cell interactions using Ly-49D+ NK cells and H-2Dd targets result in the rapid phosphorylation of Dap-12. However, Dap-12 is not phosphorylated when Ly-49D+ NK cells coexpress the inhibitory receptor, Ly-49G. These studies are novel in describing Ly-49 activation vs inhibition, where two Ly-49 receptors recognize the same class I ligand, with the dominant inhibitory receptor down-regulating phosphorylation of Dap-12, cytokine secretion, and cytotoxicity in NK cells.

Regulation of Ly49D/DAP12 Signal Transduction by Src-Family Kinases and CD45

Activating, DAP12-coupled members of the Ly-49 family of NK cell receptors help control viral infections in mice. However, the kinases and/or phosphatases mediating tyrosine phosphorylation of Ly-49D-associated DAP12 have not been elucidated. In this study, we show for the first time that Src family tyrosine kinases are physically and functionally associated with Ly-49D/DAP12 signaling in murine NK cells. Specifically, we demonstrate the following: 1) inhibition of Src family kinases suppresses DAP12 phosphorylation and downstream DAP12 signals; 2) both Fyn and Lck are capable of phosphorylating DAP12; and 3) both kinases coimmunoprecipitate with the Ly-49D/DAP12 complex in NK cells. Although we detect enhanced phosphorylation of Fyn upon Ly-49D cross-linking in NK cells, Ly-49D-mediated events in both Fyn−/− and Fyn/Lck−/− mice appear normal, reinforcing the theme of redundancy in the ability of Src family kinases to initiate activation events. In contrast to disruption of specific Src family enzymes, Ly-49D/DAP12-mediated calcium mobilization and cytokine production by CD45 null NK cells are defective. Although others have ascribed the effects of CD45 mutation solely on the suppression of Src family activity, we demonstrate in this study that DAP12 is hyperphosphorylated in CD45 null NK cells, resulting in uncoordinated tyrosine-mediated signaling upon Ly-49D ligation. Therefore, although our data are consistent with a Src kinase activity proximally within DAP12 signaling, DAP12 also appears to be a substrate of CD45, suggesting a more complex role for this phosphatase than has been reported previously.

Receptor Glycosylation Regulates Ly-49 Binding to MHC Class I

Murine NK cells express the Ly-49 family of class I MHC-binding receptors that control their ability to lyse tumor or virally infected host target cells. X-ray crystallography studies have identified two predominant contact sites (sites 1 and 2) that are involved in the binding of the inhibitory receptor, Ly-49A, to H-2Dd. Ly-49G2 (inhibitory) and Ly-49D (activating) are highly homologous to Ly-49A and also recognize H-2Dd. However, the binding of Ly-49D and G2 to H-2Dd is of lower affinity than Ly-49A. All Ly-49s contain N-glycosylation motifs; however, the importance of receptor glycosylation in Ly-49-class I interactions has not been determined. Ly-49D and G2 contain a glycosylation motif (NTT (221–223)), absent in Ly-49A, adjacent to one of the proposed binding sites for H-2Dd (site 2). The presence of a complex carbohydrate group at this critical site could interfere with class I binding. In this study, we are able to demonstrate for the first time that Ly-49D binds H-2Dd in the presence of mouse β2-microglobulin. We also demonstrate that glycosylation of the NTT (221–23) motif of Ly-49D inteferes with recognition of H-2Dd. Alteration of the Ly-49D-NTT (221–23) motif to abolish glycosylation at this site resulted in enhanced H-2Dd binding and receptor activation. Furthermore, glycosylation of Ly-49G2 at NTT (221–23) also reduces receptor binding to H-2Dd tetramers. Therefore, the addition of complex carbohydrates to the Ly-49 family of receptors may represent a mechanism by which NK cells regulate affinity for host class I ligands.

LGL-1: a potential triggering molecule on murine NK cells.

Natural killer (NK) cells mediate non‐major histocompatibility complex‐restricted lysis of tumor cells, lymphokine‐activated killing (LAK), antibody‐dependent cellular cytotoxicity (ADCC), and reverse ADCC (RADCC). LGL‐1+ cells identify a major subset (50%) of murine NK cells. Here we demonstrate that monoclonal antibodies (mAbs) to LGL‐1 consistently induce interleukin‐2‐cultured, and Corynebacterium parvum (in vivo)‐activated NK cells to induce RADCC. LGL‐1 triggering of activated NK cells coincides with enhanced LGL‐1 expression. Testing of murine mAbs to epitopes of CD2 only appears to augment RADCC induced by mAb NK‐1.1 on fresh NK cells. Immunoprecipitation of the LGL‐1 antigen reveals a highly disulfide‐linked 40‐kDa homodimer subunit that is N‐glycosylated. Therefore, LGL‐1 may be similar to other recently characterized NK‐associated antigens such as NK‐1.1, Ly‐49, and NKR‐PI. We conclude that although LGL‐1 is expressed on “resting” NK cells, enhanced surface expression following activation is usually required for it to act as a signaling molecule. J. Leukoc. Biol. 55: 362–370; 1994.

Recognition of CHO cells by inhibitory and activating Ly-49 receptors

Upon ligand recognition, members of the murine Ly‐49 receptor family can transmit inhibitory or activating signals that regulate NK cell function. Ly‐49A, G, and D have been shown to recognize the murine class I molecule H‐2Dd as a potential ligand. Recent studies also have demonstrated also that Ly‐49D+ NK cells can lyse CHO cells, although the ligand responsible for this recognition was not identified. Because allorecognition by NK cells may be important in bone‐marrow transplantation and because of the overlapping class I recognition by these receptors, recognition of CHO cells by Ly‐49G and A was investigated. Our data suggest that Ly‐49G and probably A transmit inhibitory signals in response to CHO cells. Receptor inhibition was assessed by examining NK lytic function, IFN‐γ secretion, and DAP12 phosphorylation in response to CHO cells by sorted subsets of Ly‐49D vs. G B6 NK cells. Our results suggest that CHO cells may express a common ligand(s) that is capable of engaging Ly‐49D, G, and possibly A in C576BL/6 NK cells. In addition to our findings that Ly‐49 inhibitory receptors also recognize CHO cells, activating receptors other than Ly‐49D are present in B6 mice that can lyse CHO cells.