Leonidas N. Carayannopoulos

Cutting Edge: Murine UL16-Binding Protein-Like Transcript 1: A Newly Described Transcript Encoding a High-Affinity Ligand for Murine NKG2D

Murine NKG2D is known to recognize H60 and five RAE1 variants. The human homologue recognizes both inducible MHC class I chain-related gene and constitutive (UL16-binding protein (ULBP)) ligands. Widely expressed, the latter are thought to mark transformed or infected cells for destruction by NK cells in the context of down-regulated cell surface class I (i.e., the “missing self”-response). Unlike MIC and ULBP however, mRNA for the murine ligands appears only in very limited contexts in the mature animal. In this study, we describe a NKG2D ligand termed “murine ULBP-like transcript 1 (MULT1) whose mRNA appears to be widely expressed in adult parenchyma. This molecule possesses MHC class I-like α1 and α2 domains as well as a large cytoplasmic domain. Recombinant MULT1 binds NKG2D with relatively high affinity (KD ≈ 6 nM) and low koff (∼0.006s−1). Expression of MULT1 by normally resistant RMA cells results in their susceptibility to lysis by C57BL/6 splenocytes.

Costimulation of multiple NK cell activation receptors by NKG2D

The activation of NK cells is mediated through specific interactions between activation receptors and their respective ligands. Little is known, however, about whether costimulation, which has been well characterized for T cell activation, occurs in NK cells. To study the function of NKG2D, a potential NK costimulatory receptor, we have generated two novel hamster mAbs that recognize mouse NKG2D. FACS analyses demonstrate that mouse NKG2D is expressed on all C57BL/6 IL-2-activated NK (lymphokine-activated killer (LAK)) cells, all splenic and liver NK cells, and ∼50% of splenic NKT cells. Consistent with limited polymorphism of NKG2D, its sequence is highly conserved, and the anti-NKG2D mAbs react with NK cells from a large number of different mouse strains. In chromium release assays, we show that stimulation of NK cells with anti-NKG2D mAb can redirect lysis. Also, enhanced lysis of transfected tumor targets expressing NKG2D ligand could be inhibited by addition of anti-NKG2D mAb. Interestingly, stimulation of LAK cells via NKG2D alone does not lead to cytokine release. However, stimulation of LAK via both an NK activation receptor (e.g., CD16, NK1.1, or Ly-49D) and NKG2D leads to augmentation of cytokine release compared with stimulation through the activation receptor alone. These results demonstrate that NKG2D has the ability to costimulate multiple NK activation receptors.

Costimulation through NKG2D Enhances Murine CD8+ CTL Function: Similarities and Differences between NKG2D and CD28 Costimulation

Multiple studies have demonstrated that the NK cell activating receptor NKG2D can function as a costimulatory receptor for both mouse and human CD8+ T cells. However, it has recently been suggested that stimulation through NKG2D is insufficient for costimulation of CD8+ T cells. To aid in the delineation of NKG2D function in CTL responses, we investigated whether stimulation of NKG2D by the natural ligand RAE1ε was able to costimulate effector functions of a murine CTL line generated from DUC18 TCR transgenic mice. We found that NKG2D was able to costimulate DUC CTL responses and did so in a manner similar to CD28 costimulation. The T cells exhibited increased proliferation, IFN-γ release, and cytotoxicity when presented antigenic peptide by P815 cells expressing RAE1ε or B7-1 compared with untransfected P815. In addition, both RAE1ε and B7-1 enhanced Ag-independent IFN-γ secretion in response to IL-12 and IL-18 by DUC CTL. However, only costimulation through CD28 allowed for DUC CTL survival upon secondary stimulation, whereas ligation of NKG2D, but not CD28, induced DUC CTL to form an immune synapse with target cells in the absence of TCR stimulation. Understanding the outcomes of these differences may allow for a better understanding of T cell costimulation in general.

Ligands for murine NKG2D display heterogeneous binding behavior

NKG2D transmits stimulatory signals to natural killer cells and other hematopoietic cells, leading to enhanced proliferation, cytokine secretion and target killing. Murine and human NKG2D eachrecognize five known class I‐related molecules with distinct primary structures. Here, we used surface plasmon resonance to examine the binding of murine NKG2D to its cognate ligands: RAE‐1B6 (a newly described C57BL/6J variant of RAE‐1), RAE‐1δ (common to BALB and C57BL6/J), and H60 (expressed in BALB, but not C57BL/6J). While RAE‐1B6 and H60 display relatively high affinities for NKG2D with KD in the 20‐30 nM range and koff in the 0.03 s–1 to 0.06 s–1 range (t1/2 approximately 10–20 s); the RAE‐1δ variant binds with a lower affinity: KD of approximately 750 nM. Furthermore, RAE‐1δ displays biphasic kinetics with dominant koff of approximately 0.2 s–1 (t1/2 approximately 3 s), partially explaining the lower affinity. Thus, H60 and RAE‐1B6 bind NKG2D with almost identical kinetics while sharing only 20% amino acid sequence identity; whereas other RAE‐1 molecules demonstrate faster dissociationand lower affinities than RAE‐1B6 despite sharing 90% sequence identity. C57BL/6J mice, although not expressing the H60 gene product, retain a high‐affinity ligand for NKG2D in the form of RAE‐1B6.

IFN-Dependent Down-Regulation of the NKG2D Ligand H60 on Tumors

In this study, we show that IFN-γ or IFN-α reduce expression of H60 on 3′-methylcholanthrene (MCA) sarcomas from 129/Sv mice. As determined by flow cytometry using either NKG2D tetramers or NKG2D ligand-specific mAb, H60 was identified as the NKG2D ligand most frequently expressed on these sarcomas, and its expression was selectively down-regulated by either IFN-γ or IFN-α in a manner that was dose- and time-dependent and reversible. Down-regulation occurred at the transcript level and was STAT1-dependent. It also had functional consequences. IFN-γ-treated MCA sarcomas with high levels of H60 were resistant to killing by IL-2-activated NK cells. Resistance was not solely dependent on enhanced MHC class I expression but rather also required H60 down-regulation. IFN-γ-treated tumor cells also displayed diminished capacity to down-regulate NKG2D on freshly isolated NK cells. Transplanted tumor cells reisolated from immunocompetent mice displayed reduced H60 expression and increased MHC class I expression compared with tumor cells that were either left unmanipulated or reisolated from mice treated with neutralizing IFN-γ-specific mAb. This report thus represents the first demonstration that certain cytokines and specifically the IFNs regulate expression of specific NKG2D ligands on murine tumors. This process most likely helps to specify the type of immune effector cell populations that participate in host-protective antitumor responses.

Zoonotic orthopoxviruses encode a high-affinity antagonist of NKG2D

NK and T lymphocytes express both activating and inhibiting receptors for various members of the major histocompatibility complex class I superfamily (MHCISF). To evade immunologic cytotoxicity, many viruses interfere with the function of these receptors, generally by altering the displayed profile of MHCISF proteins on host cells. Using a structurally constrained hidden Markov model, we discovered an orthopoxvirus protein, itself distantly class I–like, that acts as a competitive antagonist of the NKG2D activating receptor. This orthopoxvirus MHC class I–like protein (OMCP) is conserved among cowpox and monkeypox viruses, secreted by infected cells, and bound with high affinity by NKG2D of rodents and humans (KD ∼ 30 and 0.2 nM, respectively). OMCP blocks recognition of host-encoded ligands and inhibits NKG2D-dependent killing by NK cells. This finding represents a novel mechanism for viral interference with NKG2D and sheds light on intercellular recognition events underlying innate immunity against emerging orthopoxviruses.

Immunodeficient Mouse Strains Display Marked Variability in Growth of Human Melanoma Lung Metastases

Purpose: Immunodeficient mice serve as critical hosts for transplantation of xenogeneic cells for in vivo analysis of various biological processes. Because investigators typically select one or two immunodeficient mouse strains as recipients, no comprehensive study has been published documenting differences in human tumor engraftment. Taking advantage of the increased metastatic potential of RhoC-expressing human (A375) melanoma cells, we evaluate four immunodeficient mouse strains: severe combined immunodeficiency (scid), nonobese diabetic (NOD)-scid, NOD-scid β2mnull, and NOD-scid IL2Rγnull as xenograft tumor recipients. Experimental Design: Bioluminescence, magnetic resonance imaging, and histopathology were used to monitor serial tumor growth. Natural killer (NK) cell function was examined in each mouse strain using standard 51Chromium release assays. Results: Melanoma metastases growth is delayed and variable in scid and NOD-scid mice. In contrast, NOD-scid β2mnull and NOD-scid IL2Rγnull mice show rapid tumor engraftment, although tumor growth is variable in NOD-scid β2mnull mice. NK cells were detected in all strains except NOD-scid IL2Rγnull, and in vitro activated scid, NOD-scid, and NOD-scid β2mnull NK cells kill human melanoma lines and primary melanoma cells. Expression of human NKG2D ligands MHC class I chain–related A and B molecules renders melanoma susceptible to murine NK cell–mediated cytotoxicity and killing is inhibited by antibody blockade of murine NKG2D. Conclusions: Murine NKG2D recognition of MICA/B is an important receptor-ligand interaction used by NK cells in immunodeficient strains to limit engraftment of human tumors. The absolute NK deficiency in NOD-scid IL2Rγnull animals makes this strain an excellent recipient of melanoma and potentially other human malignancies.

Differential Susceptibility of RAE-1 Isoforms to Mouse Cytomegalovirus

ABSTRACT The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1δ, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1δ and RAE-1γ in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1δ form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1δ compared to RAE-1γ but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1δ. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.

Cutting edge: FcR-like 5 on innate B cells is targeted by a poxvirus MHC class I-like immunoevasin.

Under selective pressure from host immunity, viruses have retained genes encoding immunoevasins, molecules interfering with host viral recognition and clearance. Due to their binding specificities, immunoevasins can be exploited as affinity labels to identify host-encoded molecules of previously unsuspected importance in defense against the relevant class of virus. We previously described an orthopoxvirus MHC class I-like protein (OMCP) that binds with high affinity to the activating receptor NKG2D on NK and T cell subsets, implicating NKG2D in antiorthopoxvirus immunity. In this study, we report that OMCP also binds in an NKG2D-independent manner to B cells and monocytes/macrophages. We identify murine FcR-like 5 (FCRL5), an orphan immunoregulatory protein highly expressed by innate B lymphocytes, as a specific receptor for OMCP. The three N-terminal Ig domains of FCRL5 are required for OMCP binding. The targeting of FCRL5 by an orthopoxvirus immunoevasin strongly implicates it in contributing to host defense against zoonotic orthopoxviruses.

Markers of Nonselective and Specific NK Cell Activation

NK cell activation is controlled by the integration of signals from cytokine receptors and germline–encoded activation and inhibitory receptors. NK cells undergo two distinct phases of activation during murine CMV (MCMV) infection: a nonselective phase mediated by proinflammatory cytokines and a specific phase driven by signaling through Ly49H, an NK cell activation receptor that recognizes infected cells. We sought to delineate cell surface markers that could distinguish NK cells that had been activated nonselectively from those that had been specifically activated through NK cell receptors. We demonstrated that stem cell Ag 1 (Sca-1) is highly upregulated during viral infections (to an even greater extent than CD69) and serves as a novel marker of early, nonselective NK cell activation. Indeed, a greater proportion of Sca-1+ NK cells produced IFN-γ compared with Sca-1− NK cells during MCMV infection. In contrast to the universal upregulation of Sca-1 (as well as KLRG1) on NK cells early during MCMV infection, differential expression of Sca-1, as well as CD27 and KLRG1, was observed on Ly49H+ and Ly49H− NK cells late during MCMV infection. Persistently elevated levels of KLRG1 in the context of downregulation of Sca-1 and CD27 were observed on NK cells that expressed Ly49H. Furthermore, the differential expression patterns of these cell surface markers were dependent on Ly49H recognition of its ligand and did not occur solely as a result of cellular proliferation. These findings demonstrate that a combination of Sca-1, CD27, and KLRG1 can distinguish NK cells nonselectively activated by cytokines from those specifically stimulated through activation receptors.