The NKG2D ligand ULBP4 is not expressed by human monocytes.

The C-type lectin-like receptor NKG2D contributes to the immunosurveillance of virally infected and malignant cells by cytotoxic lymphocytes. A peculiar and puzzling feature of the NKG2D-based immunorecognition system is the high number of ligands for this single immunoreceptor. In humans, there are a total of eight NKG2D ligands (NKG2DL) comprising two members of the MIC (MICA, MICB) and six members of the ULBP family of glycoproteins (ULBP1 to ULBP6). While MICA has been extensively studied with regard to its biochemistry, cellular expression and function, very little is known about the NKG2DL ULBP4. This is, at least in part, due to its rather restricted expression by very few cell lines and tissues. Recently, constitutive ULBP4 expression by human monocytes was reported, questioning the view of tissue-restricted ULBP4 expression. Here, we scrutinized ULBP4 expression by human peripheral blood mononuclear cells and monocytes by analyzing ULBP4 transcripts and ULBP4 surface expression. In contrast to MICA, there was no ULBP4 expression detectable, neither by freshly isolated monocytes nor by PAMP-activated monocytes. However, a commercial antibody erroneously indicated surface ULBP4 on monocytes due to a non-ULBP4-specific binding activity, emphasizing the critical importance of validated reagents for life sciences. Collectively, our data show that ULBP4 is not expressed by monocytes, and likely also not by other peripheral blood immune cells, and therefore exhibits an expression pattern rather distinct from other human NKG2DL.

Arming cytotoxic lymphocytes for cancer immunotherapy by means of the NKG2D/NKG2D-ligand system.

INTRODUCTION: The activating NKG2D receptor plays a central role in the immune recognition and elimination of abnormal self-cells by cytotoxic lymphocytes. NKG2D binding to cell stress-inducible ligands (NKG2DL) up-regulated on cancer cells facilitates their immunorecognition. Yet tumor cells utilize various escape mechanisms to avert NKG2D-based immunosurveillance. Hence, therapeutic strategies targeting the potent NKG2D/NKG2DL axis and such immune escape mechanisms become increasingly attractive in cancer therapy. AREAS COVERED: This perspective provides a brief introduction into the NKG2D/NKG2DL axis and its relevance for cancer immune surveillance. Subsequently, the most advanced therapeutic approaches targeting the NKG2D system are presented focusing on NKG2D-CAR engineered immune cells and antibody-mediated strategies to inhibit NKG2DL shedding by tumors. EXPERT OPINION: Thus far, NKG2D-CAR engineered lymphocytes represent the most advanced therapeutic approach utilizing the NKG2D system. Similarly to other tumor-targeting CAR approaches, NKG2D-CAR cells demonstrate powerful on-target activity, but may also cause off-tumor toxicities or lose efficacy, if NKG2DL expression by tumors is reduced. However, NKG2D-CAR cells also act on the tumor microenvironment curtailing its immunosuppressive properties, thus providing an independent therapeutic benefit. The potency of tumoricidal NKG2D-expressing lymphocytes can be further boosted by enhancing NKG2DL expression through small molecules and therapeutic antibodies inhibiting tumor-associated shedding of NKG2DL.

Impairment of NKG2D-Mediated Tumor Immunity by TGF-ß.

Transforming growth factor-ß (TGF-ß) suppresses innate and adaptive immune responses via multiple mechanisms. TGF-ß also importantly contributes to the formation of an immunosuppressive tumor microenvironment thereby promoting tumor growth. Amongst others, TGF-ß impairs tumor recognition by cytotoxic lymphocytes via NKG2D. NKG2D is a homodimeric C-type lectin-like receptor expressed on virtually all human NK cells and cytotoxic T cells, and stimulates their effector functions upon engagement by NKG2D ligands (NKG2DL). While NKG2DL are mostly absent from healthy cells, their expression is induced by cellular stress and malignant transformation, and, accordingly, frequently detected on various tumor cells. Hence, the NKG2D axis is thought to play a decisive role in cancer immunosurveillance and, obviously, often is compromised in clinically apparent tumors. There is mounting evidence that TGF-ß, produced by tumor cells and immune cells in the tumor microenvironment, plays a key role in blunting the NKG2D-mediated tumor surveillance. Here, we review the current knowledge on the impairment of NKG2D-mediated cancer immunity through TGF-ß and discuss therapeutic approaches aiming at counteracting this major immune escape pathway. By reducing tumor-associated expression of NKG2DL and blinding cytotoxic lymphocytes through down-regulation of NKG2D, TGF-ß is acting upon both sides of the NKG2D axis severely compromising NKG2D-mediated tumor rejection. Consequently, novel therapies targeting the TGF-ß pathway are expected to reinvigorate NKG2D-mediated tumor elimination and thereby to improve the survival of cancer patients.

The NKG2D axis: an emerging target in cancer immunotherapy.

INTRODUCTION: The immunoreceptor NKG2D belongs to the best-characterized activating receptors of cytotoxic lymphocytes. NKG2D binds to a variety of cell surface glycoproteins distantly related to MHC class I molecules, termed NKG2D ligands (NKG2DL). NKG2DL are inducibly expressed upon cellular stress, viral infection or malignant transformation thus marking 'stressed' or 'harmful' cells for clearance through NKG2D+ lymphocytes. However, certain viruses and many tumors employ various strategies to escape from NKG2D-mediated immunosurveillance. Areas covered: Expression and regulation of both NKG2D and NKG2DL, especially at sites of immune responses or in the tumor microenvironment, as well as mechanisms of NKG2D escape strategies, as their understanding is key for harnessing the NKG2D/NKG2DL axis for immunotherapy. Studies documenting the importance of the NKG2D/NKG2DL axis for cancer immunosurveillance. Therapeutic approaches targeting the NKG2D/NKG2DL axis in cancer. Expert opinion: The selective expression of NKG2DL on malignant cells together with the strong activating potency of NKG2D renders the NKG2D/NKG2DL axis a prime target for immunotherapies. Based on a thorough understanding of the NKG2D/NKG2DL system as well as of the most relevant escape strategies of tumors, the diligent and thoughtful design of novel treatment modalities harnessing the NKG2D/NKG2DL axis holds great promise for the future therapy of cancer.

Precursor-Based Selective Methyl Labeling of Cell-Free Synthesized Proteins.

NMR studies of large proteins are complicated by pronounced spectral overlap and large line width. Reducing complexity by [13C, 1H] selective labeling of l-Val, l-Leu, and/or l-Ile residues in combination with optional perdeuteration is therefore commonly approached by supplying labeled amino acid precursors into bacterial expression cultures, although often compromised by high label costs, precursor instability, and label scrambling. Cell-free expression combines efficient production of membrane proteins with significant advantages for protein labeling such as small reaction volumes, defined amino acid pools, and reliable label incorporation. While amino acid specific isotopic labeling of proteins is routine application, the amino acid methyl side-chain labeling was so far difficult as appropriately labeled amino acids are hardly available. On the basis of recent proteome analyses of cell-free lysates, we have developed a competitive strategy for efficient methyl labeling of proteins based on conversion of supplied precursors. Pathway complexity of methyl side-chain labeling was reduced by implementing the promiscuous aminotransferase IlvE catalyzing the selective l-Leu, l-Val, or l-Ile biosynthesis from specific ketoacid precursors. Precursor-based l-Leu and l-Val synthesis was demonstrated with the cell-free labeling of peptidyl-prolyl cis/trans isomerase cyclophilin D and of the proton pump proteorhodopsin. The strategy is fast and cost-effective and enables the straightforward methyl side-chain labeling of individual amino acid types. It can easily be applied to any cell-free synthesized protein.