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Systemic lupus erythematosus (SLE) is a severe autoimmune disease of unknown etiology. The major histocompatibility complex (MHC) class I-related chain A (MICA) and B (MICB) are stress-inducible cell surface molecules. MICA and MICB label malfunctioning cells for their recognition by cytotoxic lymphocytes such as natural killer (NK) cells. Alterations in this recognition have been found in SLE. MICA/MICB can be shed from the cell surface, subsequently acting either as a soluble decoy receptor (sMICA/sMICB) or in CD4+ T-cell expansion. Conversely, NK cells are frequently defective in SLE and lower NK cell numbers have been reported in patients with active SLE. However, these cells are also thought to exert regulatory functions and to prevent autoimmunity. We therefore investigated whether, and how, plasma membrane and soluble MICA/B are modulated in SLE and whether they influence NK cell activity, in order to better understand how MICA/B may participate in disease development. We report significantly elevated concentrations of circulating sMICA/B in SLE patients compared with healthy individuals or a control patient group. In SLE patients, sMICA concentrations were significantly higher in patients positive for anti-SSB and anti-RNP autoantibodies. In order to study the mechanism and the potential source of sMICA, we analyzed circulating sMICA concentration in Behcet patients before and after interferon (IFN)-α therapy: no modulation was observed, suggesting that IFN-α is not intrinsically crucial for sMICA release in vivo. We also show that monocytes and neutrophils stimulated in vitro with cytokines or extracellular chromatin up-regulate plasma membrane MICA expression, without releasing sMICA. Importantly, in peripheral blood mononuclear cells from healthy individuals stimulated in vitro by cell-free chromatin, NK cells up-regulate CD69 and CD107 in a monocyte-dependent manner and at least partly via MICA-NKG2D interaction, whereas NK cells were exhausted in SLE patients. In conclusion, sMICA concentrations are elevated in SLE patients, whereas plasma membrane MICA is up-regulated in response to some lupus stimuli and triggers NK cell activation. Those results suggest the requirement for a tight control in vivo and highlight the complex role of the MICA/sMICA system in SLE.
Functional responses of natural killer (NK) cells including eradication of “harmful” cells and modulation of immune responses are regulated by a broad variety of activating and inhibitory NK receptors. Whereas the leukocyte receptor complex (LRC) encodes for NK receptors of the immunoglobulin superfamily, genes of C-type lectin-like NK receptors are clustered in the mammalian natural killer gene complex (NKC). Besides the thoroughly studied C-type lectin-like receptors NKG2D, CD94/NKG2x, and members of the murine Ly49 subfamily, the NKC also encodes for NK receptors of the less characterized NKRP1 subfamily. The prototypic mouse NKRP1 receptor is Nkrp1c (also known as NK1.1), while human members of the NKRP1 subfamily are NKRP1A, NKp80, and NKp65. The latter are not straight homologs of mouse NKRP1 receptors, but share distinct subfamily-specific traits classifying them as members of the NKRP1 subfamily. Ligands of the human NKPR1 receptors are likewise C-type lectin-like glycoproteins belonging to the CLEC2 subfamily (i.e., LLT1, AICL, and KACL), and are encoded in the NKC in tight genetic linkage to their respective receptors. Similarly, certain members of the mouse NKRP1 subfamily interact with genetically coupled CLEC2 glycoproteins, while the reasons for this intriguing tight genetic linkage remain unknown. Recent studies provided new and unique insights into the expression, interaction, and signaling of NKRP1 receptors and their ligands, thereby substantially advancing our understanding of their function and biology. Here, we review our current knowledge on NKRP1 receptors and their genetically linked CLEC2 ligands with an emphasis on the human receptor/ligand pairs NKRP1A-LLT1, NKp80-AICL, and NKp65-KACL.
Transforming growth factor-β (TGF-β) suppresses innate and adaptive immune responses via multiple mechanisms. TGF-β also importantly contributes to the formation of an immunosuppressive tumor microenvironment thereby promoting tumor growth. Amongst others, TGF-β 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-β, 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-β 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-β is acting upon both sides of the NKG2D axis severely compromising NKG2D-mediated tumor rejection. Consequently, novel therapies targeting the TGF-β pathway are expected to reinvigorate NKG2D-mediated tumor elimination and thereby to improve the survival of cancer patients.
Natural Killer Gene Complex (NKC)–encoded C-type lectin-like receptors (CTLRs) are expressed on various immune cells including T cells, NK cells and myeloid cells and thereby contribute to the orchestration of cellular immune responses. Some NKC-encoded CTLRs are grouped into the C-type lectin family 2 (CLEC2 family) and interact with genetically linked CTLRs of the NKRP1 family. While many CLEC2 family members are expressed by hematopoietic cells (e.g. CD69 (CLEC2C)), others such as the keratinocyte-associated KACL (CLEC2A) are specifically expressed by other tissues. Here we provide the first characterization of the orphan gene CLEC2L. In contrast to other CLEC2 family members, CLEC2L is conserved among mammals and located outside of the NKC. We show that CLEC2L-encoded CTLRs are expressed as non-glycosylated, disulfide-linked homodimers at the cell surface. CLEC2L expression is fairly tissue-restricted with a predominant expression in the brain. Thus CLEC2L-encoded CTLRs were designated BACL (brain-associated C-type lectin). Combining in situ hybridization and immunohistochemistry, we show that BACL is expressed by neurons in the CNS, with a pronounced expression by Purkinje cells. Notably, the CLEC2L locus is adjacent to another orphan CTLR gene (KLRG2), but reporter cell assays did neither indicate interaction of BACL with the KLRG2 ectodomain nor with human NK cell lines or lymphocytes. Along these lines, growth of BACL-expressing tumor cell lines in immunocompetent mice did not provide evidence for an immune-related function of BACL. Altogether, the CLEC2L gene encodes a homodimeric cell surface CTLR that stands out among CLEC2 family members by its conservation in mammals, its biochemical properties and the predominant expression in the brain. Future studies will have to reveal insights into the functional relevance of BACL in the context of its neuronal expression.
Rhabdomyosarcoma (RMS), the most common cancer of connective tissues in pediatrics, is often resistant to conventional therapies. One underlying mechanism of this resistance is the overexpression of Inhibitor of Apoptosis (IAP) proteins, leading to a dysfunctional cell death program within tumor cells. Smac mimetics (SM) are small molecules that can reactivate the cell death program by antagonizing IAP proteins and thereby compensating their overexpression. Here, we report that SM sensitize two RMS cell lines (RD and RH30) toward natural killer (NK) cell-mediated killing on the one hand, and increase the cytotoxic potential of NK cells on the other. The SM-induced sensitization of RH30 cells toward NK cell-mediated killing is significantly reduced through blocking tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on NK cells prior to coculture. In addition, the presence of zVAD.fmk, a pancaspase inhibitor, rescues tumor cells from the increase in killing, indicating an apoptosis-dependent cell death. On the NK cell side, the presence of SM in addition to IL-2 during the ex vivo expansion leads to an increase in their cytotoxic activity against RH30 cells. This effect is mainly TNFα-dependent and partially mediated by NK cell activation, which is associated with transcriptional upregulation of NF-κB target genes such as IκBα and RelB. Taken together, our findings implicate that SM represent a novel double-hit strategy, sensitizing tumor and activating NK cells with one single drug.
Immunosuppression is a typical hallmark of cancer and frequently includes perturbations of the NKG2D tumor recognition system as well as impaired signaling by other activating NK cell receptors. Several in vitro studies suggested that sustained engagement of the NKG2D receptor, as it is occurring in the tumor microenvironment, not only impairs expression and function of NKG2D but also impacts signaling by other activating NK receptors. Here, we made use of a transgenic mouse model of ubiquitous NKG2D ligand expression (H2-Kb-MICA mice) to investigate consequences of chronic NKG2D engagement in vivo for functional responsiveness by other activating NK receptors such as NKp46 and Ly49D. Unexpectedly, we found no evidence for an impairment of NKp46 expression and function in H2-Kb-MICA mice, as anticipated from previous in vitro experiments. However, we observed a marked downregulation and dysfunction of the activating receptor Ly49D in activated NK cells from H2-Kb-MICA mice. Ly49D shares the adaptor proteins DAP10 and DAP12 with NKG2D possibly explaining the collateral impairment of Ly49D function in situations of chronic NKG2D engagement. Altogether, our results demonstrate that persistent engagement of NKG2D in vivo, as often observed in tumors, can selectively impair functions of unrelated NK receptors and thereby compromise NK responsiveness to third-party antigens.
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.
NKG2D is a potent activating immunoreceptor expressed on nearly all cytotoxic lymphocytes promoting their cytotoxicity against self-cells expressing NKG2D ligands (NKG2DLs). NKG2DLs are MHC class I-like glycoproteins that usually are not expressed on “healthy” cells. Rather, their surface expression is induced by various forms of cellular stress, viral infection, or malignant transformation. Hence, cell surface NKG2DLs mark “dangerous” cells for elimination by cytotoxic lymphocytes and therefore can be considered as “kill-me” signals. In addition, NKG2DLs are up-regulated on activated leukocytes, which facilitates containment of immune responses. While the NKG2D receptor is conserved among mammals, NKG2DL genes have rapidly diversified during mammalian speciation, likely due to strong selective pressures exerted by species-specific pathogens. Consequently, NKG2DL genes are not conserved in man and mice, although their NKG2D-binding domains maintained structural homology. Human NKG2DLs comprise two members of the MIC (MICA/MICB) and six members of the ULBP family of glycoproteins (ULBP1–6) with MICA representing the best-studied human NKG2DLs by far. Many of these studies implicate a role of MICA in various malignant, infectious, or autoimmune diseases. However, conclusions from these studies often were limited in default of supporting in vivo experiments. Here, we report a MICA transgenic (MICAgen) mouse model that replicates central features of human MICA expression and function and, therefore, constitutes a novel tool to critically assess and extend conclusions from previous in vitro studies on MICA. Similarly to humans, MICA transcripts are broadly present in organs of MICAgen mice, while MICA glycoproteins are barely detectable. Upon activation, hematopoietic cells up-regulate and proteolytically shed surface MICA. Shed soluble MICA (sMICA) is also present in plasma of MICAgen mice but affects neither surface NKG2D expression of circulating NK cells nor their functional recognition of MICA-expressing tumor cells. Accordingly, MICAgen mice also show a delayed growth of MICA-expressing B16F10 tumors, not accompanied by an emergence of MICA-specific antibodies. Such immunotolerance for the xenoantigen MICA ideally suits MICAgen mice for anti-MICA-based immunotherapies. Altogether, MICAgen mice represent a valuable model to study regulation, function, disease relevance, and therapeutic targeting of MICA in vivo.
Stress-induced cell surface expression of MHC class I-related glycoproteins of the MIC and ULBP families allows for immune recognition of dangerous “self cells” by human cytotoxic lymphocytes via the NKG2D receptor. With two MIC molecules (MICA and MICB) and six ULBP molecules (ULBP1–6), there are a total of eight human NKG2D ligands (NKG2DL). Since the discovery of the NKG2D–NKG2DL system, the cause for both redundancy and diversity of NKG2DL has been a major and ongoing matter of debate. NKG2DL diversity has been attributed, among others, to the selective pressure by viral immunoevasins, to diverse regulation of expression, to differential tissue expression as well as to variations in receptor interactions. Here, we critically review the current state of knowledge on the poorly studied human NKG2DL ULBP4. Summarizing available facts and previous studies, we picture ULBP4 as a peculiar ULBP family member distinct from other ULBP family members by various aspects. In addition, we provide novel experimental evidence suggesting that cellular processing gives rise to mature ULBP4 glycoproteins different to previous reports. Finally, we report on the proteolytic release of soluble ULBP4 and discuss these results in the light of known mechanisms for generation of soluble NKG2DL.
A point mutation in the Ncr1 signal peptide impairs the development of innate lymphoid cell subsets
(2018)
NKp46 (CD335) is a surface receptor shared by both human and mouse natural killer (NK) cells and innate lymphoid cells (ILCs) that transduces activating signals necessary to eliminate virus-infected cells and tumors. Here, we describe a spontaneous point mutation of cysteine to arginine (C14R) in the signal peptide of the NKp46 protein in congenic Ly5.1 mice and the newly generated NCRB6C14R strain. Ly5.1C14R NK cells expressed similar levels of Ncr1 mRNA as C57BL/6, but showed impaired surface NKp46 and reduced ability to control melanoma tumors in vivo. Expression of the mutant NKp46C14R in 293T cells showed that NKp46 protein trafficking to the cell surface was compromised. Although Ly5.1C14R mice had normal number of NK cells, they showed an increased number of early maturation stage NK cells. CD49a+ILC1s were also increased but these cells lacked the expression of TRAIL. ILC3s that expressed NKp46 were not detectable and were not apparent when examined by T-bet expression. Thus, the C14R mutation reveals that NKp46 is important for NK cell and ILC differentiation, maturation and function.
Background: The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation.
Methods: Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening. Tumor spheroids were monitored for NK cell infiltration and cytotoxicity by flow cytometry. Infiltrated NK cells, could be recovered by magnetic cell separation.
Results: The tumor spheroids were stable over several days with minor alterations in phenotypic appearance. The tumor spheroids expressed high levels of cellular ligands for the natural killer (NK) group 2D receptor (NKG2D), mediating spheroid destruction by primary human NK cells. Interestingly, destruction of a three-dimensional tumor spheroid took much longer when compared to the parental monolayer cultures. Moreover, destruction of tumor spheroids was accompanied by infiltration of a fraction of NK cells, which could be recovered at high purity.
Conclusion: Tumor spheroids represent a versatile in vivo-like model system to study cytotoxicity and infiltration of immune cells in high-throughput screening. This system might proof useful for the investigation of the modulatory potential of soluble factors and cells of the tumor microenvironment on immune cell activity as well as profiling of patient-/donor-derived immune cells to personalize cellular immunotherapy.
Malignant gliomas are intrinsic brain tumors with a dismal prognosis. They are well-adapted to hypoxic conditions and poorly immunogenic. NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DL).
Here we evaluated the impact of miRNA on the expression of NKG2DL in glioma cells including stem-like glioma cells. Three of the candidate miRNA predicted to target NKG2DL were expressed in various glioma cell lines as well as in glioblastomas in vivo: miR-20a, miR-93 and miR-106b. LNA inhibitor-mediated miRNA silencing up-regulated cell surface NKG2DL expression, which translated into increased susceptibility to NK cell-mediated lysis. This effect was reversed by neutralizing NKG2D antibodies, confirming that enhanced lysis upon miRNA silencing was mediated through the NKG2D system. Hypoxia, a hallmark of glioblastomas in vivo, down-regulated the expression of NKG2DL on glioma cells, associated with reduced susceptibility to NK cell-mediated lysis. This process, however, was not mediated through any of the examined miRNA. Accordingly, both hypoxia and the expression of miRNA targeting NKG2DL may contribute to the immune evasion of glioma cells at the level of the NKG2D recognition pathway. Targeting miRNA may therefore represent a novel approach to increase the immunogenicity of glioblastoma.
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.
Cellular cytotoxicity is the hallmark of NK cells mediating both elimination of virus-infected or malignant cells, and modulation of immune responses. NK cytotoxicity is triggered upon ligation of various activating NK cell receptors. Among these is the C-type lectin-like receptor NKp80 which is encoded in the human Natural Killer Gene Complex (NKC) adjacent to its ligand, activation-induced C-type lectin (AICL). NKp80-AICL interaction promotes cytolysis of malignant myeloid cells, but also stimulates the mutual crosstalk between NK cells and monocytes.
While many activating NK cell receptors pair with ITAM-bearing adaptors, we recently reported that NKp80 signals via a hemITAM-like sequence in its cytoplasmic domain. Here we molecularly dissect the NKp80 hemITAM and demonstrate that two non-consensus amino acids, in particular arginine 6, critically impair both hemITAM phosphorylation and Syk recruitment. Impaired Syk recruitment results in a substantial attenuation of cytotoxic responses upon NKp80 ligation. Reconstituting the hemITAM consensus or Syk overexpression resulted in robust NKp80-mediated responsiveness. Collectively, our data provide a molecular rationale for the restrained activation potential of NKp80 and illustrate how subtle alterations in signaling motifs determine subsequent cellular responses. They also suggest that non-consensus alterations in the NKp80 hemITAM, as commonly present among mammalian NKp80 sequences, may have evolved to dampen NKp80-mediated cytotoxic responses toward AICL-expressing cells.
Background: The activating NK receptor NKp80 triggers cytotoxicity by human NK cells via a cytoplasmic hemITAM sequence.
Results: A non-consensus hemITAM residue impairs the capacity of NKp80 to recruit Syk kinase and to trigger cytotoxicity.
Conclusion: Unlike typical hemITAM receptors, NKp80 does not efficiently recruit Syk kinase resulting in attenuated effector responses.
Significance: An attenuated cytotoxic responsiveness critically impacts on the immunomodulatory function of NKp80.
Highlights:
• Assessment of body composition parameters in a large cohort of patients with HCC undergoing TACE.
• Fully automated artificial intelligence-based quantitative 3D volumetry of abdominal cavity tissue composition.
• Skeletal muscle volume and related parameters were independent prognostic factors in patients with HCC undergoing TACE.
Background & Aims: Body composition assessment (BCA) parameters have recently been identified as relevant prognostic factors for patients with hepatocellular carcinoma (HCC). Herein, we aimed to investigate the role of BCA parameters for prognosis prediction in patients with HCC undergoing transarterial chemoembolization (TACE).
Methods: This retrospective multicenter study included a total of 754 treatment-naïve patients with HCC who underwent TACE at six tertiary care centers between 2010–2020. Fully automated artificial intelligence-based quantitative 3D volumetry of abdominal cavity tissue composition was performed to assess skeletal muscle volume (SM), total adipose tissue (TAT), intra- and intermuscular adipose tissue, visceral adipose tissue, and subcutaneous adipose tissue (SAT) on pre-intervention computed tomography scans. BCA parameters were normalized to the slice number of the abdominal cavity. We assessed the influence of BCA parameters on median overall survival and performed multivariate analysis including established estimates of survival.
Results: Univariate survival analysis revealed that impaired median overall survival was predicted by low SM (p <0.001), high TAT volume (p = 0.013), and high SAT volume (p = 0.006). In multivariate survival analysis, SM remained an independent prognostic factor (p = 0.039), while TAT and SAT volumes no longer showed predictive ability. This predictive role of SM was confirmed in a subgroup analysis of patients with BCLC stage B.
Conclusions: SM is an independent prognostic factor for survival prediction. Thus, the integration of SM into novel scoring systems could potentially improve survival prediction and clinical decision-making. Fully automated approaches are needed to foster the implementation of this imaging biomarker into daily routine.
Impact and implications: Body composition assessment parameters, especially skeletal muscle volume, have been identified as relevant prognostic factors for many diseases and treatments. In this study, skeletal muscle volume has been identified as an independent prognostic factor for patients with hepatocellular carcinoma undergoing transarterial chemoembolization. Therefore, skeletal muscle volume as a metaparameter could play a role as an opportunistic biomarker in holistic patient assessment and be integrated into decision support systems. Workflow integration with artificial intelligence is essential for automated, quantitative body composition assessment, enabling broad availability in multidisciplinary case discussions.