Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disorder associated with bone fragility and susceptibility to fractures after minimal trauma. OI type V has an autosomal-dominant pattern of inheritance and is not caused by mutations in the type I collagen genes COL1A1 and COL1A2. The most remarkable and pathognomonic feature, observed in ~65% of affected individuals, is a predisposition to develop hyperplastic callus after fractures or surgical interventions. To identify the molecular cause of OI type V, we performed whole-exome sequencing in a female with OI type V and her unaffected parents and searched for de novo mutations. We found a heterozygous de novo mutation in the 5'-untranslated region of IFITM5 (the gene encoding Interferon induced transmembrane protein 5), 14 bp upstream of the annotated translation initiation codon (c.-14C>T). Subsequently, we identified an identical heterozygous de novo mutation in a second individual with OI type V by Sanger sequencing, thereby confirming that this is the causal mutation for the phenotype. IFITM5 is a protein that is highly enriched in osteoblasts and has a putative function in bone formation and osteoblast maturation. The mutation c.-14C>T introduces an upstream start codon that is in frame with the reference open-reading frame of IFITM5 and is embedded into a stronger Kozak consensus sequence for translation initiation than the annotated start codon. In vitro, eukaryotic cells were able to recognize this start codon, and they used it instead of the reference translation initiation signal. This suggests that five amino acids (Met-Ala-Leu-Glu-Pro) are added to the N terminus and alter IFITM5 function in individuals with the mutation.

Ciguatoxins (CTXs) are marine toxins that cause ciguatera fish poisoning, a debilitating disease dominated by sensory and neurological disturbances that include cold allodynia and various painful symptoms as well as long-lasting pruritus. Although CTXs are known as the most potent mammalian sodium channel activator toxins, the etiology of many of its neurosensory symptoms remains unresolved. We recently described that local application of 1 nM Pacific Ciguatoxin-1 (P-CTX-1) into the skin of human subjects induces a long-lasting, painful axon reflex flare and that CTXs are particularly effective in releasing calcitonin-gene related peptide (CGRP) from nerve terminals. In this study, we used mouse and rat skin preparations and enzyme-linked immunosorbent assays (ELISA) to study the molecular mechanism by which P-CTX-1 induces CGRP release. We show that P-CTX-1 induces CGRP release more effectively in mouse as compared to rat skin, exhibiting EC50 concentrations in the low nanomolar range. P-CTX-1-induced CGRP release from skin is dependent on extracellular calcium and sodium, but independent from the activation of various thermosensory transient receptor potential (TRP) ion channels. In contrast, lidocaine and tetrodotoxin (TTX) reduce CGRP release by 53-75%, with the remaining fraction involving L-type and T-type voltage-gated calcium channels (VGCC). Using transgenic mice, we revealed that the TTX-resistant voltage-gated sodium channel (VGSC) NaV1.9, but not NaV1.8 or NaV1.7 alone and the combined activation of the TTX-sensitive VGSC subtypes NaV1.7 and NaV1.1 carry the largest part of the P-CTX-1-caused CGRP release of 42% and 34%, respectively. Given the contribution of CGRP to nociceptive and itch sensing pathways, our findings contribute to a better understanding of sensory symptoms of acute and chronic ciguatera that may help in the identification of potential therapeutics.

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD2 using the CliniMACS Prodigy® system using a recently described "all-in-one" lentiviral vector combining constitutive anti-GD2 CAR expression and inducible IL-18. Starting with 0.84 x 108 and 0.91 x 108 T cells after enrichment of CD4+ and CD8+ we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 109 and 3.6 x 109 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD2-CAR mediated activation after co-cultivation with GD2-expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD2-expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4+ and CD8+ T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD2-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD2-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.

Teeth are composed of many tissues, covered by an inflexible and obdurate enamel. Unlike most other tissues, teeth become extremely cold sensitive when inflamed. The mechanisms of this cold sensation are not understood. Here, we clarify the molecular and cellular components of the dental cold sensing system and show that sensory transduction of cold stimuli in teeth requires odontoblasts. TRPC5 is a cold sensor in healthy teeth and, with TRPA1, is sufficient for cold sensing. The odontoblast appears as the direct site of TRPC5 cold transduction and provides a mechanism for prolonged cold sensing via TRPC5's relative sensitivity to intracellular calcium and lack of desensitization. Our data provide concrete functional evidence that equipping odontoblasts with the cold-sensor TRPC5 expands traditional odontoblast functions and renders it a previously unknown integral cellular component of the dental cold sensing system.

A class of Group III muscle afferent neurons has branching sensory terminals in the connective tissue between layers of mouse abdominal muscles ("CT3 muscle afferents"). These sensory endings are both mechanosensitive and metabosensitive. In the present study, responses of CT3 afferents to lactate ions and changes in temperature were recorded. Raising muscle temperature from 32.7°C to 37°C had no consistent effects on CT3 afferent basal firing rate or responses to either von Frey hair stimulation or to an applied load. Superfusion with lactate ions (15 mM, pH 7.4) was associated with an increase in firing from 6 ± 0.7 Hz to 11.7 ± 6.7 Hz (14 units, n = 13, P < 0.05, P = 0.0484) but with considerable variability in the nature and latency of response. Reducing the concentration of extracellular divalent cations, which mimicked the chelating effects of lactate, did not increase firing. Raised concentrations of divalent cations (to compensate for chelation) did not block excitatory effects of lactate on CT3 afferents, suggesting that effects via ASIC3 were not involved. Messenger RNA for the G-protein coupled receptor, hydroxyl carboxylic acid receptor 1 (HCAR1) was detected in dorsal root ganglia and HCAR1-like immunoreactivity was present in spinal afferent nerve cell bodies retrogradely labeled from mouse abdominal muscles. HCAR1-like immunoreactivity was also present in axons in mouse abdominal muscles. This raises the possibility that some effects of lactate on group III muscle afferents may be mediated by HCAR1.

Immunosuppressive therapy or T-cell depletion in transplant patients can cause uncontrolled growth of Epstein-Barr virus (EBV)-infected B cells resulting in post-transplant lymphoproliferative disease (PTLD). Current treatment options do not distinguish between healthy and malignant B cells and are thereby often limited by severe side effects in the already immunocompromised patients. To specifically target EBV-infected B cells, we developed a novel peptide-selective chimeric antigen receptor (CAR) based on the monoclonal antibody TÜ165 which recognizes an Epstein-Barr nuclear antigen (EBNA)-3C-derived peptide in HLA-B*35 context in a T-cell receptor (TCR)-like manner. In order to attract additional immune cells to proximity of PTLD cells, based on the TÜ165 CAR, we moreover generated T cells redirected for universal cytokine-mediated killing (TRUCKs), which induce interleukin (IL)-12 release on target contact.

We demonstrate a novel dual strategy against inflammation and pain through body-wide desensitization of nociceptors via TRPA1. Attenuation of experimental colitis by capsazepine (CPZ) has long been attributed to its antagonistic action on TRPV1 and associated inhibition of neurogenic inflammation. In contrast, we found that CPZ exerts its anti-inflammatory effects via profound desensitization of TRPA1. Micromolar CPZ induced calcium influx in isolated dorsal root ganglion (DRG) neurons from wild-type (WT) but not TRPA1-deficient mice. CPZ-induced calcium transients in human TRPA1-expressing HEK293t cells were blocked by the selective TRPA1 antagonists HC 030031 and A967079 and involved three cysteine residues in the N-terminal domain. Intriguingly, both colonic enemas and drinking water with CPZ led to profound systemic hypoalgesia in WT and TRPV1(-/-) but not TRPA1(-/-) mice. These findings may guide the development of a novel class of disease-modifying drugs with anti-inflammatory and anti-nociceptive effects.

Mesenchymal stromal cells (MSCs) are used in over 800 clinical trials mainly due to their immune inhibitory activity. Umbilical cord (UC), the second leading source of clinically used MSCs, is usually cut in small tissue pieces. Subsequent cultivation leads to a continuous outgrowth of MSC explant monolayers (MSC-EMs) for months. Currently, the first MSC-EM culture takes approximately 2 weeks to grow out, which is then expanded and applied to patients. The initiating tissue pieces are then discarded. However, when UC pieces are transferred to new culture dishes, MSC-EMs continue to grow out. In case the functional integrity of these cells is maintained, later induced cultures could also be expanded and used for cell therapy. This would drastically increase the number of available cells for each patient. To test the functionality of MSC-EMs from early and late induction time points, we compared the first cultures to those initiated after 2 months by investigating their clonality and immunomodulatory capacity.

Previously the effect of the pruritogens, such as histamine and chloroquine, was tested in 11 inbred mouse strains, and this study aimed to identify resistant and sensitive strains, consistent with the observation that underlies the large variability in human populations. In the present study, we used the low responder C3H/HeJ (C3H) and the more sensitive C57BL/6J (C57) strain to find out if resistance and sensitivity to develop pruritus is restricted to only histamine and chloroquine or extends to other known pruritogens as well. We tested five additional commonly known pruritogens. We established dose-response relationships by injecting four concentrations of the pruritogens in the range of 0.3, 1, 3, and ten-fold in the nuchal fold. Then we assessed the scratching behavior for 30 min after injection with an automated custom-designed device based on the bilateral implantation of mini-magnets in the hind paws and on single cages placed within a magnetic coil. We found that the resistance to pruritogens is a general phenotype of the C3H strain and extends to all pruritogens tested, including not only histamine and chloroquine, but also endothelin, trypsin, 5-HT (serotonin), the short peptide SLIGRL, and Lysophosphatidic acid (LPA). C57 was more sensitive to all pruritogens and, in contrast to C3H, dose-response relationships were evident for some of the pruritogens. In general, comparable peak scratch responses were observed for the 0.3-fold concentrations of the pruritogens in C57 whereas C3H required at least the ten-fold concentration and still displayed only between 5 and 33% of the scratch responses observed in C57 for the respective pruritogen. The general resistance to pruritogens and the low level of scratching behavior found in the C3H strain is an interesting trait and represents a model for the study of the heritability of itch. It is accompanied in C3H with a higher sensitivity in assays of nociception.

Loss-of-function mutations of Na(V)1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of Na(V)1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na(V)1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na(V)1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na(V)1.7 inhibitors and significantly reduced in Na(V)1.7(-/-) mice. To validate the use of the model for profiling Na(V)1.7 inhibitors, we determined the Na(V) selectivity and tested the efficacy of the reported Na(V)1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited Na(V)1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na(V)1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited Na(V) channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na(V)1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na(V)1.7 inhibitors.

Bone morphogenetic protein 1 (BMP1) is an astacin metalloprotease with important cellular functions and diverse substrates, including extracellular-matrix proteins and antagonists of some TGFβ superfamily members. Combining whole-exome sequencing and filtering for homozygous stretches of identified variants, we found a homozygous causative BMP1 mutation, c.34G>C, in a consanguineous family affected by increased bone mineral density and multiple recurrent fractures. The mutation is located within the BMP1 signal peptide and leads to impaired secretion and an alteration in posttranslational modification. We also characterize a zebrafish bone mutant harboring lesions in bmp1a, demonstrating conservation of BMP1 function in osteogenesis across species. Genetic, biochemical, and histological analyses of this mutant and a comparison to a second, similar locus reveal that Bmp1a is critically required for mature-collagen generation, downstream of osteoblast maturation, in bone. We thus define the molecular and cellular bases of BMP1-dependent osteogenesis and show the importance of this protein for bone formation and stability.

Immune cell therapeutics are increasingly applied in oncology. Especially chimeric antigen receptor (CAR) T cells are successfully used to treat several B cell malignancies. Efforts to engineer CAR T cells for improved activity against solid tumors include co-delivery of pro-inflammatory cytokines in addition to CARs, via either constitutive cytokine expression or inducible cytokine expression triggered by CAR recognition of its target antigen-so-called "T cells redirected for universal cytokine-mediated killing" (TRUCKs) or fourth-generation CARs. Here, we tested the hypothesis that TRUCK principles could be expanded to improve anticancer functions of NK cells. A comparison of the functionality of inducible promoters responsive to NFAT or NFκB in NK cells showed that, in contrast to T cells, the inclusion of NFκB-responsive elements within the inducible promoter construct was essential for CAR-inducible expression of the transgene. We demonstrated that GD2CAR-specific activation induced a tight NFκB-promoter-driven cytokine release in NK-92 and primary NK cells together with an enhanced cytotoxic capacity against GD2+ target cells, also shown by increased secretion of cytolytic cytokines. The data demonstrate biologically relevant differences between T and NK cells that are important when clinically translating the TRUCK concept to NK cells for the treatment of solid malignancies.

Genetically modified T cells expressing chimeric antigen receptors (CARs) so far have mostly failed in the treatment of solid tumors owing to a number of limitations, including an immunosuppressive tumor microenvironment and insufficient CAR T cell activation and persistence. Next-generation approaches using CAR T cells that secrete transgenic immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently being explored. As TRUCKs were engineered by the transduction of T cells with two separate vectors, we developed a lentiviral modular "all-in-one" vector system that combines constitutive CAR expression and inducible nuclear factor of activated T cells (NFAT)-driven transgene expression for more efficient production of TRUCKs. Activation of the GD2-specific CAR via GD2+ target cells induced NFAT promoter-driven cytokine release in primary human T cells, and indicated a tight linkage of CAR-specific activation and transgene expression that was further improved by a modified NFATsyn promoter. As proof-of-concept, we showed that T cells containing the "all-in-one" vector system secrete the immunomodulatory cytokines interleukin (IL)12 or IL18 upon co-cultivation with primary human GD2+ tumor cells, resulting in enhanced effector cell properties and increased monocyte recruitment. This highlights the potential of our system to simplify application of TRUCK-modified T cells in solid tumor therapy.

Adoptive immunotherapy with ex vivo expanded, polyspecific regulatory T cells (Tregs) is a promising treatment for graft-versus-host disease. Animal transplantation models used by us and others have demonstrated that the adoptive transfer of allospecific Tregs offers greater protection from graft rejection than that of polyclonal Tregs. This finding is in contrast to those of autoimmune models, where adoptive transfer of polyspecific Tregs had very limited effects, while antigen-specific Tregs were promising. However, antigen-specific Tregs in autoimmunity cannot be isolated in sufficient numbers. Chimeric antigen receptors (CARs) can modify T cells and redirect their specificity toward needed antigens and are currently clinically used in leukemia patients. A major benefit of CAR technology is its "off-the-shelf" usability in a translational setting in contrast to major histocompatibility complex (MHC)-restricted T cell receptors. We used CAR technology to redirect T cell specificity toward insulin and redirect T effector cells (Teffs) to Tregs by Foxp3 transduction. Our data demonstrate that our converted, insulin-specific CAR Tregs (cTregs) were functional stable, suppressive and long-lived in vivo. This is a proof of concept for both redirection of T cell specificity and conversion of Teffs to cTregs.

Currently available behavioral assays to quantify normal cold sensitivity, cold hypersensitivity and cold hyperalgesia in mice have betimes created conflicting results in the literature. Some only capture a limited spectrum of thermal experiences, others are prone to experimenter bias or are not sensitive enough to detect the contribution of ion channels to cold sensing because in mice smaller alterations in cold nociception do not manifest as frank behavioral changes. To overcome current limitations we have designed a novel device that is automated, provides a high degree of freedom, i.e. thermal choice, and eliminates experimenter bias. The device represents a thermal gradient assay designed as a circular running track. It allows discerning exploratory behavior from thermal selection behavior and provides increased accuracy by providing measured values in duplicate and by removing edge artifacts. Our custom-designed automated offline analysis by a blob detection algorithm is devoid of movement artifacts, removes light reflection artifacts and provides an internal quality control parameter which we validated. The assay delivers discrete information on a large range of parameters extracted from the occupancy of thermally defined zones such as preference temperature and skew of the distribution. We demonstrate that the assay allows increasingly accurate phenotyping of thermal sensitivity in transgenic mice by disclosing yet unrecognized details on the phenotypes of TRPM8-, TRPA1- and TRPM8/A1-deficient mice.

Detection and adaptation to cold temperature is crucial to survival. Cold sensing in the innocuous range of cold (>10-15 °C) in the mammalian peripheral nervous system is thought to rely primarily on transient receptor potential (TRP) ion channels, most notably the menthol receptor, TRPM8. Here we report that TRP cation channel, subfamily C member 5 (TRPC5), but not TRPC1/TRPC5 heteromeric channels, are highly cold sensitive in the temperature range 37-25 °C. We found that TRPC5 is present in mouse and human sensory neurons of dorsal root ganglia, a substantial number of peripheral nerves including intraepithelial endings, and in the dorsal lamina of the spinal cord that receives sensory input from the skin, consistent with a potential TRPC5 function as an innocuous cold transducer in nociceptive and thermosensory nerve endings. Although deletion of TRPC5 in 129S1/SvImJ mice resulted in no temperature-sensitive behavioral changes, TRPM8 and/or other menthol-sensitive channels appear to underpin a much larger component of noxious cold sensing after TRPC5 deletion and a shift in mechanosensitive C-fiber subtypes. These findings demonstrate that highly cold-sensitive TRPC5 channels are a molecular component for detection and regional adaptation to cold temperatures in the peripheral nervous system that is distinct from noxious cold sensing.

Damage-sensing nociceptors in the skin provide an indispensable protective function thanks to their specialized ability to detect and transmit hot temperatures that would block or inflict irreversible damage in other mammalian neurons. Here we show that the exceptional capacity of skin C-fiber nociceptors to encode noxiously hot temperatures depends on two tetrodotoxin (TTX)-resistant sodium channel α-subunits: NaV1.8 and NaV1.9. We demonstrate that NaV1.9, which is commonly considered an amplifier of subthreshold depolarizations at 20°C, undergoes a large gain of function when temperatures rise to the pain threshold. We also show that this gain of function renders NaV1.9 capable of generating action potentials with a clear inflection point and positive overshoot. In the skin, heat-resistant nociceptors appear as two distinct types with unique and possibly specialized features: one is blocked by TTX and relies on NaV1.9, and the second type is insensitive to TTX and composed of both NaV1.8 and NaV1.9. Independent of rapidly gated TTX-sensitive NaV channels that form the action potential at pain threshold, NaV1.8 is required in all heat-resistant nociceptors to encode temperatures higher than ∼46°C, whereas NaV1.9 is crucial for shaping the action potential upstroke and keeping the NaV1.8 voltage threshold within reach.

Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. After mutations in all known OI genes had been excluded by Sanger sequencing, we applied next-generation sequencing to analyze the exome of a single individual who has a severe form of the disease and whose parents are second cousins. A total of 26,922 variations from the human reference genome sequence were subjected to several filtering steps. In addition, we extracted the genotypes of all dbSNP130-annotated SNPs from the exome sequencing data and used these 299,494 genotypes as markers for the genome-wide identification of homozygous regions. A single homozygous truncating mutation, affecting SERPINF1 on chromosome 17p13.3, that was embedded into a homozygous stretch of 2.99 Mb remained. The mutation was also homozygous in the affected brother of the index patient. Subsequently, we identified homozygosity for two different truncating SERPINF1 mutations in two unrelated patients with OI and parental consanguinity. All four individuals with SERPINF1 mutations have severe OI. Fractures of long bones and severe vertebral compression fractures with resulting deformities were observed as early as the first year of life in these individuals. Collagen analyses with cultured dermal fibroblasts displayed no evidence for impaired collagen folding, posttranslational modification, or secretion. SERPINF1 encodes pigment epithelium-derived factor (PEDF), a secreted glycoprotein of the serpin superfamily. PEDF is a multifunctional protein and one of the strongest inhibitors of angiogenesis currently known in humans. Our data provide genetic evidence for PEDF involvement in human bone homeostasis.

To fulfill its role in protein biogenesis, the endoplasmic reticulum (ER) depends on the Hsp70-type molecular chaperone BiP, which requires a constant ATP supply. However, the carrier that catalyzes ATP uptake into the ER was unknown. Here, we report that our screen of gene expression datasets for member(s) of the family of solute carriers that are co-expressed with BiP and are ER membrane proteins identifies SLC35B1 as a potential candidate. Heterologous expression of SLC35B1 in E. coli reveals that SLC35B1 is highly specific for ATP and ADP and acts in antiport mode. Moreover, depletion of SLC35B1 from HeLa cells reduces ER ATP levels and, as a consequence, BiP activity. Thus, human SLC35B1 may provide ATP to the ER and was named AXER (ATP/ADP exchanger in the ER membrane). Furthermore, we propose an ER to cytosol low energy response regulatory axis (termed lowER) that appears as central for maintaining ER ATP supply.

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.