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The serum level of the soluble Receptor for Advanced Glycation End-products (sRAGE) is a promising blood biomarker for the development, severity, and progression of chronic obstructive pulmonary disease (COPD). However, cigarette smoking causes a nearly instant drop in circulating sRAGE levels, strongly impacting on the variability in sRAGE levels. In the current study, we investigated the possible mechanism behind the sudden drop in sRAGE upon smoking. We showed that the number of activated neutrophils in blood significantly increases within two hours upon smoking three cigarettes within one hour. Furthermore, an increased expression of the leukocyte activation marker CD11b, which is a known ligand for RAGE, was observed upon smoking. Additionally, the in vitro activation of neutrophils increased their capacity to bind sRAGE. Together, these data indicate that smoking activates neutrophils in the circulation with concomitant upregulation of the RAGE ligand CD11b, leading to reduced levels of sRAGE in serum.

BACKGROUND: There are few clinically useful circulating biomarkers of lung function and lung disease. We hypothesized that genome-wide association studies (GWAS) of circulating proteins in conjunction with GWAS of pulmonary traits represents a clinically relevant approach to identifying causal proteins and therapeutically useful insights into mechanisms related to lung function and disease. STUDY QUESTION: Can an integrative genomic strategy using GWAS of plasma soluble receptor for advanced glycation end-products (sRAGE) levels in conjunction with GWAS of lung function traits identify putatively causal relations of sRAGE to lung function? STUDY DESIGN AND METHODS: Plasma sRAGE levels were measured in 6,861 Framingham Heart Study participants and GWAS of sRAGE was conducted to identify protein quantitative trait loci (pQTL), including cis-pQTL variants at the sRAGE protein-coding gene locus (AGER). We integrated sRAGE pQTL variants with variants from GWAS of lung traits. Colocalization of sRAGE pQTL variants with lung trait GWAS variants was conducted, and Mendelian randomization was performed using sRAGE cis-pQTL variants to infer causality of sRAGE for pulmonary traits. Cross-sectional and longitudinal protein-trait association analyses were conducted for sRAGE in relation to lung traits. RESULTS: Colocalization identified shared genetic signals for sRAGE with lung traits. Mendelian randomization analyses suggested protective causal relations of sRAGE to several pulmonary traits. Protein-trait association analyses demonstrated higher sRAGE levels to be cross-sectionally and longitudinally associated with preserved lung function. INTERPRETATION: sRAGE is produced by type I alveolar cells, and it acts as a decoy receptor to block the inflammatory cascade. Our integrative genomics approach provides evidence for sRAGE as a causal and protective biomarker of lung function, and the pattern of associations is suggestive of a protective role of sRAGE against restrictive lung physiology. We speculate that targeting the AGER/sRAGE axis may be therapeutically beneficial for the treatment and prevention of inflammation-related lung disease.

A candidate‐based inducible eQTL study was performed in occasional smokers, identifying rs3212855 flanking the FPR as novel cigarette smoke‐induced eQTL. In addition, rs3212855 was associated with lower lung function. Lastly, CRISPR‐Cas9‐downregulated FPR1 alveolar epithelial cells were protected against the harmful effects of cigarette smoke on epithelial repair., Background and objective Cigarette smoking is one of the most prevalent causes of preventable deaths worldwide, leading to chronic diseases, including chronic obstructive pulmonary disease (COPD). Cigarette smoke is known to induce significant transcriptional modifications throughout the respiratory tract. However, it is largely unknown how genetic profiles influence the smoking‐related transcriptional changes and how changes in gene expression translate into altered alveolar epithelial repair responses. Methods We performed a candidate‐based acute cigarette smoke‐induced eQTL study, investigating the association between SNP and differential gene expression of FPR family members in bronchial epithelial cells isolated 24 h after smoking and after 48 h without smoking. The effects FPR1 on lung epithelial integrity and repair upon damage in the presence and absence of cigarette smoke were studied in CRISPR‐Cas9‐generated lung epithelial knockout cells. Results One significant (FDR 2‐fold change in gene expression. The minor allele of rs3212855 was associated with significantly higher gene expression of FPR1, FPR2 and FPR3 upon smoking. Importantly, the minor allele of rs3212855 was also associated with lower lung function. Alveolar epithelial FPR1 knockout cells were protected against CSE‐induced reduction in repair capacity upon wounding. Conclusion We identified a novel smoking‐related inducible eQTL that is associated with a smoke‐induced increase in the expression of FPR1, FPR2 and FPR3, and with lowered lung function. in vitro FPR1 down‐regulation protects against smoke‐induced reduction in lung epithelial repair.

In earlier studies, galectin-9 (Gal-9) was identified as a multifaceted player in both adaptive and innate immunity. Further, Gal-9 had direct cytotoxic and tumor-selective activity towards cancer cell lines of various origins. In the current study, we identified that treatment with Gal-9 triggered pronounced membrane alterations in cancer cells. Specifically, phosphatidyl serine (PS) was rapidly externalized, and the anti-phagocytic regulator, CD47, was downregulated within minutes. In line with this, treatment of mixed neutrophil/tumor cell cultures with Gal-9 triggered trogocytosis and augmented antibody-dependent cellular phagocytosis of cancer cells. Interestingly, this pro-trogocytic effect was also due to the Gal-9-mediated activation of neutrophils with upregulation of adhesion markers and mobilization of gelatinase, secretory, and specific granules. These activation events were accompanied by a decrease in cancer cell adhesion in mixed cultures of leukocytes and cancer cells. Further, prominent cytotoxicity was detected when leukocytes were mixed with pre-adhered cancer cells, which was abrogated when neutrophils were depleted. Taken together, Gal-9 treatment potently activated neutrophil-mediated anticancer immunity, resulting in the elimination of epithelial cancer cells.

CD47 is a prominent new target in cancer immunotherapy, with antagonistic antibodies currently being evaluated in clinical trials. For effective evaluation of this strategy it is crucial to identify which patients are suited for CD47-targeted therapy. In this respect, expression of the pro-phagocytic signal SLAMF7 on both macrophages and cancer cells was recently reported to be a requisite for CD47 antibody-mediated phagocytosis. Here, we demonstrate that in fact SLAMF7 expression on cancer cells is not required and does not impact on CD47 antibody therapy. Moreover, SLAMF7 also does not impact on phagocytosis induction by CD20 antibody rituximab nor associates with overall survival of Diffuse Large B-Cell Lymphoma patients. In contrast, expression of CD47 negatively impacts on overall and progression free survival. In conclusion, cancer cell expression of SLAMF7 is not required for phagocytosis and, in contrast to CD47 expression, should not be used as selection criterion for CD47-targeted therapy., CD47 is a promising new target in cancer immunotherapy and recently the pro-phagocytic signal SLAMF7 has been shown to have a crucial role in phagocytosis induced by CD47-blocking antibody in hematological tumors. In this study, the authors demonstrate that SLAMF7 expressed by cancer cells is not required for phagocytosis suggesting that, in contrast to CD47 expression, SLAMF7 should not be used as selection criterion for CD47-targeted therapy.

Elevated activation of the autophagy pathway is currently thought to be one of the survival mechanisms allowing therapy-resistant cancer cells to escape elimination, including for cytarabine (AraC)-resistant acute myeloid leukemia (AML) patients. Consequently, the use of autophagy inhibitors such as chloroquine (CQ) is being explored for the re-sensitization of AraC-resistant cells. In our study, no difference in the activity of the autophagy pathway was detected when comparing AraC-Res AML cell lines to parental AraC-sensitive AML cell lines. Furthermore, treatment with autophagy inhibitors CQ, 3-Methyladenine (3-MA), and bafilomycin A1 (BafA1) did not re-sensitize AraC-Res AML cell lines to AraC treatment. However, in parental AraC-sensitive AML cells, treatment with AraC did activate autophagy and, correspondingly, combination of AraC with autophagy inhibitors strongly reduced cell viability. Notably, the combination of these drugs also yielded the highest level of cell death in a panel of patient-derived AML samples even though not being additive. Furthermore, there was no difference in the cytotoxic effect of autophagy inhibition during AraC treatment in matched de novo and relapse samples with differential sensitivity to AraC. Thus, inhibition of autophagy may improve AraC efficacy in AML patients, but does not seem warranted for the treatment of AML patients that have relapsed with AraC-resistant disease.

Autophagy is a crucial recycling process that is increasingly being recognized as an important factor in cancer initiation, cancer (stem) cell maintenance as well as the development of resistance to cancer therapy in both solid and hematological malignancies. Furthermore, it is being recognized that autophagy also plays a crucial and sometimes opposing role in the complex cancer microenvironment. For instance, autophagy in stromal cells such as fibroblasts contributes to tumorigenesis by generating and supplying nutrients to cancerous cells. Reversely, autophagy in immune cells appears to contribute to tumor‐localized immune responses and among others regulates antigen presentation to and by immune cells. Autophagy also directly regulates T and natural killer cell activity and is required for mounting T‐cell memory responses. Thus, within the tumor microenvironment autophagy has a multifaceted role that, depending on the context, may help drive tumorigenesis or may help to support anticancer immune responses. This multifaceted role should be taken into account when designing autophagy‐based cancer therapeutics. In this review, we provide an overview of the diverse facets of autophagy in cancer cells and nonmalignant cells in the cancer microenvironment. Second, we will attempt to integrate and provide a unified view of how these various aspects can be therapeutically exploited for cancer therapy.

The aetiology of rheumatoid arthritis (RA) is unknown, but citrullination of proteins is thought to be an initiating event. In addition, it is increasingly evident that the lung can be a potential site for the generation of autoimmune triggers before the development of joint disease. Here, we identified that serum levels of galectin-9 (Gal-9), a pleiotropic immunomodulatory protein, are elevated in RA patients, and are even further increased in patients with comorbid bronchiectasis, a lung disease caused by chronic inflammation. The serum concentrations of Gal-9 correlate with C-reactive protein levels and DAS-28 score. Gal-9 activated polymorphonuclear leukocytes (granulocytes) in vitro, which was characterized by increased cytokine secretion, migration, and survival. Further, granulocytes treated with Gal-9 upregulated expression of peptidyl arginine deiminase 4 (PAD-4), a key enzyme required for RA-associated citrullination of proteins. Correspondingly, treatment with Gal-9 triggered citrullination of intracellular granulocyte proteins that are known contributors to RA pathogenesis (i.e., myeloperoxidase, alpha-enolase, MMP-9, lactoferrin). In conclusion, this study identifies for the first time an immunomodulatory protein, Gal-9, that triggers activation of granulocytes leading to increased PAD-4 expression and generation of citrullinated autoantigens. This pathway may represent a potentially important mechanism for development of RA.

The therapeutic effect of anti-cancer monoclonal antibodies stems from their capacity to opsonize targeted cancer cells with subsequent phagocytic removal, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) or induction of complement-mediated cytotoxicity (CDC). The major immune effector cells involved in these processes are natural killer (NK) cells and granulocytes. The latter and most prevalent blood cell population contributes to phagocytosis, but is not effective in inducing ADCC. Here, we report that targeted delivery of the tumoricidal protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to granulocyte marker C-type lectin-like molecule-1 (CLL1), using fusion protein CLL1:TRAIL, equips granulocytes with high levels of TRAIL. Upon CLL1-selective binding of this fusion protein, granulocytes acquire additional TRAIL-mediated cytotoxic activity that, importantly, potentiates antibody-mediated cytotoxicity of clinically used therapeutic antibodies (e.g., rituximab, cetuximab). Thus, CLL1:TRAIL could be used as an adjuvant to optimize the clinical potential of anticancer antibody therapy by augmenting tumoricidal activity of granulocytes.

Here, we report on a novel bispecific antibody-derivative, designated RTX-CD47, with unique capacity for CD20-directed inhibition of CD47-SIRP alpha "don't eat me" signaling. RTX-CD47 comprises a CD20-targeting scFv antibody fragment derived from rituximab fused in tandem to a CD47-blocking scFv. Single agent treatment with RTX-CD47 triggered significant phagocytic removal of CD20(pos)/CD47(pos) malignant B-cells, but not of CD20(neg)/CD47(pos) cells, and required no pro-phagocytic FcR-mediated signaling. Importantly, treatment with RTX-CD47 synergistically enhanced the phagocytic elimination of primary malignant B cells by autologous phagocytic effector cells as induced by therapeutic anticancer antibodies daratumumab (anti-CD38), alemtuzumab (anti-CD52) and obinutuzumab (anti-CD20). In conclusion, RTX-CD47 blocks CD47 "don't eat me" signaling by cancer cells in a CD20-directed manner with essentially no activity towards CD20(neg)/CD47(pos) cells and enhances the activity of therapeutic anticancer antibodies directed to B-cell malignancies.

Since their discovery, antibodies have been viewed as ideal candidates or "magic bullets" for use in targeted therapy in the fields of cancer, autoimmunity, and chronic inflammatory disorders. A wave of antibody-dedicated research followed, which resulted in the clinical approval of a first generation of monoclonal antibodies for cancer therapy such as rituximab (1997) and cetuximab (2004), and infliximab (2002) for the treatment of autoimmune diseases. More recently, the development of antibodies that prevent checkpoint-mediated inhibition of T cell responses invigorated the field of cancer immunotherapy. Such antibodies induced unprecedented long-term remissions in patients with advanced stage malignancies, most notably melanoma and lung cancer, that do not respond to conventional therapies. In this review, we will recapitulate the development of antibody-based therapy, and detail recent advances and new functions, particularly in the field of cancer immunotherapy. With the advent of recombinant DNA engineering, a number of rationally designed molecular formats of antibodies and antibody-derived agents have become available, and we will discuss various molecular formats including antibodies with improved effector functions, bispecific antibodies, antibody-drug conjugates, antibody-cytokine fusion proteins, and T cells genetically modified with chimeric antigen receptors. With these exciting advances, new antibody-based treatment options will likely enter clinical practice and pave the way toward more successful control of malignant diseases.

BACKGROUND: In certain cancers, expression of CXCL16 and its receptor CXCR6 associate with lymphocyte infiltration, possibly aiding anti-tumour immune response. In other cancers, CXCL16 and CXCR6 associate with pro-metastatic activity. In the current study, we aimed to characterise the role of CXCL16, sCXCL16, and CXCR6 in ovarian cancer (OC).METHODS: CXCL16/CXCR6 expression was analysed on tissue microarray containing 306 OC patient samples. Pre-treatment serum sCXCL16 was determined in 118 patients using ELISA. In vitro, (primary) OC cells were treated with an ADAM-10/ADAM-17 inhibitor (TAPI-2) and an ADAM-10-specific inhibitor (GI254023x), whereupon CXCL16 levels were evaluated on the cell membrane (immunofluorescent analysis, western blots) and in culture supernatants (ELISA). In addition, cell migration was assessed using scratch assays.RESULTS: sCXCL16 independently predicted for poor survival (hazard ratio=2.28, 95% confidence interval=1.29-4.02, P=0.005), whereas neither CXCL16 nor CXCR6 expression correlated with survival. Further, CXCL16/CXCR6 expression and serum sCXCL16 levels did not associate with lymphocyte infiltration. In vitro inhibition of both ADAM-17 and ADAM-10, but especially the latter, decreased CXCL16 membrane shedding and strongly reduced cell migration of A2780 and cultured primary OC-derived malignant cells.CONCLUSIONS: High serum sCXCL16 is a prognostic marker for poor survival of OC patients, possibly reflecting ADAM-10 and ADAM-17 pro-metastatic activity. Therefore, serum sCXCL16 levels may be a pseudomarker that identifies patients with highly metastatic tumours.

The endoplasmic reticulum (ER) chaperone protein, calreticulin (CRT), is essential for proper glycoprotein folding and maintaining cellular calcium homeostasis. During ER stress, CRT is overexpressed as part of the unfolded protein response (UPR). In addition, CRT can be released as a damage-associated molecular pattern (DAMP) molecule that may interact with pathogen-associated molecular patterns (PAMPs) during the innate immune response. One such PAMP is lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall. In this report, we show that recombinant and native human placental CRT strongly interacts with LPS in solution, solid phase, and the surface of gram-negative and gram-positive bacteria. Furthermore, LPS induces oilgomerization of CRT with a disappearance of the monomeric form. The application of recombinant CRT (rCRT) to size exclusion and anion exchange chromatography shows an atypical heterogeneous elution profile, indicating that LPS affects the conformation and ionic charge of CRT. Interestingly, LPS bound to CRT is detected in sera of bronchiectasis patients with chronic bacterial infections. By ELISA, rCRT dose-dependently bound to solid phase LPS via the N- and C-domain globular head region of CRT and the C-domain alone. The specific interaction of CRT with LPS may be important in PAMP innate immunity.

Antibodies that block PD-L1/PD-1 immune checkpoints restore the activity of functionally-impaired antitumor T cells. These antibodies show unprecedented clinical benefit in various advanced cancers, particularly in melanoma. However, only a subset of cancer patients responds to current PD-L1/PD-1-blocking strategies, highlighting the need for further advancements in PD-L1/PD-1-based immunotherapy. Here, we report on a novel approach designed to combine PD-L1 checkpoint inhibition with the tumor-selective induction of apoptosis by TNF-related Apoptosis Inducing Ligand (TRAIL). In brief, a new bifunctional fusion protein, designated anti-PD-L1:TRAIL, was constructed comprising a PD-L1-blocking antibody fragment genetically fused to the extracellular domain of the pro-apoptotic tumoricidal protein TRAIL. Treatment of PD-L1-expressing cancer cells with anti-PD-L1:TRAIL induced PD-L1-directed TRAIL-mediated cancer cell death. Treatment of T cells with anti-PD-L1:TRAIL augmented T cell activation, as evidenced by increased proliferation, secretion of IFN gamma and enhanced killing of cancer cell lines and primary patient-derived cancer cells in mixed T cell/cancer cell culture experiments. Of note, elevated levels of IFN gamma further upregulated PD-L1 on cancer cells and simultaneously sensitized cancer cells to TRAIL-mediated apoptosis by anti-PD-L1:TRAIL. Additionally, anti-PD-L1:TRAIL converted immunosuppressive PD-L1-expressing myeloid cells into pro-apoptotic effector cells that triggered TRAIL-mediated cancer cell death. In conclusion, combining PD-L1 checkpoint inhibition with TRAIL-mediated induction of apoptosis using anti-PD-L1:TRAIL yields promising multi-fold and mutually reinforcing anticancer activity that may be exploited to enhance the efficacy of therapeutic PD-L1/PD-1 checkpoint inhibition.

TO THE EDITOR In recent years, a regulatory role has emerged for the glycan-binding protein galectin-9 (Gal-9) in normal physiology and pathology (reviewed by Wiersma et al. (2011). In melanoma and other malignancies, the available data suggest that Gal-9 has a tumor-suppressor function, with loss of Gal-9 being closely associated with metastatic progression (Kageshita et al., 2002; Irie et al., 2005; Yamauchi et al., 2006; Liang et al., 2008). In particular, melanocytic nevi and primary melanoma lesions highly express Gal-9, whereas metastatic melanoma lesions have no or minimal expression of Gal-9 (Kageshita et al., 2002). Furthermore, ectopic expression of Gal-9 abrogates the formation of metastases by Gal-9-deficient B16F10 murine melanoma cells (Nobumoto et al., 2008). Similarly, treatment of Gal-9-deficient B16F10 cells with a recombinant form of Gal-9, designated Gal-9(0), strongly reduced metastasis formation (Nobumoto et al., 2008). This anti-metastatic activity of Gal-9(0) on B16F10 has been attributed mainly to inhibition of melanoma cell adhesion to endothelial cells and/or extracellular matrix components, such as collagen type I (collagen-I; Nobumoto et al., 2008). The data presented in the current letter suggest that within the 1-h time frame of adhesion-type assays, treatment with Gal-9(0) triggers early apoptotic cellular changes. In line with earlier findings, Gal-9(0) inhibits the adhesion of B16F10 and 7 human melanoma cell lines to collagen-I (Figure 1a). However, the morphology of Gal-9(0)-treated cells that had adhered to collagen-I-coated wells resembled that of dying cells (Figure 1b; illustrated for B16F10). Subsequent analysis of this melanoma cell line panel, as well as primary patient–derived malignant melanoma cells for the early apoptotic marker phosphatidyl serine (PS), revealed that treatment with Gal-9(0) induced ∼90% cell death within the time frame used in the adhesion assay (Figure 1c). Early apoptotic PS exposure was followed by apoptotic cell death within 24 h of treatment, as evidenced by loss of viability (Supplementary Figure S1a online), the presence of late apoptotic Annexin-V/PI double-positive cells (Supplementary Figure S1b and c online), and an increase in DNA fragmentation (Supplementary Figure S1d online). In primary human melanocytes, Gal-9(0) also triggered PS exposure, albeit to a lesser extent (Figure 1c; ∼55%). More importantly, the viability of these normal cells was not negatively affected after 24 h (Supplementary Figure S1a online). Thus, Gal-9(0) induces rapid apoptotic cell death in melanoma cells, but not in normal human melanocytes. PS exposure induced by Gal-9(0) was fully dependent on the glycan-binding specificity of Gal-9(0), as it was selectively blocked by the competitive Gal-9 inhibitor alpha-lactose but not by the irrelevant carbohydrate sucrose (Figure 1d). Sensitivity to Gal-9(0) did not or only weakly correlated with expression of endogenous Gal-9 (Supplementary Figure S2 online; r2=0.250). Time-course analysis in five of the human melanoma cell lines demonstrated that treatment with Gal-9(0) induced PS exposure in >60% of melanoma cells within 5 minutes of treatment (Figure 1e). Furthermore, the extent of PS exposure closely correlated with the inhibitory effect of Gal-9(0) on collagen I binding (Figure 1f, MM-RU; r2=0.693). Together, these data suggest that the biological effect of Gal-9(0) in adhesion assays is mediated, at least partly, through the induction of cell death. It is noteworthy that pan-caspase inhibition failed to block the anti-adhesive and apoptotic activity of Gal-9(0; Supplementary Figure S1e and f online). Thus, Gal-9(0)-mediated melanoma apoptosis does not require caspase activation, which is in line with e.g. reports on gal-1-mediated cell death of T cells (Hahn et al., 2004). Figure 1 Galectin-9 (Gal-9(0)) rapidly induces apoptosis in serum-free conditions. (a). Adhesion of B16F10 and a panel of human melanoma cell lines to collagen-I-coated wells is inhibited by recombinant Gal-9. In brief, 3 × 104 melanoma cells were added ... Adhesion assays are typically performed in serum-free conditions, whereas in normal physiological situations the presence of serum and/or plasma components may affect the biological activity of Gal-9. Indeed, it is well established that Gal-9 can interact with serum components (Cederfur et al., 2008). Therefore, the biological activity of Gal-9(0) was next evaluated in the presence of 10% fetal calf serum (FCS), the standard serum additive in cell death assays. The inclusion of 10% FCS in these apoptosis experiments completely abrogated the morphological changes in melanoma cells, with induction of PS exposure by Gal-9(0) being abrogated in six of the seven cell lines tested (Figure 2a). Similarly, PS exposure by Gal-9(0) was also strongly inhibited in the primary melanoma cells (Figure 2a). Indeed, FCS dose dependently inhibited PS exposure induced by Gal-9(0) (illustrated for cell line A2058 in Figure 2b) and blocked the binding of Gal-9(0) to A2058 cells (Figure 2c). When using dialysed FCS or heat-inactivated FCS, the activity of Gal-9(0) was still inhibited (Figure 2d). Thus, the inhibitory component present in FCS is not heat labile (i.e., complement factors) and is >10 kDa in size. Notably, FCS did not inhibit Gal-9(0) activity toward SK-MEL-28 cells (Figure 2a), which suggests that the inhibitory effect of FCS is not merely due to binding of a serum component to Gal-9(0). Possibly, a serum component may shield the receptor(s) of Gal-9 on most melanoma cells. On Sk-MEL-28 cells, Gal-9(0) may interact with an alternative receptor not subject to binding/inhibition by FCS. Figure 2 Cell death induction by galectin-9 (Gal-9(0)) is blocked by fetal calf serum (FCS) but not by human pooled plasma. (a) A panel of melanoma cells and primary patient–derived melanoma cells (n=1) were treated for 1 h with Gal-9(0) in standard ... Although FCS is the standard additive in in vitro cell death assays, a better approximation of physiological settings is the addition of human plasma. Importantly, 10% human pooled plasma did not abrogate Gal-9(0)-induced PS exposure in A2058 cells, with only a slight reduction in PS exposure compared with serum-free conditions (Figure 2d). Similar results were obtained in six melanoma cell lines and in primary patient–derived melanoma cells (Figure 2e). These experiments suggest that an important biological effect of Gal-9(0) on human melanoma cells is the induction of apoptosis. This biological effect of Gal-9(0) is masked by as yet unidentified components present in FCS, but is unmasked in serum-free conditions or when human plasma is used. Indeed, in standard FCS-containing culture conditions, apoptotic cell death of the melanoma cell line MM-RU was only detected after 72 h of treatment with Gal-9 (Kageshita et al., 2002). The use of FCS may similarly mask the biological activity of other gal family members. In this respect, gal-2, -3, -4, and -8 interact with various serum components (Cederfur et al., 2008). Notably, plasma levels of several of the gal family members are increased in malignancy (Barrow et al., 2011). The use of human pooled serum/plasma instead of FCS for in vitro biological assays with members of the Gal family therefore appears prudent. In conclusion, recombinant Gal-9 has a hitherto unrecognized cytotoxic effect toward human melanoma cells, which further highlights its potential therapeutic applicability for the treatment of human metastatic melanoma.

In recent years, an important role has emerged for the glycan-binding protein Galectin-9 (Gal-9) in health and disease. In normal physiology, Gal-9 seems to be a pivotal modulator of T-cell immunity by inducing apoptosis in specific T-cell subpopulations. Because these T-cell populations are associated with autoimmunity, inflammatory disease, and graft rejection, it was postulated that application of exogenous Gal-9 may limit pathogenic T-cell activity. Indeed, treatment with recombinant Gal-9 ameliorates disease activity in various preclinical models of autoimmunity and allograft graft rejection. In many solid cancers, the loss of Gal-9 expression is closely associated with metastatic progression. In line with this observation, treatment with recombinant Gal-9 prevents metastatic spread in various preclinical cancer models. In addition, various hematological malignancies are sensitive to apoptotic elimination by recombinant Gal-9. Here, we review the biology and physiological role of this versatile lectin and discuss the therapeutic potential of Gal-9 in various diseases, including autoimmunity, asthma, infection, and cancer.

Calreticulin is a pleiotropic molecule that normally resides in the lumen of the endoplasmatic reticulum (ER). Here it has various functions, ranging from regulation of calcium homeostasis to ensuring proper protein folding. More recently, calreticulin gained especial interest for its extracellular functions, where it has direct immunomodulatory activity. In this respect, calreticulin activates dendritic cells (DCs) and macrophages. In addition, certain anti-cancer therapies induce the translocation of calreticulin from the ER to the cell surface of dying cancer cells, where calreticulin dictates the immunogenicity of these cells. Interestingly, treatment with Tumor Necrosis Factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL) also induces membrane calreticulin exposure on cancer cells. As shown here, calreticulin directly interacts with TRAIL and its receptor (TRAILR)-signaling complex, as well as with other TNF family members. Of note, TRAIL is a well known immunomodulatory molecule, and is expressed on the surface of natural killer T-cells (NK T-cells). Therefore, calreticulin may have an as yet unrecognized wide(r) impact on immunity, with the TNF ligand family modulating virtually all aspects of the immune response.

Oncogenic mutation of KRAS (Kirsten rat sarcoma viral oncogene homolog) in colorectal cancer (CRC) confers resistance to both chemotherapy and EGFR (epidermal growth factor receptor)-targeted therapy. We uncovered that KRAS mutant (KRASmut) CRC is uniquely sensitive to treatment with recombinant LGALS9/Galectin-9 (rLGALS9), a recently established regulator of epithelial polarity. Upon treatment of CRC cells, rLGALS9 rapidly internalizes via early- and late-endosomes and accumulates in the lysosomal compartment. Treatment with rLGALS9 is accompanied by induction of frustrated autophagy in KRASmut CRC, but not in CRC with BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations (BRAFmut). In KRASmut CRC, rLGALS9 acts as a lysosomal inhibitor that inhibits autophagosome-lysosome fusion, leading to autophagosome accumulation, excessive lysosomal swelling and cell death. This antitumor activity of rLGALS9 directly correlates with elevated basal autophagic flux in KRASmut cancer cells. Thus, rLGALS9 has potent antitumor activity towards refractory KRASmut CRC cells that may be exploitable for therapeutic use.

Recently, a small subset of T cells that expresses the B cell marker CD20 has been identified in healthy volunteers and in patients with rheumatoid arthritis and multiple sclerosis. The origin of these CD20-positive T cells as well as their relevance in human disease remains unclear. Here, we identified that after functional B cell/T cell interaction CD20 molecules are transferred to the cell surface of T cells by trogocytosis together with the established trogocytosis marker HLA-DR. Further, the presence of CD20 on isolated CD20(+) T cells remained stable for up to 48h of ex vivo culture. These CD20(+) T cells almost exclusively produced IFN gamma (similar to 70% vs. similar to 20% in the CD20(+) T cell population) and were predominantly (CD8(+)) effector memory T cells (similar to 60-70%). This IFN gamma producing and effector memory phenotype was also determined for CD20(+) T cells as detected in the peripheral blood and ascitic fluids of ovarian cancer (OC) patients. In the latter, the percentage of CD20(+) T cells was further strongly increased (from similar to 6% in peripheral blood to 23% in ascitic fluid). Taken together, the data presented here indicate that CD20 is transferred to T cells upon intimate T cell/B cell interaction. Further, CD20(+) T cells are of memory and IFN gamma producing phenotype and are present in increased amounts in ascitic fluid of OC patients.