IL-15Rα membrane anchorage in either cis or trans is required for stabilization of IL-15 and optimal signaling.

Interleukin (IL)-15 plays an important role in the communication between immune cells. It delivers its signal through different modes involving three receptor chains: IL-15Rα, IL-2Rß and IL-2Rγc. The combination of the different chains result in the formation of IL-15Rα/IL-2Rß/γc trimeric or IL-2Rß/γc dimeric receptors. In this study, we have investigated the role of the IL-15Rα chain in stabilizing the cytokine in the IL-2Rß/γc dimeric receptor. By analyzing the key amino acid residues of IL-15 facing IL-2Rß, we provide evidence of differential interfaces in the presence or in the absence of membrane-anchored IL-15Rα. Moreover, we found that the anchorage of IL-15Rα to the cell surface regardless its mode of presentation - i.e. cis or trans - is crucial for complete signaling. These observations show how the cells can finely modulate the intensity of cytokine signaling through the quality and the level of expression of the receptor chains.

Rational modification, synthesis and biological evaluation of N-substituted phthalazinone derivatives designed to target interleukine-15 protein.

Interleukin (IL)-15 is a pleiotropic cytokine structurally close to IL-2 and sharing with the IL-2Rß and γc receptor (R) subunits. IL-15 plays important roles in innate and adaptative immunity, supporting the activation and proliferation of NK, NK-T, and CD8+ T cells. Over-expression of IL-15 has been shown to participate to the development of inflammatory and autoimmune diseases and diverse T cell malignancies. This study is in continuity of our previous work through which a family of small-molecule inhibitors impeding IL-15/IL-2Rß interaction with sub-micromolar activity has been identified using pharmacophore-based virtual screening and hit optimization methods. With the aim to improve the efficacy and selectivity of our lead inhibitor, specific modifications have been introduced on the basis of optimized SAR and modelisation. The new series of compounds generated have been evaluated for their capacity to inhibit the proliferation as well as the down-stream signaling of IL-15-dependent cells and to bind to IL-15.

Two-Chains Tissue Plasminogen Activator Unifies Met and NMDA Receptor Signalling to Control Neuronal Survival.

Tissue-type plasminogen activator (tPA) plays roles in the development and the plasticity of the nervous system. Here, we demonstrate in neurons, that by opposition to the single chain form (sc-tPA), the two-chains form of tPA (tc-tPA) activates the MET receptor, leading to the recruitment of N-Methyl-d-Aspartate receptors (NMDARs) and to the endocytosis and proteasome-dependent degradation of NMDARs containing the GluN2B subunit. Accordingly, tc-tPA down-regulated GluN2B-NMDAR-driven signalling, a process prevented by blockers of HGFR/MET and mimicked by its agonists, leading to a modulation of neuronal death. Thus, our present study unmasks a new mechanism of action of tPA, with its two-chains form mediating a crosstalk between MET and the GluN2B subunit of NMDARs to control neuronal survival.

Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases.

Sialidases (SAs) hydrolyze sialyl residues from glycoconjugates of the eukaryotic cell surface and are virulence factors expressed by pathogenic bacteria, viruses, and parasites. The catalytic domains of SAs are often flanked with carbohydrate-binding module(s) previously shown to bind sialosides and to enhance enzymatic catalytic efficiency. Herein, non-hydrolyzable multivalent thiosialosides were designed as probes and inhibitors of V. cholerae, T. cruzi, and S. pneumoniae (NanA) sialidases. NanA was truncated from the catalytic and lectinic domains (NanA-L and NanA-C) to probe their respective roles upon interacting with sialylated surfaces and the synthetically designed di- and polymeric thiosialosides. The NanA-L domain was shown to fully drive NanA binding, improving affinity for the thiosialylated surface and compounds by more than two orders of magnitude. Importantly, each thiosialoside grafted onto the polymer was also shown to reduce NanA and NanA-C catalytic activity with efficiency that was 3000-fold higher than that of the monovalent thiosialoside reference. These results extend the concept of multivalency for designing potent bacterial and parasitic sialidase inhibitors.

Cutting Edge: Differential Fine-Tuning of IL-2- and IL-15-Dependent Functions by Targeting Their Common IL-2/15Rß/γc Receptor.

Interleukin 2 and IL-15 are two closely related cytokines, displaying important functions in the immune system. They share the heterodimeric CD122/CD132 receptor to deliver their signals within target cells. Their specificity of action is conferred by their α receptor chains, IL-2Rα and IL-15Rα. By combining an increased affinity for CD122 and an impaired recruitment of CD132, we have generated an original molecule named IL-2Rß/γ (CD122/CD132) inhibitor (BiG), targeting the CD122/CD132 receptor. BiG efficiently inhibited IL-15- and IL-2-dependent functions of primary cells, including CD8 T and NK cells, in vitro and in vivo. We also report a differential dynamic of action of these cytokines by highlighting a major role played by the IL-2Rα receptor. Interestingly, due to the presence of IL-2Rα, BiG had no impact on IL-2-dependent regulatory T cell proliferation. Thus, by acting as a fine switch in the immune system, BiG emphasizes the differential roles of these two cytokines.

A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM.

Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).

Syndecan-1 regulates the biological activities of interleukin-34.

IL-34 is a challenging cytokine sharing functional similarities with M-CSF through M-CSFR activation. It also plays a singular role that has recently been explained in the brain, through a binding to the receptor protein tyrosine phosphatase RPTPß/ζ. The aim of this paper was to look for alternative binding of IL-34 on other cell types. Myeloid cells (HL-60, U-937, THP-1) were used as cells intrinsically expressing M-CSFR, and M-CSFR was expressed in TF-1 and HEK293 cells. IL-34 binding was studied by Scatchard and binding inhibition assays, using 125I-radiolabelled cytokines, and surface plasmon resonance. M-CSFR activation was analysed by Western blot after glycosaminoglycans abrasion, syndecan-1 overexpression or repression and addition of a blocking anti-syndecan antibody. M-CSF and IL-34 induced different patterns of M-CSFR phosphorylations, suggesting the existence of alternative binding for IL-34. Binding experiments and chondroitinase treatment confirmed low affinity binding to chondroitin sulphate chains on cells lacking both M-CSFR and RPTPß/ζ. Amongst the proteoglycans with chondroitin sulphate chains, syndecan-1 was able to modulate the IL-34-induced M-CSFR signalling pathways. Interestingly, IL-34 induced the migration of syndecan-1 expressing cells. Indeed, IL-34 significantly increased the migration of THP-1 and M2a macrophages that was inhibited by addition of a blocking anti-syndecan-1 antibody. This paper provides evidence of alternative binding of IL-34 to chondroitin sulphates and syndecan-1 at the cell surface that modulates M-CSFR activation. In addition, IL-34-induced myeloid cell migration is a syndecan-1 dependent mechanism.

IL-34 and M-CSF form a novel heteromeric cytokine and regulate the M-CSF receptor activation and localization.

Interleukin-34 (IL-34) is a newly-discovered homodimeric cytokine that regulates, like Macrophage Colony-Stimulating Factor (M-CSF), the differentiation of the myeloid lineage through M-CSF receptor (M-CSFR) signaling pathways. To date, both cytokines have been considered as competitive cytokines with regard to the M-CSFR. The aim of the present work was to study the functional relationships of these cytokines on cells expressing the M-CSFR. We demonstrate that simultaneous addition of M-CSF and IL-34 led to a specific activation pattern on the M-CSFR, with higher phosphorylation of the tyrosine residues at low concentrations. Similarly, both cytokines showed an additive effect on cellular proliferation or viability. In addition, BIAcore experiments demonstrated that M-CSF binds to IL-34, and molecular docking studies predicted the formation of a heteromeric M-CSF/IL-34 cytokine. A proximity ligation assay confirmed this interaction between the cytokines. Finally, co-expression of the M-CSFR and its ligands differentially regulated M-CSFR trafficking into the cell. This study establishes a new foundation for the understanding of the functional relationship between IL-34 and M-CSF, and gives a new vision for the development of therapeutic approaches targeting the IL-34/M-CSF/M-CSFR axis.

Tumor targeting of the IL-15 superagonist RLI by an anti-GD2 antibody strongly enhances its antitumor potency.

Immunocytokines (ICKs) targeting cytokines to the tumor environment using antibodies directed against a tumor-associated antigen often have a higher therapeutic index than the corresponding unconjugated cytokines. Various ICKs displaying significant antitumoral effects in several murine tumor models have already been developed, and some of them, in particular interleukin (IL)-2-based ICKs, are in Phase II clinical trials. Although sharing common biological activities with IL-2 in vitro, IL-15 is now considered as having a better potential in antitumor immunotherapeutical strategies and has been shown to be less toxic than IL-2 in preclinical studies. We previously developed the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. RLI showed better biological activities than IL-15 in vitro as well as higher antitumoral effects in vivo in murine and human cancer models. Here, we investigated, in the context of an ICK, the effect of associating RLI with an antibody targeting the GD2 ganglioside, a validated tumoral target expressed on many neurectodermal tumors. Anti-GD2-RLI fully retained the cytokine potential of RLI and the antibody effector functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity). It displayed strong antitumor activities in two syngeneic cancer models in immunocompetent mice (subcutaneous EL4 and metastatic NXS2). Its therapeutic potency was higher than those of RLI and anti-GD2 alone or in combination. We suggest that this is related to its bifunctional (cytokine and antibody) nature.

Counteracting Interleukin-15 to Elucidate Its Modes of Action in Physiology and Pathology.

Interleukin (IL)-15 belongs to the common gamma-dependent cytokine family, along with IL-2, IL-4, IL-7, IL-9, and IL-21. IL-15 is crucial for the homeostasis of Natural Killer (NK) and memory CD8 T cells, and to fight against cancer progression. However, dysregulations of IL-15 expression could occur and participate in the emergence of autoimmune inflammatory diseases as well as hematological malignancies. It is therefore important to understand the different modes of action of IL-15 to decrease its harmful action in pathology without affecting its beneficial effects in the immune system. In this review, we present the different approaches used by researchers to inhibit the action of IL-15, from most broad to the most selective. Indeed, it appears that it is important to selectively target the mode of action of the cytokine rather than the cytokine itself as they are involved in numerous biological processes.

Selective Targeting of IL-15Rα Is Sufficient to Reduce Inflammation.

Cytokines are crucial molecules for maintaining the proper functioning of the immune system. Nevertheless, a dysregulation of cytokine expression could be involved in the pathogenesis of autoimmune diseases. Interleukin (IL)-15 is a key factor for natural killer cells (NK) and CD8 T cells homeostasis, necessary to fight cancer and infections but could also be considered as a pro-inflammatory cytokine involved in autoimmune inflammatory disease, including rheumatoid arthritis, psoriasis, along with tumor necrosis factor alpha (TNF-α), IL-6, and IL-1ß. The molecular mechanisms by which IL-15 exerts its inflammatory function in these diseases are still unclear. In this study, we generated an IL-15-derived molecule called NANTIL-15 (New ANTagonist of IL-15), designed to selectively inhibit the action of IL-15 through the high-affinity trimeric IL-15Rα/IL-2Rß/γc receptor while leaving IL-15 signaling through the dimeric IL-2Rß/γc receptor unaffected. Administrating of NANTIL-15 in healthy mice did not affect the IL-15-dependent cell populations such as NK and CD8 T cells. In contrast, we found that NANTIL-15 efficiently reduced signs of inflammation in a collagen-induced arthritis model. These observations demonstrate that the inflammatory properties of IL-15 are linked to its action through the trimeric IL-15Rα/IL-2Rß/γc receptor, highlighting the interest of selectively targeting this receptor.

hnRNP-A1 binds to the IRES of MELOE-1 antigen to promote MELOE-1 translation in stressed melanoma cells.

The major challenge in antigen-specific immunotherapy of cancer is to select the most relevant tumor antigens to target. To this aim, understanding their mode of expression by tumor cells is critical. We previously identified a melanoma-specific antigen, melanoma-overexpressed antigen 1 (MELOE-1)-coded for by a long noncoding RNA-whose internal ribosomal entry sequence (IRES)-dependent translation is restricted to tumor cells. This restricted expression is associated with the presence of a broad-specific T-cell repertoire that is involved in tumor immunosurveillance in melanoma patients. In the present work, we explored the translation control of MELOE-1 and provide evidence that heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) binds to the MELOE-1 IRES and acts as an IRES trans-activating factor (ITAF) to promote the translation of MELOE-1 in melanoma cells. In addition, we showed that endoplasmic reticulum (ER) stress induced by thapsigargin, which promotes hnRNP-A1 cytoplasmic translocation, enhances MELOE-1 translation and recognition of melanoma cells by a MELOE-1-specific T-cell clone. These findings suggest that pharmacological stimulation of stress pathways may enhance the efficacy of immunotherapies targeting stress-induced tumor antigens such as MELOE-1.

Thrombolysis by PLAT/tPA increases serum free IGF1 leading to a decrease of deleterious autophagy following brain ischemia.

Cerebral ischemia is a pathology involving a cascade of cellular mechanisms, leading to the deregulation of proteostasis, including macroautophagy/autophagy, and finally to neuronal death. If it is now accepted that cerebral ischemia induces autophagy, the effect of thrombolysis/energy recovery on proteostasis remains unknown. Here, we investigated the effect of thrombolysis by PLAT/tPA (plasminogen activator, tissue) on autophagy and neuronal death. In two in vitro models of hypoxia reperfusion and an in vivo model of thromboembolic stroke with thrombolysis by PLAT/tPA, we found that ischemia enhances neuronal deleterious autophagy. Interestingly, PLAT/tPA decreases autophagy to mediate neuroprotection by modulating the PI3K-AKT-MTOR pathways both in vitro and in vivo. We identified IGF1R (insulin-like growth factor I receptor; a tyrosine kinase receptor) as the effective receptor and showed in vitro, in vivo and in human stroke patients and that PLAT/tPA is able to degrade IGFBP3 (insulin-like growth factor binding protein 3) to increase IGF1 (insulin-like growth factor 1) bioavailability and thus IGF1R activation.Abbreviations: AKT/protein kinase B: thymoma viral proto-oncogene 1; EGFR: epidermal growth factor receptor; Hx: hypoxia; IGF1: insulin-like growth factor 1; IGF1R: insulin-like growth factor I receptor; IGFBP3: insulin-like growth factor binding protein 3; Ka: Kainate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OGD: oxygen and glucose deprivation; OGDreox: oxygen and glucose deprivation + reoxygentation; PepA: pepstatin A1; PI3K: phosphoinositide 3-kinase; PLAT/tPA: plasminogen activator, tissue; PPP: picropodophyllin; SCH77: SCH772984; ULK1: unc-51 like kinase 1; Wort: wortmannin.

Definition and characterization of an inhibitor for interleukin-31.

Interleukin-31 (IL-31) is a recently described T cell-derived cytokine, mainly produced by T helper type 2 cells and related to the IL-6 cytokine family according to its structure and receptor. IL-31 is the ligand for a heterodimeric receptor composed of a gp130-like receptor (GPL) associated with the oncostatin M receptor (OSMR). A link between IL-31 and atopic dermatitis was shown by studying the phenotype of IL-31 transgenic mice and IL-31 gene haplotypes in patients suffering from dermatitis. In this study, we generated a potent IL-31 antagonist formed by external portions of OSMR and GPL fused with a linker. This fusion protein, OSMR-L-GPL, consisting of 720 amino acids, counteracted the binding of IL-31 to its membrane receptor complex and the subsequent signaling events involving the STATs and MAPK pathways. Neutralizing effects were found in IL-31-sensitive cell lines, including brain-derived cells and primary cultures of keratinocytes.

Phosphorothioate-modified TLR9 ligands protect cancer cells against TRAIL-induced apoptosis.

Hypomethylated CpG oligodeoxynucleotides (CpG ODNs) target TLR9 expressed by immune cells and are currently being evaluated as adjuvants in clinical trials. However, TLR signaling can promote some tumor growth and immune evasion, such as in multiple myeloma (MM). Therefore, deciphering the effects of CpG ODNs on cancer cells will help in preventing these adverse effects and in designing future clinical trials. TLR activation induces multiple signaling pathways, notably NF-kappaB that has been involved in the resistance to TRAIL. Thus, we wondered if CpG ODNs could modulate TRAIL-induced apoptosis in different models of tumors. Here, we show that TLR9+ (NCI-H929, NAN6, KMM1) and TLR9- MM cells (MM1S) were protected by CpG ODNs against recombinant TRAIL-induced apoptosis. By using two fully human, agonist mAbs directed against TRAIL receptors DR4 and DR5 (mapatumumab and lexatumumab, respectively), we show that the protection was restricted to DR5-induced apoptosis. Similar results were observed for two colon cancer (C45 and Colo205) and two breast cancer cell lines (HCC1569 and Cal51). The protection of CpG ODNs was mediated by its nuclease-resistant phosphorothioate backbone independent of TLR9. We next demonstrated by surface plasmon resonance that phosphorothioate-modified CpG ODNs directly bound to either TRAIL or lexatumumab and then decreased their binding to DR5. Finally, NK cell lysis of a DR5-sensitive MM cell line (NCI-H929) through TRAIL was partially inhibited by phosphorothioate-modified CpG ODNs. In conclusion, our results suggest that the phosphorothioate modification of CpG ODNs could dampen the clinical efficacy of CpG ODN-based adjuvants by altering TRAIL/TRAIL receptor interaction.

Factor VIII-von Willebrand factor complex inhibits osteoclastogenesis and controls cell survival.

Factor VIII-von Willebrand factor (FVIII.vWF) complex, a molecule involved in coagulation, can be physically associated with osteoprotegerin (OPG). OPG is an anti-osteoclastic protein and a soluble receptor for the proapoptotic protein TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), suggesting a potential role of FVIII.vWF complex in bone and cancer biology. We, thus, assessed the effects of FVIII.vWF complex on osteoclastogenesis and cell survival. We first evidenced that FVIII.vWF complex inhibited RANKL-induced osteoclastogenesis and enhanced the inhibitory effect of OPG. Interestingly, we revealed by surface plasmon resonance that FVIII.vWF complex bound to RANKL, whereas recombinant FVIII and vWF did not. By modeling, we showed that the OPG binding domain to the A1 domain of vWF was closely located and partially overlapped to its binding site to RANKL. Then, we demonstrated that FVIII.vWF complex cancelled the inhibitory activity of OPG on TRAIL-induced apoptosis and characterized interactions between these molecules. The present work evidenced a direct activity of FVIII.vWF complex on osteoclasts and on induced cell apoptosis, pointing out its potential involvement in physiological bone remodeling or in bone damages associated with severe hemophilia and cancer development.

Discovery of a Small-Molecule Inhibitor of Interleukin 15: Pharmacophore-Based Virtual Screening and Hit Optimization.

Interleukin (IL)-15 is a pleiotropic cytokine, which is structurally close to IL-2 and shares with it the IL-2 ß and γ receptor (R) subunits. By promoting the activation and proliferation of NK, NK-T, and CD8+ T cells, IL-15 plays important roles in innate and adaptative immunity. Moreover, the association of high levels of IL-15 expression with inflammatory and autoimmune diseases has led to the development of various antagonistic approaches targeting IL-15. This study is an original approach aimed at discovering small-molecule inhibitors impeding IL-15/IL-15R interaction. A pharmacophore and docking-based virtual screening of compound libraries led to the selection of 240 high-scoring compounds, 36 of which were found to bind IL-15, to inhibit the binding of IL-15 to the IL-2Rß chain or the proliferation of IL-15-dependent cells or both. One of them was selected as a hit and optimized by a structure-activity relationship approach, leading to the first small-molecule IL-15 inhibitor with sub-micromolar activity.

Affinity transfer to the archaeal extremophilic Sac7d protein by insertion of a CDR.

Artificially transforming a scaffold protein into binders often consists of introducing diversity into its natural binding region by directed mutagenesis. We have previously developed the archaeal extremophilic Sac7d protein as a scaffold to derive affinity reagents (Affitins) by randomization of only a flat surface, or a flat surface and two short loops with natural lengths. Short loops are believed to contribute to stability of extremophilic proteins, and loop extension has been reported detrimental for the thermal and chemical stabilities of mesophilic proteins. In this work, we wanted to evaluate the possibility of designing target-binding proteins based on Sac7d by using a complementary determining region (CDR). To this aim, we inserted into three different loops a 10 residues CDR from the cAb-Lys3 anti-lysozyme camel antibody. The chimeras obtained were as stable as wild-type (WT) Sac7d at extreme pH and their structural integrity was supported. Chimeras were thermally stable, but with T(m)s from 60.9 to 66.3°C (cf. 91°C for Sac7d) which shows that loop extension is detrimental for thermal stability of Sac7d. The loop 3 enabled anti-lysozyme activity. These results pave the way for the use of CDR(s) from antibodies and/or extended randomized loop(s) to increase the potential of binding of Affitins.

Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability.

As a useful reagent for biotechnological applications, a scaffold protein needs to be as stable as possible to ensure longer lifetimes. We have developed archaeal extremophilic proteins from the "7 kDa DNA-binding" family as scaffolds to derive affinity proteins (Affitins). In this study, we evaluated a rational structure/sequence-guided approach to stabilize an Affitin derived from Sac7d by transferring its human IgG binding site onto the framework of the more thermally stable Sso7d homolog. The chimera obtained was functional, well expressed in Escherichia coli, but less thermally stable than the original Affitin (T(m) = 74.2 °C vs. T(m) = 80.4 °C). Two single mutations described as thermally stabilizing wild type Sso7d were introduced into chimeras. Only the double mutation nearly restored thermal stability (T(m) = 76.9 °C). Interestingly, the chimera and its double mutant were stable from pH 0 up to at least pH 13. Our results show that it is possible to increase further the stability of Affitins toward alkaline conditions (+2 pH units) while conserving their advantageous properties. As Affitins are based on a growing family of homologs from archaeal extremophiles, we conclude that this approach offers new potential for their improvement, which will be useful in demanding biotechnological applications.

Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse.

Rituximab (RTX), a chimeric IgG1 monoclonal antibody directed against the CD20 antigen, has revolutionized the treatment of B-cell malignancies. Nevertheless, the relapsed/refractory rates are still high. One strategy to increase the clinical effectiveness of RTX is based on antibody-cytokine fusion protein (immunocytokine; ICK) vectorizing together at the tumor site the antibody effector activities and the cytokine co-signal required for the generation of cytotoxic cellular immunity. Such ICKs linking various antibody formats to interleukin (IL)-2 are currently being investigated in clinical trials and have shown promising results in cancer therapies. IL-15, a structurally-related cytokine, is now considered as having a better potential than IL-2 in antitumor immunotherapeutic strategies. We have previously engineered the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. Compared with IL-15, RLI displayed better biological activities in vitro and higher antitumor effects in vivo in murine and human cancer models. In this study, we investigated the advantages of fusing RLI to RTX. Anti-CD20-RLI kept its binding capacity to CD20, CD16 and IL-15 receptor and therefore fully retained both antibody effector functions (ADCC and CDC), and the cytokine potential of RLI. In a severe combined immunodeficiency (SCID) mouse model of disseminated residual lymphoma, anti-CD20-RLI was found to induce long-term survival of 90% of mice up to at least 120 days whereas RLI and RTX, alone or in combination, just delayed the disease onset (100% of death at 28, 40 and 51 days respectively). These findings suggest that such ICK could improve the clinical efficacy of RTX, particularly in patients with refractory B-cell lymphoma.