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The anti-CD20 monoclonal antibody (mAb) rituximab has been used successfully for lymphoma therapy for more than 10 years. Although several direct mechanisms by which anti-CD20 mAbs act have been characterized in vitro, their specific role in clinical efficacy is still debated. Little is known about the possible antitumor immune response that they may induce in patients, despite clinical data suggesting a “vaccinal” effect. We show here that an initial treatment with anti-CD20 induces protection against human CD20-expressing tumor cells and allows immunocompetent mice to survive tumor challenge. This long-lasting protection requires the presence of the Fc portion of the anti-CD20 mAb and is achieved through the induction of a cellular immune response. Only CD4+ cells were needed at the beginning of the treatment, but both CD4+ and CD8+ cells were required after tumor challenge to achieve protection. Finally, we show that interleukin-2 treatment, given after tumor challenge, improves the overall survival rate, compared with that obtained by anti-CD20 treatment alone. These findings demonstrate that anti-CD20 mAbs exert therapeutic effects through the induction of an adaptive cellular immune response, aside from any direct mechanisms involving effectors from innate immunity.

Epstein-Barr virus (EBV) latently infects and immortalizes B lymphocytes and causes lymphoproliferative malignancies. We show here that the EBV nuclear antigen EBNA2 induces expression of the 2 chains of the interleukin-18 receptor (IL-18R) in Burkitt lymphoma (BL) cell lines and in nontransformed B cells. Activation of IL-18R expression by EBNA2 is independent of its interaction with the transcriptional repressor RBPJκ. It occurs in the absence of any other viral protein but requires de novo synthesis of cellular proteins. IL-18R induction is a highly specific function of EBNA2, because neither other EBV latent proteins nor the cellular proteins c-myc or Notch can exert this effect. Using cDNA microarray expression profiling, we find that the IL-18 receptor expressed in EBV-infected BL cells has signaling capacity, because IL-18 significantly modified gene expression. We report that EBNA2 expression is associated with IL-18R expression in vivo in EBV-positive B-lymphomas from AIDS patients. (Blood. 2005;105:1632-1639)

International audience; Immature dendritic cells (DCs) reside in Interstitial tissues (Int-DC) or in the epidermis, where they capture antigen and, thereafter, mature and migrate to draining lymph nodes (LNs), where they present processed antigen to T cells. We have Identified Int-DCs that express both TRANCE (tumor necrosis factor-related activation-induced cytokine) and RANK (receptor activator of NF-kappaB) and have generated these cells from CD34(+) human progenitor cells using macrophage colony-stimulating factor (M-CSF). These CD34(+)-derived Int-DCs, which are related to macrophages, are long-lived, but addition of soluble RANK leads to significant reduction of cell viability and BcI-2 expression. This suggests that constitutive TRANCE-RANK interaction is responsible for CD34(+)-derived Int-DC longevity. Conversely, CD1a(+) DCs express only RANK and are short-lived. However, they can be rescued from cell death either by recombinant soluble TRANCE or by CD34(+)-derived Int-DCs. CD34(+)-derived Int-DCs mature in response to lipopolysaccharide (LPS) plus CD40 ligand (L) and become capable of CCL21/CCL19-mediated chemotaxis and naive T-cell activation. Upon maturation, they lose TRANCE, making them, like CD1a(+) DCs, dependent on exogenous TRANCE for survival. These findings provide evidence that TRANCE and RANK play important roles in the homeostasis of DCs.

Interleukin 17 (IL-17) is a proinflammatory cytokine produced by activated CD4+ memory T cells. We previously showed that IL-17 increased the growth rate of human cervical tumors transplanted into athymic nude mice. To address the possible role of T cells in the biologic activity of IL-17 for tumor control, we grafted 2 murine hematopoietic immunogenic tumors (P815 and J558L) transfected with a complementary DNA encoding murine IL-17 into syngeneic immunocompetent mice. We found that growth of the 2 IL-17–producing tumors was significantly inhibited compared with that of mock-transfected tumors. In contrast to the antitumor activity of IL-17 observed in immunocompetent mice, we observed no difference in the in vivo growth of IL-17–transfected or mock-transfected P815 cells (P815–IL-17 and P815-Neo, respectively) transplanted into nude mice. We then showed that IL-17 increased generation of specific cytolytic T lymphocytes (CTLs) directed against the immunodominant antigens from P815 called A, B, C, D, and E, since all mice injected with P815–IL-17 developed a P815-specific CTL response, whereas only 6 of 16 mice immunized with P815-Neo had a specific CTL response against the antigens. The induction of CTLs was associated with establishment of a tumor-protective immunity. These experiments suggest that T lymphocytes are involved in the antitumor activity of IL-17. Therefore, IL-17, like other cytokines, appears to be a pleiotropic cytokine with possible protumor or antitumor effects on tumor development, which often depends on the immunogenicity of tumor models.

Neutrophils isolated from cancer patients treated with granulocyte colony-stimulating factor (G-CSF) express high levels of Fc gamma RI. They exhibited an efficient killing of GD2+ neuroblastoma cells in the presence of an antidisialoganglioside (GD2) mouse monoclonal antibody (MoAb; 7A4, IgG3 kappa). However, this cytotoxicity was totally blocked by human monomeric IgG. In contrast, a bispecific antibody (7A4 bis 22/MDX-260), prepared by chemically linking an F(ab') fragment of 7A4 with an F(ab') fragment of an anti-Fc gamma RI MoAb, 22, which binds outside the Fc binding domain, triggered antibody-dependent cell cytotoxicity, even when neutrophils were preincubated with human monomeric IgG. F(ab')2 22 MoAb abrogated the MDX-260 killing without affecting that of 7A4. The 3G8 MoAb, directed against the Fc gamma RIII binding site, did not inhibit the cytotoxicity induced by either antibody. Thus, these results indicate that G-CSF-activated neutrophils exert their cytotoxic effect against neuroblastoma cells through Fc gamma RI and not Fc gamma RIII, and that the saturation of the high affinity Fc gamma RI by monomeric IgG can be overcome by the use of bispecific antibodies binding epitopes outside the IgG Fc gamma RI binding site. A combined administration of such bispecific antibodies and G-CSF may be, therefore, an efficient therapeutic approach to trigger tumor lysis by cytotoxic neutrophils in vivo.

Disparate findings have been reported as to whether human immunodeficiency virus (HIV) affects cytokine production by macrophages (MA). We investigated production of different cytokines and of macrophage inflammatory protein (MIP)-1alpha by HIV-1Ba-L-or HIV-1Ada-infected blood-derived MA. Relative to controls, only MIP-1alpha levels increased twofold to > 10-fold in supernatants 2 to 3 weeks postinfection (PI), at the time of maximum virus production; levels of the other chemokines (RANTES, interleukin (IL)-8) and cytokines (IL-1alpha, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1) investigated were not affected. MIP-1alpha mRNA signal assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) was, however, only occasionally greater in cells from infected cultures relative to controls. MIP-1alpha levels in supernatants remained in the same range as in control cultures when more than 10 mmol/L Zidovudine was added 24 hours PI, which indicates involvement of virus replication in the effect. Anti-MIP-1alpha antibody labeling identified a 10% to 25% subset of MA, strongly expressing HLA-DR and CD4, and also stained by anti-IL-6 and anti-TNF-alpha antibodies. Two weeks PI, dual staining showed that the majority of the 5% to 20% cells that were p24+ belonged to the MIP-1alpha+ population, which may define a MA subset capable to better sustain HIV replication. MIP-1alpha induced by HIV replication in MA might play a role in the pathophysiology of HIV infection; in impaired hematopoiesis; or as a CD4+ and CD8+ lymphocyte chemoattractant, by recruiting either or both HIV-susceptible and cytotoxic T lymphocytes to virus replication sites.

Abstract 2755 Background: Primary Central Nervous lymphomas (PCNSL), that comprise primary cerebral lymphoma (PCL) and primary intraocular lymphoma (PIOL), are typically CD20+ diffuse large B-cell lymphomas that have no detectable disease outside the brain or eye. Rituximab (RTX), an anti-CD20 antibody, has demonstrated encouraging clinical benefit in systemic B-cell lymphomas as well as PCL and PIOL, however, the role of RTX in treatment of PCNSL/PIOL remains controversial, and these highly aggressive malignancies are often incurable with available therapies. Therefore, additional treatment options are needed. Ublituximab (UTX) is a novel, glycoengineered chimeric anti-CD20 monoclonal antibody (mAb) that has a high affinity for FcγRIIIa (CD16) receptors, and therefore greater ADCC activity than RTX (Le Garff-Tavernier et al., 2011). Herein, we assess the antitumor effects of UTX compared to RTX in murine models of PCL and PIOL. Methods: The murine lymphoma B-cell line A20.IIA-GFP-hCD20 (H-2d) was injected into the right cerebral striatum (PCL model) or the vitreous (PIOL model) of adult BALB/c mice (H-2d); 7 days later, single doses of UTX were injected either into the tumor site intracerebrally (PCL) or intravitreously (PIOL), or at distance of the tumor site (intrathecally, PCL). RTX was used as a reference compound. Survival was monitored for injected mice for up to 100 days, and flow cytometric analyses were performed to assess tumor growth and T-cell infiltration. Results: In PCL and PIOL models, single doses of UTX had a marked antitumor effect more pronounced than that obtained with an equivalent dose of RTX. In the PCL model, there was an overall survival (OS) advantage with intracerebral injections of UTX compared to RTX (50% vs. 10%, n=10 in each group, p=0.0028). The reduction in tumor cells was correlated with an increased proportion of CD8+ T cells. Moreover, intrathecal injections of UTX increased OS compared to buffer solution. In the PIOL model, the absolute number of tumor cells analyzed 8 days after treatment had decreased more significantly (p=0.027) with UTX compared to the RTX group. This finding again confirmed the superiority of UTX in this setting. Conclusions: These results confirm that the novel, third-generation mAb, UTX has a sustained and greater antitumor effect than RTX on primary cerebral and intraocular lymphomas when assessed in vivo. Additional experiments evaluating the combination of UTX + methotrexate are ongoing. Clinical trials to evaluate UTX as an innovative therapeutic approach to treat primary cerebral and intraocular B-cell lymphomas are currently being evaluated. Disclosures: Jacquet: LFB Biotechnologies: Employment. Fridman:LFB Biotechnologies: Membership on an entity's Board of Directors or advisory committees. Sportelli:TG Therapeutics, Inc.: Employment, Equity Ownership. Urbain:LFB Biotechnologies: Employment.

Human neutrophils express two types of low affinity receptors for IgG, Fc gamma RII or CD32 and Fc gamma RIIIB or CD16. Human serum contains soluble CD16 (sCD16), which is produced by proteolysis of neutrophil Fc gamma RIIIB, the cleavage site being located close to the cell surface. In order to assess the functional roles of sCD16, we have produced, in eukaryotic cells, a recombinant sCD16 containing the extracellular region of Fc gamma RIIIB. Purified sCD16, of molecular mass of 48 kD, bound human IgG1 and IgG3 but not IgG2, IgG4, or F(ab')2. It inhibited, in a time and dose-dependent fashion, proliferation and IgM and IgG production of human peripheral blood mononuclear cells (PBMC) stimulated by pokeweed mitogen (PWM) in vitro. FACS analysis showed that biotinylated sCD16 bound specifically to a fraction (35%) of PBMC, which corresponds to monocytes and to subsets of B and T lymphocytes. Moreover, sCD16 did not modify the staining of PBMC by FITC-coupled PWM. Thus, the biologic function(s) of sCD16 on PWM-induced responses are exerted through direct and specific interaction(s) with mononuclear blood cells and not with PWM. In conclusion, neutrophils may play a regulatory role on immune responses via the production of soluble forms of CD16 with cell-binding and antiproliferative capacities.

Abstract 1166 Poster Board I-188 Introduction: The complement system consists of several serum proteins and cell membrane receptors that can be activated by three possible pathways leading to the production of pro-inflammatory factors such as the anaphylatoxins C3a and C5a and the formation of the membrane attack complex. The complement system has been implicated in the pathophysiology of several immune diseases and we previously reported that it was activated after allogeneic haematopoietic stem cell transplantation (HSCT) both in humans and mice. It's activation after allogeneic HSCT following a myeloablative conditioning could predict the development of gastrointestinal (GI) GVHD in humans. We now present updated data with long term follow up on an enlarged series of patients who did or did not activate complement after allogeneic HSCT. We also discuss the preliminary results of the inhibition of complement activation on experimental models of GVHD. Materials and methods: Complement activation was determined by measurment of complement proteins before and once a week up to 3 months after allogeneic HSCT in 34 patients allografted for diverse haematological malignancies in our institution following conventional myeloablative conditioning. Results were correlated to the clinical evolution of allogeneic HSCT. Preclinically, inhibition of mouse complement activation by C1 inhibitor esterase or an anti-C5 monoclonal antibody was tested in a parent (C57BL/6, H-2b) to F1 [(C57BL/6xDBA2), H-bd] GVHD mouse model. Lethally irradiated (9.75 to 11 Gy) mice reconstituted with either syngeneic or allogeneic bone marrow cells and splenocytes (107 and 13 to 20 ×106 cells/recipient, respectively) were treated with complement inhibitors between day-7 to Day 21 or day 1 to Day 28 post-HSCT. Clinical Results: Fifteen (44%) patients showed an activation of the classical pathway, as defined by a decrease of C3 and C4 proteins below normal values and of at least 50% of their pre-HSCT levels. Activation occurred during the 4 first weeks following HSCT in 11 patients, between 6 and 8 weeks in 2 cases and later, after withdrawal of immunosuppression, in 2 patients. Pre-transplant characteristics (age at transplant, sex, underlying haematological disease, conditioning regimen, use of ATG, type of donor and CMV risk) of patients who showed an activation of complement were comparable to those who did not. We confirmed that activation of complement was significantly associated with acute GI GVHD (80 % in the activated group versus 5.3% in the non activated, p=0.0028) and occurred from 1 to 3 weeks before the appearance of clinical GVHD. Patients in the activated group had a significantly increased incidence of capillary leak syndrome concomitant to the conditioning toxicity or GVHD (66.7% in the activated group vs 10.5% in the non activated, p=0.019). There was no significant difference between the groups in terms of skin or hepatic acute GVHD, TMA, VOD and chronic GVHD. In a landmark analysis by day 100 post-HSCT, overall survival was significantly impaired in the group of patients with complement activation (p=0.008) because of increased toxicity related mortality (p=0.02) and relapse (p=0.01) risks in comparison to the patients without complement activation. Preclinical Results: Complement inhibitors capable of blocking the first pathway (C1 inhibitor esterase) or terminal complement activation (anti-C5 mAb) were used in a mouse model of GVHD. These inhibitors were administrated before and/or during the phase of activation of complement which starts on day 4 post-HSCT. However, GVHD clinical signs and mortality did not appear to be improved following complement inhibitor administration in this mouse model. Conclusion: Activation of complement after allogeneic HSCT following a myeloablative conditioning appears as a predictive marker of acute GI GVHD and a pejorative prognostic factor of transplant outcome. However, inhibition of complement activation in a mouse experimental model of allogeneic HSCT did not allow the control of GVHD. It is not clear, therefore, that this mouse model is useful as a model of human GVHD. Disclosures: No relevant conflicts of interest to declare.

The Complement system consists of several serum proteins and receptors that belong to the innate immune response. It is activated by three possible pathways leading to the production of pro-inflammatory factors and the formation of the membrane attack complex. As it is known to be implicated in the physiopathology of several immune-mediated diseases, we investigated its role in GVHD in both humans and a mouse model. Thirty nine patients allografted for diverse haematological malignancies in our institution following myeloablative conditioning (n=22) or reduced intensity conditioning (RIC) (n=17) entered prospectively the study. Extensive exploration of the Complement system was carried out before and sequentially after allogeneic haematopoietic stem cell transplantation (HSCT). An activation of the classical pathway, as defined by a decrease of C3 and C4 proteins below normal values and of at least 50% of their pre-HSCT levels, was observed in 10/39 patients. Such activation was found in 9/22 (40%) patients who received myeloablative conditioning and in 1/17 (5.8%) patients allografted with RIC. This phenomenon occurred during the 4 first weeks after HSCT in 7 patients, at two months in one case and after withdrawal of immunosuppression in 2 patients. We observed a strong association between Complement activation and the development of acute gastrointestinal (GI) GVHD. Nine of the 10 patients who activated the Complement system (90%) developed GI GVHD within two weeks following the activation. By contrast only 4 of the 29 who did not activate the system, all of whom had received a RIC, developed GI GVHD (13.8%) (p