CD20 Ab Research Articles

Autoantibodies against IgLON5: Two new cases

Objective: To dissect the relative pathogenic relevance of myelinspecific antibodies (ab) from myelin-specific B cells in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE). Background: B cells and B cell-derived ab may both play a pathogenic role in CNS autoimmune disease. Transgenic mice in which a majority of B cells recognize myelin oligodendrocyte glycoprotein (MOG) and plasma cells secrete high titres of MOG-specific ab (Th mice) experience a fulminant course of actively-induced EAE. Double-transgenic mice which further contain myelin-reactive T cells (Th mice x 2D2) spontaneously develop EAE. We utilized Th x 2D2 mice in combination with B cell-depleting anti (a)-CD20 ab and adoptive transfer of serum or ab preparations into 2D2 recipients to dissect the relative pathogenic contribution of myelin-reactive B cells from myelin-reactive ab. Methods: Th x 2D2 mice were injected with 0.2 mg a-CD20/week starting at the age of 4 weeks. Serum from Th mice immunized with rMOG 1–117 or myelin-reactive 8.18C5 ab was transferred i.v. into naive 2D2 recipients. Serum from WT mice immunized with MOG p35-55, or an isotype control ab served as control, respectively. In-vivo proliferation of CD4 T cells was determined by BrdU assay. In-vitro, bone-marrow derived monocytes were co-cultured with CFSE-labelled T cells in the presence of 8.18C5 or an isotype control ab. T cell proliferation was determined by flow cytometry. Results: In Th x 2D2 mice, a-CD20 treatment did not interfere with development of encephalitogenic T cells or incidence/severity of spontaneous EAE. While all peripheral compartments were efficiently depleted of B cells, a-CD20 did not affect constitutive secretion of a-MOG ab. Serum from Th mice containing high titres of pathogenic a-MOG ab triggered spontaneous EAE when transferred into naive 2D2 recipients. Further dissecting the role of pathogenic ab, transfer of purified 8.18C5 ab to naive 2D2 recipients led to in-vivo proliferation of T cells and similarly triggered spontaneous EAE. In co-culture with monocytes, myelin-reactive ab enhanced T cell proliferation at low antigen concentrations of rMOG 1–117. Conclusion: Our data indicate that a-MOG ab contribute to CNS autoimmune disease independent of myelin-reactive B cells. Besides promoting CNS demyelination a-MOG ab may enhance myelinrecognition of antigen-presenting cells by opsonisation resulting in accentuated activation of myelin-reactive T cells.

Influences of the CD20 Ab Rituximab On the Renal Transplant Recipients and Renal Grafts 5 to 8 Years After Immunologically High-Risk Renal Transplantation.

Influences of the CD20 Ab Rituximab On the Renal Transplant Recipients and Renal Grafts 5 to 8 Years After Immunologically High-Risk Renal Transplantation.

Regulatory T cells and myeloid-derived suppressor cells in the tumor microenvironment undergo Fas-dependent cell death during IL-2/αCD40 therapy.

Fas ligand expression in certain tumors has been proposed to contribute to immunosuppression and poor prognosis. However, immunotherapeutic approaches may elicit the Fas-mediated elimination of immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) within tumors that represent major obstacles for cancer immunotherapy. Previously, we showed that IL-2 and agonistic CD40 Ab (αCD40) elicited synergistic antitumor responses coincident with the efficient removal of Tregs and MDSCs. We demonstrate in this study in two murine tumor models that Treg and MDSC loss within the tumor microenvironment after IL-2/αCD40 occurs through a Fas-dependent cell death pathway. Among tumor-infiltrating leukocytes, CD8(+) T cells, neutrophils, and immature myeloid cells expressed Fas ligand after treatment. Fas was expressed by tumor-associated Tregs and immature myeloid cells, including MDSCs. Tregs and MDSCs in the tumor microenvironment expressed active caspases after IL-2/αCD40 therapy and, in contrast with effector T cells, Tregs significantly downregulated Bcl-2 expression. In contrast, Tregs and MDSCs proliferated and expanded in the spleen after treatment. Adoptive transfer of Fas-deficient Tregs or MDSCs into wild-type, Treg-, or MDSC-depleted hosts resulted in the persistence of Tregs or MDSCs and the loss of antitumor efficacy in response to IL-2/αCD40. These results demonstrate the importance of Fas-mediated Treg/MDSC removal for successful antitumor immunotherapy. Our results suggest that immunotherapeutic strategies that include exploiting Treg and MDSC susceptibility to Fas-mediated apoptosis hold promise for treatment of cancer.

Targeted nanoparticle delivery overcomes off-target immunostimulatory effects of oligonucleotides and improves therapeutic efficacy in chronic lymphocytic leukemia

AbstractSeveral RNA-targeted therapeutics, including antisense oligonucleotides (ONs), small interfering RNAs, and miRNAs, constitute immunostimulatory CpG motifs as an integral part of their design. The limited success with free antisense ONs in hematologic malignancies in recent clinical trials has been attributed to the CpG motif–mediated, TLR-induced prosurvival effects and inefficient target modulation in desired cells. In an attempt to diminish their off-target prosurvival and proinflammatory effects and specific delivery, as a proof of principle, in the present study, we developed an Ab-targeted liposomal delivery strategy using a clinically relevant CD20 Ab (rituximab)–conjugated lipopolyplex nanoparticle (RIT-INP)– and Bcl-2–targeted antisense G3139 as archetypical antisense therapeutics. The adverse immunostimulatory responses were abrogated by selective B cell–targeted delivery and early endosomal compartmentalization of G3139-encapsulated RIT-INPs, resulting in reduced NF-κB activation, robust Bcl-2 down-regulation, and enhanced sensitivity to fludarabine-induced cytotoxicity. Furthermore, significant in vivo therapeutic efficacy was noted after RIT-INP–G3139 administration in a disseminated xenograft leukemia model. The results of the present study demonstrate that CD20-targeted delivery overcomes the immunostimulatory properties of CpG-containing ON therapeutics and improves efficient gene silencing and in vivo therapeutic efficacy for B-cell malignancies. The broader implications of similar approaches in overcoming immunostimulatory properties of RNA-directed therapeutics in hematologic malignancies are also discussed.

Mimicking an Induced Self Phenotype by Coating Lymphomas with the NKp30 Ligand B7-H6 Promotes NK Cell Cytotoxicity

Induced self expression of the NKp30 ligand B7-H6 facilitates NK cell-mediated elimination of stressed cells. A fusion protein consisting of the ectodomain of B7-H6 and the CD20 single-chain fragment variable 7D8 was generated to mimic an induced self phenotype required for NK cell-mediated target cell elimination. B7-H6:7D8 had bifunctional properties as reflected by its ability to simultaneously bind to the CD20 Ag and to the NKp30 receptor. B7-H6:7D8 bound by CD20(+) lymphoma cells activated human NK cells and triggered degranulation. Consequently, the immunoligand B7-H6:7D8 induced killing of lymphoma-derived cell lines as well as fresh tumor cells from chronic lymphocytic leukemia or lymphoma patients. B7-H6:7D8 was active at nanomolar concentrations in a strictly Ag-specific manner and required interaction with both CD20 and NKp30. Remarkably, NK cell cytotoxicity was further augmented by concomitant activation of Fcγ receptor IIIa or NK group 2 member D. Thus, B7-H6:7D8 acted synergistically with the CD20 Ab rituximab and the immunoligand ULBP2:7D8, which was similarly designed as B7-H6:7D8 but engaging the NK group 2 member D receptor. In conclusion, to our knowledge, B7-H6:7D8 represents the first Ab-based molecule stimulating NKp30-mediated NK cell cytotoxicity for therapeutic purposes and provides proof of concept that Ag-specific NKp30 engagement may represent an innovative strategy to enhance antitumoral NK cell cytotoxicity.

The Dual Effects of B Cell Depletion on Antigen-Specific T Cells in BDC2.5NOD Mice

B cells play a critical role in the pathogenesis of autoimmune diabetes. To investigate the mechanisms by which B cell depletion therapy attenuates islet β cell loss and particularly to examine the effect of B cells on both diabetogenic and regulatory Ag-specific T cells, we generated a transgenic BDC2.5NOD mouse expressing human CD20 on B cells. This allowed us to deplete B cells for defined time periods and investigate the effect of B cell depletion on Ag-specific BDC2.5 T cells. We depleted B cells with anti-human CD20 Ab using a multiple injection protocol. We studied two time points, before and after B cell regeneration, to examine the effect on BDC2.5 T cell phenotype and functions that included antigenic response, cytokine profile, diabetogenicity, and suppressive function of regulatory T (T(reg)) cells. We found unexpectedly that B cell depletion induced transient aggressive behavior in BDC2.5 diabetogenic T cells and reduction in T(reg) cell number and function during the depletion period. However, after B cell reconstitution, we found that more regenerated B cells, particularly in the CD1d(-) fraction, expressed immune regulatory function. Our results suggest that the regenerated B cells are likely to be responsible for the therapeutic effect after B cell depletion. Our preclinical study also provides direct evidence that B cells regulate both pathogenic and T(reg) cell function, and this knowledge could explain the increased T cell responses to islet Ag after rituximab therapy in diabetic patients in a recent report and will be useful in design of future clinical protocols.

Targeting of the non-mutated tumor antigen HER2/neu to mature dendritic cells induces an integrated immune response that protects against breast cancer in mice

IntroductionGiven their relative simplicity of manufacture and ability to be injected repeatedly, vaccines in a protein format are attractive for breast and other cancers. However, soluble human epidermal growth factor receptor (HER2)/neu protein as a vaccine has not been immunogenic. When protein is directly targeted to antigen uptake receptors, such as DEC205 (DEC), efficient processing and presentation of antigen take place. The aim of this study was to determine the immunogenicity of a HER2 protein vaccine that directly targets to DEC+ dendritic cells (DCs) in a mouse breast cancer model.MethodsWe genetically engineered the HER2 extracellular domain into a monoclonal antibody specific for DEC (DEC-HER2). Mice of various genetic backgrounds were immunized with DEC-HER2 in combination with DC maturation stimuli (poly IC ± CD40 Ab). Vaccine-induced T cell immunity was determined by analyzing the ability of CD4+/CD8+ T cell to produce interferon (IFN)-gamma and proliferate upon antigen rechallenge. Sera were assessed for the presence of antigen specific antibody (Ab). For vaccine efficacy, FVB/N mice were immunized with DEC-HER2 in combination with poly IC and protection against neu-expressing mammary tumors was assessed. Protection mechanisms and tumor-specific T cell responses were also evaluated.ResultsWe demonstrate that DEC-HER2 fusion mAb, but not Ctrl Ig-HER2, elicits strong, broad and multifunctional CD4+ T cell immunity, CD8+ T cell responses, and humoral immunity specific for HER2 antigen. Cross-reactivity to rat neu protein was also observed. Importantly, mice xeno-primed with DEC-HER2 were protected from a neu-expressing mammary tumor challenge. Both CD4+ and CD8+ T cells mediated the tumor protection. Robust anti-tumor T cell immunity was detected in tumor protected mice.ConclusionsImmunization of mice with HER2 protein vaccine targeting DEC+ DCs in vivo induced high levels of T- and B-cell immunity. Non-targeted HER2 protein was poorly immunogenic for CD4+ and CD8+ T cells. This vaccination approach provided long-term survival benefit for mice challenged with neu-expressing tumor following as little as 2.7 μg of HER2 protein incorporated in the vaccine. Vaccine-induced CD4+ and CD8+ T cells were both essential for tumor protection. This immunization strategy demonstrates great potential towards the development of vaccines for breast cancer patients.

Abstract 5596: Enhanced anti-tumor effect of combination therapy with anti-CD40 antibody and the mTOR kinase inhibitor AZD8055

Abstract mTOR signaling, which plays a central role in controlling cell cell proliferation, survival, mobility and angiogenesis, is considered an important target for new anticancer drugs development. The involvement of the CD40/CD154 signaling pathway has also been reported to play an important role in regulating anti-tumor immune responses. In this study, using a first-in-class orally bioavailable mTOR kinase inhibitor, AZD8055, which, unlike rapamycin, can inhibit both mTORC1 and mTORC2, we evaluated the anti-tumor efficacy of mTOR kinase inhibitor treatment alone in a murine model of metastatic renal cell carcinoma (Renca) and compared biological responses achieved by administering AZD8055 as a single agent to those obtained in combination with agonistic anti-CD40 antibody. In vitro survival assays showed that Renca cell apoptosis could be induced by AZD8055. In vivo, although both agents administered alone had some anti-tumor efficacy, the combination of AZD8055 and CD40 Ab induced a significant anti-tumor response in both intra-renal and intra-splenic inoculations model of renal cell carcinoma, as compared to single agent treatments. The combination treatment, also resulted in an increase and activation of tumor- and liver-associated macrophages, NK cells and CD8 T cells in vivo. AZD8055/anti-CD40 treatment also increased the level of systemic Th1 cytokines, including IL-12, IFN-γ, TNFα. IFN-γ production by CD8+ T cells and TNFα production by macrophages were also found to be elevated in the AZD8055/anti-CD40 combination treated-group, compared with any single treatment group. Furthermore, the expression of Th1-associated chemokines, RANTES and IP10, were significantly induced in the combination treated-group. These data suggest that the combination of mTOR kinase targeting agents with immunotherapy could be an area of further development in renal cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5596.

Anti-CD40 monoclonal antibody synergizes with CTLA4-Ig in promoting long-term graft survival in murine models of transplantation.

Blockade of the CD40/CD154 signaling pathway using anti-CD154 Abs has shown promise in attenuating the alloimmune response and promoting long-term graft survival in murine model systems, although side effects observed in humans have hampered its progression through clinical trials. Appropriately designed anti-CD40 Abs may provide a suitable alternative. We investigated two isoforms of a novel monoclonal rat anti-mouse CD40 Ab (7E1) for characteristics and effects mirroring those of anti-CD154: 7E1-G1 (an IgG1 isotype); and 7E1-G2b (an IgG2b isotype). In vitro proliferation assays to measure the agonist properties of the two anti-CD40 Abs revealed similar responses when plate bound. However, when present as a soluble stimulus, 7E1-G1 but not 7E1-G2b led to proliferation. 7E1-G2b was as effective as anti-CD154 when administered in vivo in concert with CTLA4-Ig in promoting both allogeneic bone marrow chimerism and skin graft survival, whereas 7E1-G1 was not. The protection observed with 7E1-G2b was not due to depletion of CD40-bearing APCs. These data suggest that an appropriately designed anti-CD40 Ab can promote graft survival as well as anti-CD154, making 7E1-G2b an attractive substitute in mouse models of costimulation blockade-based tolerance regimens.

Locally Administered TLR7 Agonists Drive Systemic Antitumor Immune Responses That Are Enhanced by Anti-CD40 Immunotherapy

Topical application of tumors with the TLR7 agonist imiquimod is an effective adjunct treatment for a range of primary dermatological cancers. However, for therapy to be effective against a broad range of solid tumor types, it must promote a strong systemic antitumor response that targets metastases in addition to primary tumor. We therefore investigated the potential of locally delivered imiquimod to stimulate an effective systemic antitumor response in a murine model of malignant mesothelioma (AB1-HA) with primary and distal tumors (dual tumor). Persistent delivery of imiquimod into primary tumor significantly retarded tumor growth in all treated mice compared with vehicle control. This local antitumor immune response required both CD8 T cells and NK cells, but not CD4 T cells, and was reliant on type I IFN induction. In vivo CTL studies and Ly6A/E staining of lymphocytes suggested that local imiquimod treatment had indeed induced a systemic, Ag-specific CD8 response. However, notably this response was not sufficient to retard the growth of an untreated distal tumor. Because local imiquimod treatment did not induce significant CD4 T cell responses, we investigated the efficacy of combining imiquimod with agonistic CD40 Ab (as a surrogate for CD4 T cell help). Combination of locally delivered imiquimod with systemic anti-CD40 immunotherapy not only significantly enhanced the local antitumor response, with 30% complete resolution, but it was also effective at significantly retarding growth of distal tumor. These results demonstrate that antitumor responses induced by locally delivered TLR7 agonists can be harnessed systemically for treating distal tumor.

Depletion of B Cells in Murine Lupus: Efficacy and Resistance

In mice, genetic deletion of B cells strongly suppresses systemic autoimmunity, providing a rationale for depleting B cells to treat autoimmunity. In fact, B cell depletion with rituximab is approved for rheumatoid arthritis patients, and clinical trials are underway for systemic lupus erythematosus. Yet, basic questions concerning mechanism, pathologic effect, and extent of B cell depletion cannot be easily studied in humans. To better understand how B cell depletion affects autoimmunity, we have generated a transgenic mouse expressing human CD20 on B cells in an autoimmune-prone MRL/MpJ-Fas(lpr) (MRL/lpr) background. Using high doses of a murine anti-human CD20 mAb, we were able to achieve significant depletion of B cells, which in turn markedly ameliorated clinical and histologic disease as well as antinuclear Ab and serum autoantibody levels. However, we also found that B cells were quite refractory to depletion in autoimmune-prone strains compared with non-autoimmune-prone strains. This was true with multiple anti-CD20 Abs, including a new anti-mouse CD20 Ab, and in several different autoimmune-prone strains. Thus, whereas successful B cell depletion is a promising therapy for lupus, at least some patients might be resistant to the therapy as a byproduct of the autoimmune condition itself.

The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique Epitopes on CD20

We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.

Stimulation through CD40 on Mouse and Human Renal Cell Carcinomas Triggers Cytokine Production, Leukocyte Recruitment, and Antitumor Responses that Can Be Independent of Host CD40 Expression

CD40, a member of the TNFR superfamily, is expressed on a variety of host immune cells, as well as some tumors. In this study, we show that stimulation of CD40 expressed on both mouse and human renal carcinoma cells (RCCs) triggers biological effects in vitro and in vivo. Treatment of the CD40+ Renca mouse RCC tumor cells in vitro with an agonistic anti-CD40 Ab induced strong expression of the genes and proteins for GM-CSF and MCP-1, and induced potent chemotactic activity. Similarly, administration of alphaCD40 to both wild-type and CD40-/- mice bearing Renca tumors resulted in substantial amounts of TNF-alpha and MCP-1 in the serum, increased the number of total splenocytes and MHC class II+ CD11c+ leukocytes, and when combined with IFN-gamma, inhibited the progression of established Renca tumors in vivo in both wild-type and CD40-/- mice. Similarly, treatment of CD40+ A704 and ACHN human RCC lines with mouse anti-human CD40 Ab induced strong expression of genes and proteins for MCP-1, IL-8, and GM-CSF in vitro and in vivo. Finally, in SCID mice, the numbers of ACHN pulmonary metastases were dramatically reduced by treatment with species-specific human CD40 Ab. These results show that CD40 stimulation of CD40+ tumor cells can enhance immune responses and result in antitumor activity.

A New Human CD20 Antibody for Improved Killing of CD20-Transgenic T Cells for Allogeneic Stem Cell Transplantation.

Allogeneic stem cell transplantation together with donor lymphocyte infusion represents the best curative option for many haematological malignancies. Donor T lymphocytes play a central role in the elimination of leukemic cells (Graft versus Leukemia, GvL) and acceptance of the graft. Unfortunately, these donor T cells also respond to the patients' own tissue, resulting in Graft versus Host Disease (GvHD).In a promising strategy to control GvHD, while at the same time maximally utilizing the GvL effect, genetically-modified donor T cells, expressing a suicide gene, are selectively eliminated in vivo in case of severe GvHD. Genetic transfer of the human CD20 molecule to donor T cells prior to transplantation permits selective elimination via CD20-reactive reagents, such as the widely used chimeric Ab rituximab (RTX). RTX activates complement (CDC), and recruits effector cells (ADCC) for elimination of CD20-positive cells.Using a novel experimental system we previously showed that the efficiency of RTX-mediated killing of CD20-positive T cells clearly depends on the level of CD20 expression. As a consequence, RTX is not able to kill all genetically modified CD20+ T cells.Here we investigated whether a novel fully human CD20 Ab, HuMab 7D8, would be better in activating complement and/or effector cells. Our results show HuMab 7D8 to be more powerful in activating complement in CDC-assays. To obtain a similar percentage cell kill compared with RTX, a 100-fold lower dose of HuMab 7D8 was needed and a 4 times lower human serum concentration. To study the effect of the number of CD20 molecules per cell we used CEM-CD20 clones (N=26), all with different CD20-expression levels, in an assay in which all other parameters were kept constant. The results show that HuMab 7D8 is significantly stronger in inducing CD20-mediated cell kill compared to RTX. Especially cells expressing very low levels of CD20, which are completely resistant to RTX-induced CDC, are lysed with high efficiency by HuMab 7D8 and complement. In contrast to RTX, HuMab 7D8 was able to eliminate 100% of transduced T cells in the presence of human serum, and effector cells. At present, studies with CD20-luciferase positive human T cells are conducted in mice to study the in vivo effect of HuMab 7D8 and RTX. These results demonstrate that the CD20 molecule represents an attractive suicide molecule and CD20-transgenic T cells can with high efficiency be eliminated by HuMab 7D8.

High, Stable and Homogenous CD20 Expression for Efficient Rituximab-Induced Elimination of CD20+ Alloreactive Donor T Cells in the Novel CD20/Rituximab ‘Suicide' System.

A promising strategy to control Graft versus Host disease (GvHD) in allogeneic stem cell transplantation is the use of donor T cells, genetically modified to express the Herpes Simplex Thymidine Kinase (HSVtk) gene. The HSVtk+ donor T cells can be selectively eliminated by ganciclovir (GCV) in vivo. Despite the encouraging clinical results limitations have been observed with this system. The viral HSVtk gene is immunogenic, GCV-activity is limited to dividing cells, and aberrant splicing of the HSVtk mRNA occurs. The human CD20 molecule, in combination with the humanized CD20 Ab Rituximab (RTX), was recently proposed as a novel ‘suicide' system (Introna et al., Hum Gene Ther 2000). CD20 is non-immunogenic and both resting and dividing CD20+ cells can be eliminated in vivo by RTX, which triggers the complement system and recruits effector cells. However, for a safe and effective clinical application a high, stable and homogenous CD20 expression by human T cells is required. We constructed different CD20-encoding retroviral vectors. To enhance CD20 expression we included the WPRE element, which is widely used to increase transgene expression. Chromatin insulators separate differentially regulated loci, shield promoters from neighboring regulatory elements and protect against transcriptional silencing. Thus, insulators may protect against insertional mutagenesis and provide a more homogenous and stable expression of the transgene. We therefore included insulators into the vectors.For this, CEM T cells were transduced with the different CD20-vectors at an MOI of 1 and a mean transduction efficiency of 22.9±5.1% was obtained. Results showed a significantly more homogenous CD20 expression for cells containing the insulators. Also after CD20 magnetic bead selection the CD20+ insulator containing cells had a more homogenous CD20 expression pattern. Surprisingly, the CD20 expression pattern became 2-fold more homogenous after continuous culturing for 3 months. In contrast, 60% of the cells without insulators lost CD20 expression. Inclusion of the WPRE element increased the CD20 expression level. CD20-vectors containing both the insulator and the WPRE element gave the highest, most homogenous and stable CD20 expression in CEM T cells.In addition, we generated individual CEM-CD20 clones with different CD20 expression levels, to analyze the efficacy of killing by RTX in a complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and a combination of both assays. The CDC assay showed that RTX-mediated kill depends on the level of CD20 expression. We observed a clear, significant correlation between number of CD20 molecules per cells and the extent of CDC (p<0.001). In a newly developed ADCC assay the CEM-CD20 clones were equally sensitive to cytokine-activated PBMCs in the presence of RTX (49.0±10.6% survival), independent of the CD20 expression level. Combination of both effector mechanisms resulted in an even better cell kill than that with either of the assays alone, suggesting that CDC and ADCC exhibit an additive effect.In summary, this is the first time that the combination of insulators and the WPRE element results in a higher, more homogeneous and stable retroviral transgene expression in T cells, which makes the T cells more sensitive to RTX. We also show for the first time that the level of CD20 expression is important for efficient CDC and not for ADCC and that CDC and ADCC synergize in Rituximab-mediated kill of CD20+ cells.

Combinations of IFN-gamma and IL-4 induce distinct profiles of dendritic cell-associated immunoregulatory properties.

Interferon gamma (IFN-gamma) and interleukin-4 (IL-4) are not only generated during cell-mediated immunity (CMI) and humoral immunity (HI), but are also generated by innate immune cells in response to pathogenic factors. How these cytokines differentially effect the development of dendritic cell (DC)-associated immunoregulatory properties from progenitor cells during innate immunity is unresolved. To address this we have utilized a homogeneous DC progenitor-like cell line, MTHC-D2, as a model to examine cytokine-induced maturation of DCs. By 6 h IFN-gamma induced genes that are important for antiviral activity and development of CMI, whereas IL-4 induced genes involved in cellular adhesion, uptake of extracellular antigen, suppression of cytotoxic T-cell responses, and that repair the extracellular matrix. By 48 h the cytokine stimulus had induced many properties characteristic of immature DCs; however, these were differentially effected by IFN-gamma and IL-4. IFN-gamma induced the greatest levels of costimulatory/ activation marker expression, and the highest levels of T-cell proliferation, whereas IL-4 induced the greatest levels of phagocytic activity. Stimulation of the cells with CD40 Ab enhanced the levels of costimulatory marker expression and T-cell stimulatory capacity of cells exposed to IFN-gamma, but had little effect on cells exposed to IL-4 in the absence of IFN-gamma.

Short-circuiting long-lived humoral immunity by the heightened engagement of CD40.

Agonistic alpha CD40 Ab's have been shown to be potent immune adjuvants for both cell- and humoral-mediated immunity. While enhancing short-lived humoral immunity, the administration of a CD40 agonist during thymus-dependent immune responses ablates germinal center formation, prematurely terminates the humoral immune response, blocks the generation of B cell memory, and prevents the generation of long-lived bone marrow plasma cells. Interestingly, some of these effects of heightened CD40 engagement could be mimicked by enhancing the magnitude of antigen-specific T cell help. Taken together, these studies demonstrate that as the magnitude of CD40 signaling intensifies, the fate of antigen-reactive B cells can be dramatically altered. These are the first studies to describe the multifaceted function of CD40 in determining the fate of antigen-reactive B cells and provide novel insights into how CD40 agonists can short-circuit humoral immunity.

Anti-CD40 antibody induces antitumor and antimetastatic effects: the role of NK cells.

We assessed the effect of the stimulatory anti-CD40 Ab on NK cell activation in vivo and the therapeutic potential of activated NK cells in tumor-bearing mice. Single-dose i.p. injection of the anti-CD40 Ab resulted in production of IL-12 and IFN-gamma in vivo, followed by a dramatic increase in NK cell cytolytic activity in PBLs. NK cell activation by anti-CD40 Ab was also observed in CD40 ligand knockout mice. Because NK cells express CD40 ligand but not CD40, our results suggest that NK activation is mediated by increased cytokine production upon CD40 ligation of APCs. Treatment of tumor-bearing mice with anti-CD40 Ab resulted in substantial antitumor and antimetastatic effects in three tumor models. Depletion of NK cells with anti-asialo GM1 Ab reduced or abrogated the observed antitumor effects in all the tested models. These results indicate that a stimulatory CD40 Ab indirectly activates NK cells, which can produce significant antitumor and antimetastatic effects.

CD40/CD40L Interactions and Cytokines Regulate HIV Replication in B Cellsin Vitro

Using ourin vitromodel of normal B cell infection, we investigated whether cellular interactions and/or cytokines directing the B cell response also regulate HIV replication. Phorbol esters and CD40 Ab plus IL4, added prior to infection, substantially increased subsequent viral replication. Postinfection, IL2 with or without IL4 and, to a lesser extent, CD40/CD40L interactions enhanced viral replication. In contrast, IL10 down-regulated HIV replication induced by cytokines, without affecting spontaneous or CD40 Ab-induced replication. Both enhancing and inhibitory effects of cytokines on viral replication were independent of their ability to modulate B cell proliferation. Thus, these two phenomena seem to be independently regulated in human B cells.

Activated T cells can induce high levels of CTLA-4 expression on B cells.

Engagement of the TCR/CD3 complex together with ligation of CD28 by its counterstructures B7-1 (CD80) and B7-2 (CD86) on APC are required for mitogenic T cell activation. After activation, T cells not only express B7-1 and B7-2 molecules, but a second receptor for the B7 ligands, CTLA-4, can be found on their surfaces. We here show that B cells can be induced to express CTLA-4 on the plasma membrane. Similar to what has been reported for T cells, CTLA-4 expression on B cells was transient. Purified B cells did not express CTLA-4 when mitogenically activated with alpha IgM and CD40 Ab, but did express the molecule when cultured in the presence of membranes from activated T cells, which suggests that induction of CTLA-4 expression on B cells was dependent on direct cell-cell contact of B lymphocytes and activated T cells. CTLA-4 molecules isolated from either T or B cells were biochemically indistinguishable. Moreover, because the ability of chimeric B7-1/Ig proteins to bind to activated B cells was correlated with CTLA-4 expression levels on these cells, we conclude that B cell-expressed CTLA-4 has ligand binding capacity. These data suggest that costimulatory receptors and their specific ligands not only play a role in T cell stimulation, but contribute in a direct fashion to the regulation of B cell responses.