CD3 mAb Research Articles

Regulatory B cell production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice

Current therapies for non-Hodgkin lymphoma commonly include CD20 mAb to deplete tumor cells. However, the response is not durable in a substantial proportion of patients. Herein, we report our studies in mice testing the hypothesis that heterogeneity in endogenous tissue CD20+ B cell depletion influences in vivo lymphoma therapy. Using highly effective CD20 mAbs that efficiently deplete endogenous mature B cells and homologous CD20+ primary lymphoma cells through monocyte- and antibody-dependent mechanisms, we found that lymphoma depletion and survival were reduced when endogenous host B cells were not depleted, particularly a rare IL-10-producing B cell subset (B10 cells) known to regulate inflammation and autoimmunity. Even small numbers of adoptively transferred B10 cells dramatically suppressed CD20 mAb-mediated lymphoma depletion by inhibiting mAb-mediated monocyte activation and effector function through IL-10-dependent mechanisms. However, the activation of innate effector cells using a TLR3 agonist that did not activate B10 cells overcame the negative regulatory effects of endogenous B10 cells and enhanced lymphoma depletion during CD20 immunotherapy in vivo. Thus, we conclude that endogenous B10 cells are potent negative regulators of innate immunity, with even small numbers of residual B10 cells able to inhibit lymphoma depletion by CD20 mAbs. Consequently, B10 cell removal could provide a way to optimize CD20 mAb-mediated clearance of malignant B cells in patients with non-Hodgkin lymphoma.

Loss of CD20 and Bound CD20 Antibody from Opsonized B Cells Occurs More Rapidly Because of Trogocytosis Mediated by Fc Receptor-Expressing Effector Cells Than Direct Internalization by the B Cells

We previously reported that 1 h after infusion of CD20 mAb rituximab in patients with chronic lymphocytic leukemia (CLL), >80% of CD20 was removed from circulating B cells, and we replicated this finding, based on in vitro models. This reaction occurs via an endocytic process called shaving/trogocytosis, mediated by FcγR on acceptor cells including monocytes/macrophages, which remove and internalize rituximab-CD20 immune complexes from B cells. Beers et al. reported that CD20 mAb-induced antigenic modulation occurs as a result of internalization of B cell-bound mAb-CD20 complexes by the B cells themselves, with internalization of ∼40% observed after 2 h at 37°C. These findings raise fundamental questions regarding the relative importance of shaving versus internalization in promoting CD20 loss and have substantial implications for the design of mAb-based cancer therapies. Therefore, we performed direct comparisons, based on flow cytometry, to determine the relative rates and extent of shaving versus internalization. B cells, from cell lines, from patients with CLL, and from normal donors, were opsonized with CD20 mAbs rituximab or ofatumumab and incubated for varying times and then reacted with acceptor THP-1 monocytes to promote shaving. We find that shaving induces considerably greater loss of CD20 and bound mAb from opsonized B cells in much shorter time periods (75-90% in <45 min) than is observed for internalization. Both shaving/trogocytosis and internalization could contribute to CD20 loss when CLL patients receive rituximab therapy, but shaving should occur more rapidly and is most likely to be the key mechanism of CD20 loss.

A glycoengineered anti‐CD19 antibody with potent antibody‐dependent cellular cytotoxicity activity in vitro and lymphoma growth inhibition in vivo

Human cluster of differentiation (CD) antigen 19 is a B cell-specific surface antigen and an attractive target for therapeutic monoclonal antibody (mAb) approaches to treat malignancies of B cell origin. MEDI-551 is an affinity-optimized and afucosylated CD19 mAb with enhanced antibody-dependent cellular cytotoxicity (ADCC). The results from in vitro ADCC assays with Natural Killer cells as effector cells, demonstrate that MEDI-551 is effective at lower mAb doses than rituximab with multiple cell lines as well as primary chronic lymphocytic leukaemia and acute lymphoblastic leukaemia samples. Targeting CD19 with MEDI-551 was also effective in several severe combined immunodeficiency lymphoma models. Furthermore, the combination of MEDI-551 with rituximab resulted in prolonged suppression of tumour growth, demonstrating that therapeutic mAbs with overlapping effector function can be combined for greater tumour growth inhibition. Together, the data demonstrate that MEDI-551 has potent antitumour activity in preclinical models of B cell malignancies. The results also suggest that the combination of the ADCC-enhanced CD19 mAb with an anti-CD20 mAb could be a novel approach for the treatment of B cell lymphomas.

Interaction with FcγRIIB Is Critical for the Agonistic Activity of Anti-CD40 Monoclonal Antibody

A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.

Insight into the roles of hypoxanthine and thydimine on cultivating antibody-producing CHO cells: cell growth, antibody production and long-term stability

The potential of hypoxanthine and thymidine (H&T) to promote growth of CHO cells and production of monoclonal antibody (mAb) was explored in this study. It was demonstrated that H&T stimulated the initial cell growth and enhanced volumetric production of anti-human CD20 mAb by 22%, mainly through the elevated integrated viable cell concentration (IVCC). The moderate alteration in cell cycle distribution might partially account for the increased cell growth. Subsequent long-term stability studies indicated that H&T did not accelerate decay kinetics in mAb productivity. Specifically, cells under both nucleic acids-replete (H&T supplementation) and nucleic acids-hungry (methotrexate treatment) culture conditions showed similar stable mAb production during the first 2 months, followed by a gradual decline in the specific production rate (q(mAb)) with a 40% drop at the fourth month. In addition, the decreased transcript level of intracellular heavy chain (HC) of anti-human CD20 mAb correlated well with the decreased q(mAb). Furthermore, genomic mutation rate regarding the loss-of-function occurrence of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was determined, showing that H&T repressed the HPRT spontaneous mutation rate while methotrexate (MTX) provoked the mutation rate. Collectively, our data illustrated that H&T as potential medium additives promoted both initial cell growth and volumetric production of mAb, while not affecting the long-term stability of antibody-producing CHO cells.

Suppression of Allograft Rejection by Tim-1-Fc through Cross-Linking with a Novel Tim-1 Binding Partner on T Cells

Engagement of T-cell immunoglobulin mucin (Tim)-1 on T cells with its ligand, Tim-4, on antigen presenting cells delivers positive costimulatory signals to T cells. However, the molecular mechanisms for Tim-1-mediated regulation of T-cell activation and differentiation are relatively poorly understood. Here we investigated the role of Tim-1 in T-cell responses and allograft rejection using recombinant human Tim-1 extracellular domain and IgG1-Fc fusion proteins (Tim-1-Fc). In vitro assays confirmed that Tim-1-Fc selectively binds to CD4+ effector T cells, but not dendritic cells or natural regulatory T cells (nTregs). Tim-1-Fc was able to inhibit the responses of purified CD4+ T cells that do not express Tim-4 to stimulation by anti-CD3/CD28 mAbs, and this inhibition was associated with reduced AKT and ERK1/2 phosphorylation, but it had no influence on nTregs. Moreover, Tim-1-Fc inhibited the proliferation of CD4+ T cells stimulated by allogeneic dendritic cells. Treatment of recipient mice with Tim-1-Fc significantly prolonged cardiac allograft survival in a fully MHC-mismatched strain combination, which was associated with impaired Th1 response and preserved Th2 and nTregs function. Importantly, the frequency of Foxp3+ cells in splenic CD4+ T cells was increased, thus shifting the balance toward regulators, even though Tim-1-Fc did not induce Foxp3 expression in CD4+CD25− T cells directly. These results indicate that Tim-1-Fc can inhibit T-cell responses through an unknown Tim-1 binding partner on T cells, and it is a promising immunosuppressive agent for preventing allograft rejection.

Penetration of antibody‐opsonized cells by the membrane attack complex of complement promotes Ca2+influx and induces streamers

We have reported that during complement-mediated cytolysis of B cells promoted by the CD20 mAbs rituximab or ofatumumab (OFA), long, thin structures that we call streamers (≥ 3 cell diameters) are rapidly generated and grow out from the cell surface. Streamers appear before cells are killed and contain opsonizing mAbs and membrane lipids. By exploiting the differential Ca(2+) requirements of discrete steps in the complement cascade, we determined that mAb-opsonized cells first tagged with C3b using C5-depleted serum are killed on addition of serum and EDTA, but the cells do not produce streamers. Also, cells first opsonized with OFA are lysed in serum containing Mg-EGTA by the alternative complement pathway but streamers are not produced. These findings indicate that Ca(2+) influx is necessary for streamer formation. Other mAbs that promote complement-mediated cytolysis also induce streamers on target cells. Streamer-like structures called nanotubes have been reported in several cellular systems, and are thought to promote intercellular communication/signaling. We tested whether this signaling could influence the susceptibility of neighboring cells contacted by streamers to complement attack and found that complement-mediated cytolysis of OFA-opsonized cells increases the resistance of unopsonized indicator cell populations to subsequent lysis when these cells are exposed to OFA and complement.

Chemoimmunotherapy with O-FC in previously untreated patients with chronic lymphocytic leukemia

AbstractWe conducted an international phase 2 trial to evaluate 2 dose levels of ofatumumab, a human CD20 mAb, combined with fludarabine and cyclophosphamide (O-FC) as frontline therapy for chronic lymphocytic leukemia (CLL). Patients with active CLL were randomized to ofatumumab 500 mg (n = 31) or 1000 mg (n = 30) day 1, with fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 days 2-4, course 1; days 1-3, courses 2-6; every 4 weeks for 6 courses. The first ofatumumab dose was 300 mg for both cohorts. The median age was 56 years; 13% of patients had a 17p deletion; 64% had β2-microglobulin > 3.5 mg/L. Based on the 1996 National Cancer Institute Working Group (NCI-WG) guidelines, the complete response (CR) rate as assessed by an independent review committee was 32% for the 500-mg and 50% for the 1000-mg cohort; the overall response (OR) rate was 77% and 73%, respectively. Based on univariable regression analyses, β2-microglobulin and the number of O-FC courses were significantly correlated (P < .05) with CR and OR rates and progression-free survival (PFS). The most frequent Common Terminology Criteria (CTC) grade 3-4 investigator-reported adverse events were neutropenia (48%), thrombocytopenia (15%), anemia (13%), and infection (8%). O-FC is active and safe in treatment-naive patients with CLL, including high-risk patients. This trial was registered at www.clinicaltrials.gov as NCT00410163.

Target-induced natural killer cell loss as a measure of NK cell responses

The interaction of natural killer cells with susceptible target cells triggers NK cell activation, eliciting not only NK cell cytotoxicity and cytokine secretion, but also NK cell death. This study shows that following target cell interaction there is a substantial loss of NK cells, the extent of which correlates with measures of NK cell cytotoxicity assessed by the target cell release of 51Cr and by the externalisation of the lysosomal marker LAMP-1 (CD107a) which is assessed on the remaining NK cells. This is the case for the killing of K562 (natural killing) and the CD20 mAb (Rituximab)-mediated killing of RAJI cells and autologous B cells (antibody-dependent cell cytotoxicity). This target-induced NK loss (TINKL) provides a sensitive and specific measure of NK cell responses appropriate to a clinical laboratory setting.

103 Gain of Chromosome 2p25.3 Involving the ACP1 Gene is Frequent in Chronic Lymphocytic Leukemia

S120 with high-risk disease features. Full Abstract: Introduction: Chemoimmunotherapy with CD20 mAb is standard frontline treatment for physically fit patients with CLL. Ofatumumab, a human mAb, binds to a membrane-proximal epitope composed of both the smalland large-loop domains of CD20. The pivotal international study of ofatumumab monotherapy demonstrated high overall response rates (ORR) of 47%–58% in patients with fludarabine-refractory CLL. This international, randomized, phase II trial evaluated efficacy and safety of 2 dose levels of ofatumumab in combination with fludarabine and cyclophosphamide as frontline therapy for CLL. Methods: Previously untreated patients with active CLL were randomized to ofatumumab 500 mg or 1000 mg day 1, with fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 days 2–4, course 1; days 1–3, courses 2–6; every 4 weeks for 6 courses. All patients received ofatumumab 300 mg for course 1. The primary endpoint was complete response (CR) rate, as assessed by an Independent Review Committee using the 1996 NCI-WG criteria. Results: 61 patients were randomized (500 mg, n 31; 1000 mg, n 30). The median (range) age was 56 (38–73) years; 13% of patients had 17p deletion; 64% had 2-microglobulin 3.5 mg/L. CR rate (95% confidence interval [CI]) was 32% (17%–51%) for 500 mg and 50% (31%–69%) for 1000 mg cohort; ORR (95% CI) was 77% (59%–90%) and 73% (54%–88%), respectively. Median progression-free survival (PFS) and overall survival (OS) have not been reached with current follow-up (median 8 months). Based on univariable regression analyses, 2-microglobulin and number of O-FC courses completed were ignificantly correlated (P 0.05) with CR, ORR and PFS. Most ommon investigator-reported adverse events (CTC grade 3–4) ere neutropenia (48%), thrombocytopenia (15%), anemia (13%) nd infection (8%). Conclusion: O-FC, at the ofatumumab dose evels evaluated, was active in previously untreated patients with LL, including in patients with high-risk features.

Abstract 3628: The forced interaction of α4 integrin and CD44 by the novel peptide HYD1 may be a determinant of HYD1-induced cell death in multiple myeloma

Abstract Our laboratory has previously shown that treatment with the novel d-amino acid peptide HYD1 (kikmviswkg) induces necrotic cell death in multiple myeloma (MM) cell lines. Furthermore, we have implicated the adhesion receptor VLA-4 as being involved in this process via shRNA silencing strategies. However, reducing α4 integrin only demonstrated partial protection indicating that other components of the HYD1 binding complex may be required for cell death. Thus, in the present study, we investigated the possible role of other adhesion molecules and/or complexes that may contribute to HYD1-induced cell death. Using an unbiased approach, we performed a total membrane pull-down with biotin conjugated HYD1 and NeutrAvidin beads on NCI-H929 MM cells. Samples were then loaded on an SDS-PAGE gel and analyzed by Mass Spectrometry. Data were then mined with the Scaffold 3 proteome software to determine pertinent hits. The adhesion receptor CD44 was determined to have the most peptide match hits and was confirmed as a valid hit via Western Blot analysis. Preliminary data using ELISA based binding studies with biotin-HYD1 as bait shows that recombinant CD44 directly binds to biotin-HYD1 in a concentration dependent manner. In order to identify complexes involved in HYD1-induced cell death, membrane immunoprecipitation assays were performed using CD44 and α4 integrin mAbs in both the presence and absence of HYD1. While Western Blot analysis confirmed basal levels of complex interactions between α4 integrin and CD44, the complex was significantly enhanced in the presence of HYD1. These data not only indicate a possible role of CD44 in HYD1-induced cell death, but also the ability of HYD1 to induce interactions among adhesion molecules and complexes. Further studies, including CD44shRNA targeting strategies, as well as looking at other adhesion molecules will be needed in order to confirm the role of individual cell surface receptors identified in the biotin-HYD1 complex in mediating HYD1 induced cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3628. doi:10.1158/1538-7445.AM2011-3628

Abstract 4569: Cell death-inducing ICAM-1 antibody has broad and potent anti-myeloma activity in vivo

Abstract We previously developed combined target and drug discovery methodology enabling isolation of tumor cell death-inducing antibodies from the human antibody library n-CoDeR®. Among several antibodies targeting different tumor cell associated receptors with documented cell death-inducing properties, antibodies specific for intercellular adhesion molecule-1 (ICAM-1) – a receptor not previously associated with tumor cell death- were obtained. We here characterize the ICAM-1 specific antibody BI-505 with respect to in vivo anti-tumor activity in different well-established tumor disease models, and explore mechanisms underlying its anti-tumor activity. We demonstrate in different tumor xenograft models that BI-505 is equally or more efficacious in preventing CD20 positive B cell tumor growth and in pro-longing tumor-bearing mouse survival compared to CD20 mAb Rituximab in vivo. Moreover, BI-505 showed significant anti-tumor activity against a panel of CD20 negative myeloma tumors in vivo, and BI-505 conferred enhanced mouse survival compared to the proteasome inhibitor bortesomib – approved for treatment of relapsed refractory multiple myeloma – in an experimental model of advanced multiple myeloma. Preliminary data indicate that the BI-505 epitope is highly expressed on myeloma cells in patients with multiple myeloma. The differential in vitro and in vivo anti-tumor activities of BI-505 Fc-variants correlated with binding to recombinant human FcγRIIIa and mouse FcγRIV receptors, respectively. These differences could not be explained by altered antigen-binding properties as all Fc-variants retained near identical EC50 values for binding to recombinant or cell surface expressed ICAM-1 protein. These observations are consistent with cell death and FcγR-dependent anti-tumor immunity being important mechanisms underlying BI-505 therapeutic activity. Collectively, our results demonstrate proof-of-principle for our human antibody based combined target and drug discovery methodology, and provide a rationale for further pre-clinical and clinical evaluation of BI-505 in the treatment of MM. An open multicenter phase I dose-escalation clinical trial with BI-505 in relapsed/refractory MM has recently started in the United States and Sweden. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4569. doi:10.1158/1538-7445.AM2011-4569

Abstract 4567: Polyubiquitin K63-related CD137 signalsomes in T cells stimulated with agonist anti-CD137 monoclonal antibodies

Abstract Agonist anti-CD137 monoclonal antibodies are employed to enhance CD8-mediated antitumor immunity both in mouse models and in clinical trials. A panel of agonist anti-human CD137 mAb was prepared recognizing three different epitopes on the CD137 surface glycoprotein. All mAb costimulated T cell activation irrespectively of interference or not with natural ligand-binding. Interestingly, CD137 perturbation with agonist mAb results in internalization towards a vesicular endosomal compartment derived from the plasma membrane. Such internalization was observed on CD137 transfectants in 293 cellsas well as on primary activated T cells from humans and mice. In vivo treatment with anti-CD137 mAb of immunodeficient mice harboring activated human or mouse T cells results in internalization of CD137 to this vesicular compartment. CD137 is known to associate with TRAF2 at the plasma membrane. TRAF-2 is an adaptor protein endowed with E3 ubiquitin ligase activity that synthesizes k63-polyubiquitin chains. K63 polyubiquitin, as opposed to K48 polyubiquitin, constitute a docking site for downtream signaling proteins that lead to NF-kB and AP-1 activation. CD137 vesicles are decorated with k63-polyubiquitin and CD137 stimulation leads to the formation of K63-polyub containing complexes. Therefore CD137 stimulation under therapy like conditions with mAbs leads to the assembly of an intracellular signaling compartment that we term as CD137 -signalsomes Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4567. doi:10.1158/1538-7445.AM2011-4567

Regulatory B lymphocyte production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice (156.27)

Abstract Non-Hodgkin’s lymphoma (NHL) is a heterogeneous group of malignancies that represents ~4% of all cancers. Rituximab, a chimeric CD20 monoclonal antibody (mAb) that effectively treats half of NHL patients variably depletes non-malignant tissue B cells. Whether endogenous or remnant B cells affect CD20 mAb-induced lymphoma depletion is unknown. To determine whether endogenous B cells can influence in vivo lymphoma therapy in mice, the therapeutic effectiveness of a potent mouse anti-mouse CD20 mAb was compared in wild type and CD20-deficient (CD20-/-) mice given homologous CD20+ primary lymphoma cells. CD20 mAb efficiently depleted endogenous mature B cells and lymphoma cells in wild type mice and prolonged survival significantly. By contrast, endogenous B cells are not depleted in CD20-/- mice, including a rare IL-10-producing regulatory B cell subset (B10 cells). Furthermore, the persistence of endogenous B cells significantly inhibited the anti-tumor effects of CD20 mAb in CD20-/- mice. Even small numbers of adoptively transferred CD20-/- B10 cells dramatically suppressed lymphoma depletion in wild type mice by inhibiting monocyte activation and effector function through IL-10-dependent mechanisms. Thus, endogenous B10 cells are potent negative regulators of innate immunity, with even small numbers of residual B10 cells inhibiting lymphoma depletion by CD20 mAb. This may provide a new explanation for treatment resistance in some lymphoma patients.

B cell depletion impairs both CD4+ and CD8+ T cell activation during acute viral infection in mice (52.33)

Abstract Therapeutic B cell depletion in mice using CD20 mAb significantly reduces CD4+ T cell activation in response to model and self-antigens, but not CD8+ T cell activation. However, the effects of B cell depletion on acute viral infections and the consequences of long-term B cell depletion on immune system function are unknown. To assess this, B cells were depleted in wild type mice using a potent CD20 mAb that depletes &amp;gt;98% of tissue and circulating mature B cells. Short-term (2 week) B cell depletion did not affect tissue T cell numbers. However, when B cells were depleted 1 week before LCMV-Armstrong infection, splenic CD44hiCD62Llo CD4+ and CD8+ effector-memory T cell numbers were reduced by 51-57%. The numbers of LCMV-specific CD4+ T cells producing IFNγ and TNFα were reduced by 64% and 80%, respectively. Virus-specific CD8+ T cells producing IFNγ and TNFα were also reduced by &amp;gt;56%. Even without pathogen challenge, similar changes were observed in mice treated chronically with CD20 mAb for 6 months, where CD4+ and CD8+ effector-memory T cell numbers were reduced by 76% and 39%, respectively. Chronic B cell depletion also reduced IFNγ- and TNFα-producing CD4+ T cell numbers by 76% and 35%. Nonetheless, naïve T cell numbers were not affected by either short-term or chronic B cell depletion. Thus, B cells are required for optimal CD4+ and CD8+ T cell activation, expansion, and function in vivo, particularly during cellular responses to pathogens.

Differential gene expression and suppressive properties of ex vivo-generated vs. naturally-occurring regulatory T-cells (168.25)

Abstract We have recently reported a novel ex vivo method to convert large numbers of CD4+Foxp3- T-cells to induced regulatory T-cells (iTregs; CD4+Foxp3+) via their incubation with plate-bound CD3 mAb, IL-2, TGFβ and retinoic acid (RA) for 4 days. These iTregs possess enhanced immune-suppressive activity in vitro and in vivo when compared to naturally-occurring Tregs (nTregs; CD4+Foxp3+). The objectives of this study were to: a) quantify and compare mRNA transcripts for genes known to be associated with Treg function using flow-purified iTregs and nTregs obtained from Foxp3GFP reporter mice and b) evaluate the stability of iTreg-mediated suppression in vitro and in vivo. We found 8 genes associated with iTregs that were significantly up-regulated when compared with nTregs with CCL4 (132-fold), Granzyme A (18-fold) and Runx1 (14-fold) showing the most dramatic increases. We also observed significant downregulation of 19, Treg-associated genes with IL-18 (600-fold), CD7 (300-fold), and IL-7 (100-fold) showing the most dramatic reductions in iTregs vs. nTregs. In addition, we found that when ex vivo-generated iTregs were “rested” by culturing iTregs for an additional 3 days in the absence of CD3 mAb, TGFβ and RA but in the presence of IL-2, Foxp3 expression, proliferative activity and in vitro suppressive activity were maintained. Unexpectedly, we found that these cells were more potent than nTregs in reversing preexisting colitis in a mouse model of chronic gut inflammation.

The effect of B-cell depletion in Theiler's viral model of multiple sclerosis (107.17)

Abstract Introduction: B cell depletion (BCD) has been utilized to treat relapsing-remitting multiple sclerosis (MS), but it is also not clear whether BCD ameliorates disability progression. We used an anti-mouse CD20 mAb (5D2), and tested its effects in Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TIDD), a viral CNS infection considered the best experimental animal model of disability progression in MS. Methods: SJL mice were injected intraperitoneally with 5D2 15 days before intracerebral infection of TMEV BeAn strain. 5D2 bioactivity was assessed by flow cytometry assay of CD19+ cells in peripheral blood mononuclear cells (PBMC). Mouse disability was measured by Rotarod, and viral load was measured by real-time reverse transcriptase PCR (RT-PCR) for TMEV RNA. Antibody levels were determined in mouse sera and CSF by ELISA, and in CNS tissues by real-time RT-PCR for IgG RNA. Results: Bioactivity of 5D2 was confirmed by nearly absent CD19+ cells in the peripheral blood from animals with persistent BCD. Antibody responses in both CNS and peripheral system were suppressed during 5D2 treatment. Relatively higher viral loads were detected in 5D2-treated mice than in PBS animals, and the viral levels correlated negatively to IgG production in the brain. 5D2 caused marked worsening of the early encephalitis, possibly from deletion of a B cell phenotype which downregulates inflammation.

Neonatal naive CD4+ T cells have increased calcium flux and higher expression of miR-181a relative to adult naive CD4+ T cells (153.39)

Abstract Neonates are highly susceptible to severe infection by a variety of intracellular pathogens, including M. tuberculosis and T. gondii. A large body of evidence suggests that a limitation in T helper 1 (Th1) responses accounts for much of this susceptibility. Our lab has previously defined a number of limitations in neonatal naive CD4+ T cells obtained from umbilical cord blood that restrict Th1 generation in vitro. These include decreased expression by naive CD4+ T cells of CD40 ligand and IFN-γ. Unexpectedly, we have also observed increased calcium flux in neonatal cells in response to CD3 mAb cross-linking. Because of the diverse molecular differences between neonatal and adult naive CD4+ T cells, we investigated differences in microRNA expression. miR-181a, which has been shown to regulate calcium flux positively in murine T cells, was expressed at higher levels in neonatal naive CD4+ T cells, consistent with increased calcium flux. Its predicted mRNA targets are expressed at lower levels in neonatal naive CD4+ T cells by gene set enrichment analysis (GSEA). Others have shown that over-expression of miR-181a results in increased phosphorylation of Erk1/2 after CD3 mAb crosslinking, and we are examining the activity of the Erk pathway in neonatal and adult naive CD4+ T cells. Also in progress are studies using a stable, lentiviral system to determine the effects of miR-181a over-expression and knockdown in human CD4 T cells focusing on validated murine mRNA targets.

CD180 functions in activation, survival and cycling of B chronic lymphocytic leukaemia cells

We previously showed that approximately 60% of B chronic lymphocytic leukaemia (B-CLL) cells express surface CD180, an orphan receptor of the Toll-like receptor family. Here we investigated the ability of anti-CD180 monoclonal antibody (mAb) to induce activation, cell cycling, survival and signalling in B-CLL cells and normal B cells. Upon addition of anti-CD180 mAb, alone or in combination with anti-CD40 mAb or recombinant IL-4 (rIL-4), expression of CD86, Ki-67, uptake of DiOC(6) , phosphorylation of signalling protein kinases and Ca(2+) flux were measured in B-CLL cells from untreated patients and normal B cells from age-matched volunteers. Normal B cells and approximately 50% of CD180(+) B-CLL clones responded to CD180 ligation by activation, cycling and increased survival comparable with, or superior to, those induced by anti-CD40 mAb or rIL-4 (Responder B-CLL). Non-responder CD180(+) B-CLL clones failed to respond to CD180 mAb and responded poorly to CD40 mAb and rIL-4. Anti-CD180 mAb induced phosphorylation of ZAP70/Syk, Erk, p38MAPK and Akt in normal B cells and Responder B-CLL cells. In contrast, Erk, p38MAPK and Akt were not phosphorylated in Non-responder B-CLL cells indicating a block in signalling and possible anergy. CD180 may provide powerful expansion and survival signals for Responder B-CLL cells and have an important prognostic value.

Renal Excretion of Recombinant Immunotoxins Containing Pseudomonas Exotoxin

Recombinant immunotoxins BL22 (CAT-3888) and LMB-2, composed of Fv fragments of anti-CD22 and CD25 MAbs, respectively, have produced major responses in patients with hematologic malignancies, and are also associated with renal toxicity, particularly with BL22. Characterization of the renal excretion of recombinant immunotoxins, which have 2-4 h half-lives in plasma, has not been reported in humans. To study the renal excretion of recombinant immunotoxins, urine from patients treated with BL22 was collected and the recombinant protein visualized after trichloroacetic acid (TCA) precipitation or anion exchange chromatography. BL22 viewed by immunoblot was found in the urine of patients within 8 h after dosing as an intact protein, and progressively degraded to fragments of <20 kDa within 1 day. We studied the stability of BL22 and LMB-2 added to urine at different time points and pH. When exposed to urine ex vivo, BL22 time-dependent proteolysis was similar to that observed in treated patients. By N-terminal sequencing, proteolysis was documented at positions 348-349 and 350-351 of BL22, and 339-340 and 341-342 of LMB-2, and other proteolytic sites were observed as well. Our data suggest that BL22 is excreted into the urine in a potentially cytotoxic form, even after its plasma level declines, and may remain intact long enough to cause renal toxicity.