42 MAPKs. Moreover, our data suggests that PLD plays animportant role in the activation of NFAT and NF-κB transcrip-tion factors, in T cell proliferation and in the production ofcytokines, IL-2 and IFN-γ. Overall, our results point to a pivotalrole for PLD in the intracellular signaling cascade activated byTCR and inTcell functional responses. Thus, our results suggestthat PLD might be a suitable target for the treatment ofinflammatory and autoimmune diseases and for prophylaxis oftransplant rejection.

doi:10.1016/j.clim.2008.03.091

Immuno-oncologySunday, June 8

2:45 pm–4:45 pm

OR.85. Leukocyte Responses during RecombinantHuman Interleukin-21 Treatment of Patients withStage IV MelanomaKresten Skak,1 Klaus Stensgaard Frederiksen,1 Jenny Lau,2

Ulrik Mouritzen,3 Grant McArthur,4 Ian Davis,5 Birte K.Skrumsager,5 Mary Sartor,2 Rune Overgaard,3 DortheLundsgaard,1 Lasse Tengbjerg Hansen.3 1Novo Nordisk A/S,Maaloev, Denmark; 2Westmead Hospital, Sydney, NSW,Australia; 3Novo Nordisk A/S, Bagsvaerd, Denmark; 4CancerTrials Australia, Melbourne, VIC, Australia; 5Austin Health,Heidelberg, VIC, Australia

Interleukin-21 is a class I cytokine with effects on severalleukocyte subsets. Leukocyte responses were monitored in anopen-label phase 2a trial of intravenous recombinant human IL-21 (rIL-21) treatment of patients with stage IV melanomaadministered as 3-9 cycles of daily dosing for 5 days followed by9 days of rest. Peripheral blood leukocyte responses weremonitored by flow cytometry, effector molecule qRT-PCR, andserum sCD25 (sIL-2Rα) analysis. On Day 4 of treatment, amarked drop in number of NK-cells and both CD4+ and CD8+ T-cells was observed. Between dosing, a rebound effect beyondbaseline levels was observed. CD8+ T-cells and NK-cells hadincreased surface expression of CD25 (IL-2Rα), CD69, thelymph-node homing marker CD62L and increased mRNAexpression of the effector genes, IFN-γ, granzyme B, andperforin. In accordance with immune activation, increasedserum levels of sCD25 were observed as early as 24-hours post-dosing. No changes in activation markers on CD4+ T-cells wereobserved; this suggests that separatemechanisms drive the rIL-21-mediated leukocyte redistribution andactivation of effectorfunctions in CD8+ T-cells and NK-cells. In contrast to lympho-cytes, increases in numbers ofmonocytes and plasma cellswereobserved. In particular, a 6-fold increase was observed in theFcγRIII+ monocyte subset, which was associatedwith increasedexpression of the ADCC-mediating FcγRIII (CD16). These datademonstrate that administration of rIL-21 to melanomapatients induces a variety of leukocyte responses related toeffector functions that warrant further investigations inrelation to anti-tumour effects of this cytokine.

doi:10.1016/j.clim.2008.03.092

OR.86. Accumulation of Tumor Infiltrating FOXP3+CD4+ Regulatory T Cells in Tumors and Not inPeripheral Blood in Patients with MelanomaMojgan Ahmadzadeh,1 Aloisio Felipe-Silva,2 Maria Merino,2

Steven Rosenberg.1 1NCI/NIH, Bethesda, MD; 2NCI,Bethesda, MD

Studies of regulatory T cells in human tumors have beendifficult and functional studies have been limited due to lowcell viability and purity. Using a novel functional assay, weexamined tumor infiltrating T cells in the single cell suspen-sions of enzymatically digested tumors to enumerate regula-tory T cells. We observed more than four-fold increase in thefrequency of FOXP3+ CD4+ T cells in tumors compared toperipheral blood of patients and healthy donors. To distinguishbetween activated and regulatory Tcells, we stimulated tumorinfiltrating Tcells in the tumor digests with PMA and ionomycinfor 6 hours followed by intracellular cytokine and FOXP3 co-staining. Our findings revealed that, similar to regulatory Tcells in peripheral blood of healthy individuals, tumorinfiltrating FOXP3+ CD4+ T cells were unable to produce IL-2and IFN-g in contrast to FOXP3- Tcells. Using the combinationof functional and phenotypic parameters, we concluded thattumor infiltrating FOXP3+ CD4+ T cells exhibited the char-acteristics of regulatory T cells. We then utilized FOXP3expression as a marker to distinguish regulatory T cells in theintratumoral and peritumoral sections of 18 melanoma tumorsusing immunohistochemical analysis. We found that there wasa significantly higher percentage of FOXP3+CD4+ regulatory Tcells intratumorally than peritumorally. These findings revealselective accumulation of regulatory T cells in the closeproximity of tumors and it further highlights that the frequencyof regulatory T cells in the peripheral blood are notrepresentative of the tumor microenvironment.

doi:10.1016/j.clim.2008.03.093

OR.87. Optimizing Anti-tumor Immunity: ReversingSystemic iNKT Cell Immunoregulatory Defects inMurine Prostate CancerMichael Nowak, Simo Arredouani, Martin Sanda, StevenBalk, Mark Exley. Beth Israel Deaconess Medical Center,Boston, MA

Breaking tolerance to self-antigens represents a promisingtreatment approach, especially for non-critical organ can-cers. Invariant natural killer T cells (iNKT) recognizeglycolipids presented on CD1 molecules by dendritic andother cell types, thereby regulating immune reactions.Importantly, in multiples types of model and human cancer,we and others have found both numerical aswell as functionaliNKT defects. In particular, decreased iNKT numbers wereaccompanied by reversible functional defects: decreasedIFN-γ production by iNKT in advanced prostate cancer,resulting in a detrimental Th2 cytokine profile of iNKT insuch patients. We analyzed numerical and functional iNKTdefects in the murine TRAMP prostate cancer model. In thismodel, probasin-directed expression of SV40 Tantigen drivesthe sequential development of prostatic intraepithelial

S35Abstracts

dysplasia, locally invasive tumors, and metastatic disease.Tumor-bearing TRAMP mice revealed systemic decreases iniNKT numbers. Interestingly, the age-dependent increase iniNKT of normal mice was not seen in frankly tumor-bearingTRAMP mice. iNKTwere detectable in the tumor and draininglymph nodes. Uponα-GalCer stimulation, iNKTof TRAMPmiceexhibited a strong Th2 bias both in vitro and in vivo comparedto WT littermates. Since we showed that the latter iNKTdefects are reversible in vitro, we are investigating reversingthese defects in the TRAMP model in vivo. iNKT adoptivetransfer fromWTmice demonstrated establishment of stablecirculating iNKT. Anti-tumor effects are being monitored. Wewill also examine tumor progression in TRAMP mice adop-tively-transferred with in vitro expanded iNKT, analogous toour cancer clinical trial protocol.

doi:10.1016/j.clim.2008.03.094

OR.88. IL-7/IL-7R alpha-Fc Immune Therapy for NonSmall Cell Lung CancerAsa Andersson,1 Anshu Buttan,1 Raj Batra,2 Li Zhu,1 StevenDubinett,1 Sherven Sharmax.1 1University of California LosAngeles, Los Angeles, CA; 2Veteran's Affairs Greater LosAngeles Healthcare System, Los Angeles, CA

T lymphocyte and antigen presenting cell (APC) activitiesare dysregulated in cancer patients. To rescue Tcell activitiesand improve Tcell-APC interactions we are evaluating a chi-mericmolecule in amurine non small cell lung cancer (NSCLC)model. Interleukin 7 (IL-7) is essential for lymphopoiesis, Tcell homeostasis, T cell maintenance, promotion of T cellcytolytic and innate responses. The biological effects of IL-7 ismediated through the high affinity IL-7 receptor (IL-7R)complex that is composed of the ligand binding IL-7Rα chainand the common shared γ chain. Compared to IL-7, IL-7/IL-7Rα-Fc has a longer half life that improves bioavailability andIL-7 mediated T cell stimulation. The Fc portion of themolecule is designed to promote the interactions with APC Fcreceptors. We find that both in vitro and in vivo; IL-7/IL-7Rα-Fc increased proliferation of CD4 and CD8 T lymphocytes,induced high levels of IL-12, IFNγ, CXCL9 and CXCL10 as wellas augmented T cell markers of activation and memory. Theenhancement in these effectors correlated with the anti-tumor benefit of IL-7/IL-7Rα-Fc in an established murineNSCLC in vivo. Our results demonstrate that the IL-7/IL-7Rα-Fc molecule helps to restore and maintain T cell anti tumoractivities in NSCLC, by augmenting T cell activation andmemory phenotype and impacting proinflammatory cyto-kines. This provides a strong rationale for further evaluationof IL-7/IL-7Rα-Fc for the potential translation to lung cancerpatients.

doi:10.1016/j.clim.2008.03.095

OR.89. Domain 4 of ILY Sensitizes Cancer AntibodyTherapy Through Abrogating Human CD59 FunctionWeiguo Hu, Gongxiong Wu, Jose Halperin, Xuebin Qin.Harvard Medical School, Cambridge, MA

CD59 is a key complement regulatory protein that inhibitsassembly of themembrane attack complex through binding toC8 and C9. Complement-dependent cytotoxicity (CDC) iscritical for antibody-based cancer therapy. However, thetherapeutic efficacy of specific anti-cancer antibodies ishampered by the expression of CD59, which is upregulated inmany cancer cells. For example, increased expression ofCD59 is responsible for the resistance of some lymphomas totreatment with Rituximab, a therapeutic monoclonal anti-body against the B-cell specific CD20 antigen. Recognition ofthe role of CD59 in resistance to cancer immunotherapy hasled to the search of a CD59 inhibitor. Intermedilysin (ILY), acytolytic pore-forming toxin secreted by Streptococcusintermedius, lyses human cells exclusively because thetoxin binds to human CD59 (hCD59) with high specificity.Specificity for hCD59 derives from binding of ILY's domain 4(ILY4) to amino acids 42-58 in hCD59, which also participate inbinding to C8 and C9. We hypothesized that truncated ILY4would abrogate hCD59 function and thereby increase anti-body-mediated CDC in cancer cells. We report here thatrecombinant ILY4 (114 residues) expressed in E. coli.,specifically blocks hCD59 function. Treatment of Rituximabresistant B lymphoma cells with ILY4 increased CDC with anIC50≈ 33 nM in vitro. In a nude mouse xenograft model of thesame Rituximab resistant B lymphoma cells, ILY4 (2.5 ug/gbody weight) significantly decreased tumor size without anyapparent toxicity. We conclude that ILY4 may represent aninnovative adjuvant for cancer immunotherapy in generaland for antibody-resistant cancers in particular.

doi:10.1016/j.clim.2008.03.096

OR.90. Characterization of B LymphocytesHarbored by Germ Cell TumorsSimon Willis,1 Kimberly L. Shampain,1 Scott Rodig,2 ScottMallozzi,1 Stefany Almendinger,1 Jeffrey Bruce,3 KevinO'Connor.1 1Harvard Medical School/Brigham & Women'sHosptial, Boston, MA; 2Harvard Medical School/Brigham &Women's Hosptial, Boston, MA; 3Columbia University Collegeof Physicians and Surgeons, New York, NY

Intracranial germ cell tumors (germinomas) are a hetero-geneous group of malignant brain lesions that almostexclusively affect children and young adults. Testiculargerm cell tumors, termed seminomas, comprise 30–40% ofall testicular tumors. Studies performed over twenty yearsago established that many germ cell tumors commonly harboran immune cell infiltrate that includes substantial numbers ofB cells. Yet, little is known about the role of the immunesystem in this tumor family. Thus, to gain a deeper under-standing of the role the B cells play in this tumor tissue, weexamined the molecular characteristics of the B cell antigenreceptor in tumor-associated B cells. Germinomas, semino-mas and control tissues were sectioned then tissue regionsand individual B and plasma cells were isolated through lasercapture micro-dissection. Immunoglobulin gene transcriptswere used to determine the variable region sequences.Analysis of these sequences revealed clear evidence ofaffinity maturation in that the cardinal features of an antigendriven B cell response were present: significant somatic

S36 Abstracts