T.106. Selective Expression of Latency Associated Peptide (LAP) and IL-1 Receptor Type I/II...
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to be the most effective for attack prevention. Rituximab proved to be effective in one NMO case, resistant to pulse cyclophosphamide (CYC) therapy. Another SLE associated NMO patient, unresponsive to high-dose oral and intravenous corticosteroids, intravenous immunoglobulin, mycopheno- late mofetil, tacrolimus, low-dose daily oral cyclophospha- mide and rituximab, was successfully treated with immunoablative dose CYC. In our two NMO associated SLE cases neurological manifestations anticipated other SLE symptoms, and despite combined high dose corticosteroid/ azathioprine treatment neurological relapses returned reg- ularly. 0,5 g/m2 body-surface monthly parenteral CYC therapy was given, followed by quarterly maintenance therapy. In both patients induction phase ended in January 2009. Until this time none of the patient had new neurological symptoms, one patient experienced improving in her neurological functions. In some cases, as we observed, not only immunoablative, but also traditional dose CYC can prevent flares in SLE associated NMO cases. doi:10.1016/j.clim.2009.03.236 T.104. Selective Depletion of Alloreactive T Cells Leads to Elimination of Graft-Versus-Host Reactivity and Stimulate Graft-Versus-Leukemia and Graft-Versus-Myeloma Effect Eva Matejkova, Ivana Buresova, Jana Muzikova, Jaroslav Michalek. Masaryk University, Brno, Czech Republic Graft-versus-host disease (GVHD) is a severe complication of allogeneic HSCT. The major role in GVHD play the alloreactive clones of donor T cells leading to host tissue damage. Selective depletion (SD) is a strategy to eliminate host-reactive donor T cells from hematopoietic stem cell allografts to prevent GVHD while conserving useful donor immune functions. We selectively depleted host-reactive donor T cells from PBMC using an anti-CD25 immunotoxin. Irradiated PBMC from cancer patients and healthy donor PBMC were used as responder cells in primary mixed leukocyte reaction (MLR). To prepare GVL/GVM-specific T cells, we depleted alloreactive T cells with anti-CD25 IT. Remaining T cells were repeatedly stimulated with purified tumor cells from the same patient. Tumor-reactive donor Tcells were then purified by FACS based on CD4CD25 and CD8CD25 production and their specificity was tested in non-radioactive cytotoxicity test. We performed 22 MLRs and (15 leukemia and 7 multiple myeloma). SD with the anti-CD25 IT led to significant depletion (99.2- 100%, median 99.7%) of alloreactive donor T cells, while GVL/GVM effect was well preserved: 1.1- 9.6% (median 7.46%) in leukemia and 0.2- 1.1% (median 0.6%) in myeloma patients. GVH reactivation of donor cells was negligible 0.14- 0.34% (median 0.17%), resp. 0- 0.3% (median 0.1%) even after repeated stimulation with patient's non-tumor PBMC. Thus, it is possible to selectively deplete donor alloreactive Tcells with the anti-CD25 IT. In leukemia patients a strong GVL reactivity was noticed, but GVM specific T cell clones seem to be less effective in their antitumor activity. doi:10.1016/j.clim.2009.03.237 T.105. Adjuvanticity of Mycobacterium W for the Oncofetal Antigen hCG Anjali Gautam, Rahul Pal, Om Singh. National Institute of Immunology, New Delhi, India Human chorionic gonadotropin (hCG) is known to be ectopically expressed by a variety of trophoblastic and non- trophoblastic cancers and can act as an autocrine growth promoter. The presence of hCG (or its subunits) is associated with increased invasiveness, chemo-and radio-resistance and poor prognosis. This study focused on assessment of immunogenicity and evaluation of efficacy of anti-hCG vaccination on hCG-secreting tumors by employing Myco- bacterium w (Mw) as an adjuvant along with a prototypic vaccine formulation (βhCG-TT adsorbed on aluminium hydroxide). Incorporation of Mw led to significantly enhanced anti-hCG antibody titres in mice of diverse genetic background (H-2d, H-2b, H-2k, H-2q). Interestingly, signifi- cant increases in IgG2a and IgG2b antibody levels were observed, providing insight into the differences in the T cell responses induced by mycobacterium. Mw-supplemented formulations elicited higher titres of biologically active antibodies which more potently inhibited receptor-hCG interaction; in all instances, antibodies exhibited high affinity for hCG. Further, elicited antibodies were reactive towards the surface of human colorectal carcinoma and non- small cell lung carcinoma cell lines. Anti-hCG antibodies were capable of inducing cytocidal effects even in the absence of complement. Taken together, our findings suggest that inclusion of Mycobacterium w in anti-hCG vaccine formulations can enhance adjuvanticity (even in low responder murine strains) for the generation of specific, therapeutic antibodies, along with providing the expected up-modulation of general immunity via non-specific mechanisms. doi:10.1016/j.clim.2009.03.238 T.106. Selective Expression of Latency Associated Peptide (LAP) and IL-1 Receptor Type I/II (CD121a/ CD121b) on Activated Human FOXP3+ Regulatory T Cells Allows for Their Purification from Expansion Cultures for Potential Immunotherapy Dat Tran, Ethan Shevach. NIAID/NIH, Bethesda, MD While adoptive transfer of regulatory T cells (Foxp3 + Tregs) has proven to be efficacious in the prevention and treatment of autoimmune diseases and graft versus host disease in rodents, a major obstacle for the use of Treg immunotherapy in humans is the difficulty of obtaining a highly purified preparation following ex vivo expansion. Inevitably the expanded Treg culture is contaminated with over 50% of FOXP3-effector T cells. Latency associated peptide (LAP) and IL-1 receptor type I and II (CD121a/ CD121b) have been identified as unique cell surface markers that distinguish activated Tregs from activated FOXP3-and FOXP3 + non-Tregs. After in vitro expansion of bead purified human CD25 + cells with a starting FOXP3 purity of 50-85%, it is feasible to sort expanded FOXP3+Tregs from non-Tregs S81 Abstracts
Transcript of T.106. Selective Expression of Latency Associated Peptide (LAP) and IL-1 Receptor Type I/II...
S81Abstracts
to be the most effective for attack prevention. Rituximabproved to be effective in one NMO case, resistant to pulsecyclophosphamide (CYC) therapy. Another SLE associatedNMO patient, unresponsive to high-dose oral and intravenouscorticosteroids, intravenous immunoglobulin, mycopheno-late mofetil, tacrolimus, low-dose daily oral cyclophospha-mide and rituximab, was successfully treated withimmunoablative dose CYC. In our two NMO associated SLEcases neurological manifestations anticipated other SLEsymptoms, and despite combined high dose corticosteroid/azathioprine treatment neurological relapses returned reg-ularly. 0,5 g/m2 body-surface monthly parenteral CYCtherapy was given, followed by quarterly maintenancetherapy. In both patients induction phase ended in January2009. Until this time none of the patient had newneurological symptoms, one patient experienced improvingin her neurological functions. In some cases, as we observed,not only immunoablative, but also traditional dose CYC canprevent flares in SLE associated NMO cases.
doi:10.1016/j.clim.2009.03.236
T.104. Selective Depletion of Alloreactive T CellsLeads to Elimination of Graft-Versus-Host Reactivityand Stimulate Graft-Versus-Leukemia andGraft-Versus-Myeloma EffectEva Matejkova, Ivana Buresova, Jana Muzikova, JaroslavMichalek. Masaryk University, Brno, Czech Republic
Graft-versus-host disease (GVHD) is a severe complicationof allogeneic HSCT. The major role in GVHD play thealloreactive clones of donor T cells leading to host tissuedamage. Selective depletion (SD) is a strategy to eliminatehost-reactive donor T cells from hematopoietic stem cellallografts to prevent GVHD while conserving useful donorimmune functions. We selectively depleted host-reactivedonor T cells from PBMC using an anti-CD25 immunotoxin.Irradiated PBMC from cancer patients and healthy donor PBMCwere used as responder cells in primary mixed leukocytereaction (MLR). To prepare GVL/GVM-specific T cells, wedepleted alloreactive T cells with anti-CD25 IT. Remaining Tcells were repeatedly stimulated with purified tumor cellsfrom the same patient. Tumor-reactive donor Tcells were thenpurified by FACS based on CD4CD25 and CD8CD25 productionand their specificity was tested in non-radioactive cytotoxicitytest. We performed 22 MLRs and (15 leukemia and 7 multiplemyeloma). SDwith the anti-CD25 IT led to significant depletion(99.2- 100%, median 99.7%) of alloreactive donor Tcells, whileGVL/GVM effect was well preserved: 1.1- 9.6% (median 7.46%)in leukemia and 0.2- 1.1% (median 0.6%) in myeloma patients.GVH reactivation of donor cells was negligible 0.14- 0.34%(median 0.17%), resp. 0- 0.3% (median 0.1%) even afterrepeated stimulation with patient's non-tumor PBMC. Thus, itis possible to selectively deplete donor alloreactive Tcells withthe anti-CD25 IT. In leukemia patients a strong GVL reactivitywas noticed, but GVM specific T cell clones seem to be lesseffective in their antitumor activity.
doi:10.1016/j.clim.2009.03.237
T.105. Adjuvanticity of Mycobacterium W for theOncofetal Antigen hCGAnjali Gautam, Rahul Pal, Om Singh. National Institute ofImmunology, New Delhi, India
Human chorionic gonadotropin (hCG) is known to beectopically expressed by a variety of trophoblastic and non-trophoblastic cancers and can act as an autocrine growthpromoter. The presence of hCG (or its subunits) is associatedwith increased invasiveness, chemo-and radio-resistanceand poor prognosis. This study focused on assessment ofimmunogenicity and evaluation of efficacy of anti-hCGvaccination on hCG-secreting tumors by employing Myco-bacterium w (Mw) as an adjuvant along with a prototypicvaccine formulation (βhCG-TT adsorbed on aluminiumhydroxide). Incorporation of Mw led to significantlyenhanced anti-hCG antibody titres in mice of diverse geneticbackground (H-2d, H-2b, H-2k, H-2q). Interestingly, signifi-cant increases in IgG2a and IgG2b antibody levels wereobserved, providing insight into the differences in the T cellresponses induced by mycobacterium. Mw-supplementedformulations elicited higher titres of biologically activeantibodies which more potently inhibited receptor-hCGinteraction; in all instances, antibodies exhibited highaffinity for hCG. Further, elicited antibodies were reactivetowards the surface of human colorectal carcinoma and non-small cell lung carcinoma cell lines. Anti-hCG antibodieswere capable of inducing cytocidal effects even in theabsence of complement. Taken together, our findingssuggest that inclusion of Mycobacterium w in anti-hCGvaccine formulations can enhance adjuvanticity (even in lowresponder murine strains) for the generation of specific,therapeutic antibodies, along with providing the expectedup-modulation of general immunity via non-specificmechanisms.
doi:10.1016/j.clim.2009.03.238
T.106. Selective Expression of Latency AssociatedPeptide (LAP) and IL-1 Receptor Type I/II (CD121a/CD121b) on Activated Human FOXP3+ Regulatory TCells Allows for Their Purification from ExpansionCultures for Potential ImmunotherapyDat Tran, Ethan Shevach. NIAID/NIH, Bethesda, MD
While adoptive transfer of regulatory T cells (Foxp3+Tregs) has proven to be efficacious in the prevention andtreatment of autoimmune diseases and graft versus hostdisease in rodents, a major obstacle for the use of Tregimmunotherapy in humans is the difficulty of obtaining ahighly purified preparation following ex vivo expansion.Inevitably the expanded Treg culture is contaminated withover 50% of FOXP3-effector T cells. Latency associatedpeptide (LAP) and IL-1 receptor type I and II (CD121a/CD121b) have been identified as unique cell surface markersthat distinguish activated Tregs from activated FOXP3-andFOXP3+non-Tregs. After in vitro expansion of bead purifiedhuman CD25+cells with a starting FOXP3 purity of 50-85%, itis feasible to sort expanded FOXP3+Tregs from non-Tregs
S82 Abstracts
using magnetic bead cell separation techniques based onexpression of these three markers. The proportion ofFOXP3+Tregs in the expansion culture ranges from 35-75%by day 21. After 48 h restimulation, 40-60% of the totalpopulation expressed LAP, CD121a or CD121b and correlatedwith FOXP3 expression. Post separation, the final productcontains N90% fully functional FOXP3+Tregs. The LAP+,CD121a+ and CD121b+FOXP3+ populations were anergic andsuppressive when compared to the negative or totalpopulations. This novel protocol should facilitate thepurification of Tregs for both cell-based therapies as wellas for detailed studies of human Treg function in health anddisease. This research was supported by the IntramuralResearch Program of the NIH, NIAID.
doi:10.1016/j.clim.2009.03.239
T.107. Copolymerized-type I Collagen DecreasesTh17 Meanwhile Increases Tregs on CIAJanette Furuzawa-Carballeda1, Angeles Galindo-Feria1,David Cruz-Robles2, Virginia Soto-Abraham2, SergioEscobar-Hernández2, Perla Macip-Rodríguez1, DeshiréAlpizar-Rodríguez1. 1Instituto Nacional de Ciencias Médicasy Nutrición Salvador Zubirán, Mexico, Mexico; 2InstitutoNacional de Cardiología Ignacio Chávez, Mexico, Mexico
Aim. To evaluate the effect of subdorsal administrationof copolymerized-Type I collagen (Pol-Clg) on collagen-induced arthritis (CIA). Methods. Toxicity model: Twentyone male DBA/1 mice were treated with 100 μl of (a)placebo, (b)Pol-Clg (c)methotrexate (2.5 mg/kg) and (d)b+c weekly/month. Early arthritis model: Forty eight age-matched mice were immunized intradermally at the baseof the tail with 100 μg of chicken CII emulsified incomplete Freund's adjuvant. At 21 day, mice were boostedwith 100 μg of CII in incomplete Freund's adjuvant.Treatments a-d were administered same day, weekly/month. Established Arthritis model: Arthritis was inducedto 48 mice. Treatments were administered 2 weeks afterboost, weekly/month. Clinimorphometric scores. Theywere assessed before immunization and thereafter weekly.Weight and temperature were determined. Inflammationof the four paws was scored as follows: 0-3 (no inflamma-tion-severe inflammation of whole paw or ankylosis).Inflammatory infiltrates were evaluated by H and E. Th1,Th2, Treg y Th17 subsets were evaluated by flowcytometry on spleen cells. Results. Incidence of CIA wasof 100% by day 28 in CII challenged mice. Clinimorpho-metric analysis showed a down-regulation of inflammationpost administration of b, c, and d treatments (pb0.05).Histology analysis showed that CIA-mice group hadextensive bone erosion, pannus and severe focal inflam-matory infiltrates vs. b, c, and d treatments. Only, Pol-Clgtreatment down-regulated Th17 and up-regulated Treg andTh1. Conclusion. Pol-Clg exerts a down-regulation ofautoimmune inflammation (Th17) on established CIA with-out side effects.
doi:10.1016/j.clim.2009.03.240
T.108. In Epicutaneous Immunotherapy, Applicationof Allergen on Intact Skin Result on Rapid Uptake bythe Dendritic Cells in Sensitized MiceVincent Dioszeghy1, Lucie Mondoulet1, Veronique Dhelft1,Christophe Dupont2, Pierre-Henri Benhamou1. 1DBVTechnologies, Paris, France; 2Hopital Saint Vincent de Paul,Paris, France
Background: On animal model, epicutaneous immunothe-rapy (EPIT) using a new device (Viaskin®) on intact skin is anefficient and secure alternative method of treatment ofallergy (JACI 2008, 121.793). We studied the capture of theallergen by the skin immune network during EPIT insensitized mice. Methods: Alexa488 (A488) conjugated OVAwas coated on a new device (VIASKIN®, DBV Technologies,Paris) and was applied on the back of naïve or OVA sensitizedmice 24 h after hair removing. Afferent lymph nodes,epidermis and dermis cells were isolated, immunostainedand analyzed by flow cytometry. Experiments pooling 5 miceper group were reproduced at least twice. Results: In naïvemice, allergen is captured in the epidermis by langerhanscells (LC) with 39%, 67% and 70% of CD11c+ cells positive forOVA-A488 after 6 h, 24 h and 48 h application respectively. Indermis, the capture of allergen by dendritic cells (DC) wasreduced and slower. In sensitized mice, the uptake ofallergen is quicker and more efficient. 81%, 90% and 84% ofLC in epidermis and 28%, 52% and 39% of DC in dermis werepositive for OVA-A488 after 6 h, 24 h, and 48 h respectively.Rapid decrease of DC is observed in the skin of sensitizedmice and percentages of A488 positive DC increasedsignificantly to 14% in lymph nodes after 48 h application.Conclusions: These results suggest that, when applied on theintact skin, an allergen coated on VIASKIN® is able to bequickly captured and presented by DC up to the lymph nodes.
doi:10.1016/j.clim.2009.03.241
T.109. Lipopeptide Vaccine Induces T Cell Immunityto Ocular Herpes Simplex Virus Type 1 in HumanLeukocyte Antigen-(HLA)-A⁎0201 TransgenicRabbit ModelAziz Alami Chentoufi, Gargi Dasgupta, Arfan Azeem, ZareenChoudhury, Anthony Nesburn, Lbachir Ben Mohamed.University of California Irvine, School of Medicine,Irvine, CA
Herpes simplex virus type 1 (HSV-1) first infects the eyes,and then establishes latency in the sensory neurons oftrigeminal ganglia (or TG). Periodic reactivations of HSV-1from latency cause a sight-threatening recurrent disease,known as recurrent herpes simplex keratitis (HSK). Mousemodel is inappropriate for testing the efficacy of candidatevaccines to prevent recurrent-HSK: (1) unlike humans andrabbits, repetitive and spontaneous reactivations do notoccur in mice and therefore no recurrent-HSK exists in mice.Indeed, mouse HSK is a one-time event. (2) The mouse strainsassess CD8+Tcell-based vaccines do not simulate the humanimmune system. To overcome these two major drawbacks,here we are developing a novel “humanized” HLA-transgenic
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