scholarly journals Effects of Peptide-Induced Immune Tolerance on Murine Lupus

2021 ◽  
Vol 12 ◽  
Author(s):  
Ram P. Singh ◽  
Bevra H. Hahn ◽  
David S. Bischoff
Keyword(s):  
T Cells ◽  
B Cells ◽  
Mhc Class Ii ◽  
Molecular Mechanisms ◽  
Cytotoxic T Lymphocyte ◽  
Cell Types ◽  
T Cell Subsets ◽  
Pcr Analysis

The regulation of autoimmunity and the molecular mechanisms by which different immune cells, including T cells, polymorphonuclear leukocytes (PMN-granulocytes), and B cells suppress autoimmune diseases is complex. We have shown previously that BWF1 lupus mice are protected from autoimmunity after i.v. injection or oral administration of tolerogenic doses of pCons, an artificial synthetic peptide based on sequences containing MHC class I and MHC class II determinants in the VH region of a J558-encoded BWF1 anti-DNA Ab. Several T cell subsets can transfer this tolerance. In this study, we determined the potential roles of granulocytes, B cells and regulatory T cells altered by pCons treatment in the BWF1 (NZB/NZW) mouse model of lupus. Immunophenotyping studies indicated that pCons treatment of BWF1 mice significantly increased CD4+FoxP3+ T cells, reduced the percent of B cells expressing CD19+CD5+ but increased the percent of CD19+CD1d+ regulatory B cells and increased the ability of the whole B cell population to suppress IgG anti-DNA production in vitro. pCons treatment significantly decreased the expression of CTLA-4 (cytotoxic T-lymphocyte-associated protein-4) in CD8+ T cells. In addition, peptide administration modified granulocytes so they became suppressive. We co-cultured sorted naïve B cells from mice making anti-DNA Ab (supported by addition of sorted naive CD4+ and CD8+ T cells from young auto-antibody-negative BWF1 mice) with sorted B cells or granulocytes from tolerized mice. Both tolerized granulocytes and tolerized B cells significantly suppressed the production of anti-DNA in vitro. In granulocytes from tolerized mice compared to saline-treated littermate controls, real-time PCR analysis indicated that expression of interferon-induced TNFAIP2 increased more than 2-fold while Ptdss2 and GATA1 mRNA were up-regulated more than 10-fold. In contrast, expression of these genes was significantly down-regulated in tolerized B cells. Further, another IFN-induced protein, Bcl2, was reduced in tolerized B cells as determined by Western blot analyses. In contrast, expression of FoxP3 was significantly increased in tolerized B cells. Together, these data suggest that B cells and granulocytes are altered toward suppressive functions by in vivo tolerization of BWF1 mice with pCons and it is possible these cell types participate in the clinical benefits seen in vivo.

scholarly journals Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells.

1994 ◽  
Vol 179 (2) ◽  
pp. 425-438 ◽  
Author(s):  
M P Cooke ◽  
A W Heath ◽  
K M Shokat ◽  
Y Zeng ◽  
F D Finkelman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Transgenic Mice ◽  
B Lymphocytes ◽  
Molecular Mechanisms ◽  
Interleukin 4 ◽  
Th Cells

The specificity of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of sIg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, non-tolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efficient activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the sIg signaling pathway which prevented activation of receptor-associated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this sIg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering sIg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of sIg signaling as a mechanism for controlling self-reactive B cells.

scholarly journals B Cells Directly Tolerize CD8+ T Cells

1998 ◽  
Vol 188 (11) ◽  
pp. 1977-1983 ◽  
Author(s):  
Sally R.M. Bennett ◽  
Francis R. Carbone ◽  
Tracey Toy ◽  
Jacques F.A.P. Miller ◽  
William R. Heath
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cd8 T Cells ◽  
Cytotoxic T Lymphocyte ◽  
Tolerance Induction ◽  
Cell Receptor ◽  
I Cells

This report investigates the response of CD8+ T cells to antigens presented by B cells. When C57BL/6 mice were injected with syngeneic B cells coated with the Kb-restricted ovalbumin (OVA) determinant OVA257–264, OVA-specific cytotoxic T lymphocyte (CTL) tolerance was observed. To investigate the mechanism of tolerance induction, in vitro–activated CD8+ T cells from the Kb-restricted, OVA-specific T cell receptor transgenic line OT-I (OT-I cells) were cultured for 15 h with antigen-bearing B cells, and their survival was determined. Antigen recognition led to the killing of the B cells and, surprisingly, to the death of a large proportion of the OT-I CTLs. T cell death involved Fas (CD95), since OT-I cells deficient in CD95 molecules showed preferential survival after recognition of antigen on B cells. To investigate the tolerance mechanism in vivo, naive OT-I T cells were adoptively transferred into normal mice, and these mice were coinjected with antigen-bearing B cells. In this case, OT-I cells proliferated transiently and were then lost from the secondary lymphoid compartment. These data provide the first demonstration that B cells can directly tolerize CD8+ T cells, and suggest that this occurs via CD95-mediated, activation-induced deletion.

scholarly journals The role of H-2-linked genes in helper T-cell function. III. Expression of immune response genes for trinitrophenyl conjugates of poly-L(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys in B cells and macrophages.

1978 ◽  
Vol 147 (6) ◽  
pp. 1596-1610 ◽  
Author(s):  
P Marrack ◽  
J W Kappler
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cell Function ◽  
Cell Types ◽  
High Responder ◽  
In Vitro Response ◽  
Low Responder

Using lymph node T cells from poly-L(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys[(TG)-A--L]-primed animals and B cells from animals primed with trinitrophenylated (TNP) protein or lipopolysaccharide, we have obtained anti-TNP-(TG)-A--L direct plaque-forming responses in vitro. Response to this antigen was shown to be controlled by the H-2 haplotype of the animal studied. The strain distribution of in vitro response was very similar to that previously reported by others for in vivo secondary IgG responses to (TG)-A--L. We investigated the cell types expressing the Ir gene(s) for (TG)-A--L in our cultures. F1, high responder x low responder mice were primed with (TG)-A--L. Their T cells were active in stimulating anti-TNP-(TG)-A--L responses of high responder but not low responder B cells and macrophages (MPHI), even though both preparations of B cells and Mphi were obtained from mice congenic at H-2 with one of the parents of the F1. For three low responder strains tested, of the H-2h2, H-2k, and H-2f haplotypes, the anti-TNP-(TG)-A--L response of low responder B cells and Mphis in the presence of high responder, F1 T cells could not be improved by the addition of high responder, antigen-bearing Mphis to the cultures. In one strain of the H-2a haplotype, it was shown that neither the B cells nor Mphis could be functional in anti-TNP-(TG)-A--L responses. Our results therefore suggested the Ir genes for anti-TNP-(TG)-A--L responses were expressed at least in B cells in all the low responder strains we studied, and, in mice of the H-2a haplotype, in Mphis too.

scholarly journals A DNA Vaccine Coding for the Brucella Outer Membrane Protein 31 Confers Protection against B. melitensis and B. ovis Infection by Eliciting a Specific Cytotoxic Response

2005 ◽  
Vol 73 (10) ◽  
pp. 6537-6546 ◽  
Author(s):  
Juliana Cassataro ◽  
Carlos A. Velikovsky ◽  
Silvia de la Barrera ◽  
Silvia M. Estein ◽  
Laura Bruno ◽  
...  
Keyword(s):  
T Cells ◽  
Outer Membrane ◽  
Dna Vaccine ◽  
Cytotoxic T Lymphocyte ◽  
Protective Efficacy ◽  
Outer Membrane Proteins ◽  
Humoral Response ◽  
T Cell Subsets

ABSTRACT The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The outer membrane proteins (Omps) of Brucella spp. have been extensively characterized as potential immunogenic and protective antigens. This study was conducted to evaluate the immunogenicity and protective efficacy of the B. melitensis Omp31 gene cloned in the pCI plasmid (pCIOmp31). Immunization of BALB/c mice with pCIOmp31 conferred protection against B. ovis and B. melitensis infection. Mice vaccinated with pCIOmp31 developed a very weak humoral response, and in vitro stimulation of their splenocytes with recombinant Omp31 did not induced the secretion of gamma interferon. Splenocytes from Omp31-vaccinated animals induced a specific cytotoxic-T-lymphocyte activity, which leads to the in vitro lysis of Brucella-infected macrophages. pCIOmp31 immunization elicited mainly CD8+ T cells, which mediate cytotoxicity via perforins, but also CD4+ T cells, which mediate lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets showed that the pCIOmp31-induced protection against Brucella infection is mediated predominantly by CD8+ T cells, although CD4+T cells also contribute. Our results demonstrate that the Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8+ T cells that eliminate Brucella-infected cells via the perforin pathway.

scholarly journals IL-21 Signaling in Immunity

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 224 ◽  
Author(s):  
Warren J. Leonard ◽  
Chi-Keung Wan
Keyword(s):  
T Cells ◽  
Molecular Mechanisms ◽  
Immune Cell ◽  
Cell Types ◽  
Clinical Settings ◽  
Type I ◽  
Next Generation Sequencing Technology ◽  
Wide Range

IL-21 is a type I cytokine produced by T cells and natural killer T cells that has pleiotropic actions on a wide range of immune and non-immune cell types. Since its discovery in 2000, extensive studies on the biological actions of IL-21 have been performed in vitro and in vivo. Recent reports describing patients with primary immunodeficiency caused by mutations of IL21 or IL21R have further deepened our knowledge of the role of this cytokine in host defense. Elucidation of the molecular mechanisms that mediate IL-21’s actions has provided the rationale for targeting IL-21 and IL-21 downstream mediators for therapeutic purposes. The use of next-generation sequencing technology has provided further insights into the complexity of IL-21 signaling and has identified transcription factors and co-factors involved in mediating the actions of this cytokine. In this review, we discuss recent advances in the biology and signaling of IL-21 and how this knowledge can be potentially translated into clinical settings.

IgG-Derived Tregitope Peptides Suppress T Cell Responses in Vitro and in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 677-677
Author(s):  
Anne S. De Groot ◽  
Leonard Moise ◽  
Yan Su ◽  
Julie A McMurry ◽  
William D Martin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Responses ◽  
Mhc Class Ii ◽  
Tolerance Induction ◽  
Elispot Response ◽  
Cytokines And Chemokines

Abstract We have identified a set of putative natural T regulatory epitopes (Tregitopes) which, when co-administered with an antigen, cause the expansion of antigen-specific adaptive Tregs in vitro and in vivo. They have the following characteristics: they bind, in most cases, with high affinity to multiple MHC class II molecules and, when co-administered with antigen, they suppress effector T cell immune responses to the antigen and up-regulate Treg associated cytokines and chemokines. T cells responding to Tregitopes exhibit a T regulatory phenotype (CD4+/CD25hiFoxP3+). To test whether Tregitopes derived from immunoglobulin (Ig) suppress immune responses to antigen co-administered in vivo, we performed two types of experiments. In the first, we dosed three groups of HLA DR4 mice every other week for six weeks with either a peptide antigen (pAg) alone, pAg with murine Fc, or pAg with mTregitope289, the murine homolog of the human Tregitope289. Mice were sacrificed and spleens harvested for assay. While the mice dosed with murine Fc demonstrated a reduced IL-4 ELIspot response to pAg, remarkably, the reduction was even greater in the mice treated with Tregitope. In a second model, C57Bl/6 mice were injected with LPS-stimulated B cells that were pulsed with either ovalbumin (OVA) alone, mTregitopes 167 and 289 or with OVA together with the two mTregitopes. One week later, mice were challenged with OVA 323–339 peptide in adjuvant. Two weeks after challenge, draining lymph nodes were harvested and LN cells stimulated with OVA 323–339 for measurement of T-cell proliferation by thymidine incorporation and by IFN-γ secretion by ELIspot. The mice receiving B cells previously pulsed with OVA alone demonstrated a robust IFN-gamma response to OVA re-stimulation. In contrast, the mice receiving B cells previously pulsed with OVA + Tregitopes demonstrated a comparatively reduced response. When sera were assayed for anti-OVA antibodies by ELISA, antibody response to OVA also declined following treatment with B cells co-administered with Tregitope. The mechanism of suppression appears to be due to the induction of antigen-specific adaptive tolerance induction (De Groot AS et al. Activation of natural regulatory T cells by IgG Fc-derived Peptide “Tregitopes” Blood112: in press, 2008).

Inhibition of Histone Deacetylase 6 (HDAC6) Disrupts the Tolerogenic STAT3 Signaling Pathway and Augments Antitumor Immune Responses in Mantle Cell Lymphoma (MCL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3724-3724
Author(s):  
Hongwei Wang ◽  
Fengdong Cheng ◽  
Zi Wang ◽  
Maritza Lienlaf ◽  
Patricio Perez-Villarroel ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Mhc Class Ii ◽  
Dominant Role ◽  
Histone Deacetylase 6 ◽  
Stat3 Phosphorylation ◽  
Anergic T Cells ◽  
Hodgkin's Lymphomas

Abstract Abstract 3724 MCL is an aggressive and incurable subtype of B-cell Non-Hodgkin's lymphomas. Although patients often respond to first-line chemotherapy plus monoclonal antibodies, relapse and decrease response to further lines of treatment eventually occurs. Manipulation of the immune system to unleash its specificity and long-lasting protective effect might provide a unique opportunity to induce more durable responses or prevent relapse in MCL. Previous studies by our group have shown that HDAC6 is required for production of the immunosuppressive cytokine, IL-10 in antigen-presenting cells (APCs) and that genetic or pharmacologic disruption of HDAC6 in these cells triggers potent antigen-specific T-cell responses. Given the role of IL-10 in suppressing the immunogenicity of B-cells, we asked therefore whether inhibition of HDAC6 with the isotype-selective inhibitor, Tubastatin-A (Tub-A) could reverse the tolerogenic properties of malignant B-cells in a murine model of MCL1. First, in vitro treatment of FC-muMCL1 cells with Tub-A resulted in increased acetylation of a-tubulin, a known HDAC6 target. Treated B-cells also displayed an enhanced expression of MHC class II and the co-stimulatory molecules B7.1, B7.2 and CD40 relative to untreated cells. Such changes resulted in more immunogenic MCL cells able to effectively activate naïve antigen-specific CD4+ T-cells and more importantly, capable of restoring the responsiveness of anergic T-cells from lymphoma-bearing mice. Second, in vivo treatment of FC-muMCL1-bearing C57BL/6 mice with Tub-A resulted in lymphoma rejection. This antitumor effect was not observed in immunodeficient (SCID) C57BL/6 mice treated with Tub-A, pointing to the immunological effects triggered by HDAC6 inhibition in B-cells as playing a dominant role in Tub-A induced anti-MCL activity. Third, mechanistically we have found that HDAC6 physically interacts with STAT3 and it is required for STAT3 phosphorylation and recruitment to the IL-10 gene promoter. Furthermore, Tub-A treated MCL cells displayed a diminished STAT3 phosphorylation and abrogation of IL-10 gene transcriptional activity. Taken together, a concerted regulatory mechanism involving HDAC6 and STAT3 influence the immunogenicity of MCL cells. Targeting HDAC6 with specific inhibitors to disrupt the tolerogenic STAT3/IL-10 axis represents a novel strategy to trigger effective anti-MCL immunity. Disclosures: Martin: Millennium Pharmaceuticals, Inc.: Speakers Bureau.

scholarly journals Involvement of GANP in B Cell Activation in T Cell-dependent Antigen Response

2002 ◽  
Vol 9 (3) ◽  
pp. 169-172 ◽  
Author(s):  
Nobuo Sakaguchi ◽  
Satoru Fujimura ◽  
Kazuhiko Kuwahara
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
B Cell Differentiation ◽  
Region Gene

Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210 kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Agin vivoand in B cells stimulated with anti-CD40 monoclonal antibodyin vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cellsin vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.

scholarly journals Glycosphingolipids as Novel Targets for T-Cell Suppression by the B Subunit of Recombinant Heat-Labile Enterotoxin

1998 ◽  
Vol 66 (4) ◽  
pp. 1299-1308 ◽  
Author(s):  
Robert L. Truitt ◽  
Carrie Hanke ◽  
Jay Radke ◽  
Reinhold Mueller ◽  
Joseph T. Barbieri
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Mammalian Cells ◽  
Cytotoxic T Lymphocyte ◽  
Fluorescein Isothiocyanate ◽  
T Cell Subsets ◽  
Activated T Cells ◽  
Heat Labile

ABSTRACT Heat-labile enterotoxin subunit B (LTB) is a noncatalytic protein derived from Escherichia coli that binds to ganglioside GM1, a glycosphingolipid on the surface of mammalian cells. In this study, the effects of recombinant LTB (rLTB) on murine lymphocytes were examined in vitro. T and B cells readily bound fluorescein isothiocyanate-labeled rLTB. CD8+ T cells bound twice as much as CD4+ T cells and B cells. Exposure of T-cell subsets and B cells to rLTB abrogated mitogen-driven proliferation. CD8+ T cells were more susceptible to rLTB than either CD4+ T cells or B cells. There were differences in the sensitivity of lymphocytes from various strains of mice to rLTB. This was attributed to qualitative and quantitative differences in the CD4+ T cells. rLTB induced apoptosis in both T-cell subsets, but the level was significantly higher in CD8+ T cells. Apoptosis peaked at around 8 h after exposure to rLTB and incubation at 37°C. Binding to ganglioside GM1 was essential for suppression, since rLTB/G33D, a mutant which does not bind GM1, failed to inhibit proliferation or induce apoptosis. Naive T cells, which were acutely sensitive to rLTB, became more resistant after activation. Conversely, activated T cells regained their sensitivity to rLTB when they reverted back to a resting state. A 1-h pulse with rLTB was sufficient to inhibit T-cell proliferation and cytotoxic-T-lymphocyte generation in primary mixed lymphocyte reaction cultures. CD8+ T cells were preferentially depleted in these cultures. rLTB also induced functional modifications in T cells as indicated by inhibition of gamma interferon secretion after polyclonal activation. Thus, rLTB may have immunomodulatory properties independent of its ability to induce apoptosis.

696 Brentuximab vedotin, a CD30-directed antibody-drug conjugate, selectively depletes activated Tregs in vitro and in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A738-A738
Author(s):  
Bryan Grogan ◽  
Reice James ◽  
Michelle Ulrich ◽  
Shyra Gardai ◽  
Ryan Heiser ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Efflux Pump ◽  
Apoptotic Cell ◽  
T Cell Subsets ◽  
Memory Cells ◽  
Antibody Drug Conjugate ◽  
Selective Depletion

BackgroundRegulatory T cells (Tregs) play an important role in maintaining immune homeostasis, preventing excessive inflammation in normal tissues. In cancer, Tregs hamper anti-tumor immunosurveillance and facilitate immune evasion. Selective targeting of intratumoral Tregs is a potentially promising treatment approach. Orthogonal evaluation of tumor-infiltrating lymphocytes (TILs) in solid tumors in mice and humans have identified CCR8, and several tumor necrosis family receptors (TNFRs), including TNFSFR8 (CD30), as receptors differentially upregulated on intratumoral Tregs compared to normal tissue Tregs and other intratumoral T cells, making these intriguing therapeutic targets.Brentuximab vedotin (BV) is approved for classical Hodgkin lymphoma (cHL) across multiple lines of therapy including frontline use in stage III/IV cHL in combination with doxorubicin, vinblastine, and dacarbazine. BV is also approved for certain CD30-expressing T-cell lymphomas. BV is comprised of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE).The activity of BV in lymphomas is thought to primarily result from tumor directed intracellular MMAE release, leading to mitotic arrest and apoptotic cell death.The role CD30 plays in normal immune function is unclear, with both costimulatory and proapoptotic roles described. CD30 is transiently upregulated following activation of memory T cells and expression has been linked to highly activated/suppressive IRF4+ effector Tregs.MethodsHere we evaluated the activity of BV on CD30-expressing T cell subsets in vitro and in vivo.ResultsTreatment of enriched T cell subsets with clinically relevant concentrations of BV drove selective depletion of CD30-expressing Tregs > CD30-expressingCD4+ T memory cells, with minimal effects on CD30-expressing CD8+ T memory cells. In a humanized xeno-GVHD model, treatment with BV selectively depleted Tregs resulting in accelerated wasting and robust T cell expansion. The observed differential activity on Tregs is likely attributable to significant increases in CD30 expression and reduced efflux pump activity relative to other T cell subsets. Interestingly, blockade of CD25 signaling prevents CD30 expression on T cell subsets without impacting proliferation, suggesting a link between CD25, the high affinity IL-2 receptor, and CD30 expression.ConclusionsTogether, these data suggest that BV may have an immunomodulatory effect through selective depletion of highly suppressive CD30-expressing Tregs.AcknowledgementsThe authors would like to thank Michael Harrison, PharmD for their assistance in abstract preparation.Ethics ApprovalAnimals studies were approved by and conducted in accordance with Seattle Genetics Institutional Care and Use Committee protocol #SGE-024.

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