Human Effector Memory T Helper Cells Engage with Mouse Macrophages and Cause Graft-versus-Host–Like Pathology in Skin of Humanized Mice Used in a Nonclinical Immunization Study
2017; Elsevier BV; Volume: 187; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2017.02.015
ISSN1525-2191
AutoresBalasai Sundarasetty, Valery Volk, Sebastian J. Theobald, Susanne Rittinghausen, Dirk Schaudien, Vanessa Neuhaus, Constança Figueiredo, Andreas Schneider, Laura Gerasch, Adele Mucci, Thomas Moritz, Constantin von Kaisenberg, Loukia M. Spineli, Katherina Sewald, Armin Braun, Henning Weigt, Arnold Ganser, Renata Stripecke,
Tópico(s)Immunotherapy and Immune Responses
ResumoHumanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced–dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4+ T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft-versus-host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4+ cells near F4/80+ mouse macrophages around hair follicles. In spleen, CD4+ cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2–type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8+ T cells from mice with GVH reacted against the pp65 antigen in vitro. These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice. Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced–dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4+ T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft-versus-host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4+ cells near F4/80+ mouse macrophages around hair follicles. In spleen, CD4+ cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2–type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8+ T cells from mice with GVH reacted against the pp65 antigen in vitro. These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice. Novel biomedical products for human use, such as monoclonal antibodies, engineered T cells expressing chimeric antigen receptors, and novel vaccines, are overdue in their translation to clinics, partly because of the limitations of appropriate human-specific preclinical models to assess their immunological efficacy and safety. Small animal models composed of immune-deficient mouse models reconstituted with human hematopoietic stem cells (Hu-HSCs) are therefore excellent options for such studies. Hu-HSC mice can potentially replace costly and demanding studies that use nonhuman primates to characterize the spatiotemporal immunological events in vivo.1Shultz L.D. Ishikawa F. Greiner D.L. Humanized mice in translational biomedical research.Nat Rev Immunol. 2007; 7: 118-130Crossref PubMed Scopus (1023) Google Scholar, 2Shultz L.D. Brehm M.A. Garcia-Martinez J.V. Greiner D.L. Humanized mice for immune system investigation: progress, promise and challenges.Nat Rev Immunol. 2012; 12: 786-798Crossref PubMed Scopus (666) Google Scholar Several highly immunodeficient mouse strains have been developed and improved in the past decade, such as the nonobese diabetic (NOD)/severe combined immunodeficiency (SCID)/IL-2Rγcnull and NOD/Rag1null/IL-2Rγcnull (NRG) mice, which completely lack lymphocytes (T, B, and natural killer cells) and therefore support robust engraftment of Hu-HSCs. An important reason for the success of Hu-HSC engraftment in the NOD-derived mutant strains is the signal regulatory protein α-CD47 receptor–ligand interaction, which provides the do not eat me signal to mouse macrophages, avoiding the rejection of the human hematopoietic system by murine phagocytosis.3Yamauchi T. Takenaka K. Urata S. Shima T. Kikushige Y. Tokuyama T. Iwamoto C. Nishihara M. Iwasaki H. Miyamoto T. Honma N. Nakao M. Matozaki T. Akashi K. Polymorphic Sirpa is the genetic determinant for NOD-based mouse lines to achieve efficient human cell engraftment.Blood. 2013; 121: 1316-1325Crossref PubMed Scopus (94) Google Scholar Development of human myeloid and lymphoid cells in the murine lymphatic tissues and subsequent homing in several organ systems has been extensively characterized in Hu-HSC mice.2Shultz L.D. Brehm M.A. Garcia-Martinez J.V. Greiner D.L. Humanized mice for immune system investigation: progress, promise and challenges.Nat Rev Immunol. 2012; 12: 786-798Crossref PubMed Scopus (666) Google Scholar However, the interplay between the human lymphocytes and the murine myeloid cells, particularly because they are completely mismatched for the major histocompatibility complex, is still not clearly understood. Most studies using these models are performed for approximately 15 to 20 weeks after HSC transplantation (HSCT). At this time, the human T-cell maturation observed in the lymphatic tissues is still incomplete, probably because of the low levels of mature human professional antigen-presenting cells (APCs), such as dendritic cells (DCs) in murine lymphatic tissues. We have previously shown significantly accelerated development of the human adaptive immune responses in Hu-HSC mice transplanted with peripheral blood or cord blood–derived CD34+ cells that were then administered with autologously engineered induced DCs (iDCs).4Daenthanasanmak A. Salguero G. Sundarasetty B.S. Waskow C. Cosgun K.N. Guzman C.A. Riese P. Gerasch L. Schneider A. Ingendoh A. Messerle M. Gabaev I. Woelk B. Ruggiero E. Schmidt M. von Kalle C. Figueiredo C. Eiz-Vesper B. von Kaisenberg C. Ganser A. Stripecke R. Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV.Mol Ther Methods Clin Dev. 2015; 1: 14060Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 5Salguero G. Daenthanasanmak A. Munz C. Raykova A. Guzman C.A. Riese P. Figueiredo C. Langer F. Schneider A. Macke L. Sundarasetty B.S. Witte T. Ganser A. Stripecke R. Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation.J Immunol. 2014; 192: 4636-4647Crossref PubMed Scopus (41) Google Scholar These iDCs consisted of monocytes that were transduced with lentiviral vectors and coexpressed granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon α (IFN-α), and were endogenously loaded with the immune dominant antigen pp65 from the human cytomegalovirus. In vitro, the transduced monocytes showed autonomous DC differentiation, maturation, and activation of T cells.6Daenthanasanmak A. Salguero G. Borchers S. Figueiredo C. Jacobs R. Sundarasetty B.S. Schneider A. Schambach A. Eiz-Vesper B. Blasczyk R. Weissinger E.M. Ganser A. Stripecke R. Integrase-defective lentiviral vectors encoding cytokines induce differentiation of human dendritic cells and stimulate multivalent immune responses in vitro and in vivo.Vaccine. 2012; 30: 5118-5131Crossref PubMed Scopus (19) Google Scholar Thus, iDCs tested in Hu-HSC mice served as a proof-of-concept model for a cellular vaccine currently being developed in our laboratory for translation into immunization of immunocompromised patients at high risk of human cytomegalovirus reactivation after HSCT.7Sundarasetty B.S. Kloess S. Oberschmidt O. Naundorf S. Kuehlcke K. Daenthanasanmak A. Gerasch L. Figueiredo C. Blasczyk R. Ruggiero E. Fronza R. Schmidt M. von Kalle C. Rothe M. Ganser A. Koehl U. Stripecke R. Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation.J Transl Med. 2015; 13: 240Crossref PubMed Scopus (13) Google Scholar We demonstrated remarkable effects once iDCs self-differentiated in vivo, maintaining high viability for up to 2 weeks. iDCs migrated effectively to the lymph node Anlage, mediating T- and B-cell activation, expansion, and antigen-specific immune responses. iDCs also augmented the thymic and extrathymic development of naïve T cells. These results supported the hypothesis that the human lymphocytes required supplementation with human iDCs for their improved major histocompatibility complex–dependent maturation in lymphopenic Hu-HSC mouse models. We thus obtained scientific advice from the German office of the European Medicines Agency regarding further nonclinical in vivo testing of iDCs for clinical development. The recommendation was to evaluate potential occurrence of immune pathologies or malignancies in a long-term model [ie, to extend the experimental observation time of the Hu-HSC model from 10 to 26 weeks (ie, 6 months) after the first iDC immunization]. In addition, studies should be performed under good laboratory practices. Therefore, in collaboration with a contract research organization, we run pilot feasibility studies of long-term analyses of human immune reconstitution after iDC administration. NRG mice were transplanted with highly pure CD34+ cells from two cord blood (CB) donors and showed robust engraftment of human CD45+ cells. A cohort of mice was prime boosted with cryopreserved/thawed induced dendritic cells expressing pp65 (iDCpp65) and kept under observation for 26 weeks. Longitudinal analyses of the blood samples were performed to follow the development and maturation of human T cells. The mice were continuously monitored for any signs of pathologies. At the end point of the studies, as expected, we documented the infiltration and maturation of human T cells in the lymphatic tissues, but no systemic graft-versus-host (GVH) disease. We report herein that three of nine mice immunized with iDCpp65 showed GVH-like signs on the skin (GVH-S). The pathology was associated with high infiltration of human CD4+ cells in tissues and detection of human T helper-2 (Th2) type cytokines in plasma. Concurrently, accumulation of mouse macrophages was observed in affected areas of skin and surrounding human CD4+ T cells in the spleen. These observations supported the hypothesis that human cytokines can activate mouse macrophages in humanized mice, which we then confirmed with an in vitro assay. Therefore, our results serve as a note of caution when using long-term Hu-HSC mice as nonclinical models to predict pathologies that can be caused by adoptive human immune cells. Even so, the efficacy of iDCpp65 was further extended, as human pp65-reactive long-term memory T cells could be detected even 6 months after the first iDCpp65 immunization. The HEK-293 (human embryonic kidney-293) cell line encoding the simian virus 40 (SV40) large T antigen (heretofore, 293T cells) was used for the production of lentiviral vectors. 293T cells were cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (Life Technologies/Thermo Fisher Sceintific, Darmstadt, Germany) supplemented with 10% fetal bovine serum (HyClone/GE Healthcare Life Sciences, Logan, UT). K562 cells expressing HLA-A*02.01 (KA2) and KA2 coexpressing CMV phosphoprotein 65 (pp65) expression were described4Daenthanasanmak A. Salguero G. Sundarasetty B.S. Waskow C. Cosgun K.N. Guzman C.A. Riese P. Gerasch L. Schneider A. Ingendoh A. Messerle M. Gabaev I. Woelk B. Ruggiero E. Schmidt M. von Kalle C. Figueiredo C. Eiz-Vesper B. von Kaisenberg C. Ganser A. Stripecke R. Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV.Mol Ther Methods Clin Dev. 2015; 1: 14060Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 5Salguero G. Daenthanasanmak A. Munz C. Raykova A. Guzman C.A. Riese P. Figueiredo C. Langer F. Schneider A. Macke L. Sundarasetty B.S. Witte T. Ganser A. Stripecke R. Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation.J Immunol. 2014; 192: 4636-4647Crossref PubMed Scopus (41) Google Scholar and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1 mg/mL geneticin (Life Technologies GmbH). RAW264.7 (mouse macrophage cell line) was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. A third-generation self-inactivating integrase-defective lentiviral vector (IDLV) coexpressing GM-CSF, IFN-α, and pp65 was generated and titrated by quantifying the p24 HIV-I core protein by ELISA, as previously described.4Daenthanasanmak A. Salguero G. Sundarasetty B.S. Waskow C. Cosgun K.N. Guzman C.A. Riese P. Gerasch L. Schneider A. Ingendoh A. Messerle M. Gabaev I. Woelk B. Ruggiero E. Schmidt M. von Kalle C. Figueiredo C. Eiz-Vesper B. von Kaisenberg C. Ganser A. Stripecke R. Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV.Mol Ther Methods Clin Dev. 2015; 1: 14060Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 7Sundarasetty B.S. Kloess S. Oberschmidt O. Naundorf S. Kuehlcke K. Daenthanasanmak A. Gerasch L. Figueiredo C. Blasczyk R. Ruggiero E. Fronza R. Schmidt M. von Kalle C. Rothe M. Ganser A. Koehl U. Stripecke R. Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation.J Transl Med. 2015; 13: 240Crossref PubMed Scopus (13) Google Scholar Umbilical CB units were obtained under informed consent of the mothers in accordance with study protocols approved by Hannover Medical School Ethics Review Board. Mononuclear cells were isolated by Ficoll gradient centrifugation, CD34+ cells were selected after two rounds of immune-magnetic bead isolation (Miltenyi Biotech, Bergisch-Gladbach, Germany), and both CD34+ and CD34− fractions were cryopreserved. A bank of CD34+ cells established in our laboratory was used for pretesting and selection of the CB units that resulted into reproducible engraftment and persistency of human CD45+ cells (>20% huCD45+) and reconstitution of lymphocytes (CD3+, CD4+, CD8+, CD19+) in HIS mice for at least 15 weeks after HSCT. Two units positive for HLA-A*02.01 and showing persistency of huCD45+ in peripheral blood lymphocytes (PBL) and tissues for at least 15 weeks were chosen. CD14+ monocytes were isolated from the CD34− fractions for the production of iDCpp65, as described.4Daenthanasanmak A. Salguero G. Sundarasetty B.S. Waskow C. Cosgun K.N. Guzman C.A. Riese P. Gerasch L. Schneider A. Ingendoh A. Messerle M. Gabaev I. Woelk B. Ruggiero E. Schmidt M. von Kalle C. Figueiredo C. Eiz-Vesper B. von Kaisenberg C. Ganser A. Stripecke R. Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV.Mol Ther Methods Clin Dev. 2015; 1: 14060Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 5Salguero G. Daenthanasanmak A. Munz C. Raykova A. Guzman C.A. Riese P. Figueiredo C. Langer F. Schneider A. Macke L. Sundarasetty B.S. Witte T. Ganser A. Stripecke R. Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation.J Immunol. 2014; 192: 4636-4647Crossref PubMed Scopus (41) Google Scholar Briefly, CD14+ cells were isolated using CD14 isolation beads (Miltenyi Biotech). The monocytes were preconditioned with recombinant human GM-CSF and IL-4 (50 ng/mL each; Cellgenix, Freiburg, Germany) in X-VIVO 15 medium (Lonza GmbH, Cologne, Germany) for 8 hours before transduction in an incubator at 37°C and 5% CO2. The 2.5 μg/mL p24 equivalent of the ID-LV-G2α-pp65 vector was used to transduce 5 × 106 monocytes in the presence of 5 μg/mL protamine sulfate (Valeant, Eschborn, Germany) for 16 hours. After transduction, monocytes were harvested, resuspended in freezing medium containing 15.5% human albumin, 10% dimethyl sulfoxide, and 5% glucose, and cryopreserved at −152°C. Before immunization, the transduced cells (iDCpp65) were thawed, resuspended in phosphate-buffered saline (PBS), washed twice with PBS, and the viable cell counts were determined by trypan blue exclusion. A total of 5 × 105 viable cells were injected s.c. near the inguinal region as bilateral prime and boost administrations performed at 10 and 11 weeks after HSCT (described further below). Cryopreserved iDCpp65 was thawed and cultured for 7 days in X-VIVO 15 medium (Lonza) without the exogenous addition of cytokines. Surface marker expression was analyzed by flow cytometry using the monoclonal antibodies conjugated with fluorochromes: fluorescein isothiocyanate–conjugated anti-CD14 (Miltenyi Biotec), phosphatidylethanolamine-conjugated anti-CD80, PerCP-conjugated anti–HLA-DR, and APC-conjugated anti-CD86 (BD Biosciences, Heidelberg, Germany). Nontransduced monocytes and monocytes cultured in X-VIVO 15 with exogenous addition of GM-CSF and IFN-α (conventional IFN-α DCs) were used as negative and positive controls, respectively. Acquisitions and analyses were performed using an LSRII Flow Cytometer (BD Biosciences) and FlowJo analysis software version 7.6.4 (Tree Star Inc., Ashland, OR). The IDLV copy numbers in transduced monocytes were quantified by real-time quantitative PCR, as previously described.7Sundarasetty B.S. Kloess S. Oberschmidt O. Naundorf S. Kuehlcke K. Daenthanasanmak A. Gerasch L. Figueiredo C. Blasczyk R. Ruggiero E. Fronza R. Schmidt M. von Kalle C. Rothe M. Ganser A. Koehl U. Stripecke R. Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation.J Transl Med. 2015; 13: 240Crossref PubMed Scopus (13) Google Scholar Samples and data were analyzed with StepOnePlus Real-Time PCR system (Applied Biosystems, Life Technologies, Darmstadt, Germany). All procedures involving mice were reviewed and approved by the Lower Saxony and followed the guidelines provided by the Animal Facility at the Hannover Medical School. NOD.Cg-Rag1tm1Mom Il-2rgtm1Wjl (NOD.Rag1−/−.IL-2rγ−/−, NRG) mice were obtained from The Jackson Laboratory (Bar Harbor, MN) and bred and maintained under pathogen-free conditions in an IVC system (BioZone, Margate, UK) at Hannover Medical School. HSCT was performed as previously described.4Daenthanasanmak A. Salguero G. Sundarasetty B.S. Waskow C. Cosgun K.N. Guzman C.A. Riese P. Gerasch L. Schneider A. Ingendoh A. Messerle M. Gabaev I. Woelk B. Ruggiero E. Schmidt M. von Kalle C. Figueiredo C. Eiz-Vesper B. von Kaisenberg C. Ganser A. Stripecke R. Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV.Mol Ther Methods Clin Dev. 2015; 1: 14060Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 5Salguero G. Daenthanasanmak A. Munz C. Raykova A. Guzman C.A. Riese P. Figueiredo C. Langer F. Schneider A. Macke L. Sundarasetty B.S. Witte T. Ganser A. Stripecke R. Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation.J Immunol. 2014; 192: 4636-4647Crossref PubMed Scopus (41) Google Scholar Shortly, 0.2 × 106 CD34+ stem cells were injected through the tail vein into 4-week-old irradiated NRG mice (450 cGy; Gammacell 3000; Best Theratonics, Ottawa, Canada). Peripheral blood was collected from surviving mice at weeks 10, 15, 20, 25, 30, 35, and 36, and engraftment of human lymphocytes was analyzed by flow cytometry. At signs of debilitating pathology or at the end point analyses (36 weeks after HSCT), mice were sacrificed for collection of tissues. For flow cytometry analyses, blood, plasma, thymus, spleen, peripheral lymph nodes, mesenteric lymph nodes, and bone marrow specimens were processed as single-cell suspensions and the following antibodies were used: PB-conjugated anti-CD45, APC-conjugated anti-CD3, Alexa700-conjugated anti-CD19, APCCy7-conjugated anti-CD4, PCy7-conjugated anti-CD8, fluorescein isothiocyanate–conjugated anti-CD45RA, and PCy5-conjugated anti-CD62L. T-cell subpopulations were defined as naïve (N) for CD45RA+CD62L+, as effector memory (EM) for CD45RA−CD62L− and central memory (CM) for CD45RA−CD62L+ cells. Analyses were performed in LSRII flow cytometer (BD Biosciences) using FlowJo software. We performed a complete necropsy on all euthanized animals. Organs and pathological lesions were fixed in 3.7% formaldehyde buffer (Carl Roth GmbH + Co KG, Karlsruhe, Germany) for 24 hours. The tissue material was then transferred to 70% ethanol for further fixation up to 48 hours. All macroscopic lesions, predefined parts of liver, spleen, kidney, colon, lung, brain, skin, and eyes with eyelids, were embedded in paraffin. Sections (3 μm thick) from all organs were prepared and routinely stained with hematoxylin and eosin. Additional skin sections were stained with periodic acid–Schiff, and liver sections were stained with Prussian blue. For histopathological evaluation of the skin, the grading of the occurrence of apoptotic cells was set as follows: very slight, up to two cells per high-power field (HPF); slight, up to five cells per HPF. The grading for the inflammatory reaction was very slight, with up to 10 inflammatory cells per HPF; slight, with up to 50 inflammatory cells per HPF; and moderate, with >50 inflammatory cells per HPF. For immunohistochemical detection of the chosen markers, paraffin slides (3 μm thick) were sectioned from the histology wax blocks with the tissue material and mounted onto glass slides. Paraffin slides were then dewaxed and subjected to antigen retrieval methods, which had been validated for each of the primary antibodies for mouse and human tissues. Heat-induced antigen retrieval was performed in a citrate-buffered solution. All slides were rinsed with Tris-buffered saline (pH 7.6) plus 0.01% Tween 20 (Merck KGaA, Darmstadt, Germany; 8.22184). Slides were incubated for 20 minutes at 21°C in normal goat serum (Vector Laboratories Inc., Burlingame, CA; S-1000) and then incubated with the primary antibody for 1 hour at 21°C or 24 hours at 4°C, respectively. As secondary antibodies, a biotin-SP–conjugated AffiniPure goat anti-mouse IgG (111-065-100), a goat anti-rabbit IgG (111-065-144), or a goat anti-rat IgG (705-066-147), which were all purchased from Jackson Immunoresearch (West Grove, PA), were applied for a 30-minute incubation time at 21°C. Immunostaining was performed with a routine method using alkaline phosphatase streptavidin-biotin (Vector Laboratories Inc.; S-5100) and Fast Red as chromogen (Fast Red substrate pack; BioGenex, Freemont, CA; HK182-5K). The primary antibodies used for immunohistochemical analyses are listed in Table 1. The slides were finally counterstained with Bluing Reagent (Thermo Scientific, Braunschweig, Germany; 7301). Coverslipping was performed using Aquatex aqueous mounting medium (Merck KGaA, Darmstadt, Germany; 1.08562). Sample permeabilization, antibody concentrations, antibody reactions, and staining procedures were optimized for each antibody to get clear and specific immunohistochemical signals. We used a bright-field microscope for evaluation of the slides.Table 1Antibodies Used for Immunohistochemical AnalysisAntibodyClone numberManufacturerCatalog numberAnti-CD1a010Dako (Glostrup, Denmark)M3571Anti-CD20EP459YAbcam (Cambridge, UK)ab78237Anti-CD3SP7Biogenex (Fremont, CA)CI597Anti-CD79-aHM57DakoM705Anti-human CD11c5D11Cell Marque (Rocklin, CA)111M-18Anti-human CD4EPR6855Abcam (Cambridge, UK)ab133616Anti-human CD68PG-M1(3)DakoM0876Anti-human CD8144BAbcamab17147Anti-human glycophorin APolyclonalAbcamab129024Anti-human HLA-DREP96Epitomics (Burlingame, CA)AC-0088RUOAnti-human nuclear antigen antibody235-1Abcamab191181Anti-langerinEPR15863Abcamab192027Anti-mast cell tryptase10D11Novocastra, Leica Microsystems (UK) Ltd (Milton Keynes, UK)NCL-MCTRYPAnti-mouse F4/80F4/80AbD Serotec (Oxford, UK)MCA497RAnti-myeloperoxidasePolyclonalAbcamab9535Anti-PGP9.5PolyclonalDakoZ5116Anti-S100PolyclonalDakoZ0311 Open table in a new tab Plasma was separated from peripheral blood and level of human Th1/Th2 cytokines was performed by fluorescent bead–based 14-plex Luminex assay, according to the manufacturer's instructions. The assay measured the following cytokines: GM-CSF, IL-4, tumor necrosis factor-α, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), IL-10, IL-1b, IL-5, IFN-γ, IL-7, IL-2, and IL-12p70 (Merck Millipore, Billerica, MA). Similarly, mouse cytokines in the plasma were analyzed for the following cytokines: GM-CSF, tumor necrosis factor-α, IL-6, IL-8, IL-10, IL-5, IFN-γ, and IL-12p70, analyzed by multiplex assay (U-Plex; Meso Scale Discovery, Rockville, MD) and single plex MSD assay for MCP-1. RAW246.7 cells were seeded in a 6-well plate (1 × 106 cells/well per 1 mL), and recombinant human MCP-1, IL-6, IL-8, and IL-10 (50 ng/mL each; all cytokines are from PeproTech GmbH, Hamburg, Germany) were added to the wells in various combinations. Three hours after the cytokine treatment, cells were harvested and phoshpoSTAT5 (pSTAT5) up-regulation was analyzed by intracellular staining. Cells were washed once with PBS (Biochrom GmbH, Berlin, Germany) and the cell pellet was resuspended in 100 μL 4% paraformaldehyde (Carl Roth GmbH + Co KG) and incubated at room temperature for 30 minutes. Cells were then washed with MACS buffer (Miltenyi Biotec) and the pellet was resuspended in 1 mL of ice-cold methanol (Th. Grayner, Renningen, Germany) and further incubated on ice for 45 minutes. Cells are washed twice with 3 mL of PBS (Biochrom). The cells were then resuspended in 100 μL of PBS and stained with 2 μL of APC-conjugated human-mouse-phospho-stat5-y694-antibody (clone SRBCZX; eBioscience, Frankfurt, Germany) for 45 minutes at room temperature in the dark. Cells are then washed twice with PBS supplemented with 0.2% of bovine serum albumin and pSTAT5 was analyzed in FACS Calibur (BD Biosciences, Heidelberg, Germany). Nontreated cells and cells treated with mouse GM-CSF were used as negative and positive controls for the assay, respectively. Data were analyzed using FlowJo and Graphpad Prism 5.01 (GraphPad Software, Inc., La Jolla, CA). Lymphocytes recovered from lymph nodes and corresponding to >90% human T cells were cryopreserved and used for functional T-cell assays, essentially as described.4Daenthanasanmak A. Salguero G. Sundarasetty B.S. Waskow C. Cosgun K.N. Guzman C.A. Riese P. Gerasch L. Schneider A. Ingendoh A. Messerle M. Gabaev I. Woelk B. Ruggiero E. Schmidt M. von Kalle C. Figueiredo C. Eiz-Vesper B. von Kaisenberg C. Ganser A. Stripecke R. Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV.Mol Ther Methods Clin Dev. 2015; 1: 14060Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The thawed lymphocytes were first homeostatically activated with human anti–CD2/CD3/CD28-conjugated Anti-Biotin MACSiBead Particles (Miltenyi Biotec) in a bead/cell ratio of 1:2 and cultured in X-VIVO 15 medium for 2 days in the presence of huIL-2 (25 IU/mL) plus huIL-7 and huIL-15 (5 ng/mL each). Subsequently, T cells were further in vitro stimulated with pp65 antigen by co-culture with iDCpp65 in a DC/T cell ratio of 1:10 for additional 7 days. T cells stimulated with iDCs expressing no antigen were used as controls. For analyses of pp65-reactive IFN-γ production, expanded T cells (50,000 cells) were seeded on a 96-well ELISPOT plate coated with anti-human IFN-γ (Mabtech, Nacka Strand, Sweden) and incubated overnight in the presence of K562 cells expressing HLA-A02*01 (heretofore artificial antigen-presenting cells or aAPC). aAPC either alone, loaded with irrelevant peptides (WT1; aAPC+WT1), pp65 overlapping peptide pool (aAPC+pp65; Miltenyi Biotec), or aAPC endogenously expressing pp65 (aAPC/pp65) were incubated with T cells expanded (see above). Twenty-four hours later, wells were washed, and a biotin-conjugated anti-human IFN-γ monoclonal antibody was added. After incubation of plates with alkaline phosphatase–conjugated streptavidin, spots were developed using NBT/5-bromo-4-chloro-3-indolyl phosphate liquid substrate and analyzed in an AELVIS ELISPOT reader (AELVIS, Hannover, Germany). T cells recovered from lymph nodes (LNs) were also analyzed by intracellular staining for detection of antigen-specific IFN-γ secretion, as described.4Daenthanasanmak A. Salguero G. Sundarasetty B.S. Waskow C. Cosgun K.N. Guzman C.A. Riese P. Gerasch L. Schneider A. Ingendoh A. Messerle M. Gabaev I. Woelk B. Ruggiero E. Schmidt M. von Kalle C. Figueiredo C. Eiz-Vesper B. von Kaisenberg C. Ganser A. Stripecke R. Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV.Mol Ther Methods Clin Dev. 2015; 1: 14060Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar As a negative control for gating, nonstimulated lymphocytes were used. T cells expanded homeostatically
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