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. 2015 Jan 7;1:14060.
doi: 10.1038/mtm.2014.60. eCollection 2015.

Engineered Dendritic Cells From Cord Blood and Adult Blood Accelerate Effector T Cell Immune Reconstitution Against HCMV

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Free PMC article

Engineered Dendritic Cells From Cord Blood and Adult Blood Accelerate Effector T Cell Immune Reconstitution Against HCMV

Anusara Daenthanasanmak et al. Mol Ther Methods Clin Dev. .
Free PMC article

Abstract

Human cytomegalovirus (HCMV) harmfully impacts survival after peripheral blood hematopoietic stem cell transplantation (PB-HSCT). Delayed immune reconstitution after cord blood (CB)-HSCT leads to even higher HCMV-related morbidity and mortality. Towards a feasible dendritic cell therapy to accelerate de novo immunity against HCMV, we validated a tricistronic integrase-defective lentiviral vector (coexpressing GM-CSF, IFN-α, and HCMV pp65 antigen) capable to directly induce self-differentiation of PB and CB monocytes into dendritic cells processing pp65 ("SmyleDCpp65"). In vitro, SmyleDCpp65 resisted HCMV infection, activated CD4(+) and CD8(+) T cells and expanded functional pp65-specific memory cytotoxic T lymphocytes (CTLs). CD34(+) cells obtained from PB and CB were transplanted into irradiated NOD.Rag1(-/-).IL2γc(-/-) mice. Donor-derived SmyleDCpp65 administration after PB-HSCT stimulated peripheral immune effects: lymph node remodeling, expansion of polyclonal effector memory CD8(+) T cells in blood, spleen and bone marrow, and pp65-reactive CTL and IgG responses. SmyleDCpp65 administration after CB-HSCT significantly stimulated thymopoiesis. Expanded frequencies of CD4(+)/CD8(+) T cell precursors containing increased levels of T-cell receptor excision circles in thymus correlated with peripheral expansion of effector memory CTL responses against pp65. The comparative in vivo modeling for PB and CB-HSCT provided dynamic and spatial information regarding human T and B cell reconstitution. In vivo potency supports future clinical development of SmyleDCpp65.

Figures

Figure 1
Figure 1
Generation and analyses of SmyleDC and SmyleDCpp65 from peripheral blood mononuclear cells of healthy donors. (a) Lentiviral vector backbones. Scheme of the chimeric multicistronic LV-G2α and LV-G2α-pp65 vectors. The open reading frames in the bicistronic LV vector encoded for GM-CSF and IFN-α and tricistronic lentiviral (LV) vector encoded for GM-CSF, IFN-α, and HCMV-pp65. Each gene was separated by a 2A element from the porcine teschovirus (P2A) upstream of human IFN-α and F2A (foot and mouth disease virus) upstream of HCMV-pp65. ID-LV-G2α and ID-LV-G2α-pp65 were used to generate SmyleDC and SmyleDCpp65 by transducing 5 × 106 preconditioned monocytes (multiplicity of infection of 5) for 16 hours. The cells were washed, cultured, and analyzed on days 7, 14, and 21. (b) Cell recovery. Total viable SmyleDC (light grey) and SmyleDCpp65 (dark grey) recovered from DC cultures on days 7, 14, and 21 were determined as absolute number relative to input of transduced monocytes. (c) DC Stability. Flow cytometry analysis was used to determine frequency of cells that were double positive for CD86+/HLA-DR+ on days 7, 14, and 21. (d) Absolute numbers of cells positive for pp65 expression analyzed by intracellular staining on days 7, 14, and 21. (e) Expression of relevant DC immunophenotypic markers (CD11c, HLA-DR, HLA-ABC, CD80, CD86, CD14, CD123, and CD83) determined as percentages of positive cells on day 7. (f) Secreted cytokines detectable in SmyleDC and SmyleDCpp65 cultures. Supernatants were collected on day 7 (IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, MCP-1, TNF-α) and transgenic cytokines (GM-CSF and IFN-α) were measured using cytokine bead arrays. All analyses were performed as independent triplicates with monocytes obtained from three different donors.
Figure 2
Figure 2
Infection of dendritic cells (DCs) with human cytomegalovirus (HCMV) in vitro. (a) Flow cytometry analyses for detection of HCMV/GFP+ infected cells at 0–10 day postinfection (d.p.i.) control human fibroblast (HF) cells (gray square, left graph) and different types of lentivirus-induced DC; SmyleDC (grey circle), SmyleDCpp65 (dark triangle), and SmartDC (dark square). Multiplicity of infection of 1 was used in the assay. (b) Detection of newly released HCMV virions in cell supernatants. Supernatants from each infected cell culture were collected on 0, 2, 4, 6, 8, and 10 d.p.i and evaluated for the presence of infectious particles by plaque assay. Control infected HF cells supernatants (left graph) and DC supernatants (right graph) are shown. Titers are represented as particle forming units (pfu)/ml. (c) Flow cytometry analyses of CD80+ cells comparing noninfected DCs (mock) with HCMV-infected DCs showing dynamic changes in CD80 expression on different d.p.i.
Figure 3
Figure 3
Stimulation of T cell with SmyleDC and SmyleDCpp65 in vitro. (a) Short stimulation of T cells measured by interferon (IFN)-γ ICS. CD3+ T cells obtained from HCMV seropositive donors (n = 3) were either not stimulated, stimulated with pp65 peptide, with SmyleDC or with SmyleDCpp65 in vitro for 16 hours at T cell to DC ratio of 30:1. Representative CD4+/IFN-γ+ and CD8+/IFN-γ+ FACS analyses from one donor determined by IFN-γ intracellular staining are shown as a density plot. Bar graph: Frequencies of IFN-γ+ producing CD4+ (white) and CD8+ (black) T cells were calculated from triplicate independent experiments. Student t-test was used for calculation of P < 0.05. (b) Long stimulation of CD8+ T cell expansion in T cell microculture. Selected CD8+ T cells were stimulated with SmyleDC or SmyleDCpp65 in vitro at T cell and DC ratio of 10:1 for two weekly cycles in the presence of irradiated autologous feeder cells plus recombinant IL-2, IL-7, and IL-15 cytokines. T cells stimulated with feeders and cytokines were used as controls. Left bar graph: viable T cells stimulated with either cytokine and feeder cells, SmyleDC or SmyleDCpp65 were counted and plotted as fold expansion relative to input (1 × 106 cells). pp65 specificity measured by pentamer staining. Expanded T cells were stained with pentamers reactive against pp65 epitopes. Representative pp65-restricted CD8+/A*0201+ and CD8+/B*0702+ analyses of T cells expanded from one donor determined by flow cytometry analyses. Right bar graph: frequencies of T cells restricted to A*0201-NLVPMVATA: white/B*0702-TPRVTGGGAM: black. Student t-test was used for calculation of P < 0.05. (c) Cytotoxic activity of CD8+ expanded T cells cocultured with K562 target cells controls or expressing pp65 epitopes and presented through HLA -A*02 or B*07 context (at different effector: target (E:T) ratios (20, 10, 5, and 1:1) for 4 hours. LDH release measured by coupled enzymatic assay was used to quantify K562 target lysis. T cells homeostatically expanded with SmyleDC were cocultured with K562 cells (−/+ expressing pp65) were used as cytotoxicity controls.
Figure 4
Figure 4
NRG mice transplanted with adult CD34+ HSC and immunized with SmyleDC or SmyleDCpp65. (a) Experimental scheme of immunization and monitoring. Four-week-old irradiated NRG mice (450 cGy) were transplanted with 5 × 105 PB CD34+ stem cells isolated from G-CSF mobilized blood. On weeks 10 and 11 after hematopoietic stem cell transplantation (HSCT), mice were immunized subcutaneously on the hind flanks with either 5 × 105 SmyleDC or SmyleDCpp65. Kinetics of human hematopoietic reconstitution in blood was determined by flow cytometry at week 10 (before immunization) and week 13 (after immunization). Mice were sacrificed and blood, plasma, spleen, bone marrow and lymph nodes were collected 20 weeks after transplantation. For all analyses, SmyleDC (light grey, n = 5) and SmyleDCpp65 (dark grey, n = 5) were compared. (b) Bar graphs showing concentrations of human cytokines measured by cytokine bead arrays detected in mice plasma (pg/ml) IL-4, IL-12p70, IL-10, IL-5, IL-6, IL-8, IL-1β, TNF-α, IFN-γ, GM-CSF, and MCP-1 were. * represents asymptotic significance analyzed by Kolmogorov–Smirnov test. (c) Detection of lymph nodes. Frequency as percentage of mice in the cohort with detectable LN at any location or at different body locations for each group. (d) Kinetics of human lymphocytes reconstitution in PB determined by flow cytometry. Frequencies of human CD45+, CD3+ in CD45+, CD4+ in CD45+, CD8+ in CD45+, and CD19+ in CD45+ were determined on weeks 10, 13, and 20. Black dashed lines represent observed levels of human cells in historical PB-HSCT control group (no immunization, 20 weeks after HSCT). Box plot indicating median and error bars indicating ranges are shown and statistical analyses were determined by post hoc test; P < 0.05 was considered significant. (e) Box plot indicating median and error bars indicating ranges of absolute counts (right) and frequencies (left) of human lymphocytes detected in spleen; CD45+, CD3+ in CD45+, CD4+ in CD45+, CD8+ in CD45+, and CD19+ in CD45+ cells determined by flow cytometry.
Figure 5
Figure 5
Functional effects of SmyleDCpp65 immunization after PB-HSCT. All Box plots indicate median and error bars indicate ranges of analyses performed 20 weeks after HSCT, SmyleDC (light grey, n = 5), and SmyleDCpp65 (dark grey, n = 5) were compared. (a) Upper panel: frequency of human lymphocytes and T cell subsets in bone marrow; CD45+, CD3+ in CD45+, CD4+ in CD45+, CD8+ in CD45+, and CD19+ in CD45+. Lower panels: subsets of T cells in CD4+ and CD8+ populations classified as naive, central memory (CM), and effector memory (EM). Analysis of variance with post hoc analysis test was used to determined significance, P < 0.05. (b) T cell responses against pp65. CD4+ or CD8+ T cells were sorted from splenocytes explanted from SmyleDCpp65-immunized NRG mice (n = 3) and pooled. T cells were homeostatically activated with CD2/CD3/CD28 beads for 48 hours followed by 7 days ex vivo expansion with SmyleDCpp65. 50,000 T cells were restimulated overnight with either HCMV-pp65 overlapping peptide pool or without peptide on anti-IFN-γ-coated ELISPOT plates. Bars represent number of IFN-γ-positive spots. (c) Human immunoglobulin (Ig) levels of IgM, IgA, IgG1, IgG2, IgG3, and IgG4 (ng/ml) detectable by bead arrays assay in plasma of NRG mice immunized with SmyleDC (n = 5) or SmyleDCpp65 (n = 5) in comparison with plasma obtained from human donors (n = 3). (d) Anti-pp65 reactivity of human IgM and IgG in plasma of mice immunized with SmyleDC or SmyleDCpp65 measured by ELISA. Human HCMV seropositive donors were used as positive controls (n = 3).
Figure 6
Figure 6
Mice transplanted with CB CD34+ HSC and immunized with SmyleDCpp65. (a) Scheme of immunization and monitoring. Four-week-old irradiated NRG mice transplanted with 1.5 × 105 CB CD34+ cells immunized with SmyleDCpp65 four times on weeks 6, 7, 10, and 11 (dark grey, n = 9) or two times on weeks 10 and 11 (light gray, n = 12). Nonimmunized mice (no DC, white, n = 12) were used as controls. Kinetics of hematopoietic reconstitution in blood was determined by flow cytometry on weeks 10 and 12. Mice were sacrificed and blood, plasma, spleen, and thymus were collected 16 weeks after transplantation. All box plots indicate median and error bars indicate ranges of analyses. (b) Human cytokines detected in mice plasma. Level of cytokines (pg/ml) IL-10, IFN-γ, GM-CSF, TNF-α, and MCP-1 was measured by cytokine bead arrays. (c) Detection of lymph nodes. Frequency as percentage of mice in the cohort with detectable LN at any location or at different body locations for each group. (d) Macroscopic examination of thymus. Enlarged thymus was observed in SmyleDCpp65-immunized mice. (e) Left panels show representative CD4+/CD8+ gating strategy. Absolute cell counts in thymus. Analyses of thymocytes at different developmental stages; DP; CD45+/CD4+/CD8+, CD4SP; CD45+/CD4+/CD8-, CD8SP; CD45+/CD4-/CD8+, CD3/TCRαβ; CD45+/CD3hi/TCRαβ+. (f) T cell receptor excision circles (TREC) analysis of thymocytes by PCR. Frequency of TRECs in blood T cells from healthy donors (45–60 years) was set to 1 to calculate the relative frequency of TRECs in CB T cells and thymocytes from HSC-NRG mice. TREC value from human CB and PB were used as controls. Analysis of variance with post hoc test was used for statistical analyses; P < 0.05 considered significant. (g) Upper panels show representative gating strategy for detection of CD31+ thymic CD4+ and CD8+ naive T cell. Lower panel shows absolute cell counts in spleen of CD31+ thymic and CD31 central CD4+ and CD8+ naive T cells. (h) Frequency of lymphocytes in blood 16 weeks after HSCT; CD45+, CD3+ in CD45+, CD4+ in CD45+, CD8+ in CD45+, and CD19+ in CD45+. (i) Absolute counts of human lymphocytes in spleen 16 weeks after HSCT; CD45+, CD3+ in CD45+, CD4+ in CD45+, CD8+ in CD45+, and CD19+ in CD45. (j) Analyses of CD4+ subsets and (k) analyses of CD8+ subsets in blood on weeks 10, 12, and 16 after HSCT determined as frequencies of naive (N), CM, and EM T cells.
Figure 7
Figure 7
Functional effects of SmyleDCpp65 after CB-HSCT. All box plots indicate median and error bars indicate ranges of analyses performed 16 weeks after hematopoietic stem cell transplantation. (a) Absolute counts of CD4+ and CD8+ T cell subsets naive, CM, and EMT cells in spleen. Analysis of variance with post hoc test was used for statistical analyses; P < 0.05 considered significant. (b) T cell expansion in microculture. T cells explanted from lymph nodes (n = 4) were homeostatically activated with CD2/CD3/CD28 beads for 48 hours prior to ex vivo stimulation with SmyleDCpp65 for 7 days. Fold expansion was determined by number of expanded T cells on day 7 relative to cell input. (c) T cell responses against pp65 measured with interferon (IFN)-γ intracellular staining. Harvested T cells were either not stimulated with peptides (baseline control) or restimulated overnight in vitro in 96-wells plate with a pp65 overlapping peptide pool and a nonrelevant tyrosinase related protein (TRP)-2 peptide pool prior to IFN-γ ICS. Fold production was determined by % of IFN-γ+ T cells detected in peptide pulsed group/% of IFN-γ+ T cells from nonstimulated group. (d) ELISPOT assay (n = 2). 50,000 T cells were restimulated overnight with either human cytomegalovirus (HCMV)-pp65 overlapping peptide pool or TRP2 peptide pool on anti-IFN-γ-coated ELISPOT plate. Bars represent average numbers of IFN-γ-positive spots. (e) Human immunoglobulins (ng/ml); IgM, IgA, IgG1, IgG2, IgG3, and IgG4 detectable by bead arrays assay in plasma of NRG mice. (f) Anti-pp65 reactivity of human IgM and IgG in plasma of mice was measured by ELISA. Serum from HCMV seropositive donors were used as positive controls (n = 5).
Figure 8
Figure 8
Analyses of T cell polyclonality by sequencing of genes encoding for the T cell receptor (TCR)-α and -β chains. (a,b) Individual plots represent number of sequences V genes, J genes, CDR3-nt clonotypes, and CDR3-aa clonotypes obtained from splenocytes of humanized mice (controls, n = 2; immunized with SmyleDC/pp65 generated with two vectors, n = 9 or with SmyleDCpp65 generated with the tricistronic vector, n = 2) in comparison with the PBMNC of stem cell donors (n = 2). (c,d) Upper panels: Graphic representation and sequence of the ten most frequent retrieved CDR3 sequences for TCR-α and -β chains. From red to violet the first and the tenth most predominant sequence are represented, grey indicates the remaining sequences. Larger grey areas represent higher polyclonality of a sample. Lower panel: detectable shared CDR3-aa sequences among different humanized mice that were immunized with SmyleDC/pp65 or SmyleDCpp65. aa, amino acid; CDR3, complementarity determining region-3; J, joining; nt, nucleotide; V, variable.

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