Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity

Abstract

Macrophage-derived chemokine (MDC) is a potent chemoattractant for antigen-specific T lymphocytes. We hypothesized that Adenovirus- (Ad-) transduced dendritic cells (DCs) overexpressing MDC would enhance the T cell-mediated humoral immune response specific for antigens presented by the DC. We challenged two strains of mice with lethal Pseudomonas aeruginosa infection 3 weeks after immunization with AdMDC-modified DCs pulsed with heat-killed P. aeruginosa. MDC-expressing DCs specifically attracted T lymphocytes and preserved typical DC surface phenotypes without growth factors in vitro. Mice immunized with AdMDC/Pseudomonas/DCs developed high levels of serum anti-Pseudomonas Ab's and were protected from a lethal respiratory challenge with Pseudomonas. The in vivo protective immunity required CD4(+) T cells, B cells, and IL-4, but not CD8(+) T cells and IL-12. AdMDC/DCs pulsed with Pseudomonas yielded significant but not absolute cross-protection against different strains of P. aeruginosa. Pseudomonas-pulsed AdMDC/DCs protected mice from Pseudomonas but not Escherichia coli and vice versa; this microbe-specific protection correlated with microbe-specific induction of CD4(+) T cell proliferation and IL-4 secretion. Based on these observations, AdMDC-modified DCs pulsed with a killed bacteria may be a useful approach to vaccination against infectious disorders.

Figure 1

RT-PCR analysis for exogenous and endogenous MDC mRNA expression in genetically modified DC. Total cellular RNA (1 μg) from DCs transduced with AdMDC, AdNull, or PBS alone (naive control) were reverse transcribed using the oligo(dT)₁₈ primer. Serial 1:10 dilution of RT-generated cDNA were used as a template to amplify exogenous MDC, endogenous MDC, and GAPDH cDNA fragments by PCR. Samples were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide.

Figure 2

Chemotaxis of human and murine T lymphocytes to conditioned media of genetically modified DCs. (a) CEM cells. Human T lymphocyte CEM cells placed in the upper chamber of a transwell chamber were assayed for chemotaxis in response to serial dilution of supernatant from DCs transduced with AdMDC, AdNull, or PBS alone (naive control) in the lower chamber. (b) EL4 cells. The study was similar to that in a, but the murine T lymphocyte EL4 cell line was used. (c) Suppression of AdMDC-mediated chemotaxis. CEM and EL4 cells were assayed for chemotaxis in response to 1:2 dilution of supernatant from AdMDC-modified DCs. Where indicated, anti–MDC neutralizing Ab or mouse IgG control Ab was added to the supernatant in the lower chambers at 10 μg/ml at the initiation of assay or cells were placed to the upper chamber in the presence of recombinant (r) MDC or TARC at 100 ng/ml. For all parts, the number of cells migrating to the lower chamber at 37°C for 4 hours was counted by FACS analysis. Migration index was calculated as the number of cells migrating to the conditioned media over the number of cells migrating to control medium. Results represent the mean ± SE (n = 3 per data point).

Figure 3

Chemoattracting activity of AdMDC-modified DCs for CCR4-expressing cells in vivo. To demonstrate the in vivo function of AdMDC-modified DCs, C57BL/6 mice were intravenously injected with AdMDC- or AdNull-modified DCs. Genetically modified DCs were labeled with PKH26 (red fluorescence) before the injection for in vivo cell tracking. After 4 days, spleens were dissected, and frozen spleen sections were stained using rabbit anti-mouse CCR4 Ab followed by visualization with anti-rabbit IgG Ab labeled with FITC (green fluorescence). Nuclei were stained with DAPI (blue fluorescence).

Figure 4

Surface phenotype of DCs genetically modified with MDC differentiated under growth factor–withdrawal conditions. DCs generated from bone marrow of C57BL/6 mice with GM-CSF and IL-4 were transduced with AdMDC or AdNull at MOI of 100 for 4 hours. Transduced cells were then washed and cultured in complete RPMI-1640 media without GM-CSF and IL-4 for 5 days. Cells before and after a withdrawal of cytokines were analyzed by two-color flow cytometry for the expression of (a) I-A^b (MHC class II) and CD40 or (b) CD80 (B7-1) and CD86 (B7-2). The percentage of cells in each quadrant is listed.

Figure 5

Immunization with syngenic AdMDC-modified DCs pulsed with P. aeruginosa protects mice against lethal P. aeruginosa respiratory challenge. Mice were immunized intravenously with AdMDC-modified DCs pulsed with heat-killed P. aeruginosa strain PAO1 (DC/MDC + PA), AdNull-modified DCs pulsed with heat-killed P. aeruginosa strain PAO1 (DC/AdNull + PA), or AdMDC-modified DCs alone (DC/MDC) at 10⁵ DCs per mouse. Controls included naive mice without any immunization (no immunization). After 3 weeks, 2 × 10⁵ CFU P. aeruginosa (PAO1) enmeshed in agar beads was administered intratracheally. Survival was recorded as the percentage of surviving animals (n = 10 mice per group). (a) C57BL/6 mice. (b) BALB/c mice.

Figure 6

In vivo Pseudomonas-specific Ab responses of mice immunized with AdMDC-modified DCs pulsed with Pseudomonas. (a–f) Titer of anti-Pseudomonas Ab in serum. C57BL/6 mice were immunized with heat-killed P. aeruginosa-pulsed (PAO1 strain) and AdMDC-modified (or AdNull-modified) DCs. Two weeks after immunization, each isotype of anti-Pseudomonas (PAO1 strain) Ab was assessed in serum using a standard ELISA protocol. Data are presented as mean ± SE (n = 3 mice per data point) of end-point titers. (a) IgM, (b) IgG1, (c) IgG2a, (d) IgG2b, (e) IgG3, (f) IgA. (g) Passive transfer of serum. Three weeks after immunization, serum was collected and pooled from C57BL/6 mice immunized as in a–f. Each recipient C57BL/6 mouse received 100 μl of heat-inactivated (at 56°C for 30 minutes) serum intravenously. After 6 hours, recipient animals were challenged by intratracheal injection with 2 × 10⁵ CFU P. aeruginosa strain PAO1 enmeshed in agar beads (day 0). Controls included mice without any infusion of serum. Survival was recorded as the percentage of surviving animals (n = 10 mice per group).

Figure 7

Immunization with AdMDC-modified DCs pulsed with P. aeruginosa protects mice against lethal P. aeruginosa infection by Th2-dominant immunity. The experiments were carried out in a fashion identical to that in Figures 5 and 6 unless otherwise noted. All respiratory challenges were with P. aeruginosa PAO1 3 weeks after the immunization (c–e) or 1 week after the transfer of splenocytes (a and b). Survival was recorded as the percentage of surviving animals (n = 10 mice per group). (a) Adoptive transfer of spleen cells. Splenocytes from C57BL/6 mice immunized as described in Figure 5 were collected 2 weeks after immunization and transferred intravenously to nonimmunized recipients. (b) CD4⁺ or CD8⁺ splenocyte transfer. Two weeks after the immunization, splenocytes were collected from C57BL/6 mice immunized with AdMDC-modified DCs pulsed with P. aeruginosa. After separation into CD4⁺ or CD8⁺ cells using magnetic beads, each cell fraction (5 × 10⁶ CD4⁺ or CD8⁺ cells) or 5 × 10⁷ total splenocytes were transferred intravenously to nonimmunized recipients. (c) Role of CD4⁺ T cells, CD8⁺ T cells, and B cells. CD4⁺ T cell–deficient, CD8⁺ T cell–deficient, B cell–deficient, or wild-type C57BL/6 mice were immunized with AdMDC-modified DCs pulsed with Pseudomonas. (d) MHC class II presentation of DCs. Wild-type C57BL/6 mice were immunized with AdMDC-modified DCs pulsed with Pseudomonas using DCs prepared from MHC class I–deficient, class II–deficient, or wild-type C57BL/6 mice. (e) Th2-dominant response of immunization. IL-4-deficient, IL-12-deficient, or wild-type C57BL/6 mice were immunized with AdMDC-modified DCs pulsed with Pseudomonas.

Figure 8

Immunization with AdMDC/DCs induces protective immunity specific to the pathogen used for pulsing DCs. (a) P. aeruginosa challenge. C57BL/6 mice were immunized with AdMDC-modified DCs pulsed with heat-killed P. aeruginosa or E. coli (25922 strain). After 3 weeks, immunized animals were infected intratracheally with 2 × 10⁵ CFU P. aeruginosa PAO1 enmeshed in agar beads. (b) E. coli challenge. Immunization of mice was identical to that in a, but 10⁸ CFU E. coli (25922 strain) were used for the intratracheal infection. For both parts, controls included mice without any immunization. Survival was recorded as the percentage of surviving animals (n = 10 mice per group).

Figure 9

Cross-protective immunity of AdMDC-modified DCs against laboratory or clinical strains of P. aeruginosa. (a and b) Immunization resulting from AdMDC-modified DCs pulsed with laboratory strains of Pseudomonas. C57BL/6 mice were immunized with AdMDC-modified DCs pulsed with heat-killed P. aeruginosa laboratory strains PAO1, PAO-NP, PAK, or PA103 at 10⁵ DCs per mouse. Three weeks after immunization, mice were challenged intratracheally with 2 × 10⁵ CFU P. aeruginosa laboratory PAO1 strain (a) or clinical PA514 strain (b) enmeshed in agar beads. (c and d) Immunization resulting from AdMDC-modified DCs pulsed with clinical strains of Pseudomonas. C57BL/6 mice were immunized with AdMDC-modified DCs pulsed with heat-killed P. aeruginosa clinical strains PA514, PA515, PA516, or PA517 at 10⁵ DCs per mouse. Three weeks after immunization, mice were challenged intratracheally with 2 × 10⁵ CFU P. aeruginosa laboratory PAO1 strain (d) or clinical PA514 strain (c) enmeshed in agar beads. In all parts, controls included naive mice without any immunization. Survival was recorded as the percentage of surviving animals (n = 10 mice per group).

Figure 10

Responses of CD4⁺ T cells from immunized mice in a microbe-specific manner. (a–d) Proliferation. C57BL/6 mice were immunized with AdMDC-modified DCs pulsed with P. aeruginosa PAO1 strain or E. coli. Three weeks after immunization, splenic CD4⁺ T cells were isolated from immunized or nonimmunized mice using the magnetic beads. In 96-well culture plates, 5 × 10⁵ CD4⁺ T cells were stimulated with 5 × 10⁴ DCs that had been pulsed with heat-killed P. aeruginosa strain PAO1 (a), heat-killed P. aeruginosa strain PA103 (b), heat-killed E. coli (c), or without any microbes (d), and irradiated. In (a–d), controls included DC culture without CD4⁺ T cells. The number of viable cells was determined using the MTS assay, as described in Methods. The data are presented as the mean percentage increase over baseline of duplicate wells. (e) IL-4 secretion. The culture medium was collected 4 days after the initiation of the coculture described above, and the levels of murine IL-4 were assayed by ELISA. The data are presented as means ± SE (n = 3 per data point).