Immune Netw.  2011 Jun;11(3):182-189. 10.4110/in.2011.11.3.182.

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

• Park JS
• Park SY
• Cho HI
• Sohn HJ
• Kim TG

• KMID: 2150706
• DOI: http://doi.org/10.4110/in.2011.11.3.182

Abstract

BACKGROUND
Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells.
METHODS
To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-gamma ELISPOT assay, cytotoxicity assay and tetramer staining.
RESULTS
DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen.
CONCLUSION
Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

Keyword

HCMV-pp65; HIV-1 Tat; Dendritic cells; Cytotoxic T lymphocyte

MeSH Terms

B-Lymphocytes
Cytomegalovirus
Dendritic Cells
Enzyme-Linked Immunospot Assay
HIV
Humans
Immunotherapy
T-Lymphocytes
T-Lymphocytes, Cytotoxic
tat Gene Products, Human Immunodeficiency Virus
Vaccines
Vaccines
tat Gene Products, Human Immunodeficiency Virus

Figure

• 

  Figure 1 Characterization of recombinant TAT/pp65 fusion protein. (A) Schematic structure of the produced fusion proteins. Proteins used in this study were pp65, pp65 fused with Tat, N-terminal pp65 (pp65N), C-terminal pp65 (pp65C), N-terminal pp65 with Tat (Tat/pp65N) and C-terminal pp65 with Tat (Tat/pp65C). (B) Production of fusion proteins. The protein concentration was estimated by the Bradford procedure and amount of produced proteins was compared. (C) Confirmation of the purified fusion proteins. The size of purified proteins was examined using Commassie blue staining after SDS-PAGE; pp65 N (lane 1), Tat/pp65 N (lane 2), and pp65 C (lane 3), Tat/pp65 C (lane 4), pp65 N (lane 5), and Tat/pp65 N (lane 6).

• 

  Figure 2 Transduction of protein fused with Tat. (A) Transduction efficacy of protein fused with Tat. DCs were incubated with GFP, or Tat/GFP for 2 hr, following maturation for 1 day. Then, GFP expression was observed using FACS analysis and was compared between DCs (black line) and DCs with GFP or Tat/GFP (filled grey) (B) Effect on cell survival after transduction of Tat/pp65 N&C protein. DCs were incubated with pp65 N&C or Tat/pp65 N&C protein for 2 h and then stained with annexin 5-PE and 7-AAD. To induce apoptosis, mock DCs were cultured with 1µg/ml of actinomycin D (AcD).

• 

  Figure 3 Enhancement of T cell stimulation by DCs pulsed with Tat/pp65. (A) Stimulation of IFN-γ producing cells by various concentrations of the recombinant pp65 N&C or Tat/pp65 N&C protein. Immature DCs were treated with 1, 0.1, 0.01µM of each protein for 2 h and then incubated for 1 day in the presence of TNF-α, PGE2 and LPS for maturation. DCs were directly added to 1×104 PBMC/well of 96 well plates and frequency of IFN-γ producing cells was measured by the ELISPOT assay. (B) CD4+ or CD8+ T cells responses by Tat/pp65N&C protein. CD4+ or CD8+ T cells were separated from PBMC with MACS microbeads and stimulated with DCs pulsed with pp65N&C or Tat/pp65N&C. Frequency of IFN-γ producing cells was measured by the ELISPOT assay.

• 

  Figure 4 In vitro generation of pp65-specific CTL by DCs pulsed with Tat-pp65 N&C. (A) IFN-γ production by pp65-specific CTL. After PBL was stimulated with DCs pulsed with pp65 N&C or Tat/pp65 N&C protein three times, the generated CTL was simulated with LCL pulsed with pp65 N&C or Tat/pp65 N&C protein. Then IFN-γ producing cells were analyzed using ELISPOT assay. (B) The cytotoxic activity of the generated CTL. The generated CTL were incubated with LCL pulsed with pp65 N&C (dotted lines) and Tat/pp65 N&C (solid lines) labeling with 51Cr as target. The amount of released-51Cr was measured by γ-counter. (C) pp65-specific epitopes. HCMVpp65-specific T cells were generated by stimulating PBMC with 0.1µM pp65 N&C and Tat/pp65 N&C-pulsed DCs 1 week after the second stimulation (14 days). The pp65-specific T cells were stained with pp65 HLA-A2402-specific tetramer and anti-CD8 antibody and analyzed by FACS. The percentage in each FACS profile represents tetramer-positive and CD8+ T cells.

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