点击显示 收起
1Graduate School of Public Health and 2School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; 3Immunex Corporation, Seattle, Washington; 4University of Oklahoma Health Sciences Center, Oklahoma City
Received 2 August 2002; revised 3 October 2002; electronically published 19 December 2002.
Proteins may serve as ideal CD8+ T cell immunogens for human immunodeficiency virus type 1 (HIV-1) if they can be delivered to and processed through the human leukocyte antigen class I pathway. This study shows that human blood monocytederived dendritic cells loaded with liposome-complexed HIV-1 proteins and matured with CD40 ligand can prime CD8+ T cells to HIV-1 in vitro. Whole HIV-1 protein in liposome may be an effective immunogen for HIV-1 vaccine protocols.
Presented in part: Dendritic Cells at the Host-Pathogen Interface Conference, sponsored by the National Institute of Allergy and Infectious Diseases, Warrenton, Virginia, 7 May 2002.
Informed consent was obtained from the study subjects, and this study was done in accordance with experimentation guidelines of the US Department of Health and Human Services and the Institutional Review Board of the University of Pittsburgh.
Potential conflict of interest: E.K.T. is an employee of Immunex Corporation, who supplied the CD40 ligand for this study.
Financial support: National Institutes of Health (grants R01 AI41870, U01 AI37984, U01 AI35041, R01 CA24553, and T32 AI 07487).
Present affiliation: Department of Immunology, Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut.
Reprints or correspondence: Dr. Charles R. Rinaldo, Jr., A427 Crabtree Hall, Graduate School of Public Health, 130 DeSoto St., University of Pittsburgh, Pittsburgh, PA 15261 ().
Generation of antigen-specific, primary CD8+ cytotoxic T lymphocyte (CTL) responses represents a challenge to the development of protein-based vaccines for prevention of human immunodeficiency virus type 1 (HIV-1) infection. The conventional mechanism for antigenic processing of extracellular proteins is through an endocytic pathway for presentation by major histocompatibility complex (MHC) class II molecules to CD4+ T cells [1]. One potential strategy to induce vigorous MHC class Irestricted CTL responses with viral proteins involves specific targeting to the cytosol of antigen-presenting cells (APCs), particularly dendritic cells (DCs), with carriers such as liposome for accessing alternative MHC class I antigen-processing pathways (termed "cross-presentation") [2]. We have previously demonstrated that lipofectin, a cationic liposome, provides a means of delivering exogenous proteins in vitro to human DCs for more efficient induction of HIV-1 specific, memory CD8+ CTL responses than protein alone [3]. The present study investigated whether DCs loaded with protein-liposome complexes can prime antiHIV-1 CD8+ T cell responses.
Materials and methods. To obtain immature DCs (iDCs), CD14+ monocytes were positive selected from peripheral blood mononuclear cells (PBMC) of healthy donors using anti-CD14 monoclonal antibody (MAb)coated magnetic microbeads (Miltenyi) and cultured in AIM V medium (GIBCO) containing 1000 U/mL of recombinant interleukin (IL)4 and 1000 U/mL recombinant granulocyte-monocyte colony stimulating factor (GM-CSF; Schering-Plough) [4]. The number of viable DCs was determined by typical DC morphology in trypan blue dyestained preparations. The purity of the DCs was 90% on the basis of the expression of HLA-DR molecules and lack of expression of lineage markers by flow cytometry [4]. The maturation state of the DCs was determined by expression of CD80, CD83, and CD86.
The toxicity of lipofectin, composed of DOTMA (N-[1-{2,3-dioleyloxy} propyl]N, N, N-trimethylammonium chloride)/DOPE (dioleoyl-phosphotidylethanolamine) (GIBCO), was determined by loading iDCs with graded doses at 37°C for 0, 2, 4, and 8 h. The viability of the DCs by trypan blue dye exclusion was >90% when lipofectin was added at 15 g/mL for up to 8 h, and decreased at higher concentrations of liposome. Therefore, 15 g/mL lipofectin was used in this study.
The vaccinia virus (VV) constructs contained gag p24 or p55 for BH10 and HxB2 strains of HIV-1 (VV-Gag p24 and VV-Gag p55; Therion Biologics). VV-Env was vPE11 containing the env coding region of HIV-1 BH10 minus the signal sequence (B. Moss, National Institutes of Health , Bethesda, MD). The NYCBH strain of VV (VV-Vac; Therion) was used as control virus. Synthetic peptides representing previously identified HLA class Irestricted Gag and Env epitopes were prepared by the University of Pittsburgh Peptide Synthesis Facility.
For preparation of APCs, 15 g/mL lipofectin was mixed with 20 g/mL HIV-1 p24 Gag or gp160 Env (IIIB; Protein Sciences) or p55 Gag (SF2; Chiron; AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH) and incubated for 10 min at room temperature [3]. The lipofectin-protein mixture was added to 1 × 106 iDCs on day 7. The cells were incubated for 4 h at 37°C in 5% CO2 and treated with human CD40L trimer (12.5 g/mL; Immunex) for 2 days to induce phenotypic and functional maturation (defined as mature DCs ). Control samples were APCs only and APCs loaded with either liposome or HIV-1 protein. In some experiments, the iDCs were obtained from plastic adherent PBMC and matured with monocyte-conditioned medium (MCM) [4].
After incubation, the cells were washed and left untreated, or treated with psoralen (10 g/mL; Calbiochem) and UV light (PUV) [5]. Cryopreserved PBMC were primed with antigen-loaded, autologous mDCs for 1 week and then stimulated with antigen-loaded, autologous PBMC as APCs for weeks 25 at an effector cell-to-APC ratio of 10 : 1 in RPMI 1640 medium containing antibiotics, 15% heat inactivated fetal calf serum, and recombinant human interleukin (IL)2 (100 U/mL; Chiron). The effector cell-APC mixtures were seeded into round-bottom microtiter wells, with fresh medium and IL-2 added every 34 days.
Antigen stimulated cells were harvested and washed after 4 or 5 weeks when there were adequate T cells for CTL assay. In some experiments, CD8+ and CD4+ T cells were enriched to 98% and 90% purity, respectively, from cultured cells by negative selection using immunomagnetic beads (Dynal) [5]. The targets were autologous B-LCLs infected with the VV vectors or loaded with synthetic HIV-1 peptides (100 g/mL) and labeled with 51Cr [4]. PBMC were typed for HLA class I by high resolution, reference strand mediated conformation analysis. To block CTL reactivity, target cells were incubated with anti-HLA class I (5 g/mL; Accurate Chemical) or anti-HLA class II MAbs (5 g/mL; Beckman Coulter) for 45 min at 37°C before adding to the effector cells. The CTL lysis assay was done in triplicate at effector/target (E/T) cell ratios of 40, 20, and 10. Specific lysis was calculated as 100 × [(experimental copies per minute {cpm} - spontaneous cpm)/(maximum cpm - spontaneous cpm)]. The percentage of antigen-specific lysis was calculated as (percentage lysis of VV-Gag or VV-Env infected or HIV-1 peptide loaded targets) - (percentage lysis of VV-vac infected or mock treated targets). The mean (±SE) lysis of the uninfected and VV-vac infected targets by the antigen-stimulated effectors at the various E/T ratios was 8% ± 3% (n = 18) and 7% ± 1% (n = 27), respectively. The coefficient of variation for cpm among replicate CTL values was 7% ± 1%. A positive CTL response was defined as 10% antigen-specific lysis.
Results. Expression of costimulatory molecules CD80 and CD86, HLA-DR, and the Ig superfamily, DC surface maturation marker, CD83, was up-regulated on CD40L-treated DCs as compared to non-CD40L-treated DCs, indicating that the CD40L treatment induced DC maturation [4, 5]. Loading of iDCs or mDCs with either liposome, p24 Gag, or their combination did not alter expression of these surface molecules (data not shown).
We next established the parameters for in vitro priming by confirming the work of Zarling et al. [6], which used 1 week of priming of PBMC with HIV-1 Gag peptide-loaded, MCM-matured DCs, followed by weekly, sequential stimulations with Gag peptide-loaded PBMC to induce anti-Gag CD8+ CTL responses (data not shown). Priming with peptide-loaded iDCs did not induce CTL reactivity. In subsequent experiments, we used CD40L-matured DCs, because this treatment resulted in a more consistent maturation of the DCs [4].
Stimulation of PBMC from an HIV-1negative subject with autologous mDCs that had been loaded with HIV-1 Gag p55 protein-liposome induced greater levels of antiHIV-1 CD8+ T cell responses than mDCs loaded with HIV-1 Gag p55 protein alone (). Similar priming was elicited by PUV-treated mDCs that had been loaded with Gag p55-liposome (). The lytic reactivity was HLA class I restricted as shown by blocking of lysis by antiHLA class I but not antiHLA class II MAbs (). To date, we have observed that CTLs specific for Gag or Env can be consistently primed by autologous mDCs loaded with these HIV-1 recombinant protein-liposome complexes in multiple PBMC samples obtained from 7 different subjects (data not shown).
fig.ommitted
Figure 1. Priming of human immunodeficiency virus type 1 (HIV-1)specific CD8+ cytotoxic T lymphocyte (CTL) responses by different antigen-presenting cells (APCs). Priming of HIV-1specific CTL in peripheral blood mononuclear cells (PBMC) from a HLA A*0201, B*27 subject by 1 week of stimulation with autologous mature dendritic cells (mDCs; either untreated or psoralen UV treated), monocytes, or PBMC loaded with Gag p55-liposome, followed by 4 sequential weekly stimulations with autologous PBMC loaded with the same antigen (A). Results are the mean of triplicate values of antigen-specific lysis. The results show positive CTL responses (10% specific lysis) to autologous B cell lines loaded with known CD8+ T cell epitopes for HLA A*0201 p24151159 (TLNAWVKVV), and B*27 p24263272 (KRWIILGLNK) and p171927 (IRLRPGGKK), but not for HLA A*0201 p177785 (SLYNTVATL). The CTL activity was blocked by anti-HLA class I monoclonal antibody (MAb) but not anti-HLA class II MAb (B). E/T, effector/target cell ratio.
The HLA class I specificity and breadth of this primary CD8+ T cell response was further demonstrated by killing of autologous targets expressing 3 of 4 HLA A*0201 and HLA B*27 HIV-1 peptides (). We confirmed the specificity of CD8+ T cells primed by mDC loaded with either Gag p24 or p55 and liposome for p24151-159 (TLNAWVKVV) in 5 of 6 HLA A*0201 subjects. In further experiments, priming of CTLs from another HLA A*0201 person with mDCs loaded with gp160-liposome induced CTLs specific for HLA A*0201 epitopes gp120311320 (RGPGRAFVTI) and gp41747755 (RLVNGSLAL), but not gp120192199 (KLTSCNTSN) or gp41818-827 (SLLNATDIAV) (data not shown). Similarly, CTLs from an HLA A*03 B*07 person that were primed by mDCs loaded with Gag p24- and Env gp160-liposome complexes were specific for HLA B*07 p24179187 (ATPQDLNTM) and HLA A*03 gp41775785 (RLRDLLLIVTR) epitopes, but not for the HLA B*07 p24148156 (SPRTNAWV) epitope (data not shown).
We showed that Gag p55-liposome loaded mDCs more efficiently primed HIV-1specific CD8+ CTLs than did monocytes or PBMC for recognition of targets expressing 4 different Gag epitopes (). Furthermore, mDCs loaded with Gag p55-liposome were superior to iDCs for priming of CTL specific for targets expressing naturally processed Gag proteins (i.e., VV-p24 and VV-p55), as well as targets loaded with a known HLA A*0201 p24 epitope (p24151-159) ().
fig.ommitted
Figure 2. Priming of human immunodeficiency virus (HIV)-1specific cytotoxic T lymphocyte (CTL) responses by immature dendritic cells (iDCs) and mature (m) DCs loaded with HIV-1 protein-liposome (lipo) complexes. Peripheral blood mononuclear cells (PBMC) from an HLA A*0201 subject were primed for HIV-1specific CTL by 1 week of stimulation with autologous iDCs and mDCs loaded with Gag p24-lipo complexes, followed by 4 sequential weekly stimulations with autologous PBMC loaded with the same antigen. Targets were autologous B cell ines infected with vaccinia virus expressing Gag p55 or p24, or loaded with an HIV-1 peptide (p24151159 ) representing a known CTL epitope for HLA A*0201. Results are the mean of triplicate values of antigen-specific lysis. E/T, effector/target cell ratio.
Discussion. We were consistently able to prime antiHIV-1 CD8+ T cell responses with mDCs loaded with HIV-1 protein-liposome complexes but not with mDCs loaded with HIV-1 proteins alone. In vitro priming of CD8+ T cells to HIV-1 was first demonstrated by Engleman et al. [7, 8] who showed that multiple, sequential stimulations of naive CD8+ T cells with autologous or allogeneic, HLA class Imatched DCs freshly isolated from blood samples and loaded with HIV-1 Gag and Env peptides, could activate HIV-1specific CTL. More recently, monocyte-derived iDCs transduced with an env- and nef-deleted HIV-1 vector pseudotyped by VSV G protein [9], HIV gag-encoded mRNA-transfected DCs [10], or iDCs that were matured with MCM or CD40L and loaded with HIV-1 peptides representing HLA class I epitopes [6, 11], have been used to prime antiHIV-1 CTL in vitro. There is only one report of in vitro priming of CD8+ T cells with whole HIV-1 protein, where Nef-specific CD8+ T cells were primed by repetitive stimulation of PBMC with -irradiated iDCs loaded with HIV-1 Nef protein [12]. In this case, optimal T cell priming required addition of interferon (IFN).
We found that priming of antiHIV-1 CD8+ T cell responses in vitro required liposome as a delivery vehicle. We believe that this was due, in part, to the unique fusogenic property of the DOPE portion of lipofectin that enhances access of exogenous protein from the endosomes/lysosomes into the cytosol of DCs by destabilizing the endosomes and allowing the release of HIV-1 protein into the cytosol [13]. Moreover, a single stimulation of PBMC for one week with HIV-1 protein-liposome loaded mDCs, followed by multiple, weekly stimulations with protein-liposome loaded PBMC, was sufficient to induce a primary, antiHIV-1 CD8+ T cell response. Initial stimulation with antigen-loaded mDCs was essential for priming, as iDCs, monocytes or PBMC loaded with liposome-complexed HIV-1 protein were poor primers of HIV-1 specific T cells. The mDCs expressed enhanced levels of costimulatory and HLA class II molecules, and these were not altered by addition of liposome-protein complexes. Maturation of DCs with CD40L increases surface expression of HLA and costimulatory molecules and release of cytokines including IL-12 p70 (when combined with IFN- stimulation) and IL-15 from mDCs that enhance antigen-specific CD8+ T cell responses [14]. Such costimulatory CD40/CD40L interactions lower the threshold of antigen required to activate primary T cell responses, and may augment survival of antigen-activated T cells.
Our data, that PUV-treated mDCs could prime antiHIV-1 CD8+ T cell responses, are concordant with our previous findings among HIV-1infected persons [4]. The PUV-treated DCs are likely undergoing apoptosis, which suggest that viable DCs are not essential for triggering of T cell priming. This may relate to the high levels of costimulatory molecule expression that is retained on PUV-treated DCs (X.-L.H., Z.F., and C.R.R., unpublished data).
Priming of CD8+ T cells with mDCs loaded with HIV-1 proteins and liposome elicited CTL reactivity against multiple HIV-1 HLA class I epitopes. The levels of lysis were within the range expected for CTL primed by a multi-epitope protein immunogen, using single epitope, individual peptides as targets. Lack of recognition of some HIV-1 peptides by the HIV-1 protein-primed T cells could reflect immunodominance among the peptides for their HLA class I alleles, differences in proteasomal cleavage and heterogeneity of peptide binding due to polymorphism of HLA class I alleles [2].
We reported elsewhere activation of HIV-1 specific, CD8+ T cells from HIV-1infected persons in vitro by cross-presentation of liposome-complexed HIV-1 proteins by DCs [3]. These studies suggest that complexing of HIV-1 protein with liposome promotes processing of the protein through an alternative, cytosolic pathway for cross-presentation of peptides by HLA class I molecules to both naive and memory CD8+ T cells. Our studies, together with a recent report showing in vitro priming of Gag-specific CD4+ T cells by mDCs loaded with uncomplexed HIV-1 p24 protein [15], suggest that a protein-based antigen approach could be used to prime or boost both antiHIV-1 CD8+ and CD4+ T cell activity in prophylactic and therapeutic vaccine protocols.
Acknowledgments
We thank K. Picha (Immunex; Seattle, WA) for assistance in providing the CD40L, S. Narula (Schering-Plough; Kenilworth, NJ) for providing hIL-4 and hGM-CSF, and J. Malenka for administrative assistance.
References
1.
Watts C, Amigorena S. Antigen traffic pathways in dendritic cells. Traffic 2000; 1:3127.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
van Kooten C, Banchereau J. CD40-CD40 ligand. J Leuko Biol 2000; 67:217.
15.