Introduction

Cytotoxic T lymphocytes (CTL) are important effectors of anti-tumor immunity^1,2 and can be best induced by the potent antigen-presenting cells, dendritic cells (DC)^3,4. Methods to engineer presentation of the target antigens by DC for use as a tumor vaccine include pulsing DC with peptides corresponding to known immunogenic epitopes^5,6, loading DC with purified target protein⁴, and introducing the gene encoding the antigen into DC by transduction with viral vectors⁷ or direct transfection with nucleotides⁸. In settings in which immunogenic epitopes have been identified, peptide-based immunization strategies represent an attractive modality. Most clinical trials have employed this approach, resulting in epitope-specific T cell responses and occasional regression of metastatic nodules^5,6. However, clinical responses are generally infrequent and, when they occur, often fail to correlate with the magnitude of the measured T cell response⁹. In part, this may reflect the preferential induction following peptide stimulation of low-avidity T cells that fail to recognize the more limited epitope density on tumor cells resulting from processing of endogenously expressed antigen^10,11. The effectiveness of peptide-based strategies may also be limited by rapid peptide dissociation from Class I molecules or turnover of surface Class I molecules, resulting in an abbreviated period of antigen presentation¹² in vivo. The alternative use of recombinant proteins to load DC is often hindered by difficulty in obtaining sufficient quantity and purity of recombinant proteins for clinical trials. The use of DNA-based or viral vectors to modify stimulator cells to express the target antigen can be technically challenging and may require the use of promoter sequences or vector constructs that can lead to problems related to vector immunogenicity¹³, oncogenesis due to expression of pro-oncogenic antigens, and the rare possibility of productive insertional mutagenesis¹⁴.

Many of these shortcomings can be addressed through the use of RNA-transfected DC. In vitro transcribed RNA, complexed to a lipid or cationic vehicle or by electroporation¹⁵, can be transfected directly into DC, obviating the requirement for viral vector-mediated transduction¹⁶. Since genomic integration is not a prerequisite for antigen expression following RNA transfection, potential concerns related to expression of an oncogenic protein are avoided. In vitro and in vivo studies have shown that RNA-transfected DC can be used to stimulate antigen-specific CTL¹⁶ and such a strategy may be superior to DNA-transfected DC⁸. However, current studies using RNA transfection are limited by the low efficiency of transfection into mature DC and may result in suboptimal T cell priming.

DC can be procured in two functional states, as immature and mature DC³. Mature DC express high levels of MHC and costimulatory molecules (e.g., CD80, CD83) and have been shown to be potent immune stimulators in vivo¹⁷. By contrast, immature DC express low levels of MHC and costimulatory molecules, provide less potent stimulation, and have the potential to tolerize rather than immunize¹⁸. For these reasons, it is generally believed that antigen-loaded mature DC are better suited to tumor vaccination than immature DC⁴.

Lipid-based reagents to date have resulted in consistently low to undetectable levels of RNA transfection into DC^8,15,19. Recently, RNA electroporation has provided an efficient strategy for transfecting monocytes and immature DC¹⁵. However, in these studies, attempts to obtain mature DC from immature RNA-electroporated populations by subsequent exposure to a maturation cocktail for 48 h yielded a significant proportion of immature CD83-negative DC (ca. 40%) and nonviable cells (15–30%)¹⁵. An added obstacle has also been the demonstration that, for several tumor antigens, the tumor-derived immunodominant epitope is poorly processed by the predominant proteasome expressed in mature dendritic cells²⁰.

In this study, we developed a strategy to transfect RNA into mature DC with high efficiency. Since the generation of an effective anti-tumor response by vaccination is predicated on the capacity to elicit not only large numbers of antigen-specific T cells but also T cells of sufficiently high affinity to recognize endogenous antigen expressed by tumor cells, we targeted the prototypic T-cell-defined tumor-associated antigen, MART-1/Melan A, a self protein expressed in melanoma cells as well as in normal melanocytes⁹. As a consequence of a T cell repertoire that has been shaped by the endogenous normal expression of MART-1 in peripheral tissues, MART-1-specific CTL isolated in vitro often exhibit low-affinity interactions^21,22. We characterize the induction of tumor-associated antigen-specific CTL using RNA-transfected mature DC and compare this modality in rigorous fashion with the current standard vaccination strategy that uses peptide-pulsed DC.

Results

Transfection of Mature DC with in Vitro Synthesized mRNA Can Be Achieved with High Efficiency

We quantitated the efficiency of RNA transfection by FACS analysis of GFP expression using in vitro transcribed GFP RNA. Following a 48-h exposure to maturation cocktail (TNF-, IL-1, IL-6, and PGE₂), we transfected DC by co-incubation of Transmessenger reagent with varying concentrations of in vitro synthesized GFP mRNA (0.25 to 1.0 g). Flow cytometric analysis of GFP expression 3 h after transfection (Fig. 1A) demonstrated an average transfection rate of 47% (range 31–61%) with maximal efficiency obtained with 0.50 g of mRNA. Costaining of DC with PE-conjugated anti-CD83 confirmed that the majority (>90%) of cells expressing GFP were mature (CD83⁺) DC and not the residual small population of immature (CD83^-) DC (Fig. 1B).

Figure 1.

Transfection of mature dendritic cells using GFP mRNA. (A) Mature DC (3 10⁵) were transfected with 0, 0.25, 0.50, or 1.0 g of GFP RNA and evaluated by flow cytometry for expression of GFP. Optimal transfection efficiencies (>50%) were achieved when 0.50 g of RNA was admixed with Transmessenger reagent and co-incubated with mature DC. (B) Greater than 90% of transfected (GFP⁺) DC costain with CD83, confirming direct transfection of mature DC under these conditions.

Full figure and legend (110K)

As the transfection reagents are potentially toxic, we optimized the time of exposure to the reagents for viability and transfection efficiency. Co-incubation with transfection reagent for 45 min yielded transfection efficiencies of up to 61% with a viability of 92%. Longer incubation times, up to 90 min, improved transfection efficiencies by only 5–10% and increased the fraction of nonviable cells to >20%.

RNA-Transfected Mature DC Are Capable of Presenting Conventional Antigen Epitope

Studies using isolated proteasomes demonstrate that, in contrast to standard proteasomes expressed in tumor cells and immature DC, immunoproteasomes that are predominantly expressed in mature DC do not generate the conventional tumor-associated epitopes for a number of tumor antigens, including the MART-1 epitope M27²⁰. However, it is unclear if mature DC may still be capable of generating tumor-derived epitopes through the minor population of standard proteasomes present. To evaluate epitope presentation by mature and immature DC transfected with MART-1 RNA, we used KK M27- and RE M27-specific CTL clones to detect the conventional tumor antigenic epitope of MART-1 (M27) in chromium release assays. We transfected mature DC with MART-1 RNA, sorted on the basis of CD83 expression to obtain a uniform population of mature (CD83⁺) DC. We used immature DC transfected with MART-1 RNA and mature DC transfected with whole cell RNA from the MART-1-negative melanoma cell line, A375, as additional targets and M27 peptide-pulsed mature DC as positive control. We observed specific lysis of both MART-1 RNA-transfected immature and mature DC (30 and 48%, respectively, Fig. 2A), demonstrating, contrary to prediction, that mature DC were capable of presenting the conventional tumor-associated M27 epitope of MART-1. Preservation of the conventional epitope processing was also demonstrated for antigens gp100 and tyrosinase when we transfected those two antigens into mature DC. We detected presentation of HLA-A2 restricted G280 epitope from gp100 and T369 epitope by using CTL clones MB G280 and CT T369 (data not shown). Although there was variation among the antigens, the antigen presentation could be reproducibly detected in the mature DC transfected with the above antigens.

Figure 2.

Presentation of MART-1 M27 epitope in mature DC following RNA transfection and generation of MART-1-specific CTL using RNA-transfected DC. (A) Following RNA transfection, M27-specific CTL clones were used to screen for presentation of the MART-1-derived M27 epitope by immature and mature DC transfected with MART-1 RNA. Unmanipulated mature DC and mature DC pulsed with M27 peptide were used, respectively, as negative and positive controls. Significant specific lysis of MART-1 RNA-transfected DC was observed (47%), demonstrating proper presentation of the tumor-derived M27 peptide epitope by mature DC. (B) CD8-enriched PBMCs were stimulated in parallel cultures using as stimulator cells mature DC transfected with RNA from the MART-1-negative A375 melanoma cell line (DC-375), mature DC pulsed with 20 M M27 peptide (DC-M27 peptide), or mature DC transfected with in vitro synthesized MART-1 RNA (DC-MART-1-RNA). After two cycles of in vitro stimulation, the cytotoxic capacity of each culture was assayed in a ⁵¹Cr-release assay using unpulsed or M27-peptide-pulsed T2 targets at an effector-to-target ratio of 20:1. T cells generated in cultures stimulated with MART-1 RNA-transfected DC and M27-peptide-pulsed DC exhibit significant specific lytic capacity. (C) Quantitation of MART-1-specific CD8⁺ T cells in bulk culture after two cycles of in vitro stimulation was done by staining with the PE-conjugated M27 peptide MHC tetramer. A significantly greater fraction of MART-1-specific CTL is seen in cultures stimulated with mature DC transfected with MART RNA than with mature DC pulsed with M27 peptide (tetramer⁺, CD8⁺ population of 3.43% vs 1.08%, respectively). These results are representative of five separate chromium-release and three separate tetramer-analysis experiments.

Full figure and legend (164K)

In Vitro Stimulation with MART-1 RNA-Transfected Mature DC Results in a Greater Frequency of Antigen-Specific CTL Compared to M27 Peptide-Pulsed Mature DC

For the purposes of comparison, we selected a peptide concentration of 20 M for pulsing mature DC since it is in the range of commonly used peptide concentrations known to be effective in stimulating CTL responses against MART-1^6,11. At this concentration, peptide-pulsed DC are maximally susceptible to lysis by M27-specific CTL, and lower M27 peptide concentrations (2 and 0.2 M) fail to stimulate detectable CTL activity (data not shown). We stimulated CD8⁺ cells isolated from HLA-A2⁺ donors in separate parallel cultures with MART-1 RNA-DC, M27 peptide-DC, or A375 whole cell RNA-DC for two cycles as described under Materials and Methods. Eight days after the second in vitro stimulation, we tested T cell cultures for specific cytotoxicity against M27-peptide-pulsed T2 cells in ⁵¹Cr-release assays. At an effector-to-target ratio of 20:1, T cell cultures generated following MART-1 RNA-DC stimulation demonstrated greater specific lysis compared to T cells generated following M27 peptide-DC stimulation (56 15% vs 39 5%) (Fig. 2B). We quantified the frequency of MART-1 (M27)-peptide-specific T cells in these cultures after two cycles of in vitro stimulation by tetramer analysis and revealed a frequency of MART-1-specific CTL following stimulation with RNA-transfected DC that was greater than three times higher than that of the M27 peptide-DC-generated cultures (3.43 0.9% vs 1.08 0.3%, Fig. 2C).

Duration of Antigen Presentation by MART-1 RNA DC Is Prolonged Compared to That of M27 Peptide-Pulsed DC

We examined the duration of antigen presentation as a possible explanation for increased CTL frequency. At 24-h intervals following RNA transfection or peptide pulsing, we evaluated the susceptibility of DC to lysis by the KK M27 CTL clone, as a measure of antigen presentation, serially in ⁵¹Cr-release assays. For M27-peptide-pulsed DC, we observed maximal lysis (55%) at the first time point (24 h) followed by a steady decline to approximately 10% by 96 h. By contrast, for MART-1 RNA-transfected DC, specific lysis remained essentially unchanged at 25–35% for the 96 h of assessment (Fig. 3). Thus RNA transfection provided for a significantly prolonged period of antigen presentation compared to peptide pulsing.

Figure 3.

Duration of antigen presentation. Mature DC were either transfected with A375 negative control RNA () or Mart-1 RNA () or pulsed with 20 mM M27 peptide () and maintained in AIM-V medium containing 500 U/ml GM-CSF, 500 U/ml IL-4, and 10 ng/ml TNF-. A portion of the cells was labeled with ⁵¹Cr at each time point and used as target for KK M27 CTL clone at the effector-to-target ratio of 20:1. The curves are derived from two experiments. Although increased lysis of M27-peptide-pulsed targets is seen initially (52 vs 32%), sustained susceptibility to lysis is observed only with MART-1 RNA-transfected targets beyond 48 h.

Full figure and legend (77K)

CTL Clones Stimulated with RNA-Transfected Mature DC Represent High-Avidity Tumor-Reactive T Cell Clones

Data from our lab and others have shown that the majority of CTL generated using peptide-pulsed DC display lower T cell receptor avidity and absent tumor reactivity^10,11, possibly as a consequence of deletion of high-avidity CTL when exposed to supraphysiologic densities of the peptide epitope on antigen-presenting cells. We postulated that RNA-transfected DC might provide for a more physiologic display of peptide MHC leading to the induction of T cells with higher T cell receptor affinity.

We stained T cells generated using MART-1 RNA-transfected or M27-peptide-pulsed mature DC (see Materials and Methods) with M27-peptide MHC tetramer and anti-CD8–FITC. Previous studies have demonstrated that, in general, the intensity of tetramer staining correlates with T cell affinity¹¹. In this study, a 1/2 to 1 log₁₀ greater intensity of tetramer staining was associated with the population of T cells generated with RNA-transfected DC compared to peptide-pulsed DC (Fig. 2C and data not shown). In two experiments, the frequency of the CTLs generated with the RNA-transfected DC is 3.43%, higher than that of 1.08% for the peptide-pulsed DC. To characterize further T cell affinity, we sorted tetramer-positive cells from these cultures and cloned them at limiting dilution into 96-well plates. At an E:T of 10:1, T cell clones isolated following MART-1 RNA-DC stimulation generally displayed higher specific lytic activity compared with those isolated from peptide-DC stimulation (48 vs 33%, P < 0.01, Fig. 4A). We further compared the avidity of CTL clones from both groups in a peptide dose titration ⁵¹Cr-release assay. Results are presented as the peptide dose (nM) required to sensitize targets to 50% lysis (P₅₀). Overall, CTL clones generated using MART-1 RNA-DC demonstrated a decreased peptide dose requirement for specific lysis that was about 1 log₁₀ lower than that required for lysis by CTL clones generated using peptide-pulsed DC (P < 0.01, Fig. 4B). This decreased peptide dose requirement is coincident with increased tumor reactivity and confirms that CTL generated using MART-1 RNA-DC exhibit a higher avidity interaction with its cognate target.

Figure 4.

Induction of high-affinity tumor-reactive MART-1-specific CTL following stimulation with MART-1 RNA-transfected mature DC. (A) Eight representative clones each from cultures stimulated with M27-peptide-pulsed DC or MART-1 RNA-transfected DC were analyzed for specific reactivity to MART-1 + tumor (526). Significantly greater lysis of antigen-positive tumor (526) with minimal lysis of antigen-negative tumor (375) is seen with MART-1-specific CTL clones isolated from cultures stimulated with RNA-transfected DC (MART-1 RNA-CTL) compared with those isolated from cultures stimulated with peptide-pulsed DC (M27 peptide-CTL) (48 vs 33% lysis, P < 0.01). (B) Eight MART-1 RNA-CTL and 8 M27 peptide-CTL clones were tested for target affinity in a Cr-release assay using targets pulsed with decreasing concentrations of peptide (peptide dose titration analysis). Results are presented as the peptide dose (nM) required to sensitize targets to 50% lysis (P₅₀). On average, CTL clones generated using MART-1 RNA-DC demonstrated a decreased peptide dose requirement for specific lysis compared to CTL clones generated using peptide-pulsed DC (2.60 nM vs 26.85 nM, respectively, P < 0.01). Results are representative of three separate experiments.

Full figure and legend (138K)

Discussion

In contrast to peptide-based approaches, RNA transfection of dendritic cells represents a method to augment T cell responses against antigens in settings in which immunogenic epitopes have not been defined, facilitating broader application of vaccination strategies for the treatment of patients with cancer. In this study, we demonstrate, for the first time, that mature DC can be efficiently transfected with RNA encoding a tumor antigen to induce tumor-reactive CTL responses in vitro that are greater, in both frequency and tumor avidity, than the responses induced by peptide-pulsed DC.

Preferential use of mature DC over immature DC as antigen-presenting cells for eliciting responses in the setting of low T cell precursor frequency, typical of many T-cell-defined antigens with shared expression in normal tissues, is based on features related to up-regulated expression of costimulatory ligands on mature DC and the capacity to induce rather than tolerize T cell responses¹⁸. In this study we report the strategy of transfecting mature DC directly for use as antigen-presenting cells in contrast to the alternative strategy of introducing RNA into immature DC followed by DC maturation. Since transfected RNA degrades over time, maturation after transfection would result in shortened duration of antigen presentation, leading to inferior T cell stimulation. However, engineering mature DC to express the target antigen of interest through RNA transfection has been limited by the reportedly poor transfection efficiency of RNA into mature DC^16,23 and the demonstration, in at least one study, that immunoproteasomes, which are up-regulated in DC as they mature, fail to process several tumor-associated antigens (e.g., MART-1, gp100) into the conventional epitopes presented by immunoproteasome-poor tumor cells²⁰, thus raising the concern that T cells induced following stimulation by mature DC may not recognize tumor cells due to differential epitope presentation.

In addressing both concerns, we demonstrate that transfection efficiencies up to 60% (mean of 47%) can be achieved under defined conditions, with excellent posttransfection DC viability. Furthermore, CD8⁺ T cells generated using mature DC transfected with MART-1 RNA recognize antigen-positive tumor cells in a specific and robust manner, despite reports that would suggest the contrary.

In reconciling these latter results, we suggest that the proteolytic pattern of in vitro isolated immunoproteasomes and standard proteasomes may not necessarily reflect the functional state of these proteasomes within cells and/or that, despite immunoproteasome up-regulation, CD83⁺ mature DC continue to express sufficient levels of the standard proteasomes to produce the conventional tumor-derived MART-1 (M27) epitope or the mature DC have an alternative pathway of antigen processing. In any event, RNA-transfected mature DC demonstrate the capacity to generate a quantitatively and qualitatively more robust response compared to peptide-pulsed DC, two attributes that may predict favorable outcomes in a vaccine-induced response⁶.

The greater frequency of antigen-specific T cells achieved using RNA-transfected compared to peptide-pulsed DC may be related to the duration of antigen presentation. Transfected RNA provides a prolonged period of continuous internal peptide supply, thus enabling longer lasting antigen presentation than peptide-pulsed DC. Na¨ve CD8⁺ T cells undergo 7 to 10 cell divisions in a preprogrammed fashion upon initial antigen encounter²⁴, but the peak of T cell response achieved (in vivo) is influenced by the duration of antigen persistence^25,26,27. An augmented T cell frequency achieved through prolonged presentation of immunogen may translate into more effective therapy. In a recent study, for example, increased in vivo stability of an exogenously pulsed tumor-derived peptide (by linking the epitope to a cell-penetrating peptide) resulted in enhanced tumor immunoprotection compared to the use of unmodified peptide¹².

In addition to the quantitative difference between the two types of DC, RNA-transfected DC-generated CTLs were more potent in tumor reactivity as observed by the increased levels of tetramer staining intensity, increased specific tumor lytic activity, and lower peptide dose requirement to sensitize killing of target cells. When supraphysiologic concentrations of peptide are used, as with exogenously pulsed antigen-presenting cells, preferential induction of lower avidity T cells occurs both in vitro and in vivo, likely as a consequence of deletion of high-affinity and expansion of lower affinity T cells, in contrast to RNA transfection, which provides more physiologic, endogenous presentation of the target epitope. The method described in this paper provides an efficient means of reproducibly delivering RNA to a uniform population of mature DC with minimal cytotoxicity in a manner that translates into robust antigen presentation. Furthermore, this study provides a rigorous comparison of the efficacy of tumor-associated antigen-specific CTL induction between RNA-transfected DC and peptide-pulsed DC (the current standard) and demonstrates superiority with the use of RNA-transfected DC in not only the frequency of antigen-specific CTL generated, but also the functional avidity at the clonal level. Since one major concern with respect to the use of peptide-pulsed or any other antigen-specific vaccine is the inadvertent induction of low-avidity, non-tumor-reactive CTL, identifying strategies that increase the degree of functional T cell avidity can be critical to the success of tumor vaccines.

Materials and Methods

Cell Lines

Melanoma cell lines A375 (gift from S. Rosenberg) and Mel 526 (gift from M. Lotze) were maintained in RPMI with Hepes (25 mM),L-glutamine (4 mM), penicillin (50 U/ml), streptomycin (50 mg/ml), sodium pyruvate (10 mM), nonessential amino acids (1 mM), and 10% fetal bovine serum (Hyclone, UT, USA). Both lines express the HLA-A2 allele, but only Mel 526 expresses the MART-1 antigen. The T2 cell line is a TAP-deficient T–B cell hybrid expressing the HLA-A2 allele.

KK M27 and RE M27 CTL clones were generated and maintained in our lab as previously described¹¹. These clones recognize the immunodominant M27-35 HLA-A2-restricted epitope of the melanoma-associated antigen MART-1/Melan A and can specifically lyse HLA-A2⁺ melanoma cells expressing MART-1.

Generation of Human DC from Peripheral Blood Mononuclear Cells (PBMC)

PBMC obtained by density-gradient centrifugation were resuspended in serum-free AIM-V medium (Life Technologies, Grand Island, NY, USA) and plated onto six-well plates at 10 10⁶ cells/well. After the nonadherent cells were removed in 2 h, the adherent cells were cultured in AIM-V medium with 500 U/ml human rIL-4 (R&D Systems, Minneapolis, MN, USA) and 800 U/ml human rGM-CSF (Immunex, Seattle, WA, USA). On days 2 and 4, IL-4 and GM-CSF were added. On day 6, DC underwent maturation by exposure to IL-1 at 2 ng/ml, IL-6 at 1000 U/ml, TNF- at 10 ng/ml (R&D Systems), and PGE-2 at 1 g/ml (Sigma) for an additional 2 days. The mature DC population contained more than 90% CD83⁺ DC on day 8 as determined by FACS analysis.

In Vitro Synthesis of RNA

The expression vector encoding the MART-1 antigen, pGEM4Z/MART/A64, was constructed by replacing the EGFP gene on pGEM4Z/GFP/A64¹⁶ at the XbaI and EcoRI sites with the full-length Mart-1 cDNA (gift from T. Boon) using the following PCR primers: 5'-TTATTCTAGAAGCAGTCTTCATACACG-3' and 5'-TTATCTTAAGGATTAGTACTGCTAGCG-3'.

In vitro synthesis of mRNA was carried out as described¹⁶ using 4 g of linearized plasmid containing either the EGFP or the MART-1 gene as template for in vitro transcription with the T7 promoter per the manufacturer's instructions (mMessage mMachine Kit; Ambion, Austin, TX, USA). Polyadenylation was carried out using 20 g of RNA with 500 U poly(A) polymerase (United States Biochemical, Cleveland, OH, USA). Following reprecipitation, RNA was dissolved in DEPC-treated water and polyadenylation verified on a 1% agarose gel.

Transfection of DC

RNA transfection into DC was carried out using a nonlipid cationic reagent (Transmessenger Transfection Reagent; Qiagen, Chatsworth, CA, USA). For transfection of mature DC, 25 l of Transfection buffer was mixed with 1 l of Enhancer reagent and 0.5 g of RNA in 1 l. After incubation for 5 min at room temperature, 3 l of Transmessenger Transfection Reagent was added and incubation continued for 10 min more at room temperature. Mature DC were harvested, washed twice with PBS, and resuspended in AIM-V medium, and 3 10⁵ DC in 125 l were added to each transfection mixture. Whole-cell RNA was isolated from melanoma cell line A375 as negative control RNA using the RNA Easy kit (Qiagen). After incubation for 45 min at 37°C, the DC were washed twice with PBS before use. For comparison, immature DC were harvested after 6 days of culture, and 3 10⁵ cells were plated in each well of 12-well plates and transfected with 1 g RNA as per the manufacturer's instruction.

CTL Stimulation

CD8⁺ cells were purified to >98% purity from PBMC by immunomagnetic enrichment (Dynabeads, Dynal, Oslo, Norway). DC used in CTL stimulation were either transfected with RNA or pulsed with 20 M MART-1 (M27) peptide (AAGIGILTV; Multiple Peptide Systems, San Diego, CA, USA) for 4 h. The peptide concentration of 20 M was selected because it is in the range of commonly used peptide concentrations shown to be effective in stimulating CTL responses^6,11. At this concentration, peptide-pulsed DC are maximally susceptible to lysis by M27-specific CTL and lower M27 peptide concentrations (2 and 0.2 M) fail to stimulate detectable CTL activity (our unpublished data). The CD8⁺ T cells were incubated with DC at ratio of 10:1 in AIM-V medium in the presence of 10 ng/ml IL-7. IL-2 (20 U/ml) was added on day 3 and then every 3 days. T cells were restimulated using the same conditions on day 11 and assays performed 8–10 days after the second stimulation.

T Cell Assays

In vitro cytotoxicity assay

Cytotoxicity assays were performed by standard ⁵¹Cr-release assay. Target cells (T2 or DC) were pulsed with 20 M M27 peptide in AIM-V medium for 4 h in the presence of ⁵¹Cr, washed, and added to 96-well plates at 2000 cells/well for use as target cells. Effector cells were added at an effector-to-target ratio of 10:1.

IFN- release assay

CTLs (4 10⁴) were incubated with 10⁴ DC in 200 l medium at 37°C. After 24 h, 30 l of supernatant from each well was collected and assayed for IFN- release by ELISA (Endogen).

Quantification of M27 epitope-specific CTL with tetramer and cloning of tetramer-positive cells

For tetramer staining, 5 10⁵ T cells were incubated with PE-conjugated M27 tetramer at room temperature. After 30 min, FITC-conjugated anti-CD8 antibody (Caltag) and Cychrome-conjugated anti-CD14, 19, and 56 were added for another 30 min at 4°C. The cells were then washed twice with PBS and DAPI was added. DAPI-negative; CD14-, 19-, and 56-negative; CD8 FITC⁺; tetramer-PE⁺ cells were enumerated, sorted on a Vantage FACS sorter (Becton–Dickinson, Rutherford, NJ, USA), and plated into 96-well plates for cloning as described¹¹. On day 14, cloned cells were tested for their cytotoxicity against 1000 Mel 526 cells in a ⁵¹Cr-release assay. Clones demonstrating specific lysis were expanded for subsequent analysis.

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Acknowledgements

This work was supported by an NIH training grant (X.L.), NIH R37 CA 33084, P01 CA18029 (P.D.G.), Leukemia and Lymphoma Society FND 7040-03 (P.D.G.), the Cancer Research Institute (C.B., C.Y.), the Italian Ministry of Health (C.B.), and a Damon Runyon Cancer Research/Eli Lilly Investigator Award (C.Y.). We also wish to acknowledge the technical assistance of John Zach Reilly and Justine Tarnawska.