Mature Dendritic Cells Prime Functionally Superior Melan-A-Specific CD8ⴙ Lymphocytes as Compared with Nonprofessional APC1 Mariolina Salio,* Dawn Shepherd,* P. Rod Dunbar,* Michael Palmowski,* Kristine Murphy,† Lijun Wu,† and Vincenzo Cerundolo2* Priming of melan-A26/27–35-specific CTL occurs only in a fraction of late stage melanoma patients, whereas during the early stages of the disease and in healthy volunteers, melan-A CTL have functional and phenotypic markers consistent with a naive phenotype. To study the requirements for expansion of naive melan-A CTL from healthy donors, we set up an in vitro priming protocol and, using tetramer assays, we demonstrate that the activity and phenotype of the expanded melan-A CTL are profoundly influenced by the type of APC used. Priming by nonprofessional APC leads to expansion of melan-A CTL with reduced cytolytic activity and low level of IFN-␥ secretion. In contrast, mature dendritic cells (DC) expand cytolytic and IFN-␥-producing melan-A CTL. Priming by mature DC is also efficient at low peptide concentration and requires only one round of stimulation. Finally, we observed that a significant fraction of CD45ROⴙ melan-A CTL primed by mature DC expresses high levels of the homing receptor CD62L, whereas CTL primed by nonprofessional APC express CD62L in lower percentages and at lower levels. These results suggest that suboptimal priming by nonprofessional APC could account for the presence in vivo of dysfunctional cells and strongly support the immunotherapeutic use of mature DC for expansion of effector and memory Ag-specific CTL. The Journal of Immunology, 2001, 167: 1188 –1197.
A
number of tumor Ags recognized by human CTL have been described in the past years (1). Among the known melanoma Ags, melan-A/melanoma Ag recognized by T cell 1 is probably the best studied, and it has become a useful target for immunotherapy (2, 3). The melan-A gene, expressed by normal melanocytes and most melanomas, encodes for an Ag recognized by tumor-reactive HLA-A*0201 (A2)-restricted CTL (4, 5). The development of HLA-A2 tetrameric complexes (6), refolded around the immunodominant melan-A26/27–35 epitope, has enabled ex vivo detection of tumor-specific CTL in a significant proportion of A2-positive melanoma patients and in healthy volunteers (7– 10). We have recently demonstrated that in late stage melanoma patients, melan-A-specific CTL acquire markers and activity consistent with a memory phenotype (10). In contrast, melan-A-specific CTL detectable in healthy volunteers and early stage melanoma patients (stages I and II) have a naive phenotype, as defined by their surface markers (CD28⫹, CD62L, CCR7⫹, CD45RA⫹, and CD45RO⫺) and inability to secrete IFN-␥ in ex vivo assays (8, 10). These results indicate a lack of CTL activation in early stage melanoma patients, and suggest that priming of melan-A-specific CTL is a late phenomenon, often associated with the presence of a large tumor burden.
*Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, U.K.; and †Millennium Pharmaceuticals, Cambridge, MA 02139 Received for publication March 8, 2001. Accepted for publication May 17, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by funds from the Cancer Research Campaign (U.K.) and the Cancer Research Institute (USA). 2 Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, U.K. E-mail address:
[email protected].
Copyright © 2001 by The American Association of Immunologists
In addition, some melanoma patients have tumor-specific CTL with an unusual phenotype, largely unresponsive to the cognate Ag and mitogenic stimulation, suggesting a state of anergy (9). Activated CTL with dysfunctional phenotypes have also been described in HIV (11)- and hepatitis C virus (12, 13)-infected patients. However, some patients develop specific and efficacious antitumor responses, which correlate with disappearance of the tumor, as shown by Molldrem and colleagues (14). Although several possibilities may account for the presence in vivo of Ag-specific T cells with a dysfunctional phenotype, including suboptimal priming by nonprofessional APC or lack of T cell help (15), the mechanisms are still undefined. As CTL priming occurs only in some tumor patients (10, 14) and others have anergic CTL (9), identification of the factors that might correlate with priming and may influence the expansion of functional tumor-specific CTL will have important clinical applications. Therefore, we set up an experimental system to study the requirements for priming the melan-A naive T cell population present in healthy donors’ PBMC, combining in vitro priming and tetramer analysis to detect the Ag-specific cells. Dendritic cells (DC)3 are the most potent APCs described to date, unique in their ability to efficiently prime both CD4⫹ helper and CD8⫹ cytotoxic T cell responses (16). Immature DC reside in peripheral tissues, where they exert a sentinel function for incoming pathogens. Upon encounter with Ags in the context of an inflammatory stimulus, they undergo a process of maturation that ultimately enhances severalfold their APC function and leads to their migration to the draining lymph nodes, where priming of naive T cells occurs (17). We compared the ability of professional (immature and mature DC) and nonprofessional APC (monocytes, B cells, and melanoma cells) to prime melan-A-specific naive T cells. We report that mature DC are unique in their ability to elicit
3
Abbreviation used in this paper: DC, dendritic cell. 0022-1767/01/$02.00
The Journal of Immunology both effector and memory cells, whereas nonprofessional APC expand CTL with a blunted effector function. In addition, mature DC are extremely potent in priming a naive CTL population at low antigenic dose and after one round of stimulation only. These findings have important implications for the development of vaccination strategies.
Materials and Methods Cell lines and cultures The medium used throughout was RPMI 1640 supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% pyruvate, 50 g/ml kanamycin, 5 ⫻ 10⫺5 M 2-ME (Life Technologies Laboratories, Grand Island, NY), and 10% FCS (HyClone Laboratories, Logan, UT). JY is an HLAA2⫹ lymphoblastoid cell line. Melanoma lines Na8 (HLA-A2⫹melan-A⫺), MZ2 (HLA-A2⫺melan-A⫹), and D10 (HLA-A2⫹melan-A⫹) were a gift of E. Padovan (Kantonspital, Basel, Switzerland). Recombinant human IL-2 and IL-4 were produced in our laboratory, as described (18).
Peptides and tetramers
1189 lyzed on a FACSCalibur (BD Biosciences) using CellQuest Software. Lymphocytes were gated according to forward light scatter/side light scatter profile, and dead cells were excluded by propidium iodide staining. Expression of melan-A and HLA-A2 was tested using mouse mAbs A103 (Novocastra, Newcastle, U.K.) and BB7.2 (ATCC), respectively, followed by a PE-conjugated affinity-purified goat anti-mouse Ab (Southern Biotechnology Associates). The profile of melanoma and DC was assessed by staining with the following mAbs: W6/32 (anti class I; ATCC); L243 (anti class II; ATCC); CD11a (HB202; ATCC); CD11b (HB24; ATCC); CD11c, CD83 (Immunotech, Westbrook, ME); CD80, CD86, ICAM-1 (BD PharMingen); and LFA-3 (HB205; ATCC). For intracellular cytokine detection, 106 cells were labeled with melan-A tetramer and subsequently stimulated with 20 M melan-A peptide in RPMI 1640 –10% FCS or left unstimulated (11). Control cells were either unstimulated or treated with 10⫺6 M PMA (Sigma) and 0.5 M ionomycin (Sigma). Brefeldin A (Sigma) was added at a final concentration of 5 M during the second hour of stimulation. Cells were harvested after a total of 6 h, washed, fixed, and permeabilized in FACS permeabilizing buffer (BD Biosciences), according to the manufacturer’s instructions. Staining was performed with anti-CD8-PerCP (BD Biosciences), anti-IFN-␥-FITC, and anti-IL-2-allophycocyanin (BD PharMingen).
Melan-A26 –35 ELAGIGILTV is a recently defined analogue of the 26 –35 epitope with an improved HLA-A2-binding affinity (19). Influenza matrix58 – 66 peptide GILGFVFTL (20) was used as control. Peptides were purchased from Sigma-Genosys (The Woodlands, TX) and were HPLC purified. Melan-A-HLA-A2 tetrameric complexes were synthesized, as previously described (6, 21). Briefly, the HLA-A*0201 H chain cDNA was modified by substitution of the transmembrane and cytosolic regions with a sequence encoding the biotin holoenzyme synthetase biotinylation recognition site. This modified HLA-A*0201 and 2-microglobulin were synthesized in a prokaryotic expression system (pET; R&D Systems, Minneapolis, MN), purified from bacterial inclusion bodies, and allowed to refold with the peptide by dilution. Refolded complexes were purified by fast protein liquid chromatography and biotinylated using biotin holoenzyme synthetase (Avidity, Denver, CO), then combined with PE- or allophycocyanin-labeled streptavidin (Sigma, St. Louis, MO) at a 4:1 molar ratio to form tetramers. Tetramers were checked against positive CTL clones, and background levels of staining (⬍0.02%) were defined by staining the PBMC of HLA-A2-negative healthy donors.
Cytolytic activity was assessed using a chromium release assay. JY EBV were labeled with 51Cr for 90 min at 37°C and washed twice. Labeled cells were peptide pulsed for 1 h, washed, and added (5000 cells/well) to graded numbers of effector cells. Chromium release was measured in the supernatant, which was harvested after 5 h of incubation at 37°C. Total release was determined in the presence of 5% Triton X-100. The percentage of specific lysis was calculated as follows: 100 ⫻ (experimental ⫺ spontaneous release)/(total ⫺ spontaneous release). Each value was calculated as the average of triplicates. The high background against unpulsed targets in DC-primed cultures is due to a strong reaction against FCS proteins (the DC were prepared in FCS) and is not observed when the EBV target line is grown in human serum or when an HLA-A2-negative EBV line, K562 or 221, is used as target (data not shown).
Generation and stimulation of DC
Priming of naive melan-A-specific CTL by professional APC
Blood was purchased from the U.K. National Blood Service (Bristol, U.K.) and screened for HLA-A2 expression by FACS analysis. Monocytes were purified from healthy donors’ PBMC by positive sorting using anti-CD14conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The recovered cells were ⬎99% CD14⫹, as determined by flow cytometry with the anti-CD14 Ab TIB228 (American Type Culture Collection (ATCC), Manassas, VA). DC were generated as previously described (22) by culturing monocytes in RPMI 1640 –10% FCS supplemented with 50 ng/ml GM-CSF (Leucomax; Novartis Pharmaceuticals, Basel, Switzerland) and 1000 U/ml IL-4 for 5 days. Cells (3 ⫻ 105/ml) were stimulated by addition of either 1 g/ml LPS (from Salmonella abortus equi; Sigma), 50 ng/ml TNF-␣ (R&D Systems), or CD40L-transfected J558 cells (at 1:5 ratio, provided by P. Lane, Medical Research Council Center for Immune Regulation, Birmingham, U.K.) (23).
Cytolytic activity
Results Melan-A tetramer⫹ cells can be detected in the peripheral blood of ⬃50% of HLA-A2⫹ healthy blood donors. These cells have a naive phenotype, as defined by their surface markers and their inability to secrete IFN-␥ in ex vivo assays (data not shown) (8, 10). Consistent with these findings, nonprofessional APC, such as autologous PBMC,
T cell priming APC were irradiated (3000 rad) and pulsed for 3 h with melan-A26 –35 peptide in serum-free medium. Cells were thoroughly washed and incubated with autologous PBMC at a 1:5 ratio in RPMI 1640 –5% human serum. Human rIL-2 was added from day 4 to 7 at 10 U/ml. Cells were then expanded with 500 U/ml IL-2 and analyzed at days 10 –15. In some experiments, human rIL-12 (R&D Systems) was added at the beginning of the cultures (5 ng/ml).
FACS analysis Cells were stained in PBS with PE- or allophycocyanin-labeled melan-A tetramer at 37°C for 20 min, washed at room temperature, and incubated on ice with one of the following Abs: CD8-FITC (Dako, Carpenteria, CA), CD8-allophycocyanin (BD PharMingen, San Diego, CA), CD8-PerCP (BD Biosciences, Mountain View, CA), CD27-FITC (BD PharMingen), CD45RO-FITC (BD PharMingen), CD62L-FITC (BD PharMingen). CCR7 Ab (clone 7H12; Millennium Pharmaceuticals, Cambridge, MA) (24) was used at 5 g/ml, followed by goat anti-mouse IgG2b-PE (Southern Biotechnology Associates, Birmingham, AL). The samples were ana-
FIGURE 1. Nonprofessional APC expand memory, but not naive cells. PBMC were pulsed with different concentrations of influenza matrix58 – 66 (f) or melan-A26 –35 (䡺) peptides and incubated with autologous responder PBMC at a 1:5 ratio. After 10 days, cultures were stained with the relevant tetramer PE and CD8-FITC, and percentages of tetramer⫹CD8⫹ CTL are reported in the graph. One representative experiment of three is shown; all three donors expanded melan-A CTL at 100 g/ml peptide, whereas the threshold for influenza matrix CTL expansion was 10 –100 ng/ml peptide.
1190 expand melan-A-specific CTL only when pulsed with high concentrations of melanA26 –35 peptide (100 g/ml, Fig. 1). Conversely, expansion of influenza matrix-specific CTL from a memory cell population is detectable, pulsing PBMC with as little as 100 ng/ml peptide. Expansion of melan-A-specific CTL can be significantly enhanced using monocyte-derived DC as APC. As shown in Fig. 2A, one round of stimulation with melan-A peptide-pulsed immature (Fig. 2Ab) and mature (Fig. 2Ad) DC allows expansion of a consistent population of melan-A tetramer⫹CD8⫹ T cells. No expansion is observed using unpulsed DC (Fig. 2A, a and c). In all 10 donors tested, mature DC were always more potent than immature DC in expanding melan-A tetramer⫹ CTL. In addition, LPS-matured DC could expand naive melan-A-specific T cells at very low peptide concentrations (0.08%
PRIMING OF MELAN-A-SPECIFIC CTL BY MATURE DC Tet⫹ cells with 1 ng/ml peptide), whereas immature DC required 10-fold more peptide to elicit a similar expansion (Fig. 2B). Priming of naive melan-A-specific CTL by allogeneic nonprofessional APC To address whether an appropriate cytokine milieu could improve the ability of nonprofessional APC to prime naive melan-A-specific CTL, we first investigated Ag presentation in the context of an allogeneic reaction. Several groups have suggested the use of HLA-A2-matched allogeneic melanomas for in vitro expansion of CTL lines, to be used for adoptive immunotherapy of melanoma (25–27). Initial experiments were performed with the allogeneic HLA-A2⫹ melan-A-negative melanoma line Na8, pulsed with different doses of melan-A peptide. Despite lack of expression of the costimulatory molecules required for priming naive T cells (Fig. 3A), allogeneic melanoma cells were capable of inducing a significant expansion of melan-A tetramer⫹ CTL (Fig. 3B). The ability of allogeneic cells to prime melan-A-specific CTL was not restricted to melanoma, as expansion was observed upon stimulation by peptide-pulsed HLA-A2⫹ allogeneic monocytes and, albeit less efficiently, B cells (Fig. 3B). Autologous B cells or monocytes did not induce any expansion. Further controls were performed to rule out the possibility that the expansion of melan-A-specific CTL observed upon stimulation by allogeneic cells was merely due to bystander activation. As shown in Fig. 4, the expansion was Ag and MHC specific, because it was induced only by HLA-A2⫹ and melan-A⫹ (or peptidepulsed) melanomas (Fig. 4, D–F). No expansion was induced by HLA-A2⫺ melan-A⫹ melanomas (A and B) or unpulsed HLAA2⫹ stimulators (Fig. 4C). These results demonstrate that nonprofessional APC pulsed with low concentration of peptide can efficiently and specifically prime a naive T cell population in the context of a strong allogeneic immune reaction. Phenotypic and functional analysis of melan-A-specific CTL expanded by different types of APC
FIGURE 2. Efficient expansion of melan-A-specific CTL from healthy donors’ PBMC by autologous DC. A, Immature (panels a and b) or LPSmatured (panels c and d) DC were left unpulsed (panels a and c) or pulsed with 100 ng/ml melan-A26 –35 peptide (panels b and d) and incubated with autologous PBL at a 1:5 ratio. After 10 days, cultures were stained with melan-A tetramer-PE and CD8-FITC. Percentages of melan-A⫹CD8⫹ CTL are reported in each panel. One representative experiment of five is shown. B, Immature (䡺 and E) and mature DC (f and F) of two different donors were pulsed with different concentrations of melan-A peptide and incubated with autologous PBL at a 1:5 ratio. After 10 days, cultures were stained as described in A. Among the 10 donors tested, the extent of expansion induced by immature DC pulsed with 100 ng/ml peptide varied between 0.01 and 0.5%, depending on the donor and the percentages of mature DC already present in the cultures. The range of expansion observed with mature DC at the same peptide dose was 0.2–1.5%. In all experiments, mature DC induced a greater expansion of melan-A-specific CTL than immature DC.
It is becoming clear that different priming conditions can drive the expansion of Ag-specific cells with different phenotypic and functional activities (28, 29). We therefore compared the phenotype of melan-A-specific CTL expanded from PBMC by autologous LPSmatured DC or allogeneic melanoma cells. At day 10, cultures were harvested and stained for markers of effector and memory cells (Fig. 5A). Tetramer⫹ CTL were CD27⫹ (Fig. 5A, b and f), CD45RO⫹ (Fig. 5A, c and g), CD56⫺, and mostly CD57⫺ and CD28⫹ (data not shown). Interestingly, we found that in cultures expanded by mature DC, CD62L, a marker of naive and a subset of memory T cells, was expressed in a higher percentage of cells and at higher levels (compare Fig 5A, d and h). Only a small fraction of tetramer⫹ CTL was CCR7⫹ (Fig. 5A, c and g), and most of the CD62L⫹ CTL did not express CCR7 (Fig. 5A, d and h). When the same cultures were restimulated with the melan-A peptide to assess the Ag responsiveness of the tetramer⫹ cells, we found that CTL expanded by mature DC (Fig. 5Ba) were able to produce higher levels of IFN-␥ and in a higher proportion than melanoma-primed CTL (Fig. 5Bc). The percentage of IFN-␥-producing CTL expanded by melanoma cells varied from 33% (Fig. 5Bc) to 0% (Table I). Indeed, lack of IFN-␥ production by melanoma-primed CTL was observed in ⬃50% of the donors (data not shown). Only a small fraction of tetramer⫹ cells produced IL-2 in response to Ag. Over 95% of the cells in both cultures secreted cytokines in response to PMA and ionomycin (Fig. 5B, b and d). As shown in Table I, both DC matured by LPS and by CD40LJ558L transfectants were able to expand CD62L⫹tetramer⫹ cells
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FIGURE 3. Expansion of melan-A-specific CTL from healthy donors’ PBMC by allogeneic nonprofessional APC. A, Expression of HLA, adhesion, and costimulatory molecules on LPS-mature DC (top panels) and the Na8 melanoma (bottom panels). B, Allogeneic HLA-A2⫹ monocytes (f), B cells (F), Na8 melanoma (Œ), or autologous monocytes (䡺), B cells (E), PBL (‚) were pulsed with different concentrations of melan-A peptide and incubated with responder PBL at a 1:5 ratio. After 10 days, cultures were stained with melan-A tetramer-PE and CD8-FITC, and percentages of melan-A⫹CD8⫹ CTL are reported in the graph. Melan-A expansion induced by autologous DC is shown in Fig. 2B (circles).
as well as IFN-␥-producing cells. Despite comparable maturation to LPS- and CD40L-treated DC (Fig. 6), TNF-␣-matured DC induced intermediate CTL expansion and only minimal type 1 polarization, confirming previous reports (30). Immature DC and DC exposed to J558L-mock transfectants expressed B7.2 and CD83 on 40 and 20% of the cells, respectively (Fig. 6), which could account for their high levels of priming. However, melan-A-specific CTL expanded under these conditions hardly produced any IFN-␥, most likely due to lack of polarizing cytokines in the cocultures. CTL expanded by the allogeneic melanomas Na8 and SKmel-29 did not secrete IFN-␥ in response to Ag, nor did they express high levels of CD62L, as compared with DC-primed CTL. Finally, the cytolytic activity of melan-A-specific CTL was assessed. We observed that only cells primed by mature DC were
able to kill peptide-pulsed HLA-A2⫹ target cells (Fig. 7). Control cells were influenza matrix-specific CTL expanded by virus-infected immature DC, which killed peptide-pulsed targets despite comparable low frequencies of tetramer⫹ cells to CTL primed by melanoma cells. Melanoma-primed CTL acquired cytolytic activity upon a second in vitro restimulation (data not shown), ruling out the possibility that the cells expanded by nonprofessional APC were anergic. In addition, despite lack of killing and poor IFN-␥ secretion, melanoma-primed CTL promptly down-regulated the TCR and CD8 and up-regulated CD69 (data not shown) in response to Ag. This response was comparable with that of DC-primed CTL. We conclude that mature DC can prime Ag-specific CTL with the characteristics of both memory and effector cells after only one
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PRIMING OF MELAN-A-SPECIFIC CTL BY MATURE DC
FIGURE 4. Expansion of melan-A CTL by allogeneic melanoma cells is Ag and MHC specific. Melanoma MZ2 (A and B) is HLA-A2⫺ and melan-A⫹. Melanoma Na8 (C and D) is HLA-A2⫹ and melan-A⫺. Melanoma D10 (E and F) is HLA-A2⫹ and melan-A⫹. Cells were left unpulsed (A, C, and E) or were pulsed with 100 ng/ml melan-A26 –35 peptide and incubated with healthy donor HLA-A2⫹ PBMC at a 1:5 ratio. After 10 days, cultures were stained with melan-A tetramer-PE and CD8-allophycocyanin. Percentages of melan-A⫹CD8⫹ CTL are reported in each panel. One representative experiment of three is shown.
round of stimulation. Conversely, despite efficient CTL expansion by allogeneic tumor lines, these CTL have no cytolytic activity and only limited IFN-␥ secretion capacity and require further rounds of stimulation to develop full effector activity. IL-12 improves the priming capacity of nonprofessional APC Mature DC secrete bioactive IL-12 and consequently promote type 1-polarized immune responses (30, 31). Therefore, we tested whether
exogenous IL-12 was capable of improving priming of melan-A CTL induced by peptide-pulsed nonprofessional APC. As shown in Fig. 8, and confirming data shown in Fig. 1, only a minimal expansion of melan-A-specific CTL was observed and a high peptide concentration was required (Fig. 8Aa). The expression of CD45RO on 72% of the tetramer⫹ cells was taken as evidence of priming, because the starting population was all CD45RO⫺ (data not shown). When 5 ng/ml IL-12 was added, a higher degree of expansion was observed (Fig. 8Ae),
FIGURE 5. Phenotypic and functional analysis of melan-A⫹ CTL. A, LPS-matured DC (a– d) and the melanoma Na8 (e– h) were pulsed with 100 ng/ml melanA26 –35 peptide and incubated with PBL (autologous to the DC) at a 1:5 ratio for 10 days. Cells were stained with melan-A tetramer-PE and CD8-allophycocyanin (a and e) or with melan-A tetramer-allophycocyanin and CD8-PerCP (data not shown). Melan-A⫹CD8⫹ CTL were gated, and expression of the following markers was analyzed: CD27-FITC (b and f), CD45RO-FITC and CCR7-PE (c and g), CD62L-FITC and CCR7-PE (d and h). Percentages of positive cells are reported in the relevant quadrants. Quadrants are set according to the marker distribution in the ungated population (data not shown), and therefore differ for each marker. B, The same cells were stimulated with melan-A peptide (a and c) or with PMA and ionomycin (b and d), as described in Materials and Methods. Intracellular staining for IFN-␥-FITC and IL-2-allophycocyanin on tetramer⫹CD8⫹ cells is shown. Cells in a and b were primed with LPS DC; in c and d, with Na8 melanoma. One representative experiment of six is shown.
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Table I. Phenotypic and functional analysis of melan-A-specific CTL expanded with different types of APCa
Stimulus
Unstimulated PBL Immature DC LPS-DC TNF-␣-DC CD40L-DC J558L-DC Na8 melanoma SKmel-29
% Melan-A⫹ % Tet⫹CD62L⫹ CD8⫹ (MFI)
0.01 0.48 0.68 0.23 1.8 0.5 0.68 0.57
100 (137) 41 (130) 35 (141) 23 (87) 30 (79) 20 (56) 12 (46) 6 (50)
% Tet⫹IFN-␥⫹ (MFI)
0 9 (127) 29 (135) 13 (48) 31 (133) 6 (42) 4 (49) 0
a DC were matured for 36 h with one of the following stimuli: LPS (1 g/ml), TNF-␣ (50 ng/ml), J558L-CD40L, or J558L mock control at a 1:5 ratio. Autologous immature DC, mature DC, or allogeneic melanoma cells were pulsed with 100 ng/ml of melan-A26 –35 peptide, irradiated, and incubated with PBL at a 1:5 ratio. After 16 days, cultures were stained with melan-A tetramer-PE, CD62L-FITC, and CD8-allophycocyanin; percentages of tetramer-positive CTL and distribution of CD62L are reported. On the same day, part of the cultures were tetramer labeled and stimulated with 10 g/ml melan-A26 –35 peptide to detect IFN-␥-producing cells, as described in Materials and Methods. Percentages of tetramer-positive IFN-␥-producing cells are reported. MFI, Mean fluorescence intensity.
together with a 10-fold lower threshold for priming, in each of three donors tested (data not shown). Percentages of melan-A tetramer⫹CD62L⫹ cells were comparable in the presence or absence of exogenous IL-12 (data not shown). Melan-Aspecific CTL from IL-12-treated cultures acquired the capacity to secrete high levels of IFN-␥ in response to a subsequent stimulation with the peptide (Fig. 8Af) and PMA and ionomycin
FIGURE 6. Expression of B7.2 and CD83 on DC used for T cell priming. Immature DC or cells treated for 36 h with LPS, TNF-␣, J558LCD40L transfectant, and J558L-control were stained for B7.2 (CD86, left panels, solid lines) or CD83 expression (right panels, solid lines). The profile of the negative control is overlaid on each histogram (dotted lines). The same cells were used for the experiment shown in Table I.
(Fig. 8Ag), whereas CTL from untreated cultures were unresponsive to both stimuli (Fig. 8A, b and c). Both cultures were equally able to down-regulate TCR and CD8 and up-regulate CD69 in response to Ag (data not shown). We conclude that the cytokine milieu provided by mature DC, and partially mimicked by the addition of IL-12, is fundamental in priming CTL precursors to become fully functional.
Discussion We have set up an in vitro priming system, followed by tetramer analysis to study the requirements for optimal expansion and activation of Ag-specific CTL from naive precursors, and to assess whether suboptimal priming events may result in cells with dysfunctional or anergic phenotypes. These issues are of great importance, because recent reports have described the presence of cells with blunted functions in HIV (11), hepatitis C virus (12, 13), and in some melanoma patients (9). In addition, in our cohort of melanoma patients, we have shown that tumor-specific responses appear too late to be able to control disease (10). Therefore, successful immunotherapeutic strategies should not only expand tumor-specific CTL in substantial numbers but should also ensure that such cells are functional in vivo. Our in vitro priming model is based on the expansion of melanA-specific CTL from healthy donors, as we have recently shown that a large proportion of HLA-A2⫹ individuals have detectable melan-A precursors with phenotypic and functional markers of naive cells (8, 10). The reason for such an elevated precursor frequency for melan-A26/27–35-specific CTL in the general population is currently unknown. It has been suggested that these cells could be cross-reactive, having been originally primed against a microbial epitope (32). Although this possibility cannot be ruled out, we find it unlikely because these cells are phenotypically and functionally naive (8, 10). In this study, we show that, unlike the expansion of memory recall CTL, priming of naive melan-A-specific CTL by nonprofessional APC requires high doses of peptide (Fig. 1). Naive
FIGURE 7. Cytolytic activity of melan-A⫹ CTL. Cultures expanded by LPS-matured DC (squares) or Na8 melanoma cells (triangles) were tested at day 20 in a 51Cr release assay against JY, an HLA-A2⫹ EBV line, pulsed with 10 g/ml melan-A peptide (filled symbols), or unpulsed (open symbols). As controls, influenza matrix58 – 66-specific CTL were expanded from the same donor by immature DC virus infected and tested against JY EBV pulsed with 10 g/ml of the relevant peptide (F) or unpulsed (E). The percentages of tetramer⫹CD8⫹ cells in each culture at the time of the assay are shown in brackets. One experiment representative of three is shown.
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PRIMING OF MELAN-A-SPECIFIC CTL BY MATURE DC
FIGURE 8. IL-12 improves the priming capacity of nonprofessional APC. Melan-A⫹ CTL were expanded by PBMC pulsed with 100 g/ml peptide in the presence (e– h) or absence (a– d) of 5 ng/ml IL-12. After 10 days, cells were stimulated with melan-A peptide (b, and f) or with PMA and ionomycin (c, d, g, and h), as described in Materials and Methods. Intracellular staining for IFN-␥-FITC and IL-2-allophycocyanin on tetramer⫹CD8⫹ cells (b, c, f, and g) or on the whole population (d and h) is shown. The lower level of both tetramer and CD8 expression (a and e) is a consequence of cell activation.
melan-A precursors can be efficiently primed by low doses of Ag only when DC are used as APC (Fig. 2). LPS-matured DC prime CTL when pulsed with concentrations of melan-A peptide as low as 1 ng/ml, whereas the threshold for expansion by nonprofessional APC is 10 –100 g/ml. Mature DC are more potent than immature DC in expanding melan-A-specific CTL (Fig. 2). Consistent with previous reports (30), different maturation stimuli show different potencies (Table I), with CD40L being the strongest and TNF-␣ the weakest. CTL primed by LPS- or CD40L-matured DC produce high levels of IFN-␥ upon Ag restimulation (Fig. 5 and Table I), consistent with the strong type 1-polarizing capacity of mature DC, due to type I IFN and bioactive IL-12 secretion (30, 31). Conversely, melan-A-specific CTL expanded by nonprofessional APC are largely unpolarized and unable to secrete cytokines in response to both Ag and mitogen (Fig. 8). However, these cells are peptide specific because they are able to down-regulate TCR and coreceptor expression and to up-regulate CD69 expression in response to Ag stimulation (data not shown). The lack of cytokine production may therefore be due to priming in an unpolarized milieu (33). These findings should be taken into account when designing vaccination protocols based upon injection of peptides. Peptide vaccination trials have in some cases shown increased levels of peptide-specific CTL in the peripheral blood; however, responses have been partial and transient (34 –36). In an attempt to improve CTL priming by nonprofessional APC, we studied the effect of IL-12, as mature DC secrete bioactive IL-12 (31). In addition, it has been reported that IL-12, administered at appropriate dose and schedule, might be useful in promoting more effective tumor Ag-specific T cell responses, by driving a type 1 polarization (37–39). Addition of IL-12 to nonprofessional APC during the priming phase resulted in a greater expansion of tetramer⫹ CTL and in cells able to secrete IFN-␥ in response to the melan-A peptide (Fig. 8). These results confirm the strong immunomodulatory activity of rIL-12 (40) and suggest that an appropriate cytokine milieu allows priming of naive CTL by nonprofessional APC.
Along this line, we investigated whether Ag presentation in the context of an allogeneic reaction could lead to CTL priming by nonprofessional APC. Presumably, the high frequency of responding alloreactive T cells (41) provides the stimulatory cytokine milieu necessary for priming a naive population. Indeed, to obviate the need for generating tailored autologous tumor lines, the use of allogeneic HLA-matched melanoma lines has been proposed for the in vitro expansion of CTL for adoptive immunotherapy (25, 26), and several clinical trials have investigated the use of allogeneic vaccines (27). We showed that allogeneic HLA-A2⫹-matched cells are capable of efficiently expanding melan-A-specific CTL in an Ag-specific reaction (Figs. 3 and 4). It remains unclear whether secretion of soluble factors during the mixed lymphocyte reaction may entirely account for the priming of naive melan-A CTL in this experimental system. However, despite efficient melan-A-specific CTL expansion with allogeneic tumor lines, our results show that these cells have no cytolytic activity and only a limited capacity to secrete IFN-␥, requiring further rounds of stimulation to develop full effector activity (Figs. 5 and 7, and data not shown). These results suggest that if priming by nonprofessional APC occurred in melanoma patients, the outcome could be a CTL population with blunted effector functions, as indeed has been reported by Lee and colleagues (9). The recent report that allogeneic bone marrow transplantation in chronic myelogenous leukemia patients induces efficacious antitumor responses does not contradict our findings, as all recipients followed in that study were 100% donor chimeras and one could envision continuous restimulation of the donor’s cells by the host (14). To improve cytolytic activity and IFN-␥ production of tumorprimed CTL, one could envision inclusion of immunostimulatory cytokines in the priming phase (42, 43). However, in preliminary experiments, addition of exogenous IL-12 to allogeneic cells resulted in a dramatic inhibition of the expansion of melan-A tetramer⫹ CTL (M. Salio, unpublished results). This observation is consistent with reports
The Journal of Immunology in the literature describing a toxic effect of high doses of IL-12 and suppression of allogeneic responses (44, 45). Upon Ag encounter, CD8⫹ T lymphocytes can differentiate to become cytotoxic T cells, capable of killing virus-infected or transformed cells. Some T cells generated during the primary immune response can survive for years as memory cells and will provide a rapid first defense upon reencounter with the Ag (46). Recent data suggest that DC control T cell responses along a linear differentiation pathway, depending on the duration of TCR signaling and cytokine stimulation (47). Memory and effector cells can be differentiated according to surface marker expression, although unequivocal classifications do not exist. Hamann et al. (48, 49) proposed a model in which naive T cells (CD45RA⫹CD45RO⫺ CD27⫹CD28⫹CD62L⫹) will progressively mature into terminally differentiated effectors (CD45RA⫹CD27⫺CD28⫺) through a memory intermediate pool (CD45RO⫹CD27⫹CD28⫹). More recently, two subsets of memory cells have been identified on the basis of expression of CCR7, a chemokine receptor that controls homing to secondary lymphoid organs (50). A first subset, called central memory, expresses CCR7 and CD62L and lacks immediate effector function, which is a characteristic of the effector memory subset, CCR7⫺CD62L⫺ (51). Using mature DC in our in vitro priming system, we obtain Ag-specific lymphocytes with markers characteristic of memory cells early in their differentiation pathway (CD45RO⫹CD27⫹ CD28⫹/⫺), able to secrete IFN-␥ upon Ag stimulation and endowed with cytotoxic activity. In addition, mature DC are able to prime a subpopulation of tetramer⫹ CTL that express CD62L, but only in a small percentage CCR7, differing from the central memory cells described by Sallusto et al. (51). CD62L⫹CCR7⫺ CTL specific for influenza matrix protein or CMV can also be expanded by mature DC from a memory population (M. Salio, unpublished results). In addition, melan-A-specific CTL with a similar phenotype (CD45RO⫹CD62L⫹CCR7⫺) are detectable ex vivo in melanoma patients’ PBMC (P. R. Dunbar, unpublished results). Indeed, simultaneous staining of PBMC with CD45RO, CD62L, CCR7, and CD27 reveals a high heterogeneity in the distribution of these markers (data not shown) (24), rendering difficult an unequivocal classification of the different memory populations. Secretion of IL-12 and type I IFN by mature DC could maintain CD62L expression by our in vitro primed cells, as previously reported (52, 53). However, we did not observe any difference in the percentage of tetramer⫹CD62L⫹ cells upon addition of IL-12 (data not shown). Alternatively, DC may selectively modulate the activity of the metalloproteinase responsible for CD62L shedding upon activation (54). The reason for the different level of expression of CD62L and CCR7 on the tetramer⫹ cells remains unclear. The L-selectin adhesion molecule (CD62L) plays a primary role in mediating the initial interaction of leukocytes with the endothelium of the high endothelial venules (55). This step is followed by CCR7-induced integrin activation and firm arrest at sites of extravasation (56). As high levels of chemokine receptor occupancy are required for integrin activation (57), the CD62L⫹CCR7⫺ in vitro primed melanA-specific CTL may not be able to home to the lymph node, but may rather express other chemokine receptors and be recruited to sites of inflammation (58). Alternatively, these cells could express the recently described CCR10 receptor, which binds to the same CCR7 ligands, EBV-induced molecule 1 ligand chemokine and secondary lymphoid tissue chemokine (59). In addition, they may represent recently activated cells that have not yet regained the ability to traffic through lymphoid organs, as suggested by Campbell and colleagues (24).
1195 Great progress has been achieved in the tumor immunology field with the identification of T and B cell responses directed against tumor-specific antigenic proteins (1, 60), and immunotherapeutic strategies are now focused on how to optimally activate antitumor CTL, without inducing anergy or cell death. Given the central role of DC in the initiation of immune responses, there has been considerable interest in their adjuvant properties to prime and boost antitumor and antiviral responses (61, 62). Pilot DC vaccination trials have shown induction of antitumor responses and some clinical benefit (63– 66). It has become clear that the immunostimulatory properties of the DC are strictly dependent on their maturation state. Ag-loaded mature DC enhance CD4 and CD8 Ag-specific T cell responses, both in vivo and in vitro (67–70). Conversely, injection of immature DC inhibits immune responses and induces IL-10-producing regulatory T cells (71–73). In addition, the ability of DC matured with different stimuli and for different length of time to regulate T cell differentiation and tissue-specific homing, further underlines their plasticity and makes them a versatile immunotherapeutic tool (29, 30). Recently, two groups have shown cross-priming of naive CD8 T cells against melanoma Ags using DC loaded with killed melanoma cells, a process that required several rounds of stimulation (74, 75). By tetramer analysis, we have for the first time reported a thorough quantification of the priming efficacy of DC as compared with nonprofessional APC. Our findings that mature DC efficiently expand after only one round of stimulation a CTL population with effector and memory functions have important implications for the development of vaccination strategies and support the use of Ag-loaded mature DC in human trials.
Acknowledgments We thank Hal Drakesmith, Mark M. Davis, and Andrew McMichael for reading the manuscript and helpful discussions.
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